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Rahman L, Williams A, Wu D, Halappanavar S. Polyethylene Terephthalate Microplastics Generated from Disposable Water Bottles Induce Interferon Signaling Pathways in Mouse Lung Epithelial Cells. NANOMATERIALS (BASEL, SWITZERLAND) 2024; 14:1287. [PMID: 39120391 PMCID: PMC11314056 DOI: 10.3390/nano14151287] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/20/2024] [Revised: 07/26/2024] [Accepted: 07/28/2024] [Indexed: 08/10/2024]
Abstract
Microplastics (MPs) are present in ambient air in a respirable size fraction; however, their potential impact on human health via inhalation routes is not well documented. In the present study, methods for a lab-scale generation of MPs from regularly used and littered plastic articles were optimized. The toxicity of 11 different types of MPs, both commercially purchased and in-lab prepared MPs, was investigated in lung epithelial cells using cell viability, immune and inflammatory response, and genotoxicity endpoints. The underlying mechanisms were identified by microarray analysis. Although laborious, the laboratory-scale methods generated a sufficient quantity of well characterized MPs for toxicity testing. Of the 11 MPs tested, the small sized polyethylene terephthalate (PETE) MPs prepared from disposable water bottles induced the maximum toxicity. Specifically, the smaller size PETE MPs induced a robust activation of the interferon signaling pathway, implying that PETE MPs are perceived by cells by similar mechanisms as those employed to recognize pathogens. The PETE MPs of heterogenous size and shapes induced cell injury, triggering cell death, inflammatory cascade, and DNA damage, hallmark in vitro events indicative of potential in vivo tissue injury. The study establishes toxicity of specific types of plastic materials in micron and nano size.
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Affiliation(s)
- Luna Rahman
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON K1A 0K9, Canada; (L.R.); (A.W.); (D.W.)
| | - Andrew Williams
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON K1A 0K9, Canada; (L.R.); (A.W.); (D.W.)
| | - Dongmei Wu
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON K1A 0K9, Canada; (L.R.); (A.W.); (D.W.)
| | - Sabina Halappanavar
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON K1A 0K9, Canada; (L.R.); (A.W.); (D.W.)
- Department of Biology, University of Ottawa, Ottawa, ON K1N 6N5, Canada
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Poulsen SS, Bengtson S, Williams A, Jacobsen NR, Troelsen JT, Halappanavar S, Vogel U. A transcriptomic overview of lung and liver changes one day after pulmonary exposure to graphene and graphene oxide. Toxicol Appl Pharmacol 2020; 410:115343. [PMID: 33227293 DOI: 10.1016/j.taap.2020.115343] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2020] [Revised: 11/13/2020] [Accepted: 11/19/2020] [Indexed: 02/07/2023]
Abstract
Hazard evaluation of graphene-based materials (GBM) is still in its early stage and it is slowed by their large diversity in the physicochemical properties. This study explores transcriptomic differences in the lung and liver after pulmonary exposure to two GBM with similar physical properties, but different surface chemistry. Female C57BL/6 mice were exposed by a single intratracheal instillation of 0, 18, 54 or 162 μg/mouse of graphene oxide (GO) or reduced graphene oxide (rGO). Pulmonary and hepatic changes in the transcriptome were profiled to identify commonly and uniquely perturbed functions and pathways by GO and rGO. These changes were then related to previously analyzed toxicity endpoints. GO exposure induced more differentially expressed genes, affected more functions, and perturbed more pathways compared to rGO, both in lung and liver tissues. The largest differences were observed for the pulmonary innate immune response and acute phase response, and for hepatic lipid homeostasis, which were strongly induced after GO exposure. These changes collective indicate a potential for atherosclerotic changes after GO, but not rGO exposure. As GO and rGO are physically similar, the higher level of hydroxyl groups on the surface of GO is likely the main reason for the observed differences. GO exposure also uniquely induced changes in the transcriptome related to fibrosis, whereas both GBM induced similar changes related to Reactive Oxygen Species production and genotoxicity. The differences in transcriptomic responses between the two GBM types can be used to understand how physicochemical properties influence biological responses and enable hazard evaluation of GBM and hazard ranking of GO and rGO, both in relation to each other and to other nanomaterials.
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Affiliation(s)
- Sarah S Poulsen
- National Research Centre for the Working Environment, Copenhagen Ø, Denmark
| | - Stefan Bengtson
- National Research Centre for the Working Environment, Copenhagen Ø, Denmark; Department of Science and Environment, Roskilde University, Roskilde, Denmark
| | - Andrew Williams
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario K1A 0K9, Canada
| | - Nicklas R Jacobsen
- National Research Centre for the Working Environment, Copenhagen Ø, Denmark
| | - Jesper T Troelsen
- Department of Science and Environment, Roskilde University, Roskilde, Denmark
| | - Sabina Halappanavar
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario K1A 0K9, Canada
| | - Ulla Vogel
- National Research Centre for the Working Environment, Copenhagen Ø, Denmark; Department of Health Technology, Technical University of Denmark, Kgs. Lyngby, Denmark.
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Khamesipour A, Kagaris D. Speeding up the discovery of combinations of differentially expressed genes for disease prediction and classification. COMPUTER METHODS AND PROGRAMS IN BIOMEDICINE 2019; 170:69-80. [PMID: 30712605 DOI: 10.1016/j.cmpb.2019.01.004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/20/2018] [Revised: 01/11/2019] [Accepted: 01/11/2019] [Indexed: 06/09/2023]
Abstract
BACKGROUND AND OBJECTIVE Finding combinations (i.e., pairs, or more generally, q-tuples with q ≥ 2) of genes whose behavior as a group differs significantly between two classes has received a lot of attention in the quest for the discovery of simple, accurate, and easily interpretable decision rules for disease classification and prediction. For example, the Top Scoring Pair (TSP) method seeks to find pairs of genes so that the probability of the reversal of the relative ranking of the expression levels of the genes in the two classes is maximized. The computational cost of finding a q-tuple of genes that scores highest under a given metric is O(Gq), where G is the total number of genes. This cost is often problematic or prohibitive in practice (even for q=2), as the number of genes G is often in the order of tens of thousands. METHODS In this paper, we show that this computational cost can be significantly reduced by excluding from consideration genes whose behavior is almost identical in the two classes and therefore their inclusion in any q-tuple is rather non-informative. Our criterion for the exclusion of genes is supported by a statistically robust metric, the Area Under the Curve (AUC) of the corresponding Receiver Operating Characteristic (ROC) curve. By filtering out genes whose AUC value is below a user-chosen threshold, as determined by a procedure that we describe in the paper, dramatic reductions in the run times are obtained while maintaining the same classification accuracy. RESULTS We have experimentally verified the gains of this approach on several case studies involving ovarian, colon, leukemia, breast and prostate cancers, and diffuse large b-cell lymphoma. CONCLUSIONS The proposed method is not only faster (for example, we observed an average 78.65% reduction over the run time of TSP) while maintaining the same classification accuracy, but it can even result in better classification accuracy due to its inherent ability to avoid the so-called "pivot" (non-informative) genes that may intrude in q-tuples chosen otherwise.
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Affiliation(s)
| | - Dimitri Kagaris
- ECE Dept., Southern Illinois University, Carbondale, IL 62901, USA.
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Desaulniers D, Khan N, Cummings-Lorbetskie C, Leingartner K, Xiao GH, Williams A, Yauk CL. Effects of cross-fostering and developmental exposure to mixtures of environmental contaminants on hepatic gene expression in prepubertal 21 days old and adult male Sprague-Dawley rats. JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH. PART A 2019; 82:1-27. [PMID: 30744511 DOI: 10.1080/15287394.2018.1542360] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/31/2018] [Revised: 10/24/2018] [Accepted: 10/26/2018] [Indexed: 06/09/2023]
Abstract
The notion that adverse health effects produced by exposure to environmental contaminants (EC) may be modulated by the presence of non-chemical stressors is gaining attention. Previously, our lab demonstrated that cross-fostering (adoption of a litter at birth) acted as a non-chemical stressor that amplified the influence of developmental exposure to EC on the glucocorticoid stress-response in adult rats. Using liver from the same rats, the aim of the current study was to investigate whether cross-fostering might also modulate EC-induced alterations in hepatic gene expression profiles. During pregnancy and nursing, Sprague-Dawley dams were fed cookies laced with corn oil (control, C) or a chemical mixture (M) composed of polychlorinated biphenyls (PCB), organochlorine pesticides (OCP), and methylmercury (MeHg), at 1 mg/kg/day. This mixture simulated the contaminant profile reported in maternal human blood. At birth, some control and M treated litters were cross-fostered to form two additional groups with different biological/nursing mothers (CC and MM). The hepatic transcriptome was analyzed by DNA microarray in male offspring at postnatal days 21 and 78-86. Mixture exposure altered the expression of detoxification and energy metabolism genes in both age groups, but with different sets of genes affected at day 21 and 78-86. Cross-fostering modulated the effects of M on gene expression pattern (MM vs M), as well as expression of energy metabolism genes between control groups (CC vs C). In conclusion, while describing short and long-term effects of developmental exposure to EC on hepatic transcriptomes, these cross-fostering results further support the consideration of non-chemical stressors in EC risk assessments.
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Affiliation(s)
- D Desaulniers
- a Health Canada, Healthy Environments and Consumer Safety Branch , Environmental Health Science and Research Bureau , Ottawa , Ontario , Canada
| | - N Khan
- a Health Canada, Healthy Environments and Consumer Safety Branch , Environmental Health Science and Research Bureau , Ottawa , Ontario , Canada
| | - C Cummings-Lorbetskie
- a Health Canada, Healthy Environments and Consumer Safety Branch , Environmental Health Science and Research Bureau , Ottawa , Ontario , Canada
| | - K Leingartner
- a Health Canada, Healthy Environments and Consumer Safety Branch , Environmental Health Science and Research Bureau , Ottawa , Ontario , Canada
| | - G-H Xiao
- a Health Canada, Healthy Environments and Consumer Safety Branch , Environmental Health Science and Research Bureau , Ottawa , Ontario , Canada
| | - A Williams
- a Health Canada, Healthy Environments and Consumer Safety Branch , Environmental Health Science and Research Bureau , Ottawa , Ontario , Canada
| | - C L Yauk
- a Health Canada, Healthy Environments and Consumer Safety Branch , Environmental Health Science and Research Bureau , Ottawa , Ontario , Canada
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Su Y, Xiao X, Ling H, Huang N, Liu F, Su W, Zhang Y, Xu L, Muhammad K, Que Y. A dynamic degradome landscape on miRNAs and their predicted targets in sugarcane caused by Sporisorium scitamineum stress. BMC Genomics 2019; 20:57. [PMID: 30658590 PMCID: PMC6339412 DOI: 10.1186/s12864-018-5400-8] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2018] [Accepted: 12/20/2018] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Sugarcane smut is a fungal disease caused by Sporisorium scitamineum. Cultivation of smut-resistant sugarcane varieties is the most effective way to control this disease. The interaction between sugarcane and S. scitamineum is a complex network system. However, to date, there is no report on the identification of microRNA (miRNA) target genes of sugarcane in response to smut pathogen infection by degradome technology. RESULTS TaqMan qRT-PCR detection and enzyme activity determination showed that S. scitamineum rapidly proliferated and incurred significant enzyme activity changes in the reactive oxygen species metabolic pathway and phenylpropanoid metabolic pathway at 2 d and 5 d after inoculation, which was the best time points to study target gene degradation during sugarcane and S. scitamineum interaction. A total of 122.33 Mb of raw data was obtained from degradome sequencing analysis of YC05-179 (smut-resistant) and ROC22 (smut-susceptible) after inoculation. The Q30 of each sample was > 93%, and the sequence used for degradation site analysis exactly matched the sugarcane reference sequence. A total of 309 target genes were predicted in sugarcane, corresponding to 97 known miRNAs and 112 novel miRNAs, and 337 degradation sites, suggesting that miRNAs can efficiently direct cleavage at multiple sites in the predicted target mRNAs. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the predicted target genes were involved in various regulatory processes, such as signal transduction mechanisms, inorganic ion transport and metabolism, defense mechanisms, translation, posttranslational modifications, energy production and conversion, and glycerolipid metabolism. qRT-PCR analysis of the expression level of 13 predicted target genes and their corresponding miRNAs revealed that there was no obvious negative regulatory relationship between miRNAs and their target genes. In addition, a number of putative resistance-related target genes regulated by miRNA-mediated cleavage were accumulated in sugarcane during S. scitamineum infection, suggesting that feedback regulation of miRNAs may be involved in the response of sugarcane to S. scitamineum infection. CONCLUSIONS This study elucidates the underlying response of sugarcane to S. scitamineum infection, and also provides a resource for miRNAs and their predicted target genes for smut resistance improvement in sugarcane.
