Suraj Peri

Human protein reference database as a discovery resource for proteomics

The rapid pace at which genomic and proteomic data is being generated necessitates the development of tools and resources for managing data that allow integration of information from disparate sources. The Human Protein Reference Database (http://www.hprd.org) is a web-based resource based on open source technologies for protein information about several aspects of human proteins including protein-protein interactions, post-translational modifications, enzyme-substrate relationships and disease associations. This information was derived manually by a critical reading of the published literature by expert biologists and through bioinformatics analyses of the protein sequence. This database will assist in biomedical discoveries by serving as a resource of genomic and proteomic information and providing an integrated view of sequence, structure, function and protein networks in health and disease.

GPMAW--a software tool for analyzing proteins and peptides.

General Protein/Mass Analysis for Windows (GPMAW) is a valuable piece of software for any molecular biologist, biochemist or mass spectrometrist wishing to analyze protein or peptide sequences. All steps from the acquisition of protein sequence from a built-in web interface, to proteolytic digests, theoretical peptide fragmentation, detailed annotation of sequences and secondary structure prediction, can be performed rapidly and intuitively without first having to spend days reading manuals.

A reassessment of the translation initiation codon in vertebrates

More than two decades ago Marilyn Kozak proposed the scanning model of translation initiation, whereby translation is initiated at the first AUG codon that is in a particular context. In this article, we re-examine the context of initiator codons using a large dataset of curated human transcripts. We find that more than 40% of transcripts contain AUG codons upstream of the actual start codon and that most authentic AUGs contain three or more mismatches from the consensus sequence, CCACCaugG. Also, in a large fraction of transcripts, the sequences surrounding the initiator codon deviate more from the consensus than those surrounding upstream AUGs, indicating that translation initiation from downstream AUGs is more common than generally believed.

Cellular oxidative stress response controls the antiviral and apoptotic programs in dengue virus-infected dendritic cells.

Dengue virus (DENV) is a re-emerging arthropod borne flavivirus that infects more than 300 million people worldwide, leading to 50,000 deaths annually. Because dendritic cells (DC) in the skin and blood are the first target cells for DENV, we sought to investigate the early molecular events involved in the host response to the virus in primary human monocyte-derived dendritic cells (Mo-DC). Using a genome-wide transcriptome analysis of DENV2-infected human Mo-DC, three major responses were identified within hours of infection - the activation of IRF3/7/STAT1 and NF-κB-driven antiviral and inflammatory networks, as well as the stimulation of an oxidative stress response that included the stimulation of an Nrf2-dependent antioxidant gene transcriptional program. DENV2 infection resulted in the intracellular accumulation of reactive oxygen species (ROS) that was dependent on NADPH-oxidase (NOX). A decrease in ROS levels through chemical or genetic inhibition of the NOX-complex dampened the innate immune responses to DENV infection and facilitated DENV replication; ROS were also essential in driving mitochondrial apoptosis in infected Mo-DC. In addition to stimulating innate immune responses to DENV, increased ROS led to the activation of bystander Mo-DC which up-regulated maturation/activation markers and were less susceptible to viral replication. We have identified a critical role for the transcription factor Nrf2 in limiting both antiviral and cell death responses to the virus by feedback modulation of oxidative stress. Silencing of Nrf2 by RNA interference increased DENV-associated immune and apoptotic responses. Taken together, these data demonstrate that the level of oxidative stress is critical to the control of both antiviral and apoptotic programs in DENV-infected human Mo-DC and highlight the importance of redox homeostasis in the outcome of DENV infection.

