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Breast and lung cancers are the leading causes of cancer-related deaths in the world. Although considerable progress has been made in the field of cancer therapy, quest to discover potent, safe and cost-effective alternatives especially from natural sources is being pursued. Snake venom, which is a treasure trove of various peptides and proteins including natural toxins that specifically target tissues and receptors in the envenomated victims. Many such proteins are being explored for their therapeutic potential against various diseases including cancers. Here, we report the mechanism of cytotoxic activity of crude venom and a purified protein, Cytotoxin from the monocled cobra (Naja kaouthia), an elapid snake with neurotoxic venom prominently found in the North-East India. The crude venom showed significant cytotoxicity against breast (MCF-7and MDA-MB-231) and lung (A549, NCI-H522) cancer cell lines. Bioassay-guided fractionation using RP-HPLC showed highest cytotoxic activity in peak P9. Liquid chromatography-tandem mass spectrometry (ESI-LC-MS/MS) analysis was employed and the fraction is identified as Cytotoxin 10 which showed comparable cytotoxicity against the experimental cell lines. Cytotoxin 10 also exhibited apoptosis in MCF-7 and A549 cell lines using AO/EtBr and flow cytometry analysis. Expressions of apoptosis related proteins e.g. Bax, Bcl-2, Caspase-7 and PARP were also studied following Cytotoxin 10 treatment in both cell lines. Molecular docking experiments performed to investigate the interactions between Cytotoxin 10 and the apoptotic proteins revealed favourable binding scores compared to their corresponding inhibitors. Interestingly, Cytotoxin 10 inhibited migration and adhesion in a time and dose-dependent manner in both MCF-7 and A549 cells. This is the first report elucidating the mechanism of cytotoxic activity of Cytotoxin 10 purified from Naja kaouthia venom of North-East India origin and could pave the way for development of potential therapeutic strategies against breast and lung cancer., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Robin Doley reports financial support was provided by Tezpur University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study explores the potential of natural bioactive peptides from animal venoms as targeted anti-cancer agents with reduced toxicity. Initially, we screened a broad collection of animal venoms for their antiproliferative activity against cancer cell lines. From this collection, we selected venoms from Naja anchietae and Naja senegalensis due to their promising activity. Utilizing reverse- phase high-performance liquid chromatography (RP HPLC), mass spectrometry (MALDI-TOF MS and MALDI-TOF TOF MSMS), and Edman degradation sequencing, we isolated and characterized three peptides named CTNanc1, CTNanc2, and CTNanc3 from Naja anchietae , and three others named CTNsen1, CTNsen2, and CTNsen3 from Naja senegalensis , each with a molecular weight of around 7 kDa. These purified peptides demonstrated inhibition of U87 glioblastoma cell proliferation, but not of U251 and T98G cells, in cell viability assays. To assess the impact of these treatments on cell viability, apoptosis, and necrosis, flow cytometry assays were conducted on U87 cells at 72 h. The results showed a decrease in cell viability and an increase in dead cells, suggesting that the treatments not only promote apoptosis, but may also lead to increased necrosis or late-stage apoptosis as the exposure time increases. These findings suggest that these peptides could be developed as leads for cancer therapy.

Introduction: Acute lung injury (ALI) as one kind of acute pulmonary inflammatory disorder, manifests primarily as damage to alveolar epithelial cells and microvascular endothelial cells. Activation of the complement system is a common pathological mechanism in ALI induced by diverse factors, with the complement alternative pathway assuming a pivotal role. Baicalin, a flavonoid derived from the root of Scutellaria baicalensis Georgi, exhibits noteworthy biological activities. The present study attempted the interventional effects and underlying mechanisms of baicalin in microangiopathy in ALI induced by complement alternative pathway activation., Methods: Activation of the complement alternative pathway by cobra venom factor (CVF). HMEC cells were pretreated with baicalin and then exposed to complement activation products. The expression of inflammatory mediators was detected by ELISA, and the intranuclear transcriptional activity of NF-κB was assessed by a dual fluorescent kinase reporter gene assay kit. Before establishing the ALI mouse model, baicalin or PDTC was gavaged for 7 d. CVF was injected into the tail vein to establish the ALI model. The levels of inflammatory mediators in BALF and serum were determined by ELISA. HE staining and immunohistochemistry evaluated pathological changes, complement activation product deposition, and NF-κB p65 phosphorylation in lung tissue., Results: Baicalin reduced complement alternative activation product-induced expression of HMEC cells adhesion molecules (ICAM-1, VCAM-1, E-selectin) and cytokines (IL-6, TNF-α) as well as upregulation of NF-κB intranuclear transcriptional activity. Baicalin intervention reduced the number of inflammatory cells and protein content in the BALF and decreased the levels of IL-6, TNF-α, and ICAM-1 in serum and IL-6, TNF-α, ICAM-1, and P-selectin in BLAF. In addition, baicalin attenuated inflammatory cell infiltration in the lung of ALI mice and reduced the deposition of complement activation products (C5a, C5b-9) and phosphorylation of NF-κB p65 in lung tissue., Conclusion: Baicalin relieves complement alternative pathway activation-induced lung inflammation by inhibition of NF-κB pathway, delaying the progression of ALI., (© 2024. The Author(s).)

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels that play a role in neurotransmission and pain sensation. The snake venom-derived peptides, mambalgins, exhibit potent analgesic effects in rodents by inhibiting central ASIC1a and peripheral ASIC1b. Despite their distinct species- and subtype-dependent pharmacology, previous structure-function studies have focussed on the mambalgin interaction with ASIC1a. Currently, the specific channel residues responsible for this pharmacological profile, and the mambalgin pharmacophore at ASIC1b remain unknown. Here we identify non-conserved residues at the ASIC1 subunit interface that drive differences in the mambalgin pharmacology from rat ASIC1a to ASIC1b, some of which likely do not make peptide binding interactions. Additionally, an amino acid variation below the core binding site explains potency differences between rat and human ASIC1. Two regions within the palm domain, which contribute to subtype-dependent effects for mambalgins, play key roles in ASIC gating, consistent with subtype-specific differences in the peptides mechanism. Lastly, there is a shared primary mambalgin pharmacophore for ASIC1a and ASIC1b activity, with certain peripheral peptide residues showing variant-specific significance for potency. Through our broad mutagenesis studies across various species and subtype variants, we gain a more comprehensive understanding of the pharmacophore and the intricate molecular interactions that underlie ligand specificity. These insights pave the way for the development of more potent and targeted peptide analogues required to advance our understating of human ASIC1 function and its role in disease., (© 2024. The Author(s).)

Background: Current immunotherapies with unexpected severe side effects and treatment resistance have not resulted in the desired outcomes for patients with melanoma, and there is a need to discover more effective medications. Cytotoxin (CTX) from Cobra Venom has been established to have favorable cytolytic activity and antitumor efficacy and is regarded as a promising novel anticancer agent. However, amphiphilic CTX with excellent anionic phosphatidylserine lipid-binding ability may also damage normal cells., Methods: We developed pH-responsive liposomes with a high CTX load (CTX@PSL) for targeted acidic-stimuli release of drugs in the tumor microenvironment. The morphology, size, zeta potential, drug-release kinetics, and preservation stability were characterized. Cell uptake, apoptosis-promoting effects, and cytotoxicity were assessed using MTT assay and flow cytometry. Finally, the tissue distribution and antitumor effects of CTX@PSL were systematically assessed using an in vivo imaging system., Results: CTX@PSL exhibited high drug entrapment efficiency, drug loading, stability, and a rapid release profile under acidic conditions. These nanoparticles, irregularly spherical in shape and small in size, can effectively accumulate at tumor sites (six times higher than free CTX) and are rapidly internalized into cancer cells (2.5-fold higher cell uptake efficiency). CTX@PSL displayed significantly stronger cytotoxicity (IC 50 0.25 μg/mL) and increased apoptosis in than the other formulations (apoptosis rate 71.78±1.70%). CTX@PSL showed considerably better tumor inhibition efficacy than free CTX or conventional liposomes (tumor inhibition rate 79.78±5.93%)., Conclusion: Our results suggest that CTX@PSL improves tumor-site accumulation and intracellular uptake for sustained and targeted CTX release. By combining the advantages of CTX and stimuli-responsive nanotechnology, the novel CTX@PSL nanoformulation is a promising therapeutic candidate for cancer treatment., Competing Interests: The authors declare no conflicts of interest., (© 2024 Lin et al.)

