Kandasamy Ravi

High definition profiling of mammalian DNA methylation by array capture and single molecule bisulfite sequencing

DNA methylation stabilizes developmentally programmed gene expression states. Aberrant methylation is associated with disease progression and is a common feature of cancer genomes. Presently, few methods enable quantitative, large-scale, single-base resolution mapping of DNA methylation states in desired regions of a complex mammalian genome. Here, we present an approach that combines array-based hybrid selection and massively parallel bisulfite sequencing to profile DNA methylation in genomic regions spanning hundreds of thousands of bases. This single molecule strategy enables methylation variable positions to be quantitatively examined with high sampling precision. Using bisulfite capture, we assessed methylation patterns across 324 randomly selected CpG islands (CGI) representing more than 25,000 CpG sites. A single lane of Illumina sequencing permitted methylation states to be definitively called for >90% of target sties. The accuracy of the hybrid-selection approach was verified using conventional bisulfite capillary sequencing of cloned PCR products amplified from a subset of the selected regions. This confirmed that even partially methylated states could be successfully called. A comparison of human primary and cancer cells revealed multiple differentially methylated regions. More than 25% of islands showed complex methylation patterns either with partial methylation states defining the entire CGI or with contrasting methylation states appearing in specific regional blocks within the island. We observed that transitions in methylation state often correlate with genomic landmarks, including transcriptional start sites and intron-exon junctions. Methylation, along with specific histone marks, was enriched in exonic regions, suggesting that chromatin states can foreshadow the content of mature mRNAs.

Genome-wide copy number analysis of single cells

Copy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells in which information regarding genetic heterogeneity is lost. Here we present a protocol that allows for the genome-wide copy number analysis of single nuclei isolated from mixed populations of cells. Single-nucleus sequencing (SNS), combines flow sorting of single nuclei on the basis of DNA content and whole-genome amplification (WGA); this is followed by next-generation sequencing to quantize genomic intervals in a genome-wide manner. Multiplexing of single cells is discussed. In addition, we outline informatic approaches that correct for biases inherent in the WGA procedure and allow for accurate determination of copy number profiles. All together, the protocol takes ∼3 d from flow cytometry to sequence-ready DNA libraries.

BRCA1 Tumor Suppression Depends on BRCT Phosphoprotein Binding, But Not Its E3 Ligase Activity

The properties of breast cancer susceptibility protein required for tumor suppression have been explored. Germline mutations of the breast cancer 1 (BRCA1) gene are a major cause of familial breast and ovarian cancer. The BRCA1 protein displays E3 ubiquitin ligase activity, and this enzymatic function is thought to be required for tumor suppression. To test this hypothesis, we generated mice that express an enzymatically defective Brca1. We found that this mutant Brca1 prevents tumor formation to the same degree as does wild-type Brca1 in three different genetically engineered mouse (GEM) models of cancer. In contrast, a mutation that ablates phosphoprotein recognition by the BRCA C terminus (BRCT) domains of BRCA1 elicits tumors in each of the three GEM models. Thus, BRCT phosphoprotein recognition, but not the E3 ligase activity, is required for BRCA1 tumor suppression.

Nitric Oxide Coordinates Cell Proliferation and Cell Movements During Early Development of Xenopus

The establishment of a vertebrate body plan during embryogenesis is achieved through precise coordination of cell proliferation and morphogenetic cell movements. Here we show that nitric oxide (NO) suppresses cell division and facilitates cell movements during early development of Xenopus, such that inhibition of NO synthase (NOS) increases proliferation in the neuroectoderm and suppresses convergent extension in the axial mesoderm and neuroectoderm. NO controls cell division and cell movement through two separate signaling pathways. Both rely on RhoA-ROCK signaling but can be distinguished by the involvement of either guanylate cyclase or the planar cell polarity regulator Dishevelled. Through the cGMP-dependent pathway, NO suppresses cell division by negatively regulating RhoA and controlling the nuclear distribution of ROCK and p21WAF1. Through the cGMP-independent pathway, NO facilitates cell movement by regulating the intracellular distribution and level of Dishevelled and the activity of RhoA, thereby controlling the activity of ROCK and regulating actin cytoskeleton remodeling and cell polarization. Concurrent control by NO helps ensure that the crucial processes of cell proliferation and morphogenetic movements are coordinated during early development.

Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation

Methylation of CpG islands associated with genes can affect the expression of the proximal gene, and methylation of non-associated CpG islands correlates to genomic instability. This epigenetic modification has been shown to be important in many pathologies, from development and disease to cancer. We report the development of a novel high-resolution microarray that detects the methylation status of over 25 000 CpG islands in the human genome. Experiments were performed to demonstrate low system noise in the methodology and that the array probes have a high signal to noise ratio. Methylation measurements between different cell lines were validated demonstrating the accuracy of measurement. We then identified alterations in CpG islands, both those associated with gene promoters, as well as non-promoter-associated islands in a set of breast and ovarian tumors. We demonstrate that this methodology accurately identifies methylation profiles in cancer and in principle it can differentiate any CpG methylation alterations and can be adapted to analyze other species.

Mass Spectroscopy and Molecular Modeling Predict Endothelial Nitric Oxide Synthase Dimer Collapse by Hydrogen Peroxide Through Zinc Tetrathiolate Metal-Binding Site Disruption

Endothelial nitric oxide synthase (eNOS) is inhibited by hydrogen peroxide (H(2)O(2)), but the mechanism has not been determined. Thus, the purpose of this study was to delineate the mechanism by which H(2)O(2) inhibits eNOS activity. Using mass spectroscopy, we found that the tetrathiolate cysteine residues 94 and 99 were susceptible to oxidation by H(2)O(2). Molecular modeling predicted that these cysteic acid modifications would disrupt the van der Waals interactions and the hydrogen bonding network mediated by the tetrathiolate cysteines 94 and 99 resulting in changes in quaternary structure, zinc release, and dimer collapse. Using recombinant human eNOS (heNOS) to test the predictions of the molecular modeling we found that H(2)O(2) caused disruption of the heNOS dimer and this was accompanied by zinc release and decreased NO generation. We also found that H(2)O(2) increased the oxidation of tetrahydrobiopterin (BH(4)) to dihydrobiopterin (BH(2)), whereas preincubation of heNOS with excess BH(4) prevented the destruction of zinc tetrathiolate and dimer collapse and preserved activity. Interestingly, we found that the dimmer-stabilizing effect of BH(4) is due to its ability to act as a catalase mimetic. Further, we confirmed that, in ovine aortic endothelial cells, H(2)O(2) could also induce dimer collapse and that increasing cellular BH(4) levels could maintain eNOS in its dimeric form and NO signaling when cells were challenged with H(2)O(2). This study links the inhibitory action of H(2)O(2) on heNOS through the destruction of zinc tetrathiolate metal-binding site and dimer collapse both in vitro and in vivo.

GTP cyclohydrolase I expression is regulated by nitric oxide: role of cyclic AMP

Our previous studies have demonstrated that nitric oxide (NO) leads to nitric oxide synthase (NOS) uncoupling and an increase in NOS-derived superoxide. However, the cause of this uncoupling has not been adequately resolved. The pteridine cofactor tetrahydrobiopterin (BH(4)) is a critical determinant of endothelial NOS (eNOS) activity and coupling, and GTP cyclohydrolase I (GCH1) is the rate-limiting enzyme in its generation. Thus the initial purpose of this study was to determine whether decreases in BH(4) could underlie, at least in part, the NO-mediated uncoupling of eNOS we have observed both in vitro and in vivo. Initially we evaluated the effect of inhaled NO levels on GCH1 expression and BH(4) levels in the intact lamb. Contrary to our hypothesis, we found that there was a significant increase in both plasma BH4 levels and peripheral lung GCH1 protein levels. Furthermore, in vitro, we found that exposure to the NO donor spermine NONOate (SPNONO) led to an increase in GCH1 protein and BH(4) levels in both COS-7 and pulmonary arterial endothelial cells. However, SPNONO treatment also caused a significant increase in phospho-cAMP response element binding protein (CREB) levels, as detected by Western blot analysis, and significantly increased cAMP levels, as detected by enzyme immunoassay. Furthermore, utilizing GCH1 promoter fragments fused to a luciferase reporter gene, we found that GCH1 promoter activity was enhanced by SPNONO in a CREB-dependent manner, and electromobility shift assays revealed an NO-dependent increase in the nuclear binding of CREB. These data suggest that NO increases BH(4) levels through a cAMP/CREB-mediated increase in GCH1 transcription and that the eNOS uncoupling associated with exogenous NO does not involved reduced BH(4) levels.

