High-throughput virtual screening, pharmacophore modelling and antagonist effects of small molecule inhibitors against cytotoxin-induced cytotoxicity.

Cobra venom cytotoxins (CTX) cause dermonecrosis in envenomed patients who suffered from limb amputations due to the limitation of serotherapy-based antivenoms. This study aimed to identify small molecule inhibitors against CTX. A structure-based high-throughput virtual screening (HTVS) was conducted based on a conserved CTX, using the Natural Product Activity and Species Source (NPASS) screening library. The hits were valerenic acid, 1-oxo-2H-isoquinoline-4-carboxylic acid, acenaphthene, and 5-bromopyrrole-2-carboxamide, which interacted with contemporary antivenom binding site A and functional loops I-III of CTX, respectively, in molecular docking studies. Furthermore, molecular dynamic simulations were performed along with analysis of ligand fitness through their pharmacophore and pharmacokinetics properties. The antagonist effects of these hits on CTX-induced cytotoxicity were examined in human keratinocytes (HaCaT). Despite having a low binding affinity (KD = 14.45 × 10-4 M), acenaphthene demonstrated a significant increase of cell viability at 6 h and 24 h in experimental envenomed HaCaT. It also demonstrated the highest neutralization potency against CTX with a median effective concentration (EC50) of 0.05 mL/mg. Acenaphthene interacted with the functional loop II, which is the crucial cytotoxic site of CTX. It has an aromatic ring as its primary pharmacophoric feature, commonly used for rational drug design. In conclusion, acenaphthene could be a promising lead compound as a small molecule inhibitor.Communicated by Ramaswamy H. Sarma.

Unravelling the Gastroprotective Potential of Kefir: Exploring Antioxidant Effects in Preventing Gastric Ulcers.

The present study was conducted to evaluate the protective effect of milk kefir against NSAID-induced gastric ulcers. Male Swiss mice were divided into three groups: control (Vehicle; UHT milk at a dose of 0.3 mL/100 g), proton pump inhibitor (PPI; lansoprazole 30 mg/kg), and 4% milk kefir (Kefir; 0.3 mL/100 g). After 14 days of treatment, gastric ulcer was induced by oral administration of indomethacin (40 mg/kg). Reactive oxygen species (ROS), nitric oxide (NO), DNA content, cellular apoptosis, IL-10 and TNF-α levels, and myeloperoxidase (MPO) enzyme activity were determined. The interaction networks between NADPH oxidase 2 and kefir peptides 1-35 were determined using the Residue Interaction Network Generator (RING) webserver. Pretreatment with kefir for 14 days prevented gastric lesions. In addition, kefir administration reduced ROS production, DNA fragmentation, apoptosis, and TNF-α systemic levels. Simultaneously, kefir increased NO bioavailability in gastric cells and IL-10 systemic levels. A total of 35 kefir peptides showed affinity with NADPH oxidase 2. These findings suggest that the gastroprotective effect of kefir is due to its antioxidant and anti-inflammatory properties. Kefir could be a promising natural therapy for gastric ulcers, opening new perspectives for future research.

Unveiling the functional epitopes of cobra venom cytotoxin by immunoinformatics and epitope-omic analyses.

Approximate 70% of cobra venom is composed of cytotoxin (CTX), which is responsible for the dermonecrotic symptoms of cobra envenomation. However, CTX is generally low in immunogenicity, and the antivenom is ineffective in attenuating its in vivo toxicity. Furthermore, little is known about its epitope properties for empirical antivenom therapy. This study aimed to determine the epitope sequences of CTX using the immunoinformatic analyses and epitope-omics profiling. A conserved CTX was used in this study to determine its T-cell and B-cell epitope sequences using immunoinformatic tools and molecular docking simulation with different Human Leukocyte Antigens (HLAs). The potential T-cell and B-cell epitopes were 'KLVPLFY,' 'CPAGKNLCY,' 'MFMVSTPTK,' and 'DVCPKNSLL.' Molecular docking simulations disclosed that the HLA-B62 supertype exhibited the greatest binding affinity towards cobra venom cytotoxin. The namely L7, G18, K19, N20, M25, K33, V43, C44, K46, N47, and S48 of CTX exhibited prominent intermolecular interactions with HLA-B62. The multi-enzymatic-limited-digestion/liquid chromatography-mass spectrometry (MELD/LC-MS) also revealed three potential epitope sequences as 'LVPLFYK,' 'MFMVS,' and 'TVPVKR'. From different epitope mapping approaches, we concluded four potential epitope sites of CTX as 'KLVPLFYK', 'AGKNL', 'MFMVSTPKVPV' and 'DVCPKNSLL'. Site-directed mutagenesis of these epitopes confirmed their locations at the functional loops of CTX. These epitope sequences are crucial to CTX's structural folding and cytotoxicity. The results concluded the epitopes that resided within the functional loops constituted potential targets to fabricate synthetic epitopes for CTX-targeted antivenom production.

