Search Results (17)

Background/Objectives: Hypertension represents a leading contributor to cardiovascular disorders and premature mortality. Given the pervasive nature of adverse effects associated with current angiotensin-converting enzyme inhibitors (ACEIs), there is a significant interest in identifying novel bioactive lead compounds from natural sources. This study identifies, for the first time, three novel angiotensin-converting enzyme (ACE) inhibitory peptides released from Bungarus multicinctus (BM) via simulated gastrointestinal digestion (SGD). Methods: Active fractions were enriched by ultrafiltration and subjected to stability assessment. The peptide sequences were then determined using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) and bioinformatics tools, followed by chemical synthesis. Finally, the inhibitory mechanism was investigated using kinetic analysis and molecular docking. Results: The intestinal digest exhibited potent ACE inhibition, with the <5 kDa fraction achieving 79% inhibition at 1 mg/mL and demonstrating favorable stability under varying temperatures, pH, and ionic strengths. Molecular docking revealed strong binding (affinity < −9.9 kcal/mol) of the peptides PPSPPRW, WGFTKF, and PSLFPPRL to key ACE residues—Tyr523, His513, and Arg522—via hydrogen and hydrophobic interactions. Enzyme kinetics characterized PPSPPRW and WGFTKF as competitive inhibitors, and PSLFPPRL as mixed type. The peptides demonstrated acceptable cell viability at lower concentrations, establishing a preliminary safety window for therapeutic application. Conclusions: These findings establish BM as a valuable source of stable, bioactive ACE-inhibitory peptides (ACEIPs) acting as promising lead compounds for antihypertensive therapies. Full article

Chinese coral snakes (Sinomicrurus) are highly neglected regarding their venom profiles and harm to humans, which impedes our ability to deeply understand their biological properties and explore their medicinal potential. In this study, we performed a comparative analysis to reveal the venom profiles of two Chinese coral snakes in terms of their venom yields, proteomic profiles, and immunorecognition by commercial antivenoms. The results showed that Sinomicrurus kelloggi expels more venom (lyophilized venom mass) than Sinomicrurus maccelellandi but possesses a similar solid venom content. These interspecific differences in venom yield were influenced by the snout–vent length. The venoms of these two species varied in their electrophoretic profiles, as well as in the presence or absence and relative abundance of protein families. They exhibited a 3-FTx-predominant phenotype, where the S. maccelellandi venom was dominated by 3-FTx (32.43%), SVMP (23.63%), PLA₂ (19.88%), and SVSP (12.61%), while the S. kelloggi venom was dominated by 3-FTx (65.81%), LAAO (11.35%), and AMP (10.09%). While both the commercial Naja atra and Bungarus multicinctus antivenoms could immunorecognize these two Chinese coral snake venoms, the N. atra antivenom possessed a higher neutralization capability than the B. multicinctus antivenom for both species of coral snakes. Our findings show significant interspecific variations in the venom profiles of these Sinomicrurus snakes for the first time. We suggest screening or preparing specific antivenoms with high efficiency for the clinical treatment of envenomation caused by these snakes. Full article

Snakebite envenomings continue to represent a major public health concern in Taiwan because of the presence of various venomous snakes whose habitats intersect with human activities. This review provides a comprehensive analysis of the clinical characteristics, complications, and management strategies associated with snakebite envenomings in Taiwan. Taiwan is inhabited by six principal venomous snakes: Trimeresurus stejnegeri stejnegeri, Protobothrops mucrosquamatus, Deinagkistrodon acutus, Daboia siamensis, Naja atra, and Bungarus multicinctus, each presenting distinct clinical challenges. The clinical manifestations vary from local symptoms such as pain, swelling, and necrosis to systemic complications including neurotoxicity, coagulopathy, and organ failure, depending on the species. Notable complications arising from these snakebite envenomings include necrotizing soft tissue infection, compartment syndrome, respiratory failure, and acute kidney injury, often necessitating intensive medical interventions. This review highlights the critical importance of early diagnosis, the prompt administration of antivenoms, and multidisciplinary care to improve patient outcomes and reduce healthcare costs. Future research is encouraged to enhance treatment efficacy, improve public awareness, and develop targeted prevention strategies. By identifying gaps in current knowledge and practice, this work contributes to the global literature on envenoming management and serves as a foundation for advancing clinical protocols and reducing snakebite-related morbidity and mortality in Taiwan. Full article

