High-density Oligonucleotide Microarrays Research Articles

Global genomic analysis reveals rapid control of a robust innate response in SIV-infected sooty mangabeys

Natural SIV infection of sooty mangabeys (SMs) is nonprogressive despite chronic virus replication. Strikingly, it is characterized by low levels of immune activation, while pathogenic SIV infection of rhesus macaques (RMs) is associated with chronic immune activation. To elucidate the mechanisms underlying this intriguing phenotype, we used high-density oligonucleotide microarrays to longitudinally assess host gene expression in SIV-infected SMs and RMs. We found that acute SIV infection of SMs was consistently associated with a robust innate immune response, including widespread upregulation of IFN-stimulated genes (ISGs) in blood and lymph nodes. While SMs exhibited a rapid resolution of ISG expression and immune activation, both responses were observed chronically in RMs. Systems biology analysis indicated that expression of the lymphocyte inhibitory receptor LAG3, a marker of T cell exhaustion, correlated with immune activation in SIV-infected RMs but not SMs. Our findings suggest that active immune regulatory mechanisms, rather than intrinsically attenuated innate immune responses, underlie the low levels of immune activation characteristic of SMs chronically infected with SIV.

ABC and GCB DLBCLs Display Unique Biologically Distinct and Clinically Relevant Epigenetic Signatures.

Abstract 619Diffuse Large B-cell Lymphoma (DLBCL) is a complex biological entity with heterogeneous genetic, biological and clinical features. Gene expression profiling studies have attempted to resolve some of this heterogeneity. For example, DLBCL patients harboring gene signatures associated with expression of germinal center B-cell genes (GCB) or activated B-cell genes (ABC) vary in their response rate to standard chemo-immunotherapy regimens. Since epigenetic gene regulation can play a fundamental role in determining the phenotype of normal and malignant tissues we asked whether ABC and GCB DLBCLs also display unique epigenetic signatures that might be clinically useful and further explain the biology of these tumors. For this purpose we examined the DNA methylation level of 50,000 cytosine residues distributed among 14,000 gene promoters in a cohort of 159 patients with DLBCL, all of whom were uniformly treated with R-CHOP, using the HELP assay and high-density oligonucleotide microarrays. For a subset of these patients (n=69), we also performed Affymetrix gene expression profiling. First, a Bayesian predictor of ABC/GCB subtypes was trained from a published expression profiling study of 203 DLBCL patients. The predictor was then applied to our cohort of 69 patients. At a probability cutoff of 0.9, 20 patients were classified as ABC, 40 were classified as GCB and 9 could not be classified (i.e. “type III DLBCL”). As expected from published studies, the differences in progression free survival (PFS) and overall survival (OS) of these ABC vs. GCB patients thus determined was highly significant, with p=0.0026 (log rank) and p=0.043 (log rank) respectively, with a much worse prognosis for ABC patients compared to GCB ones. We then asked whether the ABC and GCB subtypes could be predicted from the DNA methylation profiles of the same 69 patients. By performing a t-test we found that 239 genes were differentially methylated between ABC and GCB (p<0.0001) and also displayed >30% difference in methylation level. This DNA methylation signature was incorporated into a new Bayesian predictor, which we showed to predict ABC and GCB DLBCL subtypes from DNA methylation profiles with a 91% accuracy. Using a cross-validation procedure, we estimated that the classification performance on independent cases to be ∼87%. The predicted ABC and GCB cases retained the clinical predictive power of the gene expression profile when applied to the remaining 90 patients that did not have gene expression profiles, confirming its clinical relevance (difference in PFS p=0.0148, log rank). Gene set enrichment analysis showed that the ABC DNA methylation signature was enriched in genes involved in antigen dependent B and T-cell responses and in TNF inflammatory responses (p<1.01E-4 and <6.01E-4 respectively). A computational analysis of promoter DNA sequences of the genes involved in this signature revealed over-representation of binding sites for transcription factors including MYB, STAT5A, MAZ, and JUN and Sp1; many of these factors have a known role in B cell development and function. The role of Sp1 in these tumors is under further examination. Among the 239 genes that were differentially methylated and the 411 genes that were differentially expressed between ABC and GCB there was an overlap of 16 genes (greater than expected by chance Fisher Exact p=0.005). A predictor based on the methylation profiles of these 16 genes was on its own 92% accurate in identifying ABC vs. GCB cases among our cohort of DLBCLs. Although there was a general trend for inverse correlation in expression between the 239 differentially methylated genes, these 16 overlapping genes displayed marked and extreme inverse correlation. This was validated by single locus quantitative methylation sequencing (MassArray) and QPCR. The 16 genes included genes known to play critical roles in B-cell differentiation, proliferation and metabolism but not previously implicated in DLBCL. Gain and loss of function assays of a subset of these genes in ABC and GCB DLBCL cell lines show that they have tumor suppressor functions in DLBCL. Our results demonstrate for the first time that ABC and GCB DLBCLs are epigenetically distinct diseases; they identify new biological differences and candidate tumor suppressor genes between these tumors and demonstrate that a DNA methylation classifier can be used to distinguish GCB and ABC DLBCL subtypes. Disclosures:No relevant conflicts of interest to declare.