Selecting antibacterial aptamers against the BamA protein in Pseudomonas aeruginosa by incorporating genetic algorithm to optimise computational screening method.

Antibiotic resistance is one of the biggest threats to global health resulting in an increasing number of people suffering from severe illnesses or dying due to infections that were once easily curable with antibiotics. Pseudomonas aeruginosa is a major pathogen that has rapidly developed antibiotic resistance and WHO has categorised this pathogen under the critical list. DNA aptamers can act as a potential candidate for novel antimicrobial agents. In this study, we demonstrated that an existing aptamer is able to affect the growth of P. aeruginosa. A computational screen for aptamers that could bind to a well-conserved and essential outer membrane protein, BamA in Gram-negative bacteria was conducted. Molecular docking of about 100 functional DNA aptamers with BamA protein was performed via both local and global docking approaches. Additionally, genetic algorithm analysis was carried out to rank the aptamers based on their binding affinity. The top hits of aptamers with good binding to BamA protein were synthesised to investigate their in vitro antibacterial activity. Among all aptamers, Apt31, which is known to bind to an antitumor, Daunomycin, exhibited the highest HADDOCK score and resulted in a significant (p < 0.05) reduction in P. aeruginosa growth. Apt31 also induced membrane disruption that resulted in DNA leakage. Hence, computational screening may result in the identification of aptamers that bind to the desired active site with high affinity.

Ensemble-based molecular docking and spectrofluorometric analysis of interaction between cytotoxin and tumor necrosis factor receptor 1.

Cytotoxin (CTX) is a three-finger toxin presents predominantly in cobra venom. The functional site of the toxin is located at its three hydrophobic loop tips. Its actual mechanism of cytotoxicity remains inconclusive as few conflicting hypotheses have been proposed in addition to direct cytolytic effects. The present work investigated the interaction between CTX and death receptor families via ensemble-based molecular docking and fluorescence titration analysis. Multiple sequence alignments of different CTX isoforms obtained a conserved CTX sequence. The three-dimensional structure of the conserved CTX was later determined using homology modelling, and its quality was validated. Ensemble-based molecular docking of CTX was performed with different death receptors, such as Fas-ligand and tumor necrosis factor receptor families. Our results showed that tumor necrosis factor receptor 1 (TNFR1) was the best receptor interacting with CTX attributed to the interaction of all three functional loops and evinced with low HADDOCK, Z-score and RMSD value. The interaction between CTX and TNFR1 was also supported by a concentration-dependent reduction of fluorescence intensity with increasing binding affinity. The possible intermolecular interactions between CTX and TNFR1 were Van der Waals forces and hydrogen bonding. Our findings suggest a possibility that CTX triggers apoptosis cell death through non-covalent interactions with TNFR1.Communicated by Ramaswamy H. Sarma.

The antioxidant, wound healing properties and proteomic analysis of water extracts from the tropical cyanobacteria, Nostoc NIES-2111_MUM004.

