Amarendra Narayan Misra

Frequently asked questions about in vivo chlorophyll fluorescence: practical issues

The aim of this educational review is to provide practical information on the hardware, methodology, and the hands on application of chlorophyll (Chl) a fluorescence technology. We present the paper in a question and answer format like frequently asked questions. Although nearly all information on the application of Chl a fluorescence can be found in the literature, it is not always easily accessible. This paper is primarily aimed at scientists who have some experience with the application of Chl a fluorescence but are still in the process of discovering what it all means and how it can be used. Topics discussed are (among other things) the kind of information that can be obtained using different fluorescence techniques, the interpretation of Chl a fluorescence signals, specific applications of these techniques, and practical advice on different subjects, such as on the length of dark adaptation before measurement of the Chl a fluorescence transient. The paper also provides the physiological background for some of the applied procedures. It also serves as a source of reference for experienced scientists.

In silico subtractive genomics for target identification in human bacterial pathogens

Target identification is the first step in the drug and vaccine discovery process; in silico subtractive genomics is widely used in this process. Using this approach, in recent years, a large number of targets have been identified for bacterial pathogens that are either drug resistant or for which no suitable vaccine is available; most such reports concern a specific pathogen. The in silico method reduces the time as well as the cost of target screening. Although a powerful technique that can be applied to a wide range of pathogens, there are many pitfalls in the analysis and interpretation of the data. We review this approach, including targets that have been identified with this technique and various other aspects, including advantages and disadvantages. We also discuss our own experiences using this technology. Drug Dev Res 72: 162–177, 2011.

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.

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.

Chlorophyll Fluorescence in Plant Biology

Several molecules absorb light energy which they emit after a time difference (lifetime) as radiation energy. Molecules remain at a low energy level or the ground electronic singlet state (So) or the lowest vibrational level at room temperature (Noomnarm and Clegg, 2009). On absorption of a photon, the molecule is excited from So to the first electronic excited singlet state S1 within < 10-15 s-1 (Figure 1). These molecules can also be transferred to higher energy levels (S2 to Sn) also. These excited state molecules can relax to the S1 electronic state via vibrational relaxation within 10-12 s-1. The molecule will ultimately relax to the So state through photon emission, which is called fluorescence emission. Also here, the energy of the emitted photon must equal the changes in the energy levels.

Action and target sites of nitric oxide in chloroplasts

Nitric oxide (NO) is an important signalling molecule in plants under physiological and stress conditions. Here we review the influence of NO on chloroplasts which can be directly induced by interaction with the photosynthetic apparatus by influencing photophosphorylation, electron transport activity and oxido-reduction state of the Mn clusters of the oxygen-evolving complex or by changes in gene expression. The influence of NO-induced changes in the photosynthetic apparatus on its functions and sensitivity to stress factors are discussed.

Sodium Chloride Salt Stress Induced Changes in Thylakoid Pigment-Protein Complexes, Photosystem II Activity and Thermoluminescence Glow Peaks

In the present study, mung bean (Vigna radiata L.) - a salt susceptible and Indian mustard (Brassica juncea L.) - a salt resistant crop was studied to find out the differences in stress responses of these crops. Seedlings were grown in water soaked cotton under continuous illumination of 35 μmole m-2 s-1 at 26 ± 1 °C. Salinity treatment of 0, 0.5 and 1.0% (w/v) was given to the seedlings at 6 day Photosynthetic pigment content and PS II electron transport activity was reduced under salinity in both mung bean and Indian mustard. The pigment protein pattern of both the crops were similar. Ratio analysis of B and Q thermoluminescence (TL) glow peaks suggested that S2QA- charge recombination was relatively more affected than S2/3QB- charge recombinations

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.

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; (c) to summarize the principles of current technologies used to assay CAT and APX in plants; (d) to appraise available literature on the modulation of CAT and APX in plants under major abiotic stresses; and finally, (e) to consider a brief cross-talk on the CAT and APX, and this also highlights the aspects unexplored so far.

Photoelectron transport ability of chloroplast thylakoid membranes treated with NO donor SNP: Changes in flash oxygen evolution and chlorophyll fluorescence

The nitric oxide (NO) donor sodium nitroprusside (SNP) is frequently used in plant science in vivo. The present in vitro study reveals its effects on the photosynthetic oxygen evolution and the chlorophyll fluorescence directly on isolated pea thylakoid membranes. It was found that even at very low amounts of SNP (chlorophyll/SNP molar ratio∼67:1), the SNP-donated NO stimulates with more than 50% the overall photosystem II electron transport rate and diminishes the evolution of molecular oxygen. It was also found that the target site for SNP-donated NO is the donor side of photosystem II. Compared with other NO-donors used in plant science, SNP seems to be the only one exhibiting stimulation of electron transport through photosystem II.