Indian green pit vipers: A lesser-known snake group of north-east India.

Green pit vipers are one of the most widely distributed group of venomous snakes in south-east Asia. In Indian, green pit vipers are found in the Northern and North-eastern states spreading across eastern and central India and one of the lesser studied venoms. High morphological similarity among them has been a long-established challenge for species identification, however, a total of six species of Indian green pit viper belonging to genus Trimeresurus, Popeia and Viridovipera has been reported from North-east India. Biochemical and biological studies have revealed that venom exhibits substantial variation in protein expression level along with functional variability. The symptoms of envenomation are painful swelling at bite site, bleeding, necrosis along with systemic toxicity such as prolonged coagulopathy. Clinical data of green pit viper envenomated patients from Demow community health centre, Assam advocated against the use of Indian polyvalent antivenom pressing the need for a suitable antivenom for the treatment of green pit viper envenomation. To design effective and specific antivenom for green pit vipers, unveiling the proteome profile of these snakes is needed. In this study, a comparative venomic of green pit vipers of Northern and North-eastern India, their clinical manifestation as well as treatment protocol has been reviewed.

Identification of poorly immunodepleted phospholipase A2 (PLA2) proteins of Bungarus fasciatus venom from Assam, India and evaluation of Indian polyvalent antivenom using third-generation antivenomics.

Bungarus fasciatus also referred to as the Banded krait is a snake which possesses venom and belongs to the Elapidae family. It is widely distributed across the Indian subcontinent and South East Asian countries and is responsible for numerous snakebites in the population. B. fasciatus possesses a neurotoxic venom and envenomation by the snake results in significant morbidity and occasional morbidity in the victim if not treated appropriately. In this study, the efficacy of Indian polyvalent antivenom (Premium Serums polyvalent antivenom) was evaluated against the venom of B. fasciatus from Guwahati, Assam (India) employing the Third-generation antivenomics technique followed by identification of venom proteins from three poorly immunodepleted peaks (P5, P6 and P7) using LC-MS/MS analysis. Seven proteins were identified from the three peaks and all these venom proteins belonged to the phospholipase A2 (PLA2) superfamily. The identified PLA2 proteins were corroborated by the in vitro enzymatic activities (PLA2 and Anticoagulant activity) exhibited by the three peaks and previous reports of pathological manifestation in the envenomated victims. Neutralization of enzymatic activities by Premium Serums polyvalent antivenom was also assessed in vitro for crude venom, P5, P6 and P7 which revealed moderate to poor inhibition. Inclusion of venom proteins/peptides, which are non-immunodepleted or poorly immunodepleted, into the immunization mixture of venom used for antivenom production may help in enhancing the efficacy of the polyvalent antivenom.

Reverse vaccinology and immunoinformatics approach to design a chimeric epitope vaccine against Orientia tsutsugamushi.

Scrub typhus is a vector-borne infectious disease caused by Orientia tsutsugamushi and it is reportedly associated with up to 20 % of hospitalized cases of febrile illnesses. The major challenge of vaccine development is the lack of identified antigens that can induce both heterotypic and homotypic immunity including the production of antibodies, cytotoxic T lymphocyte, and helper T lymphocytes. We employed a comprehensive immunoinformatic prediction algorithm to identify immunogenic epitopes of the 56-kDa type-specific cell membrane surface antigen and surface cell antigen A of O. tsutsugamushi to select potential candidates for developing vaccines and diagnostic assays. We identified 35 linear and 29 continuous immunogenic B-cell epitopes and 51 and 27 strong-binding T-cell epitopes of major histocompatibility complex class I and class II molecules, respectively, in the conserved and variable regions of the 56-kDa type-specific surface antigen. The predicted B- and T-cell epitopes were used to develop immunogenic multi-epitope candidate vaccines and showed to elicit a broad-range of immune protection. A stable interactions between the multi-epitope vaccines and the host fibronectin protein were observed using docking and simulation methods. Molecular dynamics simulation studies demonstrated that the multi-epitope vaccine constructs and fibronectin docked models were stable during simulation time. Furthermore, the multi-epitope vaccine exhibited properties such as antigenicity, non-allergenicity and ability to induce interferon gamma production and had strong associations with their respective human leukocyte antigen alleles of world-wide population coverage. A correlation of immune simulations and the in-silico predicted immunogenic potential of multi-epitope vaccines implicate for further investigations to accelerate designing of epitope-based vaccine candidates and chimeric antigens for development of serological diagnostic assays for scrub typhus.

