Fangs and foliage: Unearthing the haemotoxic secrets of cannabis-dwelling rattlesnakes.

Despite a recent surge in high-throughput venom research that has enabled many species to be studied, some snake venoms remain understudied. The long-tailed rattlesnakes (Crotalus ericsmithi, C. lannomi, and C. stejnegeri) are one group where such research lags, largely owing to the rarity of these snakes and the hazardous areas, ripe with drug (marijuana and opium) production, they inhabit in Mexico. To fill this knowledge gap, we used multiple functional assays to examine the coagulotoxic (including across different plasma types), neurotoxic, and myotoxic activity of the venom of the long-tailed rattlesnakes. All crude venoms were shown to be potently anticoagulant on human plasma, which we discovered was not due to the destruction of fibrinogen, except for C. stejnegeri displaying minor fibrinogen destruction activity. All venoms exhibited anticoagulant activity on rat, avian, and amphibian plasmas, with C. ericsmithi being the most potent. We determined the mechanism of anticoagulant activity by C. ericsmithi and C. lannomi venoms to be phospholipid destruction and inhibition of multiple coagulation factors, leading to a net disruption of the clotting cascade. In the chick biventer assay, C. ericsmithi and C. lannomi did not exhibit neurotoxic activity but displayed potential weak myotoxic activity. BIRMEX® (Faboterápico Polivalente Antiviperino) antivenom was not effective in neutralising this venom effect. Overall, this study provides an in-depth investigation of venom function of understudied long-tailed rattlesnakes and provides a springboard for future venom and ecology research on the group.

Investigating skeletal muscle biomarkers for the early detection of Australian myotoxic snake envenoming: an animal model pilot study.

INTRODUCTION: Myotoxicity is an important toxidrome that can occur with envenoming from multiple Australian snake types. Early antivenom administration is an important strategy to reduce the incidence and severity of myotoxicity. The current gold standard biomarker, serum creatine kinase activity, does not rise early enough to facilitate early antivenom administration. Several other skeletal muscle biomarkers have shown promise in other animal models and scenarios. The aim of this study was to examine the predictive values of six skeletal muscle biomarkers in a rat model of Australian snake myotoxicity. METHODS: Sprague-Dawley rats were anaesthetised and administered either Pseudechis porphyriacus (red-bellied black snake) or Notechis scutatus (tiger snake) venom, or normal saline via intramuscular injection. Blood samples were collected. Assays were performed for serum creatine kinase skeletal muscle troponin-I concentration, skeletal muscle troponin-C concentration, myoglobin activity, skeletal muscle myosin light chain-1 concentration, and creatine kinase-MM activity. Serum markers were plotted against time, with comparison of area under the concentration (or activity)-time curve. The predictive values of six skeletal muscle biomarkers were examined using receiver operating characteristic curves. RESULTS: There was no difference in area under the serum creatine kinase activity-time curve between venom and control groups. Serum creatine kinase-MM activity rose early in the venom treated rats, which had a significantly greater area under the serum activity-time curve. No difference in area under the serum concentration-time curve was demonstrated for the other biomarkers. Creatine kinase-MM activity had a superior predictive values than creatine kinase activity at 0-4 hours and 0-10 hours after venom administration, as indicated by area under the receiver operating characteristic curves (95 per cent confidence intervals) of 0.91 (0.78-1.00) and 0.88 (0.73-1.00) versus 0.79 (0.63-0.95) and 0.66 (0.51-0.80). DISCUSSION: The limitations of serum creatine kinase activity in early detection of myotoxicity were demonstrated in this rat model. CONCLUSION: Serum creatine kinase-MM activity was superior for early detection of Australian myotoxic snake envenoming.

A Comparison of the Efficacy of Antivenoms and Varespladib against the In Vitro Pre-Synaptic Neurotoxicity of Thai and Javanese Russell's Viper (Daboia spp.) Venoms.

