Report from the 27th (Virtual) Meeting on Toxinology, “Toxins: Mr Hyde or Dr Jekyll?”, Organized by the French Society of Toxinology, 9–10 December 2021

The French Society of Toxinology (SFET) organized its 27th annual meeting on 9–10 December 2021 as a virtual meeting (e-RT27). The central theme of this meeting was “Toxins: Mr Hyde or Dr Jekyll?”, emphasizing the latest findings on plant, fungal, algal, animal and bacterial toxins during 10 lectures, 15 oral communications (shorter lectures) and 20 posters shared by ca. 80 participants. The abstracts of lectures and posters, as well as the winners of the best oral communication and poster awards, are presented in this report.


Acknowledgments
We warmly acknowledge the contribution of all those people who work daily at ensuring the national and international shinning of the French Society of Toxinology (SFET) and those who made the 27th Meeting on Toxinology a success. We also address special thanks to our sponsors who, this year again, supported our meeting ( Figure 1).

Acknowledgments
We warmly acknowledge the contribution of all those people who work daily at ensuring the national and international shinning of the French Society of Toxinology (SFET) and those who made the 27th Meeting on Toxinology a success. We also address special thanks to our sponsors who, this year again, supported our meeting ( Figure 1).

Preface
Toxins are biologically active substances produced by most kinds of living or isms, bacteria, fungi, plants and animals. They present a vast diversity of molecular s tures and target a wide variety of receptors involved in a range of physiological proce As toxins were selected during evolution to acquire/improve their disabling/lethal e they display finely tuned functional properties often associated with high affinities selectivity. Moreover, toxins are valuable tools to unravel cellular processes due to extreme specificity for cell surface and/or intracellular targets. Therefore, toxins are attractive compounds because of their Janus-like character, being both poisons and r dies, and as such, they have been primarily investigated not only for the light they throw on fundamental physiological processes but also for their potential therapeuti plications.
This 27th Meeting on Toxinology of the SFET was held on 9-10 December 2021 virtual meeting (e-RT27). The central theme selected for this meeting, "Toxins: Mr H or Dr Jekyll?", gave rise to three thematic sessions: the first one on plant toxins, algal to and mycotoxins; the second one on animal toxins; and the third one on bacterial to all sessions were aimed at emphasizing the latest findings on this topic. Apart from central theme, a "miscellaneous" session was dedicated to recent results obtained in inology. Ten speakers from seven countries (Australia, Brazil, Burkina Faso, France, many, the Netherlands and the United States of America) were invited as internati experts to present their work, and other researchers and students presented theirs thro 15 shorter lectures and 20 posters. Of the ca. 80 participants who registered, 38% foreigners, a value highlighting the international attractiveness of the SFET meetings Owing to a donation from MDPI-Toxins, two prizes of EUR 300 each were awa to the best oral communication and the best poster ( Figure 2), both selected from a by all the participants to the meeting.  for mosquitocidal or malariacidal biomolecules. In this study, we firstly compared genes from arthropod predators encoding insect-specific sodium, potassium and calcium channel blockers for their ability to improve the efficacy of Metarhizium against wild-caught, insecticide-resistant anophelines. Toxins expressed under the control of a hemolymphspecific promoter increased fungal lethality to mosquitoes at spore dosages as low as one conidium per mosquito. One of the most potent, the EPA-approved Hybrid (Ca 2+ /K + channel blocker), was studied for pre-lethal effects. These included reduced blood-feeding behavior, with almost 100% of insects infected with ca. six spores unable to transmit malaria within five days post-infection, surpassing the World Health Organization threshold for successful vector control agents. Furthermore, recombinant strains co-expressing Hybrid toxin and AaIT (Na + channel blocker) produced synergistic effects, requiring 45% fewer spores to kill half of the mosquitoes in five days as single toxin strains. Secondly, through a semifield trial in a MosquitoSphere (a contained, near-natural environment) in Soumousso, a region of Burkina Faso where malaria is endemic, we confirmed the proof of our concept in the field. The expression of Hybrid toxin increased fungal lethality and the likelihood that insecticide-resistant mosquitoes would be eliminated from this site. In summary, our results identified a repertoire of toxins with different modes of action that improve the utility of entomopathogens as a technology that is compatible with existing insecticide-based control methods.

Bee Venom and Its Component Apamin Have Anti-Parkinsonian Properties in Animal Models of Parkinson's Disease
Marianne Amalric 1, *, Nathalie Turle-Lorenzo 1 , Christophe Melon 2 , Nicolas Maurice 2 1 Laboratoire de Neurosciences Cognitives (LNC) UMR 7291, CNRS, Aix-Marseille Univ., Marseille, France. 2 Institut de Biologie du Développement de Marseille (IBDM) UMR 7288, NeuroMarseille, Aix-Marseille Univ., CNRS, Marseille, France. * Correspondence: marianne.amalric@univ-amu.fr Abstract: Parkinson's disease (PD) is an age-related neurological disorder that affects more than 6.3 million people worldwide. PD is characterized by progressive loss of dopaminergic (DA) neurons in the substantia nigra (SN), which leads to a wide range of debilitating motor, cognitive and neuropsychiatric symptoms. L-DOPA therapy is effective on motor symptoms and motor fluctuations but leads over the years to dyskinesia. Calcium-activated potassium channels (SK channels) have recently emerged as alternative therapeutic targets because they regulate the neuronal firing of midbrain dopamine neurons. SK channels (in particular SK 2 /SK 3 subtypes) are highly expressed in the basal ganglia (BG). These brain structures are dysregulated by DA neuronal degeneration. Potassium channels are privileged targets because they regulate neuronal excitability in order to maintain balance in the input-output circuits of the BG. Our study aimed to assess the behavioral effects of apamin, a selective SK 2 /SK 3 channel blocker peptide extracted from bee venom, and compare them with those produced by bee venom, in pharmacological and 6-hydroxydopamine (6-OHDA) lesional models of PD in rats. Both bee venom and apamin reversed haloperidol-induced catalepsy, a model of akinetic symptoms, while a co-treatment with the SK opener CYPPA prevented this antiakinetic effect. After extensive unilateral 6-OHDA nigrostriatal lesions, acute and subchronic administration