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Affiliation(s)
- Yachun Su
- Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, 350002 China
- Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou, 350002 China
| | - Xinhuan Xiao
- Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, 350002 China
| | - Hui Ling
- Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, 350002 China
| | - Ning Huang
- Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, 350002 China
| | - Feng Liu
- Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, 350002 China
| | - Weihua Su
- Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, 350002 China
| | - Yuye Zhang
- Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, 350002 China
| | - Liping Xu
- Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, 350002 China
| | - Khushi Muhammad
- Department of Genetics, Hazara University, Mansehra, 21300 Pakistan
| | - Youxiong Que
- Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, 350002 China
- Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou, 350002 China
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Affiliation(s)
- C. J. Brien
- Phenomics and Bioinformatics Research Centre; University of South Australia and University of Adelaide; GPO Box 2471 Adelaide South Australia 5001 Australia
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Rahman L, Wu D, Johnston M, William A, Halappanavar S. Toxicogenomics analysis of mouse lung responses following exposure to titanium dioxide nanomaterials reveal their disease potential at high doses. Mutagenesis 2016; 32:59-76. [PMID: 27760801 PMCID: PMC5180171 DOI: 10.1093/mutage/gew048] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
Titanium dioxide nanoparticles (TiO2NPs) induce lung inflammation in experimental animals. In this study, we conducted a comprehensive toxicogenomic analysis of lung responses in mice exposed to six individual TiO2NPs exhibiting different sizes (8, 20 and 300nm), crystalline structure (anatase, rutile or anatase/rutile) and surface modifications (hydrophobic or hydrophilic) to investigate whether the mechanisms leading to TiO2NP-induced lung inflammation are property specific. A detailed histopathological analysis was conducted to investigate the long-term disease implications of acute exposure to TiO2NPs. C57BL/6 mice were exposed to 18, 54, 162 or 486 µg of TiO2NPs/mouse via single intratracheal instillation. Controls were exposed to dispersion medium only. Bronchoalveolar lavage fluid (BALF) and lung tissue were sampled on 1, 28 and 90 days post-exposure. Although all TiO2NPs induced lung inflammation as measured by the neutrophil influx in BALF, rutile-type TiO2NPs induced higher inflammation with the hydrophilic rutile TiO2NP showing the maximum increase. Accordingly, the rutile TiO2NPs induced higher number of differentially expressed genes. Histopathological analysis of lung sections on Day 90 post-exposure showed increased collagen staining and fibrosis-like changes following exposure to the rutile TiO2NPs at the highest dose tested. Among the anatase, the smallest TiO2NP of 8nm showed the maximum response. The anatase TiO2NP of 300nm was the least responsive of all. The results suggest that the severity of lung inflammation is property specific; however, the underlying mechanisms (genes and pathways perturbed) leading to inflammation were the same for all particle types. While the particle size clearly influenced the overall acute lung responses, a combination of small size, crystalline structure and hydrophilic surface contributed to the long-term pathological effects observed at the highest dose (486 µg/mouse). Although the dose at which the pathological changes were observed is considered physiologically high, the study highlights the disease potential of certain TiO2NPs of specific properties.
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Affiliation(s)
- Luna Rahman
- Environmental Health Science and Research Bureau, Health Canada, Tunney's Pasture Bldg. 8, Ottawa, Ontario K1A 0K9, Canada and
| | - Dongmei Wu
- Environmental Health Science and Research Bureau, Health Canada, Tunney's Pasture Bldg. 8, Ottawa, Ontario K1A 0K9, Canada and
| | - Michael Johnston
- Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Ontario K1A 0K9, Canada
| | - Andrew William
- Environmental Health Science and Research Bureau, Health Canada, Tunney's Pasture Bldg. 8, Ottawa, Ontario K1A 0K9, Canada and
| | - Sabina Halappanavar
- Environmental Health Science and Research Bureau, Health Canada, Tunney's Pasture Bldg. 8, Ottawa, Ontario K1A 0K9, Canada and
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Shvedova AA, Kisin ER, Yanamala N, Farcas MT, Menas AL, Williams A, Fournier PM, Reynolds JS, Gutkin DW, Star A, Reiner RS, Halappanavar S, Kagan VE. Gender differences in murine pulmonary responses elicited by cellulose nanocrystals. Part Fibre Toxicol 2016; 13:28. [PMID: 27278671 PMCID: PMC4898310 DOI: 10.1186/s12989-016-0140-x] [Citation(s) in RCA: 59] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2016] [Accepted: 06/02/2016] [Indexed: 12/31/2022] Open
Abstract
Background Cellulose-based materials have been used for centuries to manufacture different goods derived from forestry and agricultural sources. In the growing field of nanocellulose applications, its uniquely engineered properties are instrumental for inventive products coming to competitive markets. Due to their high aspect ratio and stiffness, it is speculated that cellulose nanocrystals (CNC) may cause similar pulmonary toxicity as carbon nanotubes and asbestos, thus posing a potential negative impact on public health and the environment. Methods The present study was undertaken to investigate the pulmonary outcomes induced by repeated exposure to respirable CNC. C57BL/6 female and male mice were exposed by pharyngeal aspiration to CNC (40 μg/mouse) 2 times a week for 3 weeks. Several biochemical endpoints and pathophysiological outcomes along with gene expression changes were evaluated and compared in the lungs of male and female mice. Results Exposure to respirable CNC caused pulmonary inflammation and damage, induced oxidative stress, elevated TGF-β and collagen levels in lung, and impaired pulmonary functions. Notably, these effects were markedly more pronounced in females compared to male mice. Moreover, sex differences in responses to pulmonary exposure to CNC were also detected at the level of global mRNA expression as well as in inflammatory cytokine/chemokine activity. Conclusions Overall, our results indicate that there are considerable differences in responses to respirable CNC based on gender with a higher pulmonary toxicity observed in female mice. Electronic supplementary material The online version of this article (doi:10.1186/s12989-016-0140-x) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Anna A Shvedova
- Health Effects Laboratory Division, NIOSH, Exposure Assessment Branch, 1095 Willowdale Road, Morgantown, WV, 26505, USA. .,Department of Physiology & Pharmacology, School of Medicine/WVU, Morgantown, WV, USA.
| | - Elena R Kisin
- Health Effects Laboratory Division, NIOSH, Exposure Assessment Branch, 1095 Willowdale Road, Morgantown, WV, 26505, USA
| | - Naveena Yanamala
- Health Effects Laboratory Division, NIOSH, Exposure Assessment Branch, 1095 Willowdale Road, Morgantown, WV, 26505, USA
| | - Mariana T Farcas
- Health Effects Laboratory Division, NIOSH, Exposure Assessment Branch, 1095 Willowdale Road, Morgantown, WV, 26505, USA
| | - Autumn L Menas
- Health Effects Laboratory Division, NIOSH, Exposure Assessment Branch, 1095 Willowdale Road, Morgantown, WV, 26505, USA
| | - Andrew Williams
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, ON, K1A 0 K9, Canada
| | - Philip M Fournier
- Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA
| | - Jeffrey S Reynolds
- Engineering and Controls Technology Branch, NIOSH/CDC, Morgantown, WV, USA
| | - Dmitriy W Gutkin
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA
| | - Alexander Star
- Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA
| | - Richard S Reiner
- Forest Products Laboratory, USDA Forest Service, Madison, WI, USA
| | - Sabina Halappanavar
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, ON, K1A 0 K9, Canada
| | - Valerian E Kagan
- Free Radical Center, University of Pittsburgh, Pittsburgh, PA, USA.,Department of Environmental & Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA
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Bruning O, Rauwerda H, Dekker RJ, de Leeuw WC, Wackers PFK, Ensink WA, Jonker MJ, Breit TM. Valuable lessons-learned in transcriptomics experimentation. Transcription 2016; 6:51-5. [PMID: 26098945 PMCID: PMC4581358 DOI: 10.1080/21541264.2015.1064195] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
We have collected several valuable lessons that will help improve transcriptomics experimentation. These lessons relate to experiment design, execution, and analysis. The cautions, but also the pointers, may help biologists avoid common pitfalls in transcriptomics experimentation and achieve better results with their transcriptome studies.
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Affiliation(s)
- Oskar Bruning
- a RNA Biology & Applied Bioinformatics research group; Swammerdam Institute for Life Sciences; Faculty of Science; University of Amsterdam ; Amsterdam , the Netherlands
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Confounding Factors in the Transcriptome Analysis of an In-Vivo Exposure Experiment. PLoS One 2016; 11:e0145252. [PMID: 26789003 PMCID: PMC4720430 DOI: 10.1371/journal.pone.0145252] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2015] [Accepted: 11/30/2015] [Indexed: 11/19/2022] Open
Abstract
Confounding factors In transcriptomics experimentation, confounding factors frequently exist alongside the intended experimental factors and can severely influence the outcome of a transcriptome analysis. Confounding factors are regularly discussed in methodological literature, but their actual, practical impact on the outcome and interpretation of transcriptomics experiments is, to our knowledge, not documented. For instance, in-vivo experimental factors; like Individual, Sample-Composition and Time-of-Day are potentially formidable confounding factors. To study these confounding factors, we designed an extensive in-vivo transcriptome experiment (n = 264) with UVR exposure of murine skin containing six consecutive samples from each individual mouse (n = 64). Analysis Approach Evaluation of the confounding factors: Sample-Composition, Time-of-Day, Handling-Stress, and Individual-Mouse resulted in the identification of many genes that were affected by them. These genes sometimes showed over 30-fold expression differences. The most prominent confounding factor was Sample-Composition caused by mouse-dependent skin composition differences, sampling variation and/or influx/efflux of mobile cells. Although we can only evaluate these effects for known cell type specifically expressed genes in our complex heterogeneous samples, it is clear that the observed variations also affect the cumulative expression levels of many other non-cell-type-specific genes. ANOVA ANOVA analysis can only attempt to neutralize the effects of the well-defined confounding factors, such as Individual-Mouse, on the experimental factors UV-Dose and Recovery-Time. Also, by definition, ANOVA only yields reproducible gene-expression differences, but we found that these differences were very small compared to the fold changes induced by the confounding factors, questioning the biological relevance of these ANOVA-detected differences. Furthermore, it turned out that many of the differentially expressed genes found by ANOVA were also present in the gene clusters associated with the confounding factors. Conclusion Hence our overall conclusion is that confounding factors have a major impact on the outcome of in-vivo transcriptomics experiments. Thus the set-up, analysis, and interpretation of such experiments should be approached with the utmost prudence.
