Testing for mean and correlation changes in microarray experiments: an application for pathway analysis

<p>Abstract</p> <p>Background</p> <p>Microarray experiments examine the change in transcript levels of tens of thousands of genes simultaneously. To derive meaningful data, biologists investigate the response of genes within specific pathways. Pathways are comprised of genes that interact to carry out a particular biological function. Existing methods for analyzing pathways focus on detecting changes in the mean or over-representation of the number of differentially expressed genes relative to the total of genes within the pathway. The issue of how to incorporate the influence of correlation among the genes is not generally addressed.</p> <p>Results</p> <p>In this paper, we propose a non-parametric rank test for analyzing pathways that takes into account the correlation among the genes and compared two existing methods, Global and Gene Set Enrichment Analysis (GSEA), using two publicly available data sets. A simulation study was conducted to demonstrate the advantage of the rank test method.</p> <p>Conclusions</p> <p>The data indicate the advantages of the rank test. The method can distinguish significant changes in pathways due to either correlations or changes in the mean or both. From the simulation study the rank test out performed Global and GSEA. The greatest gain in performance was for the sample size case which makes the application of the rank test ideal for microarray experiments.</p

Residential back taxes and revitalization: a study of Winnipeg's Spence neighbourhood

report: ii, 28 pp.; ill., digital fileThe City of Winnipeg considers the Spence neighbourhood a Major Rehabilitation Area. Socio-economic conditions have deteriorated and the number of boarded-up and abandoned homes has increased. Community groups are calling for revitalization as the conditions escalate toward irreversible decay. Unfortunately, there are numerous barriers to urban revitalization; one obstacle for the redevelopment of homes in this area is the City of Winnipeg’s stringent tax policy. Many of the abandoned units have back taxes owing and are left vacant for up to five years before the city claims title to the property. If a private homeowner is in tax arrears and wants to give or sell the home for a nominal amount to a non-profit group, the city stresses that back taxes still have to be paid. Once the five-year tax sale process is completed, homes are easy to acquire from the city. The purpose of this investigation is to illustrate how detrimental a five-year waiting period can be for the already neglected housing stock and the perception of the neighbourhood, as well as its role in accelerating urban decay.Institute of Urban Studie

Cross-platform analysis of global microRNA expression technologies

<p>Abstract</p> <p>Background</p> <p>Although analysis of microRNAs (miRNAs) by DNA microarrays is gaining in popularity, these new technologies have not been adequately validated. We examined within and between platform reproducibility of four miRNA array technologies alongside TaqMan PCR arrays.</p> <p>Results</p> <p>Two distinct pools of reference materials were selected in order to maximize differences in miRNA content. Filtering for miRNA that yielded signal above background revealed 54 miRNA probes (matched by sequence) across all platforms. Using this probeset as well as all probes that were present on an individual platform, within-platform analyses revealed Spearman correlations of >0.9 for most platforms. Comparing between platforms, rank analysis of the log ratios of the two reference pools also revealed high correlation (range 0.663-0.949). Spearman rank correlation and concordance correlation coefficients for miRNA arrays against TaqMan qRT-PCR arrays were similar for all of the technologies. Platform performances were similar to those of previous cross-platform exercises on mRNA and miRNA microarray technologies.</p> <p>Conclusions</p> <p>These data indicate that miRNA microarray platforms generated highly reproducible data and can be recommended for the study of changes in miRNA expression.</p

Gene expression analysis of livers from female B6C3F1 mice exposed to carcinogenic and non-carcinogenic doses of furan, with or without bromodeoxyuridine (BrdU) treatment

AbstractStandard methodology for identifying chemical carcinogens is both time-consuming and resource intensive. Researchers are actively investigating how new technologies can be used to identify chemical carcinogens in a more rapid and cost-effective manner. Here we performed a toxicogenomic case study of the liver carcinogen furan. Full study and mode of action details were previously published in the Journal of Toxicology and Applied Pharmacology. Female B6C3F1 mice were sub-chronically treated with two non-carcinogenic (1 and 2mg/kg bw) and two carcinogenic (4 and 8mg/kg bw) doses of furan for 21days. Half of the mice in each dose group were also treated with 0.02% bromodeoxyuridine (BrdU) for five days prior to sacrifice [13]. Agilent gene expression microarrays were used to measure changes in liver gene and long non-coding RNA expression (published in Toxicological Sciences). Here we describe the experimental and quality control details for the microarray data. We also provide the R code used to analyze the raw data files, produce fold change and false discovery rate (FDR) adjusted p values for each gene, and construct hierarchical clustering between datasets

