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In silico mining in expressed sequences of Neurospora crassa for identification and abundance of microsatellites.

In the present study, 3217 UniGene sequences of Neurospora crassa downloaded from the National Center for Biotechnology Information (NCBI) were mined for the identification of microsatellites or simple sequence repeats (SSRs). A total of 287 SSRs detected gives density of 1SSR/14.6 kb of 4187.86 kb sequences mined suggests that only 250 (7.8%) of sequences contained SSRs. Depending on the repeat units, the length of SSRs ranged from 14 to 17 bp for mono-, 14 to 48 bp for di-, 18 to 90 bp for tri-, 24 to 48 bp for tetra-, 30 for penta- and 42 to 48 bp for hexa-nucleotide repeats. Tri-nucleotide repeats were the most frequent repeat type (88.8%) followed by di-nucleotide repeats (5.9%). An attempt was also made with the help of bioinformatics approach to find out primer pairs for identified SSRs and primers were found only for 239 sequences. But, this part needs experimental validation. Annotation of SSRs containing sequences was also carried out.

Computational Biology↗

The NEIBank project for ocular genomics: data-mining gene expression in human and rodent eye tissues.

NEIBank is a project to gather and organize genomic resources for eye research. The first phase of this project covers the construction and sequence analysis of cDNA libraries from human and animal model eye tissues to develop an overview of the repertoire of genes expressed in the eye and a resource of cDNA clones for further studies. The sequence data are grouped and identified using the tools of bioinformatics and the results are displayed through a web site where they can be interrogated by keyword search, chromosome location, by Blast (sequence comparison) or by alignment on completed genomes. Many novel proteins and novel splice forms of known genes have already emerged from analysis of the accumulating data. This review provides an overview of the current state of the database for human eye tissues, with specific comparisons to some parallel data from mouse and rat, and with illustrative examples of the kinds of insights and discoveries these data can produce. One of the major themes that emerges is that at the molecular level human eye tissues have significant differences from those of rodents, encompassing species specific genes, alternative splice forms and great variation in levels of gene expression. These point to specific adaptations and mechanisms in the human eye and emphasize that care needs to be taken in the application of appropriate animal model systems.

Amino Acid Sequence↗

The UCSC Archaeal Genome Browser.

As more archaeal genomes are sequenced, effective research and analysis tools are needed to integrate the diverse information available for any given locus. The feature-rich UCSC Genome Browser, created originally to annotate the human genome, can be applied to any sequenced organism. We have created a UCSC Archaeal Genome Browser, available at http://archaea.ucsc.edu/, currently with 26 archaeal genomes. It displays G/C content, gene and operon annotation from multiple sources, sequence motifs (promoters and Shine-Dalgarno), microarray data, multi-genome alignments and protein conservation across phylogenetic and habitat categories. We encourage submission of new experimental and bioinformatic analysis from contributors. The purpose of this tool is to aid biological discovery and facilitate greater collaboration within the archaeal research community.

Archaeal Proteins↗

ABCdb: an online resource for ABC transporter repertories from sequenced archaeal and bacterial genomes.

The ATP-binding cassette (ABC) transporters are one of the major classes of active transporters. They are widespread in archaea, bacteria, and eukaryota, indicating that they have arisen early in evolution. They are involved in many essential physiological processes, but the majority import or export a wide variety of compounds across cellular membranes. These systems share a common architecture composed of four (exporters) or five (importers) domains. To identify and reconstruct functional ABC transporters encoded by archaeal and bacterial genomes, we have developed a bioinformatic strategy. Cross-reference to the transport classification system is used to predict the type of compound transported. A high quality of annotation is achieved by manual verification of the predictions. However, in order to face the rapid increase in the number of published genomes, we also include analyses of genomes issuing directly from the automated strategy. Querying the database (http://www-abcdb.biotoul.fr) allows to easily retrieve ABC transporter repertories and related data. Additional query tools have been developed for the analysis of the ABC family from both functional and evolutionary perspectives.

ATP-Binding Cassette Transporters↗

New bioinformatics tools for viral genome analyses at Viral Bioinformatics--Canada.

Viruses are much smaller than prokaryotes and eukaryotes, and it is now practical to sequence closely related members of virus families, strains, or even different isolates recovered during the course of an outbreak. However, comparative analysis of viral genomes requires the development of novel bioinformatics tools that allow us to align, edit, compare and interact with these genomes at all levels, from whole genome, to gene family, to single nucleotide polymorphisms. Comparative viral genomics can lead to the identification of the core characteristics that define a virus family, as well as the unique properties of viral species or isolates that contribute to variations in pathogenesis. This paper describes a number of tools, mainly developed for Viral Bioinformatics--Canada, that can be used for annotation and comparative genomic analysis of poxviruses. Nonetheless, these tools are also broadly applicable to other virus families.

