Selective blockade of Cav1.2 (α1C) versus Cav1.3 (α1D) L-type calcium channels by the black mamba toxin calciseptine.

L-type voltage-gated calcium channels are involved in multiple physiological functions. Currently available antagonists do not discriminate between L-type channel isoforms. Importantly, no selective blocker is available to dissect the role of L-type isoforms Cav1.2 and Cav1.3 that are concomitantly co-expressed in the heart, neuroendocrine and neuronal cells. Here we show that calciseptine, a snake toxin purified from mamba venom, selectively blocks Cav1.2 -mediated L-type calcium currents (ICaL) at concentrations leaving Cav1.3-mediated ICaL unaffected in both native cardiac myocytes and HEK-293T cells expressing recombinant Cav1.2 and Cav1.3 channels. Functionally, calciseptine potently inhibits cardiac contraction without altering the pacemaker activity in sino-atrial node cells, underscoring differential roles of Cav1.2- and Cav1.3 in cardiac contractility and automaticity. In summary, calciseptine is a selective L-type Cav1.2 Ca2+ channel blocker and should be a valuable tool to dissect the role of these L-channel isoforms.

[Pathomorphological characteristics of sequelae of the black mamba bite]. / Patomorfologicheskaya kharakteristika posledstvii ukusa chernoi mamby.

Pathomorphological changes in internal organs due to the toxic effects of the black mamba venom are nonspecific in nature and presume its neurotoxic and cardiotoxic effects with the development of the DIC syndrome, pulmonary edema, and brain edema in the terminal period. In forensic medicine, the development of specific diagnostic criteria, as well as an algorithm for detecting toxins of the black mamba venom in biological fluids and tissues of internal organs can become a promising topic for scientific research.

Captive Black Mamba (Dendroaspis Polylepis) Bite Leading to Respiratory Failure.

BACKGROUND: Of the 8000-10,000 snake envenomations evaluated in U.S. emergency departments (ED) annually, approximately 1% are due to non-native snakes. We describe a 26-year-old man who was bitten by his captive black mamba (Dendroaspis polylepis) as he was packing it up for transport to another snake collector. CASE REPORT: The patient presented to the ED 1 h after being bitten on the forearm, complaining of left arm pain, oral paresthesias, and dyspnea. His vital signs: heart rate 96 beats/min, blood pressure 167/101 mm Hg, temperature 36.7°C (97.9°F), respiratory rate 20 breaths/min, and room air oxygen saturation 100%. Two mildly tender puncture wounds without swelling or ecchymosis were found on the posterior aspect of the forearm. Over the ensuing 30 min his dyspnea worsened, and he developed objective weakness. He was intubated and placed on mechanical ventilation. He was treated with atropine 2 mg for bronchorrhea. Five vials of South African Vaccine Producers (Johannesburg, South Africa) polyvalent antivenom were administered 2.5 h post-bite and the patient was admitted to the intensive care unit. He was extubated 18 h post-envenomation and discharged the following day. He has remained asymptomatic since leaving the hospital. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: The primary manifestations of D. polylepis envenomings are neurological. Initial signs may include paresthesias, dysarthria, dysphagia, and ptosis. Progressive descending paralysis leading to respiratory failure develops within 60 min. Muscarinic features are frequently observed. Cardiotoxicity and hematologic laboratory abnormalities may be present. Although pain is common, significant local tissue injury does not occur. In addition to supportive care, several non-native antivenoms are indicated for D. polylepis envenomations. Black mamba envenomings differ from the native snakebites with which U.S. physicians are familiar. Rapid, progressive neurological toxicity and muscarinic features are most common. Treatment consists of supportive care and appropriate antivenom administration.

Technical note: MAMBA-Multi-pAradigM voxel-Based Analysis: A computational cookbot.

