Expression Analysis Systematic Explorer Research Articles

Identification of activated pathways in lung adenocarcinoma based on network strategy.

Lung adenocarcinoma has increased incidence over the past years and is the cause for almost 50% of deaths attributable to lung cancer. The objective of this paper is to identify activated pathways associated with lung adenocarcinoma based on gene co-expression network analysis. Kyoto Encyclopedia of Genes and Genomes pathway analysis of dysregulated genes was performed based on Expression Analysis Systematic Explorer test to illuminate the biological pathways. Co-expression networks of lung adenocarcinoma in different tumor Stages (IA, IB, IIA, IIB, IIIA, IIIB, and IV) were constructed by Empirical Bayes approach to reweight gene pair scores. Pathway activity analysis was conducted to compute the distribution of pathways in different stages and to identify "activated" pathways in lung adenocarcinoma. We evaluated 211 dysregulated genes between lung adenocarcinoma patients and normal controls. Pathway activity analysis was performed and P values of pathways, which obtained from co-expression networks (Stage IA, IB, IIA, IIB, IIIA, IIIB, and IV), were calculated. Cell cycle, progesterone-mediated oocyte maturation, and oocyte meiosis were activated during all stages in lung adenocarcinoma. We successfully identified three activated pathways (cell cycle, progesterone-mediated oocyte maturation, and oocyte meiosis) in different Stages (IA, IB, IIA, IIB, IIIA, IIIB, and IV) of lung adenocarcinoma.

Comparative analysis of regulatory roles of P38 signaling pathway in 8 types liver cell during liver regeneration

P38MAPK signaling pathway was closely related to cell proliferation, apoptosis, cell differentiation, cell survival, cell death, and so on. However, the regulatory mechanism of P38MAPK signaling pathway in liver regeneration (LR) was unclear. In order to further reveal the roles of P38MAPK signaling pathway in rat liver regeneration, Ingenuity Pathway Analysis (IPA) software and related data sites were used to construct P38MAPK signaling pathway, and the pathway was confirmed by relevant documents literature. The expression changes of P38MAPK signaling pathway-related gene in eight type cells were further analyzed by Rat Genome 230 2.0 Array, and the results showed that 95 genes in P38MAPK signaling pathway had significant changes. H-cluster analysis showed that hepatocyte cell (HC), pit cell (PC), oval cell (OC) and biliary epithelial cell (BEC) are clustered together; and the same as Kupffer cell (KC), sinusoidal endothelial cell (SEC), dendritic cell (DC) and hepatic stellate cell (HSC). IPA software and expression analysis systematic explorer (EASE) were applied to functional enrichment analysis, and the results showed that P38MAPK signaling pathway was mainly involved in apoptosis, cell death, cell proliferation, cell survival, cell viability, activation, cell cycle progression, necrosis, synthesis of DNA and other physical activity during LR. In conclusion, P38MAPK signaling pathway regulated various physiological activities of LR through multiple signaling pathways.

Systematic tracking of disrupted modules identifies significant genes and pathways in hepatocellular carcinoma.

The objective of the present study is to identify significant genes and pathways associated with hepatocellular carcinoma (HCC) by systematically tracking the dysregulated modules of re-weighted protein-protein interaction (PPI) networks. Firstly, normal and HCC PPI networks were inferred and re-weighted based on Pearson correlation coefficient. Next, modules in the PPI networks were explored by a clique-merging algorithm, and disrupted modules were identified utilizing a maximum weight bipartite matching in non-increasing order. Then, the gene compositions of the disrupted modules were studied and compared with differentially expressed (DE) genes, and pathway enrichment analysis for these genes was performed based on Expression Analysis Systematic Explorer. Finally, validations of significant genes in HCC were conducted using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. The present study evaluated 394 disrupted module pairs, which comprised 236 dysregulated genes. When the dysregulated genes were compared with 211 DE genes, a total of 26 common genes [including phospholipase C beta 1, cytochrome P450 (CYP) 2C8 and CYP2B6] were obtained. Furthermore, 6 of these 26 common genes were validated by RT-qPCR. Pathway enrichment analysis of dysregulated genes demonstrated that neuroactive ligand-receptor interaction, purine and drug metabolism, and metabolism of xenobiotics mediated by CYP were significantly disrupted pathways. In conclusion, the present study greatly improved the understanding of HCC in a systematic manner and provided potential biomarkers for early detection and novel therapeutic methods.