Base Sequence↗

The GATO gene annotation tool for research laboratories.

Large-scale genome projects have generated a rapidly increasing number of DNA sequences. Therefore, development of computational methods to rapidly analyze these sequences is essential for progress in genomic research. Here we present an automatic annotation system for preliminary analysis of DNA sequences. The gene annotation tool (GATO) is a Bioinformatics pipeline designed to facilitate routine functional annotation and easy access to annotated genes. It was designed in view of the frequent need of genomic researchers to access data pertaining to a common set of genes. In the GATO system, annotation is generated by querying some of the Web-accessible resources and the information is stored in a local database, which keeps a record of all previous annotation results. GATO may be accessed from everywhere through the internet or may be run locally if a large number of sequences are going to be annotated. It is implemented in PHP and Perl and may be run on any suitable Web server. Usually, installation and application of annotation systems require experience and are time consuming, but GATO is simple and practical, allowing anyone with basic skills in informatics to access it without any special training. GATO can be downloaded at [http://mariwork.iq.usp.br/gato/]. Minimum computer free space required is 2 MB.

Biomedical Research↗

[Hidden Markov model used in protein sequence analysis].

Hidden Markov model (HMM) used in the research of protein is a new field of bioinformatics. Nowadays large amount of data about protein sequences and structures have been obtained. Traditional methods of protein analysis are no longer used. Biologists have updated their research methods with computer technology and statistics, which can deal with large amount of data. HMM can be used to distinguish protein sequence with the same characteristics. A family of protein from SCOP database was selected, through which a HMM model representing the family was obtained, and then the model was utilized to analyze protein sequences. Results indicate that HMM can express particular family of protein, and recognize the given protein sequences of the family from many sequences.

Algorithms↗

ESTs from brain and testis of White Leghorn and red junglefowl: annotation, bioinformatic classification of unknown transcripts and analysis of expression levels.

We report the generation, assembly and annotation of expressed sequence tags (ESTs) from four chicken cDNA libraries, constructed from brain and testis tissue dissected from red junglefowl and White Leghorn. 21,285 5'-end ESTs were generated and assembled into 2,813 contigs and 9,737 singletons, giving 12,549 tentative unique transcripts. The transcripts were annotated using BLAST by matching to known chicken genes or to putative homologues in other species using the major gene/protein databases. The results for these similarity searches are available on www.sbc.su.se/~arve/chicken. 4,129 (32.9%) of the transcripts remained without a significant match to gene/protein databases, a proportion of unmatched transcripts similar to earlier non-mammalian EST studies. To estimate how many of these transcripts may represent novel genes, they were studied for the presence of coding sequence. It was shown that most of the unique chicken transcripts do not contain coding parts of genes, but it was estimated that at least 400 of the transcripts contain coding sequence, indicating that 3.2% of avian genes belong to previously unknown gene families. Further BLAST search against dbEST left 1,649 (13.1%) of the transcripts unmatched to any library. The number of completely unmatched transcripts containing coding sequence was estimated at 180, giving a measure of the number of putative novel chicken genes identified in this study. 84.3% of the identified transcripts were found only in testis tissue, which has been poorly studied in earlier chicken EST studies. Large differences in expression levels were found between the brain and testis libraries for a large number of transcripts, and among the 525 most frequently represented transcripts, there were at least 20 transcripts with significant difference in expression levels between red junglefowl and White Leghorn.

Animals↗

Bioinformatics identification and validation of pyroptosis-related gene for ischemic stroke.