BACKGROUND: Voxel-Based (VB) analysis embraces a multifaceted ensemble of sophisticated techniques, lying at the boundary between image processing and statistical modeling, that allow for a frequentist inference of pathophysiological properties anchored to an anatomical reference. VB methods has been widely adopted in neuroimaging studies and, more recently, they are gaining momentum in radiation oncology research. However, the price for the power of VB analysis is the complexity of the underlying mathematics and algorithms. PURPOSE: In this paper, we present the Multi-pAradigM voxel-Based Analysis (MAMBA) toolbox, which is intended for a flexible application of VB analysis in a wide variety of scenarios in medical imaging and radiation oncology. METHODS: The MAMBA toolbox is implemented in Matlab. It provides open-source functions to compute VB statistical models of the input data, according to a great variety of regression schemes, and to derive VB maps of the observed significance level, performing a non-parametric permutation inference. The toolbox allows for including VB and global outcomes, as well as an arbitrary amount of VB and global Explanatory Variables (EVs). In addition, the Matlab Parallel Computing Toolbox is exploited to take advantage of the perfect parallelizability of most workloads. RESULTS: The use of MAMBA was demonstrated by means of several realistic examples on a synthetic dataset mimicking a radiation oncology scenario. CONCLUSION: MAMBA is an open-source toolbox, freely available for academic and non-commercial purposes. It is designed to make state-of-the-art VB analysis accessible to research scientists without the programming resources needed to build from scratch their own software solutions. At the same time, the source code is handed out for more experienced users to complement their own tools, also customizing user-defined models. MAMBA guarantees high generality and flexibility in the design of the statistical models, significantly expanding on the features of available free tools for VB analysis. The presented toolbox aims at increasing the reach of VB studies as well as the sharing of research results.

The MaMBA facility as a testbed for bioregenerative life support systems.

The Moon and Mars Base Analog (MaMBA) is a concept for an extraterrestrial habitat developed at the Center of Applied Space Technology and Microgravity (ZARM) in Bremen, Germany. The long-term goal of the associated project is to create a technologically functioning prototype for a base on the Moon and on Mars. One key aspect of developing such a prototype base is the integration of a bioregenerative life support system (BLSS) and its testing under realistic conditions. A long-duration mission to Mars, in particular, will require BLSS with a reliability that can hardly be reached without extensive testing, starting well in advance of the mission. Standards exist for comparing the capabilities of various BLSS, which strongly focus on technological aspects. These, we argue, should be complemented with the use of facilities that enable investigations and optimization of BLSS prototypes with regard to their requirements on logistics, training, recovery from failure and contamination, and other constraints imposed when humans are in the loop. Such facilities, however, are lacking. The purpose of this paper is to present the MaMBA facility and its potential usages that may help close this gap. We describe how a BLSS (or parts of a BLSS) can be integrated into the current existing mock-up at the ZARM for relatively low-cost investigations of human factors affecting the BLSS. The MaMBA facility is available through collaborations as a test platform for characterizing, benchmarking, and testing BLSS under nominal and off-nominal conditions.

A Combined Bioassay and Nanofractionation Approach to Investigate the Anticoagulant Toxins of Mamba and Cobra Venoms and Their Inhibition by Varespladib.

Envenomation by elapid snakes primarily results in neurotoxic symptoms and, consequently, are the primary focus of therapeutic research concerning such venoms. However, mounting evidence suggests these venoms can additionally cause coagulopathic symptoms, as demonstrated by some Asian elapids and African spitting cobras. This study sought to investigate the coagulopathic potential of venoms from medically important elapids of the genera Naja (true cobras), Hemachatus (rinkhals), and Dendroaspis (mambas). Crude venoms were bioassayed for coagulant effects using a plasma coagulation assay before RPLC/MS was used to separate and identify venom toxins in parallel with a nanofractionation module. Subsequently, coagulation bioassays were performed on the nanofractionated toxins, along with in-solution tryptic digestion and proteomics analysis. These experiments were then repeated on both crude venoms and on the nanofractionated venom toxins with the addition of either the phospholipase A2 (PLA2) inhibitor varespladib or the snake venom metalloproteinase (SVMP) inhibitor marimastat. Our results demonstrate that various African elapid venoms have an anticoagulant effect, and that this activity is significantly reduced for cobra venoms by the addition of varespladib, though this inhibitor had no effect against anticoagulation caused by mamba venoms. Marimastat showed limited capacity to reduce anticoagulation in elapids, affecting only N. haje and H. haemachatus venom at higher doses. Proteomic analysis of nanofractionated toxins revealed that the anticoagulant toxins in cobra venoms were both acidic and basic PLA2s, while the causative toxins in mamba venoms remain uncertain. This implies that while PLA2 inhibitors such as varespladib and metalloproteinase inhibitors such as marimastat are viable candidates for novel snakebite treatments, they are not likely to be effective against mamba envenomings.