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Decan N, Wu D, Williams A, Bernatchez S, Johnston M, Hill M, Halappanavar S. Characterization of in vitro genotoxic, cytotoxic and transcriptomic responses following exposures to amorphous silica of different sizes. MUTATION RESEARCH. GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2016; 796:8-22. [PMID: 26778505 DOI: 10.1016/j.mrgentox.2015.11.011] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/18/2015] [Revised: 11/18/2015] [Accepted: 11/23/2015] [Indexed: 11/25/2022]
Abstract
The objectives of the present study were to investigate the underlying mechanisms of genetic and cellular toxicity induced by silica nanoparticles (SiNPs) and determine if such toxicity is influenced by particle size. Commercially available amorphous SiNPs (12 nm, 5-10 nm, and 10-15 nm) and micrometer sized (SiP2 μm) silica were characterised for size, chemical composition, and aggregation state. Mouse lung epithelial (FE1) cells derived from Muta™Mouse were exposed to various concentrations (12.5, 25, 50, 100 μg/ml) of SiNPs and SiP2 μm. Cellular viability, clonogenic potential, oxidative stress, micronucleus formation, and mutant frequency were measured at different post-exposure time points. Cellular internalization of particles was assessed using nanoscale hyperspectral microscopy. Biological pathway and functional perturbations were assessed using DNA microarrays. Detailed characterization of particles confirmed their size, purity, and uniform dispersion in the exposure medium. Decreased cellular viability was observed acutely at 24h at concentrations higher than 25 μg/ml for all particle types, with SiNPs being the most sensitive; loss of viability was surface area dependent at the lowest concentration tested. However, only SiNP12 showed poor long-term survival. A size-dependent increase in micronucleus formation was also observed for SiNPs. In contrast to the viability results, SiP2 μm exhibited the highest potential to induce oxidative stress compared to the SiNPs at all tested concentrations. Gene ontology and biological pathway analysis revealed significant changes in the expression of genes implicated in lysosomal functions in SiNP12-treated cells, which appear closely associated with higher SiNP12 internalization and lysosomal rearrangements in the cytoplasm of these cells. These results suggest that SiNPs induce cellular and genetic toxicity in a size-dependent manner and that the observed toxicity may be the results of higher particle internalization of smaller SiNP and subsequent lysosomal overload.
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Affiliation(s)
- Nathalie Decan
- Environmental Health Science and Research Bureau, Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Canada
| | - Dongmei Wu
- Environmental Health Science and Research Bureau, Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Canada
| | - Andrew Williams
- Environmental Health Science and Research Bureau, Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Canada
| | - Stéphane Bernatchez
- New Substances Assessment and Control Bureau, Safe Environments Directorate, Health Canada, Ottawa, Canada
| | - Michael Johnston
- Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, Ottawa, Canada
| | - Myriam Hill
- New Substances Assessment and Control Bureau, Safe Environments Directorate, Health Canada, Ottawa, Canada
| | - Sabina Halappanavar
- Environmental Health Science and Research Bureau, Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Canada.
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Webster AF, Zumbo P, Fostel J, Gandara J, Hester SD, Recio L, Williams A, Wood CE, Yauk CL, Mason CE. Mining the Archives: A Cross-Platform Analysis of Gene Expression Profiles in Archival Formalin-Fixed Paraffin-Embedded Tissues. Toxicol Sci 2015; 148:460-72. [PMID: 26361796 PMCID: PMC4659533 DOI: 10.1093/toxsci/kfv195] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022] Open
Abstract
Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a potentially invaluable resource for transcriptomic research. However, use of FFPE samples in genomic studies has been limited by technical challenges resulting from nucleic acid degradation. Here we evaluated gene expression profiles derived from fresh-frozen (FRO) and FFPE mouse liver tissues preserved in formalin for different amounts of time using 2 DNA microarray protocols and 2 whole-transcriptome sequencing (RNA-seq) library preparation methodologies. The ribo-depletion protocol outperformed the other methods by having the highest correlations of differentially expressed genes (DEGs), and best overlap of pathways, between FRO and FFPE groups. The effect of sample time in formalin (18 h or 3 weeks) on gene expression profiles indicated that test article treatment, not preservation method, was the main driver of gene expression profiles. Meta- and pathway analyses indicated that biological responses were generally consistent for 18 h and 3 week FFPE samples compared with FRO samples. However, clear erosion of signal intensity with time in formalin was evident, and DEG numbers differed by platform and preservation method. Lastly, we investigated the effect of time in paraffin on genomic profiles. Ribo-depletion RNA-seq analysis of 8-, 19-, and 26-year-old control blocks resulted in comparable quality metrics, including expected distributions of mapped reads to exonic, untranslated region, intronic, and ribosomal fractions of the transcriptome. Overall, our results indicate that FFPE samples are appropriate for use in genomic studies in which frozen samples are not available, and that ribo-depletion RNA-seq is the preferred method for this type of analysis in archival and long-aged FFPE samples.
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Affiliation(s)
- A Francina Webster
- *Environmental Health Science and Research Bureau, Health Canada, Ottawa K1A 0K9, Canada; Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa K1S 5B6, Canada
| | - Paul Zumbo
- Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York 10065
| | - Jennifer Fostel
- National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
| | - Jorge Gandara
- Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York 10065
| | - Susan D Hester
- Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, North Carolina 27709
| | - Leslie Recio
- ILS, Inc., PO Box 13501, Research Triangle Park, North Carolina 27709
| | - Andrew Williams
- *Environmental Health Science and Research Bureau, Health Canada, Ottawa K1A 0K9, Canada
| | - Charles E Wood
- Office of Research and Development, US Environmental Protection Agency, Research Triangle Park, North Carolina 27709
| | - Carole L Yauk
- *Environmental Health Science and Research Bureau, Health Canada, Ottawa K1A 0K9, Canada;
| | - Christopher E Mason
- Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York 10065; The Feil Family Brain and Mind Research Institute (BMRI), 413 East 69th Street, New York, New York 10021; and The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, 1305 York Avenue, New York, New York 10065
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13
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Halappanavar S, Saber AT, Decan N, Jensen KA, Wu D, Jacobsen NR, Guo C, Rogowski J, Koponen IK, Levin M, Madsen AM, Atluri R, Snitka V, Birkedal RK, Rickerby D, Williams A, Wallin H, Yauk CL, Vogel U. Transcriptional profiling identifies physicochemical properties of nanomaterials that are determinants of the in vivo pulmonary response. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2015; 56:245-64. [PMID: 25504612 DOI: 10.1002/em.21936] [Citation(s) in RCA: 47] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/07/2014] [Revised: 11/19/2014] [Accepted: 11/21/2014] [Indexed: 05/28/2023]
Abstract
We applied transcriptional profiling to elucidate the mechanisms associated with pulmonary responses to titanium dioxide (TiO2 ) nanoparticles (NPs) of different sizes and surface coatings, and to determine if these responses are modified by NP size, surface area, surface modification, and embedding in paint matrices. Adult C57BL/6 mice were exposed via single intratracheal instillations to free forms of TiO2 NPs (10, 20.6, or 38 nm in diameter) with different surface coatings, or TiO2 NPs embedded in paint matrices. Controls were exposed to dispersion medium devoid of NPs. TiO2 NPs were characterized for size, surface area, chemical impurities, and agglomeration state in the exposure medium. Pulmonary transcriptional profiles were generated using microarrays from tissues collected one and 28 d postexposure. Property-specific pathway effects were identified. Pulmonary protein levels of specific inflammatory cytokines and chemokines were confirmed by ELISA. The data were collapsed to 659 differentially expressed genes (P ≤ 0.05; fold change ≥ 1.5). Unsupervised hierarchical clustering of these genes revealed that TiO2 NPs clustered mainly by postexposure timepoint followed by particle type. A pathway-based meta-analysis showed that the combination of smaller size, large deposited surface area, and surface amidation contributes to TiO2 NP gene expression response. Embedding of TiO2 NP in paint dampens the overall transcriptional effects. The magnitude of the expression changes associated with pulmonary inflammation differed across all particles; however, the underlying pathway perturbations leading to inflammation were similar, suggesting a generalized mechanism-of-action for all TiO2 NPs. Thus, transcriptional profiling is an effective tool to determine the property-specific biological/toxicity responses induced by nanomaterials.
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Affiliation(s)
- Sabina Halappanavar
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, Canada
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14
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Heegaard NHH, Østergaard O, Bahl JMC, Overgaard M, Beck HC, Rasmussen LM, Larsen MR. Important options available--from start to finish--for translating proteomics results to clinical chemistry. Proteomics Clin Appl 2015; 9:235-52. [PMID: 25472910 DOI: 10.1002/prca.201400137] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2014] [Revised: 11/11/2014] [Accepted: 11/26/2014] [Indexed: 12/20/2022]
Abstract
In the realm of clinical chemistry, the field of clinical proteomics, that is, the application of proteomic methods for understanding mechanisms and enabling diagnosis, prediction, measurement of activity, and treatment response in disease, is first and foremost a discovery and research tool that feeds assay development downstream. Putative new assay candidates generated by proteomics discovery projects compete with well-established assays with known indications, well-described performance, and of known value in specific clinical settings. Careful attention to the many options available in the design, execution, and interpretation of clinical proteomics studies is thus necessary for translation into clinical practice. We here review and discuss important options associated with clinical proteomics endeavors stretching from the planning phases to the final use in clinical chemistry.
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Affiliation(s)
- Niels H H Heegaard
- Department of Clinical Biochemistry, Immunology & Genetics, Statens Serum Institut, Copenhagen, Denmark; Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark
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15
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MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs. Toxicol Appl Pharmacol 2014; 284:16-32. [PMID: 25554681 DOI: 10.1016/j.taap.2014.12.011] [Citation(s) in RCA: 131] [Impact Index Per Article: 11.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2014] [Revised: 12/08/2014] [Accepted: 12/18/2014] [Indexed: 11/20/2022]
Abstract
Multi-walled carbon nanotubes (MWCNTs) are an inhomogeneous group of nanomaterials that vary in lengths, shapes and types of metal contamination, which makes hazard evaluation difficult. Here we present a toxicogenomic analysis of female C57BL/6 mouse lungs following a single intratracheal instillation of 0, 18, 54 or 162 μg/mouse of a small, curled (CNT(Small), 0.8 ± 0.1 μm in length) or large, thick MWCNT (CNT(Large), 4 ± 0.4 μm in length). The two MWCNTs were extensively characterized by SEM and TEM imaging, thermogravimetric analysis, and Brunauer-Emmett-Teller surface area analysis. Lung tissues were harvested 24h, 3 days and 28 days post-exposure. DNA microarrays were used to analyze gene expression, in parallel with analysis of bronchoalveolar lavage fluid, lung histology, DNA damage (comet assay) and the presence of reactive oxygen species (dichlorodihydrofluorescein assay), to profile and characterize related pulmonary endpoints. Overall changes in global transcription following exposure to CNT(Small) or CNT(Large) were similar. Both MWCNTs elicited strong acute phase and inflammatory responses that peaked at day 3, persisted up to 28 days, and were characterized by increased cellular influx in bronchoalveolar lavage fluid, interstitial pneumonia and gene expression changes. However, CNT(Large) elicited an earlier onset of inflammation and DNA damage, and induced more fibrosis and a unique fibrotic gene expression signature at day 28, compared to CNT(Small). The results indicate that the extent of change at the molecular level during early response phases following an acute exposure is greater in mice exposed to CNT(Large), which may eventually lead to the different responses observed at day 28.