BACKGROUND: Ischemic stroke (IS) is one of the common and frequent diseases with extremely high lethality and disability in the world, and there is no effective treatment at present. This study aimed to screen hub genes involved in cerebral ischemia/reperfusion injury (CIRI) and pyroptosis, and explore promising intervention targets. METHODS: CIRI-related genes (GSE202659 and GSE131193) and pyroptosis-related genes (PRGs) in mice were obtained from the Gene Expression Omnibus (GEO) and GeneCards database. We screened for LASSO regression to construct a prognostic model of GSE131193 and PRGs and examined by GSE137482. The functional enrichment analysis of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) were performed on pyroptosis-related differentially expressed genes (PRDEGs) of GSE202659.The key modules for CIRI and pyroptosis were identified by Weight Gene Co-expression Network Analysis (WGCNA). Subsequently, Protein-protein Interaction (PPI) network and the Cytoscape was constructed to screen out hub genes. Used the starBase to predict miRNA interacting with hub genes and constructed mRNA-miRNA-lncRNA interaction networks. CIRI-related Molecular Subtypes were constructed for hub genes. The relationship between immune cells and hub genes was verified via CIBERSORT. Finally, we selected C57BL/6 mice to construct models to confirm hub genes by enzyme linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), western blot, and Immunofluorescence. RESULTS: A total of 272 PRGs and 35 PRDEGs were screened. An eight-gene risk prediction models were established (AUC = 0.868). GO, KEGG, GSEA and GSVA analyses revealed that PRDEGs were mainly involved in positive regulation of cytokine production, and NOD-like receptor signaling pathway. And then, seven hub genes (Irf1, Icam1, Tlr2, Tnf, Cebpb, Il1rn, and Casp8) were identified by PPI. Icam1, Tnf, Cebpb, Il1rn, and Casp8 had high expression profiles in Cluster2 by hierarchical clustering. The immune infiltration analysis results showed that among the hub genes, Cebpb, Il1rn, and Casp8, showed a significant positive correlation with the degree of NK.Actived, and Icam1 showed a significant negative correlation with B.Cells.Memory. The results of animal experiments significantly demonstrated an upregulation of Irf1, Icam1, Tlr2, Cebpb, and Il1rn. CONCLUSION: Our finding indicated that Irf1, Icam1, Tlr2, Cebpb, and Il1rn are hub genes associated with pyroptosis, and these genes are all associated with different immune cells, so as to provide new targets for the prevention and treatment of IS from the perspective of pyroptosis.

Pyroptosis↗

Peptidomic and proteomic analyses of the systemic immune response of Drosophila.

Insects have developed an efficient host defense against microorganisms, which involves humoral and cellular mechanisms. Numerous data highlight similarities between defense responses of insects and innate immunity of mammals. The fruit fly, Drosophila melanogaster, is a favorable model system for the analysis of the first line defense against microorganisms. Taking advantages of improvements in mass spectrometry (MS), two-dimensional (2D) gel electrophoresis and bioinformatics, differential analyses of blood content (hemolymph) from immune-challenged versus control Drosophila were performed. Two strategies were developed: (i) peptidomic analyses through matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and high performance liquid chromatography for molecules below 15 kDa, and (ii) proteomic studies based on 2D gel electrophoresis, MALDI-TOF fingerprinting and database searches, for compounds of greater molecular masses. The peptidomic strategy led to the detection of a large number of peptides induced in the hemolymph of challenged flies as compared to controls. Of these, 28 were characterized, amongst which were antimicrobial peptides. The 2D gel electrophoresis strategy led to the detection of 70 spots differentially regulated by at least fivefold after microbial infection. This approach yielded the identity of a series of proteins that were related to the Drosophila immune response, such as proteases, protease inhibitors, prophenoloxydase-activating enzymes, serpins and a Gram-negative binding protein-like protein. This strategy also brought to light new candidates with a potential function in the immune response (odorant-binding protein, peptidylglycine alpha-hydroxylating monooxygenase and transferrin). Interestingly, several molecules resulting from the cleavage of proteins were detected after a fungal infection. Together, peptidomic and proteomic analyses represent new tools to characterize molecules involved in the innate immune reactions of Drosophila.

Animals↗

Search for potential vaccine candidate open reading frames in the Bacillus anthracis virulence plasmid pXO1: in silico and in vitro screening.

A genomic analysis of the Bacillus anthracis virulence plasmid pXO1, aimed at identifying potential vaccine candidates and virulence-related genes, was carried out. The 143 previously defined open reading frames (ORFs) (R. T. Okinaka, K. Cloud, O. Hampton, A. R. Hoffmaster, K. K. Hill, P. Keim, T. M. Koehler, G. Lamke, S. Kumano, J. Mahillon, D. Manter, Y. Martinez, D. Ricke, R. Svensson, and P. J. Jackson, J. Bacteriol. 181:6509-6515, 1999) were subjected to extensive sequence similarity searches (with the nonredundant and unfinished microbial genome databases), as well as motif, cellular location, and domain analyses. A comparative genomics analysis was conducted with the related genomes of Bacillus subtilis, Bacillus halodurans, and Bacillus cereus and the pBtoxis plasmid of Bacillus thuringiensis var. israeliensis. As a result, the percentage of ORFs with clues about their functions increased from approximately 30% (as previously reported) to more than 60%. The bioinformatics analysis permitted identification of novel genes with putative relevance for pathogenesis and virulence. Based on our analyses, 11 putative proteins were chosen as targets for functional genomics studies. A rapid and efficient functional screening method was developed, in which PCR-amplified full-length linear DNA products of the selected ORFs were transcribed and directly translated in vitro and their immunogenicities were assessed on the basis of their reactivities with hyperimmune anti-B. anthracis antisera. Of the 11 ORFs selected for analysis, 9 were successfully expressed as full-length polypeptides, and 3 of these were found to be antigenic and to have immunogenic potential. The latter ORFs are currently being evaluated to determine their vaccine potential.