Cyanobacteria bioactive compounds are chemical treasure troves for product discovery and development. The wound healing effects and antioxidant capacities of water extracts from Nostoc NIES-2111_MUM004 were evaluated via in vitro wound scratch assay and three antioxidant assays respectively. Results showed that the water extracts were protein-rich and exhibited good antioxidant properties in ABTS radical scavenging (11.27 ± 0.205 mg TAE g-1 extract), Ferric reducing antioxidant power (1652.71 ± 110.71 mg TAE g-1 extract) and ß-carotene bleaching assay (354.90 ± 31.80 mg TAE g-1 extract). Also, extracts were non-cytotoxic in concentrations up to 250 µg/mL as reflected in cytotoxicity assay. Importantly, water extracts showed considerable proliferation and migration activity at 125 µg/mL with wound closure rate as high as 42.67%. Statistical correlation revealed no significant relationship (p > 0.05) between protein fraction and the wound healing properties, confirming that phycobiliproteins were not solely responsible for wound healing activities. Subsequent Q-TOF-LCMS analysis identified six protein families involved in enhancing the proliferation and migration of epithelial cells. These findings are antecedent in the uncovering of continuous supplies of bioactive compounds from new and sustainable sources. Ultimately, enriching the microalgae menu for applications in pharmaceutical, nutraceutical and cosmeceuticals.

A digital module-based experiential learning in protein biochemistry during the COVID-19 pandemic paradigm.

Experiential learning is compromised in meeting the educational demands of our students during the challenging time of the COVID-19 pandemic. A more inclusive, flexible, and objective-oriented experiential learning environment is required. In this context, module-based experiential learning that is executable on a digital platform was designed. The learning module focused on protein biochemistry, contained a combination of asynchronous and synchronous activities categorized into 'Knowledge Hub' and 'Lab-based Movie', across 5 weeks. Digital and module-based experiential learning provides equitable, inclusive, and flexible access to students at remote locations. Furthermore, it is an objective-oriented and highly organized experiential learning framework that encourages students to engage and participate more in the learning process.

The myth of cobra venom cytotoxin: More than just direct cytolytic actions.

Cobra venom cytotoxin (CTX) is a non-enzymatic three-finger toxin that constitutes 40-60% of cobra venom. Thus, it plays an important role in the pathophysiology of cobra envenomation, especially in local dermonecrosis. The three-finger hydrophobic loops of CTX determine the cytotoxicity. Nevertheless, the actual mechanisms of cytotoxicity are not fully elucidated as they involve not only cytolytic actions but also intracellular signalling-mediated cell death pathways. Furthermore, the possible transition cell death pattern remains to be explored. The actual molecular mechanisms require further studies to unveil the relationship between different CTXs from different cobra species and cell types which may result in differential cell death patterns. Here, we discuss the biophysical interaction of CTX with the cell membrane involving four binding modes: electrostatic interaction, hydrophobic partitioning, isotropic phase, and oligomerisation. Oligomerisation of CTX causes pore formation in the membrane lipid bilayer. Additionally, the CTX-induced apoptotic pathway can be executed via death receptor-mediated extrinsic pathways and mitochondrial-mediated intrinsic pathways. We also discuss lysosomal-mediated necrosis and the occurrence of necroptosis following CTX action. Collectively, we provided an insight into concentration-dependent transition of cell death pattern which involves different mechanistic actions. This contributes a new direction for further investigation of cytotoxic pathways activated by the CTXs for future development of biotherapeutics targeting pathological effects caused by CTX.

The effects of Naja sumatrana venom cytotoxin, sumaCTX on alteration of the secretome in MCF-7 breast cancer cells following membrane permeabilization.

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.

The influence of spouses and their driving roles in self-regulation: A qualitative exploration of driving reduction and cessation practices amongst married older adults.

INTRODUCTION: There is growing evidence to suggest the importance of self-regulatory practices amongst older adults to sustain mobility. However, the decision to self-regulate driving is a complex interplay between an individual's preference and the influence of their social networks including spouse. To our best knowledge, the influence of an older adult's spouse on their decisions during driving transition has not been explored. MATERIALS AND METHODS: This qualitative descriptive study was conducted amongst married older adults aged 60 years and above. All interview responses were transcribed verbatim and examined using thematic approach and interpretative description method. RESULTS: A total of 11 married couples were interviewed. Three major themes emerged: [1] Our roles in driving; [2] Challenges to continue driving; and, [3] Our driving strategies to ensure continued driving. Older couples adopted driving strategies and regulated their driving patterns to ensure they continued to drive safely. Male partners often took the active driving role as the principal drivers, while the females adopted a more passive role, including being the passenger to accompany the principal drivers or becoming the co-driver to help in navigation. Other coping strategies include sharing the driving duties as well as using public transportation or mixed mode transportation. DISCUSSION: Our findings suggest spouse play a significant role in their partners' decision to self-regulate driving. This underscores a need to recognise the importance of interdependency amongst couples and its impact on their driving decisions and outcomes.

