Krishna P. Singh

Understanding the mechanism of atovaquone drug resistance in Plasmodium falciparum cytochrome b mutation Y268S using computational methods.

The rapid appearance of resistant malarial parasites after introduction of atovaquone (ATQ) drug has prompted the search for new drugs as even single point mutations in the active site of Cytochrome b protein can rapidly render ATQ ineffective. The presence of Y268 mutations in the Cytochrome b (Cyt b) protein is previously suggested to be responsible for the ATQ resistance in Plasmodium falciparum (P. falciparum). In this study, we examined the resistance mechanism against ATQ in P. falciparum through computational methods. Here, we reported a reliable protein model of Cyt bc1 complex containing Cyt b and the Iron-Sulphur Protein (ISP) of P. falciparum using composite modeling method by combining threading, ab initio modeling and atomic-level structure refinement approaches. The molecular dynamics simulations suggest that Y268S mutation causes ATQ resistance by reducing hydrophobic interactions between Cyt bc1 protein complex and ATQ. Moreover, the important histidine contact of ATQ with the ISP chain is also lost due to Y268S mutation. We noticed the induced mutation alters the arrangement of active site residues in a fashion that enforces ATQ to find its new stable binding site far away from the wild-type binding pocket. The MM-PBSA calculations also shows that the binding affinity of ATQ with Cyt bc1 complex is enough to hold it at this new site that ultimately leads to the ATQ resistance.

Sequence-based approach for rapid identification of cross-clade CD8+ T-cell vaccine candidates from all high-risk HPV strains

Human papilloma virus (HPV) is the primary etiological agent responsible for cervical cancer in women. Although in total 16 high-risk HPV strains have been identified so far. Currently available commercial vaccines are designed by targeting mainly HPV16 and HPV18 viral strains as these are the most common strains associated with cervical cancer. Because of the high level of antigenic specificity of HPV capsid antigens, the currently available vaccines are not suitable to provide cross-protection from all other high-risk HPV strains. Due to increasing reports of cervical cancer cases from other HPV high-risk strains other than HPV16 and 18, it is crucial to design vaccine that generate reasonable CD8+ T-cell responses for possibly all the high-risk strains. With this aim, we have developed a computational workflow to identify conserved cross-clade CD8+ T-cell HPV vaccine candidates by considering E1, E2, E6 and E7 proteins from all the high-risk HPV strains. We have identified a set of 14 immunogenic conserved peptide fragments that are supposed to provide protection against infection from any of the high-risk HPV strains across globe.

Nanoparticles: Applications, Toxicology and Safety Aspects

Nanotechnology has a global socioeconomic significance. On the brighter side, Nanoparticles (NPs) offer extraordinary technical competencies which allow them to perform enormously novel developments in science and industries. Whereas, on the darker side, just the same novel qualities of nanoparticles can concurrently evoke undesired features, which sometimes lead to adverse and harmful interactions with exposed organisms. Workers involved in manufacturing and handling of NPs in all countries face new hazards from these nanomaterials. The occupational safety and health associations have taken schemes to spot the gaps between awareness and practices. These international agencies formulate the guidelines for handling nano materials and fix their occupational exposure limits. In this chapter authors discussed the source and role of NPs in different areas, NPs induced toxicity, their interaction with different biomolecules, as well as the safety and handling guidelines of NPs in occupational and laboratory areas.

Aryl Hydrocarbon Receptor Activation Contributes to Benzanthrone-Induced Hyperpigmentation via Modulation of Melanogenic Signaling Pathways

Benzanthrone (BA), an oxidized polycyclic aromatic hydrocarbon (PAH), has been found to be a potential health threat to occupational workers involved in dye manufacturing factories. It has been observed that occupational workers become exposed to BA either during manufacturing, pulverization, or storage and developed various kinds of skin diseases like contact dermatitis, itching, erythema, roughness, and foremost, hyperpigmentation. It has been shown that some environmental organic pollutants (POPs) like dioxins, furans, and polychlorinated biphenyls (PCBs) may act as ligands for the aryl hydrocarbon receptor (AhR) and regulate hyperpigmentation. Here, we hypothesized that BA may also act as a ligand for AhR and possibly regulate the melanogenic pathway to induced hyperpigmentation. Our computation results indicate that BA has a high binding affinity toward AhR for the initiation of melanogenic signaling. Following the in silico predictions, we used primary mouse melanocytes (PMMs) and confirmed that exposure to BA (5, 10, and 25 μM) resulted in an increase in AhR expression, tyrosinase activity, and melanin synthesis. Moreover, to study the physiological relevance of these findings, C57BL/6 mice were topically exposed to BA, and enhanced pigmentation and melanin synthesis were observed. Furthermore, the study was extended to assess the mechanistic aspects involved in BA-induced hyperpigmentation in PMMs as well as in mouse skin. Our results suggest that BA exposure initiates AhR signaling and increases tyrosinase enzyme activity and melanin synthesis. Moreover, the genes that regulate the melanin synthesis, such as TRP-1, TRP-2 and the transcription factor MITF, were also found to be increased. Thus, altogether, we suggest that BA-AhR interactions are critical for BA-induced hyperpigmentation.

