Bharat B. Chattoo, Ph.D.
Professor, Faculty of Science M.S.University of Baroda, India
Biology of plant infection by Magnaporthe oryzae
The rice blast disease caused by the ascomycetous fungus Magnaporthe oryzae is a major constraint in rice production. Rice-M.oryzae is also emerging as a good model patho-system to investigate how the fungus invades and propagates within the host. Identification and characterisation of genes critical for fungal pathogenesis provides opportunities to explore their use as possible targets for development of strategies for combating fungal infection and to better understand the complex process of host-pathogen interaction.
We have used insertional mutagenesis and RNAi based approaches to identify pathogenesis related genes in this fungus. A large number of mutants were isolated using Agrobacterium tumefaciens mediated transformation (ATMT). Characterisation of several interesting mutants is in progress. We have identified a novel gene, MGA1, required for the development of appressoria. The mutant mga1 is unable to infect and is impaired in glycogen and lipid mobilization required for appressorium development. The glycerol content in the mycelia of the mutant was significantly lower as compared to wild type and it was unable to tolerate hyperosmotic stress. A novel ABC transporter was identified in this fungus. The abc4 mutant did not form functional appressoria, was non-pathogenic and showed increased sensitivity to certain antifungal molecules implying the role of ABC4 in multidrug resistance (MDR). Another mutant MoSUMO (MGG_05737) was isolated using a Split Marker technique; the mutant showed defects in growth, germination and infection. Immuno-fluorescence microscopy revealed that MoSumo is localized to septa in mycelia and nucleus as well as septa in spores. Two Dimensional Gel Electrophoresis showed differences in patterns of protein expression between Wild Type B157 and MoΔSumo mutant. We also isolated and charaterised mutants in MoALR2 (MGG_08843) and MoMNR2 (MGG_09884). Our results indicate that both MoALR2 and MoMNR2 are Mg2+ transporters, and the reduction in the levels of CorA transporters caused defects in surface hydrophobicity, cell wall stress tolerance, sporulation, appressorium formation and infection are mediated through changes in the key signaling cascades in the knock-down transformants. (Work supported by the Department of Biotechnology, Government of India)
Gokul Das, Ph.D.
Co-Director, Breast Disease Site Research Group, Roswell Park Cancer Institute, Buffalo, NY
Probing Estrogen Receptor−Tumor Suppressor p53 Interaction in Cancer: From Basic Research to Clinical Trial
Tumor suppressor p53 and estrogen receptor have opposite roles in the onset and progression of breast cancer. p53 responds to a variety of cellular of stresses by restricting the proliferation and survival of abnormal cells. Estrogen receptor plays an important role in normal mammary gland development and the preservation of adult mammary gland function; however, when deregulated it becomes abnormally pro-proliferative and greatly contributes to breast tumorigenesis. The biological actions of estrogens are mediated by two genetically distinct estrogen receptors (ERs): ER alpha and ER beta. In addition to its expression in several ER alpha-positive breast cancers and normal mammary cells, ER beta is usually present in ER alpha-negative cancers including triple-negative breast cancer. In spite of genetically being wild type, why p53 is functionally debilitated in breast cancer has remained unclear. Our recent finding that ER alpha binds directly to p53 and inhibits its function has provided a novel mechanism for inactivating genetically wild type p53 in human cancer. Using a combination of proliferation and apoptosis assays, RNAi technology, quantitative chromatin immunoprecipitation (qChIP), and quantitative real-time PCR (qRT-PCR), in situ proximity ligation assay (PLA), and protein expression analysis in patient tissue micro array (TMA), we have demonstrated binding of ER alpha to p53 and have delineated the domains on both the proteins necessary for the interaction. Importantly, ionizing radiation inhibits the ER-p53 interaction in vivo both in human cancer cells and human breast tumor xenografts in mice. In addition, antiestrogenstamoxifen and faslodex/fulvestrant (ICI 182780) disrupt the ER-p53 interaction and counteract the repressive effect of ER alpha on p53, whereas 17β-estradiol (E2) enhances the interaction. Intriguingly, E2 has diametrically opposite effects on corepressor recruitment to a p53-target gene promoter versus a prototypic ERE-containing promoter. Thus, we have uncovered a novel mechanism by which estrogen could be providing a strong proliferative advantage to cells by dual mechanisms: enhancing expression of ERE-containing pro-proliferative genes while at the same time inhibiting transcription of p53-dependent anti-proliferative genes. Consistently, ER alpha enhances cell cycle progression and inhibits apoptosis of breast cancer cells. Correlating with these observations, our retrospective clinical study shows that presence of wild type p53 in ER-positive breast tumors is associated with better response to tamoxifen therapy. These data suggest ER alpha-p53 interaction could be one of the mechanisms underlying resistance to tamoxifen therapy, a major clinical challenge encountered in breast cancer patients. We have launched a prospective clinical trial to analyze ER-p53 interaction in breast cancer patient tumors at Roswell Park Cancer Institute. Our more recent finding that ER beta has opposite functions depending on the mutational status of p53 in breast cancer cells is significant in understanding the hard-to-treat triple-negative breast cancer and in developing novel therapeutic strategies against it. Our integrated approach to analyze ER-p53 interaction at the basic, translational, and clinical research levels has major implications in the diagnosis, prognosis, and treatment of breast cancer.
Pandiaraj Manickam, Niroj Kumar Sethy, Kalpana Bhargava, Vepa Kameswararao and Karunakaran Chandran
Designing electrochemical label free immunosensors for cytochrome c using nanocomposites functionalized screen printed electrodes
Release of cytochrome c (cyt c) from mitochondria into cytosol is a hallmark of apoptosis, used as a biomarker of mitochondrial dependent pathway of cell death (Kluck et al. 1997; Green et al. 1998). We have previously reported cytochrome c reductase (CcR) based biosensors for the measurement of mitochondrial cyt c release (Pandiaraj et al. 2013). Here, we describe the development of novel label-free, immunosensor for cyt c utilizing its specific monoclonal antibody. Two types of nanocomposite modified immunosensing platforms were used for the immobilization of anti-cyt c; (i) Self-assembled monolayer (SAM) functionalized gold nanoparticles (GNP) in conducting polypyrrole (PPy) modified screen printed electrodes (SPE) (ii) Carbon nanotubes (CNT) incorporated PPy on SPE. The nanotopologies of the modified electrodes were confirmed by scanning electron microscopy (SEM). Cyclic voltammetry, electrochemical impedance spectroscopy (EIS) were used for probing the electrochemical properties of the nanocomposite modified electrodes. Method for cyt c quantification is based on the direct electron transfer between Fe3+/Fe2+-heme of cyt c selectively bound to anti-cyt c modified electrode. The Faradaic current response of these nanoimmunosensor increases with increase in cyt c concentration. The procedure for cyt c detection was also optimized (pH, incubation times, and characteristics of electrodes) to improve the analytical characteristics of immunosensors. The analytical performance of anti-cyt c biofunctionalized GNP-PPy nanocomposite platform (detection limit 0.5 nM; linear range: 0.5 nM–2 μM) was better than the CNT-PPy (detection limit 2 nM; linear range: 2 nM-500nM). The detection limits were well below the normal physiological concentration range (Karunakaran et al. 2008). The proposed method does not require any signal amplification or labeled secondary antibodies contrast to widespread ELISA and Western blot. The immunosensors results in simple and rapid measurement of cyt c and has great potential to become an inexpensive and portable device for conventional clinical immunoassays.