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1.  Genes related to emphysema are enriched for ubiquitination pathways 
BMC Pulmonary Medicine  2014;14(1):187.
Background
Increased small airway resistance and decreased lung elasticity contribute to the airflow limitation in chronic obstructive pulmonary disease (COPD). The lesion that corresponds to loss of lung elasticity is emphysema; the small airway obstruction is due to inflammatory narrowing and obliteration. Despite their convergence in altered physiology, different mechanisms contribute to these processes. The relationships between gene expression and these specific phenotypes may be more revealing than comparison with lung function.
Methods
We measured the ratio of alveolar surface area to lung volume (SA/V) in lung tissue from 43 smokers. Two samples from 21 subjects, in which SA/V differed by >49 cm2/mL were profiled to select genes whose expression correlated with SA/V. Significant genes were tested for replication in the 22 remaining subjects.
Results
The level of expression of 181 transcripts was related to SA/V ( p < 0.05). When these genes were tested in the 22 remaining subjects as a replication, thirty of the 181 genes remained significantly associated with SA/V (P < 0.05) and the direction of association was the same in 164/181. Pathway and network analysis revealed enrichment of genes involved in protein ubiquitination, and western blotting showed altered expression of genes involved in protein ubiquitination in obstructed individuals.
Conclusion
This study implicates modified protein ubiquitination and degradation as a potentially important pathway in the pathogenesis of emphysema.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2466-14-187) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2466-14-187
PMCID: PMC4280711  PMID: 25432663
Pulmonary emphysema; Surface area to lung volume ratio; Gene expression; Transcriptional analysis; mRNA; Cigarette smoking; Protein ubquitination
2.  Gene expression profiling following NRF2 and KEAP1 siRNA knockdown in human lung fibroblasts identifies CCL11/Eotaxin-1 as a novel NRF2 regulated gene 
Respiratory Research  2012;13(1):92.
Background
Oxidative Stress contributes to the pathogenesis of many diseases. The NRF2/KEAP1 axis is a key transcriptional regulator of the anti-oxidant response in cells. Nrf2 knockout mice have implicated this pathway in regulating inflammatory airway diseases such as asthma and COPD. To better understand the role the NRF2 pathway has on respiratory disease we have taken a novel approach to define NRF2 dependent gene expression in a relevant lung system.
Methods
Normal human lung fibroblasts were transfected with siRNA specific for NRF2 or KEAP1. Gene expression changes were measured at 30 and 48 hours using a custom Affymetrix Gene array. Changes in Eotaxin-1 gene expression and protein secretion were further measured under various inflammatory conditions with siRNAs and pharmacological tools.
Results
An anti-correlated gene set (inversely regulated by NRF2 and KEAP1 RNAi) that reflects specific NRF2 regulated genes was identified. Gene annotations show that NRF2-mediated oxidative stress response is the most significantly regulated pathway, followed by heme metabolism, metabolism of xenobiotics by Cytochrome P450 and O-glycan biosynthesis. Unexpectedly the key eosinophil chemokine Eotaxin-1/CCL11 was found to be up-regulated when NRF2 was inhibited and down-regulated when KEAP1 was inhibited. This transcriptional regulation leads to modulation of Eotaxin-1 secretion from human lung fibroblasts under basal and inflammatory conditions, and is specific to Eotaxin-1 as NRF2 or KEAP1 knockdown had no effect on the secretion of a set of other chemokines and cytokines. Furthermore, the known NRF2 small molecule activators CDDO and Sulphoraphane can also dose dependently inhibit Eotaxin-1 release from human lung fibroblasts.
Conclusions
These data uncover a previously unknown role for NRF2 in regulating Eotaxin-1 expression and further the mechanistic understanding of this pathway in modulating inflammatory lung disease.
doi:10.1186/1465-9921-13-92
PMCID: PMC3546844  PMID: 23061798
Asthma; NRF2; KEAP1; Oxidative stress; Eotaxin regulation; Microarray profiling
3.  Systems analysis of eleven rodent disease models reveals an inflammatome signature and key drivers 
A common inflammatome signature, as well as disease-specific expression patterns, was identified from 11 different rodent inflammatory disease models. Causal regulatory networks and the drivers of the inflammatome signature were uncovered and validated.