The heterogeneity in venom composition and potency in disparate Eastern Russell's viper (Daboia siamensis) populations has repercussions for the efficacy of antivenoms. This is particularly pronounced in geographical areas in which the venom of the local species has not been well studied and locally produced antivenoms are unavailable. In such cases, alternative therapies following envenoming, which are not limited by species specificity, may be employed to complement antivenoms. We studied the neuromuscular activity of D. siamensis venom from Thailand and Java (Indonesia) and the ability of Thai antivenoms and/or Varespladib to prevent or reverse these effects. Both Thai and Javanese D. siamensis venoms displayed potent pre-synaptic neurotoxicity but weak myotoxicity in the chick biventer cervicis nerve-muscle preparation. Whilst the neurotoxicity induced by both venoms was abolished by the prior administration of Thai D. siamensis monovalent antivenom or pre-incubation with Varespladib, Thai neuro-polyvalent antivenom only produced partial protection when added prior to venom. Pre-synaptic neurotoxicity was not reversed by the post-venom addition of either antivenom 30 or 60 min after either venom. Varespladib, when added 60 min after venom, prevented further inhibition of indirect twitches. However, the subsequent addition of additional concentrations of Varespladib did not result in further recovery from neurotoxicity. The combination of Thai monovalent antivenom and Varespladib, added 60 min after venom, resulted in additional recovery of twitches caused by either Thai or Javanese venoms compared with antivenom alone. In conclusion, we have shown that Varespladib can prevent and partially reverse the pre-synaptic neurotoxicity induced by either Thai or Javanese D. siamensis venoms. The efficacy of Thai D. siamensis monovalent antivenom in reversing pre-synaptic neurotoxicity was significantly enhanced by its co-administration with Varespladib. Further work is required to establish the efficacy of Varespladib as a primary or adjunct therapy in human envenoming.

Development of a Biosensor to Detect Venom of Malayan Krait (Bungarus candidus).

Malayan krait (Bungarus candidus) envenoming is a cause of significant morbidity and mortality in many Southeast Asian countries. If intubation and specific antivenom administration are delayed, the most significant life-threatening outcome may be the inhibition of neuromuscular transmission and subsequent respiratory failure. It is recommended that krait-envenomed victims without indications of neurotoxicity, e.g., skeletal muscle weakness or ptosis, immediately receive 10 vials of antivenom. However, the administration of excess antivenom may lead to hypersensitivity or serum sickness. Therefore, monitoring venom concentrations in patients could be used as an indicator for snake antivenom treatment. In this study, we aimed to develop a screen-printed gold electrode (SPGE) biosensor to detect B. candidus venom in experimentally envenomed rats. The gold electrodes were coated with monovalent Malayan krait IgG antivenom and used as venom detection biosensors. Electrochemical impedance spectrometry (EIS) and square wave voltammetry (SWV) measurements were performed to detect the electrical characterization between B. candidus venom and monovalent IgG antivenom in the biosensor. The EIS measurements showed increases in charge transfer resistance (Rct) following IgG immobilization and incubation with B. candidus venom solution (0.1-0.4 mg/mL); thus, the antibody was immobilized on the electrode surface and venom was successfully detected. The lowest current signal was detected by SWV measurement in rat plasma collected 30 min following B. candidus experimental envenoming, indicating the highest level of venom concentration in blood circulation (4.3 ± 0.7 µg/mL). The present study demonstrates the ability of the SPGE biosensor to detect B. candidus venom in plasma from experimentally envenomed rats. The technology obtained in this work may be developed as a detection tool for use along with the standard treatment of Malayan krait envenoming.

Career intentions and satisfaction influences in early career Australian physiotherapists.

BACKGROUND: The current workforce does not meet the demand for physiotherapy services in Australia. Future demand is predicted to expand driven primarily by the aging population. Previous research describes significant attrition and short career intentions of junior physiotherapists. OBJECTIVE: This study explored factors associated with physiotherapy graduates' early career intentions and satisfaction. METHOD: Four cohorts of student physiotherapists completed two online surveys designed specifically for this study assessing their immediate and future career intentions and satisfaction. Surveys were completed after undergraduate training (Student Survey) and 2 years later (Practitioner Survey). Question formats included single or multiple select, Likert scale, and free-text responses. Responses were analyzed via descriptive statistics and content and relational analysis. RESULTS: Despite most early career practitioners (83%) reporting career satisfaction, 27% intended to pursue long-term physiotherapy careers (>20 years) and 15% intended to work for 5 years or less. Fewer (11%) reported a longer career intention and 26% a shorter career intention compared to their student survey. Extrinsic occupational factors, such as support, were mentioned as influential in increasing intended future career length since course completion. CONCLUSION: This study found some evidence of factors contributing to shorter career intentions of early career physiotherapists. Specific support of early career physiotherapists may encourage longer career intentions and help build future workforce capacity.

