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International audience; Scorpion envenomation is a serious health problem in tropical and subtropical zones. The access to scorpion antivenom is sometimes limited in availability and specificity. The classical production process is cumbersome, from the hyper-immunization of the horses to the IgG digestion and purification of the F(ab)'(2) antibody fragments. The production of recombinant antibody fragments in Escherichia coli is a popular trend due to the ability of this microbial host to produce correctly folded proteins. Small recombinant antibody fragments, such as single-chain variable fragments (scFv) and nanobodies (VHH), have been constructed to recognize and neutralize the neurotoxins responsible for the envenomation symptoms in humans. They are the focus of interest of the most recent studies and are proposed as potentially new generation of pharmaceuticals for their use in immunotherapy against scorpion stings of the Buthidae family. This literature review comprises the current status on the scorpion antivenom market and the analyses of cross-reactivity of commercial scorpion anti-serum against non-specific scorpion venoms. Recent studies on the production of new recombinant scFv and nanobodies will be presented, with a focus on the Androctonus and Centruroides scorpion species. Protein engineering-based technology could be the key to obtaining the next generation of therapeutics capable of neutralizing and cross-reacting against several types of scorpion venoms.

Leishmaniasis is the 3rd most challenging vector-borne disease after malaria and lymphatic filariasis. Currently, no vaccine candidate is approved or marketed against leishmaniasis due to difficulties in eliciting broad immune responses when using sub-unit vaccines. The aim of this work was the design of a particulate sub-unit vaccine for vaccination against leishmaniasis. The poly (D,L-lactide) nanoparticles (PLA-NPs) were developed in order to efficiently adsorb a recombinant L. major histone H2B (L. major H2B) and to boost its immunogenicity. Firstly, a study was focused on the production of well-formed nanoparticles by the nanoprecipitation method without using a surfactant and on the antigen adsorption process under mild conditions. The set-up preparation method permitted to obtain H2B-adsorbed nanoparticles H2B/PLA (adsorption capacity of about 2.8% (w/w)) with a narrow size distribution (287 nm) and a positive zeta potential (30.9 mV). Secondly, an in vitro release assay performed at 37 °C, pH 7.4, showed a continuous release of the adsorbed H2B for almost 21 days (30%) from day 7. The immune response of H2B/PLA was investigated and compared to H2B + CpG7909 as a standard adjuvant. The humoral response intensity (IgG) was substantially similar between both formulations. Interestingly, when challenged with the standard parasite strain (GLC94) isolated from a human lesion of cutaneous leishmaniasis, mice showed a significant reduction in footpad swelling compared to unvaccinated ones, and no deaths occurred until week 17th. Taken together, these results demonstrate that PLA-NPs represent a stable, cost-effective delivery system adjuvant for use in vaccination against leishmaniasis.

The purpose of this study was to determine the phylogenetic relationships among the primary betanodavirus strains circulating in Tunisian coastal waters. A survey was conducted to investigate nodavirus infections at 15 European sea bass Dicentrarchus labrax and gilthead sea bream Sparus aurata farming sites located along the northern and eastern coasts of Tunisia. The primary objective of the study was to create epidemiological awareness of these infections by determining phylogenetic relationships between the main betanodavirus strains circulating during the period 2012-2019, using RNA1 and/or RNA2 genome segments. Approximately 40% (118 of 294) tissue pools tested were positive for betanodavirus. Positive pools were distributed across all of the sampling sites. While fish mortalities were always correlated with the presence of virus in sea bass, a severe outbreak was also identified in sea bream larvae in 2019. Phylogenetic analysis revealed that almost all Tunisian strains from both sea bass and sea bream irrespective of outbreaks clustered within the RGNNV genotype. It is noteworthy that samples collected during the 2019 outbreak from sea bream contained both RNA1 and RNA2 fragments belonging to the RGNNV and SJNNV genotype, respectively, an indication of viral genome reassortment. To our knowledge, this is the first report of reassortant betanodavirus in Tunisia.