The knee joint has long been considered a closed system. The pathological effects of joint diseases on distant organs have not been investigated. Herein, our clinical data showed that post-traumatic joint damage, combined with joint bleeding (hemarthrosis), exhibits a worse liver function compared with healthy control. With mouse model, hemarthrosis induces both cartilage degeneration and remote liver damage. Next, we found that hemarthrosis induces the upregulation in ratio and differentiation towards Th17 cells of CD4 + T cells in peripheral blood and spleen. Deletion of CD4 + T cells reverses hemarthrosis-induced liver damage. Degeneration of cartilage matrix induced by hemarthrosis upregulates serological type II collagen (COL II), which activates CD4 + T cells. Systemic application of a COL II antibody blocks the activation. Furthermore, bulk RNAseq and single-cell qPCR analysis revealed that the cartilage Akt pathway is inhibited by blood treatment. Intra-articular application of Akt activator blocks the cartilage degeneration and thus protects against the liver impairment in mouse and pig models. Taken together, our study revealed a pathological joint-liver axis mediated by matrikine-activated CD4 + T cells, which refreshes the organ-crosstalk axis and provides a new treatment target for hemarthrosis-related disease. Intra-articular bleeding induces cartilage degradation through down-reulation of cartilage Akt pathway. During this process, the soluble COL II released from the damaged cartilage can activate peripheral CD4 + T cells, differention into Th17 cells and secretion of IL-17, which consequently induces liver impairment. Intra-articular application of sc79 (inhibitor of Akt pathway) can prevent the cartilage damage as well as its peripheral influences., (© 2024. The Author(s).)

Hepatocellular carcinoma and bacterial resistance are major health burdens nowadays. Thus, providing new therapies that overcome that resistance is of great interest, particularly those derived from nature rather than chemotherapeutics to avoid cytotoxicity on normal cells. Venomous animals are among the natural sources that assisted in the discovery of novel therapeutic regimens. L-amino acid oxidase Nh-LAAO (140 kDa), purified from Egyptian Naja haje venom by a successive two-step chromatography protocol, has an optimal pH and temperature of 8 and 37 °C. Under standard assay conditions, Nh-LAAO exhibited the highest specificity toward L-Arg, L-Met and L-Leu, with K m and V max values of 3.5 mM and 10.4 μmol/min/ml, respectively. Among the metal ions, Ca +2 , Na + , and K + ions are activators, whereas Fe +2 inhibited LAAO activity. PMSF and EDTA slightly inhibited the Nh-LAAO activity. In addition, Nh-LAAO showed antibacterial and antifungal activities, particularly against Gentamicin-resistant P. aeruginosa and E. coli strains with MIC of 18 ± 2 μg/ml, as well as F. proliferatum and A. parasiticus among the selected human pathogenic strains. Furthermore, Nh-LAAO exhibited anti-proliferative activity against cancer HepG2 and Huh7 cells with IC50 of 79.37 and 60.11 μg/ml, respectively, with no detectable effect on normal WI-38 cells. Consequently, the apoptosis % of the HepG2 and Huh7 cells were 12 ± 1 and 34.5 ± 2.5 %, respectively, upon Nh-LAAO treatment. Further, the Nh-LAAO arrested the HepG2 and Huh7 cell cycles in the G0/G1 phase. Thus, the powerful selective cytotoxicity of L-amino acid oxidase opens up the possibility as a good candidate for clinical cancer therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 A 2 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., (© 2024. The Author(s).)

Snake venom L-amino acid oxidases (LAAOs) are flavoenzymes with diverse physiological and pharmacological effects. These enzymes are found to showcase anticoagulant, antiplatelet, cytotoxicity and other biological effects in bite victims. However, the exact mechanism through which they exhibit several biological properties is not yet fully understood. The current study focussed on the purification of cobra venom LAAO and the functional characterization of purified LAAO. A novel L-amino acid oxidase NNLAAO70 with a molecular weight ~70 kDa was purified from the venom of an Indian spectacled cobra ( Naja naja ). NNLAAO70 showed high substrate specificity for L-His, L-Leu, and L-Arg during its LAAO activity. It inhibited adenosine di-phosphate (ADP) and collagen-induced platelet aggregation process in a dosedependent manner. About 60% inhibition of collagen-induced and 40% inhibition of ADP-induced platelet aggregation was observed with a 40 μg/ml dose of NNLAAO70. NNLAAO70 exhibited bactericidal activity on Bacillus subtilis, Escherichia coli, Bacillus megaterium, and Pseudomonas fluorescens . NNLAAO70 also showed cytotoxicity on A549 cells in vitro . It showed severe bactericidal activity on P. fluorescens and lysed 55% of cells. NNLAAO70 also exhibited drastic cytotoxicity on A549 cells. At 1 lg/ml dosage, it demonstrated a 60% reduction in A549 viability and induced apoptosis upon 24-h incubation. H 2 O 2 released during oxidative deamination reactions played a major role in NNLAAO70-induced cytotoxicity. NNLAAO70 significantly increased intracellular reactive oxygen species (ROS) levels in A549 cells by six fold when compared to untreated cells. Oxidative stress-mediated cell injury is the primary cause of NNLAAO70-induced apoptosis in A549 cells and prolonged oxidative stress caused DNA fragmentation and activated cellular secondary necrosis.

Previously, it was shown that the non-conventional toxin WTX from the venom of the cobra Naja kaouthia, when administered intravenously, caused a decrease in blood pressure (BP) and an increase in heart rate (HR) in rats [13]. To identify the site of the toxin molecule responsible for these effects, we studied the influence of synthetic peptide fragments of the WTX on BP and HR in normotensive male Sprague-Dawley rats under general anesthesia induced by Telazol and Xylazine. It was found that peptides corresponding to the WTX central polypeptide loop, stabilized by a disulfide bond, at intravenous injection at concentrations from 0.1 to 1.0 mg/mL caused a dose-dependent decrease in BP, with the HR increasing only in the first 5-10 min after administration. Thus, WTX fragments corresponding to the central polypeptide loop reproduce the decrease in blood pressure caused by the toxin., (© 2023. The Author(s).)

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.

Colon cancer ranks fourth in mortality. This cancer is still an important clinical challenge worldwide due to its high prevalence and poor prognosis. Proteomic studies revealed that snake venom is a diverse and variable mixture of enzymatic and non-enzymatic proteins and peptides. Despite the toxic effects of these molecules, several proteins and peptides have been isolated that have practical applications and appear to induce apoptosis and prevent cell metastasis. In this study, we worked on cytotoxic effects and anticancer activity of Naja naja oxiana (Iranian Caspian cobra) snake venom components on HT-29 cell line colon cancer. Separated Fraction-5 by FPLC indicated the high cytotoxicity on HT-29 cell line colon cancer by MTT test. Further isolation of F5 by HPLC showed that the purified peak 2, nominated as Oxineur that contains a cytotoxic effect on HT-29 cells and reduces cell viability at 8 μg/ml to 4% in 24 h. Oxineur has the least cytotoxic effect on HEK-293 normal cells. Further studies on Oxineur peptide confirmed the apoptotic effects with high expression of CASP9 gene and DNA fragmentation in cancerous cells. The partial sequence of Oxineur revealed 71% homology with the neurotoxin II from Naja naja oxiana. Since our target molecule is a peptide in the molecular weight range of 7 kDa, it has potentially a therapeutic value., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Arteriogenesis, the growth of natural bypass blood vessels, can compensate for the loss of arteries caused by vascular occlusive diseases. Accordingly, it is a major goal to identify the drugs promoting this innate immune system-driven process in patients aiming to save their tissues and life. Here, we studied the impact of the Cobra venom factor (CVF), which is a C3-like complement-activating protein that induces depletion of the complement in the circulation in a murine hind limb model of arteriogenesis. Arteriogenesis was induced in C57BL/6J mice by femoral artery ligation (FAL). The administration of a single dose of CVF (12.5 µg) 24 h prior to FAL significantly enhanced the perfusion recovery 7 days after FAL, as shown by Laser Doppler imaging. Immunofluorescence analyses demonstrated an elevated number of proliferating (BrdU + ) vascular cells, along with an increased luminal diameter of the grown collateral vessels. Flow cytometric analyses of the blood samples isolated 3 h after FAL revealed an elevated number of neutrophils and platelet-neutrophil aggregates. Giemsa stains displayed augmented mast cell recruitment and activation in the perivascular space of the growing collaterals 8 h after FAL. Seven days after FAL, we found more CD68 + /MRC-1 + M2-like polarized pro-arteriogenic macrophages around growing collaterals. These data indicate that a single dose of CVF boosts arteriogenesis by catalyzing the innate immune reactions, relevant for collateral vessel growth.

