Bioconjugation of Meldrum's acid activated furan: A detergent compatible assay for protein quantitation.

A simple yet efficient assay for the quantitation of proteins ranging from plasma proteins to purified proteins from whole cell lysate, based on the bioconjugation reaction between protein and Meldrum's acid Activated Furan (MAF) is described. This easy to use, sensitive method is based on the conjugation of amine functionalities present on the protein with MAF to form the corresponding Donor Acceptor Stenhouse Adducts (DASAs) with characteristic absorption in the visible region. The reaction is rapid as well as reproducible and shows a proportionate increase in color change over a broad range of protein concentration. The assay was found to be sensitive up to 0.125 mg/mL concentration of the protein and was compatible with most of the commonly employed detergents and isolation protocols which makes it ideal for the estimation of protein samples containing detergents. Another striking feature of this protocol is its tolerance towards other major interference contributors such as chelating agents, reducing agents, carbohydrates and protease inhibitors.

Ginger extract activates caspase independent paraptosis in cancer cells via ER stress, mitochondrial dysfunction, AIF translocation and DNA damage.

The rhizome of ginger (Zingiber officinale) a common culinary agent is also known for its medicinal activity. We have earlier reported that pure 6-shogaol, an important component of ginger induces paraptosis in triple negative breast cancer (MDA-MB-231) and non small cell lung (A549) cancer cells. However, the chemopreventive potential of the whole ginger extract in food remains to be elucidated. Here, we demonstrate for the first time that ginger extract (GE) triggers similar anticancer activity/paraptosis against the same cell lines but through different molecular mechanisms. Q-TOF LC-MS analysis of the extract showed the presence of several other metabolites along with 6-shogaol and 6-gingerol. GE induces cytoplasmic vacuolation through ER stress and dilation of the ER. Drastic decrease in the mitochondrial membrane potential and ATP production along with the excess generation of ROS contributed to mitochondrial dysfunction. Consequently, GE caused the translocation of apoptosis inducing factor to the nucleus leading to the fragmentation of DNA. Taken together, these show a novel mechanism for ginger extract induced cancer cell death that can be of potential interest for cancer preventive strategies.

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.

Mechanistic understanding of Phenyllactic acid mediated inhibition of quorum sensing and biofilm development in Pseudomonas aeruginosa.

Pseudomonas aeruginosa depends on its quorum sensing (QS) system for its virulence factors' production and biofilm formation. Biofilms of P. aeruginosa on the surface of indwelling catheters are often resistant to antibiotic therapy. Alternative approaches that employ QS inhibitors alone or in combination with antibiotics are being developed to tackle P. aeruginosa infections. Here, we have studied the mechanism of action of 3-Phenyllactic acid (PLA), a QS inhibitory compound produced by Lactobacillus species, against P. aeruginosa PAO1. Our study revealed that PLA inhibited the expression of virulence factors such as pyocyanin, protease, and rhamnolipids that are involved in the biofilm formation of P. aeruginosa PAO1. Swarming motility, another important criterion for biofilm formation of P. aeruginosa PAO1, was also inhibited by PLA. Gene expression, mass spectrometric, functional complementation assays, and in silico data indicated that the quorum quenching and biofilm inhibitory activities of PLA are attributed to its ability to interact with P. aeruginosa QS receptors. PLA antagonistically binds to QS receptors RhlR and PqsR with a higher affinity than its cognate ligands N-butyryl-L-homoserine lactone (C4-HSL) and 2-heptyl-3,4-dihydroxyquinoline (PQS; Pseudomonas quinolone signal). Using an in vivo intraperitoneal catheter-associated medaka fish infection model, we proved that PLA inhibited the initial attachment of P. aeruginosa PAO1 on implanted catheter tubes. Our in vitro and in vivo results revealed the potential of PLA as anti-biofilm compound against P. aeruginosa.

Identification and characterization of a library of surfactins and fengycins from a marine endophytic Bacillus sp.

