Development and validation of multiplex-PCR assay to simultaneously detect favourable alleles of shrunken2, opaque2, crtRB1 and lcyE genes in marker-assisted selection for maize biofortification

Journal of Plant Biochemistry and Biotechnology - Sweet corn has emerged as a popular vegetable worldwide. Commercial shrunken2 (sh2)-based sweet corn lacks lysine, tryptophan and provitamin-A,...     

Development and validation of multiplex-PCR assay for simultaneous detection of rare alleles of <Emphasis Type="Italic">crtRB1</Emphasis> and <Emphasis Type="Italic">lcyE</Emphasis> governing higher accumulation of provitamin A in maize

Vitamin A deficiency is a widely prevalent health disorder among millions of people worldwide. Introgression of crtRB1 and lcyE favourable alleles that enhance concentration of provitamin A in maize endosperm have been employed in maize biofortification programmes. To make marker-assisted selection (MAS) more effective, we have developed rapid and convenient multiplex-polymerase chain reaction (PCR) assay to simultaneously discover the allelic combinations among the segregants. Validation of the multiplex assay was done in two backcross-derived populations developed using elite inbreds viz., HKI193-1 and HKI193-2 carrying unfavourable alleles of crtRB1 (296 bp) and lcyE (300 bp) and HarvestPlus inbreds viz., HP704-22 and HP704-23 possessing favourable alleles of crtRB1 (543 bp) and lcyE (650 bp). We also standardized the uniplex-PCR assays for both the genes that gave robust and reproducible results in sub-tropical populations. Gel profiles of BC₁F₁, BC₂F₁ and BC₂F₂ revealed that these assays identified the backcross progenies homo-or hetero-zygous for the favourable- or unfavourable-alleles. Multiplex-PCR assay also precisely confirmed the results of individual uniplex assays in different backcross generations. Cost and time analyses showed that multiplex-PCR assay has potential to save 41% of cost, and 50% of time compared to two uniplex assays in a MAS programme. It has also saved 50% of the manpower. The multiplex assay possesses significant advantage over uniplex assays and enhances the efficiency of selection. This is the first report of development and validation of multiplex-PCR assay of crtRB1 and lcyE for utilization in maize biofortification programme.     

Biochemical and histological changes during in vitro rooting of microcuttings of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.) Wettst.

The present study was conducted to investigate the biochemical changes vis-à-vis histological changes during adventitious rooting of microcuttings of Bacopa monnieri (L.) Wettst. The rooting in these microcuttings was induced on basal MS medium and medium supplemented with different concentrations of indole-3-acetic acid and indole-3-butyric acid (IBA). Presence of lower auxin concentration (1.0 µM) in the medium enhanced rooting and significantly improved number of roots per shoot but maximum root length was observed on basal MS medium. Histological studies were conducted to identify different phases of rooting in these microcuttings. The root meristemoids with distinct polarity become visible after 3 days and mark the beginning of in vitro root initiation phase. It was followed by primordia elongation, root emergence and visible rooting on the 5th day of culture on medium supplemented with auxins. Biochemical studies were also conducted from basal portions of microcuttings cultured on MS medium supplemented with 1.0 µM IBA and control (basal MS medium) from 0 to 7 days. Total carbohydrate content was lower during initial periods (up to day 1) and was found to increase during root initiation and primordia development, which reflects high energy demands for active cell divisions. A significantly higher level of phenols was recorded in microcuttings on medium supplemented with IBA. Polyphenol oxidase, peroxidase (POX), ascorbate peroxidase activities were also found to vary during different phases of rhizogenesis. Early phases were also marked with the lower activities of POX and IAAO. This study revealed significant role of enzymes, sugars and phenols during different phases of rooting.     

Synthesis and antifungal activity of 1,2,3-triazole containing fluconazole analogues

Fluconazole based novel mimics containing 1,2,3-triazole were designed and synthesized as antifungal agents. Their antifungal activities were evaluated in vitro by measuring the minimal inhibitory concentrations (MICs). Compounds 12, 15, and 16 were found to be more potent against Candida fungal pathogens than control drugs fluconazole and amphotericin B. The studies presented here provide structural modification of fluconazole to give 1,2,3-trazole containing molecules. Furthermore, these molecules were evaluated in vivo against Candida albicans intravenous challenge in Swiss mice and antiproliferative activities were tested against human hepatocellular carcinoma Hep3B and human epithelial carcinoma A431. It was found that compound 12 resulted in 97.4% reduction in fungal load in mice and did not show any profound proliferative effect at lower dose (0.001 mg/ml).     

