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.

Comparative pangenomic analysis of predominant human vaginal lactobacilli strains towards population-specific adaptation: understanding the role in sustaining a balanced and healthy vaginal microenvironment.

The vaginal microenvironment of healthy women has a predominance of Lactobacillus crispatus, L. iners, L. gasseri, and L. jensenii. The genomic repertoire of the strains of each of the species associated with the key attributes thereby regulating a healthy vaginal environment needs a substantial understanding.We studied all available human strains of the four lactobacilli across different countries, isolated from vaginal and urinal sources through phylogenetic and pangenomic approaches. The findings showed that L. iners has the highest retention of core genes, and L. crispatus has more gene gain in the evolutionary stratum. Interestingly, L. gasseri and L. jensenii demonstrated major population-specific gene-cluster gain/loss associated with bacteriocin synthesis, iron chelating, adherence, zinc and ATP binding proteins, and hydrolase activity. Gene ontology enrichment analysis revealed that L. crispatus strains showed greater enrichment of functions related to plasma membrane integrity, biosurfactant, hydrogen peroxide synthesis, and iron sequestration as an ancestral derived core function, while bacteriocin and organic acid biosynthesis are strain-specific accessory enriched functions. L. jensenii showed greater enrichment of functions related to adherence, aggregation, and exopolysaccharide synthesis. Notably, the key functionalities are heterogeneously enriched in some specific strains of L. iners and L. gasseri.This study shed light on the genomic features and their variability that provides advantageous attributes to predominant vaginal Lactobacillus species maintaining vaginal homeostasis. These findings evoke the need to consider region-specific candidate strains of Lactobacillus to formulate prophylactic measures against vaginal dysbiosis for women's health.

Recent advances in understanding of multifaceted changes in the vaginal microenvironment: implications in vaginal health and therapeutics.

The vagina endures multifaceted changes from neonatal to menopausal phases due to hormonal flux, metabolite deposition, and microbial colonization. These features have important implications in women's health. Several pre-factors show dynamic characteristics according to the phases that shift the vaginal microbiota from anaerobes to aerobes which is a hallmark of healthy vaginal environment. These factors include oestrogen levels, glycogen deposition, and vaginal microstructure. In the adult phase, Lactobacillus is highly dominant and regulates pH, adherence, aggregation, immune modulation, synthesis of bacteriocins, and biosurfactants (BSs) which are antagonistic to pathogens. Maternal factors are protective by favouring the colonization of lactobacilli in the vagina in the neonatal phase, which diminishes with age. The dominance of lactobacilli and dysbiosis in the adult phase depends on intrinsic and extrinsic factors in women, which vary between ethnicities. Recent developments in probiotics used against vaginal microbiome dysbiosis have shown great promise in restoring the normal microbiota including preventing the loss of beneficial bacteria. However, further in-depth studies are warranted to ensure long-term protection by probiotics. This review highlights various aspects of the vaginal microenvironment in different phases of growth and diverse ethnicities. Furthermore, it discusses future trends for formulating more effective population-specific probiotics and implications of paraprobiotics and postbiotics as effective therapeutics.

Genome analysis of SARS-CoV-2 isolates from a population reveals the rapid selective sweep of a haplotype carrying many pre-existing and new mutations.

To understand the mechanism underlying the evolution of SARS-CoV-2 in a population, we sequenced 92 viral genomes from Assam, India. Analysis of these and database sequences revealed a complete selective sweep of a haplotype in Assam carrying 13 pre-existing variants, including a high leap in frequency of a variant on ORF8, which is involved in immune evasion. A comparative study between sequences of same lineage and similar time frames in and outside Assam showed that 10 of the 13 pre-existing variants had a frequency ranging from 96 to 99%, and the remaining 3 had a low frequency outside Assam. Using a phylogenetic approach to infer sequential occurrences of variants we found that the variant Phe120del on ORF8, which had a low frequency (1.75%) outside Assam, is at the base of the phylogenetic tree of variants and became totally fixed (100%) in Assam population. Based on this observation, we inferred that the variant on ORF8 had a selective advantage, so it carried the haplotype to reach the100% frequency. The haplotype also carried 32 pre-existing variants at a frequency from 1.00 to 80.00% outside Assam. Those of these variants that are more closely linked to the S-protein locus, which often carries advantageous mutations and is tightly linked to the ORF8 locus, retained higher frequencies, while the less tightly linked variants showed lower frequencies, likely due to recombination among co- circulating variants in Assam. The ratios of non-synonymous substitutions to synonymous substitutions suggested that some genes such as those coding for the S-protein and non-structural proteins underwent positive selection while others were subject to purifying selection during their evolution in Assam. Furthermore, we observed negative correlation of the Ct value of qRT-PCR of the patients with abundant ORF6 transcripts, suggesting that ORF6 can be used as a marker for estimating viral titer. In conclusion, our in-depth analysis of SARS-CoV-2 genomes in a regional population reveals the mechanism and dynamics of viral evolution.

