| PMC Ids | Title | Abstract | Authors | Journal | Publication Date | Product Version |
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| pmc2238789 | Towards a pathway definition of Parkinson’s disease: a complex disorder with links to cancer, diabetes and inflammation | We have previously established a first whole genome transcriptomic profile of sporadic Parkinson’s disease (PD). After extensive brain tissue-based validation combined with cycles of iterative data analysis and by focusing on the most comparable cases of the cohort, we have refined our analysis and established a list of 892 highly dysregulated priority genes that are considered to form the core of the diseased Parkinsonian metabolic network. The substantia nigra pathways, now under scrutiny, contain more than 100 genes whose association with PD is known from the literature. Of those, more than 40 genes belong to the highly significantly dysregulated group identified in our dataset. Apart from the complete list of 892 priority genes, we present pathways revealing PD ‘hub’ as well as ‘peripheral’ network genes. The latter include Lewy body components or interact with known PD genes. Biological associations of PD with cancer, diabetes and inflammation are discussed and interactions of the priority genes with several drugs are provided. Our study illustrates the value of rigorous clinico-pathological correlation when analysing high-throughput data to make optimal use of the histopathological phenome, or morphonome which currently serves as the key diagnostic reference for most human diseases. The need for systematic human tissue banking, following the highest possible professional and ethical standard to enable sustainability, becomes evident. | [ Linda B.Moran, Manuel B.Graeber] | Neurogenetics | 2008-1-12 | 3.0 |
| pmc3266890 | Microarray Analysis of HIV Resistant Female Sex Workers Reveal a Gene Expression Signature Pattern Reminiscent of a Lowered Immune Activation State | To identify novel biomarkers for HIV-1 resistance, including pathways that may be critical in anti-HIV-1 vaccine design, we carried out a gene expression analysis on blood samples obtained from HIV-1 highly exposed seronegatives (HESN) from a commercial sex worker cohort in Nairobi and compared their profiles to HIV-1 negative controls. Whole blood samples were collected from 43 HIV-1 resistant sex workers and a similar number of controls. Total RNA was extracted and hybridized to the Affymetrix HUG 133 Plus 2.0 micro arrays (Affymetrix, Santa Clara CA). Output data was analysed through ArrayAssist software (Agilent, San Jose CA). More than 2,274 probe sets were differentially expressed in the HESN as compared to the control group (fold change ≥1.3; p value ≤0.0001, FDR <0.05). Unsupervised hierarchical clustering of the differentially expressed genes readily distinguished HESNs from controls. Pathway analysis through the KEGG signaling database revealed a majority of the impacted pathways (13 of 15, 87%) had genes that were significantly down regulated. The most down expressed pathways were glycolysis/gluconeogenesis, pentose phosphate, phosphatidyl inositol, natural killer cell cytotoxicity and T-cell receptor signaling. Ribosomal protein synthesis and tight junction genes were up regulated. We infer that the hallmark of HIV-1 resistance is down regulation of genes in key signaling pathways that HIV-1 depends on for infection. | [ Elijah M.Songok, MaLuo, BenLiang, PaulMclaren, NadineKaefer, WinnieApidi, GenevieveBoucher, JoshuaKimani, CharlesWachihi, RafickSekaly, KeithFowke, Blake T.Ball, Francis A.Plummer] | PLoS ONE | 2012-1-26 | |
| pmc2917033 | Cooperative signaling between Wnt1 and integrin-linked kinase induces accelerated breast tumor development | Breast cancer is genetically and clinically a heterogeneous disease. However, the exact contribution of different cell types and oncogenic mutations to this heterogeneity are not well understood. Recently, we discovered an interaction between Wnt and integrin-linked kinase (ILK) within the signaling cascade that regulates cell growth and survival. Interestingly, mammary-specific expression of either one of these proteins has been shown to promote mammary tumorigenesis. In light of our recent findings and to investigate the potential interaction between Wnt and ILK proteins during mammary tumor formation and progression, we established a transgenic mouse model that expresses both Wnt and ILK in mammary epithelial cells.MethodsA novel transgenic mouse model with mammary-specific expression of both Wnt1 and ILK was generated by crossing the two previously characterized mouse models, MMTV-Wnt1 and MMTV-ILK. The resulting MMTV-Wnt/ILK mice were closely monitored for tumor development and growth, as well as for the tumor onset. The molecular phenotypes of both tumors and premalignant mammary glands were investigated by using biochemical and global gene-expression analysis approaches.ResultsA significant acceleration in mammary tumor incidence and growth was observed in the MMTV-Wnt/ILK mice. Pre-neoplastic mammary glands also display lobuloalveolar hyperplasia and an increase in ductal epithelium proliferation. Apart from elevated expression of Wnt/ILK targets, such as β-catenin and cyclin D1, gene-expression profiling identified the surprising activation of the FOXA1 transcription factor. Upregulation of FOXA1, which is also known as the molecular marker of differentiated mammary luminal cells, was consistent with the expansion of the enriched luminal progenitor population or CD29loCD24hiCD61+ cells in MMTV-Wnt/ILK tumors.ConclusionsThese results show cooperation between Wnt1 and ILK transgenes during mammary carcinogenesis, leading to changes in a transcriptional network, which could dictate a specific breast cancer phenotype with enhanced growth dynamics. The MMTV-Wnt/ILK can be used as a model to identify further the genes downstream of the estrogen receptor-β/FOXA1 and to investigate the mechanisms targeting the expansion of the luminal progenitor cells leading to hyperplasia and tumorigenesis. | [ ArushaOloumi, MykolaMaidan, Frances ELock, HowardTearle, StevenMcKinney, William JMuller, Samuel AJRAparicio, ShoukatDedhar] | Breast Cancer Research : BCR | 2010-6-21 | |
| pmc2696328 | Effect of the Methoxychlor Metabolite HPTE on the Rat Ovarian Granulosa Cell Transcriptome In Vitro | Ovarian granulosa cells play a central role in steroidogenesis, which is critical for female reproduction. Follicle-stimulating hormone (FSH) promotes cyclic adenosine monophosphate (cAMP)-mediated signaling to regulate granulosa cell steroidogenesis. We have shown previously that 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) inhibits FSH- and dibutyryl cAMP-stimulated steroidogenesis and affects the messenger RNA levels of steroidogenic pathway enzymes in rat granulosa cells. However, HPTE showed a differential effect in FSH- and cAMP-stimulated cells in that HPTE more completely blocked FSH- when compared to cAMP-driven steroidogenesis. The objective of this study was to analyze the effects of HPTE on global gene expression profiles in untreated granulosa cells and those challenged with FSH or cAMP. Granulosa cells from immature rats were cultured with 0, 1, 5, or 10μM HPTE in the presence or absence of either 3 ng FSH/ml or 1mM cAMP for 48 h. Total RNA was isolated for real-time quantitative PCR and microarray analysis using the GeneChip Rat Genome 230 2.0 and ArrayAssist Microarray Suite. An investigation of changes in gene expression across all HPTE treatments showed that HPTE altered more genes in FSH- (∼670 genes) than in cAMP-stimulated cells (∼366 genes). Analysis confirmed that HPTE more effectively inhibited FSH- than cAMP-induced steroid pathway gene expression and steroidogenesis. Furthermore, expression patterns of novel genes regulating signal transduction, transport, cell cycle, adhesion, differentiation, motility and growth, apoptosis, development, and metabolism were all altered by HPTE. This study further established that HPTE exerts differential effects within the granulosa cell steroidogenic pathway and revealed that these effects include broader changes in gene expression. | [ Craig N.Harvey, MahmoudEsmail, QiWang, Andrew I.Brooks, RobZachow, MehmetUzumcu] | Toxicological Sciences | 2009-5-4 | |
| pmc1762020 | Heterogeneity of mammary lesions represent molecular differences | Human breast cancer is a heterogeneous disease, histopathologically, molecularly and phenotypically. The molecular basis of this heterogeneity is not well understood. We have used a mouse model of DCIS that consists of unique lines of mammary intraepithelial neoplasia (MIN) outgrowths, the premalignant lesion in the mouse that progress to invasive carcinoma, to understand the molecular changes that are characteristic to certain phenotypes. Each MIN-O line has distinguishable morphologies, metastatic potentials and estrogen dependencies.MethodsWe utilized oligonucleotide expression arrays and high resolution array comparative genomic hybridization (aCGH) to investigate whole genome expression patterns and whole genome aberrations in both the MIN-O and tumor from four different MIN-O lines that each have different phenotypes. From the whole genome analysis at 35 kb resolution, we found that chromosome 1, 2, 10, and 11 were frequently associated with whole chromosome gains in the MIN-Os. In particular, two MIN-O lines had the majority of the chromosome gains. Although we did not find any whole chromosome loss, we identified 3 recurring chromosome losses (2F1-2, 3E4, 17E2) and two chromosome copy number gains on chromosome 11. These interstitial deletions and duplications were verified with a custom made array designed to interrogate the specific regions at approximately 550 bp resolution.ResultsWe demonstrated that expression and genomic changes are present in the early premalignant lesions and that these molecular profiles can be correlated to phenotype (metastasis and estrogen responsiveness). We also identified expression changes associated with genomic instability. Progression to invasive carcinoma was associated with few additional changes in gene expression and genomic organization. Therefore, in the MIN-O mice, early premalignant lesions have the major molecular and genetic changes required and these changes have important phenotypic significance. In contrast, the changes that occur in the transition to invasive carcinoma are subtle, with few consistent changes and no association with phenotype.ConclusionWe propose that the early lesions carry the important genetic changes that reflect the major phenotypic information, while additional genetic changes that accumulate in the invasive carcinoma are less associated with the overall phenotype. | [ RuriaNamba, Jeannie EMaglione, Ryan RDavis, Colin ABaron, StephenieLiu, Condie ECarmack, Lawrence JTYoung, Alexander DBorowsky, Robert DCardiff, Jeffrey PGregg] | BMC Cancer | 2006-12-5 | |
| pmc2798864 | Comprehensive genomic screens identify a role for PLZF-RARα as a positive regulator of cell proliferation via direct regulation of c-MYC | The t(11;17)(q23;q21) translocation is associated with a retinoic acid (RA)–insensitive form of acute promyelocytic leukemia (APL), involving the production of reciprocal fusion proteins, promyelocytic leukemia zinc finger–retinoic acid receptor α (PLZF-RARα) and RARα-PLZF. Using a combination of chromatin immunoprecipitation promotor arrays (ChIP-chip) and gene expression profiling, we identify novel, direct target genes of PLZF-RARα that tend to be repressed in APL compared with other myeloid leukemias, supporting the role of PLZF-RARα as an aberrant repressor in APL. In primary murine hematopoietic progenitors, PLZF-RARα promotes cell growth, and represses Dusp6 and Cdkn2d, while inducing c-Myc expression, consistent with its role in leukemogenesis. PLZF-RARα binds to a region of the c-MYC promoter overlapping a functional PLZF site and antagonizes PLZF-mediated repression, suggesting that PLZF-RARα may act as a dominant-negative version of PLZF by affecting the regulation of shared targets. RA induced the differentiation of PLZF-RARα–transformed murine hematopoietic cells and reduced the frequency of clonogenic progenitors, concomitant with c-Myc down-regulation. Surviving RA-treated cells retained the ability to be replated and this was associated with sustained c-Myc expression and repression of Dusp6, suggesting a role for these genes in maintaining a self-renewal pathway triggered by PLZF-RARα. | [ Kim L.Rice, ItsasoHormaeche, SergeiDoulatov, Jared M.Flatow, DavidGrimwade, Ken I.Mills, MagdalenaLeiva, JulienAblain, CharutaAmbardekar, Melanie J.McConnell, John E.Dick, Jonathan D.Licht] | Blood | | |
| pmc2697450 | Dose-dependent transitions in Nrf2-mediated adaptive response and related stress responses to hypochlorous acid in mouse macrophages | Hypochlorous acid (HOCl) is potentially an important source of cellular oxidative stress. Human HOCl exposure can occur from chlorine gas inhalation or from endogenous sources of HOCl, such as respiratory burst by phagocytes. Transcription factor Nrf2 is a key regulator of cellular redox status and serves as a primary source of defense against oxidative stress. We recently demonstrated that HOCl activates Nrf2-mediated antioxidant response in cultured mouse macrophages in a biphasic manner. In an effort to determine whether Nrf2 pathways overlap with other stress pathways, gene expression profiling was performed in RAW 264.7 macrophages exposed to HOCl using whole genome mouse microarrays. Benchmark dose (BMD) analysis on gene expression data revealed that Nrf2-mediated antioxidant response and protein ubiquitination were the most sensitive biological pathways that were activated in response to low concentrations of HOCl (< 0.35 mM). Genes involved in chromatin architecture maintenance and DNA-dependent transcription were also sensitive to very low doses. Moderate concentrations of HOCl (0.35 to 1.4 mM) caused maximal activation of the Nrf2-pathway and innate immune response genes, such as IL-1β, IL-6, IL-10 and chemokines. At even higher concentrations of HOCl (2.8 to 3.5 mM) there was a loss of Nrf2-target gene expression with increased expression of numerous heat shock and histone cluster genes, AP-1-family genes, cFos and Fra1 and DNA damage-inducible Gadd45 genes. These findings confirm an Nrf2-centric mechanism of action of HOCl in mouse macrophages and provide evidence of interactions between Nrf2, inflammatory, and other stress pathways. | [ Courtney G.Woods, JingqiFu, PengXue, YongyongHou, Linda J.Pluta, LonglongYang, QiangZhang, Russell S.Thomas, Melvin E.Andersen, JingboPi] | Toxicology and applied pharmacology | 2009-4-17 | |
| pmc2725770 | Constitutively Active Protein Kinase A Qualitatively Mimics the Effects of Follicle-Stimulating Hormone on Granulosa Cell Differentiation | Activation of the protein kinase A (PKA) signaling system is necessary for FSH-induced granulosa cell differentiation, but it is not known whether activation of PKA is sufficient to account for the complex pattern of gene expression that occurs during this process. We addressed this question by infecting granulosa cells with a lentiviral vector that directs the expression of a constitutively active mutant of PKA (PKA-CQR) and compared the cellular responses to PKA-CQR with cells stimulated by FSH. Expression of PKA-CQR in undifferentiated granulosa cells resulted in the induction of both estrogen and progesterone production in the absence of cAMP. The stimulatory effects of both PKA-CQR and FSH on estrogen and progesterone production were suppressed by the PKA inhibitor H-89 and were mimicked by PKA-selective cAMP agonists. mRNA levels for P450scc and 3β-HSD were induced to a similar extent by FSH and PKA-CQR, whereas mRNA levels for P450arom and the LHr were induced to a greater extent by FSH. Microarray analysis of gene expression profiles revealed that the majority of genes appeared to be comparably regulated by FSH and PKA-CQR but that some genes appear to be induced to a greater extent by FSH than by PKA-CQR. These results indicate that the PKA signaling pathway is sufficient to account for the induction of most genes (as identified by microarray analysis), including those of the progesterone biosynthetic pathway during granulosa cell differentiation. However, optimal induction of aromatase, the LHr, and other genes by FSH appears to require activation of additional signaling pathways. | [ RosalbaEscamilla-Hernandez, LyndaLittle-Ihrig, Kyle E.Orwig, JunmingYue, UmaChandran, Anthony J.Zeleznik] | Molecular Endocrinology | 2008-6-5 | |
| pmc2974173 | GENE EXPRESSION PROFILING AND DIFFERENTIATION ASSESSMENT IN PRIMARY HUMAN HEPATOCYTE CULTURES, ESTABLISHED HEPATOMA CELL LINES, AND HUMAN LIVER TISSUES | Frequently, primary hepatocytes are used as an in vitro model for the liver in vivo. However, the culture conditions reported vary considerably, with associated variability in performance. In this study, we characterized the differentiation character of primary human hepatocytes cultured using a highly defined, serum-free two-dimensional sandwich system, one that configures hepatocytes with collagen I as the substratum together with a dilute extracellular matrix (Matrigel) overlay combined with a defined serum-free medium containing nanomolar levels of dexamethasone. Gap junctional communication, indicated by immunochemical detection of connexin 32 protein, was markedly enhanced in hepatocytes cultured in the Matrigel sandwich configuration. Whole genome expression profiling enabled direct comparison of liver tissues to hepatocytes and to the hepatoma-derived cell lines, HepG2 and Huh7. PANTHER database analyses were used to identify biological processes that were comparatively overrepresented among probe sets expressed in the in vitro systems. The robustness of the primary hepatocyte cultures was reflected by the extent of unchanged expression character when compared directly to liver, with more than 77% of the probe sets unchanged in all overrepresented categories, representing such genes as C/EBPα, HNF4α, CYP2D6, and ABCB1. In contrast, HepG2 and Huh7 cells were unchanged from the liver tissues for fewer than 48% and 55% of these probe sets, respectively. Further, hierarchical clustering of the hepatocytes, but not the cell lines, shifted from donor-specific to treatment-specific when the probe sets were filtered to focus on phenobarbital-inducible genes, indicative of the highly differentiated nature of the hepatocytes when cultured in a highly defined 2-dimensional sandwich system. | [ Katy M.Olsavsky, Jeanine L.Page, Mary C.Johnson, HelmutZarbl, Stephen C.Strom, Curtis J.Omiecinski] | Toxicology and applied pharmacology | 2007-4-21 | |
| pmc1913576 | Attributing Effects of Aqueous C60 Nano-Aggregates to Tetrahydrofuran Decomposition Products in Larval Zebrafish by Assessment of Gene Expression | C60 is a highly insoluble nanoparticle that can form colloidal suspended aggregates in water, which may lead to environmental exposure in aquatic organisms. Previous research has indicated toxicity from C60 aggregate; however, effects could be because of tetrahydrofuran (THF) vehicle used to prepare aggregates.ObjectiveOur goal was to investigate changes in survival and gene expression in larval zebrafish Danio rerio after exposure to aggregates of C60 prepared by two methods: a) stirring and sonication of C60 in water (C60–water); and b) suspension of C60 in THF followed by rotovaping, resuspension in water, and sparging with nitrogen gas (THF–C60).ResultsSurvival of larval zebrafish was reduced in THF–C60 and THF–water but not in C60–water. The greatest differences in gene expression were observed in fish exposed to THF–C60 and most (182) of these genes were similarly expressed in fish exposed to THF–water. Significant up-regulation (3- to 7-fold) of genes involved in controlling oxidative damage was observed after exposure to THF–C60 and THF–water. Analyses of THF–C60 and THF–water by gas chromatography–mass spectrometry did not detect THF but found THF oxidation products γ-butyrolactone and tetrahydro-2-furanol. Toxicity of γ-butyrolactone (72-hr lethal concentration predicted to kill 50% was 47 ppm) indicated effects in THF treatments can result from γ-butyrolactone toxicity.ConclusionThis research is the first to link toxic effects directly to a THF degradation product (γ-butyrolactone) rather than to C60 and may explain toxicity attributed to C60 in other investigations. The present work was first presented at the meeting “Overcoming Obstacles to Effective Research Design in Nanotoxicology” held 24–26 April 2006 in Cambridge, Massachusetts, USA. | [ Theodore B.Henry, Fu-MinMenn, James T.Fleming, JohnWilgus, Robert N.Compton, Gary S.Sayler] | Environmental Health Perspectives | 2007-2-21 | |
| pmc2648076 | The Arabidopsis MYB5 Transcription Factor Regulates Mucilage Synthesis, Seed Coat Development, and Trichome Morphogenesis[W][OA] | The Arabidopsis thaliana MYB5 gene is expressed in trichomes and seeds, including the seed coat. Constitutive expression of MYB5 resulted in the formation of more small trichomes and ectopic trichomes and a reduction in total leaf trichome numbers and branching. A myb5 mutant displayed minimal changes in trichome morphology, while a myb23 mutant produced increased numbers of small trichomes and two-branched trichomes. A myb5 myb23 double mutant developed more small rosette trichomes and two-branched trichomes than the single mutants. These results indicate that MYB5 and MYB23 regulate trichome extension and branching. The seed coat epidermal cells of myb5 and myb5 myb23 were irregular in shape, developed flattened columellae, and produced less mucilage than those of the wild type. Among the downregulated genes identified in the myb5 seeds using microarray analysis were ABE1 and ABE4 (α/β fold hydrolase/esterase genes), MYBL2, and GLABRA2. The same genes were also downregulated in transparent testa glabra1 (ttg1) seeds, suggesting that MYB5 collaborates with TTG1 in seed coat development. These genes were upregulated in leaves and roots by ectopically expressed MYB5. The MYBL2, ABE1, and ABE4 promoters were active in seeds, including seed coats, and the latter two also in trichomes. Models of the MYB5 regulatory networks involved in seed coat and trichome development are presented. | [ Song FengLi, Olga NicolaouMilliken, HanhPham, RegSeyit, RossNapoli, JeremyPreston, Anna M.Koltunow, Roger W.Parish] | The Plant Cell | | |
| pmc2576257 | Genome-wide identification, organization and phylogenetic analysis of Dicer-like, Argonaute and RNA-dependent RNA Polymerase gene families and their expression analysis during reproductive development and stress in rice | Important developmental processes in both plants and animals are partly regulated by genes whose expression is modulated at the post-transcriptional level by processes such as RNA interference (RNAi). Dicers, Argonautes and RNA-dependent RNA polymerases (RDR) form the core components that facilitate gene silencing and have been implicated in the initiation and maintenance of the trigger RNA molecules, central to process of RNAi. Investigations in eukaryotes have revealed that these proteins are encoded by variable number of genes with plants showing relatively higher number in each gene family. To date, no systematic expression profiling of these genes in any of the organisms has been reported.ResultsIn this study, we provide a complete analysis of rice Dicer-like, Argonaute and RDR gene families including gene structure, genomic localization and phylogenetic relatedness among gene family members. We also present microarray-based expression profiling of these genes during 14 stages of reproductive and 5 stages of vegetative development and in response to cold, salt and dehydration stress. We have identified 8 Dicer-like (OsDCLs), 19 Argonaute (OsAGOs) and 5 RNA-dependent RNA polymerase (OsRDRs) genes in rice. Based on phylogeny, each of these genes families have been categorized into four subgroups. Although most of the genes express both in vegetative and reproductive organs, 2 OsDCLs, 14 OsAGOs and 3 OsRDRs were found to express specifically/preferentially during stages of reproductive development. Of these, 2 OsAGOs exhibited preferential up-regulation in seeds. One of the Argonautes (OsAGO2) also showed specific up-regulation in response to cold, salt and dehydration stress.ConclusionThis investigation has identified 23 rice genes belonging to DCL, Argonaute and RDR gene families that could potentially be involved in reproductive development-specific gene regulatory mechanisms. These data provide an insight into probable domains of activity of these genes and a basis for further, more detailed investigations aimed at understanding the contribution of individual components of RNA silencing machinery during reproductive phase of plant development. | [ MeenuKapoor, RitaArora, TenishaLama, AashimaNijhawan, Jitendra PKhurana, Akhilesh KTyagi, SanjayKapoor] | BMC Genomics | 2008-10-1 | |
| pmc3164642 | Significant Effects of Antiretroviral Therapy on Global Gene Expression in Brain Tissues of Patients with HIV-1-Associated Neurocognitive Disorders | Antiretroviral therapy (ART) has reduced morbidity and mortality in HIV-1 infection; however HIV-1-associated neurocognitive disorders (HAND) persist despite treatment. The reasons for the limited efficacy of ART in the brain are unknown. Here we used functional genomics to determine ART effectiveness in the brain and to identify molecular signatures of HAND under ART. We performed genome-wide microarray analysis using Affymetrix U133 Plus 2.0 Arrays, real-time PCR, and immunohistochemistry in brain tissues from seven treated and eight untreated HAND patients and six uninfected controls. We also determined brain virus burdens by real-time PCR. Treated and untreated HAND brains had distinct gene expression profiles with ART transcriptomes clustering with HIV-1-negative controls. The molecular disease profile of untreated HAND showed dysregulated expression of 1470 genes at p<0.05, with activation of antiviral and immune responses and suppression of synaptic transmission and neurogenesis. The overall brain transcriptome changes in these patients were independent of histological manifestation of HIV-1 encephalitis and brain virus burdens. Depending on treatment compliance, brain transcriptomes from patients on ART had 83% to 93% fewer dysregulated genes and significantly lower dysregulation of biological pathways compared to untreated patients, with particular improvement indicated for nervous system functions. However a core of about 100 genes remained similarly dysregulated in both treated and untreated patient brain tissues. These genes participate in adaptive immune responses, and in interferon, cell cycle, and myelin pathways. Fluctuations of cellular gene expression in the brain correlated in Pearson's formula analysis with plasma but not brain virus burden. Our results define for the first time an aberrant genome-wide brain transcriptome of untreated HAND and they suggest that antiretroviral treatment can be broadly effective in reducing pathophysiological changes in the brain associated with HAND. Aberrantly expressed transcripts common to untreated and treated HAND may contribute to neurocognitive changes defying ART. | [ AlejandraBorjabad, SusanMorgello, WeiChao, Seon-YoungKim, Andrew I.Brooks, JacintaMurray, Mary JanePotash, David J.Volsky] | PLoS Pathogens | 2011-9-1 | |
| pmc2810986 | Comparison of gene expression profiles between human and mouse monocyte subsets | Blood of both humans and mice contains 2 main monocyte subsets. Here, we investigated the extent of their similarity using a microarray approach. Approximately 270 genes in humans and 550 genes in mice were differentially expressed between subsets by 2-fold or more. More than 130 of these gene expression differences were conserved between mouse and human monocyte subsets. We confirmed numerous of these differences at the cell surface protein level. Despite overall conservation, some molecules were conversely expressed between the 2 species' subsets, including CD36, CD9, and TREM-1. Other differences included a prominent peroxisome proliferator-activated receptor γ (PPARγ) signature in mouse monocytes, which is absent in humans, and strikingly opposed patterns of receptors involved in uptake of apoptotic cells and other phagocytic cargo between human and mouse monocyte subsets. Thus, whereas human and mouse monocyte subsets are far more broadly conserved than currently recognized, important differences between the species deserve consideration when models of human disease are studied in mice. | [ Molly A.Ingersoll, RainerSpanbroek, ClaudioLottaz, Emmanuel L.Gautier, MarionFrankenberger, ReinhardHoffmann, RolandLang, MuzlifahHaniffa, MatthewCollin, FrankTacke, Andreas J. R.Habenicht, LoemsZiegler-Heitbrock, Gwendalyn J.Randolph] | Blood | | |
| pmc2754828 | EMT tumorigenesis in the mouse mammary gland | The term EMT (epithelial–mesenchymal transition) is used in many settings. This term is used to describe the mechanisms facilitating cellular repositioning and redeployment during embryonic development and tissue reconstruction after injury. Recently, EMT has also been applied to potential mechanisms for malignant progression and has appeared as a specific diagnostic category of tumors. In mice, most ‘EMT’ tumors have a spindle cell phenotype. The definition of EMT is controversial because spindle cell tumors are not common in humans, especially in human breast cancers. Spindle cell tumors of the mouse mammary gland have been observed for many years where they are usually classified as sarcomas or carcinosarcomas. Genetically engineered mice develop mammary spindle cell tumors that appear to arise in the epithelium and undergo EMT. To better understand the origin and evolution of these spindle cell tumors in progression and metastases, seven cohorts of spindle cell tumors from the archives of the University of California, Davis Mutant Mouse Pathology Laboratory were studied. This study provides experimental and immunohistochemical evidence of EMT showing that dual epithelial and mesenchymal staining of tumor spindle cells identifies some, but not all, EMT-type tumors in the mouse. This suggests that potential EMT tumors are best designated EMT-phenotype tumors. | [ PatriziaDamonte, Jeffrey PGregg, Alexander DBorowsky, Blaine AKeister, Robert DCardiff] | Laboratory investigation; a journal of technical methods and pathology | 2007-10-8 | |
| pmc3107560 | Gene Expression Profiles of HIV-1-Infected Glia and Brain: Toward Better Understanding of the Role of Astrocytes in HIV-1-Associated Neurocognitive Disorders | Astrocytes are the major cellular component of the central nervous system (CNS), and they play multiple roles in brain development, normal brain function, and CNS responses to pathogens and injury. The functional versatility of astrocytes is linked to their ability to respond to a wide array of biological stimuli through finely orchestrated changes in cellular gene expression. Dysregulation of gene expression programs, generally by chronic exposure to pathogenic stimuli, may lead to dysfunction of astrocytes and contribute to neuropathogenesis. Here, we review studies that employ functional genomics to characterize the effects of HIV-1 and viral pathogenic proteins on cellular gene expression in astrocytes in vitro. We also present the first microarray analysis of primary mouse astrocytes exposed to HIV-1 in culture. In spite of different experimental conditions and microarray platforms used, comparison of the astrocyte array data sets reveals several common gene-regulatory changes that may underlie responses of these cells to HIV-1 and its proteins. We also compared the transcriptional profiles of astrocytes with those obtained in analyses of brain tissues of patients with HIV-1 dementia and macaques infected with simian immunodeficiency virus (SIV). Notably, many of the gene characteristics of responses to HIV-1 in cultured astrocytes were also altered in HIV-1 or SIV-infected brains. Functional genomics, in conjunction with other approaches, may help clarify the role of astrocytes in HIV-1 neuropathogenesis. | [ AlejandraBorjabad, Andrew I.Brooks, David J.Volsky] | Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology | 2009-8-21 | 5.5.1 |
| pmc2685508 | Characterization of a preclinical model of chronic ischemic wound | Chronic ischemic wounds presenting at wound clinics are heterogeneous with respect to etiology, age of the wound, and other factors complicating wound healing. In addition, there are ethical challenges associated with collecting repeated biopsies from a patient to develop an understanding of the temporal dynamics of the mechanisms underlying chronic wounds. The need for a preclinical model of ischemic wound is therefore compelling. The porcine model is widely accepted as an excellent preclinical model for human wounds. A full-thickness bipedicle flap approach was adopted to cause skin ischemia. Closure of excisional wounds placed on ischemic tissue was severely impaired resulting in chronic wounds. Histologically, ischemic wounds suffered from impaired re-epithelialization, delayed macrophage recruitment and poorer endothelial cell abundance and organization. Compared with the pair-matched nonischemic wound, unique aspects of the ischemic wound biology were examined on days 3, 7, 14, and 28 by systematic screening of the wound tissue transcriptome using high-density porcine GeneChips. Ischemia markedly potentiated the expression of arginase-1, a cytosolic enzyme that metabolizes the precursor of nitric oxide l-arginine. Ischemia also induced the SOD2 in the wound tissue perhaps as survival response of the challenged tissue. Human chronic wounds also demonstrated elevated expression of SOD2 and arginase-1. This study provides a thorough database that may serve as a valuable reference tool to develop novel hypotheses aiming to elucidate the biology of ischemic chronic wounds in a preclinical setting. | [ SashwatiRoy, SabyasachiBiswas, SavitaKhanna, GayleGordillo, ValerieBergdall, JeanneGreen, Clay B.Marsh, Lisa J.Gould, Chandan K.Sen] | Physiological Genomics | 2009-3-17 | 5.1 |
| pmc2494843 | Characterization of the acute temporal changes in excisional murine cutaneous wound inflammation by screening of the wound-edge transcriptome | This work represents a maiden effort to systematically screen the transcriptome of the healing wound-edge tissue temporally using high-density GeneChips. Changes during the acute inflammatory phase of murine excisional wounds were characterized histologically. Sets of genes that significantly changed in expression during healing could be segregated into the following five sets: up-early (6–24 h; cytokine-cytokine receptor interaction pathway), up-intermediary (12–96 h; leukocyte-endothelial interaction pathway), up-late (48–96 h; cell-cycle pathway), down-early (6–12 h; purine metabolism) and down-intermediary (12–96 h; oxidative phosphorylation pathway). Results from microarray and real-time PCR analyses were consistent. Results listing all genes that were significantly changed at any specific time point were further mined for cell-type (neutrophils, macrophages, endothelial, fibroblasts, and pluripotent stem cells) specificity. Candidate genes were also clustered on the basis of their functional annotation, linking them to inflammation, angiogenesis, reactive oxygen species (ROS), or extracellular matrix (ECM) categories. Rapid induction of genes encoding NADPH oxidase subunits and downregulation of catalase in response to wounding is consistent with the fact that low levels of endogenous H2O2 is required for wound healing. Angiogenic genes, previously not connected to cutaneous wound healing, that were induced in the healing wound-edge included adiponectin, epiregulin, angiomotin, Nogo, and VEGF-B. This study provides a digested database that may serve as a valuable reference tool to develop novel hypotheses aiming to elucidate the biology of cutaneous wound healing comprehensively. | [ SashwatiRoy, SavitaKhanna, CameronRink, SabyasachiBiswas, Chandan K.Sen] | Physiological Genomics | 2008-5-6 | 5.1 |
| pmc2270353 | Neuronal pentraxin II is highly upregulated in Parkinson’s disease and a novel component of Lewy bodies | Neuronal pentraxin II (NPTX2) is the most highly upregulated gene in the Parkinsonian substantia nigra based on our whole genome expression profiling results. We show here that it is a novel component of Lewy bodies and Lewy neurites in sporadic Parkinson’s disease (PD). NPTX2 is also known as the neuronal activity-regulated protein (Narp), which is secreted and involved in long-term neuronal plasticity. Narp further regulates AMPA receptors which have been found to mediate highly selective non-apoptotic cell death of dopaminergic neurons. NPTX2/Narp is found in close association with alpha-synuclein aggregates in both substantia nigra and cerebral cortex in PD but unlike alpha-synuclein gene expression, which is down-regulated in the Parkinsonian nigra, NPTX2 could represent a driver of the disease process. In view of its profound (>800%) upregulation and its established role in synaptic plasticity as well as dopaminergic nerve cell death, NPTX2 is a very interesting novel player which is likely to be involved in the pathway dysregulation which underlies PD.The online version of this article (doi:10.1007/s00401-007-0309-3) contains supplementary material, which is available to authorized users. | [ Linda B.Moran, LorraineHickey, Gregory J.Michael, MariaDerkacs, Lynne M.Christian, Michail E.Kalaitzakis, Ronald K. B.Pearce, Manuel B.Graeber] | Acta Neuropathologica | 2007-11-7 | 5.5 |
