ESCC ATLAS: A population wide compendium of biomarkers for Esophageal Squamous Cell Carcinoma.

Esophageal cancer (EC) is the eighth most aggressive malignancy and its treatment remains a challenge due to the lack of biomarkers that can facilitate early detection. EC is identified in two major histological forms namely - Adenocarcinoma (EAC) and Squamous cell carcinoma (ESCC), each showing differences in the incidence among populations that are geographically separated. Hence the detection of potential drug target and biomarkers demands a population-centric understanding of the molecular and cellular mechanisms of EC. To provide an adequate impetus to the biomarker discovery for ESCC, which is the most prevalent esophageal cancer worldwide, here we have developed ESCC ATLAS, a manually curated database that integrates genetic, epigenetic, transcriptomic, and proteomic ESCC-related genes from the published literature. It consists of 3475 genes associated to molecular signatures such as, altered transcription (2600), altered translation (560), contain copy number variation/structural variations (233), SNPs (102), altered DNA methylation (82), Histone modifications (16) and miRNA based regulation (261). We provide a user-friendly web interface ( http://www.esccatlas.org , freely accessible for academic, non-profit users) that facilitates the exploration and the analysis of genes among different populations. We anticipate it to be a valuable resource for the population specific investigation and biomarker discovery for ESCC.

Intronic SNP in ESR1 encoding human estrogen receptor alpha is associated with brain ESR1 mRNA isoform expression and behavioral traits.

Genetic variants of ESR1 have been implicated in multiple diseases, including behavioral disorders, but causative variants remain uncertain. We have searched for regulatory variants affecting ESR1 expression in human brain, measuring allelic ESR1 mRNA expression in human brain tissues with marker SNPs in exon4 representing ESR1-008 (or ESRα-36), and in the 3'UTR of ESR1-203, two main ESR1 isoforms in brain. In prefrontal cortex from subjects with bipolar disorder, schizophrenia, and controls (n = 35 each; Stanley Foundation brain bank), allelic ESR1 mRNA ratios deviated from unity up to tenfold at the exon4 marker SNP, with large allelic ratios observed primarily in bipolar and schizophrenic subjects. SNP scanning and targeted sequencing identified rs2144025, associated with large allelic mRNA ratios (p = 1.6E10-6). Moreover, rs2144025 was significantly associated with ESR1 mRNA levels in the Brain eQTL Almanac and in brain regions in the Genotype-Tissue Expression project. In four GWAS cohorts, rs2104425 was significantly associated with behavioral traits, including: hypomanic episodes in female bipolar disorder subjects (GAIN bipolar disorder study; p = 0.0004), comorbid psychological symptoms in both males and females with attention deficit hyperactivity disorder (GAIN ADHD, p = 0.00002), psychological diagnoses in female children (eMERGE study of childhood health, subject age ≥9, p = 0.0009), and traits in schizophrenia (e.g., grandiose delusions, GAIN schizophrenia, p = 0.0004). The first common ESR1 variant (MAF 12-33% across races) linked to regulatory functions, rs2144025 appears conditionally to affect ESR1 mRNA expression in the brain and modulate traits in behavioral disorders.

Allele-Selective Transcriptome Recruitment to Polysomes Primed for Translation: Protein-Coding and Noncoding RNAs, and RNA Isoforms.

mRNA translation into proteins is highly regulated, but the role of mRNA isoforms, noncoding RNAs (ncRNAs), and genetic variants remains poorly understood. mRNA levels on polysomes have been shown to correlate well with expressed protein levels, pointing to polysomal loading as a critical factor. To study regulation and genetic factors of protein translation we measured levels and allelic ratios of mRNAs and ncRNAs (including microRNAs) in lymphoblast cell lines (LCL) and in polysomal fractions. We first used targeted assays to measure polysomal loading of mRNA alleles, confirming reported genetic effects on translation of OPRM1 and NAT1, and detecting no effect of rs1045642 (3435C>T) in ABCB1 (MDR1) on polysomal loading while supporting previous results showing increased mRNA turnover of the 3435T allele. Use of high-throughput sequencing of complete transcript profiles (RNA-Seq) in three LCLs revealed significant differences in polysomal loading of individual RNA classes and isoforms. Correlated polysomal distribution between protein-coding and non-coding RNAs suggests interactions between them. Allele-selective polysome recruitment revealed strong genetic influence for multiple RNAs, attributable either to differential expression of RNA isoforms or to differential loading onto polysomes, the latter defining a direct genetic effect on translation. Genes identified by different allelic RNA ratios between cytosol and polysomes were enriched with published expression quantitative trait loci (eQTLs) affecting RNA functions, and associations with clinical phenotypes. Polysomal RNA-Seq combined with allelic ratio analysis provides a powerful approach to study polysomal RNA recruitment and regulatory variants affecting protein translation.