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16
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Abstract
Exposure to nanoparticles has been associated with inflammation-related progression of atherosclerosis. To examine nanoparticle-induced cardiac effects in more detail, we characterized heart gene expression profiles alongside plasma proteins associated with cardiovascular disease in C57BL/6 mice intratracheally instilled with vehicle or 0.162 mg Printex 90 carbon black nanoparticles (CBNPs). Mice were killed 1, 3, and 28 days after the exposure and expression profiles were derived using DNA microarrays. Cardiac gene expression was unperturbed by CBNP exposure in two independent experiments, despite substantive changes in pulmonary and hepatic gene expression. MicroRNAs were not affected. Plasma levels of cell adhesion molecules (sE-selectin, sICAM-1, sVCAM-1) and total PAI-1 were immediately increased up to day 3, whereas Apo-A1 and Apo-E were marginally decreased on day 1. These data suggest that though adverse cardiovascular effects are likely following CBNP exposure, these effects are unlikely to be mediated by major direct effects on cardiac gene expression.
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17
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Webster AF, Williams A, Recio L, Yauk CL. Bromodeoxyuridine (BrdU) treatment to measure hepatocellular proliferation does not mask furan-induced gene expression changes in mouse liver. Toxicology 2014; 323:26-31. [PMID: 24910943 DOI: 10.1016/j.tox.2014.06.002] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2014] [Revised: 05/23/2014] [Accepted: 06/04/2014] [Indexed: 11/16/2022]
Abstract
Bromodeoxyuridine (BrdU) is a synthetic nucleoside used to detect cellular proliferation. BrdU incorporates in the place of thymine but pairs with guanine, thereby increasing the risk of transition mutations in dividing cells. Given its mutagenicity, standard practice is to use a second cohort of animals for parallel toxicogenomics studies; however, the impact of BrdU on global gene expression is unknown. To test this, we performed a case study to determine whether the molecular mode of action of furan, a liver carcinogen, could be detected in BrdU-treated samples. We measure global hepatic gene expression using Agilent DNA microarrays in female B6C3F1 mice that were sub-chronically exposed to 0, 1, 4, or 8mg/kg bodyweight (bw) per day furan either in the presence (+BrdU) or absence (-BrdU) of BrdU. Exposure to 0.02% BrdU in drinking water for five days resulted in minimal gene expression changes. A comparison of +BrdU versus -BrdU control mice revealed only 11 probes with fold change≥1.5 and false discovery rate (FDR) corrected p≤0.05. The same comparison in the high dose group yielded only 3 differentially expressed probes. Differentially expressed gene lists generated for furan-treated versus control mice and were compared for the -BrdU and +BrdU groups. The high dose of furan had 452 shared probes and 27 and 90 unique probes for -BrdU and +BrdU groups, respectively. These differences did not impact hierarchical clustering. Further, they did not impair detection of the previously reported furan mode of action, which was well represented in the BrdU-treated samples. Taken together, we demonstrate that BrdU treatment does not mask important furan-induced transcriptional changes. We suggest that BrdU-treated mice could be used for toxicogenomic analysis, which would generally halve the number of rodents required for toxicogenomics studies. However, we also recommend that this type of case study be repeated for other chemicals before the use of BrdU-treated animals in omics studies becomes common practice.
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Affiliation(s)
- Anna Francina Webster
- Environmental Health Science and Research Bureau, Health Canada, Ottawa K1A 0K9, Canada; Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa K1S 5B6, Canada.
| | - Andrew Williams
- Environmental Health Science and Research Bureau, Health Canada, Ottawa K1A 0K9, Canada.
| | - Leslie Recio
- ILS, Inc., P.O. Box 13501, Research Triangle Park, NC 27709, USA.
| | - Carole L Yauk
- Environmental Health Science and Research Bureau, Health Canada, Ottawa K1A 0K9, Canada.
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18
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Sordillo J, Raby BA. Gene expression profiling in asthma. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2014; 795:157-81. [PMID: 24162908 DOI: 10.1007/978-1-4614-8603-9_10] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [MESH Headings] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Affiliation(s)
- Joanne Sordillo
- Channing Division of Network Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, MA, 02115, USA,
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19
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Glass A, Henning J, Karopka T, Scheel T, Bansemer S, Koczan D, Gierl L, Rolfs A, Gimsa U. Representation of Individual Gene Expression in Completely Pooled mRNA Samples. Biosci Biotechnol Biochem 2014; 69:1098-103. [PMID: 15973040 DOI: 10.1271/bbb.69.1098] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Designing microarray experiments, scientists are often confronted with the question of pooling due to financial constraints, but discussion of the validity of pooling tends toward a sub-pooling recommendation. Since complete pooling protocols can be considered part of sub-pooling designs, gene expression data from three complete pooling experiments were analyzed. Data from complete pooled versus individual mRNA samples of rat brain tissue were compared to answer the question whether the pooled sample represents individual samples in small-sized experiments. Our analytic approach provided clear results concerning the Affymetrix MAS 5.0 signal and detection call parameters. Despite a strong similarity of arrays within experimental groups, the individual signals were evidently not appropriately represented in the pooled sample, with slightly more than half of all the genes considered. Our analysis reveals problems in cases of small complete pooling designs with less than six subjects pooled.
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Affiliation(s)
- Anne Glass
- Institute for Medical Informatics and Biometry, University of Rostock, Medical Faculty, Germany.
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20
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Yang Y, Fear J, Hu J, Haecker I, Zhou L, Renne R, Bloom D, McIntyre LM. Leveraging biological replicates to improve analysis in ChIP-seq experiments. Comput Struct Biotechnol J 2014; 9:e201401002. [PMID: 24688750 PMCID: PMC3962196 DOI: 10.5936/csbj.201401002] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2013] [Revised: 01/17/2014] [Accepted: 01/17/2014] [Indexed: 12/27/2022] Open
Abstract
ChIP-seq experiments identify genome-wide profiles of DNA-binding molecules including transcription factors, enzymes and epigenetic marks. Biological replicates are critical for reliable site discovery and are required for the deposition of data in the ENCODE and modENCODE projects. While early reports suggested two replicates were sufficient, the widespread application of the technique has led to emerging consensus that the technique is noisy and that increasing replication may be worthwhile. Additional biological replicates also allow for quantitative assessment of differences between conditions. To date it has remained controversial about how to confirm peak identification and to determine signal strength across biological replicates, particularly when the number of replicates is greater than two. Using objective metrics, we evaluate the consistency of biological replicates in ChIP-seq experiments with more than two replicates. We compare several approaches for binding site determination, including two popular but disparate peak callers, CisGenome and MACS2. Here we propose read coverage as a quantitative measurement of signal strength for estimating sample concordance. Determining binding based on genomic features, such as promoters, is also examined. We find that increasing the number of biological replicates increases the reliability of peak identification. Critically, binding sites with strong biological evidence may be missed if researchers rely on only two biological replicates. When more than two replicates are performed, a simple majority rule (>50% of samples identify a peak) identifies peaks more reliably in all biological replicates than the absolute concordance of peak identification between any two replicates, further demonstrating the utility of increasing replicate numbers in ChIP-seq experiments.
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Affiliation(s)
- Yajie Yang
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA ; UF Genetics Institute, University of Florida, Gainesville, Florida, USA
| | - Justin Fear
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA ; UF Genetics Institute, University of Florida, Gainesville, Florida, USA
| | - Jianhong Hu
- Human Genome Sequencing Center, Baylor College of Medicine, Houston, Texas, USA
| | - Irina Haecker
- Department of Applied Entomology, University of Giessen, Giessen, Germany
| | - Lei Zhou
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA
| | - Rolf Renne
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA ; UF Genetics Institute, University of Florida, Gainesville, Florida, USA ; UF Shands Cancer Center, University of Florida, Gainesville, Florida, USA
| | - David Bloom
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA
| | - Lauren M McIntyre
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, USA ; UF Genetics Institute, University of Florida, Gainesville, Florida, USA
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21
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Ahmed FE. Microarray RNA transcriptional profiling: Part I. Platforms, experimental design and standardization. Expert Rev Mol Diagn 2014; 6:535-50. [PMID: 16824028 DOI: 10.1586/14737159.6.4.535] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
This review summarizes, in a balanced and comprehensive manner, the various components of microarrays and their types, substrate architecture, platforms for microarray probe implementation, standardizations and confounders. The review is intended to familiarize the beginner with the principles of experimental design and the selection of an appropriate microarray platform. This parallel technology has revolutionized transcriptomic approaches to data profiling and has a major role in the identification of expressed genes, classification and diagnosis studies. The technology is still evolving and guidelines for standardization and reporting have been developed and are being improved.
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Affiliation(s)
- Farid E Ahmed
- Leo W Jenkins Cancer Center, Department of Radiation Oncology, LSB 014, The Brody School of Medicine at East Carolina University, Greenville, NC 27858, USA.
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22
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Farhat A, Buick JK, Williams A, Yauk CL, O'Brien JM, Crump D, Williams KL, Chiu S, Kennedy SW. Tris(1,3-dichloro-2-propyl) phosphate perturbs the expression of genes involved in immune response and lipid and steroid metabolism in chicken embryos. Toxicol Appl Pharmacol 2014; 275:104-12. [PMID: 24407104 DOI: 10.1016/j.taap.2013.12.020] [Citation(s) in RCA: 70] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2013] [Revised: 11/27/2013] [Accepted: 12/24/2013] [Indexed: 11/17/2022]
Abstract
We previously demonstrated that in ovo exposure to the flame retardant tris(1,3-dichloro-2-propyl) phosphate (TDCPP) decreased plasma thyroxine levels, reduced growth parameters, and decreased gallbladder size in chicken embryos. In the current study DNA microarrays were used to evaluate global mRNA expression in liver tissue of male chicken embryos that exhibited the above mentioned effects. Injected doses were dimethyl sulfoxide vehicle control, 7.6 or 45 μg TDCPP/g egg. TDCPP caused significant changes in the expression of five genes at the low dose and 47 genes at the high dose (False Discovery Rate p ≤ 0.1, fold change ≥ 1.5). The gene expression analysis suggested a compromised immune function, a state of cholestatic liver/biliary fibrosis, and disrupted lipid and steroid metabolism. Circulating bile acid levels were elevated, which is an indication of liver dysfunction, and plasma cholesterol levels were reduced; however, hepatic bile acid and cholesterol levels were unaltered. Interactome analyses identified apolipoprotein E, hepatocyte nuclear factor 4 alpha, and peroxisome proliferator-activated receptor alpha as key regulatory molecules involved in the effects of TDCPP. Our results demonstrate a targeted effect of TDCPP toxicity on lipid metabolism, including cholesterol, that helps explain the aforementioned phenotypic effects, as chicken embryos are highly dependent on yolk lipids for growth and maintenance throughout development. Finally, our results are in concordance with the literature that describes TDCPP as a cancer-causing agent, since the majority of dysregulated genes were involved in cancer pathways.
