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Abstract Local tissue damage following snakebite envenoming remains a poorly researched area. To develop better strategies to treat snakebites, it is critical to understand the mechanisms through which venom toxins induce envenomation effects including local tissue damage. Here, we demonstrate how the venoms of two medically important Indian snakes (Russell's viper and cobra) affect human skeletal muscle using a cultured human myoblast cell line. The data suggest that both venoms affect the viability of myoblasts. Russell’s viper venom reduced the total number of cells, their migration, and the area of focal adhesions. It also suppressed myogenic differentiation and induced muscle atrophy. While cobra venom decreased the viability, it did not largely affect cell migration and focal adhesions. Cobra venom affected the formation of myotubes and induced atrophy. Cobra venom-induced atrophy could not be reversed by small molecule inhibitors such as varespladib (a phospholipase A2 inhibitor) and prinomastat (a metalloprotease inhibitor), and soluble activin type IIb receptor (a molecule used to promote regeneration of skeletal muscle), although the antivenom (raised against the Indian ‘Big Four’ snakes) has attenuated the effects. However, all these molecules rescued the myotubes from Russell’s viper venom-induced atrophy. This study demonstrates key steps in the muscle regeneration process that are affected by both Indian Russell’s viper and cobra venoms and offers insights into the potential causes of clinical features displayed in envenomed victims. Further research is required to investigate the molecular mechanisms of venom-induced myotoxicity under in vivo settings and develop better therapies for snakebite-induced muscle damage.

Snakebite envenoming and its resulting complications are serious threats to the health of vulnerable people living in rural areas of developing countries. The knowledge of the heterogeneity of symptoms associated with snakebite envenoming and their management strategies is vital to treat such life-threatening complications to save lives. Russell’s viper envenomation induces a diverse range of clinical manifestations from commonly recognised haemotoxic and local effects to several rare conditions that are often not reported. The lack of awareness about these unusual manifestations can affect prompt diagnosis, appropriate therapeutic approaches, and positive outcomes for patients. Here, we report pulmonary thromboembolism that developed in three patients following Russell’s viper envenomation and demonstrate their common clinical features and diagnostic and therapeutic approaches used. All patients showed clinical signs of local (oedema) and systemic (blood coagulation disturbances) envenomation, which were treated using polyvalent antivenom. They exhibited elevated heart rates, breathlessness, and reduced oxygen saturation, which are non-specific but core parameters in the diagnosis of pulmonary embolism. The recognition of pulmonary embolism was also achieved by an electrocardiogram, which showed sinus tachycardia and computed tomography and echocardiogram scans further confirmed this condition. Anti-coagulant treatment using low-molecular-weight heparin offered clinical benefits in these patients. In summary, this report reinforces the broad spectrum of previously unreported consequences of Russell’s viper envenomation. The constant updating of healthcare professionals and the dissemination of major lessons learned in the clinical management of snakebite envenoming through scientific documentation and educational programs are necessary to mitigate the adverse impacts of venomous snakebites in vulnerable communities.

BackgroundIndia suffers ~58,000 annual deaths due to snakebites. The 'Big Four' snakes (Russell's viper, Indian cobra, common krait, and saw-scaled viper) that are responsible for most bites cause diverse clinical effects. Delayed treatment increases the risk of serious complications and treatment costs. Although government hospitals offer free treatment for snakebites in India, most patients opt for private healthcare, which is an out-of-pocket expense as they often lack health insurance coverage. This study aims to analyse snakebite treatment costs in private tertiary care hospitals in Tamil Nadu, India and identifies the key factors contributing to treatment costs.Methodology/principal findingsThe treatment cost details for 913 snakebite victims were collected from 10 private tertiary care hospitals across Tamil Nadu. The data were classified into hospital, pharmacy, investigation, and laboratory costs, and analysed to determine various factors that contribute to the costs. The results demonstrate that the average treatment costs vary widely for different snakes. The hospital and pharmacy costs are higher than investigation and laboratory costs for all snakebites. Notably, Russell's viper bites cost significantly more than the bites from other snakes. Overall, the type of snake, nature of complications, specialist treatments required, and arrival time to hospitals were identified as some of the key factors for higher treatment costs.Conclusions/significanceThese data demonstrate that ~80% of snakebite patients can be treated with INR 100,000 (~GBP 1000 or USD 1200) or less. This study emphasises the urgent need to improve rural medical care by providing appropriate training for healthcare professionals and essential resources to facilitate early assessment of patients, administer the initial dose of antivenom and refer the patients to tertiary care only when needed. Moreover, the outcome of this study forms a basis for developing appropriate policies to regulate snakebite treatment costs and provide affordable medical insurance for vulnerable communities.

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Antimicrobial peptides have been developed based on plant-derived molecular scaffolds for the treatment of infectious diseases. Chenopodin is an abundant seed storage protein in quinoa, an Andean plant with high nutritional and therapeutic properties. Here, we used computer- and physicochemical-based strategies and designed four peptides derived from the primary structure of Chenopodin. Two peptides reproduce natural fragments of 14 amino acids from Chenopodin, named Chen1 and Chen2, and two engineered peptides of the same length were designed based on the Chen1 sequence. The two amino acids of Chen1 containing amide side chains were replaced by arginine (ChenR) or tryptophan (ChenW) to generate engineered cationic and hydrophobic peptides. The evaluation of these 14-mer peptides on Staphylococcus aureus and Escherichia coli showed that Chen1 does not have antibacterial activity up to 512 µM against these strains, while other peptides exhibited antibacterial effects at lower concentrations. The chemical substitutions of glutamine and asparagine by amino acids with cationic or aromatic side chains significantly favoured their antibacterial effects. These peptides did not show significant hemolytic activity. The fluorescence microscopy analysis highlighted the membranolytic nature of Chenopodin-derived peptides. Using molecular dynamic simulations, we found that a pore is formed when multiple peptides are assembled in the membrane. Whereas, some of them form secondary structures when interacting with the membrane, allowing water translocations during the simulations. Finally, Chen2 and ChenR significantly reduced SARS-CoV-2 infection. These findings demonstrate that Chenopodin is a highly useful template for the design, engineering, and manufacturing of non-toxic, antibacterial, and antiviral peptides.

Envenomings by Russell's viper (Daboia russelii), a species of high medical importance in India and other Asian countries, commonly result in hemorrhage, coagulopathies, necrosis, and acute kidney injury. Although bleeding complications are frequently reported following viper envenomings, thrombotic events occur rarely (reported only in coronary and carotid arteries) with serious consequences. For the first time, we report three serious cases of peripheral arterial thrombosis following Russell's viper bites and their diagnostic, clinical management, and mechanistic insights. These patients developed occlusive thrombi in their peripheral arteries and symptoms despite antivenom treatment. In addition to clinical features, computed tomography angiography was used to diagnose arterial thrombosis and ascertain its precise locations. They were treated using thrombectomy or amputation in one case that presented with gangrenous digits. Mechanistic insights into the pathology through investigations revealed the procoagulant actions of Russell's viper venom in standard clotting tests as well as in rotational thromboelastometry analysis. Notably, Russell's viper venom inhibited agonist-induced platelet activation. The procoagulant effects of Russell's viper venom were inhibited by a matrix metalloprotease inhibitor, marimastat, although a phospholipase A2 inhibitor (varespladib) did not show any inhibitory effects. Russell's viper venom induced pulmonary thrombosis when injected intravenously in mice and thrombi in the microvasculature and affected skeletal muscle when administered locally. These data emphasize the significance of peripheral arterial thrombosis in snakebite victims and provide awareness, mechanisms, and robust strategies for clinicians to tackle this issue in patients.