Toxicogenomic analysis of susceptibility to inhaled urban particulate matter in mice with chronic lung inflammation

<p>Abstract</p> <p>Background</p> <p>Individuals with chronic lung disease are at increased risk of adverse health effects from airborne particulate matter. Characterization of underlying pollutant-phenotype interactions may require comprehensive strategies. Here, a toxicogenomic approach was used to investigate how inflammation modifies the pulmonary response to urban particulate matter.</p> <p>Results</p> <p>Transgenic mice with constitutive pulmonary overexpression of tumour necrosis factor (TNF)-α under the control of the surfactant protein C promoter and wildtype littermates (C57BL/6 background) were exposed by inhalation for 4 h to particulate matter (0 or 42 mg/m³EHC-6802) and euthanized 0 or 24 h post-exposure. The low alveolar dose of particles (16 μg) did not provoke an inflammatory response in the lungs of wildtype mice, nor exacerbate the chronic inflammation in TNF animals. Real-time PCR confirmed particle-dependent increases of CYP1A1 (30–100%), endothelin-1 (20–40%), and metallothionein-II (20–40%) mRNA in wildtype and TNF mice (p < 0.05), validating delivery of a biologically-effective dose. Despite detection of striking genotype-related differences, including activation of immune and inflammatory pathways consistent with the TNF-induced pathology, and time-related effects attributable to stress from nose-only exposure, microarray analysis failed to identify effects of the inhaled particles. Remarkably, the presence of chronic inflammation did not measurably amplify the transcriptional response to particulate matter.</p> <p>Conclusion</p> <p>Our data support the hypothesis that health effects of acute exposure to urban particles are dominated by activation of specific physiological response cascades rather than widespread changes in gene expression.</p

Evaluating Volatile Organic Compound Emissions from Cross-Laminated Timber Bonded with a Soy-Based Adhesive

Volatile organic compound (VOC) emissions from indoor sources are large determinants of the indoor air quality (IAQ) and occupant health. Cross-laminated timber (CLT) is a panelized engineered wood product often left exposed as an interior surface finish. As a certified structural building product, CLT is currently exempt from meeting VOC emission limits for composite wood products and confirming emissions through California Department of Public Health (CDPH) Standard Method testing. In this study, small chamber testing was conducted to evaluate VOC emissions from three laboratory-produced CLT samples: One bonded with a new soy-based cold-set adhesive; a second bonded with a commercially available polyurethane (PUR) adhesive; and the third assembled without adhesive using dowels. A fourth commercially-produced eight-month-old sample bonded with melamine formaldehyde (MF) adhesive was also tested. All four samples were produced with Douglas-fir. The test results for the three laboratory-produced samples demonstrated VOC emissions compliance with the reference standard. The commercially-produced and aged CLT sample bonded with MF adhesive did not meet the acceptance criterion for formaldehyde of ≤9.0 µg/m3. The estimated indoor air concentration of formaldehyde in an office with the MF sample was 54.4 µg/m3; the results for the soy, PUR, and dowel samples were all at or below 2.5 µg/m3

Meta-analysis of transcriptomic responses as a means to identify pulmonary disease outcomes for engineered nanomaterials

BACKGROUND: The increasing use of engineered nanomaterials (ENMs) of varying physical and chemical characteristics poses a great challenge for screening and assessing the potential pathology induced by these materials, necessitating novel toxicological approaches. Toxicogenomics measures changes in mRNA levels in cells and tissues following exposure to toxic substances. The resulting information on altered gene expression profiles, associated pathways, and the doses at which these changes occur, are used to identify the underlying mechanisms of toxicity and to predict disease outcomes. We evaluated the applicability of toxicogenomics data in identifying potential lung-specific (genomic datasets are currently available from experiments where mice have been exposed to various ENMs through this common route of exposure) disease outcomes following exposure to ENMs. METHODS: Seven toxicogenomics studies describing mouse pulmonary responses over time following intra-tracheal exposure to increasing doses of carbon nanotubes (CNTs), carbon black, and titanium dioxide (TiO(2)) nanoparticles of varying properties were examined to understand underlying mechanisms of toxicity. mRNA profiles from these studies were compared to the publicly available datasets of 15 other mouse models of lung injury/diseases induced by various agents including bleomycin, ovalbumin, TNFα, lipopolysaccharide, bacterial infection, and welding fumes to delineate the implications of ENM-perturbed biological processes to disease pathogenesis in lungs. RESULTS: The meta-analysis revealed two distinct clusters—one driven by TiO(2) and the other by CNTs. Unsupervised clustering of the genes showing significant expression changes revealed that CNT response clustered with bleomycin injury and bacterial infection models, both of which are known to induce lung fibrosis, in a post-exposure-time dependent manner, irrespective of the CNT’s physical-chemical properties. TiO(2) samples clustered separately from CNTs and disease models. CONCLUSIONS: These results indicate that in the absence of apical toxicity data, a tiered strategy beginning with short term, in vivo tissue transcriptomics profiling can effectively and efficiently screen new ENMs that have a higher probability of inducing pulmonary pathogenesis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12989-016-0137-5) contains supplementary material, which is available to authorized users

