Safe, efficient, and economically beneficial remediation of arsenic-contaminated soil: possible strategies for increasing arsenic tolerance and accumulation in non-edible economically important native plants.

Anthropogenic activities, geological processes, and biogenic sources have led to the enhanced concentration of arsenic (As), a toxic metalloid in water and soil. Non-edible, economically important plants can be employed for safe As phytoremediation in addition to generating extra income. However, these plants may get affected by stressful local environmental conditions. Native plant species are adapted to local environmental conditions and hence overcome this problem. Native non-edible economic plant species which show high As tolerance and accumulation are promising candidate for safe, efficient, and economically beneficial phytoremediation of As-contaminated sites. The current review discusses the potential of native economic plant species that can be used in As phytoremediation programme. However, since their phytoremediation potential is moderate, possible strategies for increasing As  olerance and accumulation, especially genetic modification, have been discussed in detail. Knowledge gained from the review can be used for the development of As tolerance and accumulation in non-edible economic native plants.

Effect of arsenate toxicity on antioxidant enzymes and expression of nicotianamine synthase in contrasting genotypes of bioenergy crop Ricinus communis.

Arsenic (As) is a toxic environmental pollutant. Growing Ricinus communis (castor) on As-contaminated land has the advantage that in addition to revegetation of contaminated land, it can produce bioenergy. To date, As tolerance mechanisms of this plant are not fully understood. In our previous study, we screened tolerant and sensitive genotypes of castor and reported higher total As concentration, enhanced reactive oxygen species (ROS) generation, and oxidative stress in sensitive genotypes of castor GCH 2 and GCH 4 in comparison to tolerant genotypes WM and DCH 177. In the present study, we compared the activity, isoenzyme profile, and gene expression of ROS-scavenging enzymes, proline content, and expression of nicotianamine synthase genes (RcNAS1, RcNAS2, and RcNAS3) in As-tolerant and As-sensitive genotypes of castor. SOD and GPX activity increased significantly in roots of tolerant genotype WM but remained the same or decreased in sensitive genotype GCH 2 and GCH 4 at 200 µM arsenate [As(V)] treatment indicating their important role in As tolerance in castor. CAT activity and proline content increased in sensitive genotypes but remained the same in tolerant genotypes due to As(V) treatment. APX activity showed no significant change in roots and leaves of both tolerant and sensitive genotypes. NAS genes (RcNAS1, RcNAS2, and RcNAS3) encode enzymes that catalyze trimerization of S-adenosylmethionine to form nicotianamine and are critical for metal chelation and heavy metal tolerance. Differential responses of RcNAS1, RcNAS2, and RcNAS3 genes in WM and GCH 2 due to As(V) treatment suggest their role in As(V) tolerance.

Differential responses of thiol metabolism and genes involved in arsenic detoxification in tolerant and sensitive genotypes of bioenergy crop Ricinus communis.

Castor, a non-food, dedicated bioenergy crop, has immense potential to be used for phytoremediation/revegetation of heavy metal contaminated sites. In the previous study, we identified arsenate [As(V)]-tolerant (WM) and As(V)-sensitive (GCH 2) genotypes of castor (Ricinus communis L.) with differential accumulation and tolerance of arsenic [As]. The role of thiols in As(V) toxicity and tolerance mechanism in the castor plant is not fully understood. On the one hand, thiol-dependent reduction of As(V) to As(III) by arsenate reductase (AR) makes it capable of reacting with thiol groups of protein leading to disturbed metabolic pathways; on the other hand, reduction of As(V) to arsenite [As(III)] by AR and then complexation of As(III) with phytochelatins (PCs) and compartmentalization of As(III)-PC complex are considered as the major detoxification mechanisms of As(V). In our study, the expression of RcAR increased in leaves and roots of As(V)-tolerant castor genotype WM but decreased in sensitive genotype GCH 2 due to 200 µM As(V) treatment. The activity of glutathione reductase (GR) increased significantly in the tolerant genotype, whereas it remained same in the sensitive genotype. GSH/GSSH ratio declined substantially in the sensitive genotype. The increased expression of phytochelatin synthase 1 isoform 1 (RcPCS1X1) in roots, RcPCS1X2 and metallothionein type 2 (RcMT2) in leaves, and c-type ABC transporter (RcABCC) in roots and leaves of WM was observed, but the expression of these genes declined or remained the same in GCH 2. Overall, our results suggest the essential roles of GR, RcAR, RcPCS1, RcMT2, and RcABCC in the tolerance of WM castor genotype to As(V) toxicity.