Individual and community-level impacts of the unarmed all-women Black Mamba Anti-Poaching Unit.

In a conservation setting where escalating tension has been the norm, the unarmed primarily female-composed Black Mambas Anti-Poaching Unit (BMAPU) in South Africa are a successful counterpoint in that they have decimated poaching using only diplomacy and patrols. We sought to understand if the BMAPU is achieving its secondary goal of influencing support for conservation and wildlife among the nearby populace via community-based conservation actions including outreach and environmental education. We also determined the impact that the program has on the Mambas themselves. Using a mixed-methods survey we conducted structured in-person interviews with 120 community members from four communities where the women of the BMAPU live, and among all the women that were on active duty in the BMAPU at the time of the surveys. We found that all participants in the BMAPU program reported improved self and community perception of their societal role as financial providers, as well as their sense of agency and self-efficacy, relative to before becoming rangers. We also found that having BMAPU rangers living in the communities by itself did not contribute to community-level support for wildlife conservation or protected areas. However, one community with both a large-scale children's conservation education program and an equitable distribution of financial benefits paid by the nearby conservation concessionaires was significantly more supportive of wildlife conservation and protected areas than the other three. Further research to parse the relative contributions of the two contributing factors of education and financial benefit would help clarify their relative contributions. From this study, we conclude that a combination of child-focused conservation education programs and equitable distribution of financial benefits leads to increased community support for wildlife, conservation, and protected natural areas, and decreases support for poaching.

MT9, a natural peptide from black mamba venom antagonizes the muscarinic type 2 receptor and reverses the M2R-agonist-induced relaxation in rat and human arteries.

All five muscarinic receptors have important physiological roles. The endothelial M2 and M3 subtypes regulate arterial tone through direct coupling to Gq or Gi/o proteins. Yet, we lack selective pharmacological drugs to assess the respective contribution of muscarinic receptors to a given function. We used mamba snake venoms to identify a selective M2R ligand to investigate its contribution to arterial contractions. Using a bio-guided screening binding assay, we isolated MT9 from the black mamba venom, a three-finger toxin active on the M2R subtype. After sequencing and chemical synthesis of MT9, we characterized its structure by X-ray diffraction and determined its pharmacological characteristics by binding assays, functional tests, and ex vivo experiments on rat and human arteries. Although MT9 belongs to the three-finger fold toxins family, it is phylogenetically apart from the previously discovered muscarinic toxins, suggesting that two groups of peptides evolved independently and in a convergent way to target muscarinic receptors. The affinity of MT9 for the M2R is 100 times stronger than that for the four other muscarinic receptors. It also antagonizes the M2R/Gi pathways in cell-based assays. MT9 acts as a non-competitive antagonist against acetylcholine or arecaine, with low nM potency, for the activation of isolated rat mesenteric arteries. These results were confirmed on human internal mammary arteries. In conclusion, MT9 is the first fully characterized M2R-specific natural toxin. It should provide a tool for further understanding of the effect of M2R in various arteries and may position itself as a new drug candidate in cardio-vascular diseases.

Case Report: Safe Tourniquet Removal in Black Mamba (Dendroaspis polylepis) Bites.

The black mamba is known for its notorious potent neurotoxic venom. For this reason, their bites are often erroneously treated in the field with the application of a tourniquet in the hope of delaying systemic spread of the venom. Observational studies have shown that inappropriate tourniquet application is a common, harmful practice. An arterial tourniquet is not a recommended first aid measure because of the risk of limb ischemia and gangrene. When inappropriately applied, the rapid removal of the tourniquet in the emergency department may precipitate a life-threatening venom and metabolic toxin rush, leading to respiratory arrest. We present two cases of black mamba bites in Gauteng, South Africa, where gradual tourniquet removal was used to avoid a venom rush and rapid respiratory paralysis. Venom and metabolic toxin rush with potentially fatal respiratory muscle paralysis may be averted by gradual, cautious removal of field-applied tourniquets with concomitant antivenom administration.