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Affiliation(s)
- Amani Farhat
- Department of Biology, University of Ottawa, Ottawa, ON K1N 6N5, Canada; National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3, Canada
| | - Julie K Buick
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON K1A 0K9, Canada
| | - Andrew Williams
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON K1A 0K9, Canada
| | - Carole L Yauk
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON K1A 0K9, Canada
| | - Jason M O'Brien
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON K1A 0K9, Canada
| | - Doug Crump
- National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3, Canada
| | - Kim L Williams
- National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3, Canada
| | - Suzanne Chiu
- National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3, Canada
| | - Sean W Kennedy
- Department of Biology, University of Ottawa, Ottawa, ON K1N 6N5, Canada; National Wildlife Research Centre, Environment Canada, Ottawa, ON K1A 0H3, Canada.
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23
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Søs Poulsen S, Jacobsen NR, Labib S, Wu D, Husain M, Williams A, Bøgelund JP, Andersen O, Købler C, Mølhave K, Kyjovska ZO, Saber AT, Wallin H, Yauk CL, Vogel U, Halappanavar S. Transcriptomic analysis reveals novel mechanistic insight into murine biological responses to multi-walled carbon nanotubes in lungs and cultured lung epithelial cells. PLoS One 2013; 8:e80452. [PMID: 24260392 PMCID: PMC3834097 DOI: 10.1371/journal.pone.0080452] [Citation(s) in RCA: 69] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2013] [Accepted: 10/03/2013] [Indexed: 12/22/2022] Open
Abstract
There is great interest in substituting animal work with in vitro experimentation in human health risk assessment; however, there are only few comparisons of in vitro and in vivo biological responses to engineered nanomaterials. We used high-content genomics tools to compare in vivo pulmonary responses of multiwalled carbon nanotubes (MWCNT) to those in vitro in cultured lung epithelial cells (FE1) at the global transcriptomic level. Primary size, surface area and other properties of MWCNT- XNRI -7 (Mitsui7) were characterized using DLS, SEM and TEM. Mice were exposed via a single intratracheal instillation to 18, 54, or 162 μg of Mitsui7/mouse. FE1 cells were incubated with 12.5, 25 and 100 μg/ml of Mitsui7. Tissue and cell samples were collected at 24 hours post-exposure. DNA microarrays were employed to establish mechanistic differences and similarities between the two models. Microarray results were confirmed using gene-specific RT-qPCR. Bronchoalveolar lavage (BAL) fluid was assessed for indications of inflammation in vivo. A strong dose-dependent activation of acute phase and inflammation response was observed in mouse lungs reflective mainly of an inflammatory response as observed in BAL. In vitro, a wide variety of core cellular functions were affected including transcription, cell cycle, and cellular growth and proliferation. Oxidative stress, fibrosis and inflammation processes were altered in both models. Although there were similarities observed between the two models at the pathway-level, the specific genes altered under these pathways were different, suggesting that the underlying mechanisms of responses are different in cells in culture and the lung tissue. Our results suggest that careful consideration should be given in selecting relevant endpoints when substituting animal with in vitro testing.
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Affiliation(s)
- Sarah Søs Poulsen
- National Research Centre for the Working Environment, Copenhagen, Denmark
- Department of Science, Systems and Models, Roskilde University, Roskilde, Denmark
| | | | - Sarah Labib
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | - Dongmei Wu
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | - Mainul Husain
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | - Andrew Williams
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | | | - Ole Andersen
- Department of Science, Systems and Models, Roskilde University, Roskilde, Denmark
| | - Carsten Købler
- Department of Micro- and Nanotechnology, Technical University of Denmark, Kgs. Lyngby, Denmark
| | - Kristian Mølhave
- Department of Micro- and Nanotechnology, Technical University of Denmark, Kgs. Lyngby, Denmark
| | - Zdenka O. Kyjovska
- National Research Centre for the Working Environment, Copenhagen, Denmark
| | - Anne T. Saber
- National Research Centre for the Working Environment, Copenhagen, Denmark
| | - Håkan Wallin
- National Research Centre for the Working Environment, Copenhagen, Denmark
- Department of Public Health, University of Copenhagen, Copenhagen, Denmark
| | - Carole L. Yauk
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
| | - Ulla Vogel
- National Research Centre for the Working Environment, Copenhagen, Denmark
- Department of Micro- and Nanotechnology, Technical University of Denmark, Kgs. Lyngby, Denmark
| | - Sabina Halappanavar
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada
- * E-mail:
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24
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Malik AI, Rowan-Carroll A, Williams A, Lemieux CL, Long AS, Arlt VM, Phillips DH, White PA, Yauk CL. Hepatic genotoxicity and toxicogenomic responses in Muta™Mouse males treated with dibenz[a,h]anthracene. Mutagenesis 2013; 28:543-54. [PMID: 23793610 DOI: 10.1093/mutage/get031] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Dibenz[a,h]anthracene (DB[a,h]A) is a polycyclic aromatic hydrocarbon that is a by-product of combustion and a potent carcinogen. Few studies have investigated the effects of DB[a,h]A on mRNA and microRNA expression to dissect the mechanisms involved in carcinogenesis. In this study, mature male mice (Muta(™)Mouse) were exposed to 6.25, 12.5 and 25mg/kg/day DB[a,h]A by oral gavage for 28 consecutive days. Results were compared with mice similarly exposed to benzo[a]pyrene (B[a]P) in our previous work. Liver DNA adduct levels and lacZ mutant frequency increased dose dependently for both chemicals. Aryl hydrocarbon receptor (AhR) potency was greater for DB[a,h]A than B[a]P using the chemical-activated luciferase expression assay. Microarray analysis revealed 19 up-regulated and 22 down-regulated genes (false discovery rate-adjusted P ≤ 0.05; fold change ≥ 1.5) following treatment with 6.25 mg/kg/day DB[a,h]A. Thirteen transcripts were up-regulated and 32 down-regulated in the 12.5mg/kg/day group. The 25mg/kg/day dose had major effects on mRNA expression with 135 up-regulated and 104 down-regulated genes. Overall, perturbations were greater for DB[a,h]A than for B[a]P; in vitro chemical-activated luciferase expression supports that this may be driven by the AhR. Many of the DB[a,h]A-affected genes are implicated in cancer and are essential in vital biological functions including circadian rhythm, glucose metabolism, lipid metabolism, immune response, cell cycle and apoptosis. Although a number of functional groups were similarly affected by B[a]P and DB[a,h]A, in general the responses generated by each chemical were quite distinct. Commonalities included a DNA damage response leading to induction of cell cycle arrest and apoptosis in both Tp53-dependent and Tp53-independent manners. MicroRNA expression was identical for both chemicals, with only miR-34a showing a dose-dependent increase in treated mice.
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Affiliation(s)
- Amal I Malik
- Environmental Health Science and Research Bureau, Health Canada, 50 Colombine Driveway, Ottawa, Ontario K1A 0K9, Canada
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Maertens RM, White PA, Williams A, Yauk CL. A global toxicogenomic analysis investigating the mechanistic differences between tobacco and marijuana smoke condensates in vitro. Toxicology 2013; 308:60-73. [PMID: 23542559 DOI: 10.1016/j.tox.2013.03.008] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2013] [Revised: 03/19/2013] [Accepted: 03/20/2013] [Indexed: 12/15/2022]
Abstract
Like tobacco smoking, habitual marijuana smoking causes numerous adverse pulmonary effects. However, the mechanisms of action involved, especially as compared to tobacco smoke, are still unclear. To uncover putative modes of action, this study employed a toxicogenomics approach to compare the toxicological pathways perturbed following exposure to marijuana and tobacco smoke condensate in vitro. Condensates of mainstream smoke from hand-rolled tobacco and marijuana cigarettes were similarly prepared using identical smoking conditions. Murine lung epithelial cells were exposed to low, medium and high concentrations of the smoke condensates for 6h. RNA was extracted immediately or after a 4h recovery period and hybridized to mouse whole genome microarrays. Tobacco smoke condensate (TSC) exposure was associated with changes in xenobiotic metabolism, oxidative stress, inflammation, and DNA damage response. These same pathways were also significantly affected following marijuana smoke condensate (MSC) exposure. Although the effects of the condensates were largely similar, dose-response analysis indicates that the MSC is substantially more potent than TSC. In addition, steroid biosynthesis, apoptosis, and inflammation pathways were more significantly affected following MSC exposure, whereas M phase cell cycle pathways were more significantly affected following TSC exposure. MSC exposure also appeared to elicit more severe oxidative stress than TSC exposure, which may account for the greater cytotoxicity of MSC. This study shows that in general MSC impacts many of the same molecular processes as TSC. However, subtle pathway differences can provide insight into the differential toxicities of the two complex mixtures.
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Affiliation(s)
- Rebecca M Maertens
- Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, ON, Canada.
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26
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Husain M, Saber AT, Guo C, Jacobsen NR, Jensen KA, Yauk CL, Williams A, Vogel U, Wallin H, Halappanavar S. Pulmonary instillation of low doses of titanium dioxide nanoparticles in mice leads to particle retention and gene expression changes in the absence of inflammation. Toxicol Appl Pharmacol 2013; 269:250-62. [DOI: 10.1016/j.taap.2013.03.018] [Citation(s) in RCA: 85] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2013] [Revised: 03/11/2013] [Accepted: 03/21/2013] [Indexed: 01/20/2023]
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Telaar A, Repsilber D, Nürnberg G. Biomarker discovery: classification using pooled samples. Comput Stat 2013. [DOI: 10.1007/s00180-011-0302-0] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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Robinson JF, Piersma AH. Toxicogenomic approaches in developmental toxicology testing. Methods Mol Biol 2013; 947:451-73. [PMID: 23138921 DOI: 10.1007/978-1-62703-131-8_31] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
The emergence of toxicogenomic applications provides new tools to characterize, classify, and potentially predict teratogens. However, due to the vast number of experimental and statistical procedural steps, toxicogenomic studies are challenging. Here, we guide researchers through the basic framework of conducting toxicogenomic investigations in the field of developmental toxicology, providing examples of biological and technical factors that may influence response and interpretation. Furthermore, we review current, diverse applications of toxicogenomic-based approaches in teratology testing, including exposure-response characterization (dose and duration), chemical classification studies, and cross-model comparisons study designs. This review is intended to guide scientists through the challenging and complex structure of conducting toxicogenomic analyses, while considering the many applications of using toxicogenomics in study designs and the future of these types of "omics" approaches in developmental toxicology.
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Affiliation(s)
- Joshua F Robinson
- Laboratory for Health Protection Research-National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
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29
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Franiak-Pietryga I, Sałagacka A, Maciejewski H, Błoński JZ, Borowiec M, Mirowski M, Robak T, Korycka-Wołowiec A. Apoptotic gene expression under influence of fludarabine and cladribine in chronic lymphocytic leukemia-microarray study. Pharmacol Rep 2012; 64:412-20. [PMID: 22661193 DOI: 10.1016/s1734-1140(12)70782-x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2011] [Revised: 11/24/2011] [Indexed: 11/19/2022]
Abstract
BACKGROUND A deep insight into gene expression profiling (GEP) is a key to understanding the background of disease. It can lead to identification of diagnostic and prognostic factors and then to a selection of the most appropriate therapy. The aim of this study was to evaluate differences in apoptotic gene expression in chronic lymphocytic leukemia (CLL) cells influenced by fludarabine (FA) or cladribine (2-CdA). METHODS GEP was performed in cells obtained from 10 untreated CLL patients and cultured in vitro with FA or 2-CdA. Ninety-three selected apoptotic genes were analyzed using 384 TaqMan® Low Density Arrays in pooled RNA. RESULTS Relevant results were found in a set of 27 genes, however, the most striking differences between FA and 2-CdA were observed in the following 5 genes: BAD, TNFRSF21, DAPK1, CARD 6 and CARD 9. CONCLUSION We have found some differences in apoptotic gene expression between FAand 2-CdA. These findings give prominence to genes qualifying for further studies currently conducted in our Department.
