Report from the 26th Meeting on Toxinology, "Bioengineering of Toxins", Organized by the French Society of Toxinology (SFET) and Held in Paris, France, 4-5 December 2019.

This 26th edition of the annual Meeting on Toxinology (RT26) of the SFET (http://sfet.asso.fr/international) was held at the Institut Pasteur of Paris on 4-5 December 2019 [...].


Preface
This 26th edition of the annual Meeting on Toxinology (RT26) of the SFET (http://sfet.asso.fr/ international) was held at the Institut Pasteur of Paris on 4-5 December 2019. The central theme selected for this meeting, "Bioengineering of Toxins", gave rise to two thematic sessions: one on animal and plant toxins (one of our "core" themes), and a second one on bacterial toxins in honour of Dr. Michel R. Popoff (Institut Pasteur, Paris, France), both sessions being aimed at emphasizing the latest findings on their respective topics. Nine speakers from eight countries (Belgium, Denmark, France, Germany, Russia, Singapore, the United Kingdom, and the United States of America) were invited as international experts to present their work, and other researchers and students presented theirs through 23 shorter lectures and 27 posters. Of the~80 participants who registered,~40% were foreigners (Algeria, Belgium, Denmark, France, Germany, Italy, the Netherlands, Russia, Singapore, the United Kingdom, and the United States of America), thereby highlighting the international attractiveness of the SFET meetings. For this RT26, the SFET aimed to ensure a fair balance between participants interested in toxins from the animal/plant versus bacterial kingdoms.
Owing to a donation from MDPI Toxins, two prizes of 250 EUR each were awarded to the best oral communication and the best poster, both selected by a jury of 10 persons. Various useful or amusing goodies, generously offered by our sponsors, were distributed to all presenters.
Last but not least, we warmly thank the Editors of MDPI Toxins for permitting the publication of a Special Issue focused on the "Bioengineering of Toxins" and gathering this meeting report, along with peer-reviewed original articles and reviews. We hope that this Special Issue will be attractive to all, including those colleagues who could not attend the RT26 meeting, and that it will represent a comprehensive source of information for researchers and students in the field of Toxinology. Abstract: Voltage-gated sodium (NaV) channels play crucial roles in a range of (patho)physiological processes. Much interest has arisen within the pharmaceutical industry in pursuing these channels as analgesic targets following the overwhelming evidence that the NaV channel subtypes NaV1.7-NaV1.9 are involved in nociception. More recently, NaV1.1, NaV1.3 and NaV1. 6 have also been identified as involved in pain pathways. Venom-derived, disulfide-rich peptide toxins, isolated from spiders and cone snails, have been used extensively as probes to investigate these channels and have attracted much interest as drug leads for pharmaceutical development. However, few peptide drug leads have made it as drugs, due to their unfavourable physiochemical attributes including being poor in vivo pharmacokinetics, and having rapid proteolytic cleavage and limited oral bioavailability. The present work aims to bridge the gap in the development pipeline between drug leads and drugs candidates by downsizing these larger venom-derived NaV inhibitors into smaller, more "drug-like" molecules. As a start, a 13 amino acid, voltage-gated, sodium (NaV) channel inhibitor peptide, Pn, containing two disulfide bridges, was designed using a chimeric approach. This approach was based on a common pharmacophore deduced from the sequence and secondary structural homology of two NaV inhibitors: Conus kinoshitai toxin KIIIA, a 14 residue cone snail peptide with three disulfide bonds, and Phoneutria nigriventer toxin 1, a 78 residue spider toxin with seven disulfide bonds. As with the parent peptides, this novel NaV channel inhibitor was active on NaV1.2. Through the generation of three series of peptide mutants, we investigated the role of key residues and cyclization, and their influence on NaV inhibition and subtype selectivity. Cyclic PnCS1, a ten-residue peptide cyclized via a disulfide Toxins 2020, 12, 31 6 of 30 bond, exhibited increased inhibitory activity toward therapeutically relevant NaV channel subtypes, including NaV1.7 and NaV1.9, while displaying remarkable serum stability. Using sophisticated peptide engineering of small cyclic peptide design to aid in the determination of what drives the subtype selectivity and molecular interactions of these downsized inhibitors across NaV subtypes, we designed a series of small, stable and novel NaV probes based on PnCS1. These analogous display interesting subtype selectivity and potency in vitro, coupled with exciting in vivo analgesic activity, rendering these peptides potential analgesic drug candidates. Furthermore, we