With the continued growth of human populations, rural urbanisation and habitat degradation are on the rise, resulting in the displacement of native wildlife and an increase in human-wildlife conflicts. The presence of human habitation and waste often attracts rodents and thereby, snakes, leading to increased snake sightings in homes. To address this problem, snake handlers, who are volunteers that remove and relocate snakes away from human development areas, are called upon. However, snake removal is a high-risk task that poses a risk of envenomation, particularly when dealing with spitting snakes. Several cobra species have the ability to spit venom. If the venom enters a person's eye, it can result in ophthalmic envenomation, which can have serious consequences for their eyesight. Therefore, snake handlers should take precautions, wear suitable eye protection, and use appropriate tools to ensure their safety and that of the snake. In this case, an experienced snake handler was called to remove a spitting cobra, but they were ill-equipped. During the removal, the venom was sprayed across the handler's face, and some of it entered their eye, resulting in ophthalmic envenomation. The handler promptly irrigated their eye, but medical treatment was still necessary. This report highlights the risks and consequences of ophthalmic injury and the importance of wearing appropriate eye protection and taking due care when dealing with venomous species, particularly those that can spit venom. It serves as a reminder that accidents can happen at any time and experienced snake handlers are not exempt from the risks.

Snakebite envenoming (SBE) predominantly affects rural impoverished communities that have limited access to immediate healthcare. These communities often hold numerous myths/misbeliefs about snakes and SBE. Moreover, healthcare professionals who practice in rural regions often work in unstable situations with limited medical infrastructure and therefore, lack sufficient knowledge/experience and confidence in the clinical management of SBE. Due to the lack of reliable statistics on the true burden of SBE, developing health policies for this condition by relevant authorities may be difficult. Hence, it is critical to improve awareness about SBE among rural communities, healthcare professionals and health authorities using robust multifaceted community health education approaches. Here, we describe the design, development, implementation, and impact of distinctive community health education approaches that we used in India and Brazil. A wide range of educational tools including information leaflets, posters, pocket guides, learning materials for healthcare professionals and short/long video documentaries were developed in local languages and used to engage with target communities through direct assemblies as well as mass/traditional and social media. Notably, we used diverse methods to determine the impact of our programs in improving awareness, treatment-seeking behaviour, and clinical practice. The people-centred approaches that we used were inclusive and highly impactful in instigating fundamental changes in the management of SBE among rural communities. The resources and approaches presented in this article can be easily adapted for wider use in other countries in order to collectively reduce SBE-induced deaths, disabilities and socioeconomic ramifications.

The interactions between specific snake venom toxins and muscle constituents are the major cause of severe muscle damage that often result in amputations and subsequent socioeconomic ramifications for snakebite victims and/or their families. Therefore, improving our understanding of venom-induced muscle damage and determining the underlying mechanisms of muscle degeneration/regeneration following snakebites is critical to developing better strategies to tackle this issue. Here, we analysed intramuscular bleeding and thrombosis in muscle injuries induced by two different snake venom toxins (CAMP—Crotalus atrox metalloprotease (a PIII metalloprotease from the venom of this snake) and a three-finger toxin (CTX, a cardiotoxin from the venom of Naja pallida)). Classically, these toxins represent diverse scenarios characterised by persistent muscle damage (CAMP) and successful regeneration (CTX) following acute damage, as normally observed in envenomation by most vipers and some elapid snakes of Asian, Australasian, and African origin, respectively. Our immunohistochemical analysis confirmed that both CAMP and CTX induced extensive muscle destruction on day 5, although the effects of CTX were reversed over time. We identified the presence of fibrinogen and P-selectin exposure inside the damaged muscle sections, suggesting signs of bleeding and the formation of platelet aggregates/microthrombi in tissues, respectively. Intriguingly, CAMP causes integrin shedding but does not affect any blood clotting parameters, whereas CTX significantly extends the clotting time and has no impact on integrin shedding. The rates of fibrinogen clearance and reduction in microthrombi were greater in CTX-treated muscle compared to CAMP-treated muscle. Together, these findings reveal novel aspects of venom-induced muscle damage and highlight the relevance of haemostatic events such as bleeding and thrombosis for muscle regeneration and provide useful mechanistic insights for developing better therapeutic interventions.

Envenomation by the Indian ornamental tarantula (Poecilotheria regalis) is medically relevant to humans, both in its native India and worldwide, where they are kept as pets. Muscle-related symptoms such as cramps and pain are commonly reported in humans following envenomation by this species. There is no specific treatment, including antivenom, for its envenomation. Moreover, the scientific knowledge of the impact of this venom on skeletal muscle function is highly limited. Therefore, we carried out this study to better understand the myotoxic properties of Poecilotheria regalis venom by determining its effects in cultured myoblasts and in the tibialis anterior muscle in mice. While there was no effect found on undifferentiated myoblasts, the venom affected differentiated multinucleated myotubes resulting in the reduction of fusion and atrophy of myotubes. Similarly, intramuscular administration of this venom in the tibialis anterior muscle in mice resulted in extensive muscle damage on day 5. However, by day 10, the regeneration was evident, and the regeneration process continued until day 20. Nevertheless, some tissue abnormalities including reduced dystrophin expression and microthrombi presence were observed on day 20. Overall, this study demonstrates the ability of this venom to induce significant muscle damage and affect its regeneration in the early stages. These data provide novel mechanistic insights into this venom-induced muscle damage and guide future studies to isolate and characterise individual toxic component(s) that induce muscle damage and their significance in developing better therapeutics.

Snakebite envenomation (SBE) is a life-threatening medical emergency with a high mortality rate. Common secondary complications following SBE, such as wound infections, are significant due to their impact on worsening local tissue damage and causing systemic infection. Antivenoms are not effective to treat wound infections following SBE. Moreover, in several rural clinical settings, broad-spectrum antibiotics are often used without clear guidelines or based on limited laboratory data, resulting in undesirable side effects and exacerbated treatment costs. Therefore, robust antibiotic strategies should be developed to tackle this critical issue. Currently, there is limited information available on the bacterial profiles of SBE-induced infections and antibiotic susceptibility. Hence, it is essential to improve the knowledge of bacterial profiles and their antibiotic sensitivity in SBE victims to develop better treatment strategies. This study aimed to address this issue by examining the bacterial profiles of SBE victims with a specific focus on Russell’s viper envenomation. The most frequently found bacteria in the bites of SBE victims were Staphylococcus aureus, Klebsiella sp., Escherichia coli, and Pseudomonas aeruginosa. Linezolid, clindamycin, colistin, meropenem, and amikacin were some of the most effective antibiotics for commonly grown bacteria in SBE victims. Similarly, ciprofloxacin, ampicillin, amoxiclave, cefixime, and tetracyclin were the least effective antibiotics for common bacteria found in the wound swabs of SBE victims. These data provide robust guidance for infection management following SBE and offer useful insights to aid in designing effective treatment protocols for SBE with serious wound infections in rural areas where laboratory facilities may not be readily available.