Cytotoxicity of snake venom enzymatic toxins: phospholipase A2 and l-amino acid oxidase.

The phospholipase A2 (PLA2) and l-amino acid oxidase (LAAO) are two major enzymes found in the venoms from most snake species. These enzymes have been structurally and functionally characterised for their pharmacological activities. Both PLA2 and LAAO from different venoms demonstrate considerable cytotoxic effects on cancer cells via induction of apoptosis, cell cycle arrest and suppression of proliferation. These enzymes produce more pronounced cytotoxic effects in cancer cells than normal cells, thus they can be potential sources as chemotherapeutic agents. It is proposed that PLA2 and LAAO contribute to an elevated oxidative stress due to their catalytic actions, for instance, the ability of PLA2 to produce reactive oxygen species during lipolysis and formation of H2O2 from LAAO catalytic activity which consequently lead to cell death. Nonetheless, the cell-death signalling pathways associated with exposure to these enzymatic toxins are not fully elucidated yet. Here in this review, we will discuss the cytotoxic effects of PLA2 and LAAO in relationship to their catalytic mechanisms and the underlying mechanisms of cytotoxic actions.

Arctium lappa L. root extract induces cell death via mitochondrial-mediated caspase-dependent apoptosis in Jurkat human leukemic T cells.

Arctium lappa L. is a perennial herb traditionally consumed to improve well-being. It has been widely reported for its antioxidant properties; however, very little is known for its exact mechanisms underlying the anticancer activity. This study aimed to investigate the mechanisms of anticancer action for different A. lappa root extracts. Arctium lappa root was extracted with ethanol, hexane and ethyl acetate, then examined for in vitro anticancer activity against cancerous HeLa, MCF-7, Jurkat cell lines and non-cancerous 3T3 cell lines. Induction of apoptosis was determined by cellular morphological changes, mitochondrial membrane potential (ΔΨm), caspase-3/7 activity and DNA fragmentation. The active compounds present in the most potent root extracts were identified by LC-ESI-MS. Among all the extracts, ethyl acetate root extract has the highest potency with IC50 of 102.2 ± 42.4 µg/ml, followed by ethanolic root extract in Jurkat T cells, at 24 h. None of the extracts were cytotoxic against 3T3 cells, suggesting that the extracts were selective against cancerous cells only. Both ethyl acetate and ethanolic root extracts exhibited significant morphological changes in Jurkat T cells, including the detachment from adjacent cells, appearance of apoptotic bodies and cells shrinkage. The extracts treated cells also displayed an increase in caspase-3/7 activity and alteration in mitochondrial membrane potential. Only ethyl acetate root extract at IC50 induced DNA fragmentation in Jurkat T cells. LC-ESI-MS analysis of the extract revealed the presence of 8 compounds, of which only 6 compounds with various biological activities reported. These findings suggest that the ethyl acetate extract of A. lappa had strong anticancer potential and induced intrinsic apoptosis via loss of ΔΨm and activation of caspase-3/7 This study can provide new insight to the discovery of new promising lead compound in chemopreventive and chemotherapeutic strategies.

Exendin-4 from Heloderma suspectum venom: From discovery to its latest application as type II diabetes combatant.