of bee venom and apamin reduced forelimb asymmetry in the cylinder test and apomorphine-induced rotations revealing an antiparkinsonian action on motor symptoms. In another rat model of partial bilateral DA nigrostriatal lesions, apamin also reduced deficits revealed on anxiety, social interaction and visuospatial memory. The neural substrates of these effects were investigated by in vivo electrophysiological recordings of neuronal activity of the BG output structure substantia nigra pars reticulata (SNr). Bee venom restored the balance between the inhibitory and excitatory influence exerted by the trans-striatal direct and indirect pathways that were disrupted by the pharmacological blockade of DA receptors. These Abstract: Recent advances in organoid technology have proven this system to be a valuable tool in understanding human organ development and pathologies. These adult stem cellderived cultures closely recapitulate the structural and functional properties of their organ of origin. Here, we expand the organoid technology toolbox by describing a protocol to culture non-mammalian organoids derived from a snake venom gland. The complexity of venom production, composition and function remain largely unknown for many species. Organoids derived from an Aspidelaps lubricus venom gland can be long-term expanded and histologically resemble the gland. Expression of typical venom-related transcripts (threefinger toxins and Kunitz-type protease inhibitors) can be detected in proliferating organoids with RNA sequencing. Single-cell RNA sequencing reveals distinct venom-expressing cell types, as well as proliferating cells with features of mammalian stem cells. By using mass spectrometry, we identified peptides in the culture medium supernatant that match the composition of the crude venom of the same species. Venom gland organoids furthermore consist of specialized secretory cells visible by transmission electron microscopy. The system enables the investigation of venom production and function on a cellular level in controlled conditions and without the need for experimental animals. This study describes the adaption of organoid technology to a non-mammalian species, providing a model to understand the complexity of the snake venom gland. Abstract: Venomous animals may cause severe medical complications and untimely death, but their venoms are also sources of molecules acting on the nervous, cardiovascular, immune and other systems. Animal toxins are often used as pharmacological tools for the validation of therapeutic targets, but they are also used as cosmeceuticals and diagnostic tools in the design of new therapeutic agents and to improve drug libraries. Some drugs used in the therapy of many disorders, such as diabetes and cardiovascular diseases, were developed based on the molecular structures of animal toxins. Captopril was the first animal toxin-based drug approved for human use. Scorpion neurotoxins are known to be responsible for the pathological manifestations of scorpionism. They are a threat to human health but may serve as leads for the development of new therapies against current and emerging diseases. The scorpion toxin chlorotoxin is undergoing clinical phase trials as a fluorescent molecular probe that paints tumors, while the CPP-Ts peptide is a potential intranuclear delivery tool targeting cancer cells. The bioprospection of neurotoxins that block KV1.3 potassium channels may lead to the development of drugs to treat autoimmune diseases since these channels are found in macrophages, platelets and T cells. The overexpression of KV1.3 in lymphocytes is related to diseases such as atherosclerosis, some types of cancers, obesity and autoimmune diseases such as Crohn's disease, dermatitis, psoriasis and rheumatoid arthritis, among others. Related to snake toxins, a heterologous fibrin sealant, comprising a cryoprecipitate rich in fibrinogen extracted from the blood of buffaloes in association with a serine protease from rattlesnake, is a biodegradable biological product that reduces surgical time, promotes faster postoperative recovery, is highly adhesive and can also be used as an adjuvant in suture procedures and as a carrier for drug delivery. The use of biotechnological tools, such as the heterologous expression of peptides and proteins, enables the production of biologically active molecules in sufficient quantity for the evaluation and development of new medicines. Recently, a serine protease was recombinantly expressed with functional and structural integrity and showed fibrinogenolytic activity and inhibition against the hEAG1 channel, highly expressed in tumor cells, in a mechanism independent of its catalytic activity. Non-PEGylated and PEGylated forms of this enzyme share similar kinetic and functional characteristics, and the latter showed no evidence of immunogenicity or cytotoxicity, even at high concentrations. Given the examples, approaches to improve the druggability of animal toxins are fruitful fields for future research. Abstract: Muscarinic acetylcholine receptors are prototypical class-A GPCRs that are distributed throughout the human body and play critical roles in the maintenance of fundamental human physiology by responding to the neurotransmitter acetylcholine. Five muscarinic receptor subtypes (M1R-M5R) have been identified in humans with distinct G protein coupling preferences, distribution profile and physiological roles. With their conserved orthosteric ligand-binding site and the presence of an allosteric ligand-binding site at the extracellular region, muscarinic receptors were extensively studied as a model system for the subtype-selective targeting at a GPCR through small molecule allosteric modulators. Animals have evolved venoms to hunt prey and/or run from predators. These venoms have been a rich source to isolate natural products that modulate numerous protein functions, including GPCRs. Peptide and small protein toxins are emerging modalities because of their superior selectivity and stability. Muscarinic toxin belongs to the three-finger toxin family and is one of the best-characterized peptide toxins produced by venomous snakes. A member of muscarinic toxin, MT7, shows extremely high selectivity to M1R over other muscarinic receptors and elicits an inhibitory effect. We solved the crystal structure of the M1R-MT7 complex and revealed the exquisite design of this toxin to exclusively fit into M1R by selectively engaging at the allosteric site with residues unique to M1R. By using the structural information and the surface display platform, we succeeded in converting the subtype selectivity of MT7 to another muscarinic receptor, M2R. Furthermore, we obtained conformational selective three-finger proteins exclusively targeting the active conformation of muscarinic receptors. Our data indicate a promise of repurposing this natural toxin scaffold to a broader range of target systems.

