Alternative recombinant sources of antivenoms have been successfully generated. The application of such strategies requires the characterization of the venoms for the development of specific neutralizing molecules against the toxic components. Five toxic peptides to mammals from the Mexican scorpion Centruroides villegasi were isolated by chromatographic procedures by means of gel filtration on Sephadex G-50, followed by ion-exchange columns on carboxy-methyl-cellulose (CMC) resins and finally purified by high-performance chromatography (HPLC) columns. Their primary structures were determined by Edman degradation. They contain 66 amino acids and are maintained well packed by four disulfide bridges, with molecular mass from 7511.3 to 7750.1 Da. They are all relatively toxic and deadly to mice and show high sequence identity with known peptides that are specific modifiers of the gating mechanisms of Na+ ion channels of type beta-toxin (β-ScTx). They were named Cv1 to Cv5 and used to test their recognition by single-chain variable fragments (scFv) of antibodies, using surface plasmon resonance. Three different scFvs generated in our laboratory (10FG2, HV, LR) were tested for recognizing the various new peptides described here, paving the way for the development of a novel type of scorpion antivenom.

UNASSIGNED: The expansion of the territory of human habitation leads to inevitable interference in the natural range of distribution of one or another species of animals, some of which may be dangerous for human life. Scorpions-the Arachnida class and order Scorpiones-can be considered as such typical representatives. Scorpions of the Buthidae family pose a particular danger to humans. However, LD50 has not yet been defined for many species of this family, in particular, new representatives of the genus Leiurus. Leiurus macroctenus is a newly described species of scorpion distributed in Oman, and the toxicity of its venom is still unknown. Estimating the LD50 of the venom is the first and most important step in creating the antivenom and understanding the medical significance of the researched animal species. The purpose of this study was to determine the lethal dose (LD100), the maximum tolerated dose (LD0), and the average lethal dose (LD50) in rats when using Leiurus macroctenus scorpion venom.
UNASSIGNED: 15 sexually mature scorpions were used in the study, which were kept in the same conditions and milked by a common method (electric milking). For the study, 60 male rats were used, which were injected intramuscularly with 0.5 ml of venom solution with a gradual increase in the dose (5 groups, 10 rats in each), and 10 rats were injected intramuscularly with physiological solution as control group. LD calculations were done using probit analysis method in the modification of the method by V.B. Prozorovsky. The LD0 of Leiurus macroctenus scorpion venom under the conditions of intramuscular injection was 0.02 mg/kg, LD100 was 0.13 mg/kg, and LD50 was 0.08 ± 0.01 mg/kg.
UNASSIGNED: The analysis of scientific publications and other sources of information gives reason to believe that Leiurus macroctenus has one of the highest values of LD50 not only among scorpions but also among all arthropods in the world. All these point to the significant clinical importance of this species of scorpion and require further research that will concern the toxic effect of its venom on various organ systems. Determining the LD50 of the venom for new scorpion species is crucial for creating effective antivenoms and understanding the medical implications of envenomation by this species.

Heloderma horridum horridum, a venomous reptile native to America, has a venom with potential applications in treating type II diabetes. In this work, H. h. horridum venom was extracted, lyophilized, and characterized using enzymatic assays for hyaluronidase, phospholipase, and protease. Proteomic analysis of the venom was conducted employing bottom-up/shotgun approaches, SDS-PAGE, high-pH reversed-phase chromatography, and fractionation of tryptic peptides using nano-LC-MS/MS. The proteins found in H. h. horridum venom were reviewed according to the classification of the transcriptome previously reported. The proteomic approach identified 101 enzymes, 36 other proteins, 15 protein inhibitors, 11 host defense proteins, and 1 toxin, including novel venom components such as calcium-binding proteins, phospholipase A2 inhibitors, serpins, cathepsin, subtilases, carboxypeptidase-like, aminopeptidases, glycoside hydrolases, thioredoxin transferases, acid ceramidase-like, enolase, multicopper oxidases, phosphoglucose isomerase (PGI), fructose-1,6-bisphosphatase class 1, pentraxin-related, peptidylglycine α-hydroxylating monooxygenase/peptidyl-hydroxyglycine α-amidating lyase, carbonic anhydrase, acetylcholinesterase, dipeptidylpeptidase, and lysozymes. These findings contribute to understanding the venomous nature of H. h. horridum and highlight its potential as a source of bioactive compounds. Data are available via PRoteomeXchange with the identifier PXD052417.

