Transient Hematotoxicity After Emerald Horned Pitviper (Ophryacus smaragdinus) Envenomation: A Case Report.

BACKGROUND: A minority of snake envenomations in the United States involve non-native snakes. In this report, we describe what we believe is the first documented human envenoming from an emerald horned pitviper, Ophryacus smaragdinus. CASE REPORT: A previously healthy 36-year-old woman was bitten on her left index finger by a captive emerald horned pitviper she was medicating at work. Swelling to the entire hand was present on emergency department arrival. She had no systemic symptoms and her initial laboratory studies were unremarkable. The affected limb was elevated. We administered five vials of Antivipmyn TRIⓇ (Bioclon), which specifically lists Ophryacus among the envenomations for which it is indicated. She developed pruritus and was treated with IV diphenhydramine and famotidine. Her swelling improved, but her repeat laboratory studies were notable for a platelet count of 102 K/µL and a fibrinogen level of 116 mg/dL. She declined additional antivenom because of the previous allergic reaction. She was admitted for further monitoring and pain control. Subsequent laboratory tests were better, but a small hemorrhagic bleb developed at the bite site. She was discharged the next day and followed up as an outpatient. Her swelling had resolved, her bleb had healed, and her laboratory studies continued to improve. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Emergency physicians may be required to treat bites from non-native snakes. Many of these bites will warrant treatment with non-U.S. Food and Drug Administration-approved antivenoms. Consultation with a regional poison center or medical toxicologist may be necessary to procure the proper antivenom.

Whole Snake Genomes from Eighteen Families of Snakes (Serpentes: Caenophidia) and Their Applications to Systematics.

We present genome assemblies for 18 snake species representing 18 families (Serpentes: Caenophidia): Acrochordus granulatus, Aparallactus werneri, Boaedon fuliginosus, Calamaria suluensis, Cerberus rynchops, Grayia smithii, Imantodes cenchoa, Mimophis mahfalensis, Oxyrhabdium leporinum, Pareas carinatus, Psammodynastes pulverulentus, Pseudoxenodon macrops, Pseudoxyrhopus heterurus, Sibynophis collaris, Stegonotus admiraltiensis, Toxicocalamus goodenoughensis, Trimeresurus albolabris, and Tropidonophis doriae. From these new genome assemblies, we extracted thousands of loci commonly used in systematic and phylogenomic studies on snakes, including target-capture datasets composed of UCEs and AHEs, as well as traditional Sanger loci. Phylogenies inferred from the two target-capture loci datasets were identical with each other, and strongly congruent with previously published snake phylogenies. To show additional utility of these non-model genomes for investigative evolutionary research, we mined the genome assemblies of two New Guinea island endemics in our dataset (Stegonotus admiraltiensis and Tropidonophis doriae) for the ATP1a3 gene, a thoroughly researched indicator of resistance to toad toxin ingestion by squamates. We find that both these snakes possess the genotype for toad toxin resistance despite their endemism to New Guinea, a region absent of any toads until the human-mediated introduction of Cane Toads in the 1930s. These species possess identical substitutions that suggest the same bufotoxin resistance as their Australian congenerics (Stegonotus cucullatus and Tropidonophis mairii) which forage on invasive Cane Toads. Herein, we show the utility of short-read high coverage genomes, as well as improving the deficit of available squamate genomes with associated voucher specimens.

Characterization of plant produced VHH antibodies against cobra venom toxins for antivenom therapy.

Cobra (Naja kaouthia) venom contains many toxins including α-neurotoxin (αNTX) and phospholipase A2 (PLA2), which can cause neurodegeneration, respiratory failure, and even death. The traditional antivenom derived from animal serum faces many challenges and limitations. Heavy-chain-only antibodies (HCAb), fusing VHH with human IgG Fc region, offer advantages in tissue penetration, antigen binding, and extended half-life. This research involved the construction and transient expression of two types of VHH-FC which are specific to α-Neurotoxin (VHH-αNTX-FC) and Phospholipase A2 (VHH-PLA2-FC) in Nicotiana benthamiana leaves. The recombinant HCAbs were incubated for up to six days to optimize expression levels followed by purification by affinity chromatography and characterization using LC/Q-TOF mass spectrometry (MS). Purified proteins demonstrated over 92 % sequence coverage and an average mass of around 82 kDa with a high-mannose N-glycan profile. An antigen binding assay showed that the VHH-αNTX-Fc has a greater ability to bind to crude venom than VHH-PLA2-Fc.

