Phytomedicine for neurodegenerative diseases: The road ahead.

Neurodegenerative disorders (NDs) are among the most common causes of death across the globe. NDs are characterized by progressive damage to CNS neurons, leading to defects in specific brain functions such as memory, cognition, and movement. The most common NDs are Parkinson's, Alzheimer's, Huntington's, and amyotrophic lateral sclerosis (ALS). Despite extensive research, no therapeutics or medications against NDs have been proven to be effective. The current treatment of NDs involving symptom-based targeting of the disease pathogenesis has certain limitations, such as drug resistance, adverse side effects, poor blood-brain barrier permeability, and poor bioavailability of drugs. Some studies have shown that plant-derived natural compounds hold tremendous promise for treating and preventing NDs. Therefore, the primary objective of this review article is to critically analyze the properties and potency of some of the most studied phytomedicines, such as quercetin, curcumin, epigallocatechin gallate (EGCG), apigenin, and cannabinoids, and highlight their advantages and limitations for developing next-generation alternative treatments against NDs. Further extensive research on pre-clinical and clinical studies for developing plant-based drugs against NDs from bench to bedside is warranted.

A Correlation Study to Comprehend the SAR-COV-2 Viral Load, Antiviral Antibody Titer, and Severity of COVID-19 Symptoms Post-infection Amongst the Vaccinated Population in Kamrup District of Assam, Northeast India.

BACKGROUND: As per the recommendation of the United States Food and Drug Administration, more research is needed to determine the antibody titer against COVID-19 vaccination. OBJECTIVE: The study aimed to understand the relationship between the antibody titer to the demographics, infection severity, and cycle threshold (CT) values of confirmed COVID-19 patients. METHODS: Initially, we obtained consent from 185 populations and included sixty RT-PCRpositive COVID-19 patients from Kamrup District in the Northeast State of Assam, India. The vaccination status was recorded and tested for the level of serum immunoglobulin (IgG). The CT values, gender, and clinical symptoms-based scoring (CSBS) correlated with their IgG value. RESULTS: Around 48% of participants gained an antibody titer more than the threshold value and showed CT values between 18-25. Moreover, the maximum distributed score above the average was found between the CT values 18-25. CONCLUSION: The IgG titer value differs significantly amongst the vaccinated population, which may depend upon their genetic and demographic variability.

Therapeutic Potential of Bioactive Compounds from Edible Mushrooms to Attenuate SARS-CoV-2 Infection and Some Complications of Coronavirus Disease (COVID-19).

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a highly infectious positive RNA virus, has spread from its epicenter to other countries with increased mortality and morbidity. Its expansion has hampered humankind's social, economic, and health realms to a large extent. Globally, investigations are underway to understand the complex pathophysiology of coronavirus disease (COVID-19) induced by SARS-CoV-2. Though numerous therapeutic strategies have been introduced to combat COVID-19, none are fully proven or comprehensive, as several key issues and challenges remain unresolved. At present, natural products have gained significant momentum in treating metabolic disorders. Mushrooms have often proved to be the precursor of various therapeutic molecules or drug prototypes. The plentiful bioactive macromolecules in edible mushrooms, like polysaccharides, proteins, and other secondary metabolites (such as flavonoids, polyphenols, etc.), have been used to treat multiple diseases, including viral infections, by traditional healers and the medical fraternity. Some edible mushrooms with a high proportion of therapeutic molecules are known as medicinal mushrooms. In this review, an attempt has been made to highlight the exploration of bioactive molecules in mushrooms to combat the various pathophysiological complications of COVID-19. This review presents an in-depth and critical analysis of the current therapies against COVID-19 versus the potential of natural anti-infective, antiviral, anti-inflammatory, and antithrombotic products derived from a wide range of easily sourced mushrooms and their bioactive molecules.

A Novel Therapeutic Formulation for the Improved Treatment of Indian Red Scorpion (Mesobuthus tamulus) Venom-Induced Toxicity-Tested in Caenorhabditis elegans and Rodent Models.

