Comparative functional characterization and in vitro immunological cross-reactivity studies on Daboia russelii and Craspedocephalus malabaricus venom.

BACKGROUND: Snake venom is a complex mixture of organic and inorganic constituents, including proteins and peptides. Several studies showed that antivenom efficacy differs due to intra- and inter-species venom variation. METHODS: In the current study, comparative functional characterization of major enzymatic proteins present in Craspedocephalus malabaricus and Daboia russelii venom was investigated through various in vitro and immunological cross-reactivity assays. RESULTS: The enzymatic assays revealed that hyaluronidase and phospholipase A2 activities were markedly higher in D. russelii. By contrast, fibrinogenolytic, fibrin clotting and L-amino acid oxidase activities were higher in C. malabaricus venom. ELISA results suggested that all the antivenoms had lower binding potential towards C. malabaricus venom. For D. russelii venom, the endpoint titration value was observed at 1:72 900 for all the antivenoms. In the case of C. malabaricus venom, the endpoint titration value was 1:2700, except for Biological E (1:8100). All these results, along with the avidity assays, indicate the strength of venom-antivenom interactions. Similarly, the western blot results suggest that all the antivenoms showed varied efficacies in binding and detecting the venom antigenic epitopes in both species. CONCLUSIONS: The results highlight the need for species-specific antivenom to better manage snakebite victims.

The Need for Next-Generation Antivenom for Snakebite Envenomation in India.

The limitations posed by currently available antivenoms have emphasized the need for alternative treatments to counteract snakebite envenomation. Even though exact epidemiological data are lacking, reports have indicated that most global snakebite deaths are reported in India. Among the many problems associated with snakebite envenomation, issues related to the availability of safer and more efficient antivenoms are of primary concern. Since India has the highest number of global snakebite deaths, efforts should be made to reduce the burden associated with snakebite envenoming. Alternative methods, including aptamers, camel antivenoms, phage display techniques for generating high-affinity antibodies and antibody fragments, small-molecule inhibitors, and natural products, are currently being investigated for their effectiveness. These alternative methods have shown promise in vitro, but their in vivo effectiveness should also be evaluated. In this review, the issues associated with Indian polyvalent antivenoms in neutralizing venom components from geographically distant species are discussed in detail. In a nutshell, this review gives an overview of the current drawbacks of using animal-derived antivenoms and several alternative strategies that are currently being widely explored.

Venomics and antivenomics of Indian spectacled cobra (Naja naja) from the Western Ghats.

Venom proteome profiling of Naja naja from the Western Ghats region in Kerala was achieved through SDS-PAGE and RP-HPLC followed by Q-TOF LC-MS/MS analysis, incorporating PEAKS and Novor assisted de novo sequencing methodologies. A total of 115 proteins distributed across 17 different enzymatic and non-enzymatic venom protein families were identified through conventional and 39 peptides through homology-driven proteomics approaches. Fourteen peptides derived through de novo complements the Mascot data indicating the importance of homology-driven approaches in improving protein sequence information. Among the protein families identified, glutathione peroxidase and endonuclease were reported for the first time in the Indian cobra venom. Immunological cross-reactivity assessed using Indian polyvalent antivenoms suggested that VINS showed better EC50 (2.48 µg/mL) value than that of PSAV (6.04 µg/mL) and Virchow (6.03 µg/mL) antivenoms. Western blotting experiments indicated that all the antivenoms elicited poor binding specificities, especially towards low molecular mass proteins. Second-generation antivenomics studies revealed that VINS antivenom was less efficient to detect many low molecular mass proteins such as three-finger toxins and Kunitz-type serine protease Inhibitors. Taken together, the present study enabled a large-scale characterization of the venom proteome of Naja naja from the Western Ghats and emphasized the need for developing more efficient antivenoms.

Identification of carbonylated proteins from monocytic cells under diabetes-induced stress conditions.

Diabetes is a metabolic disorder characterized by the presence of elevated glucose in the blood and enhanced oxidative stress. It affects the cellular homeostasis that leads to the development of micro-and macro-vascular complications. Monocytes are the primary immune cells present in the circulatory system. Under high-glucose conditions, the cells undergo oxidative stress and secrete reactive oxygen species. The enhanced release of reactive species is known to modify biomolecules like proteins and nucleic acids. Protein carbonylation, one of the most harmful and irreversible protein modifications, is considered as a key player in the progression of diabetes and associated complications. Hence, the present study explores the identification of carbonylated proteins from the monocytes under diabetic stress and determination of their site of modification. Combined avidin affinity chromatography and bottom-up proteomics experiments identified 13 consistently expressed carbonylated proteins. Most of the identified proteins were reported to have altered functions under diabetic conditions that contribute to the development of diabetes-associated inflammation and complications. We were able to determine oxidative stress-induced modifications on Lys, Val, Ile, Cys, Thr and Asp residues.

Delineating the venom toxin arsenal of Malabar pit viper (Trimeresurus malabaricus) from the Western Ghats of India and evaluating its immunological cross-reactivity and in vitro cytotoxicity.