Overexpression of CD44 is associated with the occurrence and migration of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a potentially fatal disease and the incidence is increasing annually. In order to diagnose and treat NSCLC effectively, greater understanding of its molecular mechanism is required. In the present study, 36 NSCLC tissues and 10 normal tissues were selected. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to analyze the CD44 mRNA expression level in NSCLC tissue and DNA sequencing was performed to further verify the CD44 expression level. Differentially expressed genes between tumor tissues and controls were determined by DNA sequencing and the Gene_act_net between CD44 and its associated genes was constructed. Gene Ontology (GO) term enrichment analysis of the differentially expressed genes was performed by the Biological Networks Gene Ontology tool. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed based on the Expression Analysis Systematic Explorer test applied in the Database for Annotation, Visualization and Integrated Discovery. RT-qPCR results showed that CD34 was overexpressed in 21 of the 36 NSCLC tissues (58.3%). The Gene_act_net indicated that there were 20 differentially expressed genes with 17 upregulated and 3 downregulated. Among them, CD44, MET, ERBB2, EGFR, AKT1, IQGAP1 and STAT3 were associated with the occurrence and migration of NSCLC. In KEGG pathway analysis, extracellular matrix-receptor interaction and hematopoietic cell lineage pathways were the most affected by overexpressed CD44; and thus may be important in the development and migration of NSCLC. In conclusion, CD44 was overexpressed in NSCLC and the overexpression was associated with the occurrence of NSCLC and migration of NSCLC cells.

A novel application of breath hydrogen and methane monitoring in nutritional science

s / Journal of Nutrition & Intermediary Metabolism 1 (2014) 1e55 14 I296V; rs10246939) and grouped by haplotype. Serum and red cell folate levels (chemiluminescent immunoassay) and estimated folate intake (food frequency questionnaires) were determined. 6-n-propylthiouracil solutions were used to determine bitter taste phenotype (“taster”/”nontaster”). Differences between groups were assessed by t-tests, c2 tests and RR of AP. Results: TAS2R38 haplotype significantly, but only partially predicted taster phenotype (r 1⁄4 0.43, c21⁄435.5, p 50% improvement of symptoms, was assessed. Difference between the groups was tested via Fishers exact test. Results: Of 157 women with IBS, 56 (35%) had a concurrent diagnosis of endometriosis. The median age of those with endometriosis was 32 as opposed to those without where median age was 39 (p 1⁄4 0.014). However, response was reported in 71% with endometriosis compared with 47% of those without endometriosis (p 1⁄4 0.004). Conclusions: The high rate of response to a LFD suggests that this approach should be added to the repertoire of management strategies for endometriosis. Why the response was poorer in those without endometriosis was not clear. Funding source(s): N/A. POSTPRANDIAL NUTRIGENOMIC PATHWAY ANALYSIS OF ADIPOSE TISSUE IN MEN WITH METABOLIC SYNDROME (METS) A.L. Dordevic , M.P. Bonham , A.E. Larsen , P. Gran , A.J. Sinclair , J.B. Jowett , N. Konstantopoulos , D. Cameron-Smith . 1 School of Exercise & Nutritional Science, Deakin University, Australia; Department Nutrition & Dietetics, Monash University, Australia; Metbolic Research Unit, Deakin University, Australia; Genomic & Systems Biology, Baker IDI, VIC, Australia; 5 Liggins Institute, Auckland University, NZ, New Zealand E-mail: aimee.dordevic@monash.edu (A.L. Dordevic) Background/Aims: Profiling the transcriptomic regulation of adipose tissue (AT) by dietary manipulation in MetS will elucidate therapeutic and nutritional targets for chronic disease management. Methods: Seventeen men (8 control; 9 MetS) were recruited to participate in a controlled randomised, single-blinded crossover single meal study. Participants consumed either a saturated fatty acid (FA)-rich breakfast or an unsaturated FA-rich breakfast. AT biopsies were taken at baseline (0 h), and 4 h following each test meal. Global gene expression profiling of the AT was performed using Illumina HumanWG-6 v3 microarray chips followed by gene set enrichment analysis (GSEA) using DAVID software. Pathways were considered significant with Expression Analysis Systematic Explorer (EASE) score < 0.05 and a false discovery rate (FDR) value < 0.05. Results: Two pathways that were differentially regulated in MetS compared to control AT at baseline were also enriched by the following meal consumption. The Lysosome pathway was up-regulated (p < 0.001) and the Ribosome pathway was down-regulated (p < 0.001) in MetS AT compared with controls at baseline. Control AT exhibited enrichment of the Lysosome pathway with the SFA meal (p1⁄4 0.001) and MetS AT showed enrichment of the Ribosome pathway after the UFA meal (p 1⁄4 0.02). Conclusions: Lysosome and Ribosome pathways are impaired in MetS AT and can be manipulated by alterations in meal composition. This finding is important in the discovery of potential nutritional targets for chronic metabolic disease. Funding source(s): Dairy Health & Nutrition Consortium (DHNC). REDUCED FODMAPS IN GLUTEN-FREE GRAINS MAY EXPLAIN THE IMPROVED SYMPTOMS IN PEOPLE WITH IBS FOLLOWING A GLUTENFREE DIET J.G. Muir , J. Mills , D. Suter , F. Bekes , K. Liels , C.K. Yao , P.R. Gibson . Department of Gastroenterology, Central Clinical School, Monash University, Australia; George Weston Foods Ltd, North Ryde, NSW, Australia E-mail: Jane.Muir@monash.edu (J.G. Muir) Background/Aims: Individuals with IBS blame gluten-containing grains for triggering gastrointestinal symptoms. Another component of grains that requires consideration is the FODMAPs mostly fructans and galacto-oligsaccharides (GOS). We aim to analyse the gluten and FODMAP content of 22