Nano-risk Science: application of toxicogenomics in an adverse outcome pathway framework for risk assessment of multi-walled carbon nanotubes

BACKGROUND: A diverse class of engineered nanomaterials (ENMs) exhibiting a wide array of physical-chemical properties that are associated with toxicological effects in experimental animals is in commercial use. However, an integrated framework for human health risk assessment (HHRA) of ENMs has yet to be established. Rodent 2-year cancer bioassays, clinical chemistry, and histopathological endpoints are still considered the ‘gold standard’ for detecting substance-induced toxicity in animal models. However, the use of data derived from alternative toxicological tools, such as genome-wide expression profiling and in vitro high-throughput assays, are gaining acceptance by the regulatory community for hazard identification and for understanding the underlying mode-of-action. Here, we conducted a case study to evaluate the application of global gene expression data in deriving pathway-based points of departure (PODs) for multi-walled carbon nanotube (MWCNT)-induced lung fibrosis, a non-cancer endpoint of regulatory importance. METHODS: Gene expression profiles from the lungs of mice exposed to three individual MWCNTs with different physical-chemical properties were used within the framework of an adverse outcome pathway (AOP) for lung fibrosis to identify key biological events linking MWCNT exposure to lung fibrosis. Significantly perturbed pathways were categorized along the key events described in the AOP. Benchmark doses (BMDs) were calculated for each perturbed pathway and were used to derive transcriptional BMDs for each MWCNT. RESULTS: Similar biological pathways were perturbed by the different MWCNT types across the doses and post-exposure time points studied. The pathway BMD values showed a time-dependent trend, with lower BMDs for pathways perturbed at the earlier post-exposure time points (24 h, 3d). The transcriptional BMDs were compared to the apical BMDs derived by the National Institute for Occupational Safety and Health (NIOSH) using alveolar septal thickness and fibrotic lesions endpoints. We found that regardless of the type of MWCNT, the BMD values for pathways associated with fibrosis were 14.0–30.4 μg/mouse, which are comparable to the BMDs derived by NIOSH for MWCNT-induced lung fibrotic lesions (21.0–27.1 μg/mouse). CONCLUSIONS: The results demonstrate that transcriptomic data can be used to as an effective mechanism-based method to derive acceptable levels of exposure to nanomaterials in product development when epidemiological data are unavailable. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12989-016-0125-9) contains supplementary material, which is available to authorized users

DNA methylation changes from primary cultures through senescence-bypass in Syrian hamster fetal cells initially exposed to benzo[a]pyrene

Current chemical testing strategies are limited in their ability to detect non-genotoxic carcinogens (NGTxC). Epigenetic anomalies develop during carcinogenesis regardless of whether the molecular initiating event is associated with genotoxic (GTxC) or NGTxC events; therefore, epigenetic markers may be harnessed to develop new approach methodologies that improve the detection of both types of carcinogens. This study used Syrian hamster fetal cells to establish the chronology of carcinogen-induced DNA methylation changes from primary cells until senescence-bypass as an essential carcinogenic step. Cells exposed to solvent control for 7 days were compared to naïve primary cultures, to cells exposed for 7 days to benzo[a]pyrene, and to cells at the subsequent transformation stages: normal colonies, morphologically transformed colonies, senescence, senescence-bypass, and sustained proliferation in vitro. DNA methylation changes identified by reduced representation bisulphite sequencing were minimal at day-7. Profound DNA methylation changes arose during cellular senescence and some of these early differentially methylated regions (DMRs) were preserved through the final sustained proliferation stage. A set of these DMRs (e.g., Pou4f1, Aifm3, B3galnt2, Bhlhe22, Gja8, Klf17, and L1l) were validated by pyrosequencing and their reproducibility was confirmed across multiple clones obtained from a different laboratory. These DNA methylation changes could serve as biomarkers to enhance objectivity and mechanistic understanding of cell transformation and could be used to predict senescence-bypass and chemical carcinogenicity