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John Quackenbush

Publications and source records attributed to John Quackenbush.

At least 37 records · Page 2Linked to original sources
PubMed2006

Data standards: a call to action.

Access to data is something that every molecular biologist takes for granted nowadays, but data alone is of little use unless it is made available in a useable form through the development and global uptake of data standards. The challenge of standards development has been taken up by grass-roots movements working within several different branches of the biomedical research community. Many of these initiatives are proving extremely successful; for example, the Gene Ontology, which provides a controlled vocabulary for describing the properties of gene products, the Microarray Gene Expression Data Society's standards for describing microarray experiments, and the emerging standards developed by the Proteomics Standards Initiative are gaining broad acceptance. Standards development now faces its greatest ever challenge--the integration of diverse data types to fulfill the goals of systems biology. Now is the time for the communities that are developing these standards, the funding bodies that have invested so heavily in high-throughput data generation, and the publishers of biomedical research papers to cooperate fully to make the goals of integrated data analysis a reality.

Animals↗
PubMed2006

Inferring steady state single-cell gene expression distributions from analysis of mesoscopic samples.

BACKGROUND: A great deal of interest has been generated by systems biology approaches that attempt to develop quantitative, predictive models of cellular processes. However, the starting point for all cellular gene expression, the transcription of RNA, has not been described and measured in a population of living cells. RESULTS: Here we present a simple model for transcript levels based on Poisson statistics and provide supporting experimental evidence for genes known to be expressed at high, moderate, and low levels. CONCLUSION: Although the model describes a microscopic process occurring at the level of an individual cell, the supporting data we provide uses a small number of cells where the echoes of the underlying stochastic processes can be seen. Not only do these data confirm our model, but this general strategy opens up a potential new approach, Mesoscopic Biology, that can be used to assess the natural variability of processes occurring at the cellular level in biological systems.

Base Sequence↗
PubMed2006

Transcript annotation in FANTOM3: mouse gene catalog based on physical cDNAs.

The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species.

Animals↗
PubMed2005

Molecular processes during fat cell development revealed by gene expression profiling and functional annotation.

BACKGROUND: Large-scale transcription profiling of cell models and model organisms can identify novel molecular components involved in fat cell development. Detailed characterization of the sequences of identified gene products has not been done and global mechanisms have not been investigated. We evaluated the extent to which molecular processes can be revealed by expression profiling and functional annotation of genes that are differentially expressed during fat cell development. RESULTS: Mouse microarrays with more than 27,000 elements were developed, and transcriptional profiles of 3T3-L1 cells (pre-adipocyte cells) were monitored during differentiation. In total, 780 differentially expressed expressed sequence tags (ESTs) were subjected to in-depth bioinformatics analyses. The analysis of 3'-untranslated region sequences from 395 ESTs showed that 71% of the differentially expressed genes could be regulated by microRNAs. A molecular atlas of fat cell development was then constructed by de novo functional annotation on a sequence segment/domain-wise basis of 659 protein sequences, and subsequent mapping onto known pathways, possible cellular roles, and subcellular localizations. Key enzymes in 27 out of 36 investigated metabolic pathways were regulated at the transcriptional level, typically at the rate-limiting steps in these pathways. Also, coexpressed genes rarely shared consensus transcription-factor binding sites, and were typically not clustered in adjacent chromosomal regions, but were instead widely dispersed throughout the genome. CONCLUSIONS: Large-scale transcription profiling in conjunction with sophisticated bioinformatics analyses can provide not only a list of novel players in a particular setting but also a global view on biological processes and molecular networks.

3T3-L1 Cells↗
PubMed2005

Extracting meaning from functional genomics experiments.

The completion of draft genome sequences for human, mouse, rat, and an increasing number of other species, has provided us with preliminary gene catalogues for many organisms of medical and scientific interests. Interpreting these gene lists in the context of the organism's underlying biology, however, remains difficult. The development of DNA microarrays provided one potential source of data to help interpret gene function; by profiling global patterns of gene expression across diverse conditions, it was hoped that we might be able to develop insight into biological function. But the power of these functional genomics assays, as well as assays in proteomics and metabolomics, is that they primarily give us lists of differentially expressed genes that can be correlated with particular phenotypic states, but which remain difficult to link mechanistically to the biology driving the phenotype.

Animals↗
PubMed2005

MeSHer: identifying biological concepts in microarray assays based on PubMed references and MeSH terms.