Identification of therapeutic target genes with DNA microarray in multiple myeloma cell line treated by IKKβ/NF-κB inhibitor.

To explore the mechanism of resistance to IKKβ inhibitor in multiple myeloma (MM) cells and uncover novel therapeutic targets for MM. We downloaded the microarray data (GSE8476) from GEO (Gene Expression Omnibus) database. The data were derived from the human MM cells lines (L363 cells) treated with IKKβ inhibitor MLN120b (MLN) for eight, 12 and 24 hours. Furthermore, we applied the Search Tool for the Retrieval of Interacting Genes (STRING) and Expression Analysis Systematic Explorer (EASE) database to construct protein-protein interaction networks and identified over-represented pathway among DEGs (differentially expressed genes). We obtained 108 DGEs in 8h vs. 12h group and 101 ones in 8h vs. 24h group. Most of DGEs were found to be involved in biological regulation. The significant pathways were Ig A pathway and the CAMs pathways. In addition, 24 common DGEs were found in the networks of the two groups such as ICAM 3 and SELL. Intercellular adhesion molecule 3 and SELL may be potential targets in multiple myeloma treatment in the future.

A time course study about gene expression of post-thermal injury with DNA microarray.

Burn injury is one of the most common and devastating forms of trauma in daily life. However, the exact sequence of events after burn injury remains unknown. This study aims to investigate gene expression alterations after burn injury. Microarray data set GSE8056 was downloaded from the Gene Expression Omnibus (GEO) database, including 12 samples, equally distributed in four groups: normal skin tissue as control and damaged tissues 1-3days after burn (early period); 4-7days after burn (middle period); and more than 7days after burn (late period). Packages in R language were utilized to pre-process the data and filter out the differentially expressed genes (DEGs). Functional annotation of all three groups of DEGs was conducted by using clusters of orthologous groups analysis. The DEGs shared by all three groups were picked out and analyzed with STRING to set up a protein-protein interaction network. CFinder was chosen to implement module analysis, and expression analysis systematic explorer was then adopted to reveal the dysfunctional pathways for each module. A total of 727, 782, and 445 DEGs were identified in the early, middle, and late period after burn, and 234 DEGs were identified as continually differentially expressed throughout all time periods, including genes encoding proinflammatory cytokines, such as interleukin (IL)-6, IL-8, and IL-1β, and genes associated with cell proliferation. Three modules associated with cell proliferation and inflammatory responses were generated from the protein-protein interaction network. Our findings are beneficial for understanding the progression of the wound healing response after burn.