Do personal and behavioural characteristics of physiotherapy students predict performance during training and course completion?

BACKGROUND: Specific personal and behavioural characteristics are required for competent health care practice. Research investigating relationships between these characteristics and course performance of health professions students is expanding, yet little research is conducted within the undergraduate physiotherapy student population. This study aimed to explore the relationships between personality, approaches to learning, and coping strategies of undergraduate physiotherapy students and their performance in academic, clinical and in-course assessment tasks and course progression. METHODS: Participants from six cohorts of undergraduate physiotherapy students (commencing years 2012-2017, 66% response rate) completed questionnaires measuring personality (NEO-FFI-3), approaches to learning (RASI) and coping strategies (Brief COPE). Correlation and multiple regression analysis were conducted to investigate relationships between scores on written examinations, in-course assessment tasks and assessments of clinical performance. Mann-Whitney U test was used to compare subgroups on these measures in those who completed or did not complete the course. RESULTS: Conscientiousness and a strategic approach to learning predicted higher scores in written examinations, and for most clinical and in-course assessments with conscientiousness being a stronger predictor. A lack of purpose (surface) learning approach was predictive of lower clinical placement scores. Non-course completers had higher scores for lack of purpose (surface) approach to learning and lower scores for the coping strategies of support seeking and humour. CONCLUSIONS: This study confirms the importance of conscientiousness and a strategic learning approach on the academic and clinical performance of undergraduate physiotherapy students. Identifying learners with a surface learning approach and low support seeking coping strategies could assist in providing support to students at risk of poor performance and minimising attrition.

In Vitro Efficacy of Antivenom and Varespladib in Neutralising Chinese Russell's Viper (Daboia siamensis) Venom Toxicity.

The venom of the Russell's viper (Daboia siamensis) contains neurotoxic and myotoxic phospholipase A2 toxins which can cause irreversible damage to motor nerve terminals. Due to the time delay between envenoming and antivenom administration, antivenoms may have limited efficacy against some of these venom components. Hence, there is a need for adjunct treatments to circumvent these limitations. In this study, we examined the efficacy of Chinese D. siamensis antivenom alone, and in combination with a PLA2 inhibitor, Varespladib, in reversing the in vitro neuromuscular blockade in the chick biventer cervicis nerve-muscle preparation. Pre-synaptic neurotoxicity and myotoxicity were not reversed by the addition of Chinese D. siamensis antivenom 30 or 60 min after venom (10 µg/mL). The prior addition of Varespladib prevented the neurotoxic and myotoxic activity of venom (10 µg/mL) and was also able to prevent further reductions in neuromuscular block and muscle twitches when added 60 min after venom. The addition of the combination of Varespladib and antivenom 60 min after venom failed to produce further improvements than Varespladib alone. This demonstrates that the window of time in which antivenom remains effective is relatively short compared to Varespladib and small-molecule inhibitors may be effective in abrogating some activities of Chinese D. siamensis venom.

Neuroprotective effect of phospholipase A2 from Malaysian Naja sumatrana venom against H2O2-induced cell damage and apoptosis.