The venom protein components of Malabar pit viper (Trimeresurus malabaricus) were identified by combining SDS-PAGE and ion-exchange chromatography pre-fractionation techniques with LC-MS/MS incorporating Novor and PEAKS-assisted de novo sequencing strategies. Total 97 proteins that belong to 16 protein families such as L-amino acid oxidase, metalloprotease, serine protease, phospholipase A2, 5'-nucleotidase, C-type lectins/snaclecs and disintegrin were recognized from the venom of a single exemplar species. Of the 97 proteins, eighteen were identified through de novo approaches. Immunological cross-reactivity assessed through ELISA and western blot indicate that the Indian antivenoms binds less effectively to Malabar pit viper venom components compared to that of Russell's viper venom. The in vitro cell viability assays suggest that compared to the normal cells, MPV venom induces concentration dependent cell death in various cancer cells. Moreover, crude venom resulted in chromatin condensation and apoptotic bodies implying the induction of apoptosis. Taken together, the present study enabled in dissecting the venom proteome of Trimeresurus malabaricus and revealed the immuno-cross-reactivity profiles of commercially available Indian polyvalent antivenoms that, in turn, is expected to provide valuable insights on the need in improving antivenom preparations against its bite.

Mass spectrometry-assisted venom profiling of Hypnale hypnale found in the Western Ghats of India incorporating de novo sequencing approaches.

Hypnale hypnale (hump-nosed pit viper) is considered to be one among the medically important venomous snake species of India and Sri Lanka. In the present study, venom proteome profiling of a single Hypnale hypnale from Western Ghats of India was achieved using SDS-PAGE based protein separation followed by LC-MS/MS analysis. The identities of the proteins that were not established using the Mascot search were determined through de novo sequencing tools such as Novor followed by MS-BLAST based sequence similarity search algorithm and PEAKS proteomics software. The combined proteomics analysis revealed a total of 37 proteins belonging to nine different snake venom families, in which 7 proteins were exclusively identified through de novo strategies. The enzymatic and non-enzymatic venom protein families identified include serine proteases, metalloproteases, phospholipase A2, thrombin-like enzymes, phospholipase B, C-type lectins/snaclecs, disintegrins, cysteine rich secretory proteins and nerve growth factor. Among these, disintegrins, nerve growth factor, phospholipase B and cysteine rich secretory protein families were identified for the first time in HPV venom. This could possibly explain the regiospecific venom variation seen across snake species. Taken together, the venom proteome profiling on Indian Hypnale hypnale venom correlates with the clinical manifestations often seen in the envenomed victims.

Identification, purification, biochemical and mass spectrometric characterization of novel phycobiliproteins from a marine red alga, Centroceras clavulatum.

Phycobilisomes are light-harvesting protein complexes and are widely distributed in red algae and cyanobacteria. Each phycobilisome contains highly fluorescent protein components called phycobiliproteins. Based upon the distinct physiochemical properties, phycobiliproteins are classified as allophycocyanin, phycocyanin, phycoerythrin and phycoerythrocyanin. In the present study, we describe purification and structural characterization of a novel phycocyanin and phycoerythrin isolated from a marine red macroalga, Centroceras clavulatum. The absorbance and fluorescence studies indicated that the purified proteins belong to R-Phycocyanin (R-PC) and R-Phycoerythrin (R-PE). The single bands under native-polyacrylamide gel electrophoresis revealed the intact molecular weights of R-PC and R-PE as 110kDa and 250kDa. The polypeptide compositions of the two proteins were demonstrated by SDS-PAGE. The result showed that R-PC contains two bands at 17 and 21kDa and were identified as α and ß subunits through mass spectrometry based proteomics experiments. SDS-PAGE of R-PE showed three distinct bands at 18, 19 and 35kDa and was subsequently identified as α, ß and γ subunits. The near-complete amino acid sequences of α and ß subunits of R-PC and R-PE were derived from mass spectrometric data combined with Mascot software and multiple de novo sequencing tools followed by homology search and manual validation.

Weight control interventions improve therapeutic efficacy of dacarbazine in melanoma by reversing obesity-induced drug resistance.

BACKGROUND: Obesity-related cellular, metabolic, and molecular alterations have been shown to increase cancer risk and tumor progression and are associated with poorer therapeutic outcome in cancer patients. However, the impact of obesity and weight-control interventions on the therapeutic response in melanoma is poorly understood. METHODS: High fat diet (HFD)-induced obese mouse model was used in this study to evaluate the outcome of dacarbazine (DTIC) therapy in melanoma. We employed LC-MS/MS to determine the quantity of the drug in tumor, and in various tissues. Unique in vitro approach was used to complement in vivo findings by culturing melanoma cells in either conditioned medium (CM) obtained from differentiated adipocytes or in serum collected from experimental mice. RESULTS: We report that diet-induced obesity impairs the outcome of DTIC therapy and reduces overall survival in tumor-bearing mice. We provide evidence that obesity restricts the accessibility of DTIC to tumor tissue. Critically, upon curtailing adiposity, accumulation and efficacy of DTIC is significantly improved. Moreover, using appropriate in vitro approaches, we show that melanoma cells exhibit a drug-resistant phenotype when cultured in serum collected from diet-induced obese mice or in CM collected from 3T3-L1 adipocytes. The impaired therapeutic response to DTIC in obese state is mediated by fatty acid synthase (FASN), caveolin-1 (Cav-1), and P-glycoprotein (P-gp). The response to DTIC and overall survival were improved upon employing weight control interventions in the tumor-bearing HFD-fed (obese) mice. CONCLUSIONS: This study indicates that obesity not only supports rapid melanoma progression but also impairs the outcome of chemotherapy, which can be improved upon employing weight control interventions. From clinically relevant point of view, our study exemplifies the importance of lifestyle interventions in the treatment of obesity-promoted cancers.