In this study, we report the innovative application of whole-cell patch-clamp electrophysiology in assessing broad-spectrum neutralisation by three different antivenoms, of venoms from the medically significant scorpion genus Centruroides. Envenomations by as many as 21 species from the Centruroides genus result in up to 300,000 envenomations per year in Mexico, which poses significant and potentially life-threatening pathophysiology. We first evaluated the in vitro manifestation of envenomation against two human voltage-gated sodium (hNaV) channel subtypes: hNaV1.4 and hNaV1.5, which are primarily expressed in skeletal muscles and cardiomyocytes, respectively. The neutralisation of venom activity was then characterised for three different antivenoms using a direct competition model against the more potent target, hNaV1.4. While broad-spectrum neutralisation was identified, variation in neutralisation arose for Centruroides elegans, C. limpidus, C. noxius and C. suffusus venoms, despite the presence of a number of these venoms within the immunising mixture. This raises questions regarding the truly \"broad\" neutralisation capacity of the antivenoms. This study not only extends previous validation of the in vitro investigation of antivenom efficacy utilising the whole-cell patch-clamp technique but also underscores the potential of this animal-free model in exploring cross-reactivity, experimental scalability, and most importantly, informing clinical management practices regarding the administration of antivenom in Mexico.

UNASSIGNED: Medicinal leeches (Hirudo spp.) have been used for therapeutic purposes in humans since ancient times. Because of their growth conditions, leeches carry certain bacteria and endosymbionts (e.g., Aeromonas spp). In both leech farms and hirudotherapy clinics, there are no reliable antiseptics that can be used with leeches. This study aimed to determine whether methylene blue (MB) is a safe antiseptic for medicinal leeches and assess its safe usage.
UNASSIGNED: This study evaluated the efficacy of MB by determining lethal concentrations (LC), effective concentrations (EC), and lethal times (LT) for the medicinal leech Hirudo verbena Carena, 1820. A total of 570 H. verbana specimens obtained from a local farm were used in this study. Eighteen different concentrations of MB (between 1 ppm and 512 ppm) were tested.
UNASSIGNED: The LC50 and EC50 values for H. verbana were determined to be 60.381 (53.674-66.636) ppm and 2.013 (1.789-2.221) ppm, respectively. The LT50 durations for MB concentrations of 32 and 512 ppm were calculated as 212.92 h (138.43 h-1485.78 h) and 17.82 h (8.08 h-23.90 h), respectively.
UNASSIGNED: The results show that MB concentrations between 2 and 19 ppm can be safely used as antiseptics in hirudotherapy clinics and leech farms to address bacterial concerns caused by medicinal leeches.
UNASSIGNED: Tıbbi sülükler (Hirudo spp.) eski çağlardan beri insanlarda tedavi amaçlı kullanılmaktadır. Sülükler, büyüme koşulları nedeniyle bazı bakterileri ve endosimbiontları (örneğin; Aeromonas spp.) taşırlar. Hem sülük çiftliklerinde hem de hirudoterapi kliniklerinde sülüklerle birlikte kullanılabilecek güvenilir antiseptikler bulunmamaktadır. Bu çalışmanın amacı, metilen mavisinin (MB) tıbbi sülükler için güvenli bir antiseptik olup olmadığını belirlemek ve güvenli kullanımını değerlendirmektir.
UNASSIGNED: Bu çalışmada, tıbbi sülük Hirudo verbana Carena, 1820 için ölümcül konsantrasyonlar (LC), etkili konsantrasyonlar (EC) ve ölümcül süreler (LT) belirlenerek MB’nin etkinliği değerlendirilmiştir. Bu çalışmada yerel bir çiftlikten elde edilen toplam 570 H. verbana örneği kullanılmıştır. On sekiz farklı MB konsantrasyonu (1 ppm ile 512 ppm arasında) test edilmiştir.
UNASSIGNED: H. verbana için LC50 ve EC50 değerleri sırasıyla 60.381 (53.674-66.636) ppm ve 2.013 (1.789-2.221) ppm olarak belirlenmiştir. 32 ve 512 ppm MB konsantrasyonları için LT50 süreleri sırasıyla 212.92 saat (138.43 saat-1485.78 saat) ve 17.82 saat (8.08 saat-23.90 saat) olarak hesaplanmıştır.
UNASSIGNED: Sonuçlar, 2 ila 19 ppm arasındaki MB konsantrasyonlarının, tıbbi sülüklerin neden olduğu bakteriyel endişeleri gidermek için hirudoterapi kliniklerinde ve sülük çiftliklerinde antiseptik olarak güvenle kullanılabileceğini göstermektedir.