Improving in vivo assays in snake venom and antivenom research: A community discussion.

On the 26 th January 2023, a free to attend, 'improving in vivo snake venom research: a community discussion' meeting was held virtually. This webinar brought together researchers from around the world to discuss current neutralisation of venom lethality mouse assays that are used globally to assess the efficacy of therapies for snakebite envenoming. The assay's strengths and weaknesses were highlighted, and we discussed what improvements could be made to refine and reduce animal testing, whilst supporting preclinical antivenom and drug discovery for snakebite envenoming. This report summarises the issues highlighted, the discussions held, with additional commentary on key perspectives provided by the authors.

Mass spectrometric analysis of Odonthobuthus Doriae scorpion venom and its non-neutralized fractions after interaction with commercial antivenom.

It is believed that antivenoms play a crucial role in neutralizing venoms. However, uncontrolled clinical effects appear in patients stung by scorpions after the injection of antivenom. In this research, non-neutralized components of the venom of the Iranian scorpion Odonthobuthus doriae were analyzed after interacting with the commercial antivenom available in the market. The venom and antivenom interaction was performed, then centrifuged, and the supernatant was analyzed by high-performance liquid chromatography (HPLC). Two peaks of Odonthobuthus doriae venom were observed in the chromatogram of the supernatant. Two components were isolated by HPLC and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) instruments. Peptide sequencing was done by Liquid Chromatography Quadrupole Time-of-Flight Tandem Mass Spectrometry (LC-Q-TOF MS/MS). Results indicate that the components of scorpion venom mainly have a molecular weight below 10 kDa, consisting of toxic peptides that disrupt the function of sodium and potassium channels. The MALDI-TOF MS results show that two toxic peptides with molecular masses of 6941 Da and 6396 Da were not neutralized by the antivenom. According to the MS/MS sequencing data, the components have been related to peptides A0A5P8U2Q6_MESEU and A0A0U4FP89_ODODO, which belong to the sodium and potassium channels toxins family, respectively.

Detection of direct oral anticoagulants with the diluted Russel's viper venom time.

INTRODUCTION: The activity of direct oral anticoagulants (DOAC) is important in acute clinical situations. Recent studies have suggested a strong influence of DOAC on the diluted Russel's Viper Venom Time (dRVVT). Therefore, it may be a suitable screening parameter for antithrombotic plasma activity of different DOAC. This prospective study aims to evaluate the sensitivity and specificity of dRVVT to detect residual DOAC activity at recommended plasma level thresholds. METHODS: A total of 80 patients were recruited, with 20 each treated with one of the four approved DOAC (apixaban, edoxaban, rivaroxaban or dabigatran), respectively. Blood plasma was collected before (baseline), at plasma peak time, and 6 and 12 h after DOAC. DRVVT was measured using the screen (LA1) and confirm (LA2) assay for lupus anticoagulant and compared with DOAC plasma levels. A reference range was calculated based on the dRVVT values of 61 healthy blood donors. RESULTS: All DOAC significantly prolonged the dRVVT especially at higher DOAC plasma levels. The LA1 time ≥41 s had a sensitivity ≥98% to detect edoxaban, dabigatran and rivaroxaban plasma levels ≥30 ng/mL but it was only 87% for apixaban. Sensitivity was ≥98% for all DOAC with the LA2 assay ≥36 s. The negative predictive value of a DOAC plasma level <30 ng/mL and dRVVT LA2 <36 s was 99%. CONCLUSIONS: The dRVVT confirm assay (LA2) reliably detects residual DOAC plasma levels ≥30 ng/mL and could be useful to rapidly rule out relevant DOAC activity in emergency situations and to guide treatment decisions.

Emerging anticancer potential and mechanisms of snake venom toxins: A review.