Gene expression profiles of patients with cerebral hematoma following spontaneous intracerebral hemorrhage.

The present study aimed to investigate the gene functions and expression profiles in perihematomal (PH) brain regions following spontaneous intracerebral hemorrhage. The gene expression profiles were downloaded from the Gene Expression Omnibus database under accession number GSE24265, which includes 11 brain samples from different regions, including four samples from PH areas, four from contralateral grey matter (CG) and three from contralateral white matter (CW). The gene expression profiles were pre-processed and the differentially expressed genes (DEGs) between PH and CG tissue, and PH and CW tissue were identified using R packages. The expression of genes in different tissues was analyzed by hierarchical clustering. Then, the interaction network between the DEGs was constructed using String software. Finally, Gene Ontology was performed and pathway analysis was conducted using FuncAssociate and Expression Analysis Systematic Explorer to identify the gene function. As a result, 399 DEGs were obtained between PH and CG, and 756 DEGs were identified between PH and CW. There were 35 common DEGs between the two groups. These DEGs may be involved in PH edema by regulating the calcium signaling pathway [calcium channel, voltage-dependent, T-type, α1I subunit, Ca2+/calmodulin-dependent protein kinase II α (CAMK2A), ryanodine receptor 2 (RYR2) and inositol 1,4,5-trisphosphate receptor, type 1 (ITPR1)], cell proliferation (sphingosine kinase 1), neuron differentiation (Ephrin-A5) or extracellular matrix-receptor interaction [collagen, type I, α 2, laminin B1 (LAMB1), syndecan 2, fibronectin 1 and integrin α5 (ITGA5)]. A number of genes may cooperate to participate in the same pathway, such as ITPR1-RYR2, CAMK2A-RYR2 and ITGA5-LAMB1 interaction pairs. The present study provides several potential targets to decrease hematoma expansion and alleviate neuronal cell death following spontaneous intracerebral hemorrhage.

Identification of differentially expressed microRNAs in metastatic melanoma using next-generation sequencing technology

In this study, we investigated differentially expressed microRNAs (miRNAs or miRs) and their functions in metastatic melanoma using next-generation sequencing technology. The GSE36236 data set was downloaded from the Gene Expression Omnibus (GEO) database and 4 primary cutaneous melanoma samples (used as controls) and 3 metastatic melanoma samples were selected from 31 samples for further analysis. Firstly, the differentially expressed miRNAs were screened by limma package in R language. Secondly, the target genes of the miRNAs were retrieved with TargetScanHuman 6.2, and the interactions among these genes were identified by String and an interaction network was established. Finally, functional and pathway analyses were performed for the genes in the network using Expression Analysis Systematic Explorer (EASE). A total of 4 differentially expressed miRNAs (hsa-miR-146, hsa-miR-27, hsa-miR-877 and hsa-miR-186) were obtained between the metastatic melanoma and primary cutaneous melanoma samples. We predicted 101 high-confidence target genes of hsa-miR-27 and obtained a network with 41 interactions. Finally, functional and pathway analyses revealed that the genes in the network were significantly enriched at the transcriptional level. Differentially expressed miRNAs were identified in the metastatic melanoma compared with the primary cutaneous melanoma samples and the target genes of hsa-miR-27 were found to be significantly enriched at the transcriptional level. The results presented in our study may prove helpful in the diagnosis and treatment of metastatic melanoma.

