S. Ramaswamy, Ph.D.
CEO of c-CAMP, Dean, inStem, NCBS, Bangalore, India
Discovery, engineering and applications of Blue Fish Protein with Red Fluorescence
Swagatha Ghosh, Chi-Li Yu, Daniel Ferraro, Sai Sudha, Wayne Schaefer, David T Gibson and S. Ramaswamy
Fluorescent proteins and their applications have revolutionized our understanding of biology significantly. In spite of several years since the discovery of the classic GFP, proteins of this class are used as the standard flag bearers. We have recently discovered a protein from the fish Sanders vitrius that shows interesting fluorescent properties – including a 280 nm stoke shift and infrared emission. The crystal structure of the wild type protein shows that it is a tetramer. We have engineered mutations to make a monomer with very similar fluorescent properties. We have used this protein for tissue imaging as well as for in cell-fluorescence successfully
Rajgopal Srinivasan, Ph.D.
Principal Scientist & Head Bio IT R&D, TCS Innovation Labs, India
Interpretation of Genomic Variation – Identifying Rare Variations Leading to Disease
Genome sequencing technologies are generating an abundance of data on human genetic variations. A big challenge lies in interpreting the functional relevance of such variations, especially in clinical settings. A first step in understanding the clinical relevance of genetic variations is to annotate the variants for region of occurrence, degree of conservation both within and across species, pattern of variation across related individuals, novelty of the variation and know effects of related variations. Several tools already exist for this purpose. However, these tools have their strengths and weaknesses. A second issue is the development of algorithms, which, given a rich annotation of variants are able to prioritize the variants as being relevant to the phenotype under investigation.
In my talk I will detail work that has been done in our labs to address both of the above problems. I will also illustrate the application of these tools that helped identify a rare mutation in the ATM gene leading to a diagnosis of AT in two infants.
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.
![Delegate Talk: Inefficient NETosis: Cause for Predisposition to Recurrent Infections in Type 2 Diabetes @ Acharya Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/gtm.jpg)
Manjunath Joshi, Apoorva Lad, Bharat Prasad Alevoor, Aswath Balakrishnan, Lingadakai Ramachandra and Kapaettu Satyamoorthy
Pathological conditions during Type 2 Diabetes (T2D) are associated with elevated risk for common community acquired infections due to poor glycemic control. Multiple studies have indicated specific defects in innate and adaptive immune function in diabetic subjects. Neutrophils play an important role in eliminating pathogens as an active constituent of innate immune system. Apart from canonically known phagocytosis mechanism, neutrophils are endowed with a unique ability to produce extracellular traps (NETs) to kill pathogens by expelling DNA coated with bactericidal proteins and histone. NETosis is stimulated by diverse bacteria and their products, fungi, protozoans, cytokines, phorbol esters and by activated platelets. Considering deregulation of metabolic and immune response pathways during pathological state of diabetes and NETosis as a potential mechanism for killing bacteria, we therefore, investigated whether hyperglycemic conditions modulate formation of neutrophil NETs and attempted to identify underlying immunoregulatory mechanisms. Freshly isolated neutrophils from normal individuals were cultured in absence or presence of high glucose (different concentrations) for 24 hours and activated with either LPS (2 mg/ml) or PMA (20 ng/ml) or IL-6 (20 ng/ml) for 3 hours. NETs were visualized and quantified by addition of DNA binding dye SYTOX green using fluorescence microscope and fluorimetry. NETs were quantified in Normal and diabetic subjects. Serum IL-6 levels were measured using ELISA technique. NETs bound elasatse were quantified in normal and diabetic subjects in presence or absence of DNase. Bacterial killing assays were performed upon infecting E.coli with activated neutrophils from normal and diabetic subjects. Microscopy and fluorimetry analysis suggested dramatic impairment in NETs formation under high glucose conditions. Extracellular DNA lattices formed in hyperglycemic conditions were short lived and unstable leading to rapid disintegration. Subsequent, time course experiments showed that NETs production was delayed in hyperglycemic conditions. To validate our findings more closely to clinical conditions, we investigated the neutrophil activation and NETs formation in diabetic patients. Upon stimulation with LPS for three hours, neutrophils from diabetic subjects responded weakly to LPS and lesser NETs were formed; whereas, neutrophils from normal individuals showed robust release of NETs. In few patients we found short and imperfect NETs in basal conditions suggesting constitutive activation of neutrophils in diabetic subjects. Interestingly, NETs bound elastase activity was reduced in diabetes subjects when compared to non-diabetic individuals, indicating a dysfunction of one of the important protein component of NETs during diabetes. Neutrophils from diabetic subjects released higher levels of IL-6 without any stimulation suggesting an existence of constitutively activated pro-inflammatory state. IL-6 induced NETs formation and was abrogated by high glucose. Weobserved that glycolysis inhibitor 2-DG resensitize the high glucose attenuated LPS and IL-6 induced NETs. a) NETs are influenced by glucose homeostasis, b) IL-6 as potent inducer of energy dependent NETs formation and c) hyperglycemia mimics a state of constitutively active pro-inflammatory condition in neutrophils leading to reduced response to external stimuli making diabetic subjects susceptible for infections.