An endophytic bacterial strain from a marine green alga, Ulva lactuca, was isolated and identified by 16S rRNA gene sequencing method. The bacterial isolate was found to secrete two major families of cyclic depsilipopeptides, surfactins, and fengycins. Sequencing of the isolated lipopeptides was carried out using the MSn data obtained from an electrospray ionization (ESI) ion trap mass spectrometer coupled to an HPLC system. The assigned sequences were confirmed by a chemical derivatization approach involving esterification followed by mass spectrometric analysis. Distinction of leucine residues from isoleucine was established through a combined electron transfer dissociation-collision-induced dissociation (ETD-CID) method. The fengycins described in this study were found to cause significant delay of growth of two plants, Vigna radiata (mung bean) and Oryza sativa (rice). To the best of our knowledge, this is the first study describing identification and characterization of cyclic peptides from an endophytic Bacillus sp. isolated from marine algae.

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.

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.

Sensitization of hepatocellular carcinoma cells towards doxorubicin and sorafenib is facilitated by glucosedependent alterations in reactive oxygen species, P-glycoprotein and DKK4.

Altered glucose uptake and metabolism is the key characteristic of cancer cells including hepatocellular carcinoma (HCC). However, role of glucose availability in chemotherapeutic outcome of HCC is unclear. The present study investigates the effect of glucose facilitated sensitization of HCC cells towards doxorubicin (DOX) and sorafenib (SORA). In HCC cells, we observed that hyperglycemic culture condition (HG) is associated with increased sensitivity towards DOX and SORA. P-glycoprotein (P-gp), a transporter involved in drug efflux, was elevated in HCC cells in NG, rendering them less susceptible to DOX and SORA. Further, this study demonstrated that knockdown of dickkopf protein 4 (DKK4), a Wnt antagonist protein, causes enhanced glucose uptake and reduction in P-gp level rendering HCC cells in NG sensitive to DOX and SORA. Moreover, HG elevates the level of intracellular reactive oxygen species (ROS), which regulates P-gp. Alteration in intracellular ROS did not directly affect regulation of DKK4 in HCC cells. Functional assays suggest that alterations in DKK4 and P-gp level in HCC cells are dependent on glucose availability and changes in ROS level because of enhanced glucose utilization, respectively. Collectively, the present study highlights direct involvement of glucose-induced ROS, DKK4 and P-gp in altering the sensitivity of HCC cells towards DOX and SORA.

Combination of Repurposed Drug Diosmin with Amoxicillin-Clavulanic acid Causes Synergistic Inhibition of Mycobacterial Growth.

Effective therapeutic regimens for the treatment of tuberculosis (TB) are limited. They are comprised of multiple drugs that inhibit the essential cellular pathways in Mycobacterium tuberculosis (Mtb). The present study investigates an approach which enables a combination of Amoxicillin-Clavulanic acid (AMC) and a repurposed drug for its synergistic effect towards TB treatment. We identified Diosmin (DIO), by targeting the active site residues of L,D-transpeptidase (Ldt) enzymes involved in Mtb cell wall biosynthesis by using a structure-based drug design method. DIO is rapidly converted into aglycone form Diosmetin (DMT) after oral administration. Binding of DIO or DMT towards Ldt enzymes was studied using molecular docking and bioassay techniques. Combination of DIO (or DMT) and AMC exhibited higher mycobactericidal activity against Mycobacterium marinum as compared to individual drugs. Scanning electron microscopy study of M. marinum treated with AMC-DIO and AMC-DMT showed marked cellular leakage. M. marinum infected Drosophila melanogaster fly model showed an increased fly survival of ~60% upon treatment with a combination of AMC and DIO (or DMT). Finally, the enhanced in vitro antimicrobial activity of AMC-DIO was validated against Mtb H37Ra and a MDR clinical isolate. Our results demonstrate the potential for AMC and DIO (or DMT) as a synergistic combination for the treatment of TB.

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.

Clove Bud Oil Modulates Pathogenicity Phenotypes of the Opportunistic Human Pathogen Pseudomonas aeruginosa.