| pmc2972244 | Sp1 acetylation is associated with loss of DNA binding at promoters associated with cell cycle arrest and cell death in a colon cell line | Butyrate, a known histone deacetylase inhibitor (HDACi) and product of fibre fermentation, is postulated to mediate the protective effect of dietary fibre against colon cancer. The transcription factor Sp1 is a target of acetylation and is known to be associated with class I HDACs, including HDAC1. Sp1 is a ubiquitous transcription factor and Sp1-regulated genes include those involved in cell cycle regulation, apoptosis and lipogenesis: all major pathways in cancer development. The only known acetylated residue of Sp1 is lysine703 which resides in the DNA binding domain. Here we show that acetylated Sp1 loses p21- and bak-promoter -binding function in vitro. Furthermore treatment with a panel of HDAC inhibitors showed clustering of activities for a subset of inhibitors, causing G2 cell cycle arrest, Sp1 acetylation, p21 and Bak over-expression, all with very similar EC50 concentrations. These HDACi activities were not distributed according to the molecular class of compound. In order to mimic loss of binding, an siRNA strategy was used to reduce Sp1 expression. This resulted in altered expression of multiple elements of the p53/p21 pathway. Taken together our data suggest a mechanistic model for the chemopreventive actions of butyrate in colon epithelial cells, and provide new insight into the differential activities some classes of HDAC inhibitors. | [ Jennifer SWaby, HaridasanChirakkal, ChenWeiYu, Gareth JGriffiths, Roderick SPBenson, Colin DBingle, Bernard MCorfe] | Molecular Cancer | 2010-10-15 | |
| pmc2817434 | The Evolution of Reduced Microbial Killing | Bacteria engage in a never-ending arms race in which they compete for limited resources and niche space. The outcome of this intense interaction is the evolution of a powerful arsenal of biological weapons. Perhaps the most studied of these are colicins, plasmid-based toxins produced by and active against Escherichia coli. The present study was designed to explore the molecular responses of a colicin-producing strain during serial transfer evolution. What evolutionary changes occur when colicins are produced with no target present? Can killing ability be maintained in the absence of a target? To address these, and other, questions, colicinogenic strains and a noncolicinogenic ancestor were evolved for 253 generations. Samples were taken throughout the experiment and tested for killing ability. By the 38th transfer, a decreased killing ability and an increase in fitness were observed in the colicin-producing strains. Surprisingly, DNA sequence determination of the colicin plasmids revealed no changes in plasmid sequences. However, a set of chromosomally encoded loci experienced changes in gene expression that were positively associated with the reduction in killing. The most significant expression changes were observed in DNA repair genes (which were downregulated in the evolved strains), Mg ion uptake genes (which were upregulated), and late prophage genes (which were upregulated). These results indicate a fine-tuned response to the evolutionary pressures of colicin production, with far more genes involved than had been anticipated. | [ Jan A. C.Vriezen, MichaelValliere, Margaret A.Riley] | Genome Biology and Evolution | 2009-10-20 | 5.5.1 |
| pmc2724701 | Differentially expressed genes between drought-tolerant and drought-sensitive barley genotypes in response to drought stress during the reproductive stage | Drought tolerance is a key trait for increasing and stabilizing barley productivity in dry areas worldwide. Identification of the genes responsible for drought tolerance in barley (Hordeum vulgare L.) will facilitate understanding of the molecular mechanisms of drought tolerance, and also facilitate the genetic improvement of barley through marker-assisted selection or gene transformation. To monitor the changes in gene expression at the transcriptional level in barley leaves during the reproductive stage under drought conditions, the 22K Affymetrix Barley 1 microarray was used to screen two drought-tolerant barley genotypes, Martin and Hordeum spontaneum 41-1 (HS41-1), and one drought-sensitive genotype Moroc9-75. Seventeen genes were expressed exclusively in the two drought-tolerant genotypes under drought stress, and their encoded proteins may play significant roles in enhancing drought tolerance through controlling stomatal closure via carbon metabolism (NADP malic enzyme, NADP-ME, and pyruvate dehydrogenase, PDH), synthesizing the osmoprotectant glycine-betaine (C-4 sterol methyl oxidase, CSMO), generating protectants against reactive-oxygen-species scavenging (aldehyde dehydrogenase,ALDH, ascorbate-dependent oxidoreductase, ADOR), and stabilizing membranes and proteins (heat-shock protein 17.8, HSP17.8, and dehydrin 3, DHN3). Moreover, 17 genes were abundantly expressed in Martin and HS41-1 compared with Moroc9-75 under both drought and control conditions. These genes were possibly constitutively expressed in drought-tolerant genotypes. Among them, seven known annotated genes might enhance drought tolerance through signalling [such as calcium-dependent protein kinase (CDPK) and membrane steroid binding protein (MSBP)], anti-senescence (G2 pea dark accumulated protein, GDA2), and detoxification (glutathione S-transferase, GST) pathways. In addition, 18 genes, including those encoding Δl-pyrroline-5-carboxylate synthetase (P5CS), protein phosphatase 2C-like protein (PP2C), and several chaperones, were differentially expressed in all genotypes under drought; thus they were more likely to be general drought-responsive genes in barley. These results could provide new insights into further understanding of drought-tolerance mechanisms in barley. | [ PeiguoGuo, MichaelBaum, StefaniaGrando, SalvatoreCeccarelli, GuihuaBai, RonghuaLi, Mariavon Korff, Rajeev K.Varshney, AndreasGraner, JanValkoun] | Journal of Experimental Botany | 2009-6-26 | |
| pmc544504 | Cytosolic Ascorbate Peroxidase 1 Is a Central Component of the Reactive Oxygen Gene Network of ArabidopsisW⃞ | Reactive oxygen species (ROS), such as O2− and H2O2, play a key role in plant metabolism, cellular signaling, and defense. In leaf cells, the chloroplast is considered to be a focal point of ROS metabolism. It is a major producer of O2− and H2O2 during photosynthesis, and it contains a large array of ROS-scavenging mechanisms that have been extensively studied. By contrast, the function of the cytosolic ROS-scavenging mechanisms of leaf cells is largely unknown. In this study, we demonstrate that in the absence of the cytosolic H2O2-scavenging enzyme ascorbate peroxidase 1 (APX1), the entire chloroplastic H2O2-scavenging system of Arabidopsis thaliana collapses, H2O2 levels increase, and protein oxidation occurs. We further identify specific proteins oxidized in APX1-deficient plants and characterize the signaling events that ensue in knockout-Apx1 plants in response to a moderate level of light stress. Using a dominant-negative approach, we demonstrate that heat shock transcription factors play a central role in the early sensing of H2O2 stress in plants. Using knockout plants for the NADPH oxidase D protein (knockout-RbohD), we demonstrate that RbohD might be required for ROS signal amplification during light stress. Our study points to a key role for the cytosol in protecting the chloroplast during light stress and provides evidence for cross-compartment protection of thylakoid and stromal/mitochondrial APXs by cytosolic APX1. | [ SholpanDavletova, LudmilaRizhsky, HongjianLiang, ZhongShengqiang, David J.Oliver, JesseCoutu, VladimirShulaev, KarenSchlauch, RonMittler] | The Plant Cell | | |
| pmc2570144 | Mammary Epithelial-Specific Disruption of Focal Adhesion Kinase Retards Tumor Formation and Metastasis in a Transgenic Mouse Model of Human Breast Cancer | Focal adhesion kinase (FAK) is a central regulator of the focal adhesion, influencing cell proliferation, survival, and migration. Despite evidence demonstrating FAK overexpression in human cancer, its role in tumor initiation and progression is not well understood. Using Cre/LoxP technology to specifically knockout FAK in the mammary epithelium, we showed that FAK is not required for tumor initiation but is required for tumor progression. The mechanistic underpinnings of these results suggested that FAK regulates clinically relevant gene signatures and multiple signaling complexes associated with tumor progression and metastasis, such as Src, ERK, and p130Cas. Furthermore, a systems-level analysis identified FAK as a major regulator of the tumor transcriptome, influencing genes associated with adhesion and growth factor signaling pathways, and their cross talk. Additionally, FAK was shown to down-regulate the expression of clinically relevant proliferation- and metastasis-associated gene signatures, as well as an enriched group of genes associated with the G2 and G2/M phases of the cell cycle. Computational analysis of transcription factor-binding sites within ontology-enriched or clustered gene sets suggested that the differentially expressed proliferation- and metastasis-associated genes in FAK-null cells were regulated through a common set of transcription factors, including p53. Therefore, FAK acts as a primary node in the activated signaling network in transformed motile cells and is a prime candidate for novel therapeutic interventions to treat aggressive human breast cancers. | [ Paolo P.Provenzano, David R.Inman, Kevin W.Eliceiri, Hilary E.Beggs, Patricia J.Keely] | The American Journal of Pathology | | |
| pmc2220043 | The PcG Gene Sfmbt2 is Paternally Expressed in Extraembryonic Tissues | Genomic imprinting has dramatic effects on placental development, as has been clearly observed in interspecific hybrid, somatic cell nuclear transfer, and uniparental embryos. In fact, the earliest defects in uniparental embryos are evident first in the extraembryonic trophoblast. We performed a microarray comparison of gynogenetic and androgenetic mouse blastocysts, which are predisposed to placental pathologies, to identify imprinted genes. In addition to identifying a large number of known imprinted genes, we discovered that the Polycomb group (PcG) gene Sfmbt2 is imprinted. Sfmbt2 is expressed preferentially from the paternal allele in early embryos, and in later stage extraembryonic tissues. A CpG island spanning the transcriptional start site is differentially methylated on the maternal allele in e14.5 placenta. Sfmbt2 is located on proximal chromosome 2, in a region known to be imprinted, but for which no genes had been identified until now. This possibly identifies a new imprinted domain within the murine genome. We further demonstrate that murine SFMBT2 protein interacts with the transcription factor YY1, similar to the Drosophila PHO-RC. | [ AnastasiaKuzmin, ZhimingHan, Michael C.Golding, Mellissa R.W.Mann, Keith E.Latham, SusannahVarmuza] | Gene expression patterns : GEP | 2007-10-9 | |
| pmc3112077 | Analysis of anther transcriptomes to identify genes contributing to meiosis and male gametophyte development in rice | In flowering plants, the anther is the site of male gametophyte development. Two major events in the development of the male germline are meiosis and the asymmetric division in the male gametophyte that gives rise to the vegetative and generative cells, and the following mitotic division in the generative cell that produces two sperm cells. Anther transcriptomes have been analyzed in many plant species at progressive stages of development by using microarray and sequence-by synthesis-technologies to identify genes that regulate anther development. Here we report a comprehensive analysis of rice anther transcriptomes at four distinct stages, focusing on identifying regulatory components that contribute to male meiosis and germline development. Further, these transcriptomes have been compared with the transcriptomes of 10 stages of rice vegetative and seed development to identify genes that express specifically during anther development.ResultsTranscriptome profiling of four stages of anther development in rice including pre-meiotic (PMA), meiotic (MA), anthers at single-celled (SCP) and tri-nucleate pollen (TPA) revealed about 22,000 genes expressing in at least one of the anther developmental stages, with the highest number in MA (18,090) and the lowest (15,465) in TPA. Comparison of these transcriptome profiles to an in-house generated microarray-based transcriptomics database comprising of 10 stages/tissues of vegetative as well as reproductive development in rice resulted in the identification of 1,000 genes specifically expressed in anther stages. From this sub-set, 453 genes were specific to TPA, while 78 and 184 genes were expressed specifically in MA and SCP, respectively. The expression pattern of selected genes has been validated using real time PCR and in situ hybridizations. Gene ontology and pathway analysis of stage-specific genes revealed that those encoding transcription factors and components of protein folding, sorting and degradation pathway genes dominated in MA, whereas in TPA, those coding for cell structure and signal transduction components were in abundance. Interestingly, about 50% of the genes with anther-specific expression have not been annotated so far.ConclusionsNot only have we provided the transcriptome constituents of four landmark stages of anther development in rice but we have also identified genes that express exclusively in these stages. It is likely that many of these candidates may therefore contribute to specific aspects of anther and/or male gametophyte development in rice. In addition, the gene sets that have been produced will assist the plant reproductive community in building a deeper understanding of underlying regulatory networks and in selecting gene candidates for functional validation. | [ PriyankaDeveshwar, William DBovill, RitaSharma, Jason AAble, SanjayKapoor] | BMC Plant Biology | 2011-5-9 | 5.5 |
| pmc3111392 | The testosterone-dependent and independent transcriptional networks in the hypothalamus of Gpr54 and Kiss1 knockout male mice are not fully equivalent | Humans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin do not progress through puberty, caused by a failure to release GnRH. The transcriptional networks regulated by these proteins in the hypothalamus have yet to be explored by genome-wide methods.ResultsWe show here, using 1 million exon mouse arrays (Exon 1.0 Affymetrix) and quantitative polymerase chain reaction (QPCR) validation to analyse microdissected hypothalamic tissue from Gpr54 and Kiss1 knockout mice, the extent of transcriptional regulation in the hypothalamus. The sensitivity to detect important transcript differences in microdissected RNA was confirmed by the observation of counter-regulation of Kiss1 expression in Gpr54 knockouts and confirmed by immunohistochemistry (IHC). Since Gpr54 and Kiss1 knockout animals are effectively pre-pubertal with low testosterone (T) levels, we also determined which of the validated transcripts were T-responsive and which varied according to genotype alone. We observed four types of transcriptional regulation (i) genotype only dependent regulation, (ii) T only dependent regulation, (iii) genotype and T-dependent regulation with interaction between these variables, (iv) genotype and T-dependent regulation with no interaction between these variables. The results implicate for the first time several transcription factors (e.g. Npas4, Esr2), proteases (Klk1b22), and the orphan 10-transmembrane transporter TMEM144 in the biology of GPR54/kisspeptin function in the hypothalamus. We show for the neuronal activity regulated transcription factor NPAS4, that distinct protein over-expression is seen in the hypothalamus and hippocampus in Gpr54 knockout mice. This links for the first time the hypothalamic-gonadal axis with this important regulator of inhibitory synapse formation. Similarly we confirm TMEM144 up-regulation in the hypothalamus by RNA in situ hybridization and western blot.ConclusionsTaken together, global transcriptional profiling shows that loss of GPR54 and kisspeptin are not fully equivalent in the mouse hypothalamus. | [ Leah MPrentice, Xavierd'Anglemont de Tassigny, StevenMcKinney, TeresaRuiz de Algara, DamianYap, GulisaTurashvili, StevenPoon, MargaretSutcliffe, PatAllard, AngelaBurleigh, JohnFee, David GHuntsman, William HColledge, Samuel AJAparicio] | BMC Genomics | 2011-4-28 | |
| pmc2948007 | Molecular Signatures of the Primitive Prostate Stem Cell Niche Reveal Novel Mesenchymal-Epithelial Signaling Pathways | Signals between stem cells and stroma are important in establishing the stem cell niche. However, very little is known about the regulation of any mammalian stem cell niche as pure isolates of stem cells and their adjacent mesenchyme are not readily available. The prostate offers a unique model to study signals between stem cells and their adjacent stroma as in the embryonic prostate stem cell niche, the urogenital sinus mesenchyme is easily separated from the epithelial stem cells. Here we investigate the distinctive molecular signals of these two stem cell compartments in a mammalian system.Methodology/Principal FindingsWe isolated fetal murine urogenital sinus epithelium and urogenital sinus mesenchyme and determined their differentially expressed genes. To distinguish transcripts that are shared by other developing epithelial/mesenchymal compartments from those that pertain to the prostate stem cell niche, we also determined the global gene expression of epidermis and dermis of the same embryos. Our analysis indicates that several of the key transcriptional components that are predicted to be active in the embryonic prostate stem cell niche regulate processes such as self-renewal (e.g., E2f and Ap2), lipid metabolism (e.g., Srebp1) and cell migration (e.g., Areb6 and Rreb1). Several of the enriched promoter binding motifs are shared between the prostate epithelial/mesenchymal compartments and their epidermis/dermis counterparts, indicating their likely relevance in epithelial/mesenchymal signaling in primitive cellular compartments. Based on differential gene expression we also defined ligand-receptor interactions that may be part of the molecular interplay of the embryonic prostate stem cell niche.Conclusions/SignificanceWe provide a comprehensive description of the transcriptional program of the major regulators that are likely to control the cellular interactions in the embryonic prostatic stem cell niche, many of which may be common to mammalian niches in general. This study provides a comprehensive source for further studies of mesenchymal/epithelial interactions in the prostate stem cell niche. The elucidation of pathways in the normal primitive niche may provide greater insight into mechanisms subverted during abnormal proliferative and oncogenic processes. Understanding these events may result in the development of specific targeted therapies for prostatic diseases such as benign prostatic hypertrophy and carcinomas. | [ RoyBlum, RashmiGupta, Patricia E.Burger, Christopher S.Ontiveros, Sarah N.Salm, XiaozhongXiong, AlexanderKamb, HolgerWesche, LisaMarshall, GeneCutler, XiangyunWang, JiriZavadil, DavidMoscatelli, E. LynetteWilson] | PLoS ONE | 2010-9-30 | |
| pmc2844426 | Mutations in CHMP2B in Lower Motor Neuron Predominant Amyotrophic Lateral Sclerosis (ALS) | Amyotrophic lateral sclerosis (ALS), a common late-onset neurodegenerative disease, is associated with fronto-temporal dementia (FTD) in 3–10% of patients. A mutation in CHMP2B was recently identified in a Danish pedigree with autosomal dominant FTD. Subsequently, two unrelated patients with familial ALS, one of whom also showed features of FTD, were shown to carry missense mutations in CHMP2B. The initial aim of this study was to determine whether mutations in CHMP2B contribute more broadly to ALS pathogenesis.Methodology/Principal FindingsSequencing of CHMP2B in 433 ALS cases from the North of England identified 4 cases carrying 3 missense mutations, including one novel mutation, p.Thr104Asn, none of which were present in 500 neurologically normal controls. Analysis of clinical and neuropathological data of these 4 cases showed a phenotype consistent with the lower motor neuron predominant (progressive muscular atrophy (PMA)) variant of ALS. Only one had a recognised family history of ALS and none had clinically apparent dementia. Microarray analysis of motor neurons from CHMP2B cases, compared to controls, showed a distinct gene expression signature with significant differential expression predicting disassembly of cell structure; increased calcium concentration in the ER lumen; decrease in the availability of ATP; down-regulation of the classical and p38 MAPK signalling pathways, reduction in autophagy initiation and a global repression of translation. Transfection of mutant CHMP2B into HEK-293 and COS-7 cells resulted in the formation of large cytoplasmic vacuoles, aberrant lysosomal localisation demonstrated by CD63 staining and impairment of autophagy indicated by increased levels of LC3-II protein. These changes were absent in control cells transfected with wild-type CHMP2B.Conclusions/SignificanceWe conclude that in a population drawn from North of England pathogenic CHMP2B mutations are found in approximately 1% of cases of ALS and 10% of those with lower motor neuron predominant ALS.We provide a body of evidence indicating the likely pathogenicity of the reported gene alterations. However, absolute confirmation of pathogenicity requires further evidence, including documentation of familial transmission in ALS pedigrees which might be most fruitfully explored in cases with a LMN predominant phenotype. | [ Laura E.Cox, LauraFerraiuolo, Emily F.Goodall, Paul R.Heath, AdrianHigginbottom, HeatherMortiboys, Hannah C.Hollinger, Judith A.Hartley, AliceBrockington, Christine E.Burness, Karen E.Morrison, Stephen B.Wharton, Andrew J.Grierson, Paul G.Ince, JanineKirby, Pamela J.Shaw] | PLoS ONE | 2010-3-24 | |
| pmc2825440 | Human Negative Elongation Factor Activates Transcription and Regulates Alternative Transcription Initiation* | The human negative elongation factor (NELF) is a four-subunit protein complex that inhibits the movement of RNA polymerase II (RNAPII) at an early elongation stage in vitro. NELF-mediated stalling of RNAPII also attenuates transcription of a number of inducible genes in human cells. To obtain a genome-wide understanding of human NELF-mediated transcriptional regulation in vivo, we carried out an exon array study in T47D breast cancer cells with transient small interfering RNA knockdown of individual NELF subunits. Upon depletion of NELF-A, -C, or -E, the vast majority of NELF-regulated genes were down-regulated. Many of the down-regulated genes encode proteins that play key roles in cell cycle progression. Consequently, NELF knockdown resulted in significant reduction in DNA synthesis and cell proliferation. Chromatin immunoprecipitation showed that NELF knockdown led to dissociation of RNAPII from the promoter-proximal region of the cell cycle-regulating genes. This was accompanied by decreased histone modifications associated with active transcription initiation (H3K9Ac) and elongation (H3K36Me3), as well as reduced recruitment of the general transcription factor TFIIB and increased overall histone occupancy at a subset of the down-regulated promoters. Lastly, our study indicates that NELF regulates alternative transcription initiation of BSG (Basigin) gene by differentially influencing RNAPII density at the two neighboring exons at the 5′ end of the gene. Taken together, our data suggest a diverse transcriptional consequence of NELF-mediated RNAPII pausing in the human genome. | [ JianlongSun, RongLi] | The Journal of Biological Chemistry | 2009-12-22 | |
| pmc2684642 | Molecular Signatures of Prostate Stem Cells Reveal Novel Signaling Pathways and Provide Insights into Prostate Cancer | The global gene expression profiles of adult and fetal murine prostate stem cells were determined to define common and unique regulators whose misexpression might play a role in the development of prostate cancer.Methodology/Principal FindingsA distinctive core of transcriptional regulators common to both fetal and adult primitive prostate cells was identified as well as molecules that are exclusive to each population. Elements common to fetal and adult prostate stem cells include expression profiles of Wnt, Shh and other pathways identified in stem cells of other organs, signatures of the aryl-hydrocarbon receptor, and up-regulation of components of the aldehyde dehydrogenase/retinoic acid receptor axis. There is also a significant lipid metabolism signature, marked by overexpression of lipid metabolizing enzymes and the presence of the binding motif for Srebp1. The fetal stem cell population, characterized by more rapid proliferation and self-renewal, expresses regulators of the cell cycle, such as E2f, Nfy, Tead2 and Ap2, at elevated levels, while adult stem cells show a signature in which TGF-β has a prominent role. Finally, comparison of the signatures of primitive prostate cells with previously described profiles of human prostate tumors identified stem cell molecules and pathways with deregulated expression in prostate tumors including chromatin modifiers and the oncogene, Erg.Conclusions/SignificanceOur data indicate that adult prostate stem or progenitor cells may acquire characteristics of self-renewing primitive fetal prostate cells during oncogenesis and suggest that aberrant activation of components of prostate stem cell pathways may contribute to the development of prostate tumors. | [ RoyBlum, RashmiGupta, Patricia E.Burger, Christopher S.Ontiveros, Sarah N.Salm, XiaozhongXiong, AlexanderKamb, HolgerWesche, LisaMarshall, GeneCutler, XiangyunWang, JiriZavadil, DavidMoscatelli, E. LynetteWilson] | PLoS ONE | 2009-5-29 | |
| pmc2743568 | Profiling of Dynamically Changed Gene Expression in Dorsal Root Ganglia Post Peripheral Nerve injury and A Critical Role of Injury-Induced Glial Fibrillary Acetic Protein in Maintenance of Pain Behaviors | To explore cellular changes in sensory neurons after nerve injury and identify potential target genes contributing to different stages of neuropathic pain development, we used Affymetrix oligo arrays to profile gene expression patterns in L5/6 dorsal root ganglia (DRG) from the neuropathic pain model of left L5/6 spinal nerve ligation at different stages of neuropathic pain development. Our data indicated that nerve injury induced changes in expression of genes with similar biological functions in a temporal specific manner that correlates with particular stages of neuropathic pain development, indicating dynamic neuroplasticity in the DRG in response to peripheral nerve injury and during neuropathic pain development. Data from post-array validation indicated that there was a temporal correlation between injury-induced expression of the glial fibrillary acidic protein (GFAP), a marker for activated astrocytes, and neuropathic pain development. Spinal nerve ligation injury in GFAP knockout mice resulted in neuropathic pain states with similar onset, but a shortened duration compared with that in age, and gender-matched wild-type littermates. Intrathecal GFAP antisense oligonucleotide treatment in injured rats with neuropathic pain states reversed injury-induced behavioral hypersensitivity and GFAP upregulation in DRG and spinal cord. Together, these findings indicate that injury-induced GFAP upregulation not only serves as a marker for astrocyte activation, but it may play a critical, but yet identified, role in the maintenance of neuropathic pain states. | [ Doo-sikKim, Katherine W.Figueroa, Kang-WuLi, AminBoroujerdi, TimYolo, Z. DavidLuo] | Pain | | |
| pmc2637607 | Glucose and Auxin Signaling Interaction in Controlling Arabidopsis thaliana Seedlings Root Growth and Development | Plant root growth and development is highly plastic and can adapt to many environmental conditions. Sugar signaling has been shown to affect root growth and development by interacting with phytohormones such as gibberellins, cytokinin and abscisic acid. Auxin signaling and transport has been earlier shown to be controlling plant root length, number of lateral roots, root hair and root growth direction.Principal FindingsIncreasing concentration of glucose not only controls root length, root hair and number of lateral roots but can also modulate root growth direction. Since root growth and development is also controlled by auxin, whole genome transcript profiling was done to find out the extent of interaction between glucose and auxin response pathways. Glucose alone could transcriptionally regulate 376 (62%) genes out of 604 genes affected by IAA. Presence of glucose could also modulate the extent of regulation 2 fold or more of almost 63% genes induced or repressed by IAA. Interestingly, glucose could affect induction or repression of IAA affected genes (35%) even if glucose alone had no significant effect on the transcription of these genes itself. Glucose could affect auxin biosynthetic YUCCA genes family members, auxin transporter PIN proteins, receptor TIR1 and members of a number of gene families including AUX/IAA, GH3 and SAUR involved in auxin signaling. Arabidopsis auxin receptor tir1 and response mutants, axr2, axr3 and slr1 not only display a defect in glucose induced change in root length, root hair elongation and lateral root production but also accentuate glucose induced increase in root growth randomization from vertical suggesting glucose effects on plant root growth and development are mediated by auxin signaling components.ConclusionOur findings implicate an important role of the glucose interacting with auxin signaling and transport machinery to control seedling root growth and development in changing nutrient conditions. | [ Bhuwaneshwar S.Mishra, ManjulSingh, PriyankaAggrawal, AshveryaLaxmi] | PLoS ONE | 2009-2-18 | |
| pmc2614561 | Lysophosphatidylcholine acyltransferase 1 (LPCAT1) overexpression in human colorectal cancer | The alteration of the choline metabolite profile is a well-established characteristic of cancer cells. In colorectal cancer (CRC), phosphatidylcholine is the most prominent phospholipid. In the present study, we report that lysophosphatidylcholine acyltransferase 1 (LPCAT1; NM_024830.3), the enzyme that converts lysophosphatidylcholine into phosphatidylcholine, was highly overexpressed in colorectal adenocarcinomas when compared to normal mucosas. Our microarray transcription profiling study showed a significant (p<10−8) transcript overexpression in 168 colorectal adenocarcinomas when compared to ten normal mucosas. Immunohistochemical analysis of colon tumors with a polyclonal antibody to LPCAT1 confirmed the upregulation of the LPCAT1 protein. Overexpression of LPCAT1 in COS7 cells localized the protein to the endoplasmic reticulum and the mitochondria and increased LPCAT1 specific activity 38-fold. In cultured cells, overexpressed LPCAT1 enhanced the incorporation of [14C]palmitate into phosphatidylcholine. COS7 cells transfected with LPCAT1 showed no growth rate alteration, in contrast to the colon cancer cell line SW480, which significantly (p<10−5) increased its growth rate by 17%. We conclude that LPCAT1 may contribute to total choline metabolite accumulation via phosphatidylcholine remodeling, thereby altering the CRC lipid profile, a characteristic of malignancy. | [ FranciscoMansilla, Kerry-Annda Costa, ShuliWang, MogensKruhøffer, Tal M.Lewin, Torben F.Ørntoft, Rosalind A.Coleman, KarinBirkenkamp-Demtröder] | Journal of molecular medicine (Berlin, Germany) | 2008-10-31 | |
| pmc3147292 | Aryl Hydrocarbon Receptor-Mediated Down-Regulation of Sox9b Causes Jaw Malformation in Zebrafish Embryos | Exposure to environmental contaminants can disrupt normal development of the early vertebrate skeleton. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) impairs craniofacial skeletal development across many vertebrate species, and its effects are especially prominent in early life stages of fish. TCDD activates the aryl hydrocarbon receptor, a transcription factor that mediates most if not all TCDD responses. We investigated the transcriptional response in the developing zebrafish jaw after TCDD exposure using DNA microarrays. Zebrafish larvae were exposed to TCDD at 96 h after fertilization, and jaw cartilage tissue was harvested for microarray analysis at 1, 2, 4, and 12 h after exposure. Numerous chondrogenic transcripts were misregulated by TCDD in the jaw. Comparison of transcripts altered by TCDD in jaw with transcripts altered in embryonic heart showed that the transcriptional responses in the jaw and the heart were strikingly different. Sox9b, a critical chondrogenic transcription factor, was the most significantly reduced transcript in the jaw. We hypothesized that the TCDD reduction of sox9b expression plays an integral role in affecting the formation of the embryonic jaw. Morpholino knockdown of sox9b expression demonstrated that partial reduction of sox9b expression alone was sufficient to produce a TCDD-like jaw phenotype. Loss of a single copy of the sox9b gene in sox9b(+/−) heterozygotes increased sensitivity to jaw malformation by TCDD. Finally, embryos injected with sox9b mRNA and then exposed to TCDD blocked TCDD-induced jaw toxicity in approximately 14% of sox9b-injected embryos. These results suggest that reduced sox9b expression in TCDD-exposed zebrafish embryos contributes to jaw malformation. | [ Kong M.Xiong, Richard E.Peterson, WarrenHeideman] | Molecular pharmacology | 2008-9-10 | |
| pmc2495035 | Meta-Analysis of Microarray Studies Reveals a Novel Hematopoietic Progenitor Cell Signature and Demonstrates Feasibility of Inter-Platform Data Integration | Microarray-based studies of global gene expression (GE) have resulted in a large amount of data that can be mined for further insights into disease and physiology. Meta-analysis of these data is hampered by technical limitations due to many different platforms, gene annotations and probes used in different studies. We tested the feasibility of conducting a meta-analysis of GE studies to determine a transcriptional signature of hematopoietic progenitor and stem cells. Data from studies that used normal bone marrow-derived hematopoietic progenitors was integrated using both RefSeq and UniGene identifiers. We observed that in spite of variability introduced by experimental conditions and different microarray platforms, our meta-analytical approach can distinguish biologically distinct normal tissues by clustering them based on their cell of origin. When studied in terms of disease states, GE studies of leukemias and myelodysplasia progenitors tend to cluster with normal progenitors and remain distinct from other normal tissues, further validating the discriminatory power of this meta-analysis. Furthermore, analysis of 57 normal hematopoietic stem and progenitor cell GE samples was used to determine a gene expression signature characteristic of these cells. Genes that were most uniformly expressed in progenitors and at the same time differentially expressed when compared to other normal tissues were found to be involved in important biological processes such as cell cycle regulation and hematopoiesis. Validation studies using a different microarray platform demonstrated the enrichment of several genes such as SMARCE, Septin 6 and others not previously implicated in hematopoiesis. Most interestingly, alpha-integrin, the only common stemness gene discovered in a recent comparative murine analysis (Science 302(5644):393) was also enriched in our dataset, demonstrating the usefulness of this analytical approach. | [ DavendraSohal, AndrewYeatts, KennyYe, AndreaPellagatti, LiZhou, PerryPahanish, YongkaiMo, TusharBhagat, JohnMariadason, JacquelineBoultwood, AriMelnick, JohnGreally, AmitVerma] | PLoS ONE | 2008-8-13 | |
| pmc2547001 | A Gene Signature-Based Approach Identifies mTOR as a Regulator of p73▿ | Although genomic technologies have advanced the characterization of gene regulatory networks downstream of transcription factors, the identification of pathways upstream of these transcription factors has been more challenging. In this study we present a gene signature-based approach for connecting signaling pathways to transcription factors, as exemplified by p73. We generated a p73 gene signature by integrating whole-genome chromatin immunoprecipitation and expression profiling. The p73 signature was linked to corresponding signatures produced by drug candidates, using the in silico Connectivity Map resource, to identify drugs that would induce p73 activity. Of the pharmaceutical agents identified, there was enrichment for direct or indirect inhibitors of mammalian Target of Rapamycin (mTOR) signaling. Treatment of both primary cells and cancer cell lines with rapamycin, metformin, and pyrvinium resulted in an increase in p73 levels, as did RNA interference-mediated knockdown of mTOR. Further, a subset of genes associated with insulin response or autophagy exhibited mTOR-mediated, p73-dependent expression. Thus, downstream gene signatures can be used to identify upstream regulators of transcription factor activity, and in doing so, we identified a new link between mTOR, p73, and p73-regulated genes associated with autophagy and metabolic pathways. | [ Jennifer M.Rosenbluth, Deborah J.Mays, Maria F.Pino, Luo JiaTang, Jennifer A.Pietenpol] | Molecular and Cellular Biology | 2008-8-4 | |
| pmc2492871 | Increased cell motility and invasion upon knockdown of lipolysis stimulated lipoprotein receptor (LSR) in SW780 bladder cancer cells | Mechanisms underlying the malignant development in bladder cancer are still not well understood. Lipolysis stimulated lipoprotein receptor (LSR) has previously been found to be upregulated by P53. Furthermore, we have previously found LSR to be differentially expressed in bladder cancer. Here we investigated the role of LSR in bladder cancer.MethodsA time course siRNA knock down experiment was performed to investigate the functional role of LSR in SW780 bladder cancer cells. Since LSR was previously shown to be regulated by P53, siRNA against TP53 was included in the experimental setup. We used Affymetrix GeneChips for measuring gene expression changes and we used Ingenuity Pathway Analysis to investigate the relationship among differentially expressed genes upon siRNA knockdown.ResultsBy Ingenuity Pathway analysis of the microarray data from the different timepoints we identified six gene networks containing genes mainly related to the functional categories "cancer", "cell death", and "cellular movement". We determined that genes annotated to the functional category "cellular movement" including "invasion" and "cell motility" were highly significantly overrepresented. A matrigel assay showed that 24 h after transfection the invasion capacity was significantly increased 3-fold (p < 0.02) in LSR-siRNA transfected cells, and 2.7-fold (p < 0.02) in TP53-siRNA transfected cells compared to controls. After 48 h the motility capacity was significantly increased 3.5-fold (p < 0.004) in LSR-siRNA transfected cells, and 4.7-fold (p < 0.002) in TP53-siRNA transfected cells compared to controls.ConclusionWe conclude that LSR may impair bladder cancer cells from gaining invasive properties. | [ MaleneHerbsleb, KarinBirkenkamp-Demtroder, ThomasThykjaer, CarstenWiuf, Anne-Mette KHein, Torben FØrntoft, LarsDyrskjøt] | BMC Medical Genomics | 2008-7-22 | |