Representative inflammatome gene signatures, as well as disease model-specific gene signatures, were identified from 12 gene expression profiling data sets derived from 9 different tissues isolated from 11 rodent inflammatory disease models.The inflammatome signature is highly enriched for immune response-related genes, disease causal genes, and drug targets.Regulatory relationships among the inflammatome signature genes were examined in over 70 causal networks derived from a number of large-scale genetic studies of multiple diseases, and the potential key drivers were uncovered and validated prospectively.Over 70% of the inflammatome signature genes and over 50% of the key driver genes have not been reported in previous studies of common signatures in inflammatory conditions.
Common inflammatome gene signatures as well as disease-specific signatures were identified by analyzing 12 expression profiling data sets derived from 9 different tissues isolated from 11 rodent inflammatory disease models. The inflammatome signature significantly overlaps with known drug targets and co-expressed gene modules linked to metabolic disorders and cancer. A large proportion of genes in this signature are tightly connected in tissue-specific Bayesian networks (BNs) built from multiple independent mouse and human cohorts. Both the inflammatome signature and the corresponding consensus BNs are highly enriched for immune response-related genes supported as causal for adiposity, adipokine, diabetes, aortic lesion, bone, muscle, and cholesterol traits, suggesting the causal nature of the inflammatome for a variety of diseases. Integration of this inflammatome signature with the BNs uncovered 151 key drivers that appeared to be more biologically important than the non-drivers in terms of their impact on disease phenotypes. The identification of this inflammatome signature, its network architecture, and key drivers not only highlights the shared etiology but also pinpoints potential targets for intervention of various common diseases.
doi:10.1038/msb.2012.24
PMCID: PMC3421440  PMID: 22806142
Bayesian network; co-expression network; inflammatome; inflammatory diseases; key regulators
4.  Strategic Applications of Gene Expression: From Drug Discovery/Development to Bedside 
The AAPS Journal  2013;15(2):427-437.
ABSTRACT
Gene expression is useful for identifying the molecular signature of a disease and for correlating a pharmacodynamic marker with the dose-dependent cellular responses to exposure of a drug. Gene expression offers utility to guide drug discovery by illustrating engagement of the desired cellular pathways/networks, as well as avoidance of acting on the toxicological pathways. Successful employment of gene-expression signatures in the later stages of drug development depends on their linkage to clinically meaningful phenotypic characteristics and requires a biologically meaningful mechanism combined with a stringent statistical rigor. Much of the success in clinical drug development is hinged on predefining the signature genes for their fitness for purposes of application. Specific examples are highlighted to illustrate the breadth and depth of the potential utility of gene-expression signatures in drug discovery and clinical development to targeted therapeutics at the bedside.
doi:10.1208/s12248-012-9447-1
PMCID: PMC3675744  PMID: 23319288
clinical molecular signatures; molecular signatures of disease; signature genes; target engagement; toxicological pathways
5.  Chronic immune activation is a distinguishing feature of liver and PBMC gene signatures from HCV/HIV coinfected patients and may contribute to hepatic fibrogenesis 
Virology  2012;430(1):43-52.
Hepatitis C virus/human immunodeficiency virus (HCV/HIV) coinfected patients demonstrate accelerated progression to severe liver injury in comparison to HCV monoinfected patients, although the underlying mechanisms are unclear owing to infection of separate tissue compartments with two distinct viral pathogens. Microarray analysis of paired liver biopsy and peripheral blood mononuclear cell (PBMC) specimens from HCV/HIV coinfected and HCV monoinfected patients identified a gene expression signature associated with increased inflammation and immune activation that was present only in liver and PBMC samples from coinfected patients. We also identified in these samples liver- and PBMC-specific signatures enriched with fibrogenic/hepatic stellate activation and proinflammatory genes, respectively. Finally, Bayesian networks were constructed by assimilating these data with existing data from liver and PBMC samples from other cohorts, augmenting enrichment of biologically important pathways and further indicating that chronic immune activation in HCV/HIV coinfection may exacerbate liver disease progression in coinfected patients.
doi:10.1016/j.virol.2012.04.011
PMCID: PMC3371131  PMID: 22608059
hepatitis C virus; human immunodeficiency virus; HCV/HIV coinfection; liver; peripheral blood; hepatic fibrosis; systems biology; gene expression; pathogenesis
7.  Integrative Analysis of a Cross-Loci Regulation Network Identifies App as a Gene Regulating Insulin Secretion from Pancreatic Islets 
PLoS Genetics  2012;8(12):e1003107.
