The scorpion toxin BeKm-1 blocks hERG cardiac potassium channels using an indispensable arginine residue.

BeKm-1 is a peptide toxin from scorpion venom that blocks the pore of the potassium channel hERG (Kv11.1) in the human heart. Although individual protein structures have been resolved, the structure of the complex between hERG and BeKm-1 is unknown. Here, we used molecular dynamics and ensemble docking, guided by previous double-mutant cycle analysis data, to obtain an in silico model of the hERG-BeKm-1 complex. Adding to the previous mutagenesis study of BeKm-1, our model uncovers the key role of residue Arg20, which forms three interactions (a salt bridge and hydrogen bonds) with the channel vestibule simultaneously. Replacement of this residue even by lysine weakens the interactions significantly. In accordance, the recombinantly produced BeKm-1R20K mutant exhibited dramatically decreased activity on hERG. Our model may be useful for future drug design attempts.

Divergence in toxin antigenicity and venom enzymes in Tityus melici, a medically important scorpion, despite transcriptomic and phylogenetic affinities with problematic Brazilian species.

The Brazilian scorpion Tityus melici, native to Minas Gerais and Bahia, is morphologically related to Tityus serrulatus, the most medically significant species in Brazil. Despite inhabiting scorpion-envenomation endemic regions, T. melici venom remains unexplored. This work evaluates T. melici venom composition and function using transcriptomics, enzymatic activities, and in vivo and in vitro immunological analyses. Next-Generation Sequencing unveiled 86 components putatively involved in venom toxicity: 39 toxins, 28 metalloproteases, seven disulfide isomerases, six hyaluronidases, three phospholipases and three amidating enzymes. T. serrulatus showed the highest number of toxin matches with 80-100 % sequence similarity. T. melici is of medical importance as it has a venom LD50 of 0.85 mg/kg in mice. We demonstrated venom phospholipase A2 activity, and elevated hyaluronidase and metalloprotease activities compared to T. serrulatus, paralleling our transcriptomic findings. Comparison of transcriptional levels for T. serrulatus and T. melici venom metalloenzymes suggests species-specific expression patterns in Tityus. Despite close phylogenetic association with T. serrulatus inferred from COI sequences and toxin similarities, partial neutralization of T. melici venom toxicity was achieved when using the anti-T. serrulatus antivenom, implying antigenic divergence among their toxins. We suggest that the Brazilian therapeutic scorpion antivenom could be improved to effectively neutralize T. melici venom.

Genome-wide identification, structural homology analysis, and evolutionary diversification of the phospholipase D gene family in the venom gland of three scorpion species.

BACKGROUND: Venom phospholipase D (PLDs), dermonecrotic toxins like, are the major molecules in the crude venom of scorpions, which are mainly responsible for lethality and dermonecrotic lesions during scorpion envenoming. The purpose of this study was fivefold: First, to identify transcripts coding for venom PLDs by transcriptomic analysis of the venom glands from Androctonus crassicauda, Hottentotta saulcyi, and Hemiscorpius lepturus; second, to classify them by sequence similarity to known PLDs and motif extraction method; third, to characterize scorpion PLDs; fourth to structural homology analysis with known dermonecrotic toxins; and fifth to investigate phylogenetic relationships of the PLD proteins. RESULTS: We found that the venom gland of scorpions encodes two PLD isoforms: PLD1 ScoTox-beta and PLD2 ScoTox-alpha I. Two highly conserved regions shared by all PLD1s beta are GAN and HPCDC (HX2PCDC), and the most important conserved regions shared by all PLD2s alpha are two copies of the HKDG (HxKx4Dx6G) motif. We found that PLD1 beta is a 31-43 kDa acidic protein containing signal sequences, and PLD2 alpha is a 128 kDa basic protein without known signal sequences. The gene structures of PLD1 beta and PLD2 alpha contain 6 and 21 exons, respectively. Significant structural homology and similarities were found between the modeled PLD1 ScoTox-beta and the crystal structure of dermonecrotic toxins from Loxosceles intermedia. CONCLUSIONS: This is the first report on identifying PLDs from A. crassicauda and H. saulcyi venom glands. Our work provides valuable insights into the diversity of scorpion PLD genes and could be helpful in future studies on recombinant antivenoms production.