Animals↗

Bioinformatics and reanalysis of subtracted expressed sequence tags from the human ciliary body: Identification of novel biological functions.

PURPOSE: The ciliary body is largely known for its major roles in the regulation of aqueous humor secretion, intraocular pressure, and accommodation of the lens. In this review article we applied bioinformatics to re-examine hundreds of expressed sequence tags (ESTs) previously isolated by subtractive hybridization from a human ciliary body library [1]. The DNA sequences of these clones have been recently added to the web site of NEIBank. METHODS: DNA sequence comparisons of subtracted ESTs were performed against all entries in the last available release of the non-redundant database containing GenBank, EMBL, DDBJ and PDB sequences using the BlastN program accessed through NCBI's BLAST services on the internet (NCBI). Sequences were also compared and mapped using the Blast search program provided through the Internet by the Human Genome Project (UCSC). RESULTS: A total number of 284 independent ESTs were classified in 17 functional groups. Analysis of their relationships allowed to define the expression of five major groups of known genes: (i) protein synthesis, folding, secretion and degradation (20%); (ii) energy supply and biosynthesis (12%); (iii) contractility and cytoskeleton structure (6%); (iv) cellular signaling and cell cycle regulation (7%); and (v) nerve cell related tasks (2%), including neuropeptide processing and putative non-visual phototransduction and circadian rhythm control. The largest group contain unidentified sequences, a total of 105 sequences, accounting for 37% of ESTs. The unidentified sequences show similarity to genomic non-coding regions, or genes of unknown function. CONCLUSIONS: The most highly represented EST, correspond to myocilin, a gene involved in glaucoma. The data also confirms the secretory functions of the ciliary epithelium, and its high metabolism; the presence of a neuroendocrine peptidergic system presumably involved in the regulation of the intraocular pressure and/or aqueous humor secretion. Additional genes may be related to a non-visual phototransduction cascade and/or to circadian rhythms. Overall this initial group of subtracted ESTs can lead to uncover novel physiological functions of the ciliary body in normal and in disease, as well as novel candidate genes for ocular diseases.

Ciliary Body↗

The human proteome organization (HUPO) and environmental health.

The Human Proteome Organization, or HUPO, was formed to promote research and large-scale analysis of the human proteome. By consolidating national proteome organizations into an international body, HUPO will coordinate international initiatives, biological resources, protocols, standards and data for studying the human proteome. HUPO has identified five key areas to advance study of the human proteome, specifically in bioinformatics, new technologies, the plasma proteome, cell models, and a public antibody initiative. Consideration of three major issue areas may help develop HUPO's strategy for human proteome study. First is the need to distinguish the value of high throughput platforms from discovery platforms in proteomics. Second is the importance for international planning on integrating both transcriptome and proteome data and databases. Last is that effects of the environment from chemical, physical, and biological exposures alter the expression and structure of the proteome, which become manifest in long-term adverse health effects and disease. Environmental health research stands to greatly benefit from the shared resources, data, and vision of the HUPO organization as a valuable resource in exploiting knowledge of the human proteome toward improving public health.

Computational Biology↗

A combination of chemical derivatisation and improved bioinformatic tools optimises protein identification for proteomics.

The identification of individual protein species within an organism's proteome has been optimised by increasing the information produced from mass spectral analysis through the chemical derivatisation of tryptic peptides and the development of new software tools. Peptide fragments are subjected to two forms of derivatisation. First, lysine residues are converted to homoarginine moieties by guanidination. This procedure has two advantages, first, it usually identifies the C-terminal amino acid of the tryptic peptide and also greatly increases the total information content of the mass spectrum by improving the signal response of C-terminal lysine fragments. Second, an Edman-type phenylthiocarbamoyl (PTC) modification is carried out on the N-terminal amino acid. The renders the first peptide bond highly susceptible to cleavage during mass spectrometry (MS) analysis and consequently allows the ready identification of the N-terminal residue. The utility of the procedure has been demonstrated by developing novel bioinformatic tools to exploit the additional mass spectral data in the identification of proteome proteins from the yeast Saccharomyces cerevisiae. With this combination of novel chemistry and bioinformatics, it should be possible to identify unambiguously any yeast protein spot or band from either two-dimensional or one-dimensional electropheretograms.

