Real-time Expression Profiling Research Articles

Characterization and overexpression of sterol Δ22-desaturase, a key enzyme modulates the biosyntheses of stigmasterol and withanolides in Withania somnifera (L.) Dunal

Withanolides constitute an extensive and vital class of metabolites displaying wide array of structural and therapeutic properties with unique side-chain modifications. These show diversified scaffolds and are promising pharmaceutical molecules with well documented anti-inflammatory and anti-cancer properties. Sterols are dynamic class of compounds and essential molecules having structural and functional significance. These contribute to the synthesis of withanolides by providing structural precursors. In this context, we have characterized sterol Δ22-desaturase from Withania somnifera and also functionally validating it by confirming its desaturase nature in conjunction with quantitative real-time expression profiling and metabolite evaluation. Further, transgenic hairy roots of W. somnifera displayed a higher accumulation of stigmasterol and withanolides. The increase in chemical constituents was concomitant with an increased gene copy number predicted via Southern blotting. Additionally, transgenic lines of tobacco over-expressing WsCYP710A11 displayed a substantial increase in its expression, corroborating well with enhanced stigmasterol content. Characterization of CYP710A11 from W. somnifera and its homologous transgenic expression has demonstrated its role in the regulation of withanolides biosynthesis. It also exhibited a differential transcriptional profile in response to exogenous elicitations. These empirical findings suggest the crucial role of CYP710A11 in stigmasterol biosynthesis. This in turn has implications for the overproduction of withanolides via pathway channelling.

Quantitative real-time expression profiling of aquaporins-isoforms and growth response of Brassica juncea under arsenite stress

The present study analyzed the expression level of aquaporins of plasma membrane intrinsic protein (PIP) class in response to arsenite (AsIII) exposure of 100 μM from 0.5 h to 8 days in Brassica juncea. The expression levels of most of the PIPs were down-regulated during the course of AsIII exposure. This led to decrease in total water content of plants, which in turn hampered seedling growth. The level of reactive oxygen species (superoxide radicals and hydrogen peroxide), lipid peroxidation and root oxidizability increased significantly upon exposure to AsIII as compared to that of control leading to an increase in cell death. The study proposes that the down-regulation of PIPs happened presumably to regulate AsIII levels, which, however, occurred at the cost of reduced growth, disturbed water balance and induced oxidative stress.

Molecular Subtype Characterization of Formalin-Fixed, Paraffin-Embedded Diffuse Large B-Cell Lymphoma Samples on the Iceplex® System.

AbstractAbstract 2705 Introduction:Molecular characterization of diffuse large B-cell lymphoma (DLBCL) provides important prognostic, and potentially therapeutic, information. Although the original classification of DLBCL into germinal center B cell-like (GCB) and activated B cell-like (ABC) subtypes was based on microarray gene expression profiling, this technique is not currently feasible in a clinical setting. Surrogate studies to assign classification, such as immunohistochemistry, have shown variability between laboratories and may not yield reliable results. Therefore, we have developed a novel multiplex real-time assay on the automated ICEPlex® System which allows differentiation between germinal center B cell-like (GCB) and activated B cell-like (ABC) subtypes of DLBCL from formalin-fixed, paraffin-embedded (FFPE) specimens. Materials and Methods:A 17-gene panel was constructed from genes utilized previously in DLBCL subtype classification (Wright classifier and Lossos outcome predictor). Primers were designed for this panel, and two reference genes, using extension tag amplification (ETA). This method allows amplification from FFPE tissue with a template size less than 100 nucleotides. Thirty de novo DLBCL clinical samples, with paired frozen tissue and FFPE tissue, from patients at our institution were selected. Gene expression profiling using microarrays was performed on frozen tissue, and molecular subtype was assigned based on Wright classification. The ICEPlex® assay was performed on isolated RNA from two cells lines, one GCB subtype and one ABC subtype. The ICEPlex® assay was also completed on RNA extracted from three DLBCL clinical samples, and molecular subtype was determined for each sample. Analysis of FFPE clinical samples and cell lines is underway using the multiplex assay on the ICEPlex® instrument for determination of molecular subtype. Results:The real-time 19-plex expression profiling assay for DLBCL molecular subtyping was developed and optimized on the ICEPlex® System. Samples from two cell lines and from three clinical samples with frozen tissue were analyzed. The assay identified the molecular subtype of both cell lines. All three clinical samples were classified into the identical molecular subtype by the multiplex assay and the microarray gene expression profile. Results from the profiling of additional FFPE clinical samples and cell lines using the multiplex assay will be presented. Conclusion:A novel 19-plex quantitative real-time expression profiling assay designed for DLBCL molecular classification into GCB or ABC subtypes was developed. This assay correctly assigned DLBCL cell lines and clinical samples into the correct subtype based on microarray gene expression profiling as the gold standard. Further work involves assessment of additional clinical DLBCL FFPE samples. This potentially robust multiplex assay may provide rapid molecular classification that is feasible for routine laboratory use in a clinical setting. * ICEPlex® System is for Research Use Only. Not for clinical diagnostic use. Disclosures:Nolling:PrimeraDx: Employment. Kong:PrimeraDx: Employment.