UNLABELLED: MeSHer uses a simple statistical approach to identify biological concepts in the form of Medical Subject Headings (MeSH terms) obtained from the PubMed database that are significantly overrepresented within the identified gene set relative to those associated with the overall collection of genes on the underlying DNA microarray platform. As a demonstration, we apply this approach to gene lists acquired from a published study of the effects of angiotensin II (Ang II) treatment on cardiac gene expression and demonstrate that this approach can aid in the interpretation of the resulting 'significant' gene set. AVAILABILITY: The software is available at http://www.tm4.org. SUPPLEMENTARY INFORMATION: Results from the analysis of significant genes from the published Ang II study.

Artificial Intelligence↗
PubMed2005

Molecular staging for survival prediction of colorectal cancer patients.

PURPOSE: The Dukes' staging system is the gold standard for predicting colorectal cancer prognosis; however, accurate classification of intermediate-stage cases is problematic. We hypothesized that molecular fingerprints could provide more accurate staging and potentially assist in directing adjuvant therapy. METHODS: A 32,000 cDNA microarray was used to evaluate 78 human colon cancer specimens, and these results were correlated with survival. Molecular classifiers were produced to predict outcome. RESULTS: Molecular staging, based on 43 core genes, was 90% accurate (93% sensitivity, 84% specificity) in predicting 36-month overall survival in 78 patients. This result was significantly better than Dukes' staging (P = .03878), discriminated patients into significantly different groups by survival time (P < .001, log-rank test), and was significantly different from chance (P < .001, 1,000 permutations). Furthermore, the classifier was able to discriminate a survival difference in an independent test set from Denmark. Molecular staging identifies patient prognosis (as represented by 36-month survival) more accurately than the traditional clinical staging, particularly for intermediate Dukes' stage B and C patients. The classifier was based on a core set of 43 genes, including osteopontin and neuregulin, which have biologic significance for this disease. CONCLUSION: These data support further evaluation of molecular staging to discriminate good from poor prognosis patients, with the potential to direct adjuvant therapy.

Adult↗
PubMed2005

CGHAnalyzer: a stand-alone software package for cancer genome analysis using array-based DNA copy number data.

SUMMARY: This synopsis provides an overview of array-based comparative genomic hybridization data display, abstraction and analysis using CGHAnalyzer, a software suite, designed specifically for this purpose. CGHAnalyzer can be used to simultaneously load copy number data from multiple platforms, query and describe large, heterogeneous datasets and export results. Additionally, CGHAnalyzer employs a host of algorithms for microarray analysis that include hierarchical clustering and class differentiation. AVAILABILITY: CGHAnalyzer, the accompanying manual, documentation and sample data are available for download at http://acgh.afcri.upenn.edu. This is a Java-based application built in the framework of the TIGR MeV that can run on Microsoft Windows, Macintosh OSX and a variety of Unix-based platforms. It requires the installation of the free Java Runtime Environment 1.4.1 (or more recent) (http://www.java.sun.com).

Algorithms↗
PubMed2005

Multiple-laboratory comparison of microarray platforms.

Microarray technology is a powerful tool for measuring RNA expression for thousands of genes at once. Various studies have been published comparing competing platforms with mixed results: some find agreement, others do not. As the number of researchers starting to use microarrays and the number of cross-platform meta-analysis studies rapidly increases, appropriate platform assessments become more important. Here we present results from a comparison study that offers important improvements over those previously described in the literature. In particular, we noticed that none of the previously published papers consider differences between labs. For this study, a consortium of ten laboratories from the Washington, DC-Baltimore, USA, area was formed to compare data obtained from three widely used platforms using identical RNA samples. We used appropriate statistical analysis to demonstrate that there are relatively large differences in data obtained in labs using the same platform, but that the results from the best-performing labs agree rather well.

Baltimore↗
PubMed2005

Independence and reproducibility across microarray platforms.

Microarrays have been widely used for the analysis of gene expression, but the issue of reproducibility across platforms has yet to be fully resolved. To address this apparent problem, we compared gene expression between two microarray platforms: the short oligonucleotide Affymetrix Mouse Genome 430 2.0 GeneChip and a spotted cDNA array using a mouse model of angiotensin II-induced hypertension. RNA extracted from treated mice was analyzed using Affymetrix and cDNA platforms and then by quantitative RT-PCR (qRT-PCR) for validation of specific genes. For the 11,710 genes present on both arrays, we assessed the relative impact of experimental treatment and platform on measured expression and found that biological treatment had a far greater impact on measured expression than did platform for more than 90% of genes, a result validated by qRT-PCR. In the small number of cases in which platforms yielded discrepant results, qRT-PCR generally did not confirm either set of data, suggesting that sequence-specific effects may make expression predictions difficult to make using any technique.

Angiotensin II↗
PubMed2005

A robust method for the amplification of RNA in the sense orientation.

