Nandan Deshpande

ONCOMINE: A Cancer Microarray Database and Integrated Data-Mining Platform

DNA microarray technology has led to an explosion of oncogenomic analyses, generating a wealth of data and uncovering the complex gene expression patterns of cancer. Unfortunately, due to the lack of a unifying bioinformatic resource, the majority of these data sit stagnant and disjointed following publication, massively underutilized by the cancer research community. Here, we present ONCOMINE, a cancer microarray database and web-based data-mining platform aimed at facilitating discovery from genome-wide expression analyses. To date, ONCOMINE contains 65 gene expression datasets comprising nearly 48 million gene expression measurements form over 4700 microarray experiments. Differential expression analyses comparing most major types of cancer with respective normal tissues as well as a variety of cancer subtypes and clinical-based and pathology-based analyses are available for exploration. Data can be queried and visualized for a selected gene across all analyses or for multiple genes in a selected analysis. Furthermore, gene sets can be limited to clinically important annotations including secreted, kinase, membrane, and known gene-drug target pairs to facilitate the discovery of novel biomarkers and therapeutic targets.

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.

Analysis of the human protein interactome and comparison with yeast, worm and fly interaction datasets.

We present the first analysis of the human proteome with regard to interactions between proteins. We also compare the human interactome with the available interaction datasets from yeast (Saccharomyces cerevisiae), worm (Caenorhabditis elegans) and fly (Drosophila melanogaster). Of >70,000 binary interactions, only 42 were common to human, worm and fly, and only 16 were common to all four datasets. An additional 36 interactions were common to fly and worm but were not observed in humans, although a coimmunoprecipitation assay showed that 9 of the interactions do occur in humans. A re-examination of the connectivity of essential genes in yeast and humans indicated that the available data do not support the presumption that the number of interaction partners can accurately predict whether a gene is essential. Finally, we found that proteins encoded by genes mutated in inherited genetic disorders are likely to interact with proteins known to cause similar disorders, suggesting the existence of disease subnetworks. The human interaction map constructed from our analysis should facilitate an integrative systems biology approach to elucidating the cellular networks that contribute to health and disease states.

Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma

BackgroundMycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.ResultsHere we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei.ConclusionsThe data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

Protein glycosylation pathways in filamentous fungi

Glycosylation of proteins is important for protein stability, secretion, and localization. In this study, we have investigated the glycan synthesis pathways of 12 filamentous fungi including those of medical/agricultural/industrial importance for which genomes have been recently sequenced. We have adopted a systems biology approach to combine the results from comparative genomics techniques with high confidence information on the enzymes and fungal glycan structures, reported in the literature. From this, we have developed a composite representation of the glycan synthesis pathways in filamentous fungi (both N- and O-linked). The N-glycosylation pathway in the cytoplasm and endoplasmic reticulum was found to be highly conserved evolutionarily across all the filamentous fungi considered in the study. In the final stages of N-glycan synthesis in the Golgi, filamentous fungi follow the high mannose pathway as in Saccharomyces cerevisiae, but the level of glycan mannosylation is reduced. Highly specialized N-glycan structures with galactofuranose residues, phosphodiesters, and other insufficiently trimmed structures have also been identified in the filamentous fungi. O-Linked glycosylation in filamentous fungi was seen to be highly conserved with many mannosyltransferases that are similar to those in S. cerevisiae. However, highly variable and diverse O-linked glycans also exist. We have developed a web resource for presenting the compiled data with user-friendly query options, which can be accessed at www.fungalglycans.org. This resource can assist attempts to remodel glycosylation of recombinant proteins expressed in filamentous fungal hosts.

GlycoSpectrumScan : fishing glycopeptides from MS spectra of protease digests of human colostrum sIgA

With the emergence of glycoproteomics, there is a need to develop bioinformatic tools to identify glycopeptides in protease digests of glycoproteins. GlycoSpectrumScan is a web-based tool that identifies the glycoheterogeneity on a peptide from mass spectrometric data. Two experimental data sets are required as inputs: (1) oligosaccharide compositions of the N- and/or O-linked glycans present in the sample and (2) in silico derived peptide masses of proteolytically digested proteins with a potential number of N- and/or O-glycosylation sites. GlycoSpectrumScan uses MS data, rather than MS/MS data, to identify glycopeptides and determine the relative distribution of N- and O-glycoforms at each site. It is functional for assigning monosaccharide compositions on glycopeptides with single and multiple sites of glycosylation. The algorithm allows the input of raw mass data, including multiply charged ions, making it applicable for both ESI and MALDI data from all mass spectrometer platforms. Manual analysis time for identifying glycosylation heterogeneity at each site on glycoprotein(s) is substantially decreased. The application of this tool to characterize the N- and O-linked glycopeptides from human secretory IgA (sIgA), consisting of secretory component (7 N-linked sites), IgA1 (2 N-linked, <or=5 O-linked sites), IgA2 (4 N-linked sites) and J-chain (1 N-linked site) is described. GlycoSpectrumScan is freely available at www.glycospectrumscan.org .