Indian Red Scorpion (Mesobuthus tamulus) stings are a neglected public health problem in tropical and sub-tropical countries, including India. The drawbacks of conventional therapies using commercial anti-scorpion antivenom (ASA) and α1-adrenoreceptor antagonists (AAA) have prompted us to search for an adequate formulation to improve treatment against M. tamulus stings. Novel therapeutic drug formulations (TDF) of low doses of commercial ASA, AAA, and ascorbic acid have remarkably improved in neutralising the in vivo toxic effects of M. tamulus venom (MTV) tested in Caenorhabditis elegans and Wistar strain albino rats in vivo models. The neutralisation of MTV-induced production of free radicals, alteration of the mitochondrial transmembrane potential, and upregulated expression of genes involved in apoptosis, detoxification, and stress response in C. elegans by TDF surpassed the same effect shown by individual components of the TDF. Further, TDF efficiently neutralized the MTV-induced increase in blood glucose level within 30 to 60 min post-treatment, organ tissue damage, necrosis, and pulmonary oedema in Wistar rats, indicating its clinical application for effecting treating M. tamulus envenomation. This study demonstrates for the first time that C. elegans can be a model organism for screening the neutralization potency of the drug molecules against a neurotoxic scorpion venom.

A retrospective analysis of epidemiology, clinical features of envenomation, and in-patient management of snakebites in a model secondary hospital of Assam, North-east India.

Assam, a Northeastern State of India, is inhabited by several venomous snake species causing substantial morbidity and mortality. The data on the epidemiology of snakebites and their management is underreported in this region. Hence, a secondary health-based retrospective study was carried out at Demow Model Hospital, Sivasagar, Assam, to evaluate the clinical and epidemiological profile of snakebite cases reported in this rural hospital and their management. Snakebites occurring between April 2018 to August 2022 were reviewed based on socio-demographic details of the patient, clinical symptoms, and treatment using a standard questionnaire. Out of the 1011 registered snakebite cases, 139 patients (13.7%) counted for venomous bites, among which 92 patients (66.19%) accounted for viper bites (green pit viper and Salazar's pit viper), and 30 patients (21.5%) were bitten by elapid snakes (Indian monocled Cobra, banded krait, and greater/lesser black krait). A maximum number of snakebite cases (80.5%) were reported from the interior rural villages and documented from July to September (51.3%). Elapid snake envenomed patients, except one, were successfully treated with commercial antivenom, neostigmine, and glycopyrrolate. Because commercial polyvalent antivenom against "Big Four" venomous snakes of India showed poor neutralization of pit-vipers envenomation; therefore, pit-viper bite patients were treated with repurposed drugs magnesium sulfate and glycerin compression dressing. Adverse serum reactions were reported only in 3 (11.1%) cases. The preventive measures and facilities adopted at the Demow Model Hospital significantly reduce snakebite death and morbidity; therefore, they can be s practised across various states in India as a prototype.

Computational and in vitro analyses to identify the anticoagulant regions of Echicetin, a snake venom anticoagulant C-type lectin (snaclec): possibility to develop anticoagulant peptide therapeutics?

Snake venom C-type lectins (Snaclecs) display anticoagulant and platelet-modulating activities; however, their interaction with the critical components of blood coagulation factors was unknown. Computational analysis revealed that Echicetin (Snaclec from Echis carinatus venom) interacted with heavy chain of thrombin, and heavy and light chains of factor Xa (FXa). Based on FXa and thrombin binding regions of Echicetin, the two synthetic peptides (1A and 1B) were designed. The in silico binding studies of the peptides with thrombin and FXa showed that peptide 1B interacted with both heavy and light chains of thrombin and, peptide 1A interacted with only heavy chain of thrombin. Similarly, peptide 1B interacted with both heavy and light chains of FXa; however, peptide 1A interacted only with heavy chain of FXa. Alanine screening predicted the hot-spots residues for peptide 1A (Aspartic acid6, Valine8, Valine9, and Tyrosine17 with FXa, and Isoleucine14, Lysine15 with thrombin) and peptide 1B (Valine16 with FXa). Spectrofluorometric interaction study showed a lower Kd value for peptide 1B binding with both FXa and thrombin than peptide 1A, indicating higher binding strength of the former peptide. The circular dichroism spectroscopy also established the interaction between thrombin and the custom peptides. The in vitro study demonstrated higher anticoagulant activity of peptide 1B than peptide 1A due to higher inhibition of thrombin and FXa. Inhibition of anticoagulant activity of the peptides by respective anti-peptide antibodies corroborates our hypothesis that peptides 1A and 1B represent the anticoagulant regions of Echicetin and may be developed as antithrombotic peptide drug prototypes.Communicated by Ramaswamy H. Sarma.