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MESH Headings
- Antineoplastic Agents/pharmacology
- Apoptosis/drug effects
- Apoptosis/genetics
- Cladribine/pharmacology
- DNA, Complementary/genetics
- Humans
- Leukemia, Lymphocytic, Chronic, B-Cell/blood
- Leukemia, Lymphocytic, Chronic, B-Cell/genetics
- Leukemia, Lymphocytic, Chronic, B-Cell/pathology
- Leukocytes, Mononuclear/drug effects
- Leukocytes, Mononuclear/pathology
- Oligonucleotide Array Sequence Analysis
- Real-Time Polymerase Chain Reaction
- Transcriptome
- Tumor Cells, Cultured
- Vidarabine/analogs & derivatives
- Vidarabine/pharmacology
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Affiliation(s)
- Ida Franiak-Pietryga
- Department of Hematology, Medical University of Lodz, Ciołkowskiego 2, PL 93-510 Łódź, Poland
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30
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Bordeaux JM, Lorenz WW, Dean JFD. Biomarker genes highlight intraspecific and interspecific variations in the responses of Pinus taeda L. and Pinus radiata D. Don to Sirex noctilio F. acid gland secretions. TREE PHYSIOLOGY 2012; 32:1302-1312. [PMID: 23042767 DOI: 10.1093/treephys/tps091] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/01/2023]
Abstract
Sirex noctilio F., a Eurasian horntail woodwasp recently introduced into North America, oviposits in pines and other conifers and in the process spreads a phytopathogenic fungus that serves as a food source for its larvae. During oviposition the woodwasp also deposits mucus produced in its acid (venom) gland that alters pine defense responses and facilitates infection by the fungus. A 26,496-feature loblolly pine cDNA microarray was used to survey gene expression of pine tissue responding to S. noctilio venom. Six genes were selected for further assessment by quantitative real-time polymerase chain reaction (qRT-PCR), including one that encoded an apparent PR-4 protein and another that encoded a thaumatin-like protein. Expression of both was strongly induced in response to venom, while expression of an apparent actin gene (ACT1) was stable in response to the venom. The pattern of gene response was similar in Pinus taeda L. and Pinus radiata D. Don, but the magnitude of response in P. radiata was significantly stronger for each of the induced genes. The magnitude of the biomarker gene response to venom also varied according to genotype within these two species. The qRT-PCR assay was used to demonstrate that the primary bioactive component in S. noctilio venom is a polypeptide.
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Affiliation(s)
- John Michael Bordeaux
- Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA 30602, USA
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31
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Cassone CG, Taylor JJ, O'Brien JM, Williams A, Yauk CL, Crump D, Kennedy SW. Transcriptional profiles in the cerebral hemisphere of chicken embryos following in ovo perfluorohexane sulfonate exposure. Toxicol Sci 2012; 129:380-91. [PMID: 22790973 DOI: 10.1093/toxsci/kfs219] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
In a recent egg injection study, we showed that in ovo exposure to perfluorohexane sulfonate (PFHxS) affects the pipping success of developing chicken (Gallus gallus domesticus) embryos. We also found evidence of thyroid hormone (TH) pathway interference at multiple levels of biological organization (i.e., somatic growth, messenger RNA expression, and circulating free thyroxine levels). Based on these findings, we hypothesize that PFHxS exposure interferes with TH-dependent neurodevelopmental pathways. This study investigates global transcriptional profiles in cerebral hemispheres of chicken embryos following exposure to a solvent control, 890 or 38,000 ng PFHxS/g egg (n = 4-5 per group); doses that lead to the adverse effects indicated above. PFHxS significantly alters the expression (≥ 1.5-fold, p ≤ 0.001) of 11 transcripts at the low dose (890 ng/g) and 101 transcripts at the high dose (38,000 ng/g). Functional enrichment analysis shows that PFHxS affects genes involved in tissue development and morphology, cellular assembly and organization, and cell-to-cell signaling. Pathway and interactome analyses suggest that genes may be affected through several potential regulatory molecules, including integrin receptors, myelocytomatosis viral oncogene, and CCAAT/enhancer-binding protein. This study identifies key functional and regulatory modes of PFHxS action involving TH-dependent and -independent neurodevelopmental pathways. Some of these TH-dependent mechanisms that occur during embryonic development include tight junction formation, signal transduction, and integrin signaling, whereas TH-independent mechanisms include gap junction intercellular communication.
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Affiliation(s)
- Cristina G Cassone
- Environment Canada, National Wildlife Research Centre, Ottawa, Ontario, Canada K1A 0H3
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32
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Intra- and inter-individual variance of gene expression in clinical studies. PLoS One 2012; 7:e38650. [PMID: 22723873 PMCID: PMC3377725 DOI: 10.1371/journal.pone.0038650] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2011] [Accepted: 05/11/2012] [Indexed: 01/29/2023] Open
Abstract
Background Variance in microarray studies has been widely discussed as a critical topic on the identification of differentially expressed genes; however, few studies have addressed the influence of estimating variance. Methodology/Principal Findings To break intra- and inter-individual variance in clinical studies down to three levels–technical, anatomic, and individual–we designed experiments and algorithms to investigate three forms of variances. As a case study, a group of “inter-individual variable genes” were identified to exemplify the influence of underestimated variance on the statistical and biological aspects in identification of differentially expressed genes. Our results showed that inadequate estimation of variance inevitably led to the inclusion of non-statistically significant genes into those listed as significant, thereby interfering with the correct prediction of biological functions. Applying a higher cutoff value of fold changes in the selection of significant genes reduces/eliminates the effects of underestimated variance. Conclusions/Significance Our data demonstrated that correct variance evaluation is critical in selecting significant genes. If the degree of variance is underestimated, “noisy” genes are falsely identified as differentially expressed genes. These genes are the noise associated with biological interpretation, reducing the biological significance of the gene set. Our results also indicate that applying a higher number of fold change as the selection criteria reduces/eliminates the differences between distinct estimations of variance.
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Erickson HS. Measuring molecular biomarkers in epidemiologic studies: laboratory techniques and biospecimen considerations. Stat Med 2012; 31:2400-13. [PMID: 22593027 DOI: 10.1002/sim.4485] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2011] [Revised: 11/05/2011] [Accepted: 11/14/2011] [Indexed: 12/20/2022]
Abstract
The future of personalized medicine depends on the ability to efficiently and rapidly elucidate a reliable set of disease-specific molecular biomarkers. High-throughput molecular biomarker analysis methods have been developed to identify disease risk, diagnostic, prognostic, and therapeutic targets in human clinical samples. Currently, high throughput screening allows us to analyze thousands of markers from one sample or one marker from thousands of samples and will eventually allow us to analyze thousands of markers from thousands of samples. Unfortunately, the inherent nature of current high throughput methodologies, clinical specimens, and cost of analysis is often prohibitive for extensive high throughput biomarker analysis. This review summarizes the current state of high throughput biomarker screening of clinical specimens applicable to genetic epidemiology and longitudinal population-based studies with a focus on considerations related to biospecimens, laboratory techniques, and sample pooling.
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Affiliation(s)
- Heidi S Erickson
- Department of Thoracic/Head and Neck Medical Oncology, UT MD Anderson Cancer Center, Houston, TX 77030, USA.
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Chauhan V, Howland M, Mendenhall A, O'Hara S, Stocki TJ, McNamee JP, Wilkins RC. Effects of alpha particle radiation on gene expression in human pulmonary epithelial cells. Int J Hyg Environ Health 2012; 215:522-35. [PMID: 22608759 DOI: 10.1016/j.ijheh.2012.04.004] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2011] [Revised: 03/27/2012] [Accepted: 04/15/2012] [Indexed: 01/11/2023]
Abstract
The general public receives approximately half of its exposure to natural radiation through alpha (α)-particles from radon ((222)Rn) gas and its decay progeny. Epidemiological studies have found a positive correlation between exposure to (222)Rn and lung carcinogenesis. An understanding of the transcriptional responses involved in these effects remains limited. In this study, genomic technology was employed to mine for subtle changes in gene expression that may be representative of an altered physiological state. Human lung epithelial cells were exposed to 0, 0.03, 0.3 and 0.9Gy of α-particle radiation. Microarray analysis was employed to determine transcript expression levels 4h and 24h after exposure. A total of 590 genes were shown to be differentially expressed in the α-particle radiated samples (false discovery rate (FDR)≤0.05). Sub-set of these transcripts were time-responsive, dose-responsive and both time- and dose-responsive. Pathway analysis showed functions related to cell cycle arrest, and DNA replication, recombination and repair (FDR≤0.05). The canonical pathways associated with these genes were in relation to pyrimidine metabolism, G2/M damage checkpoint regulation and p53 signaling (FDR≤0.05). Overall, this gene expression profile suggests that α-particle radiation inhibits DNA synthesis and subsequent mitosis, and causes cell cycle arrest.
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Affiliation(s)
- Vinita Chauhan
- Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario, Canada K1A 0K9. Vinita
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35
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Prasad SS, Russell M, Nowakowska M, Williams A, Yauk C. Gene expression analysis to identify molecular correlates of pre- and post-conditioning derived neuroprotection. J Mol Neurosci 2012; 47:322-39. [PMID: 22467039 DOI: 10.1007/s12031-012-9751-3] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2011] [Accepted: 03/08/2012] [Indexed: 12/16/2022]
Abstract
Mild ischaemic exposures before or after severe injurious ischaemia that elicit neuroprotective responses are referred to as preconditioning and post-conditioning. The corresponding molecular mechanisms of neuroprotection are not completely understood. Identification of the genes and associated pathways of corresponding neuroprotection would provide insight into neuronal survival, potential therapeutic approaches and assessments of therapies for stroke. The objectives of this study were to use global gene expression approach to infer the molecular mechanisms in pre- and post-conditioning-derived neuroprotection in cortical neurons following oxygen and glucose deprivation (OGD) in vitro and then to apply these findings to predict corresponding functional pathways. To this end, microarray analysis was applied to rat cortical neurons with or without the pre- and post-conditioning treatments at 3-h post-reperfusion, and differentially expressed transcripts were subjected to statistical, hierarchical clustering and pathway analyses. The expression patterns of 3,431 genes altered under all conditions of ischaemia (with and without pre- or post-conditioning). We identified 1,595 genes that were commonly regulated within both the pre- and post-conditioning treatments. Cluster analysis revealed that transcription profiles clustered tightly within controls, non-conditioned OGD and neuroprotected groups. Two clusters defining neuroprotective conditions associated with up- and downregulated genes were evident. The five most upregulated genes within the neuroprotective clusters were Tagln, Nes, Ptrf, Vim and Adamts9, and the five most downregulated genes were Slc7a3, Bex1, Brunol4, Nrxn3 and Cpne4. Pathway analysis revealed that the intracellular and second messenger signalling pathways in addition to cell death were predominantly associated with downregulated pre- and post-conditioning associated genes, suggesting that modulation of cell death and signal transduction pathways plays a role in the neuroprotection. A high degree of similarity in the pathways associated with the differentially expressed genes in the pre- and post-conditioning treatments suggests that similar molecular mechanisms may mediate their neuroprotective effects.
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Affiliation(s)
- Shiv S Prasad
- Genomics Laboratories, Biologics and Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, Ottawa, ON, K1A 0K9, Canada.