Unraveling the Reaction Mechanism of Russell's Viper Venom Factor X Activator: A Paradigm for the Reactivity of Zinc Metalloproteinases?

Snake venom metalloproteinases (SVMPs) are important drug targets against snakebite envenoming, the neglected tropical disease with the highest mortality worldwide. Here, we focus on Russell's viper (Daboia russelii), one of the "big four" snakes of the Indian subcontinent that, together, are responsible for ca. 50,000 fatalities annually. The "Russell's viper venom factor X activator" (RVV-X), a highly toxic metalloproteinase, activates the blood coagulation factor X (FX), leading to the prey's abnormal blood clotting and death. Given its tremendous public health impact, the WHO recognized an urgent need to develop efficient, heat-stable, and affordable-for-all small-molecule inhibitors, for which a deep understanding of the mechanisms of action of snake's principal toxins is fundamental. In this study, we determine the catalytic mechanism of RVV-X by using a density functional theory/molecular mechanics (DFT:MM) methodology to calculate its free energy profile. The results showed that the catalytic process takes place via two steps. The first step involves a nucleophilic attack by an in situ generated hydroxide ion on the substrate carbonyl, yielding an activation barrier of 17.7 kcal·mol-1, while the second step corresponds to protonation of the peptide nitrogen and peptide bond cleavage with an energy barrier of 23.1 kcal·mol-1. Our study shows a unique role played by Zn2+ in catalysis by lowering the pKa of the Zn2+-bound water molecule, enough to permit the swift formation of the hydroxide nucleophile through barrierless deprotonation by the formally much less basic Glu140. Without the Zn2+ cofactor, this step would be rate-limiting.

Comprehensive bacterial-metabolite profiles of Hawaijar, Bekang, and Akhone: a comparative study on traditional fermented soybeans of north-east India.

Preparation of traditionally fermented soybeans varies across ethnicities with distinct tastes, flavour, and nutritional values. The fermented soybean varieties Hawaijar, Bekang, and Akhone of north-east India are associated with diverse ethnic groups from Manipur, Mizoram, and Nagaland, respectively. These varieties differ in substrate and traditional practice that exerts differential bacterial-metabolite profile, which needs an in-depth analysis i. Culture-dependent and independent techniques investigated the bacterial diversity of the fermented soybean varieties. Gas chromatography and mass spectroscopy (GC-MS) studied these varieties' metabolite profiles. The common dominant bacterial genera detected in Hawaijar, Bekang, and Akhone were Bacillus, Ignatzschinaria, and Corynebacterium, with the presence of Brevibacillus and Staphylococcus exclusively in Hawaijar and Oceanobacillus in Bekang and Akhone. The metabolite analysis identified a higher abundance of essential amino acids, amino and nucleotide sugars, and vitamins in Hawaijar, short-chain fatty acids in Bekang, polyunsaturated fatty acids in Akhone and Hawaijar, and prebiotics in Akhone. The bacteria-metabolite correlation analysis predicted four distinct bacterial clusters associated with the differential synthesis of the functional metabolites. While B. subtilis is ubiquitous, cluster-1 comprised B. thermoamylovorans/B. amyloliquefaciens, cluster-2 comprised B. tropicus, cluster-3 comprised B. megaterium/B. borstelensis, and cluster-4 comprised B. rugosus. To the best of our knowledge, this is the first comparative study on traditional fermented soybean varieties of north-east India linking bacterial-metabolite profiles which may help in designing starters for desired functionalities in the future.

Characterization of lipopeptide biosurfactant produced by a carbazole-degrading bacterium Roseomonas cervicalis: The role of biosurfactant in carbazole solubilisation.