Proteome analysis of Daboia russelii venom, a medically important snake from the Indian sub-continent.

Daboia russelii is a category-I medically important snake throughout the Indian sub-continent contributing to majority of snakebite incidences in this part of the world. As such, extensive studies on its venom composition and search of efficient and appropriate interventions for its treatment become crucial. In this study, the proteome of Daboia russelii venom from Tanore, Rajshahi, Bangladesh was profiled using a combination of chromatographic and mass spectrometric techniques. A total of 37 different proteins belonging to 11 different snake venom protein families were detected. Proteomics analysis revealed the presence of major phospholipase A2 toxins. Daboiatoxin (both A and B subunits), the main lethal PLA2 toxin in the venom of Daboia siamensis (Myanmar viper) which is neurotoxic, myotoxic and cytotoxic was detected. Presence of Daboxin P, which is a major protein in the venom of Indian Daboia russelii with strong anticoagulant activity, was also observed. Inconsistent distribution of such lethal toxins in the venom of same species calls for more investigations of snake venoms from lesser explored regions and formulation of better alternatives to the current antivenom therapy for efficient treatment.

Daboxin P, a phospholipase A2 of Indian Daboia russelii venom, modulates thrombin-mediated platelet aggregation.

Daboxin P, reported earlier from the venom of Daboia russellii, disturbs the blood coagulation cascade by targeting factor X and factor Xa. The present study exhibits that Daboxin P also inhibits platelet aggregation induced by various agonists. The thrombin-induced platelet aggregation was inhibited maximum whereas inhibition of collagen-induced platelet aggregation was found to be 50% and no inhibition of adenosine diphosphate (ADP) and arachidonic acid-induced aggregation was observed. Daboxin P dose-dependently inhibited the thrombin-induced platelet aggregation with Anti-Aggregation 50 (AD50 ) dose of 55.166 nM and also reduced the thrombin-mediated calcium influx. In-silico interaction studies suggested that Daboxin P binds to thrombin and blocks its interaction with its receptor on the platelet surface. Quenching of thrombin's emission spectrum by Daboxin P and electrophoretic profiles of pull-down assay further reveals the binding between Daboxin P and thrombin. Thus, the present study demonstrates that Daboxin P inhibits thrombin-induced platelet aggregation by binding to thrombin.

Multilevel Comparison of Indian Naja Venoms and Their Cross-Reactivity with Indian Polyvalent Antivenoms.

Snake envenoming is caused by many biological species, rather than a single infectious agent, each with a multiplicity of toxins in their venom. Hence, developing effective treatments is challenging, especially in biodiverse and biogeographically complex countries such as India. The present study represents the first genus-wide proteomics analysis of venom composition across Naja species (N. naja, N. oxiana, and N. kaouthia) found in mainland India. Venom proteomes were consistent between individuals from the same localities in terms of the toxin families present, but not in the relative abundance of those in the venom. There appears to be more compositional variation among N. naja from different locations than among N. kaouthia. Immunoblotting and in vitro neutralization assays indicated cross-reactivity with Indian polyvalent antivenom, in which antibodies raised against N. naja are present. However, we observed ineffective neutralization of PLA2 activities of N. naja venoms from locations distant from the source of immunizing venoms. Antivenom immunoprofiling by antivenomics revealed differential antigenicity of venoms from N. kaouthia and N. oxiana, and poor reactivity towards 3FTxs and PLA2s. Moreover, there was considerable variation between antivenoms from different manufacturers. These data indicate that improvements to antivenom manufacturing in India are highly desirable.

Multiple protein-patterned surface plasmon resonance biochip for the detection of human immunoglobulin-G.