Earlier studies from our laboratory have demonstrated that clove bud oil (CBO) attenuates expression of certain virulence factors of Pseudomonas aeruginosa PAO1. Here, we probe more deeply into the effect of CBO on four pseudomonal proteases - elastase A, elastase B, protease IV and alkaline protease - each known to play key roles in disease pathogenesis. CBO inhibited the activity of these proteases present in the bacterial culture supernatant. Zymography studies indicated that these proteases can activate host matrix metalloproteases (MMPs) to establish infection, through conversion of pro-MMP-2 to active MMP-2. PAO1 is a predominant pathogen in burn wound infections and we show the modulatory effect of CBO on MMPs in an in vitro model of burn injury. Furthermore, CBO induced dose-dependent neutrophil extracellular trap formation in human neutrophils. CBO also increased the survival of C. elegans infected with PAO1, establishing an anti-infective role in a whole animal model of pathogenesis. LC-MS/MS analysis indicated that CBO treatment elicited a significant reduction of signalling molecules (Acyl-Homoserine-Lactone) involved in quorum sensing regulation. Our observations demonstrate that CBO attenuates key virulence mechanisms of this important human pathogen, while concomitantly enhancing host innate immunomodulatory functions, with potential implications for topical therapy against antibiotic-resistant infections.

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.

Structural characterization of an unusually stable cyclic peptide, kalata B2 from Oldenlandia affinis.

Kalata peptides are isolated from an African medicinal plant, Oldenlandia affinis, an aqueous decoction of which can be ingested to accelerate uterine contraction during childbirth. The closely packed disulfide core of kalata peptides confers unusual stability against thermal, chemical, and enzymatic degradation. The molecular arrangement may hamper NMR-assisted disulfide connectivity assignment. We have combined NMR with high-resolution mass spectrometry (MS) and MS/MS of native and chemically derivatized kalata B2 to determine its amino acid sequence and disulfide connectivity. Infrared multiphoton dissociation establishes the disulfide bond linkages in kalata B2 as I-IV, II-V and III-VI.

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.

Strategy to enhance efficacy of doxorubicin in solid tumor cells by methyl-ß-cyclodextrin: Involvement of p53 and Fas receptor ligand complex.

Doxorubicin (DOX) is one of the preferred drugs for treating breast and liver cancers. However, its clinical application is limited due to severe side effects and the accompanying drug resistance. In this context, we investigated the effect on therapeutic efficacy of DOX by cholesterol depleting agent methyl-ß-cyclodextrin (MCD), and explored the involvement of p53. MCD sensitizes MCF-7 and Hepa1-6 cells to DOX, Combination of MCD and marginal dose of DOX reduces the cell viability, and promoted apoptosis through induction of pro-apoptotic protein, Bax, activation of caspase-8 and caspase-7, down regulation of anti-apoptotic protein Bcl-2 and finally promoting PARP cleavage. Mechanistically, sensitization to DOX by MCD was due to the induction of FasR/FasL pathway through p53 activation. Furthermore, inhibition of p53 by pharmacological inhibitor pifithrin-α (PFT-α) or its specific siRNA attenuated p53 function and down-regulated FasR/FasL, thereby preventing cell death. Animal experiments were performed using C57BL/6J mouse isografted with Hepa1-6 cells. Tumor growth was retarded and survival increased in mice administered MCD together with DOX to as compared to either agent alone. Collectively, these results suggest that MCD enhances the sensitivity to DOX for which wild type p53 is an important determinant.

De novo sequencing and disulfide mapping of a bromotryptophan-containing conotoxin by Fourier transform ion cyclotron resonance mass spectrometry.

T-1-family conotoxins belong to the T-superfamily and are composed of 10-17 amino acids. They share a common cysteine framework and disulfide connectivity and exhibit unusual posttranslational modifications, such as tryptophan bromination, glutamic acid carboxylation, and threonine glycosylation. We have isolated and characterized a novel peptide, Mo1274, containing 11 amino acids, that shows the same cysteine pattern, -CC-CC, and disulfide linkage as those of the T-1-family members. The complete sequence, GNWCCSARVCC, in which W denotes bromotryptophan, was derived from MS-based de novo sequencing. The FT-ICR MS/MS techniques of electron capture dissociation (ECD), infrared multiphoton dissociation, and collision-induced dissociation served to detect and localize the tryptophan bromination. The bromine contributes a distinctive isotopic distribution in all fragments that contain bromotryptophan. ECD fragmentation results in the loss of bromine and return to the normal isotopic distribution. Disulfide connectivity of Mo1274, between cysteine pairs 1-3 and 2-4, was determined by mass spectrometry in combination with chemical derivatization employing tris(2-carboxyethyl)phosphine, followed by differential alkylation with N-ethylmaleimide and iodoacetamide. The ECD spectra of the native and partially modified peptide reveal a loss of bromine in a process that requires the presence of a disulfide bond.