The clinical course and treatment of black mamba (Dendroaspis polylepis) envenomations: a narrative review.

CONTEXT: The black mamba (Dendroaspis polylepis) is, due to its extremely toxic venom, one of the most dangerous snake species in Sub-Saharan Africa. A D. polylepis bite is a medical emergency and requires adequate action to prevent severe complications. However, there are no comprehensive reviews available based on clinical cases, and no readily accessible guidelines for standardized treatment. Therefore, we aim to provide an overview regarding the currently available clinical literature on D. polylepis envenomations; in order to promote knowledge on symptomatology and treatment options. METHODS: We searched for cases reporting humans bitten by D. polylepis in PubMed, Embase, Scopus, and Sabinet. We searched the reference lists of all eligible articles for additional articles. After quality assessment, 29 cases were included in this review. We used descriptive analysis to create an overview of the collected parameters. DISCUSSION: Among the included case reports and case series, D. polylepis envenomations most frequently resulted in decreased respiratory function, sweating and paralysis. The onset of symptoms usually occurred within 60 minutes. Neurological symptoms occurred more often than symptoms of autonomic dysfunction. In the reported cases most patients (26/29) received antivenom and most survived (25/29). We recommend the reporting of additional structured case reports to improve future analyses on the clinical course of envenomations, in order to improve public health response to D. polylepis envenomations.

Unusual quaternary structure of a homodimeric synergistic-type toxin from mamba snake venom defines its molecular evolution.

Snake venoms are complex mixtures of enzymes and nonenzymatic proteins that have evolved to immobilize and kill prey animals or deter predators. Among them, three-finger toxins (3FTxs) belong to the largest superfamily of nonenzymatic proteins. They share a common structure of three ß-stranded loops extending like fingers from a central core containing all four conserved disulfide bonds. Most 3FTxs are monomers and through subtle changes in their amino acid sequences, they interact with different receptors, ion channels and enzymes to exhibit a wide variety of biological effects. The 3FTxs have further expanded their pharmacological space through covalent or noncovalent dimerization. Synergistic-type toxins (SynTxs) isolated from the deadly mamba venoms, although nontoxic, have been known to enhance the toxicity of other venom proteins. However, the details of three-dimensional structure and molecular mechanism of activity of this unusual class of 3FTxs are unclear. We determined the first three-dimensional structure of a SynTx isolated from Dendroaspis jamesoni jamesoni (Jameson's mamba) venom. The SynTx forms a unique homodimer that is held together by an interchain disulfide bond. The dimeric interface is elaborate and encompasses loops II and III. In addition to the inter-subunit disulfide bond, the hydrogen bonds and hydrophobic interactions between the monomers contribute to the dimer formation. Besides, two sulfate ions that mediate interactions between the monomers. This unique quaternary structure is evolved through noncovalent homodimers such as κ-bungarotoxins. This novel dimerization further enhances the diversity in structure and function of 3FTxs.

Mechanisms Responsible for the Anticoagulant Properties of Neurotoxic Dendroaspis Venoms: A Viscoelastic Analysis.

