Unravelling the key steps impairing the metabolic state of Xanthomonas cells undergoing programmed cell death.

Programmed cell death (PCD) has been reported in Xanthomonas axonopodis pv. glycines (Xag) wild type earlier and was indirectly shown to be induced by metabolic stress; however, deciphering the key proteins regulating the metabolic stress remained unrevealed. In this study, transcriptomic and proteomic analyses were performed to investigate the prominent pathways, having a role in the induction of metabolic stress in Xag cells undergoing PCD. A comprehensive analysis of transcriptome and proteome data revealed the major involvement of metabolic pathways related to branched chain amino acid degradation, such as acyl-CoA dehydrogenase and energy-yielding, ubiquinol:cytochrome c oxidoreductase complex, in Xag cells undergoing PCD. Consequently, oxidative stress response genes showed major upregulation in Xag cells in PCD-inducing medium; however, no such upregulation was observed at the protein level, indicative of depleted protein levels under excessive stress conditions. Activation of stress response and DNA repair proteins was also observed in Xag cells grown in PCD-inducing medium, which is indicative of excessive cellular damage. Thus, the findings indicate that programmed cell death in Xag is an outcome of metabolic stress in nutrient condition not suitable for a plant pathogen like Xanthomonas, which is more acclimatised with altogether a different nutritional requirement predominantly having an enriched carbohydrate source.

Programmed cell death in Xanthomonas axonopodis pv. glycines is associated with modulation of gene expression resulting in altered states of motility, biofilm and virulence.

One of the foremost report of apoptosis-like programmed cell death (PCD) came from Xanthomonas axonopodis pv. glycines (Xag), which displayed rapid post-exponential cell death in PCD inducing media (PIM) but not in a non-inducing media (PNIM). The current study aims to decipher for the first time, the advantages of the existence of PCD in this phytopathogenic microorganism. Analysis of RNA-seq under inducing and non-inducing conditions, revealed differential expression of a number of genes related to key physiology of Xag, such as, motility, xanthan biosynthesis and export as well as virulence. A PCD negative mutant Xag M42 displayed diminished virulence and a contrasting transcriptome pattern. In vitro experiments revealed that under PCD inducing condition, Xag produced negligible xanthan gum as well as extracellular amylase, displayed enhanced swarming motility, released copious e-DNA and formed scanty biofilm. Lack of 'diffusible signalling factor' production was eliminated as possible reason for PCD-induction. Altogether, it appears that, in planta existence of the pathogen metabolically resembles PNIM, and on being transferred to PIM, the cells experience oxidative stress and circumvents it by adopting PCD as an altruistic response. Survival of the remaining population is encouraged by upregulating motility, detachment from the fragile biofilm to achieve dispersal.

Gamma radiation processing for extending shelf-life and ensuring quality of minimally processed ready-to-eat onions.

Onions are always in high demand owing to various culinary as well as health protective properties and these days there is increased consumer preference for ready-to-eat or ready-to-cook onions. In this context, the current study was aimed to extend the keeping quality of minimally processed onions for an extended period while ensuring microbial safety as well as sprouting inhibition through an integrated approach. The optimized combinatorial approach included gamma radiation treatment (Dmin60 Gy), minimal processing (de-skinning and scooping) and packaging in trays wrapped with polypropylene (PP; 10 µm thick) film followed by storage at low temperature (4-6 °C, relative humidity RH 65-70%). The parameters like shelf life, physico-chemical (colorimetry, moisture), organoleptic and nutritional properties were comprehensively assessed and found to be well retained up to 30 days with moisture loss of ≤ 5% and overall acceptability rating of 7 on 9-point hedonic scale. Microbiological analyses confirmed absence of Salmonella spp in these stored onions thus ensuring microbial safety. Nutritional profiling including carbohydrate, protein, fat, energy, and ash content revealed no significant change due to the processing as well as during storage. Thus, the radiation processing of freshly harvested bulbs followed by minimal processing, packing in formulated package and storage under low temperature conditions were found acceptable up to 30 days in the ready-to-eat form. Current findings provide credible evidences ascertaining extended shelf-life as well ensuring microbial safety of processed onions for commercial utilization by the food industries.