A transcriptome analysis identifies molecular effectors of unconjugated bilirubin in human neuroblastoma SH-SY5Y cells

BackgroundThe deposition of unconjugated bilirubin (UCB) in selected regions of the brain results in irreversible neuronal damage, or Bilirubin Encephalopathy (BE). Although UCB impairs a large number of cellular functions in other tissues, the basic mechanisms of neurotoxicity have not yet been fully clarified. While cells can accumulate UCB by passive diffusion, cell protection may involve multiple mechanisms including the extrusion of the pigment as well as pro-survival homeostatic responses that are still unknown.ResultsTranscriptome changes induced by UCB exposure in SH-SY5Y neuroblastoma cell line were examined by high density oligonucleotide microarrays. Two-hundred and thirty genes were induced after 24 hours. A Gene Ontology (GO) analysis showed that at least 50 genes were directly involved in the endoplasmic reticulum (ER) stress response. Validation of selected ER stress genes is shown by quantitative RT-PCR. Analysis of XBP1 splicing and DDIT3/CHOP subcellular localization is presented.ConclusionThese results show for the first time that UCB exposure induces ER stress response as major intracellular homeostasis in surviving neuroblastoma cells in vitro.

A nutrigenomic analysis of intestinal response to partial soybean meal replacement in diets for juvenile Atlantic halibut, Hippoglossus hippoglossus, L.

Aquaculture feeds for carnivorous finfish species have been dependent upon the use of fish meal as the major source of dietary protein; however, the increasing demands upon the finite quantity of this high-quality protein source requires that feeds become increasingly comprised of alternative plant and/or animal protein. Soybean meal has been has been used to partially replace fish meal in the diets of several fish but it is known to cause enteritis in Atlantic salmon, Salmo salar. We have compared two groups of juvenile (207.2 ± 6.6 g) Atlantic halibut, Hippoglossus hippoglossus, L., fed diets containing fish meal (FM; control) or 30% soybean meal (SBM; experimental) as a protein source for 3 weeks. No detectable difference in feed intake or palatability was evident with the SBM diet relative to the FM diet. Histological examination of the distal intestine was performed to examine leukocyte infiltration of the lamina propria and other changes in morphology commonly observed with soybean-induced enteritis of salmonids. No significant difference was found between fish fed the FM and SBM diets. Global gene expression profiling performed using a high-density oligonucleotide microarray containing 9260 unique features, printed in quadruplicate, from Atlantic halibut revealed subtle underlying changes in the expression of several immune genes and genes involved in muscle formation, lipid transport, xenobiotic detoxification, digestion and intermediary metabolism. These results indicate that SBM can be used successfully as a replacement for animal protein in diet for juvenile Atlantic halibut, although long-term effects on the immune system may ensue.