Complex diseases result from molecular changes induced by multiple genetic factors and the environment. To derive a systems view of how genetic loci interact in the context of tissue-specific molecular networks, we constructed an F2 intercross comprised of >500 mice from diabetes-resistant (B6) and diabetes-susceptible (BTBR) mouse strains made genetically obese by the Leptinob/ob mutation (Lepob). High-density genotypes, diabetes-related clinical traits, and whole-transcriptome expression profiling in five tissues (white adipose, liver, pancreatic islets, hypothalamus, and gastrocnemius muscle) were determined for all mice. We performed an integrative analysis to investigate the inter-relationship among genetic factors, expression traits, and plasma insulin, a hallmark diabetes trait. Among five tissues under study, there are extensive protein–protein interactions between genes responding to different loci in adipose and pancreatic islets that potentially jointly participated in the regulation of plasma insulin. We developed a novel ranking scheme based on cross-loci protein-protein network topology and gene expression to assess each gene's potential to regulate plasma insulin. Unique candidate genes were identified in adipose tissue and islets. In islets, the Alzheimer's gene App was identified as a top candidate regulator. Islets from 17-week-old, but not 10-week-old, App knockout mice showed increased insulin secretion in response to glucose or a membrane-permeant cAMP analog, in agreement with the predictions of the network model. Our result provides a novel hypothesis on the mechanism for the connection between two aging-related diseases: Alzheimer's disease and type 2 diabetes.
Author Summary
Alzheimer's disease and type 2 diabetes are two common aging-related diseases. Numerous studies have shown that the two diseases are associated. However, the mechanisms of such connection are not clear. Both diseases are complex diseases that are induced by multiple genetic factors and the environment. To understand the molecular network regulated by complex genetic factors causing type 2 diabetes, we constructed an F2 intercross comprised of >500 mice from diabetes-resistant and diabetic mouse strains. We measured genotypes, clinical traits, and expression profiling in five tissues for each mouse. We then performed an integrative analysis to investigate the inter-relationship among genetic factors, expression traits, and plasma insulin, a hallmark diabetes trait, and developed a novel method for inferring key regulators for regulating plasma insulin. In islets, the Alzheimer's gene App was identified as a top candidate regulator. Islets from 17-week-old, but not 10-week-old, App knockout mice showed increased insulin secretion in response to glucose, in agreement with the predictions of the network model. Our result provides a novel hypothesis on the mechanism for the connection between two aging-related diseases: Alzheimer's disease and type 2 diabetes.
doi:10.1371/journal.pgen.1003107
PMCID: PMC3516550  PMID: 23236292
8.  Transcriptional profiling of vaccine‐induced immune responses in humans and non‐human primates 
Microbial biotechnology  2012;5(2):177-187.
Summary
There is an urgent need for pre‐clinical and clinical biomarkers predictive of vaccine immunogenicity, efficacy and safety to reduce the risks and costs associated with vaccine development. Results emerging from immunoprofiling studies in non‐human primates and humans demonstrate clearly that (i) type and duration of immune memory are largely determined by the magnitude and complexity of the innate immune signals and (ii) genetic signatures highly predictive of B‐cell and T‐cell responses can be identified for specific vaccines. For vaccines with similar composition, e.g. live attenuated viral vaccines, these signatures share common patterns. Signatures predictive of vaccine efficacy have been identified in a few experimental challenge studies. This review aims to give an overview of the current literature on immunoprofiling studies in humans and also presents some of our own data on profiling of licensed and experimental vaccines in non‐human primates.