Infections caused by multi-drug-resistant (MDR) bacteria are a global threat to human health. As venoms are the source of biochemically diverse bioactive proteins and peptides, we investigated the antimicrobial activity and murine skin infection model-based wound healing efficacy of a 13 kDa protein. The active component PaTx-II was isolated from the venom of Pseudechis australis (Australian King Brown or Mulga Snake). PaTx-II inhibited the growth of Gram-positive bacteria in vitro, with moderate potency (MICs of 25 µM) observed against S. aureus, E. aerogenes, and P. vulgaris. The antibiotic activity of PaTx-II was associated with the disruption of membrane integrity, pore formation, and lysis of bacterial cells, as evidenced by scanning and transmission microscopy. However, these effects were not observed with mammalian cells, and PaTx-II exhibited minimal cytotoxicity (CC50 > 1000 µM) toward skin/lung cells. Antimicrobial efficacy was then determined using a murine model of S. aureus skin infection. Topical application of PaTx-II (0.5 mg/kg) cleared S. aureus with concomitant increased vascularization and re-epithelialization, promoting wound healing. As small proteins and peptides can possess immunomodulatory effects to enhance microbial clearance, cytokines and collagen from the wound tissue samples were analyzed by immunoblots and immunoassays. The amounts of type I collagen in PaTx-II-treated sites were elevated compared to the vehicle controls, suggesting a potential role for collagen in facilitating the maturation of the dermal matrix during wound healing. Levels of the proinflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and interleukin-10 (IL-10), factors known to promote neovascularization, were substantially reduced by PaTx-II treatment. Further studies that characterize the contributions towards efficacy imparted by in vitro antimicrobial and immunomodulatory activity with PaTx-II are warranted.

Annexin A1 (ANXA1) is an endogenous protein, which plays a central function in the modulation of inflammation. While the functions of ANXA1 and its exogenous peptidomimetics, N-Acetyl 2-26 ANXA1-derived peptide (ANXA1Ac2-26), in the modulation of immunological responses of neutrophils and monocytes have been investigated in detail, their effects on the modulation of platelet reactivity, haemostasis, thrombosis, and platelet-mediated inflammation remain largely unknown. Here, we demonstrate that the deletion of Anxa1 in mice upregulates the expression of its receptor, formyl peptide receptor 2/3 (Fpr2/3, orthologue of human FPR2/ALX). As a result, the addition of ANXA1Ac2-26 to platelets exerts an activatory role in platelets, as characterised by its ability to increase the levels of fibrinogen binding and the exposure of P-selectin on the surface. Moreover, ANXA1Ac2-26 increased the development of platelet-leukocyte aggregates in whole blood. The experiments carried out using a pharmacological inhibitor (WRW4) for FPR2/ALX, and platelets isolated from Fpr2/3-deficient mice ascertained that the actions of ANXA1Ac2-26 are largely mediated through Fpr2/3 in platelets. Together, this study demonstrates that in addition to its ability to modulate inflammatory responses via leukocytes, ANXA1 modulates platelet function, which may influence thrombosis, haemostasis, and platelet-mediated inflammation under various pathophysiological settings.

Adult skeletal muscle has robust regenerative capabilities due to the presence of a resident stem cell population called satellite cells. Muscle injury leads to these normally quiescent cells becoming molecularly and metabolically activated and embarking on a program of proliferation, migration, differentiation, and fusion culminating in the repair of damaged tissue. These processes are highly coordinated by paracrine signaling events that drive cytoskeletal rearrangement and cell-cell communication. Pannexins are a family of transmembrane channel proteins that mediate paracrine signaling by ATP release. It is known that Pannexin1 (Panx1) is expressed in skeletal muscle, however, the role of Panx1 during skeletal muscle development and regeneration remains poorly understood. Here we show that Panx1 is expressed on the surface of myoblasts and its expression is rapidly increased upon induction of differentiation and that Panx1–/– mice exhibit impaired muscle regeneration after injury. Panx1–/– myoblasts activate the myogenic differentiation program normally, but display marked deficits in migration and fusion. Mechanistically, we show that Panx1 activates P2 class purinergic receptors, which in turn mediate a lipid signaling cascade in myoblasts. This signaling induces bleb-driven amoeboid movement that in turn supports myoblast migration and fusion. Finally, we show that Panx1 is involved in the regulation of cell-matrix interaction through the induction of ADAMTS (Disintegrin-like and Metalloprotease domain with Thrombospondin-type 5) proteins that help remodel the extracellular matrix. These studies reveal a novel role for lipid-based signaling pathways activated by Panx1 in the coordination of myoblast activities essential for skeletal muscle regeneration.

India suffers the highest incidence of snakebite envenomation (SBE) in the world. Rural communities within India and other countries have long-held cultural beliefs surrounding snakes and SBE treatments, with snake statues present in numerous Hindu temples. While most cultural beliefs are well respected and do not affect anyone, some people worship live venomous snakes without any safety precautions. Moreover, they practice various inappropriate first aid and traditional treatments that exacerbate SBE-induced complications. We report an unusual case of SBE on the tongue of a patient who was bitten while worshipping Russell’s viper following the advice of an astrologer based on the appearance of a snake in the patient’s dream. Following the bite, the tongue was deeply incised by the priest as a first aid to mitigate SBE-induced complications. The patient suffered profuse bleeding and swelling of the tongue resulting in difficulties in intubating them. The patient regained consciousness after antivenom administration, intranasal ventilation, and blood removal from the mouth. The tongue underwent extensive surgery to restore movement and function. This report advises caution to those undertaking the extremely risky practice of worshipping live snakes and emphasises the urgent need to develop and enforce policies to mitigate such actions and educate rural communities.

Micrurus is a medically relevant genus of venomous snakes composed of 85 species. Bites caused by coral snakes are rare, but they are usually associated with very severe and life-threatening clinical manifestations. Ecuador is a highly biodiverse country with a complex natural environment, which is home to approximately 20% of identified Micrurus species. Additionally, it is on the list of Latin American countries with the highest number of snakebites. However, there is no local antivenom available against the Ecuadorian snake venoms, and the biochemistry of these venoms has been poorly explored. Only a limited number of samples collected in the country from the Viperidae family were recently characterised. Therefore, this study addressed the compositional patterns of two coral snake venoms from Ecuador, M. helleri and M. mipartitus, using venomics strategies, integrating sample fractionation, gel electrophoresis, and mass spectrometry. Chromatographic and electrophoretic profiles of these snake venoms revealed interspecific variability, which was ascertained by mass spectrometry. The two venoms followed the recently recognised dichotomic toxin expression trends displayed by Micrurus species: M. helleri venom contains a high proportion (72%) of phospholipase A2, whereas M. mipartitus venom is dominated by three-finger toxins (63%). A few additional protein families were also detected in these venoms. Overall, these results provide the first comprehensive views on the composition of two Ecuadorian coral snake venoms and expand the knowledge of Micrurus venom phenotypes. These findings open novel perspectives to further research the functional aspects of these biological cocktails of PLA2s and 3FTxs and stress the need for the preclinical evaluation of the currently used antivenoms for therapeutic purposes in Ecuador.