Keywords
Keywords: muscarinic acetylcholine receptor; protein engineering; three-finger toxin

Synthetic AB-Toxins for Targeted Pharmacological Modulation of Cancer and Immune Cells
Holger Barth * University of Ulm Medical Center, Institute of Pharmacology and Toxicology, Albert-Einstein-Allee 11, 89081 Ulm, Germany. * Correspondence: holger.barth@uni-ulm.de Abstract: Bacterial AB-toxins are highly toxic proteins because of their unique modular structure: these toxins bind to mammalian cells by a specific receptor binding (B) subunit and deliver their enzymatically active (A) subunit into the host cell cytosol via their intrinsic translocation (T) subunit. In the cytosol, the A subunit modifies its specific cellular substrate, which alters cellular structures and/or functions and is the reason for the clinical symptoms of the particular toxin-associated disease. Because of their intracellular, highly specific and extremely potent mode of action, some AB-toxins serve as valuable molecular tools in pharmacology and cell biology for the targeted modulation of cell functions and are used as drugs. In past years, we and others used the non-toxic B and T subunits of various toxins to deliver pharmacologically active proteins (e.g., enzymes) and peptides into the cytosol of cells and developed cell type-selective transport systems to modulate functions of immune and cancer cells. However, for some cell types, there are no selective AB toxins available by nature, and therefore, we aimed to develop "synthetic" AB-toxins. In one approach, supramolecular transporter systems were generated, where avidin with four biotin-binding sites serves as a central binding platform for three biotinylated binding peptides that selectively bind to receptors on target cells and trigger internalization of the transporter molecule, and biotinylated cargo molecules that act as "A subunit" in the cytosol. Proofof-concept was provided by using clostridial C3 Rho-inhibitor as cargo enzyme and the peptide somatostatin as a ligand to selectively target human lung cancer cells in vitro and in the HET/CAM model for a human xenograft lung tumor that overexpresses somatostatin receptor. This strategy also served for the generation of a novel cell type-selective Rhoinhibitor for human neutrophils. This universal "LEGO-like" modular approach represents a promising platform for the generation of cell-type selective synthetic AB-toxins for the targeted pharmacological modulation of cell functions.
Keywords: bacterial AB-toxin; cellular uptake; targeted drug delivery; receptor binding; membrane translocation; avidin-biotin technology; (semi)synthetic AB-type molecule to suggest that antitoxin drugs should be developed to treat common public health bacterial infections as an alternative to antibiotics. In these respects, we developed two phenotypic screenings of small chemical molecules in 2005 and 2015; the first was against ricin toxin, and the second (in collaboration with Emmanuel Lemichez, Institut Pasteur) was against the cytotoxic necrotizing factor CNF1, produced by uropathogenic and extra-intestinal strains of Escherichia coli. The first screening was designed to select molecules capable of rescuing cells from ricin-induced toxicity. The second screening was designed to select molecules inhibiting the degradation of CNF1-induced activated Rac1. Overall, the anti-ricin screening identified three original inhibitors of intracellular trafficking. Retro-1 and Retro-2 act on the retrograde trafficking pathway (collaboration with Ludger Johannes, Institut Curie). In particular, Retro-2 interferes with the ERES protein Sec16A and the circulation of syntaxin-5 along the anterograde and retrograde routes. ABMA acts on the endolysosomal pathway by the accumulation of late endosomes and alteration of the autophagic flux. The CNF1 screening identified C910 as an original inhibitor of early endosome sorting function. Most interestingly, all four inhibitors displayed a broad spectrum of inhibition against a series of bacterial toxins, but also viruses, bacteria and parasites exploiting these pathways for intoxication or infection of cells. There are at least eight examples published or under the submission of in vivo protection of mice by these compounds against intoxications and/or infections. Compound optimization by medicinal chemistry was performed. The challenges of turning these molecules into drugs are discussed. Nevertheless, 16 years of research by us and others showed that intracellular trafficking pathways might be interesting druggable targets to fight intracellular toxins and pathogens. Abstract: The detection of the activities of pathogen-encoded virulence factors by the innate immune system has emerged as a new paradigm of pathogen recognition. Much remains to be determined regarding the molecular components contributing to this defense mechanism in mammals and its importance during infection. Our team showed the central role of the IL-1β signaling axis in controlling the Escherichia coli burden in the blood in response to the sensing of the RhoGTPase-activating toxin CNF1. Using the CNF1 toxin, we provided evidence of the role of the NLRP3 inflammasome in sensing the activity of bacterial toxins and virulence factors that activates host RhoGTPases. We demonstrated that this activation relies on monitoring of the toxin activity on the RhoGTPase Rac2. We also showed that the NLRP3 inflammasome is activated by a signaling cascade involving the P21-activated kinases (Pak)-1/2. The Pak1-mediated phosphorylation of threonine-659 of NLRP3 was necessary for NLRP3-Nek7 interaction, inflammasome activation and IL-1ß cytokine maturation. Furthermore, inhibition of the Pak1-NLRP3 axis diminished the bacterial clearance of CNF1-expressing E. coli during bacteremia. Altogether, our results established Pak1/2 as critical regulators of the NLRP3 inflammasome and revealed the role of the Pak1-NLRP3 signaling axis in vivo during bacteremia.

Abstract:
The isoquinoline alkaloids (IA) is a large chemical group of natural compounds with various structures and acting on multiple pharmacological targets of therapeutical interest, including ion channels, such as voltage-gated Na+ (Nav) and Ca2+ (Cav) channels. These ion channels are established molecular targets for the development of therapeutical agents in cardiovascular diseases and particularly arrhythmia. Here, we screened 62 IA from an in-house vegetal chemical library, using a model of excitable cells, the rat pituitary GH3b6 cells, which endogenously express both Nav and Cav channels, to find new blockers for these channels. Moreover, we demonstrated that Nav activation by selective neurotoxins induced an intracellular Ca 2+ concentration [Ca 2+ ] i elevation mediated by Cav channels, highlighting crosstalk between Nav and Cav channels that we used for pharmacological studies. We first tested these IA for their abilities to inhibit batrachotoxin (BTX)-induced depolarization using fluorescent voltage-sensor probes (VSP) and identified two oxoaporphines, namely liriodenine and oxostephanine, which abolished BTX-induced VSP signal with IC 50 values in the micromolar range. While the blocking activity of liriodenine was already reported, the activity of oxostephanine on Nav channels has never been described yet. Interestingly, oxostephanine differs from liriodenine only by a methoxy group on the benzyl part of the skeleton. This subclass of IA might constitute a new group of ligands of Nav channels. We confirmed the blocking effect of both molecules in a Na+ influx assay using the Na + fluorescent probe ANG-2. Since liriodenine is also known to block Cav channels, we hypothesized that oxostephanine probably targets Cav channels. Thus, we investigated the effects of both IA on L-type Cav channels (LTCC) expressed in GH3b6 cells. In order to activate LTCC, we used a chemical depolarization with KCl or the agonist Bay K8644 and monitored [Ca 2+ ] i change with the fura-2 probe. Our results showed that liriodenine and oxostephanine induced a concentration-dependent inhibition of KCl-and Bay K8644-evoked Ca 2+ responses, with similar IC 50 values in the micromolar range. In addition, this interaction of liriodenine and oxostephanine on LTCC was also highlighted by their ability to inhibit veratridine (VTD)-and BTX-induced [Ca 2+ ] i elevation.