Chlorotoxin binds to both matrix metalloproteinase 2 and neuropilin 1.

Chlorotoxin (CTX), a scorpion venom-derived 36-residue miniprotein, binds to and is taken up selectively by glioblastoma cells. Previous studies provided controversial results concerning target protein(s) of CTX. These included CLC3 chloride channel, matrix metalloproteinase 2 (MMP-2), regulators of MMP-2, annexin A2, and neuropilin 1 (NRP1). The present study aimed at clarifying which of the proposed binding partners can really interact with CTX using biochemical methods and recombinant proteins. For this purpose, we established two new binding assays based on anchoring the tested proteins to microbeads and quantifying the binding of CTX by flow cytometry. Screening of His-tagged proteins anchored to cobalt-coated beads indicated strong interaction of CTX with MMP-2 and NRP1, whereas binding to annexin A2 was not confirmed. Similar results were obtained with fluorophore-labeled CTX and CTX-displaying phages. Affinity of CTX to MMP-2 and NRP1 was assessed by the "immunoglobulin-coated bead" test, in which the proteins were anchored to beads by specific antibodies. This assay yielded highly reproducible data using both direct titration and displacement approach. The affinities of labeled and unlabeled CTX appeared to be similar for both MMP-2 and NRP1 with estimated KD values of 0.5 to 0.7 µM. Contrary to previous reports, we found that CTX does not inhibit the activity of MMP-2 and that CTX not only with free carboxyl end but also with carboxamide terminal end binds to NRP1. We conclude that the presented robust assays could also be applied for affinity-improving studies of CTX to its genuine targets using phage display libraries.

Production of recombinant scorpion antivenoms in E. coli: current state and perspectives.

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. KEY POINTS: • Commercial antivenoms consist of predominantly purified equine F(ab)'2fragments. • Nanobody-based antivenom can neutralize Androctonus venoms and have a low immunogenicity. • Affinity maturation and directed evolution are used to obtain potent scFv families against Centruroides scorpions.

An overview of Tityus cisandinus scorpion venom: Transcriptome and mass fingerprinting reveal conserved toxin homologs across the Amazon region and novel lipolytic components.

Tityus cisandinus, a neglected medically important scorpion in Ecuadorian and Peruvian Amazonia, belongs to a complex of species related to the eastern Amazon endemic Tityus obscurus, spanning a distribution of ca. 4000 km. Despite high morbidity and mortality rates, no effective scorpion antivenom is currently available in the Amazon region. Knowledge of the structural/functional relationships between T. cisandinus venom components and those from related Amazonian species is crucial for designing region-specific therapeutic antivenoms. In this work, we carried out the first venom gland transcriptomic study of an Amazonian scorpion outside Brazil, T. cisandinus. We also fingerprinted its total venom through MALDI-TOF MS, which supported our transcriptomic findings. We identified and calculated the expression level of 94 components: 60 toxins, 25 metalloproteases, five disulfide isomerases, three amidating enzymes, one hyaluronidase, and also uncovered transcripts encoding novel lipolytic beta subunits produced by New World buthid scorpions. This study demonstrates the high similarity between T. cisandinus and T. obscurus venoms, reinforcing the existence of a neglected complex of genetically and toxinologically related Amazonian scorpions of medical importance. Finally, we demonstrated the low recognition of currently available therapeutic sera against T. cisandinus and T. obscurus venoms, and concluded that these should be improved to protect against envenomation by Amazonian Tityus spp.

Characterization of a Family of Scorpion Toxins Modulating Ca2+-Activated Cl- Current in Vascular Myocytes.

The pharmacology of calcium-activated chloride current is not well developed. Peptides from scorpion venom present potent pharmacological actions on ionic conductance used to characterize the function of channels but can also be helpful to develop organic pharmacological tools. Using electrophysiological recording coupled with calcium measurement, we tested the potent effect of peptides extracted from Leuirus quinquestratus quinquestratus venom on the calcium-activated chloride current expressed in smooth muscle cells freshly dissociated from rat portal veins. We identified one peptide which selectively inhibited the chloride conductance without effects on either calcium signaling or calcium and potassium currents expressed in this cell type. The synthetic peptide had the same affinity, but the chemical modification of the amino acid sequence altered the efficiency to inhibit the calcium-activated chloride conductance.