Snake venom nerve growth factor-inspired designing of novel peptide therapeutics for the prevention of paraquat-induced apoptosis, neurodegeneration, and alteration of metabolic pathway genes in the rat pheochromocytoma PC-12 cell.

Neurodegenerative disorders (ND), associated with the progressive loss of neurons, oxidative stress-mediated production of reactive oxygen species (ROS), and mitochondrial dysfunction, can be treated with synthetic peptides possessing innate neurotrophic effects and neuroprotective activity. Computational analysis of two small synthetic peptides (trideca-neuropeptide, TNP; heptadeca-neuropeptide, HNP) developed from the nerve growth factors from snake venoms predicted their significant interaction with the human TrkA receptor (TrkA). In silico results were validated by an in vitro binding study of the FITC-conjugated custom peptides to rat pheochromocytoma PC-12 cell TrkA receptors. Pre-treatment of PC-12 cells with TNP and HNP induced neuritogenesis and significantly reduced the paraquat (PT)-induced cellular toxicity, the release of lactate dehydrogenase from the cell cytoplasm, production of intracellular ROS, restored the level of antioxidants, prevented alteration of mitochondrial transmembrane potential (ΔΨm) and adenosine triphosphate (ATP) production, and inhibited cellular apoptosis. These peptides lack in vitro cytotoxicity, haemolytic activity, and platelet-modulating properties and do not interfere with the blood coagulation system. Functional proteomic analyses demonstrated the reversal of PT-induced upregulated and downregulated metabolic pathway genes in PC-12 cells that were pre-treated with HNP and revealed the metabolic pathways regulated by HNP to induce neuritogenesis and confer protection against PT-induced neuronal damage in PC-12. The quantitative RT-PCR analysis confirmed that the PT-induced increased and decreased expression of critical pro-apoptotic and anti-apoptotic genes had been restored in the PC-12 cells pre-treated with the custom peptides. A network gene expression profile was proposed to elucidate the molecular interactions among the regulatory proteins for HNP to salvage the PT-induced damage. Taken together, our results show how the peptides can rescue PT-induced oxidative stress, mitochondrial dysfunction, and cellular death and suggest new opportunities for developing neuroprotective drugs.

Mushroom mycetism - A neglected and challenging medical emergency in the Indian subcontinent: A road map for its prevention and treatment.

Edible mushrooms, a class of macroscopic fungi, serve as delicious and nutritious food supplements around the world. Nevertheless, accidental consumption of poisonous mushrooms that results in fatality or severe illness is typical in all countries, especially among the tribal indigenous communities that forage wild mushrooms for food. In the Indian subcontinent, mushroom poisoning cases are underreported and neglected. Different classes of toxins, characterized from the poisonous mushrooms found globally, show variable clinical symptoms post-consumption. Although the Indian subcontinent is a biodiversity hotspot and home to different classes of fungi and mushrooms, many species of poisonous mushrooms and their toxins, have yet to be identified and characterized. No epidemiological studies or retrospective analyses of mushroom poisoning cases have been reported from the poison control centers in the Indian subcontinent. Nevertheless, some limited clinical and epidemiological data is available from India and Nepal, and therefore, we critically analyse the mushroom poisoning scenario in these countries, and discuss the mushroom toxins that are likely responsible for the post-ingestion toxicities. We also correlate the clinical manifestations of mushroom intoxication in India and Nepal with the pharmacological properties of the prevalent mushroom toxins in these countries. Our limited study of mushroom poisoning demonstrates that the adverse pharmacological effects of amatoxin, one of the deadliest mushroom toxins, are responsible for the highest mortality and morbidity in India and Nepal. Further, no specific antidote is available to treat mushroom intoxication in the region, and systemic and supportive care is all that is available for in-patient management of cases of severe poisoning. We also suggest a roadmap for the prevention and specific treatment against mushroom poisoning in the Indian subcontinent.

In vitro laboratory analyses of commercial anti-scorpion (Mesobuthus tamulus) antivenoms reveal their quality and safety but the prevalence of a low proportion of venom-specific antibodies.