AIM: Characterization of biosurfactant produced by a carbazole-degrading bacterium Roseomonas cervicalis and proteomic analysis of intracellular proteins of bacterium while growing on glucose and carbazole medium. METHODS AND RESULTS: The bacterium R. cervicalis was isolated from a soil sample contaminated with crude petroleum oil. PCR amplification ascertained the existence of some hydrocarbon-degrading catabolic genes (alkB and PAH-RHDα, C12O, and C23O) in the bacterial genome. GC-MS and RP-HPLC analyses demonstrated 62% and 60% carbazole degradation, respectively, by R. cervicalis 144 h post-incubation at 37℃ and pH 6.5. Due to the paucity of protein databases, expressions of only 29 and 14 intracellular proteins were explicitly recognized and quantitated by mass spectrometry analysis when R. cervicalis was grown in carbazole and glucose medium, respectively. FTIR, NMR and HR-MS/MS analyses demonstrated the lipopeptide nature of the purified biosurfactant produced by R. cervicalis. The biosurfactant is also presumed to assist in the solubilization of carbazole. CONCLUSION: The isolated R. cervicalis strain is a potential candidate for the bioremediation of carbazole in petroleum-oil-contaminated sites. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the promising R. cervicalis strain proficient in carbazole biodegradation.

Potential clinical applications of phytopharmaceuticals for the in-patient management of coagulopathies in COVID-19.

Thrombotic complications occur in many cardiovascular pathologies and have been demonstrated in COVID-19. The currently used antithrombotic drugs are not free of adverse reactions, and COVID-19 patients in particular, when treated with a therapeutic dose of an anticoagulant do not receive mortality benefits. The clinical management of COVID-19 is one of the most difficult tasks for clinicians, and the search for safe, potent, and effective antithrombotic drugs may benefit from exploring naturally bioactive molecules from plant sources. This review describes recent advances in understanding the antithrombotic potential of herbal drug prototypes and points to their future clinical use as potent antithrombotic drugs. Although natural products are perceived to be safe, their clinical and therapeutic applications are not always apparent or accepted. More in-depth studies are necessary to demonstrate the clinical usefulness of plant-derived, bioactive compounds. In addition, holistic approaches in systematic investigations and the identification of antithrombotic mechanisms of the herbal bioactive molecule(s) need to be conducted in pre-clinical studies. Moreover, rigorous studies are needed to compare the potency of herbal drugs to that of competitor chemical antithrombotic drugs, and to examine their interactions with Western antithrombotic medicines. We have also proposed a road map to improve the commercialization of phytopharmaceuticals.

Transcriptomic and functional proteomics analyses to unveil the common and unique pathway(s) of neuritogenesis induced by Russell's viper venom nerve growth factor in rat pheochromocytoma neuronal cells.

Background: The snake venom nerve growth factor (NGF)-induced signal transduction mechanism has never been explored.Research design and methods: Homology modeling and molecular dynamic studies of the interaction between Russell's viper venom NGF (RVV-NGFa) and mammalian tropomyosin-receptor kinase A (TrkA) was done by computational analysis. Transcriptomic and quantitative tandem mass spectrometry analyses determined the expression of intracellular genes and proteins, respectively, in RVV-NGFa-treated PC-12 neuronal cells. Small synthetic inhibitors of the signal transduction pathways were used to validate the major signaling cascades of neuritogenesis by RVV-NGFa.Results: A comparative computational analysis predicted the binding of RVV-NGFa, mouse 2.5S-NGF (conventional neurotrophin), and Nn-α-elapitoxin-1 (non-conventional neurotrophin) to different domains of the TrkA receptor in PC-12 cells. The transcriptomic and quantitative proteomic analyses in unison showed differential expressions of common and unique genes and intracellular proteins, respectively, in RVV-NGFa-treated cells compared to control (untreated) mouse 2.5S-NGF and Nn-α-elapitoxin-1-treated PC-12 cells. The RVV-NGFa primarily triggered the mitogen-activated protein kinase-1 (MAPK1) signaling pathway for inducing neuritogenesis; however, 36 pathways of neuritogenesis were uniquely expressed in RVV-NGFa-treated PC-12 cells compared to mouse 2.5S NGF or Nn-α-elapitoxin-1 treated cells.Conclusion: The common and unique intracellular signaling pathways of neuritogenesis by classical and non-classical neurotrophins were identified.

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.

Triple-negative breast cancer-derived microvesicles transfer microRNA221 to the recipient cells and thereby promote epithelial-to-mesenchymal transition.