In conclusion, our data showed that liriodenine and oxostephanine, two oxoaporphine alkaloids, inhibit Nav and Cav channels with similar potency. The oxoaporphine skeleton might bring an interesting pharmacophore for structure-function relationship studies for designing more selective ligands toward Cav and Nav channels and for developing new antiarrhythmic therapeutical leads. Abstract: Spider gomesin peptides target melanoma cells of BRAF-mutation with minimum effect on non-transformed neonatal foreskin fibroblasts. We hypothesized that the selectivity of gomesin peptides towards melanoma of BRAF mutation could be influenced by interactions with lipids or cholesterol present in cell membranes. In order to elucidate upon this, we employed a multidisciplinary approach, including CRISPR/Cas9 genomewide screening, to identify key players underlying the antiproliferative mechanisms of gomesins. We also used a combination of fluorescence spectroscopy to measure membrane dipole changes, electrical impedance spectroscopy (EIS) with tethered bilayer membranes (tBLMs) to quantify alterations in membrane conductance and membrane thickness, and cell viability assays. Gomesin and variants showed weak binding to POPC membranes alone. However, the presence of POPS and cholesterol significantly improved the binding of the peptides and lessened membrane disruption. In addition, the cytotoxicity of gomesin was blunted by increasing concentrations of cholesterol in both melanoma cells and fibroblasts. Conversely, cholesterol depletion potentiated the cytotoxicity of peptides in fibroblasts to almost the levels originally observed in melanoma cells. In conclusion, we postulate a specific role of cholesterol in the selective cytotoxic profile of gomesin in melanoma of BRAF mutation. Abstract: Pseudomonas aeruginosa is a ubiquitous and opportunistic Gram-negative bacterium. It is a leading cause of nosocomial infections and is responsible for acute and chronic infections thanks to a broad panel of virulence factors. Among them, the Type 3 Secretion System (T3SS) plays an essential role in the pathogenicity of the bacterium. This system pierces the cell membrane and allows the delivery of toxins into the host cytoplasm. Four T3SS effectors have been identified-ExoS, ExoU, ExoT and ExoY. Most strains inject two or three of these effectors simultaneously, with ExoU and ExoS being mutually exclusive. Each T3SS effector is inactive when injected and requires a host cell factor to be activated. Strains injecting ExoU toxin induce the most severe pathologies because the damage caused by the toxin is rapid and irreversible, leaving no time for proper clinical management. Its mechanism of action is well described. Inside host cells, ExoU is a phospholipase that interacts with a specific lipid at the cytosolic side of the plasma membrane, allowing its activation and leading to cell necrosis. However, several aspects of ExoU activation and its trafficking in human cells upon injection remain elusive. Thanks to genetic approaches and analyses by microscopy, we identified a favored anterograde transport, led by the co-chaperone DNAJC5, which targets ExoU to the plasma membrane. Inactivation of DNAJC5 gene disrupts ExoU-dependent toxicity in vitro and in vivo. Unlike ExoU, the role of ExoY in the pathogenicity of P. aeruginosa is still controversial. This toxin is a nucleotidyl cyclase that interacts with the eukaryotic filamentous actin (F-actin) to be activated and thereby to massively convert nucleotide triphosphates into their cyclic form. It was reported that ExoS toxicity to the epithelial cells is enhanced in the absence of ExoY and that in many ExoU+ strains, ExoY is not active. Thus, ExoY seems to limit the action of other toxins. We will discuss our advances in exploring the role of ExoY by studying its effects in infected cells. Abstract: The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. Bordetella pertussis, the causative agent of whooping cough, produces an adenylate cyclase (CyaA) toxin that plays an essential role in the early stages of respiratory tract colonization. CyaA displays a unique intoxication pathway of human cells via a direct translocation of its catalytic domain (AC) across the plasma membrane. Once in the cytosol, AC impairs the physiology of immune cells, leading to cell death. We showed that the P454 peptide (CyaA residues 454-484) is able to translocate across membranes and interact with calmodulin. The key residues involved in membrane-active and calmodulin-binding properties were identified. The mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. We propose that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin ( Figure 3). Trapping of CyaA by the calmodulin:P454 interaction in the cytosol may assist the entry of AC by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells. The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. Bordetella pertussis, the causative agent of whooping cough, produces an adenylate cyclase (CyaA) toxin that plays an essential role in the early stages of respiratory tract colonization. CyaA displays a unique intoxication pathway of human cells via a direct translocation of its catalytic domain (AC) across the plasma membrane. Once in the cytosol, AC impairs the physiology of immune cells, leading to cell death. We showed that the P454 peptide (CyaA residues 454-484) is able to translocate across membranes and interact with calmodulin. The key residues involved in membrane-active and calmodulin-binding properties were identified. The mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. We propose that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin (Figure 3). Trapping of CyaA by the calmodulin:P454 interaction in the cytosol may assist the entry of AC by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells.   