Animal venoms are intricate and teem with potential for groundbreaking medical advancements. Although traditional methods for purifying venom proteins are effective, they usually require complicated, multi-step processes that lead to lower yields. Our study introduces an efficient, one-step technique for extracting venom-derived proteins through reverse-phase high-performance liquid chromatography (RP-HPLC). We carefully optimized the RP-HPLC process, focusing on the gradient elution conditions and the strategic use of our columns’ stationary phase characteristics, to enhance the effectiveness of our separations. This enabled us to efficiently isolate six venom proteins: melittin (2.846 kDa) from Apis mellifera with a yield of 4.5% and homogeneity of 99%; α-cobratoxin (7.821 kDa) from Naja kaouthia with a yield of 15% and homogeneity of 99%; α-bungarotoxin (7.983 kDa) from Bungarus multicinctus with a yield of 7% and purity of 99%; calciseptine (7.035 kDa) from Dendroaspis polylepis with a yield of 6% and homogeneity of 95%; notexin (13.593 kDa) from Notechis scutatus with a yield of 10% and homogeneity of 95%; and CVFm (150 kDa) from Naja melanoleuca with a yield of 0.8% and homogeneity of 94%. These were all accomplished in one step. This breakthrough simplifies the purification of venom peptides and proteins, making the process more feasible and economical. It paves the way for developing new drugs and promising treatments that are both more effective and precisely targeted. Full article

The oral cavity of snakes serves as a habitat for various microorganisms, some of which may include potential zoonotic pathogens posing risks to hosts and causing wound infections in snakebite victims. Clinical studies on snakebite cases in Taiwan have identified specific pathogens, such as Enterococcus faecalis (Gram-positive), Morganella morganii, Aeromonas hydrophila, and Pseudomonas aeruginosa (Gram-negative). However, the prevalence of these bacteria in the oral cavity of wild snakes remains largely unknown. This study investigated the occurrence of these bacteria in six wild snake species (Naja atra, Bungarus multicinctus, Trimeresurus stejnegeri, Protobothrops mucrosquamatus, Boiga kraepelini, and Elaphe taeniura friesi) from southern Taiwan, along with factors influencing their presence. Oropharyngeal swab samples were collected from a substantial number of wild-caught snakes (n = 1104), followed by DNA extraction, polymerase chain reaction, and gel electrophoresis. The band positions of samples were compared with positive and negative controls to determine the presence of target bacteria in each sample. The overall occurrence rates were 67.4% for E. faecalis, 31.5% for M. morganii, 8.2% for A. hydrophila, and 7.7% for P. aeruginosa. Among snake species, B. kraepelini exhibited dominance in E. faecalis (93.4%), A. hydrophila (17.1%), and P. aeruginosa (14.5%), while male N. atra showed dominance in M. morganii (51.3%). The occurrence of E. faecalis was lowest in winter. The results of multiple logistic regression analyses suggest that factors such as species, sex, temperature, season, and coexisting pathogens may have a significant impact on the occurrence of target bacteria. These findings have implications for wildlife medicine and snakebite management. Full article

Snakebite is considered a concerning issue and a neglected tropical disease. Three-finger toxins (3FTxs) in snake venoms primarily cause neurotoxic effects since they have high affinity for nicotinic acetylcholine receptors (nAChRs). Their small molecular size makes 3FTxs weakly immunogenic and therefore not appropriately targeted by current antivenoms. This study aims at presenting and applying an analytical method for investigating the therapeutic potential of the acetylcholine-binding protein (AChBP), an efficient nAChR mimic that can capture 3FTxs, for alternative treatment of elapid snakebites. In this analytical methodology, snake venom toxins were separated and characterised using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS) and high-throughput venomics. By subsequent nanofractionation analytics, binding profiling of toxins to the AChBP was achieved with a post-column plate reader-based fluorescence-enhancement ligand displacement bioassay. The integrated method was established and applied to profiling venoms of six elapid snakes (Naja mossambica, Ophiophagus hannah, Dendroaspis polylepis, Naja kaouthia, Naja haje and Bungarus multicinctus). The methodology demonstrated that the AChBP is able to effectively bind long-chain 3FTxs with relatively high affinity, but has low or no binding affinity towards short-chain 3FTxs, and as such provides an efficient analytical platform to investigate binding affinity of 3FTxs to the AChBP and mutants thereof and to rapidly identify bound toxins. Full article