Type II diabetes mellitus (T2DM) is a chronic non-communicable disease due to abnormal insulin actions causing uncontrolled hyperglycaemia. The treatment for T2DM, for instance, metformin and incretin mimetic, mainly focuses on the restoration of insulin sensitivity and secretion. Exendin-4 is a short incretin-mimetic peptide consisting of 39 amino acids. It is discovered in the venom of Heloderma suspectum as a full agonist for the glucagon-like peptide 1 (GLP-1) receptor and produces insulinotropic effects. It is more resistant to enzymatic degradation by dipeptidyl-peptidase-4 and has a longer half-life than the endogenous GLP-1; thus, it is further developed as an incretin hormone analogue used to treat T2DM. The helical region of the peptide first interacts with the extracellular N-terminal domain (NTD) of GLP-1 receptor while the C-terminal extension containing the tryptophan cage further enhances its binding affinity. After binding to the NTD of the receptor, it may cause the receptor to switch from its auto-inhibited state of the receptor to its auto-activated state. Exendin-4 enhances the physiological functions of ß-cells and the up-regulation of GLP-1 receptors, thus reducing the plasma glucose levels. Moreover, exendin-4 has also been found to ameliorate neuropathy, nephropathy and ventricular remodelling. The therapeutic effects of exendin-4 have also been extrapolated into several clinical trials. Although exendin-4 has a reasonable subcutaneous bioavailability, its half-life is rather short. Therefore, several modifications have been undertaken to improve its pharmacokinetics and insulinotropic potency. This review focuses on the pharmacology of exendin-4 and the structure-function relationships of exendin-4 with GLP-1 receptor. The review also highlights some challenges and future directions in the improvement of exendin-4 as an anti-diabetic drug.

Venomics of Tropidolaemus wagleri, the sexually dimorphic temple pit viper: Unveiling a deeply conserved atypical toxin arsenal.

Tropidolaemus wagleri (temple pit viper) is a medically important snake in Southeast Asia. It displays distinct sexual dimorphism and prey specificity, however its venomics and inter-sex venom variation have not been thoroughly investigated. Applying reverse-phase HPLC, we demonstrated that the venom profiles were not significantly affected by sex and geographical locality (Peninsular Malaya, insular Penang, insular Sumatra) of the snakes. Essentially, venoms of both sexes share comparable intravenous median lethal dose (LD50) (0.56-0.63 µg/g) and cause neurotoxic envenomation in mice. LCMS/MS identified six waglerin forms as the predominant lethal principles, comprising 38.2% of total venom proteins. Fourteen other toxin-protein families identified include phospholipase A2, serine proteinase, snaclec and metalloproteinase. In mice, HPLC fractions containing these proteins showed insignificant contribution to the overall venom lethality. Besides, the unique elution pattern of approximately 34.5% of non-lethal, low molecular mass proteins (3-5 kDa) on HPLC could be potential biomarker for this primitive crotalid species. Together, the study unveiled the venom proteome of T. wagleri that is atypical among many pit vipers as it comprises abundant neurotoxic peptides (waglerins) but little hemotoxic proteinases. The findings also revealed that the venom is relatively well conserved intraspecifically despite the drastic morphological differences between sexes.

Unveiling the elusive and exotic: Venomics of the Malayan blue coral snake (Calliophis bivirgata flaviceps).

The venom proteome of the Malayan blue coral snake, Calliophis bivirgata flaviceps from west Malaysia was investigated by 1D-SDS-PAGE and shotgun-LCMS/MS. A total of 23 proteins belonging to 11 protein families were detected from the venom proteome. For the toxin proteins, the venom consists mainly of phospholipase A2 (41.1%), cytotoxin (22.6%), SVMPs (18.7%) and vespryns (14.6%). However, in contrast to the venoms of New World coral snakes and most elapids, there was no post-synaptic α-neurotoxin detected. The proteome also revealed a relatively high level of phosphodiesterase (1.3%), which may be associated with the reported high level of adenosine in the venom. Also detected were 5'-nucleotidase (0.3%), hyaluronidase (0.1%) and cysteine-type endopeptide inhibitor (0.6%). Enzymatic studies confirmed the presence of phospholipase A2, phosphodiesterase, 5'-nucleotidase and acetylcholinesterase activities but not l-amino acid oxidase activity. The venom exhibited moderate cytotoxic activity against CRL-2648 fibroblast cell lines (IC50=62.14±0.87 µg/mL) and myotoxicity in mice, presumably due to the action of its cytotoxin or its synergistic action with phospholipase A2. Interestingly, the venom lethality could be cross-neutralized by a neurotoxic bivalent antivenom from Taiwan. Together, the findings provide insights into the composition and functions of the venom of this exotic oriental elapid snake. BIOLOGICAL SIGNIFICANCE: While venoms of the New World coral snake have been extensively studied, literature pertaining to the Old World or Asiatic coral snake venoms remains lacking. This could be partly due to the inaccessibility to the venom of this rare species and infrequent cases of envenomation reported. This study identified and profiled the venom proteome of the Malayan blue coral snake (C. b. flaviceps) through SDS-PAGE and a high-resolution nano-LCMS/MS method, detailing the types and abundance of proteins found in the venom. The biological and toxic activities of the venom were also investigated, offering functional correlation to the venom proteome studied. Of note, the venom contains a unique toxin profile predominated with phospholipase A2 and cytotoxin with no detectable post-synaptic neurotoxin. The venom is moderately lethal to mice and the fatal effect could be cross-neutralized by a heterologous elapid bivalent antivenom from Taiwan. The findings enrich snake toxin databases and provide insights into the composition and pathogenesis of the venom of this exotic species.