Mesobuthus tamulus (Indian Red Scorpion) sting is a severe but neglected health issue in India. The accomplishment of in-patient scorpion sting management is highly dependent on the safety, efficacy, and homogeneity of scorpion antivenom preparation. Therefore, in this study, the above qualities of commercial anti-scorpion antivenoms manufactured in India were assessed by in vitro laboratory analyses. Biophysical characterization of venom by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, size exclusion chromatography, and proteomics analysis demonstrated that anti-scorpion antivenoms (ASAs) mostly contain F(ab')2 molecules with a trace amount of undigested immunoglobulin (Ig) G. The physicochemical characterization, electron microscopy, and dynamic light scattering studies revealed that ASAs were prepared according to the guidelines of World Health Organization (WHO), and were devoid of aggregate content and virus particles. ASAs did not show IgE contamination and bacterial endotoxin but demonstrated moderate complement activation properties, which may have adverse effects in treated patients. Spectrofluorometric and atomic force microscopy analyses showed poor binding of venom with commercial ASAs. The percent of antibodies raised against the venom toxins in commercial ASAs was determined at the range of 5.3-6.3%, which is a reason for their poor efficacy. This study advocates the importance of in vitro laboratory analyses for assessing commercial antivenom's quality and safety parameters before their pre-clinical research and clinical use to treat Indian red scorpion sting.

A comparison of two different analytical workflows to determine the venom proteome composition of Naja kaouthia from North-East India and immunological profiling of venom against commercial antivenoms.

The Indian monocled cobra (Naja kaouthia) is one of the most prevalent venomous snakes in northeast India (NEI) and is the cause of many fatalities. The composition of NEI N. kaouthia venom (NkV) was deciphered using two different proteomic approaches: (i) 1D SDS-PAGE coupled to label-free quantification of protein bands using stringent identification criteria and (ii) reversed-phase high-performance liquid chromatography (RP-HPLC) followed by quantification based on area under the RP-HPLC peaks. The proteomic data from both strategies were compared. Proteomic analyses from both workflows identified 32 proteins (toxins) distributed over 10-14 snake venom protein families in NEI NkV. The relative abundances of the venom proteins determined from the analytical workflows coincided with the densitometry band intensities of the NEI NkV. Phospholipase A2 (13.1-16.0%) and three-finger toxins (58.5-64.2%) represented the most abundant enzymatic and non-enzymatic proteins in NEI NkV, respectively. Immuno-cross-reactivity studies by enzyme-linked immunoassay and immunoblot analyses pointed to the poor efficacy of commercial PAVs in recognizing the low molecular mass (<15 kDa) toxins of NEI NkV. Spectrofluorometric titration determined the presence of NEI NkV-specific antibodies in commercial PAV, at a level that was higher than that previously reported for eastern India NkV-specific antibodies in commercial antivenom.

Assessment of quality and pre-clinical efficacy of a newly developed polyvalent antivenom against the medically important snakes of Sri Lanka.

Snake envenomation is a severe problem in Sri Lanka (SL) and Indian polyvalent antivenom (PAV) is mostly used for treating snakebite albeit due to geographical variation in venom composition, Indian PAV shows poor efficacy in neutralizing the lethality and toxicity of venom from the same species of snakes in SL. Therefore, the quality and in vivo venom neutralization potency of a country-specific PAV produced against the venom of the five most medically important snakes of SL (Daboia russelii, Echis carinatus, Hypnale hypnale, Naja naja, Bungarus caeruleus) was assessed. LC-MS/MS analysis of two batches of PAV showed the presence of 88.7-97.2% IgG and traces of other plasma proteins. The tested PAVs contained minor amounts of undigested IgG and F(ab')2 aggregates, showed complement activation, were devoid of IgE, endotoxin, and content of preservative was below the threshold level. Immunological cross-reactivity and in vitro neutralization of enzymatic activities, pharmacological properties demonstrated superior efficacy of SL PAV compared to Indian PAV against SL snake venoms. The in vivo neutralization study showed that the tested PAVs are potent to neutralize the lethality and venom-induced toxicity of SL snake venoms. Therefore, our study suggests that introduction of SL-specific PAV will improve snakebite management in SL.

The application of laboratory-based analytical tools and techniques for the quality assessment and improvement of commercial antivenoms used in the treatment of snakebite envenomation.

