Biomphalaria glabrata transcriptome: cDNA microarray profiling identifies resistant- and susceptible-specific gene expression in haemocytes from snail strains exposed to Schistosoma mansoni

PubMed Central

Lockyer, Anne E; Spinks, Jenny; Kane, Richard A; Hoffmann, Karl F; Fitzpatrick, Jennifer M; Rollinson, David; Noble, Leslie R; Jones, Catherine S

2008-01-01

Background Biomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. B. glabrata/S. mansoni provides a useful model system for investigating the intimate interactions between host and parasite. Examining differential gene expression between S. mansoni-exposed schistosome-resistant and susceptible snail lines will identify genes and pathways that may be involved in snail defences. Results We have developed a 2053 element cDNA microarray for B. glabrata containing clones from ORESTES (Open Reading frame ESTs) libraries, suppression subtractive hybridization (SSH) libraries and clones identified in previous expression studies. Snail haemocyte RNA, extracted from parasite-challenged resistant and susceptible snails, 2 to 24 h post-exposure to S. mansoni, was hybridized to the custom made cDNA microarray and 98 differentially expressed genes or gene clusters were identified, 94 resistant-associated and 4 susceptible-associated. Quantitative PCR analysis verified the cDNA microarray results for representative transcripts. Differentially expressed genes were annotated and clustered using gene ontology (GO) terminology and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis. 61% of the identified differentially expressed genes have no known function including the 4 susceptible strain-specific transcripts. Resistant strain-specific expression of genes implicated in innate immunity of invertebrates was identified, including hydrolytic enzymes such as cathepsin L, a cysteine proteinase involved in lysis of phagocytosed particles; metabolic enzymes such as ornithine decarboxylase, the rate-limiting enzyme in the production of polyamines, important in inflammation and infection processes, as well as scavenging damaging free radicals produced during production of reactive oxygen species; stress response genes such as HSP70; proteins involved in signalling, such as importin 7 and copine 1, cytoplasmic intermediate filament (IF) protein and transcription enzymes such as elongation factor 1α and EF-2. Conclusion Production of the first cDNA microarray for profiling gene expression in B. glabrata provides a foundation for expanding our understanding of pathways and genes involved in the snail internal defence system (IDS). We demonstrate resistant strain-specific expression of genes potentially associated with the snail IDS, ranging from signalling and inflammation responses through to lysis of proteinacous products (encapsulated sporocysts or phagocytosed parasite components) and processing/degradation of these targeted products by ubiquitination. PMID:19114004

Biomphalaria glabrata transcriptome: cDNA microarray profiling identifies resistant- and susceptible-specific gene expression in haemocytes from snail strains exposed to Schistosoma mansoni.

PubMed

Lockyer, Anne E; Spinks, Jenny; Kane, Richard A; Hoffmann, Karl F; Fitzpatrick, Jennifer M; Rollinson, David; Noble, Leslie R; Jones, Catherine S

2008-12-29

Biomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. B. glabrata/S. mansoni provides a useful model system for investigating the intimate interactions between host and parasite. Examining differential gene expression between S. mansoni-exposed schistosome-resistant and susceptible snail lines will identify genes and pathways that may be involved in snail defences. We have developed a 2053 element cDNA microarray for B. glabrata containing clones from ORESTES (Open Reading frame ESTs) libraries, suppression subtractive hybridization (SSH) libraries and clones identified in previous expression studies. Snail haemocyte RNA, extracted from parasite-challenged resistant and susceptible snails, 2 to 24 h post-exposure to S. mansoni, was hybridized to the custom made cDNA microarray and 98 differentially expressed genes or gene clusters were identified, 94 resistant-associated and 4 susceptible-associated. Quantitative PCR analysis verified the cDNA microarray results for representative transcripts. Differentially expressed genes were annotated and clustered using gene ontology (GO) terminology and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis. 61% of the identified differentially expressed genes have no known function including the 4 susceptible strain-specific transcripts. Resistant strain-specific expression of genes implicated in innate immunity of invertebrates was identified, including hydrolytic enzymes such as cathepsin L, a cysteine proteinase involved in lysis of phagocytosed particles; metabolic enzymes such as ornithine decarboxylase, the rate-limiting enzyme in the production of polyamines, important in inflammation and infection processes, as well as scavenging damaging free radicals produced during production of reactive oxygen species; stress response genes such as HSP70; proteins involved in signalling, such as importin 7 and copine 1, cytoplasmic intermediate filament (IF) protein and transcription enzymes such as elongation factor 1alpha and EF-2. Production of the first cDNA microarray for profiling gene expression in B. glabrata provides a foundation for expanding our understanding of pathways and genes involved in the snail internal defence system (IDS). We demonstrate resistant strain-specific expression of genes potentially associated with the snail IDS, ranging from signalling and inflammation responses through to lysis of proteinacous products (encapsulated sporocysts or phagocytosed parasite components) and processing/degradation of these targeted products by ubiquitination.

Meta-analysis of pathway enrichment: combining independent and dependent omics data sets.

PubMed

Kaever, Alexander; Landesfeind, Manuel; Feussner, Kirstin; Morgenstern, Burkhard; Feussner, Ivo; Meinicke, Peter

2014-01-01

A major challenge in current systems biology is the combination and integrative analysis of large data sets obtained from different high-throughput omics platforms, such as mass spectrometry based Metabolomics and Proteomics or DNA microarray or RNA-seq-based Transcriptomics. Especially in the case of non-targeted Metabolomics experiments, where it is often impossible to unambiguously map ion features from mass spectrometry analysis to metabolites, the integration of more reliable omics technologies is highly desirable. A popular method for the knowledge-based interpretation of single data sets is the (Gene) Set Enrichment Analysis. In order to combine the results from different analyses, we introduce a methodical framework for the meta-analysis of p-values obtained from Pathway Enrichment Analysis (Set Enrichment Analysis based on pathways) of multiple dependent or independent data sets from different omics platforms. For dependent data sets, e.g. obtained from the same biological samples, the framework utilizes a covariance estimation procedure based on the nonsignificant pathways in single data set enrichment analysis. The framework is evaluated and applied in the joint analysis of Metabolomics mass spectrometry and Transcriptomics DNA microarray data in the context of plant wounding. In extensive studies of simulated data set dependence, the introduced correlation could be fully reconstructed by means of the covariance estimation based on pathway enrichment. By restricting the range of p-values of pathways considered in the estimation, the overestimation of correlation, which is introduced by the significant pathways, could be reduced. When applying the proposed methods to the real data sets, the meta-analysis was shown not only to be a powerful tool to investigate the correlation between different data sets and summarize the results of multiple analyses but also to distinguish experiment-specific key pathways.

CLIC, a tool for expanding biological pathways based on co-expression across thousands of datasets

PubMed Central

Li, Yang; Liu, Jun S.; Mootha, Vamsi K.

2017-01-01

In recent years, there has been a huge rise in the number of publicly available transcriptional profiling datasets. These massive compendia comprise billions of measurements and provide a special opportunity to predict the function of unstudied genes based on co-expression to well-studied pathways. Such analyses can be very challenging, however, since biological pathways are modular and may exhibit co-expression only in specific contexts. To overcome these challenges we introduce CLIC, CLustering by Inferred Co-expression. CLIC accepts as input a pathway consisting of two or more genes. It then uses a Bayesian partition model to simultaneously partition the input gene set into coherent co-expressed modules (CEMs), while assigning the posterior probability for each dataset in support of each CEM. CLIC then expands each CEM by scanning the transcriptome for additional co-expressed genes, quantified by an integrated log-likelihood ratio (LLR) score weighted for each dataset. As a byproduct, CLIC automatically learns the conditions (datasets) within which a CEM is operative. We implemented CLIC using a compendium of 1774 mouse microarray datasets (28628 microarrays) or 1887 human microarray datasets (45158 microarrays). CLIC analysis reveals that of 910 canonical biological pathways, 30% consist of strongly co-expressed gene modules for which new members are predicted. For example, CLIC predicts a functional connection between protein C7orf55 (FMC1) and the mitochondrial ATP synthase complex that we have experimentally validated. CLIC is freely available at www.gene-clic.org. We anticipate that CLIC will be valuable both for revealing new components of biological pathways as well as the conditions in which they are active. PMID:28719601

Microarray analysis of potential genes in the pathogenesis of recurrent oral ulcer.

PubMed

Han, Jingying; He, Zhiwei; Li, Kun; Hou, Lu

2015-01-01

Recurrent oral ulcer seriously threatens patients' daily life and health. This study investigated potential genes and pathways that participate in the pathogenesis of recurrent oral ulcer by high throughput bioinformatic analysis. RT-PCR and Western blot were applied to further verify screened interleukins effect. Recurrent oral ulcer related genes were collected from websites and papers, and further found out from Human Genome 280 6.0 microarray data. Each pathway of recurrent oral ulcer related genes were got through chip hybridization. RT-PCR was applied to test four recurrent oral ulcer related genes to verify the microarray data. Data transformation, scatter plot, clustering analysis, and expression pattern analysis were used to analyze recurrent oral ulcer related gene expression changes. Recurrent oral ulcer gene microarray was successfully established. Microarray showed that 551 genes involved in recurrent oral ulcer activity and 196 genes were recurrent oral ulcer related genes. Of them, 76 genes up-regulated, 62 genes down-regulated, and 58 genes up-/down-regulated. Total expression level up-regulated 752 times (60%) and down-regulated 485 times (40%). IL-2 plays an important role in the occurrence, development and recurrence of recurrent oral ulcer on the mRNA and protein levels. Gene microarray can be used to analyze potential genes and pathways in recurrent oral ulcer. IL-2 may be involved in the pathogenesis of recurrent oral ulcer.

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data.

PubMed

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R; Del Río-Navarro, Blanca E; Mendoza-Vargas, Alfredo; Sánchez, Filiberto; Ochoa-Leyva, Adrian

2017-01-01

In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6-10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments.

Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples.

PubMed

Wimmer, Isabella; Tröscher, Anna R; Brunner, Florian; Rubino, Stephen J; Bien, Christian G; Weiner, Howard L; Lassmann, Hans; Bauer, Jan

2018-04-20

Formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources commonly used in pathology. However, formalin fixation modifies nucleic acids challenging the isolation of high-quality RNA for genetic profiling. Here, we assessed feasibility and reliability of microarray studies analysing transcriptome data from fresh, fresh-frozen (FF) and FFPE tissues. We show that reproducible microarray data can be generated from only 2 ng FFPE-derived RNA. For RNA quality assessment, fragment size distribution (DV200) and qPCR proved most suitable. During RNA isolation, extending tissue lysis time to 10 hours reduced high-molecular-weight species, while additional incubation at 70 °C markedly increased RNA yields. Since FF- and FFPE-derived microarrays constitute different data entities, we used indirect measures to investigate gene signal variation and relative gene expression. Whole-genome analyses revealed high concordance rates, while reviewing on single-genes basis showed higher data variation in FFPE than FF arrays. Using an experimental model, gene set enrichment analysis (GSEA) of FFPE-derived microarrays and fresh tissue-derived RNA-Seq datasets yielded similarly affected pathways confirming the applicability of FFPE tissue in global gene expression analysis. Our study provides a workflow comprising RNA isolation, quality assessment and microarray profiling using minimal RNA input, thus enabling hypothesis-generating pathway analyses from limited amounts of precious, pathologically significant FFPE tissues.

In silico Pathway Activation Network Decomposition Analysis (iPANDA) as a method for biomarker development.

PubMed

Ozerov, Ivan V; Lezhnina, Ksenia V; Izumchenko, Evgeny; Artemov, Artem V; Medintsev, Sergey; Vanhaelen, Quentin; Aliper, Alexander; Vijg, Jan; Osipov, Andreyan N; Labat, Ivan; West, Michael D; Buzdin, Anton; Cantor, Charles R; Nikolsky, Yuri; Borisov, Nikolay; Irincheeva, Irina; Khokhlovich, Edward; Sidransky, David; Camargo, Miguel Luiz; Zhavoronkov, Alex

2016-11-16

Signalling pathway activation analysis is a powerful approach for extracting biologically relevant features from large-scale transcriptomic and proteomic data. However, modern pathway-based methods often fail to provide stable pathway signatures of a specific phenotype or reliable disease biomarkers. In the present study, we introduce the in silico Pathway Activation Network Decomposition Analysis (iPANDA) as a scalable robust method for biomarker identification using gene expression data. The iPANDA method combines precalculated gene coexpression data with gene importance factors based on the degree of differential gene expression and pathway topology decomposition for obtaining pathway activation scores. Using Microarray Analysis Quality Control (MAQC) data sets and pretreatment data on Taxol-based neoadjuvant breast cancer therapy from multiple sources, we demonstrate that iPANDA provides significant noise reduction in transcriptomic data and identifies highly robust sets of biologically relevant pathway signatures. We successfully apply iPANDA for stratifying breast cancer patients according to their sensitivity to neoadjuvant therapy.