Construction of Drug Network Based on Side Effects and Its Application for Drug Repositioning

Drugs with similar side-effect profiles may share similar therapeutic properties through related mechanisms of action. In this study, a drug-drug network was constructed based on the similarities between their clinical side effects. The indications of a drug may be inferred by the enriched FDA-approved functions of its neighbouring drugs in the network. We systematically screened new indications for 1234 drugs with more than 2 network neighbours, 36.87% of the drugs achieved a performance score of Normalized Discounted Cumulative Gain in the top 5 positions (NDCG@5)≥0.7, which means most of the known FDA-approved indications were well predicted at the top 5 positions. In particular, drugs for diabetes, obesity, laxatives and antimycobacterials had extremely high performance with more than 80% of them achieving NDCG@5≥0.7. Additionally, by manually checking the predicted 1858 drug-indication pairs with Expression Analysis Systematic Explorer (EASE) score≤10−5 (EASE score is a rigorously modified Fisher exact test p value), we found that 80.73% of such pairs could be verified by preclinical/clinical studies or scientific literature. Furthermore, our method could be extended to predict drugs not covered in the network. We took 98 external drugs not covered in the network as the test sample set. Based on our similarity criteria using side effects, we identified 41 drugs with significant similarities to other drugs in the network. Among them, 36.59% of the drugs achieved NDCG@5≥0.7. In all of the 106 drug-indication pairs with an EASE score≤0.05, 50.94% of them are supported by FDA approval or preclinical/clinical studies. In summary, our method which is based on the indications enriched by network neighbors may provide new clues for drug repositioning using side effects.

FRI0198 Expressions of immune response related genes were normalised after tocilizumab treatment in rheumatoid arthritis (RA) patients

BackgroundTocilizumab (TCZ), a humanised anti-interleukin (IL)6 receptor antibody, was shown to be therapeutically effective in RA patients but the biological effect of TCZ on the immune response was not fully...

Gene expression profiles reveal significant differences between rat liver cancer and liver regeneration

Rapid cell proliferation and growth occur in both liver cancer (LC) and liver regeneration (LR). Does it imply that LC and LR share some similar molecular mechanisms? To elucidate the intrinsic similarities and differences between the above two processes at transcriptional level, rat models of diethylnitrosamine-induced LC and 2/3 partial hepatectomy-induced LR were separately established. Then Rat Genome 230 2.0 Array was used to detect gene expression profiles of liver tissues obtained from the above two models, and bioinformatics methods, such as hierarchical clustering, k-means clustering and Expression Analysis Systematic Explorer (EASE), were applied to uncover the correlation between gene expression changes and physiological activities in LC and LR. Subsequently expression changes of six selected genes were confirmed by real-time quantitative RT-PCR. As a result, the expressions of 909 genes were found significantly changed during LC occurrence and 948 genes in LR. The expression profiles of the above two events were extremely different, and their expression patterns were classified into 6 clusters. Based on the correlation between expression patterns and functional profiles, we found that drug/toxin metabolism and oxidation reduction were induced in LC, but decreased in LR at the transcription level, while lipid, steroid and chemical homeostasis were remarkably repressed in LC but induced in LR; inflammation/immune response and apoptosis were not obvious or weaker in LC than in LR, whereas the activities of angiogenesis and cell adhesion/migration in LC were much stronger.

NEASE: a method for gene ontology subclassification of high-throughput gene expression data

High-throughput technologies can identify genes whose expression profiles correlate with specific phenotypes; however, placing these genes into a biological context remains challenging. To help address this issue, we developed nested Expression Analysis Systematic Explorer (nEASE). nEASE complements traditional gene ontology enrichment approaches by determining statistically enriched gene ontology subterms within a list of genes based on co-annotation. Here, we overview an open-source software version of the nEASE algorithm. nEASE can be used either stand-alone or as part of a pathway discovery pipeline. nEASE is implemented within the Multiple Experiment Viewer software package available at http://www.tm4.org/mev. Supplementary data are available at Bioinformatics online.