show that our design strategy can also be used to design inhibitors of voltage-gated calcium channels. These peptides represent the smallest cyclic peptidic ion channel modulators to date and are promising templates for the development of toxin-based therapeutic agents. Abstract: Nicotinic acetylcholine receptors (nAChRs) are targeted by a number of toxins. The best known are α-neurotoxins and α-conotoxins, from the Elapidae snakes and Conus mollusks, respectively. However, the multiplicity of nAChR subtypes requires the discovery of new subtype-specific ligands, and very often these compounds are present in animal venoms in extremely low amounts, insufficient for extensive study of biological activity. Larger quantities can be prepared by peptide synthesis or heterologous expression in bacteria. Our studies on the biological activity of scorpion venoms revealed their anticholinergic activity, for which the already-known toxins OSK-1 from Orthochirus scrobiculosus, spinoxin from Heterometrus spinifer and HelaTx1 from H. laoticus were responsible. All of them are blockers of voltage-gated potassium channels. For detailed biological activity studies, the toxins were prepared either by peptide synthesis (spinoxin and HelaTx1) or by heterologous expression in Escherichia coli (charybdotoxin, hongotoxin-1, kaliotoxin-1 and agitoxin-2). Investigation of these toxins revealed their micromolar and sub-micromolar affinities towards muscle-type Torpedo nAChR. The most active compounds (OSK-1 and spinoxin), in competition with α-bungarotoxin, showed IC 50 of about 0.5 µM. Similar blocking efficacy was revealed in the functional test on mouse muscle-type nAChR, expressed in Xenopus oocytes. The affinity of all tested scorpion toxins to the human neuronal α7 receptor was significantly lower. While scorpion toxins and conotoxins possessing several disulfides require the correct closure of disulfide bonds after synthesis, a linear peptide azemiopsin from Azemiops feae venom is much easier to synthesize. The synthetic azemiopsin efficiently competed with α-bungarotoxin for binding to the Torpedo muscle-type nAChR (IC 50 = 0.18 µM) and with lower efficiency to the human neuronal α7 nAChR (IC 50 = 22 µM). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing the human muscle-type nAChR, and was more potent against the adult, ε-subunit-containing form (EC 50 = 0.44 µM) than the fetal, γ-subunit-containing form (EC 50 = 1.56 µM). There are numerous data about the presence of transcripts for three-finger toxins in the venom glands of Viperidae snakes. However, there are no data about the putative biological activity of viper three-finger toxins. By heterologous expression in E. coli we prepared two toxins, TFT-AF and TFT-VN, the amino acid sequences of which were deduced from cDNA sequences cloned from venom glands of the vipers A. feae and Vipera nikolskii, respectively. The study of their biological activity showed that the viper three-finger toxins are antagonists of neuronal as well as muscle-type nAChRs.

Abstract:
The contributions of post-translational modifications to the folding and activity of proteins are still poorly understood. The adenylate cyclase toxin, CyaA, is a major virulence factor of Bordetella pertussis, the causative agent of whooping cough, and plays an essential role in the early stages of respiratory tract colonization. CyaA is produced as an inactive pro-toxin, which is post-translationally acylated in the bacterial cytosol to yield the active CyaA toxin, able to intoxicate and impair the physiology of immune cells. However, the relationships between post-translational modification and the folding and cytotoxic activities of CyaA remain elusive. Here, using a combination of biophysical approaches, including SEC-SAXS, HDX-MS and SR-CD, we show that calcium-induced disorder-to-order transitions and acylation are involved in CyaA secretion and folding into a compact and functional state. Our data sheds light on the complex relationship between post-translational modifications, structural disorder and protein folding. We propose a refolding model that is dependent on calcium and driven by local and distal acylation-dependent interactions within CyaA. Coupling calcium-binding and acylation-driven folding is likely also pertinent for other toxins produced by many Gram-negative bacterial pathogens. Abstract: All venom proteins evolved from physiological proteins; however, the mechanisms of that transition are not known. The canonical way of thinking about this matter relies heavily on gene duplication to provide the relaxed selection constraints that are deemed necessary for the gene to acquire a new function. However, several recent studies have challenged that paradigm, providing examples of single gene recruitment. In our research, we looked into some of the major toxin families of reptiles and discovered that all of them evolve from families with widespread copy number variation.