Viral infections are linked to a variety of human diseases. Despite the achievements made in drug and vaccine development, several viruses still lack preventive vaccines and efficient antiviral compounds. Thus, developing novel antiviral agents is of great concern, particularly the natural products that are promising candidates for such discoveries. In this study, we have purified an approximately 15 kDa basic phospholipase A2 (PLA2) enzyme from the Egyptian cobra Naja haje haje venom. The purified N. haje PLA2 showed a specific activity of 22 units/mg protein against 6 units/mg protein for the whole crude venom with 3.67-fold purification. The antiviral activity of purified N. haje PLA2 has been investigated in vitro against bovine coronavirus (BCoV) and simian rotavirus (RV SA-11). Our results showed that the CC 50 of PLA2 were 33.6 and 29 µg/ml against MDBK and MA104 cell lines, respectively. Antiviral analysis of N. haje PLA2 showed an inhibition of BCoV and RV SA-11 infections with a therapeutic index equal to 33.6 and 16, respectively. Moreover, N. haje PLA2 decreased the BCoV and RV SA-11 titers by 4.25 log 10 TCID 50 and 2.5 log 10 TCID 50 , respectively. Thus, this research suggests the potential antiviral activity of purified N. haje PLA2 against BCoV and RV SA-11 infections in vitro., (© 2022. The Author(s).)

Coral snake venoms from the Micrurus genus are a natural library of components with multiple targets, yet are poorly explored. In Brazil, 34 Micrurus species are currently described, and just a few have been investigated for their venom activities. Micrurus venoms are composed mainly of phospholipases A 2 and three-finger toxins, which are responsible for neuromuscular blockade-the main envenomation outcome in humans. Beyond these two major toxin families, minor components are also important for the global venom activity, including Kunitz-peptides, serine proteases, 5' nucleotidases, among others. In the present study, we used the two-microelectrode voltage clamp technique to explore the crude venom activities of five different Micrurus species from the south and southeast of Brazil: M. altirostris , M. corallinus , M. frontalis , M. carvalhoi and M. decoratus . All five venoms induced full inhibition of the muscle-type α1β1δε nAChR with different levels of reversibility. We found M. altirostris and M. frontalis venoms acting as partial inhibitors of the neuronal-type α7 nAChR with an interesting subsequent potentiation after one washout. We discovered that M. altirostris and M. corallinus venoms modulate the α1β2 GABA A R. Interestingly, the screening on K V 1.3 showed that all five Micrurus venoms act as inhibitors, being totally reversible after the washout. Since this activity seems to be conserved among different species, we hypothesized that the Micrurus venoms may rely on potassium channel inhibitory activity as an important feature of their envenomation strategy. Finally, tests on Na V 1.2 and Na V 1.4 showed that these channels do not seem to be targeted by Micrurus venoms. In summary, the venoms tested are multifunctional, each of them acting on at least two different types of targets.

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/G i 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., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Diverse physiological phenotypes in a neuronal population can broaden the range of computational capabilities within a brain region. The avian cochlear nucleus angularis (NA) contains a heterogeneous population of neurons whose variation in intrinsic properties results in electrophysiological phenotypes with a range of sensitivities to temporally modulated input. The low-threshold potassium conductance (G KLT ) is a key feature of neurons involved in fine temporal structure coding for sound localization, but a role for these channels in intensity or spectrotemporal coding has not been established. To determine whether G KLT affects the phenotypical variation and temporal properties of NA neurons, we applied dendrotoxin-I (DTX), a potent antagonist of Kv1-type potassium channels, to chick brain stem slices in vitro during whole cell patch-clamp recordings. We found a cell-type specific subset of NA neurons that was sensitive to DTX: single-spiking NA neurons were most profoundly affected, as well as a subset of tonic-firing neurons. Both tonic I (phasic onset bursting) and tonic II (delayed firing) neurons showed DTX sensitivity in their firing rate and phenotypical firing pattern. Tonic III neurons were unaffected. Spike time reliability and fluctuation sensitivity measured in DTX-sensitive NA neurons was also reduced with DTX. Finally, DTX reduced spike threshold adaptation in these neurons, suggesting that G KLT contributes to the temporal properties that allow coding of rapid changes in the inputs to NA neurons. These results suggest that variation in Kv1 channel expression may be a key factor in functional diversity in the avian cochlear nucleus. NEW & NOTEWORTHY The dendrotoxin-sensitive voltage-gated potassium conductance typically associated with neuronal coincidence detection in the timing pathway for sound localization is demonstrated to affect spiking patterns and temporal input sensitivity in the intensity pathway in the avian auditory brain stem. The Kv1-family channels appear to be present in a subset of cochlear nucleus angularis neurons, regulate spike threshold dynamics underlying high-pass membrane filtering, and contribute to intrinsic firing diversity.

Naja sumatrana venom cytotoxin (sumaCTX) is a basic protein which belongs to three-finger toxin family. It has been shown to induce caspase-dependent, mitochondrial-mediated apoptosis in MCF-7 cells at lower concentrations. This study aimed to investigate the alteration of secretome in MCF-7 cells following membrane permeabilization by high concentrations of sumaCTX, using label-free quantitative (LFQ) approach. The degree of membrane permeabilization of sumaCTX was determined by lactate dehydrogenase (LDH) assay and calcein-propidium iodide (PI) assays. LDH and calcein-PI assays revealed time-dependent membrane permeabilization within a narrow concentration range. However, as toxin concentrations increased, prolonged exposure of MCF-7 cells to sumaCTX did not promote the progression of membrane permeabilization. The secretome analyses showed that membrane permeabilization was an event preceding the release of intracellular proteins. Bioinformatics analyses of the LFQ secretome revealed the presence of 105 significantly distinguished proteins involved in metabolism, structural supports, inflammatory responses, and necroptosis in MCF-7 cells treated with 29.8 μg/mL of sumaCTX. Necroptosis was presumably an initial stress response in MCF-7 cells when exposed to high sumaCTX concentration. Collectively, sumaCTX-induced the loss of membrane integrity in a concentration-dependent manner, whereby the cell death pattern of MCF-7 cells transformed from apoptosis to necroptosis with increasing toxin concentrations., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Hepatocellular carcinoma (HCC) is the most common primary liver cancer in adults, the fifth most common malignancy worldwide and the third leading cause of cancer related death. An alternative to the surgical treatments and drugs, such as sorafenib, commonly used in medicine is necessary to overcome this public health problem. In this study, we determine the anticancer effect on HCC of Moroccan cobra Naja haje venom and its fraction obtained by gel filtration chromatography against Huh7.5 cancer cell line. Cells were grown together with WI38 human fibroblast cells, LX2 human hepatic stellate cell line, and human endothelial cells (HUVEC) in MCTS (multi-cellular tumor spheroids) models. The hepatotoxicity of venom and its fractions were also evaluated using the normal hepatocytes cell line (Fa2N-4 cells). Our results showed that an anti HCC activity of Moroccan cobra Naja haje venom and, more specifically, the F7 fraction of gel filtration chromatography exhibited the greatest anti-hepatocellular carcinoma effect by decreasing the size of MCTS. This effect is associated with a low toxicity against normal hepatocytes. These results strongly suggest that the F7 fraction of Moroccan cobra Naja haje venom obtained by gel filtration chromatography possesses the ability to inhibit cancer cells proliferation. More research is needed to identify the specific molecule(s) responsible for the anticancer effect and investigate their mechanism of action.