Decoding the signature of molecular mechanism involved in mutation associated resistance to 1, 3-benzothiazin-4-ones (Btzs) based DprE1 inhibitors using BTZ043 as a reference drug

ABSTRACT

Different resistant strains of M. tuberculosis (Mtb) highlight the urgent need of novel anti-tubercular drugs. In mycobacteria, decaprenyl-phosphoryl-β-D-ribose 2’-oxidase (DprE1) is an appealing enzyme to target as it is involved in the biosynthesis of cell wall component arabinogalactan.1, 3-benzothiazin-4-ones (BTZs) based drugs are promising irreversible inhibitors of DprE1. However, a single point mutation of Cys387Ser in DprE1 results in the development of resistance to these drugs. Herein, we made an effort to decode the molecular mechanism of Cys387Ser DprE1 mutation associated resistance in Mtb against BTZs using different in silico techniques. Since the 3D crystal structure of mutant Cys387Ser protein is not yet been solved, thus the homology model was also developed using 4P8N as a template protein with 99.8% homology with the target protein. The computational results suggested that the factors like HOMO–LUMO energy gap, Burgi-Dunitz angle and distance support the covalent inhibition of wild DprE1 by 1, 3-benzothiazin-4-ones class of drugs, using BTZ043 as a reference drug and the same factors support the cause of resistance in case of Cys387Ser mutation. On the basis of these results, it was concluded that BTZ043 can efficiently inhibit the wild type DprE1 than mutant DprE1.     

Pharmacophore modeling and molecular dynamics approach to identify putative DNA Gyrase B inhibitors for resistant tuberculosis

One of the major mechanisms followed by the therapeutic agents to target the causative organism of TB, mycobacterium tuberculosis (Mtb), involves disruption of the replication cycle of the pathogen DNA. The process involves two steps that occur simultaneously, ie, breakage and reunion of DNA at gyrase A (GyrA) domain and ATP hydrolysis at gyrase B (GyrB) domain. Current therapy for multi-drug resistant TB involves FDA approved, Fluoroquinolone-based antibiotics, which act by targeting the replication process at GyrA domain. However, resistance against fluoroquinolones due to mutations in the GyrA domain has limited the use of this therapy and shifted the focus of the research community on the GyrB domain. Thus, this study involves in silico designing of chemotherapeutic agents for resistant TB by targeting GyrB domain. In the current study, a pharmacophore model for GyrB domain was generated using reported inhibitors. It was utilized as a query search against three commercial databases to identify GyrB domain inhibitors. Additionally, a qualitative Hip-Hop pharmacophore model for GyrA was also developed on the basis of some marketed fluoroquinolone-based GyrA inhibitors, to remove non-selective gyrase inhibitors obtained in virtual screening. Further, molecular dynamic simulations were carried out to determine the stability of the obtained molecules in complex with both the domains. Finally, Molecular mechanics with generalized Born and surface area solvation score was calculated to determine the binding affinity of obtained molecule with both domains to determine the selectivity of the obtained molecules that resulted in seven putative specific inhibitors of GyrB domain.     

Anti-diabetic vanadyl complexes reduced Alzheimer’s disease pathology independent of amyloid plaque deposition

Association of Alzheimer's disease(AD) with cerebral glucose hypometabolism, likely due to impairments of insulin signaling,has been reported recently, with encouraging results when additional insulin is provided to AD patients. Here, we tested the potential effects of the anti-diabetic vanadium, vanadyl(IV) acetylacetonate(VAC), on AD in vitro and in vivo models. The experimental results showed that VAC at sub-micromolar concentrations improved the viability of neural cells with or without increased β-amyloid(Aβ) burden; and in APP/PS1 transgenic mice, VAC treatment(0.1 mmol kg-1 d-1) preserved cognitive function and attenuated neuron loss, but did not reduce brain Aβ plaques. Further studies revealed that VAC attenuated Aβpathogenesis by(i) activation of the PPARγ-AMPK signal transduction pathway, leading to improved glucose and energy metabolism;(ii) up-regulation of the expression of glucose-regulated protein 75(Grp75), thus suppressing p53-mediated neuronal apoptosis under Aβ-related stresses; and(iii) decreasing toxic soluble Aβ peptides. Overall, our work suggested that vanadyl complexes may have great potential for effective therapeutic treatment of AD.