Frequency and spectrum of chlorophyll mutations and induced variabilityin ricebean (Vigna umbellata Thunb, Ohwi and Ohashi)

A comparative study of frequency and spectrum of chlorophyll mutations induced by physical (gamma rays) and chemical mutagens (EMS) in relation to the effect of mutations in M1 plant, induction of mutants in M2 and variability studies in M3 generation was made in two ricebean (Vigna umbellata Thunb, Ohwi and Ohashi) varieties; one local (Totru Local) and one widely cultivated one (BRS-1) during 2005-2008. The treatments included three doses each of gamma rays (30, 40 and 50 kR) and EMS (0.50, 0.60 and 0.70%). Both gamma rays and EMS proved to be hazardous for germination and plant survival at higher dose and the comparative reduction in these characters was higher under EMS treatments than gamma rays. The frequencies and spectrum of five different kind of induced chlorophyll mutations was in the order; viridis > xantha = albina > chlorina > albo-viridis in BRS-1 and viridis > xantha > albina > chlorina = albo-viridis in Totru Local. EMS was found to be more effective in inducing maximum frequencies of chlorophyll mutants than gamma rays. Highest frequency of chlorophyll mutants were found under O.50% EMS in BRS-1 and 0.70% EMS in Totru Local. Genotypic differences were seen as highest frequency of mutations was observed in Totru Local which was more sensitive to mutagenic treatments than BRS-1. The coefficients of variation for all quantitative characters studied in M3 generation were of higher magnitude compared to control in both the cultivars. The lower doses (30kR in gamma rays and 0.50% in EMS) were found to be the most important doses for inducing desirable variability in ricebean and three traits i.e. pods per cluster, seeds per pod and pod length showed high heritability coupled with high genetic advance in both BRS-1 and Totru Local indicating that breeding for these traits can be achieved by phenotypic selection.

Radiation induced variability and gene effects for polygenic traits in ricebean (Vigna umbellata Thunb, Ohwi and Ohashi).

Background and Objective: Concentration of sucrose in sugarcane was affected by several enzymes, one of which is pyrophosphate-dependent phosphofructokinase (PFP). The PFP is an enzyme that catalyzed the conversion of fructose 6-phosphate (F6P) and pyrophosphate (PPi) to fructose 1,6 bisphosphate and inorganic phosphate (Pi) reversibly. The PFP activity is inversely correlated to sucrose content in sugarcane. In this study, insert RNAi pfp gene to inhibit PFP protein so, sucrose content in sugarcane increased. The study objective is to obtain an efficient protocol of Agrobacterium-mediated transformation of sugarcane was needed. Materials and Methods: This study used A. tumefaciens strain GV3101 containing expression vector of pART27. Calluses from sugarcane cv. Bululawang (BL) and cv. PS862 were used as explants. Callus induction of sugarcane leaf roll was performed in solid MS medium supplemented with 3 mg LG1 2,4-D and 0.5 mg LG1 kinetin. Treatments of co-cultivation were 24 and 72 h whereas, age of callus was 7th and 10th weeks old. Selection was done in MS medium supplemented with kanamycin 100 mg LG1. Planlets which have kanamycin resistant were analyzed by PCR. Results: The study results showed that the highest transformation efficiency was obtained from treatment of co-cultivation 72 h and 7th weeks old of callus whereas, 7th weeks old of callus as an target for transformation have better transformation efficiency than 10th weeks old. Many planlets show the positive PCR. Conclusion: Co-cultivation duration and difference age of callus affected the success of RNAi pfp gene transformation of sugarcane mediated by A. tumefaciens. An efficient protocol was obtained at use of co-cultivation 72 h and 7th weeks old callus.

Comparative mutagenic effectiveness and efficiency of physical and chemical mutagen and induced variability in ricebean (Vigna umbellata Thunb, Ohwi and Ohashi)

Healthy and pure seeds of two varieties of ricebean viz., BRS-1 and Totru Local were treated with gamma rays (30, 40 and 50 kR) and ethyl methane sulphonate (EMS) (0.50, 0.60 and 0.70%). A steady reduction in germination and subsequent survival of the treated population and seedling height reduction was observed with the increasing dose/concentration of mutagens in both the cultivars regardless of the mutagens used. EMS was almost four times more effective and two times more efficient than gamma-rays and both mutagenic effectiveness and efficiency decreased with an increasing doses/concentrations of mutagens. The coefficient of variation for all the quantitative characters were of higher magnitude compared to control in both the cultivars. The lower doses (30kR in gamma rays and 0.50% in EMS) were found to be the most important doses for inducing desirable variability in ricebean and two traits i.e. pods per cluster and seeds per pod showed high heritability coupled with high genetic advance in both BRS-1 and Totru Local indicating that breeding for these traits can be achieved by phenotypic selection.