In silico Pathway Activation Network Decomposition Analysis (iPANDA) as a method for biomarker development

PubMed Central

Ozerov, Ivan V.; Lezhnina, Ksenia V.; Izumchenko, Evgeny; Artemov, Artem V.; Medintsev, Sergey; Vanhaelen, Quentin; Aliper, Alexander; Vijg, Jan; Osipov, Andreyan N.; Labat, Ivan; West, Michael D.; Buzdin, Anton; Cantor, Charles R.; Nikolsky, Yuri; Borisov, Nikolay; Irincheeva, Irina; Khokhlovich, Edward; Sidransky, David; Camargo, Miguel Luiz; Zhavoronkov, Alex

2016-01-01

Signalling pathway activation analysis is a powerful approach for extracting biologically relevant features from large-scale transcriptomic and proteomic data. However, modern pathway-based methods often fail to provide stable pathway signatures of a specific phenotype or reliable disease biomarkers. In the present study, we introduce the in silico Pathway Activation Network Decomposition Analysis (iPANDA) as a scalable robust method for biomarker identification using gene expression data. The iPANDA method combines precalculated gene coexpression data with gene importance factors based on the degree of differential gene expression and pathway topology decomposition for obtaining pathway activation scores. Using Microarray Analysis Quality Control (MAQC) data sets and pretreatment data on Taxol-based neoadjuvant breast cancer therapy from multiple sources, we demonstrate that iPANDA provides significant noise reduction in transcriptomic data and identifies highly robust sets of biologically relevant pathway signatures. We successfully apply iPANDA for stratifying breast cancer patients according to their sensitivity to neoadjuvant therapy. PMID:27848968

Microarray Analysis of Long Noncoding RNAs in Female Diabetic Peripheral Neuropathy Patients.

PubMed

Luo, Lin; Ji, Lin-Dan; Cai, Jiang-Jia; Feng, Mei; Zhou, Mi; Hu, Su-Pei; Xu, Jin; Zhou, Wen-Hua

2018-01-01

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus (DM). Because of its controversial pathogenesis, DPN is still not diagnosed or managed properly in most patients. In this study, human lncRNA microarrays were used to identify the differentially expressed lncRNAs in DM and DPN patients, and some of the discovered lncRNAs were further validated in additional 78 samples by quantitative realtime PCR (qRT-PCR). The microarray analysis identified 446 and 1327 differentially expressed lncRNAs in DM and DPN, respectively. The KEGG pathway analysis further revealed that the differentially expressed lncRNA-coexpressed mRNAs between DPN and DM groups were significantly enriched in the MAPK signaling pathway. The lncRNA/mRNA coexpression network indicated that BDNF and TRAF2 correlated with 6 lncRNAs. The qRT-PCR confirmed the initial microarray results. These findings demonstrated that the interplay between lncRNAs and mRNA may be involved in the pathogenesis of DPN, especially the neurotrophin-MAPK signaling pathway, thus providing relevant information for future studies. © 2018 The Author(s). Published by S. Karger AG, Basel.

Endothelial Inflammatory Transcriptional Responses Induced by Plasma Following Inhalation of Diesel Emissions

PubMed Central

Schisler, Jonathan C.; Ronnebaum, Sarah M.; Madden, Michael; Channell, Meghan M.; Campen, Matthew J.; Willis, Monte S.

2016-01-01

Background Air pollution, especially emissions derived from traffic sources, is associated with adverse cardiovascular outcomes. However, it remains unclear how inhaled factors drive extrapulmonary pathology. Objectives Previously, we found that canonical inflammatory response transcripts were elevated in cultured endothelial cells treated with plasma obtained after exposure compared with pre-exposure samples or filtered air (sham) exposures. While the findings confirmed the presence of bioactive factor(s) in the plasma after diesel inhalation, we wanted to better examine the complete genomic response to investigate 1) major responsive transcripts and 2) collected response pathways and ontogeny that may help to refine this method and inform the pathogenesis. Methods We assayed endothelial RNA with gene expression microarrays, examining the responses of cultured endothelial cells to plasma obtained from 6 healthy human subjects exposed to 100 μg/m3 diesel exhaust or filtered air for 2 h on separate occasions. In addition to pre-exposure baseline samples, we investigated samples obtained immediately-post and 24h-post exposure. Results Microarray analysis of the coronary artery endothelial cells challenged with plasma identified 855 probes that changed over time following diesel exhaust exposure. Over-representation analysis identified inflammatory cytokine pathways were upregulated both at the 2 and 24 h condition. Novel pathways related to FOX transcription factors and secreted extracellular factors were also identified in the microarray analysis. Conclusions These outcomes are consistent with our recent findings that plasma contains bioactive and inflammatory factors following pollutant inhalation. The specific study design implicates a novel pathway related to inflammatory blood borne components that may drive the extrapulmonary toxicity of ambient air pollutants. PMID:25942053

Reliable pre-eclampsia pathways based on multiple independent microarray data sets.

PubMed

Kawasaki, Kaoru; Kondoh, Eiji; Chigusa, Yoshitsugu; Ujita, Mari; Murakami, Ryusuke; Mogami, Haruta; Brown, J B; Okuno, Yasushi; Konishi, Ikuo

2015-02-01

Pre-eclampsia is a multifactorial disorder characterized by heterogeneous clinical manifestations. Gene expression profiling of preeclamptic placenta have provided different and even opposite results, partly due to data compromised by various experimental artefacts. Here we aimed to identify reliable pre-eclampsia-specific pathways using multiple independent microarray data sets. Gene expression data of control and preeclamptic placentas were obtained from Gene Expression Omnibus. Single-sample gene-set enrichment analysis was performed to generate gene-set activation scores of 9707 pathways obtained from the Molecular Signatures Database. Candidate pathways were identified by t-test-based screening using data sets, GSE10588, GSE14722 and GSE25906. Additionally, recursive feature elimination was applied to arrive at a further reduced set of pathways. To assess the validity of the pre-eclampsia pathways, a statistically-validated protocol was executed using five data sets including two independent other validation data sets, GSE30186, GSE44711. Quantitative real-time PCR was performed for genes in a panel of potential pre-eclampsia pathways using placentas of 20 women with normal or severe preeclamptic singleton pregnancies (n = 10, respectively). A panel of ten pathways were found to discriminate women with pre-eclampsia from controls with high accuracy. Among these were pathways not previously associated with pre-eclampsia, such as the GABA receptor pathway, as well as pathways that have already been linked to pre-eclampsia, such as the glutathione and CDKN1C pathways. mRNA expression of GABRA3 (GABA receptor pathway), GCLC and GCLM (glutathione metabolic pathway), and CDKN1C was significantly reduced in the preeclamptic placentas. In conclusion, ten accurate and reliable pre-eclampsia pathways were identified based on multiple independent microarray data sets. A pathway-based classification may be a worthwhile approach to elucidate the pathogenesis of pre-eclampsia. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Microarray RNA expression analysis of cerebral white matter lesions reveals changes in multiple functional pathways.

PubMed

Simpson, Julie E; Hosny, Ola; Wharton, Stephen B; Heath, Paul R; Holden, Hazel; Fernando, Malee S; Matthews, Fiona; Forster, Gill; O'Brien, John T; Barber, Robert; Kalaria, Raj N; Brayne, Carol; Shaw, Pamela J; Lewis, Claire E; Ince, Paul G

2009-02-01

White matter lesions (WML) in brain aging are linked to dementia and depression. Ischemia contributes to their pathogenesis but other mechanisms may contribute. We used RNA microarray analysis with functional pathway grouping as an unbiased approach to investigate evidence for additional pathogenetic mechanisms. WML were identified by MRI and pathology in brains donated to the Medical Research Council Cognitive Function and Ageing Study Cognitive Function and Aging Study. RNA was extracted to compare WML with nonlesional white matter samples from cases with lesions (WM[L]), and from cases with no lesions (WM[C]) using RNA microarray and pathway analysis. Functional pathways were validated for selected genes by quantitative real-time polymerase chain reaction and immunocytochemistry. We identified 8 major pathways in which multiple genes showed altered RNA transcription (immune regulation, cell cycle, apoptosis, proteolysis, ion transport, cell structure, electron transport, metabolism) among 502 genes that were differentially expressed in WML compared to WM[C]. In WM[L], 409 genes were altered involving the same pathways. Genes selected to validate this microarray data all showed the expected changes in RNA levels and immunohistochemical expression of protein. WML represent areas with a complex molecular phenotype. From this and previous evidence, WML may arise through tissue ischemia but may also reflect the contribution of additional factors like blood-brain barrier dysfunction. Differential expression of genes in WM[L] compared to WM[C] indicate a "field effect" in the seemingly normal surrounding white matter.

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data

PubMed Central

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R.; del Río-Navarro, Blanca E.; Mendoza-Vargas, Alfredo; Sánchez, Filiberto

2017-01-01

Background In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. Methods We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6–10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). Results From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Discussion Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments. PMID:29230367

The pathogenesis shared between abdominal aortic aneurysms and intracranial aneurysms: a microarray analysis.

PubMed

Wang, Wen; Li, Hao; Zhao, Zheng; Wang, Haoyuan; Zhang, Dong; Zhang, Yan; Lan, Qing; Wang, Jiangfei; Cao, Yong; Zhao, Jizong

2018-04-01

Abdominal aortic aneurysms (AAAs) and intracranial saccular aneurysms (IAs) are the most common types of aneurysms. This study was to investigate the common pathogenesis shared between these two kinds of aneurysms. We collected 12 IAs samples and 12 control arteries from the Beijing Tiantan Hospital and performed microarray analysis. In addition, we utilized the microarray datasets of IAs and AAAs from the Gene Expression Omnibus (GEO), in combination with our microarray results, to generate messenger RNA expression profiles for both AAAs and IAs in our study. Functional exploration and protein-protein interaction (PPI) analysis were performed. A total of 727 common genes were differentially expressed (404 was upregulated; 323 was downregulated) for both AAAs and IAs. The GO and pathway analyses showed that the common dysregulated genes were mainly enriched in vascular smooth muscle contraction, muscle contraction, immune response, defense response, cell activation, IL-6 signaling and chemokine signaling pathways, etc. The further protein-protein analysis identified 35 hub nodes, including TNF, IL6, MAPK13, and CCL5. These hub node genes were enriched in inflammatory response, positive regulation of IL-6 production, chemokine signaling pathway, and T/B cell receptor signaling pathway. Our study will gain new insight into the molecular mechanisms for the pathogenesis of both types of aneurysms and provide new therapeutic targets for the patients harboring AAAs and IAs.

New Statistics for Testing Differential Expression of Pathways from Microarray Data

NASA Astrophysics Data System (ADS)

Siu, Hoicheong; Dong, Hua; Jin, Li; Xiong, Momiao

Exploring biological meaning from microarray data is very important but remains a great challenge. Here, we developed three new statistics: linear combination test, quadratic test and de-correlation test to identify differentially expressed pathways from gene expression profile. We apply our statistics to two rheumatoid arthritis datasets. Notably, our results reveal three significant pathways and 275 genes in common in two datasets. The pathways we found are meaningful to uncover the disease mechanisms of rheumatoid arthritis, which implies that our statistics are a powerful tool in functional analysis of gene expression data.

Pathway analysis of high-throughput biological data within a Bayesian network framework.

PubMed

Isci, Senol; Ozturk, Cengizhan; Jones, Jon; Otu, Hasan H

2011-06-15

Most current approaches to high-throughput biological data (HTBD) analysis either perform individual gene/protein analysis or, gene/protein set enrichment analysis for a list of biologically relevant molecules. Bayesian Networks (BNs) capture linear and non-linear interactions, handle stochastic events accounting for noise, and focus on local interactions, which can be related to causal inference. Here, we describe for the first time an algorithm that models biological pathways as BNs and identifies pathways that best explain given HTBD by scoring fitness of each network. Proposed method takes into account the connectivity and relatedness between nodes of the pathway through factoring pathway topology in its model. Our simulations using synthetic data demonstrated robustness of our approach. We tested proposed method, Bayesian Pathway Analysis (BPA), on human microarray data regarding renal cell carcinoma (RCC) and compared our results with gene set enrichment analysis. BPA was able to find broader and more specific pathways related to RCC. Accompanying BPA software (BPAS) package is freely available for academic use at http://bumil.boun.edu.tr/bpa.