R.A73 Enterococcus faecium (HG937697) was tested for investigating anti-nodaviral activity and immunomodulatory properties on E-11 fish cells. Non R.A73-related toxicity was confirmed. Likewise, in-vitro inhibition of nodavirus replication and gene expression of nodavirus capsid protein gene in addition to CCR3, IL-10 and TNFα genes were evaluated using qPCR. A preventive treatment of E-11 cells by the bacterial suspension generated a remarkable reduction in virus loads (RQ values from 0.1 to 0.9) at day 7. Interestingly, R.A73 E. faecium showed a significant gene expression upregulation of proinflammatory IL-10 cytokine whereas it did not significantly enhance CCR3 nor TNF-α immune gene expression in E-11 cells. Therefore, add-on its probiotic potential, R.A73 E. faecium is able to stimulate the immune system in the preventive phase throughout the differential expression of main inflammatory genes. This study provides the first evidence that R.A73 E. faecium strain can prevents effectively nodavirus replication, in vitro.

Rapid and specific detection of pathogenic bacteria in fecal samples is of critical importance for the diagnosis of neonatal diarrhea in veterinary clinics. Nanobodies are a promising tool for the treatment and diagnosis of infectious diseases due to their unique recognition properties. In this study, we report the design of a nanobody-based magnetofluorescent immunoassay for the sensitive detection of pathogenic Escherichia coli F17-positive strains (E. coli F17). For this, a camel was immunized with purified F17A protein from F17 fimbriae and a nanobody library was constructed by phage display. Two specific anti-F17A nanobodies (Nbs) were selected to design the bioassay. The first one (Nb1) was conjugated to magnetic beads (MBs) to form a complex capable of efficiently capturing the target bacteria. A second horseradish peroxidase (HRP)-conjugated nanobody (Nb4) was used for detection by oxidizing o-phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (DAP). Our results show that the immunoassay recognizes E. coli F17 with high specificity and sensitivity, with a detection limit of 1.8 CFU/mL in only 90 min. Furthermore, we showed that the immunoassay can be applied to fecal samples without pretreatment and remains stable for at least one month when stored at 4 °C.

Scorpion envenoming is a severe health problem in many regions causing significant clinical toxic effects and fatalities. In the Middle East/North Africa (MENA) region, Buthidae scorpion stings are responsible for devastating toxic outcomes in human. The only available specific immunotherapeutic treatment is based on IgG fragments of animal origin. To overcome the limitations of classical immunotherapy, we have demonstrated the in vivo efficacy of NbF12-10 bispecific nanobody at preclinical level. Nanobodies were developed against BotI analogues belonging to a distinct structural and antigenic group of scorpion toxins, occurring in the MENA region. From Buthus occitanus tunetanus venom, BotI-like toxin was purified. The 41 N-terminal amino acid residues were sequenced, and the LD50 was estimated at 40 ng/mouse. The BotI-like toxin was used for dromedary immunization. An immune VHH library was constructed, and after screening, two nanobodies were selected with nanomolar and sub-nanomolar affinity and recognizing an overlapping epitope. NbBotI-01 was able to neutralize 50% of the lethal effect of 13 LD50 BotI-like toxins in mice when injected by i.c.v route, whereas NbBotI-17 neutralized 50% of the lethal effect of 7 LD50. Interestingly, NbBotI-01 completely reduced the lethal effect of the 2 LD50 of BotG50 when injected at 1:4 molar ratio excess. More interestingly, an equimolar mixture of NbBotI-01 with NbF12-10 neutralized completely the lethal effect of 7 and 5 LD50 of BotG50 or AahG50, at 1:4 and 1:2 molar ratio, respectively. Hence, NbBotI-01 and NbF12-10 display synergic effects, leading to a novel therapeutic candidate for treating Buthus occitanus scorpion stings in the MENA region.

Tenascin-C (TNC) and tenascin-W (TNW), large hexameric glycoproteins overexpressed in the tumor microenvironment, are useful tumor biomarkers for theranostic applications. For now, polyclonal and monoclonal antibodies, as well as aptamers targeting TNC and TNW have been developed. However, the immunostaining sensitivity of antibodies is very heterogenous. The main aim of this study was to generate antibodies in dromedary that detect TNC and TNW, respectively. We show that immune sera from immunized dromedaries are able to specifically bind native TNC and TNW by ELISA and also to detect TNC and TNW in matrix tracks of mammary tumors by immunostaining. Furthermore, we demonstrate that purified IgG subtypes are able to interact specifically with TNC or TNW by ELISA and immunostaining. These camelid antibodies are a good basis to develop tools for the detection of TNC and TNW in the tumor microenvironment and could potentially have a broader application for early diagnosis of solid cancers.