Gene expression analysis of the biocontrol fungus Trichoderma harzianum in the presence of tomato plants, chitin, or glucose using a high-density oligonucleotide microarray.

BackgroundIt has recently been shown that the Trichoderma fungal species used for biocontrol of plant diseases are capable of interacting with plant roots directly, behaving as symbiotic microorganisms. With a view to providing further information at transcriptomic level about the early response of Trichoderma to a host plant, we developed a high-density oligonucleotide (HDO) microarray encompassing 14,081 Expressed Sequence Tag (EST)-based transcripts from eight Trichoderma spp. and 9,121 genome-derived transcripts of T. reesei, and we have used this microarray to examine the gene expression of T. harzianum either alone or in the presence of tomato plants, chitin, or glucose.ResultsGlobal microarray analysis revealed 1,617 probe sets showing differential expression in T. harzianum mycelia under at least one of the culture conditions tested as compared with one another. Hierarchical clustering and heat map representation showed that the expression patterns obtained in glucose medium clustered separately from the expression patterns observed in the presence of tomato plants and chitin. Annotations using the Blast2GO suite identified 85 of the 257 transcripts whose probe sets afforded up-regulated expression in response to tomato plants. Some of these transcripts were predicted to encode proteins related to Trichoderma-host (fungus or plant) associations, such as Sm1/Elp1 protein, proteases P6281 and PRA1, enchochitinase CHIT42, or QID74 protein, although previously uncharacterized genes were also identified, including those responsible for the possible biosynthesis of nitric oxide, xenobiotic detoxification, mycelium development, or those related to the formation of infection structures in plant tissues.ConclusionThe effectiveness of the Trichoderma HDO microarray to detect different gene responses under different growth conditions in the fungus T. harzianum strongly indicates that this tool should be useful for further assays that include different stages of plant colonization, as well as for expression studies in other Trichoderma spp. represented on it. Using this microarray, we have been able to define a number of genes probably involved in the transcriptional response of T. harzianum within the first hours of contact with tomato plant roots, which may provide new insights into the mechanisms and roles of this fungus in the Trichoderma-plant interaction.

Induction of Epithelial Mesenchimal Transition and Vasculogenesis in the Lenses of Dbl Oncogene Transgenic Mice

BackgroundThe Dbl family of proteins represents a large group of proto-oncogenes involved in cell growth regulation. The numerous domains that are present in many Dbl family proteins suggest that they act to integrate multiple inputs in complicated signaling networks involving the Rho GTPases. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders and neoplastic transformation. We generated transgenic mice introducing the cDNA of Dbl oncogene linked to the metallothionein promoter into the germ line of FVB mice and found that onco-Dbl expression in mouse lenses affected proliferation, migration and differentiation of lens epithelial cells.ResultsWe used high density oligonucleotide microarray to define the transcriptional profile induced by Dbl in the lenses of 2 days, 2 weeks, and 6 weeks old transgenic mice. We observed modulation of genes encoding proteins promoting epithelial-mesenchymal transition (EMT), such as down-regulation of epithelial cell markers and up-regulation of fibroblast markers. Genes encoding proteins involved in the positive regulation of apoptosis were markedly down regulated while anti-apoptotic genes were strongly up-regulated. Finally, several genes encoding proteins involved in the process of angiogenesis were up-regulated. These observations were validated by histological and immunohistochemical examination of the transgenic lenses where vascularization can be readily observed.ConclusionOnco-Dbl expression in mouse lens correlated with modulation of genes involved in the regulation of EMT, apoptosis and vasculogenesis leading to disruption of the lens architecture, epithelial cell proliferation, and aberrant angiogenesis. We conclude that onco-Dbl has a potentially important, previously unreported, capacity to dramatically alter epithelial cell migration, replication, polarization and differentiation and to induce vascularization of an epithelial tissue.