Abstract: IKr current, a major component of cardiac repolarization, is mediated by human Ether-à-go-go-Related Gene (hERG, Kv11.1) potassium channels. The blockage of these channels by pharmacological compounds is associated with drug-induced long QT syndrome (LQTS), which is a life-threatening disorder characterized by ventricular arrhythmias and defects in cardiac repolarization that can be illustrated using cardiomyocytes derived from human-induced pluripotent stem cells (hiPS-CMs). This study was meant to assess the modification in hiPS-CMs excitability and contractile properties by BeKm-1, a natural scorpion venom peptide that selectively interacts with the extracellular face of Abstract: Snake envenoming is a major but neglected human tropical disease. Among venomous snakes, kraits (snakes of the Bungarus genus) are medically important venomous species that cause reversible peripheral neuroparalysis. Hospitalization and use of antivenoms derived from an animal immunized with Bungarus venoms are the primary therapies that prevent death from early-onset respiratory paralysis. There is a general consensus that additional and non-expensive treatments, which can be delivered even long after the snakebite, are needed. Traumatic or toxic degeneration of peripheral motor neurons, with ensuing neuroparalysis, is characterized by the activation of a pro-regenerative intercellular signaling program. A major player is the intercellular signaling axis consisting of the chemokine CXCL12a, produced by perisynaptic Schwann cells and acting on the CXCR4 receptor expressed on the damaged neuronal axons. The CXCR4 agonist NUCC-390 was recently found to promote axonal growth. Here, we tested its efficacy on the neuroparalysis induced by the venoms of three major krait species, i.e., Bungarus caeruleus, B. multicinctus and B. candidus that are prevalent in Asia. These venoms cause a complete degeneration of motor axon terminals. Functional recovery of the neuromuscular junction was assessed by electrophysiological recordings and by imaging. We report that NUCC-390 administration to venom-injected mice greatly accelerates the recovery from paralysis. These data candidate NUCC-390 to be tested as novel therapeutics to reduce death by respiratory deficits and to improve the recovery of normal neuromuscular physiology, thus reducing the human and hospital costs of envenoming. NUCC-390 can be administered any time after the snakebite and has great potential of becoming a non-expensive addition to the currently available antivenom treatments whose efficacy is limited to a short period af-ter snakebite. This drug is expected to decrease the long and expensive post-snakebite mechanical ventilation phase. Abstract: Current pit-viper antivenoms for human use in Mexico have shown a low level of protection against one neurotoxin found in some rattlesnakes (Crotalus spp.), crotamine. This toxin has a low molecular weight (~5 kDa) and is well known for its spastic paralysis symptom provoked in mice with no evidence of neutralization. Recently, it was reported that crotamine is the major toxin found in some rattlesnake venoms such as C. molossus nigrescens (~50%) and C. oreganus helleri (62%). On the other hand, sphingomyelinase D (SMD) is a highly immunogenic enzyme (MW:~30 kDa) found in Loxosceles spp. spider venom. In this study, we aimed to neutralize the crotamine-induced main symptom of paralysis by employing, as immunogens, two rattlesnake venoms and a novel recombinant fusion protein made from crotamine and SMD, used as a carrier. Methods-Crotamine cDNA was synthetized from venom gland mRNA of a C. m. nigrescens individual from Mexico, while one plasmid containing L. reclusa SMD was available in the lab. We cloned the sphingomyelinase D and crotamine in tandem into the expression vector pQE30 to transform competent Origami Escherichia coli cells for the production of the protein. By using the recombinant protein and the whole venoms of C. o. helleri and C. m. nigrescens, we performed separate immunization protocols in rabbits to measure the antibody recognition to crotamine (ELISA and Western blot) and the neutralization capacity in mice against the main toxin-induced paralysis symptom. Results-The fusion protein was obtained at~10 mg/L of bacterial culture with the expected 37.5 kDa molecular mass as analyzed by SDS-PAGE and Western-blot. The recombinant protein and the two whole crotamineenriched venoms generated antibodies with cross-reactivity against crotamine from up to seven species. The three experimental antivenoms were able to neutralize the paralysis symptom provoked by crotamine in the mice model. Discussion/Conclusion-We showed, for the first time, the neutralization of the crotamine-induced spastic paralysis symptom by three experimental antivenoms. The recombinant SMD-Crotamine fusion protein as well as crotamine-enriched venoms could be good candidate immunogens for the improvement of Mexican antivenoms. Abstract: Hepatocellular carcinoma (HCC) is the most common primary liver cancer in adults, the fifth most common malignancy worldwide and the third leading cause of cancer-related death. An alternative to the surgical treatments and drugs, such as sorafenib, commonly used in medicine, is necessary to overcome this public health problem. In this study, we determine the anticancer effect on HCC of Moroccan cobra Naja haje venom and its fraction obtained by gel filtration chromatography against Huh7.5 cancer cell line. Cells were grown together with WI38 human fibroblast cells, LX2 human hepatic stellate cell line, and human endothelial cells (HUVEC) in MCTS (multi-cellular tumor spheroids) models. The hepatotoxicity of venom and its fractions were also evaluated using the normal hepatocytes cell line (Fa2N-4 cells). Our results showed that an anti HCC activity of N. haje venom and, more specifically, the F7 fraction of gel filtration chromatography exhibited the greatest anti-hepatocellular carcinoma effect by decreasing the size of MCTS. This effect is associated with low toxicity against normal hepatocytes. These results strongly suggest that the F7 fraction of N. haje venom obtained by gel filtration chromatography possesses the ability to inhibit cancer cell proliferation. More research is needed to identify the specific molecule(s) responsible for the anticancer effect and investigate their mechanism of action. The hypothalamic-pituitary-adrenal (HPA) axis activity was evaluated by measurement of adrenocorticotropic (ACTH) and corticosterone plasma hormones levels as well as by analysis of CD68 immunohistochemistry staining. The inflammation was assessed by the measurement of the serum level of the proinflammatory cytokines, IL-17 and IL-6, evaluation of neutrophil infiltration and oxidative/nitrosative stress markers (Nitrites, MDA, H2O2, CAT and GSH). Glucose level and activities of AST and ALT as well as histopathological analysis of hepatic tissue, were also performed in the two groups of animals (1 HALO and 18 HALO). Obtained results showed a high increase of serum ACTH and corticosterone levels as well as positive immunostaining of CD68 cells during the dark period indicating activation of the HPA axis associated with local inflammation. The results also showed day-night variations, with significantly high levels of nitrite, hydrogen peroxide, myeloperoxidase and lipid peroxidation during the daytime compared with the nighttime. Significant variations in catalase activity and GSH levels are also observed with the highest evening values. Furthermore, an increase in IL-6 levels was observed during the active phase, while no differences were observed in the IL-17 levels between day and night times. These results indicate also that Aah venom induced a time-dependent increase of metabolic parameters during the dark phase and severe hepatocellular injury.