Release of an encrypted, highly potent ACE-inhibitory peptide by enzymatic hydrolysis of moth bean (Vigna aconitifolia) protein.

Aim: Dietary approaches for the regulation of blood pressure are the need of the hour. Hence, identifying the foods possessing such activity is gaining importance. With this aim, moth bean (Vigna aconitifolia), an underutilized pulse, was explored for the presence of antihypertensive activity in terms of angiotensin converting enzyme (ACE)-inhibition bioactivity. Methods: Defatted moth bean protein concentrate was hydrolyzed by using different proteases including Alcalase, papain, and trypsin, to identify the enzyme producing highly potent ACE inhibitory peptides. The hydrolysate showing the highest ACE inhibitory activity was further fractionated using an ultrafiltration membrane (10, 3 and 1 kDa) based on ACE inhibitory activity. The active fraction was further subjected to the ion-exchange chromatography followed by RP-HPLC and LC-MS/MS analysis for the enrichment and identification of ACE inhibitory peptides. Finally, based on the bioinformatic analysis, few peptides were synthesized and evaluated for ACE inhibitory activity, followed by docking study and molecular dynamic simulation of a peptide with the highest ACE inhibitory activity. Results and discussion: Out of the three proteases, Alcalase-derived hydrolysate showed the highest (~59%) ACE inhibition activity. Molecular weight-based fractionation revealed that <1 kDa fraction possessed the highest ACE inhibitory activity. Activity guided separation of 1 kDa fraction using ion-exchange chromatography, RP-HPLC and LC-MS/MS showed the presence of about 45 peptides. Based on the bioinformatic analysis, 15 peptides were synthesized and evaluated for ACE inhibitory activity. Among these, a novel octapeptide FPPPKVIQ showed the highest ACE inhibitory activity (93.4%) with an IC50 of 0.24 µM. This peptide retained about 59% activity post gastrointestinal digestion simulation. A Dixon plot as well as docking studies revealed the uncompetitive inhibitory nature of this peptide with a Ki value of 0.81 µM. Molecular dynamic simulation studies till 100 ns ensured the stability of the ACE-peptide complex. Conclusion: Thus, present study identified a novel potent ACE inhibitory peptide from moth bean that can be incorporated in a functional dietary formulation for regulation of hypertension.

Alpha-cadinol as a potential ACE-inhibitory volatile compound identified from Phaseolus vulgaris L. through in vitro and in silico analysis.

Hypertension is a major risk factor of cardiovascular diseases, which is mainly caused due to over activation of renin-angiotensin system. The angiotensin converting enzyme (ACE), which is involved in formation of angiotensin II from angiotensin I, causes the blood vessels to constrict, in turn leading to hypertension. The current study was initiated to understand the role of bioactive volatile compounds from Phaseolus vulgaris L. (common bean), in ACE enzyme inhibition. Beans aqueous extract (BAE) showed maximum ACE inhibition of 88.4 ± 0.8% in comparison to other commonly consumed vegetables like spinach and garlic. The head space gas chromatography-mass spectrometry analysis showed the presence of a number of terpenes and terpenoids, which were present prominently in BAE. In silico molecular docking studies indicated that among the other volatile compounds, alpha-cadinol (-7.27 kcal/mol) and ar-tumerone (-6.44 kcal/mol) have the maximum binding affinity with the active site of ACE, as compared to that of captopril (-6.41 kcal/mol). The molecular dynamic simulation in biological environment, showed that alpha-cadinol forms a stable complex with ACE, with average binding energy of -42 kJ/mol. The ACE:alpha-cadinol complex was found to be stable mainly due to the hydrophobic interactions of alpha-cadinol with active site residues (Tyr523 and Phe457) of ACE. The in silico drug-likeness analysis showed that alpha-cadinol is appropriate for human system with no predicted hepatotoxicity or mutagenicity (AMES toxicity).Communicated by Ramaswamy H. Sarma.