Scorpion α-toxins are neurotoxins that target the fast inactivation mechanism of voltage-gated sodium (Na

The extracellular matrix molecule Tenascin-C (TNC) promotes cancer and chronic inflammation by multiple mechanisms. Recently, TNC was shown to promote an immune suppressive tumor microenvironment (TME) through binding soluble chemoattracting factors, thus retaining leukocytes in the stroma. TNC also binds to fibronectin (FN) and other molecules, raising the question of a potential common TNC binding mechanism. By sequence comparison of two TNC-interacting domains in FN, the fifth (FN5) and thirteenth (FN13) fibronectin type III domains we identified a MAtrix REgulating MOtif "MAREMO" or M-motif that is highly conserved amongst vertebrates. By sequence analysis, structural modeling and functional analysis we found also putative M-motifs in TNC itself. We showed by negative staining electron microscopic imaging that the M-motif in FN mediates interactions with FN as well as with TNC. We generated two M-motif mimetic peptides P5 and P13 resembling the M-motif in FN5 and FN13, respectively. By using structural information we modelled binding of these M-motif mimetics revealing a putative MAREMO binding site MBS in FN5 and TN3, respectively overlapping with the M-motif. We further demonstrated that the M-motif mimetic peptides blocked several functions of TNC, such as binding of TNC to FN, cell rounding on a mixed FN/TNC substratum, FN matrix expression and subsequent assembly, TNC-induced signaling and gene expression, TNC chemokine binding and dendritic cell retention, thus providing novel opportunities to inhibit TNC actions. Our results suggest that targeting the MAREMO/MBS interaction could be exploited for reducing inflammation and matrix functions in cancer and fibrosis.

Background Whole-genome sequencing using high throughput technologies has revolutionized and speeded up the scientific investigation of bacterial genetics, biochemistry, and molecular biology. Lactic acid bacteria (LABs) have been extensively used in fermentation and more recently as probiotics in food products that promote health. Genome sequencing and functional genomics investigations of LABs varieties provide rapid and important information about their diversity and their evolution, revealing a significant molecular basis. This study investigated the whole genome sequences of the Enterococcus faecium strain (HG937697), isolated from the mucus of freshwater fish in Tunisian dams. Genomic DNA was extracted using the Quick-GDNA kit and sequenced using the Illumina HiSeq2500 system. Sequences quality assessment was performed using FastQC software. The complete genome annotation was carried out with the Rapid Annotation using Subsystem Technology (RAST) web server then NCBI PGAAP. Results The Enterococcus faecium R.A73 assembled in 28 contigs consisting of 2,935,283 bps. The genome annotation revealed 2884 genes in total including 2834 coding sequences and 50 RNAs containing 3 rRNAs (one rRNA 16 s, one rRNA 23 s and one rRNA 5 s) and 47 tRNAs. Twenty-two genes implicated in bacteriocin production are identified within the Enterococcus faecium R.A73 strain. Conclusion Data obtained provide insights to further investigate the effective strategy for testing this Enterococcus faecium R.A73 strain in the industrial manufacturing process. Studying their metabolism with bioinformatics tools represents the future challenge and contribution to improving the utilization of the multi-purpose bacteria in food.