ESTExplorer: an expressed sequence tag (EST) assembly and annotation platform

The analysis of expressed sequence tag (EST) datasets offers a rapid and cost-effective approach to elucidate the transcriptome of an organism, but requiring several computational methods for assembly and annotation. ESTExplorer is a comprehensive workflow system for EST data management and analysis. The pipeline uses a ‘distributed control approach’ in which the most appropriate bioinformatics tools are implemented over different dedicated processors. Species-specific repeat masking and conceptual translation are in-built. ESTExplorer accepts a set of ESTs in FASTA format which can be analysed using programs selected by the user. After pre-processing and assembly, the dataset is annotated at the nucleotide and protein levels, following conceptual translation. Users may optionally provide ESTExplorer with assembled contigs for annotation purposes. Functionally annotated contigs/ESTs can be analysed individually. The overall outputs are gene ontologies, protein functional identifications in terms of mapping to protein domains and metabolic pathways. ESTExplorer has been applied successfully to annotate large EST datasets from parasitic nematodes and to identify novel genes as potential targets for parasite intervention. ESTExplorer runs on a Linux cluster and is freely available for the academic community at http://estexplorer.biolinfo.org.

The pathogenic potential of Campylobacter concisus strains associated with chronic intestinal diseases.

Campylobacter concisus has garnered increasing attention due to its association with intestinal disease, thus, the pathogenic potential of strains isolated from different intestinal diseases was investigated. A method to isolate C. concisus was developed and the ability of eight strains from chronic and acute intestinal diseases to adhere to and invade intestinal epithelial cells was determined. Features associated with bacterial invasion were investigated using comparative genomic analyses and the effect of C. concisus on host protein expression was examined using proteomics. Our isolation method from intestinal biopsies resulted in the isolation of three C. concisus strains from children with Crohns disease or chronic gastroenteritis. Four C. concisus strains from patients with chronic intestinal diseases can attach to and invade host cells using mechanisms such as chemoattraction to mucin, aggregation, flagellum-mediated attachment, “membrane ruffling”, cell penetration and damage. C. concisus strains isolated from patients with chronic intestinal diseases have significantly higher invasive potential than those from acute intestinal diseases. Investigation of the cause of this increased pathogenic potential revealed a plasmid to be responsible. 78 and 47 proteins were upregulated and downregulated in cells infected with C. concisus, respectively. Functional analysis of these proteins showed that C. concisus infection regulated processes related to interleukin-12 production, proteasome activation and NF-κB activation. Infection with all eight C. concisus strains resulted in host cells producing high levels of interleukin-12, however, only strains capable of invading host cells resulted in interferon-γ production as confirmed by ELISA. These findings considerably support the emergence of C. concisus as an intestinal pathogen, but more significantly, provide novel insights into the host immune response and an explanation for the heterogeneity observed in the outcome of C. concisus infection. Moreover, response to infection with invasive strains has substantial similarities to that observed in the inflamed mucosa of Crohns disease patients.

Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E

RNA sequencing studies have identified hundreds of non‐coding RNAs in bacteria, including regulatory small RNA (sRNA). However, our understanding of sRNA function has lagged behind their identification due to a lack of tools for the high‐throughput analysis of RNA–RNA interactions in bacteria. Here we demonstrate that in vivo sRNA–mRNA duplexes can be recovered using UV‐crosslinking, ligation and sequencing of hybrids (CLASH). Many sRNAs recruit the endoribonuclease, RNase E, to facilitate processing of mRNAs. We were able to recover base‐paired sRNA–mRNA duplexes in association with RNase E, allowing proximity‐dependent ligation and sequencing of cognate sRNA–mRNA pairs as chimeric reads. We verified that this approach captures bona fide sRNA–mRNA interactions. Clustering analyses identified novel sRNA seed regions and sets of potentially co‐regulated target mRNAs. We identified multiple mRNA targets for the pathotype‐specific sRNA Esr41, which was shown to regulate colicin sensitivity and iron transport in E. coli. Numerous sRNA interactions were also identified with non‐coding RNAs, including sRNAs and tRNAs, demonstrating the high complexity of the sRNA interactome.

Sequencing and Validation of the Genome of a Campylobacter concisus Reveals Intra-Species Diversity

Campylobacter concisus is an emerging pathogen of the human gastrointestinal tract. Its role in different diseases remains a subject of debate; this may be due to strain to strain genetic variation. Here, we sequence and analyze the genome of a C. concisus from a biopsy of a child with Crohns disease (UNSWCD); the second such genome for this species. A 1.8 Mb genome was assembled with paired-end reads from a next-generation sequencer. This genome is smaller than the 2.1 Mb C. concisus reference BAA-1457. While 1593 genes were conserved across UNSWCD and BAA-1457, 138 genes from UNSWCD and 281 from BAA-1457 were unique when compared against the other. To further validate the genome assembly and annotation, comprehensive shotgun proteomics was performed. This confirmed 78% of open reading frames in UNSWCD and, importantly, provided evidence of expression for 217 proteins previously defined as ‘hypothetical’ in Campylobacter. Substantial functional differences were observed between the UNSWCD and the reference strain. Enrichment analysis revealed differences in membrane proteins, response to stimulus, molecular transport and electron carriers. Synteny maps for the 281 genes not present in UNSWCD identified seven functionally associated gene clusters. These included one associated with the CRISPR family and another which encoded multiple restriction endonucleases; these genes are all involved in resistance to phage attack. Many of the observed differences are consistent with UNSWCD having adapted to greater surface interaction with host cells, as opposed to BAA-1457 which may prefer a free-living environment.