Simulation study to assess the effectiveness of gamma radiation for inactivation of viruses on food packaging material.

The recent COVID-19 pandemic spread across the globe has raised the concern about the possible transmission of viruses through food packaging material during domestic and international trade. Therefore, mitigation strategies are needed to address these safety issues. Preliminary in-silico study showed that interactions between food packaging material and viral surface proteins were possibly hydrophobic in nature with most favourable interaction having a binding free energy of -5.24 kcal/mol. Since these interactions can cause viruses to adsorb on the food packets and get transmitted during supply chain, it is necessary to inactivate the viruses. In this context, efficacy of gamma irradiation in inactivating the viruses on the food packaging material was assessed. For this simulation study P1 (virulent) bacteriophage of E. coli was used as a model system. Gamma irradiation of food packets at an absorbed dose >8 kGy was found to completely inactivate the infectivity of P1(virulent) bacteriophage when co-cultured with E. coli host and assayed for viral plaque formation. Reduction in infectivity of P1(vir) phage was more prominent at ambient temperature (25 ± 2 °C) as compared to cold temperature (6 ± 2 °C) when assayed after storage (one week). Gamma irradiation (2 kGy) completely inactivated the virus particles on food packets when stored for 1 week at both the above temperatures. It is thus proposed that gamma irradiation (2 kGy) can possibly be integrated as a final treatment of the packaged food products to rule out the possibility of viral transmission. However, the efficacy of radiation processing against different pathogenic viruses needs to be determined prior to actual commercial deployment.

Anti-proliferative effect and underlying mechanism of ethoxy-substituted phylloquinone (vitamin K1 derivative) from Spinacia oleracea leaf and enhancement of its extractability using radiation technology.

In our previous studies, a novel antimutagenic compound, 2-ethoxy-3-(3,7,11,15-tetramethylhexadec-2-ethyl) naphthaquinone-1,4-dione (ethoxy-substituted phylloquinone; ESP) from spinach was characterized and mechanism contributing to its antimutagenicity was deduced. In the current study, anti-proliferative activity of ESP was assessed in lung cancer (A549) cells using MTT [3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide], clonogenic assays and cell cycle analysis. ESP treatment showed selective cytotoxicity against lung cancer cells and no cytotoxicity in normal lung (WI38) cells. Cell cycle analysis revealed that ESP treatment arrests A549 cell population in G2-M phase. In-silico analysis indicated positive drug-likeness features of ESP. Molecular docking showed H-bonding and hydrophobic interactions between ESP and B-DNA dodecamer residues at minor groove. SWATH-MS (Sequential Window Acquisition of All Theoretical Mass Spectra) based proteomic analysis indicated down-regulation of proteins involved in EGFR signaling, NEDDylation and other metabolic pathways and up-regulation of tumor suppressor (STAT1 and NDRG1) proteins. Treatment of spinach powder with gamma radiation (5-20 kGy) from cobalt (Co-60) enhanced the extractability of ESP up to 4.4-fold at the highest dose of 20 kGy. Scanning electron microscopy of spinach powder displayed decrease in smoothness and compactness with increase in radiation dose attributing to its enhanced extractability. Increase in the extractability of ESP with increasing radiation doses as measured by fluorescence intensity and dry weight basis was strongly correlated. Nonetheless, radiation treatment did not affect the functionality of ESP in terms of anti-proliferative and antimutagenic activities. Current findings thus highlight broad spectrum bioactivity of ESP from spinach, its underlying mechanism and applicability of radiation technology in enhancing extractability. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03264-6.

Molecular events confirming antimutagenicity to abscisic acid derived from a floral honey establishing its functional relevance.