We discuss what therapeutic regimen might be acceptable/successful in the first clinical trial of genetically engineered pig kidney or heart transplantation. As regimens based on a calcineurin inhibitor or CTLA4-Ig have proved unsuccessful, the regimen we administer to baboons is based on induction therapy with antithymocyte globulin, an anti-CD20 mAb (Rituximab), and cobra venom factor, with maintenance therapy based on blockade of the CD40/CD154 costimulation pathway (with an anti-CD40 mAb), with rapamycin, and a corticosteroid. An anti-inflammatory agent (etanercept) is administered for the first 2 wk, and adjuvant therapy includes prophylaxis against thrombotic complications, anemia, cytomegalovirus, and pneumocystis. Using this regimen, although antibody-mediated rejection certainly can occur, we have documented no definite evidence of an adaptive immune response to the pig xenograft. This regimen could also form the basis for the first clinical trial, except that cobra venom factor will be replaced by a clinically approved agent, for example, a C1-esterase inhibitor. However, none of the agents that block the CD40/CD154 pathway are yet approved for clinical use, and so this hurdle remains to be overcome. The role of anti-inflammatory agents remains unproven. The major difference between this suggested regimen and those used in allotransplantation is the replacement of a calcineurin inhibitor with a costimulation blockade agent, but this does not appear to increase the complications of the regimen., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Acid-sensing ion channels (ASICs) are proton-gated cationic channels involved in pain and other processes, underscoring the potential therapeutic value of specific inhibitors such as the three-finger toxin mambalgin-1 (Mamb-1) from snake venom. A low-resolution structure of the human-ASIC1a/Mamb-1 complex obtained by cryo-electron microscopy has been recently reported, implementing the structure of the chicken-ASIC1/Mamb-1 complex previously published. Here we combine structure-activity relationship of both the rat ASIC1a channel and the Mamb-1 toxin with a molecular dynamics simulation to obtain a detailed picture at the level of side-chain interactions of the binding of Mamb-1 on rat ASIC1a channels and of its inhibition mechanism. Fingers I and II of Mamb-1 but not the core of the toxin are required for interaction with the thumb domain of ASIC1a, and Lys-8 of finger I potentially interacts with Tyr-358 in the thumb domain. Mamb-1 does not interfere directly with the pH sensor as previously suggested, but locks by several contacts a key hinge between α4 and α5 helices in the thumb domain of ASIC1a to prevent channel opening. Our results provide an improved model of inhibition of mammalian ASIC1a channels by Mamb-1 and clues for further development of optimized ASIC blockers., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background and Aims: Due to the lack of an effective vaccine and complexity of the control measures against vectors and reservoir hosts, the control of leishmaniasis depends primarily on chemotherapy. This study was aimed to assess the snake venom, Naja naja oxiana fraction 11(NNOVF11) on Leishmania infantum and its broad mode of action., Methods: A wide range of in vitro advanced assays including high-performance liquid chromatography (HPLC), MTT (3-[4, 5-Dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide; Thiazolyl blue), macrophage assays, quantitative real-time polymerase chain reaction (qPCR), flow cytometry and enzyme- linked immunosorbent assay (ELISA) on L. infantum promastigote and amastigote stages were used. IC 50 values of L. infantum stages, CC 50 value, and apoptosis were also analyzed., Results: The NNOV-F11 demonstrated strong antileishmanial activity against L. infantum stages in a dose-dependent manner compared to the untreated control group. Interleukin (IL)-12, TNF-α, and iNOS genes expression as the indicators of T helper(h)1 response significantly increased; in contrast, the expression level of IL-10, as the representative of Th2 response significantly decreased (p < 0.001). Reactive oxygen species (ROS) detection showed a significant increase (p < 0.001) after treatment with different concentrations of NNOV-F11, unlike arginase (ARG) activity, which displayed a significant reduction (p < 0.001)., Conclusion: NNOV-F11 possessed a potent inhibitory effect on L. infantum stages with the multifunctional and broad mode of actions, which promoted the immunomodulatory role, induced ROS production, stimulated apoptotic-like mechanisms, and inhibited L-ARG activity, which collectively led to the parasite death. Further studies are crucial to assess the effect of the NNOV-F11 on animal models or clinical settings., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Multiple sclerosis (MS) is considered a chronic disease of the central nervous system, with a strong neurodegenerative component. The exact mechanism of MS is not clear. However, the therapeutic strategies for controlling MS are based on immune modulation and inflammation control. Regarding this, the present study was conducted to investigate the influence of snake venom on the suppression of the immune system after the induction of experimental autoimmune encephalomyelitis (EAE) in mice. For this purpose, C57BL/6 female mice, divided into three groups, were selected to be induced by EAE. Groups 2 and 3 received flank injection with the emulsion of myelin oligodendrocyte glycoprotein (MOG 35-55), as well as complete Freund adjuvant, followed by the administration of pertussis toxin. Furthermore, the treatment group, as an immune-modulator, received cobra venom (CV) after EAE induction. The mice were then evaluated daily based on clinical symptoms, weight changes (within 26 days), histopathological analysis, and serum levels of interleukin 27 (IL-27) for neurological motor deficits. The clinical signs of MOG-EAE in C57BL/6 mice began 9-14 days post-immunization. Histopathological results also revealed that CV-treated EAE mice, compared to the untreated EAE group, witnessed a significant reduction in the intensity of inflammatory cells in parenchymal sections. Furthermore, the increase of IL-27 levels was significant in the CV-treated group (P=0.001), compared with those in the EAE and control groups. Based on results obtained in the present study, it may be concluded that Naja naja oxiana snake venom is a potential immunomodulatory agent that can be effective in the treatment of MS., (Copyright © 2021, Author(s). Published by Kowsar.)

In complement-driven thrombotic microangiopathies, failure to regulate complement activation leads to end-organ damage. The modified Ham (mHam) test measures complement-mediated killing of a nucleated cell in vitro but lacks a confirmatory assay and reliable positive controls. We demonstrate that C5b-9 accumulation on the surface of TF1 PIGAnull cells correlates with cell killing in the mHam. We also show that Sialidase treatment of cells or addition of Shiga toxin 1 to human serum serve as a more reliable positive control for the mHam than cobra venom factor or lipopolysaccharide. Simultaneously performing the mHam and measuring C5b-9 accumulation either in GVB ++ or GVB 0 MgEGTA buffer with the addition of complement pathway specific inhibitors (anti-C5 antibody or a factor D inhibitor, ACH-145951) can be used to localize defects in complement regulation. As more targeted complement inhibitors become available, these assays may aid in the selection of personalized treatments for patients with complement-mediated diseases., (Copyright © 2020. Published by Elsevier Inc.)

This is the first report showing unique neuritogenesis potency of Indian Cobra N. naja venom long-chain α-neurotoxin (Nn-α-elapitoxin-1) exhibiting no sequence similarity to conventional nerve growth factor, by high-affinity binding to its tyrosine kinase A (TrkA) receptor of rat pheochromocytoma (PC-12) cells without requiring low-affinity receptor p 75 NTR. The binding residues between Nn-α-elapitoxin-1 and mammalian TrkA receptor are predicted by in silico analysis. This binding results in a time-dependent internalization of TrkA receptor into the cytoplasm of PC-12 cells. The transcriptomic analysis has demonstrated the differential expression of 445 genes; 38 and 32 genes are up-regulated and down-regulated, respectively in the PC-12 cells post-treatment with Nn-α-elapitoxin-1. Global proteomic analysis in concurrence with transcriptomic data has also demonstrated that in addition to expression of a large number of common intracellular proteins in the control and Nn-α-elapitoxin-1-treated PC-12 cells, the latter cells also showed the expression of uniquely up-regulated and down-regulated intracellular proteins involved in diverse cellular functions. Altogether, the data from transcriptomics, proteomics, and inhibition of downstream signaling pathways by specific inhibitors, and the immunoblot analysis of major regulators of signaling pathways of neuritogenesis unambiguously demonstrate that, similar to mouse 2.5S-nerve growth factor, the activation of mitogen activated protein kinase/extracellular signal-regulated kinase is the major signaling pathway for neuritogenesis by Nn-α-elapitoxin-1. Nonetheless, fibroblast growth factor signaling and heterotrimeric G-protein signaling pathways were found to be uniquely expressed in Nn-α-elapitoxin-1-treated PC-12 cells and not in mouse 2.5S-nerve growth factor -treated cells. The TrkA binding region of Nn-α-elapitoxin-1 may be developed as a peptide-based drug prototype for the treatment of major central neurodegenerative diseases. Read the Editorial Highlight for this article on page 599., (© 2020 International Society for Neurochemistry.)

Analysis of the presynaptic action potential's (AP syn ) role in synaptic facilitation in hippocampal pyramidal neurons has been difficult due to size limitations of axons. We overcame these size barriers by combining high-resolution optical recordings of membrane potential, exocytosis, and Ca 2+ in cultured hippocampal neurons. These recordings revealed a critical and selective role for K v 1 channel inactivation in synaptic facilitation of excitatory hippocampal neurons. Presynaptic K v 1 channel inactivation was mediated by the K v β1 subunit and had a surprisingly rapid onset that was readily apparent even in brief physiological stimulation paradigms including paired-pulse stimulation. Genetic depletion of K v β1 blocked all broadening of the AP syn during high-frequency stimulation and eliminated synaptic facilitation without altering the initial probability of vesicle release. Thus, using all quantitative optical measurements of presynaptic physiology, we reveal a critical role for presynaptic K v channels in synaptic facilitation at presynaptic terminals of the hippocampus upstream of the exocytic machinery., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Breast cancer (BC) is among the leading causes of mortality from cancer in women. Many of the available anticancer drugs have various side effects. Therefore, researchers are seeking novel anticancer agents particularly from natural compounds and in this regard, snake venom is still one of the main sources of drug discovery. Previous studies showed potential anticancer effects of Cytotoxin II (CTII) from Naja naja oxiana against the different types of cancers. In this study, a pET-SUMO-CTII vector was transformed into SHuffle® T7 Express, an Escherichia coli strain, for recombinant protein expression (rCTII) and the cytotoxic effects of this protein was assessed in MCF-7 cells. The flow cytometry assay was applied to measure the apoptosis and cell cycle. Also, mRNA levels of the Bax, Bcl2, P53, caspase-3, caspase-8, caspase-9, caspase-10, matrix metalloproteinases (MMP)-3, and MMP-9 were analyzed by quantitative real-time PCR to determine the underlying cellular pathways affected by rCTII. The results of this study showed that treatment with 4 μg mL -1 of rCTII enhanced apoptosis through the intrinsic and extrinsic pathways. Also, the increase of the cells' proportion in the sub-G1 phase as well as a reduction in S phase was observed. In addition, the expression of MMP-3 and MMP-9 was decreased in the treated group in comparison to the control group that may contribute to the reduced migratory ability of tumor cells. These experimental results indicate that rCTII has anti-proliferative potential, and so this protein could be a potential drug for BC therapy in combination with other drugs., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