Bioinformatics analysis of differentially expressed gene profiles associated with systemic lupus erythematosus

PubMed Central

Wu, Chengjiang; Zhao, Yangjing; Lin, Yu; Yang, Xinxin; Yan, Meina; Min, Yujiao; Pan, Zihui; Xia, Sheng; Shao, Qixiang

2018-01-01

DNA microarray and high-throughput sequencing have been widely used to identify the differentially expressed genes (DEGs) in systemic lupus erythematosus (SLE). However, the big data from gene microarrays are also challenging to work with in terms of analysis and processing. The presents study combined data from the microarray expression profile (GSE65391) and bioinformatics analysis to identify the key genes and cellular pathways in SLE. Gene ontology (GO) and cellular pathway enrichment analyses of DEGs were performed to investigate significantly enriched pathways. A protein-protein interaction network was constructed to determine the key genes in the occurrence and development of SLE. A total of 310 DEGs were identified in SLE, including 193 upregulated genes and 117 downregulated genes. GO analysis revealed that the most significant biological process of DEGs was immune system process. Kyoto Encyclopedia of Genes and Genome pathway analysis showed that these DEGs were enriched in signaling pathways associated with the immune system, including the RIG-I-like receptor signaling pathway, intestinal immune network for IgA production, antigen processing and presentation and the toll-like receptor signaling pathway. The current study screened the top 10 genes with higher degrees as hub genes, which included 2′-5′-oligoadenylate synthetase 1, MX dynamin like GTPase 2, interferon induced protein with tetratricopeptide repeats 1, interferon regulatory factor 7, interferon induced with helicase C domain 1, signal transducer and activator of transcription 1, ISG15 ubiquitin-like modifier, DExD/H-box helicase 58, interferon induced protein with tetratricopeptide repeats 3 and 2′-5′-oligoadenylate synthetase 2. Module analysis revealed that these hub genes were also involved in the RIG-I-like receptor signaling, cytosolic DNA-sensing, toll-like receptor signaling and ribosome biogenesis pathways. In addition, these hub genes, from different probe sets, exhibited significant co-expressed tendency in multi-experiment microarray datasets (P<0.01). In conclusion, these key genes and cellular pathways may improve the current understanding of the underlying mechanism of development of SLE. These key genes may be potential biomarkers of diagnosis, therapy and prognosis for SLE. PMID:29257335

Methods to study legionella transcriptome in vitro and in vivo.

PubMed

Faucher, Sebastien P; Shuman, Howard A

2013-01-01

The study of transcriptome responses can provide insight into the regulatory pathways and genetic factors that contribute to a specific phenotype. For bacterial pathogens, it can identify putative new virulence systems and shed light on the mechanisms underlying the regulation of virulence factors. Microarrays have been previously used to study gene regulation in Legionella pneumophila. In the past few years a sharp reduction of the costs associated with microarray experiments together with the availability of relatively inexpensive custom-designed commercial microarrays has made microarray technology an accessible tool for the majority of researchers. Here we describe the methodologies to conduct microarray experiments from in vitro and in vivo samples.

Expression profiling and pathway analysis of Krüppel-like factor 4 in mouse embryonic fibroblasts

PubMed Central

Hagos, Engda G; Ghaleb, Amr M; Kumar, Amrita; Neish, Andrew S; Yang, Vincent W

2011-01-01

Background: Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4. Methods: Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels. Results: One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data. Conclusions: These data are not only consistent with previous functional studies of KLF4's role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4's functions. PMID:21892412

Multi-Tissue Computational Modeling Analyzes Pathophysiology of Type 2 Diabetes in MKR Mice

PubMed Central

Kumar, Amit; Harrelson, Thomas; Lewis, Nathan E.; Gallagher, Emily J.; LeRoith, Derek; Shiloach, Joseph; Betenbaugh, Michael J.

2014-01-01

Computational models using metabolic reconstructions for in silico simulation of metabolic disorders such as type 2 diabetes mellitus (T2DM) can provide a better understanding of disease pathophysiology and avoid high experimentation costs. There is a limited amount of computational work, using metabolic reconstructions, performed in this field for the better understanding of T2DM. In this study, a new algorithm for generating tissue-specific metabolic models is presented, along with the resulting multi-confidence level (MCL) multi-tissue model. The effect of T2DM on liver, muscle, and fat in MKR mice was first studied by microarray analysis and subsequently the changes in gene expression of frank T2DM MKR mice versus healthy mice were applied to the multi-tissue model to test the effect. Using the first multi-tissue genome-scale model of all metabolic pathways in T2DM, we found out that branched-chain amino acids' degradation and fatty acids oxidation pathway is downregulated in T2DM MKR mice. Microarray data showed low expression of genes in MKR mice versus healthy mice in the degradation of branched-chain amino acids and fatty-acid oxidation pathways. In addition, the flux balance analysis using the MCL multi-tissue model showed that the degradation pathways of branched-chain amino acid and fatty acid oxidation were significantly downregulated in MKR mice versus healthy mice. Validation of the model was performed using data derived from the literature regarding T2DM. Microarray data was used in conjunction with the model to predict fluxes of various other metabolic pathways in the T2DM mouse model and alterations in a number of pathways were detected. The Type 2 Diabetes MCL multi-tissue model may explain the high level of branched-chain amino acids and free fatty acids in plasma of Type 2 Diabetic subjects from a metabolic fluxes perspective. PMID:25029527

Transcript Profiling Identifies Dynamic Gene Expression Patterns and an Important Role for Nrf2/Keap1 Pathway in the Developing Mouse Esophagus

PubMed Central

Li, Haiyan; Hu, Yuhui; Tevebaugh, Whitney; Yamamoto, Masayuki; Que, Jianwen; Chen, Xiaoxin

2012-01-01

Background and Aims Morphological changes during human and mouse esophageal development have been well characterized. However, changes at the molecular level in the course of esophageal morphogenesis remain unclear. This study aims to globally profile critical genes and signaling pathways during the development of mouse esophagus. By using microarray analysis this study also aims to determine how the Nrf2/Keap1 pathway regulates the morphogenesis of the esophageal epithelium. Methods Gene expression microarrays were used to survey gene expression in the esophagus at three critical phases: specification, metaplasia and maturation. The esophagi were isolated from wild-type, Nrf2−/−, Keap1−/−, or Nrf2−/−Keap1−/− embryos or young adult mice. Array data were statistically analyzed for differentially expressed genes and pathways. Histochemical and immunohistochemical staining were used to verify potential involvement of the Wnt pathway, Pparβ/δ and the PI3K/Akt pathway in the development of esophageal epithelium. Results Dynamic gene expression patterns accompanied the morphological changes of the developing esophagus at critical phases. Particularly, the Nrf2/Keap1 pathway had a baseline activity in the metaplasia phase and was further activated in the maturation phase. The Wnt pathway was active early and became inactive later in the metaplasia phase. In addition, Keap1−/− mice showed increased expression of Nrf2 downstream targets and genes involved in keratinization. Microarray and immunostaining data also suggested that esophageal hyperkeratosis in the Keap1−/− mice was due to activation of Pparβ/δ and the PI3K/Akt pathway. Conclusions Morphological changes of the esophageal epithelium are associated with dynamic changes in gene expression. Nrf2/Keap1 pathway activity is required for maturation of mouse esophageal epithelium. PMID:22567161

Direct labeling of serum proteins by fluorescent dye for antibody microarray.

PubMed

Klimushina, M V; Gumanova, N G; Metelskaya, V A

2017-05-06

Analysis of serum proteome by antibody microarray is used to identify novel biomarkers and to study signaling pathways including protein phosphorylation and protein-protein interactions. Labeling of serum proteins is important for optimal performance of the antibody microarray. Proper choice of fluorescent label and optimal concentration of protein loaded on the microarray ensure good quality of imaging that can be reliably scanned and processed by the software. We have optimized direct serum protein labeling using fluorescent dye Arrayit Green 540 (Arrayit Corporation, USA) for antibody microarray. Optimized procedure produces high quality images that can be readily scanned and used for statistical analysis of protein composition of the serum. Copyright © 2017 Elsevier Inc. All rights reserved.

Global Microarray Analysis of Carbohydrate Use in Alkaliphilic Hemicellulolytic Bacterium Bacillus sp. N16-5

PubMed Central

Song, Yajian; Xue, Yanfen; Ma, Yanhe

2013-01-01

The alkaliphilic hemicellulolytic bacterium Bacillus sp. N16-5 has a broad substrate spectrum and exhibits the capacity to utilize complex carbohydrates such as galactomannan, xylan, and pectin. In the monosaccharide mixture, sequential utilization by Bacillus sp. N16-5 was observed. Glucose appeared to be its preferential monosaccharide, followed by fructose, mannose, arabinose, xylose, and galactose. Global transcription profiles of the strain were determined separately for growth on six monosaccharides (glucose, fructose, mannose, galactose, arabinose, and xylose) and four polysaccharides (galactomannan, xylan, pectin, and sodium carboxymethylcellulose) using one-color microarrays. Numerous genes potentially related to polysaccharide degradation, sugar transport, and monosaccharide metabolism were found to respond to a specific substrate. Putative gene clusters for different carbohydrates were identified according to transcriptional patterns and genome annotation. Identification and analysis of these gene clusters contributed to pathway reconstruction for carbohydrate utilization in Bacillus sp. N16-5. Several genes encoding putative sugar transporters were highly expressed during growth on specific sugars, suggesting their functional roles. Two phosphoenolpyruvate-dependent phosphotransferase systems were identified as candidate transporters for mannose and fructose, and a major facilitator superfamily transporter was identified as a candidate transporter for arabinose and xylose. Five carbohydrate uptake transporter 1 family ATP-binding cassette transporters were predicted to participate in the uptake of hemicellulose and pectin degradation products. Collectively, microarray data improved the pathway reconstruction involved in carbohydrate utilization of Bacillus sp. N16-5 and revealed that the organism precisely regulates gene transcription in response to fluctuations in energy resources. PMID:23326578

Identifying molecular features for prostate cancer with Gleason 7 based on microarray gene expression profiles.

PubMed

Bălăcescu, Loredana; Bălăcescu, O; Crişan, N; Fetica, B; Petruţ, B; Bungărdean, Cătălina; Rus, Meda; Tudoran, Oana; Meurice, G; Irimie, Al; Dragoş, N; Berindan-Neagoe, Ioana

2011-01-01

Prostate cancer represents the first leading cause of cancer among western male population, with different clinical behavior ranging from indolent to metastatic disease. Although many molecules and deregulated pathways are known, the molecular mechanisms involved in the development of prostate cancer are not fully understood. The aim of this study was to explore the molecular variation underlying the prostate cancer, based on microarray analysis and bioinformatics approaches. Normal and prostate cancer tissues were collected by macrodissection from prostatectomy pieces. All prostate cancer specimens used in our study were Gleason score 7. Gene expression microarray (Agilent Technologies) was used for Whole Human Genome evaluation. The bioinformatics and functional analysis were based on Limma and Ingenuity software. The microarray analysis identified 1119 differentially expressed genes between prostate cancer and normal prostate, which were up- or down-regulated at least 2-fold. P-values were adjusted for multiple testing using Benjamini-Hochberg method with a false discovery rate of 0.01. These genes were analyzed with Ingenuity Pathway Analysis software and were established 23 genetic networks. Our microarray results provide new information regarding the molecular networks in prostate cancer stratified as Gleason 7. These data highlighted gene expression profiles for better understanding of prostate cancer progression.

Time-series RNA-seq analysis package (TRAP) and its application to the analysis of rice, Oryza sativa L. ssp. Japonica, upon drought stress.

PubMed

Jo, Kyuri; Kwon, Hawk-Bin; Kim, Sun

2014-06-01

Measuring expression levels of genes at the whole genome level can be useful for many purposes, especially for revealing biological pathways underlying specific phenotype conditions. When gene expression is measured over a time period, we have opportunities to understand how organisms react to stress conditions over time. Thus many biologists routinely measure whole genome level gene expressions at multiple time points. However, there are several technical difficulties for analyzing such whole genome expression data. In addition, these days gene expression data is often measured by using RNA-sequencing rather than microarray technologies and then analysis of expression data is much more complicated since the analysis process should start with mapping short reads and produce differentially activated pathways and also possibly interactions among pathways. In addition, many useful tools for analyzing microarray gene expression data are not applicable for the RNA-seq data. Thus a comprehensive package for analyzing time series transcriptome data is much needed. In this article, we present a comprehensive package, Time-series RNA-seq Analysis Package (TRAP), integrating all necessary tasks such as mapping short reads, measuring gene expression levels, finding differentially expressed genes (DEGs), clustering and pathway analysis for time-series data in a single environment. In addition to implementing useful algorithms that are not available for RNA-seq data, we extended existing pathway analysis methods, ORA and SPIA, for time series analysis and estimates statistical values for combined dataset by an advanced metric. TRAP also produces visual summary of pathway interactions. Gene expression change labeling, a practical clustering method used in TRAP, enables more accurate interpretation of the data when combined with pathway analysis. We applied our methods on a real dataset for the analysis of rice (Oryza sativa L. Japonica nipponbare) upon drought stress. The result showed that TRAP was able to detect pathways more accurately than several existing methods. TRAP is available at http://biohealth.snu.ac.kr/software/TRAP/. Copyright © 2014 Elsevier Inc. All rights reserved.