However, the transition from physiological gene to venom gene seems to happen via change of function followed by duplication, not the other way around as is commonly assumed. It is unlikely that all venom genes in all the lineages of venomous animals have evolved that way, but it is abundantly clear that the currently established paradigms cannot satisfyingly fit these results, necessitating the creation of more sophisticated models of gene evolution, as well as prompting new research agendas to validate them. Abstract: The pentameric ACh-binding proteins (AChBP) from water snails are soluble structural and pharmacological surrogates of the extracellular, ligand-binding domain of nicotinic ACh receptors (nAChRs) [1]. As such, they offer pertinent models for studying the modes of binding of nicotinic effectors and the associated conformational changes, and correlate them with the functional alteration of the nAChR channel. The crystal structures of AChBP complexes revealed that nicotinic agonists and competitive antagonists bind primarily within a nest of aromatic side chains, contributed by loops C and F, which are located on opposing faces of each subunit interface, and induce a range of loop C conformations that modulate the size and shape of the binding pocket [2][3][4]. The macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Binding and voltage-clamp recordings on muscle-type and neuronal nAChRs revealed subnanomolar affinities dictated by slow dissociation, potent antagonism, and varying levels of nAChR subtype selectivity. The crystal structures of the complexes showed that common AChBP determinants imbedded within the aromatic nest confer high-affinity binding to the toxins, while the distinctive determinants brought about by loop F and located within the nest, or extending outside the nest towards apical, radial or 'membrane' subsites of the interface, dictate either broad or narrow nAChR subtype selectivity by the toxins [5−7]. Based on these data, new organic compounds aimed at pinpointing the minimal chemical motif that dictates antagonism were designed, synthetized and analyzed relative to nAChRs and AChBP [8]. Structural analysis of the complexes showed that the spiroimine core common to these compounds is the major component of their mode of binding, while the surrounding substituents are involved in nAChR subtype specificity. These data identify distinctive ligands, functional determinants and binding sites for the design of new drugs targeting disease-associated nAChR subtypes.
Keywords: AChBP; antagonism; macrocylic imine toxin; nicotinic ACh receptor; structurefunction relationships Abstract: Blood vessel cells express voltage-gated Na + channels (Nav channels) and their activation induces a Ca 2+ response mediated by Na + /Ca 2+ exchangers (NCX) in Ca 2+ entry mode. Nevertheless, the physiological role of Nav channels in vascular tissue is still controversial. The aim of our study was to identify the Nav channel subtypes in resistance artery and to define their contribution to the regulation of their vasomotricity by physiological and pharmacological approaches. To this end, we used mesenteric arteries (MA), as a suitable model of resistance artery from 5-month-old mice (C57Bl6/J, male and female). Our RT-qPCR data showed the expression of three transcripts encoding Nav1.2 (scn2a), Nav1.3 (scn3a) and Nav1.5 (scn5a) in MA. Presence of Nav channels in these arteries was confirmed by histoimmunostaining. Surprisingly, the activation of Nav channel by veratridine (VTD) induced the vasorelaxation of MA, monitored by wire myography. This VTD-induced vasorelaxation was totally abolished by tetrodotoxine (300 µM), L-NNA (a NO synthase inhibitor), indicating that the activation of TTX-sensitive Nav channels mediates the stimulation of the eNO-synthase (eNOS). Next, we investigated NCX's implication in this pathway. We established the gene expression profile of NCX in murine MA by RT-qPCR, revealing the detection slc8a1 and slc8a2, encoding NCX1 and NCX2.