Entrapment of enzyme in the presence of proline: effective approach to enhance activity and stability of horseradish peroxidase.

In this report, activity and stability of horseradish peroxidase (HRP) entrapped in polyacrylamide gel in the presence of proline (HEP) are compared with that of enzyme entrapped in absence of proline (HE). Within polyacrylamide (8%) beads, 80% entrapment yield for peroxidase was observed in the presence as well as absence of proline. The HEP (1.5 M proline) showed 170% higher activity compared to HE. HEP also showed significant increase in K M, V max and K cat. At 8th cycle of use, HEP retained 40% of its activity, whereas HE retained only 10% of activity. In addition, in comparison with HE, HEP showed increased storage stability and thermo-stability. HEP showed higher activity compared to HE over an extensive range of pH (4-8), temperature (30-80 °C) and inhibitors such as NaN3, Cd2+ and Pb2+. Our results suggest that peroxidase entrapment in polyacrylamide gel in the presence of proline can be a useful approach for increasing activity and stability of horseradish peroxidase.

Differential responses of growth, photosynthesis, oxidative stress, metals accumulation and NRAMP genes in contrasting Ricinus communis genotypes under arsenic stress.

Effect of arsenate [As(V)] on biomass, photosynthetic rate, stomatal conductance, transpiration, oxidative stress, accumulation of As, Fe, Zn, Cu and Mn and expression of NRAMP genes was investigated in As(V) tolerant and sensitive genotypes of bioenergy crop Ricinus communis. As(V) treatments (100 and 200 µM) led to significant reduction in root and leaf biomass, photosynthetic rate, stomatal conductance and transpiration in GCH 2 and GCH 4 genotypes but no significant change or increase was observed in WM and DCH 177 genotypes. No significant difference was observed in hydrogen peroxide content and lipid peroxidation in As(V)-treated tolerant genotypes compared to control, whereas these parameters enhanced significantly in As(V)-treated sensitive genotypes. GCH 2 accumulated around two times As in leaves and showed significant reduction in concentration of Zn and Mn in the leaves and roots due to 200 µM As(V) treatment compared to WM. NRAMP genes are critical for uptake and distribution of essential divalent metal cations, photosynthesis and controlled production of reactive oxygen species in plants. RcNRAMP2, RcNRAMP3 and RcNRAMP5 genes showed differential expression in response to 200 µM As(V) in GCH 2 and WM suggesting that NRAMP genes are associated with differential responses of WM and GCH 2 genotypes to As(V) stress.

Catalase and ascorbate peroxidase-representative H2O2-detoxifying heme enzymes in plants.

Plants have to counteract unavoidable stress-caused anomalies such as oxidative stress to sustain their lives and serve heterotrophic organisms including humans. Among major enzymatic antioxidants, catalase (CAT; EC 1.11.1.6) and ascorbate peroxidase (APX; EC 1.11.1.11) are representative heme enzymes meant for metabolizing stress-provoked reactive oxygen species (ROS; such as H2O2) and controlling their potential impacts on cellular metabolism and functions. CAT mainly occurs in peroxisomes and catalyzes the dismutation reaction without requiring any reductant; whereas, APX has a higher affinity for H2O2 and utilizes ascorbate (AsA) as specific electron donor for the reduction of H2O2 into H2O in organelles including chloroplasts, cytosol, mitochondria, and peroxisomes. Literature is extensive on the glutathione-associated H2O2-metabolizing systems in plants. However, discussion is meager or scattered in the literature available on the biochemical and genomic characterization as well as techniques for the assays of CAT and APX and their modulation in plants under abiotic stresses. This paper aims (a) to introduce oxidative stress-causative factors and highlights their relationship with abiotic stresses in plants; (b) to overview structure, occurrence, and significance of CAT and APX in plants;

Exoproteome and secretome derived broad spectrum novel drug and vaccine candidates in Vibrio cholerae targeted by Piper betel derived compounds.

