Development of Normalized Cdna Library from Fusarium Wilt Infected Roots of a Tolerant Banana Genotype 'Calcutta-4' Musa acuminata ssp. burmannicoides

Management of the most devasting disease, Fusarium wilt of banana, caused by the fungus Fusarium oxysporum f. sp., cubense, is a challenge to the plant pathologist and the banana grower. Currently, genomics is providing the way for understanding plant defense mechanism, having acquired an important place in crop improvement. To identify the relevant genes and to understand the defense mechanism induced during Fusarium wilt infection, a normalized cDNA library was constructed from infected root samples of a tolerant banana genotype, Musa acuminata spp. burmannicoides 'Calcutta-4', by duplex specific nuclease (DSN) based normalization, using the SMART (switching mechanism at 5' end of RNA transcript) full-length cDNA construction method. Sequencing and analysis of 600 clones revealed 392 non-redundant clones. In all, of 88% of the sequences were annotated using Musa genome database, and the remaining 12% were identified as novel loci not annotated. We observed several resistance genes, ROS scavenging genes and genes involved in ubiquitin-proteosome pathway in this study. These genes may have a possible role against Foc infection. These sequences would enrich the EST data developed against specific stress, which is an indispensable tool for predicting functional genes and understanding the defense mechanism

Laboratory Information Management Software for genotyping workflows: Applications in high throughput crop genotyping

Background With the advances in DNA sequencer-based technologies, it has become possible to automate several steps of the genotyping process leading to increased throughput. To efficiently handle the large amounts of genotypic data generated and help with quality control, there is a strong need for a software system that can help with the tracking of samples and capture and management of data at different steps of the process. Such systems, while serving to manage the workflow precisely, also encourage good laboratory practice by standardizing protocols, recording and annotating data from every step of the workflow. Results A laboratory information management system (LIMS) has been designed and implemented at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) that meets the requirements of a moderately high throughput molecular genotyping facility. The application is designed as modules and is simple to learn and use. The application leads the user through each step of the process from starting an experiment to the storing of output data from the genotype detection step with auto-binning of alleles; thus ensuring that every DNA sample is handled in an identical manner and all the necessary data are captured. The application keeps track of DNA samples and generated data. Data entry into the system is through the use of forms for file uploads. The LIMS provides functions to trace back to the electrophoresis gel files or sample source for any genotypic data and for repeating experiments. The LIMS is being presently used for the capture of high throughput SSR (simple-sequence repeat) genotyping data from the legume (chickpea, groundnut and pigeonpea) and cereal (sorghum and millets) crops of importance in the semi-arid tropics. Conclusion A laboratory information management system is available that has been found useful in the management of microsatellite genotype data in a moderately high throughput genotyping laboratory. The application with source code is freely available for academic users and can be downloaded from http://www.icrisat.org/gt-bt/lims/lims.asp

Laboratory Information Management Software for genotyping workflows: applications in high throughput crop genotyping

BACKGROUND: With the advances in DNA sequencer-based technologies, it has become possible to automate several steps of the genotyping process leading to increased throughput. To efficiently handle the large amounts of genotypic data generated and help with quality control, there is a strong need for a software system that can help with the tracking of samples and capture and management of data at different steps of the process. Such systems, while serving to manage the workflow precisely, also encourage good laboratory practice by standardizing protocols, recording and annotating data from every step of the workflow. RESULTS: A laboratory information management system (LIMS) has been designed and implemented at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) that meets the requirements of a moderately high throughput molecular genotyping facility. The application is designed as modules and is simple to learn and use. The application leads the user through each step of the process from starting an experiment to the storing of output data from the genotype detection step with auto-binning of alleles; thus ensuring that every DNA sample is handled in an identical manner and all the necessary data are captured. The application keeps track of DNA samples and generated data. Data entry into the system is through the use of forms for file uploads. The LIMS provides functions to trace back to the electrophoresis gel files or sample source for any genotypic data and for repeating experiments. The LIMS is being presently used for the capture of high throughput SSR (simple-sequence repeat) genotyping data from the legume (chickpea, groundnut and pigeonpea) and cereal (sorghum and millets) crops of importance in the semi-arid tropics. CONCLUSION: A laboratory information management system is available that has been found useful in the management of microsatellite genotype data in a moderately high throughput genotyping laboratory. The application with source code is freely available for academic users and can be downloaded from

Effect of xenobiotic compounds on steroidogenesis in humans

Gonadal steroids are crucial hormones responsible for the proper functioning and growth of the body. The sex hormones are produced in the adrenal glands and gonads by a process called steroidogenesis. Steroidogenesis is an enzymatic reaction where cholesterol gets converted to active steroid hormones in the respective gonads and play a dominant role in determining the primary and secondary characteristics of organisms. Studies has revealed that highly divergent groups of xenobiotic compounds are known to obstruct with steroidogenesis and cause endocrine-disrupting effects. Environmental contaminants such as DDT and PCBs are known to affect steroidogenesis. Chemicals such as azole fungicides and antifungal drugs is known to function as powerful inhibitors of steroidogenic enzymes, resulting in endocrine disruption. With the increasing various hormonal disorders and decreased fertility rate due to stress and improper lifestyle, understanding the role and environmental impact of sex hormones on humans helps to manage and lead a healthy life. This review highlights the biosynthesis, functional mechanism of estrogen, progesterone and testosterone hormones including the effects of its varying levels and the influence of endocrine disrupting compounds (EDCs) on the steroidogenesis process

Cloning, Expression and Characterization of NAD Kinase from Staphylococcus aureus Involved in the Formation of NADP (H): A Key Molecule in the Maintaining of Redox Status and Biofilm Formation

Background:Staphylococcus aureus has the ability to form biofilms on any niches, a key pathogenic factor of this organism and this phenomenon is directly related to the concentration of NADPH. The formation of NADP is catalyzed by NAD kinase (NADK) and this gene of S. aureus ATCC 12600 was cloned, sequenced, expressed and characterized. Materials and Methods: The NADK gene was polymerase chain reaction amplified from the chromosomal DNA of S. aureus ATCC 12600 and cloned in pQE 30 vector, sequenced and expressed in Escherichia coli DH5α. The pure protein was obtained by passing through nickel metal chelate agarose column. The enzyme kinetics of the enzyme and biofilm assay of the S. aureus was carried out in both aerobic and anaerobic conditions. The kinetics was further confirmed by the ability of the substrates to dock to the NADK structure. Results: The recombinant NADK exhibited single band with a molecular weight of 31kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence (GenBank: JN645814) revealed presence of only one kind of NADK in all S. aureus strains. The enzyme exhibited very high affinity for NAD compared to adenosine triphosphate concurring with the docking results. A root-mean-square deviation value 14.039Š observed when NADK structure was superimposed with its human counterpart suggesting very low homology. In anaerobic conditions, higher biofilm units were found with decreased NADK activity. Conclusion: The results of this study suggest increased NADPH concentration in S. aureus plays a vital role in the biofilm formation and survival of this pathogen in any environmental conditions