In presence of the NCX inhibitor, KB-R7943, the relaxation induced by VTD was almost abolished. Altogether, our data highlight for the first time the role of Nav channels in the vasorelaxation response in murine MA. The activation of Nav channels induces Na + entry and the subsequent membrane depolarization, which both trigger Ca 2+ entry through NCX. This possible Nav channels-NCX-NOS cross-talk reflects the link between Na + and Ca 2+ homeostasis in vascular cells. Abstract: Cryptophycin-1 is a toxin naturally produced by filamentous cyanobacteria as a secondary metabolite. Its toxicity is due to tubulin inhibitor effects and to the cell cycle stop in the G2/M phase. Thus, it was examined as novel anticancer agent and it reached clinical phase II in 2002. However, the total synthesis of cryptophycin faces stereoselectivity issues and its yield is only moderate (<13%), and unsuitable for industrial production 1 . These considerations provide a rationale to investigate alternative solutions, like stressing the environmental conditions of the culture (ATCC53789) in order to find the optimal condition for metabolite over-production (more than 0.52 mg/L of culture) 2 . Here, we examine the effect of light photoperiod, light wavelength, light intensity and media composition.
To examine the effect of light photoperiod, we tested constant and partial light (24:0, 16:8 Light:Dark), while, for light wavelength, color films were applied to test the effect of red and blue light. Low, medium and high light intensity experiments were performed in order to test the effect of photoinhibition.
To look at medium composition, the nitrogen concentration was examined to test the implication of heterocysts, a N-fixing cell type. The culture was cultivated in an incubator at 25 • C and 120 rpm. For a 10 day period, the biomass of a 20 mL sample was extracted every second day, and then metabolite concentration was calculated through HPLC. Lastly, the "growth/time" and "metabolite concentration/time" curves were drawn. Remarkably, photoperiod has an opposite effect on the two curves. Regarding growth, the optimal period was 16:8 > 24:0, however, the inverse occurred for cryptophycin production. It is shown that even if the presence of non-light is needed for cell growth, metabolite production is higher under constant light conditions. Regarding the nitrogen concentration, and therefore the implication of heterocysts, it was noticed that they were not involved in the depsipeptide expression process. Moreover, the differentiation of light wavelength has a major effect on the culture, as the red filter contributed to a noticeable increase in culture growth. At last, the microorganism prefers medium light intensities for both growth and metabolite expression, as 80 µE was the optimal. These stress-test results contribute to the formation of the optimal culture conditions for metabolite overproduction, which would further the research and development of a novel potential anticancer drug. Abstract: A few animals have evolved molecular resistance to alpha-neurotoxins, most famously the mongoose and cobra. The resistance consists of one or two base-pair changes in the ligand-binding of the nicotinic acetylcholine receptor (nAChR) with possible posttranslational modification of the receptor protein. This leads to reduced binding of snake alpha neurotoxins to the receptor without a major effect on the physiological binding of acetylcholine. We have sequenced the ligand-binding domain of the acetylcholine in a wide range of vertebrate taxa and looked for evidence of resistance-related changes in the coding sequence. We find that several snake lineages and lizards, but not birds, have evolved such changes. The lack of resistance-related changes in birds is surprising, given the prevalence of ophiophagy in the species examined. Functional studies in embryos confirmed that the chicken is six times more susceptible to cobra venom than Pogona vitticeps, which is putatively resistant, based on our sequencing of its nAChR. Bioinformatics analysis showed evidence that putative resistance-related sites in the nAChR are under positive selection.

Abstract: Clostridium perfringens causes a broad spectrum of diseases in animals and humans.