The Chinese Cobra (Naja atra) is an elapid snake of major medical importance in southern China. Although previous studies have shown that postsynaptic neurotoxins account for 11-23% of N. atra venom, envenomed patients do not display marked signs of neurotoxicity. We have previously shown that the lack of clinical neurotoxicity following snake envenoming by some species with 'neurotoxic' venoms may be related to the high prevalence of short-chain postsynaptic neurotoxins in these venoms. In this study, we describe the isolation and characterization of α-Elapitoxin-Na1a (α-EPTX-Na1a; 6949 Da), a short-chain postsynaptic neurotoxin, which accounts for approximately 9% of N. atra crude venom. α-EPTX-Na1a (30-300 nM) produced concentration-dependent inhibition of indirect-twitches, with a t 90 value of 17 ± 2 min at 300 nM, and abolished contractile responses to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The prior addition of either Chinese N. atra monovalent antivenom (0.3 U/ml) or Australian polyvalent snake antivenom (2.4 U/ml), prevented the in vitro neurotoxic effects of α-EPTX-Na1a (30 nM). Addition of each of these antivenoms at the t 90 time point partially reversed the in vitro neurotoxicity caused by α-EPTX-Na1a (30 nM). The inhibition of indirect twitches by α-EPTX-Na1a (30 nM) was not reversed by repeatedly washing the tissue. α-EPTX-Na1a displayed pseudo-irreversible antagonism of concentration-response curves to carbachol with a pA 2 value of 8.21. De novo protein sequencing of α-EPTX-Na1a revealed a typical short-chain postsynaptic neurotoxin profile of 62 amino acids which shared >98% amino acid sequence similarity with short-chain postsynaptic neurotoxins from other Naja species. When compared to short-chain neurotoxins isolated from cobras in China, α-EPTX-Na1a contained novel residues K47Q (i.e. lysine to glutamine), N48T (i.e. asparagine to threonine) and G49A (i.e. glycine to alanine). In conclusion, α-EPTX-Na1a is a potent, pseudo-irreversible, short-chain neurotoxin. The high prevalence of α-EPTX-Na1a in Chinese N. atra venom is likely to explain the mild neurotoxicity experienced by envenomed patients., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Venoms act with remarkable specificity upon a broad diversity of physiological targets. Venoms are composed of proteins, peptides, and small molecules, providing the foundation for the development of novel therapeutics. This study assessed the effect of venom from the red-bellied black snake ( Pseudechis porphyriacus ) on human primary leukocytes using bead-based flow cytometry, mixed lymphocyte reaction, and cell viability assays. We show that venom treatment had a significant immunosuppressive effect, inhibiting the secretion of interleukin (IL)-2 and tumor necrosis factor (TNF) from purified human T cells by 90% or greater following stimulation with mitogen (phorbol 12-myristate 13-acetate and ionomycin) or via cluster of differentiation (CD) receptors, CD3/CD28. In contrast, venom treatment did not inhibit TNF or IL-6 release from antigen-presenting cells stimulated with lipopolysaccharide. The reduced cytokine release from T cells was not associated with inhibition of T cell proliferation or reduction of cell viability, consistent with an anti-inflammatory mechanism unrelated to the cell cycle. Deconvolution of the venom using reverse-phase HPLC identified four fractions responsible for the observed immunosuppressive activity. These data suggest that compounds from P. porphyriacus venom may be potential drug leads for T cell-associated conditions such as graft versus host disease, rheumatoid arthritis, and inflammatory bowel disease.

Animal venoms are small natural mixtures highly enriched in bioactive components. They are known to target at least two important pharmacological classes of cell surface receptors: ion channels and G protein coupled receptors. Since sperm cells express a wide variety of ion channels and membrane receptors, required for the control of cell motility and acrosome reaction, two functions that are defective in infertility issues, animal venoms should contain interesting compounds capable of modulating these two essential physiological functions. Herein, we screened for bioactive compounds from the venom of the Egyptian black snake Walterinnesia aegyptia (Wa) that possess the property to activate sperm motility in vitro from male mice OF1. Using RP-HPLC and cation exchange chromatography, we identified a new toxin of 6389.89 Da (termed walterospermin) that activates sperm motility. Walterospermin was de novo sequenced using a combination of matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF MS/MS) and liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF MS/MS) following reduction, alkylation, and enzymatic proteolytic digestion with trypsin, chymotrypsin or V8 protease. The peptide is 57 amino acid residues long and contains three disulfide bridges and was found to be identical to the previously cloned Wa Kunitz-type protease inhibitor II (Wa Kln-II) sequence. Moreover, it has strong homology with several other hitherto cloned Elapidae and Viperidae snake toxins suggesting that it belongs to a family of compounds able to regulate sperm function. The synthetic peptide shows promising activation of sperm motility from a variety of species, including humans. Its fluorescently-labelled analog predominantly marks the flagellum, a localization in agreement with a receptor that controls motility function.

The evolution of an aquatic lifestyle from land dwelling venomous elapids is a radical ecological modification, bringing about many evolutionary changes from morphology to diet. Diet is an important ecological facet which can play a key role in regulating functional traits such as venom composition and prey-specific targeting of venom. In addition to predating upon novel prey (e.g., fish, fish eggs and invertebrates), the venoms of aquatic elapids also face the challenge of increased prey-escape potential in the aquatic environment. Thus, despite the independent radiation into an aquatic niche on four separate occasions, the venoms of aquatic elapids are evolving under convergent selection pressures. Utilising a biolayer interferometry binding assay, this study set out to elucidate whether crude venoms from representative aquatic elapids were target-specific to the orthosteric site of postsynaptic nicotinic acetylcholine receptor mimotopes of fish compared to other terrestrial prey types. Representatives of the four aquatic lineages were: aquatic coral snakes representative was Micrurus surinamensis ;, sea kraits representative was Laticauda colubrina; sea snakes representatives were two Aipysurus spp. and eight Hydrophis spp; and water cobras representative was Naja annulata . No prey-specific differences in crude venom binding were observed from any species tested, except for Aipysurus laevis, which showed slight evidence of prey-potency differences. For Hydrophis caerulescens , H. peronii , H. schistosus and M. surinamensis , there was a lack of binding to the orthosteric site of any target lineage. Subsequent testing on the in vitro chick-biventer cervicis muscle preparation suggested that, while the venoms of these species bound postsynaptically, they bound to allosteric sites rather than orthosteric. Allosteric binding is potentially a weaker but faster-acting form of neurotoxicity and we hypothesise that the switch to allosteric binding is likely due to selection pressures related to prey-escape potential. This research has potentially opened up the possibility of a new functional class of toxins which have never been assessed previously while shedding light on the selection pressures shaping venom evolution.

Cysteine-Rich Secretory Proteins (CRISP) are widespread in snake venoms and known to target ion channels. More recently, CRISPs have been shown to mediate inflammatory responses. Involvement of potential receptor in CRISP-induced inflammatory reactions, however, remains unknown. A CRISP protein named as Nk-CRISP, was isolated from the venom of Naja kaouthia. The molecular mass of the purified protein was found to be ~25 kDa and the primary sequence was determined by MALDI TOF-TOF. The involvement of this protein in proinflammatory effects were evaluated in THP-1 macrophage-like cells. Nk-CRISP treated cells induced up-regulation of several inflammatory marker genes in dose dependent manner. Toll like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex are known to play crucial role in recognition of damage/pathogen-associated molecular patterns and activation of innate immune response. Therefore, we hypothesized that snake venom CRISP could also modulate the innate immune response via TLR4-MD2 complex. In-silico molecular docking study of cobra CRISP with TLR4-MD2 receptor complex reveals CRISP engages its cysteine-rich domain (CRD) to interact with complex. Inhibition of TLR4 signalling pathway using CLI-095 confirmed the role of TLR4 in Nk-CRISP induced inflammatory responses. Collectively, these findings imply that TLR4 initiates proinflammatory signalling following recognition of cobra CRISP and alteration of TLR4 receptor might improve or control CRISP induced inflammation., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Acid-sensing ion channels (ASICs) are proton-gated cation channels that are involved in diverse neuronal processes including pain sensing. The peptide toxin Mambalgin1 (Mamba1) from black mamba snake venom can reversibly inhibit the conductance of ASICs, causing an analgesic effect. However, the detailed mechanism by which Mamba1 inhibits ASIC1s, especially how Mamba1 binding to the extracellular domain affects the conformational changes of the transmembrane domain of ASICs remains elusive. Here, we present single-particle cryo-EM structures of human ASIC1a (hASIC1a) and the hASIC1a-Mamba1 complex at resolutions of 3.56 and 3.90 Å, respectively. The structures revealed the inhibited conformation of hASIC1a upon Mamba1 binding. The combination of the structural and physiological data indicates that Mamba1 preferentially binds hASIC1a in a closed state and reduces the proton sensitivity of the channel, representing a closed-state trapping mechanism., Competing Interests: DS, SL, SL, MZ, FY, MW, PS, TW, MP, SC, XZ, LZ, CT, LL No competing interests declared, (© 2020, Sun et al.)