The triple-negative phenotype is the most prevalent form of human breast cancer worldwide and is characterized by poor survival, high aggressiveness, and recurrence. Microvesicles (MV) are shredded plasma membrane components and critically mediate cell-cell communication, but can also induce cancer proliferation and metastasis. Previous studies have revealed that protease-activated receptor 2 (PAR2) contributes significantly to human triple-negative breast cancer (TNBC) progression by releasing nano-size MV and promoting cell proliferation, migration, and invasion. MV isolated from highly aggressive human TNBC cells impart metastatic potential to nonmetastatic cells. Over-expression of microRNA221 (miR221) has also been reported to enhance the metastatic potential of human TNBC, but miR221's relationship to PAR2-induced MV is unclear. Here, using isolated MV, immunoblotting, quantitative RT-PCR, FACS analysis, and enzymatic assays, we show that miR221 is translocated via human TNBC-derived MV, which upon fusion with recipient cells, enhance their proliferation, survival, and metastasis both in vitro and in vivo by inducing the epithelial-to-mesenchymal transition (EMT). Administration of anti-miR221 significantly impaired MV-induced expression of the mesenchymal markers Snail, Slug, N-cadherin, and vimentin in the recipient cells, whereas restoring expression of the epithelial marker E-cadherin. We also demonstrate that MV-associated miR221 targets phosphatase and tensin homolog (PTEN) in the recipient cells, followed by AKT Ser/Thr kinase (AKT)/NF-κB activation, which promotes EMT. Moreover, elevated miR221 levels in MV derived from human TNBC patients' blood could induce cell proliferation and metastasis in recipient cells. In summary, miR221 transfer from TNBC cells via PAR2-derived MV induces EMT and enhances the malignant potential of recipient cells.

Transcriptomic, proteomic, and biochemical analyses reveal a novel neuritogenesis mechanism of Naja naja venom α-elapitoxin post binding to TrkA receptor of rat pheochromocytoma cells.

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 p75NTR. 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.

Mass spectrometric analysis to unravel the venom proteome composition of Indian snakes: opening new avenues in clinical research.

INTRODUCTION: The 'Big Four' venomous snakes - Daboia russelii, Naja naja, Bungarus caeruleus, and Echis carinatus - are primarily responsible for the majority of snake envenomation in India. Several other lesser-known venomous snake species also inflict severe envenomation in the country. AREAS COVERED: A comprehensive analysis of the venom proteome composition of the 'Big Four' and other medically important venomous snakes of India and the effect of regional variation in venom composition on immunorecognition and/or neutralization by commercial antivenom was undertaken by searching the literature (from 1985 to date) available in large public databases. Further, mass spectrometric identification of poorly immunogenic toxins of snake venom (against which commercial polyvalent antivenom contains a significantly lower proportion of antibodies) and its impact on antivenom therapy against snakebite are discussed. The application of mass spectrometry to identify protein (toxin) complexes as well as drug prototypes from Indian snake venoms and the clinical importance of such studies are also highlighted. EXPERT OPINION: Further detailed clinical and proteomic research is warranted to better understand the effects of regional snake venom composition on the clinical manifestation of envenomation and antivenom therapy and to improve the production of antibodies against poorly immunogenic venom components.

A simple, cost-effective, and rapid separation process for the isolation of anticoagulant active fraction from the fruit extract of Momordica charantia: Characterization of bioactive components and anticoagulant mechanism of active fraction in a mouse model.

A simple, rapid, and cost-effective process for the separation of an active anticoagulant fraction from the aqueous fruit extract of Momordica charantia by using rice husk as adsorbed is described. The in vitro anticoagulant activity of active anticoagulant fraction was comparable to commercial anticoagulants heparin and warfarin. Phytochemical analysis revealed the presence of alkaloids, flavonoids, and phytols in the active anticoagulant fraction, nevertheless; it was devoid of glycosides, triterpenoids, tannins, saponins, steroids, and carbohydrates. By gas chromatography with mass spectrometry analysis, decanoic acid, 1,2,3-propanetriyl ester (22.3%), dodecanoic acid, 1,2,3-propanetriyl ester-d5 (17.3%), dodecenoic acid, 1,2,3-propanetriyl ester (12.5%), and 4-B-methylandrostane 2,3-diol-1,17-dione (11.4%) were identified as the most abundant constituents of active anticoagulant fraction. Presence of αß-fibrinogenase enzyme was identified by biochemical assay but not by liquid chromatography with tandem mass spectrometry analysis suggesting presence of a novel protease enzyme in this fraction. The active anticoagulant fraction demonstrated biding to fibrinogen but not to thrombin or Factor Xa, inhibited the collagen/ADP-induced mammalian platelet aggregation, showed in vitro thrombolytic activity, noncytotoxic to mammalian cells, showed in vivo plasma defibrinogenation and anticoagulant activities, and inhibited k-carrageen-induced thrombus formation in the tails of mice. Therefore, active anticoagulant fraction (an herbal drug) may find therapeutic application for the prevention and/or treatment of hyperfibrinogenemia/thrombosis-associated cardiovascular disorders.