Depending on the production of four major toxins, it is classified into five toxinotypes, A-E. The most important of the 12 minor toxins produced is the C. perfringens enterotoxin (CPE), the second largest cause of most cases of bacterial food-borne illnesses and antibiotic-associated diarrhea. This makes CPE an important analyte in clinical differential diagnostics. In contrast, CPE is also explored as a potential anticancer agent. CPE is a 35 kDa β, pore-forming toxin classified into the aerolysin family, which oligomerizes upon receptor recognition and subsequently forms cation-selective pores in the membrane of epithelial cells, thereby inducing cell death. Here, we aim at designing a capture structure specific for functionally active CPE, which will be implemented into a rapid detection system. The C-terminal 15 kDa domain of the CPE constitutes the receptor-binding domain (CPE-RBD), which recognizes claudins (CLDN), a family of 20-27 kDa tetraspanin proteins forming tight junctions between epithelial cells. Robust binding of CPE to CLDN-3 and-4 and weaker binding to CLDN-1,-6,-7,-8,-9,-14 and-19 has been reported. We explored CLDN-1,-3,-4 and-19 as CPE capture structure candidates. Various fusion proteins and truncation mutants of CLDNs were recombinantly expressed in eukaryotic cell lines and E. coli. The isolated 15 kDa CPE-RBD served as prey or bait in various pull-down assays and co-immunoprecipitations with CLDN mutants. We identified CLDN-4 among CLDN-1,-3,-4 and-19 as the best binder and, in contrast to previous reports, determined full-length CLDN-4 as being essential for high-affinity binding to CPE-RBD, as well as full-length recombinant CPE. Various CLDN-4 constructs were explored and tested for expression and isolation to obtain a soluble, pure and functionally folded CLDN-4. The binding kinetics of CPE-RBD to CLDN mutants were determined by SPR experiments. The optimization of detergent allowed its spotting on a gold-chip to serve in a rapid detection system. The optimal CLDN-4 capture structure, comprising four transmembrane domains, was isolated in acceptable yield and good purity and displayed sub-nanomolar binding affinity constants towards CPE. Currently, this structure is being implemented into a rapid detection system. Abstract: Malaria remains a major concern for health organizations around the world. In 2017, the World Health Organization reported more than 219 million cases and 435,000 deaths. With 87 countries affected, more than 800 million people are at risk of infection. The emergence and transmission of resistances to most antimalarial drugs is a real worry. Thus, the need for new therapeutic candidates is an absolute necessity [1]. In recent years, animal venoms and secretions have sparked a growing interest in scientists. In fact, toad venoms constitute a rich source of molecules, mainly bufadienolides, with many potential therapeutic activities [2]. The objective of this on-going project is to develop a bio-guided fractionation process and the subsequent discovery of new drug candidates against malaria from toad venom. Raw extract characterization: Multiple Bufo species will be considered during this work. Up to now, two species have been studied: Rhinella marina and Bufo bufo. The extraction process from the air-dried gland secretions of the Bufo toads consists of an ultrasonication-assisted solvent extraction. Two solvents have been tested: methanol and acetonitrile. The venom composition is subject to variability between batches depending on the animal's habitat and its diet. After each extraction, the raw extracts are analyzed by TLC and LC-MS to provide an overview of the compounds present in the sample. Fractionation process: during this step, flash chromatography is considered as the first approach, to obtain rough fractions that will also be analyzed by TLC and LC-MS and then biologically studied. Flash chromatography offers a fast and simple separation process that can be applied to complex natural products. In the first fractionation round, three to four fractions are obtained. The following step will consist of producing subfractions of the fractions displaying interesting therapeutic properties. For this purpose, further preparative techniques will be considered such as flash chromatography and semi-preparative HPLC. Biological activity: each raw extract and the subsequently obtained fractions are tested for their antiplasmodial activity (3D7 and W2 strains) using the pLDH assay and microscopic evaluation. Their cytotoxicities are also assessed on a panel of human cell lines. A parallel project aims to evaluate the effect of the above-mentioned extracts and fractions on several human melanoma cell lines that have developed a resistance to targeted therapies. The samples that display antiplasmodial activities and/or cytotoxic activities against melanoma cells will be further analyzed (LC-MS) and structurally characterized by NMR analysis ( 1 H-NMR, 13 C-NMR, COSY).