Natural dietary products of health promoting and disease preventive functional relevance are gaining significant prominence. Current investigation was aimed to decipher the underlying molecular mechanism responsible for the antimutagenic action contributing to functional relevance of floral honey ('Pongammia pinnata', Karanj honey) derived abscisic acid (ABA) against ethyl methanesulfonate (EMS) induced mutagenesis. Differential expression of proteins under different treatment conditions was ascertained by 2D gel electrophoresis. Selectively up-regulated characterized using MALDI-TOF MS/MS were identified as polyribonucleotide nucleotidyl transferse (PNPase), LPS-assembly lipoprotein (LptE), Outer membrane Usher protein (HtrE), ATP-dependent DNA helicase (RecG), and Phosphomethyl pyrimidine synthase (ThiC). Antimutagenicity exerted by ABA against EMS was ∼78% in wild type E. coli MG1655 strain however, in E. coli MG1655 ΔthiC, ΔpnpA, ΔrecG, and ΔhtrE this activity was found to be ∼60, 10, 9 and 10%, respectively. Proteomic analysis and antimutagenicity studies using E. coli single gene knockout strains thus indicated about the possible role of thiC, htrE, lptE, recG and pnp in observed antimutagenicity. Cyclic voltametry as well as competition kinetics through pulse radiolysis confirmed lack of antioxidant capacity in abscisic acid apparently ruling out the possibility of scavenging of electrophilic intermediates generated by ethyl methanesulfonate. It is proposed that ABA is exerting antimutagenicity through its involvement at the cellular level leading to physiological adaptation, strengthening of cell wall proteins and up-regulation of the repair proteins. This study provides a novel dimension to the functional role of abscisic acid from its nutraceutical perspective.

Control of post-harvest storage losses in water chestnut (Trapa bispinosa Roxburg) fruits by natural functional herbal coating and gamma radiation processing.

Water chestnut (Trapa bispinosa Roxburg) has short shelf life even after drying due to insect infestations resulting in huge economic losses to farmers. Study was performed including coating with Aloe-vera gel, air drying, LDPE packaging and gamma irradiation (≤ 1 kGy) were evaluated for their efficacy in controlling storage losses. The study was performed with complete randomized design up to 6-month storage period. Physico-chemical properties analysis indicated, weight loss (0.11-0.88%), damage (upto 6.5%) and lesser grain borer infestation (up to 16 no. per packet) during storage at different intervals. Moisture content ranged (9.25-10.10%), protein (8.82-8.89%), fat (0.90-1.00%), carbohydrate (76.83-76.89%), total ash (3.11-4.18%), total sugar (5.83-5.89%), reducing sugar (1.84-1.88%), non-reducing sugar (3.98-4.02%), amylose (66.54-66.64%), in-vitro starch digestibility (3.14-3.18%), phenolics (0.14-0.18%), acidity (0.11-0.12%) and ascorbic acid (41.66-50.00 mg/100 g). These treatments were not found to affect the quality attributes significantly however; it helps in controlling insect infestations at ambient condition. The sensory score of the product, 'Sev' was ranked as like slightly to moderately range. It could be concluded that water chestnut with combinations of 50% aloe-vera gel +0.75 kGy gamma radiation treatment could be stored up to 6 months with retention of quality attributes.

Secondary product from strawberry (Fragaria ananassa) fruit for extended preservation and value addition.

Strawberry is highly delicate and perishable fruit prone to microbial spoilage. To address these issues, secondary product(s) using strawberry pulp was prepared by adding sweetener (jaggery or sucrose) and anti-browning agent (citrate) to reduce the extent of drying induced sourness and discoloration, respectively. Subsequently, these products were LDPE packed and radiation processed (≤ 5 kGy) to ensure microbiological safety during ambient temperature storage. Physical, biochemical, functional (antioxidant and antimutagenic) and organoleptic properties (including flavor compounds) were found to be either retained or enhanced compared to dehydrated strawberry pulp. The product termed as dehydrated strawberry roll having jaggery and citrate (DSRJ) was found to be more acceptable than that having sucrose and citrate (DSRS) till 5 months. Scanning electron microscopy indicated better surface quality of DSRJ than DSRS. Electrochemical property when analyzed using cyclic voltammetry displayed strong correlation to radical scavenging antioxidant assays with marginal storage associated shift in the peak potential. GC-MS analysis indicated retention of most of the flavor compounds in DSRJ during storage. Thus, DSRJ, a value-added product could provide a solution to control post-harvest losses of the fruit by processing to a more stable form and ensuring availability beyond the season. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13197-021-05171-9.