A Fluorescent Peptide Toxin for Selective Visualization of the Voltage-Gated Potassium Channel KV1.3.

Upregulation of the voltage-gated potassium channel KV1.3 is implicated in a range of autoimmune and neuroinflammatory diseases, including rheumatoid arthritis, psoriasis, multiple sclerosis, and type I diabetes. Understanding the expression, localization, and trafficking of KV1.3 in normal and disease states is key to developing targeted immunomodulatory therapies. HsTX1[R14A], an analogue of a 34-residue peptide toxin from the scorpion Heterometrus spinifer, binds KV1.3 with high affinity (IC50 of 45 pM) and selectivity (2000-fold for KV1.3 over KV1.1). We have synthesized a fluorescent analogue of HsTX1[R14A] by N-terminal conjugation of a Cy5 tag. Electrophysiology assays show that Cy5-HsTX1[R14A] retains activity against KV1.3 (IC50 ∼ 0.9 nM) and selectivity over a range of other potassium channels (KV1.2, KV1.4, KV1.5, KV1.6, KCa1.1 and KCa3.1), as well as selectivity against heteromeric channels assembled from KV1.3/KV1.5 tandem dimers. Live imaging of CHO cells expressing green fluorescent protein-tagged KV1.3 shows co-localization of Cy5-HsTX1[R14A] and KV1.3 fluorescence signals at the cell membrane. Moreover, flow cytometry demonstrated that Cy5-HsTX1[R14A] can detect KV1.3-expressing CHO cells. Stimulation of mouse microglia by lipopolysaccharide, which enhances membrane expression of KV1.3, was associated with increased staining by Cy5-HsTX1[R14A], demonstrating that it can be used to identify KV1.3 in disease-relevant models of inflammation. Furthermore, the biodistribution of Cy5-HsTX1[R14A] could be monitored using ex vivo fluorescence imaging of organs in mice dosed subcutaneously with the peptide. These results illustrate the utility of Cy5-HsTX1[R14A] as a tool for visualizing KV1.3, with broad applicability in fundamental investigations of KV1.3 biology, and the validation of novel disease indications where KV1.3 inhibition may be of therapeutic value.

Smp24, a Scorpion-Venom Peptide, Exhibits Potent Antitumor Effects against Hepatoma HepG2 Cells via Multi-Mechanisms In Vivo and In Vitro.

Scorpion-venom-derived peptides have become a promising anticancer agent due to their cytotoxicity against tumor cells via multiple mechanisms. The suppressive effect of the cationic antimicrobial peptide Smp24, which is derived from the venom of Scorpio Maurus palmatus, on the proliferation of the hepatoma cell line HepG2 has been reported earlier. However, its mode of action against HepG2 hepatoma cells remains unclear. In the current research, Smp24 was discovered to suppress the viability of HepG2 cells while having a minor effect on normal LO2 cells. Moreover, endocytosis and pore formation were demonstrated to be involved in the uptake of Smp24 into HepG2 cells, which subsequently interacted with the mitochondrial membrane and caused the decrease in its potential, cytoskeleton reorganization, ROS accumulation, mitochondrial dysfunction, and alteration of apoptosis- and autophagy-related signaling pathways. The protecting activity of Smp24 in the HepG2 xenograft mice model was also demonstrated. Therefore, our data suggest that the antitumor effect of Smp24 is closely related to the induction of cell apoptosis, cycle arrest, and autophagy via cell membrane disruption and mitochondrial dysfunction, suggesting a potential alternative in hepatocellular carcinoma treatment.

Differential venom gland gene expression analysis of juvenile and adult scorpions Androctonus crassicauda.