Keywords
Keywords: antiplasmodial; toad; venom Abstract: Scorpion venom is known to cause the liberation of neurotransmitters and the release of several inflammatory mediators, like cytokines, eicosanoids, nitric oxide and reactive oxygen species (ROS). These latter are important contributors to heart failure and disease. However, little is known about how circadian rhythms, or rhythm desynchrony, are involved in these key pathologic stress responses. The aim of this study is to investigate the role of the circadian rhythm on the cardiac oxidative stress pathways, and indicate how free radical biology coincides with the pathogenesis of the cardiovascular system. Twelve NMRI mice were envenomed with a sublethal dose of Androctonus australis hector venom (Aah) (0.75 mg/kg, s.c) during light (at 1 HALO, n = 6) and dark phases (at 18 HALO, n = 6) in order to investigate the circadian variations in pro-oxidant parameters, antioxidant defenses and lipid peroxidation. Evaluation of the myeloperoxidase activity as a quantitative assessment of neutrophil infiltration, vascular permeability, as well as a histopathological analysis of cardiac tissue, was also performed in the two groups (1 HALO and 18 HALO). Higher levels of nitrite (p < 0.0001), hydrogen peroxide (p < 0.0001) and lipid peroxidation (p < 0.0001) were detected in evening-excised hearts, associated with a lower (p < 0.05) myeloperoxidase activity. For the antioxidant defenses, the catalase activity increased during the light phase, while depletion in GSH concentration was observed at the dark phase. Moreover, a greater extravasation of Evans blue (p < 0.01) was detected in the myocardial homogenates of the dark phase group, as compared to light phase hearts. The histopathological alterations were similar in the two phases. In conclusion, a higher oxidative stress seems to be operative in the mouse heart during the middle of the dark phase. An imbalance of antioxidant defences, and/or a higher radical generation and unsaturation degree of bio-membranes lipids, may be hypothesized to favour myocardial oxidative stress at the motor activity phase in mice. This is an entirely new frontier of investigation, leading to new understanding and avenues for treating heart disease. Abstract: Organ dysfunction during scorpion envenomation could be attributed to the activation of a complex inflammatory process, characterized by several inflammatory mediators releasing vasoactive mediators, such as histamine. Immunotherapy constitutes the specific treatment although different approaches have been developed to treat the deleterious effects of the venom. Nevertheless, the use of mammalian antivenoms, which are F(ab')2 immunoglobulin fragments purified from the blood of hyperimmunized horses with scorpion venom, may cause adverse effects due to the host's immune system activation. In addition to immunotherapy, symptomatic treatment is currently administered (analgesics, antipyretics, antihypertensives, anticonvulsants and steroids). The aim of the current study is to develop an appropriate therapy for severe envenomation cases. We evaluate the effects of egg yolk antibodies (IgYs) purified from hyperimmunized chicken with Androctonus australis hector (Aah) scorpion venom, alone or associated with a histamine H4-receptor antagonist (JNJ-7777120), against the pulmonary and splenic inflammatory response and tissue alteration induced by Aah scorpion venom. The egg yolk antibodies and the histamine H4-receptor antagonist were administered thirty minutes after the experimental envenomation. The inflammation response was evaluated 24 h after venom injection by the estimation of vascular permeability changes, infiltration of inflammatory cells, oxidative stress markers, and histological analysis, as well as metabolic enzyme release in mice sera.

Keywords
The results showed that scorpion venom induced inflammatory disorders characterized by an increase in inflammatory cell infiltration and levels of reactive oxygen/nitrogen species, lipid peroxidation, and a decreased antioxidant defense. Moreover, significant alterations in the pulmonary and the splenic tissues were also observed. The administration of the IgYs antibodies fragments to mice after venom inoculation resulted in a decrease of leukocyte infiltration as well as a decrease in the vascular permeability amount. A marked reduction in oxygen species levels, membrane lipids peroxidation, and an increase in antioxidant levels with decreased pulmonary and splenic tissue alteration, were also observed. The association of IgYs fragments and the histamine H4-receptor antagonist resulted in more significant reduction of inflammatory and oxidative stress markers. In addition, a reduction in the perturbation of the lung and spleen tissue structure and metabolic enzyme levels was observed after the addition of these two treatments. These results indicate that the immunotherapy with histamine H4-receptor antagonist exhibits potent therapeutic effects against scorpion venom-induced inflammation response and oxidative/nitrosative stress in pulmonary and splenic tissues and offers the possibility of the use of IgYs antibodies associated to the histamine H4-receptor antagonist in the treatment of scorpion venom-induced immune-inflammatory disorders.