Comprehensive transcriptome analysis unravels the existence of crucial genes regulating primary metabolism during adventitious root formation in Petunia hybrida.

PubMed

Ahkami, Amirhossein; Scholz, Uwe; Steuernagel, Burkhard; Strickert, Marc; Haensch, Klaus-Thomas; Druege, Uwe; Reinhardt, Didier; Nouri, Eva; von Wirén, Nicolaus; Franken, Philipp; Hajirezaei, Mohammad-Reza

2014-01-01

To identify specific genes determining the initiation and formation of adventitious roots (AR), a microarray-based transcriptome analysis in the stem base of the cuttings of Petunia hybrida (line W115) was conducted. A microarray carrying 24,816 unique, non-redundant annotated sequences was hybridized to probes derived from different stages of AR formation. After exclusion of wound-responsive and root-regulated genes, 1,354 of them were identified which were significantly and specifically induced during various phases of AR formation. Based on a recent physiological model distinguishing three metabolic phases in AR formation, the present paper focuses on the response of genes related to particular metabolic pathways. Key genes involved in primary carbohydrate metabolism such as those mediating apoplastic sucrose unloading were induced at the early sink establishment phase of AR formation. Transcriptome changes also pointed to a possible role of trehalose metabolism and SnRK1 (sucrose non-fermenting 1- related protein kinase) in sugar sensing during this early step of AR formation. Symplastic sucrose unloading and nucleotide biosynthesis were the major processes induced during the later recovery and maintenance phases. Moreover, transcripts involved in peroxisomal beta-oxidation were up-regulated during different phases of AR formation. In addition to metabolic pathways, the analysis revealed the activation of cell division at the two later phases and in particular the induction of G1-specific genes in the maintenance phase. Furthermore, results point towards a specific demand for certain mineral nutrients starting in the recovery phase.

Comprehensive Transcriptome Analysis Unravels the Existence of Crucial Genes Regulating Primary Metabolism during Adventitious Root Formation in Petunia hybrida

PubMed Central

Ahkami, Amirhossein; Scholz, Uwe; Steuernagel, Burkhard; Strickert, Marc; Haensch, Klaus-Thomas; Druege, Uwe; Reinhardt, Didier; Nouri, Eva; von Wirén, Nicolaus; Franken, Philipp; Hajirezaei, Mohammad-Reza

2014-01-01

To identify specific genes determining the initiation and formation of adventitious roots (AR), a microarray-based transcriptome analysis in the stem base of the cuttings of Petunia hybrida (line W115) was conducted. A microarray carrying 24,816 unique, non-redundant annotated sequences was hybridized to probes derived from different stages of AR formation. After exclusion of wound-responsive and root-regulated genes, 1,354 of them were identified which were significantly and specifically induced during various phases of AR formation. Based on a recent physiological model distinguishing three metabolic phases in AR formation, the present paper focuses on the response of genes related to particular metabolic pathways. Key genes involved in primary carbohydrate metabolism such as those mediating apoplastic sucrose unloading were induced at the early sink establishment phase of AR formation. Transcriptome changes also pointed to a possible role of trehalose metabolism and SnRK1 (sucrose non-fermenting 1- related protein kinase) in sugar sensing during this early step of AR formation. Symplastic sucrose unloading and nucleotide biosynthesis were the major processes induced during the later recovery and maintenance phases. Moreover, transcripts involved in peroxisomal beta-oxidation were up-regulated during different phases of AR formation. In addition to metabolic pathways, the analysis revealed the activation of cell division at the two later phases and in particular the induction of G1-specific genes in the maintenance phase. Furthermore, results point towards a specific demand for certain mineral nutrients starting in the recovery phase. PMID:24978694

Development of DNA Microarrays for Metabolic Pathway and Bioprocess Monitoring

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gregory Stephanopoulos

Transcriptional profiling experiments utilizing DNA microarrays to study the intracellular accumulation of PHB in Synechocystis has proved difficult in large part because strains that show significant differences in PHB which would justify global analysis of gene expression have not been isolated.

Characteristic Markers of the WNT Signaling Pathways Are Differentially Expressed in Osteoarthritic Cartilage

PubMed Central

Dehne, T.; Lindahl, A.; Brittberg, M.; Pruss, A.; Ringe, J.; Sittinger, M.; Karlsson, C.

2012-01-01

Objective: It is well known that expression of markers for WNT signaling is dysregulated in osteoarthritic (OA) bone. However, it is still not fully known if the expression of these markers also is affected in OA cartilage. The aim of this study was therefore to examine this issue. Methods: Human cartilage biopsies from OA and control donors were subjected to genome-wide oligonucleotide microarrays. Genes involved in WNT signaling were selected using the BioRetis database, KEGG pathway analysis was searched using DAVID software tools, and cluster analysis was performed using Genesis software. Results from the microarray analysis were verified using quantitative real-time PCR and immunohistochemistry. In order to study the impact of cytokines for the dysregulated WNT signaling, OA and control chondrocytes were stimulated with interleukin-1 and analyzed with real-time PCR for their expression of WNT-related genes. Results: Several WNT markers displayed a significantly altered expression in OA compared to normal cartilage. Interestingly, inhibitors of the canonical and planar cell polarity WNT signaling pathways displayed significantly increased expression in OA cartilage, while the Ca2+/WNT signaling pathway was activated. Both real-time PCR and immunohistochemistry verified the microarray results. Real-time PCR analysis demonstrated that interleukin-1 upregulated expression of important WNT markers. Conclusions: WNT signaling is significantly affected in OA cartilage. The result suggests that both the canonical and planar cell polarity WNT signaling pathways were partly inhibited while the Ca2+/WNT pathway was activated in OA cartilage. PMID:26069618

The application of DNA microarrays in gene expression analysis.

PubMed

van Hal, N L; Vorst, O; van Houwelingen, A M; Kok, E J; Peijnenburg, A; Aharoni, A; van Tunen, A J; Keijer, J

2000-03-31

DNA microarray technology is a new and powerful technology that will substantially increase the speed of molecular biological research. This paper gives a survey of DNA microarray technology and its use in gene expression studies. The technical aspects and their potential improvements are discussed. These comprise array manufacturing and design, array hybridisation, scanning, and data handling. Furthermore, it is discussed how DNA microarrays can be applied in the working fields of: safety, functionality and health of food and gene discovery and pathway engineering in plants.

Transcriptional Profiling of Caulobacter crescentus during Growth on Complex and Minimal Media

PubMed Central

Hottes, Alison K.; Meewan, Maliwan; Yang, Desiree; Arana, Naomi; Romero, Pedro; McAdams, Harley H.; Stephens, Craig

2004-01-01

Microarray analysis was used to examine gene expression in the freshwater oligotrophic bacterium Caulobacter crescentus during growth on three standard laboratory media, including peptone-yeast extract medium (PYE) and minimal salts medium with glucose or xylose as the carbon source. Nearly 400 genes (approximately 10% of the genome) varied significantly in expression between at least two of these media. The differentially expressed genes included many encoding transport systems, most notably diverse TonB-dependent outer membrane channels of unknown substrate specificity. Amino acid degradation pathways constituted the largest class of genes induced in PYE. In contrast, many of the genes upregulated in minimal media encoded enzymes for synthesis of amino acids, including incorporation of ammonia and sulfate into glutamate and cysteine. Glucose availability induced expression of genes encoding enzymes of the Entner-Doudoroff pathway, which was demonstrated here through mutational analysis to be essential in C. crescentus for growth on glucose. Xylose induced expression of genes encoding several hydrolytic exoenzymes as well as an operon that may encode a novel pathway for xylose catabolism. A conserved DNA motif upstream of many xylose-induced genes was identified and shown to confer xylose-specific expression. Xylose is an abundant component of xylan in plant cell walls, and the microarray data suggest that in addition to serving as a carbon source for growth of C. crescentus, this pentose may be interpreted as a signal to produce enzymes associated with plant polymer degradation. PMID:14973021

Comparative analysis of gene expression profiles of hip articular cartilage between non-traumatic necrosis and osteoarthritis.

PubMed

Wang, Wenyu; Liu, Yang; Hao, Jingcan; Zheng, Shuyu; Wen, Yan; Xiao, Xiao; He, Awen; Fan, Qianrui; Zhang, Feng; Liu, Ruiyu

2016-10-10

Hip cartilage destruction is consistently observed in the non-traumatic osteonecrosis of femoral head (NOFH) and accelerates its bone necrosis. The molecular mechanism underlying the cartilage damage of NOFH remains elusive. In this study, we conducted a systematically comparative study of gene expression profiles between NOFH and osteoarthritis (OA). Hip articular cartilage specimens were collected from 12 NOFH patients and 12 controls with traumatic femoral neck fracture for microarray (n=4) and quantitative real-time PCR validation experiments (n=8). Gene expression profiling of articular cartilage was performed using Agilent Human 4×44K Microarray chip. The accuracy of microarray experiment was further validated by qRT-PCR. Gene expression results of OA hip cartilage were derived from previously published study. Significance Analysis of Microarrays (SAM) software was applied for identifying differently expressed genes. Gene ontology (GO) and pathway enrichment analysis were conducted by Gene Set Enrichment Analysis software and DAVID tool, respectively. Totally, 27 differently expressed genes were identified for NOFH. Comparing the gene expression profiles of NOFH cartilage and OA cartilage detected 8 common differently expressed genes, including COL5A1, OGN, ANGPTL4, CRIP1, NFIL3, METRNL, ID2 and STEAP1. GO comparative analysis identified 10 common significant GO terms, mainly implicated in apoptosis and development process. Pathway comparative analysis observed that ECM-receptor interaction pathway and focal adhesion pathway were enriched in the differently expressed genes of both NOFH and hip OA. In conclusion, we identified a set of differently expressed genes, GO and pathways for NOFH articular destruction, some of which were also involved in the hip OA. Our study results may help to reveal the pathogenetic similarities and differences of cartilage damage of NOFH and hip OA. Copyright © 2016 Elsevier B.V. All rights reserved.

Testicular Lumicrine Factors Regulate ERK, STAT, and NFKB Pathways in the Initial Segment of the Rat Epididymis to Prevent Apoptosis1

PubMed Central

Xu, Bingfang; Abdel-Fattah, Rana; Yang, Ling; Crenshaw, Sallie A.; Black, Michael B.; Hinton, Barry T.

2011-01-01

The initial segment of the epididymis is vital for male fertility; therefore, it is important to understand the mechanisms that regulate this important region. Deprival of testicular luminal fluid factors/lumicrine factors from the epididymis results in a wave of apoptosis in the initial segment. In this study, a combination of protein array and microarray analyses was used to examine the early changes in downstream signal transduction pathways following loss of lumicrine factors. We discovered the following cascade of events leading to the loss of protection and eventual apoptosis: in the first 6 h after loss of lumicrine factors, down-regulation of the ERK pathway components was observed at the mRNA expression and protein activity levels. Microarray analysis revealed that mRNA levels of several key components of the ERK pathway, Dusp6, Dusp5, and Etv5, decreased sharply, while the analysis from the protein array revealed a decline in the activities of MAP2K1/2 and MAPK1. Immunostaining of phospho-MAPK3/1 indicated that down-regulation of the ERK pathway was specific to the epithelial cells of the initial segment. Subsequently, after 12 h of loss of lumicrine factors, levels of mRNA expression of STAT and NFKB pathway components increased, mRNA levels of several genes encoding cell cycle inhibitors increased, and levels of protein expression of several proapoptotic phosphatases increased. Finally, after 18 h of loss of protection from lumicrine factors, apoptosis was observed. In conclusion, testicular lumicrine factors protect the cells of the initial segment by activating the ERK pathway, repressing STAT and NFKB pathways, and thereby preventing apoptosis. PMID:21311037

An integrated bioinformatics approach to improve two-color microarray quality-control: impact on biological conclusions.

PubMed

van Haaften, Rachel I M; Luceri, Cristina; van Erk, Arie; Evelo, Chris T A

2009-06-01

Omics technology used for large-scale measurements of gene expression is rapidly evolving. This work pointed out the need of an extensive bioinformatics analyses for array quality assessment before and after gene expression clustering and pathway analysis. A study focused on the effect of red wine polyphenols on rat colon mucosa was used to test the impact of quality control and normalisation steps on the biological conclusions. The integration of data visualization, pathway analysis and clustering revealed an artifact problem that was solved with an adapted normalisation. We propose a possible point to point standard analysis procedure, based on a combination of clustering and data visualization for the analysis of microarray data.