Background: Whole-genome sequencing using high throughput technologies has revolutionized and speeded up the scientific investigation of bacterial genetics, biochemistry, and molecular biology. Lactic acid bacteria (LABs) have been extensively used in fermentation and more recently as probiotics in food products that promote health. Genome sequencing and functional genomics investigations of LABs varieties provide rapid and important information about their diversity and their evolution, revealing a significant molecular basis.This study investigates the whole genome sequences of the Enterococcus faecium genome (HG937697), isolated from the mucus of freshwater fish in Tunisian dams. Genomic DNA was extracted using the Quick-GDNA kit (Zymoresearch) and sequenced using the Illumina HiSeq2500 system. Sequences quality assessment was performed using FastQC software. The complete genome annotation was carried out with the RAST web server then NCBI PGAAP. Results: The Enterococcus faecium R.A73 assembled in 28 contigs consisting of 2,935,283 bps. The genome annotation revealed 2,884 genes in total including 2,834 coding sequences and 50 RNAs containing 3 rRNAs (one rRNA 16s, one rRNA 23s and one rRNA 5s) and 47 tRNAs. Twenty-two genes implicated in bacteriocin production are identified within the Enterococcus faecium R.A73 genome.Conclusions: Data obtained provide insights to further investigate the effective strategy for testing this Enterococcus faecium R.A73 strain in the industrial manufacturing process. Studying their metabolism with bioinformatics tools represents the future challenge and contribution to improving the utilization of the multi-purpose bacteria in food.

New Delhi Metallo-&beta, lactamase-1 (NDM-1) is the most prevalent type of metallo-&beta, lactamase, able to hydrolyze almost all antibiotics of the &beta, lactam group, leading to multidrug-resistant bacteria. To date, there are no clinically relevant inhibitors to fight NDM-1. The use of dromedary polyclonal antibody inhibitors against NDM-1 represents a promising new class of molecules with inhibitory activity. In the current study, immunoreactivities of dromedary Immunoglobulin G (IgG) isotypes containing heavy-chain and conventional antibodies were tested after successful immunization of dromedary using increasing amounts of the recombinant NDM-1 enzyme. Inhibition kinetic assays, performed using a spectrophotometric method with nitrocefin as a reporter substrate, demonstrated that IgG1, IgG2, and IgG3 were able to inhibit not only the hydrolytic activity of NDM-1 but also Verona integron-encoded metallo-&beta, lactamase (VIM-1) (subclass B1) and L1 metallo-&beta, lactamase (L1) (subclass B3) with inhibitory concentration (IC50) values ranging from 100 to 0.04 &mu, M. Investigations on the ability of IgG subclasses to reduce the growth of recombinant Escherichia coli BL21(DE3)/codon plus cells containing the recombinant plasmid expressing NDM-1, L1, or VIM-1 showed that the addition of IgGs (4 and 8 mg/L) to the cell culture was unable to restore the susceptibility of carbapenems. Interestingly, IgGs were able to interact with NDM-1, L1, and VIM-1 when tested on the periplasm extract of each cultured strain. The inhibitory concentration was in the micromolar range for all &beta, lactams tested. A visualization of the 3D structural basis using the three enzyme Protein Data Bank (PDB) files supports preliminarily the recorded inhibition of the three MBLs.

Lactic acid bacteria (LAB) are traditionally used in food and feed bio-industries as they have health-promoting probiotic effect for their host and currently considered safe for human and animal consumption. Recently, we isolated 177 strains from freshwater fishes of dams of Tunisia exhibiting antimicrobial activities against various food-borne pathogens and generated a laboratory biobank to be characterized. Herein, we investigated whether MALDI-TOF could assist rapid identification of LAB genus. First, a lactic microflora-selective Man Rogosa Sharpe medium was used. Isolates were further screened according to the antimicrobial activity, using well-difusion agar. In total, four major genera have been well identified and species frequency has been estimated (74% Enterococcus, 24% Leuconostoc, 3% Lactococcus, and 2% Vagococcus). Eighteen isolates was further analyzed using MALDI-TOF. Comparative analysis of spectral fingerprints to six referenced MALDI-TOF fingerprints was carried out from the recently developed USC library ( www.spectrabank.org ). The intra- and inter-specific phyloproteomic relationships among strains were compared to available phylogenetic data based on 16S rDNA genes. This study showed that MALDI-TOF MS is a potent and reliable rapid method for both discrimination and identification of LABs. This highlights the insight of proteomic approach into the screening of food-derived beneficial microorganisms.