In India, most snakebite envenomation (SBE) incidents are caused by the “Big Four” snakes which include Russell’s viper, common krait, Indian cobra, and saw-scaled viper. Their common envenomation effects include neurotoxicity, myotoxicity, and coagulopathy. However, they also induce rare complications such as priapism, pseudoaneurysm, and sialolithiasis. Ocular manifestations such as optic neuritis develop rarely following envenomations by non-spitting snakes and they may cause temporary vision changes and blindness if untreated. While optic neuritis following Indian cobra envenomation has been reported previously, this was not encountered in victims of common kraits. Hence, for the first time, we report optic neuritis developed in a victim following envenomation by a common krait and compare its clinical features and diagnostic and therapeutic methods used with another case of optic neuritis in a victim of an Indian cobra bite. Both patients received antivenom treatment and made an initial recovery; however, optic neuritis developed several days later. The condition was diagnosed using ophthalmic examination together with computed tomography and/or magnetic resonance imaging methods. Due to very similar clinical features, both patients received intravenous corticosteroids which restored their vision and successfully treated optic neuritis. This case report suggests that the optic neuritis developed in a common krait envenomation is comparable to the one developed following a cobra bite, and therefore, the same diagnostic and therapeutic approaches can be used. This study also raises awareness of this rare complication and provides guidance for the diagnosis and treatment of SBE-induced optic neuritis.

Following a bite from a juvenile Russell's viper (Daboia russelii), a priapism (painful erection) developed rapidly in a 16-year-old male and only subsided after administration of antivenom 3 hours later. Potential mechanisms for this snakebite-induced priapism are unclear but likely due to venom toxins causing nitric oxide (NO) release and subsequent vasodilation of endothelium in the corpus cavernosum, although the possible involvement of other mechanisms cannot be ruled out. We strongly believe that this unusual case report may lead to further scientific research in order to improve the clinical understanding of the pathophysiology of envenomation due to Russell's viper bites. Although it is too early to speculate, further research may also discover the possibilities of developing venom-based candidate molecules to treat sexual dysfunction in males and females.

Activin/myostatin signalling acts to induce skeletal muscle atrophy in adult mammals by inhibiting protein synthesis as well as promoting protein and organelle turnover. Numerous strategies have been successfully developed to attenuate the signalling properties of these molecules, which result in augmenting muscle growth. However, these molecules, in particular activin, play major roles in tissue homeostasis in numerous organs of the mammalian body. We have recently shown that although the attenuation of activin/myostatin results in robust muscle growth, it also has a detrimental impact on the testis. Here, we aimed to discover the long-term consequences of a brief period of exposure to muscle growth-promoting molecules in the testis. We demonstrate that muscle hypertrophy promoted by a soluble activin type IIB ligand trap (sActRIIB) is a short-lived phenomenon. In stark contrast, short-term treatment with sActRIIB results in immediate impact on the testis, which persists after the sessions of the intervention. Gene array analysis identified an expansion in aberrant gene expression over time in the testis, initiated by a brief exposure to muscle growth-promoting molecules. The impact on the testis results in decreased organ size as well as quantitative and qualitative impact on sperm. Finally, we have used a drug-repurposing strategy to exploit the gene expression data to identify a compound – N6-methyladenosine – that may protect the testis from the impact of the muscle growth-promoting regime. This work indicates the potential long-term harmful effects of strategies aimed at promoting muscle growth by attenuating activin/myostatin signalling. Furthermore, we have identified a molecule that could, in the future, be used to overcome the detrimental impact of sActRIIB treatment on the testis.

The lack of public awareness surrounding the dangers of snakebite envenomation (SBE) is one of the most critical factors contributing to SBE-induced complications, and subsequently exacerbating the number of deaths and disabilities resulting from SBE. In this study, we deployed a multifaceted community education programme to educate students, healthcare professionals and members of the public in rural areas of Tamil Nadu, India about the dangers of SBE, appropriate first aid measures and the 'do's and don'ts' following a snakebite. An assessment of prior knowledge within these communities identified several misconceptions concerning snakes and SBE. Using a combination of direct engagement (estimated to reach over 200,000 people), information leaflets (200,000 distributed), posters, video documentaries, media and social media (>2.8 million engagements), over the course of one year (January to December 2019) we reached over 3 million people in rural Tamil Nadu (around 8% of population). Evaluation of community-based assemblies indicated that at least 90% of attendees were able to recall the key messages at the end of the events, and at least 85% were able to recall the key messages even after 12 months. Due to high demand, a one-day symposium was organised to provide clinical knowledge and training on SBE to 250 healthcare professionals in rural Tamil Nadu. Notably, an assessment of patient data (291 victims) collected from a snakebite referral hospital over the same 12-month period (2019) indicated that arrival time at hospital following a snakebite was significantly faster and the effective first aid measures were administered to patients who were aware of our activities compared to those that were not. Overall, our approach provides a framework on how to educate rural communities about the dangers of SBE and thereby, mitigate delayed SBE treatment leading to an overall reduction in SBE-induced mortality, morbidity, treatment costs and other socio-economic ramifications.

Russell’s viper (Daboia russelii), one of the ‘Big Four’ venomous snakes in India, is responsible for the majority of snakebite-induced deaths and permanent disabilities. Russell’s viper bites are known to induce bleeding/clotting abnormalities, as well as myotoxic, nephrotoxic, cytotoxic and neurotoxic envenomation effects. In addition, they have been reported to induce rare envenomation effects such as priapism, sialolithiasis and splenic rupture. However, Russell’s viper bite-induced pseudoaneurysm (PA) has not been previously reported. PA or false aneurysm is a rare phenomenon that occurs in arteries following traumatic injuries including some animal bites, and it can become a life-threatening condition if not treated promptly. Here, we document two clinical cases of Russell’s viper bites where PA has developed, despite antivenom treatment. Notably, a non-surgical procedure, ultrasound-guided compression (USGC), either alone, or in combination with thrombin was effectively used in both the cases to treat the PA. Following this procedure and additional measures, the patients made complete recoveries without the recurrence of PA which were confirmed by subsequent examination and ultrasound scans. These data demonstrate the development of PA as a rare complication following Russell’s viper bites and the effective use of a simple, non-surgical procedure, USGC for the successful treatment of PA. These results will create awareness among healthcare professionals on the development of PA and the use of USGC in snakebite victims following bites from Russell’s vipers, as well as other viper bites.