A portable surface plasmon resonance (SPR) measurement prototype integrated with a multiple protein-patterned SPR biochip is introduced for label-free and selective detection of human immunoglobulin-G (H-IgG). The polyclonal anti-H-IgG antibodies derived from goat, rabbit, and mouse were immobilized through polydimethylsiloxane (PDMS) microchannels to fabricate the patterned SPR biochip. The PDMS surface was functionalized using 3-aminopropyltrimethoxysilane and bonded to carbodiimide-activated gold substrates to construct irreversibly bonded hydrophilic microfluidic chip at room temperature. For SPR measurement, a custom-made system is developed with a high angular scanning accuracy of 0.005° and a wide scanning range of 30°-80° that avoids the conventional requirement of expensive goniometric stages and detector arrays. The SPR biochip immobilized with 750 µg/mL goat anti-H-IgG demonstrated detection of H-IgG with a detection limits of 15 µg/mL, and linear response through a wide concentration range (15-225 µg/mL) of high coefficient of determination (R2  = 0.99661). The selectivity of the sensor was investigated by exposing them to two different non-specific targets (bovine serum albumin and polyvalent antivenom). The results indicate negligible sensor response towards nonspecific targets (0.25° for 30 µg/mL bovine serum albumin (BSA) and 0.25° for 30 µg/mL polyvalent antivenom) in comparison to H-IgG (1.5° for 30 µg/mL).

Proteome Decomplexation of Trimeresurus erythrurus Venom from Mizoram, India.

Green pit vipers are the largest group of venomous vipers in tropical and subtropical Asia, which are responsible for most of the bite cases across this region. Among the green pit vipers of the Indian subcontinent, Trimeresurus erythrurus is the most prevalent; however, limited knowledge is available about its venomics. Proteome decomplexation of T. erythrurus venom using mass spectrometry revealed a blend of 53 different proteins/peptides belonging to 10 snake venom protein families. Phospholipase A2 and snake venom serine proteases were found to be the major enzymatic families, and Snaclec was the major nonenzymatic family in this venom. These protein families might be responsible for consumptive coagulopathy in victims. Along with these, snake venom metalloproteases, l-amino acid oxidases, disintegrins, and cysteine-rich secretory proteins were also found, which might be responsible for inducing painful edema, tissue necrosis, blistering, and defibrination in patients. Protein belonging to C-type lectins, C-type natriuretic peptides, and glutaminyl-peptide cyclotransfreases were also observed as trace proteins. The crude venom shows platelet aggregation in the absence of any agonist, suggesting their role in alterations in platelet functions. This study is the first proteomic analysis of T. erythrurus venom, contributing an overview of different snake venom proteins/peptides responsible for various pathophysiological disorders obtained in patients. Data are available via ProteomeXchange with the identifier PXD038311.

Recurrent neurotoxity in Naja kaouthia envenomation: A case report from Assam, India.

A 35 year old, male patient, bitten by Naja kaouthia with mild pain was admitted in Demow Government Community Health Centre. After 90 min post bite he developed neurotoxic symptoms. As per standard protocol, the patient was treated with 25 vials of antivenom and two doses of glycopyrrolate and neostigmine. Subsequently, he was seemingly devoid of any neurotoxic symptoms and showed signs of recovery. However, after 70 h, the neurotoxic symptoms recurred, and the patient was again treated with an additional 10 vials of ASV along with one dose of glycopyrrolate and neostigmine. Subsequently, the patient recovered completely from all the other symptoms of envenomation. This is the first report of recurrence of neurotoxic symptoms in a patient envenomed by Naja kaouthia in Assam, India and supports the need for greater attention and careful documentation of management of snakebite in the region.

Glycome Profiling and Bioprospecting Potential of the Himalayan Buddhist Handmade Paper of Tawang Region of Arunachal Pradesh.