In contrast to comprehensively investigated antibacterial activity of snake venoms, namely crude venoms and their selected components, little is known about antifungal properties of elapid snake venoms. In the present study, the proteome of two venoms of red spitting cobra Naja pallida (NPV) and Mozambique spitting cobra Naja mossambica (NMV) was characterized using LC-MS/MS approach, and the antifungal activity of crude venoms against three Candida species was established. A complex response to venom treatment was revealed. NPV and NMV, when used at relatively high concentrations, decreased cell viability of C. albicans and C. tropicalis , affected cell cycle of C. albicans , inhibited C. tropicalis -based biofilm formation and promoted oxidative stress in C. albicans , C. glabrata and C. tropicalis cells. NPV and NMV also modulated ammonia pulses during colony development and aging in three Candida species. All these observations provide evidence that NPV and NMV may diminish selected pathogenic features of Candida species. However, NPV and NMV also promoted the secretion of extracellular phospholipases that may facilitate Candida pathogenicity and limit their usefulness as anti-candidal agents. In conclusion, antifungal activity of snake venoms should be studied with great caution and a plethora of pathogenic biomarkers should be considered in the future experiments.

The role of the ASIC1a in evoking the exercise pressor reflex in rats with simulated peripheral artery disease is unknown. This prompted us to determine whether ASIC1a plays a role in evoking the exaggerated exercise pressor reflex in decerebrated rats with simulated peripheral artery disease. To simulate peripheral artery disease, we ligated the left femoral artery 72 h before the experiment. The right femoral artery was freely perfused and used as a control. To test our hypothesis, we measured the effect of injecting two ASIC1a blockers into the arterial supply of the triceps surae muscles with and without the femoral artery ligated on the reflex pressor responses to 1 ) static contraction of the triceps surae muscles, 2 ) calcaneal tendon stretch, and 3 ) intra-arterial injection of diprotonated phosphate (pH 6.0). We found that the ASIC1a blockers psalmotoxin-1 (200 ng/kg) and mambalgin-1 (6.5 μg/kg) decreased the pressor responses to static contraction as well as the peak pressor responses to injection of diprotonated phosphate when these responses were evoked from the freely perfused hindlimb. In contrast, ASIC1a blockers only decreased the peak pressor responses evoked by injection of diprotonated phosphate in the hindlimb circulation with simulated peripheral artery disease. This inhibitory effect was less than the one measured from the healthy hindlimb. Independently of the hindlimb of interest, ASIC1a blockers had no effect on the pressor responses to tendon stretch. Our results do not support the hypothesis that ASIC1a play a role in evoking the exercise pressor reflex arising from a hindlimb with simulated peripheral artery disease. NEW & NOTEWORTHY The role of ASIC1a in evoking the metabolic component of the exercise pressor reflex in peripheral artery disease is unknown. Using a within-rat experimental design, we found that the contribution of ASIC1a decreased in a rat model of peripheral artery disease. These results have key implications to help finding better treatments and improve morbidity, quality of life, and mortality in patients with peripheral artery disease.

Background: Neutrophils are the first line defense cells of the innate immunity. As a final defense, they discharge their de-condensed chromatin/DNA fibers, the NETs (Neutrophil Extracellular Traps), by a process called NETosis. Two types of NETosis have been currently described: the suicidal/delayed/classical-type, which is ROS dependent that results in the ejection of nuclear DNA, and the vital/rapid/early-type, which may or may not require ROS but, eject nuclear/mitochondrial DNA or both. Thus, Echis carinatus and Naja naja venoms are comparatively studied for their NET inducing property., Methods: Formation of NETs, cell viability, ROS, and Ca 2+ levels are estimated. An in vivo toxicity study and possible cellular signaling have been addressed using immunoblots and pharmacological inhibitors., Results: E. carinatus and N. naja venoms respectively induce suicidal and vital NETosis. E. carinatus venom induces NETosis by activating NOX and PAD-4 enzymes in a ROS dependent manner via PKC/ERK/JNK signaling axis, while N. naja venom does it by activating PAD-4 enzyme, but independent of ROS requirement and as well as PKC/ERK/JNK activation., Conclusion: For the first time our study demonstrates the distinct action of E. carinatus and N. naja venoms on the process of NETosis. NETosis being a newly explored area in snake venom pharmacodynamics, it is important to study its impact on the various pathophysiological properties induced by snake venoms., Significance: Understanding the varied actions of snake venoms on neutrophils/blood cells and the role of DNase are likely to provide insights for better management of snakebite pathophysiology., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2020. Published by Elsevier B.V.)

Mitochondrial dysfunction is implicated in a variety of neurodegenerative diseases of the nervous system. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a regulator of mitochondrial function in multiple cell types. In sensory neurons, AMP-activated protein kinase (AMPK) augments PGC-1α activity and this pathway is depressed in diabetes leading to mitochondrial dysfunction and neurodegeneration. Antimuscarinic drugs targeting the muscarinic acetylcholine type 1 receptor (M 1 R) prevent/reverse neurodegeneration by inducing nerve regeneration in rodent models of diabetes and chemotherapy-induced peripheral neuropathy (CIPN). Ca 2+ /calmodulin-dependent protein kinase kinase β (CaMKKβ) is an upstream regulator of AMPK activity. We hypothesized that antimuscarinic drugs modulate CaMKKβ to enhance activity of AMPK, and PGC-1α, increase mitochondrial function and thus protect from neurodegeneration. We used the specific M 1 R antagonist muscarinic toxin 7 (MT7) to manipulate muscarinic signaling in the dorsal root ganglia (DRG) neurons of normal rats or rats with streptozotocin-induced diabetes. DRG neurons treated with MT7 (100 nM) or a selective muscarinic antagonist, pirenzepine (1 μM), for 24 h showed increased neurite outgrowth that was blocked by the CaMKK inhibitor STO-609 (1 μM) or short hairpin RNA to CaMKKβ. MT7 enhanced AMPK phosphorylation which was blocked by STO-609 (1 μM). PGC-1α reporter activity was augmented up to 2-fold (p < 0.05) by MT7 and blocked by STO-609. Mitochondrial maximal respiration and spare respiratory capacity were elevated after 3 h of exposure to MT7 (p < 0.05). Diabetes and CIPN induced a significant (p < 0.05) decrease in corneal nerve density which was corrected by topical delivery of MT7. We reveal a novel M 1 R-modulated, CaMKKβ-dependent pathway in neurons that represents a therapeutic target to enhance nerve repair in two of the most common forms of peripheral neuropathy.

We aimed at establishing a sensitive and robust assay for estimation of systemic complement activation at complement component C3 level in mouse and human plasma samples. In order to capture the activation products iC3b and C3dg in a specific and physiological relevant manner we utilized a construct consisting of the iC3b/C3dg-binding site of human complement receptor 2 (CR2) attached to an Fc-part of mouse IgG. This construct binds C3dg and iC3b from both mice and humans. We purified the CR2-IgG construct from mouse B myeloma cell line supernatants, J558L-CR2-IgG, by protein G affinity chromatography. The CR2-IgG construct was used for capturing C3 fragments in microtiter wells and an anti-mouse or an anti-human-C3 antibody was used for detection of bound C3 fragments. Initially we tested the specificity of the assays with the use of purified C3 fragments. Further, with the use of the CR2-based assay, we measured an up to three-fold higher signal in activated mouse serum as compared to non-activated mouse serum, whereas activated serum from a C3 knock-out mouse gave no signal. We tested in vivo generated samples from a mouse experiment; complement activation was induced by injecting cobra venom factor or heat aggregated IgG into C57bl6 mice, followed by withdrawal of EDTA blood samples at different time points and measurement of iC3b/C3dg. We observed a clear time-dependent distinction in signals between samples with expected high and low complement activation. Furthermore, with the use of the assay for human C3 fragments, we observed that patients with systemic lupus erythematosus (SLE) ( n = 144) had significantly higher iC3b/C3dg levels as compared to healthy individuals ( n = 144) ( p < 0.0001). We present two functional immunoassays, that are able to measure systemic levels of the C3-activation products iC3b and C3dg in mice and humans. To our knowledge, these are the first assays for complement activation that use a physiological relevant capture construct such as CR2. These assays will be a relevant tool when investigating mouse models and human diseases involving the complement system., (Copyright © 2020 Halkjær, Troldborg, Pedersen, Jensen, Hansen, Hansen, Bjerre, Østergaard and Thiel.)