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Kerr KF. Optimality criteria for the design of 2-color microarray studies. Stat Appl Genet Mol Biol 2012; 11:Article 10. [PMID: 22499679 DOI: 10.1515/1544-6115.1583] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
We discuss the definition and application of design criteria for evaluating the efficiency of 2-color microarray designs. First, we point out that design optimality criteria are defined differently for the regression and block design settings. This has caused some confusion in the literature and warrants clarification. Linear models for microarray data analysis have equivalent formulations as ANOVA or regression models. However, this equivalence does not extend to design criteria. We discuss optimality criterion, and argue against applying regression-style D-optimality to the microarray design problem. We further disfavor E- and D-optimality (as defined in block design) because they are not attuned to scientific questions of interest.
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Paquette MA, Dong H, Gagné R, Williams A, Malowany M, Wade MG, Yauk CL. Thyroid hormone-regulated gene expression in juvenile mouse liver: identification of thyroid response elements using microarray profiling and in silico analyses. BMC Genomics 2011; 12:634. [PMID: 22206413 PMCID: PMC3340398 DOI: 10.1186/1471-2164-12-634] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2011] [Accepted: 12/29/2011] [Indexed: 01/26/2023] Open
Abstract
Background Disruption of thyroid hormone signalling can alter growth, development and energy metabolism. Thyroid hormones exert their effects through interactions with thyroid receptors that directly bind thyroid response elements and can alter transcriptional activity of target genes. The effects of short-term thyroid hormone perturbation on hepatic mRNA transcription in juvenile mice were evaluated, with the goal of identifying genes containing active thyroid response elements. Thyroid hormone disruption was induced from postnatal day 12 to 15 by adding goitrogens to dams' drinking water (hypothyroid). A subgroup of thyroid hormone-disrupted pups received intraperitoneal injections of replacement thyroid hormones four hours prior to sacrifice (replacement). An additional group received only thyroid hormones four hours prior to sacrifice (hyperthyroid). Hepatic mRNA was extracted and hybridized to Agilent mouse microarrays. Results Transcriptional profiling enabled the identification of 28 genes that appeared to be under direct thyroid hormone-regulation. The regulatory regions of the genome adjacent to these genes were examined for half-site sequences that resemble known thyroid response elements. A bioinformatics search identified 33 thyroid response elements in the promoter regions of 13 different genes thought to be directly regulated by thyroid hormones. Thyroid response elements found in the promoter regions of Tor1a, 2310003H01Rik, Hect3d and Slc25a45 were further validated by confirming that the thyroid receptor is associated with these sequences in vivo and that it can bind directly to these sequences in vitro. Three different arrangements of thyroid response elements were identified. Some of these thyroid response elements were located far up-stream (> 7 kb) of the transcription start site of the regulated gene. Conclusions Transcriptional profiling of thyroid hormone disrupted animals coupled with a novel bioinformatics search revealed new thyroid response elements associated with genes previously unknown to be responsive to thyroid hormone. The work provides insight into thyroid response element sequence motif characteristics.
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Affiliation(s)
- Martin A Paquette
- Environmental Health Sciences and Research Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, 50 Colombine Driveway, Ottawa, Ontario K1A 0K9, Canada
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O'Brien JM, Austin AJ, Williams A, Yauk CL, Crump D, Kennedy SW. Technical-grade perfluorooctane sulfonate alters the expression of more transcripts in cultured chicken embryonic hepatocytes than linear perfluorooctane sulfonate. ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY 2011; 30:2846-2859. [PMID: 21994020 DOI: 10.1002/etc.700] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/31/2023]
Abstract
Recently it was discovered that the perfluorooctane sulfonate (PFOS) detected in wildlife, such as fish-eating birds, had a greater proportion of linear PFOS (L-PFOS) than the manufactured technical product (T-PFOS), which contains linear and branched isomers. This suggests toxicological studies based on T-PFOS data may inaccurately assess exposure risk to wildlife. To determine whether PFOS effects were influenced by isomer content, we compared the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either L-PFOS or T-PFOS using Agilent microarrays. At equal concentrations (10 µM), T-PFOS altered the expression of more transcripts (340, >1.5-fold change, p < 0.05) compared with L-PFOS (130 transcripts). Higher concentrations of L-PFOS (40 µM) were also less transcriptionally disruptive (217 transcripts) than T-PFOS at 10 µM. Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, hepatic system development, and cellular growth and proliferation. Pathway and interactome analysis suggested that genes may be affected through the RXR receptor, oxidative stress response, TP53 signaling, MYC signaling, Wnt/β-catenin signaling, and PPARγ and SREBP receptors. In all functional categories and pathways examined, the response elicited by T-PFOS was greater than that of L-PFOS. These data show that T-PFOS elicits a greater transcriptional response in CEH than L-PFOS alone and demonstrates the importance of considering the isomer-specific toxicological properties of PFOS when assessing exposure risk.
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Affiliation(s)
- Jason M O'Brien
- Centre for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, Ottawa, Ontario, Canada.
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Jackson P, Halappanavar S, Hougaard KS, Williams A, Madsen AM, Lamson JS, Andersen O, Yauk C, Wallin H, Vogel U. Maternal inhalation of surface-coated nanosized titanium dioxide (UV-Titan) in C57BL/6 mice: effects in prenatally exposed offspring on hepatic DNA damage and gene expression. Nanotoxicology 2011; 7:85-96. [PMID: 22117692 DOI: 10.3109/17435390.2011.633715] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
We investigated effects of maternal pulmonary exposure to titanium dioxide (UV-Titan) on prenatally exposed offspring. Time-mated mice (C57BL/6BomTac) were inhalation exposed (1 h/day to 42 mg UV-Titan/m(3) aerosolised powder or filtered air) during gestation days (GDs) 8-18. We evaluated DNA strand breaks using the comet assay in bronchoalveolar lavage (BAL) cells and livers of the time-mated mice (5 and 26-27 days after inhalation exposure), and in livers of the offspring (post-natal days (PND) 2 and 22). We also analysed hepatic gene expression in newborns using DNA microarrays. UV-Titan exposure did not induce DNA strand breaks in time-mated mice or their offspring. Transcriptional profiling of newborn livers revealed changes in the gene expression related to the retinoic acid signalling pathway in the females, while gene expression in male offspring was unaffected. Changes may be a secondary response to maternal inflammation although no direct link was evident through gene expression analysis.
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Affiliation(s)
- Petra Jackson
- National Research Centre for the Working Environment, DK- 2100 Copenhagen Ø, Denmark
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40
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Reynier F, Petit F, Paye M, Turrel-Davin F, Imbert PE, Hot A, Mougin B, Miossec P. Importance of correlation between gene expression levels: application to the type I interferon signature in rheumatoid arthritis. PLoS One 2011; 6:e24828. [PMID: 22043277 PMCID: PMC3197194 DOI: 10.1371/journal.pone.0024828] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2011] [Accepted: 08/19/2011] [Indexed: 11/24/2022] Open
Abstract
Background The analysis of gene expression data shows that many genes display similarity in their expression profiles suggesting some co-regulation. Here, we investigated the co-expression patterns in gene expression data and proposed a correlation-based research method to stratify individuals. Methodology/Principal Findings Using blood from rheumatoid arthritis (RA) patients, we investigated the gene expression profiles from whole blood using Affymetrix microarray technology. Co-expressed genes were analyzed by a biclustering method, followed by gene ontology analysis of the relevant biclusters. Taking the type I interferon (IFN) pathway as an example, a classification algorithm was developed from the 102 RA patients and extended to 10 systemic lupus erythematosus (SLE) patients and 100 healthy volunteers to further characterize individuals. We developed a correlation-based algorithm referred to as Classification Algorithm Based on a Biological Signature (CABS), an alternative to other approaches focused specifically on the expression levels. This algorithm applied to the expression of 35 IFN-related genes showed that the IFN signature presented a heterogeneous expression between RA, SLE and healthy controls which could reflect the level of global IFN signature activation. Moreover, the monitoring of the IFN-related genes during the anti-TNF treatment identified changes in type I IFN gene activity induced in RA patients. Conclusions In conclusion, we have proposed an original method to analyze genes sharing an expression pattern and a biological function showing that the activation levels of a biological signature could be characterized by its overall state of correlation.
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Affiliation(s)
- Frédéric Reynier
- Joint Unit Hospices Civils de Lyon - bioMérieux, Hôpital Edouard Herriot, Lyon, France
| | - Fabien Petit
- Joint Unit Hospices Civils de Lyon - bioMérieux, Hôpital Edouard Herriot, Lyon, France
| | - Malick Paye
- Joint Unit Hospices Civils de Lyon - bioMérieux, Hôpital Edouard Herriot, Lyon, France
| | - Fanny Turrel-Davin
- Joint Unit Hospices Civils de Lyon - bioMérieux, Hôpital Edouard Herriot, Lyon, France
| | | | - Arnaud Hot
- Joint Unit Hospices Civils de Lyon - bioMérieux, Hôpital Edouard Herriot, Lyon, France
| | - Bruno Mougin
- Joint Unit Hospices Civils de Lyon - bioMérieux, Hôpital Edouard Herriot, Lyon, France
| | - Pierre Miossec
- Joint Unit Hospices Civils de Lyon - bioMérieux, Hôpital Edouard Herriot, Lyon, France
- Department of Clinical Immunology and Rheumatology, and immunogenomics and Inflammation Research Unit EA 4130, University of Lyon, Hôpital Edouard Herriot, Lyon, France
- * E-mail:
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Jackson P, Hougaard KS, Vogel U, Wu D, Casavant L, Williams A, Wade M, Yauk CL, Wallin H, Halappanavar S. Exposure of pregnant mice to carbon black by intratracheal instillation: toxicogenomic effects in dams and offspring. Mutat Res 2011; 745:73-83. [PMID: 22001195 DOI: 10.1016/j.mrgentox.2011.09.018] [Citation(s) in RCA: 84] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2011] [Accepted: 09/27/2011] [Indexed: 01/05/2023]
Abstract
Exposure to nanomaterials (NM) during sensitive developmental stages may predispose organisms to diseases later in life. However, direct translocation of NM from mother to fetus through the placenta is limited. The present study tests the hypothesis that pulmonary exposure to NM and NM-induced response, such as inflammation during gestation, leads to secondary effects in the fetus. Time-mated C57BL/6BomTac mice were exposed by intratracheal instillation to vehicle (Nanopure water) or one of three concentrations (2.75, 13.5 or 67 μg in 40 μl Nanopure water) of carbon black Printex 90 (CB) on gestational days 7, 10, 15 and 18, to final cumulative doses of 11, 54 or 268 μg/animal. Samples from a subset of male and female newborns were collected on postnatal day 2 (4 days after the last maternal exposure) and from dams 26 to 27 days post-exposure (post-weaning period). Histopathology, DNA microarrays, pathway-specific RT-PCR arrays, focussed RT-PCR, and tissue protein analysis were employed to characterize pulmonary response in dams exposed to CB during pregnancy. Hepatic gene expression in newborns was interpreted in light of the observed biological responses and gene expression changes arising in the lungs of dams following CB exposure. Although retention of CB particles was observed in dams from both the medium and the high dose groups, neutrophil-marked inflammation and altered expression of several cytokines and chemokines, both at the transcriptional and tissue protein levels, was significant only in the high dose group. Analysis of newborn livers by DNA microarrays revealed that female offspring were more sensitive to maternal exposure than male offspring. Cellular signalling, inflammation, cell cycle and lipid metabolism were among the biological pathways affected in female offspring. Males, however, responded with subtle changes in metabolism-related genes. Further investigation is required to determine the long-term health consequences of the gene expression changes in offspring and response to environmental stresses.