Modern analytical size exclusion chromatography (SEC) is a suitable technique to separate venom toxin families according to their size characteristics. In this study, a method was developed to separate intact venom toxins from Bungarus multicinctus and Daboia russelii venoms via analytical SEC using volatile, non-salt-containing eluents for post-column mass spectrometry, coagulation bioassaying and high-throughput venomics. Two venoms were used to demonstrate the method developed. While the venom of Bungaurs multicinctus is known to exert anticoagulant effects on plasma, in this study, we showed the existence of both procoagulant toxins and anticoagulant toxins. For Daboia russelii venom, the method revealed characteristic procoagulant effects, with a 90 kDa mass toxin detected and matched with the Factor X-activating procoagulant heterotrimeric glycoprotein named RVV-X. The strong procoagulant effects for this toxin show that it was most likely eluted from size exclusion chromatography non-denatured. In conclusion, the separation of snake venom by size gave the opportunity to separate some specific toxin families from each other non-denatured, test these for functional bioactivities, detect the eluting mass on-line via mass spectrometry and identify the eluted toxins using high-throughput venomics. Full article

A venomous snake’s oral cavity may harbor pathogenic microorganisms that cause secondary infection at the wound site after being bitten. We collected oral samples from 37 individuals belonging to seven species of wild venomous snakes in Taiwan, including Naja atra (Na), Bungarus multicinctus (Bm), Protobothrops mucrosquamatus (Pm), Trimeresurus stejnegeri (Ts), Daboia siamensis (Ds), Deinagkistrodon acutus (Da), and alpine Trimeresurus gracilis (Tg). Bacterial species were identified using full-length 16S rRNA amplicon sequencing analysis, and this is the first study using this technique to investigate the oral microbiota of multiple Taiwanese snake species. Up to 1064 bacterial species were identified from the snake’s oral cavities, with 24 pathogenic and 24 non-pathogenic species among the most abundant ones. The most abundant oral bacterial species detected in our study were different from those found in previous studies, which varied by snake species, collection sites, sampling tissues, culture dependence, and analysis methods. Multivariate analysis revealed that the oral bacterial species compositions in Na, Bm, and Pm each were significantly different from the other species, whereas those among Ts, Ds, Da, and Tg showed fewer differences. Herein, we reveal the microbial diversity in multiple species of wild snakes and provide potential therapeutic implications regarding empiric antibiotic selection for wildlife medicine and snakebite management. Full article

Protozoal infections are a world-wide problem. The toxicity and somewhat low effectiveness of the existing drugs require the search for new ways of protozoa suppression. Snake venom contains structurally diverse components manifesting antiprotozoal activity; for example, those in cobra venom are cytotoxins. In this work, we aimed to characterize a novel antiprotozoal component(s) in the Bungarus multicinctus krait venom using the ciliate Tetrahymena pyriformis as a model organism. To determine the toxicity of the substances under study, surviving ciliates were registered automatically by an original BioLaT-3.2 instrument. The krait venom was separated by three-step liquid chromatography and the toxicity of the obtained fractions against T. pyriformis was analyzed. As a result, 21 kDa protein toxic to Tetrahymena was isolated and its amino acid sequence was determined by MALDI TOF MS and high-resolution mass spectrometry. It was found that antiprotozoal activity was manifested by β-bungarotoxin (β-Bgt) differing from the known toxins by two amino acid residues. Inactivation of β-Bgt phospholipolytic activity with p-bromophenacyl bromide did not change its antiprotozoal activity. Thus, this is the first demonstration of the antiprotozoal activity of β-Bgt, which is shown to be independent of its phospholipolytic activity. Full article

Venom-induced thrombocytopenia (VIT) is one of the most important hemotoxic effects of a snakebite, which is often associated with venom-induced consumptive coagulopathy (VICC). Refractory thrombocytopenia without significant coagulation abnormalities has also been reported after envenomation by some viperid snakes; however, the mechanisms are not well understood and therapeutic strategies are lacking. Here, we found that patients injured by Daboia siamensis or Agkistrodon halys snakes, who were resistant to standard antivenom treatment, had developed coagulopathy-independent thrombocytopenia. Venoms from these viperid snakes, rather than from the elapid snake (Bungarus multicinctus), induced platelet surface expression of neuraminidase-1 (NEU-1), and significantly increased the desialylation of the glycoproteins on human platelets. The desialylated platelets caused by viperid snake venoms were further internalized by macrophages, which resulted in reduced platelet numbers in peripheral blood. Importantly, neuraminidase inhibitor significantly decreased viper venom-induced platelet desialylation, therefore inhibiting platelet phagocytosis by macrophages, and alleviating venom-induced thrombocytopenia. Collectively, these findings support an important role for desialylated platelet clearance in the progression of viper envenomation-induced, coagulopathy-independent thrombocytopenia. Our study demonstrates that the neuraminidase inhibitor may be a potential therapy or adjuvant therapy to treat snakebite-induced thrombocytopenia. Full article