Annotation and &amp;lt;i&amp;gt;in silico&amp;lt;/i&amp;gt; localization of the Affymetrix GeneChip Porcine Genome Array

Abstract. Expression microarrays including the Affymetrix GeneChip Porcine Genome Arrays are valuable tools for studying genes and functional networks relevant for the expression of complex traits and the responsiveness of the organism to various treatments. An updated annotation and, for the first time, localization on the porcine physical genome map of »Affymetrix GeneChip Porcine Genome Array probe sets« was made through a workflow of 3 pipelines of comparisions addressing various NCBI (National Center for Biotechnology Information) and EnsEMBL (Ensembl project) databases. »BLAST« (Basic Local Alignment Search Tool) comparisons of Affymetrix probe set consensus sequences with the EnsEMBL Sscrofa 9 cDNA database provided 23 799 probe sets with hits. After annotation 19 730 gene symbols were obtained using the data management system BioMart. Comparison of the Affymetrix probe set consensus sequences with the porcine genome sequence (EnsEMBL Sscrofa 9 LatestGP database) revealed 23 298 probe sets with BLAST hits. In the third pipeline in addition to EnsEMBL Sscrofa 9 cDNA and genomic sequence databases also human, mouse and pig NCBI reference sequence RNA databases were interrogated in an integrated approach where also a threshold of bit score &gt;50 or &gt;90 % identity over &gt;100 bp was applied in order to filter questionable annotations and localizations. Gene symbols and gene names were queried from HGNC (human genome organization (HUGO) gene nomenclature committee), EASE (EASE: the Expression Analysis Systematic Explorer) and Entrez Gene revealing 20 269 annotated probe sets. 20 467 probe sets were in silico mapped addressing various sources: EnsEMBL Sscrofa 9 LatestGP, pre-EnsEMBL Sscrofa 8.52 LatestGP, NCBI pig reference sequence RNA and genomic databases and PigQTLdb (Pig Quantitative Trait Locus [QTL] database). Using the new annotation and localization data in functional genomics studies will facilitate improving the understanding of the control of quantitative traits in pigs.

Axonal Transport Proteomics Reveals Mobilization of Translation Machinery to the Lesion Site in Injured Sciatic Nerve

Investigations of the molecular mechanisms underlying responses to nerve injury have highlighted the importance of axonal transport systems. To obtain a comprehensive view of the protein ensembles associated with axonal transport in injured axons, we analyzed the protein compositions of axoplasm concentrated at ligatures following crush injury of rat sciatic nerve. LC-MS/MS analyses of iTRAQ-labeled peptides from axoplasm distal and proximal to the ligation sites revealed protein ensembles transported in both anterograde and retrograde directions. Variability of replicates did not allow straightforward assignment of proteins to functional transport categories; hence, we performed principal component analysis and factor analysis with subsequent clustering to determine the most prominent injury-related transported proteins. This strategy circumvented experimental variability and allowed the extraction of biologically meaningful information from the quantitative neuroproteomics experiments. 299 proteins were highlighted by principal component analysis and factor analysis, 145 of which correlate with retrograde and 154 of which correlate with anterograde transport after injury. The analyses reveal extensive changes in both anterograde and retrograde transport proteomes in injured peripheral axons and emphasize the importance of RNA binding and translational machineries in the axonal response to injury.

Testicular gene expression profiling following 2-methoxyethanol and 2-ethoxyethanol exposure in male rats reveals abnormal expression of the actin binding protein cortactin in degenerating spermatocytes