Keywords
In conclusion, the daily variation of the HPA axis activation and inflammatory response appears to be closely related to the circadian moment of envenomation. Future studies should investigate the molecular mechanisms resulting in this circadian rhythmicity. Abstract: Voltage-gated sodium channels (Na v ) play critical functional roles by controlling, in excitable and non-excitable cells, the action potential initiation/propagation, cell motility, and proliferation. In humans, there are nine subtypes of Na v channels-1.1 to 1.9-with Na v 1.4 being the muscle subtype. Na v channel dysfunctions are associated with neurological, cardiovascular, muscular and psychiatric disorders. Marine phytoplankton and freshwater cyanobacteria are able to synthesize a series of neurotoxins targeting Na v channels, among them, saxitoxin, a potent site-1 pore blocker classified as a chemical bioweapon because of its high lethality for humans. Receptor binding assays are suitable analytical techniques for ligand screening owing to their extremely high resolution and sensitivity, their fast analysis and easy automation, which facilitates the development of high-throughput protocols. There is, however, a lack of toxin-tracers for Na v toxins detection. The marine cone snail of the genus Conus produces a series of conotoxins for prey hunting, among them, µ-conotoxin peptides, with an exquisite affinity for Na v channels. Wild-type and biotinylated synthesis of five µ-conotoxins was performed using a Protein Technologies Prelude synthesizer. Biotinylation was performed on-column after Fmoc deprotection of the N-terminal residue of the µ-conotoxins. After refolding, the resulting conopeptides were purified by reverse-phase HPLC. The affinity of each µ-conotoxin for the Na v 1.4 channel was determined. Two biotinylated µ-conopeptides with high affinity for Na v channels were identified as potential toxin tracers for the detection of site-1 Na v pore blockers by ligand binding assay. Abstract: Characterizing the distribution of drug receptors through optical imaging can be challenging, as the availability of suitable fluorescent probes is limited. Over history, natural toxins have benefited science through an ever-growing source of biologically active peptides, many of which show great potential as probes to study the distribution of their associated receptors. Here, we aim at developing a synthetic, fluorescent analog of α-bungarotoxin (α-BgTx), a highly potent, selective and irreversible inhibitor of the muscle-type nicotinic receptors (mnAChR), through the addition of a Cy5 tag. We first performed functional validation of this Cy5-α-BgTx analog by high-throughput patch-clamp (SyncroPatch364, Nanion), looking at the inhibition of acetylcholine-mediated currents on TE671 cells. It, therefore, appeared that the addition of the Cy5 tag still generates a peptide with nanomolar affinity. In order to further ensure that natural binding properties of the peptide were preserved, we synthesized a peptide analog of the α-BgTx binding site on the mnAChR (BSpep). Preincubation of Cy5-α-BgTx with BSpep resulted in the capture of the α-BgTx and complete blockage of its inhibitory effect. The fluorescent toxin was then used to study the distribution of mnAChR on fixed TE671 cells, allowing us to visualize both its intra and extracellular distribution. Thus, we were able to synthesize a functional, synthetic analog of α-BgTx that can be used to localize mnAChRs, highlighting despite their phylogenetical proximity, Androctonus australis is the heart of 365 publications (Pubmed), whereas A. mauritanicus is only considered in five of them. In this work, we aim at comparing the venom profiles of these scorpions at the protein level. A first macroscopic overview of the protein content of each venom was evaluated by 1D SDS-PAGE and showed no notable difference. During the second time, the crude venoms of the two scorpions were analyzed with bottom-up proteomics. Toxins were classically reduced to remove the disulfide bonds, alkylated to avoid their reformation and finally digested with trypsin. The LC-MS/MS spectra were realized with M-Class UPLC coupled to a Q-Exactive spectrometer. Data analysis was performed with the bioinformatics dedicated software "Peaks X+", with the use of an extracted database from Uniprot built with the keyword "Buthidae". As preliminary results, A. mauritanicus presents 163 Top proteins against 142 for A. australis with this database. They share 100 peptides and 63 proteins in common. A table with the comparison of the classification of proteins in each species is presented below ( Table 1). In the light of the protein classification, some groups of non-toxins are unexplored in the scorpion venoms, such as the Kunitz-type family and Cysteine-rich venom proteins. Therefore, the proteomic approaches can still open perspectives for the study of these groups, which is relevant to highlight them for future explorations. Comparing a wellstudied scorpion species with another less studied but phylogenetically close is interesting to highlight the evolution, diversity and complexity of venom.  . However, until now, this approach has not been applied to venomic studies, which may be useful to define the venom composition. The generation of a higher number of peptides per protein during the MELD digestion increases the quality of protein sequencing and, consequently, identification. Herein we applied the MELD strategy for the venomics of two snake species: Echis ocellatus (EoV) and Dendroaspis polylepis (DpV). Ten micrograms of each venom were reduced/alkylated followed by two different digestion protocols: (1) trypsin and (2) MELD (Trypsin, GluC and Chymotrypsin). The digested materials were analyzed in a Q-Exactive™ Plus Mass Spectrometer with protein identification performed by Peaks Studio X+ using the Uniprot and transcriptomic databases. MELD showed more peptides/proteins identified for both venoms compared to the trypsin protocol. In EoV, 82.8% 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 coverage of 26.2% for toxins, 39.9% for non-toxins and 33.9% for CC, while for trypsin, we obtained 23.3% for toxins, 37.4% for non-toxins and 39.3% for CC. MELD was able to identify new components in both venoms. Fifty-one percent of the EoV were metalloproteinases (MP), while DpV showed a high content of nerve growth factor (22%), which was not identified using the transcriptome database. The highest number of mass spectra was obtained for a metalloproteinase (tr|Q2UXQ4) for E. ocellatus, in which the MELD approach led to four times more mass spectra. For D. polylepis, dendrotoxin I (P00979) showed the highest number of mass spectra, and the trypsin-based approach yields two times more mass spectra. As the number of mass spectra gives a rough evaluation of the toxin concentration, the two cited toxins are among the most produced in the venoms. 