Animal-derived venom, like snake venom, has been proven to be valuable natural resources for the drug development. Previously, snake venom was mainly investigated in its pharmacological activities in regulating coagulation, vasodilation, and cardiovascular function, and several marketed cardiovascular drugs were successfully developed from snake venom. In recent years, snake venom fractions have been demonstrated with anticancer properties of inducing apoptotic and autophagic cell death, restraining proliferation, suppressing angiogenesis, inhibiting cell adhesion and migration, improving immunity, and so on. A number of active anticancer enzymes and peptides have been identified from snake venom toxins, such as L-amino acid oxidases (LAAOs), phospholipase A2 (PLA2), metalloproteinases (MPs), three-finger toxins (3FTxs), serine proteinases (SPs), disintegrins, C-type lectin-like proteins (CTLPs), cell-penetrating peptides, cysteine-rich secretory proteins (CRISPs). In this review, we focus on summarizing these snake venom-derived anticancer components on their anticancer activities and underlying mechanisms. We will also discuss their potential to be developed as anticancer drugs in the future.

Seasonality in Crotalus durissus venom.

Rattlesnakes belonging to the genus Crotalus are widely distributed throughout the Americas. In Brazil, symptoms commonly associated with envenomation by Crotalus durissus collilineatus include myalgia, rhabdomyolysis, renal failure, neurotoxicity, and progressive paralysis, which are related to the protein composition of this venom. Snake venom composition exhibits compositional variability that may reflect geographic distribution, age, captivity, diet, sex, and even individual genetics. Although seasonality is also considered a possible source of variation, there are few reports of such variability in snake venom. In this work, venoms of the same eight C. durissus collilineatus were extracted every three months for two years, to analyze seasonal changes in composition and activities. To this end, venom composition was analyzed by protein quantification, SDS-PAGE, and HPLC, and the LAAO, PLA2 and coagulant activities were measured. Venoms of these C. d. collilineatus showed minor seasonal differences in venom activities and no composition differences were found. LAAO and coagulant activities displayed a pattern of seasonal change, while PLA2 activity seemed to have no seasonality tendency. Also, there are sexual differences, in which males seem to be more stable than females in regard to some activities. Individual variability occurs even in seasonal variation of activities, highlighting the importance of controlling circumstances of venom extraction before comparing results between groups of snakes.

Novel Scorpion Toxin ω-Buthitoxin-Hf1a Selectively Inhibits Calcium Influx via CaV3.3 and CaV3.2 and Alleviates Allodynia in a Mouse Model of Acute Postsurgical Pain.

Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 µM) and CaV3.3 (IC50 = 0.49 µM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.

Snake Eyes: Characterization of Topical Ocular Exposures from Rattlesnakes in Arizona.

Patients occasionally present with reports of ocular exposure to fluids from rattlesnakes, claiming or suspecting the substance to be venom. This study set out to evaluate and characterize reported cases of suspected venom-induced ophthalmia in humans. A retrospective review of rattlesnake exposures reported to the Arizona Poison and Drug Information Center over a 24-year period was conducted for ocular exposures. Recorded information included patient demographics, clinical course, laboratory results, and treatments. Documentation regarding interactions between patients and snakes was reviewed by Arizona Poison and Drug Information Center herpetologists to evaluate what substance was expelled from the snake resulting in ocular exposure. Our review of rattlesnake encounters found a total of 26 ocular exposure cases. Patient demographics were largely intentional interactions and involved the male sex. Symptoms ranged from asymptomatic to minor effects with 46.2% managed from home and treated with fluid irrigation. A review of cases by herpetologists concluded the exposure patients commonly experienced was to snake musk. Kinematics of venom expulsion by rattlesnakes conclude the venom gland must be compressed, fangs erected to ≥60o, and fang sheath compressed against the roof of the mouth for venom expulsion. Evidence suggests the chance of venom "spitting" by rattlesnakes is close to zero. Rattlesnakes are documented to forcefully expel airborne malodorous "musk" defensively. An important distinction to remember is musk has a foul odor and is usually colorless, while venom is comparatively odorless and yellow. Rattlesnake venom-induced ophthalmia is a rare event as venom expulsion requires the kinematics of feeding or defensive bites. If the rattlesnake is not in the process of biting or otherwise contacting some other object with its mouth, it is more biologically plausible patients are being exposed to snake musk as a deterrent. Whether it's venom or musk, topical exposure to the eyes should prompt immediate irrigation.

Presentation, treatment profiles, and outcome of snake bite patients presented in emergency department at a tertiary hospital in Eastern Nepal.

