Conserved abundance and topological features in chromatin-remodeling protein interaction networks.

The study of conserved protein interaction networks seeks to better understand the evolution and regulation of protein interactions. Here, we present a quantitative proteomic analysis of 18 orthologous baits from three distinct chromatin-remodeling complexes in Saccharomyces cerevisiae and Homo sapiens. We demonstrate that abundance levels of orthologous proteins correlate strongly between the two organisms and both networks have highly similar topologies. We therefore used the protein abundances in one species to cross-predict missing protein abundance levels in the other species. Lastly, we identified a novel conserved low-abundance subnetwork further demonstrating the value of quantitative analysis of networks.

Analysis of cancer metabolism with high-throughput technologies.

BACKGROUND: Recent advances in genomics and proteomics have allowed us to study the nuances of the Warburg effect--a long-standing puzzle in cancer energy metabolism--at an unprecedented level of detail. While modern next-generation sequencing technologies are extremely powerful, the lack of appropriate data analysis tools makes this study difficult. To meet this challenge, we developed a novel application for comparative analysis of gene expression and visualization of RNA-Seq data. RESULTS: We analyzed two biological samples (normal human brain tissue and human cancer cell lines) with high-energy, metabolic requirements. We calculated digital topology and the copy number of every expressed transcript. We observed subtle but remarkable qualitative and quantitative differences between the citric acid (TCA) cycle and glycolysis pathways. We found that in the first three steps of the TCA cycle, digital expression of aconitase 2 (ACO2) in the brain exceeded both citrate synthase (CS) and isocitrate dehydrogenase 2 (IDH2), while in cancer cells this trend was quite the opposite. In the glycolysis pathway, all genes showed higher expression levels in cancer cell lines; and most notably, digital gene expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase (ENO) were considerably increased when compared to the brain sample. CONCLUSIONS: The variations we observed should affect the rates and quantities of ATP production. We expect that the developed tool will provide insights into the subtleties related to the causality between the Warburg effect and neoplastic transformation. Even though we focused on well-known and extensively studied metabolic pathways, the data analysis and visualization pipeline that we developed is particularly valuable as it is global and pathway-independent.

Rat toxicogenomic study reveals analytical consistency across microarray platforms.

To validate and extend the findings of the MicroArray Quality Control (MAQC) project, a biologically relevant toxicogenomics data set was generated using 36 RNA samples from rats treated with three chemicals (aristolochic acid, riddelliine and comfrey) and each sample was hybridized to four microarray platforms. The MAQC project assessed concordance in intersite and cross-platform comparisons and the impact of gene selection methods on the reproducibility of profiling data in terms of differentially expressed genes using distinct reference RNA samples. The real-world toxicogenomic data set reported here showed high concordance in intersite and cross-platform comparisons. Further, gene lists generated by fold-change ranking were more reproducible than those obtained by t-test P value or Significance Analysis of Microarrays. Finally, gene lists generated by fold-change ranking with a nonstringent P-value cutoff showed increased consistency in Gene Ontology terms and pathways, and hence the biological impact of chemical exposure could be reliably deduced from all platforms analyzed.

Using RNA sample titrations to assess microarray platform performance and normalization techniques.

We have assessed the utility of RNA titration samples for evaluating microarray platform performance and the impact of different normalization methods on the results obtained. As part of the MicroArray Quality Control project, we investigated the performance of five commercial microarray platforms using two independent RNA samples and two titration mixtures of these samples. Focusing on 12,091 genes common across all platforms, we determined the ability of each platform to detect the correct titration response across the samples. Global deviations from the response predicted by the titration ratios were observed. These differences could be explained by variations in relative amounts of messenger RNA as a fraction of total RNA between the two independent samples. Overall, both the qualitative and quantitative correspondence across platforms was high. In summary, titration samples may be regarded as a valuable tool, not only for assessing microarray platform performance and different analysis methods, but also for determining some underlying biological features of the samples.

The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements.

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings.

The balance of reproducibility, sensitivity, and specificity of lists of differentially expressed genes in microarray studies.

BACKGROUND: Reproducibility is a fundamental requirement in scientific experiments. Some recent publications have claimed that microarrays are unreliable because lists of differentially expressed genes (DEGs) are not reproducible in similar experiments. Meanwhile, new statistical methods for identifying DEGs continue to appear in the scientific literature. The resultant variety of existing and emerging methods exacerbates confusion and continuing debate in the microarray community on the appropriate choice of methods for identifying reliable DEG lists. RESULTS: Using the data sets generated by the MicroArray Quality Control (MAQC) project, we investigated the impact on the reproducibility of DEG lists of a few widely used gene selection procedures. We present comprehensive results from inter-site comparisons using the same microarray platform, cross-platform comparisons using multiple microarray platforms, and comparisons between microarray results and those from TaqMan - the widely regarded "standard" gene expression platform. Our results demonstrate that (1) previously reported discordance between DEG lists could simply result from ranking and selecting DEGs solely by statistical significance (P) derived from widely used simple t-tests; (2) when fold change (FC) is used as the ranking criterion with a non-stringent P-value cutoff filtering, the DEG lists become much more reproducible, especially when fewer genes are selected as differentially expressed, as is the case in most microarray studies; and (3) the instability of short DEG lists solely based on P-value ranking is an expected mathematical consequence of the high variability of the t-values; the more stringent the P-value threshold, the less reproducible the DEG list is. These observations are also consistent with results from extensive simulation calculations. CONCLUSION: We recommend the use of FC-ranking plus a non-stringent P cutoff as a straightforward and baseline practice in order to generate more reproducible DEG lists. Specifically, the P-value cutoff should not be stringent (too small) and FC should be as large as possible. Our results provide practical guidance to choose the appropriate FC and P-value cutoffs when selecting a given number of DEGs. The FC criterion enhances reproducibility, whereas the P criterion balances sensitivity and specificity.