Genome-based expression profiles study for the pathogenesis of pelvic organ prolapse: an array of 33 genes model

To explore the potential molecular mechanisms contributing to the pathogenesis of pelvic organ prolapse (POP) with the aid of high-density oligonucleotide microarrays. We compared microarray gene expression profiles in pelvic connective tissue from women with POP and nonprolapse controls. The round ligament and uterosacral ligament tissues were removed from each subject at the time of laparoscopic hysterectomy. RNA was then extracted, and all labeled samples were hybridized to ABI Human Genome Survey Microarray version 2.0 (Applied Biosystems, CA, USA). The Mahalanobis distance in hierarchical cluster analysis revealed a model of 33 genes, which contained high expressions of round and uterosacral ligaments from women with POP. According to gene ontology, the expressions of mitochondrial genes encoding ribosomal protein were upregulated. Genes involved in potential interactions with mitochondrial electron transport, nucleosome assembly, cell cycle, and apoptosis were also upregulated. As a result, defective mitochondrial translation caused by ribosomal protein contributes to the potential molecular etiology of POP. Such changes, jointly termed "remodeling of pelvic connective tissue", can constitute an important long-term consequence of POP. Our results support the use of genome-based expression profiling as a commonplace platform for diagnostic tests of POP.

Anti-oncogenic and pro-differentiation effects of clorgyline, a monoamine oxidase A inhibitor, on high grade prostate cancer cells

BackgroundMonoamine oxidase A (MAO-A), a mitochondrial enzyme that degrades monoamines including neurotransmitters, is highly expressed in basal cells of the normal human prostatic epithelium and in poorly differentiated (Gleason grades 4 and 5), aggressive prostate cancer (PCa). Clorgyline, an MAO-A inhibitor, induces secretory differentiation of normal prostate cells. We examined the effects of clorgyline on the transcriptional program of epithelial cells cultured from high grade PCa (E-CA).MethodsWe systematically assessed gene expression changes induced by clorgyline in E-CA cells using high-density oligonucleotide microarrays. Genes differentially expressed in treated and control cells were identified by Significance Analysis of Microarrays. Expression of genes of interest was validated by quantitative real-time polymerase chain reaction.ResultsThe expression of 156 genes was significantly increased by clorgyline at all time points over the time course of 6 – 96 hr identified by Significance Analysis of Microarrays (SAM). The list is enriched with genes repressed in 7 of 12 oncogenic pathway signatures compiled from the literature. In addition, genes downregulated ≥ 2-fold by clorgyline were significantly enriched with those upregulated by key oncogenes including beta-catenin and ERBB2, indicating an anti-oncogenic effect of clorgyline. Another striking effect of clorgyline was the induction of androgen receptor (AR) and classic AR target genes such as prostate-specific antigen together with other secretory epithelial cell-specific genes, suggesting that clorgyline promotes differentiation of cancer cells. Moreover, clorgyline downregulated EZH2, a critical component of the Polycomb Group (PcG) complex that represses the expression of differentiation-related genes. Indeed, many genes in the PcG repression signature that predicts PCa outcome were upregulated by clorgyline, suggesting that the differentiation-promoting effect of clorgyline may be mediated by its downregulation of EZH2.ConclusionOur results suggest that inhibitors of MAO-A, already in clinical use to treat depression, may have potential application as therapeutic PCa drugs by inhibiting oncogenic pathway activity and promoting differentiation.

A Genomewide Assessment of Inbreeding Depression: Gene Number, Function, and Mode of Action

Although the genetic basis of inbreeding depression is still being debated, most fitness effects are thought to be the result of increased homozygosity for recessive or partially recessive deleterious alleles rather than the loss of overdominant genes. It is unknown how many loci are associated with inbreeding depression, the genes or gene pathways involved, or their mode of action. To uncover genes associated with variation in fitness following inbreeding, we generated a set of inbred lines of Drosophila melanogaster for which only the third chromosome varied among lines and measured male competitive reproductive success among these lines to estimate inbreeding depression. Male competitive reproductive success for different lines validated our prediction that equally inbred lines show variation in inbreeding depression. To begin to assess the molecular basis of inbreeding depression for male competitive reproductive success, we detected variation in whole-genome gene expression across these inbred lines with commercially available high-density oligonucleotide microarrays. A total of 567 genes were differentially expressed among these inbred lines, indicating that inbreeding directly or indirectly affects a large number of genes: genes that are disproportionately involved in metabolism, stress and defense responses. Subsequently, we generated a set of outbred lines by crossing the highest inbreeding depression lines to each other and contrasted gene expression between parental inbred lines and F(1) hybrids with transcript abundance as a quantitative phenotype to determine the mode of action of the genes associated with inbreeding depression. Although our results indicated that approximately 75% of all genes involved in inbreeding depression were additive, partially additive, or dominant, about 25% of all genes expressed patterns of overdominance. These results should be viewed with caution given that they may be confounded by issues of statistical inference or associative overdominance.