Consensus strategy in genes prioritization and combined bioinformatics analysis for preeclampsia pathogenesis.

PubMed

Tejera, Eduardo; Cruz-Monteagudo, Maykel; Burgos, Germán; Sánchez, María-Eugenia; Sánchez-Rodríguez, Aminael; Pérez-Castillo, Yunierkis; Borges, Fernanda; Cordeiro, Maria Natália Dias Soeiro; Paz-Y-Miño, César; Rebelo, Irene

2017-08-08

Preeclampsia is a multifactorial disease with unknown pathogenesis. Even when recent studies explored this disease using several bioinformatics tools, the main objective was not directed to pathogenesis. Additionally, consensus prioritization was proved to be highly efficient in the recognition of genes-disease association. However, not information is available about the consensus ability to early recognize genes directly involved in pathogenesis. Therefore our aim in this study is to apply several theoretical approaches to explore preeclampsia; specifically those genes directly involved in the pathogenesis. We firstly evaluated the consensus between 12 prioritization strategies to early recognize pathogenic genes related to preeclampsia. A communality analysis in the protein-protein interaction network of previously selected genes was done including further enrichment analysis. The enrichment analysis includes metabolic pathways as well as gene ontology. Microarray data was also collected and used in order to confirm our results or as a strategy to weight the previously enriched pathways. The consensus prioritized gene list was rationally filtered to 476 genes using several criteria. The communality analysis showed an enrichment of communities connected with VEGF-signaling pathway. This pathway is also enriched considering the microarray data. Our result point to VEGF, FLT1 and KDR as relevant pathogenic genes, as well as those connected with NO metabolism. Our results revealed that consensus strategy improve the detection and initial enrichment of pathogenic genes, at least in preeclampsia condition. Moreover the combination of the first percent of the prioritized genes with protein-protein interaction network followed by communality analysis reduces the gene space. This approach actually identifies well known genes related with pathogenesis. However, genes like HSP90, PAK2, CD247 and others included in the first 1% of the prioritized list need to be further explored in preeclampsia pathogenesis through experimental approaches.

RECOVERING FILTER-BASED MICROARRAY DATA FOR PATHWAYS ANALYSIS USING A MULTIPOINT ALIGNMENT STRATEGY

EPA Science Inventory

The use of commercial microarrays are rapidly becoming the method of choice for profiling gene expression and assessing various disease states. Research Genetics has provided a series of well defined biological and software tools to the research community for these analyses. Th...

New insights about host response to smallpox using microarray data.

PubMed

Esteves, Gustavo H; Simoes, Ana C Q; Souza, Estevao; Dias, Rodrigo A; Ospina, Raydonal; Venancio, Thiago M

2007-08-24

Smallpox is a lethal disease that was endemic in many parts of the world until eradicated by massive immunization. Due to its lethality, there are serious concerns about its use as a bioweapon. Here we analyze publicly available microarray data to further understand survival of smallpox infected macaques, using systems biology approaches. Our goal is to improve the knowledge about the progression of this disease. We used KEGG pathways annotations to define groups of genes (or modules), and subsequently compared them to macaque survival times. This technique provided additional insights about the host response to this disease, such as increased expression of the cytokines and ECM receptors in the individuals with higher survival times. These results could indicate that these gene groups could influence an effective response from the host to smallpox. Macaques with higher survival times clearly express some specific pathways previously unidentified using regular gene-by-gene approaches. Our work also shows how third party analysis of public datasets can be important to support new hypotheses to relevant biological problems.

Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma

PubMed Central

Yan, Deng; Yi, Song; Chiu, Wang Chi; Qin, Liu Gui; Kin, Wong Hoi; Kwok Hung, Chung Tony; Linxiao, Han; Wai, Choy Kwong; Yi, Sui; Tao, Yang; Tao, Tang

2017-01-01

Objective This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. Methods Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). Results Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification (P=0.009) or deletion (P=0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly (P=1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. Conclusion Chromosomal CNVs may contribute to their transcript expression in cervical cancer. PMID:29312578

Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma.

PubMed

Yan, Deng; Yi, Song; Chiu, Wang Chi; Qin, Liu Gui; Kin, Wong Hoi; Kwok Hung, Chung Tony; Linxiao, Han; Wai, Choy Kwong; Yi, Sui; Tao, Yang; Tao, Tang

2017-12-12

This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification ( P =0.009) or deletion ( P =0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly ( P =1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. Chromosomal CNVs may contribute to their transcript expression in cervical cancer.

Large-scale integrative network-based analysis identifies common pathways disrupted by copy number alterations across cancers

PubMed Central

2013-01-01

Background Many large-scale studies analyzed high-throughput genomic data to identify altered pathways essential to the development and progression of specific types of cancer. However, no previous study has been extended to provide a comprehensive analysis of pathways disrupted by copy number alterations across different human cancers. Towards this goal, we propose a network-based method to integrate copy number alteration data with human protein-protein interaction networks and pathway databases to identify pathways that are commonly disrupted in many different types of cancer. Results We applied our approach to a data set of 2,172 cancer patients across 16 different types of cancers, and discovered a set of commonly disrupted pathways, which are likely essential for tumor formation in majority of the cancers. We also identified pathways that are only disrupted in specific cancer types, providing molecular markers for different human cancers. Analysis with independent microarray gene expression datasets confirms that the commonly disrupted pathways can be used to identify patient subgroups with significantly different survival outcomes. We also provide a network view of disrupted pathways to explain how copy number alterations affect pathways that regulate cell growth, cycle, and differentiation for tumorigenesis. Conclusions In this work, we demonstrated that the network-based integrative analysis can help to identify pathways disrupted by copy number alterations across 16 types of human cancers, which are not readily identifiable by conventional overrepresentation-based and other pathway-based methods. All the results and source code are available at http://compbio.cs.umn.edu/NetPathID/. PMID:23822816

Developmental effects of tobacco smoke exposure during human embryonic stem cell differentiation are mediated through the transforming growth factor-β superfamily member, Nodal

PubMed Central

Liszewski, Walter; Ritner, Carissa; Aurigui, Julian; Wong, Sharon S. Y.; Hussain, Naveed; Krueger, Winfried; Oncken, Cheryl; Bernstein, Harold S.

2012-01-01

While the pathologies associated with in utero smoke exposure are well established, their underlying molecular mechanisms are incompletely understood. We differentiated human embryonic stem cells in the presence of physiological concentrations of tobacco smoke and nicotine. Using post hoc microarray analysis, quantitative PCR, and immunoblot analysis, we demonstrated that tobacco smoke has lineage- and stage-specific effects on human embryonic stem cell differentiation, through both nicotine-dependent and -independent pathways. We show that three major stem cell pluripotency/differentiation pathways, Notch, canonical Wnt, and transforming growth factor-β, are affected by smoke exposure, and that Nodal signaling through SMAD2 is specifically impacted by effects on Lefty1, Nodal, and FoxH1. These events are associated with upregulation of microRNA-302a, a post-transcriptional silencer of Lefty1. The described studies provide insight into the mechanisms by which tobacco smoke influences fetal development at the cellular level, and identify specific transcriptional, post-transcriptional, and signaling pathways by which this likely occurs. PMID:22381624

Wnt/β-catenin signaling pathway inhibits the proliferation and apoptosis of U87 glioma cells via different mechanisms

PubMed Central

Gao, Liyang; Chen, Bing; Li, Jinhong; Yang, Fan; Cen, Xuecheng; Liao, Zhuangbing; Long, Xiao’ao

2017-01-01

The Wnt signaling pathway is necessary for the development of the central nervous system and is associated with tumorigenesis in various cancers. However, the mechanism of the Wnt signaling pathway in glioma cells has yet to be elucidated. Small-molecule Wnt modulators such as ICG-001 and AZD2858 were used to inhibit and stimulate the Wnt/β-catenin signaling pathway. Techniques including cell proliferation assay, colony formation assay, Matrigel cell invasion assay, cell cycle assay and Genechip microarray were used. Gene Ontology Enrichment Analysis and Gene Set Enrichment Analysis have enriched many biological processes and signaling pathways. Both the inhibiting and stimulating Wnt/β-catenin signaling pathways could influence the cell cycle, moreover, reduce the proliferation and survival of U87 glioma cells. However, Affymetrix expression microarray indicated that biological processes and networks of signaling pathways between stimulating and inhibiting the Wnt/β-catenin signaling pathway largely differ. We propose that Wnt/β-catenin signaling pathway might prove to be a valuable therapeutic target for glioma. PMID:28837560

A novel approach for discovering condition-specific correlations of gene expressions within biological pathways by using cloud computing technology.

PubMed

Chang, Tzu-Hao; Wu, Shih-Lin; Wang, Wei-Jen; Horng, Jorng-Tzong; Chang, Cheng-Wei

2014-01-01

Microarrays are widely used to assess gene expressions. Most microarray studies focus primarily on identifying differential gene expressions between conditions (e.g., cancer versus normal cells), for discovering the major factors that cause diseases. Because previous studies have not identified the correlations of differential gene expression between conditions, crucial but abnormal regulations that cause diseases might have been disregarded. This paper proposes an approach for discovering the condition-specific correlations of gene expressions within biological pathways. Because analyzing gene expression correlations is time consuming, an Apache Hadoop cloud computing platform was implemented. Three microarray data sets of breast cancer were collected from the Gene Expression Omnibus, and pathway information from the Kyoto Encyclopedia of Genes and Genomes was applied for discovering meaningful biological correlations. The results showed that adopting the Hadoop platform considerably decreased the computation time. Several correlations of differential gene expressions were discovered between the relapse and nonrelapse breast cancer samples, and most of them were involved in cancer regulation and cancer-related pathways. The results showed that breast cancer recurrence might be highly associated with the abnormal regulations of these gene pairs, rather than with their individual expression levels. The proposed method was computationally efficient and reliable, and stable results were obtained when different data sets were used. The proposed method is effective in identifying meaningful biological regulation patterns between conditions.

Molecular classification of benign prostatic hyperplasia: A gene expression profiling study in a rat model.

PubMed

Hata, Junya; Satoh, Yuichi; Akaihata, Hidenori; Hiraki, Hiroyuki; Ogawa, Soichiro; Haga, Nobuhiro; Ishibashi, Kei; Aikawa, Ken; Kojima, Yoshiyuki

2016-07-01

To characterize the molecular features of benign prostatic hyperplasia by carrying out a gene expression profiling analysis in a rat model. Fetal urogenital sinus isolated from 20-day-old male rat embryo was implanted into a pubertal male rat ventral prostate. The implanted urogenital sinus grew time-dependently, and the pathological findings at 3 weeks after implantation showed epithelial hyperplasia as well as stromal hyperplasia. Whole-genome oligonucleotide microarray analysis utilizing approximately 30 000 oligonucleotide probes was carried out using prostate specimens during the prostate growth process (3 weeks after implantation). Microarray analyses showed 926 upregulated (>2-fold change, P < 0.01) and 3217 downregulated genes (<0.5-fold change, P < 0.01) in benign prostatic hyperplasia specimens compared with normal prostate. Gene ontology analyses of upregulated genes showed predominant genetic themes of involvement in development (162 genes, P = 2.01 × 10(-4) ), response to stimulus (163 genes, P = 7.37 × 10(-13) ) and growth (32 genes, P = 1.93 × 10(-5) ). When we used both normal prostate and non-transplanted urogenital sinuses as controls to identify benign prostatic hyperplasia-specific genes, 507 and 406 genes were upregulated and downregulated, respectively. Functional network and pathway analyses showed that genes associated with apoptosis modulation by heat shock protein 70, interleukin-1, interleukin-2 and interleukin-5 signaling pathways, KIT signaling pathway, and secretin-like G-protein-coupled receptors, class B, were relatively activated during the growth process in the benign prostatic hyperplasia specimens. In contrast, genes associated with cholesterol biosynthesis were relatively inactivated. Our microarray analyses of the benign prostatic hyperplasia model rat might aid in clarifying the molecular mechanism of benign prostatic hyperplasia progression, and identifying molecular targets for benign prostatic hyperplasia treatment. © 2016 The Japanese Urological Association.