Wound infections after venomous snakebites are clinically important. Information regarding the nature and antibiotic susceptibilities of snake oral bacterial flora could support empiric antibiotic therapy. Wild venomous snakes were collected from southern Taiwan: a total of 30 each of Bungarus multicinctus, Naja atra, Protobothrops mucrosquamatus, and Trimeresurus stejnegeri; 3 Deinagkistrodon acutus; and 4 Daboia siamensis. The species and antibiotic susceptibilities of their oral bacteria were determined. Aerobic gram-negative bacteria, especially Pseudomonas aeruginosa and Proteus vulgaris, were the most abundant. Proteus vulgaris were more abundant in B. multicinctus, N. atra, and P. mucrosquamatus than in T. stejnegeri (40%, 43.3%, and 40% vs. 13.3%, respectively). The gram-negative species were less susceptible to first- and second-generation cephalosporins and ampicillin-sulbactam than to third-generation cephalosporins, fluoroquinolones, carbapenems, or piperacillin-tazobactam. The most abundant aerobic gram-positive species cultured was Enterococcus faecalis, which was more abundant in N. atra than in other snakes (p < 0.001) and was highly susceptible to ampicillin, high-level gentamicin, penicillin, teicoplanin, and vancomycin. Bacteroides fragilis and Clostridium species were the most common anaerobic bacteria. The anaerobic organisms were highly susceptible to metronidazole and piperacillin. As a reference for empiric antimicrobial therapy, third-generation cephalosporins, fluoroquinolones, carbapenems, or piperacillin-tazobactam can be initiated in venomous snakebites wound infections. Full article

α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of Bungarus multicinctus snake. Like most large toxins, chemical synthesis of α-bungarotoxin is challenging, explaining why all previous reports use purified or recombinant α-bungarotoxin. However, only chemical synthesis allows easy insertion of non-natural amino acids or new chemical functionalities. Herein, we describe a procedure for the chemical synthesis of a fluorescent-tagged α-bungarotoxin. The full-length peptide was designed to include an alkyne function at the amino-terminus through the addition of a pentynoic acid linker. Chemical synthesis of α-bungarotoxin requires hydrazide-based coupling of three peptide fragments in successive steps. After completion of the oxidative folding, an azide-modified Cy5 fluorophore was coupled by click chemistry onto the toxin. Next, we determined the efficacy of the fluorescent-tagged α-bungarotoxin to block acetylcholine (ACh)-mediated currents in response to muscle nicotinic receptor activation in TE671 cells. Using automated patch-clamp recordings, we demonstrate that fluorescent synthetic α-bungarotoxin has the expected nanomolar affinity for the nicotinic receptor. The blocking effect of fluorescent α-bungarotoxin could be displaced by incubation with a 20-mer peptide mimicking the α-bungarotoxin binding site. In addition, TE671 cells could be labelled with fluorescent toxin, as witnessed by confocal microscopy, and this labelling was partially displaced by the 20-mer competitive peptide. We thus demonstrate that synthetic fluorescent-tagged α-bungarotoxin preserves excellent properties for binding onto muscle nicotinic receptors. Full article

Snake envenomation is a serious public health issue in many tropical and subtropical countries. Accurate diagnosis and immediate antivenom treatment are critical for effective management. However, the venom concentration in the victims’ plasma is usually low, representing one of the bottlenecks in developing clinically applicable assays for venom detection and snakebite diagnosis. In this study, we attempted to develop a simple method for rapid enrichment of venom proteins from human plasma to facilitate detection. Our experiments showed that several major protein components of both Naja atra (N. atra) and Bungarus multicinctus (B. multicinctus) venoms have higher isoelectric point (pI) values relative to high-abundance human plasma proteins and could be separated via strong cation exchange–high-performance liquid chromatography (SCX-HPLC). Based on this principle, we developed an SCX tip column-based protocol for rapid enrichment of N. atra and B. multicinctus venom proteins from human plasma. Application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) led to the identification of cytotoxin and beta-bungarotoxin as the major proteins enriched by the SCX tip column in each venom sample. The entire process of venom enrichment could be completed within 10–15 min. Combination of this method with our previously developed lateral flow strip assays (rapid test) significantly enhanced the sensitivity of the rapid test, mainly via depletion of the plasma protein background, as well as increase in venom protein concentration. Notably, the SCX tip column-based enrichment method has the potential to efficiently enrich other Elapidae snake venoms containing proteins with higher pI values, thereby facilitating venom detection with other assays. This simple and rapid sample preparation method should aid in improving the clinical utility of diagnostic assays for snakebite. Full article