The glycol ether solvents 2-methoxyethanol (2-ME) and 2-ethoxyethanol (2-EE) produce testicular toxicity characterized by spermatocyte degeneration, while a similar glycol ether, 2-butoxyethanol (2-BE), has no testicular effects. The goal of the current study was to better understand the mechanism of glycol ether testicular toxicity through gene expression profiling and functional classification of differentially expressed genes. Male rats were administered 2-ME (150 and 50 mg/kg/day), 2-EE (500 mg/kg/day), 2-BE (125 mg/kg/day), or vehicle for 3 days, and testes were collected for histopathological and gene expression analysis. Histopathological changes in the testes were observed only in animals given 150 mg/kg/day 2-ME, consisting of degeneration and necrosis of spermatocytes and reductions in spermatocyte numbers. Microarray analysis of testicular samples from these animals revealed a large number of differentially expressed genes from animals exposed to 2-EE or to 50 mg/kg or 150 mg/kg 2-ME (>900 each at >1.5-fold changed), compared to 28 genes from 2-BE treated animals. Expression Analysis Systematic Explorer (EASE) analysis of these genes demonstrated statistical enrichment in genes in categories including protein transport, endocytosis, protein kinase activity, cell cycle, and meiosis. Quantitative PCR confirmation of select genes confirmed increased expression of the actin binding protein cortactin and the transcription factor Wilm's tumor 1 (Wt1) following 2-ME exposure. Increased localization of cortactin in abnormal spermatocytes was also observed by immunohistochemistry, consistent with a possible role for this protein in the mechanism of toxicity.

Analysis of gene expression profiles in HeLa cells in response to overexpression or siRNA-mediated depletion of NASP

BackgroundNASP (Nuclear Autoantigenic Sperm Protein) is a linker histone chaperone required for normal cell division. Changes in NASP expression significantly affect cell growth and development; loss of gene function results in embryonic lethality. However, the mechanism by which NASP exerts its effects in the cell cycle is not understood. To understand the pathways and networks that may involve NASP function, we evaluated gene expression in HeLa cells in which NASP was either overexpressed or depleted by siRNA.MethodsTotal RNA from HeLa cells overexpressing NASP or depleted of NASP by siRNA treatment was converted to cRNA with incorporation of Cy5-CTP (experimental samples), or Cy3-CTP (control samples). The labeled cRNA samples were hybridized to whole human genome microarrays (Agilent Technologies, Wilmington, Delaware, USA). Various gene expression analysis techniques were employed: Significance Analysis of Microarrays (SAM), Expression Analysis Systematic Explorer (EASE), and Ingenuity Pathways Analysis (IPA).ResultsFrom approximately 36 thousand genes present in a total human genome microarray, we identified a set of 47 up-regulated and 7 down-regulated genes as a result of NASP overexpression. Similarly we identified a set of 56 up-regulated and 71 down-regulated genes as a result of NASP siRNA treatment. Gene ontology, molecular network and canonical pathway analysis of NASP overexpression demonstrated that the most significant changes were in proteins participating in organismal injury, immune response, and cellular growth and cancer pathways (major "hubs": TNF, FOS, EGR1, NFκB, IRF7, STAT1, IL6). Depletion of NASP elicited the changed expression of proteins involved in DNA replication, repair and development, followed by reproductive system disease, and cancer and cell cycle pathways (major "hubs": E2F8, TP53, FGF, FSH, FST, hCG, NFκB, TRAF6).ConclusionThis study has demonstrated that NASP belongs to a network of genes and gene functions that are critical for cell survival. We have confirmed the previously reported interactions between NASP and HSP90, HSP70, histone H1, histone H3, and TRAF6. Overexpression and depletion of NASP identified overlapping networks that included TNF as a core protein, confirming that both high and low levels of NASP are detrimental to cell cycle progression. Networks with cancer-related functions had the highest significance, however reproductive networks containing follistatin and FSH were also significantly affected, which confirmed NASP's important role in reproductive tissues. This study revealed that, despite some overlap, each response was associated with a unique gene signature and placed NASP in important cell regulatory networks.

Brief Bout of Exercise Alters Gene Expression in Peripheral Blood Mononuclear Cells of Early- and Late-Pubertal Males