The paper and pulp industry (PPI) is one of the largest industries that contribute to the growing economy of the world. While wood remains the primary raw material of the PPIs, the demand for paper has also grown alongside the expanding global population, leading to deforestation and ecological imbalance. Wood-based paper production is associated with enormous utilization of water resources and the release of different wastes and untreated sludge that degrades the quality of the environment and makes it unsafe for living creatures. In line with this, the indigenous handmade paper making from the bark of Daphne papyracea, Wall. ex G. Don by the Monpa tribe of Arunachal Pradesh, India is considered as a potential alternative to non-wood fiber. This study discusses the species distribution modeling of D. papyracea, community-based production of the paper, and glycome profiling of the paper by plant cell wall glycan-directed monoclonal antibodies. The algorithms used for ecological and geographical modeling indicated the maximum predictive distribution of the plant toward the western parts of Arunachal Pradesh. It was also found that the suitable distribution of D. papyracea was largely affected by the precipitation and temperature variables. Plant cell walls are primarily made up of cellulose, hemicellulose, lignin, pectin, and glycoproteins. Non-cellulosic cell wall glycans contribute significantly to various physical properties such as density, crystallinity, and tensile strength of plant cell walls. Therefore, a detailed analysis of non-cellulosic cell wall glycan through glycome profiling and glycosyl residue composition analysis is important for the polymeric composition and commercial processing of D. papyracea paper. ELISA-based glycome profiling results demonstrated that major classes of cell wall glycans such as xylan, arabinogalactans, and rhamnogalacturonan-I were present on D. papyracea paper. The presence of these polymers in the Himalayan Buddhist handmade paper of Arunachal Pradesh is correlated with its high tensile strength. The results of this study imply that non-cellulosic cell wall glycans are required for the production of high-quality paper. To summarize, immediate action is required to strengthen the centuries-old practice of handmade paper, which can be achieved through education, workshops, technical know-how, and effective marketing aid to entrepreneurs.

Venom of several Indian green pit vipers: Comparison of biochemical activities and cross-reactivity with antivenoms.

Green pit vipers, a name that can refer to several unrelated species, comprise a large group of venomous snakes found across the humid areas of tropical and sub-tropical Asia, and are responsible for most of the bite cases across this region. In India, green pit vipers belonging to several genera are prevalent in the northern and north-eastern hilly region, unrelated to species present in the peninsular region. In the present study, crude venom of representative species of green pit vipers present in the north and north-eastern hilly region of India (Trimeresurus erythrurus, T. septentrionalis, Viridovipera medoensis, and Popiea popieorum) were characterized to elucidate venom composition and venom variation. Profiling of crude venoms using SDS-PAGE and RP-HPLC methods revealed quantitative differences among the species. Further, in vitro biochemical assays reveal variable levels of phospholipase activity, coagulation activity, thrombin-like activity, fibrinogenolytic and haemolytic activity. This correlates with the pseudo-procoagulant effects on the haemostatic system of victims, which causes consumptive coagulopathy, frequently observed in patients bitten by green pit vipers. The immunoreactivity of Indian polyvalent antivenom and Thai green pit viper antivenom towards crude venoms were also evaluated by western blotting and inhibition of biochemical activities. The results exhibited poor efficacy of Indian polyvalent antivenom in neutralizing the venom toxins of crude venoms; however, Thai green pit viper antivenin (raised against the venom of Trimeresurus allbolabris, not present in India) showed higher immunoreactivity towards congeneric venoms tested. Analysis of green pit viper bite patients records from a community health centre in Assam, India, further revealed the inability of Indian polyvalent antivenom to reverse the extended coagulopathy featured.

Immunoinformatics mapping of potential epitopes in SARS-CoV-2 structural proteins.

All approved coronavirus disease 2019 (COVID-19) vaccines in current use are safe, effective, and reduce the risk of severe illness. Although data on the immunological presentation of patients with COVID-19 is limited, increasing experimental evidence supports the significant contribution of B and T cells towards the resolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Despite the availability of several COVID-19 vaccines with high efficacy, more effective vaccines are still needed to protect against the new variants of SARS-CoV-2. Employing a comprehensive immunoinformatic prediction algorithm and leveraging the genetic closeness with SARS-CoV, we have predicted potential immune epitopes in the structural proteins of SARS-CoV-2. The S and N proteins of SARS-CoV-2 and SARS-CoVs are main targets of antibody detection and have motivated us to design four multi-epitope vaccines which were based on our predicted B- and T-cell epitopes of SARS-CoV-2 structural proteins. The cardinal epitopes selected for the vaccine constructs are predicted to possess antigenic, non-allergenic, and cytokine-inducing properties. Additionally, some of the predicted epitopes have been experimentally validated in published papers. Furthermore, we used the C-ImmSim server to predict effective immune responses induced by the epitope-based vaccines. Taken together, the immune epitopes predicted in this study provide a platform for future experimental validations which may facilitate the development of effective vaccine candidates and epitope-based serological diagnostic assays.