Duchenne Muscular Dystrophy is a devastating disease caused by the absence of a functional rod-shaped cytoplasmic protein called dystrophin. Several avenues are being developed aimed to restore dystrophin expression in boys affected by this X-linked disease. However, its complete cure is likely to need combinational approaches which may include regimes aimed at restoring muscle mass. Augmenting muscle growth through the manipulation of the Myostatin/Activin signalling axis has received much attention. However, we have recently shown that while manipulation of this axis in wild type mice using the sActRIIB ligand trap indeed results in muscle growth, it also had a detrimental impact on the testis. Here we examined the impact of administering a powerful Myostatin/Activin antagonist in two mouse models of Duchenne Muscular Dystrophy. We report that whilst the impact on muscle growth was not always positive, both models showed attenuated testis development. Sperm number, motility and ultrastructure were significantly affected by the sActRIIB treatment. Our report suggests that interventions based on Myostatin/Activin should investigate off-target effects on tissues as well as muscle.

Cardiovascular diseases (CVDs) are a primary cause of deaths worldwide. Thrombotic diseases, specifically stroke and coronary heart diseases, account for around 85% of CVDs-induced deaths. Platelets (small circulating blood cells) are responsible for the prevention of excessive bleeding upon vascular injury, through blood clotting (haemostasis). However, unnecessary activation of platelets under pathological conditions, such as upon the rupture of atherosclerotic plaques, results in thrombus formation (thrombosis), which can cause life threatening conditions such as stroke or heart attack. Therefore, antiplatelet medications are usually prescribed for people who are at a high risk of thrombotic diseases. The currently used antiplatelet drugs are associated with major side effects such as excessive bleeding, and some patients are resistant to these drugs. Therefore, numerous studies have been conducted to develop new antiplatelet agents and notably, to establish the relationship between edible plants, specifically fruits, vegetables and spices, and cardiovascular health. Indeed, healthy and balanced diets have proven to be effective for the prevention of CVDs in diverse settings. A high intake of fruits and vegetables in regular diet is associated with lower risks for stroke and coronary heart diseases because of their plethora of phytochemical constituents. In this review, we discuss the impacts of commonly used selected edible plants (specifically vegetables, fruits and spices) and/or their isolated compounds on the modulation of platelet function, haemostasis and thrombosis.

Snakebite-induced acute kidney injury (AKI) is frequently observed in patients following bites from vipers such as Russell’s viper (Daboia russelii) in India. Currently, the levels of serum creatinine are mainly used as a marker to determine the necessity for renal replacement therapy (RRT) (haemodialysis) in severe cases of AKI. However, it takes up to 48 h to ascertain a distinct change in creatinine levels compared to its baseline level upon admission. The time lost between admission and the 48 h timepoint significantly affects the clinical management of snakebite victims. Moreover, early diagnosis of AKI and decision on the necessity for RRT in snakebite victims is critical in saving lives, reducing long-term complications, and minimising treatment costs arising from expensive haemodialysis. Neutrophil gelatinase–associated lipocalin (NGAL) has been recently studied as a robust early marker for AKI in non-snakebite patients. However, its suitability for clinical use in snakebite victims has not been rigorously established. Here, we demonstrate the clinical significance of plasma NGAL as a robust marker for RRT following AKI using a large cohort (309) of Russell’s viper victims without any pre-existing health conditions. NGAL levels upon admission are positively correlated with creatinine levels at 48 h in different stages of AKI. Overall, NGAL acts as a robust early marker to ascertain the need for RRT following Russell’s viper bites. The quantification of NGAL can be recommended as a routine test in hospitals that treat snakebites to decide on RRT at early time points instead of waiting for 48 h to confirm the increase in creatinine levels. The diagnostic use of NGAL in Russell’s viper victims with pre-existing comorbidities and for other vipers should be evaluated in future studies.

1,8-cineole, a monoterpenoid is a major component of eucalyptus oil and has been proven to possess numerous beneficial effects in humans. Notably, 1,8-cineole is the primary active ingredient of a clinically approved drug, Soledum® which is being mainly used for the maintenance of sinus and respiratory health. Due to its clinically valuable properties, 1,8-cineole has gained significant scientific interest over the recent years specifically to investigate its anti-inflammatory and antioxidant effects. However, the impact of 1,8-cineole on the modulation of platelet activation, thrombosis and haemostasis was not fully established. Therefore, in this study, we demonstrate the effects of 1,8-cineole on agonists-induced platelet activation, thrombus formation under arterial flow conditions and haemostasis in mice. 1,8-cineole largely inhibits platelet activation stimulated by glycoprotein VI (GPVI) agonists such as collagen and cross-linked collagen-related peptide (CRP-XL), while it displays minimal inhibitory effects on thrombin or ADP-induced platelet aggregation. It inhibited inside-out signalling to integrin αIIbβ3 and outside-in signalling triggered by the same integrin as well as granule secretion and intracellular calcium mobilisation in platelets. 1,8-cineole affected thrombus formation on collagen-coated surface under arterial flow conditions and displayed a minimal effect on haemostasis of mice at a lower concentration of 6.25 µM. Notably, 1,8-cineole was found to be non-toxic to platelets up to 50 µM concentration. The investigation on the molecular mechanisms through which 1,8-cineole inhibits platelet function suggests that this compound affects signalling mediated by various molecules such as AKT, Syk, LAT, and cAMP in platelets. Based on these results, we conclude that 1,8-cineole may act as a potential therapeutic agent to control unwarranted platelet reactivity under various pathophysiological settings.

Abstract The constant increase in cardiovascular disease rate coupled with significant drawbacks of existing therapies emphasise the necessity to improve therapeutic strategies. Natural flavonoids exert innumerable pharmacological effects in humans. Here, we demonstrate the effects of chrysin, a natural flavonoid found largely in honey and passionflower on the modulation of platelet function, haemostasis and thrombosis. Chrysin displayed significant inhibitory effects on isolated platelets, however, its activity was substantially reduced under physiological conditions. In order to increase the efficacy of chrysin, a sulfur derivative (thio-chrysin), and ruthenium-complexes (Ru-chrysin and Ru-thio-chrysin) were synthesised and their effects on the modulation of platelet function were evaluated. Indeed, Ru-thio-chrysin displayed a 4-fold greater inhibition of platelet function and thrombus formation in vitro than chrysin under physiologically relevant conditions such as in platelet-rich plasma and whole blood. Notably, Ru-thio-chrysin exhibited similar efficacy to chrysin in the modulation of haemostasis in mice. Increased bioavailability and cell permeability of Ru-thio-chrysin compared to chrysin were found to be the basis for its enhanced activity. Together, these results demonstrate that Ru-thio-coupled natural compounds such as chrysin may serve as promising templates for the development of novel anti-thrombotic agents.