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. Abstract: Scorpion envenomation (SE) triggers granulopoiesis, resulting in heightened production of neutrophils that, in turn, exacerbate tissue damage. However, the mechanisms that sustain their production and recruitment to the injured tissue are unclear. Experimental evidence suggests that alarmines, inter alia adenosine, can modulate proliferation and differentiation of hematopoietic progenitors as well as mature neutrophils mobilization. Indeed, adenosine accumulates in the extracellular space in response to cell damage and can modulate immune cells through its transmembrane receptors A1, A2A, A2B, and A3. However, despite increasing data incriminating adenosine in many models of inflammation, little is known about its effect in the inflammatory response caused by the venom of Androctonus australis hector (Aah). Aim-In this study, we focused on deciphering molecular signals that promote neutrophil production and recruitment after SE by targeting adenosine as a potential candidate. Methods-Using a mouse model of envenomation and a pharmacological strategy, we prevented adenosine signaling by 1,3,7-triméthylxanthine, a nonselective inhibitor of adenosine A1 and A2A receptors. We first characterized the effect of this treatment on granulopoiesis in the bone marrow and then evaluated the expanse of inflammation to various tissues, mainly of the cardiorespiratory system. To this end, medullar MPO activity was assessed as a marker of progenitor cells proliferation and differentiation. In parallel, differential count of leucocytes and histopathological analysis of heart and lungs were carried out. Results-Inhibition of the action of adenosine helped to attenuate venom-induced granulopoiesis and reduced the accumulation of neutrophils in the blood. The damages caused by neutrophils in the lungs and heart were improved to a large extent. Conclusion-These results highlight the involvement of adenosine in the inflammatory response induced after SE via its A1 and A2A receptors and could help identify a new target to modulate inflammation and prevent cardiorespiratory failure in envenomed patients. Abstract: Snake venoms contain a wide range of components that are able to initiate or inhibit several steps of coagulation or platelet aggregation. Two thrombin-like enzymes, VLCV and VLCII (45 and 60 kDa, respectively) with fibrinogenolytic and pro-aggregating activities, are purified from Vipera lebetina venom. These molecules of interest are involved in platelet adhesion to fibrinogen by activating signaling pathways via αIIbβ3 integrins and thus binding to fibrinogen. Results also showed that both molecules present a high pro-aggregating effect with a similar mechanism of thrombin. The incubation of heparin with VLCV or VLCII has no inhibition on induced platelet aggregation by these molecules, suggesting that both enzymes have similar functional characteristics. The use of PMSF also has no effect on the activity of both molecules, indicating that the induced platelet aggregation by VLCV or VLCII involves an independent mechanism of catalytic activity.

Keywords
Results also indicate that both molecules VLCII and VLCV present a mechanism of action involving the thrombin and/or ADP pathways via PAR1/PAR4/P2Y12 receptors, as well as the αIIbβ3 integrin, leading to adhesion and platelets activation. Both thrombin-like VLCII and VLCV could be used as potential targets for the development of new drugs.

Abstract:
The neuro-immuno-inflammatory response triggered by neurotoxins is a key event in the pathogenesis of scorpion envenomation. In the present study, this response was evaluated in cardiac, pulmonary and brain tissues of intoxicated mice with kaliotoxin; a neurotoxin derived from Androctonus australis hector scorpion venom. The involvement of serotoninergic and histaminergic pathways in the systemic inflammatory response following kaliotoxin administration was also investigated. Obtained results revealed that the injected kaliotoxin by intracerebroventricular route induces an important immuneinflammatory response in brain, cardiac and pulmonary tissues. This response is mainly characterized by local features such as edema formation, inflammatory cell infiltration and imbalanced redox status. These effects are correlated with severe tissue alterations and a concomitant increase in metabolic enzymes in sera. Pretreatment of mice with antagonists of serotonin (5HT) and histamine (H1) receptors markedly attenuated these alterations in all the studied tissues. Serotonin and histamine-specific receptors seem to be the main pharmacological targets involved in the neural and systemic inflammatory processes. These findings could help to understand better the role of serotonin and histamine in scorpion venom-induced inflammatory response and pave the way to new therapies targeting 5HT and H1 receptors in order to attenuate the induced neuro-immuno-inflammatory disturbances that may be encountered in severe grades of scorpion envenoming. Abstract: Dysfunctions of voltage-gated sodium channels (Nav) have been associated with many pathologies such as cardiac diseases, neuropathic pain and epilepsy. In order to study the role of these channels in diseases or to restore their function, selective molecules targeting ion channels are needed. Only a few molecules selective to a single sodium channel isoform have been discovered. This makes these channel types important targets in drug discovery. Animal venoms, and especially spider venoms, contain various peptides that target ion channels. By screening spider venoms on Nav isoforms, we aimed to identify new peptidic toxins targeting specifically one of them. We performed a screening of about thirty spider venoms on Nav1.3, Nav1.4, Nav1.5 and Nav1.6 isoforms using the automated patch-clamp (APC) technique (SyncroPatch364, Nanion). All venoms were preliminarily fractionated in 64 fractions and tested on each Nav. Fractions of interest are those that reduce sodium peak current (by at least 30%), slow-down inactivation or increase late sodium current. False-positive fractions were excluded based on the detection of material in HPLC or mass spectrometry. The primary screening identified 220 fractions active on at least one isoform. Some of these fractions were then selected for purification and tested again on the automatic patch-clamp (74 for Nav1.3, 42 for Nav1.4, 28 for Nav1.5 and 23 for Nav1.6). The most interesting peptides were sequenced, synthesized and characterized. This study suggests that among a large number of toxins present in venoms, close to 50% of them target sodium channels with specificity for each sodium channel isoform. Abstract: Gambierol, characterized by a transfused octacyclic polyether core, was first isolated and chemically typified from cultured Gambierdiscus toxicus dinoflagellates collected in French