| pmc2494702 | Identification of downstream genetic pathways of Tbx1 in the second heart field | Tbx1, a T-box transcription factor, and an important gene for velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) in humans, causes outflow tract (OFT) heart defects when inactivated in the mouse. Tbx1 is expressed in the second heart field (SHF) and is required in this tissue for OFT development. To identify Tbx1 regulated genetic pathways in the SHF, we performed gene expression profiling of the caudal pharyngeal region in Tbx1-/- and wild type embryos. Isl1, a key marker for the SHF, as well as Hod and Nkx2-6, were downregulated in Tbx1-/- mutants, while genes required for cardiac morphogenesis, such as Raldh2, Gata4, and Tbx5, as well as a subset of muscle contractile genes, signifying myocardial differentiation, were ectopically expressed. Pan-mesodermal ablation of Tbx1 resulted in similar gene expression changes, suggesting cell-autonomous roles of Tbx1 in regulating these genes. Opposite expression changes concomitant with SHF-derived cardiac defects occurred in TBX1 gain-of-function mutants, indicating that appropriate levels of Tbx1 are required for heart development. When taken together, our studies show that Tbx1 acts upstream in a genetic network that positively regulates SHF cell proliferation and negatively regulates differentiation, cell-autonomously in the caudal pharyngeal region. | [ JunLiao, Vimla S.Aggarwal, SonjaNowotschin, AlexeiBondarev, ShariLipner, Bernice E.Morrow] | Developmental biology | 2008-2-13 | |
| pmc2963074 | A distinct population of clonogenic and multipotent murine follicular keratinocytes residing in the upper isthmus | The bulge region of adult murine hair follicles harbors epidermal stem cells with multipotent capacity; however, the restricted contributions of these cells under homeostatic conditions indicates that additional stem or progenitor cell populations may be required to maintain squamous and sebaceous lineages. We have identified a distinct population of murine hair follicle keratinocytes residing in the upper isthmus (UI) between the infundibulum and bulge regions that are distinguished by low α6 integrin levels and are negative for CD34 and Sca-1. Purified UI cells give rise to long-term, stable epidermal, follicular and sebaceous lineages and can self-renew in vivo. These cells are non-quiescent and possess a unique transcript profile compared with bulge stem cells and may represent a distinct reservoir of epidermal stem or progenitor cells. | [ Uffe BirkJensen, XiaohongYan, CharlotteTriel, Seung-HyunWoo, RikkeChristensen, David M.Owens] | Journal of cell science | 2008-2-5 | |
| pmc2932699 | Identification of Novel Retinal Target Genes of Thyroid Hormone in the Human WERI Cells by Expression Microarray Analysis | Using the human WERI-Rb1 cell line as a model system, we performed a genome-wide search for retinal target genes of thyroid hormone (TH) via expression microarray analysis followed by quantitative real-time RT-PCR verification. We identified 12 novel retinal targets of TH, including 10 up-regulated genes (OPN1MW, OPN1LW, TIMP3, RP1L1, GNGT2, CRX, ARR3, GCAP1, IMPDH1, and PDE6C) and 2 down-regulated genes (GNGT1 and GNB3). In addition, we found a number of novel TH targets that are not currently known to be retinal genes. This is the first report of human retinal targets regulated by thyroid hormone. | [ YanLiu, LiFu, Ding-GengChen, Samir S.Deeb] | Vision research | 2007-7-25 | |
| pmc2359975 | Differential expression of DHHC9 in microsatellite stable and instable human colorectal cancer subgroups | Microarray analysis on pooled samples has previously identified ZDHHC9 (DHHC9) to be upregulated in colon adenocarcinoma compared to normal colon mucosa. Analyses of 168 samples from proximal and distal adenocarcinomas using U133plus2.0 microarrays validated these findings, showing a significant two-fold (log 2) upregulation of DHHC9 transcript (P<10−6). The upregulation was more striking in microsatellite stable (MSS), than in microsatellite instable (MSI), tumours. Genes known to interact with DHHC9 as H-Ras or N-Ras did not show expression differences between MSS and MSI. Immunohistochemical analysis was performed on 60 colon adenocarcinomas, previously analysed on microarrays, as well as on tissue microarrays with 40 stage I–IV tumours and 46 tumours from different organ sites. DHHC9 protein was strongly expressed in MSS compared to MSI tumours, readily detectable in premalignant lesions, compared to the rare expression seen in normal mucosa. DHHC9 was specific for tumours of the gastrointestinal tract and localised to the Golgi apparatus, in vitro and in vivo. Overexpression of DHHC9 decreased the proliferation of SW480 and CaCo2 MSS cell lines significantly. In conclusion, DHHC9 is a gastrointestinal-related protein highly expressed in MSS colon tumours. The palmitoyl transferase activity, modifying N-Ras and H-Ras, suggests DHHC9 as a target for anticancer drug design. | [ FMansilla, KBirkenkamp-Demtroder, MKruhøffer, F BSørensen, C LAndersen, PLaiho, L AAaltonen, H WVerspaget, T FØrntoft] | British Journal of Cancer | 2007-05-22 | |
| pmc1698855 | Tumor Cell Plasticity in Uveal Melanoma | The histological detection of laminin-rich vasculogenic mimicry patterns in human primary uveal melanomas is associated with death from metastases. We therefore hypothesized that highly invasive uveal melanoma cells forming vasculogenic mimicry patterns after exposure to a laminin-rich three-dimensional microenvironment would differentially express genes associated with invasive and metastatic behavior. However, we discovered that genes associated with differentiation (GDF15 and ATF3) and suppression of proliferation (CDKNa1/p21) were up-regulated in highly invasive uveal melanoma cells forming vasculogenic mimicry patterns, and genes associated with promotion of invasive and metastatic behavior such as CD44, CCNE2 (cyclin E2), THBS1 (thrombospondin 1), and CSPG2 (chondroitin sulfate proteoglycan; versican) were down-regulated. After forming vasculogenic mimicry patterns, uveal melanoma cells invaded only short distances, failed to replicate, and changed morphologically from the invasive epithelioid to the indolent spindle A phenotype. In human tissue samples, uveal melanoma cells within vasculogenic mimicry patterns assumed the spindle A morphology, and the expression of Ki67 was significantly reduced in adjacent melanoma cells. Thus, the generation of vasculogenic mimicry patterns is accompanied by dampening of the invasive and metastatic uveal melanoma genotype and phenotype and underscores the plasticity of these cells in response to cues from the microenvironment. | [ RobertFolberg, ZaremaArbieva, JonasMoses, AminHayee, ToneSandal, ShriHariKadkol, Amy Y.Lin, KlaraValyi-Nagy, SumanSetty, LuLeach, PatriciaChévez-Barrios, PeterLarsen, DibyenMajumdar, JacobPe’er, Andrew J.Maniotis] | The American Journal of Pathology | | |
| pmc3206077 | Mll5 Is Required for Normal Spermatogenesis | Mll5 is currently a member of the Mll family of SET domain histone methyltransferase proteins but studies have also showed that it could be part of the SET3 branch of proteins. Recently, constitutive knock out animal studies have shown that Mll5 is required for proper haematopoietic stem cell differentiation, and loss of Mll5 results in synthetic lethality for genome de-methylation. Mll5 deficient male mice are infertile and here we analyse the consequences of Mll5 deficiency for spermatogenesis.Methodology/Principal FindingsMll5 deficient male mice, but not female mice, are infertile. Here we show using RNA in-situ hybridization that Mll5 is expressed in the germ cells of the testes of wild type mice. Consistent with the expression of Mll5, we demonstrate by electron microscopy, video microscopy and in vitro fertilisation techniques that Mll5 deficient mice have defects in terminal maturation and packaging of sperm. The defects seen include detachment of the acrosomal cap and impaired excess cytoplasm removal. Functional tests of sperm motility show a lack of progressive motility of spermatozoa from Mll5 deficient animals. None of these defects could be rescued by in vitro fertilization. Using microarray analysis we show that transcripts implicated in spermatogenesis are dysregulated.Conclusions/SignificanceOur data demonstrate a clear role of Mll5 in mammalian spermatogenesis at the level of terminal differentiation providing further support for its classification in the SET3 branch of proteins. Moreover, this study identifies Tlk2, Utx, Gpr64, Sult4a1, Rap2ip, Vstm2 and HoxA10 as possible Mll5 targets that together may account for the observed spermatozoa maturation defects. | [ Damian B.Yap, David C.Walker, Leah M.Prentice, StevenMcKinney, GulisaTurashvili, KatrinMooslehner-Allen, Teresa Ruizde Algara, JohnFee, Xavier d'Anglemontde Tassigny, William H.Colledge, SamuelAparicio] | PLoS ONE | 2011-11-1 | |
| pmc3197591 | Cross-Platform Array Screening Identifies COL1A2, THBS1, TNFRSF10D and UCHL1 as Genes Frequently Silenced by Methylation in Melanoma | Epigenetic regulation of tumor suppressor genes (TSGs) has been shown to play a central role in melanomagenesis. By integrating gene expression and methylation array analysis we identified novel candidate genes frequently methylated in melanoma. We validated the methylation status of the most promising genes using highly sensitive Sequenom Epityper assays in a large panel of melanoma cell lines and resected melanomas, and compared the findings with those from cultured melanocytes. We found transcript levels of UCHL1, COL1A2, THBS1 and TNFRSF10D were inversely correlated with promoter methylation. For THBS1 and UCHL1 the effect of this methylation on expression was confirmed at the protein level. Identification of these candidate TSGs and future research designed to understand how their silencing is related to melanoma development will increase our understanding of the etiology of this cancer and may provide tools for its early diagnosis. | [ Vanessa F.Bonazzi, Derek J.Nancarrow, Mitchell S.Stark, Ralf J.Moser, Glen M.Boyle, Lauren G.Aoude, ChristopherSchmidt, Nicholas K.Hayward] | PLoS ONE | 2011-10-20 | 1 |
| pmc3182759 | Cortical Gene Expression in Spinal Cord Injury and Repair: Insight into the Functional Complexity of the Neural Regeneration Program | Traumatic spinal cord injury (SCI) results in the formation of a fibrous scar acting as a growth barrier for regenerating axons at the lesion site. We have previously shown (Klapka et al., 2005) that transient suppression of the inhibitory lesion scar in rat spinal cord leads to long distance axon regeneration, retrograde rescue of axotomized cortical motoneurons, and improvement of locomotor function. Here we applied a systemic approach to investigate for the first time specific and dynamic alterations in the cortical gene expression profile following both thoracic SCI and regeneration-promoting anti-scarring treatment (AST). In order to monitor cortical gene expression we carried out microarray analyses using total RNA isolated from layer V/VI of rat sensorimotor cortex at 1–60 days post-operation (dpo). We demonstrate that cortical neurons respond to injury by massive changes in gene expression, starting as early as 1 dpo. AST, in turn, results in profound modifications of the lesion-induced expression profile. The treatment attenuates SCI-triggered transcriptional changes of genes related to inhibition of axon growth and impairment of cell survival, while upregulating the expression of genes associated with axon outgrowth, cell protection, and neural development. Thus, AST not only modifies the local environment impeding spinal cord regeneration by reduction of fibrous scarring in the injured spinal cord, but, in addition, strikingly changes the intrinsic capacity of cortical pyramidal neurons toward enhanced cell maintenance and axonal regeneration. | [ FabianKruse, FrankBosse, Christina F.Vogelaar, NicoleBrazda, PatrickKüry, MarciaGasis, Hans W.Müller] | Frontiers in Molecular Neuroscience | 2011-9-29 | 4.2.0 |
| pmc3160043 | The MYB80 Transcription Factor Is Required for Pollen Development and the Regulation of Tapetal Programmed Cell Death in Arabidopsis thaliana[W][OA] | This work examines downstream targets of MYB80 and shows that an aspartic protease regulated by MYB80 is involved in the timing of programmed cell death of the tapetum in the Arabidopsis anther. | [ Huy AnhPhan, SylvanaIacuone, Song F.Li, Roger W.Parish] | The Plant Cell | 2011-6-14 | |
| pmc3123171 | A combination of transcriptome and methylation analyses reveals embryologically-relevant candidate genes in MRKH patients | The Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is present in at least 1 out of 4,500 female live births and is the second most common cause for primary amenorrhea. It is characterized by vaginal and uterine aplasia in an XX individual with normal secondary characteristics. It has long been considered a sporadic anomaly, but familial clustering occurs. Several candidate genes have been studied although no single factor has yet been identified. Cases of discordant monozygotic twins suggest that the involvement of epigenetic factors is more likely.MethodsDifferences in gene expression and methylation patterns of uterine tissue between eight MRKH patients and eight controls were identified using whole-genome microarray analyses. Results obtained by expression and methylation arrays were confirmed by qRT-PCR and pyrosequencing.ResultsWe delineated 293 differentially expressed and 194 differentially methylated genes of which nine overlap in both groups. These nine genes are mainly embryologically relevant for the development of the female genital tract.ConclusionOur study used, for the first time, a combined whole-genome expression and methylation approach to reveal the etiology of the MRKH syndrome. The findings suggest that either deficient estrogen receptors or the ectopic expression of certain HOXA genes might lead to abnormal development of the female reproductive tract. In utero exposure to endocrine disruptors or abnormally high maternal hormone levels might cause ectopic expression or anterior transformation of HOXA genes. It is, however, also possible that different factors influence the anti-Mullerian hormone promoter activity during embryological development causing regression of the Müllerian ducts. Thus, our data stimulate new research directions to decipher the pathogenic basis of MRKH syndrome. | [ KatharinaRall, GianmariaBarresi, MichaelWalter, SvenPoths, KarinaHaebig, KarinSchaeferhoff, BirgittSchoenfisch, OlafRiess, DiethelmWallwiener, MichaelBonin, SaraBrucker] | Orphanet Journal of Rare Diseases | 2011-5-28 | 4.0 |
| pmc3091051 | Silencing of Soybean Seed Storage Proteins Results in a Rebalanced Protein Composition Preserving Seed Protein Content without Major Collateral Changes in the Metabolome and Transcriptome[W][OA] | The ontogeny of seed structure and the accumulation of seed storage substances is the result of a determinant genetic program. Using RNA interference, the synthesis of soybean (Glycine max) glycinin and conglycinin storage proteins has been suppressed. The storage protein knockdown (SP−) seeds are overtly identical to the wild type, maturing to similar size and weight, and in developmental ontogeny. The SP− seeds rebalance the proteome, maintaining wild-type levels of protein and storage triglycerides. The SP− soybeans were evaluated with systems biology techniques of proteomics, metabolomics, and transcriptomics using both microarray and next-generation sequencing transcript sequencing (RNA-Seq). Proteomic analysis shows that rebalancing of protein content largely results from the selective increase in the accumulation of only a few proteins. The rebalancing of protein composition occurs with small alterations to the seed’s transcriptome and metabolome. The selectivity of the rebalancing was further tested by introgressing into the SP− line a green fluorescent protein (GFP) glycinin allele mimic and quantifying the resulting accumulation of GFP. The GFP accumulation was similar to the parental GFP-expressing line, showing that the GFP glycinin gene mimic does not participate in proteome rebalancing. The results show that soybeans make large adjustments to the proteome during seed filling and compensate for the shortage of major proteins with the increased selective accumulation of other proteins that maintains a normal protein content. | [ Monica A.Schmidt, W. BradBarbazuk, MichaelSandford, GregMay, ZhihongSong, WenxuZhou, Basil J.Nikolau, Eliot M.Herman] | Plant Physiology | 2011-3-10 | |
| pmc3067812 | Role for Cohesin in the Formation of a Heterochromatic Domain at Fission Yeast Subtelomeres▿ | Increasing evidence implicates cohesin in the control of gene expression. Here we report the first analysis of cohesin-dependent gene regulation in fission yeast. Global expression profiling of the mis4-367 cohesin loader mutant identified a small number of upregulated and downregulated genes within subtelomeric domains (SD). These 20- to 40-kb regions between chromosome arm euchromatin and telomere-proximal heterochromatin are characterized by a combination of euchromatin (methylated lysine 4 on histone H3/methylated Tysine 9 on histone H3 [H3K4me]) and heterochromatin (H3K9me) marks. We focused our analysis on the chromosome 1 right SD, which contains several upregulated genes and is bordered on the telomere-distal side by a pair of downregulated genes. We find that the expression changes in the SD also occur in a mutant of the cohesin core component Rad21. Remarkably, mutation of Rad21 results in the depletion of Swi6 binding in the SD. In fact, the Rad21 mutation phenocopied Swi6 loss of function: both mutations led to reduced cohesin binding, reduced H3K9me, and similar gene expression changes in the SD. In particular, expression of the gene pair bordering the SD was dependent both on cohesin and on Swi6. Our data indicate that cohesin participates in the setup of a subtelomeric heterochromatin domain and controls the expression of the genes residing in that domain. | [ SoniaDheur, Sven J.Saupe, SylvieGenier, StéphanieVazquez, Jean-PaulJaverzat] | Molecular and Cellular Biology | 2010-12-28 | |
| pmc2987832 | The Parental Non-Equivalence of Imprinting Control Regions during Mammalian Development and Evolution | In mammals, imprinted gene expression results from the sex-specific methylation of imprinted control regions (ICRs) in the parental germlines. Imprinting is linked to therian reproduction, that is, the placenta and imprinting emerged at roughly the same time and potentially co-evolved. We assessed the transcriptome-wide and ontology effect of maternally versus paternally methylated ICRs at the developmental stage of setting of the chorioallantoic placenta in the mouse (8.5dpc), using two models of imprinting deficiency including completely imprint-free embryos. Paternal and maternal imprints have a similar quantitative impact on the embryonic transcriptome. However, transcriptional effects of maternal ICRs are qualitatively focused on the fetal-maternal interface, while paternal ICRs weakly affect non-convergent biological processes, with little consequence for viability at 8.5dpc. Moreover, genes regulated by maternal ICRs indirectly influence genes regulated by paternal ICRs, while the reverse is not observed. The functional dominance of maternal imprints over early embryonic development is potentially linked to selection pressures favoring methylation-dependent control of maternal over paternal ICRs. We previously hypothesized that the different methylation histories of ICRs in the maternal versus the paternal germlines may have put paternal ICRs under higher mutational pressure to lose CpGs by deamination. Using comparative genomics of 17 extant mammalian species, we show here that, while ICRs in general have been constrained to maintain more CpGs than non-imprinted sequences, the rate of CpG loss at paternal ICRs has indeed been higher than at maternal ICRs during evolution. In fact, maternal ICRs, which have the characteristics of CpG-rich promoters, have gained CpGs compared to non-imprinted CpG-rich promoters. Thus, the numerical and, during early embryonic development, functional dominance of maternal ICRs can be explained as the consequence of two orthogonal evolutionary forces: pressure to tightly regulate genes affecting the fetal-maternal interface and pressure to avoid the mutagenic environment of the paternal germline. | [ ReinerSchulz, CharlotteProudhon, Timothy H.Bestor, KathrynWoodfine, Chyuan-ShengLin, Shau-PingLin, MarinePrissette, Rebecca J.Oakey, DéborahBourc'his] | PLoS Genetics | 2010-11-18 | |
| pmc3009859 | Phosphatidylinositol 3-Kinase (PI3K) Signaling via Glycogen Synthase Kinase-3 (Gsk-3) Regulates DNA Methylation of Imprinted Loci* | Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3β, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3β in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci. | [ Anthony P.Popkie, Leigh C.Zeidner, Ashley M.Albrecht, AnthonyD'Ippolito, SigridEckardt, David E.Newsom, JoannaGroden, Bradley W.Doble, BruceAronow, K. JohnMcLaughlin, PeterWhite, Christopher J.Phiel] | The Journal of Biological Chemistry | 2010-11-3 | |
| pmc2952814 | Identification of Aluminum Responsive Genes in Al-Tolerant Soybean Line PI 416937 | Soybean is one of the most aluminum (Al) sensitive plants. The complex inheritance of Al tolerance trait has so far undermined breeding efforts to develop Al-tolerant soybeans. Discovering the genetic factors underlying the Al tolerance mechanisms would undoubtedly accelerate the pace of such endeavor. As a first step toward this goal, we analyzed the transcriptome profile in roots of Al-tolerant soybean line PI 416937 comparing Al-treated and untreated control plants using DNA microarrays. Many genes involved in transcription activation, stress response, cell metabolism and signaling were differentially expressed. Patterns of gene expression and mechanisms of Al toxicity and tolerance suggest that Cys2His2 and ADR6 transcription activators, cell wall modifying enzymes, and phytosulfokines growth factor play role in soybean Al tolerance. Our data provide insights into the molecular mechanisms of soybean Al tolerance and will have practical value in genetic improvement of Al tolerance trait. | [ DechassaDuressa, KhairySoliman, DongquanChen] | International Journal of Plant Genomics | 2010-10-3 | |
| pmc2934734 | Hand2 ensures an appropriate environment for cardiac fusion by limiting Fibronectin function | Heart formation requires the fusion of bilateral cardiomyocyte populations as they move towards the embryonic midline. The bHLH transcription factor Hand2 is essential for cardiac fusion; however, the effector genes that execute this function of Hand2 are unknown. Here, we provide in zebrafish the first evidence for a downstream component of the Hand2 pathway that mediates cardiac morphogenesis. Although hand2 is expressed in cardiomyocytes, mosaic analysis demonstrates that it plays a non-autonomous role in regulating cardiomyocyte movement. Gene expression profiles reveal heightened expression of fibronectin 1 (fn1) in hand2 mutant embryos. Reciprocally, overexpression of hand2 leads to decreased Fibronectin levels. Furthermore, reduction of fn1 function enables rescue of cardiac fusion in hand2 mutants: bilateral cardiomyocyte populations merge and exhibit improved tissue architecture, albeit without major changes in apicobasal polarity. Together, our data provide a novel example of a tissue creating a favorable environment for its morphogenesis: the Hand2 pathway establishes an appropriate environment for cardiac fusion through negative modulation of Fn1 levels. | [ Zayra V.Garavito-Aguilar, Heather E.Riley, DeborahYelon] | Development (Cambridge, England) | | |
| pmc2945943 | Differential detection of alternatively spliced variants of Ciz1 in normal and cancer cells using a custom exon-junction microarray | Ciz1 promotes initiation of mammalian DNA replication and is present within nuclear matrix associated DNA replication factories. Depletion of Ciz1 from normal and cancer cells restrains entry to S phase and inhibits cell proliferation. Several alternative splicing events with putative functional consequences have been identified and reported, but many more variants are predicted to exist based on publicly available mRNAs and expressed sequence tags.MethodsHere we report the development and validation of a custom exon and exon-junction microarray focused on the human CIZ1 gene, capable of reproducible detection of differential splice-variant expression.ResultsUsing a pair of paediatric cancer cell lines and a pool of eight normal lines as reference, the array identified expected and novel CIZ1 splicing events. One novel variant (delta 8-12) that encodes a predicted protein lacking key functional sites, was validated by quantitative RT-PCR and found to be over-represented in a range of other cancer cell lines, and over half of a panel of primary lung tumours.ConclusionsExpression of CIZ1 delta 8-12 appears to be restricted to cancer cells, and may therefore be a useful novel biomarker | [ Faisal ARahman, NaveedAziz, DawnCoverley] | BMC Cancer | 2010-9-10 | |
| pmc3012546 | During EPO or anemia challenge, erythroid progenitor cells transit through a selectively expandable proerythroblast pool | Investigations of bone marrow (BM) erythroblast development are important for clinical concerns but are hindered by progenitor cell and tissue availability. We therefore sought to more specifically define dynamics, and key regulators, of the formation of developing BM erythroid cell cohorts. A unique Kit−CD71highTer119− “stage E2” proerythroblast pool first is described, which (unlike its Kit+ “stage E1” progenitors, or maturing Ter119+ “stage E3” progeny) proved to selectively expand ∼ 7-fold on erythropoietin challenge. During short-term BM transplantation, stage E2 proerythroblasts additionally proved to be a predominantly expanded progenitor pool within spleen. This E1→E2→E3 erythroid series reproducibly formed ex vivo, enabling further characterizations. Expansion, in part, involved E1 cell hyperproliferation together with rapid E2 conversion plus E2 stage restricted BCL2 expression. Possible erythropoietin/erythropoietin receptor proerythroblast stage specific events were further investigated in mice expressing minimal erythropoietin receptor alleles. For a hypomorphic erythropoietin receptor-HM allele, major defects in erythroblast development occurred selectively at stage E2. In addition, stage E2 cells proved to interact productively with primary BM stromal cells in ways that enhanced both survival and late-stage development. Overall, findings reveal a novel transitional proerythroblast compartment that deploys unique expansion devices. | [ ArvindDev, JingFang, PradeepSathyanarayana, AnamikaPradeep, ChristineEmerson, Don M.Wojchowski] | Blood | | |
| pmc2943559 | Homeodomain only protein is required for the function of induced regulatory T cells in dendritic cell-mediated peripheral T cell unresponsiveness | Induced T regulatory (iTreg) cells can be generated by peripheral dendritic cells (DCs) that mediate T cell-unresponsiveness to re-challenge with antigen. The molecular factors required for the function of such iTreg cells remain unknown. We report a critical role for the transcription co-factor Homeodomain only protein (Hop, also know as Hopx) in iTregs cells to mediate T cell unresponsiveness in vivo. Hopx-sufficient iTreg cells down-regulate the expression of the AP-1 complex and suppress other T cells. In the absence of Hopx, iTreg cells express high levels of the AP-1 complex, proliferate and fail to mediate T cell-unresponsiveness to re-challenge with antigen. Thus, Hopx is required for the function of Treg cells induced by DCs and the promotion of DC-mediated T cell unresponsiveness in vivo. | [ DanielHawiger, Yisong Y.Wan, Elizabeth E.Eynon, Richard A.Flavell] | Nature immunology | 2010-8-29 | |
| pmc2950110 | Introduction of a human-specific deletion in mouse Cmah increases disease severity in the mdx model of Duchenne muscular dystrophy | The evolution of humans included introduction of an inactivating deletion in the CMAH gene, which eliminated biosynthesis of N-glycolylneuraminic acid from all human cells. Here we show that this human-specific sialylation change contributes to the marked discrepancy in phenotype between the mdx mouse model for Duchenne muscular dystrophy (DMD) and the human disease. Despite lacking dystrophin protein in almost all muscle cells, mdx mice show slower development, relative to overall lifespan, or reduced severity of a number of clinically relevant disease phenotypes compared to DMD patients. This is especially true for loss of ambulation, cardiac and respiratory muscle weakness, and loss of lifespan, all major phenotypes contributing to DMD morbidity and mortality. All these phenotypes occur at an earlier age or to a greater degree in mdx mice bearing a human-like mutation in the mouse Cmah gene. Altered phenotypes correlate with changes in two mechanisms; reduced strength and expression of the dystrophin-associated glycoprotein complex and increased activation of complement. Activation of complement may be driven by the increased expression of anti-Neu5Gc antibodies in Cmah−/−mdx animals and ultimately by uptake of N-glycolylneuraminic acid, a foreign glycan in humans and Cmah-deficient mice, from dietary sources. Cmah-deficient mdx mice represent a new small animal model for DMD that better approximates the human glycome and its contributions to muscular dystrophy. | [ KumaranChandrasekharan, Jung HaeYoon, YingXu, SarahdeVries, MarybethCamboni, Paulus M.L.Janssen, AjitVarki, Paul T.Martin] | Science translational medicine | | |
| pmc3005533 | Xenomitochondrial Mice: Investigation into Mitochondrial Compensatory Mechanisms | Xenomitochondrial mice, harboring evolutionarily divergent Mus terricolor mitochondrial DNA (mtDNA) on a Mus musculus domesticus nuclear background (B6NTac(129S6)-mtM. terricolor/Capt; line D7), were subjected to molecular and phenotypic analyses. No overt in vivo phenotype was identified in contrast to in vitro xenomitochondrial cybrid studies. Microarray analyses revealed differentially expressed genes in xenomitochondrial mice, though none were directly involved in mitochondrial function. qRT-PCR revealed upregulation of mt-Co2 in xenomitochondrial mice. These results illustrate that cellular compensatory mechanisms for mild mitochondrial dysfunction alter mtDNA gene expression at a proteomic and/or translational level. Understanding these mechanisms will facilitate development of therapeutics for mitochondrial disorders. | [ M.V.Cannon, D.A.Dunn, M.H.Irwin, A.I.Brooks, F.F.Bartol, I.A.Trounce, C.A.Pinkert] | Mitochondrion | 2010-7-16 | |
| pmc2945597 | Towards an Optimized Culture Medium for the Generation of Mouse Induced Pluripotent Stem Cells* | Generation of induced pluripotent stem cells from somatic cells using defined factors has potential relevant applications in regenerative medicine and biology. However, this promising technology remains inefficient and time consuming. We have devised a serum free culture medium termed iSF1 that facilitates the generation of mouse induced pluripotent stem cells. This optimization of the culture medium is sensitive to the presence of Myc in the reprogramming factors. Moreover, we could reprogram meningeal cells using only two factors Oct4/Klf4. Therefore, iSF1 represents a basal medium that may be used for mechanistic studies and testing new reprogramming approaches. | [ JiekaiChen, JingLiu, QingkaiHan, DajiangQin, JianyongXu, YouChen, JiaqiYang, HongSong, DongshanYang, MeixiuPeng, WenzhiHe, RonghuiLi, HaoWang, YiGan, KeDing, LingwenZeng, LiangxueLai, Miguel A.Esteban, DuanqingPei] | The Journal of Biological Chemistry | 2010-7-1 | |
| pmc2918996 | Elevated Cyclic AMP Levels in T Lymphocytes Transformed by Human T-Cell Lymphotropic Virus Type 1▿ | Human T-cell lymphotropic virus type 1 (HTLV-1), the cause of adult T-cell leukemia/lymphoma (ATLL), transforms CD4+ T cells to permanent growth through its transactivator Tax. HTLV-1-transformed cells share phenotypic properties with memory and regulatory T cells (T-reg). Murine T-reg-mediated suppression employs elevated cyclic AMP (cAMP) levels as a key regulator. This led us to determine cAMP levels in HTLV-1-transformed cells. We found elevated cAMP concentrations as a consistent feature of all HTLV-1-transformed cell lines, including in vitro-HTLV-1-transformed, Tax-transformed, and patient-derived cells. In transformed cells with conditional Tax expression, high cAMP levels coincided with the presence of Tax but were lost without it. However, transient ectopic expression of Tax alone was not sufficient to induce cAMP. We found specific downregulation of the cAMP-degrading phosphodiesterase 3B (PDE3B) in HTLV-1-transformed cells, which was independent of Tax in transient expression experiments. This is in line with the notion that PDE3B transcripts and cAMP levels are inversely correlated. Overexpression of PDE3B led to a decrease of cAMP in HTLV-1-transformed cells. Decreased expression of PDE3B was associated with inhibitory histone modifications at the PDE3B promoter and the PDE3B locus. In summary, Tax transformation and its continuous expression contribute to elevated cAMP levels, which may be regulated through PDE3B suppression. This shows that HTLV-1-transformed cells assume biological features of long-lived T-cell populations that potentially contribute to viral persistence. | [ Andrea K.Kress, GritSchneider, KlemensPichler, MartinaKalmer, BernhardFleckenstein, RalphGrassmann] | Journal of Virology | 2010-6-23 | |
| pmc2919000 | Molecular Characterization of In Vivo Adjuvant Activity in Ferrets Vaccinated against Influenza Virus▿ † | The 2009 H1N1 influenza pandemic has prompted a significant need for the development of efficient, single-dose, adjuvanted vaccines. Here we investigated the adjuvant potential of CpG oligodeoxynucleotide (ODN) when used with a human seasonal influenza virus vaccine in ferrets. We found that the CpG ODN-adjuvanted vaccine effectively increased antibody production and activated type I interferon (IFN) responses compared to vaccine alone. Based on these findings, pegylated IFN-α2b (PEG-IFN) was also evaluated as an adjuvant in comparison to CpG ODN and complete Freund's adjuvant (CFA). Our results showed that all three vaccines with adjuvant added prevented seasonal human A/Brisbane/59/2007 (H1N1) virus replication more effectively than did vaccine alone. Gene expression profiles indicated that, as well as upregulating IFN-stimulated genes (ISGs), CpG ODN enhanced B-cell activation and increased Toll-like receptor 4 (TLR4) and IFN regulatory factor 4 (IRF4) expression, whereas PEG-IFN augmented adaptive immunity by inducing major histocompatibility complex (MHC) transcription and Ras signaling. In contrast, the use of CFA as an adjuvant induced limited ISG expression but increased the transcription of MHC, cell adhesion molecules, and B-cell activation markers. Taken together, our results better characterize the specific molecular pathways leading to adjuvant activity in different adjuvant-mediated influenza virus vaccinations. | [ YuanFang, ThomasRowe, Alberto J.Leon, DavidBanner, AliDanesh, LuolingXu, LongsiRan, Steven E.Bosinger, YiGuan, HonglinChen, Cheryl C.Cameron, Mark J.Cameron, David J.Kelvin] | Journal of Virology | 2010-6-9 | |
| pmc2890984 | Quantitative trait locus analysis identifies Gabra3 as a regulator of behavioral despair in mice | The Tail Suspension Test (TST), which measures behavioral despair, is widely used as an animal model of human depressive disorders and antidepressant efficacy. In order to identify novel genes involved in the regulation of TST performance, we crossed an inbred strain exhibiting low immobility in the TST (RIIIS/J) with two high-immobility strains (C57BL/6J and NZB/BlNJ) to create two distinct F2 hybrid populations. All F2 offspring (n = 655) were genotyped at high density with a panel of SNP markers. Whole-genome interval mapping of the F2 populations identified statistically significant quantitative trait loci (QTLs) on mouse chromosomes (MMU) 4, 6, and X. Microarray analysis of hippocampal gene expression in the three parental strains was used to identify potential candidate genes within the MMUX QTLs identified in the NZB/BlNJ × RIIIS/J cross. Expression of Gabra3, which encodes the GABAA receptor α3 subunit, was robust in the hippocampus of B6 and RIIIS mice but absent from NZB hippocampal tissue. To verify the role of Gabra3 in regulating TST behavior in vivo, mice were treated with SB-205384, a positive modulator of the α3 subunit. SB-205384 significantly reduced TST immobility in B6 mice without affecting general activity, but it had no effect on behavior in NZB mice. This work suggests that GABRA3 regulates a behavioral endophenotype of depression and establishes this gene as a viable new target for the study and treatment of human depression.The online version of this article (doi:10.1007/s00335-010-9266-6) contains supplementary material, which is available to authorized users. | [ Brooke H.Miller, Laura E.Schultz, Bradley C.Long, Mathew T.Pletcher] | Mammalian Genome | 2010-5-29 | |