Vibrio cholerae is the causal organism of the cholera epidemic, which is mostly prevalent in developing and underdeveloped countries. However, incidences of cholera in developed countries are also alarming. Because of the emergence of new drug-resistant strains, even though several generic drugs and vaccines have been developed over time, Vibrio infections remain a global health problem that appeals for the development of novel drugs and vaccines against the pathogen. Here, applying comparative proteomic and reverse vaccinology approaches to the exoproteome and secretome of the pathogen, we have identified three candidate targets (ompU, uppP and yajC) for most of the pathogenic Vibrio strains. Two targets (uppP and yajC) are novel to Vibrio, and two targets (uppP and ompU) can be used to develop both drugs and vaccines (dual targets) against broad spectrum Vibrio serotypes. Using our novel computational approach, we have identified three peptide vaccine candidates that have high potential to induce both B- and T-cell-mediated immune responses from our identified two dual targets. These two targets were modeled and subjected to virtual screening against natural compounds derived from Piper betel. Seven compounds were identified first time from Piper betel to be highly effective to render the function of these targets to identify them as emerging potential drugs against Vibrio. Our preliminary validation suggests that these identified peptide vaccines and betel compounds are highly effective against Vibrio cholerae. Currently we are exhaustively validating these targets, candidate peptide vaccines, and betel derived lead compounds against a number of Vibrio species.

Conserved host-pathogen PPIs. Globally conserved inter-species bacterial PPIs based conserved host-pathogen interactome derived novel target in C. pseudotuberculosis, C. diphtheriae, M. tuberculosis, C. ulcerans, Y. pestis, and E. coli targeted by Piper betel compounds.

Although attempts have been made to unveil protein-protein and host-pathogen interactions based on molecular insights of important biological events and pathogenesis in various organisms, these efforts have not yet been reported in Corynebacterium pseudotuberculosis (Cp), the causative agent of Caseous Lymphadenitis (CLA). In this study, we used computational approaches to develop common conserved intra-species protein-protein interaction (PPI) networks first time for four Cp strains (Cp FRC41, Cp 316, Cp 3/99-5, and Cp P54B96) followed by development of a common conserved inter-species bacterial PPI using conserved proteins in multiple pathogens (Y. pestis, M. tuberculosis, C. diphtheriae, C. ulcerans, E. coli, and all four Cp strains) and E. Coli based experimentally validated PPI data. Furthermore, the interacting proteins in the common conserved inter-species bacterial PPI were used to generate a conserved host-pathogen interaction (HP-PPI) network considering human, goat, sheep, bovine, and horse as hosts. The HP-PPI network was validated, and acetate kinase (Ack) was identified as a novel broad spectrum target. Ceftiofur, penicillin, and two natural compounds derived from Piper betel were predicted to inhibit Ack activity. One of these Piper betel compounds found to inhibit E. coli O157:H7 growth similar to penicillin. The target specificity of these betel compounds, their effects on other studied pathogens, and other in silico results are currently being validated and the results are promising.

Tight controlled expression and secretion of Lactobacillus brevis SlpA in Lactococcus lactis.

Prokaryotes commonly present outer cell wall structures composed of a crystalline array of proteinaceous subunits, known as surface layers (S-layers). The ORF encoding the S-layer protein (SlpA) of Lactobacillus brevis was cloned into Lactococcus lactis under the transcriptional control of the xylose-inducible expression system (XIES). SlpA was secreted into the extracellular medium, as determined by immunoblotting, and assays on the kinetics of SlpA production revealed that repression of the system with glucose did not require the depletion of xylose from the medium that allows transitory ORF expression. The successful use of XIES to express S-layer proteins in the versatile and generally recognized as safe species L. lactis opens new possibilities for an efficient production and isolation of SlpA S-layer protein for its various applications in biotechnology and importantly as an antigen-carrying vehicle.

A novel comparative genomics analysis for common drug and vaccine targets in Corynebacterium pseudotuberculosis and other CMN group of human pathogens.