CHST11 gene expression and DNA methylation in breast cancer.

Our previously published data link P-selectin-reactive chondroitin sulfate structures on the surface of breast cancer cells to metastatic behavior of cells. We have shown that a particular sulfation pattern mediated by the expression of carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) correlates with P-selectin binding and aggressiveness of human breast cancer cell lines. The present study was performed to evaluate the prognostic value of CHST11 expression and determine whether aberrant DNA methylation controls CHST11 expression in breast cancer. Publicly available datasets were used to examine the association of CHST11 expression to aggressiveness and progression of breast cancer. Methylation status was analyzed using bisulfite genomic sequencing. 5-aza-2'-deoxycytidine (5AzadC) was used for DNA demethylation. Reduced representation bisulfite sequencing was performed in the CpG island of CHST11 with a minimum coverage of 10. Quantitative real-time RT-PCR was employed to confirm the expression profile of CHST11 in breast cancer cell lines. Flow cytometry was also used to confirm the expression of the CHST11 product, chondroitin sulfate A (CS-A). The expression of CHST11 was significantly higher in basal-like and Her2-amplified cell lines compared to luminal cell lines. CHST11 was also highly expressed in cancer tissues compared to normal tissues and the expression levels were significantly associated with tumor progression. We observed very low levels of DNA methylation in a CpG island of CHST11 in basal-like cells but very high levels in the same region in luminal cells. Treatment of MCF7 cells, a luminal cell line with very low expression of CHST11, with 5AzadC increased the expression of CHST11 and its immediate product, CS-A, in a dose-dependent manner. These results suggest that CHST11 may play a direct role in progression of breast cancer and that its expression is controlled by DNA methylation. Therefore, in addition to CHST11 mRNA levels, the methylation status of this gene also has potential as a prognostic biomarker.

Non-invasive analysis of intestinal development in preterm and term infants using RNA-Sequencing.

The state and development of the intestinal epithelium is vital for infant health, and increased understanding in this area has been limited by an inability to directly assess epithelial cell biology in the healthy newborn intestine. To that end, we have developed a novel, noninvasive, molecular approach that utilizes next generation RNA sequencing on stool samples containing intact epithelial cells for the purpose of quantifying intestinal gene expression. We then applied this technique to compare host gene expression in healthy term and extremely preterm infants. Bioinformatic analyses demonstrate repeatable detection of human mRNA expression, and network analysis shows immune cell function and inflammation pathways to be up-regulated in preterm infants. This study provides incontrovertible evidence that whole-genome sequencing of stool-derived RNA can be used to examine the neonatal host epithelial transcriptome in infants, which opens up opportunities for sequential monitoring of gut gene expression in response to dietary or therapeutic interventions.

A metagenomic study of diet-dependent interaction between gut microbiota and host in infants reveals differences in immune response.

BACKGROUND: Gut microbiota and the host exist in a mutualistic relationship, with the functional composition of the microbiota strongly affecting the health and well-being of the host. Thus, it is important to develop a synthetic approach to study the host transcriptome and the microbiome simultaneously. Early microbial colonization in infants is critically important for directing neonatal intestinal and immune development, and is especially attractive for studying the development of human-commensal interactions. Here we report the results from a simultaneous study of the gut microbiome and host epithelial transcriptome of three-month-old exclusively breast- and formula-fed infants. RESULTS: Variation in both host mRNA expression and the microbiome phylogenetic and functional profiles was observed between breast- and formula-fed infants. To examine the interdependent relationship between host epithelial cell gene expression and bacterial metagenomic-based profiles, the host transcriptome and functionally profiled microbiome data were subjected to novel multivariate statistical analyses. Gut microbiota metagenome virulence characteristics concurrently varied with immunity-related gene expression in epithelial cells between the formula-fed and the breast-fed infants. CONCLUSIONS: Our data provide insight into the integrated responses of the host transcriptome and microbiome to dietary substrates in the early neonatal period. We demonstrate that differences in diet can affect, via gut colonization, host expression of genes associated with the innate immune system. Furthermore, the methodology presented in this study can be adapted to assess other host-commensal and host-pathogen interactions using genomic and transcriptomic data, providing a synthetic genomics-based picture of host-commensal relationships.