BACKGROUND: The Androctonus crassicauda, belonging to the genus Androctonus of the family Buthidae, is the most venomous scorpion in Middle East countries. However, the venom gland transcriptome profile of A. crassicauda scorpion has not yet been studied. In this study, we elucidated and compared the venom gland gene expression profiles of adult and juvenile male scorpion A. crassicauda using high-throughput transcriptome sequencing. This is the first report of transcriptional analysis of the venom glands of scorpions in different growth stages, with insights into the identification of the key genes during venom gland development. RESULTS: A total of 209,951 mRNA transcripts were identified from total RNA-seq data, of which 963 transcripts were differentially expressed (DE) in adult and juvenile scorpions (p < 0.01). Overall, we identified 558 up-regulated and 405 down-regulated transcripts in the adult compared to the juvenile scorpions, of which 397 and 269 unique unigenes were annotated, respectively. GO and KEGG enrichment analyses indicated that the metabolic, thermogenesis, cytoskeleton, estrogen signaling, GnRH signaling, growth hormone signaling, and melanogenesis pathways were affected by two different growth conditions and the results suggested that the DE genes related to those pathways are important genes associated with scorpion venom gland development, in which they may be important in future studies, including Chs, Elovl, MYH, RDX, ACTN, VCL, PIP5K, PP1C, FGFR, GNAS, EGFR, CREB, CoA, PLCB, CALM, CACNA, PKA and CAMK genes. CONCLUSIONS: These findings broadened our knowledge of the differences between adult and juvenile scorpion venom and opened new perspectives on the application of comparative transcriptome analysis to identify the special key genes.

Scorpion venom polypeptide governs alveolar macrophage M1/M2 polarization to alleviate pulmonary fibrosis.

The onset of connective tissue disease-related interstitial lung disease (CTD-ILD) is generally insidious and progressive, with pulmonary fibrosis in the middle stage, and eventually respiratory failure and death. This study aimed to explore the role of scorpion venom polypeptide (SVP), the primary active constituent of the entire scorpion, in alveolar macrophages and pulmonary fibrosis. Pulmonary fibrosis mouse models were established, and then SVP and JAK inhibitor (tofacitinib) was used to treat models. Alveolar macrophages were isolated and the impacts of SVP on M1/M2 polarization and the JAK/STAT6 pathway in vitro were assessed. H&E and Masson staining revealed that SVP and tofacitinib treatment alleviated lung damage and fibrosis. They also hindered the M2-polarization of macrophages in lung tissue and declined cytokine levels associated with M2 polarization (IL-4, IL-13) and fibrosis drivers (TGF-ß, VEGF) in mice. Consistent with the trend presented by tofacitinib treatment, SVP suppressed the phosphorylation of proteins in the JAK/STAT6 pathway. In addition, the in vitro treatment of SVP on the isolated macrophages represented consistent results with in vivo experiments. The findings of the present study indicated that SVP suppressed the JAK/STAT6 signaling pathway, hindered alveolar macrophage M2-type polarization, and possessed the potential to ameliorate pulmonary fibrosis.

Iranian Mesobuthus Eupeus Crude Venom Induces Selective Toxicity in Chronic Lymphocytic Leukemia B-Lymphocytes Through Lysosomal/Mitochondrial Dysfunction and Reactive Oxygen Species Formation.

From ancient times to the present-day animal venoms had been used as medicinal and therapeutic agents. Recently it has been reported that the scorpion venom is a potential source of active and therapeutic compounds to design potent drugs against variety of cancerous cells and other diseases. The current study aimed to evaluate the selective toxicity of Iranian Mesobuthus eupeus (IMe) crude venom as a potential source of anticancer compounds on cancerous CLL B-lymphocytes and normal lymphocytes. For this purpose, we isolated cancerous CLL B-lymphocytes and normal lymphocytes from chronic lymphocytic leukemia patients and healthy volunteers. Cancerous CLL B-lymphocytes and normal lymphocytes were treated with different concentration (0, 5, 10, 20, 40 and 80 µg/ml) of IMe crude venom for 12 hours and cytotoxicity, reactive oxygen species (ROS) production, collapse of mitochondrial membrane potential (MMP) and lysosomal membrane integrity were determined. The data demonstrated the significant cytotoxic effect of IMe crude venom on cancerous CLL B-lymphocytes, with a concentration value (IC50) that inhibits 50% of the cell viability of 60 µg/ ml after 12 h of incubation. MTT assay proved that the IMe crude venom is selectively toxic to cancerous CLL B-lymphocytes, and IMe crude venom induced selective cell death via activation of ROS formation and mitochondrial/lysosomal dysfunction. These finding showed that IMe crude venom has a selective mitochondrial/lysosomal-mediated cell death effect on cancerous CLL B-lymphocytes. Therefore, the IMe crude venom and its fractions may be promising in the future anticancer drug development for treatment of CLL and variety of cancers.