Snakebite envenomation is a public health problem of high impact, particularly for the developing world. Antivenom, which contains whole or protease-digested immunoglobulin G, purified from the plasma of hyper-immunized animals (mainly horses), is the mainstay for the treatment of snakebite envenomation. The success of antivenom therapy depends upon its ability to abrogate or reduce the local and systemic toxicity of envenomation. In addition, antivenom administration must be safe for the patients. Therefore, antivenom manufacturers must ensure that these products are effective and safe in the treatment of envenomations. Antivenom efficacy and safety are determined by the physicochemical characteristics of formulations, purity of the immunoglobulin fragments and antibodies, presence of protein aggregates, endotoxin burden, preservative load, and batch to batch variation, as well as on the ability to neutralize the most important toxins of the venoms against which the antivenom is designed. In this context, recent studies have shown that laboratory-based simple analytical techniques, for example, size exclusion chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis, mass spectrometry, immunological profiling including immuno-turbidimetry and enzyme-linked immunosorbent assays, Western blotting, immune-chromatographic technique coupled to mass spectrometry analysis, reverse-phase high performance liquid chromatography, spectrofluorometric analysis, in vitro neutralization of venom enzymatic activities, and other methodologies, can be applied for the assessment of antivenom quality, safety, stability, and efficacy. This article reviews the usefulness of different analytical techniques for the quality assessment of commercial antivenoms. It is suggested that these tests should be applied for screening the quality of commercial antivenoms before their preclinical and clinical assessment.

The in vitro laboratory tests and mass spectrometry-assisted quality assessment of commercial polyvalent antivenom raised against the 'Big Four' venomous snakes of India.

India has recorded the maximum snakebite deaths in the world. Intravenous administration of polyvalent antivenom (PAV) raised against the 'Big Four' venomous snakes of India (Naja naja, Daboia russelli, Echis carinatus, and Bungarus caeraleus) is the only choice of treatment. The WHO has recommended the evaluation of quality and safety of commercial antivenom by in vitro laboratory tests prior to their pre-clinical evaluation in animal model and therapeutic use. Therefore, in this study an attempt has been made to evaluate the quality of commercial polyvalent antivenom produced in India by simple, and affordable laboratory tests. Proteomic analysis revealed that PAVs contained 78.7-94.8% IgG/F(ab')2 and small quantities of plasma proteins. The PAVs showed batch-to-batch variations with varying amounts of undigested IgG and its aggregates, and moderate complement activation. However, absence of IgE, negligible endotoxin contamination, and recommended limit of preservative (cresol) in PAVs were observed. The PAVs contain varying proportions and least amount of venom-specific antibodies against venoms of the 'Big Four' snakes from different locales of India, and against eastern India N. kaouthia venom, respectively. The importance of independent in vitro laboratory tests for the quality control and safety assessment for improving the quality of Indian commercial PAV is reinforced.

Assessment of snakebite burdens, clinical features of envenomation, and strategies to improve snakebite management in Vietnam.

The sheer paucity of scientific documentation of herpetofauna in Vietnam and the rudimentary healthcare response to snakebite have stimulated this review. Over six decades of data culled from public data bases and search engines, have been used to assess snakebite burdens, clinical features of envenomation, and strategies for snakebite management in Vietnam. In addition, biochemical and proteomic analyses to decipher venom composition, rapid analytical techniques to be used for clinical diagnosis of snakebite in Vietnam have been discussed in detail. The assessment of efficacy, safety, and quality of commercial antivenom produced in Vietnam and improvement of antivenom production to meet the national requirement has been critically examined. It is apparent that snake bite incidence in Vietnam is exacerbated by mismatch in demand and supply of antivenom therapy, insufficient medical facilities, preference for traditional healers and poor management of clinical records. The impediments arising from geographical and species-specific variation in venom composition can be overcome by the 'Omics approach', and scientific documentation of pathophysiological manifestations post envenomation. The development of next generation of therapeutics, encouraging clinical research, novel approaches and social awareness against snakebite and its treatments have been suggested to significantly reduce the snakebite mortality and morbidity in this region.

Proteomic Analysis of Sri Lanka Echis carinatus Venom: Immunological Cross-Reactivity and Enzyme Neutralization Potency of Indian Polyantivenom.

The saw-scaled viper (Echis carinatus carinatus) is a major venomous snake in Sri Lanka (SL) responsible for massive numbers of snakebites on the island; nevertheless, its venom proteome composition has never been explored. The proteome composition of SL E. c. carinatus venom (SL ECV), revealed by tandem mass spectrometry analysis, showed that it is composed of 42 enzymatic and nonenzymatic proteins belonging to 12 snake venom protein families. Snake venom metalloproteases (SVMP) and snaclec comprised the most abundant enzymatic and nonenzymatic proteins, respectively. When the composition of SL ECV was compared to the previously determined venom composition of Southern India ECV (SI ECV), 16 proteins were found in common. The SL ECV proteome composition was correlated with the clinical manifestations and pathophysiology of E. c. carinatus envenomation in SL. Polyvalent antivenom (PAV) raised in equine against the "Big Four" venomous snakes of India is typically exported to SL for snakebite treatment; however, the poor immunological cross-reactivity, partial in vitro neutralization of enzymatic activities, and some pharmacological properties, mostly shown by low molecular mass toxins (25 kDa) of SL ECV by Indian PAVs are major concerns for the effective treatment of ECV envenomation in SL.