Background: Snake bite is one of the most common animal bites in Nepal. Different species of snake cause different clinical presentations. The incidence of snakebite is very high in rural Nepal. The objectives were to assess the presenting pattern, demographic profile, outcome, and treatment profiles of snakebite victims admitted to the emergency ward. Materials and Methods: A retrospective cross-sectional study was conducted among the patients who presented in emergency department with alleged history of snake bites from 2015 to 2016. The patient's record files were reviewed and the relevant data were recorded on a self-designed proforma. Descriptive statistics were calculated using SPSS version 11.5. Results: Out of 137 snakebite victims, 73 (53.3%) were female. The mean age was 35.17 ± 18.27 years. The upper limb (59%) was the most common site for snake bites followed by the lower limb (35.1%). Fifty patients (36.2%) were bitten by snakes during night (20.00-2.59 AM). Twenty-eight (20.4%) patients presented with ptosis as the most common sign and symptom followed by diplopia (15.3%). Out of 137 patients, 39 (28.5%) were admitted, 65 (47.4%) discharged, and 12 (8.8%) patients expired. Antisnake venom was given to 30 patients among which 23 patients (76.7%) were improved. Conclusions: Snake bite is one of the major problems in rural Nepal. It can be easily managed if treatment is given properly and in a timely manner. The importance of effective first aid management and effective treatment have to be disseminated among the peoples in rural areas via social media and radio.

Ame-miR-1-3p of bee venom reduced cell viability through the AZIN1/OAZ1-ODC1-polyamines pathway and enhanced the defense ability of honeybee (Apis mellifera L.).

Bee venom serves as an essential defensive weapon for bees and also finds application as a medicinal drug. MicroRNAs (miRNAs) serve as critical regulators and have been demonstrated to perform a variety of biological functions. However, the presence of miRNAs in bee venom needs to be confirmed. Therefore, we conducted small RNA sequencing and identified 158 known miRNAs, 15 conserved miRNAs and 4 novel miRNAs. It is noteworthy that ame-miR-1-3p, the most abundant among them, accounted for over a quarter of all miRNA reads. To validate the function of ame-miR-1-3p, we screened 28 candidate target genes using transcriptome sequencing and three target gene prediction software (miRanda, PITA and TargetScan) for ame-miR-1-3p. Subsequently, we employed real-time quantitative reverse transcription PCR (qRT-PCR), Western blot and other technologies to confirm that ame-miR-1-3p inhibits the relative expression of antizyme inhibitor 1 (AZIN1) by targeting the 3' untranslated region (UTR) of AZIN1. This, in turn, caused ODC antizyme 1 (OAZ1) to bind to ornithine decarboxylase 1 (ODC1) and mark ODC1 for proteolytic destruction. The reduction in functional ODC1 ultimately resulted in a decrease in polyamine biosynthesis. Furthermore, we determined that ame-miR-1-3p accelerates cell death through the AZIN1/OAZ1-ODC1-polyamines pathway. Our studies demonstrate that ame-miR-1-3p diminishes cell viability and it may collaborate with sPLA2 to enhance the defence capabilities of honeybees (Apis mellifera L.). Collectively, these data further elucidate the defence mechanism of bee venom and expand the potential applications of bee venom in medical treatment.

A review on snake venom extracellular vesicles: Past to present.

Around 95% of snake venom is protein. Along with the soluble proteins, snake venom also contains proteins encapsulated in vesicles known as Snake Venom Extracellular Vesicles (SVEV). SVEVs are nano-sized membrane-bound vesicles released from the snake venom gland cells. The available published research works on SVEVs are minimal. Extracellular vesicles in the Snake Venom gland were initially discovered during the histopathological analysis of the Crotalus durissus terrificus snakes' venom gland. Later, various techniques were employed to isolate and characterize the SVEVs. The cargo of SVEV consists of a variety of proteins like Phospholipase A-2, C-type Lectins, L-Amino Acid Oxidase, Cysteine-Rich Secretory Proteins, Serine Proteinases, Dipeptidyl Peptidase-IV, Aminopeptidase-A, Ecto-5'-nucleotidases, Disintegrins. Proteomic data revealed the presence of some exclusive proteins in the SVEVs, and the other proteins are in varying concentrations in the SVEVs compared to their whole Venom. Interaction of SVEVs with mammalian cell lines showed the disruption of primary physiological functions leads to host immune modulation, and long-term effects of envenoming. Snakebite victim's blood showed variations in the specific Extracellular vesicle concentration. It has been hypothesized that SVEVs are responsible for long-term toxicity. The current review focuses on the various techniques adopted to isolate and characterize SVEVs and discusses the exclusiveness and variations of SVEV proteins and their role in snakebites.