International audience; Scorpion envenoming and its treatment is a public health problem in many parts of the world due to highly toxic venom polypeptides diffusing rapidly within the body of severely envenomed victims. Recently, 38 AahII-specific Nanobody sequences (Nbs) were retrieved from which the performance of NbAahII10 nanobody candidate, to neutralize the most poisonous venom compound namely AahII acting on sodium channels, was established. Herein, structural computational approach is conducted to elucidate the Nb-AahII interactions that support the biological characteristics, using Nb multiple sequence alignment (MSA) followed by modeling and molecular docking investigations (RosettaAntibody, ZDOCK software tools). Sequence and structural analysis showed two dissimilar residues of NbAahII10 CDR1 (Tyr27 and Tyr29) and an inserted polar residue Ser30 that appear to play an important role. Indeed, CDR3 region of NbAahII10 is characterized by a specific Met104 and two negatively charged residues Asp115 and Asp117. Complex dockings reveal that NbAahII17 and NbAahII38 share one common binding site on the surface of the AahII toxin divergent from the NbAahII10 one's. At least, a couple of NbAahII10 - AahII residue interactions (Gln38 - Asn44 and Arg62, His64, respectively) are mainly involved in the toxic AahII binding site. Altogether, this study gives valuable insights in the design and development of next generation of antivenom.

Graphical abstract, 7-Ketocholesterol, which is one of the earliest cholesterol oxidization products identified, is essentially formed by the auto-oxidation of cholesterol. In the body, 7-ketocholesterol is both provided by food and produced endogenously. This pro-oxidant and pro-inflammatory molecule, which can activate apoptosis and autophagy at high concentrations, is an abundant component of oxidized Low Density Lipoproteins. 7-Ketocholesterol appears to significantly contribute to the development of age-related diseases (cardiovascular diseases, age-related macular degeneration, and Alzheimer's disease), chronic inflammatory bowel diseases and to certain cancers. Recent studies have also shown that 7-ketocholesterol has anti-viral activities, including on SARS-CoV-2, which are, however, lower than those of oxysterols resulting from the oxidation of cholesterol on the side chain. Furthermore, 7-ketocholesterol is increased in the serum of moderately and severely affected COVID-19 patients. In the case of COVID-19, it can be assumed that the antiviral activity of 7-ketocholesterol could be counterbalanced by its toxic effects, including pro-oxidant, pro-inflammatory and pro-coagulant activities that might promote the induction of cell death in alveolar cells. It is therefore suggested that this oxysterol might be involved in the pathophysiology of COVID-19 by contributing to the acute respiratory distress syndrome and promoting a deleterious, even fatal outcome. Thus, 7-ketocholesterol could possibly constitute a lipid biomarker of COVID-19 outcome and counteracting its toxic effects with adjuvant therapies might have beneficial effects in COVID-19 patients.

International audience; Voltage-gated potassium (Kv) channels form cells repolarizing power and are commonly expressed in excitable cells. In non-excitable cells, Kv channels such as Kv2.1 are involved in cell differentiation and growth. Due to the involvement of Kv2.1 in several physiological processes, these channels are promising therapeutic targets. To develop Kv2.1 specific antibody-based channel modulators, we applied a novel approach and immunized a dromedary with heterologous Ltk- cells that overexpress the mouse Kv2.1 channel instead of immunizing with channel protein fragments. The advantage of this approach is that the channel is presented in its native tetrameric configuration. Using a Cell-ELISA, we demonstrated the ability of the immune serum to detect Kv2.1 channels on the surface of cells that express the channel. Then, using a Patch Clamp electrophysiology assay we explored the capability of the dromedary serum in modulating Kv2.1 currents. Cells that were incubated for 3h with serum taken at Day 51 from the start of the immunization displayed a statistically significant 2-fold reduction in current density compared to control conditions as well as cells incubated with serum from Day 0. Here we show that an immunization approach with cells overexpressing the Kv2.1 channel yields immune serum with Kv2.1 specific antibodies.

Stability and quality control of therapeutic protein formulations is a substantial part of drug development process. The objective of this study is to obtain information about stability of a recombinant human granulocyte colony stimulating factor (rhG-CSF) against various stress factors. This will play a crucial role in the finished product formulation development. In this study, rhG-CSF was exposed to various chemical and physical stress conditions at different levels in order to identify degradation pathways and the nature of impurities generated. Experiments were performed by a combination of orthogonal analytical techniques (reversed phase chromatography (RP-HPLC), size exclusion chromatography (SEC-HPLC), polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF)) to set and characterize the different degraded samples. The SEC-HPLC results suggest that the major degradation factors generating aggregated forms of the protein are basically thermal stress, freeze-thaw cycles and vortexing. Meanwhile, deamidated rhG-CSF was induced by basic pH as shown by IEF electrophoregram. As well, oxidized forms were generated increasingly with the time of exposure to hydrogen peroxide as outlined by RP-HPLC analysis. Based on these results, it was possible to define the storage and handling conditions of rhG-CSF finished product during its shelf life.

