Eosinophils are recruited to sites of inflammation via the action of a number of chemical mediators, including PAF, leukotrienes, eotaxins, ECF-A and histamine. Although many of the cell-surface receptors for these mediators have been identified, histamine-driven chemotaxis has not been conclusively attributed to any of the three known histamine receptor subtypes, suggesting the possibility of a 4th histamine-responsive receptor on eosinophils. We have identified and cloned a novel G protein-coupled receptor (GPCR), termed Pfi-013, from an IL-5 stimulated eosinophil cDNA library which is homologous to the human histamine H3 receptor, both at the sequence and gene structure level. Expression data indicates that Pfi-013 is predominantly expressed in peripheral blood leukocytes, with lower expression levels in spleen, testis and colon. Ligand-binding studies using Pfi-013 expressed in HEK-293Galpha15 cells, demonstrates specific binding to histamine with a Kd of 3.28 +/- 0.76 nM and possesses a unique rank order of potency against known histaminergic compounds in a competitive ligand-binding assay (histamine > clobenpropit > iodophenpropit > thioperamide > R-alpha-methylhistamine > cimetidine > pyrilamine). We have therefore termed this receptor human histamine H4. Chemotaxis studies on isolated human eosinophils have confirmed that histamine is chemotactic and that agonists of the known histamine receptors (H1, H2, and H3) do not induce such a response. Furthermore, studies employing histamine-receptor antagonists have shown an inhibition of chemotaxis only by the H3 antagonists clobenpropit and thioperamide. Since these compounds are also antagonists of hH4 we postulate that the receptor mediating histaminergic chemotaxis is this novel histamine H4 receptor.

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.

When expressed via an inducible promoter in human embryonic kidney 293 cells, the rat Mas-related gene D (rMrgD) receptor responded to ␤-alanine but not L-alanine by elevating intracellular [Ca 2ϩ ], stimulating phosphorylation of the mitogenactivated protein kinases known as extracellular signal-regulated kinase (ERK) 1 and ERK2 and translocating from the plasma membrane to punctate intracellular vesicles. By contrast, the related rat Mas-related gene E (rMrgE) receptor did not respond to ␤-alanine. Coexpression of rMrgD with rMrgE, which occurs in peripheral nociceptive neurons, allowed coimmunoprecipitation of the two receptors and resulted in the detection of cell surface rMrgD-rMrgE heterodimers via timeresolved fluorescence resonance energy transfer. These interactions increased the potency of ␤-alanine to phosphorylate ERK1 and ERK2 as well as maintaining the capacity of ␤-alanine to elevate intracellular [Ca 2ϩ ], which was reduced in magnitude and slowed in response with increasing times of expression of rMrgD in isolation. Associated with these effects, the presence of rMrgE restricted ␤-alanine-induced internalization of rMrgD. This is the first report of heterodimeric interactions between members of the Mas-related gene (Mrg) receptor family and indicates that interactions between rMrgD and rMrgE modulate the function of rMrgD. Because the Mrg receptors are potential therapeutic targets in pain, these results suggest that efforts to understand the function and regulation of individual Mrg family receptors may require coexpression of relevant pairs.

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.

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.

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Adequate pain control is still a significant challenge and largely unmet medical need in the 21st century. With many small molecules failing to reach required levels of potency and selectivity, drug discovery is once again turning to nature to replenish pain therapeutic pipelines. Venomous animals are frequently stereotyped as inflictors of pain and distress and have historically been vilified by mankind. Yet, ironically, the very venoms that cause pain when directly injected by the host animal may actually turn out to contain the next generation of analgesics when injected by the clinician. The last 12 months have seen dramatic discoveries of analgesic tools within venoms. Spiders, snakes and even centipedes are yielding peptides with immense therapeutic potential. Significant advances are also taking place in delivery methods that can improve bioavailability and pharmacokinetics of these exciting natural resources. Turning proteinaceous venom into pharmaceutical liquid gold is the goal of venomics and the focus of this article.

Background: The orphan GPCR MrgE is one of an extended family of GPCRs that are expressed in dorsal root ganglia (DRG). Based on these expression patterns it has been suggested that GPCRs like MrgE may play a role in nociception however, to date, no direct supporting evidence has emerged. We generated mutant mice lacking MrgE and examined the effects of deletion of this gene in three pain behavioural models. The effect of MrgE gene deletion on expression of Mrgs and genes involved in sensory neurone function was also investigated.