Mechanisms Underlying the Inhibition of KV1.3 Channel by Scorpion Toxin ImKTX58.

Voltage-gated KV1.3 channel has been reported to be a drug target for the treatment of autoimmune diseases, and specific inhibitors of Kv1.3 are potential therapeutic drugs for multiple diseases. The scorpions could produce various bioactive peptides that could inhibit KV1.3 channel. Here, we identified a new scorpion toxin polypeptide gene ImKTX58 from the venom gland cDNA library of the Chinese scorpion Isometrus maculatus Sequence alignment revealed high similarities between ImKTX58 mature peptide and previously reported KV1.3 channel blockers-LmKTX10 and ImKTX88-suggesting that ImKTX58 peptide might also be a KV1.3 channel blocker. By using electrophysiological recordings, we showed that recombinant ImKTX58 prepared by genetic engineering technologies had a highly selective inhibiting effect on KV1.3 channel. Further alanine scanning mutagenesis and computer simulation identified four amino acid residues in ImKTX58 peptide as key binding sites to KV1.3 channel by forming hydrogen bonds, salt bonds, and hydrophobic interactions. Among these four residues, 28th lysine of the ImKTX58 mature peptide was found to be the most critical amino acid residue for blocking KV1.3 channel. SIGNIFICANCE STATEMENT: In this study, we discovered a scorpion toxin gene ImKTX58 that has not been reported before in Hainan Isometrus maculatus and successfully used the prokaryotic expression system to express and purify the polypeptides encoded by this gene. Electrophysiological experiments on ImKTX58 showed that ImKTX58 has a highly selective blocking effect on KV1.3 channel over Kv1.1, Kv1.2, Kv1.5, SK2, SK3, and BK channels. These findings provide a theoretical basis for designing highly effective KV1.3 blockers to treat autoimmune and other diseases.

The Enzymatic Core of Scorpion Venoms.

Enzymes are an integral part of animal venoms. Unlike snakes, in which enzymes play a primary role in envenomation, in scorpions, their function appears to be ancillary in most species. Due to this, studies on the diversity of scorpion venom components have focused primarily on the peptides responsible for envenomation (toxins) and a few others (e.g., antimicrobials), while enzymes have been overlooked. In this work, a comprehensive study on enzyme diversity in scorpion venoms was performed by transcriptomic and proteomic techniques. Enzymes of 63 different EC types were found, belonging to 330 orthogroups. Of them, 24 ECs conform the scorpion venom enzymatic core, since they were determined to be present in all the studied scorpion species. Transferases and lyases are reported for the first time. Novel enzymes, which can play different roles in the venom, including direct toxicity, as venom spreading factors, activators of venom components, venom preservatives, or in prey pre-digestion, were described and annotated. The expression profile for transcripts coding for venom enzymes was analyzed, and shown to be similar among the studied species, while being significantly different from their expression pattern outside the telson.

CD14 regulates the metabolomic profiles of distinct macrophage subsets under steady and activated states.

Macrophages play pivotal roles during homeostasis and inflammation. They sense exogenous and endogenous molecular patterns via surface and intracellular receptors, which trigger innate immune responses. CD14 is a co-receptor for lipopolysaccharide (LPS), but also drives macrophage responses to Tityus serrulatus scorpion venom (TsV). Cellular activation is tightly coupled with metabolism that sustain their polarization and generate antimicrobial and signaling molecules. Macrophage's origin and nature of stimulus are critical for their responses, but whether these factors impact macrophage metabolism is unknown. Moreover, the regulation of intracellular metabolism by CD14 has not been assessed. Using an untargeted metabolomics approach, we determined the longitudinal metabolic responses of peritoneal (PMs) and bone marrow derived macrophages (BMDMs) stimulated with LPS and TsV for 12 h. These data revealed alterations on the relative levels of several metabolites and pathways related to amino acids, nucleotides, lipids, and vitamins. Our data suggest activation of selenoamino acid metabolism and increased abundance of selenomethionine in both cell subsets stimulated with LPS. Moreover, the results suggest a differential activity of vitamin B3 metabolism pathway in response to TsV stimulus, with differences on regulation of the relative levels of nicotinamide mononucleotide and deamino-NAD+. CD14 deficiency affects the metabolome of both cell subsets at steady state. Moreover, CD14 was required for arginine consumption in PMs stimulated with LPS, but not TsV or by BMDMs stimulated by both stimuli. Importantly, the data suggest that CD14 mediates the accumulation of lipids in both macrophage subsets stimulated with LPS, providing insights into the potential role of CD14 for the development of metabolic diseases. We conclude that macrophages acquire a spectrum of metabolic profiles that depend on the origin of these cells, the nature of the stimuli and signaling by innate immune receptors.