Functional venomics of the Sri Lankan Russell's viper (Daboia russelii) and its toxinological correlations.

UNLABELLED: The venom proteome (venomics) of the Sri Lankan Daboia russelii was elucidated using 1D SDS PAGE nano-ESI-LCMS/MS shotgun proteomics. A total of 41 different venom proteins belonging to 11 different protein families were identified. The four main protein families are phospholipase A2 (PLA2, 35.0%), snaclec (SCL, 22.4%, mainly platelet aggregation inhibitors), snake venom serine proteinase (SVSP, 16.0%, mainly Factor V activating enzyme) and snake venom metalloproteinase (SVMP, 6.9%, mainly heavy chain of Factor X activating enzyme). Other protein families that account for more than 1% of the venom protein include l-amino acid oxidase (LAAO, 5.2%), Kunitz-type serine proteinase inhibitor (KSPI, 4.6%), venom nerve growth factor (VNGF. 3.5%), 5'-nucleotidase (5'NUC, 3.0%), cysteine-rich secretory protein (CRISP, 2.0%) and phosphodiesterase (PDE, 1.3%). The venom proteome is consistent with the enzymatic and toxic activities of the venom, and it correlates with the clinical manifestations of Sri Lankan D. russelii envenomation which include hemorrhage, coagulopathy, renal failure, neuro-myotoxicity and intravascular hemolysis. The venom exhibited remarkable presypnatic neurotoxicity presumably due to the action of basic PLA2 in high abundance (35.0%). Besides, SCLs, Factor X activating enzymes (SVMPs), SVSPs, and LAAOs are potential hemotoxins (50.5%), contributing to coagulopathy and hemorrhagic syndrome in Sri Lankan D. russelii envenomation. SIGNIFICANCE: The study demonstrated the proteomic profile of the Sri Lankan Russell's viper venom, unraveling its complex composition of toxins and correlations with major toxic activities. The types, numbers, and relative abundances of toxins were reported. The venom content was dominated by the neurotoxic basic phospholipases A2 (>30% of total protein abundance) and several hemotoxic or coagulopathic protein families (approximately 50% in total). The proteome correlates with the functional and toxinological characterizations of the venom, and reflects the pathophysiological effects of envenomation by the Sri Lankan Russell's viper. The venom proteomics may serve to propel the understanding on pathogenesis and treatment strategy for envenomation by this viper in Sri Lanka. The enriched database contributed by the proteomic findings will be useful for comparing venom variations among Russell's vipers from different geographical areas.

The Effect of a Polyvalent Antivenom on the Serum Venom Antigen Levels of Naja sputatrix (Javan Spitting Cobra) Venom in Experimentally Envenomed Rabbits.

The treatment protocol of antivenom in snake envenomation remains largely empirical, partly due to the insufficient knowledge of the pharmacokinetics of snake venoms and the effects of antivenoms on the blood venom levels in victims. In this study, we investigated the effect of a polyvalent antivenom on the serum venom antigen levels of Naja sputatrix (Javan spitting cobra) venom in experimentally envenomed rabbits. Intravenous infusion of 4 ml of Neuro Polyvalent Snake Antivenom [NPAV, F(ab')2 ] at 1 hr after envenomation caused a sharp decline of the serum venom antigen levels, followed by transient resurgence an hour later. The venom antigen resurgence was unlikely to be due to the mismatch of pharmacokinetics between the F(ab')2 and venom antigens, as the terminal half-life and volume of distribution of the F(ab')2 in serum were comparable to that of venom antigens (p > 0.05). Infusion of an additional 2 ml of NPAV was able to prevent resurgence of the serum venom antigen level, resulting in a substantial decrease (67.1%) of the total amount of circulating venom antigens over time course of envenomation. Our results showed that the neutralization potency of NPAV determined by neutralization assay in mice may not be an adequate indicator of its capability to modulate venom kinetics in relation to its in vivo efficacy to neutralize venom toxicity. The findings also support the recommendation of giving high initial dose of NPAV in cobra envenomation, with repeated doses as clinically indicated in the presence of rebound antigenemia and symptom recurrence.