Genomewide mRNA profiling of esophageal squamous cell carcinoma for identification of cancer biomarkers

Esophageal squamous cell carcinoma (ESCC) is a common cancer worldwide that has a poor survival rate among patients mainly because of lack of early markers to identify this cancer. Molecular mechanisms contributing to initiation and progression of esophageal squamous cell carcinoma are still poorly understood. Development of DNA microarrays technology allows high-throughput identification of gene expression profiles in cancers. In order to identify molecules as candidates for early diagnosis and/or therapeutic targets, we analyzed mRNA expression profiles of 20 surgically resected specimens of ESCC and compared them to their adjacent normal epithelium using whole genome DNA microarrays. We observed 119 genes significantly upregulated in ESCC samples as compared to the adjacent normal epithelium. The expression of two previously unreported overexpressed genes, ORAOV2 and FAP, was validated at the protein level by immunohistochemical labeling of tissue microarrays (TMAs) and archival tissue sections. Overexpression of ORAOV2 was observed in 116/118 (98%) of ESCC cases, while FAP overexpression was in 79/116 (68%) of cases. Osteopontin, which was identified in earlier studies, was observed to be upregulated in 114/118 (97%) cases. Overall, using this approach, we have identified a number of promising novel candidates that can be validated further for their potential to serve as biomarkers for ESCC.

HBV DNA Integration and Clonal Hepatocyte Expansion in Chronic Hepatitis B Patients Considered Immune Tolerant

BACKGROUND & AIMS Chronic infection with hepatitis B virus (HBV) progresses through different phases. The first, called the immune-tolerant phase, has been associated with a lack of disease activity. We examined HBV-DNA integration, clonal hepatocyte expansion, HBV antigen expression, and HBV-specific immune responses in patients in the immune-tolerant phase to assess whether this designation is appropriate or if there is evidence of disease activity. METHODS We studied HBV-DNA integration, clonal hepatocyte expansion, and expression of hepatitis B surface antigen and core antigen in liver tissues from 26 patients with chronic HBV infection (ages, 14-39 y); 9 patients were positive for hepatitis B e antigen (HBeAg) in the immune-tolerant phase and were matched for age with 10 HBeAg-positive patients with active disease and 7 HBeAg-negative patients with active disease. Peripheral blood samples were collected and HBV-specific T cells were quantified for each group. RESULTS Detection of HBV antigens differed among groups. However, unexpectedly high numbers of HBV-DNA integrations, randomly distributed among chromosomes, were detected in all groups. Clonal hepatocyte expansion in patients considered immune tolerant also was greater than expected, potentially in response to hepatocyte turnover mediated by HBV-specific T cells, which were detected in peripheral blood cells from patients in all phases of infection. CONCLUSIONS We measured HBV-specific T cells, HBV-DNA integration, and clonal hepatocyte expansion in different disease phases of young patients with chronic hepatitis B, with emphasis on the so-called immune-tolerant phase. A high level of HBV-DNA integration and clonal hepatocyte expansion in patients considered immune tolerant indicated that hepatocarcinogenesis could be underway-even in patients with early stage chronic HBV infection. Our findings do not support the concepts that this phase is devoid of markers of disease progression or that an immune response has not been initiated. We propose that this early phase be called a high-replication, low-inflammation stage. The timing of therapeutic interventions to minimize further genetic damage to the hepatocyte population should be reconsidered.

Genome annotation of Anopheles gambiae using mass spectrometry-derived data

BackgroundA large number of animal and plant genomes have been completely sequenced over the last decade and are now publicly available. Although genomes can be rapidly sequenced, identifying protein-coding genes still remains a problematic task. Availability of protein sequence data allows direct confirmation of protein-coding genes. Mass spectrometry has recently emerged as a powerful tool for proteomic studies. Protein identification using mass spectrometry is usually carried out by searching against databases of known proteins or transcripts. This approach generally does not allow identification of proteins that have not yet been predicted or whose transcripts have not been identified.ResultsWe searched 3,967 mass spectra from 16 LC-MS/MS runs of Anopheles gambiae salivary gland homogenates against the Anopheles gambiae genome database. This allowed us to validate 23 known transcripts and 50 novel transcripts. In addition, a novel gene was identified on the basis of peptides that matched a genomic region where no gene was known and no transcript had been predicted. The amino termini of proteins encoded by two predicted transcripts were confirmed based on N-terminally acetylated peptides sequenced by tandem mass spectrometry. Finally, six sequence polymorphisms could be annotated based on experimentally obtained peptide sequences.ConclusionThe peptide sequences from this study were mapped onto the genomic sequence using the distributed annotation system available at Ensembl and can be visualized in the context of all other existing annotations. The strategy described in this paper can be used to correct and confirm genome annotations and permit discovery of novel proteins in a high-throughput manner by mass spectrometry.