| pmc2872667 | Skeletal Site-Related Variation in Human Trabecular Bone Transcriptome and Signaling | The skeletal site-specific influence of multiple genes on bone morphology is recognised, but the question as to how these influences may be exerted at the molecular and cellular level has not been explored.MethodologyTo address this question, we have compared global gene expression profiles of human trabecular bone from two different skeletal sites that experience vastly different degrees of mechanical loading, namely biopsies from iliac crest and lumbar spinal lamina.Principal FindingsIn the lumbar spine, compared to the iliac crest, the majority of the differentially expressed genes showed significantly increased levels of expression; 3406 transcripts were up- whilst 838 were down-regulated. Interestingly, all gene transcripts that have been recently demonstrated to be markers of osteocyte, as well as osteoblast and osteoclast-related genes, were markedly up-regulated in the spine. The transcriptome data is consistent with osteocyte numbers being almost identical at the two anatomical sites, but suggesting a relatively low osteocyte functional activity in the iliac crest. Similarly, osteoblast and osteoclast expression data suggested similar numbers of the cells, but presented with higher activity in the spine than iliac crest. This analysis has also led to the identification of expression of a number of transcripts, previously known and novel, which to our knowledge have never earlier been associated with bone growth and remodelling.Conclusions and SignificanceThis study provides molecular evidence explaining anatomical and micro-architectural site-related changes in bone cell function, which is predominantly attributable to alteration in cell transcriptional activity. A number of novel signaling molecules in critical pathways, which have been hitherto not known to be expressed in bone cells of mature vertebrates, were identified. | [ Satya S.Varanasi, Ole K.Olstad, Daniel C.Swan, PaulSanderson, Vigdis T.Gautvik, SjurReppe, Roger M.Francis, Kaare M.Gautvik, Harish K.Datta] | PLoS ONE | 2010-5-18 | |
| pmc2855344 | Oral Activated Charcoal Prevents Experimental Cerebral Malaria in Mice and in a Randomized Controlled Clinical Trial in Man Did Not Interfere with the Pharmacokinetics of Parenteral Artesunate | Safe, cheap and effective adjunct therapies preventing the development of, or reducing the mortality from, severe malaria could have considerable and rapid public health impact. Oral activated charcoal (oAC) is a safe and well tolerated treatment for acute poisoning, more recently shown to have significant immunomodulatory effects in man. In preparation for possible efficacy trials in human malaria, we sought to determine whether oAC would i) reduce mortality due to experimental cerebral malaria (ECM) in mice, ii) modulate immune and inflammatory responses associated with ECM, and iii) affect the pharmacokinetics of parenteral artesunate in human volunteers.Methods/Principal FindingsWe found that oAC provided significant protection against P. berghei ANKA-induced ECM, increasing overall survival time compared to untreated mice (p<0.0001; hazard ratio 16.4; 95% CI 6.73 to 40.1). Protection from ECM by oAC was associated with reduced numbers of splenic TNF+ CD4+ T cells and multifunctional IFNγ+TNF+ CD4+ and CD8+ T cells. Furthermore, we identified a whole blood gene expression signature (68 genes) associated with protection from ECM. To evaluate whether oAC might affect current best available anti-malarial treatment, we conducted a randomized controlled open label trial in 52 human volunteers (ISRCTN NR. 64793756), administering artesunate (AS) in the presence or absence of oAC. We demonstrated that co-administration of oAC was safe and well-tolerated. In the 26 subjects further analyzed, we found no interference with the pharmacokinetics of parenteral AS or its pharmacologically active metabolite dihydroartemisinin.Conclusions/SignificanceoAC protects against ECM in mice, and does not interfere with the pharmacokinetics of parenteral artesunate. If future studies succeed in establishing the efficacy of oAC in human malaria, then the characteristics of being inexpensive, well-tolerated at high doses and requiring no sophisticated storage would make oAC a relevant candidate for adjunct therapy to reduce mortality from severe malaria, or for immediate treatment of suspected severe malaria in a rural setting.Trial RegistrationControlled-Trials.com ISRCTN64793756 | [ J. Briande Souza, UduakOkomo, Neal D.Alexander, NaveedAziz, Benjamin M. J.Owens, HarparkashKaur, MomodouJasseh, SantMuangnoicharoen, Percy F.Sumariwalla, David C.Warhurst, Stephen A.Ward, David J.Conway, LuisUlloa, Kevin J.Tracey, Brian M. J.Foxwell, Paul M.Kaye, MichaelWalther] | PLoS ONE | 2010-4-15 | |
| pmc2934885 | Adrenoceptors in Brain: Cellular Gene Expression and Effects on Astrocytic Metabolism and [Ca2+]i | Recent in vivo studies have established astrocytes as a major target for locus coeruleus activation (Bekar et al., Cereb. Cortex 18, 2789–2795), renewing interest in cell culture studies on noradrenergic effects on astrocytes in primary cultures and calling for additional information about the expression of adrenoceptor subtypes on different types of brain cells. In the present communication, mRNA expression of α1-, α2- and β-adrenergic receptors and their subtypes was determined in freshly-isolated, cell marker-defined populations of astrocytes, NG2-positive cells, microglia, endothelial cells, and Thy1-positive neurons (mainly glutamatergic projection neurons) in murine cerebral cortex. Immediately after dissection of frontal, parietal and occipital cortex of 10–12-week-old transgenic mice, which combined each cell-type marker with a specific fluorescent signal, the tissue was digested, triturated and centrifuged, yielding a solution of dissociated cells of all types, which were separated by fluorescence-activated cell sorting (FACS). mRNA expression in each cell fraction was determined by microarray analysis. α1A-Receptors were unequivocally expressed in astrocytes and NG2-positive cells, but absent in other cell types, and α1B-receptors were not expressed in any cell population. Among α2-receptors only α2A-receptors were expressed, unequivocally in astrocytes and NG-positive cells, tentatively in microglia and questionably in Thy1-positive neurons and endothelial cells. β1-Receptors were unequivocally expressed in astrocytes, tentatively in microglia, and questionably in neurons and endothelial cells, whereas β2-adrenergic receptors showed tentative expression in neurons and astrocytes and unequivocal expression in other cell types. This distribution was supported by immunochemical data and its relevance established by previous studies in well-differentiated primary cultures of mouse astrocytes, showing that stimulation of α2-adrenoceptors increases glycogen formation and oxidative metabolism, the latter by a mechanism depending on intramitochondrial Ca2+, whereas α1-adrenoceptor stimulation enhances glutamate uptake, and β-adrenoceptor activation causes glycogenolysis and increased Na+,K+-ATPase activity. The Ca2+- and cAMP-mediated association between energy-consuming and energy-yielding processes is emphasized. | [ LeifHertz, DitteLovatt, Steven A.Goldman, MaikenNedergaard] | Neurochemistry international | 2010-4-7 | |
| pmc2847749 | Examination of transcriptional networks reveals an important role for TCFAP2C, SMARCA4, and EOMES in trophoblast stem cell maintenance | Trophoblast stem cells (TS cells), derived from the trophectoderm (TE) of blastocysts, require transcription factors (TFs) and external signals (FGF4, INHBA/NODAL/TGFB1) for self-renewal. While many reports have focused on TF networks that regulate embryonic stem cell (ES cell) self-renewal and pluripotency, little is know about TF networks that regulate self-renewal in TS cells. To further understand transcriptional networks in TS cells, we used chromatin immunoprecipitation with DNA microarray hybridization (ChIP-chip) analysis to investigate targets of the TFs—TCFAP2C, EOMES, ETS2, and GATA3—and a chromatin remodeling factor, SMARCA4. We then evaluated the transcriptional states of target genes using transcriptome analysis and genome-wide analysis of histone H3 acetylation (AcH3). Our results describe previously unknown transcriptional networks in TS cells, including TF occupancy of genes involved in ES cell self-renewal and pluripotency, co-occupancy of TCFAP2C, SMARCA4, and EOMES at a significant number of genes, and transcriptional regulatory circuitry within the five factors. Moreover, RNAi depletion of Tcfap2c, Smarca4, and Eomes transcripts resulted in a loss of normal colony morphology and down-regulation of TS cell–specific genes, suggesting an important role for TCFAP2C, SMARCA4, and EOMES in TS cell self-renewal. Through genome-wide mapping and global expression analysis of five TF target genes, our data provide a comprehensive analysis of transcriptional networks that regulate TS cell self-renewal. | [ Benjamin L.Kidder, StephenPalmer] | Genome Research | | |
| pmc2837405 | Innate Killing of Leishmania donovani by Macrophages of the Splenic Marginal Zone Requires IRF-7 | Highly phagocytic macrophages line the marginal zone (MZ) of the spleen and the lymph node subcapsular sinus. Although these macrophages have been attributed with a variety of functions, including the uptake and clearance of blood and lymph-borne pathogens, little is known about the effector mechanisms they employ after pathogen uptake. Here, we have combined gene expression profiling and RNAi using a stromal macrophage cell line with in situ analysis of the leishmanicidal activity of marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM) in wild type and gene targeted mice. Our data demonstrate a critical role for interferon regulatory factor-7 (IRF-7) in regulating the killing of intracellular Leishmania donovani by these specialised splenic macrophage sub-populations. This study, therefore, identifies a new role for IRF-7 as a regulator of innate microbicidal activity against this, and perhaps other, non-viral intracellular pathogens. This study also highlights the importance of selecting appropriate macrophage populations when studying pathogen interactions with this functionally diverse lineage of cells. | [ RebeccaPhillips, MattiasSvensson, NaveedAziz, AsherMaroof, NajmeeyahBrown, LynetteBeattie, NathalieSignoret, Paul M.Kaye] | PLoS Pathogens | 2010-3-12 | |
| pmc2913550 | Oxygen-sensitive outcomes and gene expression in acute ischemic stroke | Acute ischemic stroke (AIS) results in focal deprivation of blood-borne factors, one of them being oxygen. The purpose of this study was two-fold: (1) to identify therapeutic conditions for supplemental oxygen in AIS and (2) to use transcriptome-wide screening toward uncovering oxygen-sensitive mechanisms. Transient MCAO in rodents was used to delineate the therapeutic potential of normobaric (NBO, 100% O2, 1ATA) and hyperbaric oxygen (HBO, 100% O2, 2ATA) during ischemia (iNBO, iHBO) and after reperfusion (rNBO, rHBO). Stroke lesion was quantified using 4.7 T MRI at 48 h. Supplemental oxygen during AIS significantly attenuated percent stroke hemisphere lesion volume as compared with that in room air (RA) controls, whereas identical treatment immediately after reperfusion exacerbated lesion volume (RA=22.4±1.8, iNBO=9.9±3.6, iHBO=6.6±4.8, rNBO=29.8±3.6, rHBO=35.4±7.6). iNBO and iHBO corrected penumbra tissue pO2 during AIS as measured by EPR oxymetry. Unbiased query of oxygen-sensitive transcriptome in stroke-affected tissue identified 5,769 differentially expressed genes. Candidate genes were verified by real-time PCR using neurons laser-captured from the stroke-affected somatosensory cortex. Directed microarray analysis showed that supplemental oxygen limited leukocyte accumulation to the infarct site by attenuation of stroke-inducible proinflammatory chemokine response. The findings provide key information relevant to understanding oxygen-dependent molecular mechanisms in the AIS-affected brain. | [ CameronRink, SashwatiRoy, MahmoodKhan, PavanAnanth, PeriannanKuppusamy, Chandan KSen, SavitaKhanna] | Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism | 2010-02-10 | |
| pmc2849450 | Global Change of Gene Expression and Cell Physiology in YidC-Depleted Escherichia coli▿ † | YidC depletion affects membrane protein insertion and leads to a defect in the growth of the Escherichia coli cell. We analyzed global changes in gene expression upon YidC depletion to determine the importance of YidC for cellular functions using a gene chip method to compare the transcriptomes of JS71 (control) and JS7131 (yidC depletion strain). Of the more than 4,300 genes identified, 163 were upregulated and 99 were downregulated upon YidC depletion, including genes which are responsible for DNA/RNA repair; energy metabolism; various transporters, proteases and chaperones; stress response; and translation and transcription functions. Real-time PCR was performed on selected genes to confirm the results. Specifically, we found upregulation of the genes encoding the energy transduction proteins F1Fo ATP synthase and cytochrome bo3 oxidase due to perturbation in assembly when YidC was depleted. We also determined that the high-level induction of the PspA stress protein under YidC depletion conditions is roughly 10-fold higher than the activation due to the addition of protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), which dissipates the proton motive force. In addition, the gene chip data reveal the Cpx stress pathway is activated upon YidC depletion. The data show the broad physiological contribution of YidC to the bacterial cell and the considerable ramification to the cell when it is depleted. | [ PengWang, AndreasKuhn, Ross E.Dalbey] | Journal of Bacteriology | 2010-1-8 | |
| pmc2808307 | Differentially expressed alternatively spliced genes in Malignant Pleural Mesothelioma identified using massively parallel transcriptome sequencing | Analyses of Expressed Sequence Tags (ESTs) databases suggest that most human genes have multiple alternative splice variants. The alternative splicing of pre-mRNA is tightly regulated during development and in different tissue types. Changes in splicing patterns have been described in disease states. Recently, we used whole-transcriptome shotgun pryrosequencing to characterize 4 malignant pleural mesothelioma (MPM) tumors, 1 lung adenocarcinoma and 1 normal lung. We hypothesized that alternative splicing profiles might be detected in the sequencing data for the expressed genes in these samples.MethodsWe developed a software pipeline to map the transcriptome read sequences of the 4 MPM samples and 1 normal lung sample onto known exon junction sequences in the comprehensive AceView database of expressed sequences and to count how many reads map to each junction. 13,274,187 transcriptome reads generated by the Roche/454 sequencing platform for 5 samples were compared with 151,486 exon junctions from the AceView database. The exon junction expression index (EJEI) was calculated for each exon junction in each sample to measure the differential expression of alternative splicing events. Top ten exon junctions with the largest EJEI difference between the 4 mesothelioma and the normal lung sample were then examined for differential expression using Quantitative Real Time PCR (qRT-PCR) in the 5 sequenced samples. Two of the differentially expressed exon junctions (ACTG2.aAug05 and CDK4.aAug05) were further examined with qRT-PCR in additional 18 MPM and 18 normal lung specimens.ResultsWe found 70,953 exon junctions covered by at least one sequence read in at least one of the 5 samples. All 10 identified most differentially expressed exon junctions were validated as present by RT-PCR, and 8 were differentially expressed exactly as predicted by the sequence analysis. The differential expression of the AceView exon junctions for the ACTG2 and CDK4 genes were also observed to be statistically significant in an additional 18 MPM and 18 normal lung samples examined using qRT-PCR. The differential expression of these two junctions was shown to successfully classify these mesothelioma and normal lung specimens with high sensitivity (89% and 78%, respectively).ConclusionWhole-transcriptome shotgun sequencing, combined with a downstream bioinformatics pipeline, provides powerful tools for the identification of differentially expressed exon junctions resulting from alternative splice variants. The alternatively spliced genes discovered in the study could serve as useful diagnostic markers as well as potential therapeutic targets for MPM. | [ LingshengDong, Roderick VJensen, AssuntaDe Rienzo, Gavin JGordon, YanlongXu, David JSugarbaker, RaphaelBueno] | BMC Medical Genetics | 2009-12-31 | |
| pmc2795170 | Fgfr3 Is a Transcriptional Target of Ap2δ and Ash2l-Containing Histone Methyltransferase Complexes | Polycomb (PcG) and trithorax (trxG) proteins play important roles in establishing lineage-specific genetic programs through induction of chromatin modifications that lead to gene silencing or activation. Previously, we described an association between the MLL/SET1 complexes and a highly restricted, gene-specific DNA-binding protein Ap2δ that is required for recruitment of the MLL/SET1 complex to target Hoxc8 specifically. Here, we reduced levels of Ap2δ and Ash2l in the neuroblastoma cell line, Neuro2A, and analyzed their gene expression profiles using whole-genome mouse cDNA microarrays. This analysis yielded 42 genes that are potentially co-regulated by Ap2δ and Ash2l, and we have identified evolutionarily conserved Ap2-binding sites in 20 of them. To determine whether some of these were direct targets of the Ap2δ-Ash2l complex, we analyzed several promoters for the presence of Ap2δ and Ash2l by chromatin immunoprecipitation (ChIP). Among the targets we screened, we identified Fgfr3 as a direct transcriptional target of the Ap2δ-Ash2l complex. Additionally, we found that Ap2δ is necessary for the recruitment of Ash2l-containing complexes to this promoter and that this recruitment leads to trimethylation of lysine 4 of histone H3 (H3K4me3). Thus, we have identified several candidate targets of complexes containing Ap2δ and Ash2l that can be used to further elucidate their roles during development and showed that Fgfr3 is a novel direct target of these complexes. | [ Cheryl C.Tan, Martin J.Walsh, Bruce D.Gelb] | PLoS ONE | 2009-12-31 | |
| pmc2809955 | Cytokines Interleukin-1β and Tumor Necrosis Factor-α Regulate Different Transcriptional and Alternative Splicing Networks in Primary β-Cells | Cytokines contribute to pancreatic β-cell death in type 1 diabetes. This effect is mediated by complex gene networks that remain to be characterized. We presently utilized array analysis to define the global expression pattern of genes, including spliced variants, modified by the cytokines interleukin (IL)-1β + interferon (IFN)-γ and tumor necrosis factor (TNF)-α + IFN-γ in primary rat β-cells.RESEARCH DESIGN AND METHODSFluorescence-activated cell sorter–purified rat β-cells were exposed to IL-1β + IFN-γ or TNF-α + IFN-γ for 6 or 24 h, and global gene expression was analyzed by microarray. Key results were confirmed by RT-PCR, and small-interfering RNAs were used to investigate the mechanistic role of novel and relevant transcription factors identified by pathway analysis.RESULTSNearly 16,000 transcripts were detected as present in β-cells, with temporal differences in the number of genes modulated by IL-1β + IFNγ or TNF-α + IFN-γ. These cytokine combinations induced differential expression of inflammatory response genes, which is related to differential induction of IFN regulatory factor-7. Both treatments decreased the expression of genes involved in the maintenance of β-cell phenotype and growth/regeneration. Cytokines induced hypoxia-inducible factor-α, which in this context has a proapoptotic role. Cytokines also modified the expression of >20 genes involved in RNA splicing, and exon array analysis showed cytokine-induced changes in alternative splicing of >50% of the cytokine-modified genes.CONCLUSIONSThe present study doubles the number of known genes expressed in primary β-cells, modified or not by cytokines, and indicates the biological role for several novel cytokine-modified pathways in β-cells. It also shows that cytokines modify alternative splicing in β-cells, opening a new avenue of research for the field. | [ FernandaOrtis, NajibNaamane, DaisyFlamez, LaurenceLadrière, FabriceMoore, Daniel A.Cunha, Maikel L.Colli, ThomasThykjaer, KasperThorsen, Torben F.Ørntoft, Decio L.Eizirik] | Diabetes | 2009-11-23 | |
| pmc2950308 | Development of Potential Pharmacodynamic and Diagnostic Markers for Anti-IFN-α Monoclonal Antibody Trials in Systemic Lupus Erythematosus | To identify potential pharmacodynamic biomarkers to guide dose selection in clinical trials using anti-interferon-alpha (IFN-α) monoclonal antibody (mAb) therapy for systemic lupus erythematosus (SLE), we used an Affymetrix human genome array platform and identified 110 IFN-α/β-inducible transcripts significantly upregulated in whole blood (WB) of 41 SLE patients. The overexpression of these genes was confirmed prospectively in 54 additional SLE patients and allowed for the categorization of the SLE patients into groups of high, moderate, and weak overexpressers of IFN-α/β-inducible genes. This approach could potentially allow for an accurate assessment of drug target neutralization in early trials of anti-IFN-α mAb therapy for SLE. Furthermore, ex vivo stimulation of healthy donor peripheral blood mononuclear cells with SLE patient serum and subsequent neutralization with anti-IFN-α mAb or anti-IFN-α receptor mAb showed that anti-IFN-α mAb has comparable effects of neutralizing the overexpression of type I IFN-inducible genes as that of anti-IFNAR mAb. These results suggest that IFN-α, and not other members of type I IFN family in SLE patients, is mainly responsible for the induction of type I IFN-inducible genes in WB of SLE patients. Taken together, these data strengthen the view of IFN-α as a therapeutic target for SLE. | [ YihongYao, Brandon W.Higgs, ChrisMorehouse, Melissade los Reyes, WendyTrigona, PhilipBrohawn, WendyWhite, JianliangZhang, BarbaraWhite, Anthony J.Coyle, Peter A.Kiener, BahijaJallal] | Human Genomics and Proteomics : HGP | 2009-11-17 | |
| pmc2842492 | Genomic Profiling of MicroRNAs and Messenger RNAs Reveals Hormonal Regulation in MicroRNA Expression in Human Endometrium1 | MicroRNAs (miRNAs), a class of small noncoding RNAs that regulate gene expression, have fundamental roles in biological processes, including cell differentiation and proliferation. These small molecules mainly direct either target messenger RNA (mRNA) degradation or translational repression, thereby functioning as gene silencers. Epithelial cells of the uterine lumen and glands undergo cyclic changes under the influence of the sex steroid hormones estradiol-17beta and progesterone. Because the expression of miRNAs in human endometrium has been established, it is important to understand whether miRNAs have a physiological role in modulating the expression of hormonally induced genes. The studies herein establish concomitant differential miRNA and mRNA expression profiles of uterine epithelial cells purified from endometrial biopsy specimens in the late proliferative and midsecretory phases. Bioinformatics analysis of differentially expressed mRNAs revealed cell cycle regulation as the most significantly enriched pathway in the late proliferative-phase endometrial epithelium (P = 5.7 × 10−15). In addition, the WNT signaling pathway was enriched in the proliferative phase. The 12 miRNAs (MIR29B, MIR29C, MIR30B, MIR30D, MIR31, MIR193A-3P, MIR203, MIR204, MIR200C, MIR210, MIR582-5P, and MIR345) whose expression was significantly up-regulated in the midsecretory-phase samples were predicted to target many cell cycle genes. Consistent with the role of miRNAs in suppressing their target mRNA expression, the transcript abundance of predicted targets, including cyclins and cyclin-dependent kinases, as well as E2F3 (a known target of MIR210), was decreased. Thus, our findings suggest a role for miRNAs in down-regulating the expression of some cell cycle genes in the secretory-phase endometrial epithelium, thereby suppressing cell proliferation. | [ SatuKuokkanen, BoChen, LaureenOjalvo, LumieBenard, NanetteSantoro, Jeffrey W.Pollard] | Biology of Reproduction | 2009-10-28 | |
| pmc2786556 | Nitric Oxide Signaling in Pseudomonas aeruginosa Biofilms Mediates Phosphodiesterase Activity, Decreased Cyclic Di-GMP Levels, and Enhanced Dispersal▿ † | Bacteria in biofilms often undergo active dispersal events and revert to a free-swimming, planktonic state to complete the biofilm life cycle. The signaling molecule nitric oxide (NO) was previously found to trigger biofilm dispersal in the opportunistic pathogen Pseudomonas aeruginosa at low, nontoxic concentrations (N. Barraud, D. J. Hassett, S. H. Hwang, S. A. Rice, S. Kjelleberg, and J. S. Webb, J. Bacteriol. 188:7344-7353, 2006). NO was further shown to increase cell motility and susceptibility to antimicrobials. Recently, numerous studies revealed that increased degradation of the secondary messenger cyclic di-GMP (c-di-GMP) by specific phosphodiesterases (PDEs) triggers a planktonic mode of growth in eubacteria. In this study, the potential link between NO and c-di-GMP signaling was investigated by performing (i) PDE inhibitor studies, (ii) enzymatic assays to measure PDE activity, and (iii) direct quantification of intracellular c-di-GMP levels. The results suggest a role for c-di-GMP signaling in triggering the biofilm dispersal event induced by NO, as dispersal requires PDE activity and addition of NO stimulates PDE and induces the concomitant decrease in intracellular c-di-GMP levels in P. aeruginosa. Furthermore, gene expression studies indicated global responses to low, nontoxic levels of NO in P. aeruginosa biofilms, including upregulation of genes involved in motility and energy metabolism and downregulation of adhesins and virulence factors. Finally, site-directed mutagenesis of candidate genes and physiological characterization of the corresponding mutant strains uncovered that the chemotaxis transducer BdlA is involved in the biofilm dispersal response induced by NO. | [ NicolasBarraud, DavidSchleheck, JanoschKlebensberger, Jeremy S.Webb, Daniel J.Hassett, Scott A.Rice, StaffanKjelleberg] | Journal of Bacteriology | 2009-10-2 | |
| pmc2743997 | miR-155 Inhibition Sensitizes CD4+ Th Cells for TREG Mediated Suppression | In humans and mice naturally occurring CD4+CD25+ regulatory T cells (nTregs) are a thymus-derived subset of T cells, crucial for the maintenance of peripheral tolerance by controlling not only potentially autoreactive T cells but virtually all cells of the adaptive and innate immune system. Recent work using Dicer-deficient mice irrevocably demonstrated the importance of miRNAs for nTreg cell-mediated tolerance.Principal FindingsDNA-Microarray analyses of human as well as murine conventional CD4+ Th cells and nTregs revealed a strong up-regulation of mature miR-155 (microRNA-155) upon activation in both populations. Studying miR-155 expression in FoxP3-deficient scurfy mice and performing FoxP3 ChIP-Seq experiments using activated human T lymphocytes, we show that the expression and maturation of miR-155 seem to be not necessarily regulated by FoxP3. In order to address the functional relevance of elevated miR-155 levels, we transfected miR-155 inhibitors or mature miR-155 RNAs into freshly-isolated human and mouse primary CD4+ Th cells and nTregs and investigated the resulting phenotype in nTreg suppression assays. Whereas miR-155 inhibition in conventional CD4+ Th cells strengthened nTreg cell-mediated suppression, overexpression of mature miR-155 rendered these cells unresponsive to nTreg cell-mediated suppression.ConclusionInvestigation of FoxP3 downstream targets, certainly of bound and regulated miRNAs revealed the associated function between the master regulator FoxP3 and miRNAs as regulators itself. miR-155 is shown to be crucially involved in nTreg cell mediated tolerance by regulating the susceptibility of conventional human as well as murine CD4+ Th cells to nTreg cell-mediated suppression. | [ Heiko F.Stahl, TanjaFauti, NinaUllrich, TobiasBopp, JanKubach, WernerRust, PaulLabhart, VassiliAlexiadis, ChristianBecker, MathiasHafner, AndreasWeith, Martin C.Lenter, HelmutJonuleit, EdgarSchmitt, DetlevMennerich] | PLoS ONE | 2009-9-24 | |
| pmc2745576 | Identification of an Unusual Pattern of Global Gene Expression in Group B Streptococcus Grown in Human Blood | Because passage of the bacterium to blood is a crucial step in the pathogenesis of many group B Streptococcus (GBS) invasive infections, we recently conducted a whole-genome transcriptome analysis during GBS incubation ex vivo with human blood. In the current work, we sought to analyze in detail the difference in GBS gene expression that occurred in one blood sample (donor A) relative to other blood samples. We incubated GBS strain NEM316 with fresh heparinized human blood obtained from healthy volunteers, and analyzed GBS genome expression and cytokine production. Principal component analysis identified extensive clustering of the transcriptome data among all samples at time 0. In striking contrast, the whole bacterial gene expression in the donor A blood sample was significantly different from the gene expression in all other blood samples studied, both after 30 and 90 min of incubation. More genes were up-regulated in donor A blood relative to the other samples, at 30 min and 90 min. Furthermore, there was significant variation in transcript levels between donor A blood and other blood samples. Notably, genes with the highest transcript levels in donor A blood were those involved in carbohydrate metabolism. We also discovered an unusual production of proinflammatory and immunomodulatory cytokines: MIF, tPAI-1 and IL-1β were produced at higher levels in donor A blood relative to the other blood samples, whereas GM-CSF, TNF-α, IFN-γ, IL-7 and IL-10 remained at lower levels in donor A blood. Potential reasons for our observations are that the immune response of donor A significantly influenced the bacterial transcriptome, or both GBS gene expression and immune response were influenced by the metabolic status of donor A. | [ LaurentMereghetti, IzabelaSitkiewicz, Nicole M.Green, James M.Musser] | PLoS ONE | 2009-9-23 | |
| pmc2786550 | Role of the Transcriptional Regulator RamB (Rv0465c) in the Control of the Glyoxylate Cycle in Mycobacterium tuberculosis▿ † | Mycobacterium tuberculosis generally is assumed to depend on lipids as a major carbon and energy source when persisting within the host. The utilization of fatty acids requires a functional glyoxylate cycle with the key enzymes isocitrate lyase (Icl) and malate synthase. The open reading frame Rv0465c of M. tuberculosis H37Rv encodes a protein with significant sequence similarity to the transcriptional regulator RamB, which in Corynebacterium glutamicum controls the expression of several genes involved in acetate metabolism, i.e., those encoding enzymes of acetate activation and the glyoxylate cycle. We show here that the M. tuberculosis Rv0465c protein can functionally complement RamB in C. glutamicum and that it binds to the promoter regions of M. tuberculosis icl1 and Rv0465c. Construction and subsequent transcriptional and enzymatic analysis of a defined Rv0465c deletion mutant in M. tuberculosis revealed that the Rv0465c protein, now designated RamB, represses icl1 expression during growth with glucose and negatively autoregulates the expression of its own operon. Whole-genome microarray analysis of the M. tuberculosis ramB (ramBMT) mutant and the wild type furthermore showed that apart from icl1 and the ramBMT operon, the expression of all other M. tuberculosis genes involved in acetate metabolism remain unchanged in the mutant. Thus, RamBMT has a more specific regulatory function as RamB from C. glutamicum and is confined to expression control of icl1 and the ramBMT operon. | [ Julia C.Micklinghoff, Katrin J.Breitinger, MaschaSchmidt, RobertGeffers, Bernhard J.Eikmanns, Franz-ChristophBange] | Journal of Bacteriology | 2009-9-18 | |
| pmc2715097 | STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing | Signal transducer and activator of transcription (STAT) 3 is a pleiotropic transcription factor with important functions in cytokine signaling in a variety of tissues. However, the role of STAT3 in the intestinal epithelium is not well understood. We demonstrate that development of colonic inflammation is associated with the induction of STAT3 activity in intestinal epithelial cells (IECs). Studies in genetically engineered mice showed that epithelial STAT3 activation in dextran sodium sulfate colitis is dependent on interleukin (IL)-22 rather than IL-6. IL-22 was secreted by colonic CD11c+ cells in response to Toll-like receptor stimulation. Conditional knockout mice with an IEC-specific deletion of STAT3 activity were highly susceptible to experimental colitis, indicating that epithelial STAT3 regulates gut homeostasis. STAT3IEC-KO mice, upon induction of colitis, showed a striking defect of epithelial restitution. Gene chip analysis indicated that STAT3 regulates the cellular stress response, apoptosis, and pathways associated with wound healing in IECs. Consistently, both IL-22 and epithelial STAT3 were found to be important in wound-healing experiments in vivo. In summary, our data suggest that intestinal epithelial STAT3 activation regulates immune homeostasis in the gut by promoting IL-22–dependent mucosal wound healing. | [ GeethanjaliPickert, ClemensNeufert, MoritzLeppkes, YanZheng, NadineWittkopf, MoritzWarntjen, Hans-AntonLehr, SebastianHirth, BennoWeigmann, StefanWirtz, WenjunOuyang, Markus F.Neurath, ChristophBecker] | The Journal of Experimental Medicine | | |
| pmc2756131 | Early epigenetic changes and DNA damage do not predict clinical response in an overlapping schedule of 5-azacytidine and entinostat in patients with myeloid malignancies | Sequential administration of DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors has demonstrated clinical efficacy in patients with hematologic malignancies. However, the mechanism behind their clinical efficacy remains controversial. In this study, the methylation dynamics of 4 TSGs (p15INK4B, CDH-1, DAPK-1, and SOCS-1) were studied in sequential bone marrow samples from 30 patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) who completed a minimum of 4 cycles of therapy with 5-azacytidine and entinostat. Reversal of promoter methylation after therapy was observed in both clinical responders and nonresponders across all genes. There was no association between clinical response and either baseline methylation or methylation reversal in the bone marrow or purified CD34+ population, nor was there an association with change in gene expression. Transient global hypomethylation was observed in samples after treatment but was not associated with clinical response. Induction of histone H3/H4 acetylation and the DNA damage–associated variant histone γ-H2AX was observed in peripheral blood samples across all dose cohorts. In conclusion, methylation reversal of candidate TSGs during cycle 1 of therapy was not predictive of clinical response to combination “epigenetic” therapy. This trial is registered with http://www.clinicaltrials.gov under NCT00101179. | [ Tamer E.Fandy, James G.Herman, PatrickKerns, AnchaleeJiemjit, Elizabeth A.Sugar, Si-HoChoi, Allen S.Yang, TimothyAucott, TiannaDauses, RosalieOdchimar-Reissig, JonathanLicht, Melanie J.McConnell, ChrisNasrallah, Marianne K. H.Kim, WeijiaZhang, YezouSun, AnthonyMurgo, IgorEspinoza-Delgado, KatharineOteiza, IbitayoOwoeye, Lewis R.Silverman, Steven D.Gore, Hetty E.Carraway] | Blood | | |
| pmc2708714 | An integrated genome screen identifies the Wnt signaling pathway as a major target of WT1 | WT1, a critical regulator of kidney development, is a tumor suppressor for nephroblastoma but in some contexts functions as an oncogene. A limited number of direct transcriptional targets of WT1 have been identified to explain its complex roles in tumorigenesis and organogenesis. In this study we performed genome-wide screening for direct WT1 targets, using a combination of ChIP–ChIP and expression arrays. Promoter regions bound by WT1 were highly G-rich and resembled the sites for a number of other widely expressed transcription factors such as SP1, MAZ, and ZNF219. Genes directly regulated by WT1 were implicated in MAPK signaling, axon guidance, and Wnt pathways. Among directly bound and regulated genes by WT1, nine were identified in the Wnt signaling pathway, suggesting that WT1 modulates a subset of Wnt components and responsive genes by direct binding. To prove the biological importance of the interplay between WT1 and Wnt signaling, we showed that WT1 blocked the ability of Wnt8 to induce a secondary body axis during Xenopus embryonic development. WT1 inhibited TCF-mediated transcription activated by Wnt ligand, wild type and mutant, stabilized β-catenin by preventing TCF4 loading onto a promoter. This was neither due to direct binding of WT1 to the TCF binding site nor to interaction between WT1 and TCF4, but by competition of WT1 and TCF4 for CBP. WT1 interference with Wnt signaling represents an important mode of its action relevant to the suppression of tumor growth and guidance of development. | [ Marianne K.-H.Kim, Thomas J.McGarry, PilibÓ Broin, Jared M.Flatow, Aaron A.-J.Golden, Jonathan D.Licht] | Proceedings of the National Academy of Sciences of the United States of America | 2009-6-22 | |
| pmc2737991 | Transcriptional Profiling Identifies the Metabolic Phenotype of Gonococcal Biofilms▿ † | Neisseria gonorrhoeae, the etiologic agent of gonorrhea, is frequently asymptomatic in women, often leading to chronic infections. One factor contributing to this may be biofilm formation. N. gonorrhoeae can form biofilms on glass and plastic surfaces. There is also evidence that biofilm formation may occur during natural cervical infection. To further study the mechanism of gonococcal biofilm formation, we compared transcriptional profiles of N. gonorrhoeae biofilms to planktonic profiles. Biofilm RNA was extracted from N. gonorrhoeae 1291 grown for 48 h in continuous-flow chambers over glass. Planktonic RNA was extracted from the biofilm runoff. In comparing biofilm with planktonic growth, 3.8% of the genome was differentially regulated. Genes that were highly upregulated in biofilms included aniA, norB, and ccp. These genes encode enzymes that are central to anaerobic respiratory metabolism and stress tolerance. Downregulated genes included members of the nuo gene cluster, which encodes the proton-translocating NADH dehydrogenase. Furthermore, it was observed that aniA, ccp, and norB insertional mutants were attenuated for biofilm formation on glass and transformed human cervical epithelial cells. These data suggest that biofilm formation by the gonococcus may represent a response that is linked to the control of nitric oxide steady-state levels during infection of cervical epithelial cells. | [ Megan L.Falsetta, Thomas B.Bair, Shan ChiKu, Rachel N.vanden Hoven, Christopher T.Steichen, Alastair G.McEwan, Michael P.Jennings, Michael A.Apicella] | Infection and Immunity | 2009-6-15 | |