'BhAVI-23'-A spice-herb based dietary infusion possessing in-vitro anti-viral potential.

BACKGROUND: Viruses cause many life threatening human diseases. Recently, COVID-19 pandemic has challenged the health care systems worldwide. As a disease preventive approach and to bring relief to the severity of the symptoms, a infusion termed as Bhabha Anti-Viral Infusion-23 ('BhAVI-23') was conceptualized and formulated which comprised of 23 selected spices and herbals. OBJECTIVE: The present study was conducted to assess the in vitro antiviral potential of the formulation, BhaAVI-23. MATERIAL AND METHODS: The in-vitro anti-viral potential of BhAVI-23 was assessed through inhibition of HIV1 reverse transcriptase (RT) as well as through a novel P1 (virulent) bacteriphage based screening assay system. Anti-diabetic potential was assessed by non-enzymatic glycosylation of haemoglobin and the bioactive volatile components were detected through headspace gas chromatography followed by molecular docking analysis. RESULTS: The infusion displayed prominent anti-viral activity as evident from significant (57%) inhibition of the HIV1-RT as well as through reduction in the infectivity of P1 (virulent) bacteriophage. The infusion also exerted profound protection (∼64%) to non-enzymatic glycosylation of haemoglobin. Headspace gas chromatography and mass spectrometric analysis confirmed the presence of at least 47 major compounds. Docking analysis indicated possible interaction of α-pinene and eugenol with SARS-CoV spike protein. CONCLUSION: This 'BhAVI-23' infusion displayed prominent in-vitro anti-viral and anti-diabetic potential in different model systems. These attributes have relevance as diabetic patients are more prone to COVID-19 morbidity. 'BhAVI-23' opens the avenue for its potential inclusion as a supportive health care system upon due regulatory approval during the current pandemic.

Molecular mechanism of antimutagenicity by an ethoxy-substituted phylloquinone (vitamin K1 derivative) from spinach (Spinacea oleracea L.).

In our previous study, an antimutagenic compound from spinach (Spinacea oleracea L.), ethoxy-substituted phylloquinone (ESP) was isolated and characterized. The current study deals with elucidation of the possible mechanism of antimutagenicity of ESP against ethyl methanesulfonate (EMS) deploying model systems such as human lymphoblast (TK+/- or TK6) cell line (thymidine kinase gene mutation assay) and Escherichia coli MG1655 (rifampicin resistance assay). Findings of the study ruled out the possibility of direct inactivation of EMS by ESP. DAPI competitive binding assay indicated the DNA minor groove binding activity of ESP. Interestingly, ESP did not display major groove binding or intercalating abilities. Further, proteomics study using 2-D gel electrophoresis in E. coli and subsequent studies involving single gene knockout strains revealed the possible role of tnaA (tryptophanase) and dgcP (diguanylate cyclase) genes in observed antimutagenicity. These genes have been reported to be involved in indole and cyclic-di-GMP biosynthesis, respectively, which eventually lead to cell division inhibition. In case of TK+/- cell line system, ADCY genes (adenylate cyclase), a functional analogue of dgcP gene, were found to be transcriptionally up-regulated. The generation/doubling time were significantly higher in E. coli or TK+/- cells treated with ESP than control cells. The findings indicated inhibition of cell proliferation by ESP through gene regulation as a possible mechanism of antimutagenicity across the biological system. Cell division inhibition actually provides additional time for the repair of damaged DNA leading to antimutagenicity.

