DNA fingerprinting for the authentication of Ruta graveolens

Ruta graveolens is a small aromatic shrub and has been used medicinally and magically, since ancient times. In this study, random amplified polymorphic DNA (RAPD) was employed to develop reproducible markers for authentication of this species from its adulterant Euphorbia dracunculoides. The random decamer oligonucleotide primers (42) were screened for identification of genuine and adulterant samples using the DNA isolated from the dried leaf, seed and stem of both samples. Out of 42 primers, 10 gave faint band, 12 gave species-specific reproducible unique band and the remaining did not amplify the DNA. RAPD could thus, serve as a complementary tool for quality control.Key words: Adulterant, Euphorbia dracunculoides, herbal drugs, random amplified polymorphic DNA

Concentration-dependent toxicity of iron oxide nanoparticles mediated by increased oxidative stress

Iron oxide nanoparticles with unique magnetic properties have a high potential for use in several biomedical, bioengineering and in vivo applications, including tissue repair, magnetic resonance imaging, immunoassay, drug delivery, detoxification of biologic fluids, cell sorting, and hyperthermia. Although various surface modifications are being done for making these nonbiodegradable nanoparticles more biocompatible, their toxic potential is still a major concern. The current in vitro study of the interaction of superparamagnetic iron oxide nanoparticles of mean diameter 30 nm coated with Tween 80 and murine macrophage (J774) cells was undertaken to evaluate the dose- and time-dependent toxic potential, as well as investigate the role of oxidative stress in the toxicity. A 15–30 nm size range of spherical nanoparticles were characterized by transmission electron microscopy and zeta sizer. MTT assay showed >95% viability of cells in lower concentrations (25–200 μg/mL) and up to three hours of exposure, whereas at higher concentrations (300–500 μg/mL) and prolonged (six hours) exposure viability reduced to 55%–65%. Necrosis-apoptosis assay by propidium iodide and Hoechst-33342 staining revealed loss of the majority of the cells by apoptosis. H2DCFDDA assay to quantify generation of intracellular reactive oxygen species (ROS) indicated that exposure to a higher concentration of nanoparticles resulted in enhanced ROS generation, leading to cell injury and death. The cell membrane injury induced by nanoparticles studied using the lactate dehydrogenase assay, showed both concentration- and time-dependent damage. Thus, this study concluded that use of a low optimum concentration of superparamagnetic iron oxide nanoparticles is important for avoidance of oxidative stress-induced cell injury and death

Genetic diversity analysis in the Hypericum perforatum populations in the Kashmir valley by using inter-simple sequence repeats (ISSR) markers

Assessment of genetic variability among the Hypericum perforatum populations is critical to the development of effective conservation  strategies in the Kashmir valley. To obtain accurate estimates of genetic diversity among and within populations of H. perforatum, inter-simple sequence repeats (ISSR) markers were used. The study was aimed to check, whether ISSR fingerprinting may be a useful tool for studying genetic variations among H. perforatum populations in the Kashmir valley (India). A total of 15 ISSR primers were tested with the 20 genotypes of H. perforatum. The ten informative primers were selected and used to evaluate the degree of polymorphism and genetic relationships within and among all the H. perforatum populations. ISSR of 20 genotypes analysis yielded 98 fragments that could be scored, of which 71 were polymorphic, with an average of 7.1 polymorphic fragments per primer. Number of amplified fragments varied in size from 150 to 1650 bp. Percentage of polymorphism ranged from 60% to a maximum of 100%. Resolving power ranged from a minimum of 7.7 to a maximum of 14.3. Shannon indexes ranges from 0.166 to 0.389 with an average of 0.198 and Nei’s genetic diversity (h) ranges from 6.98 to 9.8. Estimated value of gene flow (Nm = 0.579) indicated that there was limited gene flow among the populations. The genetic diversity (Ht) within the population of 0.245 was clearly higher than that of among population genetic diversity (Hs= 0.115), indicating an out-crossing predominance in the studied populations. Analysis of molecular variance by ISSR markers indicated that over half of the total variation in the studied populations (58%) could be accounted for by differences among the 8 divisions, with a further 42% being accounted for by the variation among populations within a division.The dendrogram grouping the populations by unweighted pair-group method with arithmeticaverages (UPGMA) method revealed eight main clusters. In conclusion, combined analysis of ISSR markers and hypericin content is an optimal approach for further progress and breeding programs.Keywords: Hypericum perforatum (St. John's Wort), inter-simple sequence repeats (ISSR) markers, unweighted pair-group method with arithmetic averages (UPGMA), Nei’s genetic diversityAfrican Journal of Biotechnology, Vol. 13(1), pp. 18-31, 1 January, 201

Concentration-dependent toxicity of iron oxide nanoparticles mediated by increased oxidative stress