Oxidative stress is one of the factors involved in the pathogenesis of several neurodegenerative diseases. It has been reported that a secretory phospholipase A2 known as A2-EPTX-NSm1a has lower cytotoxicity in neuronal cells compared to its crude Naja sumatrana venom. In this study, A2-EPTX-NSm1a was tested for its neuroprotective activity on human neuroblastoma cells (SH-SY5Y) differentiated into cholinergic neurons against oxidative stress induced by hydrogen peroxide (H2O2). H2O2 treatment alone increased the caspase-3 and caspase-8 activities, whereas pre-treatment with A2-EPTX-NSm1a reduced the activity of these apoptosis-associated proteins. Moreover, A2-EPTX-NSm1a protects the morphology and ultrastructure of differentiated SH-SY5Y cells in the presence of H2O2. Oxidative stress increased the number of small mitochondria. Further evaluation showed the size of mitochondria with a length below 0.25 µm in oxidative stress conditions is higher than the control group, suggesting mitochondria fragmentation. Pre-treatment with A2-EPTX-NSm1a attenuated the number of mitochondria in cells with H2O2 Furthermore, A2-EPTX-NSm1a altered the expression of several neuroprotein biomarkers of GDNF, IL-8, MCP-1, TIMP-1, and TNF-R1 in cells under oxidative stress induced by H2O2. These findings indicate that anti-apoptosis with mitochondria-related protection, anti-inflammatory effect, and promote expression of important markers for cell survival may underlie the neuroprotective effect of A2-EPTX-NSm1a in cholinergic rich human cells under oxidative stress, a vital role in the neuronal disorder.

In Vitro neurotoxicity and myotoxicity of Malaysian Naja sumatrana and Naja kaouthia venoms: Neutralization by monovalent and Neuro Polyvalent Antivenoms from Thailand.

Naja sumatrana and Naja kaouthia are medically important elapids species found in Southeast Asia. Snake bite envenoming caused by these species may lead to morbidity or mortality if not treated with the appropriate antivenom. In this study, the in vitro neurotoxic and myotoxic effects N. sumatrana and N. kaouthia venoms from Malaysian specimens were assessed and compared. In addition, the neutralizing capability of Cobra Antivenom (CAV), King Cobra Antivenom (KCAV) and Neuro Polyvalent Antivenom (NPAV) from Thailand were compared. Both venoms produced concentration-dependent neurotoxic and myotoxic effects in the chick biventer cervicis nerve-muscle preparation. Based on the time to cause 90% inhibition of twitches (i.e. t90) N. kaouthia venom displayed more potent neurotoxic and myotoxic effects than N. sumatrana venom. All three of the antivenoms significantly attenuated venom-induced twitch reduction of indirectly stimulated tissues when added prior to venom. When added after N. sumatrana venom, at the t90 time point, CAV and NPAV partially restored the twitch height but has no significant effect on the reduction in twitch height caused by N. kaouthia venom. The addition of KCAV, at the t90 time point, did not reverse the attenuation of indirectly stimulated twitches caused by either venom. In addition, none of the antivenoms, when added prior to venom, prevented attenuation of directly stimulated twitches. Differences in the capability of antivenoms, especially NPAV and CAV, to reverse neurotoxicity and myotoxicity indicate that there is a need to isolate and characterize neurotoxins and myotoxins from Malaysian N. kaouthia and N. sumatrana venoms to improve neutralization capability of the antivenoms.

Histopathological Changes in the Liver, Heart and Kidneys Following Malayan Pit Viper (Calloselasma rhodostoma) Envenoming and the Neutralising Effects of Hemato Polyvalent Snake Antivenom.

Calloselasma rhodostoma (Malayan pit viper) is a medically important snake species that is widely distributed across Southeast Asia. Systemic coagulopathy causing severe haemorrhage and local tissue injury is commonly observed following C. rhodostoma envenoming. However, nephrotoxicity and congestive heart failure were previously reported in a patient who had a long length of hospital stay. In this study, we determined the effect of C. rhodostoma envenoming on cardiovascular disturbances and the associated morphological changes in the liver, heart and kidneys using animal models. We also evaluated the efficacy of Hemato polyvalent antivenom (HPAV; Queen Saovabha Memorial Institute (QSMI) of the Thai Red Cross Society, Thailand) in neutralising the histopathological effects of C. rhodostoma venom. The intravenous (i.v.) administration of C. rhodostoma venom (1000 µg/kg) caused a rapid decrease in mean arterial pressure (MAP) followed by complete cardiac collapse in anaesthetized rats. Moreover, the intraperitoneal (i.p.) administration of C. rhodostoma venom (11.1 mg/kg; 3 × LD50) for 24 h caused cellular lesions in the liver and heart tissues. C. rhodostoma venom also induced nephrotoxicity, as indicated by the presence of tubular injury, interstitial vascular congestion and inflammatory infiltration in the whole area of the kidney. The administration of HPAV, at manufacturer-recommended doses, 15 min prior to or after the addition of C. rhodostoma venom reduced the extent of the morphological changes in the liver, heart and kidneys. This study found that experimental C. rhodostoma envenoming induced cardiovascular disturbances, hepatotoxicity and nephrotoxicity. We also highlighted the potential broad utility of HPAV to neutralise the histopathological effects of C. rhodostoma venom. The early delivery of antivenom appears capable of preventing envenoming outcomes.