Snakebite is a major neglected tropical health issue that affects over 5 million people worldwide resulting in around 1.8 million envenomations and 100,000 deaths each year. Snakebite envenomation also causes innumerable morbidities, specifically loss of limbs as a result of excessive tissue/muscle damage. Snake venom metalloproteases (SVMPs) are a predominant component of viper venoms, and are involved in the degradation of basement membrane proteins (particularly collagen) surrounding the tissues around the bite site. Although their collagenolytic properties have been established, the molecular mechanisms through which SVMPs induce permanent muscle damage are poorly understood. Here, we demonstrate the purification and characterisation of an SVMP from a viper (Crotalus atrox) venom. Mass spectrometry analysis confirmed that this protein is most likely to be a group III metalloprotease (showing high similarity to VAP2A) and has been referred to as CAMP (Crotalus atrox metalloprotease). CAMP displays both collagenolytic and fibrinogenolytic activities and inhibits CRP-XL-induced platelet aggregation. To determine its effects on muscle damage, CAMP was administered into the tibialis anterior muscle of mice and its actions were compared with cardiotoxin I (a three-finger toxin) from an elapid snake (Naja pallida) venom. Extensive immunohistochemistry analyses revealed that CAMP significantly damages skeletal muscles by attacking the collagen scaffold and other important basement membrane proteins, and prevents their regeneration through disrupting the functions of satellite cells. In contrast, cardiotoxin I destroys skeletal muscle by damaging the plasma membrane, but does not impact regeneration due to its inability to affect the extracellular matrix. Overall, this study provides novel insights into the mechanisms through which SVMPs induce permanent muscle damage.

Snakebite envenomation (SBE) is a high-priority, neglected tropical disease. This devastating occupational health hazard disproportionately affects rural farming communities in tropical countries. This is exacerbated by the distribution and densities of venomous snakes, incidence of encounters, and limited access to advanced healthcare, including antivenom. Before the development of antivenom, desperation and spiritual beliefs led patients to experiment with a wide range of traditional treatments. Many of these treatments still survive today, particularly in regions where access to healthcare is limited. Plants are a major source of bioactive molecules, including several lifesaving medications that are widely used to this day. However, much of the research into the use of traditional plant treatments for SBE are limited to preliminary analysis or have focused on techniques used to confirm antibody efficacy that are not suitable for non-antibody-containing treatments. Modern drugs are developed through a robust pharmaceutical drug discovery and development process, which applies as much to SBE as it does to any other disease. This review discusses specifically why research into ethnobotanical practices has failed to identify or develop a novel treatment for SBE and proposes specific approaches that should be considered in this area of research in the future.

Exotic snakebites (i.e. from non-native species) are a rare occurrence, but they present a unique challenge to clinicians treating these patients. Poison control centers are often contacted to assist in the management and care of these medical emergencies. In this study, we analyzed case records of the two Pennsylvania poison control centers from 2004 to 2018 to describe clinical features reported as a result of exotic snakebite envenomation. For the 15-year period reviewed, 18 exotic snakebites were reported with effects ranging from mild local tissue injury to patients who were treated with mechanical ventilation due to respiratory failure. The mean age of the patients was 35 years and males accounted for 83% of the cases. Antivenom, the only specific treatment, was administered in seven of 18 patients within an average of four h of envenomation. The procurement of antivenom against these exotic species may require substantial logistical efforts due to limited stocking of this rarely used treatment. Newer, targeted, small molecule treatments that are being currently investigated may aid in the treatment of snakebites in general. However, people should be cautious when handling these exotic species, and clinicians should be aware of these bites and relevant clinical effects in order to manage these when reported.

Platelet-associated complications including thrombosis, thrombocytopenia, and haemorrhage are commonly observed during various inflammatory diseases such as psoriasis. Although several mechanisms that may contribute to the dysfunction of platelets during inflammatory diseases have been reported, knowledge on the primary molecules/mechanisms that underpin platelet-associated complications in such conditions is not fully established. Here, we report the significance of the mouse antimicrobial cathelicidin, mouse cathelicidin-related antimicrobial peptide (mCRAMP) (an orthologue of LL37 in humans), on the modulation of platelet reactivity during psoriasis using Imiquimod-induced psoriasis in mice as an inflammatory disease model for psoriasis vulgaris in humans. The activation of platelets during psoriasis is increased as evidenced by the elevated levels of fibrinogen binding and P-selectin exposure on the surface of platelets, and the level of soluble P-selectin in the plasma of psoriatic mice. The skin and plasma of psoriatic mice displayed increased levels of mCRAMP. Moreover, the plasma of psoriatic mice augmented the activation of platelets obtained from healthy mice. The effect of mCRAMP is partially mediated through formyl peptide receptor 2/3 (Fpr2/3, the orthologue to human FPR2/ALX) in platelets as a significant reduction in their activation was observed when FPR2/ALX-selective inhibitors such as WRW4 or Fpr2/3-deficient mouse platelets were used in these assays. Since the level of antimicrobial cathelicidin is increased in numerous inflammatory diseases such as psoriasis, atherosclerosis, and inflammatory bowel disease, the results of this study point towards a critical role for antimicrobial cathelicidin and FPR2/ALX in the development of platelet-related complications in such diseases.

In the field of antivenom research, development, and manufacture, it is often advised to follow the World Health Organization’s (WHO) guidelines for the production, control, and regulation of snake antivenom immunoglobulins, which recommend the use of preincubation assays to assess the efficacy of snakebite therapeutics. In these assays, venom and antivenom are mixed and incubated prior to in vivo administration to rodents, which allows for a standardizable comparison of antivenoms with similar characteristics. However, these assays are not necessarily sufficient for therapeutics with significantly different pharmacological properties than antibody-based antivenoms, such as small molecule inhibitors, nanoparticles, and other modalities. To ensure that the in vivo therapeutic utility of completely novel toxin-neutralizing molecules with no history of use in envenoming therapy and variable pharmacokinetics is properly evaluated, such molecules must also be tested in preclinical rescue assays, where rodents are first challenged with appropriate doses of venoms or toxins, followed by the administration of neutralizing modalities after an appropriate time delay to better mimic the real-life scenarios faced by human snakebite victims. Such an approach takes the venom (or toxin) toxicokinetics, the drug pharmacokinetics, and the drug pharmacodynamics into consideration. If new modalities are only assessed in preincubation assays and not subjected to evaluation in rescue assays, the publication of neutralization data may unintentionally misrepresent the actual therapeutic efficacy and suitability of the modality being tested, and thus potentially misguide strategic decision making in the research and development of novel therapies for snakebite envenoming.

Snakebite envenomation causes over 140,000 deaths every year, predominantly in developing countries. As a result, it is one of the most lethal neglected tropical diseases. It is associated with incredibly complex pathophysiology due to the vast number of unique toxins/proteins present in the venoms of diverse snake species found worldwide. Here, we report the purification and functional characteristics of a Group I (PI) metalloprotease (CAMP-2) from the venom of the western diamondback rattlesnake, Crotalus atrox. Its sensitivity to matrix metalloprotease inhibitors (batimastat and marimastat) was established using specific in vitro experiments and in silico molecular docking analysis. CAMP-2 shows high sequence homology to atroxase from the venom of Crotalus atrox and exhibits collagenolytic, fibrinogenolytic and mild haemolytic activities. It exerts a mild inhibitory effect on agonist-induced platelet aggregation in the absence of plasma proteins. Its collagenolytic activity is completely inhibited by batimastat and marimastat. Zinc chloride also inhibits the collagenolytic activity of CAMP-2 by around 75% at 50 μM, while it is partially potentiated by calcium chloride. Molecular docking studies have demonstrated that batimastat and marimastat are able to bind strongly to the active site residues of CAMP-2. This study demonstrates the impact of matrix metalloprotease inhibitors in the modulation of a purified, Group I metalloprotease activities in comparison to the whole venom. By improving our understanding of snake venom metalloproteases and their sensitivity to small molecule inhibitors, we can begin to develop novel and improved treatment strategies for snakebites.