Correlation of Venom Toxinome Composition of Indian Red Scorpion (Mesobuthus tamulus) with Clinical Manifestations of Scorpion Stings: Failure of Commercial Antivenom to Immune-Recognize the Abundance of Low Molecular Mass Toxins of This Venom.

The Indian red scorpion (Mesobuthus tamulus), with its life-threatening sting, is the world's most dangerous species of scorpion. The toxinome composition of M. tamulus venom was determined by tandem mass spectrometry (MS) analysis of venom protein bands separated by SDS-PAGE. A total of 110 venom toxins were identified from searching the MS data against the Buthidae family (taxid: 6855) of toxin entries in nonredundant protein databases. The Na+ and K+ ion channel toxins taken together are the most abundant toxins (76.7%) giving rise to the neurotoxic nature of this venom. The other minor toxin classes in the M. tamulus venom proteome are serine protease-like protein (2.9%), serine protease inhibitor (2.2%), antimicrobial peptide (2.3%), hyaluronidase (2.2%), makatoxin (2.1%), lipolysis potentiating peptides (1.2%), neurotoxin affecting Cl- channel (1%), parabutoporin (0.6%), Ca2+ channel toxins (0.8%), bradykinin potentiating peptides (0.2%), HMG CoA reductase inhibitor (0.1%), and other toxins with unknown pharmacological activity (7.7%). Several of these toxins have been shown to be promising drug candidates. M. tamulus venom does not show enzymatic activity (phospholipase A2, l-amino acid oxidase, adenosine tri-, di-, and monophosphatase, hyaluronidase, metalloproteinase, and fibrinogenolytic), in vitro hemolytic activity, interference with blood coagulation, or platelet modulation properties. The clinical manifestations post M. tamulus sting have been described in the literature and are well correlated with its venom proteome composition. An abundance of low molecular mass toxins (3-15 kDa) are responsible for exerting the major pharmacological effects of M. tamulus venom, though they are poorly immune-recognized by commercial scorpion antivenom. This is a major concern for the development of effective antivenom therapy against scorpion stings.

Quantitative proteomic analysis of venom from Southern India common krait (Bungarus caeruleus) and identification of poorly immunogenic toxins by immune-profiling against commercial antivenom.

OBJECTIVES: To study the venom proteome composition of Southern India (SI) Common Krait (Bungarus caeruleus) and immunological cross-reactivity between venom against commercial antivenom. METHODS: Proteomic analysis was done by nano LC-MS/MS and toxins were quantitated by label-free analysis. The immunological cross-reactivity of venom towards polyvalent antivenom (PAV) was assessed by ELISA, Immunoblotting, and immuno-chromatographic methods. RESULTS: A total of 57 enzymatic and non-enzymatic proteins belonging to 12 snake venom protein families were identified. The three finger toxins (3FTx) (48.3%) and phospholipase A2 (PLA2) (37.6%) represented the most abundant non-enzymatic and enzymatic proteins, respectively. ß-bungarotoxin (12.9%), a presynaptic neurotoxin, was also identified. The venom proteome composition is well correlated with its enzymatic activities, reported pharmacological properties, and clinical manifestations of krait envenomation. Immuno-cross-reactivity studies demonstrated better recognition of high molecular weight proteins (>45 kDa) of this venom by PAVs compared to low molecular weight (<15 kDa) toxins such as PLA2 and 3FTxs. CONCLUSION: The poor recognition of <15 kDa mass SI B. caeruleus venom proteins is of grave concern for the successful treatment of krait envenomation. Therefore, emphasis should be given to improve the immunization protocols and/or supplement of antibodies raised specifically against the <15 kDa toxins of this venom.

Proteomic analysis and antivenomics study of Western India Naja naja venom: correlation between venom composition and clinical manifestations of cobra bite in this region.