Bungarus multicinctus, the Chinese krait, is a highly venomous elapid snake which causes considerable morbidity and mortality in southern China. B. multicinctus venom contains pre-synaptic PLA₂ neurotoxins (i.e., β-bungarotoxins) and post-synaptic neurotoxins (i.e., α-bungarotoxins). We examined the in vitro neurotoxicity of B. multicinctus venom, and the efficacy of specific monovalent Chinese B. multicinctus antivenom, and Australian polyvalent elapid snake antivenom, against venom-induced neurotoxicity. B. multicinctus venom (1–10 μg/mL) abolished indirect twitches in the chick biventer cervicis nerve-muscle preparation as well as attenuating contractile responses to exogenous ACh and CCh, but not KCl. This indicates a post-synaptic neurotoxic action but myotoxicity was not evident. Given that post-synaptic α-neurotoxins have a more rapid onset than pre-synaptic neurotoxins, the activity of the latter in the whole venom will be masked. The prior addition of Chinese B. multicinctus antivenom (12 U/mL) or Australian polyvalent snake antivenom (15 U/mL), markedly attenuated the neurotoxic actions of B. multicinctus venom (3 μg/mL) and prevented the inhibition of contractile responses to ACh and CCh. The addition of B. multicinctus antivenom (60 U/mL), or Australian polyvalent snake antivenom (50 U/mL), at the t₉₀ time point after the addition of B. multicinctus venom (3 μg/mL), did not restore the twitch height over 180 min. The earlier addition of B. multicinctus antivenom (60 U/mL), at the t₂₀ or t₅₀ time points, also failed to prevent the neurotoxic effects of the venom but did delay the time to abolish twitches based on a comparison of t₉₀ values. Repeated washing of the preparation with physiological salt solution, commencing at the t₂₀ time point, failed to reverse the neurotoxic effects of venom or delay the time to abolish twitches. This study showed that B. multicinctus venom displays marked in vitro neurotoxicity in a skeletal muscle preparation which is not reversed by antivenom. This does not appear to be related to antivenom efficacy, but due to the irreversible/pseudo-irreversible nature of the neurotoxins. Full article

The phospholipase A₂ (PLA₂) inhibitor Varespladib (LY315920) and its orally bioavailable prodrug, methyl-Varespladib (LY333013) inhibit PLA₂ activity of a wide variety of snake venoms. In this study, the ability of these two forms of Varespladib to halt or delay lethality of potent neurotoxic snake venoms was tested in a mouse model. The venoms of Notechis scutatus, Crotalus durissus terrificus, Bungarus multicinctus, and Oxyuranus scutellatus, all of which have potent presynaptically acting neurotoxic PLA₂s of variable quaternary structure, were used to evaluate simple dosing regimens. A supralethal dose of each venom was injected subcutaneously in mice, followed by the bolus intravenous (LY315920) or oral (LY333013) administration of the inhibitors, immediately and at various time intervals after envenoming. Control mice receiving venom alone died within 3 h of envenoming. Mice injected with O. scutellatus venom and treated with LY315920 or LY333013 survived the 24 h observation period, whereas those receiving C. d. terrificus and B. multicinctus venoms survived at 3 h or 6 h with a single dose of either form of Varespladib, but not at 24 h. In contrast, mice receiving N. scutatus venom and then the inhibitors died within 3 h, similarly to the control animals injected with venom alone. LY315920 was able to reverse the severe paralytic manifestations in mice injected with venoms of O. scutellatus, B. multicinctus, and C. d. terrificus. Overall, results suggest that the two forms of Varespladib are effective in abrogating, or delaying, neurotoxic manifestations induced by some venoms whose neurotoxicity is mainly dependent on presynaptically acting PLA₂s. LY315920 is able to reverse paralytic manifestations in severely envenomed mice, but further work is needed to understand the significance of species-specific differences in animal models as they compare to clinical syndromes in human and for potential use in veterinary medicine. Full article