| pmc2711785 | A brief bout of exercise alters gene expression and distinct gene pathways in peripheral blood mononuclear cells of early- and late-pubertal females | Recent studies show that brief exercise alters circulating neutrophil and peripheral blood mononuclear cell (PBMC) gene expression, ranging from cell growth to both pro-and anti-inflammatory processes. These initial observations were made solely in males, but whether PBMC gene expression is altered by exercise in females is not known. Ten early-pubertal girls (8–11 yr old) and 10 late-pubertal girls (15–17 yr old) performed ten 2-min bouts of cycle ergometry (∼90% peak heart rate) interspersed with 1-min rest intervals. Blood was obtained at rest and after exercise, and microarrays were performed in each individual subject. RNA was hybridized to Affymetrix U133+2.0 Arrays. Exercise induced significant changes in PBMC gene expression in early (1,320 genes)- and late (877 genes)-pubertal girls. The expression of 622 genes changed similarly in both groups. Exercise influenced a variety of established gene pathways (EASE < 0.04) in both older (6 pathways) and younger girls (11 pathways). Five pathways were the same in both groups and were functionally related to inflammation, stress, and apoptosis, such as natural killer cell-mediated cytotoxicity, antigen processing and presentation, B cell receptor signaling, and apoptosis. In summary, brief exercise alters PBMC gene expression in early- and late-pubertal girls. The pattern of change involves diverse genetic pathways, consistent with a global danger-type response, perhaps readying PBMCs for a range of physiological functions from inflammation to tissue repair that would be useful following a bout of physical activity. | [ ShlomitRadom-Aizik, FrankZaldivar, Szu-YunLeu, Dan M.Cooper] | Journal of Applied Physiology | 2009-4-30 | |
| pmc2698729 | A Naturally Occurring HER2 Carboxy-Terminal Fragment Promotes Mammary Tumor Growth and Metastasis▿ † | HER2 is a tyrosine kinase receptor causally involved in cancer. A subgroup of breast cancer patients with particularly poor clinical outcomes expresses a heterogeneous collection of HER2 carboxy-terminal fragments (CTFs). However, since the CTFs lack the extracellular domain that drives dimerization and subsequent activation of full-length HER2, they are in principle expected to be inactive. Here we show that at low expression levels one of these fragments, 611-CTF, activated multiple signaling pathways because of its unanticipated ability to constitutively homodimerize. A transcriptomic analysis revealed that 611-CTF specifically controlled the expression of genes that we found to be correlated with poor prognosis in breast cancer. Among the 611-CTF-regulated genes were several that have previously been linked to metastasis, including those for MET, EPHA2, matrix metalloproteinase 1, interleukin 11, angiopoietin-like 4, and different integrins. It is thought that transgenic mice overexpressing HER2 in the mammary glands develop tumors only after acquisition of activating mutations in the transgene. In contrast, we show that expression of 611-CTF led to development of aggressive and invasive mammary tumors without the need for mutations. These results demonstrate that 611-CTF is a potent oncogene capable of promoting mammary tumor progression and metastasis. | [ KimPedersen, Pier-DavideAngelini, SirleLaos, AlbaBach-Faig, Matthew P.Cunningham, CristinaFerrer-Ramón, AntonioLuque-García, JesúsGarcía-Castillo, Josep LluisParra-Palau, MaurizioScaltriti, Santiago Ramóny Cajal, JoséBaselga, JoaquínArribas] | Molecular and Cellular Biology | 2009-4-13 | |
| pmc2856484 | Genome-Wide Expression Analysis of Intra- and Extraarticular Connective Tissue | In comparison to extraarticular ligaments and tendons, the intraarticular ligaments such as the anterior and posterior cruciates exhibit different biochemical, biomechanical, and viscoelastic properties and most importantly, differential abilities to heal after surgical repair. Little is known about the underlying basis for these differences, in large measure due to the paucity of molecular markers distinguishing different classes of tendons and ligaments. To date, there has been no systematic analysis of gene expression differences between different types of connective tissues. We used Affymetrix expression arrays to analyze the differences in gene expression levels between the anterior cruciate, posterior cruciate, and medial collateral ligaments, the patellar and Achilles tendons and the synovium. We have identified five clusters of gene cohorts displaying similar expression patterns. These clusters group into three categories including: (1) genes that are strongly expressed in all connective tissues compared to the synovium control tissue; (2) genes that distinguish intraarticular connective tissues from extraarticular connective tissues; and (3) a group of genes expressed in common by the patellar tendon and the synovium. Our analysis identifies a new marker of tendons and ligaments (fibin2), demonstrates molecular diversity between subtypes of tendons and ligaments, and indicates that the primary molecular subdivision among dense regular connective tissues is intra- versus extraarticular rather than ligament versus tendon. | [ Richard V.Pearse, DianaEsshaki, Clifford J.Tabin, Martha M.Murray] | Journal of orthopaedic research : official publication of the Orthopaedic Research Society | | |
| pmc2662880 | Population-specific gene expression in the plant pathogenic nematode Heterodera glycines exists prior to infection and during the onset of a resistant or susceptible reaction in the roots of the Glycine max genotype Peking | A single Glycine max (soybean) genotype (Peking) reacts differently to two different populations of Heterodera glycines (soybean cyst nematode) within the first twelve hours of infection during resistant (R) and susceptible (S) reactions. This suggested that H. glycines has population-specific gene expression signatures. A microarray analysis of 7539 probe sets representing 7431 transcripts on the Affymetrix® soybean GeneChip® were used to identify population-specific gene expression signatures in pre-infective second stage larva (pi-L2) prior to their infection of Peking. Other analyses focused on the infective L2 at 12hours post infection (i-L212h), and the infective sedentary stages at 3days post infection (i-L23d) and 8days post infection (i-L2/L38d).ResultsDifferential expression and false discovery rate (FDR) analyses comparing populations of pi-L2 (i.e., incompatible population, NL1-RHg to compatible population, TN8) identified 71 genes that were induced in NL1-RHg as compared to TN8. These genes included putative gland protein G23G12, putative esophageal gland protein Hgg-20 and arginine kinase. The comparative analysis of pi-L2 identified 44 genes that were suppressed in NL1-RHg as compared to TN8. These genes included a different Hgg-20 gene, an EXPB1 protein and a cuticular collagen. By 12 h, there were 7 induced genes and 0 suppressed genes in NL1-RHg. By 3d, there were 9 induced and 10 suppressed genes in NL1-RHg. Substantial changes in gene expression became evident subsequently. At 8d there were 13 induced genes in NL1-RHg. This included putative gland protein G20E03, ubiquitin extension protein, putative gland protein G30C02 and β-1,4 endoglucanase. However, 1668 genes were found to be suppressed in NL1-RHg. These genes included steroid alpha reductase, serine proteinase and a collagen protein.ConclusionThese analyses identify a genetic expression signature for these two populations both prior to and subsequently as they undergo an R or S reaction. The identification of genes like steroid alpha reductase and serine proteinase that are involved in feeding and nutritional uptake as being highly suppressed during the R response at 8d may indicate genes that the plant is targeting. The analyses also identified numerous putative parasitism genes that are differentially expressed. The 1668 genes that are suppressed in NL1-RHg, and hence induced in TN8 may represent genes that are important during the parasitic stages of H. glycines development. The potential for different arrays of putative parasitism genes to be expressed in different nematode populations may indicate how H. glycines evolve mechanisms to overcome resistance. | [ Vincent PKlink, ParsaHosseini, Margaret HMacDonald, Nadim WAlkharouf, Benjamin FMatthews] | BMC Genomics | 2009-3-16 | |
| pmc2787730 | Neuroprotective and Antiinflammatory Properties of a Novel Demethylated Curcuminoid | A demethylated derivative of curcumin (DC; 67.8% bisdemethylcurcumin, 20.7% demethylmonodemethoxycurcumin, 5.86% bisdemethoxycurcumin, 2.58% demethylcurcumin) was prepared by using a 95% extract of curcumin (C95; 72.2% curcumin, 18.8% monodemethoxycurcumin, 4.5% bisdemethoxycurcumin). DC increased glutathione and reduced reactive oxygen species (ROS) in HT4 neuronal cells. In a model of glutamate-induced death of HT4, DC was more effective than C95 in neuroprotection. The protective effects of DC were retained even when DC was withdrawn from culture media after pretreatment. DC treatment, unlike an equal dose of C95, completely spared glutamate-induced loss of cellular GSH. Both DC and C95 prevented glutamate-induced elevation of cellular ROS but failed to attenuate glutamate-induced elevation of intracellular calcium. In human microvascular endothelial cells (HMECs) challenged with TNF-α, GeneChip analysis revealed that only a subcluster of 23 TNF-α–inducible genes were uniquely sensitive to C95. In sharp contrast, 1,065 TNF-α–inducible genes were sensitive to DC but not to C95, suggesting that DC was more effective in antagonizing the effects of TNF-α on HMECs. Functional analysis identified that the genes uniquely sensitive to DC belonged in four functional categories: cytokine-receptor interaction, focal adhesion, cell adhesion, and apoptosis. Real-time PCR as well as ELISA studies demonstrated that TNF-α–inducible CXCL10 and CXCL11 expression was sensitive to DC but not to C95. Flow-cytometry studies recognized ICAM-1 and VCAM-1 as TNF-α–inducible adhesion molecules that were uniquely sensitive to DC. Taken together, DC exhibited promising neuroprotective and antiinflammatory properties that must be characterized in vivo. Antioxid. Redox Signal. 11, 449–468. | [ SavitaKhanna, Han-APark, Chandan K.Sen, TrimurtuluGolakoti, KrishanuSengupta, SomepalliVenkateswarlu, SashwatiRoy] | Antioxidants & Redox Signaling | | |
| pmc2709101 | Atypical X-Chromosome Inactivation in an X;1 Translocation Patient Demonstrating Xq28 Functional Disomy | X-chromosome inactivation is an epigenetic process used to regulate gene dosage in mammalian females by silencing genes on one X-chromosome. While the pattern of X-chromosome inactivation is typically random in normal females, abnormalities of the X-chromosome may result in skewing due to disadvantaged cell growth. We describe a female patient with an X;1 translocation [46,X,t(X;1)(q28;q21)] and unusual pattern of X-chromosome inactivation who demonstrates functional disomy of the Xq28 region distal to the translocation breakpoint. There was complete skewing of X-chromosome inactivation in the patient, along with the atypical findings of an active normal X chromosome and an inactive derivative X. Characterization of the translocation revealed that the patient’s Xq28 breakpoint interrupts the DKC1 gene. Molecular analysis of the breakpoint region revealed functional disomy of Xq28 genes distal to DKC1. We propose that atypical X-chromosome inactivation occurred in the patient due to a post-inactivation cell selection mechanism likely initiated by disruption of DKC1. As a result, the pattern of X-chromosome inactivation is opposite that of the expected for an X;autosome translocation. Therefore, we suggest the phenotypic abnormalities found in the patient are a result of functional disomy in the Xq28 region. | [ Catherine ECottrell, AnnemarieSommer, Gail DWenger, StevenBullard, TamaraBusch, Katherine NashKrahn, Andrew CLidral, Julie MGastier-Foster] | American journal of medical genetics. Part A | | |
| pmc2635423 | Hepatic function is preserved in the absence of mature microRNAs | MiRNAs are small non-coding RNA molecules that regulate gene expression through partial or complete complementarity with target mRNAs. The function of miRNAs in normal liver physiology is largely unknown. Here we address the role of Dicer1 in the differentiated liver.MethodsWe derived mice lacking Dicer1 function in hepatocytes and assessed the loss of mature miRNA by quantitative PCR. Gene expression microarray analysis was performed on liver RNA from mutant and control mice. Liver sections from mutant and control mice were examined and liver function tests were performed.ResultsMice lacking Dicer1 function in hepatocytes appeared and behaved normally. Despite the loss of mature miRNAs, hepatic function was maintained, as reflected by normal blood glucose, albumin, cholesterol, and bilirubin. However, mutant mice between 2-4 months of age exhibited progressive hepatocyte damage with elevated serum ALT and AST. Liver mass was increased in mutant mice, as were cellular markers of both proliferation and apoptosis. Microarray analysis indicated large-scale changes in gene expression, with increased expression of many miRNA targets, particularly imprinted genes.ConclusionsLoss of miRNA processing in the liver at late gestation has a remarkably mild phenotype, suggesting that miRNAs do not play an essential role in hepatic function. However, miRNA deficiency results in hepatocyte apoptosis, hepatocyte regeneration, and portal inflammation. Finally, microarray analysis of gene expression in mutant liver supports a previously hypothesized role for Dicer1 in the repression of imprinted genes. | [ Nicholas J.Hand, Zankhana R.Master, JohnLe Lay, Joshua R.Friedman] | Hepatology (Baltimore, Md.) | | |
| pmc2658541 | Dysregulation of the transcription factors SOX4, CBFB and SMARCC1 correlates with outcome of colorectal cancer | The aim of this study was to identify deregulated transcription factors (TFs) in colorectal cancer (CRC) and to evaluate their relation with the recurrence of stage II CRC and overall survival. Microarray-based transcript profiles of 20 normal mucosas and 424 CRC samples were used to identify 51 TFs displaying differential transcript levels between normal mucosa and CRC. For a subset of these we provide in vitro evidence that deregulation of the Wnt signalling pathway can lead to the alterations observed in tissues. Furthermore, in two independent cohorts of microsatellite-stable stage II cancers we found that high SOX4 transcript levels correlated with recurrence (HR 2.7; 95% CI, 1.2–6.0; P=0.01). Analyses of ∼1000 stage I–III adenocarcinomas, by immunohistochemistry, revealed that patients with tumours displaying high levels of CBFB and SMARCC1 proteins had a significantly better overall survival rate (P=0.0001 and P=0.0275, respectively) than patients with low levels. Multivariate analyses revealed that a high CBFB protein level was an independent predictor of survival. In conclusion, several of the identified TFs seem to be involved in the progression of CRC. | [ C LAndersen, L LChristensen, KThorsen, TSchepeler, F BSørensen, H WVerspaget, RSimon, MKruhøffer, L AAaltonen, SLaurberg, T FØrntoft] | British Journal of Cancer | 2009-01-20 | |
| pmc2702237 | Heat-Induced Reactivation of HSV-1 in Latent Mice: Upregulation in the TG of CD83 and Other Immune Response Genes and Their LAT-ICP0 Locus | To determine changes in host gene expression in HSV-1 latent trigeminal ganglia (TG) after hyperthermic stress.MethodsScarified corneas of 6-week-old female BALB/c mice were inoculated with either HSV-1 17Syn+ (high phenotypic reactivator) or 17ΔPst(LAT−) (low phenotypic reactivator) at 104 plaque-forming units/eye. At 28 days after infection, viral reactivation was induced in some of the infected mice with hyperthermic stress, and the mice were killed after 1 hour. Heat-treated uninfected mice served as the control. Labeled cRNA derived from TG-isolated total RNA was hybridized to 430 2.0 chips containing 14,000 mouse genes. Gene expression was confirmed by quantitative real-time PCR.ResultsThere was no difference in gene expression in the non–heat-treated mice. Gene expression in the TG of each of the heat-treated mouse groups (17Syn+, 17ΔPst(LAT−) and uninfected) yielded upregulation of more than twofold of a group of the same genes, designated as heat stress–induced gene expression. Twenty-nine genes (0.2%) were significantly upregulated (2- to 17-fold) when the heat stress–induced gene expression was subtracted from the gene expression of 17Syn+ latent TG relative to 17ΔPst(LAT−) latent TG 1 hour after mouse hyperthermic stress. Nine host adaptive immunity genes comprising Ig molecules, CD83, CD8A, ADA, and CCL8 were the largest subset upregulated, and all were confirmed by real-time PCR. Others identified included genes involved in hypothalamic-pituitary gland functions.ConclusionsHyperthermic stress–induced reactivation of the HSV-1 high phenotypic reactivator can upregulate gene expression involved in B-cell function and in T-cell function. CD83 is implicated in HSV-1 latency, suggesting it could also be involved in immune-mediated mechanisms of viral reactivation. | [ ChristianClement, Partha S.Bhattacharjee, Herbert E.Kaufman, James M.Hill] | Investigative ophthalmology & visual science | 2009-1-17 | |
| pmc2628666 | Delineation of VEGF-regulated genes and functions in the cervix of pregnant rodents by DNA microarray analysis | VEGF-regulated genes in the cervices of pregnant and non-pregnant rodents (rats and mice) were delineated by DNA microarray and Real Time PCR, after locally altering levels of or action of VEGF using VEGF agents, namely siRNA, VEGF receptor antagonist and mouse VEGF recombinant protein.MethodsTissues were analyzed by genome-wide DNA microarray analysis, Real-time and gel-based PCR, and SEM, to decipher VEGF function during cervical remodeling. Data were analyzed by EASE score (microarray) and ANOVA (Real Time PCR) followed by Scheffe's F-test for multiple comparisons.ResultsOf the 30,000 genes analyzed, about 4,200 genes were altered in expression by VEGF, i.e., expression of about 2,400 and 1,700 genes were down- and up-regulated, respectively. Based on EASE score, i.e., grouping of genes according to their biological process, cell component and molecular functions, a number of vascular- and non-vascular-related processes were found to be regulated by VEGF in the cervix, including immune response (including inflammatory), cell proliferation, protein kinase activity, and cell adhesion molecule activity. Of interest, mRNA levels of a select group of genes, known to or with potential to influence cervical remodeling were altered. For example, real time PCR analysis showed that levels of VCAM-1, a key molecule in leukocyte recruitment, endothelial adhesion, and subsequent trans-endothelial migration, were elevated about 10 folds by VEGF. Further, VEGF agents also altered mRNA levels of decorin, which is involved in cervical collagen fibrillogenesis, and expression of eNO, PLC and PKC mRNA, critical downstream mediators of VEGF. Of note, we show that VEGF may regulate cervical epithelial proliferation, as revealed by SEM.ConclusionThese data are important in that they shed new insights in VEGF's possible roles and mechanisms in cervical events near-term, including cervical remodeling. | [ Chishimba NMowa, TianboLi, SubrinaJesmin, Hans GFolkesson, Sharon EUsip, Raymond EPapka, GuichuanHou] | Reproductive Biology and Endocrinology : RB&E | 2008-12-16 | 3.4 |
| pmc2592696 | Stat3 and c-Myc Genome-Wide Promoter Occupancy in Embryonic Stem Cells | Embryonic stem (ES) cell pluripotency is regulated in part by transcription factor (TF) pathways that maintain self-renewal and inhibit differentiation. Stat3 and c-Myc TFs are essential for maintaining mouse ES cell self-renewal. c-Myc, together with Oct4, Sox2, and Klf4, is a reprogramming factor. While previous studies have investigated core transcriptional circuitry in ES cells, other TF pathways that promote ES cell pluripotency have yet to be investigated. Therefore, to further understand ES cell transcriptional networks, we used genome-wide chromatin immunoprecipitation and microarray analysis (ChIP-chip) to map Stat3 and c-Myc binding targets in ES cells. Our results show that Stat3 and c-Myc occupy a significant number of genes whose expression is highly enriched in ES cells. By comparing Stat3 and c-Myc target genes with gene expression data from undifferentiated ES cells and embryoid bodies (EBs), we found that Stat3 binds active and inactive genes in ES cells, while c-Myc binds predominantly active genes. Moreover, the transcriptional states of Stat3 and c-Myc targets are correlated with co-occupancy of pluripotency-related TFs, polycomb group proteins, and active and repressive histone modifications. We also provide evidence that Stat3 targets are differentially expressed in ES cells following removal of LIF, where culture of ES cells in the absence of LIF resulted in downregulation of Stat3 target genes enriched in ES cells, and upregulation of lineage specific Stat3 target genes. Altogether, we reveal transcriptional targets of two key pluripotency-related genes in ES cells – Stat3 and c-Myc, thus providing further insight into the ES cell transcriptional network. | [ Benjamin L.Kidder, JimYang, StephenPalmer] | PLoS ONE | 2008-12-11 | |
| pmc2662282 | Mouse Meningiocytes Express Sox2 and Yield High Efficiency of Chimeras after Nuclear Reprogramming with Exogenous Factors*S⃞ | Induced pluripotent stem cell technology, also termed iPS, is an emerging approach to reprogram cells into an embryonic stem cell-like state by viral transduction with defined combinations of factors. iPS cells share most characteristics of embryonic stem cells, counting pluripotency and self-renewal, and have so far been obtained from mouse and humans, including patients with genetic diseases. Remarkably, autologous transplantation of cell lineages derived from iPS cells will eliminate the possibility of immunological rejection, as well as current ethical issues surrounding human embryonic stem cell research. However, before iPS can be used for clinical purposes, technical problems must be overcome. Among other considerations, full and homogeneous iPS reprogramming is an important prerequisite. However, despite the fact that cells from several mouse tissues can be successfully induced to iPS, the overall efficiency of chimera formation of these clones remains low even if selection for Oct4 or Nanog expression is applied. In this report, we demonstrate that cells from the mouse meningeal membranes express elevated levels of the embryonic master regulator Sox2 and are highly amenable to iPS. Meningeal iPS clones, generated without selection, are fully and homogeneously reprogrammed based on DNA methylation analysis and 100% chimera competent. Our results define a population of somatic cells that are ready to undergo iPS, thus highlighting a very attractive cell type for iPS research and application. | [ DajiangQin, YiGan, KaifengShao, HaoWang, WenLi, TaoWang, WenzhiHe, JianyongXu, YuZhang, ZhaohuiKou, LingwenZeng, GuoqingSheng, Miguel A.Esteban, ShaorongGao, DuanqingPei] | The Journal of Biological Chemistry | | |
| pmc2612029 | mtDNA depletion confers specific gene expression profiles in human cells grown in culture and in xenograft | Interactions between the gene products encoded by the mitochondrial and nuclear genomes play critical roles in eukaryotic cellular function. However, the effects mitochondrial DNA (mtDNA) levels have on the nuclear transcriptome have not been defined under physiological conditions. In order to address this issue, we characterized the gene expression profiles of A549 lung cancer cells and their mtDNA-depleted ρ0 counterparts grown in culture and as tumor xenografts in immune-deficient mice.ResultsCultured A549 ρ0 cells were respiration-deficient and showed enhanced levels of transcripts relevant to metal homeostasis, initiation of the epithelial-mesenchymal transition, and glucuronidation pathways. Several well-established HIF-regulated transcripts showed increased or decreased abundance relative to the parental cell line. Furthermore, growth in culture versus xenograft has a significantly greater influence on expression profiles, including transcripts involved in mitochondrial structure and both aerobic and anaerobic energy metabolism. However, both in vitro and in vivo, mtDNA levels explained the majority of the variance observed in the expression of transcripts in glucuronidation, tRNA synthetase, and immune surveillance related pathways. mtDNA levels in A549 xenografts also affected the expression of genes, such as AMACR and PHYH, involved in peroxisomal lipid metabolic pathways.ConclusionWe have identified mtDNA-dependent gene expression profiles that are shared in cultured cells and in xenografts. These profiles indicate that mtDNA-depleted cells could provide informative model systems for the testing the efficacy of select classes of therapeutics, such as anti-angiogenesis agents. Furthermore, mtDNA-depleted cells grown culture and in xenografts provide a powerful means to investigate possible relationships between mitochondrial activity and gene expression profiles in normal and pathological cells. | [ DarrenMagda, PhilipLecane, JuliaPrescott, PatriciaThiemann, XuanMa, Patricia KDranchak, Donna MToleno, KrishnaRamaswamy, Kimberly DSiegmund, Joseph GHacia] | BMC Genomics | 2008-11-3 | |
| pmc2604788 | Analysis of gene expression in pancreatic islets from diet-induced obese mice | In insulin-resistant status such as obesity, failure of pancreatic islets to increase insulin secretion leads to diabetes. We sought to screen for the islet genes that facilitate islet adaptation to obesity by comparing gene expression profiles between two strains of obesity-prone inbred mice with different propensities for hyperglycemia. C57BL/6J and AKR/J were fed regular rodent chow or high-fat diet, after which islet morphology, secretory function, and gene expression were assessed. AKR/J had lower blood glucose and higher insulin levels compared with C57BL/6J mice on regular rodent chow or high-fat diet. Insulin secretion was 3.2-fold higher in AKR/J than C57BL/6J mice following intraperitoneal glucose injection. Likewise, glucose-stimulated insulin secretion from isolated islets was higher in AKR/J. Additionally, islet mass was 1.4-fold greater in AKR/J compared with C57BL/6J. To elucidate the factors associated with the differences in islet function, we analyzed the gene expression profiles in islets in AKR/J and C57BL/6J mice. Of 14,000 genes examined, 202 were upregulated and 270 were downregulated in islets from diet-induced obese AKR/J mice compared with C57BL/6J mice. Key genes involved in islet signaling and metabolism, e.g., glucagon-like peptide-1 receptor, sterol Co-A desaturase 1 and 2, and fatty acid desaturase 2 were upregulated in obese AKR/J mice. The expression of multiple extracellular matrix proteins was also increased in AKR/J mice, suggesting a role in modulation of islet mass. Functional analyses of differentially regulated genes hold promise for elucidating factors linking obesity to alterations in islet function. | [ YumiImai, Hiral R.Patel, Nicolai M.Doliba, Franz M.Matschinsky, John W.Tobias, Rexford S.Ahima] | Physiological Genomics | 2008-10-14 | |
| pmc2537432 | Histone deacetylase inhibition reduces myocardial ischemia-reperfusion injury in mice | Limitation of infarct size is a major goal of therapy for acute coronary syndromes, and research has focused on achieving rapid patency of infarct-related vessels. However, new understandings of epigenetic modifications during ischemia suggest additional targeted approaches that have not been extensively explored. Here, we show that ischemia induces histone deacetylase (HDAC) activity in the heart with deacetylation of histones H3/4 in vitro and in vivo. We show, utilizing a standard murine model of ischemia-reperfusion, that chemical HDAC inhibitors significantly reduce infarct area, even when delivered 1 h after the ischemic insult. We demonstrate that HDAC inhibitors prevent ischemia-induced activation of gene programs that include hypoxia inducible factor-1α, cell death, and vascular permeability in vivo and in vitro, thus providing potential mechanisms to explain reduced vascular leak and myocardial injury. In vitro, siRNA knockdown experiments implicate HDAC4 as a mediator of the effects in ischemic cardiac myocytes. These results demonstrate that HDAC inhibitors alter the response to ischemic injury in the heart and reduce infarct size, suggesting novel therapeutic approaches for acute coronary syndromes.—Granger, A., Abdullah, I., Huebner, F., Stout, A., Wang, T., Huebner, T., Epstein, J. A., Gruber, P. J. Histone deacetylase inhibition reduces myocardial ischemia-reperfusion injury in mice. | [ AnneGranger, IbrahimAbdullah, FaithHuebner, AndreaStout, TaoWang, ThomasHuebner, Jonathan A.Epstein, Peter J.Gruber] | The FASEB Journal | | |
| pmc2677706 | Pathogenic Anti-DNA Antibodies Modulate Gene Expression in Mesangial Cells: | Although anti-DNA antibodies have been decisively linked to the pathogenesis of lupus nephritis, the mechanisms have not been conclusively determined. Recently, we reported that anti-DNA antibodies may contribute to kidney damage by upregulation of proinflammatory genes in mesangial cells (MC), a process involving both Fc receptor dependent and independent pathways. In investigating the mechanism by which pathogenic anti-DNA antibodies modulate gene expression in MC, we found that the pathogenic anti-DNA antibody 1A3F bound to high mobility group binding protein 1 (HMGB1), an endogenous ligand for TLR2/4 and RAGE (receptor for advanced glycation end-products). Interestingly, HMGB1 treatment of MC induced a similar pattern of genes as stimulation with 1A3F. Furthermore, HMGB1 and 1A3F exhibited a synergistic proinflammatory effect in the kidney, where increased expression of HMGB1 was found in lupus patients but not in patients with other types of renal disease. TLR2/Fc and RAGE/Fc inhibited the proinflammatory effects of 1A3F on MC. Finally, we found enhanced susceptibility of lupus prone MRL-lpr/lpr (MRL/lpr) as compared to normal BALB/c derived MC to pathogenic anti-DNA antibody and LPS stimulation (in particular enhanced chemokine synthesis), in addition to significantly increased expression of TLR4. Our results suggest that gene upregulation in MC induced by nephritogenic anti-DNA antibodies is TLR2/4 and RAGE dependent. Finally, HMGB1 may act as a proinflammatory mediator in antibody induced kidney damage in systemic lupus erythematosus (SLE). | [ XiaopingQing, MilenaPitashny, David B.Thomas, FranckBarrat, Mark P.Hogarth, ChaimPutterman] | Immunology letters | 2008-9-24 | |
| pmc3259809 | Lactaturia and Loss of Sodium-dependent Lactate Uptake in the Colon of SLC5A8-deficient Mice* | SLC5A8 is a member of the sodium/glucose cotransporter family. It has been proposed that SLC5A8 might act as an apical iodide transporter in the thyroid follicular cells or as a transporter of short chain monocarboxylates. We have directly addressed the functional role of SLC5A8 in vivo by generation of SLC5A8 mutant mice. We found that SLC5A8 is responsible for the re-absorption of lactate at the apical membrane of the kidney proximal tubules and of serous salivary gland ducts. In addition, SLC5A8 mediated the uptake of lactate into colonocytes under physiological conditions. We did not find any evidence of SLC5A8 being essential for the apical iodide transport in the thyroid gland, even if the ion-cotransporter SLC26A4, causing the human Pendred syndrome, is missing. Because SLC5A8 is transcriptionally silenced in many tumors, it has been proposed that SLC5A8-mediated transport of butyrate suppresses tumor formation. Treatment of Slc5a8-/- mice with carcinogens and breeding to the Apcmin mouse line did not reveal a higher incidence of tumor formation. We conclude that SLC5A8 is instrumental in preventing lactaturia and loss of sodium-dependent uptake of lactate in the colon but does not have any apparent role in the prevention of tumor formation and growth. | [ HenningFrank, NicoleGröger, MartinDiener, ChristophBecker, ThomasBraun, ThomasBoettger] | The Journal of Biological Chemistry | | |
| pmc2519835 | Extensive Adaptive Changes Occur in the Transcriptome of Streptococcus agalactiae (Group B Streptococcus) in Response to Incubation with Human Blood | To enhance understanding of how Streptococcus agalactiae (group B streptococcus, GBS) adapts during invasive infection, we performed a whole-genome transcriptome analysis after incubation with whole human blood. Global changes occurred in the GBS transcriptome rapidly in response to blood contact following shift from growth in a rich laboratory medium. Most (83%) of the significantly altered transcripts were down-regulated after 30 minutes of incubation in blood, and all functional categories of genes were abundantly represented. We observed complex dynamic changes in the expression of transcriptional regulators and stress response genes that allow GBS to rapidly adapt to blood. The transcripts of relatively few proven virulence genes were up-regulated during the first 90 minutes. However, a key discovery was that genes encoding proteins involved in interaction with the host coagulation/fibrinolysis system and bacterial-host interactions were rapidly up-regulated. Extensive transcript changes also occurred for genes involved in carbohydrate metabolism, including multi-functional proteins and regulators putatively involved in pathogenesis. Finally, we discovered that an incubation temperature closer to that occurring in patients with severe infection and high fever (40°C) induced additional differences in the GBS transcriptome relative to normal body temperature (37°C). Taken together, the data provide extensive new information about transcriptional adaptation of GBS exposed to human blood, a crucial step during GBS pathogenesis in invasive diseases, and identify many new leads for molecular pathogenesis research. | [ LaurentMereghetti, IzabelaSitkiewicz, Nicole M.Green, James M.Musser] | PLoS ONE | 2008-9-4 | |
| pmc2729231 | High Resolution Array-Comparative Genomic Hybridization Profiling Reveals Deoxyribonucleic Acid Copy Number Alterations Associated with Medullary Thyroid Carcinoma | Context: Activating mutations in the RET protooncogene have been demonstrated in multiple endocrine neoplasia 2 and sporadic medullary thyroid carcinoma (MTC). However, the complete genetic etiology underlying MTC tumorigenesis remains unclear.Objective: Our objective was to define more precisely the chromosomal regions and uncover novel genes associated with MTC tumorigenesis.Design and Setting: In this study, we used high resolution array-based comparative genomic hybridization to define tumor-associated copy number alterations (CNA) in 30 primary MTCs: 20 sporadic tumors (50% of which harbored RET mutation), and 10 hereditary.Results: We identified 98 CNA, including 76 genomic allelic losses, two gains, and 20 copy number variations associated with MTC. Across sporadic and hereditary groups, there was a similar and overlapping pattern of predominant allelic loss. There were 29 regions containing at least 30% CNA in the 30 tumor samples. The most frequent allelic loss occurred in four loci, 7q36.1, 12p13.31, 13q12.11, and 19p13.3-11. No regions were found to be uniquely altered in the hereditary tumors. There were 21 CNA specific to sporadic MTC, with loss of 11q23.3 uniquely altered in RET negative tumors. Pathway analysis found cellular growth and proliferation as the most significant overall target, and cell death as the most significant pathway targeted in sporadic MTC.Conclusions: Our findings underscore the importance of candidate tumor suppressor genes together with RET alterations in MTCs. Despite of RET status, all MTC might share similar oncogenetic mechanisms. Dysfunction of cell proliferation and cell death may both be involved in MTC tumorigenesis. | [ LeiYe, LiberoSantarpia, Gilbert J.Cote, Adel K.El-Naggar, Robert F.Gagel] | The Journal of Clinical Endocrinology and Metabolism | 2008-9-2 | |
| pmc2500185 | Exon Expression Arrays as a Tool to Identify New Cancer Genes | Identification of genes that are causally implicated in oncogenesis is a major goal in cancer research. An estimated 10–20% of cancer-related gene mutations result in skipping of one or more exons in the encoded transcripts. Here we report on a strategy to screen in a global fashion for such exon-skipping events using PAttern based Correlation (PAC). The PAC algorithm has been used previously to identify differentially expressed splice variants between two predefined subgroups. As genetic changes in cancer are sample specific, we tested the ability of PAC to identify aberrantly expressed exons in single samples.Principal FindingsAs a proof-of-principle, we tested the PAC strategy on human cancer samples of which the complete coding sequence of eight cancer genes had been screened for mutations. PAC detected all seven exon-skipping mutants among 12 cancer cell lines. PAC also identified exon-skipping mutants in clinical cancer specimens although detection was compromised due to heterogeneous (wild-type) transcript expression. PAC reduced the number of candidate genes/exons for subsequent mutational analysis by two to three orders of magnitude and had a substantial true positive rate. Importantly, of 112 randomly selected outlier exons, sequence analysis identified two novel exon skipping events, two novel base changes and 21 previously reported base changes (SNPs).ConclusionsThe ability of PAC to enrich for mutated transcripts and to identify known and novel genetic changes confirms its suitability as a strategy to identify candidate cancer genes. | [ MiekeSchutte, FonsElstrodt, Linda B. C.Bralten, Jord H. A.Nagel, ElzaDuijm, AntoinetteHollestelle, Maartje J.Vuerhard, MarijkeWasielewski, Justine K.Peeters, Petervan der Spek, Peter A.Sillevis Smitt, Pim J.French] | PLoS ONE | 2008-8-20 | |