BACKGROUND: Small quantities of RNA (1-4 microg total RNA) available from biological samples frequently require a single round of amplification prior to analysis, but current amplification strategies have limitations that may restrict their usefulness in downstream genomic applications. The Eberwine amplification method has been extensively validated but is limited by its ability to produce only antisense RNA. Alternatives lack extensive validation and are often confounded by problems with bias or yield attributable to their greater biological and technical complexity. RESULTS: To overcome these limitations, we have developed a straightforward and robust protocol for amplification of RNA in the sense orientation. This protocol is based upon Eberwine's method but incorporates elements of more recent amplification techniques while avoiding their complexities. Our technique yields greater than 100-fold amplification, generates long transcript, and produces mRNA that is well suited for use with microarray applications. Microarrays performed with RNA amplified using this protocol demonstrate minimal amplification bias and high reproducibility. CONCLUSION: The protocol we describe here is readily adaptable for the production of sense or antisense, labeled or unlabeled RNA from intact or partially-degraded prokaryotic or eukaryotic total RNA. The method outperforms several commercial RNA amplification kits and can be used in conjunction with a variety of microarray platforms, such as cDNA arrays, oligonucleotide arrays, and Affymetrix GeneChip arrays.

Algorithms↗
PubMed2005

Global transcription profiling reveals comprehensive insights into hypoxic response in Arabidopsis.

Plants have evolved adaptation mechanisms to sense oxygen deficiency in their environments and make coordinated physiological and structural adjustments to enhance their hypoxic tolerance. To gain insight into how plants respond to low-oxygen stress, gene expression profiling using whole-genome DNA amplicon microarrays was carried out at seven time points over 24 h, in wild-type and transgenic P(SAG12):ipt Arabidopsis (Arabidopsis thaliana) plants under normoxic and hypoxic conditions. Transcript levels of genes involved in glycolysis and fermentation pathways, ethylene synthesis and perception, calcium signaling, nitrogen utilization, trehalose metabolism, and alkaloid synthesis were significantly altered in response to oxygen limitation. Analysis based on gene ontology assignments suggested a significant down-regulation of genes whose functions are associated with cell walls, nucleosome structures, water channels, and ion transporters and a significant up-regulation of genes involved in transcriptional regulation, protein kinase activity, and auxin responses under conditions of oxygen shortage. Promoter analysis on a cluster of up-regulated genes revealed a significant overrepresentation of the AtMYB2-binding motif (GT motif), a sugar response element-like motif, and a G-box-related sequence, and also identified several putative anaerobic response elements. Finally, quantitative real-time polymerase chain reactions using 29 selected genes independently verified the microarray results. This study represents one of the most comprehensive analyses conducted to date investigating hypoxia-responsive transcriptional networks in plants.

Adaptation, Physiological↗
PubMed2005

Gene discovery in Plasmodium vivax through sequencing of ESTs from mixed blood stages.

Despite the significance of Plasmodium vivax as the most widespread human malaria parasite and a major public health problem, gene expression in this parasite is poorly understood. To accelerate gene discovery and facilitate the annotation phase of the P. vivax genome project, we have undertaken a transcriptome approach to study gene expression in the mixed blood stages of a P. vivax field isolate. Using a cDNA library constructed from purified blood stages, we have obtained single-pass sequences for approximately 21,500 expressed sequence tags (ESTs), the largest number of transcript tags obtained so far for this species. Cluster analysis revealed that the library is highly redundant, resulting in 5407 clusters. Clustered ESTs were searched against public protein databases for functional annotation, and more than one-third showed a significant match, the majority of these to Plasmodium falciparum proteins. The most abundant clusters were to genes encoding ribosomal proteins and proteins involved in metabolism, consistent with the predominance of trophozoites in the field isolate sample. In spite of the scarcity of other parasite stages in the field isolate, we could identify genes that are expressed in rings, schizonts and gametocytes. This study should facilitate our understanding of the gene expression in P. vivax asexual stages and provide valuable data for gene prediction and annotation of the P. vivax genome sequence.

Animals↗
PubMed2005

Synchronous global assessment of gene and protein expression in colorectal cancer progression.

Well-established models of colorectal cancer progression are based on the idea that the disease evolves through a multistep process involving sequential genetic mutations, suggesting that progression through clinically defined stages should correlate with well-defined patterns of gene expression. The majority of studies to date, however, have assessed these processes one gene and one protein at a time. We report the first comprehensive assessment of both gene and protein expression performed in parallel across progressive stages of human colorectal neoplasia. Remarkably, despite the global nature of the gene expression assessment, very few genes could be linked with certainty to specific proteins through currently available annotations. Furthermore, the correlation of expression between identified genes and proteins was poor. Nevertheless, both produced expression signatures that differentiated normal mucosa and nonmalignant adenomas from each other and from the malignant carcinomas and both produced fairly consistent subclasses of the malignancies, suggesting that a molecular staging might be more appropriate provided that these profiles can be tied to clinical outcome. This is potentially important as clinical staging is widely used as a prognostic indicator and used in the decision to pursue adjuvant therapies.

