AVIA v2.0: annotation, visualization and impact analysis of genomic variants and genes.

UNLABELLED: As sequencing becomes cheaper and more widely available, there is a greater need to quickly and effectively analyze large-scale genomic data. While the functionality of AVIA v1.0, whose implementation was based on ANNOVAR, was comparable with other annotation web servers, AVIA v2.0 represents an enhanced web-based server that extends genomic annotations to cell-specific transcripts and protein-level functional annotations. With AVIA's improved interface, users can better visualize their data, perform comprehensive searches and categorize both coding and non-coding variants. AVAILABILITY AND IMPLEMENTATION: AVIA is freely available through the web at http://avia.abcc.ncifcrf.gov. CONTACT: Hue.Vuong@fnlcr.nih.gov SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

AVIA: an interactive web-server for annotation, visualization and impact analysis of genomic variations.

MOTIVATION: The plethora of information that emerges from large-scale genome characterization studies has triggered the development of computational frameworks and tools for efficient analysis, interpretation and visualization of genomic data. Functional annotation of genomic variations and the ability to visualize the data in the context of whole genome and/or multiple genomes has remained a challenging task. We have developed an interactive web-based tool, AVIA (Annotation, Visualization and Impact Analysis), to explore and interpret large sets of genomic variations (single nucleotide variations and insertion/deletions) and to help guide and summarize genomic experiments. The annotation, summary plots and tables are packaged and can be downloaded by the user from the email link provided. AVAILABILITY AND IMPLEMENTATION: http://avia.abcc.ncifcrf.gov.

Analysis of the Pythium ultimum transcriptome using Sanger and Pyrosequencing approaches.

BACKGROUND: Pythium species are an agriculturally important genus of plant pathogens, yet are not understood well at the molecular, genetic, or genomic level. They are closely related to other oomycete plant pathogens such as Phytophthora species and are ubiquitous in their geographic distribution and host rage. To gain a better understanding of its gene complement, we generated Expressed Sequence Tags (ESTs) from the transcriptome of Pythium ultimum DAOM BR144 (= ATCC 200006 = CBS 805.95) using two high throughput sequencing methods, Sanger-based chain termination sequencing and pyrosequencing-based sequencing-by-synthesis. RESULTS: A single half-plate pyrosequencing (454 FLX) run on adapter-ligated cDNA from a normalized cDNA population generated 90,664 reads with an average read length of 190 nucleotides following cleaning and removal of sequences shorter than 100 base pairs. After clustering and assembly, a total of 35,507 unique sequences were generated. In parallel, 9,578 reads were generated from a library constructed from the same normalized cDNA population using dideoxy chain termination Sanger sequencing, which upon clustering and assembly generated 4,689 unique sequences. A hybrid assembly of both Sanger- and pyrosequencing-derived ESTs resulted in 34,495 unique sequences with 1,110 sequences (3.2%) that were solely derived from Sanger sequencing alone. A high degree of similarity was seen between P. ultimum sequences and other sequenced plant pathogenic oomycetes with 91% of the hybrid assembly derived sequences > 500 bp having similarity to sequences from plant pathogenic Phytophthora species. An analysis of Gene Ontology assignments revealed a similar representation of molecular function ontologies in the hybrid assembly in comparison to the predicted proteomes of three Phytophthora species, suggesting a broad representation of the P. ultimum transcriptome was present in the normalized cDNA population. P. ultimum sequences with similarity to oomycete RXLR and Crinkler effectors, Kazal-like and cystatin-like protease inhibitors, and elicitins were identified. Sequences with similarity to thiamine biosynthesis enzymes that are lacking in the genome sequences of three Phytophthora species and one downy mildew were identified and could serve as useful phylogenetic markers. Furthermore, we identified 179 candidate simple sequence repeats that can be used for genotyping strains of P. ultimum. CONCLUSION: Through these two technologies, we were able to generate a robust set (approximately 10 Mb) of transcribed sequences for P. ultimum. We were able to identify known sequences present in oomycetes as well as identify novel sequences. An ample number of candidate polymorphic markers were identified in the dataset providing resources for phylogenetic and diagnostic marker development for this species. On a technical level, in spite of the depth possible with 454 FLX platform, the Sanger and pyro-based sequencing methodologies were complementary as each method generated sequences unique to each platform.