![Delegate Talk: Inflammation Induced Epigenetic Changes in Endothelial Cells: Role in Vascular Insulin Resistance @ Acharya Hall | Vallikavu | Kerala | India](http://www.amritabioquest.org/conference/2013/wp-content/uploads/sites/2/2015/02/gtm.jpg)
Aswath Balakrishnan, Kapaettu Satyamoorthy and Manjunath B Joshi
Introduction
Insulin resistance is a hall mark of metabolic disorders such as diabetes. Reduced insulin response in vasculature leads to disruption of IR/Akt/eNOS signaling pathway resulting in vasoconstriction and subsequently to cardiovascular diseases. Recent studies have demonstrated that inflammatory regulator interleukin-6 (IL-6), as one of the potential mediators that can link chronic inflammation with insulin resistance. Accumulating evidences suggest a significant role of epigenetic mechanisms such as DNA methylation in progression of metabolic disorders. Hence the present study aimed to understand the role of epigenetic mechanisms involved during IL-6 induced vascular insulin resistance and its consequences in cardiovascular diseases.
Materials and Methods
Human umbilical vein endothelial cells (HUVEC) and Human dermal microvascular endothelial cells (HDMEC) were used for this study. Endothelial cells were treated in presence or absence of IL-6 (20ng/ml) for 36 hours and followed by insulin (100nM) stimulation for 15 minutes. Using immunoblotting, cell lysates were stained for phosphor- and total Akt levels to measure insulin resistance. To investigate changes in DNA methylation, cells were treated with or without neutrophil conditioned medium (NCM) as a physiological source of inflammation or IL-6 (at various concentrations) for 36 hours. Genomic DNA was processed for HPLC analysis for methyl cytosine content and cell lysates were analyzed for DNMT1 (DNA (cytosine-5)-methyltransferase 1) and DNMT3A (DNA (cytosine-5)-methyltransferase 3A) levels using immunoblotting.
Results
Endothelial cells stimulated with insulin exhibited an increase in phosphorylation of Aktser 473 in serum free conditions but such insulin response was not observed in cells treated with IL-6, suggesting chronic exposure of endothelial cells to IL-6 leads to insulin resistance. HPLC analysis for global DNA methylation resulted in decreased levels of 5-methyl cytosine in cells treated with pro-inflammatory molecules (both by NCM and IL-6) as compared to untreated controls. Subsequently, analysis in cells treated with IL-6 showed a significant decrease in DNMT1 levels but not in DNMT3A. Other pro-inflammatory marker such as TNF-α did not exhibit such changes.
Conclusion
Our study suggests: a) Chronic treatment of endothelial cells with IL-6 results in insulin resistance b) Neutrophil conditioned medium and IL-6 decreases methyl cytosine levels c) DNMT1 but not DNMT3a levels are reduced in cells treated with IL-6.
Akhilesh Pandey, Ph.D.
Professor, Johns Hopkins University School of Medicine, Baltimore, USA
A draft map of the human proteome
We have generated a draft map of the human proteome through a systematic and comprehensive analysis of normal human adult tissues, fetal tissues and hematopoietic cells as an India-US initiative. This unique dataset was generated from 30 histologically normal adult tissues, fetal tissues and purified primary hematopoietic cells that were analyzed at high resolution in the MS mode and by HCD fragmentation in the MS/MS mode on LTQ-Orbitrap Velos/Elite mass spectrometers. This dataset was searched against a 6-frame translation of the human genome and RNA-Seq transcripts in addition to standard protein databases. In addition to confirming a large majority (>16,000) of the annotated protein-coding genes in humans, we obtained novel information at multiple levels: novel protein-coding genes, unannotated exons, novel splice sites, proof of translation of pseudogenes (i.e. genes incorrectly annotated as pseudogenes), fused genes, SNPs encoded in proteins and novel N-termini to name a few. Many proteins identified in this study were identified by proteomic methods for the first time (e.g. hypothetical proteins or proteins annotated based solely on their chromosomal location). We have generated a catalog of proteins that show a more tissue-restricted pattern of expression, which should serve as the basis for pursuing biomarkers for diseases pertaining to specific organs. This study also provides one of the largest sets of proteotypic peptides for use in developing MRM assays for human proteins. Identification of several novel protein-coding regions in the human genome underscores the importance of systematic characterization of the human proteome and accurate annotation of protein-coding genes. This comprehensive dataset will complement other global HUPO initiatives using antibody-based as well as MRM mass spectrometry-based strategies. Finally, we believe that this dataset will become a reference set for use as a spectral library as well as for interesting interrogations pertaining to biomedical as well as bioinformatics questions.