doi:10.1111/j.1751-7915.2011.00317.x
PMCID: PMC3380551  PMID: 22103427
9.  Microsomal Prostaglandin E Synthase-2 Is Not Essential For In Vivo Prostaglandin E2 Biosynthesis 
Prostaglandin E2 (PGE2) plays an important role in the normal physiology of many organ systems. Increased levels of this lipid mediator are associated with many disease states, and it potently regulates inflammatory responses. Three enzymes capable of in vitro synthesis of PGE2 from the cyclooxygenase metabolite PGH2 have been described. Here, we examine the contribution of one of these enzymes to PGE2 production, mPges-2, which encodes microsomal prostaglandin synthase-2 (mPGES-2), by generating mice homozygous for the null allele of this gene. Loss of mPges-2 expression did not result in a measurable decrease in PGE2 levels in any tissue or cell type examined from healthy mice. Taken together, analysis of the mPGES-2 deficient mouse lines does not substantiate the contention that mPGES-2 is a PGE2 synthase.
doi:10.1016/j.prostaglandins.2008.10.003
PMCID: PMC3182462  PMID: 19010439
Microsomal Prostaglandin E2 Synthase-2; Prostaglandin E2
10.  Identification and validation of genes affecting aortic lesions in mice 
The Journal of Clinical Investigation  2010;120(7):2414-2422.
Atherosclerosis represents the most significant risk factor for coronary artery disease (CAD), the leading cause of death in developed countries. To better understand the pathogenesis of atherosclerosis, we applied a likeli­hood-based model selection method to infer gene-disease causality relationships for the aortic lesion trait in a segregating mouse population demonstrating a spectrum of susceptibility to developing atherosclerotic lesions. We identified 292 genes that tested causal for aortic lesions from liver and adipose tissues of these mice, and we experimentally validated one of these candidate causal genes, complement component 3a receptor 1 (C3ar1), using a knockout mouse model. We also found that genes identified by this method overlapped with genes progressively regulated in the aortic arches of 2 mouse models of atherosclerosis during atherosclerotic lesion development. By comparing our gene set with findings from public human genome-wide association studies (GWAS) of CAD and related traits, we found that 5 genes identified by our study overlapped with published studies in humans in which they were identified as risk factors for multiple atherosclerosis-related pathologies, including myocardial infarction, serum uric acid levels, mean platelet volume, aortic root size, and heart failure. Candidate causal genes were also found to be enriched with CAD risk polymorphisms identified by the Wellcome Trust Case Control Consortium (WTCCC). Our findings therefore validate the ability of causality testing procedures to provide insights into the mechanisms underlying atherosclerosis development.
doi:10.1172/JCI42742
PMCID: PMC2898611  PMID: 20577049
11.  Obesity and genetics regulate microRNAs in islets, liver and adipose of diabetic mice 
Type 2 diabetes results from severe insulin resistance coupled with a failure of β-cells to compensate by secreting sufficient insulin. Multiple genetic loci are involved in the development of diabetes, although the effect of each gene on diabetes susceptibility is thought to be small. MicroRNAs (miRNA) are non-coding 19–22 nucleotide RNA molecules that potentially regulate the expression of thousands of genes. To understand the relationship between miRNA regulation and obesity-induced diabetes, we quantitatively profiled ~220 miRNAs in pancreatic islets, adipose tissue, and liver from diabetes-resistant (B6) and diabetes-susceptible (BTBR) mice. More than half of the miRNAs profiled were expressed in all 3 tissues, with many miRNAs in each tissue showing significant changes in response to genetic obesity. Further, several miRNAs in each tissue were differentially responsive to obesity in B6 versus BTBR mice, suggesting that they may be involved in the pathogenesis of diabetes. In liver, there were ~40 miRNAs that were down-regulated in response to obesity in B6, but not BTBR mice, indicating that genetic differences between the mouse strains play a critical role in miRNA regulation. In order to elucidate the genetic architecture of hepatic miRNA expression, we measured the expression of miRNAs in genetically obese F2 mice. Approximately 10% of the miRNAs measured showed significant linkage (miR-eQTLs), identifying loci that control miRNA abundance. Understanding the influence that obesity and genetics exert on the regulation of miRNA expression will reveal the role miRNAs play in the context of obesity-induced type 2 diabetes.