Quantitative analysis of deadenylation-independent mRNA decay by a modified MBRACE assay.

Endonuclease cleavage is the rate-limiting step in the decay of nonsense-containing human ß-globin mRNA in erythroid cells. The 5'-truncated intermediates thus generated are polyadenylated and more stable than the parent mRNA. Northern blotting is commonly used to measure the decay rate of full-length mRNA, and S1 nuclease protection is used to assay the fate of decay intermediates. We have adapted the more sensitive and facile MBRACE assay (Lasham et al., Nucleic Acids Res 38: e19, 2010) to quantitatively monitor the decay process by detecting full-length ß-globin and its decay intermediates.

DNA promoter methylation-dependent transcription of the double C2-like domain ß (DOC2B) gene regulates tumor growth in human cervical cancer.

Double C2-like domain ß (DOC2B) gene encodes for a calcium-binding protein, which is involved in neurotransmitter release, sorting, and exocytosis. We have identified the promoter region of the DOC2B gene as hypermethylated in pre-malignant, malignant cervical tissues, and cervical cancer cell lines by methylation-sensitive dimethyl sulfoxide-polymerase chain reaction and bisulfite genome sequencing; whereas, it was unmethylated in normal cervical tissues (p < 0.05). The promoter hypermethylation was inversely associated with mRNA expression in SiHa, CaSki, and HeLa cells and treatment with demethylating agent 5-aza-2-deoxycytidine restored DOC2B expression. The region -630 to +25 bp of the DOC2B gene showed robust promoter activity by a luciferase reporter assay and was inhibited by in vitro artificial methylation with Sss1 methylase prior to transient transfections. Overexpression of the DOC2B gene in SiHa cells when compared with controls showed significantly reduced colony formation, cell proliferation, induced cell cycle arrest, and repressed cell migration and invasion (p < 0.05). Ectopic expression of DOC2B resulted in anoikis-mediated cell death and repressed tumor growth in a nude mice xenograft model (p < 0.05). DOC2B expressing cells showed a significant increase in intracellular calcium level (p < 0.05), impaired AKT1 and ERK1/2 signaling, and induced actin cytoskeleton remodeling. Our results show that promoter hypermethylation and silencing of the DOC2B gene is an early and frequent event during cervical carcinogenesis and whose reduced expression due to DNA promoter methylation may lead to selective cervical tumor growth.

Mitochondrial DNA variation analysis in cervical cancer.

This study was undertaken to investigate the mitochondrial DNA (mtDNA) variation in non-malignant and malignant cervical tissue samples. We have identified 229 and 739 variations non-malignant and malignant tissues respectively distributed over 321 locations in the D-loop (50 in non-malignant and 166 in malignant; 216 variations), coding region (139 in non-malignant and 455 in malignant; 594 variations) tRNA and rRNA genes (39 in non-malignant and 119 in malignant; 158 variations). Besides, 77 novel and 34 various other disease associated variations were identified in non-malignant and malignant samples. A total of 236 tumor specific variations in 201 locations representing 30.1% in D-loop, 59.3% in coding regions and 10.6% in RNA genes were also identified. Our study shows that D loop (in 67 locations) is highly altered followed by ND5 (35 locations) region. Moreover, mtDNA alterations were significantly higher in malignant samples by two tailed Fisher's exact test (P≤0.05) with decreased mtDNA copy numbers. Bioinformatic analysis of 59 non-synonymous changes predicted several variations as damaging leading to decreased stability of the proteins. Taken together, mtDNA is highly altered in cervical cancer and functional studies are needed to be investigated to understand the consequence of these variations in cervical carcinogenesis and their potential application as biomarkers.