Abstract:
The ALERTOX-NET project is funded by the INTERREG Atlantic Area European Regional Development fund. The project aims to develop an easy-to-use detection and alert system for emerging marine toxins. The project will utilize state-of-the-art toxicity detection systems and disseminate results to all end users. Under the coordination of Prof. Luis Botana, partners from eleven centers of excellence in Spain (four centers), Portugal (one), France (one), the United Kingdom (two) and Ireland (two) bring together a wealth of experience to provide innovative solutions to achieve the project deliverables. ALERTOX-NET is an inter-laboratory collaborative effort for the development and integration of an alert system for marine toxins, considering environmental factors and linking the Seafood Industries' needs and consumers' health protection. ALERTOX-NET will grow into a cluster of excellence in seafood toxicity issues by (a) contributing data and technological innovation; (b) involving the main Regulatory Agencies in adapting the legislation to the phenomena of emerging toxins and advising/informing about new detection systems. The ALERTOX-NET objectives are to a) identify industrial needs regarding marine toxins detection; b) develop innovative toxicological alert systems for more safety seafood; (c) develop easy-to-use toxicity alert systems for the industrial sector; (d) develop the information exchange network ALERTOX-NET at the EU level. In the frame of ALERTOX-NET, we participated in the Capitalization workpackage (WP), by creating a Regional Working Group in close interaction with IFREMER French partner, inviting stakeholders to joint our network and discussing biotech views and needs with them regarding marine toxin detection. We also participated in the Identification industry needs and detection methods WP, by editing a Catalogue of Methods and Procedures focused on Emergent Marine Toxins in Europe (tetrodotoxins, palytoxins and cyclic imine toxins) in close interaction with all partners. Further, we characterized the antagonistic activity of prorocentrolide and portimine on muscle-type and neuronal nicotinic acetylcholine receptors, thereby increasing knowledge of this large family of cyclic imine toxins. Next, we initiated a novel approach for studying the impact of cyclic imine toxins on zebrafish larvae. In the Joint Design of an Easy-to-Use Toxicity Alert System WP, we launched an inter-laboratory method comparison to test the performance of our developed methods, namely the microplate-receptor binding assay (WO2012101378 A1) and the lateral flow test NeuroTorp (WO2017108115 A1), to detect cyclic imine toxins. Both methods are based on a new concept for these technologies: the high affinity of the toxins for their receptor targets. In the frame of the Industrial Pilot of the Toxicity Alert System WP, we contacted several stakeholders to inquire about their needs in the field of marine toxin detection and proposed NeuroTorp for industrial piloting.
Taking advantage of the fact that NeuroTorp is a fast and cost-effective early warning device for in-field detection of marine neurotoxins by end-users Novakits, a Biotech located at Nantes, France, will pilot the performance of this lateral flow test in field conditions with the participation of shellfish farmers for the early detection of cyclic imine toxins. The final aim of ALERTOX-NET is to propose Guidelines for the integration/implementation of the Alert system for marine toxins. Abstract: Over the past two decades, venom toxins have been explored as alternatives to opioids to treat chronic debilitating pain. Approximately 20 potential analgesic toxins, mainly from spider venom, are known to inhibit the Nav1.7 subtype of voltage-gated sodium (Nav) channels with high affinity, making them the most promising genetically validated antinociceptive targets identified so far. The present study aimed to consolidate the development of phlotoxin 1 (PhlTx1), a 34-amino acid and 3-disulfide bridge peptide of a Phlogiellus genus spider, as an antinociceptive agent by improving its affinity and selectivity for the human (h) Nav1.7 subtype. The synthetic homologue of PhlTx1 was generated and, as the natural peptide, equilibrated between two active forms on reverse-phase liquid chromatography, and exhibited potent analgesic effects in a mouse model of inflammatory pain. The effects of PhlTx1 and eight successfully synthetized alanine-substituted variants were studied (by automated whole-cell patch-clamp electrophysiology) on cell lines stably overexpressing hNav subtypes, as well as two cardiac targets, the hCav1.2 and hKv11.1 subtypes of voltage-gated calcium (Cav) and potassium (Kv) channels, respectively. PhlTx1 and D7A-PhlTx1 were shown to inhibit hNav 1.1-1.3 and 1.5-1.7 subtypes at one hundred nanomolar concentrations, while their affinities for hNav1.4 and 1.8, hCav1.2 and hKv11.1 subtypes were over micromolar concentrations. Despite similar analgesic effects in the mouse model of inflammatory pain and selectivity profiles, the affinity of D7A-PhlTx1 for the Nav1.7 subtype was at least five times higher than that of the wild-type peptide.