Neutralization of Daboxin P activities by rationally designed aptamers.

Inefficacy and associated risks of current antivenom has raised the need for alternative approaches of snakebite management. Aptamers are one such alternative which is being pursued for therapeutic interventions as well as for design of diagnostic kits due to its high specificity. Present study focussed on designing and validating nucleic acid aptamers against snake venom PLA2, a hydrolytic enzyme present in all venomous snakes. The aptamers were designed by adding nucleic acid chain on the surface of Daboxin P, a major PLA2 enzyme of Daboia russelii venom. Binding characteristics of the aptamers were confirmed by docking to Daboxin P as well as acidic and basic PLA2s from different snake species using in silico docking. The aptamers folded into different tertiary structures and bound to the active and Ca2+ binding site of PLA2 enzymes. Molecular dynamics simulation analysis of Daboxin P-aptamer complexes showed that the complexes were stable in an aqueous environment. The electrophoretic mobility shift assay further confirmed the binding of the synthetic aptamers to Daboxin P and other snake venom PLA2 enzymes. The aptamers inhibited the sPLA2 activity with an IC50 value ranging between 0.52 µM and 0.77 µM as well as the anticoagulant activity of Daboxin P. The aptamers could also inhibit the PLA2 activity of Echis carinatus crude venom and anti-coagulant activity of Bungarus caeruleus crude venom, members of big four snakes. However, the aptamers didn't inhibit fibrinogenolytic or proteolytic activity of big four venom as well as the coagulation and hemolytic activities. Thus, aptamers can be rationally designed to inhibit the biochemical and biological activities of snake venom proteins.

Bipartite molecular approach for species delimitation and resolving cryptic speciation of Exobasidium vexans within the Exobasidium genus.

Exobasidium vexans, a basidiomycete pathogen, is the causal organism of blister blight disease in tea. The molecular identification of the pathogen remains a challenge due to the limited availability of genomic data in sequence repositories and cryptic speciation within its genus Exobasidium. In this study, the nuclear internal transcribed spacer rDNA region (ITS) based DNA barcode was developed for E. vexans, to address the problem of molecular identification within the background of cryptic speciation. The isolation of E. vexans strain was confirmed through morphological studies followed by molecular identification utilizing the developed ITS barcode. Phylogenetic analysis based on Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI) confirmed the molecular identification of the pathogen as E. vexans strain. Further, BI analysis using BEAST mediated the estimation of the divergence time and evolutionary relationship of E. vexans within genus Exobasidium. The speciation process followed the Yule diversification model wherein the genus Exobasidium is approximated to have diverged in the Paleozoic era. The study thus sheds light on the molecular barcode-based species delimitation and evolutionary relationship of E. vexans within its genus Exobasidium.

In silico and in vitro neutralization of PLA2 activity of Daboxin P by butein, mimosine and bakuchiol.

Medicinal plants have always been used for snakebite treatment by traditional healers but they lack scientific evidence of action. However secondary metabolites of such plants have been explored and found to inhibit the toxic effect of venom proteins. Literature survey from 2003 to 2019 resulted in identification of 251 secondary metabolites with such properties. In silico docking studies of these metabolites with modelled structure of Daboxin P, a PLA2 from Indian Daboia russelii revealed that butein, mimosine and bakuchiol bind to Daboxin P with high affinity. Butein interacted with the catalytic triad but mimosine and bakuchiol interacted with the Ca2+ binding residues of Daboxin P. In vitro validation showed that the molecules inhibited the sPLA2 activity of Daboxin P. Interestingly, mimosine and bakuchiol could also neutralize the anti-coagulatory activity of Daboxin P. Further, it was observed that butein and mimosine could neutralize the PLA2 activity of Indian big four venoms dose dependently. On the other hand, mimosine and bakuchiol could also neutralize the pro/anti-coagulatory effect of big four crude venom. Thus, in this study, three molecules have been identified which can neutralize the PLA2 activity and pro/anti-coagulatory effect of Daboxin P as well as crude venom of big four.