BK Polyoma Virus Induces Aberrant Methylation of Host Genome to Promote Epithelial Mesenchymal Transition and Fibrosis

Although most people are infected during early childhood, BK polyoma virus (BKV) reactivates and causes disease primarily in renal transplant patients. BKV is currently the leading cause of graft loss during the first two years after renal transplant. Activation of BKV is associated with inflammation, tissue destruction and progression to fibrosis through a process involving the transformation of epithelial cells in to mesenchymal phenotype. Understanding how BKV causes these changes will help us in better prediction and management of BKV nephropathy. As the primary site of BKV tropism is renal tubular epithelial cells, it needs to device ways to promote the transition and proliferation of these terminally differentiated cells. We hypothesized that BK may activate epigenetic mechanisms to promote these changes by inducing or inhibiting key genes involved in maintaining cellular integrity and stability. DNA CpG methylation is one such epigenetic mechanism which can control gene regulation by causing changes to the DNA which inhibit the accessibility of transcription factors to gene promoters. For our experiments, we have used primary human proximal renal tubular epithelial cells (HPTC) isolated from donor kidneys or procured from ATCC. Cells were infected with BKV for different time points. Real-time expression profiling was done to identify key genes modulated by BKV. Bisulfite conversion and Methylation specific PCR was used to identify specific CpG changes induced by BKV. Sequencing of the bisulfite converted DNA was done to identify the methylation status of entire CpGs islands in the promoters of these genes. Promoter region with specific CpGs was then cloned in pGL3 luciferase reporter vector to specify the role of particular CpGs in regulation and rule out the effect of trans-acting elements. For luciferase assays, CCD1105 KIDtr and HPTC cells were transfected using Lipofectamine- LTX and Plus reagent, infected with BK virus and assayed using Dual luciferase assay. Cell cycle analysis was done using propidium iodide staining and flow cytometry. Infection of renal epithelial cells by BKV caused induction of proinflammatory genes such as Cox-2 and interleukin 1 Beta along with Vimentin which is the marker for epithelial mesenchymal transition. More importantly, it inhibited retinoblastoma (RB1) and E-cadherin (CDH1) genes. We have for the first time identified that BK inhibits these genes by causing hypermethylation of specific CpG islands in their upstream regulatory promoters. These genes are important regulators of cell-cell adhesion (CDH1) and cell cycle (RB1), and their inhibition may lead to loss of adhesion and cell proliferation respectively. Luciferase Reporter assay using cloned RB1 promoter confirmed the involvement of these CpG islands and showed progressive decrease in RB1 promoter activity with increasing BK infection. Our studies also suggest that BKV infection may lead to increased aneuploidy in these cells further contributing to cellular instability and transformation. The changes induced by BKV may ultimately lead to inflammation, cellular proliferation and progression to renal fibrosis and graft loss. Specific CpG methylation patterns induced by BKV may also serve as biomarkers to predict the initiation of BKV nephropathy and help in devising relevant control strategies.

Primary metabolism of chickpea is the initial target of wound inducing early sensed Fusarium oxysporum f. sp. ciceri race I.

BackgroundBiotrophic interaction between host and pathogen induces generation of reactive oxygen species that leads to programmed cell death of the host tissue specifically encompassing the site of infection conferring resistance to the host. However, in the present study, biotrophic relationship between Fusarium oxysporum and chickpea provided some novel insights into the classical concepts of defense signaling and disease perception where ROS (reactive oxygen species) generation followed by hypersensitive responses determined the magnitude of susceptibility or resistant potentiality of the host.Methodology/Principal FindingsMicroscopic observations detected wound mediated in planta pathogenic establishment and its gradual progression within the host vascular tissue. cDNA-AFLP showed differential expression of many defense responsive elements. Real time expression profiling also validated the early recognition of the wound inducing pathogen by the host. The interplay between fungus and host activated changes in primary metabolism, which generated defense signals in the form of sugar molecules for combating pathogenic encounter.Conclusions/SignificanceThe present study showed the limitations of hypersensitive response mediated resistance, especially when foreign encounters involved the food production as well as the translocation machinery of the host. It was also predicted from the obtained results that hypersensitivity and active species generation failed to impart host defense in compatible interaction between chickpea and Fusarium. On the contrary, the defense related gene(s) played a critical role in conferring natural resistance to the resistant host. Thus, this study suggests that natural selection is the decisive factor for selecting and segregating out the suitable type of defense mechanism to be undertaken by the host without disturbing its normal metabolism, which could deviate from the known classical defense mechanisms.

Real-time expression profiling of microRNA precursors in human cancer cell lines

Our previous study described a real-time PCR method to quantify microRNA (miRNA) precursors using SYBR green detection [T. D. Schmittgen, J. Jiang, Q. Liu and L. Yang (2004) Nucleic Acids Res., 32, e43]. The present study adapted the assay to a 384-well format and expanded it to include primers to 222 human miRNA precursors. TaqMan minor groove binder probes were used to discriminate nearly identical members of the let-7 family of miRNA isoforms. The miRNA precursor expression was profiled in 32 human cell lines from lung, breast, colorectal, hematologic, prostate, pancreatic, and head and neck cancers. Some miRNA precursors were expressed at similar levels in many of the cell lines, while others were differentially expressed. Clustering analysis of the miRNA precursor expression data revealed that most of the cell lines clustered into their respective tissues from which each cell line was ostensibly derived. miRNA precursor expression by PCR paralleled the mature miRNA expression by northern blotting for most of the conditions studied. Our study provides PCR primer sequences to all of the known human miRNA precursors as of December 2004 and provides a database of the miRNA precursor expression in many commonly used human cancer cell lines.