Pharmacokinetics of Naja sumatrana (equatorial spitting cobra) venom and its major toxins in experimentally envenomed rabbits.

BACKGROUND: The optimization of snakebite management and the use of antivenom depend greatly on the knowledge of the venom's composition as well as its pharmacokinetics. To date, however, pharmacokinetic reports on cobra venoms and their toxins are still relatively limited. In the present study, we investigated the pharmacokinetics of Naja sumatrana (Equatorial spitting cobra) venom and its major toxins (phospholipase A2, neurotoxin and cardiotoxin), following intravenous and intramuscular administration into rabbits. PRINCIPAL FINDINGS: The serum antigen concentration-time profile of the N. sumatrana venom and its major toxins injected intravenously fitted a two-compartment model of pharmacokinetics. The systemic clearance (91.3 ml/h), terminal phase half-life (13.6 h) and systemic bioavailability (41.9%) of N. sumatrana venom injected intramuscularly were similar to those of N. sputatrix venom determined in an earlier study. The venom neurotoxin and cardiotoxin reached their peak concentrations within 30 min following intramuscular injection, relatively faster than the phospholipase A2 and whole venom (Tmax=2 h and 1 h, respectively). Rapid absorption of the neurotoxin and cardiotoxin from the injection site into systemic circulation indicates fast onsets of action of these principal toxins that are responsible for the early systemic manifestation of envenoming. The more prominent role of the neurotoxin in N. sumatrana systemic envenoming is further supported by its significantly higher intramuscular bioavailability (Fi.m.=81.5%) compared to that of the phospholipase A2 (Fi.m.=68.6%) or cardiotoxin (Fi.m.=45.6%). The incomplete absorption of the phospholipase A2 and cardiotoxin may infer the toxins' affinities for tissues at the injection site and their pathological roles in local tissue damages through synergistic interactions. CONCLUSION/SIGNIFICANCE: Our results suggest that the venom neurotoxin is absorbed very rapidly and has the highest bioavailability following intramuscular injection, supporting its role as the principal toxin in systemic envenoming.

Proteomic characterization of venom of the medically important Southeast Asian Naja sumatrana (Equatorial spitting cobra).

The proteome of Naja sumatrana (Equatorial spitting cobra) venom was investigated by shotgun analysis and a combination of ion-exchange chromatography and reverse phase HPLC. Shotgun analysis revealed the presence of 39 proteins in the venom while the chromatographic approach identified 37 venom proteins. The results indicated that, like other Asiatic cobra venoms, N. sumatrana contains large number of three finger toxins and phospholipases A2, which together constitute 92.1% by weight of venom protein. However, only eight of the toxins can be considered as major venom toxins. These include two phospholipases A2, three neurotoxins (two long neurotoxins and a short neurotoxin) and three cardiotoxins. The eight major toxins have relative abundance of 1.6-27.2% venom proteins and together account for 89.8% (by weight) of total venom protein. Other venom proteins identified include Zn-metalloproteinase-disintegrin, Thaicobrin, CRISP, natriuretic peptide, complement depleting factors, cobra venom factors, venom nerve growth factor and cobra serum albumin. The proteome of N. sumatrana venom is similar to proteome of other Asiatic cobra venoms but differs from that of African spitting cobra venom. Our results confirm that the main toxic action of N. sumatrana venom is neurotoxic but the large amount of cardiotoxins and phospholipases A2 are likely to contribute significantly to the overall pathophysiological action of the venom. The differences in toxin distribution between N. sumatrana venom and African spitting cobra venoms suggest possible differences in the pathophysiological actions of N. sumatrana venom and the African spitting cobra venoms, and explain why antivenom raised against Asiatic cobra venom is not effective against African spitting cobra venoms.