| pmc2464734 | Remodeling of the Streptococcus agalactiae Transcriptome in Response to Growth Temperature | To act as a commensal bacterium and a pathogen in humans and animals, Streptococcus agalactiae (group B streptococcus, GBS) must be able to monitor and adapt to different environmental conditions. Temperature variation is a one of the most commonly encountered variables.Methodology/Principal FindingsTo understand the extent to which GBS modify gene expression in response to temperatures encountered in the various hosts, we conducted a whole genome transcriptome analysis of organisms grown at 30°C and 40°C. We identified extensive transcriptome remodeling at various stages of growth, especially in the stationary phase (significant transcript changes occurred for 25% of the genes). A large proportion of genes involved in metabolism was up-regulated at 30°C in stationary phase. Conversely, genes up-regulated at 40°C relative to 30°C include those encoding virulence factors such as hemolysins and extracellular secreted proteins with LPXTG motifs. Over-expression of hemolysins was linked to larger zones of hemolysis and enhanced hemolytic activity at 40°C. A key theme identified by our study was that genes involved in purine metabolism and iron acquisition were significantly up-regulated at 40°C.Conclusion/SignificanceGrowth of GBS in vitro at different temperatures resulted in extensive remodeling of the transcriptome, including genes encoding proven and putative virulence genes. The data provide extensive new leads for molecular pathogenesis research. | [ LaurentMereghetti, IzabelaSitkiewicz, Nicole M.Green, James M.Musser] | PLoS ONE | 2008-7-30 | |
| pmc2584582 | Osteitis Fibrosa Is Mediated by Platelet-Derived Growth Factor-A Via a Phosphoinositide 3-Kinase-Dependent Signaling Pathway in a Rat Model for Chronic Hyperparathyroidism | Abnormal secretion of PTH by the parathyroid glands contributes to a variety of common skeletal disorders. Prior studies implicate platelet-derived growth factor-A (PDGF-A) as an important mediator of selective PTH actions on bone. The present studies used targeted gene profiling and small-molecule antagonists directed against candidate gene products to elucidate the roles of specific PTH-regulated genes and signaling pathways. A group of 29 genes in rats continuously infused with PTH and cotreated with the PDGF receptor antagonist trapidil were differentially expressed compared with PTH treatment alone. Several of the identified genes were functionally clustered as regulators of fibroblast differentiation and extracellular matrix modeling, including the matrix cross-linking enzyme lysyl oxidase (LOX). Treatment with β-aminopropionitrile, an irreversible inhibitor of LOX activity, dramatically reduced diffuse mineralization but had no effect on PTH-induced fibrosis. In contrast, the receptor tyrosine kinase inhibitor Gleevec and the phosphoinositide 3-kinase inhibitor wortmannin each reduced bone marrow fibrosis. In summary, the present studies support the hypotheses that PTH-induced bone marrow fibrosis is mediated by PDGF-A via a phosphoinositide 3-kinase-dependent signaling pathway and that increased LOX gene expression plays a key role in abnormal mineralization, a hallmark of chronic hyperparathyroidism. | [ Malcolm B.Lowry, SutadaLotinun, Alexey A.Leontovich, MinzhiZhang, AvudaiappanMaran, Kristen L.Shogren, Brett K.Palama, KevinMarley, Urszula T.Iwaniec, Russell T.Turner] | Endocrinology | 2008-7-17 | |
| pmc2778036 | Merlin Is a Potent Inhibitor of Glioma Growth | Neurofibromatosis 2 (NF2) is an inherited cancer syndrome in which affected individuals develop nervous system tumors, including schwannomas, meningiomas, and ependymomas. The NF2 protein merlin (or schwannomin) is a member of the Band 4.1 superfamily of proteins, which serve as linkers between transmembrane proteins and the actin cytoskeleton. In addition to mutational inactivation of the NF2 gene in NF2-associated tumors, mutations and loss of merlin expression have also been reported in other types of cancers. In the present study, we show that merlin expression is dramatically reduced in human malignant gliomas and that reexpression of functional merlin dramatically inhibits both subcutaneous and intracranial growth of human glioma cells in mice. We further show that merlin reexpression inhibits glioma cell proliferation and promotes apoptosis in vivo. Using microarray analysis, we identify altered expression of specific molecules that play key roles in cell proliferation, survival, and motility. These merlin-induced changes of gene expression were confirmed by real-time quantitative PCR, Western blotting, and functional assays. These results indicate that reexpression of merlin correlates with activation of mammalian sterile 20-like 1/2–large tumor suppressor 2 signaling pathway and inhibition of canonical and noncanonical Wnt signals. Collectively, our results show that merlin is a potent inhibitor of high-grade human glioma. | [ Ying-Ka IngarLau, Lucas B.Murray, Sean S.Houshmandi, YinXu, David H.Gutmann, QinYu] | Cancer research | | |
| pmc3033660 | Synthesis and Anticancer Properties of Water-Soluble Zinc Ionophores | Several water-solubilized versions of the zinc ionophore 1-hydroxypyridine-2-thione (ZnHPT), synthesized as part of the present study, have been found both to increase the intracellular concentrations of free zinc and to produce an antiproliferative activity in exponential phase A549 human lung cancer cultures. Gene expression profiles of A549 cultures treated with one of these water-soluble zinc ionophores, PCI-5002, reveal the activation of stress response pathways under the control of metal-responsive transcription factor 1 (MTF-1), hypoxia-inducible transcription factor 1 (HIF-1), and heat shock transcription factors. Additional oxidative stress response and apoptotic pathways were activated in cultures grown in zinc-supplemented media. We also show that these water-soluble zinc ionophores can be given to mice at 100 μmol/kg (300 μmol/m2) with no observable toxicity and inhibit the growth of A549 lung and PC3 prostate cancer cells grown in xenograft models. Gene expression profiles of tumor specimens harvested from mice 4 h after treatment confirmed the in vivo activation of MTF-1–responsive genes. Overall, we propose that water-solubilized zinc ionophores represent a potential new class of anticancer agents. | [ DarrenMagda, PhilipLecane, ZhongWang, WeilinHu, PatriciaThiemann, XuanMa, Patricia K.Dranchak, XiaomingWang, VincentLynch, WenhaoWei, ViktorCsokai, Joseph G.Hacia, Jonathan L.Sessler] | Cancer research | | |
| pmc2519612 | Chemopreventive Doses of Methylselenocysteine Alter Circadian Rhythm in Rat Mammary Tissue | It is known that organic forms of Selenium (Se) inhibit chemically-induced rat mammary carcinogenesis, although the molecular basis remains to be elucidated. To identify signaling pathways involved in carcinogenesis that are also modulated by methylselenocysteine (MSC), we compared the global gene expression profiles in mammary tissues from pubescent female rats maintained on a selenium supplemented (3 ppm) diet with those on a standardized diet after N-nitroso-N-methylurea (NMU). While the Se enriched diet altered the steady state levels of genes involved in various cellular functions, the most dramatic effect was the coordinated changes in the expression of multiple genes that regulate circadian rhythm. Normal mammary tissue of rats fed a standardized diet showed little circadian oscillation relative to liver tissue. By contrast, mammary tissue of rats maintained on the Se enriched diet showed a progressive, time dependent increase in the expression of circadian gene Per 2 and circadian regulated transcription factor DBP. Our results further demonstrated that the expression of Per2 and DBP mRNAs were significantly decreased in mammary tumors arising in rats on the selenium enriched diet, but not in tumors of rats on the control diet, suggesting that Se-induced elevation in the expression of circadian genes was incompatible with mammary carcinogenesis. Given the previously reported role of Per2 as a tumor suppressor, these observations suggest that Per2 is an important target of MSC during chemoprevention in NMU-induced rat mammary carcinogenesis, and for the first time provide a mechanistic link between chemoprevention and circadian rhythm. | [ XunZhang, HelmutZarbl] | Cancer prevention research (Philadelphia, Pa.) | | |
| pmc2892791 | Topical Application of a Bioadhesive Black Raspberry Gel Modulates Gene Expression and Reduces Cyclooxygenase 2 Protein in Human Premalignant Oral Lesions | Reduced expression of proapoptotic and terminal differentiation genes in conjunction with increased levels of the proinflammatory and angiogenesis-inducing enzymes, cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS), correlate with malignant transformation of oral intraepithelial neoplasia (IEN). Accordingly, this study investigated the effects of a 10% (w/w) freeze-dried black raspberry gel on oral IEN histopathology, gene expression profiles, intraepithelial COX-2 and iNOS proteins, and microvascular densities. Our laboratories have shown that freeze-dried black raspberries possess antioxidant properties and also induce keratinocyte apoptosis and terminal differentiation. Oral IEN tissues were hemisected to provide samples for pretreatment diagnoses and establish baseline biochemical and molecular variables. Treatment of the remaining lesional tissue (0.5 g gel applied four times daily for 6 weeks) began 1 week after the initial biopsy. RNA was isolated from snap-frozen IEN lesions for microarray analyses, followed by quantitative reverse transcription-PCR validation. Additional epithelial gene-specific quantitative reverse transcription-PCR analyses facilitated the assessment of target tissue treatment effects. Surface epithelial COX-2 and iNOS protein levels and microvascular densities were determined by image analysis quantified immunohistochemistry. Topical berry gel application uniformly suppressed genes associated with RNA processing, growth factor recycling, and inhibition of apoptosis. Although the majority of participants showed posttreatment decreases in epithelial iNOS and COX-2 proteins, only COX-2 reductions were statistically significant. These data show that berry gel application modulated oral IEN gene expression profiles, ultimately reducing epithelial COX-2 protein. In a patient subset, berry gel application also reduced vascular densities in the superficial connective tissues and induced genes associated with keratinocyte terminal differentiation. | [ Susan R.Mallery, Jared C.Zwick, PingPei, MengTong, Peter E.Larsen, Brian S.Shumway, BoLu, Henry W.Fields, Russell J.Mumper, Gary D.Stoner] | Cancer research | | |
| pmc2438380 | The cyclin-dependent kinase inhibitor p57kip2 is a negative regulator of Schwann cell differentiation and in vitro myelination | The p57kip2 gene encodes a member of the cyclin-dependent kinase inhibitor family, proteins known to block G1/S transition during the mammalian cell cycle. We observed that expression of p57kip2 in Schwann cells of the developing and injured adult peripheral nervous system is dynamically regulated. Using gene knockdown by means of vector-based RNA interference in cultured primary Schwann cells we found that reduced levels of p57kip2 lead to cell cycle exit, actin filament stabilization, altered cell morphology and growth, and down-regulation of promyelinating markers as well as induction of myelin genes and proteins. In addition, we could demonstrate that in vitro myelination is enhanced by p57kip2-suppressed Schwann cells. Using microarray technology we found that these cellular reactions are specific to lowered p57kip2 expression levels and detected a shift of the transcriptional expression program toward the pattern known from Schwann cells in developing peripheral nerves. Because in the absence of axons primary Schwann cells normally do not display differentiation-associated reactions, we conclude that we have identified a mechanism and an important intrinsic negative regulator of myelinating glia differentiation. | [ AndréHeinen, DavidKremer, PeterGöttle, FabianKruse, BirgitHasse, HelmarLehmann, Hans PeterHartung, PatrickKüry] | Proceedings of the National Academy of Sciences of the United States of America | 2008-6-11 | |
| pmc2483357 | Arabidopsis DREB2A-Interacting Proteins Function as RING E3 Ligases and Negatively Regulate Plant Drought Stress–Responsive Gene Expression[W] | The DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN2A (DREB2A) transcription factor controls water deficit–inducible gene expression and requires posttranslational modification for its activation. The activation mechanism is not well understood; however, the stability of this protein in the nucleus was recently found to be important for its activation. Here, we report the isolation of Arabidopsis thaliana DREB2A-INTERACTING PROTEIN1 (DRIP1) and DRIP2, C3HC4 RING domain–containing proteins that interact with the DREB2A protein in the nucleus. An in vitro ubiquitination assay showed that they function as E3 ubiquitin ligases and are capable of mediating DREB2A ubiquitination. Overexpression of DRIP1 in Arabidopsis delayed the expression of DREB2A-regulated drought-responsive genes. Drought-inducible gene expression was slightly enhanced in the single T-DNA mutants of drip1-1 and drip2-1. By contrast, significantly enhanced gene expression was revealed in the drip1 drip2 double mutant under dehydration stress. Collectively, these data imply that DRIP1 and DRIP2 function negatively in the response of plants to drought stress. Moreover, overexpression of full-length DREB2A protein was more stable in drip1-1 than in the wild-type background. These results suggest that DRIP1 and DRIP2 act as novel negative regulators in drought-responsive gene expression by targeting DREB2A to 26S proteasome proteolysis. | [ FengQin, YohSakuma, Lam-Son PhanTran, KyonoshinMaruyama, SatoshiKidokoro, YasunariFujita, MikiFujita, TaishiUmezawa, YorikoSawano, Ken-ichiMiyazono, MasaruTanokura, KazuoShinozaki, KazukoYamaguchi-Shinozaki] | The Plant Cell | | |
| pmc2409018 | Functional Redundancy and New Roles for Genes of the Autonomous Floral-Promotion Pathway1[C][W][OA] | The early-flowering habit of rapid-cycling accessions of Arabidopsis (Arabidopsis thaliana) is, in part, due to the genes of the autonomous floral-promotion pathway (AP). The AP promotes flowering by repressing expression of the floral inhibitor FLOWERING LOCUS C (FLC). AP mutants are therefore late flowering due to elevated levels of FLC, and this late-flowering phenotype is eliminated by loss-of-function mutations in FLC. To further investigate the role of the AP, we created a series of double mutants. In contrast to the phenotypes of single mutants, which are largely limited to delayed flowering, a subset of AP double mutants show a range of defects in growth and development. These phenotypes include reduced size, chlorophyll content, growth rate, and fertility. Unlike the effects of the AP on flowering time, these phenotypes are FLC independent. Recent work has also shown that two AP genes, FCA and FPA, are required for the repression and, in some cases, proper DNA methylation of two transposons. We show that similar effects are seen for all AP genes tested. Microarray analysis of gene expression in AP single and double mutants, however, suggests that the AP is not likely to play a broad role in the repression of gene expression through DNA methylation: very few of the genes that have been reported to be up-regulated in DNA methylation mutants are misexpressed in AP mutants. Together, these data indicate that the genes of the AP play important and sometimes functionally redundant roles in aspects of development in addition to flowering time. | [ Kira M.Veley, Scott D.Michaels] | Plant Physiology | | |
| pmc2346728 | Identification and In Vitro Expansion of Functional Antigen-Specific CD25+ FoxP3+ Regulatory T Cells in Hepatitis C Virus Infection▿ | CD4+CD25+ regulatory T cells (CD25+ Tregs) play a key role in immune regulation. Since hepatitis C virus (HCV) persists with increased circulating CD4+CD25+ T cells and virus-specific effector T-cell dysfunction, we asked if CD4+CD25+ T cells in HCV-infected individuals are similar to natural Tregs in uninfected individuals and if they include HCV-specific Tregs using the specific Treg marker FoxP3 at the single-cell level. We report that HCV-infected patients display increased circulating FoxP3+ Tregs that are phenotypically and functionally indistinguishable from FoxP3+ Tregs in uninfected subjects. Furthermore, HCV-specific FoxP3+ Tregs were detected in HCV-seropositive persons with antigen-specific expansion, major histocompatibility complex class II/peptide tetramer binding affinity, and preferential suppression of HCV-specific CD8 T cells. Transforming growth factor β contributed to antigen-specific Treg expansion in vitro, suggesting that it may contribute to antigen-specific Treg expansion in vivo. Interestingly, FoxP3 expression was also detected in influenza virus-specific CD4 T cells. In conclusion, functionally active and virus-specific FoxP3+ Tregs are induced in HCV infection, thus providing targeted immune regulation in vivo. Detection of FoxP3 expression in non-HCV-specific CD4 T cells suggests that immune regulation through antigen-specific Treg induction extends beyond HCV. | [ HirotoshiEbinuma, NobuhiroNakamoto, YunLi, David A.Price, EmmaGostick, Bruce L.Levine, J.Tobias, William W.Kwok, Kyong-MiChang] | Journal of Virology | 2008-3-12 | |
| pmc2262897 | Effect of thyroid hormone concentration on the transcriptional response underlying induced metamorphosis in the Mexican axolotl (Ambystoma) | Thyroid hormones (TH) induce gene expression programs that orchestrate amphibian metamorphosis. In contrast to anurans, many salamanders do not undergo metamorphosis in nature. However, they can be induced to undergo metamorphosis via exposure to thyroxine (T4). We induced metamorphosis in juvenile Mexican axolotls (Ambystoma mexicanum) using 5 and 50 nM T4, collected epidermal tissue from the head at four time points (Days 0, 2, 12, 28), and used microarray analysis to quantify mRNA abundances.ResultsIndividuals reared in the higher T4 concentration initiated morphological and transcriptional changes earlier and completed metamorphosis by Day 28. In contrast, initiation of metamorphosis was delayed in the lower T4 concentration and none of the individuals completed metamorphosis by Day 28. We identified 402 genes that were statistically differentially expressed by ≥ two-fold between T4 treatments at one or more non-Day 0 sampling times. To complement this analysis, we used linear and quadratic regression to identify 542 and 709 genes that were differentially expressed by ≥ two-fold in the 5 and 50 nM T4 treatments, respectively.ConclusionWe found that T4 concentration affected the timing of gene expression and the shape of temporal gene expression profiles. However, essentially all of the identified genes were similarly affected by 5 and 50 nM T4. We discuss genes and biological processes that appear to be common to salamander and anuran metamorphosis, and also highlight clear transcriptional differences. Our results show that gene expression in axolotls is diverse and precise, and that axolotls provide new insights about amphibian metamorphosis. | [ Robert BPage, Stephen RVoss, Amy KSamuels, Jeramiah JSmith, SrikrishnaPutta, Christopher KBeachy] | BMC Genomics | 2008-2-11 | |
| pmc2245831 | Genomic Survey and Gene Expression Analysis of the Basic Leucine Zipper Transcription Factor Family in Rice1[W][OA] | The basic leucine (Leu) zipper (bZIP) proteins compose a family of transcriptional regulators present exclusively in eukaryotes. The bZIP proteins characteristically harbor a bZIP domain composed of two structural features: a DNA-binding basic region and the Leu zipper dimerization region. They have been shown to regulate diverse plant-specific phenomena, including seed maturation and germination, floral induction and development, and photomorphogenesis, and are also involved in stress and hormone signaling. We have identified 89 bZIP transcription factor-encoding genes in the rice (Oryza sativa) genome. Their chromosomal distribution and sequence analyses suggest that the bZIP transcription factor family has evolved via gene duplication. The phylogenetic relationship among rice bZIP domains as well as with bZIP domains from other plant bZIP factors suggests that homologous bZIP domains exist in plants. Similar intron/exon structural patterns were observed in the basic and hinge regions of their bZIP domains. Detailed sequence analysis has been done to identify additional conserved motifs outside the bZIP domain and to predict their DNA-binding site specificity as well as dimerization properties, which has helped classify them into different groups and subfamilies, respectively. Expression of bZIP transcription factor-encoding genes has been analyzed by full-length cDNA and expressed sequence tag-based expression profiling. This expression profiling was complemented by microarray analysis. The results indicate specific or coexpression patterns of rice bZIP transcription factors starting from floral transition to various stages of panicle and seed development. bZIP transcription factor-encoding genes in rice also displayed differential expression patterns in rice seedlings in response to abiotic stress and light irradiation. An effort has been made to link the structure and expression pattern of bZIP transcription factor-encoding genes in rice to their function, based on the information obtained from our analyses and earlier known results. This information will be important for functional characterization of bZIP transcription factors in rice. | [ AashimaNijhawan, MukeshJain, Akhilesh K.Tyagi, Jitendra P.Khurana] | Plant Physiology | | |
| pmc2268699 | Bioinformatic identification of FGF, p38-MAPK, and calcium signalling pathways associated with carcinoma in situ in the urinary bladder | Carcinoma in situ (CIS) is believed to be a precursor of invasive bladder cancer. Identification of CIS is a valuable prognostic factor since radical treatment strategies can be offered these patients before the disease becomes invasive.MethodsWe developed a pathway based classifier approach to predict presence or absence of CIS in patients suffering from non muscle invasive bladder cancer. From Ingenuity Pathway Analysis we considered four canonical signalling pathways (p38 MAPK, FGF, Calcium, and cAMP pathways) with most coherent expression of transcription factors (TFs) across samples in a set of twenty-eight non muscle invasive bladder carcinomas. These pathways contained twelve TFs in total. We used the expression of the TFs to predict presence or absence of CIS in a Leave-One-Out Cross Validation classification.ResultsWe showed that TF expression levels in three pathways (FGF, p38 MAPK, and calcium signalling) or the expression of the twelve TFs together could be used to predict presence or absence of concomitant CIS. A cluster analysis based on expression of the twelve TFs separated the samples in two main clusters: one branch contained 11 of the 15 patients without concomitant CIS and with the majority of the genes being down regulated; the other branch contained 10 of 13 patients with concomitant CIS, and here genes were mostly up regulated. The expression in the CIS group was comparable to the expression of twenty-three patients suffering from muscle-invasive bladder carcinoma. Finally, we validated our results in an independent test set and found that prediction of CIS status was possible using TF expression of the p38 MAPK pathway.ConclusionWe conclude that it is possible to use pathway analysis for molecular classification of bladder tumors. | [ MaleneHerbsleb, Ole FChristensen, ThomasThykjaer, CarstenWiuf, MichaelBorre, Torben FØrntoft, LarsDyrskjøt] | BMC Cancer | 2008-1-31 | 4.2.0 |
| pmc2148100 | Sex-Related Differences in Gene Expression in Human Skeletal Muscle | There is sexual dimorphism of skeletal muscle, the most obvious feature being the larger muscle mass of men. The molecular basis for this difference has not been clearly defined. To identify genes that might contribute to the relatively greater muscularity of men, we compared skeletal muscle gene expression profiles of 15 normal men and 15 normal women by using comprehensive oligonucleotide microarrays. Although there were sex-related differences in expression of several hundred genes, very few of the differentially expressed genes have functions that are obvious candidates for explaining the larger muscle mass of men. The men tended to have higher expression of genes encoding mitochondrial proteins, ribosomal proteins, and a few translation initiation factors. The women had >2-fold greater expression than the men (P<0.0001) of two genes that encode proteins in growth factor pathways known to be important in regulating muscle mass: growth factor receptor-bound 10 (GRB10) and activin A receptor IIB (ACVR2B). GRB10 encodes a protein that inhibits insulin-like growth factor-1 (IGF-1) signaling. ACVR2B encodes a myostatin receptor. Quantitative RT-PCR confirmed higher expression of GRB10 and ACVR2B genes in these women. In an independent microarray study of 10 men and 9 women with facioscapulohumeral dystrophy, women had higher expression of GRB10 (2.7-fold, P<0.001) and ACVR2B (1.7-fold, P<0.03). If these sex-related differences in mRNA expression lead to reduced IGF-1 activity and increased myostatin activity, they could contribute to the sex difference in muscle size. | [ StephenWelle, RabiTawil, Charles A.Thornton] | PLoS ONE | 2008-1-2 | |
| pmc2082023 | Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA | The interpretability of microarray data can be affected by sample quality. To systematically explore how RNA quality affects microarray assay performance, a set of rat liver RNA samples with a progressive change in RNA integrity was generated by thawing frozen tissue or by ex vivo incubation of fresh tissue over a time course.ResultsIncubation of tissue at 37°C for several hours had little effect on RNA integrity, but did induce changes in the transcript levels of stress response genes and immune cell markers. In contrast, thawing of tissue led to a rapid loss of RNA integrity. Probe sets identified as most sensitive to RNA degradation tended to be located more than 1000 nucleotides upstream of their transcription termini, similar to the positioning of control probe sets used to assess sample quality on Affymetrix GeneChip® arrays. Samples with RNA integrity numbers less than or equal to 7 showed a significant increase in false positives relative to undegraded liver RNA and a reduction in the detection of true positives among probe sets most sensitive to sample integrity for in silico modeled changes of 1.5-, 2-, and 4-fold.ConclusionAlthough moderate levels of RNA degradation are tolerated by microarrays with 3'-biased probe selection designs, in this study we identify a threshold beyond which decreased specificity and sensitivity can be observed that closely correlates with average target length. These results highlight the value of annotating microarray data with metrics that capture important aspects of sample quality. | [ Karol LThompson, P ScottPine, Barry ARosenzweig, YaronTurpaz, JacquesRetief] | BMC Biotechnology | 2007-9-13 | 4.0 |
| pmc1964861 | Transcriptome-wide analysis of blood vessels laser captured from human skin and chronic wound-edge tissue | Chronic wounds represent a substantial public health problem. The development of tools that would enable sophisticated scrutiny of clinical wound tissue material is highly desirable. This work presents evidence enabling rapid specific identification and laser capture of blood vessels from human tissue in a manner which lends itself to successful high-density (U133A) microarray analysis. Such screening of transcriptome followed by real-time PCR and immunohistochemical verification of candidate genes and their corresponding products were performed by using 3 mm biopsies. Of the 18,400 transcripts and variants screened, a focused set of 53 up-regulated and 24 down-regulated genes were noted in wound-derived blood vessels compared with blood vessels from intact human skin. The mean abundance of periostin in wound-site blood vessels was 96-fold higher. Periostin is known to be induced in response to vascular injury and its expression is associated with smooth muscle cell differentiation in vitro and promotes cell migration. Forty-fold higher expression of heparan sulfate 6-O-endosulfatase1 (Sulf1) was noted in wound-site vessels. Sulf1 has been recently recognized to be anti-angiogenic. During embryonic vasculogenesis, CD24 expression is down-regulated in human embryonic stem cells. Wound-site vessels had lower CD24 expression. The findings of this work provide a unique opportunity to appreciate the striking contrast in the transcriptome composition in blood vessels collected from the intact skin and from the wound-edge tissue. Sets of genes with known vascular functions but never connected to wound healing were identified to be differentially expressed in wound-derived blood vessels paving the way for innovative clinically relevant hypotheses. | [ SashwatiRoy, DarshanPatel, SavitaKhanna, Gayle M.Gordillo, SabyasachiBiswas, AvnerFriedman, Chandan K.Sen] | Proceedings of the National Academy of Sciences of the United States of America | 2007-8-29 | |
| pmc1983368 | Form-Deprivation Myopia in Chick Induces Limited Changes in Retinal Gene Expression | Evidence has implicated the retina as a principal controller of refractive development. In the present study, the retinal transcriptome was analyzed to identify alterations in gene expression and potential signaling pathways involved in form-deprivation myopia of the chick.MethodsOne-week-old white Leghorn chicks wore a unilateral image-degrading goggle for 6 hours or 3 days (n = 6 at each time). Total RNA from the retina/(retinal pigment epithelium) was used for expression profiling with chicken gene microarrays (Chicken GeneChips; Affymetrix, Santa Clara, CA). To identify gene expression level differences between goggled and contralateral nongoggled eyes, normalized microarray signal intensities were analyzed by the significance analysis of microarrays (SAM) approach. Differentially expressed genes were validated by real-time quantitative reverse transcription–polymerase chain reaction (qPCR) in independent biological replicates.ResultsSmall changes were detected in differentially expressed genes in form-deprived eyes. In chickens that had 6 hours of goggle wear, downregulation of bone morphogenetic protein 2 and connective tissue growth factor was validated. In those with 3 days of goggle wear, downregulation of bone morphogenetic protein 2, vasoactive intestinal peptide, preopro-urotensin II–related peptide and mitogen-activated protein kinase phosphatase 2 was validated, and upregulation of endothelin receptor type B and interleukin-18 was validated.ConclusionsForm-deprivation myopia, in its early stages, is associated with only minimal changes in retinal gene expression at the level of the transcriptome. While the list of validated genes is short, each merits further study for potential involvement in the signaling cascade mediating myopia development. | [ Alice M.McGlinn, Donald A.Baldwin, John W.Tobias, Murat T.Budak, Tejvir S.Khurana, Richard A.Stone] | Investigative ophthalmology & visual science | | |
| pmc1951299 | Insulin-Like Growth Factor II Receptor-Mediated Intracellular Retention of Cathepsin B Is Essential for Transformation of Endothelial Cells by Kaposi's Sarcoma-Associated Herpesvirus▿ | Kaposi's sarcoma-associated herpesvirus (KSHV) is the pathological agent of Kaposi's sarcoma (KS), a tumor characterized by aberrant proliferation of endothelial-cell-derived spindle cells. Since in many cancers tumorigenesis is associated with an increase in the activity of the cathepsin family, we studied the role of cathepsins in KS using an in vitro model of KSHV-mediated endothelial cell transformation. Small-molecule inhibitors and small interfering RNA (siRNA) targeting CTSB, but not other cathepsins, inhibited KSHV-induced postconfluent proliferation and the formation of spindle cells and foci of dermal microvascular endothelial cells. Interestingly, neither CTSB mRNA nor CTSB protein levels were induced in endothelial cells latently infected with KSHV. Secretion of CTSB was strongly diminished upon KSHV infection. Increased targeting of CTSB to endosomes was caused by the induction by KSHV of the expression of insulin-like growth factor-II receptor (IGF-IIR), a mannose-6-phosphate receptor (M6PR) that binds to cathepsins. Inhibition of IGF-IIR/M6PR expression by siRNA released CTSB for secretion. In contrast to the increased cathepsin secretion observed in most other tumors, viral inhibition of CTSB secretion via induction of an M6PR is crucial for the transformation of endothelial cells. | [ Patrick P.Rose, MatthewBogyo, Ashlee V.Moses, KlausFrüh] | Journal of Virology | 2007-5-16 | |
| pmc1868757 | Gene-resolution analysis of DNA copy number variation using oligonucleotide expression microarrays | Array-based comparative genomic hybridization (aCGH) is a high-throughput method for measuring genome-wide DNA copy number changes. Current aCGH methods have limited resolution, sensitivity and reproducibility. Microarrays for aCGH are available only for a few organisms and combination of aCGH data with expression data is cumbersome.ResultsWe present a novel method of using commercial oligonucleotide expression microarrays for aCGH, enabling DNA copy number measurements and expression profiles to be combined using the same platform. This method yields aCGH data from genomic DNA without complexity reduction at a median resolution of approximately 17,500 base pairs. Due to the well-defined nature of oligonucleotide probes, DNA amplification and deletion can be defined at the level of individual genes and can easily be combined with gene expression data.ConclusionA novel method of gene resolution analysis of copy number variation (graCNV) yields high-resolution maps of DNA copy number changes and is applicable to a broad range of organisms for which commercial oligonucleotide expression microarrays are available. Due to the standardization of oligonucleotide microarrays, graCNV results can reliably be compared between laboratories and can easily be combined with gene expression data using the same platform. | [ HerbertAuer, David LNewsom, Norma JNowak, Kirk MMcHugh, SunitaSingh, Chack-YungYu, YanYang, Gail DWenger, Julie MGastier-Foster, KarlKornacker] | BMC Genomics | 2007-4-30 | 3.4 |
| pmc1784109 | Synthesis and biologic properties of hydrophilic sapphyrins, a new class of tumor-selective inhibitors of gene expression | Sapphyrin analogues and related porphyrin-like species have attracted attention as anticancer agents due to their selective localization in various cancers, including hematologic malignancies, relative to surrounding tissues. Sapphyrins are electron affinic compounds that generate high yields of singlet oxygen formation. Although initially explored in the context of photodynamic therapy, sapphyrins have intrinsic anticancer activity that is independent of their photosensitizing properties. However, the mechanisms for their anticancer activity have not been fully elucidated.ResultsWe have prepared a series of hydrophilic sapphyrins and evaluated their effect on proliferation, uptake, and cell death in adherent human lung (A549) and prostate (PC3) cancer cell lines and in an A549 xenograft tumor model. PCI-2050, the sapphyrin derivative with the highest in vitro growth inhibitory activity, significantly lowered 5-bromo-2'-deoxyuridine incorporation in S-phase A549 cells by 60% within eight hours and increased levels of reactive oxygen species within four hours. The growth inhibition pattern of PCI-2050 in the National Cancer Institute 60 cell line screen correlated most closely using the COMPARE algorithm with known transcriptional or translational inhibitors. Gene expression analyses conducted on A549 plateau phase cultures treated with PCI-2050 uncovered wide-spread decreases in mRNA levels, which especially affected short-lived transcripts. Intriguingly, PCI-2050 increased the levels of transcripts involved in RNA processing and trafficking, transcriptional regulation, and chromatin remodeling. We propose that these changes reflect the activation of cellular processes aimed at countering the observed wide-spread reductions in transcript levels. In our A549 xenograft model, the two lead compounds, PCI-2050 and PCI-2022, showed similar tumor distributions despite differences in plasma and kidney level profiles. This provides a possible explanation for the better tolerance of PCI-2022 relative to PCI-2050.ConclusionHydrophilic sapphyrins were found to display promise as novel agents that localize to tumors, generate oxidative stress, and inhibit gene expression. | [ ZhongWang, Philip SLecane, PatriciaThiemann, QingFan, CeciliaCortez, XuanMa, DanielleTonev, DaleMiles, LouieNaumovski, Richard AMiller, DarrenMagda, Dong-GyuCho, Jonathan LSessler, Brian LPike, Samantha MYeligar, Mazen WKaraman, Joseph GHacia] | Molecular Cancer | 2007-1-19 | |