Naja kaouthia venom protein, Nk-CRISP, upregulates inflammatory gene expression in human macrophages.

Cysteine-Rich Secretory Proteins (CRISP) are widespread in snake venoms and known to target ion channels. More recently, CRISPs have been shown to mediate inflammatory responses. Involvement of potential receptor in CRISP-induced inflammatory reactions, however, remains unknown. A CRISP protein named as Nk-CRISP, was isolated from the venom of Naja kaouthia. The molecular mass of the purified protein was found to be ~25 kDa and the primary sequence was determined by MALDI TOF-TOF. The involvement of this protein in proinflammatory effects were evaluated in THP-1 macrophage-like cells. Nk-CRISP treated cells induced up-regulation of several inflammatory marker genes in dose dependent manner. Toll like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex are known to play crucial role in recognition of damage/pathogen-associated molecular patterns and activation of innate immune response. Therefore, we hypothesized that snake venom CRISP could also modulate the innate immune response via TLR4-MD2 complex. In-silico molecular docking study of cobra CRISP with TLR4-MD2 receptor complex reveals CRISP engages its cysteine-rich domain (CRD) to interact with complex. Inhibition of TLR4 signalling pathway using CLI-095 confirmed the role of TLR4 in Nk-CRISP induced inflammatory responses. Collectively, these findings imply that TLR4 initiates proinflammatory signalling following recognition of cobra CRISP and alteration of TLR4 receptor might improve or control CRISP induced inflammation.

Exploring rotavirus proteome to identify potential B- and T-cell epitope using computational immunoinformatics.

Rotavirus is the most common cause of acute gastroenteritis in infants and children worldwide. The functional correlation of B- and T-cells to long-lasting immunity against rotavirus infection in the literature is limited. In this work, a series of computational immuno-informatics approaches were applied and identified 28 linear B-cells, 26 conformational B-cell, 44 TC cell and 40 TH cell binding epitopes for structural and non-structural proteins of rotavirus. Further selection of putative B and T cell epitopes in the multi-epitope vaccine construct was carried out based on immunogenicity, conservancy, allergenicity and the helical content of predicted epitopes. An in-silico vaccine constructs was developed using an N-terminal adjuvant (RGD motif) followed by TC and TH cell epitopes and B-cell epitope with an appropriate linker. Multi-threading models of multi-epitope vaccine construct with B- and T-cell epitopes were generated and molecular dynamics simulation was performed to determine the stability of designed vaccine. Codon optimized multi-epitope vaccine antigens was expressed and affinity purified using the E. coli expression system. Further the T cell epitope presentation assay using the recombinant multi-epitope constructs and the T cell epitope predicted and identified in this study have not been investigated. Multi-epitope vaccine construct encompassing predicted B- and T-cell epitopes may help to generate long-term immune responses against rotavirus. The computational findings reported in this study may provide information in developing epitope-based vaccine and diagnostic assay for rotavirus-led diarrhea in children's.

1α, 25-dihydroxy Vitamin D3 containing fractions of Catharanthus roseus leaf aqueous extract inhibit preadipocyte differentiation and induce lipolysis in 3T3-L1 cells.

BACKGROUND: To investigate the potential of Catharanthus roseus leaf aqueous crude extract (CRACE) as a regulator of adipocyte development and function. METHODS: 3T3-L1 adipogenesis model was used to investigate the effect of CRACE on adipogenesis. 3T3-L1 preadipocytes (for adipogenic differentiation) and mature 3T3-L1 adipocytes (for adipocyte function) were treated with non-toxic doses of CRACE. The outcomes were corroborated by intracellular lipid accumulation, expression of pro-and anti-adipogenic effector molecules. To investigate CRACE mediated lipolysis, cAMP accumulation, glycerol release and phosphorylation of key effector molecules were tested in treated mature adipocytes. Finally, the extract was fractionated to identify the active molecule/s in the extract. RESULTS: CRACE significantly reduced adipocyte differentiation by modulating PPARγ expression. At early stage CRACE directly targeted Lipin1 expression and consequently impacted KLF7, subsequently expression of GATA2, CEBPα, SREBP1c were targeted, with PPARγ expression, particularly curtailed. While CRACE significantly reduced several lipogenic genes like FAS and GPD1 in mature adipocytes, concomitantly, it greatly increased lipolysis resulting in decreased lipid accumulation in mature adipocytes. The increase in lipolysis was due to decreased Akt activation, increased cAMP level, and PKA activity. The fractionation of CRACE allowed identification of two fractions with potent anti-adipogenic activity. Both the fractions contained 1α, 25-dihydroxy Vitamin D3 as major component. CONCLUSIONS: 1α, 25-dihydroxy Vitamin D3 containing CRACE can be developed into an effective anti-obesity formulation that decreases adipogenesis and increases lipid catabolism.