Small single-chain variable fragments (scFv) are promising biomolecules to inhibit and neutralize toxins and to act as antivenoms. In this work, we aimed to produce a functional scFv-6009FV in the yeast Pichia pastoris, which inhibits the pure Cn2 neurotoxin and the whole venom of Centruroides noxius. We were able to achieve yields of up to 31.6 ± 2 mg/L in flasks. Furthermore, the protein showed a structure of 6.1 % α-helix, 49.1 % β-sheet, and 44.8 % of random coil by CD. Mass spectrometry confirmed the amino acid sequence and showed no glycosylation profile for this molecule. Purified scFv-6009FV allowed us to develop anti-scFvs in rabbits, which were then used in affinity columns to purify other scFvs. Determination of its half-maximal inhibitory concentration value (IC50) was 40 % better than the scFvs produced by E. coli as a control. Finally, we found that scFv-6009FV was able to inhibit ex vivo the pure Cn2 toxin and the whole venom from C. noxius in murine rescue experiments. These results demonstrated that under the conditions assayed here, P. pastoris is suited to produce scFv-6009FV that, compared to scFvs produced by E. coli, maintains the characteristics of an antibody and neutralizes the Cn2 toxin more effectively.

Studies on the interaction sites of peptide toxins and ion channels typically involve site-directed mutations in toxins. However, natural mutant toxins exist among them, offering insights into how the evolutionary process has conserved crucial sequences for activities and molecular target selection. In this study, we present a comparative investigation using electrophysiological approaches and computational analysis between two alpha toxins from evolutionarily close scorpion species of the genus Tityus, namely, Tst3 and Ts3 from T. stigmurus and T. serrulatus, respectively. These toxins exhibit three natural substitutions near the C-terminal region, which is directly involved in the interaction between alpha toxins and Nav channels. Additionally, we characterized the activity of the Tst3 toxin on Nav1.1-Nav1.7 channels. The three natural changes between the toxins did not alter sensitivity to Nav1.4, maintaining similar intensities regarding their ability to alter opening probabilities, delay fast inactivation, and induce persistent currents. Computational analysis demonstrated a preference for the down conformation of VSD4 and a shift in the conformational equilibrium towards this state. This illustrates that the sequence of these toxins retained the necessary information, even with alterations in the interaction site region. Through electrophysiological and computational analyses, screening of the Tst3 toxin on sodium isoform revealed its classification as a classic α-NaTx with a broad spectrum of activity. It effectively delays fast inactivation across all tested isoforms. Structural analysis of molecular energetics at the interface of the VSD4-Tst3 complex further confirmed this effect.