Gene expression analysis of drug-resistant MCF-7 cells: implications for relation to extracellular matrix proteins

Since multidrug resistance is a multifactorial phenomenon, a large-scale expression analysis of drug-resistant cells by using high-density oligonucleotide microarrays may provide information about new candidate genes contributing to resistance. Extracellular matrix (ECM) is responsible for many aspects of proliferation and invasive/metastatic behavior of tumor cells. This study demonstrates alterations in gene expression levels of several ECM components, matrix metalloproteinases (MMPs), adamalysins (ADAMs and ADAMTSs) and tissue inhibitors of metalloproteinases (TIMPs) in paclitaxel, docetaxel, vincristine and doxorubicin-resistant MCF-7 cells. Resistant MCF-7 cells were developed by stepwise selection of cells in increasing concentrations of drugs. Affymetrix GeneChip Human Genome U133 Plus 2.0 Array was used for hybridizations. Statistical significance was determined by independent sample t test. The genes having altered expression levels in drug-resistant sublines were selected and filtered by volcano plots. Genes up/downregulated more than twofolds were selected and listed. Expression of 25 genes encoding ECM proteins (including collagen, finronectin and syndecan) and integrin receptor subunits were found to be upregulated in drug-resistant cells. In addition, expression levels of, 13 genes encoding MMPs, ADAMs, ADAMTSs and TIMPs (including MMP1, MMP9, ADAM9 and TIMP3) were found to be altered in drug-resistant sublines when compared with sensitive MCF-7. Based on the expression analysis profiles, this report provides a preliminary insight into the relationship between drug resistance and ECM components, which are related to invasion and metastasis. Correlation of each specific ECM component with drug resistance requires further analysis.

Defective expression of polarity protein PAR-3 gene (PARD3) in esophageal squamous cell carcinoma

The partition-defective 3 (PAR-3) protein is implicated in the formation of tight junctions at epithelial cell-cell contacts. We investigated DNA copy number aberrations in human esophageal squamous cell carcinoma (ESCC) cell lines using a high-density oligonucleotide microarray and found a homozygous deletion of PARD3 (the gene encoding PAR-3). Exogenous expression of PARD3 in ESCC cells lacking this gene enhanced the recruitment of zonula occludens 1 (ZO-1), a marker of tight junctions, to sites of cell-cell contact. Conversely, knockdown of PARD3 in ESCC cells expressing this gene caused a disruption of ZO-1 localization at cell-cell borders. A copy number loss of PARD3 was observed in 15% of primary ESCC cells. Expression of PARD3 was significantly reduced in primary ESCC tumors compared with their nontumorous counterparts, and this reduced expression was associated with positive lymph node metastasis and poor differentiation. Our results suggest that deletion and reduced expression of PARD3 may be a novel mechanism that drives the progression of ESCC.