Rescue of Escherichia coli cells from UV-induced death and filamentation by caspase-3 inhibitor

Escherichia coli cells have been observed earlier to display caspase-3-like protease activity (CLP) and undergo programmed cell death (PCD) when exposed to gamma rays. The presence of an irreversible caspase-3 inhibitor (Ac-DEVD-CMK) during irradiation was observed to increase cell survival. Since radiation is known to induce SOS response, the effect of a caspase-3 inhibitor on SOS response was studied in E. coli. UV, a well-known SOS inducer, was used in the current study. Cell filamentation in E. coli upon UV exposure was found to be inhibited by ninefold in the presence of a caspase-3 inhibitor. CLP activity was found to increase twofold in UV-exposed cells than in control (non-treated) cells. Further, bright fluorescing filaments were observed in UV-exposed E. coli cells treated with FITC-DEVD-FMK, a fluorescent dye tagged with an irreversible caspase-3 inhibitor (DEVD-FMK), indicating the presence of active CLP in these cells. Unlike caspase-3 inhibitor, a serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), was not found to improve cell survival after UV treatment. Additionally, a SOS reporter system known as SIVET (selectable in vivo expression technology) assay was performed to reconfirm the inhibition of SOS induction in the presence of caspase-3 inhibitor. SIVET assay is used to quantify cells in which the SOS response has been induced leading to a scorable permanent selectable change in the cell. The SIVET induction frequency (calculated as the ratio of SIVET-induced cells to total viable cells) increased around tenfold in UV-exposed cultures. The induction frequency was found to decrease significantly to 51 from 80% in the cells pre-incubated with caspase-3 inhibitor. On the contrary, caspase-3 inhibitor failed to improve cell survival of E. coli ΔrecA and E. coli DM49 (SOS non-inducible) cells post UV treatment. Summing together, the results indicated a possible linkage of SOS response and the PCD process in E. coli. The findings also indicated that functional SOS pathway is required for CLP-like activity; however, the exact mechanism remains to be elucidated

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Rescue of Escherichia coli cells from UV-induced death and filamentation by caspase-3 inhibitor.

Escherichia coli cells have been observed earlier to display caspase-3-like protease activity (CLP) and undergo programmed cell death (PCD) when exposed to gamma rays. The presence of an irreversible caspase-3 inhibitor (Ac-DEVD-CMK) during irradiation was observed to increase cell survival. Since radiation is known to induce SOS response, the effect of a caspase-3 inhibitor on SOS response was studied in E. coli. UV, a well-known SOS inducer, was used in the current study. Cell filamentation in E. coli upon UV exposure was found to be inhibited by ninefold in the presence of a caspase-3 inhibitor. CLP activity was found to increase twofold in UV-exposed cells than in control (non-treated) cells. Further, bright fluorescing filaments were observed in UV-exposed E. coli cells treated with FITC-DEVD-FMK, a fluorescent dye tagged with an irreversible caspase-3 inhibitor (DEVD-FMK), indicating the presence of active CLP in these cells. Unlike caspase-3 inhibitor, a serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), was not found to improve cell survival after UV treatment. Additionally, a SOS reporter system known as SIVET (selectable in vivo expression technology) assay was performed to reconfirm the inhibition of SOS induction in the presence of caspase-3 inhibitor. SIVET assay is used to quantify cells in which the SOS response has been induced leading to a scorable permanent selectable change in the cell. The SIVET induction frequency (calculated as the ratio of SIVET-induced cells to total viable cells) increased around tenfold in UV-exposed cultures. The induction frequency was found to decrease significantly to 51 from 80% in the cells pre-incubated with caspase-3 inhibitor. On the contrary, caspase-3 inhibitor failed to improve cell survival of E. coli ΔrecA and E. coli DM49 (SOS non-inducible) cells post UV treatment. Summing together, the results indicated a possible linkage of SOS response and the PCD process in E. coli. The findings also indicated that functional SOS pathway is required for CLP-like activity; however, the exact mechanism remains to be elucidated.

No induced mutagenesis in human lymphoblast cell line and bacterial systems upon their prolonged sub-culturing in irradiated food blended media.