Genome Expression Pathway Analysis Tool – Analysis and visualization of microarray gene expression data under genomic, proteomic and metabolic context

PubMed Central

Weniger, Markus; Engelmann, Julia C; Schultz, Jörg

2007-01-01

Background Regulation of gene expression is relevant to many areas of biology and medicine, in the study of treatments, diseases, and developmental stages. Microarrays can be used to measure the expression level of thousands of mRNAs at the same time, allowing insight into or comparison of different cellular conditions. The data derived out of microarray experiments is highly dimensional and often noisy, and interpretation of the results can get intricate. Although programs for the statistical analysis of microarray data exist, most of them lack an integration of analysis results and biological interpretation. Results We have developed GEPAT, Genome Expression Pathway Analysis Tool, offering an analysis of gene expression data under genomic, proteomic and metabolic context. We provide an integration of statistical methods for data import and data analysis together with a biological interpretation for subsets of probes or single probes on the chip. GEPAT imports various types of oligonucleotide and cDNA array data formats. Different normalization methods can be applied to the data, afterwards data annotation is performed. After import, GEPAT offers various statistical data analysis methods, as hierarchical, k-means and PCA clustering, a linear model based t-test or chromosomal profile comparison. The results of the analysis can be interpreted by enrichment of biological terms, pathway analysis or interaction networks. Different biological databases are included, to give various information for each probe on the chip. GEPAT offers no linear work flow, but allows the usage of any subset of probes and samples as a start for a new data analysis. GEPAT relies on established data analysis packages, offers a modular approach for an easy extension, and can be run on a computer grid to allow a large number of users. It is freely available under the LGPL open source license for academic and commercial users at . Conclusion GEPAT is a modular, scalable and professional-grade software integrating analysis and interpretation of microarray gene expression data. An installation available for academic users can be found at . PMID:17543125

Identification of embryonic pancreatic genes using Xenopus DNA microarrays.

PubMed

Hayata, Tadayoshi; Blitz, Ira L; Iwata, Nahoko; Cho, Ken W Y

2009-06-01

The pancreas is both an exocrine and endocrine endodermal organ involved in digestion and glucose homeostasis. During embryogenesis, the anlagen of the pancreas arise from dorsal and ventral evaginations of the foregut that later fuse to form a single organ. To better understand the molecular genetics of early pancreas development, we sought to isolate markers that are uniquely expressed in this tissue. Microarray analysis was performed comparing dissected pancreatic buds, liver buds, and the stomach region of tadpole stage Xenopus embryos. A total of 912 genes were found to be differentially expressed between these organs during early stages of organogenesis. K-means clustering analysis predicted 120 of these genes to be specifically enriched in the pancreas. Of these, we report on the novel expression patterns of 24 genes. Our analyses implicate the involvement of previously unsuspected signaling pathways during early pancreas development. Developmental Dynamics 238:1455-1466, 2009. (c) 2009 Wiley-Liss, Inc.

PageMan: an interactive ontology tool to generate, display, and annotate overview graphs for profiling experiments.

PubMed

Usadel, Björn; Nagel, Axel; Steinhauser, Dirk; Gibon, Yves; Bläsing, Oliver E; Redestig, Henning; Sreenivasulu, Nese; Krall, Leonard; Hannah, Matthew A; Poree, Fabien; Fernie, Alisdair R; Stitt, Mark

2006-12-18

Microarray technology has become a widely accepted and standardized tool in biology. The first microarray data analysis programs were developed to support pair-wise comparison. However, as microarray experiments have become more routine, large scale experiments have become more common, which investigate multiple time points or sets of mutants or transgenics. To extract biological information from such high-throughput expression data, it is necessary to develop efficient analytical platforms, which combine manually curated gene ontologies with efficient visualization and navigation tools. Currently, most tools focus on a few limited biological aspects, rather than offering a holistic, integrated analysis. Here we introduce PageMan, a multiplatform, user-friendly, and stand-alone software tool that annotates, investigates, and condenses high-throughput microarray data in the context of functional ontologies. It includes a GUI tool to transform different ontologies into a suitable format, enabling the user to compare and choose between different ontologies. It is equipped with several statistical modules for data analysis, including over-representation analysis and Wilcoxon statistical testing. Results are exported in a graphical format for direct use, or for further editing in graphics programs.PageMan provides a fast overview of single treatments, allows genome-level responses to be compared across several microarray experiments covering, for example, stress responses at multiple time points. This aids in searching for trait-specific changes in pathways using mutants or transgenics, analyzing development time-courses, and comparison between species. In a case study, we analyze the results of publicly available microarrays of multiple cold stress experiments using PageMan, and compare the results to a previously published meta-analysis.PageMan offers a complete user's guide, a web-based over-representation analysis as well as a tutorial, and is freely available at http://mapman.mpimp-golm.mpg.de/pageman/. PageMan allows multiple microarray experiments to be efficiently condensed into a single page graphical display. The flexible interface allows data to be quickly and easily visualized, facilitating comparisons within experiments and to published experiments, thus enabling researchers to gain a rapid overview of the biological responses in the experiments.

Nutrient control of gene expression in Drosophila: microarray analysis of starvation and sugar-dependent response

PubMed Central

Zinke, Ingo; Schütz, Christina S.; Katzenberger, Jörg D.; Bauer, Matthias; Pankratz, Michael J.

2002-01-01

We have identified genes regulated by starvation and sugar signals in Drosophila larvae using whole-genome microarrays. Based on expression profiles in the two nutrient conditions, they were organized into different categories that reflect distinct physiological pathways mediating sugar and fat metabolism, and cell growth. In the category of genes regulated in sugar-fed, but not in starved, animals, there is an upregulation of genes encoding key enzymes of the fat biosynthesis pathway and a downregulation of genes encoding lipases. The highest and earliest activated gene upon sugar ingestion is sugarbabe, a zinc finger protein that is induced in the gut and the fat body. Identification of potential targets using microarrays suggests that sugarbabe functions to repress genes involved in dietary fat breakdown and absorption. The current analysis provides a basis for studying the genetic mechanisms underlying nutrient signalling. PMID:12426388

Gene Expression Profiling Identifies Downregulation of the Neurotrophin-MAPK Signaling Pathway in Female Diabetic Peripheral Neuropathy Patients.

PubMed

Luo, Lin; Zhou, Wen-Hua; Cai, Jiang-Jia; Feng, Mei; Zhou, Mi; Hu, Su-Pei; Xu, Jin; Ji, Lin-Dan

2017-01-01

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes mellitus (DM). It is not diagnosed or managed properly in the majority of patients because its pathogenesis remains controversial. In this study, human whole genome microarrays identified 2898 and 4493 differentially expressed genes (DEGs) in DM and DPN patients, respectively. A further KEGG pathway analysis indicated that DPN and DM share four pathways, including apoptosis, B cell receptor signaling pathway, endocytosis, and Toll-like receptor signaling pathway. The DEGs identified through comparison of DPN and DM were significantly enriched in MAPK signaling pathway, NOD-like receptor signaling pathway, and neurotrophin signaling pathway, while the "neurotrophin-MAPK signaling pathway" was notably downregulated. Seven DEGs from the neurotrophin-MAPK signaling pathway were validated in additional 78 samples, and the results confirmed the initial microarray findings. These findings demonstrated that downregulation of the neurotrophin-MAPK signaling pathway may be the major mechanism of DPN pathogenesis, thus providing a potential approach for DPN treatment.

Genome‐Wide MicroRNA and Gene Analysis of Mesenchymal Stem Cell Chondrogenesis Identifies an Essential Role and Multiple Targets for miR‐140‐5p

PubMed Central

Tselepi, Maria; Gómez, Rodolfo; Woods, Steven; Hui, Wang; Smith, Graham R.; Shanley, Daryl P.; Clark, Ian M.; Young, David A.

2015-01-01

Abstract microRNAs (miRNAs) are abundantly expressed in development where they are critical determinants of cell differentiation and phenotype. Accordingly miRNAs are essential for normal skeletal development and chondrogenesis in particular. However, the question of which miRNAs are specific to the chondrocyte phenotype has not been fully addressed. Using microarray analysis of miRNA expression during mesenchymal stem cell chondrogenic differentiation and detailed examination of the role of essential differentiation factors, such as SOX9, TGF‐β, and the cell condensation phase, we characterize the repertoire of specific miRNAs involved in chondrocyte development, highlighting in particular miR‐140 and miR‐455. Further with the use of mRNA microarray data we integrate miRNA expression and mRNA expression during chondrogenesis to underline the particular importance of miR‐140, especially the ‐5p strand. We provide a detailed identification and validation of direct targets of miR‐140‐5p in both chondrogenesis and adult chondrocytes with the use of microarray and 3′UTR analysis. This emphasizes the diverse array of targets and pathways regulated by miR‐140‐5p. We are also able to confirm previous experimentally identified targets but, additionally, identify a novel positive regulation of the Wnt signaling pathway by miR‐140‐5p. Wnt signaling has a complex role in chondrogenesis and skeletal development and these findings illustrate a previously unidentified role for miR‐140‐5p in regulation of Wnt signaling in these processes. Together these developments further highlight the role of miRNAs during chondrogenesis to improve our understanding of chondrocyte development and guide cartilage tissue engineering. Stem Cells 2015;33:3266–3280 PMID:26175215

A novel approach to select differential pathways associated with hypertrophic cardiomyopathy based on gene co‑expression analysis.

PubMed

Chen, Xiao-Min; Feng, Ming-Jun; Shen, Cai-Jie; He, Bin; Du, Xian-Feng; Yu, Yi-Bo; Liu, Jing; Chu, Hui-Min

2017-07-01

The present study was designed to develop a novel method for identifying significant pathways associated with human hypertrophic cardiomyopathy (HCM), based on gene co‑expression analysis. The microarray dataset associated with HCM (E‑GEOD‑36961) was obtained from the European Molecular Biology Laboratory‑European Bioinformatics Institute database. Informative pathways were selected based on the Reactome pathway database and screening treatments. An empirical Bayes method was utilized to construct co‑expression networks for informative pathways, and a weight value was assigned to each pathway. Differential pathways were extracted based on weight threshold, which was calculated using a random model. In order to assess whether the co‑expression method was feasible, it was compared with traditional pathway enrichment analysis of differentially expressed genes, which were identified using the significance analysis of microarrays package. A total of 1,074 informative pathways were screened out for subsequent investigations and their weight values were also obtained. According to the threshold of weight value of 0.01057, 447 differential pathways, including folding of actin by chaperonin containing T‑complex protein 1 (CCT)/T‑complex protein 1 ring complex (TRiC), purine ribonucleoside monophosphate biosynthesis and ubiquinol biosynthesis, were obtained. Compared with traditional pathway enrichment analysis, the number of pathways obtained from the co‑expression approach was increased. The results of the present study demonstrated that this method may be useful to predict marker pathways for HCM. The pathways of folding of actin by CCT/TRiC and purine ribonucleoside monophosphate biosynthesis may provide evidence of the underlying molecular mechanisms of HCM, and offer novel therapeutic directions for HCM.

Enhancing interdisciplinary mathematics and biology education: a microarray data analysis course bridging these disciplines.

PubMed

Tra, Yolande V; Evans, Irene M

2010-01-01

BIO2010 put forth the goal of improving the mathematical educational background of biology students. The analysis and interpretation of microarray high-dimensional data can be very challenging and is best done by a statistician and a biologist working and teaching in a collaborative manner. We set up such a collaboration and designed a course on microarray data analysis. We started using Genome Consortium for Active Teaching (GCAT) materials and Microarray Genome and Clustering Tool software and added R statistical software along with Bioconductor packages. In response to student feedback, one microarray data set was fully analyzed in class, starting from preprocessing to gene discovery to pathway analysis using the latter software. A class project was to conduct a similar analysis where students analyzed their own data or data from a published journal paper. This exercise showed the impact that filtering, preprocessing, and different normalization methods had on gene inclusion in the final data set. We conclude that this course achieved its goals to equip students with skills to analyze data from a microarray experiment. We offer our insight about collaborative teaching as well as how other faculty might design and implement a similar interdisciplinary course.

Enhancing Interdisciplinary Mathematics and Biology Education: A Microarray Data Analysis Course Bridging These Disciplines

PubMed Central

Evans, Irene M.