Array-CGH Reveals Recurrent Genomic Changes in Merkel Cell Carcinoma Including Amplification of L-Myc

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer with poorly characterized genetics. We performed high resolution comparative genomic hybridization on 25 MCC specimens using a high-density oligonucleotide microarray. Tumors frequently carried extra copies of chromosomes 1, 3q, 5p, and 6 and lost chromosomes 3p, 4, 5q, 7, 10, and 13. MCC tumors with less genomic aberration were associated with improved survival (P=0.04). Tumors from 13 of 22 MCC patients had detectable Merkel cell polyomavirus DNA, and these tumors had fewer genomic deletions. Three regions of genomic alteration were of particular interest: a deletion of 5q12-21 occurred in 26% of tumors, a deletion of 13q14-21 was recurrent in 26% of tumors and contains the well-characterized tumor suppressor RB1, and a previously unreported focal amplification at 1p34 was present in 39% of tumors and centers on L-Myc (MYCL1). L-Myc is related to the c-Myc proto-oncogene, has transforming activity, and is amplified in the closely related small cell lung cancer. Normal skin showed no L-Myc expression, whereas 4/4 MCC specimens tested expressed L-Myc RNA in relative proportion to the DNA copy number gain. These findings suggest several genes that may contribute to MCC pathogenesis, most notably L-Myc.

SNEP: Simultaneous detection of nucleotide and expression polymorphisms using Affymetrix GeneChip

BackgroundHigh-density short oligonucleotide microarrays are useful tools for studying biodiversity, because they can be used to investigate both nucleotide and expression polymorphisms. However, when different strains (or species) produce different signal intensities after mRNA hybridization, it is not easy to determine whether the signal intensities were affected by nucleotide or expression polymorphisms. To overcome this difficulty, nucleotide and expression polymorphisms are currently examined separately.ResultsWe have developed SNEP, a new method that allows simultaneous detection of both nucleotide and expression polymorphisms. SNEP involves a robust statistical procedure based on the idea that a nucleotide polymorphism observed at the probe level can be regarded as an outlier, because the nucleotide polymorphism can reduce the hybridization signal intensity. To investigate the performance of SNEP, we used three species: barley, rice and mice. In addition to the publicly available barley data, we obtained new rice and mouse data from the strains with available genome sequences. The sensitivity and false positive rate of nucleotide polymorphism detection were estimated based on the sequence information. The robustness of expression polymorphism detection against nucleotide polymorphisms was also investigated.ConclusionSNEP performed well regardless of the genome size and showed a better performance for nucleotide polymorphism detection, when compared with other previously proposed methods. The R-software 'SNEP' is available at .

Chronically CNS-Injured Adult Sensory Neurons Gain Regenerative Competence upon a Lesion of Their Peripheral Axon

Several experimental manipulations result in axonal regeneration in the central nervous system (CNS) when applied before or at the time of injury but not when initiated after a delay, which would be clinically more relevant. As centrally injured neurons show signs of atrophy and degeneration, it raises the question whether chronically injured neurons are able to regenerate. To address this question, we used adult rodent primary sensory neurons that regenerate their central axon when their peripheral axon is cut (called conditioning) beforehand but not afterwards. We found that primary sensory neurons express regeneration-associated genes and efficiently regrow their axon in cell culture two months after a central lesion upon conditioning. Moreover, conditioning enables central axons to regenerate through a fresh lesion independent of a previous central lesion. Using in vivo imaging we demonstrated that conditioned neurons rapidly regrow their axons through a fresh central lesion. Finally, when single sensory axons were cut with a two-photon laser, they robustly regenerate within days after attaining growth competence through conditioning. We conclude that sensory neurons can acquire the intrinsic potential to regenerate their axons months after a CNS lesion, which they implement in the absence of traumatic tissue.

Gain‐of‐function mutations and copy number increases of Notch2 in diffuse large B‐cell lymphoma

Signaling through the Notch1 receptor has a pivotal role in early thymocyte development. Gain of Notch1 function results in the development of T-cell acute lymphoblastic leukemia in a number of mouse experimental models, and activating Notch1 mutations deregulate Notch1 signaling in the majority of human T-cell acute lymphoblastic leukemias. Notch2, another member of the Notch gene family, is preferentially expressed in mature B cells and is essential for marginal zone B-cell generation. Here, we report that 5 of 63 (approximately 8%) diffuse large B-cell lymphomas, a subtype of mature B-cell lymphomas, have Notch2 mutations. These mutations lead to partial or complete deletion of the proline-, glutamic acid-, serine- and threonine-rich (PEST) domain, or a single amino acid substitution at the C-terminus of Notch2 protein. Furthermore, high-density oligonucleotide microarray analysis revealed that some diffuse large B-cell lymphoma cases also have increased copies of the mutated Notch2 allele. In the Notch activation-sensitive luciferase reporter assay in vitro, mutant Notch2 receptors show increased activity compared with wild-type Notch2. These findings implicate Notch2 gain-of-function mutations in the pathogenesis of a subset of B-cell lymphomas, and suggest broader roles for Notch gene mutations in human cancers.