A central role for advanced glycation end products (AGE) and their receptor (RAGE) in the pathogenesis of multiple cancer types, including colorectal cancer (CRC) was reported. We investigated the association between CRC and rs2853807, rs77170610, rs184003, rs1035798, rs2070600, rs1800684, rs1800624, and rs1800625 RAGE gene (AGER) polymorphic variants. Study subjects comprised 293 CRC patients [186 colon cancer (CC) and 107 rectal cancer (RC)] patients), and 264 age-, gender-, BMI-, and ethnicity-matched controls. Minor allele frequency (MAF) of rs77170610 and rs1800625 were significantly lower, while MAF of rs1035798 was significantly higher in CRC patients compared to control subjects, which was associated with reduced and increased risk of CRC, respectively; MAF of the remaining variants was comparable between CRC patients and controls. Significant difference in the distribution of rs2853807 and rs77170610 genotypes was seen between CRC patients and controls, with both variants associated with decreased risk of CRC. Comparison of the distribution of minor allele-carrying genotypes in CC and RC patient subgroups revealed lack of significant difference in the distribution of these genotypes between the patient subgroups. In view of the lack of LD between rs2853807 and rs77170610 with other variants, six-locus (rs184003, rs1035798, rs2070600, rs1800684, rs1800624, rs1800625) haplotypes were constructed. Haplotype analysis did not identify any specific 6-locus AGER haplotype associated with CRC. In conclusion, AGER gene rs2853807 and rs77170610 variants rs77170610 are associated with altered risk of CRC in Tunisians, but with no discrimination between CC and RC types.

The KCNB1 gene variants were differentially associated with cancers. However, their association with colorectal cancer has not yet been explored. We investigated the contribution of the KCNB1 gene variants rs3331, rs1051295, and indel (insertion/deletion) rs11468831 Polymorphism as predictors of the treatment response in colorectal cancer patients. A retrospective study, which involved 291 Tunisian colorectal cancer patients (aged 60.0 ± 13.1 years), who were stratified into responder and non-responder groups, according to TNM stages and their responsiveness to chemotherapy based on fluorouracil. KCNB1 genotyping was performed with amplification-refractory mutation system–polymerase chain reaction, and was confirmed by Sanger sequencing. Sex-specific response was found and colorectal cancer females are less likely to achieve a positive response during the chemotherapy strategy, compared to males. Weight and body mass index, tumor size, and tumor localization are considered as predictive factors to treatment responsiveness. Carriage of rs11468831 Ins allele was significantly associated with successful therapy achievement ( p adjusted

The potency of a recombinant human erythropoietin (rhEPO) pharmaceutical preparation was evaluated by the normocythaemic mice bioassay using standardized and randomized protocol of European Pharmacopeia 5.0. Eight-week-old normocythaemic B6D2F1/c mice received single daily injections of test solutions (EPOTIN recombinant human erythropoietin, batch preparation) at different concentrations. Erythropoietin biological reference product (batch 3.3) was used as standard. The suitability of the bioassay was recorded according to gender and age. The blood sampling was performed 4 days after the last injection and reticulocytes were counted by manual and automated methods. Intra-assay precision was analyzed and Bland-Altman plot was obtained. The effect of rhEPO administration doses on the hemoglobin concentration, hematocrit and number of red blood cells in the blood stream were investigated. The dose response curve was linear in the concentration range of 20–80 IU/ml. The relative potency was estimated to 13.78 and 71.53% of the stated potency, for male and female mice, respectively. The recorded limits were compared to the required ones. This study gives valuable insights for better control of biosimilars and bioproducts.