2010-01-01

BIO2010 put forth the goal of improving the mathematical educational background of biology students. The analysis and interpretation of microarray high-dimensional data can be very challenging and is best done by a statistician and a biologist working and teaching in a collaborative manner. We set up such a collaboration and designed a course on microarray data analysis. We started using Genome Consortium for Active Teaching (GCAT) materials and Microarray Genome and Clustering Tool software and added R statistical software along with Bioconductor packages. In response to student feedback, one microarray data set was fully analyzed in class, starting from preprocessing to gene discovery to pathway analysis using the latter software. A class project was to conduct a similar analysis where students analyzed their own data or data from a published journal paper. This exercise showed the impact that filtering, preprocessing, and different normalization methods had on gene inclusion in the final data set. We conclude that this course achieved its goals to equip students with skills to analyze data from a microarray experiment. We offer our insight about collaborative teaching as well as how other faculty might design and implement a similar interdisciplinary course. PMID:20810954

Microarray analysis of peripheral blood lymphocytes from ALS patients and the SAFE detection of the KEGG ALS pathway

PubMed Central

2011-01-01

Background Sporadic amyotrophic lateral sclerosis (sALS) is a motor neuron disease with poorly understood etiology. Results of gene expression profiling studies of whole blood from ALS patients have not been validated and are difficult to relate to ALS pathogenesis because gene expression profiles depend on the relative abundance of the different cell types present in whole blood. We conducted microarray analyses using Agilent Human Whole Genome 4 × 44k Arrays on a more homogeneous cell population, namely purified peripheral blood lymphocytes (PBLs), from ALS patients and healthy controls to identify molecular signatures possibly relevant to ALS pathogenesis. Methods Differentially expressed genes were determined by LIMMA (Linear Models for MicroArray) and SAM (Significance Analysis of Microarrays) analyses. The SAFE (Significance Analysis of Function and Expression) procedure was used to identify molecular pathway perturbations. Proteasome inhibition assays were conducted on cultured peripheral blood mononuclear cells (PBMCs) from ALS patients to confirm alteration of the Ubiquitin/Proteasome System (UPS). Results For the first time, using SAFE in a global gene ontology analysis (gene set size 5-100), we show significant perturbation of the KEGG (Kyoto Encyclopedia of Genes and Genomes) ALS pathway of motor neuron degeneration in PBLs from ALS patients. This was the only KEGG disease pathway significantly upregulated among 25, and contributing genes, including SOD1, represented 54% of the encoded proteins or protein complexes of the KEGG ALS pathway. Further SAFE analysis, including gene set sizes >100, showed that only neurodegenerative diseases (4 out of 34 disease pathways) including ALS were significantly upregulated. Changes in UBR2 expression correlated inversely with time since onset of disease and directly with ALSFRS-R, implying that UBR2 was increased early in the course of ALS. Cultured PBMCs from ALS patients accumulated more ubiquitinated proteins than PBMCs from healthy controls in a serum-dependent manner confirming changes in this pathway. Conclusions Our study indicates that PBLs from sALS patients are strong responders to systemic signals or local signals acquired by cell trafficking, representing changes in gene expression similar to those present in brain and spinal cord of sALS patients. PBLs may provide a useful means to study ALS pathogenesis. PMID:22027401

Gene Expression Profiling Identifies Downregulation of the Neurotrophin-MAPK Signaling Pathway in Female Diabetic Peripheral Neuropathy Patients

PubMed Central

Luo, Lin; Zhou, Wen-Hua; Cai, Jiang-Jia; Feng, Mei; Zhou, Mi; Hu, Su-Pei

2017-01-01

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes mellitus (DM). It is not diagnosed or managed properly in the majority of patients because its pathogenesis remains controversial. In this study, human whole genome microarrays identified 2898 and 4493 differentially expressed genes (DEGs) in DM and DPN patients, respectively. A further KEGG pathway analysis indicated that DPN and DM share four pathways, including apoptosis, B cell receptor signaling pathway, endocytosis, and Toll-like receptor signaling pathway. The DEGs identified through comparison of DPN and DM were significantly enriched in MAPK signaling pathway, NOD-like receptor signaling pathway, and neurotrophin signaling pathway, while the “neurotrophin-MAPK signaling pathway” was notably downregulated. Seven DEGs from the neurotrophin-MAPK signaling pathway were validated in additional 78 samples, and the results confirmed the initial microarray findings. These findings demonstrated that downregulation of the neurotrophin-MAPK signaling pathway may be the major mechanism of DPN pathogenesis, thus providing a potential approach for DPN treatment. PMID:28900628

Transgenic increases in seed oil content are associated with the differential expression of novel Brassica-specific transcripts.

PubMed

Sharma, Nirmala; Anderson, Maureen; Kumar, Arvind; Zhang, Yan; Giblin, E Michael; Abrams, Suzanne R; Zaharia, L Irina; Taylor, David C; Fobert, Pierre R

2008-12-19

Seed oil accumulates primarily as triacylglycerol (TAG). While the biochemical pathway for TAG biosynthesis is known, its regulation remains unclear. Previous research identified microsomal diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) as controlling a rate-limiting step in the TAG biosynthesis pathway. Of note, overexpression of DGAT1 results in substantial increases in oil content and seed size. To further analyze the global consequences of manipulating DGAT1 levels during seed development, a concerted transcriptome and metabolome analysis of transgenic B. napus prototypes was performed. Using a targeted Brassica cDNA microarray, about 200 genes were differentially expressed in two independent transgenic lines analyzed. Interestingly, 24-33% of the targets showing significant changes have no matching gene in Arabidopsis although these represent only 5% of the targets on the microarray. Further analysis of some of these novel transcripts indicated that several are inducible by ABA in microspore-derived embryos. Of the 200 Arabidopsis genes implicated in lipid biology present on the microarray, 36 were found to be differentially regulated in DGAT transgenic lines. Furthermore, kinetic reverse transcriptase Polymerase Chain Reaction (k-PCR) analysis revealed up-regulation of genes encoding enzymes of the Kennedy pathway involved in assembly of TAGs. Hormone profiling indicated that levels of auxins and cytokinins varied between transgenic lines and untransformed controls, while differences in the pool sizes of ABA and catabolites were only observed at later stages of development. Our results indicate that the increased TAG accumulation observed in transgenic DGAT1 plants is associated with modest transcriptional and hormonal changes during seed development that are not limited to the TAG biosynthesis pathway. These might be associated with feedback or feed-forward effects due to altered levels of DGAT1 activity. The fact that a large fraction of significant amplicons have no matching genes in Arabidopsis compromised our ability to draw concrete inferences from the data at this stage, but has led to the identification of novel genes of potential interest.

Bioinformatics approaches for cross-species liver cancer analysis based on microarray gene expression profiling

PubMed Central

Fang, H; Tong, W; Perkins, R; Shi, L; Hong, H; Cao, X; Xie, Q; Yim, SH; Ward, JM; Pitot, HC; Dragan, YP

2005-01-01

Background The completion of the sequencing of human, mouse and rat genomes and knowledge of cross-species gene homologies enables studies of differential gene expression in animal models. These types of studies have the potential to greatly enhance our understanding of diseases such as liver cancer in humans. Genes co-expressed across multiple species are most likely to have conserved functions. We have used various bioinformatics approaches to examine microarray expression profiles from liver neoplasms that arise in albumin-SV40 transgenic rats to elucidate genes, chromosome aberrations and pathways that might be associated with human liver cancer. Results In this study, we first identified 2223 differentially expressed genes by comparing gene expression profiles for two control, two adenoma and two carcinoma samples using an F-test. These genes were subsequently mapped to the rat chromosomes using a novel visualization tool, the Chromosome Plot. Using the same plot, we further mapped the significant genes to orthologous chromosomal locations in human and mouse. Many genes expressed in rat 1q that are amplified in rat liver cancer map to the human chromosomes 10, 11 and 19 and to the mouse chromosomes 7, 17 and 19, which have been implicated in studies of human and mouse liver cancer. Using Comparative Genomics Microarray Analysis (CGMA), we identified regions of potential aberrations in human. Lastly, a pathway analysis was conducted to predict altered human pathways based on statistical analysis and extrapolation from the rat data. All of the identified pathways have been known to be important in the etiology of human liver cancer, including cell cycle control, cell growth and differentiation, apoptosis, transcriptional regulation, and protein metabolism. Conclusion The study demonstrates that the hepatic gene expression profiles from the albumin-SV40 transgenic rat model revealed genes, pathways and chromosome alterations consistent with experimental and clinical research in human liver cancer. The bioinformatics tools presented in this paper are essential for cross species extrapolation and mapping of microarray data, its analysis and interpretation. PMID:16026603

Manganese-Induced Neurotoxicity and Alterations in Gene Expression in Human Neuroblastoma SH-SY5Y Cells.

PubMed

Gandhi, Deepa; Sivanesan, Saravanadevi; Kannan, Krishnamurthi

2018-06-01

Manganese (Mn) is an essential trace element required for many physiological functions including proper biochemical and cellular functioning of the central nervous system (CNS). However, exposure to excess level of Mn through occupational settings or from environmental sources has been associated with neurotoxicity. The cellular and molecular mechanism of Mn-induced neurotoxicity remains unclear. In the current study, we investigated the effects of 30-day exposure to a sub-lethal concentration of Mn (100 μM) in human neuroblastoma cells (SH-SY5Y) using transcriptomic approach. Microarray analysis revealed differential expression of 1057 transcripts in Mn-exposed SH-SY5Y cells as compared to control cells. Gene functional annotation cluster analysis exhibited that the differentially expressed genes were associated with several biological pathways. Specifically, genes involved in neuronal pathways including neuron differentiation and development, regulation of neurogenesis, synaptic transmission, and neuronal cell death (apoptosis) were found to be significantly altered. KEGG pathway analysis showed upregulation of p53 signaling pathways and neuroactive ligand-receptor interaction pathways, and downregulation of neurotrophin signaling pathway. On the basis of the gene expression profile, possible molecular mechanisms underlying Mn-induced neuronal toxicity were predicted.

Fear conditioning leads to alteration in specific genes expression in cortical and thalamic neurons that project to the lateral amygdala.

PubMed

Katz, Ira K; Lamprecht, Raphael

2015-02-01

RNA transcription is needed for memory formation. However, the ability to identify genes whose expression is altered by learning is greatly impaired because of methodological difficulties in profiling gene expression in specific neurons involved in memory formation. Here, we report a novel approach to monitor the expression of genes after learning in neurons in specific brain pathways needed for memory formation. In this study, we aimed to monitor gene expression after fear learning. We retrogradely labeled discrete thalamic neurons that project to the lateral amygdala (LA) of rats. The labeled neurons were dissected, using laser microdissection microscopy, after fear conditioning learning or unpaired training. The RNAs from the dissected neurons were subjected to microarray analysis. The levels of selected RNAs detected by the microarray analysis to be altered by fear conditioning were also assessed by nanostring analysis. We observed that the expression of genes involved in the regulation of translation, maturation and degradation of proteins was increased 6 h after fear conditioning compared to unpaired or naïve trained rats. These genes were not expressed 24 h after training or in cortical neurons that project to the LA. The expression of genes involved in transcription regulation and neuronal development was altered after fear conditioning learning in the cortical-LA pathway. The present study provides key information on the identity of genes expressed in discrete thalamic and cortical neurons that project to the LA after fear conditioning. Such an approach could also serve to identify gene products as targets for the development of a new generation of therapeutic agents that could be aimed to functionally identified brain circuits to treat memory-related disorders. © 2014 International Society for Neurochemistry.

New insights about host response to smallpox using microarray data

PubMed Central

Esteves, Gustavo H; Simoes, Ana CQ; Souza, Estevao; Dias, Rodrigo A; Ospina, Raydonal; Venancio, Thiago M

2007-01-01

Background Smallpox is a lethal disease that was endemic in many parts of the world until eradicated by massive immunization. Due to its lethality, there are serious concerns about its use as a bioweapon. Here we analyze publicly available microarray data to further understand survival of smallpox infected macaques, using systems biology approaches. Our goal is to improve the knowledge about the progression of this disease. Results We used KEGG pathways annotations to define groups of genes (or modules), and subsequently compared them to macaque survival times. This technique provided additional insights about the host response to this disease, such as increased expression of the cytokines and ECM receptors in the individuals with higher survival times. These results could indicate that these gene groups could influence an effective response from the host to smallpox. Conclusion Macaques with higher survival times clearly express some specific pathways previously unidentified using regular gene-by-gene approaches. Our work also shows how third party analysis of public datasets can be important to support new hypotheses to relevant biological problems. PMID:17718913

Microarray profiling of human white adipose tissue after exogenous leptin injection.

PubMed

Taleb, S; Van Haaften, R; Henegar, C; Hukshorn, C; Cancello, R; Pelloux, V; Hanczar, B; Viguerie, N; Langin, D; Evelo, C; Zucker, J; Clément, K; Saris, W H M

2006-03-01

Leptin is a secreted adipocyte hormone that plays a key role in the regulation of body weight homeostasis. The leptin effect on human white adipose tissue (WAT) is still debated. The aim of this study was to assess whether the administration of polyethylene glycol-leptin (PEG-OB) in a single supraphysiological dose has transcriptional effects on genes of WAT and to identify its target genes and functional pathways in WAT. Blood samples and WAT biopsies were obtained from 10 healthy nonobese men before treatment and 72 h after the PEG-OB injection, leading to an approximate 809-fold increase in circulating leptin. The WAT gene expression profile before and after the PEG-OB injection was compared using pangenomic microarrays. Functional gene annotations based on the gene ontology of the PEG-OB regulated genes were performed using both an 'in house' automated procedure and GenMAPP (Gene Microarray Pathway Profiler), designed for viewing and analyzing gene expression data in the context of biological pathways. Statistical analysis of microarray data revealed that PEG-OB had a major down-regulated effect on WAT gene expression, as we obtained 1,822 and 100 down- and up-regulated genes, respectively. Microarray data were validated using reverse transcription quantitative PCR. Functional gene annotations of PEG-OB regulated genes revealed that the functional class related to immunity and inflammation was among the most mobilized PEG-OB pathway in WAT. These genes are mainly expressed in the cell of the stroma vascular fraction in comparison with adipocytes. Our observations support the hypothesis that leptin could act on WAT, particularly on genes related to inflammation and immunity, which may suggest a novel leptin target pathway in human WAT.