Mapping the interaction surface of scorpion ß-toxins with an insect sodium channel.

The interaction of insect-selective scorpion depressant ß-toxins (LqhIT2 and Lqh-dprIT3 from Leiurus quinquestriatus hebraeus) with the Blattella germanica sodium channel, BgNav1-1a, was investigated using site-directed mutagenesis, electrophysiological analyses, and structural modeling. Focusing on the pharmacologically defined binding site-4 of scorpion ß-toxins at the voltage-sensing domain II (VSD-II), we found that charge neutralization of D802 in VSD-II greatly enhanced the channel sensitivity to Lqh-dprIT3. This was consistent with the high sensitivity of the splice variant BgNav2-1, bearing G802, to Lqh-dprIT3, and low sensitivity of BgNav2-1 mutant, G802D, to the toxin. Further mutational and electrophysiological analyses revealed that the sensitivity of the WT = D802E < D802G < D802A < D802K channel mutants to Lqh-dprIT3 correlated with the depolarizing shifts of activation in toxin-free channels. However, the sensitivity of single mutants involving IIS4 basic residues (K4E = WT << R1E < R2E < R3E) or double mutants (D802K = K4E/D802K = R3E/D802K > R2E/D802K > R1E/D802K > WT) did not correlate with the activation shifts. Using the cryo-EM structure of the Periplaneta americana channel, NavPaS, as a template and the crystal structure of LqhIT2, we constructed structural models of LqhIT2 and Lqh-dprIT3-c in complex with BgNav1-1a. These models along with the mutational analysis suggest that depressant toxins approach the salt-bridge between R1 and D802 at VSD-II to form contacts with linkers IIS1-S2, IIS3-S4, IIIP5-P1 and IIIP2-S6. Elimination of this salt-bridge enables deeper penetration of the toxin into a VSD-II gorge to form new contacts with the channel, leading to increased channel sensitivity to Lqh-dprIT3.

CTX-CNF1 Recombinant Protein Selectively Targets Glioma Cells In Vivo.

Current strategies for glioma treatment are only partly effective because of the poor selectivity for tumoral cells. Hence, the necessity to identify novel approaches is urgent. Recent studies highlighted the effectiveness of the bacterial protein cytotoxic necrotizing factor 1 (CNF1) in reducing tumoral mass, increasing survival of glioma-bearing mice and protecting peritumoral neural tissue from dysfunction. However, native CNF1 needs to be delivered into the brain, because of its incapacity to cross the blood-brain barrier (BBB) per se, thus hampering its clinical translation. To allow a non-invasive administration of CNF1, we here developed a chimeric protein (CTX-CNF1) conjugating CNF1 with chlorotoxin (CTX), a peptide already employed in clinics due to its ability of passing the BBB and selectively binding glioma cells. After systemic administration, we found that CTX-CNF1 is able to target glioma cells and significantly prolong survival of glioma-bearing mice. Our data point out the potentiality of CTX-CNF1 as a novel effective tool to treat gliomas.

Epidemiology of scorpionism in France: nationwide scorpion exposure.