In Vitro Toxicity of Chinese Russell's Viper (Daboia siamensis) Venom and Neutralisation by Antivenoms.

Daboia siamensis (Russell's viper) is a highly venomous and medically important snake in China, as well as much of Asia. There is minimal information on the pharmacological activity of the venom of the Chinese species, and currently no commercially available specific antivenom in China. This has led to the use of non-specific antivenoms to treat D. siamensis envenomation. In this study, the in vitro neurotoxicity and myotoxicity of D. siamensis venom was examined and the efficacy of four antivenoms was investigated, including the recently developed Chinese D. siamensis monovalent antivenom (C-DsMAV) and three commercially available antivenoms (Thai D. siamensis (Thai-DsMAV) monovalent antivenom, Deinagkistrodon acutus monovalent antivenom (DaAV), and Gloydius brevicaudus monovalent antivenom (GbAV). D. siamensis venom (10-30 µg/mL) caused the concentration-dependent inhibition of indirect twitches in the chick biventer cervicis nerve muscle preparation, without abolishing contractile responses to exogenous agonists ACh or CCh, indicating pre-synaptic neurotoxicity. Myotoxicity was also evident at these concentrations with inhibition of direct twitches, an increase in baseline tension, and the partial inhibition of ACh, CCh, and KCl responses. The prior addition of C-DsMAV or Thai-DsMAV prevented the neurotoxic and myotoxic activity of D. siamensis venom (10 µg/mL). The addition of non-specific antivenoms (GbAV and DaAV) partially prevented the neurotoxic activity of venom (10 µg/mL) but failed to neutralize the myotoxic effects. We have shown that D. siamensis venom exhibits in vitro weak presynaptic neurotoxicity and myotoxicity, which can be prevented by the pre-addition of the Chinese and Thai Russell's viper antivenoms. Non-specific antivenoms were poorly efficacious. There should be further development of a monospecific antivenom against D. siamensis envenomation in China.

In Vitro Neutralization of the Myotoxicity of Australian Mulga Snake (Pseudechis australis) and Sri Lankan Russell's Viper (Daboia russelii) Venoms by Australian and Indian Polyvalent Antivenoms.

We studied the neutralisation of Sri Lankan Russell's viper (Daboia russelii) and Australian mulga snake (Pseudechis australis) venom-induced myotoxicity by Indian (Vins and Bharat) and Australian (Seqirus) polyvalent antivenoms, using the in vitro chick biventer skeletal muscle preparation. Prior addition of Bharat or Vins antivenoms abolished D. russelii venom (30 µg/mL)-mediated inhibition of direct twitches, while Australian polyvalent antivenom was not protective. Bharat antivenom prevented, while Vins and Australian polyvalent antivenoms partially prevented, the inhibition of responses to exogenous KCl. Myotoxicity of Mulga venom (10 µg/mL) was fully neutralised by the prior addition of Australian polyvalent antivenom, partially neutralised by Vins antivenom but not by Bharat antivenom. Although the myotoxicity of both venoms was partially prevented by homologous antivenoms when added 5 min after the venom, with an increasing time delay between venom and antivenom, the reversal of myotoxicity gradually decreased. However, antivenoms partially prevented myotoxicity even 60 min after venom. The effect of antivenoms on already initiated myotoxicity was comparable to physical removal of the toxins by washing the bath at similar time points, indicating that the action of the antivenoms on myotoxicity is likely to be due to trapping the toxins or steric hindrance within the circulation, not allowing the toxins to reach target sites in muscles.

Isolation and Characterization of Two Postsynaptic Neurotoxins From Indian Cobra (Naja Naja) Venom.