The webinars series form part of the regular activities of any scientific consortium that aims to strengthen research activities and foster collaborations amongst the different partners. The webinars are intended to foster the exchange of ideas, build potential collaborations across multiple disciplines and enabling the participation and sharing of knowledge in current research. The Ten Simple Rules for organizing a webinar series can be summarized as follow: The webinar coordination team assists with all the planning and logistics for hosting a webinar (Rule 1); Choosing webinar themes requires mapping of the target audience needs and interest (Rule 2); Drafting a webinar planning checklist through regular planning meetings as well as post webinar meetings (Rule 3); Decentralized webinar organization of tasks and resources through accessible shared space (Rule 4); Planing early and settling on the provisional dates and times of the webinar events along with their themes (Rule 5); Choosing and settling on convenient and user friendly webinar platform (Rule 6); Approaching and confirming potential speakers (Rule 7); Obtaining the webinar title, abstract and presenter’s biography for creating the webinar announcement through emails and social media channels (Rule 8); Allocating time for the platform orientation (Rule 9); and Keeping close-up track of webinar metrics for regular assessment and evaluation (Rule 10). These Ten Simple Rules shared with the computational biology community will help those who have not yet ventured into training through webinars to learn from our experience. In our experience, the feedback from the post-webinar surveys clearly demonstrate that webinars are an effective way to create a two-way conversation between presenters and participants via a web-based platform.

Significant progress has been made in immunological studies of scorpion toxins and several formats of antibodies directed against scorpion toxins have been reported. Some of these are commonly used in a specific treatment against envenoming; others are primarily used for immuno-biochemical characterizations. The preparation protocol of the antibody or its fragments can be substantially different from one laboratory to another, which complicates a direct comparison of the potency of the antivenom. The use of immune sera, the total immunoglobulin fraction or Fab and Fab'2 fragments as the therapeutic agent is widespread. A number of monoclonal antibodies have also been reported and used for engineering of Fv, ScFv or Fab fragments. Recently, a novel antibody format - known as nanobodies - derived from HCAbs of camelids and selected after phage display shows great potential to provide a more efficient therapy against scorpion envenoming. Subsequent bispecific derivatives have been designed and their pharmacokinetics have been studied. Distinct advantages and disadvantages have been attributed to these equine, murine or camelid antibodies and their derived fragments. Some fragments are easily amenable into next generation therapeutics after proper manufacturing and provide an ensured availability of the product to the medical community. Through examples, we will show how the comparison of the serotherapeutic effectiveness is compromised due to the absence of standardization, on the preparation of immunogens, production processes and / or nature of the products. We will report on recent advances in the field.

International audience; During scorpion envenoming, highly toxic small polypeptides of the venom diffuse rapidly within the victim, causing serious medical problems. Nanobodies (Nbs), the recombinant single-domain antigen-binding fragments of camel-specific heavy-chain only antibodies, offer special advantages in therapy over classic antibody fragments due to their robustness and smaller size, matching the size of the scorpion toxins. Recently, a potent AahII scorpion toxin-neutralizing Nb was identified. However, this NbAahII10 contains a single Cys in its first antigen-binding loop, leading to Nb dimerization upon prolonged storage. In this work, we first investigate the efficacy of NbAahII10 variants in which this Cys was substituted by Ala, Ser or Thr. Second, the NbAahII10 Cys/Ser mutant displaying the best functional properties is subsequently humanized. It is demonstrated that the maximally humanized version of NbAahII10 Cys/Ser maintains its high affinity for the antigen without conceding much on expression yield and stability. More importantly, its neutralizing capacity is preserved as all mice survive injections of seven LD(50) and 50% of mice survived nine LD(50) of the scorpion toxin. Thus, this humanized Nb is the best candidate to develop a therapy in human against the most toxic venom compound of one of the most dangerous scorpions.