INTRODUCTION: In France, 57 species of scorpions are described with a limited number of clinical studies. In this article, we report the epidemiology of scorpion sting events in mainland France and its overseas territories based on cases reported to the French poison-control centres (FPCC). MATERIAL AND METHOD: This retrospective multicentre study was conducted with data from FPCC's files about scorpion stings between January 1, 2011 and December 31, 2020. RESULT: Among 975 recorded files, 624 patients were included because they were stung by scorpions native to French territories. Most stings occurred along the Mediterranean coast in summer and indoors (in persons' homes) during the daytime. The scorpions were identified in 50% of cases. According to signs of envenoming, patients were divided into class III (2 cases; 1%), class II (51 cases; 8%), class I (444 cases; 71%) and asymptomatic stings (127 cases; 20%). Twelve pregnant women were stung and two of them had contractions, which triggered childbirth in one woman. Ten patients had local infections in the first week after the sting. One patient had venous thrombosis 2 days after the sting. Life-threatening scorpions, i.e., Tityus obscurus, Tityus sylvestris and Centruroides pococki, in French territories are limited to French Guiana and Lesser Antilles. Class II envenoming cases are recorded for Buthus occitanus, Euscorpius spp. in mainland France, and Isometrus maculatus in French Guiana, the Lesser Antilles (Guadeloupe and Martinique) and territories in the Indian Ocean (Mayotte and Réunion Island) and Pacific Ocean (French Polynesia). Only cases of local manifestation was reported for Belisarius xambeui in mainland France. CONCLUSION: Scorpion stings in French territories are frequently on the Mediterranean coast and French Guiana. Life-threatening cases are limited to T. obscurus, T. sylvestris and Centruroides pococki stings in French Guiana and Lesser Antilles.

N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site.

Recently developed fluorescent protein-scorpion toxin chimeras (FP-Tx) show blocking activities for potassium voltage-gated channels of Kv1 family and retain almost fully pharmacological profiles of the parental peptide toxins (Kuzmenkov et al., Sci Rep. 2016, 6, 33314). Here we report on N-terminally green fluorescent protein (GFP)-tagged agitoxin 2 (GFP-L2-AgTx2) with high affinity and selectivity for the binding site of Kv1.3 channel involved in the pathogenesis of various (primarily of autoimmune origin) diseases. The basis for this selectivity relates to N-terminal location of GFP, since transposition of GFP to the C-terminus of AgTx2 recovered specific interactions with the Kv1.1 and Kv1.6 binding sites. Competitive binding experiments revealed that the binding site of GFP-L2-AgTx2 overlaps that of charybdotoxin, kaliotoxin 1, and agitoxin 2, the known Kv1.3-channel pore blockers. GFP-L2-AgTx2 was demonstrated to be applicable as a fluorescent probe to search for Kv1.3 pore blockers among individual compounds and in complex mixtures, to measure blocker affinities, and to visualize Kv1.3 distribution at the plasma membrane of Kv1.3-expressing HEK293 cells. Our studies show that definite combinations of fluorescent proteins and peptide blockers can result in considerable modulation of the natural blocker-channel binding profile yielding selective fluorescent ligands of certain channels.

Genetic and toxinological divergence among populations of Tityus trivittatus Kraepelin, 1898 (Scorpiones: Buthidae) inhabiting Paraguay and Argentina.

Envenoming by scorpions in genus Tityus is a public health problem in Tropical America. One of the most medically significant species is Tityus trivittatus, which is known to occur from southwest Brazil to central-northern and eastern Argentina. In this work, we studied the lethality, composition, antigenicity, and enzymatic activity of venom from a T. trivittatus population found further north in urban areas of eastern Paraguay, where it has caused serious envenomation of children. Our results indicate that the population is of medical importance as it produces a potently toxic venom with an LD50 around 1.19 mg/kg. Venom neutralization in preliminary mouse bioassays was complete when using Brazilian anti-T. serrulatus antivenom but only partial when using Argentinean anti-T. trivittatus antivenom. Venom competitive solid-phase enzyme immunoassays and immunoblotting from Argentinean and Paraguayan T. trivittatus populations indicated that antigenic differences exist across the species range. SDS-PAGE showed variations in type and relative amounts of venom proteins between T. trivitattus samples from Argentina and Paraguay. MALDI-TOF mass spectrometry indicated that while some sodium channel toxins are shared, including ß-toxin Tt1g, others are population-specific. Proteolytic activity by zymography and peptide identification through nESI-MS/MS also point out that population-specific proteases may exist in T. trivitattus, which are postulated to be involved in the envenoming process. A time-calibrated molecular phylogeny of mitochondrial COI sequences revealed a significant (8.14%) genetic differentiation between the Argentinean and Paraguayan populations, which appeared to have diverged between the mid Miocene and early Pliocene. Altogether, toxinological and genetic evidence indicate that T. trivitattus populations from Paraguay and Argentina correspond to distinct, unique cryptic species, and suggest that further venom and taxonomic diversity exists in synanthropic southern South American Tityus than previously thought.