Left ventricular global transcriptional profiling in human end-stage dilated cardiomyopathy

We employed ABI high-density oligonucleotide microarrays containing 31,700 sixty-mer probes (representing 27,868 annotated human genes) to determine differential gene expression in idiopathic dilated cardiomyopathy (DCM). We identified 626 up-regulated and 636 down-regulated genes in DCM compared to controls. Most significant changes occurred in the tricarboxylic acid cycle, angiogenesis, and apoptotic signaling pathways, among which 32 apoptosis- and 13 MAPK activity-related genes were altered. Inorganic cation transporter, catalytic activities, energy metabolism and electron transport-related processes were among the most critically influenced pathways. Among the up-regulated genes were HTRA1 (6.9-fold), PDCD8(AIFM1) (5.2) and PRDX2 (4.4) and the down-regulated genes were NR4A2 (4.8), MX1 (4.3), LGALS9 (4), IFNA13 (4), UNC5D (3.6) and HDAC2 (3) ( p < 0.05), all of which have no clearly defined cardiac-related function yet. Gene ontology and enrichment analysis also revealed significant alterations in mitochondrial oxidative phosphorylation, metabolism and Alzheimer's disease pathways. Concordance was also confirmed for a significant number of genes and pathways in an independent validation microarray dataset. Furthermore, verification by real-time RT-PCR showed a high degree of consistency with the microarray results. Our data demonstrate an association of DCM with alterations in various cellular events and multiple yet undeciphered genes that may contribute to heart muscle disease pathways.

Ammonium Glycyrrhizinate Protects Gastric Epithelial Cells from Hydrogen Peroxide-Induced Cell Death

Glycyrrhiza uralensis has a potential for preventing or ameliorating gastric mucosal ulceration. To understand the molecular mechanism about the medicinal effect of G. uralensis, we isolated four single compounds from G. uralensis and one related compound and screened for the cellular protective effect against H(2)O(2)-induced damage in gastric epithelial AGS cells. Interestingly, we found that ammonium glycyrrhizinate (AG) prepared from glycyrrhizin dramatically protects AGS cells from H(2)O(2)-induced damage as measured by the integrity of actin cytoskeleton. AG also inhibited FeSO(4)-induced reactive oxygen radicals in a dose-dependent manner, suggesting the role for AG as a free radical scavenger. To better understand the protective role of AG at the transcriptional level, we performed genome-wide expression profiling using high-density oligonucleotide microarrays, followed by validation using RT-PCR. Among the 33,096 genes that were screened in the microarray, 936 genes were found to be differentially expressed in a statistically significant manner in the presence or absence of H(2)O(2) and AG. Among the 936 genes, 51 genes were differentially expressed at least 3-fold in response to the H(2)O(2) treatment. AG blocked the expression of genes related to apoptotic cell death (GDF15, ATF3, TNFRSF10A, NALP1) or oxidative stress path-ways (HMOX1) which was elevated in response to H(2)O(2) treatment, suggesting a potential protective role for AG in oxidative stress-induced cell death. Collectively, current results demonstrate that AG is a novel antioxidant that could be effective for the treatment of gastric diseases related to the oxidative stress-induced mucosal damage.