Acute splenic hematoma: A rare complication of snake bite.

Splenic hematoma secondary to snake bite is a potential complication due to snake envenomation and poses a significant risk to the health of the patients. Although relatively rare, this complication once diagnosed, should be initiated with timely anti-venom administration and supportive care. Clinicians must be aware of any signs of hematological abnormalities in snakebite patients, as the development of splenic hematoma can have serious implications for patient outcomes. Awareness of this potential complication and multidisciplinary collaboration among medical teams are crucial to ensuring effective management and optimal patient care in these clinical scenarios. Understanding this concern can improve patient prognosis and advance the overall approach to snakebite management in healthcare settings.

Whole transcriptome sequencing reveals the activity of the PLA2 family members in Androctonus crassicauda (Scorpionida: Buthidae) venom gland.

Phospholipase A2 is the most abundant venom gland enzyme, whose activity leads to the activation of the inflammatory response by accumulating lipid mediators. This study aimed to identify, classify, and investigate the properties of venom PLA2 isoforms. Then, the present findings were confirmed by chemically measuring the activity of PLA2. The sequences representing PLA2 annotation were extracted from the Androctonus crassicauda transcriptome dataset using BLAS searches against the local PLA2 database. We found several cDNA sequences of PLA2 classified and named by conducting multiple searches as platelet-activating factor acetylhydrolases, calcium-dependent PLA2s, calcium-independent PLA2s, and secreted PLA2s. The largest and smallest isoforms of these proteins range between approximately 70.34 kDa (iPLA2) and 17.75 kDa (cPLA2). Among sPLA2 isoforms, sPLA2GXIIA and sPLA2G3 with ORF encoding 169 and 299 amino acids are the smallest and largest secreted PLA2, respectively. These results collectively suggested that A. crassicauda venom has PLA2 activity, and the members of this protein family may have important biological roles in lipid metabolism. This study also revealed the interaction between members of PLA2s in the PPI network. The results of this study would greatly help with the classification, evolutionary relationships, and interactions between PLA2 family proteins in the gene network.

Proteomic analysis and lethality of the venom of Aegaeobuthus nigrocinctus, a scorpion of medical significance in the Middle East.

The scorpion Aegaeobuthus nigrocinctus inhabits areas in Turkey and the Levant region of the Middle East where severe/lethal envenomings have been reported. Previous research indicated its extreme venom lethality to vertebrates and distinct envenomation syndrome. We report on the composition of A. nigrocinctus venom from Lebanese specimens using nESI-MS/MS, MALDI-TOF MS, SDS-PAGE and RP-HPLC. Venom lethality in mice was also assessed (LD50 = 1.05 (0.19-1.91) mg/kg, i.p), confirming A. nigrocinctus venom toxicity from Levantine populations. Forty-seven peaks were resolved using RP-HPLC, 25 of which eluted between 20 and 40 % acetonitrile. In reducing SDS-PAGE, most predominant components were <10 kDa, with minor components at higher molecular masses of 19.6, 26.1, 46.3 and 57.7 kDa. MALDI-TOF venom fingerprinting detected 20 components within the 1,000-12,000 m/z range. Whole venom 'shotgun' bottom-up nLC-MS/MS approach, combined with in-gel tryptic digestion of SDS-PAGE bands, identified at least 67 different components belonging to 15 venom families, with ion channel-active components (K+ toxins (23); Na+ toxins (20); Cl- toxins (2)) being predominant. The sequence of a peptide (named α-KTx9.13) ortholog to Leiurus hebraeus putative α-KTx9.3 toxin was fully determined, which exhibited 81-96 % identity to other members of the α-KTx9 subfamily targeting Kv1.x and Ca2+-activated K+ channels. Chlorotoxin-like peptides were also identified. Our study underscores the medical significance of A. nigrocinctus in the region and reveals the potential value of its venom components as lead templates for biomedical applications. Future work should address whether available antivenoms in the Middle East are effective against A. nigrocinctus envenoming in the Levant area.

Beyond the venom: Exploring the antimicrobial peptides from Androctonus species of scorpion.