Introduction: Several disease processes trigger prolonged activation of the alternative complement pathway. Crosslinks between complement activation and physiologic changes in platelets and neutrophils have been identified, but how this interplay alters the hemostatic potential in humans remains undefined. We hypothesize that activation of the alternative pathway triggers a hypercoagulable state., Methods: C3/C5 convertase Cobra Venom Factor (CVF, 10 Units/mL) was employed to activate the alternative complement pathway in whole blood. Complement inhibition was completed with inhibitors for C3/C3b (Compstatin, 25 and 50 μM), C3a receptor (SB290157, 300 nM, C3aR), and C5a receptor (W54011, 6 nM, C5aR). Coagulation was assessed using native thrombelastography which produces the following: reaction time (R time); angle; maximum amplitude (MA); percent fibrinolysis at 30-min post-MA (LY30)., Results: Inhibition with C3aR and C5aR inhibitors did not alter clot formation (R time, 11.2 vs 11.6 min, P = 0.36), clot strength (MA, 52.0 vs 52.3 mm, P = 0.43), or fibrinolysis (LY30, 1.6 vs 4.0%, P = 0.19). Compstatin did not influence clot formation or clot strength but did induce a dose-dependent increase in fibrinolysis (control LY30 3.0 vs 7.8% and 12.4% for 25 and 50 μM respectively, P = 0.0002). CVF increased MA (58.0 vs 62.8 mm, P < 0.0001), decreased LY30 (2.3 vs 1.4%, P = 0.004), and increased R time (8.4 vs 9.9 min, P = 0.008). Compstatin reversed the effects of CVF, while C5a reversed only the change in LY30., Conclusions: C3 contributes to fibrinolysis, as inhibition with Compstatin enhanced fibrinolysis, and CVF cleavage of C3 decreased fibrinolysis. CVF also induced a hypercoagulable state with increased clot strength.

One of the key problems of modern infectious disease medicine is the growing number of drug-resistant and multi-drug-resistant bacterial strains. For this reason, many studies are devoted to the search for highly active antimicrobial substances that could be used in therapy against bacterial infections. As it turns out, snake venoms are a rich source of proteins that exert a strong antibacterial effect, and therefore they have become an interesting research material. We analyzed Naja ashei venom for such antibacterial properties, and we found that a specific composition of proteins can act to eliminate individual bacterial cells, as well as the entire biofilm of Staphylococcus epidermidis . In general, we used ion exchange chromatography (IEX) to obtain 10 protein fractions with different levels of complexity, which were then tested against certified and clinical strains of S. epidermidis . One of the fractions (F2) showed exceptional antimicrobial effects both alone and in combination with antibiotics. The protein composition of the obtained fractions was determined using mass spectrometry techniques, indicating a high proportion of phospholipases A 2 , three-finger toxins, and L-amino acids oxidases in F2 fraction, which are most likely responsible for the unique properties of this fraction. Moreover, we were able to identify a new group of low abundant proteins containing the Ig-like domain that have not been previously described in snake venoms., Competing Interests: The authors declare no conflict of interest.

The role of the acid-sensing ion channel 1a (ASIC1a) in evoking the exercise pressor reflex is unknown, despite the fact that ASIC1a is opened by decreases in pH in the physiological range. This fact prompted us to test the hypothesis that ASIC1a plays an important role in evoking the exercise pressor reflex in decerebrated rats with freely perfused hindlimb muscles. To test this hypothesis, we measured the effect of injecting two ASIC1a blockers into the arterial supply of the triceps surae muscles on the reflex pressor responses to four maneuvers, namely 1 ) static contraction of the triceps surae muscles (i.e., the exercise pressor reflex), 2 ) calcaneal tendon stretch, 3 ) intra-arterial injection of lactic acid, and 4 ) intra-arterial injection of diprotonated phosphate. We found that the 2 ASIC1a blockers, psalmotoxin-1 (200 ng/kg) and mambalgin-1 (6.5 μg/kg), decreased the pressor responses to static contraction as well as the peak pressor responses to injection of lactic acid and diprotonated phosphate. In contrast, neither ASIC1a blocker had any effect on the pressor responses to tendon stretch. Importantly, we found that ASIC1a blockade significantly decreased the pressor response to static contraction after a latency of at least 8 s. Our results support the hypothesis that ASIC1a plays a key role in evoking the metabolic component of the exercise pressor reflex. NEW & NOTEWORTHY The role played by acid-sensing ion channel 1a (ASIC1a) in evoking the exercise pressor reflex remains unknown. In decerebrated rats with freely perfused femoral arteries, blocking ASIC1a with psalmotoxin-1 or mambalgin-1 significantly attenuated the pressor response to static contraction, lactic acid, and diprotonated phosphate injection but had no effect on the pressor response to stretch. We conclude that ASIC1a plays a key role in evoking the exercise pressor reflex by responding to contraction-induced metabolites, such as protons.

Background: A phenomena of interest is the in vitro anticoagulant effects of neurotoxins found in elapid venoms that kill by paralysis. These enzymes include phospholipase A2 (PLA2), and it has recently been demonstrated that carbon monoxide inhibits the PLA2-dependent neurotoxin contained in Mojave rattlesnake type A venom. The purpose of this investigation was to assess if the anticoagulant activity of elapid venoms containing PLA2 and/or three finger toxins could be inhibited by carbon monoxide., Methods: Venoms collected from Bungarus multicinctus, Micrurus fulvius, and five Naja species were exposed to carbon monoxide via carbon monoxide releasing molecule-2 prior to placement into human plasma. Coagulation kinetics were assessed via thrombelastography., Results: Compared with plasma without venom addition, all venoms had significant anticoagulant effects, with a 160-fold range of concentrations having similar anticoagulant effects in a species-specific manner. Carbon monoxide significantly inhibited the anticoagulant effect of all venoms tested, but inhibition was not complete in all cases., Conclusion: Given that individual neurotoxin activity often depends on intact activity that includes anticoagulant action, it may be possible that carbon monoxide inhibits neurotoxicity. Future investigation is justified to assess such carbon monoxide mediated inhibition with purified neurotoxins in vitro and in vivo.

Background: Thalamic pain is a neuropathic pain syndrome that occurs as a result of thalamic damage. It is difficult to develop therapeutic interventions for thalamic pain because its mechanism is unclear. To better understand the pathophysiological basis of thalamic pain, we developed and characterized a new rat model of thalamic pain using a technique of microinjecting cobra venom into the ventral posterolateral nucleus (VPL) of the thalamus., Objectives: This study will establish a new thalamic pain rat model produced by administration of cobra venom to the unilateral ventral posterolateral nucleus., Study Design: This study used an experimental design in rats., Setting: The research took place in the laboratory at the Aviation General Hospital of China Medical University and Beijing Institute of Translational Medicine., Methods: Male Sprague-Dawley rats were subjected to the administration of cobra venom or saline into the left VPL. The development of mechanical hyperalgesia and changes in pain-related behaviors and motor function were measured after intrathalamic cobra venom microinjection using the von Frey test, video recording, and cylinder test, respectively. On postoperative days 7 to 35, both electroacupuncture and pregabalin (PGB) were administered to verify that the model reproduced the findings in humans. Moreover, the organizational and structural alterations of the thalamus were examined via transmission electron microscopy (TEM)., Results: The threshold for mechanical stimuli in the left facial skin was significantly decreased on day 3 after thalamic pain modeling as compared with pre-venom treatment. Furthermore, the ultrastructural alterations of neurons such as indented neuronal nuclei, damaged mitochondria and endoplasmic reticulum, and dissolved surrounding tissues were observed under TEM. Moreover, electroacupuncture treatment ameliorated mechanical hyperalgesia, pain-like behaviors, and motor dysfunction, as well as restore normal structures of neurons in the thalamic pain rat model. However, no such beneficial effects were noted when PGB was administered., Limitations: The pathophysiological features were different from the present model and the patients in clinical practice (in most cases strokes, either ischemic or hemorrhagic)., Conclusion: The cobra venom model may provide a reasonable model for investigating the mechanism of thalamic pain and for testing therapies targeting recovery and pain after thalamic lesions., Key Words: Thalamic pain, cobra venom, electroacupuncture, pregabalin, indented neuronal nuclei, damaged mitochondria, dissolved endoplasmic reticulum, golgi body.

The biochemical properties and biological activities of the venom from three individual Ophiophagus hannah (King cobra) specimens was compared. The toxicity against mice, the cytotoxicity against five cell lines, and the antioxidant activity were measured. The KV2 venom showed a higher cytotoxicity than the KV6 and the non-cytotoxic KV9 venoms. Comparative analysis of the O. hannah venom proteins was performed after 2-dimensional (2-D) denaturing gel electrophoresis and reverse phase high performance liquid chromatography (RP-HPLC). 2-D analysis by isoelectric focusing (IEF) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) resolution of the venoms revealed significant differences between all three venoms, with most spots being unique to that venom. Only 2 out of the 13-16 distinct spots were common to all three venoms, and four spots were common to KV6 and KV9. KV2 had the highest proportion of low molecular mass spots, and KV6 and KV9 appeared more related to each other than to KV9. From peptide mass mapping by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and MASCOT-based amino acid sequence database searching, the two venom proteins that were common to all three specimens are likely to be ophanin and acidic phospholipase A 2 (PLA 2 ), whilst the proteins unique to the cytotoxic KV2 venom, included three other PLA 2 proteins. The RP-HPLC pattern of KV2 was different from the other two venoms with a higher protein concentration eluting in the 31-41% (v/v) acetonitrile (ACN) fraction than for the other two venoms.