Aberrant activation of the transcription factor NF-κB, as well as uncontrolled inflammation, has been linked to autoimmune diseases, development and progression of cancer, and neurological disorders like Alzheimer’s disease. Reporter cell lines are a valuable state-of-the art tool for comparative analysis of in vitro drug screening. However, a reporter cell line for the investigation of NF-κB-driven neuroinflammation has not been available. Thus, we developed a stable neural NF-κB-reporter cell line to assess the potency of proinflammatory molecules and peptides, as well as anti-inflammatory pharmaceuticals. We used lentivirus to transduce the glioma cell line U251-MG with a tandem NF-κB reporter construct containing GFP and firefly luciferase allowing an assessment of NF-κB activity via fluorescence microscopy, flow cytometry, and luminometry. We observed a robust activation of NF-κB after exposure of the reporter cell line to tumour necrosis factor alpha (TNFα) and amyloid-β peptide [1-42] as well as to LPS derived from Salmonella minnesota and Escherichia coli. Finally, we demonstrate that the U251-NF-κB-GFP-Luc reporter cells can be used for assessing the anti-inflammatory potential of pharmaceutical compounds using Bay11-7082 and IMD0354. In summary, our newly generated cell line is a robust and cost-efficient tool to study pro- and anti-inflammatory potential of drugs and biologics in neural cells.

Platelets are anucleated blood cells that participate in a wide range of physiological and pathological functions. Their major role is mediating haemostasis and thrombosis. In addition to these classic functions, platelets have emerged as important players in the innate immune system. In particular, they interact with leukocytes, secrete pro- and anti-inflammatory factors, and express a wide range of inflammatory receptors including Toll-like receptors (TLRs), for example, Toll-like receptor 4 (TLR4). TLR4, which is the most extensively studied TLR in nucleated cells, recognises lipopolysaccharides (LPS) that are compounds of the outer surface of Gram-negative bacteria. Unlike other TLRs, TLR4 is able to signal through both the MyD88-dependent and MyD88-independent signalling pathways. Notably, despite both pathways culminating in the activation of transcription factors, TLR4 has a prominent functional impact on platelet activity, haemostasis, and thrombosis. In this review, we summarise the current knowledge on TLR4 signalling in platelets, critically discuss its impact on platelet function, and highlight the open questions in this area.

Cardiovascular diseases represent a major cause of mortality and morbidity in the world, and specifically, thrombotic conditions such as heart attacks and strokes are caused by unwarranted activation of platelets and subsequent formation of blood clots (thrombi) within the blood vessels during pathological circumstances. Therefore, platelets act as a primary therapeutic target to treat and prevent thrombotic conditions. Current treatments are limited due to intolerance, and they are associated with severe side effects such as bleeding complications. Hence, the development of novel therapeutic strategies for thrombotic diseases is an urgent priority. Flavonoids are naturally occurring plant-derived molecules that exert numerous beneficial effects in humans through modulating the functions of distinct cell types. However, naturally occurring flavonoids suffer from several issues such as poor solubility, lipophilicity, and bioavailability, which hinder their efficacy and potency. Despite these, flavonoids act as versatile templates for the design and synthesis of novel molecules for various therapeutic targets. Indeed, several synthetic flavonoids have recently been developed to improve their stability, bioavailability, and efficacy, including for the modulation of platelet function. Here, we provide insight into the actions of certain natural flavonoids along with the advantages of synthetic flavonoids in the modulation of platelet function, haemostasis, and thrombosis.

Snakebite envenoming (SBE) is a priority neglected tropical disease, which kills in excess of 100,000 people per year. Additionally, many millions of survivors also suffer through disabilities and long-term health consequences. The only treatment for SBE, antivenom, has a number of major associated problems, not least, adverse reactions and limited availability. This emphasises the necessity for urgent improvements to the management of this disease. Administration of antivenom is too frequently based on symptomatology, which results in wasting crucial time. The majority of SBE-affected regions rely on broad-spectrum polyvalent antivenoms that have a low content of case-specific efficacious immunoglobulins. Research into small molecular therapeutics such as varespladib/methyl-varespladib (PLA2 inhibitors) and batimastat/marimastat (metalloprotease inhibitors) suggest that such adjunctive treatments could be hugely beneficial to victims. Progress into toxin-specific monoclonal antibodies as well as alternative binding scaffolds such as aptamers hold much promise for future treatment strategies. SBE is not implicit during snakebite, due to venom metering. Thus, the delay between bite and symptom presentation is critical and when symptoms appear it may often already be too late to effectively treat SBE. The development of reliable diagnostical tools could therefore initiate a paradigm shift in the treatment of SBE. While the complete eradication of SBE is an impossibility, mitigation is in the pipeline, with new treatments and diagnostics rapidly emerging. Here we critically review the urgent necessity for the development of diagnostic tools and improved therapeutics to mitigate the deaths and disabilities caused by SBE.

Snakebite envenomation is an affliction currently estimated to be killing upwards of 100,000 people annually. Snakebite is associated with a diverse pathophysiology due to the magnitude of variation in venom composition that is observed worldwide. The haemolytic (i.e., lysis of red blood cells) actions of snake venoms are well documented, although the direct impact of venoms on haemoglobin is not fully understood. Here we report on the varied ability of a multitude of snake venoms to oxidise haemoglobin into methaemoglobin. Moreover, our results demonstrate that the venom of an elapid, the black necked spitting cobra, Naja nigricollis, oxidises oxyhaemoglobin (Fe2+) into methaemoglobin (Fe3+) in a time- and concentration-dependent manner that is unparalleled within the 47 viper and elapid venoms evaluated. The treatment of venom with a reducing agent, dithiothreitol (DTT) is observed to potentiate this effect at higher concentrations, and the use of denatured venom demonstrates that this effect is dependent upon the heat-sensitive proteinaceous elements of the venom. Together, our results suggest that Naja nigricollis venom appears to promote methaemoglobin production to a degree that is rare within the Elapidae family, and this activity appears to be independent of proteolytic activities of venom components on haemoglobin.