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.

Proteomic Analysis and Immuno-Profiling of Eastern India Russell's Viper ( Daboia russelii) Venom: Correlation between RVV Composition and Clinical Manifestations Post RV Bite.

The proteomes of Russell's viper venom (RVV) from Burdwan (RVV B) and Nadia (RVV N), the two districts of West Bengal, eastern India (EI), were investigated by gel-filtration chromatography (GFC) followed by tandem mass spectrometry of tryptic fragments of the fractions. A total of 73 and 69 proteins belonging to 15 snake venom protein families were identified in RVV B and RVV N, respectively, by MS/MS search against Viperidae (taxid 8689) protein entries of the nonredundant NCBI database. The minor differences in venom composition of both the EI RV were established unequivocally by their biochemical and pharmacological properties and by SDS-PAGE, gel filtration chromatography, and LC-MS/MS analyses. The composition of EI RVVs was well correlated with published reports on the pathophysiology of RV-envenomed patients from this part of the country. Venom-antivenom cross-reactivity determined by ELISA, Western blotting, and antivenomics approaches demonstrated poor recognition of low molecular mass (<20 kDa) RVV proteins by commercial polyvalent antivenoms, which was substantiated by neutralization of RVV enzymes by antivenom.

Coagulation factor VIIa-mediated protease-activated receptor 2 activation leads to ß-catenin accumulation via the AKT/GSK3ß pathway and contributes to breast cancer progression.

Cell migration and invasion are very characteristic features of cancer cells that promote metastasis, which is one of the most common causes of mortality among cancer patients. Emerging evidence has shown that coagulation factors can directly mediate cancer-associated complications either by enhancing thrombus formation or by initiating various signaling events leading to metastatic cancer progression. It is well established that, apart from its distinct role in blood coagulation, coagulation factor FVIIa enhances aggressive behaviors of breast cancer cells, but the underlying signaling mechanisms still remain elusive. To this end, we investigated FVIIa's role in the migration and invasiveness of the breast cancer cell line MDA-MB-231. Consistent with previous observations, we observed that FVIIa increased the migratory and invasive potential of these cells. We also provide molecular evidence that protease-activated receptor 2 activation followed by PI3K-AKT activation and GSK3ß inactivation is involved in these processes and that ß-catenin, a well known tumor-regulatory protein, contributes to this signaling pathway. The pivotal role of ß-catenin was further indicated by the up-regulation of its downstream targets cyclin D1, c-Myc, COX-2, MMP-7, MMP-14, and Claudin-1. ß-Catenin knockdown almost completely attenuated the FVIIa-induced enhancement of breast cancer migration and invasion. These findings provide a new perspective to counteract the invasive behavior of breast cancer, indicating that blocking PI3K-AKT pathway-dependent ß-catenin accumulation may represent a potential therapeutic approach to control breast cancer.

Protease-activated receptor 2 promotes actomyosin dependent transforming microvesicles generation from human breast cancer.

Apart from blood coagulation, coagulation proteases are involved inextricably in cancer progression/propagation via intra/inter-cellular signaling, mediated predominantly by protease-activated receptors (PARs). Microvesicles (MVs), a plasma membrane shredded component, has recently been identified as an important contributor to human breast cancer metastasis. However, the role of PAR2 in promoting MVs generation from breast cancer cells remains largely unexplored. The objective of this study is to investigate whether coagulation protease-mediated human breast cancer propagation commences via MVs and also to decipher the underlying signaling mechanism. Here, we elicited that coagulation factor-FVIIa and Trypsin activates PAR2, which governs MVs shedding from MDAMB231 cells by altering actomyosin dynamics. Treatment of cells with PAR2 activators facilitate MVs generation by activating three independent (MAPK, P38, and Rho) signaling cascades. MAPK, signals through activating MLCK followed by MLC phosphorylation to alter myosin organization whereas, P38 reorganizes actin dynamics by the sequential activation of MK2 and HSP27. RhoA-dependent ROCK-II activation again contributes to remodeling myosin II activity. Further, both our in vitro and in vivo analyses showed that these MVs potentiate invasive and migratory property to the recipient cells. Breast cancer patients blood show an elevation of TF-bearing, pro-metastatic MVs than normal. These findings give an insight into the detailed signaling mechanism involved in the production of MVs with transforming ability from PAR2-activated human breast cancer cells. Understanding these mechanistic details will certainly help to identify crucial targets for therapeutic interventions in MVs-associated human breast cancer metastasis.