BACKGROUND: Profound apprehension towards safety of irradiated food has remained a major cause behind tardy acceptance of this technology although it has immense socio-economic potential. Generation of in-depth scientific evidence will help to refute these apprehensions. With this prospective, the present study was undertaken where safety of various irradiated (Dmin up to 25 kGy) foods was evaluated through long-term exposure studies in models including human lymphoblast TK6 cell line (100 generations) and Escherichia coli MG1655 cells (exclusive sub-culturing in irradiated food medium for 3000 generations). Additionally, the Ames test, micronucleus test, comet assay, DNA sequencing and restriction profiling of phagemid DNA from E. coli cells sub-cultured in irradiated food medium were also performed. RESULTS: No induced mutagenesis was observed in these cells during long-term sub-culturing in various irradiated food medium. Also no change was observed in profiles of comet, micronucleus, restriction digestion, random amplification of polymorphic DNA as well as DNA sequences. The latter also ruled out the possibility of any silent mutation. CONCLUSION: Findings of the current study thus provided credible molecular evidence supporting the safety of irradiated foods. This would be helpful in confidence building among consumers, entrepreneurs, and strengthening the overall food irradiation program to achieve 'food safety' and 'security'. © 2017 Society of Chemical Industry.

Lack of induced mutagenesis in E. coli or human lymphoblast cell line upon long-term sub-culturing in medium from irradiated meat.

PURPOSE: Current study was aimed to enhance the confidence of consumers as well as entrepreneurs towards food irradiation program. MATERIALS AND METHODS: In this work, safety of high dose (25 kGy) irradiated meat samples (HDIMS) was ascertained by scoring mutation frequency through a long-term sub-culturing study in Escherichia coli MG1655 cells (ATCC 700926) up to 1500 generations (at 1%), 250 generations (at 5% and 10%) and human lymphoblast thymidine kinase heterozygote (TK6) cell line (ATCC CRL-8015) [at two gene loci, tk-/+ (thymidine kinase) and hprt+ (Hypoxanthine Phosphoribosyltransferase)] up to 156 generations using goat meat sample. Also these samples were assayed at further radiation doses of 10, 45 and 70 kGy at 2% concentration (in cell line), and 1% (in E. coli). Study was also performed with other meat samples such as chicken, fishes (pomfret and rohu) and shrimps by carrying out limited long-term sub-culturing trials in human lymphoblast cell line. Mutation analysis was also carried out using a novel DPAR (Differential loss of Plasmid Antibiotic Resistance) assay followed by sequencing of tcR (tetracycline resistance) gene of pBR322 plasmid isolated from E. coli cells grown for 1500 generations on HDIMS medium and RAPD (Random Amplified Polymorphic DNA) analysis of the genome. RESULTS AND CONCLUSION: None of the assays exhibited any induced mutation when analyzed at regular time intervals. RAPD analysis also did not indicate any change in its nucleotide sequence, ruling out the occurrence of any silent mutation. Thus, the present findings report absence of mutagenic effect of high dose irradiated meat samples.

Natural Predominance of Abscisic Acid in Pongammia pinnata ("Karanj") Honey Contributed to its Strong Antimutagenicity.

Various samples of raw (unprocessed) floral honey collected from different geographical locations of India were assayed for its antimutagenicity against ethyl methanesulfonate in E. coli MG1655 cells through rifampicin resistance assay. A monofloral honey ("Pongammia pinnata", local name "Karanj") displayed maximum antimutagenicity (78.0 ± 1.7; P ≤ 0.05). Solid phase extraction (using Amberlite XAD-2 resin) followed by HPLC resulted into different peaks displaying varying antimutagenicity. Peak at retention time (Rt) 27.9 min (henceforth called P28) displayed maximum antimutagenicity and was further characterized to be abscisic acid (ABA) using ESI-MS and NMR. Its antimutagenicity was reconfirmed through human lymphoblast cell line (TK6) mutation assay using thymidine kinase (tk+/-) cell line. Although ABA from this honey displayed strong antimutagenicity, it lacked any in vitro antioxidant capacity indicating noninvolvement of any radical scavenging in the observed antimutagenicity.

Purification and Characterization of the Principal Antimutagenic Bioactive as Ethoxy-Substituted Phylloquinone from Spinach (Spinacea oleracea L.) Based on Evaluation in Models Including Human Lymphoblast TK+/- Cells.