| pmc1894940 | Rofecoxib modulates multiple gene expression pathways in a clinical model of acute inflammatory pain | New insights into the biological properties of cyclooxygenase-2 (COX-2) and its response pathway challenge the hypothesis that COX-2 is simply pro-inflammatory and inhibition of COX-2 solely prevents the development of inflammation and ameliorates inflammatory pain. The present study performed a comprehensive analysis of gene/protein expression induced by a selective inhibitor of COX-2, rofecoxib, compared with a non-selective COX inhibitor, ibuprofen, and placebo in a clinical model of acute inflammatory pain (the surgical extraction of impacted third molars) using microarray analysis followed by quantitative RT-PCR verification and Western blotting. Inhibition of COX-2 modulated gene expression related to inflammation and pain, the arachidonic acid pathway, apoptosis/angiogenesis, cell adhesion and signal transduction. Compared to placebo, rofecoxib treatment increased the gene expression of ANXA3 (annexin 3), SOD2 (superoxide dismutase 2), SOCS3 (suppressor of cytokine signaling 3) and IL1RN (IL1 receptor antagonist) which are associated with inhibition of phospholipase A2 and suppression of cytokine signaling cascades, respectively. Both rofecoxib and ibuprofen treatment increased the gene expression of the pro-inflammatory mediators, IL6 and CCL2 (chemokine C-C motif ligand 2), following tissue injury compared to the placebo treatment. These results indicate a complex role for COX-2 in the inflammatory cascade in addition to the well-characterized COX-dependent pathway, as multiple pathways are also involved in rofecoxib-induced anti-inflammatory and analgesic effects at the gene expression level. These findings may also suggest an alternative hypothesis for the adverse effects attributed to selective inhibition of COX-2. | [ Xiao-MinWang, Tian-XiaWu, MayHamza, Edward S.Ramsay, Sharon M.Wahl, Raymond A.Dionne] | Pain | 2006-10-27 | |
| pmc1994650 | Altered Gene Expression in Mice Selected for High Maternal Aggression | We previously applied selective breeding on outbred mice to increase maternal aggression (maternal defense). In this study, we compared gene expression within a continuous region of the CNS involved in maternal aggression (hypothalamus and preoptic regions) between lactating selected (S) and non-selected control (C) mice (n = 6 per group). Using microarrays representing over 40,000 genes or expressed sequence tags, two statistical algorithms were used to identify significant differences in gene expression: robust multi array and the probe logarithmic intensity error method. ∼ 200 genes were identified as significant using an intersection from both techniques. A subset of genes were examined for confirmation by real-time PCR. Significant decreases were found in S mice for neurotensin and neuropeptide Y receptor Y2 (both confirmed by PCR). Significant increases were found in S mice for neuronal nitric oxide synthase (confirmed by PCR), the K+ channel subunit, Kcna1 (confirmed by PCR), corticotrophin releasing factor binding protein (just above significance using PCR; p = 0.051), and GABA A receptor subunit 1A (not confirmed by PCR, but similar direction). S mice also exhibited significantly higher levels of the neurotransmitter receptor, adenosine A1 receptor, and the transcription factors, c-Fos, and Egr-1. Interestingly, for 24 genes related to metabolism, all were significantly elevated in S mice, suggesting altered metabolism in these mice. Together, this study provides a list of candidate genes (some previously implicated in maternal aggression and some novel) that may play an important role in the production of this behavior. | [ Stephen C.Gammie, Anthony P.Auger, Heather M.Jessen, Rena J.Vanzo, Tarif A.Awad, Sharon A.Stevenson] | Genes, brain, and behavior | 2006-8-29 | |
| pmc1489790 | Reverse Engineering Antibiotic Sensitivity in a Multidrug-Resistant Pseudomonas aeruginosa Isolate | Antibiotic resistance is a pervasive and growing clinical problem. We describe an evaluation of a reverse engineering approach for identifying cellular mechanisms and genes that could be manipulated to increase antibiotic sensitivity in a resistant Pseudomonas aeruginosa isolate. We began by chemically mutating a broadly resistant isolate of P. aeruginosa and screening for mutants with increased sensitivity to the aminoglycoside amikacin, followed by performing whole-genome transcriptional profiling of the mutant and wild-type strains to characterize the global changes occurring as a result of the mutations. We then performed a series of assays to characterize the mechanisms involved in the increased sensitivity of the mutant strains. We report four primary results: (i) mutations that increase sensitivity occur at a high frequency (10−2) relative to the frequency of those that increase resistance (10−5 to 10−10) and occur at a frequency 104 higher than the frequency of a single point mutation; (ii) transcriptional profiles were altered in sensitive mutants, resulting in overall expression patterns more similar to those of the sensitive laboratory strain PAO1 than those of the parental resistant strain; (iii) genes found from transcriptional profiling had the more dramatic changes in expression-encoded functions related to cellular membrane permeability and aminoglycoside modification, both of which are known aminoglycoside resistance mechanisms; and finally, (iv) even though we did not identify the specific sites of mutation, several different follow-up MIC assays suggested that the mutations responsible for increased sensitivity differed between sensitive mutants. | [ Julie M.Struble, Ryan T.Gill] | Antimicrobial Agents and Chemotherapy | | |
| pmc1431565 | How to decide? Different methods of calculating gene expression from short oligonucleotide array data will give different results | Short oligonucleotide arrays for transcript profiling have been available for several years. Generally, raw data from these arrays are analysed with the aid of the Microarray Analysis Suite or GeneChip Operating Software (MAS or GCOS) from Affymetrix. Recently, more methods to analyse the raw data have become available. Ideally all these methods should come up with more or less the same results. We set out to evaluate the different methods and include work on our own data set, in order to test which method gives the most reliable results.ResultsCalculating gene expression with 6 different algorithms (MAS5, dChip PMMM, dChip PM, RMA, GC-RMA and PDNN) using the same (Arabidopsis) data, results in different calculated gene expression levels. Consequently, depending on the method used, different genes will be identified as differentially regulated. Surprisingly, there was only 27 to 36% overlap between the different methods. Furthermore, 47.5% of the genes/probe sets showed good correlation between the mismatch and perfect match intensities.ConclusionAfter comparing six algorithms, RMA gave the most reproducible results and showed the highest correlation coefficients with Real Time RT-PCR data on genes identified as differentially expressed by all methods. However, we were not able to verify, by Real Time RT-PCR, the microarray results for most genes that were solely calculated by RMA. Furthermore, we conclude that subtraction of the mismatch intensity from the perfect match intensity results most likely in a significant underestimation for at least 47.5% of the expression values. Not one algorithm produced significant expression values for genes present in quantities below 1 pmol. If the only purpose of the microarray experiment is to find new candidate genes, and too many genes are found, then mutual exclusion of the genes predicted by contrasting methods can be used to narrow down the list of new candidate genes by 64 to 73%. | [ Frank FMillenaar, JohnOkyere, Sean TMay, Martijnvan Zanten, Laurentius ACJVoesenek, Anton JMPeeters] | BMC Bioinformatics | 2006-3-15 | |
| pmc1346858 | Interferon Regulatory Factor 3 Is Necessary for Induction of Antiviral Genes during Human Cytomegalovirus Infection† | Viral infection activates interferon regulatory factor 3 (IRF3), a cofactor for the induction of interferon-stimulated genes (ISGs). The role of IRF3 in the activation of ISGs by human cytomegalovirus (HCMV) is controversial despite the fact that HCMV has consistently been shown to induce ISGs during infection of fibroblasts. To address the function of IRF3 in HCMV-mediated ISG induction, we monitored ISG expression and global gene expression in HCMV-infected cells in which IRF3 function had been depleted by small interfering RNA or blocked by dominant negative IRF3. A specific reduction of ISG induction was observed, whereas other transcripts were unaffected. We therefore conclude that IRF3 specifically regulates ISG induction during the initial phase of HCMV infection. | [ Victor R.DeFilippis, BridgetRobinson, Thomas M.Keck, Scott G.Hansen, Jay A.Nelson, Klaus J.Früh] | Journal of Virology | | |
| pmc2361501 | Gene expression profiling of noninvasive primary urothelial tumours using microarrays | At present, the mechanism leading to bladder cancer is still poorly understood, and our knowledge about early events in tumorigenesis is limited. This study describes the changes in gene expression occurring during the neoplastic transition from normal bladder urothelium to primary Ta tumours. Using DNA microarrays, we identified novel differentially expressed genes in Ta tumours compared to normal bladder, and genes that were altered in high-grade tumours. Among the mostly changed genes between normal bladder and Ta tumours, we found genes related to the cytoskeleton (keratin 7 and syndecan 1), and transcription (high mobility group AT-hook 1). Altered genes in high-grade tumours were related to cell cycle (cyclin-dependent kinase 4) and transcription (jun d proto-oncogene). Furthermore, we showed the presence of high keratin 7 transcript expression in bladder cancer, and Western blotting analysis revealed three major molecular isoforms of keratin 7 in the tissues. These could be detected in urine sediments from bladder tumour patients. | [ MAaboe, NMarcussen, K M-EJensen, TThykjaer, LDyrskjøt, T FØrntoft] | British Journal of Cancer | 2005-11-01 | 3.0 |
| pmc1373786 | Gene Array Profiling of Large Hypothalamic CNS Regions in Lactating and Randomly Cycling Virgin Mice | A dramatic example of neuronal and physiological plasticity in adult mammals occurs during the transition from a non-maternal to a maternal, lactating state. In this study we compared gene expression within a large continuous region of the CNS involved in maternal behaviors (hypothalamus, preoptic regions, and nucleus accumbens) between lactating (L) (postpartum Day 7) and randomly cycling virgin (V) outbred mice. Using high density oligonucleotide arrays representing 11,904 genes, two statistical algorithms were used to identify significant differences in gene expression: robust multi array (p < 0.001) (n = 92 genes) and significance analysis of microarrays using a 10% false discover rate (n = 114 genes). 27 common genes were identified as significant using both techniques. A subset of genes (n = 5) were selected and examined by real-time PCR. Our findings were consistent with previous published work. For example, neuropeptide Y (NPY) and proenkephalin were elevated in L mice, whereas POMC was decreased. Increased levels of NPY Y2 receptor and polo-like kinase and decreased levels of endothelin receptor type b in L mice are examples of novel gene expression changes not previously identified. Expression differences occurred in broad classes. Together, our findings provide possible new material on gene expression changes that may support maternal behaviors. The advantages and drawbacks of sampling large CNS regions using arrays are discussed. | [ Stephen C.Gammie, Nina S.Hasen, Tarif A.Awad, Anthony P.Auger, Heather M.Jessen, Jules B.Panksepp, Anne M.Bronikowski] | Brain research. Molecular brain research | | |
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| sciencedirectS1424390311000160 | Invasive markers identified by gene expression profiling in pancreatic cancer | BackgroundMolecular profiling has proven utility as a diagnostic and predictive tool in clinical oncology. However, a clinically relevant gene expression profile in pancreatic cancer remains elusive.MethodsPrimary and metastatic pancreatic cancer cell lines (BxPC-3 and AsPC-1), were stimulated with phorbol-12-myristate 13-acetate (PMA), a known inducer of cell invasion. Affymetrix gene expression microarray analysis was performed, comparing gene expression to unstimulated controls. Differential expression was identified using ArrayAssist, and confirmed using quantitative real-time PCR. Bioinformatic analysis was performed using Pathway Studio and GOstat. The derived gene expression was further validated in fresh frozen pancreatic tumour samples. The ability of the derived 3 gene expression markersto differentiate between pancreatic adenocarcinoma (PDAC) and other neoplasms, and its association with clinicopathological variables was examined.ResultsPMA-induced significant changes in cell line gene expression, from which distinctive 3 potential invasive markers were derived. Expression of these genes, uPA, MMP-1 and IL1-R1 was confirmed in human pancreatic tumours, and was found to differentiate PDAC from other pancreatic neoplasms. The expression of IL1-R1 in PDAC is a novel finding. We found that the expression of MMP-1 was associated with high-grade PDAC (p = 0.035, Wilcoxon rank sum).ConclusionWe have identified three potential invasive markers, uPA, MMP-1 and IL1-R1, whose gene expression may differentiate PDAC from other pancreatic neoplasms, and potentially reflect a more invasive phenotype. | [A. Rogersa, M.J. Smitha, P. Doolanc, C. Clarkec, M. Clynesc, J.F. Murphya, A. McDermottb, N. Swanb, P. Crottyb, P.F. Ridgwaya, K.C. Conlona] | Pancreatology | | |
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| sciencedirectS0925477311000396 | An ENU-induced point mutation in the mouse Btaf1 gene causes post-gastrulation embryonic lethality and protein instability | The mouse Btaf1 gene, an ortholog of yeast MOT1, encodes a highly conserved general transcription factor. The function of this SNF2-like ATPase has been studied mainly in yeast and human cells, which has revealed that it binds directly to TBP, forming the B-TFIID complex. This complex binds to core promoters of RNA polymerase II-transcribed genes and, of crucial importance, BTAF1–TBP interactions have been shown to affect the kinetics of TBP–promoter interactions. Here we report the isolation of a mouse line carrying a Btaf1 allele containing an ENU-induced point mutation that causes a substitution mutation in the BTAF1 ATPase domain. Embryos homozygous for this loss-of-function mutation appear to be morphologically normal until early somite stages, but die between embryonic days 9 and 10.5 displaying growth arrest and edema. Analyses in vitro suggest that the altered protein is less stable and, independent from this, functionally impaired in releasing of TBP from chromatin, but not in binding to TBP. | [Carolien Wansleebena 1, Léon van Gurpa, Petra de Graafb, Florence Moussonb, H.Th. Marc Timmersb, Frits Meijlinka] | Mechanisms of Development | May–June 2011 | |
| sciencedirectS1389172310003762 | Gingival and dermal fibroblasts: Their similarities and differences revealed from gene expression | Gene expression profiles in normal human gingival and dermal fibroblasts were investigated using DNA microarrays. Their fundamental characteristics were almost identical, but 5% of their genes were uniquely expressed. These results help us to choose an optimal cell source for effective fibroblast-based cell therapy that is dependent on differential gene expression profiles. | [Katsumi Ebisawa1 2 †, Ryuji Kato3 4 †, Mai Okada3 4 5, Tomotaka Sugimura3, Mazlyzam Abdul Latif5 6, Yusuke Hori7, Yuji Narita3 5 8, Minoru Ueda1, Hiroyuki Honda4, Hideaki Kagami5 9] | Journal of Bioscience and Bioengineering | March 2011 | |
| sciencedirectS0042682210006380 | Early gene expression events in ferrets in response to SARS coronavirus infection versus direct interferon-alpha2b stimulation | Type I interferons (IFNs) are essential to the clearance of viral diseases, however, a clear distinction between genes upregulated by direct virus–cell interactions and genes upregulated by secondary IFN production has not been made. Here, we investigated differential gene regulation in ferrets upon subcutaneous administration of IFN-?2b and during SARS-CoV infection. In vivo experiments revealed that IFN-?2b causes STAT1 phosphorylation and upregulation of abundant IFN response genes (IRGs), chemokine receptors, and other genes that participate in phagocytosis and leukocyte transendothelial migration. During infection with SARS-CoV not only a variety of IRGs were upregulated, but also a significantly broader range of genes involved in cell migration and inflammation. This work allowed dissection of several molecular signatures present during SARS-CoV which are part of a robust IFN antiviral response. These signatures can be useful markers to evaluate the status of IFN responses during a viral infection and specific features of different viruses. | [Ali Danesha b, Cheryl M. Camerona, Alberto J. Leónd, Longsi Rana, Luoling Xua, Yuan Fanga b, Alyson A. Kelvinc g, Thomas Rowea 1, Honglin Chend, Yi Guand, Colleen B. Jonssone, Mark J. Camerona, David J. Kelvina d f] | Virology | 5 January 2011 | |
| sciencedirectS156772491000111X | Xenomitochondrial mice: Investigation into mitochondrial compensatory mechanisms | Xenomitochondrial mice, harboring evolutionarily divergent Mus terricolor mitochondrial DNA (mtDNA) on a Mus musculus domesticus nuclear background (B6NTac(129S6)-mtM. terricolor/Capt; line D7), were subjected to molecular and phenotypic analyses. No overt in vivo phenotype was identified in contrast to in vitro xenomitochondrial cybrid studies. Microarray analyses revealed differentially expressed genes in xenomitochondrial mice, though none were directly involved in mitochondrial function. qRT-PCR revealed upregulation of mt-Co2 in xenomitochondrial mice. These results illustrate that cellular compensatory mechanisms for mild mitochondrial dysfunction alter mtDNA gene expression at a proteomic and/or translational level. Understanding these mechanisms will facilitate the development of therapeutics for mitochondrial disorders. | [M.V. Cannona b, D.A. Dunna b, M.H. Irwina, A.I. Brooksc, F.F. Bartold, I.A. Trouncee, C.A. Pinkerta b] | Mitochondrion | January 2011 | |
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| sciencedirectS019701861000118X | Adrenoceptors in brain: Cellular gene expression and effects on astrocytic metabolism and [Ca2+]i | Recent in vivo studies have established astrocytes as a major target for locus coeruleus activation (Bekar et al., 2008), renewing interest in cell culture studies on noradrenergic effects on astrocytes in primary cultures and calling for additional information about the expression of adrenoceptor subtypes on different types of brain cells. In the present communication, mRNA expression of ?1-, ?2- and ?-adrenergic receptors and their subtypes was determined in freshly isolated, cell marker-defined populations of astrocytes, NG2-positive cells, microglia, endothelial cells, and Thy1-positive neurons (mainly glutamatergic projection neurons) in murine cerebral cortex. Immediately after dissection of frontal, parietal and occipital cortex of 10–12-week-old transgenic mice, which combined each cell-type marker with a specific fluorescent signal, the tissue was digested, triturated and centrifuged, yielding a solution of dissociated cells of all types, which were separated by fluorescence-activated cell sorting (FACS). mRNA expression in each cell fraction was determined by microarray analysis. ?1A-Receptors were unequivocally expressed in astrocytes and NG2-positive cells, but absent in other cell types, and ?1B-receptors were not expressed in any cell population. Among ?2-receptors only ?2A-receptors were expressed, unequivocally in astrocytes and NG-positive cells, tentatively in microglia and questionably in Thy1-positive neurons and endothelial cells. ?1-Receptors were unequivocally expressed in astrocytes, tentatively in microglia, and questionably in neurons and endothelial cells, whereas ?2-adrenergic receptors showed tentative expression in neurons and astrocytes and unequivocal expression in other cell types. This distribution was supported by immunochemical data and its relevance established by previous studies in well-differentiated primary cultures of mouse astrocytes, showing that stimulation of ?2-adrenoceptors increases glycogen formation and oxidative metabolism, the latter by a mechanism depending on intramitochondrial Ca2+, whereas ?1-adrenoceptor stimulation enhances glutamate uptake, and ?-adrenoceptor activation causes glycogenolysis and increased Na+, K+-ATPase activity. The Ca2+- and cAMP-mediated association between energy-consuming and energy-yielding processes is emphasized. | [Leif Hertza, Ditte Lovattb c, Steven A. Goldmanc, Maiken Nedergaardb] | Neurochemistry International | November 2010 | |
| sciencedirectS0176161710002075 | Microarray analysis of endophyte-infected and endophyte-free tall fescue | Many grasses have mutualistic symbioses with fungi of the family Clavicipitaceae. Tall fescue can harbor the obligate endophyte, Neotyphodiumcoenophialum that is asexually propagated and transmitted via host seeds. Total RNA was isolated from pseudostems of known endophyte-infected (E+) and endophyte-free (E−) plants and tested in triplicate on the Affymetrix Wheat Genome Array GeneChip® and Barley1 Genome Array GeneChip®. Overall 14–15% and 17–18% of the probe sets were called present on the wheat and barley chips, respectively. In order to identify genes that were specifically differentially expressed between the E+ and E− tall fescue, a combination of both barley and wheat target sequences that were differentially expressed (greater than twofold) that were similar on both chips on both barley and wheat arrays yielded 32 probe set (genes) that were differentially expressed. Tall fescue ESTs were identified for a number of the probe sets that were differentially expressed on the barley and wheat arrays. PCR primers were designed to fescue ESTs and tested to verify the expression profile observed in the microarray experiments. Some primers confirmed the expected results, although in other cases no differences were observed between the E+ and E− plants, or the results were contrary to what was expected. Our results suggest that while some differentially expressed genes were identified by this method, the cross-species hybridization appears to have significant limitations for the transcriptome analysis of tall fescue. | [Randy D. Dinkinsa, Adam Barnesb 1, Whitney Watersb 2] | Journal of Plant Physiology | 15 September 2010 | |
| sciencedirectS0176161710001203 | Global analysis of the root hair morphogenesis transcriptome reveals new candidate genes involved in root hair formation in barley | Root hairs are long tubular outgrowths of specialized root epidermal cells that play an important role in plant nutrition and water uptake. They are also an important model in studies of higher plant cell differentiation. In contrast to the model dicot Arabidopsis thaliana, currently very little is known about the genetic and molecular basis of root hair formation in monocots, including major cereals. To elucidate candidate genes controlling this developmental process in barley, we took advantage of the recently established Affymetrix GeneChip Barley1 Genome Array to carry out global transcriptome analyses of hairless and root hair primordia-forming roots of two barely mutant lines. Expression profiling of the root-hairless mutant rhl1.a and its wild type parent variety ‘Karat’ revealed 10 genes potentially involved in the early step of root hair formation in barley. Differential expression of all identified genes was confirmed by quantitative reverse transcription-polymerase chain reaction. The genes identified encode proteins associated with the cell wall and membranes, including one gene for xyloglucan endotransglycosylase, three for peroxidase enzymes and five for arabinogalactan protein, extensin, leucine-rich-repeat protein, phosphatidylinositol phosphatidylcholine transfer protein and a RhoGTPase GDP dissociation inhibitor, respectively. The molecular function of one gene is unknown at present. The expression levels of these genes were strongly reduced in roots of the root-hairless mutant rhl1.a compared to the parent variety, while expression of all 10 genes was similar in another mutant, i.e. rhp1.b, that has lost its ability to develop full root hairs but still forms hairs blocked at the primordium stage, and its wild type relative. This clearly indicates that the new genes identified are involved in the initiation of root hair morphogenesis in barley. | [Miroslaw Kwasniewskia, Agnieszka Janiaka, Bernd Mueller-Roeberb, Iwona Szarejkoa] | Journal of Plant Physiology | 1 September 2010 | |
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| sciencedirectS002751070900373X | Temporal gene expression changes induced by a low concentration of benzo[a]pyrene diol epoxide in a normal human cell line | (±)-anti-Benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), which causes bulky-adduct DNA damage, is well-characterized as the ultimate carcinogen of benzo[a]pyrene (BaP). In this study, we have employed Affymetrix HG-U133 Plus 2.0 microarray and quantitative real-time RT-PCR methods to investigate a temporal transcriptomic response triggered by a low concentration (0.05 ?M) of BPDE at 1, 10, and 22 h after exposure in normal human cells. The differential gene expression profiles at the three time points varied greatly, and generally reflected a cellular responsive process from initiation to progression and to recovery after the BPDE-caused damage. The dynamic regulation of the genes related with cell cycle progression and cell fate exhibited a tendency from inhibition to survival, which was accordant with the cell cycle arrest and cytotoxicity data induced by the low-dose BPDE exposure. In silico comparison of the genomic data revealed that BPDE and ultraviolet induced a panel of common transcriptional responses, which might be related with a series of similar molecular processes elicited by these two DNA-damaging agents. In conclusion, this whole-genome time-course study has identified a dynamically regulated transcriptional signature after low-dose BPDE exposure, which may help to understand the complex mechanisms of mutagenesis and carcinogenesis induced by BPDE. | [Xiangyun Lu1, Jimin Shao1, Hongjuan Li2, Yingnian Yu] | Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis | 3 February 2010 | |
| sciencedirectS0981942809002447 | Syncytium gene expression in Glycine max[PI 88788] roots undergoing a resistant reaction to the parasitic nematode Heterodera glycines | The plant parasitic nematode, Heterodera glycines is the major pathogen of Glycine max (soybean). H. glycines accomplish parasitism by creating a nurse cell known as the syncytium from which it feeds. The syncytium undergoes two developmental phases. The first is a parasitism phase where feeding sites are selected, initiating the development of the syncytium. During this earlier phase (1–4 days post infection), syncytia undergoing resistant and susceptible reactions appear the same. The second phase is when the resistance response becomes evident (between 4 and 6 dpi) and is completed by 9 dpi. Analysis of the resistant reaction of G. max genotype PI 88788 (G. max[PI 88788]) to H. glycines population NL1-RHg/HG-type 7 (H. glycines[NL1-RHg/HG-type 7]) is accomplished by laser microdissection of syncytia at 3, 6 and 9 dpi. Comparative analyses are made to pericycle and their neighboring cells isolated from mock-inoculated roots. These analyses reveal induced levels of the jasmonic acid biosynthesis and 13-lipoxygenase pathways. Direct comparative analyses were also made of syncytia at 6 days post infection to those at 3 dpi (base line). The comparative analyses were done to identify localized gene expression that characterizes the resistance phase of the resistant reaction. The most highly induced pathways include components of jasmonic acid biosynthesis, 13-lipoxygenase pathway, S-adenosyl methionine pathway, phenylpropanoid biosynthesis, suberin biosynthesis, adenosylmethionine biosynthesis, ethylene biosynthesis from methionine, flavonoid biosynthesis and the methionine salvage pathway. In comparative analyses of 9 dpi to 6 dpi (base line), these pathways, along with coumarin biosynthesis, cellulose biosynthesis and homogalacturonan degradation are induced. The experiments presented here strongly implicate the jasmonic acid defense pathway as a factor involved in the localized resistant reaction of G. max[PI 88788] to H. glycines[NL1-RHg/HG-type 7]. | [Vincent P. Klinka, Parsa Hosseinib c, Prachi D. Matsyea, Nadim W. Alkharoufc, Benjamin F. Matthewsb] | Plant Physiology and Biochemistry | February–March 2010 | |
| sciencedirectS0965174809001763 | Identification of juvenile hormone target genes in the adult female mosquitoes | Many aspects of reproductive maturation in newly emerged adult female mosquitoes are controlled by juvenile hormone (JH). However, the molecular basis of this hormonal regulation remains poorly understood. In this study, we analyzed transcriptome changes in young female adults after topical application of exogenous JH. mRNA levels of 16 and 72 genes were enhanced at 3 h and 12 h after hormone administration, respectively. The observed upregulations can be also induced by the JH analogues, methoprene and pyriproxyfen, but not by farnesol, indicating that these responses are JH-specific. Among the genes activated by JH is the mosquito Krüppel homolog 1 (Kr-h1) gene. Studies in the beetle Tribolium castaneum have indicated that Kr-h1 is transcriptionally activated by JH during metamorphosis and encodes a gene expression regulator acting downstream of the methoprene-tolerant (Met) gene in the JH signaling pathway. We found that the upregulation of AaKr-h1 after eclosion relied on the function of the mosquito Met protein, suggesting that a conserved JH signaling pathway is utilized both in the metamorphosis of T. castaneum and in the adult reproductive maturation of the Aedes aegypti mosquito. This study lays a foundation for a better understanding of the mechanisms of juvenile hormone action. | [Jinsong Zhu, Jefferson M. Busche, Xing Zhang] | Insect Biochemistry and Molecular Biology | January 2010 | |
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| sciencedirectS0006291X09009000 | Profiling of anti-fibrotic signaling by hepatocyte growth factor in renal fibroblasts | Hepatocyte growth factor (HGF) is a multifunctional growth factor affecting cell proliferation and differentiation. Due to its mitogenic potential, HGF plays an important role in tubular repair and regeneration after acute renal injury. However, recent reports have shown that HGF also acts as an anti-inflammatory and anti-fibrotic factor, affecting various cell types such as renal fibroblasts and triggering tubulointerstitial fibrosis of the kidney.The present study provides evidence that HGF stimulation of renal fibroblasts results in the activation of both the Erk1/2 and the Akt pathways. As previously shown, Erk1/2 phosphorylation results in Smad-linker phosphorylation, thereby antagonizing cellular signals induced by TGF?. By siRNA mediated silencing of the Erk1/2-Smad linkage, however, we now demonstrate that Akt signaling acts as an auxiliary pathway responsible for the anti-fibrotic effects of HGF. In order to define the anti-fibrotic function of HGF we performed comprehensive expression profiling of HGF-stimulated renal fibroblasts by microarray hybridization. Functional cluster analyses and quantitative PCR assays indicate that the HGF-stimulated pathways transfer the anti-fibrotic effects in renal interstitial fibroblasts by reducing expression of extracellular matrix proteins, various chemokines, and members of the CCN family. | [Stephanie Schievenbuscha, Ingo Stracka, Melanie Schefflera, Kerstin Wennholda, Julia Maurera, Roswitha Nischtb, Hans Peter Dienesa, Margarete Odenthala] | Biochemical and Biophysical Research Communications | 17 July 2009 | |
| sciencedirectS0041008X09001513 | Dose-dependent transitions in Nrf2-mediated adaptive response and related stress responses to hypochlorous acid in mouse macrophages | Hypochlorous acid (HOCl) is potentially an important source of cellular oxidative stress. Human HOCl exposure can occur from chlorine gas inhalation or from endogenous sources of HOCl, such as respiratory burst by phagocytes. Transcription factor Nrf2 is a key regulator of cellular redox status and serves as a primary source of defense against oxidative stress. We recently demonstrated that HOCl activates Nrf2-mediated antioxidant response in cultured mouse macrophages in a biphasic manner. In an effort to determine whether Nrf2 pathways overlap with other stress pathways, gene expression profiling was performed in RAW 264.7 macrophages exposed to HOCl using whole genome mouse microarrays. Benchmark dose (BMD) analysis on gene expression data revealed that Nrf2-mediated antioxidant response and protein ubiquitination were the most sensitive biological pathways that were activated in response to low concentrations of HOCl (< 0.35 mM). Genes involved in chromatin architecture maintenance and DNA-dependent transcription were also sensitive to very low doses. Moderate concentrations of HOCl (0.35 to 1.4 mM) caused maximal activation of the Nrf2 pathway and innate immune response genes, such as IL-1?, IL-6, IL-10 and chemokines. At even higher concentrations of HOCl (2.8 to 3.5 mM) there was a loss of Nrf2-target gene expression with increased expression of numerous heat shock and histone cluster genes, AP-1-family genes, cFos and Fra1 and DNA damage-inducible Gadd45 genes. These findings confirm an Nrf2-centric mechanism of action of HOCl in mouse macrophages and provide evidence of interactions between Nrf2, inflammatory, and other stress pathways. | [Courtney G. Woodsa c, Jingqi Fub d, Peng Xueb d, Yongyong Houb d, Linda J. Plutaa, Longlong Yanga, Qiang Zhanga, Russell S. Thomasa, Melvin E. Andersena, Jingbo Pib] | Toxicology and Applied Pharmacology | 1 July 2009 | |
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| sciencedirectS0969996108003021 | The ?-chemokine CXCL14 is up-regulated in the sciatic nerve of a mouse model of Charcot–Marie–Tooth disease type 1A and alters myelin gene expression in cultured Schwann cells | At present the pathogenesis of CMT1A neuropathy, caused by the overexpression of PMP22, has not yet been entirely understood. The PMP22-overexpressing C61 mutant mouse is a suitable animal model, which mimics the human CMT1A disorder. We observed that myelin gene expression in the sciatic nerve of the C61 mouse was up-regulated at postnatal day 4 to 7 (P4–P7). When investigating the morphology of peripheral nerves in C61 and wildtype mice at early stages of postnatal development, hypermyelination could be detected in the femoral quadriceps and sciatic nerve of transgenic animals at postnatal day 7 (P7). In order to identify genes, other than Pmp22, that are modulated in sciatic nerve of P7 transgenic mice, we applied microarray technology. Amongst the regulated genes, the gene encoding the ?-chemokine CXCL14 was most prominently up-regulated. We report that Cxcl14 was expressed exclusively by Schwann cells of the sciatic nerve, as well as by cultured Schwann cells triggered to differentiate. Furthermore, in cultured Schwann cells CXCL14 modulated the expression of myelin genes and altered cell proliferation. Our findings demonstrate that early overexpression of PMP22, in a mouse model of CMT1A, results in a strong up-regulation of CXCL14, which seems to play a novel regulatory role in Schwann cell differentiation. | [Elena M. Barbariaa, Bianca Kohlb, Bettina A. Buhrena, Kerstin Hasenpusch-Theila, Fabian Krusea, Patrick Kürya, Rudolf Martinib, Hans Werner Müllera c] | Neurobiology of Disease | March 2009 | |
| sciencedirectS0006899308025560 | Genomic response of the rat brain to global ischemia and reperfusion | To identify genes that are involved in ischemia response of the brain, we have evaluated changes of gene expression in rat cerebrum after 15 min complete global ischemia, followed by reperfusion for 1 h, 6 h or 24 h. The expression profiles of ∼ 30,000 transcripts from three subjects in each group (including sham-operated controls) were monitored employing oligonucleotide microarrays. About 20,000 transcripts were detectable in rat brains. The levels of 576 transcripts (∼ 2.9%) were significantly altered in response to experimental ischemia. 419 transcripts were up- and 157 downregulated; 39 transcripts changed after 1 h reperfusion, 174 after 6 h and 462 after 24 h. Results from quantitative real-time reverse transcription PCR of 18 selected genes showed excellent agreement with the microarray data. There is surprisingly little overlap between gene regulation patterns at different reperfusion times (only seven genes displayed significant changes in transcript levels at all reperfusion times. Several genes that were previously unknown to be involved in ischemia-response have been identified. Analyses of gene ontology patterns and the most strongly regulated transcripts showed that the immediate response to an ischemia/reperfusion is mediated by the induction of specific transcription factors and stress genes. Delayed gene expression response is characterised by inflammation and immune-related genes. These results support the hypothesis that the brain's response to ischemia is an active, specific and coordinated process. | [Fabian Büttnera 1, Christian Cordesb 1, Frank Gerlachc 1, Axel Heimannb, Beat Alessandrib, Ulrich Luxemburgerd, Özlem Türecid, Thomas Hankelne, Oliver Kempskib, Thorsten Burmesterc] | Brain Research | | |