Corrigendum: Genome-wide copy number analysis of single cells.

Nat. Protoc. 7, 1024–1041 (2012); published online 3 May 2012; corrected after print 24 February 2016 In the version of this article initially published, the units for the concentration of NaCl in the NST buffer described in the Reagent Setup section were incorrect. The correct unit should be mM. The error has been corrected in the HTML and PDF versions of the article.

Mastoid cavity obliteration in open cavity type (MRM) with fibro fatty tissue and subcutaneous periosteal flaps study conducted at RIMS college Ongole ENT department 2015-2016.

A mastoid cavity resulting from a canal wall down can result in major morbidity for patients due to chronic otorhea and infection, difficulty with hearing aids and vertigo with temperature changes. Mastoid obliteration recreates the normal anatomy to certain extent avoid such morbidity. Few have the studied the quality of life benefited by this procedure. Open cavity mastiodectomy techniques applied for chronic suppurative otitis media with attico antral disease cause some cavity problems, these pts complaints of vertigo in cold weather and during swimming, other problems are difficulty in wearing hearing aids, frequent fungal infections and exuberant wax, and wide meatoplasty which is non aesthetic .and these pts need lifelong otological care for debris that accumulates due to insufficient self cleaning mechanism of mastiodectomy cavities, various obliteration techniques have been recommended to eliminate open cavity problem. Aim & objectives To study surgical results of mastoid cavity obliteration, efficacy of different methods of mastoid obliteration cavity problems and need of cavity care. Materials and methods Retrospective study was carried out in a tertiary care center involving 100 pts from 2015 June – May 2016 having csom with attico antral type of disease who underwent MRM with the cavity obliterated with fibro fatty tissues , cartilage or periosteal flaps . The indication for MRM were patients with extensive cholesteatoma ,with extra cranial complications ,patients with poor compliance for follow up, patients presented with tubo tympanic type, of csom and those with sensorineural hearing loss and intracranial complications were excluded from the study. A detailed history and through ENT examination was done in all patients. Microscopic examination of the affected ears was done to confirm the findings . The lateral oblique view of both mastoids were taken ,HRCT temporal bone was done in these cases where clinical diagnosis is not clear pure tone audiometry and tuning fork tests were done preoperatively and informed written consent was obtained . Results and conclusion In our study the minimum age of the pt was 10 years and maximum age was 55 yrs. Maximum no of pts i.e. 35% in the age group 11-20 yrs followed by 34% in the age group of 21-30 yrs . out of 100 pts male 65 females 35 the male to female ratio1.35;1 65% of the patents belong to rural area 35% belongs to urban area. 100% patients presented with ear discharge ,94% pts presented with hearig loss ,3% of cases presented with facial palsy cholesteatoma was found in all 100 cases , postero superior perforations found in 70% in 30 ears while 20% pts had attic perforation ,30 ears were found to have discharge at 3 months followup 18 ears became dry after medical treatment . At the end of 1 year, well healed cavity with intact graft was seen in 100 cases and 8 cases were advised revision surgery Incidence of discharge, debris,giddiness pain ware reduced in oblitetarating cavities healing was better in obliterating cavity . cavity oblitearing with fibrofatty tissues and flap had better and early epithelisation as compared to cartilage .