Saba Naqvi1, Mohammad Samim2, MZ Abdin3, Farhan Jalees Ahmed4, AN Maitra5, CK Prashant6, Amit K Dinda61Faculty of Engineering and Interdisciplinary Sciences, 2Department of Chemistry, 3Department of Biotechnology, Faculty of Science, 4Department of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard, Hamdard University, 5Department of Chemistry, University of Delhi, 6Department of Pathology, All India Institute of Medical Sciences, New Delhi, IndiaAbstract: Iron oxide nanoparticles with unique magnetic properties have a high potential for use in several biomedical, bioengineering and in vivo applications, including tissue repair, magnetic resonance imaging, immunoassay, drug delivery, detoxification of biologic fluids, cell sorting, and hyperthermia. Although various surface modifications are being done for making these nonbiodegradable nanoparticles more biocompatible, their toxic potential is still a major concern. The current in vitro study of the interaction of superparamagnetic iron oxide nanoparticles of mean diameter 30 nm coated with Tween 80 and murine macrophage (J774) cells was undertaken to evaluate the dose- and time-dependent toxic potential, as well as investigate the role of oxidative stress in the toxicity. A 15–30 nm size range of spherical nanoparticles were characterized by transmission electron microscopy and zeta sizer. MTT assay showed >95% viability of cells in lower concentrations (25–200 µg/mL) and up to three hours of exposure, whereas at higher concentrations (300–500 µg/mL) and prolonged (six hours) exposure viability reduced to 55%–65%. Necrosis-apoptosis assay by propidium iodide and Hoechst-33342 staining revealed loss of the majority of the cells by apoptosis. H2DCFDDA assay to quantify generation of intracellular reactive oxygen species (ROS) indicated that exposure to a higher concentration of nanoparticles resulted in enhanced ROS generation, leading to cell injury and death. The cell membrane injury induced by nanoparticles studied using the lactate dehydrogenase assay, showed both concentration- and time-dependent damage. Thus, this study concluded that use of a low optimum concentration of superparamagnetic iron oxide nanoparticles is important for avoidance of oxidative stress-induced cell injury and death.Keywords: superparamagnetic iron oxide nanoparticles, cytotoxicity, MTT assay, J774 cell lin

Evaluation of Yeast as an Expression System

477-493Developments in recombinant DNA technology have provided an alternate route for the production of proteins. Gene expression and production of proteins of interest are of great importance for basic research as well as for biomedical applications. A number of expression systems using mammalian cells, insect cells, yeast and other bacteria as host have been developed. Yeast has received attention as a suitable host for expression of many mammalian genes due to many specific characteristics. Yeast strains, Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris, have many advantages over other systems and may be the host of choice for the expression of complex proteins of therapeutic value. During the post-genomicera, the importance of these strains for the expression of heterologous genesmay enhance considerably

Rapid and efficient <em>Agrobacterium </em> mediated transformation of early scutellum derived calli of <em>indica</em> rice

20-28Rice is a staple food for humans and its demand in 2035 has been put at 852 million tons. Knowledge on genes and genome architecture helps in better understanding of growth and development mechanisms for crop improvement. Transgenic crops may offer a solution by means of higher yield and resistance to biotic and abiotic stresses. In this context, modification of Agrobacterium mediated transformation protocol for indica rice cultivar is imperative to increase transformation efficiency and reduce duration of transgenic development. Here, we developed an efficient Agrobacterium mediated transformation protocol using early scutellum derived calli of the indica rice cultivar Pusa Sugandh 2. Competency of 3, 4, 5 and 6 day old primary calli was compared with 21- day old secondary calli for Agrobacterium mediated transformation using a modified pCAMBIA 1304 harbouring GFP-GUS fusion gene driven by maize ubiquitin 1 promoter. The highest competency with stable transformation efficiency of 51% was observed for 5-6 day old primary calli. Molecular analysis confirmed stable integration of the transgene. Transgenic lines of Pusa Sugandh 2 were developed within a short period of two months using 5-6 day old primary calli

Development of Highly Sensitive Sandwich ELISA for the Early-Phase Diagnosis of Chikungunya Virus Utilizing rE2-E1 Protein