Analysis of 90 Mb of the potato genome reveals conservation of gene structures and order with tomato but divergence in repetitive sequence composition.

BACKGROUND: The Solanaceae family contains a number of important crop species including potato (Solanum tuberosum) which is grown for its underground storage organ known as a tuber. Albeit the 4th most important food crop in the world, other than a collection of approximately 220,000 Expressed Sequence Tags, limited genomic sequence information is currently available for potato and advances in potato yield and nutrition content would be greatly assisted through access to a complete genome sequence. While morphologically diverse, Solanaceae species such as potato, tomato, pepper, and eggplant share not only genes but also gene order thereby permitting highly informative comparative genomic analyses. RESULTS: In this study, we report on analysis 89.9 Mb of potato genomic sequence representing 10.2% of the genome generated through end sequencing of a potato bacterial artificial chromosome (BAC) clone library (87 Mb) and sequencing of 22 potato BAC clones (2.9 Mb). The GC content of potato is very similar to Solanum lycopersicon (tomato) and other dicotyledonous species yet distinct from the monocotyledonous grass species, Oryza sativa. Parallel analyses of repetitive sequences in potato and tomato revealed substantial differences in their abundance, 34.2% in potato versus 46.3% in tomato, which is consistent with the increased genome size per haploid genome of these two Solanum species. Specific classes and types of repetitive sequences were also differentially represented between these two species including a telomeric-related repetitive sequence, ribosomal DNA, and a number of unclassified repetitive sequences. Comparative analyses between tomato and potato at the gene level revealed a high level of conservation of gene content, genic feature, and gene order although discordances in synteny were observed. CONCLUSION: Genomic level analyses of potato and tomato confirm that gene sequence and gene order are conserved between these solanaceous species and that this conservation can be leveraged in genomic applications including cross-species annotation and genome sequencing initiatives. While tomato and potato share genic features, they differ in their repetitive sequence content and composition suggesting that repetitive sequences may have a more significant role in shaping speciation than previously reported.

The influence of hydro-alcoholic media on hypromellose matrix systems.

Hydrophilic matrices are widely used for extended release drug delivery, with hypromellose (HPMC) being a popular rate-controlling carrier. The FDA has recently issued an alert regarding the potential negative influence of alcohol on extended release dosage forms. The aim of this study was to investigate the hydroalcoholic solution effect on hydration, gel formation and drug release from HPMC matrices. None of the investigated matrix formulations (felodipine, gliclazide, and metformin hydrochloride) resulted in dose-dumping when exposed to ethanol solutions. HPMC compacts made of three different viscosity grades of Methocel showed consistent swelling and gel formation when exposed to hydroalcoholic media.

Functional polymorphisms in the transcription factor NRF2 in humans increase the risk of acute lung injury.

We recently used positional cloning to identify the transcription factor Nrf2 (NF-E2 related factor 2) as a susceptibility gene in a murine model of oxidant-induced acute lung injury (ALI). NRF2 binds to antioxidant response elements (ARE) and up-regulates protective detoxifying enzymes in response to oxidative stress. This led us to investigate NRF2 as a candidate susceptibility gene for risk of development of ALI in humans. We identified multiple single nucleotide polymorphisms (SNPs) by resequencing NRF2 in ethnically diverse subjects, and one (-617 C/A) significantly (P<0.001) diminished luciferase activity of promoter constructs containing the SNP and significantly decreased the binding affinity (P<0.001) relative to the wild type at this locus (-617 CC). In a nested case-control study, patients with the -617 A SNP had a significantly higher risk for developing ALI after major trauma (OR 6.44; 95% CI 1.34, 30.8; P=0.021) relative to patients with the wild type (-617 CC). This translational investigation provides novel insight into the molecular mechanisms of susceptibility to ALI and may help to identify patients who are predisposed to develop ALI under at risk conditions, such as trauma and sepsis. Furthermore, these findings may have important implications in other oxidative stress related illnesses.

Genome sequence of Aeromonas hydrophila ATCC 7966T: jack of all trades.