Scorpions, a seemingly primitive, stinging arthropod taxa, are known to exhibit marked diversity in their venom components. These venoms are known for their human pathology, but they are also important as models for therapeutic and drug development applications. In this study, we report a high-quality genome assembly and annotation of the striped bark scorpion, Centruroides vittatus, created with several shotgun libraries. The final assembly is 760 Mb in size, with a BUSCO score of 97.8%, a 30.85% GC, and an N50 of 2.35 Mb. We estimated 36,189 proteins with 37.32% assigned to Gene Ontology (GO) terms in our GO annotation analysis. We mapped venom toxin genes to 18 contigs and 2 scaffolds. We were also able to identify expression differences between venom gland (telson) and body tissue (carapace) with 19 sodium toxin and 14 potassium toxin genes to 18 contigs and 2 scaffolds. This assembly, along with our transcriptomic data, provides further data to investigate scorpion venom genomics.

BACKGROUND: The Mesobuthus martensii scorpions, called as \"Quanxie\", are known Chinese medicinal material base on the \"Combat poison with poison\" strategy for more than one thousand years, and still widely used to treat various diseases according to the Pharmacopoeia of the People\'s Republic of China nowadays.
OBJECTIVE: The study aims to investigate the similarity of scorpion neurotoxins at the protein level between the juvenile and adult Mesobuthus martensii scorpions as Chinese medicine materials.
METHODS: The second-, third- and fourth-instar, and adult Mesobuthus martensii scorpions were collected for the characterization of neurotoxin expression through multiple strategic proteomics, including undigested scorpion venom, endopeptidase-digested, and undigested scorpion telson extract for the sample analysis.
RESULTS: Based on the known 107 scorpion neurotoxins from the genomic and transcriptomic analysis of adult Mesobuthus martensii scorpions, the multiple strategic proteomics first revealed that neurotoxins exhibited more stability in telson extract than secreted venom. In the reported transcripts of scorpion neurotoxins, approximately 53%, 56%, 66% and 78% of neurotoxins were detected through undigested scorpion venom, the endopeptidase Arg-C-, Lys-C-digested telson extract, and undigested telson extract strategies, respectively. Nearly 79% of scorpion neurotoxins detected in third-instar Mesobuthus martensii scorpions represent the largest number of scorpion neurotoxins from proteomic analysis to date. Moreover, a total of 84% of scorpion neurotoxins were successfully identified at the protein level, and similar neurotoxin expression profiles in second-, third- and fourth-instar, and adult Mesobuthus martensii scorpions were first revealed by the multiple strategic proteomics.
CONCLUSIONS: These findings for the first time demonstrate the similar neurotoxin expression profiles between the juvenile and adult Mesobuthus martensii scorpions as Chinese medicinal material, which would serve as a paradigm for further toxin analysis from different venomous animals.

BACKGROUND: Hemiscorpius lepturus envenomation is a serious health problem in the southern provinces of Iran. The antiserum produced in Iran to counteract this scorpion venom is not entirely effective due to the risk of anaphylactic shock and other adverse effects.
METHODS: Therefore, more efficient alternatives to treat patients deserve attention, and plants are extensively good candidates to be studied. This study aimed to assess the potential of the aqueous fraction of Malva sylvestris in inhibiting the toxic effects of H. lepturus venom. Injection of sub-lethal dose of H. lepturus venom leads to severe tissue damage in vital organs including the kidney, liver, heart and intestine, after 24 hours.
RESULTS: By injecting 80 mg of the aqueous extract of M. sylvestris into the peritoneum helped treat the damaged tissues caused by H. lepturus venom in mice.
CONCLUSIONS: Thus, Malva sylvestris could serve as an alternative treatment for scorpion sting envenomation and may be used as a drug to neutralize relevant toxic effects in patients stung by H. lepturus.