The Indian Cobra (Naja naja) is among the "Big Four" responsible for most of the snakebite envenoming cases in India. Although recent proteomic studies suggest the presence of postsynaptic neurotoxins in N. naja venom, little is known about the pharmacology of these toxins. We isolated and characterized α-Elapitoxin-Nn2a (α-EPTX-Nn2a; 7020 Da) and α-Elapitoxin-Nn3a (α-EPTX-Nn3a; 7807 Da), a short-chain and long-chain postsynaptic neurotoxin, respectively, which constitute 1 and 3% of N. naja venom. α-EPTX-Nn2a (100-300 nM) and α-EPTX-Nn3a (100-300 nM) both induced concentration-dependent inhibition of indirect twitches and abolished contractile responses of tissues to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The prior incubation of tissues with Indian polyvalent antivenom (1 ml/0.6 mg) prevented the in vitro neurotoxic effects of α-EPTX-Nn2a (100 nM) and α-EPTX-Nn3a (100 nM). The addition of Indian polyvalent antivenom (1 ml/0.6 mg), at the t90 time point, could not reverse the in vitro neurotoxicity of α-EPTX-Nn2a (100 nM). The in vitro neurotoxicity of α-EPTX-Nn3a (100 nM) was partially reversed by the addition of Indian polyvalent antivenom (1 ml/0.6 mg), as well as repeated washing of the tissue. α-EPTX-Nn2a displayed non-competitive antagonism of concentration-response curves to carbachol, with a pA2 of 8.01. In contrast, α-EPTX-Nn3a showed reversible antagonism of concentration-response curves to carbachol, with a pA2 of 8.17. De novo sequencing of α-EPTX-Nn2a and α-EPTX-Nn3a showed a short-chain and long-chain postsynaptic neurotoxin, respectively, with 62 and 71 amino acids. The important observation made in this study is that antivenom can reverse the neurotoxicity of the clinically important long-chain neurotoxin, but not the short-chain neurotoxin, from N. naja venom.

The Effect of Australian and Asian Commercial Antivenoms in Reversing the Post-Synaptic Neurotoxicity of O. hannah, N. naja and N. kaouthia Venoms In Vitro.

Despite antivenoms being the only established specific treatment for neuromuscular paralysis arising from snake envenoming, their ability to reverse the post-synaptic neurotoxicity in snake envenoming is poorly understood. We investigated the ability of five commercial antivenoms i.e., King cobra monovalent, Thai cobra monovalent, Thai neuro polyvalent, Indian polyvalent and Australian polyvalent antivenoms to reverse neurotoxicity induced by the venoms of King cobra (Ophiophagus hannah, 3 µg/mL), Indian cobra (Naja naja, 5 µg/mL) and Thai cobra (Naja kaouthia, 3 µg/mL) using the in vitro chick-biventer cervicis nerve-muscle preparation. All three venoms displayed post-synaptic neurotoxicity, which was prevented by all tested antivenoms (40 µL/mL) added to the bath prior to venom. All antivenoms partially reversed the established post-synaptic neuromuscular block after the addition of the three venoms during a 180 min observation period, but to varying degrees and at different rates. The neurotoxic effects of O. hannah venom recovered to a greater magnitude (based on twitch height restoration) and faster than the neurotoxicity of N. kaouthia venom, which recovered to a lower magnitude more slowly. The recovery of post-synaptic neurotoxicity by N. naja venom was hindered due to the likely presence of cytotoxins in the venom, which cause direct muscle damage. The observations made in this study provide further evidence that the commercial antivenoms are likely to actively reverse established α-neurotoxin-mediated neuromuscular paralysis in snake envenoming, and there is cross-neutralisation with different antivenoms.

Isolation and Pharmacological Characterization of α-Elapitoxin-Oh3a, a Long-Chain Post-Synaptic Neurotoxin From King Cobra (Ophiophagus hannah) Venom.