The complete genome of Aeromonas hydrophila ATCC 7966(T) was sequenced. Aeromonas, a ubiquitous waterborne bacterium, has been placed by the Environmental Protection Agency on the Contaminant Candidate List because of its potential to cause human disease. The 4.7-Mb genome of this emerging pathogen shows a physiologically adroit organism with broad metabolic capabilities and considerable virulence potential. A large array of virulence genes, including some identified in clinical isolates of Aeromonas spp. or Vibrio spp., may confer upon this organism the ability to infect a wide range of hosts. However, two recognized virulence markers, a type III secretion system and a lateral flagellum, that are reported in other A. hydrophila strains are not identified in the sequenced isolate, ATCC 7966(T). Given the ubiquity and free-living lifestyle of this organism, there is relatively little evidence of fluidity in terms of mobile elements in the genome of this particular strain. Notable aspects of the metabolic repertoire of A. hydrophila include dissimilatory sulfate reduction and resistance mechanisms (such as thiopurine reductase, arsenate reductase, and phosphonate degradation enzymes) against toxic compounds encountered in polluted waters. These enzymes may have bioremediative as well as industrial potential. Thus, the A. hydrophila genome sequence provides valuable insights into its ability to flourish in both aquatic and host environments.

Multiple cis-elements mediate the transcriptional activation of human fra-1 by 12-O-tetradecanoylphorbol-13-acetate in bronchial epithelial cells.

Recent studies indicate a potential role for Fra-1, a heterodimeric partner of activator protein 1 (AP1), in toxicant-induced epithelial injury, repair, and cellular transformation. Here, we have investigated the transcriptional regulation of fra-1 by 12-O-tetradecanoylphorbol-13-acetate (TPA) in human bronchial epithelial (HBE) cells, which are the direct targets of inhaled toxins/carcinogens. In contrast to a transient induction by H2O2, TPA persistently activated fra-1 transcription, principally at the transcriptional level. A deletion analysis of the fra-1 promoter revealed that several cis-elements located between -105/+32 and -283/-105 bp mediate minimal and basal promoter activities, respectively. A region between -379 and -283 bp, which harbors a putative TPA response element, a GC box, and an Ets-like binding site, was required for high level TPA-inducible expression. Mutations in any of these cis-elements markedly reduced both basal and TPA-inducible expression. Thus, cooperative interactions between factors binding to multiple cis-elements of the -379/-283 promoter region appear to regulate TPA-induced fra-1 transcription in HBE cells. Consistent with this finding, electrophoretic mobility shift assays indicated the formation of multiple complexes consisting of the AP1-, Sp-, and ETS-specific family of transcription factors with the -379/-283 fragment. Members of the AP1 family distinctly regulated the fra-1 promoter. In particular, coexpression of c-Jun, Jun-D, and Fra-2 up-regulated fra-1 transcription. Chromatin immunoprecipitation assays revealed an enhanced recruitment of c-Jun, Jun-D, and Fra-2 to the endogenous fra-1 promoter upon TPA stimulation. These results underscore the regulatory role of c-Jun, Jun-D, and Fra-2 in TPA-inducible fra-1 expression in HBE cells in vivo.

Interplay between proximal and distal promoter elements is required for squamous differentiation marker induction in the bronchial epithelium: role for ESE-1, Sp1, and AP-1 proteins.