Adenoma↗
PubMed2005

Transcriptional divergence of the duplicated oxidative stress-responsive genes in the Arabidopsis genome.

Previous studies have indicated that Arabidopsis thaliana experienced a genome-wide duplication event shortly before its divergence from Brassica followed by extensive chromosomal rearrangements and deletions. While a large number of the duplicated genes have significantly diverged or lost their sister genes, we found 4222 pairs that are still highly conserved, and as a result had similar functional assignments during the annotation of the genome sequence. Using whole-genome DNA microarrays, we identified 906 duplicated gene pairs in which at least one member exhibited a significant response to oxidative stress. Among these, only 117 pairs were up- or down-regulated in both pairs and many of these exhibited dissimilar patterns of expression. Examination of the expression patterns of PAL1 and PAL2, ACD1 and ACD2, genes coding for two Hsp20s, various P450s, and electron transfer flavoproteins suggests Arabidopsis evolved a number of distinct oxidative stress response mechanisms using similar gene sets following the duplication of its genome.

Arabidopsis↗
PubMed2005

Murine macrophage transcriptional responses to Bacillus anthracis infection and intoxication.

Interactions between Bacillus anthracis and host macrophages represent critical early events in anthrax pathogenesis, but their details are not clearly understood. Here we report the first genomewide characterization of the transcriptional changes within macrophages infected with B. anthracis and the identification of several hundred host genes that were differentially expressed during this intracellular stage of infection. These loci included both genes that are known to be regulated differentially in response to many other bacterial pathogens and those that appear to be differentially regulated in response to B. anthracis but not other bacterial species that have been tested. These data provide a transcriptional basis for a variety of physiological changes observed during infection, including the induction of apoptosis caused by the infecting bacteria. The expression patterns underlying B. anthracis-induced apoptosis led us to test further the importance of one very highly induced macrophage gene, that for ornithine decarboxylase. Our data show that this enzyme plays an important and previously unrecognized role in suppressing apoptosis in B. anthracis-infected cells. We have also characterized the transcriptional response to anthrax lethal toxin in activated macrophages and found that, following toxin treatment, many of the host inflammatory response pathways are dampened. These data provide insights into B. anthracis pathogenesis as well as potential leads for the development of new diagnostic and therapeutic options.

Animals↗
PubMed2004

Cardiac transcriptional response to acute and chronic angiotensin II treatments.

Exposure of experimental animals to increased angiotensin II (ANG II) induces hypertension associated with cardiac hypertrophy, inflammation, and myocardial necrosis and fibrosis. Some of the most effective antihypertensive treatments are those that antagonize ANG II. We investigated cardiac gene expression in response to acute (24 h) and chronic (14 day) infusion of ANG II in mice; 24-h treatment induces hypertension, and 14-day treatment induces hypertension and extensive cardiac hypertrophy and necrosis. For genes differentially expressed in response to ANG II treatment, we tested for significant regulation of pathways, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Microarray Pathway Profiler (GenMAPP) databases, as well as functional classes based on Gene Ontology (GO) terms. Both acute and chronic ANG II treatments resulted in decreased expression of mitochondrial metabolic genes, notably those for the electron transport chain and Krebs-TCA cycle; chronic ANG II treatment also resulted in decreased expression of genes involved in fatty acid metabolism. In contrast, genes involved in protein translation and ribosomal activity increased expression following both acute and chronic ANG II treatments. Some classes of genes showed differential response between acute and chronic ANG II treatments. Acute treatment increased expression of genes involved in oxidative stress and amino acid metabolism, whereas chronic treatments increased cytoskeletal and extracellular matrix genes, second messenger cascades responsive to ANG II, and amyloidosis genes. Although a functional linkage between Alzheimer disease, hypertension, and high cholesterol has been previously documented in studies of brain tissue, this is the first demonstration of induction of Alzheimer disease pathways by hypertension in heart tissue. This study provides the most comprehensive available survey of gene expression changes in response to acute and chronic ANG II treatment, verifying results from disparate studies, and suggests mechanisms that provide novel insight into the etiology of hypertensive heart disease and possible therapeutic interventions that may help to mitigate its effects.

Angiotensin II↗