Using thrombelastography to gain mechanistic insights, recent investigations have identified enzymes and compounds in Naja and Crotalus species' neurotoxic venoms that are anticoagulant in nature. The neurotoxic venoms of the four extant species of Dendroaspis (the Black and green mambas) were noted to be anticoagulant in nature in human blood, but the mechanisms underlying these observations have never been explored. The venom proteomes of these venoms are unique, primarily composed of three finger toxins (3-FTx), Kunitz-type serine protease inhibitors (Kunitz-type SPI) and <7% metalloproteinases. The anticoagulant potency of the four mamba venoms available were determined in human plasma via thrombelastography; vulnerability to inhibition of anticoagulant activity to ethylenediaminetetraacetic acid (EDTA) was assessed, and inhibition of anticoagulant activity after exposure to a ruthenium (Ru)-based carbon monoxide releasing molecule (CORM-2) was quantified. Black mamba venom was the least potent by more than two orders of magnitude compared to the green mamba venoms tested; further, Black Mamba venom anticoagulant activity was not inhibited by either EDTA or CORM-2. In contrast, the anticoagulant activities of the green mamba venoms were all inhibited by EDTA to a greater or lesser extent, and all had anticoagulation inhibited with CORM-2. Critically, CORM-2-mediated inhibition was independent of carbon monoxide release, but was dependent on a putative Ru-based species formed from CORM-2. In conclusion, there was great species-specific variation in potency and mechanism(s) responsible for the anticoagulant activity of Dendroaspis venom, with perhaps all three protein classes-3-FTx, Kunitz-type SPI and metalloproteinases-playing a role in the venoms characterized.

An ecological analysis of snakes captured by C.J.P. Ionides in eastern Africa in the mid-1900s.

Historical data can clarify ecological attributes of fauna in sites that have subsequently been altered by anthropogenic activities. We used the 1960s notebooks of pioneering East African naturalist C.J.P. Ionides to extract quantitative information on captures of 484 snakes of five species (rhinoceros vipers Bitis nasicornis, black mambas Dendroaspis polylepis, Jameson's mambas D. jamesoni, water cobras Naja annulata, and eastern forest cobras N. subfulva). High capture rates suggest high abundances of all species. The relative numbers of each species collected changed over the years and differed seasonally, reflecting targeting by Ionides. Sex ratios and age-class distributions differed among species and were affected by factors such as month of collection and time of day. Habitat use was affected by species, sex and body size: for example, arboreality became less common with increasing body size in the rhinoceros viper and black mamba, and males were found in arboreal sites more often than were females. In both D. jamesoni and D. polylepis, adult males and females were recorded together in September-October, suggesting reproductive activity at this time of year. Although fragmentary, the data from Ionides' notebooks provide a unique glimpse into ecological patterns of snakes within an African landscape half a century ago.

Black Mamba Death: Venom Versus Antivenom?

We present the case of a male adult who was admitted to an emergency department after having sustained envenomation from a black mamba (Dendroaspis polylepis). According to the available history, a single fang hooked his right index finger, post venom extraction. After administering antivenom in the accident and emergency department, further vials were transfused in the intensive care unit. An urticarial rash was noted, which was thought to be related to the antivenom. The victim remained in a coma for 3 days, after which he was declared dead. A medicolegal postmortem examination was performed 4 days after death because of logistical reasons. The complexities of differentiating acute envenomation from black mamba versus early acute reactions to polyvalent antivenom administration are highlighted in this case study.

Envenomation by a western green mamba (Dendroaspis viridis) - A report of three episodes in Switzerland.

The African elapid snake genus Dendroaspis comprises four species, with D. polylepsis the most dangerous of them. D. viridis is believed to cause stronger neurotoxic symptoms than the potentially least toxic of the genus, D. angusticeps, but seems less toxic compared to either of the D. jamesoni species (D. j. jamesoni(TRAILL 1843) and D. j. kaimosae (Loveridge 1936)). We present three episodes of bites byD. viridis in the same patient, sustained on three different occasions, caused by three different and unrelated snakes. While the first bite remained oligosymptomatic with a slight tightness of the throat and speedy resolution of symptoms without specific therapy, episodes two and three resulted in the patient developing massive local swelling. However, the patient showed only minimal neurologic and systemic symptoms such as tightness of the throat and a tingling sensation of the body. Episode two resolved with fasciotomy after compartment syndrome was diagnosed with a measured intracompartmental pressure of 52 mmHg. In episode three, antivenom was administered with good resolution of symptoms. The clinical courses in this patient were remarkable as he displayed mainly local symptoms after three individual bites by a supposedly neurotoxic snake.

In vivo neutralization of dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies.