Systems analysis of a RIG-I agonist inducing broad spectrum inhibition of virus infectivity.

The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5′ triphosphate (5′ppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5′pppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, and induction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN) signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5′pppRNA, and not by IFNα-2b, that included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5′pppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5′pppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach provides transcriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5′pppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents.

Detection of Clonally Expanded Hepatocytes in Chimpanzees with Chronic Hepatitis B Virus Infection

ABSTRACT During a hepadnavirus infection, viral DNA integrates at a low rate into random sites in the host DNA, producing unique virus-cell junctions detectable by inverse nested PCR (invPCR). These junctions serve as genetic markers of individual hepatocytes, providing a means to detect their subsequent proliferation into clones of two or more hepatocytes. A previous study suggested that the livers of 2.4-year-old woodchucks (Marmota monax) chronically infected with woodchuck hepatitis virus contained at least 100,000 clones of >1,000 hepatocytes (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. USA 102:1139-1144, 2005). However, possible correlations between sites of viral-DNA integration and clonal expansion could not be explored because the woodchuck genome has not yet been sequenced. In order to further investigate this issue, we looked for similar clonal expansion of hepatocytes in the livers of chimpanzees chronically infected with hepatitis B virus (HBV). Liver samples for invPCR were collected from eight chimpanzees chronically infected with HBV for at least 20 years. Fifty clones ranging in size from ∼35 to 10,000 hepatocytes were detected using invPCR in 32 liver biopsy fragments (∼1 mg) containing, in total, ∼3 × 107 liver cells. Based on searching the analogous human genome, integration sites were found on all chromosomes except Y, ∼30% in known or predicted genes. However, no obvious association between the extent of clonal expansion and the integration site was apparent. This suggests that the integration site per se is not responsible for the outgrowth of large clones of hepatocytes.

Characterization of a Genomic Signature of Pregnancy Identified in the Breast

The objective of this study was to comprehensively compare the genomic profiles in the breast of parous and nulliparous postmenopausal women to identify genes that permanently change their expression following pregnancy. The study was designed as a two-phase approach. In the discovery phase, we compared breast genomic profiles of 37 parous with 18 nulliparous postmenopausal women. In the validation phase, confirmation of the genomic patterns observed in the discovery phase was sought in an independent set of 30 parous and 22 nulliparous postmenopausal women. RNA was hybridized to Affymetrix HG_U133 Plus 2.0 oligonucleotide arrays containing probes to 54,675 transcripts, scanned and the images analyzed using Affymetrix GCOS software. Surrogate variable analysis, logistic regression, and significance analysis of microarrays were used to identify statistically significant differences in expression of genes. The false discovery rate (FDR) approach was used to control for multiple comparisons. We found that 208 genes (305 probe sets) were differentially expressed between parous and nulliparous women in both discovery and validation phases of the study at an FDR of 10% and with at least a 1.25-fold change. These genes are involved in regulation of transcription, centrosome organization, RNA splicing, cell-cycle control, adhesion, and differentiation. The results provide initial evidence that full-term pregnancy induces long-term genomic changes in the breast. The genomic signature of pregnancy could be used as an intermediate marker to assess potential chemopreventive interventions with hormones mimicking the effects of pregnancy for prevention of breast cancer. Cancer Prev Res; 4(9); 1457–64. ©2011 AACR.