Background: Snakebite is a severe problem in the tropical countries including Indian subcontinent. Premier cases of cobra bites are being reported from western India (WI). Research design and methods: The proteome of WI N. naja venom (NnV) was deciphered by high resolution mass spectrometry analysis of venom, further fractionated by gel filtration (GF) or RP-HPLC followed by SDS-PAGE and then tandem mass spectrometric analysis of protein bands. The efficacy of commercial polyantivenom (PAV) towards WINnV was assessed by ELISA, immuno-blot, neutralization, and venom-PAV immunoaffinity chromatography studies. Results: Proteomic analysis of WINnV, GF fractions, and SDS-PAGE protein bands of RP-HPLC and GF peaks identified 14, 34, 40, and 54, distinct proteins, respectively, when searched against Elapidae database. The biochemical properties of WINnV correlated well with its proteome composition and pathophysiology of cobra envenomation, including neuroparalysis. This study also highlighted the differences in proteome composition between WINnV and previously reported Eastern India NnV. The tested antivenoms exhibited poor immuno-recognition and neutralization of low molecular mass proteins (<20 kDa), such as three-finger toxins, the major class of protein in WINnV. Conclusion: Improvements in production protocols of antivenoms is the necessity of the hour, supplemented with antibodies raised against the poorly recognized toxins.

Proteomics analysis to compare the venom composition between Naja naja and Naja kaouthia from the same geographical location of eastern India: Correlation with pathophysiology of envenomation and immunological cross-reactivity towards commercial polyantivenom.

BACKGROUND: Cobra bite is frequently reported across the Indian subcontinent and is associated with a high rate of death and morbidity. In eastern India (EI) Naja naja and Naja kaouthia are reported to be the two most abundant species of cobra. RESEARCH DESIGN AND METHODS: The venom proteome composition of N. naja (NnV) and N. kaouthia (NkV) from Burdwan districts of EI were compared by separation of venom proteins by 1D-SDS-PAGE followed by LC-MS/MS analysis of protein bands. The potency of commercial polyantivenom (PAV) was assessed by neutralization, ELISA, immuno-blot and venom-PAV immunoaffinity chromatography studies. RESULTS: Proteomic analysis identified 52 and 55 proteins for NnV and NkV, respectively, when searched against the Elapidae database. A small quantitative difference in venom composition between these two species of cobra was observed. PAVs exhibited poor cross-reactivity against low molecular mass toxins (<20 kDa) of both cobra venoms, which was substantiated by a meager neutralization of their phospholipase A2 activity. Phospholipase A2 and 3FTx, the two major classes of nonenzymatic and enzymatic proteins, respectively, were partially recognized by PAVs. CONCLUSIONS: Efforts must be made to improve immunization protocols and supplement existing antivenoms with antibodies raised against the major toxins of these venoms.

Assessment of quality, safety, and pre-clinical toxicity of an equine polyvalent anti-snake venom (Pan Africa): Determination of immunological cross-reactivity of antivenom against venom samples of Elapidae and Viperidae snakes of Africa.

Snakebite causes a large amount of morbidities and mortalities in Africa. The safety, efficacy, and homogeneity of anti-snake venoms are crucial for snakebite treatments to be effective with minimal adverse effects. We assessed the homogeneity of preparations of three different batches of Combipack snake venom antiserums (Pan Africa) [CSVAPA] by quantitatively analysing F(ab')2, IgG, and other contaminating proteins of plasma. LC-MS/MS analysis showed that approximately 92.4% of the proteins from the CSVAPA samples was IgG/F(ab')2 and the percent composition of contaminating proteins in CSVAPA varied from 0.07 to 4.6%. Batch 1 of the CSVAPA also contained a minor amount of undigested IgG and F(ab')2 aggregates. CSVAPA contained more than 60% venom-specific antibodies, showed moderate complement activation, no IgE contamination, safe level of endotoxin, and also showed pre-clinical safety. The immuno cross-reactivity of CSVAPA against 14 Viperidae and Elapidae snake venoms of Africa was tested by ELISA and immunoblotting, and the neutralization of major enzymatic venom activities, demonstrating that high molecular weight (>50 kDa) venom proteins are better recognized/neutralized compared to relatively low molecular weight (<20 kDa) venom proteins. CSVAPA at a dose of 3-12 times higher than the clinical dose did not cause deaths or adverse reaction of treated rabbits. The results suggest the satisfactory quality, safety, and efficacy of CSVAPA.