Caseous lymphadenitis is a chronic goat and sheep disease caused by Corynebacterium pseudotuberculosis (Cp) that accounts for a huge economic loss worldwide. Proper vaccination or medication is not available because of the lack of understanding of molecular biology of the pathogen. In a recent approach, four Cp (CpFrc41, Cp1002, CpC231, and CpI-19) genomes were sequenced to elucidate the molecular pathology of the bacteria. In this study, using these four genome sequences along with other eight genomes (total 12 genomes) and a novel subtractive genomics approach (first time ever applied to a veterinary pathogen), we identified potential conserved common drug and vaccine targets of these four Cp strains along with other Corybacterium, Mycobacterium and Nocardia (CMN) group of human pathogens (Corynebacterium diphtheriae and Mycobacterium tuberculosis) considering goat, sheep, bovine, horse, and human as the most affected hosts. The minimal genome of Cp1002 was found to consist of 724 genes, and 20 conserved common targets (to all Cp strains as well as CMN group of pathogens) from various metabolic pathways (13 from host-pathogen common and seven from pathogen's unique pathways) are potential targets irrespective of all hosts considered. ubiA from host-pathogen common pathway and an ABC-like transporter from unique pathways may serve dual (drug and vaccine) targets. Two Corynebacterium-specific (mscL and resB) and one broad-spectrum (rpmB) novel targets were also identified. Strain-specific targets are also discussed. Six important targets were subjected to virtual screening, and one compound was found to be potent enough to render two targets (cdc and nrdL). We are currently validating all identified targets and lead compounds.

A novel strategy of epitope design in Neisseria gonorrhoeae.

In spite of genome sequences of both human and N. gonorrhoeae in hand, vaccine for gonorrhea is yet not available. Due to availability of several host and pathogen genomes and numerous tools for in silico prediction of effective B-cell and T-cell epitopes; recent trend of vaccine designing has been shifted to peptide or epitope based vaccines that are more specific, safe, and easy to produce. In order to design and develop such a peptide vaccine against the pathogen, we adopted a novel computational approache based on sequence, structure, QSAR, and simulation methods along with fold level analysis to predict potential antigenic B-cell epitope derived T-cell epitopes from four vaccine targets of N. gonorrhoeae previously identified by us [Barh and Kumar (2009) In Silico Biology 9, 1-7]. Four epitopes, one from each protein, have been designed in such a way that each epitope is highly likely to bind maximum number of HLA molecules (comprising of both the MHC-I and II) and interacts with most frequent HLA alleles (A*0201, A*0204, B*2705, DRB1*0101, and DRB1*0401) in human population. Therefore our selected epitopes are highly potential to induce both the B-cell and T-cell mediated immune responses. Of course, these selected epitopes require further experimental validation.

Thiamin confers enhanced tolerance to oxidative stress in Arabidopsis.

Thiamin and thiamin pyrophosphate (TPP) are well known for their important roles in human nutrition and enzyme catalysis. In this work, we present new evidence for an additional role of these compounds in the protection of cells against oxidative damage. Arabidopsis (Arabidopsis thaliana) plants subjected to abiotic stress conditions, such as high light, cold, osmotic, salinity, and oxidative treatments, accumulated thiamin and TPP. Moreover, the accumulation of these compounds in plants subjected to oxidative stress was accompanied by enhanced expression of transcripts encoding thiamin biosynthetic enzymes. When supplemented with exogenous thiamin, wild-type plants displayed enhanced tolerance to oxidative stress induced by paraquat. Thiamin application was also found to protect the reactive oxygen species-sensitive ascorbate peroxidase1 mutant from oxidative stress. Thiamin-induced tolerance to oxidative stress was accompanied by decreased production of reactive oxygen species in plants, as evidenced from decreased protein carbonylation and hydrogen peroxide accumulation. Because thiamin could protect the salicylic acid induction-deficient1 mutant against oxidative stress, thiamin-induced oxidative protection is likely independent of salicylic acid signaling or accumulation. Taken together, our studies suggest that thiamin and TPP function as important stress-response molecules that alleviate oxidative stress during different abiotic stress conditions.