Overexpression of SPRR1B in bronchial epithelial cells is a marker for early metaplastic changes induced by various toxicants/carcinogens. Previously, we have shown that the transcriptional stimulation of SPRR1B expression by phorbol 12-myristate 13-acetate (PMA) is mainly mediated by a -150/-94 bp enhancer harboring two critical 12-O-tetradecanoylphorbol-13-acetate-responsive elements (TREs) and by Jun.Fra-1 dimers. Here, we show that a region between -54 and -39 bp containing an ETS-binding site (EBS) and a GC box is essential for both basal and PMA-inducible SPRR1B transcription. In vivo footprinting demonstrated binding of transcription factors to these elements. However, unlike enhancer TREs, exposure of cells to PMA did not significantly alter the footprinting pattern at these elements. Mutations that crippled both the EBS and GC box suppressed both basal and PMA-inducible SPRR1B transcription. Consistent with this, overexpression of EBS-binding proteins ESE-1 and ESE-3 significantly stimulated SPRR1B promoter activity. Furthermore, preceding SPRR1B transcription, PMA up-regulated mRNA expression of ETS family members such as ESE-1 and ESE-3. Although ESE-1 synergistically activated c-Jun- and PMA-enhanced SPRR1B transcription, coexpression of Sp1 and ESE-1 showed no synergistic or additive effect on promoter activity, indicating an obligatory role for AP-1 proteins in such regulation. In support of this notion, deletion or mutation of two functional TREs inhibited ESE-1- and Sp1-enhanced promoter activation. Thus, the interaction between ESE-1 and Sp1, and AP-1 proteins that bind to the proximal and distal promoter regions, respectively, play a critical role in the induction of squamous differentiation marker expression in bronchial epithelial cells.

BMK1 (ERK5) regulates squamous differentiation marker SPRR1B transcription in Clara-like H441 cells.

Various toxicants and carcinogens upregulate the expression of small proline-rich protein 1B (SPRR1B), a squamous differentiation marker, in bronchial epithelial cells both in vivo and in vitro. We have recently shown that phorbol 13-myristate 12-acetate (PMA)-stimulated SPRR1B transcription in Clara-like H441 cells is mainly mediated by activator protein-1 (AP-1) and c-Jun N-terminal kinase-1 (JNK1). Though mitogen-activated protein kinase (MAPK) kinase (MEK)-1/2 pathway inhibitors strongly suppressed both basal and PMA-inducible SPRR1B transcription, overexpression of dominant negative (dn) forms of extracellular signal-regulated kinase (ERK)-1 and/or -2 did not have any significant effect indicating the involvement of another ERK-like MAPK in this pathway. Here, we report for the first time the involvement of ERK5 in PMA-inducible SPRR1B transcription in H441 cells. PMA significantly induced ERK5 activation in H441 cells. Overexpression of dn-ERK5 strongly suppressed both basal and PMA-inducible SPRR1B transcription, whereas wild-type ERK5 upregulated it. Consistent with this, a mutant form of MEK-5, an upstream activator of ERK5, strongly suppressed PMA-inducible promoter activity. However, coexpression of c-Jun restored promoter activation suppressed by dn-ERK5. Thus, in addition to JNK1, the activation of MEK5-ERK5 MAPK pathway probably plays a pivotal role in transcriptional regulation of AP-1-mediated SPRR1B expression in the distal bronchiolar region.

JNK1 and AP-1 regulate PMA-inducible squamous differentiation marker expression in Clara-like H441 cells.

Exposure of distal bronchiolar region to various toxicants and pollutants suppresses Clara cell differentiation marker expression and greatly enhances the induction of squamous cell differentiation (SCD). Here, we demonstrate for the first time phorbol 13-myristate 12-acetate (PMA)-inducible expression of SCD markers, SPRRs, in Clara-like H441 cells. The transcriptional stimulation of human SPRR1B expression is mainly mediated by a -150- to -84-bp region that harbors two critical activator protein (AP)-1 sites. In unstimulated cells, the -150- to -84-bp region is weakly bound by AP-1 proteins, mainly JunD and Fra1. However, PMA prominently induced the binding of JunB and Fra1. Consistent with this, overexpression of wild-type Jun proteins upregulated the SPRR1B promoter activity. Conversely, a c-jun mutant suppressed both basal and PMA-inducible reporter gene expression. Intriguingly, overexpression of fra2 suppressed PMA-inducible reporter activity, whereas fra1 significantly enhanced basal level activity, indicating an opposing role for these proteins in SPRR1B expression in a manner similar to that observed in proximal tracheobronchial epithelial cells (BEAS-2B clone S6). Interestingly, unlike in S6 cells, a catalytically inactive c-Jun NH(2)-terminal kinase (JNK) 1 mutant significantly reduced the PMA-inducible SPRR1B promoter activity in H441 cells. Thus either temporal expression and/or spatial activation of AP-1 proteins by JNK1 might contribute to the induction of SCD in Clara cells.