Complement (C) activation can underlie the infusion reactions to liposomes and other nanoparticle-based medicines, a hypersensitivity syndrome that can be partially reproduced in animal models. However, the sensitivities and manifestations substantially differ in different species, and C activation may not be the only cause of pathophysiological changes. In order to map the species variation of C-dependent and -independent pseudoallergy (CARPA/CIPA), here we used known C activators and C activator liposomes to compare their acute hemodynamic, hematological, and biochemical effects in rats. These C activators were cobra venom factor (CVF), zymosan, AmBisome (at 2 doses), its amphotericin B-free vehicle (AmBisombo), and a PEGylated cholesterol-containing liposome (PEG-2000-chol), all having different powers to activate C in rat blood. The pathophysiological endpoints measured were blood pressure, leukocyte and platelet counts, and plasma thromboxane B2, while C activation was assessed by C3 consumption using the Pan-Specific C3 assay. The results showed strong linear correlation between C activation and systemic hypotension, pointing to a causal role of C activation in the hemodynamic changes. The observed thrombocytopenia and leukopenia followed by leukocytosis also correlated with C3 conversion in case of C activators, but not necessarily with C activation by liposomes. These findings are consistent with the double hit hypothesis of hypersensitivity reactions (HSRs), inasmuch as strong C activation can fully account for all symptoms of HSRs, but in case of no-, or weak C activators, the pathophysiological response, if any, is likely to involve other activation pathways.

Background: Cytotoxin 1 (CTX1) purified from Naja atra Cantor venom could inhibit cancer cell proliferation, but the mechanism is not clear. This study aimed to investigate the mechanism by which leukemia cells are killed by CTX1., Materials and Methods: HL-60 and KG1a cells were treated with CTX1 and the cell death was detected., Results: The viability of HL-60 and KG1a cells decreased in a dose- and time-dependent manner after treatment with CTX1. CTX1 mainly induced late apoptosis and necrosis. The cell death induced by CTX1 could be rescued by specific necroptosis inhibitor Nec-1 but not by caspase inhibitor Z-VAD-fmk in HL-60 cells. In addition, CTX1 increased lysosome membrane permeability (LMP) and release of cathepsin B., Conclusion: CTX1 could induce necroptosis in leukemia cells, and it is related to LMP increase and cathepsin release. CTX1 could be a promising anti-cancer drug for leukemia therapy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

An acidic phospholipase A 2 enzyme (NnPLA 2 -I) interacts with three finger toxins (cytotoxin and neurotoxin) from Naja naja venom to form cognate complexes to enhance its cytotoxicity towards rat L6 myogenic cells. The cytotoxicity was further enhanced in presence of trace quantity of venom nerve growth factor. The purified rat myoblast cell membrane protein showing interaction with NnPLA 2 -I was identified as vimentin by LC-MS/MS analysis. The ELISA, immunoblot and spectrofluorometric analyses showed greater binding of NnPLA 2 -I cognate complex to vimentin as compared to the binding of individual NnPLA 2 -I. The immunofluorescence and confocal microscopy studies evidenced the internalization of NnPLA 2 -I to partially differentiated myoblasts post binding with vimentin in a time-dependent manner. Pre-incubation of polyvalent antivenom with NnPLA 2 -I cognate complex demonstrated better neutralization of cytotoxicity towards L6 cells as compared to exogenous addition of polyvalent antivenom 60-240 min post treatment of L6 cells with cognate complex suggesting clinical advantage of early antivenom treatment to prevent cobra venom-induced cytotoxicity. The in silico analysis showed that 19-22 residues, inclusive of Asp48 residue, of NnPLA 2 -I preferentially binds with the rod domain (99-189 and 261-335 regions) of vimentin with a predicted free binding energy (ΔG) and dissociation constant (K D ) values of -12.86 kcal/mol and 3.67 × 10 -10  M, respectively; however, NnPLA 2 -I cognate complex showed greater binding with the same regions of vimentin indicating the pathophysiological significance of cognate complex in cobra venom-induced cytotoxicity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Natrin, a new member of the cysteine-rich secretory protein (CRISP) family purified from the snake venom of Naja naja atra, has been demonstrated to have anticancer activity. However, the underlying molecular mechanisms need further elucidation. In this study, MTT was used to evaluate cell viability. Apoptotic cells were analyzed by employing a transmission electron microscope (TEM). Metabolomic study of the metabolic perturbations caused by natrin-induced apoptosis in differentiated SMMC-7721 cells was performed for the first time by using integrative ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS). To investigate the possible mechanism in the mitochondrial pathway of natrin-induced apoptosis, we measured apoptosis-related mRNA changes using real-time fluorescent quantitative PCR (FQ-PCR). Cell proliferation was significantly inhibited after treatment with natrin in a dose-dependent manner. Principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) clearly demonstrated that metabolic profiles were affected by natrin. The results of multivariate statistical analysis showed that a total of 13 metabolites were characterized as potential biomarkers highly implicated in natrin-induced apoptosis, which corresponded to fluctuations of five pathways, including sphingolipid metabolism, fatty acid biosynthesis, fatty acid metabolism, glycerophospholipid metabolism and glycosphingolipid biosynthesis. Furthermore, natrin-induced apoptosis showed an increase in the Bax/Bcl-2 ratio in the mitochondrial pathway compared with controls. This study illustrated that rapid and holistic cell metabolomics combining molecular biological approaches might be a powerful tool for evaluating the underlying mechanisms of natrin-induced apoptosis, which would help to deepen specific insights into the anti-hepatoma mechanisms of natrin and facilitate the clinical application of natrin in the future., (Copyright © 2018. Published by Elsevier B.V.)

Cancer is a deadly disease and there is an urgent need for the development of effective and safe therapeutic agents to treat it. Snake venom is a complex mixture of bioactive proteins that represents an attractive source of novel and naturally-derived anticancer agents. Malaysia is one of the world's most biodiverse countries and is home to various venomous snake species, including cobras. Naja kaouthia , Naja sumatrana , and Ophiophagus hannah are three of the most common cobra species in Malaysia and are of medical importance. Over the past decades, snake venom has been identified as a potential source of therapeutic agents, including anti-cancer agents. This present review highlights the potential anticancer activity of the venom and purified venom protein of N. kaouthia, N. sumatrana , and O. hannah . In conclusion, this review highlights the important role of the venom from Malaysian cobras as an important resource that researchers can exploit to further investigate its potential in cancer treatment.

Snakebite is a global tropical disease that has long had huge implications for human health and well-being. Despite its long-standing medical importance, it has been the most neglected of tropical diseases. Reflective of this is that many aspects of the pathology have been underinvestigated. Snakebite by species in the Elapidae family is typically characterised by neurotoxic effects that result in flaccid paralysis. Thus, while clinically significant disturbances to the coagulation cascade have been reported, the bulk of the research to date has focused upon neurotoxins. In order to fill the knowledge gap regarding the coagulotoxic effects of elapid snake venoms, we screened 30 African and Asian venoms across eight genera using in vitro anticoagulant assays to determine the relative inhibition of the coagulation function of thrombin and the inhibition of the formation of the prothrombinase complex through competitive binding to a nonenzymatic site on Factor Xa (FXa), thereby preventing FXa from binding to Factor Va (FVa). It was revealed that African spitting cobras were the only species that were potent inhibitors of either clotting factor, but with Factor Xa inhibited at 12 times the levels of thrombin inhibition. This is consistent with at least one death on record due to hemorrhage following African spitting cobra envenomation. To determine the efficacy of antivenom in neutralising the anticoagulant venom effects, for the African spitting cobras we repeated the same 8-point dilution series with the addition of antivenom and observed the shift in the area under the curve, which revealed that the antivenom performed extremely poorly against the coagulotoxic venom effects of all species. However, additional tests with the phospholipase A₂ inhibitor LY315920 (trade name: varespladib) demonstrated a powerful neutralisation action against the coagulotoxic actions of the African spitting cobra venoms. Our research has important implications for the clinical treatment of cobra snakebites and also sheds light on the molecular mechanisms involved in coagulotoxicity within Naja . As the most coagulotoxic species are also those that produce characteristic extreme local tissue damage, future research should investigate potential synergistic actions between anticoagulant toxins and cytotoxins., Competing Interests: The authors declare no conflict of interest.