Computational modelling was performed to deduce the 3D-structure of PhlTx1 and to suggest the amino acids involved in the efficiency of the molecule. In conclusion, the present structure-activity relationship study of PhlTx1 results in a low improved affinity of the molecule for the Nav1.7 subtype, without any marked change in the molecule selectivity against the other studied ion channel subtypes. Further experiments are therefore necessary before considering the development of PhlTx1 or synthetic variants as antinociceptive drug candidates. Abstract: Envenomation is a major health problem in many regions of the world with increasing prevalence in tropical regions. Snake envenomation is characterized by various effects, such as hemorrhage, inflammation, edema and necrosis. Snakes represent the most venomous animals; their venoms are a potential source of bioactive proteins, and each of them can cause serious disturbances. Immunotherapy is the only treatment; however, it has limited efficiency, due mainly to the delay in its administration. In this study, an immunopreventive approach based on vaccine nanoformulation, using Cerastes cerastes venom encapsulated in calcium-alginate nanoparticles, was designed in order to enhance the efficiency of the immune response. An immunization schedule was undertaken in mice by intranasal route to evaluate the potential of C. cerastes venom encapsulated in calcium-alginate nanoparticles against C. cerastes envenomation. The obtained results showed that this formulation stimulated the humoral immune response by inducing the production of high levels of specific IgG antibodies, conferring immunoprotection up to 6 LD 50 . This immune response was associated to a low systemic reactogenicity. The results also showed a moderate inflammatory response characterized by the recruitment of inflammatory blood cells, low myeloperoxidase (MPO) and eosinophil peroxidase (EPO) activities and a reduction in histopathological alterations. Calcium alginate nanoparticles appear to be a promising adjuvant system against envenomation by C. cerastes as they is able to improve the development of an effective humoral response and immunoprotection against the deleterious effects of severe envenomation. Abstract: Scorpion toxins are powerful pharmacological tools to study the mechanisms of neurodegenerative diseases related to ionic channel dysfunction, such as the demyelination of the central nervous system (CNS). Several studies illustrate that kaliotoxin, a potent and highly selective blocker of the Kv1.1 and Kv1.3 potassium channels, can restore the conduction of demyelinated axons and potentiate synaptic transmission. In this study, the effect of kaliotoxin on the neuroinflammatory response was investigated in a murine demyelinating model induced by cuprizone. The obtained results showed that mice exposed to cuprizone over six weeks develop important neuro-immunological disorders characterized by severe alterations in the cerebral structure and function. These alterations were marked by a myelin degeneration, neuronal oedema and axonal loss associated with a neuro-immuno-inflammatory response. Furthermore, a low dose of kaliotoxin, injected by the intracerebroventricular route, seemed to reduce tissue alterations accompanied with a decrease in neuroinflammatory and oxidative stress markers. These data suggest that kaliotoxin is able to ameliorate neuronal conduction and reduce neuro-inflammation in the murine cuprizone-induced demyelinating model. Potassium channel blockers may represent useful therapeutic agents in demyelination-related diseases via the suppression of neuro-inflammation in the CNS.
at higher doses. At sublethal doses, animals recovered, with no apparent sequelae. We extrapolated human symptoms that may be observed in cases of shellfish intoxication, from what is currently known of the PnTX mode of action in vitro and in vivo. As there is currently no regulatory threshold for PnTXs, a risk assessment work has been carried out on PnTX G, the mostly detected PnTX in shellfish in France. A provisional acute benchmark value for human oral exposure was proposed, based on the dose-dependent symptoms observed in mice 4 . For a large portion size of shellfish consumption, the concentration of PnTX G in shellfish not expected to induce adverse effects in humans was derived and may be used as a basis for the establishment of a regulatory limit in shellfish.