Prevalent worldwide, the Androctonus scorpion genus contributes a vital role in scorpion envenoming. While diverse scorpionisms are observed because of several different species, their secretions to protect themselves have been identified as a potent source of antimicrobial peptide (AMP)-like compounds. Distinctly, the venom of these species contains around 24 different AMPs, with definite molecules studied for their therapeutic potential as antimicrobial, antifungal, antiproliferative and antiangiogenic agents. Our review focuses on the therapeutic potential of native and synthetic AMPs identified so far in the Androctonus scorpion genus, identifying research gaps in peptide therapeutics and guiding further investigations. Certain AMPs have demonstrated remarkable compatibility to be prescribed as anticancer drug to reduce cancer cell proliferation and serve as a potent antibiotic alternative. Besides, analyses were performed to explore the characteristics and affinities of peptides for membranes. Overall, the study of AMPs derived from the Androctonus scorpion genus provides valuable insights into their potential applications in medicine and drug development.

Highly conserved and extremely variable: The paradoxical pattern of toxin expression revealed by comparative venom-gland transcriptomics of Phalotris (Serpentes: Dipsadidae).

Although non-front fanged snakes account for almost two-thirds of snake diversity, most studies on venom composition and evolution focus exclusively on front-fanged species, which comprise most of the clinically relevant accidents. Comprehensive reports on venom composition of non-front fanged snakes are still scarce for several groups. In this study, we address such shortage of knowledge by providing new insights about the venom composition among species of Phalotris, a poorly studied Neotropical dipsadid genus. Phalotris are known for their specialized venom delivery system and toxic venoms, which can cause life-threatening accidents in humans. We evaluate the venom-gland transcriptome of Phalotris, comparing the following three South American species: P. reticulatus for the Araucaria Pine forests, P. lemniscatus for the Pampa grasslands, and P. mertensi for the Brazilian Cerrado. Our results indicate similar venom profiles, in which they share a high expression level of Kunitz-type inhibitors (KUNZ). On the other hand, comparative analyses revealed substantial differences in the expression levels of C-type lectins (CTL) and snake venom metalloproteinases (SVMP). The diverse set of SVMP and CTL isoforms shows signals of positive selection, and we also identified truncated forms of type III SVMPs, which resemble type II and type I SVMPs of viperids. Additionally, we identified a CNP precursor hosting a proline-rich region containing a BPP motif resembling those commonly detected in viperid venoms with hypotensive activity. Altogether, our results suggest an evolutionary history favoring high expression levels of few KUNZ isoforms in Phalotris venoms, contrasting with a highly diverse set of SVMP and CTL isoforms. Such diversity can be comparable with the venom variability observed in some viperids. Our findings highlight the extreme phenotypic diversity of non-front fanged snakes and the importance to allocate greater effort to study neglected groups of Colubroidea.

Similar neurotoxin expression profiles of traditional Chinese scorpion medicine material between juvenile and adult Mesobuthus martensii scorpions revealed by multiple strategic proteomics.

ETHNOPHARMACOLOGICAL RELEVANCE: 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 the Pharmacopoeia of the People's Republic of China nowadays. AIM OF STUDY: 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. MATERIALS AND 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. CONCLUSION: 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.

Expression of Recombinant Stonustoxin Alpha Subunit and Preparation of polyclonal antiserum for Stonustoxin Neutralization Studies.

Stonustoxin (SNTX) is a lethal protein found in stonefish venom, responsible for many of the symptoms associated with stonefish envenomation. To counter stonefish venom challenges, antivenom is a well-established and effective solution. In this study, we aimed to produce the recombinant alpha subunit protein of Stonustoxin from Synanceia horrida and prepare antibodies against it The SNTXα gene sequence was optimized for E. coli BL21 (DE3) expression and cloned into the pET17b vector. Following purification, the recombinant protein was subcutaneously injected into rabbits, and antibodies were extracted from rabbit´s serum using a G protein column As a result of codon optimization, the codon adaptation index for the SNTXα cassette increased to 0.94. SDS-PAGE analysis validated the expression of SNTXα, with a band observed at 73.5 kDa with a yield of 60 mg/l. ELISA results demonstrated rabbits antibody titers were detectable up to a 1:256,000 dilution. The isolated antibody from rabbit´s serum exhibited a concentration of 1.5 mg/ml, and its sensitivity allowed the detection of a minimum protein concentration of 9.7 ng. In the neutralization assay the purified antibody against SNTXα protected mice challenged with 2 LD50. In conclusion, our study successfully expressed the alpha subunit of Stonustoxin in a prokaryotic host, enabling the production of antibodies for potential use in developing stonefish antivenom.