The King Cobra (Ophiophagus hannah) is the world's largest venomous snake and has a widespread geographical distribution throughout Southeast Asia. Despite proteomic studies indicating the presence of postsynaptic neurotoxins in O. hannah venom, there are few pharmacological investigations of these toxins. We isolated and characterized α-elapitoxin-Oh3a (α-EPTX-Oh3a; 7,938 Da), a long-chain postsynaptic neurotoxin, which constitutes 5% of O. hannah venom. α-EPTX-Oh3a (100-300 nM) caused concentration-dependent inhibition of indirect twitches and inhibited contractile responses of tissues to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The prior incubation of tissues with Thai Red Cross Society King Cobra antivenom (1 ml/0.8 mg) prevented the in vitro neurotoxic effects of α-EPTX-Oh3a (100 nM). The addition of Thai Red Cross Society King Cobra antivenom (1 ml/0.8 mg), at the t90 time point partially reversed the in vitro neurotoxicity of α-EPTX-Oh3a (100 nM). Repeatedly washing the tissue did not allow significant recovery from the in vitro neurotoxic effects of α-EPTX-Oh3a (100 nM). α-EPTX-Oh3a demonstrated pseudo-irreversible antagonism of concentration-response curves to carbachol, with a pA2 of 8.99. De novo sequencing of α-EPTX-Oh3a showed a long-chain postsynaptic neurotoxin with 72 amino acids, sharing 100% sequence identity with Long neurotoxin OH-55. In conclusion, the antivenom is useful for reversing the clinically important long-chain α-neurotoxin-mediated neuromuscular paralysis.

Novel Neurotoxic Activity in Calliophis intestinalis Venom.

In this work, we investigated the in vitro neurotoxicity of Calliophis intestinalis venom using chick biventer cervicis neuromuscular preparations and electrophysiological analysis of voltage-gated sodium (NaV) channels expressed in HEK293 cells. We found that the indirect twitches of the neuromuscular preparations decreased over time when exposed to venom. However, the responses of these preparations to the agonists acetylcholine, carbachol, and potassium chloride were not changed after incubation with the venom. Our electrophysiological experiments show that C. intestinalis venom acts as a NaV channel antagonist-the first known from a vertebrate venom-by decreasing the peak current of NaV1.4 channels without changing the kinetics of activation or inactivation. Our proteomic results accord with earlier analyses and find that the venom contains three-finger toxins, cysteine-rich secretory proteins, kunitz peptides, phospholipase A2s, snake venom metalloproteases, and vespryns. Some of the three-finger toxins are similar to the δ-elapitoxins from the venom of the closely related Calliophis bivirgatus. However, δ-elapitoxins act as NaV channel agonists in C. bivirgatus whereas C. intestinalis venom contains NaV channel antagonists. The toxins and mechanisms responsible for the neuromuscular symptoms remain unclear as does the identity of the NaV channel antagonists. These aspects of this unusual venom require further study.

Isolation and Characterization of A2-EPTX-Nsm1a, a Secretory Phospholipase A2 from Malaysian Spitting Cobra (Naja sumatrana) Venom.

Phospholipase A2 (PLA2) toxins are one of the main toxin families found in snake venom. PLA2 toxins are associated with various detrimental effects, including neurotoxicity, myotoxicity, hemostatic disturbances, nephrotoxicity, edema, and inflammation. Although Naja sumatrana venom contains substantial quantities of PLA2 components, there is limited information on the function and activities of PLA2 toxins from the venom. In this study, a secretory PLA2 from the venom of Malaysian N. sumatrana, subsequently named A2-EPTX-Nsm1a, was isolated, purified, and characterized. A2-EPTX-Nsm1a was purified using a mass spectrometry-guided approach and multiple chromatography steps. Based on LC-MSMS, A2-EPTX-Nsm1a was found to show high sequence similarity with PLA2 from venoms of other Naja species. The PLA2 activity of A2-EPTX-Nsm1 was inhibited by 4-BPB and EDTA. A2-EPTX-Nsm1a was significantly less cytotoxic in a neuroblastoma cell line (SH-SY5Y) compared to crude venom and did not show a concentration-dependent cytotoxic activity. To our knowledge, this is the first study that characterizes and investigates the cytotoxicity of an Asp49 PLA2 isolated from Malaysian N. sumatrana venom in a human neuroblastoma cell line.

A Biochemical and Pharmacological Characterization of Phospholipase A2 and Metalloproteinase Fractions from Eastern Russell's Viper (Daboia siamensis) Venom: Two Major Components Associated with Acute Kidney Injury.