The black mamba (Dendroaspis polylepis) is one of the most feared snake species of the African savanna. It has a potent, fast-acting neurotoxic venom comprised of dendrotoxins and α-neurotoxins associated with high fatality in untreated victims. Current antivenoms are both scarce on the African continent and present a number of drawbacks as they are derived from the plasma of hyper-immunized large mammals. Here, we describe the development of an experimental recombinant antivenom by a combined toxicovenomics and phage display approach. The recombinant antivenom is based on a cocktail of fully human immunoglobulin G (IgG) monoclonal antibodies capable of neutralizing dendrotoxin-mediated neurotoxicity of black mamba whole venom in a rodent model. Our results show the potential use of fully human monoclonal IgGs against animal toxins and the first use of oligoclonal human IgG mixtures against experimental snakebite envenoming.

The medical threat of mamba envenoming in sub-Saharan Africa revealed by genus-wide analysis of venom composition, toxicity and antivenomics profiling of available antivenoms.

Mambas (genus Dendroaspis) are among the most feared of the medically important elapid snakes found in sub-Saharan Africa, but many facets of their biology, including the diversity of venom composition, remain relatively understudied. Here, we present a reconstruction of mamba phylogeny, alongside genus-wide venom gland transcriptomic and high-resolution top-down venomic analyses. Whereas the green mambas, D. viridis, D. angusticeps, D. j. jamesoni and D. j. kaimosae, express 3FTx-predominant venoms, black mamba (D. polylepis) venom is dominated by dendrotoxins I and K. The divergent terrestrial ecology of D. polylepis compared to the arboreal niche occupied by all other mambas makes it plausible that this major difference in venom composition is due to dietary variation. The pattern of intrageneric venom variability across Dendroaspis represented a valuable opportunity to investigate, in a genus-wide context, the variant toxicity of the venom, and the degree of paraspecific cross-reactivity between antivenoms and mamba venoms. To this end, the immunological profiles of the five mamba venoms were assessed against a panel of commercial antivenoms generated for the sub-Saharan Africa market. This study provides a genus-wide overview of which available antivenoms may be more efficacious in neutralising human envenomings caused by mambas, irrespective of the species responsible. The information gathered in this study lays the foundations for rationalising the notably different potency and pharmacological profiles of Dendroaspis venoms at locus resolution. This understanding will allow selection and design of toxin immunogens with a view to generating a safer and more efficacious pan-specific antivenom against any mamba envenomation. BIOLOGICAL SIGNIFICANCE: The mambas (genus Dendroaspis) comprise five especially notorious medically important venomous snakes endemic to sub-Saharan Africa. Their highly potent venoms comprise a high diversity of pharmacologically active peptides, including extremely rapid-acting neurotoxins. Previous studies on mamba venoms have focused on the biochemical and pharmacological characterisation of their most relevant toxins to rationalize the common neurological and neuromuscular symptoms of envenomings caused by these species, but there has been little work on overall venom composition or comparisons between them. Only very recently an overview of the composition of the venom of two Dendroaspis species, D. angusticeps and D. polylepis, has been unveiled through venomics approaches. Here we present the first genus-wide transcriptomic-proteomic analysis of mamba venom composition. The transcriptomic analyses described in this paper have contributed 29 (D. polylepis), 23 (D. angusticeps), 40 (D. viridis), 25 (D. j. jamesoni) and 21 (D. j. kaimosae), novel full-length toxin sequences to the non-redundant Dendroaspis sequence database. The mamba genus-wide venomic analysis demonstrated that major D. polylepis venom components are Kunitz-fold family toxins. This feature is unique in relation to the relatively conserved three-finger toxin (3FTx)-dominated venom compositions of the green mambas. Venom variation was interpreted in the context of dietary variation due to the divergent terrestrial ecology of D. polylepis compared to the arboreal niche occupied by all other mambas. Additionally, the degree of cross-reactivity conservation of mamba venoms was assessed by antivenomics against a panel of commercial antivenoms generated for the sub-Saharan Africa market. This study provides a genus-wide overview to infer which available antivenoms may be capable of neutralising human envenomings caused by mambas, irrespective of the species responsible. The information gathered in this study lays the foundations for rationalising the pharmacological profiles of mamba venoms at locus resolution. This understanding will contribute to the generation of a safer and more efficacious pan-Dendroaspis therapeutic antivenom against any mamba envenomation.