Identification of Genes With Differential Expression in Chemoresistant Epithelial Ovarian Cancer Using High-Density Oligonucleotide Microarrays

A major obstacle in treatment of epithelial ovarian cancer is chemoresistance. The aim of this study was to determine whether distinct gene expression profiles are associated with chemoresistance in epithelial ovarian carcinoma. We performed global gene expression analysis in 13 primary epithelial ovarian cancer tissues including 5 primary chemosensitive tumors and 8 primary chemoresistant tumors using Affymetrix HGU133A microarray. The gene expression patterns of chemosensitive tumors were compared with those of chemoresistant tumors using fold change. Validity of microarray results was examined by semiquantitative RT-PCR. We identified over 320 genes differentially expressed in chemoresistant epithelial ovarian cancer (> or = twofold). Upregulated genes in chemoresistant tumors included cell cycle regulating genes (TOP2A, BCAT1, CDCA8, CCNA2, CENPE), and genes with previously known mechanisms in tumorigenesis (S100A9, APOA1, RNF125, IFI16). Downregulated genes in chemoresistant tumors included genes related to cell adhesion (MUC5B, CITED2), transcription regulating genes (FOXD1, MAD1L1, PAX2), genes involving signal transduction (SOSTDC1, SNX1, SFRP1, FOXA2, PLK2), and stress protein gene (TP53AP1). These data show that gene expression profiling can discriminate primary chemoresistant from primary chemosensitive ovarian cancers. This type of molecular profiling could provide a basis for additional functional studies.

Infection and genotype remodel the entire soybean transcriptome

BackgroundHigh throughput methods, such as high density oligonucleotide microarray measurements of mRNA levels, are popular and critical to genome scale analysis and systems biology. However understanding the results of these analyses and in particular understanding the very wide range of levels of transcriptional changes observed is still a significant challenge. Many researchers still use an arbitrary cut off such as two-fold in order to identify changes that may be biologically significant. We have used a very large-scale microarray experiment involving 72 biological replicates to analyze the response of soybean plants to infection by the pathogen Phytophthora sojae and to analyze transcriptional modulation as a result of genotypic variation.ResultsWith the unprecedented level of statistical sensitivity provided by the high degree of replication, we show unambiguously that almost the entire plant genome (97 to 99% of all detectable genes) undergoes transcriptional modulation in response to infection and genetic variation. The majority of the transcriptional differences are less than two-fold in magnitude. We show that low amplitude modulation of gene expression (less than two-fold changes) is highly statistically significant and consistent across biological replicates, even for modulations of less than 20%. Our results are consistent through two different normalization methods and two different statistical analysis procedures.ConclusionOur findings demonstrate that the entire plant genome undergoes transcriptional modulation in response to infection and genetic variation. The pervasive low-magnitude remodeling of the transcriptome may be an integral component of physiological adaptation in soybean, and in all eukaryotes.

Triple X syndrome in a patient with partial lipodystrophy discovered using a high-density oligonucleotide microarray: a case report

IntroductionPatients with lipodystrophy experience selective or generalized atrophy of adipose tissue. The disruption of lipid metabolism results in an increased risk for development of metabolic syndrome and coronary artery disease. Currently, the mutations responsible for approximately half of lipodystrophy patients are known, but new techniques and examination of different types of genetic variation may identify new disease-causing mechanisms.Case presentationA 53-year-old woman of African descent was referred to a tertiary care endocrinology clinic for treatment of severe insulin resistance, treatment-resistant hypertension and dyslipidemia. After all known lipodystrophy-causing mutations were excluded by DNA sequencing, the patient was found to have triple X syndrome after an initial investigation into copy number variation using a high-density oligonucleotide microarray. The patient also had a previously unobserved duplication of 415 kilobases of chromosome 5q33.2. This is the first case report of a patient with lipodystrophy who also had triple X syndrome.ConclusionWhile we cannot make a direct link between the presence of triple X syndrome and partial lipodystrophy, if unrelated, this is an extremely rare convergence of syndromes. This patient poses an interesting possibility regarding the influence triple X syndrome may have on an individual with other underlying lipodystrophy susceptibility. Finally, impending large-scale case-control and cohort copy number variation investigations will, as a by-product, further document the prevalence of triple X syndrome in various patient groups.