Polynesia. Subsequently, distinct groups in Japan and the USA, using various strategies, achieved their total chemical synthesis. This allowed detailed studies on its mode of action. Gambierol inhibits voltage-gated K + (K V ) channels in various excitable and non-excitable cells, as well as in motor nerve terminals of the skeletal neuromuscular junction. In the present study, we investigated first the effects of nanomolar concentrations of gambierol on K + current of cultured chromaffin cells from fetal rat adrenal medulla using perforated patch-clamp current recordings. Our results show that gambierol only blocked a small component of the total K + current and affected neither calcium-activated K + (K Ca ) nor ATP-sensitive K + (K ATP ) channels, as revealed using apamin and iberiotoxin (selective K Ca channel blockers) and glibenclamid (K ATP channel blocker). After inhibiting K ATP and K Ca channel activation, the gambierol concentration blocking 50% of the K + current component (IC 50 ) was 7.6 ± 1.1 nM. The repolarizing phase of all-or-none elicited action potentials, recorded under current-clamp conditions (triggered by 1-ms depolarizing pulses), was sensitive to the action of gambierol but insensitive to the action of apamin and iberiotoxin, indicating that K Ca channels do not participate in the modulation of action potential duration triggered by short depolarizing pulses. The use of simultaneous patch-clamp and single-cell amperometry allowed controlling the membrane potential and detecting exocytosis events (with a carbon electrode polarized to +650 mV to allow the oxidation of released catecholamines). Such recordings revealed that gambierol did not modify the membrane potential following 14-s depolarizing current steps and did not significantly increase the number of exocytotic catecholamine release events with respect to controls. The addition of K Ca channel blockers (in the continuous presence of gambierol) enhanced the membrane depolarization by about 15 mV (during the 14 s current step), and at the same time, increased significantly the number of exocytotic events related to catecholamine secretion. Such enhanced depolarization induced by the K Ca channel blockers probably brings the membrane potential above the activation threshold of high-voltage activated Ca V channels, triggering both Ca 2+ influx and subsequent catecholamine secretion. These results emphasize the diversity of K V channels in chromaffin cells from fetal rat adrenal medulla and highlight the modulatory role played by K Ca channels in the control of exocytosis in the absence of splanchnic innervation. oculi muscle. In this study, we used surgical waste muscle specimens from 14 patients treated with repeated injections of either Dysport ® , abobotulinumtoxinA Ipsen or Xeomin ® incobotulinumtoxinA, Merz. These muscle specimens were compared to others, obtained in normal subjects (naïve of BoNT/A) during blepharoplasty. The morphological study was performed blinded to the BoNT/A sample treatment. Neuromuscular specimens analyzed by confocal laser scanning microscopy, using fluorescent staining and immune-labeling of presynaptic proteins, revealed that the pattern of innervation, the muscle nicotinic ACh receptors (nAChR) and the NMJs exhibited marked differences in BoNT/A-treated muscles (regardless of the toxin clinically used), with respect to controls. The control muscles were constantly innervated by a single motor axon (mono-innervated), and NMJs were relatively simple with the typical nAChR array. In contrast, most of the BoNT/A-treated muscles exhibited profuse and stable poly-neuronal innervation, with two unambiguous features: one in which multiple axons innervated a single muscle fiber, the other in which distinct motor axons converged to a unique endplate (convergent innervation). The ability to increase the proportion of poly-innervated muscle fibers may be related to either the stimulation of nerve sprouting (due to muscle inactivity caused by BoNT/A) or the absence of synapse elimination. During development, the structures composing the NMJ undergo rapid formation and elimination. In rodents, unessential synapses are eliminated. We previously reported synapse elimination in mature rodent NMJs following a single BoNT/A injection. The new findings reported here raise a number of questions about the origin and factors contributing to the plasticity changes observed and the expected detrimental functioning of NMJs and muscle fibers.
Keywords: botulinum neurotoxin type A; innervation pattern; sprouting 6.16. Synthetic Analogues of Huwentoxin-IV Spider Peptide with Altered Human Na V 1.7/Na V 1.6 Selectivity Ratios Abstract: Huwentoxin-IV (HwTx-IV), a peptide discovered in the venom of the Chinese bird spider Cyriopagopus schmidti, has been reported to be a potent antinociceptive compound due to its action on the genetically validated Na V 1.7 pain target. Using this peptide for antinociceptive applications in vivo suffers from one major drawback, namely its negative impact on the neuromuscular system. Although it was studied only recently, this effect appears to be due to an interaction between the peptide and the Na V 1.6 channel subtype located at the presynaptic level. The aim of this work was to investigate how HwTx-IV could be modified in order to alter the original human (h) Na V 1.7/Na V 1.6 selectivity ratio of 23. Nineteen HwTx-IV analogs were chemically synthesized and tested for their blocking effects on the Na + currents flowing through these two channel subtypes stably expressed in cell lines. The dose-response curves for these analogs were generated, thanks to the use of an automated patch-clamp system. Several key amino acid positions were targeted owing to the information provided by earlier structure-activity relationship (SAR) studies. Among the analogs tested, the potency of HwTx-IV E 4 K was significantly improved for hNa V 1.6, leading to a decreased hNa V 1.7/hNa V 1.6 selectivity ratio (close to 1). Similar decreased selectivity ratios, but with increased potency for both subtypes, were observed for HwTx-IV analogs that combine a substitution at position 4 with a modification of amino acid 1 or 26 (HwTx-IV E 1 G/E 4 G and HwTx-IV E 4 K/R 26 Q). In contrast, increased selectivity ratios (>46) were obtained if the E 4 K mutation was combined to an additional double substitution (R 26 A/Y 33 W) or simply by further substituting the C-terminal amidation of the peptide by a carboxylated motif, linked to a marked loss of potency on hNa V 1.6 in this latter case. These results demonstrate that it is possible to significantly modulate the selectivity ratio for these two channel subtypes in order to improve the potency of a given analog for hNa V 1.6 and/or hNa V 1.7 subtypes. In addition, selective analogs for hNa V 1.7, possessing better safety profiles, were produced to limit neuromuscular impairments.