Objective. Most snakebite deaths occur prior to hospital arrival; yet inexpensive, effective, and easy to administer out-of-hospital treatments do not exist. Acetylcholinesterase inhibitors can be therapeutic in neurotoxic envenomations when administered intravenously, but nasally delivered drugs could facilitate prehospital therapy for these patients. We tested the feasibility of this idea in experimentally envenomed mice. Methods. Mice received intraperitoneal injections of Naja naja venom 2.5 to 10 times the estimated LD50 and then received 5 μL neostigmine (0.5 mg/mL) or 5 μL normal saline by nasal administration. Animals were observed up to 12 hours and survivors were euthanized. Results. 100% of control mice died. Untreated mice injected with 2.5× LD50 Naja naja died at average 193 minutes after injection, while 10 of 15 (67%) of treated mice survived and were behaviorally normal by 6 hours (P

BackgroundSnakebite represents a significant health issue worldwide, affecting several million people each year with as many as 95,000 deaths. India is considered to be the country most affected, but much remains unknown about snakebite incidence in this country, its socio-economic impact and how snakebite management could be improved.Methods/principal findingsWe conducted a study within rural villages in Tamil Nadu, India, which combines a household survey (28,494 people) of snakebite incidence with a more detailed survey of victims in order to understand the health and socio-economic effects of the bite, the treatments obtained and their views about future improvements. Our survey suggests that snakebite incidence is higher than previously reported. 3.9% of those surveyed had suffered from snakebite and the number of deaths corresponds to 0.45% of the population. The socio-economic impact of this is very considerable in terms of the treatment costs and the long-term effects on the health and ability of survivors to work. To reduce this, the victims recommended improvements to the accessibility and affordability of antivenom treatment.ConclusionsSnakebite has a considerable and disproportionate impact on rural populations, particularly in South Asia. This study provides an incentive for researchers and the public to work together to reduce the incidence and improve the outcomes for snake bite victims and their families.

Serine proteases are major components of viper venom and target various stages of the blood coagulation system in victims and prey. A better understanding of the diversity of serine proteases and other enzymes present in snake venom will help to understand how the complexity of snake venom has evolved and will aid the development of novel therapeutics for treating snake bites.Four serine protease-encoding genes from the venom gland transcriptome of Bitis gabonica rhinoceros were amplified and sequenced. Mass spectrometry suggests the four enzymes corresponding to these genes are present in the venom of B. g. rhinoceros. Two of the enzymes, rhinocerases 2 and 3 have substitutions to two of the serine protease catalytic triad residues and are thus unlikely to be catalytically active, though they may have evolved other toxic functions. The other two enzymes, rhinocerases 4 and 5, have classical serine protease catalytic triad residues and thus are likely to be catalytically active, however they have glycine rather than the more typical aspartic acid at the base of the primary specificity pocket (position 189). Based on a detailed analysis of these sequences we suggest that alternative splicing together with individual amino acid mutations may have been involved in their evolution. Changes within amino acid segments which were previously proposed to undergo accelerated change in venom serine proteases have also been observed.Our study provides further insight into the diversity of serine protease isoforms present within snake venom and discusses their possible functions and how they may have evolved. These multiple serine protease isoforms with different substrate specificities may enhance the envenomation effects and help the snake to adapt to new habitats and diets. Our findings have potential for helping the future development of improved therapeutics for snake bites.

Snake bite is a major neglected public health issue within poor communities living in the rural areas of several countries throughout the world. An estimated 2.5 million people are bitten by snakes each year and the cost and lack of efficacy of current anti-venom therapy, together with the lack of detailed knowledge about toxic components of venom and their modes of action, and the unavailability of treatments in rural areas mean that annually there are around 125,000 deaths worldwide. In order to develop cheaper and more effective therapeutics, the toxic components of snake venom and their modes of action need to be clearly understood. One particularly poorly understood component of snake venom is aminopeptidases. These are exo-metalloproteases, which, in mammals, are involved in important physiological functions such as the maintenance of blood pressure and brain function. Although aminopeptidase activities have been reported in some snake venoms, no detailed analysis of any individual snake venom aminopeptidases has been performed so far. As is the case for mammals, snake venom aminopeptidases may also play important roles in altering the physiological functions of victims during envenomation. In order to further understand this important group of snake venom enzymes we have isolated, functionally characterised and analysed the sequence-structure relationships of an aminopeptidase from the venom of the large, highly venomous West African gaboon viper, Bitis gabonica rhinoceros.The venom of B. g. rhinoceros was fractionated by size exclusion chromatography and fractions with aminopeptidase activities were isolated. Fractions with aminopeptidase activities showed a pure protein with a molecular weight of 150 kDa on SDS-PAGE. In the absence of calcium, this purified protein had broad aminopeptidase activities against acidic, basic and neutral amino acids but in the presence of calcium, it had only acidic aminopeptidase activity (APA). Together with the functional data, mass spectrometry analysis of the purified protein confirmed this as an aminopeptidase A and thus this has been named as rhiminopeptidase A. The complete gene sequence of rhiminopeptidase A was obtained by sequencing the PCR amplified aminopeptidase A gene from the venom gland cDNA of B. g. rhinoceros. The gene codes for a predicted protein of 955 amino acids (110 kDa), which contains the key amino acids necessary for functioning as an aminopeptidase A. A structural model of rhiminopeptidase A shows the structure to consist of 4 domains: an N-terminal saddle-shaped beta domain, a mixed alpha and beta catalytic domain, a beta-sandwich domain and a C-terminal alpha helical domain.This study describes the discovery and characterisation of a novel aminopeptidase A from the venom of B. g. rhinoceros and highlights its potential biological importance. Similar to mammalian aminopeptidases, rhiminopeptidase A might be capable of playing roles in altering the blood pressure and brain function of victims. Furthermore, it could have additional effects on the biological functions of other host proteins by cleaving their N-terminal amino acids. This study points towards the importance of complete analysis of individual components of snake venom in order to develop effective therapies for snake bites.

Serine proteases are a major component of viper venoms and are thought to disrupt several distinct elements of the blood coagulation system of envenomed victims. A detailed understanding of the functions of these enzymes is important both for acquiring a fuller understanding of the pathology of envenoming and because these venom proteins have shown potential in treating blood coagulation disorders.In this study a novel, highly abundant serine protease, which we have named rhinocerase, has been isolated and characterised from the venom of Bitis gabonica rhinoceros using liquid phase isoelectric focusing and gel filtration. Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36 kDa, which reduces to 31 kDa after deglycosylation. The partial amino acid sequence shows similarity to other viper venom serine proteases, but is clearly distinct from the sequence of the only other sequenced serine protease from Bitis gabonica. Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional. It is capable of degrading alpha and beta chains of fibrinogen, dissolving plasma clots and of hydrolysing a kallikrein substrate.A novel multifunctional viper venom serine protease has been isolated and characterised. The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension. This study will form the basis for future research to understand the mechanisms of serine protease action, and examine the potential for rhinocerase to be used clinically to reduce the risk of human haemostatic disorders such as heart attacks and strokes.

The ABO blood type has been reported to be associated with several diseases such as hepatitis and malaria. Recently, some studies have reported that people with O blood type are protected against COVID-19, while people with A blood type are more susceptible to contract this disease. Here, we analysed data from 5668 COVID-19 patients along with the same number of control samples in a study population in Iraq. Our analysis confirms that people with O blood type are protected partially against COVID-19. Notably, we demonstrate that people with RhD- are more susceptible to contract COVID-19 than people with RhD+ blood type. The blood types are associated with some clinical symptoms such as headache and asthenia of COVID-19, but there is no association with other symptoms. There is no association between blood types and deaths among COVID-19 patients. This study suggests that in addition to ABO, RhD blood type influences the susceptibility to contract COVID-19. Overall, we conclude that susceptibility/protection against COVID-19 may not be determined based only on blood types among the global population as this might vary based on a number of other factors such as ethnicity, geographical locations, occupation and the level of exposure to infected people.