International audience; Envenoming following scorpion sting is a common emergency in many parts of the world. Our aim was to ameliorate the current 100-kDa horse plasma antivenom serum (PAS)-derived Fab'(2) to more quickly reach the highly diffusible scorpion toxins (7 kDa). We immunized dromedaries with toxins from Androctonus australis hector (Aah) scorpions and cloned the single-domain antibody fragments or nanobodies (15 kDa) from their B cells. Nanobodies against AahI' toxin (with AahII the most toxic compound of the venom) were retrieved from the libraries, and their AahI'-toxin neutralization was monitored in mice. Remarkably, the NbAahI'F12 fully protected mice against 100 LD(50) of AahI' administered intracerebroventricularly. Moreover, where PAS failed completely to neutralize 2 LD(50) of crude venom injected subcutaneously, the designed bispecific NbF12-10 against AahI'/AahII toxins succeeded in neutralizing 5 LD(50). Finally, in a challenge assay in which mice were subcutaneously injected with a lethal dose of scorpion venom, the subsequent intravenous injection of 85 microg of NbF12-10 protected all mice, even if the whole procedure was repeated 3 times. Furthermore, the NbF12-10 remained fully protective when mice with severe signs of envenoming were treated a few minutes before the untreated mice died.

BotXIV and LqhαIT are two structurally related long chain scorpion α-toxins that inhibit sodium current inactivation in excitable cells. However, while LqhαIT from Leiurus quinquestriatus hebraeus is classified as a true and strong insect α-toxin, BotXIV from Buthus occitanus tunetanus is characterized by moderate biological activities. To assess the possibility that structural differences between these two molecules could reflect the localization of particular functional topographies, we compared their sequences. Three structurally deviating segments located in three distinct and exposed loops were identified. They correspond to residues 8–10, 19–22, and 38–43. To evaluate their functional role, three BotXIV/LqhαIT chimeras were designed by transferring the corresponding LqhαIT sequences into BotXIV. Structural and antigenic characterizations of the resulting recombinant chimera show that BotXIV can accommodate the imposed modifications, confirming the structural flexibility of that particular α/β fold. Interestingly, substitution of residues 8–10 yields to a new electrophysiological profile of the corresponding variant, partially comparable to that one of α-like scorpion toxins. Taken together, these results suggest that even limited structural deviations can reflect functional diversity, and also that the structure–function relationships between insect α-toxins and α-like scorpion toxins are probably more complex than expected.

International audience; We have constructed a cDNA library from venom glands of the scorpion Buthus occitanus tunetanus and cloned a DNA sequence that encodes an alpha-toxin. This clone was efficiently expressed in Escherichia coli as a fusion protein with two Ig-binding (Z) domains of protein A from Staphylococcus aureus. After CNBr treatment of the fusion protein and HPLC purification, we obtained approximately 1 mg recombinant apha-toxin/l bacterial culture. The toxin, called Bot XIV, displays no toxicity towards mammals but is active towards insects as shown by its paralytic activity against Blatella germanica cockroach and by electrophysiological studies on Periplaneta americana cockroaches. The Bot XIV protein fused to two Z domains is highly immunogenic in mice and induces production of antisera that specifically recognize and neutralize highly toxic components that had been injected into mice. This fusion protein could be very useful for development of potent protective antisera against scorpion venoms.

International audience; Scorpions represent a significant threat to humans and animals in various countries throughout the world. Recently, we introduced Nanobodies (Nbs) to combat more efficiently scorpion envenoming and demonstrated the performance of NbAahIF12 and NbAahII10 to neutralize scorpion toxins of Androctonus australis hector venom. A bispecific Nb construct (NbF12-10) comprising these two Nbs is far more protective than the classic Fab'(2) based therapy and is the most efficient antivenom therapy against scorpion sting in preclinical studies. Now we investigate the biodistribution and pharmacokinetics of (99m)Tc labeled Nbs by in vivo imaging in rodents and compared these data with those of the Fab'(2) product (PAS). The pharmacodynamics of the Nbs was investigated in rats by in vivo echocardiography and it is shown that NbF12-10 prevents effectively the hemodynamic disturbances induced by a lethal dose of venom. Moreover, even a late injection of NbF12-10 restores the heart rate and brings the blood pressure to baseline values. Histology confirms that NbF12-10 prevents lung and heart lesions of treated mice after envenoming. In conjunction, in this preclinical study, we provide proof of concept that NbF12-10 prevents effectively the fatal disturbances induced by Androctonus venom, and that the Nanobody based therapeutic has a potential to substitute the classic Fab'(2) based product as immunotherapeutic in scorpion envenoming. Further clinical study using larger cohorts of animals should be considered to confirm the full protecting potential of our NbF12-10.