Acute kidney injury (AKI) following Eastern Russell's viper (Daboia siamensis) envenoming is a significant symptom in systemically envenomed victims. A number of venom components have been identified as causing the nephrotoxicity which leads to AKI. However, the precise mechanism of nephrotoxicity caused by these toxins is still unclear. In the present study, we purified two proteins from D. siamensis venom, namely RvPLA2 and RvMP. Protein identification using LCMS/MS confirmed the identity of RvPLA2 to be snake venom phospholipase A2 (SVPLA2) from Thai D. siamensis venom, whereas RvMP exhibited the presence of a factor X activator with two subunits. In vitro and in vivo pharmacological studies demonstrated myotoxicity and histopathological changes of kidney, heart, and spleen. RvPLA2 (3-10 µg/mL) caused inhibition of direct twitches of the chick biventer cervicis muscle preparation. After administration of RvPLA2 or RvMP (300 µg/kg, i.p.) for 24 h, diffuse glomerular congestion and tubular injury with minor loss of brush border were detected in envenomed mice. RvPLA2 and RvMP (300 µg/kg; i.p.) also induced congestion and tissue inflammation of heart muscle as well as diffuse congestion of mouse spleen. This study showed the significant roles of PLA2 and SVMP in snake bite envenoming caused by Thai D. siamensis and their similarities with observed clinical manifestations in envenomed victims. This study also indicated that there is a need to reevaluate the current treatment strategies for Thai D. siamensis envenoming, given the potential for irreversible nephrotoxicity.

The Dragon's Paralysing Spell: Evidence of Sodium and Calcium Ion Channel Binding Neurotoxins in Helodermatid and Varanid Lizard Venoms.

Bites from helodermatid lizards can cause pain, paresthesia, paralysis, and tachycardia, as well as other symptoms consistent with neurotoxicity. Furthermore, in vitro studies have shown that Heloderma horridum venom inhibits ion flux and blocks the electrical stimulation of skeletal muscles. Helodermatids have long been considered the only venomous lizards, but a large body of robust evidence has demonstrated venom to be a basal trait of Anguimorpha. This clade includes varanid lizards, whose bites have been reported to cause anticoagulation, pain, and occasionally paralysis and tachycardia. Despite the evolutionary novelty of these lizard venoms, their neuromuscular targets have yet to be identified, even for the iconic helodermatid lizards. Therefore, to fill this knowledge gap, the venoms of three Heloderma species (H. exasperatum, H. horridum and H. suspectum) and two Varanus species (V. salvadorii and V. varius) were investigated using Gallus gallus chick biventer cervicis nerve-muscle preparations and biolayer interferometry assays for binding to mammalian ion channels. Incubation with Heloderma venoms caused the reduction in nerve-mediated muscle twitches post initial response of avian skeletal muscle tissue preparation assays suggesting voltage-gated sodium (NaV) channel binding. Congruent with the flaccid paralysis inducing blockage of electrical stimulation in the skeletal muscle preparations, the biolayer interferometry tests with Heloderma suspectum venom revealed binding to the S3-S4 loop within voltage-sensing domain IV of the skeletal muscle channel subtype, NaV1.4. Consistent with tachycardia reported in clinical cases, the venom also bound to voltage-sensing domain IV of the cardiac smooth muscle calcium channel, CaV1.2. While Varanus varius venom did not have discernable effects in the avian tissue preparation assay at the concentration tested, in the biointerferometry assay both V. varius and V. salvadorii bound to voltage-sensing domain IV of both NaV1.4 and CaV1.2, similar to H. suspectum venom. The ability of varanid venoms to bind to mammalian ion channels but not to the avian tissue preparation suggests prey-selective actions, as did the differential potency within the Heloderma venoms for avian versus mammalian pathophysiological targets. This study thus presents the detailed characterization of Heloderma venom ion channel neurotoxicity and offers the first evidence of varanid lizard venom neurotoxicity. In addition, the data not only provide information useful to understanding the clinical effects produced by envenomations, but also reveal their utility as physiological probes, and underscore the potential utility of neglected venomous lineages in the drug design and development pipeline.