SMG6 cleavage generates metastable decay intermediates from nonsense-containing ß-globin mRNA.

mRNAs targeted by endonuclease decay generally disappear without detectable decay intermediates. The exception to this is nonsense-containing human ß-globin mRNA, where the destabilization of full-length mRNA is accompanied by the cytoplasmic accumulation of 5'-truncated transcripts in erythroid cells of transgenic mice and in transfected erythroid cell lines. The relationship of the shortened RNAs to the decay process was characterized using an inducible erythroid cell system and an assay for quantifying full-length mRNA and a truncated RNA missing 169 nucleotides from the 5' end. In cells knocked down for Upf1 a reciprocal increase in full-length and decrease in shortened RNA confirmed the role of NMD in this process. Kinetic analysis demonstrated that the 5'-truncated RNAs are metastable intermediates generated during the decay process. SMG6 previously was identified as an endonuclease involved in NMD. Consistent with involvement of SMG6 in the decay process full-length nonsense-containing ß-globin mRNA was increased and the Δ169 decay intermediate was decreased in cells knocked down for SMG6. This was reversed by complementation with siRNA-resistant SMG6, but not by SMG6 with inactivating PIN domain mutations. Importantly, none of these altered the phosphorylation state of Upf1. These data provide the first proof for accumulation of stable NMD products by SMG6 endonuclease cleavage.

Whole transcriptome RNA-Seq allelic expression in human brain.

BACKGROUND: Measuring allelic RNA expression ratios is a powerful approach for detecting cis-acting regulatory variants, RNA editing, loss of heterozygosity in cancer, copy number variation, and allele-specific epigenetic gene silencing. Whole transcriptome RNA sequencing (RNA-Seq) has emerged as a genome-wide tool for identifying allelic expression imbalance (AEI), but numerous factors bias allelic RNA ratio measurements. Here, we compare RNA-Seq allelic ratios measured in nine different human brain regions with a highly sensitive and accurate SNaPshot measure of allelic RNA ratios, identifying factors affecting reliable allelic ratio measurement. Accounting for these factors, we subsequently surveyed the variability of RNA editing across brain regions and across individuals. RESULTS: We find that RNA-Seq allelic ratios from standard alignment methods correlate poorly with SNaPshot, but applying alternative alignment strategies and correcting for observed biases significantly improves correlations. Deploying these methods on a transcriptome-wide basis in nine brain regions from a single individual, we identified genes with AEI across all regions (SLC1A3, NHP2L1) and many others with region-specific AEI. In dorsolateral prefrontal cortex (DLPFC) tissues from 14 individuals, we found evidence for frequent regulatory variants affecting RNA expression in tens to hundreds of genes, depending on stringency for assigning AEI. Further, we find that the extent and variability of RNA editing is similar across brain regions and across individuals. CONCLUSIONS: These results identify critical factors affecting allelic ratios measured by RNA-Seq and provide a foundation for using this technology to screen allelic RNA expression on a transcriptome-wide basis. Using this technology as a screening tool reveals tens to hundreds of genes harboring frequent functional variants affecting RNA expression in the human brain. With respect to RNA editing, the similarities within and between individuals leads us to conclude that this post-transcriptional process is under heavy regulatory influence to maintain an optimal degree of editing for normal biological function.

Studies on Brahma rasayana in male swiss albino mice: Chromosomal aberrations and sperm abnormalities.

Ayurveda, the Indian holistic healthcare system encompasses traditional medicines with a principle of creating harmony and maintaining balance within the natural rhythms of the body. Rasayana is one of the branches of Ayurveda frequently used as rejuvenant therapy to overcome many discomforts and prevent diseases. It has been reported that rasayanas have immunomodulatory, antioxidant and antitumor functions. However, the genotoxic potential of many rasayanas remains to be evaluated. The present study was undertaken to assess the role of Brahma rasayana(BR) on genotoxicity in vivo in a mouse test system. The older mice (9 months) were orally fed with rasayana for 8 weeks. The treated groups showed no signs of dose-dependent toxicity at the dosage levels tested. The body weight loss/gain and feed consumption were unaffected at tested doses. Furthermore, sperm abnormalities and chromosomal aberrations were insignificant in the treatment group when compared to controls. However, there was a marginal increase in sperm count in the BR treated animals. These findings clearly indicate that there are no observed adverse genotoxic effects elicited by BR in experimental animals such as mice.