Databases, Factual↗

From XML to RDF: how semantic web technologies will change the design of 'omic' standards.

With the ongoing rapid increase in both volume and diversity of 'omic' data (genomics, transcriptomics, proteomics, and others), the development and adoption of data standards is of paramount importance to realize the promise of systems biology. A recent trend in data standard development has been to use extensible markup language (XML) as the preferred mechanism to define data representations. But as illustrated here with a few examples from proteomics data, the syntactic and document-centric XML cannot achieve the level of interoperability required by the highly dynamic and integrated bioinformatics applications. In the present article, we discuss why semantic web technologies, as recommended by the World Wide Web consortium (W3C), expand current data standard technology for biological data representation and management.

Algorithms↗

In silico identification of new secretory peptide genes in Drosophila melanogaster.

Bioactive peptides play critical roles in regulating most biological processes in animals. The elucidation of the amino acid sequence of these regulatory peptides is crucial for our understanding of animal physiology. Most of the (neuro)peptides currently known were identified by purification and subsequent amino acid sequencing. With the entire genome sequence of some animals now available, it has become possible to predict novel putative peptides. In this way, BLAST (Basic Local Alignment Searching Tool) analysis of the Drosophila melanogaster genome has allowed annotation of 36 secretory peptide genes so far. Peptide precursor genes are, however, poorly predicted by this algorithm, thus prompting an alternative approach described here. With the described searching program we scanned the Drosophila genome for predicted proteins with the structural hallmarks of neuropeptide precursors. As a result, 76 additional putative secretory peptide genes were predicted in addition to the 43 annotated ones. These putative (neuro)peptide genes contain conserved motifs reminiscent of known neuropeptides from other animal species. Peptides that display sequence similarities to the mammalian vasopressin, atrial natriuretic peptide, and prolactin precursors and the invertebrate peptides orcokinin, prothoracicotropic hormones, trypsin modulating oostatic factor, and Drosophila immune induced peptides (DIMs) among others were discovered. Our data hence provide further evidence that many neuropeptide genes were already present in the ancestor of Protostomia and Deuterostomia prior to their divergence. This bioinformatic study opens perspectives for the genome-wide analysis of peptide genes in other eukaryotic model organisms.

Algorithms↗

The MGED Ontology: a resource for semantics-based description of microarray experiments.

MOTIVATION: The generation of large amounts of microarray data and the need to share these data bring challenges for both data management and annotation and highlights the need for standards. MIAME specifies the minimum information needed to describe a microarray experiment and the Microarray Gene Expression Object Model (MAGE-OM) and resulting MAGE-ML provide a mechanism to standardize data representation for data exchange, however a common terminology for data annotation is needed to support these standards. RESULTS: Here we describe the MGED Ontology (MO) developed by the Ontology Working Group of the Microarray Gene Expression Data (MGED) Society. The MO provides terms for annotating all aspects of a microarray experiment from the design of the experiment and array layout, through to the preparation of the biological sample and the protocols used to hybridize the RNA and analyze the data. The MO was developed to provide terms for annotating experiments in line with the MIAME guidelines, i.e. to provide the semantics to describe a microarray experiment according to the concepts specified in MIAME. The MO does not attempt to incorporate terms from existing ontologies, e.g. those that deal with anatomical parts or developmental stages terms, but provides a framework to reference terms in other ontologies and therefore facilitates the use of ontologies in microarray data annotation. AVAILABILITY: The MGED Ontology version.1.2.0 is available as a file in both DAML and OWL formats at http://mged.sourceforge.net/ontologies/index.php. Release notes and annotation examples are provided. The MO is also provided via the NCICB's Enterprise Vocabulary System (http://nciterms.nci.nih.gov/NCIBrowser/Dictionary.do). CONTACT: Stoeckrt@pcbi.upenn.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Computational Biology↗

The IUBMB-endorsed transporter classification system.

We here describe the transporter classification (TC) system that provides a rational means of classifying all transmembrane transport systems found in living organisms. The availability of this system provides a framework for the use of bioinformatic technologies to answer fundamental questions about the functions, mechanisms, and evolutionary pathways taken for the appearance of these systems. Such advances are discussed.

Amino Acid Sequence↗