Mohammad Islamuddin,1,2 Abuzer Ali,3 Wajihul Hasan Khan,4 Amena Ali,5 Syed Kazim Hasan,1 Mohd Abdullah,6 Kentaro Kato,2 Malik Zainul Abdin,7 Shama Parveen1 1Molecular Virology Laboratory, Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi, 110025, India; 2Laboratory of Sustainable Animal Environment, Graduate School of Agricultural Science, Tohoku University, Miyagi, Japan; 3Microbiology Laboratory, Ansari Health Center, Jamia Millia Islamia, New Delhi, 110025, India; 4Department of Pharmacognosy, College of Pharmacy, Taif University, Taif, 21944, Saudi Arabia; 5Molecular Virology Lab, Department of Microbiology, All India Institute of Medical Sciences (AIIMS), New Delhi, 110029, India; 6Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, Taif, 21944, Saudi Arabia; 7Department of Biotechnology, School of Chemical and Life Sciences, Hamdard University, New Delhi, 110026, IndiaCorrespondence: Mohammad Islamuddin; Shama Parveen, Email [email protected]; [email protected]: Chikungunya is caused by an alpha virus transmitted to humans by an infected mosquito. Infection is generally considered to be self-limiting and non-critical. Chikungunya infection may be diagnosed by severe joint pain with fever, but it is difficult to diagnose because the symptoms of chikungunya are common to many pathogens, including dengue fever. Diagnosis mainly depends on viral culture, reverse transcriptase polymerase chain reaction (RT-PCR), and IgM ELISA. Early and accurate diagnosis of the virus can be achieved by the application of PCR methods, but the high cost and the need for a thermal cycler restrict the use of such methods. On the other hand, antibody-based IgM ELISA is considered to be inexpensive, but antibodies against chikungunya virus (CHIKV) only develop after 4 days of infection, so it has limited application in the earlier diagnosis of viral infection and the management of patients. Because of these challenges, a simple antigen-based sensitive, specific, and rapid detection method is required for the early and accurate clinical diagnosis of chikungunya.Methods: The amino acid sequence of CHIKV ectodomain E1 and E2 proteins was analyzed using bioinformatics tools to determine the antigenic residues, particularly the B-cell epitopes and their characteristics. Recombinant E2-E1 CHIKV antigen was used for the development of polyclonal antibodies in hamsters and IgG was purified. Serological tests of 96 CHIKV patients were conducted by antigen-capture ELISA using primary antibodies raised against rCHIKV E2-E1 in hamsters and human anti-CHIKV antibodies.Results: We observed high specificity and sensitivity, of 100% and 95.8%, respectively, and these values demonstrate the efficiency of the test as a clinical diagnostic tool. There was no cross-reactivity with samples taken from dengue patients.Discussion: Our simple and sensitive sandwich ELISA for the early-phase detection of CHIKV infection may be used to improve the diagnosis of chikungunya.Graphical Abstract: Keywords: chikungunya, sandwich ELISA, recombinant E2-E1, B-cell epitopes, IgG, hamster antibodie

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Not AvailableRice blast is a global threat to food security with up to 50% yield losses. Panicle blast is a more severe form of rice blast and the response of rice plant to leaf and panicle blast is distinct in different genotypes. To understand the specific response of rice in panicle blast, transcriptome analysis of blast resistant cultivar Tetep, and susceptible cultivar HP2216 was carried out using RNA-Seq approach after 48, 72 and 96 h of infection with Magnaporthe oryzae along with mock inoculation. Transcriptome data analysis of infected panicle tissues revealed that 3553 genes differentially expressed in HP2216 and 2491 genes in Tetep, which must be the responsible factor behind the differential disease response. The defense responsive genes are involved mainly in defense pathways namely, hormonal regulation, synthesis of reactive oxygen species, secondary metabolites and cell wall modification. The common differentially expressed genes in both the cultivars were defense responsive transcription factors, NBS-LRR genes, kinases, pathogenesis related genes and peroxidases. In Tetep, cell wall strengthening pathway represented by PMR5, dirigent, tubulin, cell wall proteins, chitinases, and proteases was found to be specifically enriched. Additionally, many novel genes having DOMON, VWF, and PCaP1 domains which are specific to cell membrane were highly expressed only in Tetep post infection, suggesting their role in panicle blast resistance. Thus, our study shows that panicle blast resistance is a complex phenomenon contributed by early defense response through ROS production and detoxification, MAPK and LRR signaling, accumulation of antimicrobial compounds and secondary metabolites, and cell wall strengthening to prevent the entry and spread of the fungi. The present investigation provided valuable candidate genes that can unravel the mechanisms of panicle blast resistance and help in the rice blast breeding program.CABin Scheme, ICA

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Not AvailableRice blast is a global threat to food security with up to 50% yield losses. Panicle blast is a more severe form of rice blast and the response of rice plant to leaf and panicle blast is distinct in different genotypes. To understand the specific response of rice in panicle blast, transcriptome analysis of blast resistant cultivar Tetep, and susceptible cultivar HP2216 was carried out using RNA-Seq approach after 48, 72 and 96 h of infection with Magnaporthe oryzae along with mock inoculation. Transcriptome data analysis of infected panicle tissues revealed that 3553 genes differentially expressed in HP2216 and 2491 genes in Tetep, which must be the responsible factor behind the differential disease response. The defense responsive genes are involved mainly in defense pathways namely, hormonal regulation, synthesis of reactive oxygen species, secondary metabolites and cell wall modification. The common differentially expressed genes in both the cultivars were defense responsive transcription factors, NBS-LRR genes, kinases, pathogenesis related genes and peroxidases. In Tetep, cell wall strengthening pathway represented by PMR5, dirigent, tubulin, cell wall proteins, chitinases, and proteases was found to be specifically enriched. Additionally, many novel genes having DOMON, VWF, and PCaP1 domains which are specific to cell membrane were highly expressed only in Tetep post infection, suggesting their role in panicle blast resistance. Thus, our study shows that panicle blast resistance is a complex phenomenon contributed by early defense response through ROS production and detoxification, MAPK and LRR signaling, accumulation of antimicrobial compounds and secondary metabolites, and cell wall strengthening to prevent the entry and spread of the fungi. The present investigation provided valuable candidate genes that can unravel the mechanisms of panicle blast resistance and help in the rice blast breeding programNot Availabl