| sciencedirectS0045653508012368 | Comparative transcriptome analysis of arsenate and arsenite stresses in rice seedlings | The effect of arsenic (As) exposure on genome-wide expression was examined in rice (Oryza sativa L., ssp. Indica). A group of defense and stress-responsive genes, transporters, heat-shock proteins, metallothioneins, sulfate-metabolizing proteins, and regulatory genes showed differential expression in rice seedlings challenged with arsenate (AsV) and arsenite (AsIII). AsV stress led to upregulation or downregulation of an additional set of genes in comparison to AsIII. Differential expression of several genes that showed the highest contrast in a microarray analysis was validated by following the quantitative changes in the levels of individual transcripts following challenge with AsV, AsIII, Cd, Cr, and Pb. Most of the selected genes responded to challenge by heavy metals such as arsenic. However, expression of one of the cytochrome P450 genes (Os01g43740) in rice root was induced by AsV but not by other heavy metals. Similarly, one glutaredoxin (Os01g26912) is expressed specifically in the AsIII-treated shoot. | [Debasis Chakrabarty1, Prabodh Kumar Trivedi1, Prashant Misra, Manish Tiwari, Manju Shri, Devesh Shukla, Smita Kumar, Arti Rai, Ashutosh Pandey, Deepti Nigam, Rudra Dev Tripathi, Rakesh Tuli] | Chemosphere | February 2009 | |
| sciencedirectS019701860800171X | Temporal profile of estrogen-dependent gene expression in LHRH-producing GT1–7 cells | The long-term cellular effects of estrogens are mediated by nuclear estrogen receptors which act as transcription factors to regulate gene expression. Hypothalamic targets of estrogen action include luteinizing hormone-releasing hormone-secreting neurons controlling reproduction in vertebrates. Microarray analysis and qRT-PCR studies were performed on GT1–7, immortalized LHRH neurons after 17?-estradiol treatment to reveal the nature of estrogen-regulated genes and the time course of changes in their expression profile. More than 1000 transcripts showed robust responses to estrogen treatment and the majority of responding genes were up-regulated. Early-responding genes showed altered expression 0.5–2 h after estrogen exposure, whereas late-responding genes changed after 24–48 h treatment. Up-regulated genes encoded transcription factors, molecules involved in cellular movement, cell death, immune response, neurotransmitter and neuropeptide receptors, ion channels and transporters. The 17?-estradiol modulation of 12 genes – representing characteristic gene clusters – has been confirmed by qRT-PCR.Our studies highlighted diverse gene networks, cell regulatory mechanisms and metabolic pathways through which estrogen may alter gene expression in immortalized LHRH neurons. The findings also support the notion that genomic effects of estrogen targeting in vivo directly the LHRH neuronal network of mammals play an important role in the central feedback regulation of the reproductive axis by estrogen. | [Patricia Varjua, Ken C. Changb, Erik Hrabovszkya, István Merchenthalerc, Zsolt Lipositsa d] | Neurochemistry International | February 2009 | |
| sciencedirectS0014482708004321 | The LIM-homeodomain transcription factor LMX1B regulates expression of NF-kappa B target genes | LMX1B is a LIM-homeodomain transcription factor essential for development. Putative LMX1B target genes have been identified through mouse gene targeting studies, but their identity as direct LMX1B targets remains hypothetical. We describe here the first molecular characterization of LMX1B target gene regulation. Microarray analysis using a tetracycline-inducible LMX1B expression system in HeLa cells revealed that a subset of NF-?B target genes, including IL-6 and IL-8, are upregulated in LMX1B-expressing cells. Inhibition of NF-?B activity by short interfering RNA-mediated knock-down of p65 impairs, while activation of NF-?B activity by TNF-? synergizes induction of NF-?B target genes by LMX1B. Chromatin immunoprecipitation demonstrated that LMX1B binds to the proximal promoter of IL-6 and IL-8 in vivo, in the vicinity of the characterized ?B site, and that LMX1B recruitment correlates with increased NF-?B DNA association. IL-6 promoter–reporter assays showed that the ?B site and an adjacent putative LMX1B binding motif are both involved in LMX1B-mediated transcription. Expression of NF-?B target genes is affected in the kidney of Lmx1b−/− knock-out mice, thus supporting the biological relevance of our findings. Together, these data demonstrate for the first time that LMX1B directly regulates transcription of a subset of NF-?B target genes in cooperation with nuclear p50/p65 NF-?B. | [Anne Rascle 1, Tanja Neumann1, Anne-Sarah Raschta1, Astrid Neumann1, Eva Heining2, Juergen Kastner, Ralph Witzgall] | Experimental Cell Research | 1 January 2009 | |
| sciencedirectS0165247808002125 | Pathogenic anti-DNA antibodies modulate gene expression in mesangial cells: Involvement of HMGB1 in anti-DNA antibody-induced renal injury | Although anti-DNA antibodies have been decisively linked to the pathogenesis of lupus nephritis, the mechanisms have not been conclusively determined. Recently, we reported that anti-DNA antibodies may contribute to kidney damage by upregulation of proinflammatory genes in mesangial cells (MC), a process involving both Fc receptor-dependent and independent pathways. In investigating the mechanism by which pathogenic anti-DNA antibodies modulate gene expression in MC, we found that the pathogenic anti-DNA antibody 1A3F bound to high mobility group binding protein 1 (HMGB1), an endogenous ligand for TLR2/4 and RAGE (receptor for advanced glycation end products). Interestingly, HMGB1 treatment of MC induced a similar pattern of genes as stimulation with 1A3F. Furthermore, HMGB1 and 1A3F exhibited a synergistic proinflammatory effect in the kidney, where increased expression of HMGB1 was found in lupus patients but not in patients with other types of renal disease. TLR2/Fc and RAGE/Fc inhibited the proinflammatory effects of 1A3F on MC. Finally, we found enhanced susceptibility of lupus prone MRL-lpr/lpr (MRL/lpr) as compared to normal BALB/c derived MC to pathogenic anti-DNA antibody and LPS stimulation (in particular enhanced chemokine synthesis), in addition to significantly increased expression of TLR4. Our results suggest that gene upregulation in MC induced by nephritogenic anti-DNA antibodies is TLR2/4 and RAGE-dependent. Finally, HMGB1 may act as a proinflammatory mediator in antibody-induced kidney damage in systemic lupus erythematosus (SLE). | [Xiaoping Qinga, Milena Pitashnya, David B. Thomasc, Franck J. Barratd, Mark P. Hogarthe, Chaim Puttermana b] | Immunology Letters | 16 November 2008 | |
| sciencedirectS1874939908000916 | Identification of cold-inducible microRNAs in plants by transcriptome analysis | MicroRNAs are ~ 21-nt long, non-coding RNAs that play critical roles in post-transcriptional gene regulation. Even though a large number of miRNAs have been identified, annotating their functions remains a challenge. We develop a computational, transcriptome-based approach to annotating stress-inducible microRNAs in plants. With this approach, we find that nineteen microRNA genes of eleven microRNA families in Arabidopsis thaliana are up-regulated by cold stress. Our experiments validate that among the eleven microRNAs, eight are differentially induced and three are constantly expressed under low temperature. Our result expands the number of cold-inducible microRNAs from four to eight. A promoter analysis further reveals that the cold-responsive microRNA genes contain many known stress-related cis-regulatory elements in their promoters. Our analysis also indicates that many signaling pathways, such as auxin pathways, may be affected by cold-inducible microRNAs. Our approach can be applied to plant microRNAs responding to other abiotic and biotic stresses. The research demonstrates that machine learning methods, augmented by wet-lab analysis, hold a great promise for functional annotation of microRNAs. | [Xuefeng Zhoua 1, Guandong Wanga, Keita Sutohc, Jian-Kang Zhuc, Weixiong Zhanga b 1] | Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms | November 2008 | |
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| sciencedirectS0012160608010749 | Myogenin regulates a distinct genetic program in adult muscle stem cells | In contrast to the detailed understanding we have for the regulation of skeletal muscle gene expression in embryos, similar insights into postnatal muscle growth and regeneration are largely inferential or do not directly address gene regulatory mechanisms. Muscle stem cells (satellite cells) are chiefly responsible for providing new muscle during postnatal and adult life. The purpose of this study was to determine the role that the myogenic basic helix–loop–helix regulatory factor myogenin has in postnatal muscle growth and adult muscle stem cell gene expression. We found that myogenin is absolutely required for skeletal muscle development and survival until birth, but it is dispensable for postnatal life. However, Myog deletion after birth led to reduced body size implying a role for myogenin in regulating body homeostasis. Despite a lack of skeletal muscle defects in Myog-deleted mice during postnatal life and the efficient differentiation of cultured Myog-deleted adult muscle stem cells, the loss of myogenin profoundly altered the pattern of gene expression in cultured muscle stem cells and adult skeletal muscle. Remarkably, these changes in gene expression were distinct from those found in Myog-null embryonic skeletal muscle, indicating that myogenin has separate functions during postnatal life. | [Eric Meadowsa b, Jang-Hyeon Choa, Jesse M. Flynna b, William H. Kleina b] | Developmental Biology | 15 October 2008 | |
| sciencedirectS0300483X08002709 | Perfluorooctane sulfonate-induced changes in fetal rat liver gene expression | In utero exposure of laboratory rats to perfluorooctane sulfonate (PFOS, C8F17SO3−), a chemically stable surfactant that is widely disseminated in the environment and present in serum samples from wildlife and humans, is associated with decreased neonatal survival, and growth deficits as well as hepatomegaly. This hepatomegaly in newborn rats exposed to PFOS in utero resembles that observed in adults and is characterized by peroxisome proliferation and decreased liver triglycerides, both of which are suspected to be manifested through PPAR?-mediated transcriptional regulation. The purpose of the present investigation was to determine whether these changes in metabolic status are a reflection of transcriptional changes in fetal rat liver using global gene expression array analyses. Gravid Sprague–Dawley rats were administered 3 mg/kg PFOS by gavage daily from gestational day 2–20 and terminated on day 21. Although there was no treatment-related frank terata, there was a substantial effect of PFOS on the perinatal hepatic transcriptome—225 unique transcripts were identified as statistically increased and 220 decreased by PFOS exposure; few transcripts were changed by more than two-fold. Although the PPAR? transcript (Ppara) itself was not affected, there was a significant increase in expression of gene transcripts associated with hepatic peroxisomal proliferation as well as those responsible for fatty acid activation, transport and oxidation pathways (both mitochondrial and peroxisomal). Additional metabolic pathways altered by in utero PFOS exposure were a stimulation of fetal hepatic fatty acid biosynthesis and a net reduction of Cyp7a1 transcript, which is required for bile acid synthesis. There were minimal effects on the expression of thyroid-related gene transcripts. In conclusion, gene expression analysis provides strong evidence indicating transcriptional control of the altered metabolic status of neonates following PFOS exposure in utero, much of which appears to be under the influence of a functional perinatal PPAR? regulatory pathway. | [James A. Bjorka, Christopher Laub, Sue C. Changc, John L. Butenhoffc, Kendall B. Wallacea] | Toxicology | 29 September 2008 | |
| sciencedirectS0168945208001350 | Gene expression polymorphisms and ESTs associated with gravitropic response of subterranean branch meristems and growth habit in Leymus wildryes | Negatively orthogeotropic (NOGT) tiller and diageotropic (DGT) rhizome meristems develop from the same type of lateral axillary meristems and phytomer structure. Although subterranean NOGT and DGT buds appear similar, they display different responses to gravity and perhaps other cues governing branch angle and overall growth habit (GH). Leymus wildryes show remarkable variation in GH and include some of the largest native grasses in western North America. Previous studies detected GH QTLs on homoeologous regions of LG3a and LG3b controlling differences between caespitose Leymus cinereus and rhizomatous Leymus triticoides allotetraploids. Heterologous barley and wheat microarrays in conjunction with bulk segregate analysis were used to find gene expression polymorphisms associated with GH QTLs. Approximately 34% and 25% of the probe sets showed detectable signals on the barley and wheat arrays, respectively. Overall gene expression patterns of NOGT and DGT meristems were remarkably similar, consistent with the assertion that Leymus NOGT and DGT buds develop from homologous meristems. Only 28 and 27 genes on barley and wheat gene chips, respectively, showed more than twofold differential expressions between NOGT and DGT tissues. One expression polymorphism genetically mapped in the Leymus LG3 rhizome QTL region. | [Parminder Kaura, Ivan W. Mottb, Steven R. Larsonb, B. Shaun Bushmanb, Alvaro G. Hernandezc, W. Ryan Kimc, Lei Liuc, Mark A. Mikeld] | Plant Science | September 2008 | |
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| sciencedirectS001216060800078X | Identification of downstream genetic pathways of Tbx1 in the second heart field | Tbx1, a T-box transcription factor, and an important gene for velo-cardio-facial syndrome/DiGeorge syndrome (VCFS/DGS) in humans, causes outflow tract (OFT) heart defects when inactivated in the mouse. Tbx1 is expressed in the second heart field (SHF) and is required in this tissue for OFT development. To identify Tbx1 regulated genetic pathways in the SHF, we performed gene expression profiling of the caudal pharyngeal region in Tbx1−/− and wild type embryos. Isl1, a key marker for the SHF, as well as Hod and Nkx2-6, were downregulated in Tbx1−/− mutants, while genes required for cardiac morphogenesis, such as Raldh2, Gata4, and Tbx5, as well as a subset of muscle contractile genes, signifying myocardial differentiation, were ectopically expressed. Pan-mesodermal ablation of Tbx1 resulted in similar gene expression changes, suggesting cell-autonomous roles of Tbx1 in regulating these genes. Opposite expression changes concomitant with SHF-derived cardiac defects occurred in TBX1 gain-of-function mutants, indicating that appropriate levels of Tbx1 are required for heart development. When taken together, our studies show that Tbx1 acts upstream in a genetic network that positively regulates SHF cell proliferation and negatively regulates differentiation, cell-autonomously in the caudal pharyngeal region. | [Jun Liaoa 1, Vimla S. Aggarwala, Sonja Nowotschina 2, Alexei Bondareva, Shari Lipnerb, Bernice E. Morrowa] | Developmental Biology | 15 April 2008 | |
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| sciencedirectS0012160607014777 | The receptor tyrosine kinase RET regulates hindgut colonization by sacral neural crest cells | The enteric nervous system (ENS) is formed from vagal and sacral neural crest cells (NCC). Vagal NCC give rise to most of the ENS along the entire gut, whereas the contribution of sacral NCC is mainly limited to the hindgut. This, and data from heterotopic quail–chick grafting studies, suggests that vagal and sacral NCC have intrinsic differences in their ability to colonize the gut, and/or to respond to signalling cues within the gut environment. To better understand the molecular basis of these differences, we studied the expression of genes known to be essential for ENS formation, in sacral NCC within the chick hindgut. Our results demonstrate that, as in vagal NCC, Sox10, EdnrB, and Ret are expressed in sacral NCC within the gut. Since we did not detect a qualitative difference in expression of these ENS genes we performed DNA microarray analysis of vagal and sacral NCC. Of 11 key ENS genes examined from the total data set, Ret was the only gene identified as being highly differentially expressed, with a fourfold increase in expression in vagal versus sacral NCC. We also found that over-expression of RET in sacral NCC increased their ENS developmental potential such that larger numbers of cells entered the gut earlier in development, thus promoting the fate of sacral NCC towards that of vagal NCC. | [Jean-Marie Delalandea 1 2, Amanda J. Barlowa 2, Aaron J. Thomasb, Adam S. Wallacea, Nikhil Thapara, Carol A. Ericksonb, Alan J. Burnsa] | Developmental Biology | 1 January 2008 | |
| sciencedirectS1567133X07000956 | The PcG gene Sfmbt2 is paternally expressed in extraembryonic tissues | Genomic imprinting has dramatic effects on placental development, as has been clearly observed in interspecific hybrid, somatic cell nuclear transfer, and uniparental embryos. In fact, the earliest defects in uniparental embryos are evident first in the extraembryonic trophoblast. We performed a microarray comparison of gynogenetic and androgenetic mouse blastocysts, which are predisposed to placental pathologies, to identify imprinted genes. In addition to identifying a large number of known imprinted genes, we discovered that the Polycomb group (PcG) gene Sfmbt2 is imprinted. Sfmbt2 is expressed preferentially from the paternal allele in early embryos, and in later stage extraembryonic tissues. A CpG island spanning the transcriptional start site is differentially methylated on the maternal allele in e14.5 placenta. Sfmbt2 is located on proximal chromosome 2, in a region known to be imprinted, but for which no genes had been identified until now. This possibly identifies a new imprinted domain within the murine genome. We further demonstrate that murine SFMBT2 protein interacts with the transcription factor YY1, similar to the Drosophila PHO-RC. | [Anastasia Kuzmina, Zhiming Hanb, Michael C. Goldingc, Mellissa R.W. Mannc, Keith E. Lathamb, Susannah Varmuzaa] | Gene Expression Patterns | January 2008 | |
| sciencedirectS0888754307002327 | Gene expression changes in children with autism | The objective of this study was to identify gene expression differences in blood differences in children with autism (AU) and autism spectrum disorder (ASD) compared to general population controls. Transcriptional profiles were compared with age- and gender-matched, typically developing children from the general population (GP). The AU group was subdivided based on a history of developmental regression (A–R) or a history of early onset (A–E without regression). Total RNA from blood was processed on human Affymetrix microarrays. Thirty-five children with AU (17 with early onset autism and 18 with autism with regression) and 14 ASD children (who did not meet criteria for AU) were compared to 12 GP children. Unpaired t tests (corrected for multiple comparisons with a false discovery rate of 0.05) detected a number of genes that were regulated more than 1.5-fold for AU versus GP (n = 55 genes), for A–E versus GP (n = 140 genes), for A–R versus GP (n = 20 genes), and for A–R versus A–E (n = 494 genes). No genes were significantly regulated for ASD versus GP. There were 11 genes shared between the comparisons of all autism subgroups to GP (AU, A–E, and A–R versus GP) and these genes were all expressed in natural killer cells and many belonged to the KEGG natural killer cytotoxicity pathway (p = 0.02). A subset of these genes (n = 7) was tested with qRT-PCR and all genes were found to be differentially expressed (p < 0.05). We conclude that the gene expression data support emerging evidence for abnormalities in peripheral blood leukocytes in autism that could represent a genetic and/or environmental predisposition to the disorder. | [Jeffrey P. Gregga b i, Lisa Litb c, Colin A. Barona b, Irva Hertz-Picciottob d i, Wynn Walkerb c, Ryan A. Davisa, Lisa A. Croene, Sally Ozonoffb f, Robin Hansenb g i, Isaac N. Pessahb h i, Frank R. Sharpb c i] | Genomics | January 2008 | |
| sciencedirectS0076687908026165 | Chapter 16 Measuring mRNA Stability During Early Drosophila Embryogenesis | Maternal mRNAs play a major role in directing early Drosophila melanogaster development, and thus, precise posttranscriptional regulation of these messages is imperative for normal embryogenesis. Although initially abundant on egg deposition, a subset of these maternal mRNAs is targeted for destruction during the first 2 to 3 h of embryogenesis. In this chapter, we describe molecular methods to determine the kinetics and mechanisms of maternal mRNA decay in the early D. melanogaster embryo. We show how both unfertilized eggs and fertilized embryos can be used to identify maternal mRNAs destined for degradation, to explain changes in decay kinetics over time, and to uncover the molecular mechanisms of targeted maternal mRNA turnover. In the first section, we explore the methods and outcomes of measuring decay on a “gene?by?gene” basis, which involves examination of a small number of transcripts by Northern blotting, RNA dot blotting, and real?time RT?PCR. In the second section, we provide a comprehensive examination of the applications of microarray technology to study global changes in maternal mRNA decay during early development. Genome?wide surveys of maternal mRNA turnover provide a wealth of information regarding the magnitude, temporal regulation, and genetic control of maternal mRNA turnover. Methods that permit the collection and analysis of highly reproducible and statistically robust data in this developmental system are discussed. | [Jennifer L. Semotok* †, J. Timothy Westwood‡, Aaron L. Goldman* †, Ramona L. Cooperstock* †, Howard D. Lipshitz* †] | Methods in Enzymology | | |
| sciencedirectS001448270700314X | The Shwachman–Bodian–Diamond syndrome associated protein interacts with HsNip7 and its down-regulation affects gene expression at the transcriptional and translational levels | The Shwachman–Bodian–Diamond syndrome (SDS) is an autosomal disorder with pleiotropic phenotypes including pancreatic, skeletal and bone marrow deficiencies and predisposition to hematological dysfunctions. SDS has been associated to mutations in the SBDS gene, encoding a highly conserved protein that was shown to function in ribosome biogenesis in yeast. In this work, we show that SBDS is found in complexes containing the human Nip7 ortholog. Analysis of pre-rRNA processing in a stable SBDS knock-down HEK293-derivative cell line revealed accumulation of a small RNA which is a further indication of SBDS involvement in rRNA biosynthesis. Global transcription and polysome-bound mRNA profiling revealed that SBDS knock-down affects expression of critical genes involved in brain development and function, bone morphogenesis, blood cell proliferation and differentiation, and cell adhesion. Expression of a group of growth and signal transduction factors and of DNA damage response genes is also affected. In SBDS knock-down cells, 34 mRNAs showed decreased and 55 mRNAs showed increased association to polysomes, among which is a group encoding proteins involved in alternative splicing and RNA modification. These results indicate that SBDS is required for accurate expression of genes important for proper brain, skeletal, and blood cell development. | [Cédric Heslinga, Carla C. Oliveirab, Beatriz A. Castilhoc, Nilson I.T. Zanchina] | Experimental Cell Research | 10 December 2007 | |
| sciencedirectS0012160607012602 | Target gene selectivity of the myogenic basic helix–loop–helix transcription factor myogenin in embryonic muscle | The myogenic regulatory factors MyoD and myogenin are crucial for skeletal muscle development. Despite their importance, the mechanisms by which these factors selectively regulate different target genes are unclear. The purpose of the present investigation was to compare embryonic skeletal muscle from myogenin+/+ and myogenin−/− mice to identify genes whose expression was dependent on the presence of myogenin but not MyoD and to determine whether myogenin-binding sites could be found within regulatory regions of myogenin-dependent genes independent of MyoD. We identified a set of 140 muscle-expressed genes whose expression in embryonic tongue muscle of myogenin−/− mice was downregulated in the absence of myogenin, but in the presence of MyoD. Myogenin bound within conserved regulatory regions of several of the downregulated genes, but MyoD bound only to a subset of these same regions, suggesting that many downregulated genes were selective targets of myogenin. The regulatory regions activated gene expression in cultured myoblasts and fibroblasts overexpressing myogenin or MyoD, indicating that expression from exogenously introduced DNA could not recapitulate the selectivity for myogenin observed in vivo. The results identify new target genes for myogenin and show that myogenin's target gene selectivity is not based solely on binding site sequences. | [Judith K. Davie1, Jang-Hyeon Cho, Eric Meadows, Jesse M. Flynn, Jennifer R. Knapp2, William H. Klein] | Developmental Biology | 15 November 2007 | |
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| sciencedirectS0168945207001823 | Comparative transcriptome analysis of salt-tolerant wheat germplasm lines using wheat genome arrays | Salt-tolerant wheat lines W4909 and W4910 were derived from a cross between AJDAj5 (a disomic addition line carrying a pair of Eb chromosomes from Thinopyrum junceum) and PhI (a line having the PhI allele from Aegilops speltoides, which promotes homoeologous recombination). Both lines have greater salt tolerance than their parental lines, which are more salt-tolerant than the common wheat background, Chinese Spring (CS). Genomic constitution of W4909 and W4910 wheat has been estimated at 1.9% and 2.4% non-CS, respectively. Affymetrix GeneChip® Wheat Arrays were used to identify differentially expressed genes in roots and leaves of the above-mentioned five lines under salt stress at electrical conductivity (EC) of 30 dS/m. Based on expression polymorphisms in AJDAj5 and PhI, differentially expressed genes in W4909 and W4910 were attributed to one or the other parental lines when possible. The PhI parent contributed nearly twice as many expression polymorphisms as AJDAj5 to both W4909 and W4910. Nine transcripts in W4910 showed transgression gene expression significantly higher or lower than either parent. The majority of expression polymorphisms exhibited very low expression levels compared to the control and mapped to three distinct chromosomal locations, 2S, 4L and 2L. Salt treatment responsive gene expression profiles common to all five tested lines provide a short list of candidate salt-tolerance genes in wheat. A gene for tonoplast aquaporin that was transferred from PhI and a gene for putative potassium channel protein that was attributable to AJDAj5 are identified as candidate genes for the tissue salt tolerance in W4909 and W4910. | [Ivan W. Mott, Richard R.-C. Wang] | Plant Science | September 2007 | |
| sciencedirectS0042698907001897 | Identification of novel retinal target genes of thyroid hormone in the human WERI cells by expression microarray analysis | Using the human WERI-Rb1 cell line as a model system, we performed a genome-wide search for retinal target genes of thyroid hormone (TH) via expression microarray analysis followed by quantitative real-time RT-PCR verification. We identified 12 novel retinal targets of TH, including 10 up-regulated genes (OPN1MW, OPN1LW, TIMP3, RP1L1, GNGT2, CRX, ARR3, GCAP1, IMPDH1, and PDE6C) and 2 down-regulated genes (GNGT1 and GNB3). In addition, we found a number of novel TH-targets that are not currently known to be retinal genes. This is the first report of human retinal targets regulated by thyroid hormone. | [Yan Liua, Li Fua, Ding-Geng Chenb c, Samir S. Deeba] | Vision Research | August 2007 | |
| sciencedirectS0041008X07001299 | Gene expression profiling and differentiation assessment in primary human hepatocyte cultures, established hepatoma cell lines, and human liver tissues | Frequently, primary hepatocytes are used as an in vitro model for the liver in vivo. However, the culture conditions reported vary considerably, with associated variability in performance. In this study, we characterized the differentiation character of primary human hepatocytes cultured using a highly defined, serum-free two-dimensional sandwich system, one that configures hepatocytes with collagen I as the substratum together with a dilute extracellular matrix (Matrigel™) overlay combined with a defined serum-free medium containing nanomolar levels of dexamethasone. Gap junctional communication, indicated by immunochemical detection of connexin 32 protein, was markedly enhanced in hepatocytes cultured in the Matrigel sandwich configuration. Whole genome expression profiling enabled direct comparison of liver tissues to hepatocytes and to the hepatoma-derived cell lines, HepG2 and Huh7. PANTHER database analyses were used to identify biological processes that were comparatively over-represented among probe sets expressed in the in vitro systems. The robustness of the primary hepatocyte cultures was reflected by the extent of unchanged expression character when compared directly to liver, with more than 77% of the probe sets unchanged in each of the over-represented categories, representing such genes as C/EBP?, HNF4?, CYP2D6, and ABCB1. In contrast, HepG2 and Huh7 cells were unchanged from the liver tissues for fewer than 48% and 55% of these probe sets, respectively. Further, hierarchical clustering of the hepatocytes, but not the cell lines, shifted from donor-specific to treatment-specific when the probe sets were filtered to focus on phenobarbital-inducible genes, indicative of the highly differentiated nature of the hepatocytes when cultured in a highly defined two-dimensional sandwich system. | [Katy M. Olsavskya, Jeanine L. Pagea, Mary C. Johnsona, Helmut Zarblb, Stephen C. Stromc, Curtis J. Omiecinskia d] | Toxicology and Applied Pharmacology | 1 July 2007 | |
| sciencedirectS0168165607001587 | Performance of different small sample RNA amplification techniques for hybridization on Affymetrix GeneChips | A key issue in RNA amplification techniques is the preservation of original transcript abundance, however popular high-grade RNA amplification methods lack sufficient validation regarding the potential bias of gene expression profiles. This study evaluated a double-round T7-based and a PCR-based amplification protocol, using the Affymetrix GeneChip platform. Both small sample methods performed excellently in terms of yield and reproducibility (r > 0.99), and also the within-method concordance with respect to differential gene expression was as high as with standard single-round T7-based amplification. However, when comparing the overlap of all differentially expressed genes between standard and small sample methods, this was only moderate for the double-round T7 (48.7–55.0%) as well as for the PCR-based amplification protocol (51.9–58.0%). In contrast, the concordance for the top 100 genes with highest fold changes was significantly higher, indicating that both small sample methods generate reliable results when focusing on strongly regulated genes. | [Florian Wagner, Uwe Radelof] | Journal of Biotechnology | 10 May 2007 | |
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| sciencedirectS0065242307440082 | Gene Expression Assays | 1. The study of the profile of gene expression in a cell or tissue at a particular moment gives an insight into the plans of the cell for protein synthesis. Recent technological advances make it possible to analyze the expression of the entire genome in a single experiment. These “gene expression assays” complement or replace previous assays which measured the gene expression of only one gene, or a select group of genes. Within this chapter we outline the development of the gene expression assay and provide examples of the wide range of disciplines in which it is used. An overview of the current technologies is given, and includes an introduction to laser capture microdissection and linear amplification of RNA, both of which have extended the application of gene expression assays. Illustrative examples in the field of cancer and neuroscience highlight the scientific achievements. This technology has made in understanding the pathogenesis of diseases, including breast cancer, Huntington's disease, and schizophrenia. With recent advances including exon arrays to investigate alternative splicing, tiling arrays to investigate novel transcription start sites, and on?chip chromatin immunoprecipitation to investigate DNA–protein interactions, the future of gene expression assays is set to further our understanding of the complexities of gene expression. | [Janine Kirby*, Paul R. Heath*, Pamela J. Shaw*, Freddie C. Hamdy†] | Advances in Clinical Chemistry | | |
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| sciencedirectS0580951706360096 | 9 Transcript Analysis: A Microarray Approach | | [Andrew Hayes1, Juan I Castrillo1, Stephen G Oliver1, Andy Brass1 2, Leo AH Zeef1] | Methods in Microbiology | Pages 189–219 | |
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| sciencedirectS0012160606008116 | Tbx5-dependent rheostatic control of cardiac gene expression and morphogenesis | Dominant mutations in the T-box transcription factor gene TBX5 cause Holt–Oram syndrome (HOS), an inherited human disease characterized by upper limb malformations and congenital heart defects (CHDs) of variable severity. We hypothesize that minor alterations in the dosage of Tbx5 directly influences severity of CHDs. Using a mouse allelic series, we show a sensitive inverse correlation between Tbx5 dosage and abnormal cardiac morphogenesis and gene expression. The CHDs found in mice harbouring a hypomorphic allele of Tbx5 (Tbx5lox/+ mice) are less pronounced than those found in Tbx5 haploinsufficient mice (Tbx5del/+), and homozygous hypomorphic (Tbx5lox/lox) embryos have noticeably more advanced cardiac development than Tbx5 null (Tbx5del/del) embryos. Examination of target gene expression across the allelic series uncovers very fine sensitivity across the range of Tbx5 dosages, in which some genes respond dramatically differently to only 15% differences in Tbx5 mRNA levels. This analysis was expanded to a genome-wide level, which uncovered a Tbx5 dosage-sensitive genetic program involving a network of cardiac transcription factors, developmentally important cell–cell signaling molecules, and ion channel proteins. These results indicate an exquisite sensitivity of the developing heart to Tbx5 dosage and provide significant insight into the transcriptional and cellular mechanisms that are disrupted in CHDs. | [Alessandro D. Moria b c, Yonghong Zhua c, Ilyas Vahoraa, Brian Niemand e, Kazuko Koshiba-Takeuchia c, Lorinda Davidsond, Anne Pizardf, J.G. Seidmanf, Christine E. Seidmanf g, X. Josette Chend e, R. Mark Henkelmand e, Benoit G. Bruneaua b c] | Developmental Biology | 15 September 2006 | |
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| sciencedirectS0169328X05002330 | Gene array profiling of large hypothalamic CNS regions in lactating and randomly cycling virgin mice | A dramatic example of neuronal and physiological plasticity in adult mammals occurs during the transition from a non-maternal to a maternal, lactating state. In this study, we compared gene expression within a large continuous region of the CNS involved in maternal behaviors (hypothalamus, preoptic regions, and nucleus accumbens) between lactating (L) (postpartum Day 7) and randomly cycling virgin (V) outbred mice. Using high-density oligonucleotide arrays representing 11,904 genes, two statistical algorithms were used to identify significant differences in gene expression: robust multiarray (P < 0.001) (n = 92 genes) and significance analysis of microarrays using a 10% false discover rate (n = 114 genes). 27 common genes were identified as significant using both techniques. A subset of genes (n = 5) were selected and examined by real-time PCR. Our findings were consistent with previous published work. For example, neuropeptide Y (NPY) and proenkephalin were elevated in L mice, whereas POMC was decreased. Increased levels of NPY Y2 receptor and polo-like kinase and decreased levels of endothelin receptor type b in L mice are examples of novel gene expression changes not previously identified. Expression differences occurred in broad classes. Together, our findings provide possible new material on gene expression changes that may support maternal behaviors. The advantages and drawbacks of sampling large CNS regions using arrays are discussed. | [Stephen C. Gammiea b, Nina S. Hasena, Tarif A. Awadd, Anthony P. Augerb c, Heather M. Jessenc, Jules B. Pankseppb, Anne M. Bronikowskie] | Molecular Brain Research | 3 October 2005 | |
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| sciencedirectS0022282804003657 | Claudin-5 localizes to the lateral membranes of cardiomyocytes and is altered in utrophin/dystrophin-deficient cardiomyopathic mice | Remodeling of adherens junction, gap junction, and desmosomal proteins at the intercalated discs of cardiomyocytes in heart characterizes several animal models of cardiomyopathy, especially dilated cardiac myopathy (DCM). In this study, we show that the tight junction protein, claudin-5, is present in cardiac muscle and localizes to the lateral membranes of cardiomyocytes in normal mice. We further examined claudin-5 in utrophin/dystrophin-deficient (double knockout, dko) mice, a mouse model of muscular dystrophy with cardiomyopathy, and found that claudin-5 mRNA and protein levels are decreased in dko hearts as compared with normal. Intercalated disc cell junction proteins, and another tight junction protein, zonula occludens-1 (ZO-1), are not altered in the dko mouse. Ultrastructural data from dko hearts also shows that the lateral membranes of cardiomyocytes exhibit an abnormal wavy appearance. These data demonstrate that claudin-5 is specifically altered in dko hearts, suggesting that alterations of the lateral membranes of cardiomyocytes, rather than intercalated discs, are associated with cardiomyopathy in the dko mouse. | [Jamie L. Sanforda, Jonathan D. Edwardsa, Tessily A. Maysa, Bendi Gongb, Anita P. Merriamb, Jill A. Rafael-Fortneya] | Journal of Molecular and Cellular Cardiology | February 2005 | |
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