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Affiliation(s)
- Petra Jackson
- National Research Centre for the Working Environment, Copenhagen DK-2100, Denmark
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Row–column designs with minimal units. J Stat Plan Inference 2011. [DOI: 10.1016/j.jspi.2011.04.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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Halappanavar S, Jackson P, Williams A, Jensen KA, Hougaard KS, Vogel U, Yauk CL, Wallin H. Pulmonary response to surface-coated nanotitanium dioxide particles includes induction of acute phase response genes, inflammatory cascades, and changes in microRNAs: a toxicogenomic study. ENVIRONMENTAL AND MOLECULAR MUTAGENESIS 2011; 52:425-39. [PMID: 21259345 PMCID: PMC3210826 DOI: 10.1002/em.20639] [Citation(s) in RCA: 131] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/12/2010] [Revised: 10/12/2010] [Indexed: 05/17/2023]
Abstract
Titanium dioxide nanoparticles (nanoTiO(2) ) are used in various applications including in paints. NanoTiO(2) inhalation may induce pulmonary toxicity and systemic effects. However, the underlying molecular mechanisms are poorly understood. In this study, the effects of inhaled surface-coated nanoTiO(2) on pulmonary global messenger RNA (mRNA) and microRNA (miRNA) expression in mouse were characterized to provide insight into the molecular response. Female C57BL/6BomTac mice were exposed for 1 hr daily to 42.4 ± 2.9 (SEM) mg surface-coated nanoTiO(2) /m(3) for 11 consecutive days by inhalation and were sacrificed 5 days following the last exposure. Physicochemical properties of the particles were determined. Pulmonary response to nanoTiO(2) was characterized using DNA microarrays and pathway-specific PCR arrays and related to data on pulmonary inflammation from bronchial lavages. NanoTiO(2) exposure resulted in increased levels of mRNA for acute phase markers serum amyloid A-1 (Saa1) and serum amyloid A-3 (Saa3), several C-X-C and C-C motif chemokines, and cytokine tumor necrosis factor genes. Protein analysis of Saa1 and 3 showed selective upregulation of Saa3 in lung tissues. Sixteen miRNAs were induced by more than 1.2-fold (adjusted P-value < 0.05) following exposure. Real time polymerase chain reaction confirmed the upregulation of miR-1, miR-449a and revealed dramatic induction of miR-135b (60-fold). Thus, inhalation of surface-coated nanoTiO(2) results in changes in the expression of genes associated with acute phase, inflammation and immune response 5 days post exposure with concomitant changes in several miRNAs. The role of these miRNAs in pulmonary response to inhaled particles is unknown and warrants further research.
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Affiliation(s)
- Sabina Halappanavar
- Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, Canada.
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Halappanavar S, Wu D, Williams A, Kuo B, Godschalk RW, Van Schooten FJ, Yauk CL. Pulmonary gene and microRNA expression changes in mice exposed to benzo(a)pyrene by oral gavage. Toxicology 2011; 285:133-41. [DOI: 10.1016/j.tox.2011.04.011] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2011] [Revised: 04/07/2011] [Accepted: 04/20/2011] [Indexed: 12/14/2022]
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Brien CJ, Harch BD, Correll RL, Bailey RA. Multiphase Experiments with at Least One Later Laboratory Phase. I. Orthogonal Designs. JOURNAL OF AGRICULTURAL BIOLOGICAL AND ENVIRONMENTAL STATISTICS 2011. [DOI: 10.1007/s13253-011-0060-z] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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Yauk CL, Jackson K, Malowany M, Williams A. Lack of change in microRNA expression in adult mouse liver following treatment with benzo(a)pyrene despite robust mRNA transcriptional response. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2011; 722:131-9. [DOI: 10.1016/j.mrgentox.2010.02.012] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/15/2010] [Accepted: 02/20/2010] [Indexed: 12/12/2022]
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Assessment of subclinical, toxicant-induced hepatic gene expression profiles after low-dose, short-term exposures in mice. Regul Toxicol Pharmacol 2011; 60:54-72. [DOI: 10.1016/j.yrtph.2011.02.006] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2010] [Revised: 02/09/2011] [Accepted: 02/09/2011] [Indexed: 12/19/2022]
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Lorenz WW, Alba R, Yu YS, Bordeaux JM, Simões M, Dean JFD. Microarray analysis and scale-free gene networks identify candidate regulators in drought-stressed roots of loblolly pine (P. taeda L.). BMC Genomics 2011; 12:264. [PMID: 21609476 PMCID: PMC3123330 DOI: 10.1186/1471-2164-12-264] [Citation(s) in RCA: 78] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2010] [Accepted: 05/24/2011] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND Global transcriptional analysis of loblolly pine (Pinus taeda L.) is challenging due to limited molecular tools. PtGen2, a 26,496 feature cDNA microarray, was fabricated and used to assess drought-induced gene expression in loblolly pine propagule roots. Statistical analysis of differential expression and weighted gene correlation network analysis were used to identify drought-responsive genes and further characterize the molecular basis of drought tolerance in loblolly pine. RESULTS Microarrays were used to interrogate root cDNA populations obtained from 12 genotype × treatment combinations (four genotypes, three watering regimes). Comparison of drought-stressed roots with roots from the control treatment identified 2445 genes displaying at least a 1.5-fold expression difference (false discovery rate = 0.01). Genes commonly associated with drought response in pine and other plant species, as well as a number of abiotic and biotic stress-related genes, were up-regulated in drought-stressed roots. Only 76 genes were identified as differentially expressed in drought-recovered roots, indicating that the transcript population can return to the pre-drought state within 48 hours. Gene correlation analysis predicts a scale-free network topology and identifies eleven co-expression modules that ranged in size from 34 to 938 members. Network topological parameters identified a number of central nodes (hubs) including those with significant homology (E-values ≤ 2 × 10-30) to 9-cis-epoxycarotenoid dioxygenase, zeatin O-glucosyltransferase, and ABA-responsive protein. Identified hubs also include genes that have been associated previously with osmotic stress, phytohormones, enzymes that detoxify reactive oxygen species, and several genes of unknown function. CONCLUSION PtGen2 was used to evaluate transcriptome responses in loblolly pine and was leveraged to identify 2445 differentially expressed genes responding to severe drought stress in roots. Many of the genes identified are known to be up-regulated in response to osmotic stress in pine and other plant species and encode proteins involved in both signal transduction and stress tolerance. Gene expression levels returned to control values within a 48-hour recovery period in all but 76 transcripts. Correlation network analysis indicates a scale-free network topology for the pine root transcriptome and identifies central nodes that may serve as drivers of drought-responsive transcriptome dynamics in the roots of loblolly pine.
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Affiliation(s)
- W Walter Lorenz
- Warnell School of Forestry and Natural Resources, The University of Georgia, Athens, GA 30602, USA
| | - Rob Alba
- Monsanto Company, Mailstop C1N, 800 N. Lindbergh Blvd., St. Louis, MO 63167, USA
| | - Yuan-Sheng Yu
- Warnell School of Forestry and Natural Resources, The University of Georgia, Athens, GA 30602, USA
| | - John M Bordeaux
- Warnell School of Forestry and Natural Resources, The University of Georgia, Athens, GA 30602, USA
| | - Marta Simões
- Instituto de Biologia Experimental e Tecnológica (IBET)/Instituto de Tecnologia Química e Biológica-Universidade Nova de Lisboa (ITQB-UNL), Av. República (EAN) 2784-505 Oeiras, Portugal
| | - Jeffrey FD Dean
- Warnell School of Forestry and Natural Resources, The University of Georgia, Athens, GA 30602, USA
- Department of Biochemistry & Molecular Biology, The University of Georgia, Life Sciences Building, Athens, GA 30602, USA
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Wheat CW, Fescemyer HW, Kvist J, Tas E, Vera JC, Frilander MJ, Hanski I, Marden JH. Functional genomics of life history variation in a butterfly metapopulation. Mol Ecol 2011; 20:1813-28. [PMID: 21410806 DOI: 10.1111/j.1365-294x.2011.05062.x] [Citation(s) in RCA: 63] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/01/2022]
Abstract
In fragmented landscapes, small populations frequently go extinct and new ones are established with poorly understood consequences for genetic diversity and evolution of life history traits. Here, we apply functional genomic tools to an ecological model system, the well-studied metapopulation of the Glanville fritillary butterfly. We investigate how dispersal and colonization select upon existing genetic variation affecting life history traits by comparing common-garden reared 2-day adult females from new populations with those from established older populations. New-population females had higher expression of abdomen genes involved in egg provisioning and thorax genes involved in the maintenance of flight muscle proteins. Physiological studies confirmed that new-population butterflies have accelerated egg maturation, apparently regulated by higher juvenile hormone titer and angiotensin converting enzyme mRNA, as well as enhanced flight metabolism. Gene expression varied between allelic forms of two metabolic genes (Pgi and Sdhd), which themselves were associated with differences in flight metabolic rate, population age and population growth rate. These results identify likely molecular mechanisms underpinning life history variation that is maintained by extinction-colonization dynamics in metapopulations.
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Affiliation(s)
- Christopher W Wheat
- Department of Biology, 208 Mueller Lab, Pennsylvania State University, University Park, PA 16802, USA.
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Chen C, Grennan K, Badner J, Zhang D, Gershon E, Jin L, Liu C. Removing batch effects in analysis of expression microarray data: an evaluation of six batch adjustment methods. PLoS One 2011; 6:e17238. [PMID: 21386892 PMCID: PMC3046121 DOI: 10.1371/journal.pone.0017238] [Citation(s) in RCA: 338] [Impact Index Per Article: 24.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2010] [Accepted: 01/24/2011] [Indexed: 01/07/2023] Open
Abstract
The expression microarray is a frequently used approach to study gene expression on a genome-wide scale. However, the data produced by the thousands of microarray studies published annually are confounded by “batch effects,” the systematic error introduced when samples are processed in multiple batches. Although batch effects can be reduced by careful experimental design, they cannot be eliminated unless the whole study is done in a single batch. A number of programs are now available to adjust microarray data for batch effects prior to analysis. We systematically evaluated six of these programs using multiple measures of precision, accuracy and overall performance. ComBat, an Empirical Bayes method, outperformed the other five programs by most metrics. We also showed that it is essential to standardize expression data at the probe level when testing for correlation of expression profiles, due to a sizeable probe effect in microarray data that can inflate the correlation among replicates and unrelated samples.
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Affiliation(s)
- Chao Chen
- National Ministry of Education Key Laboratory of Contemporary Anthropology, Fudan University, Shanghai, People's Republic of China
- Department of Psychiatry, University of Chicago, Chicago, Illinois, United States of America
| | - Kay Grennan
- Department of Psychiatry, University of Chicago, Chicago, Illinois, United States of America
| | - Judith Badner
- Department of Psychiatry, University of Chicago, Chicago, Illinois, United States of America
| | - Dandan Zhang
- Department of Pathology, Zhejiang University, Hangzhou, People's Republic of China
| | - Elliot Gershon
- Department of Psychiatry, University of Chicago, Chicago, Illinois, United States of America
| | - Li Jin
- National Ministry of Education Key Laboratory of Contemporary Anthropology, Fudan University, Shanghai, People's Republic of China
| | - Chunyu Liu
- Department of Psychiatry, University of Chicago, Chicago, Illinois, United States of America
- * E-mail:
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