Peripheral blood mononuclear cells (PBMCs) are stimulated by exercise and contribute not only to host defense, but also to growth, repair, and disease pathogenesis. Whether PBMC gene expression is altered by exercise in children is not known. Ten early pubertal boys (8-12 y) and 10 late pubertal boys (15-18 y) performed ten 2-min bouts of strenuous, constant work rate exercise with 1-min rest intervals. PBMCs were isolated before and after exercise and microarray (Affymetrix U133 + 2 chips) analyzed. Statistical criterion to identify gene expression changes was less than 5% false discovery rate (FDR) with 95% confidence interval. One thousand two hundred forty-six genes were altered in older boys (517 up, 729 down), but only 109 were altered in the younger group (79 up, 30 down). In older boys, 13 gene pathways (using Expression Analysis Systematic Explorer, p < 0.05) were found (e.g. natural killer cell cytotoxicity, apoptosis). Epiregulin gene expression (EREG, a growth factor involved in wound healing) increased in older boys. In older boys exercise altered genes such as TBX21, GZMA, PGTDR, and CCL5 also play roles in pediatric inflammatory diseases like asthma. Sixty-six genes were changed significantly in both groups. The pattern of PBMC gene expression suggests the initiation of an immunologic "danger" signal associated with a sudden change in energy expenditure.

DIFFERENTIAL EXPRESSION OF TOLL-LIKE RECEPTOR GENES

Toll-like receptors (TLRs) are critical components of innate immunity. This study was designed to evaluate differential expression of genes for TLR and associated signal transduction molecules in critically ill patients developing sepsis compared with those with sterile inflammation. Uninfected critically ill patients with systemic inflammatory response syndrome were prospectively followed daily for development of sepsis. They were divided into two groups and compared in a case-control manner: (a) preseptic patients (n = 45) who subsequently developed sepsis, and (b) uninfected systemic inflammatory response syndrome patients (n = 45) who remained uninfected. Whole blood RNA was collected (PAXGene tube) at study entry and 1, 2, and 3 days before clinical sepsis diagnosis (or time-matched uninfected control) and analyzed via Affymetrix Hg_U133 Plus 2.0 microarrays. Genes were considered differentially expressed if they met univariate significance controlled for multiple comparisons at P < 0.005. Differentially expressed probes were uploaded into the Database for Annotation, Visualization and Integrated Discovery. The TLR pathway (Kyoto Encyclopedia of Genes and Genomes-KEGG) significance was determined via Expression Analysis Systematic Explorer (EASE) scoring. A total of 2,974 Affymetrix probes representing 2,190 unique genes were differentially expressed 1 day before sepsis diagnosis. Thirty-six probes representing 25 genes were annotated to the TLR pathway (KEGG) via the Database for Annotation, Visualization and Integrated Discovery with an EASE score at P < 0.0004. Notable TLR genes demonstrating increased expression include TLR-4 (median, 1.43-fold change), TLR-5 (2.08-fold change), and MAPK14 (1.90-fold change). An additional 11 unique genes were manually annotated into the TLR pathway based on known relevance such as TLR-8 (1.54-fold change). The total 36 genes contained 28 showing increased expression and 8 showing decreased expression. Differential gene expression was noted for TLR receptors (eight genes), TLR intracellular signal transduction cascade molecules (27 genes), and TLR-related effector molecules (one gene). The TLR and downstream signaling genes are differentially expressed in critically ill patients developing sepsis compared with those with sterile inflammation. These expression differences occur before phenotypic-based diagnosis of clinical sepsis.

Analysis of transcriptional profiles and functional clustering of global cerebellar gene expression in PCD3J mice

The Purkinje cell degeneration (PCD) mutant mouse is characterized by a degeneration of cerebellar Purkinje cells and progressive ataxia. To identify the molecular mechanisms that lead to the death of Purkinje neurons in PCD mice, we used Affymetrix microarray technology to compare cerebellar gene expression profiles in pcd3J mutant mice 14 days of age (prior to Purkinje cell loss) to unaffected littermates. Microarray analysis, Ingenuity Pathway Analysis (IPA) and expression analysis systematic explorer (EASE) software were used to identify biological and molecular pathways implicated in the progression of Purkinje cell degeneration. IPA analysis indicated that mutant pcd3J mice showed dysregulation of specific processes that may lead to Purkinje cell death, including several molecules known to control neuronal apoptosis such as Bad, CDK5 and PTEN. These findings demonstrate the usefulness of these powerful microarray analysis tools and have important implications for understanding the mechanisms of selective neuronal death and for developing therapeutic strategies to treat neurodegenerative disorders.