Proteomics of Naja kaouthia venom from North East India and assessment of Indian polyvalent antivenom by third generation antivenomics.

In the present study, venom composition, toxic effects, and immunological characteristics of Naja kaouthia venom from North East India has been studied. Using RP-HPLC, venom components were separated and proteins in the fractions were identified using ESI-LC MS/MS. Proteins identified belong to 9 different snake venom protein families. Three finger toxins and PLA2 were the most abundant protein families detected by mass spectrometry analysis. The other minor proteins families identified in the venom were kunitz-type serine inhibitors, waprin, L-amino acid oxidase, CRISP, vespyrn, nerve growth factor and metalloproteinase. This proteome composition correlated with the tested enzymatic and toxic activities of the venom. Western blot and third generation antivenomics analysis using Vins polyvalent antivenom revealed immunoreactivity towards Naja kaouthia venom of North East India. Concentration-dependent immunocapturing profile carried out using RP-HPLC displayed immunerecognition of majority of venom proteins of Naja kaouthia except few three-finger toxins. Presence of such non-immunodepleted toxins apparently may affect the performance of Vins polyvalent antivenom. Thus, inclusion of antibodies of most relevant non-immunorecognized toxins in antivenom might help to improve the quality of antivenom. BIOLOGICAL SIGNIFICANCE: Envenomings by genus Naja, represent a serious medical problem in Asian countries including North east India. In North East India, Naja kaouthia is most prevalent cobra species causing a large number of fatalities. To gain deeper insight into the spectrum of medically relevant toxins, we applied proteomics approach to unveil the proteome profile of Naja kaouthia venom. The proteomic analysis divulged the presence of two major protein families: three finger toxins and phospholipases A2. In general, polyvalent antivenom is administered for Naja kaouthia envenomings, however, this venom is not included in the immunization mixtures (only Indian Big Four venoms) for production of these polyvalent antivenoms. For the first time, third generation antivenomics approach was used to decipher maximal binding capacity of Indian polyvalent antivenom against Naja kaouthia venom. Although Vins polyvalent antivenom was effective in immunocapturing majority of venom components, however, large amount of antivenom was required to immunocapture the venom proteins. Moreover, the study revealed poor immunorecognition capacity of Vins antivenom towards four three finger toxin subtypes. This may have significant impact on antivenom efficacy in treating Naja kaouthia envenomings.

Comparative analysis of Naja kaouthia venom from North-East India and Bangladesh and its cross reactivity with Indian polyvalent antivenoms.

Naja kaouthia is one of the most prevalent medically important snakes of North East India and Bangladesh responsible for most of the bite cases. In this study, an attempt was made to decipher venom variation of Naja kaouthia venom from North East India and Bangladesh. Using multidimensional methods including reverse phase HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-PAGE) and two-dimensional gel electrophoresis (2D-PAGE), the quantitative differences in venom composition have been revealed. Moreover, tested in-vitro biochemical and biological activities also exhibited differences which could be due to venom variability. Furthermore, neutralization efficacy of commercially available Indian polyvalent antivenoms (Vins, Bharat Serum, Haffkine) was evaluated and the results displayed significant differences in neutralizing efficacy between the antivenoms. Immunoblotting experiments showed antivenom molecules cross reacted with high molecular mass components while poorly reacted towards low molecular mass proteins. Immuno-depletion study demonstrated that Vins polyvalent antivenom was poor in immunocapturing the venom proteins of both North East Indian and Bangladesh origin Naja kaouthia at the ratio of 1:16 (venom: antivenom).