doi:10.1007/s00335-009-9217-2
PMCID: PMC2879069  PMID: 19727952
12.  Regulation of Apoptosis in Myeloid Cells by Interferon Consensus Sequence–Binding Protein 
Mice with a null mutation of the gene encoding interferon consensus sequence–binding protein (ICSBP) develop a disease with marked expansion of granulocytes and macrophages that frequently progresses to a fatal blast crisis, thus resembling human chronic myelogenous leukemia (CML). One important feature of CML is decreased responsiveness of myeloid cells to apoptotic stimuli. Here we show that myeloid cells from mice deficient in ICSBP exhibit reduced spontaneous apoptosis and a significant decrease in sensitivity to apoptosis induced by DNA damage. In contrast, apoptosis in thymocytes from ICSBP-deficient mice is unaffected. We also show that overexpression of ICSBP in the human U937 monocytic cell line enhances the rate of spontaneous apoptosis and the sensitivity to apoptosis induced by etoposide, lipopolysaccharide plus ATP, or rapamycin. Programmed cell death induced by etoposide was specifically blocked by peptides inhibitory for the caspase-1 or caspase-3 subfamilies of caspases. Studies of proapoptotic genes showed that cells overexpressing ICSBP have enhanced expression of caspase-3 precursor protein. In addition, analyses of antiapoptotic genes showed that overexpression of ICSBP results in decreased expression of Bcl-XL. These data suggest that ICSBP modulates survival of myeloid cells by regulating expression of apoptosis-related genes.
PMCID: PMC2195590  PMID: 10430629
apoptosis; caspase; chronic myelogenous leukemia; interferon; interferon consensus sequence–binding protein
13.  The Histone Acetylase PCAF Is a Phorbol-Ester-Inducible Coactivator of the IRF Family That Confers Enhanced Interferon Responsiveness 
Molecular and Cellular Biology  1999;19(3):1810-1820.
Transcription factors of the interferon regulatory factor (IRF) family bind to the type I interferon (IFN)-responsive element (ISRE) and activate transcription from IFN-inducible genes. To identify cofactors that associate with IRF proteins, DNA affinity binding assays were performed with nuclear extracts prepared from tissue culture cells. The results demonstrated that the endogenous IRFs bound to the ISRE are complexed with the histone acetylases, PCAF, GCN5, and p300/CREB binding protein and that histone acetylase activities are accumulated on the IRF-ISRE complexes. By testing recombinant proteins, we show that PCAF directly binds to some but not all members of the IRF family through distinct domains of the two proteins. This interaction was functionally significant, since transfection of PCAF strongly enhanced IRF-1- and IRF-2-dependent promoter activities. Further studies showed that expression of PCAF and other histone acetylases was markedly induced in U937 cells upon phorbol ester treatment, which led to increased recruitment of PCAF to the IRF-ISRE complexes. Coinciding with the induction of histone acetylases, phorbol ester markedly enhanced IFN-α-stimulated gene expression in U937 cells. Supporting the role for PCAF in conferring IFN responsiveness, transfection of PCAF into U937 cells led to a large increase in IFN-α-inducible promoter activity. These results demonstrate that PCAF is a phorbol ester-inducible coactivator of the IRF proteins which contributes to the establishment of type I IFN responsiveness.
PMCID: PMC83974  PMID: 10022868
14.  Transcriptional profiling of vaccine-induced immune responses in humans and non-human primates 
Microbial Biotechnology  2012;5(2):177-187.
There is an urgent need for pre-clinical and clinical biomarkers predictive of vaccine immunogenicity, efficacy and safety to reduce the risks and costs associated with vaccine development. Results emerging from immunoprofiling studies in non-human primates and humans demonstrate clearly that (i) type and duration of immune memory are largely determined by the magnitude and complexity of the innate immune signals and (ii) genetic signatures highly predictive of B-cell and T-cell responses can be identified for specific vaccines. For vaccines with similar composition, e.g. live attenuated viral vaccines, these signatures share common patterns. Signatures predictive of vaccine efficacy have been identified in a few experimental challenge studies. This review aims to give an overview of the current literature on immunoprofiling studies in humans and also presents some of our own data on profiling of licensed and experimental vaccines in non-human primates.
doi:10.1111/j.1751-7915.2011.00317.x
PMCID: PMC3380551  PMID: 22103427

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