During in vitro analysis, spinach (Spinacea oleracea L.) leaf extracts displayed varying antimutagenicity when analyzed in models including human lymphoblast (TK+/-) cell line (thymidine kinase gene mutation assay) and Escherichia coli MG1655 (rifampicin resistance assay) against chemically (ethyl methanesulfonate and 5-azacytidine) induced mutagenicity. Highest antimutagenicity was displayed by the quinone extract. The principal bioactive compound exhibited fluorescence in TLC at 366 nm (termed C4) resolved at Rf 0.32 and tR 15.2 min in TLC and HPLC, respectively. On the TLC plate, three spots (C1-C3), observed at 254 nm, displayed comparatively lesser antimutagenicity. Furthermore, biochemical and spectroscopic analyses using MALDI-TOF MS and NMR indicated the nature of the potent compound (C4) as an ethoxy-substituted phylloquinone derivative [2-ethoxy-3-((E)-3,7,11,15-tetramethylhexadec-2-enyl)naphthalene-1,4-dione]. The C4 compound did not display any cytotoxicity and hence possesses significant nutraceutical-based intervention possibility to combat the onset of mutation-associated disease(s).

Potential Prophylactic Properties of Apple and Characterization of Potent Bioactive from cv. "Granny Smith" Displaying Strong Antimutagenicity in Models Including Human Lymphoblast TK6(+/-) Cell Line.

Potential prophylactic attributes in terms of antimutagenicity, antioxidant, and radioprotective properties were evaluated for 8 common apple cultivars namely "Fuji," "Golden Delicious," "Granny Smith," "Ambri Kashmiri," "Kinnaur," "Red Delicious," "Royal Gala," and "Shimla," where cultivar based significant variation was observed. Cv. "Granny Smith" displayed significantly higher and broad spectrum antimutagenicity in Escherichia coli rpoB based rifampicin resistance (Rif(R) ) assay, whereas, "Ambri Kashmiri," "Royal Gala," and "Shimla" showed lower antimutagenicity. Cultivars "Ambri Kashmiri," "Kinnaur," and "Red Delicious" exhibited strong antioxidant activity than cv. "Granny Smith" as assayed by radical scavenging, reducing potential and radioprotective property assays. The antioxidant and radioprotective properties were found to be better correlated than antimutagenicity. Suppression of error-prone DNA repair pathway (such as E. coli SOS response) was found to be one of the possible mechanisms contributing to its antimutagenicity. Phenolic extract of "Granny Smithˮ showing higher antimutagenicity was HPLC purified and the bioactive fraction (tR 35.4 min) contributing maximally (∼80%) to the observed antimutagenicity was identified as procyanidin dimer (PD) by ESI-MS/MS. The above observed antimutagenicity in bacterial assay system was well reproduced in Thymidine Kinase Mutation (TKM) assay performed using human lymphoblast cell line (TK6(+/-) ) cell line making the findings more prophylactically relevant.

Food Processing by Irradiation­­An effective technology for food safety and security.

Radiation processing of food involves controlled application of energy from ionizing radiations from radioisotopes (Cobalt-60 and Caesium-137), electron beam (≤10 MeV) or X-rays (≤5 MeV) in an irradiation chamber shielded by 1.5 - 1.8 m thick concrete walls. Food, either pre-packed or in-bulk, placed in suitable containers is sent into the chamber through an automatic conveyor. Major benefits achieved by radiation processing of food are inhibition of sprouting of tubers and bulbs, delay in ripening and senescence of fruits and vegetables, disinfestations of insect pests in agricultural commodities, destruction of microbes responsible for food spoilage, and elimination of food pathogens and parasites of public health importance. Irradiation produces very little chemical changes in food, and the changes are similar to those by other preservation methods like heat. The radiolytic products and free radicals produced are identical to those present in foods subjected to treatments such as cooking and canning. None of the changes known to occur have been found to be harmful. Radiation processing of food has been approved by various international statutory bodies and organizations to ensure 'Food Security & Safety', and overcome 'Technical barrier to International Trade' and currently is being practiced in more than 60 countries worldwide.