Microarray characterization of gene expression changes in blood during acute ethanol exposure

PubMed Central

2013-01-01

Background As part of the civil aviation safety program to define the adverse effects of ethanol on flying performance, we performed a DNA microarray analysis of human whole blood samples from a five-time point study of subjects administered ethanol orally, followed by breathalyzer analysis, to monitor blood alcohol concentration (BAC) to discover significant gene expression changes in response to the ethanol exposure. Methods Subjects were administered either orange juice or orange juice with ethanol. Blood samples were taken based on BAC and total RNA was isolated from PaxGene™ blood tubes. The amplified cDNA was used in microarray and quantitative real-time polymerase chain reaction (RT-qPCR) analyses to evaluate differential gene expression. Microarray data was analyzed in a pipeline fashion to summarize and normalize and the results evaluated for relative expression across time points with multiple methods. Candidate genes showing distinctive expression patterns in response to ethanol were clustered by pattern and further analyzed for related function, pathway membership and common transcription factor binding within and across clusters. RT-qPCR was used with representative genes to confirm relative transcript levels across time to those detected in microarrays. Results Microarray analysis of samples representing 0%, 0.04%, 0.08%, return to 0.04%, and 0.02% wt/vol BAC showed that changes in gene expression could be detected across the time course. The expression changes were verified by qRT-PCR. The candidate genes of interest (GOI) identified from the microarray analysis and clustered by expression pattern across the five BAC points showed seven coordinately expressed groups. Analysis showed function-based networks, shared transcription factor binding sites and signaling pathways for members of the clusters. These include hematological functions, innate immunity and inflammation functions, metabolic functions expected of ethanol metabolism, and pancreatic and hepatic function. Five of the seven clusters showed links to the p38 MAPK pathway. Conclusions The results of this study provide a first look at changing gene expression patterns in human blood during an acute rise in blood ethanol concentration and its depletion because of metabolism and excretion, and demonstrate that it is possible to detect changes in gene expression using total RNA isolated from whole blood. The analysis approach for this study serves as a workflow to investigate the biology linked to expression changes across a time course and from these changes, to identify target genes that could serve as biomarkers linked to pilot performance. PMID:23883607

In-vitro analysis of Quantum Molecular Resonance effects on human mesenchymal stromal cells

PubMed Central

Sella, Sabrina; Adami, Valentina; Amati, Eliana; Bernardi, Martina; Chieregato, Katia; Gatto, Pamela; Menarin, Martina; Pozzato, Alessandro; Pozzato, Gianantonio; Astori, Giuseppe

2018-01-01

Electromagnetic fields play an essential role in cellular functions interfering with cellular pathways and tissue physiology. In this context, Quantum Molecular Resonance (QMR) produces waves with a specific form at high-frequencies (4–64 MHz) and low intensity through electric fields. We evaluated the effects of QMR stimulation on bone marrow derived mesenchymal stromal cells (MSC). MSC were treated with QMR for 10 minutes for 4 consecutive days for 2 weeks at different nominal powers. Cell morphology, phenotype, multilineage differentiation, viability and proliferation were investigated. QMR effects were further investigated by cDNA microarray validated by real-time PCR. After 1 and 2 weeks of QMR treatment morphology, phenotype and multilineage differentiation were maintained and no alteration of cellular viability and proliferation were observed between treated MSC samples and controls. cDNA microarray analysis evidenced more transcriptional changes on cells treated at 40 nominal power than 80 ones. The main enrichment lists belonged to development processes, regulation of phosphorylation, regulation of cellular pathways including metabolism, kinase activity and cellular organization. Real-time PCR confirmed significant increased expression of MMP1, PLAT and ARHGAP22 genes while A2M gene showed decreased expression in treated cells compared to controls. Interestingly, differentially regulated MMP1, PLAT and A2M genes are involved in the extracellular matrix (ECM) remodelling through the fibrinolytic system that is also implicated in embryogenesis, wound healing and angiogenesis. In our model QMR-treated MSC maintained unaltered cell phenotype, viability, proliferation and the ability to differentiate into bone, cartilage and adipose tissue. Microarray analysis may suggest an involvement of QMR treatment in angiogenesis and in tissue regeneration probably through ECM remodelling. PMID:29293552

Using pathway modules as targets for assay development in xenobiotic screening

EPA Science Inventory

Toxicology and pharmaceutical research is increasingly making use of high throughout-screening (HTS) methods to assess the effects of chemicals on molecular pathways, cells and tissues. Whole-genome microarray analysis provides broad information on the response of biological syst...

Pure mechanistic analysis of additive neuroprotective effects between baicalin and jasminoidin in ischemic stroke mice.

PubMed

Wang, Peng-Qian; Liu, Qiong; Xu, Wen-Juan; Yu, Ya-Nan; Zhang, Ying-Ying; Li, Bing; Liu, Jun; Wang, Zhong

2018-06-01

Both baicalin (BA) and jasminoidin (JA) are active ingredients in Chinese herb medicine Scutellaria baicalensis and Fructus gardeniae, respectively. They have been shown to exert additive neuroprotective action in ischemic stroke models. In this study we used transcriptome analysis to explore the pure therapeutic mechanisms of BA, JA and their combination (BJ) contributing to phenotype variation and reversal of pathological processes. Mice with middle cerebral artery obstruction were treated with BA, JA, their combination (BJ), or concha margaritifera (CM). Cerebral infarct volume was examined to determine the effect of these compounds on phenotype. Using the hippocampus microarray and ingenuity pathway analysis (IPA) software, we exacted the differentially expressed genes, networks, pathways, and functions in positive-phenotype groups (BA, JA and BJ) by comparing with the negative-phenotype group (CM). In the BA, JA, and BJ groups, a total of 7, 4, and 11 specific target molecules, 1, 1, and 4 networks, 51, 59, and 18 canonical pathways and 70, 53, and 64 biological functions, respectively, were identified. Pure therapeutic mechanisms of BA and JA were mainly overlapped in specific target molecules, functions and pathways, which were related to the nervous system, inflammation and immune response. The specific mechanisms of BA and JA were associated with apoptosis and cancer-related signaling and endocrine and hormone regulation, respectively. In the BJ group, novel target profiles distinct from mono-therapies were revealed, including 11 specific target molecules, 10 functions, and 10 pathways, the majority of which were related to a virus-mediated immune response. The pure additive effects between BA and JA were based on enhanced action in virus-mediated immune response. This pure mechanistic analysis may provide a clearer outline of the target profiles of multi-target compounds and combination therapies.

Gene expression profile in cardiovascular disease and preeclampsia: a meta-analysis of the transcriptome based on raw data from human studies deposited in Gene Expression Omnibus.

PubMed

Sitras, V; Fenton, C; Acharya, G

2015-02-01

Cardiovascular disease (CVD) and preeclampsia (PE) share common clinical features. We aimed to identify common transcriptomic signatures involved in CVD and PE in humans. Meta-analysis of individual raw microarray data deposited in GEO, obtained from blood samples of patients with CVD versus controls and placental samples from women with PE versus healthy women with uncomplicated pregnancies. Annotation of cases versus control samples was taken directly from the microarray documentation. Genes that showed a significant differential expression in the majority of experiments were selected for subsequent analysis. Hypergeometric gene list analysis was performed using Bioconductor GOstats package. Bioinformatic analysis was performed in PANTHER. Seven studies in CVD and 5 studies in PE were eligible for meta-analysis. A total of 181 genes were found to be differentially expressed in microarray studies investigating gene expression in blood samples obtained from patients with CVD compared to controls and 925 genes were differentially expressed between preeclamptic and healthy placentas. Among these differentially expressed genes, 22 were common between CVD and PE. Bioinformatic analysis of these genes revealed oxidative stress, p-53 pathway feedback, inflammation mediated by chemokines and cytokines, interleukin signaling, B-cell activation, PDGF signaling, Wnt signaling, integrin signaling and Alzheimer disease pathways to be involved in the pathophysiology of both CVD and PE. Metabolism, development, response to stimulus, immune response and cell communication were the associated biologic processes in both conditions. Gene set enrichment analysis showed the following overlapping pathways between CVD and PE: TGF-β-signaling, apoptosis, graft-versus-host disease, allograft rejection, chemokine signaling, steroid hormone synthesis, type I and II diabetes mellitus, VEGF signaling, pathways in cancer, GNRH signaling, Huntingtons disease and Notch signaling. CVD and PE share same common traits in their gene expression profile indicating common pathways in their pathophysiology. Copyright © 2014 Elsevier Ltd. All rights reserved.

Gene expression profiling and pathway analysis in MCF-7 and MDA-MB-231 human breast cancer cell lines treated with dioscin

USDA-ARS?s Scientific Manuscript database

The long-term goal of our study is to understand the genetic and epigenetic mechanisms of breast cancer metastasis in human and to discover new possible genetic markers for use in clinical practice. We have used microarray technology (Human OneArray microarray, phylanxbiotech.com) to compare gene ex...

Differences in gene expression profiles and signaling pathways in rhabdomyolysis-induced acute kidney injury.

PubMed

Geng, Xiaodong; Wang, Yuanda; Hong, Quan; Yang, Jurong; Zheng, Wei; Zhang, Gang; Cai, Guangyan; Chen, Xiangmei; Wu, Di

2015-01-01

Rhabdomyolysis is a threatening syndrome because it causes the breakdown of skeletal muscle. Muscle destruction leads to the release of myoglobin, intracellular proteins, and electrolytes into the circulation. The aim of this study was to investigate the differences in gene expression profiles and signaling pathways upon rhabdomyolysis-induced acute kidney injury (AKI). In this study, we used glycerol-induced renal injury as a model of rhabdomyolysis-induced AKI. We analyzed data and relevant information from the Gene Expression Omnibus database (No: GSE44925). The gene expression data for three untreated mice were compared to data for five mice with rhabdomyolysis-induced AKI. The expression profiling of the three untreated mice and the five rhabdomyolysis-induced AKI mice was performed using microarray analysis. We examined the levels of Cyp3a13, Rela, Aldh7a1, Jun, CD14. And Cdkn1a using RT-PCR to determine the accuracy of the microarray results. The microarray analysis showed that there were 1050 downregulated and 659 upregulated genes in the rhabdomyolysis-induced AKI mice compared to the control group. The interactions of all differentially expressed genes in the Signal-Net were analyzed. Cyp3a13 and Rela had the most interactions with other genes. The data showed that Rela and Aldh7a1 were the key nodes and had important positions in the Signal-Net. The genes Jun, CD14, and Cdkn1a were also significantly upregulated. The pathway analysis classified the differentially expressed genes into 71 downregulated and 48 upregulated pathways including the PI3K/Akt, MAPK, and NF-κB signaling pathways. The results of this study indicate that the NF-κB, MAPK, PI3K/Akt, and apoptotic pathways are regulated in rhabdomyolysis-induced AKI.

Gene Expression Profiling of Gastric Cancer

PubMed Central

Marimuthu, Arivusudar; Jacob, Harrys K.C.; Jakharia, Aniruddha; Subbannayya, Yashwanth; Keerthikumar, Shivakumar; Kashyap, Manoj Kumar; Goel, Renu; Balakrishnan, Lavanya; Dwivedi, Sutopa; Pathare, Swapnali; Dikshit, Jyoti Bajpai; Maharudraiah, Jagadeesha; Singh, Sujay; Sameer Kumar, Ghantasala S; Vijayakumar, M.; Veerendra Kumar, Kariyanakatte Veeraiah; Premalatha, Chennagiri Shrinivasamurthy; Tata, Pramila; Hariharan, Ramesh; Roa, Juan Carlos; Prasad, T.S.K; Chaerkady, Raghothama; Kumar, Rekha Vijay; Pandey, Akhilesh

2015-01-01

Gastric cancer is the second leading cause of cancer death worldwide, both in men and women. A genomewide gene expression analysis was carried out to identify differentially expressed genes in gastric adenocarcinoma tissues as compared to adjacent normal tissues. We used Agilent’s whole human genome oligonucleotide microarray platform representing ~41,000 genes to carry out gene expression analysis. Two-color microarray analysis was employed to directly compare the expression of genes between tumor and normal tissues. Through this approach, we identified several previously known candidate genes along with a number of novel candidate genes in gastric cancer. Testican-1 (SPOCK1) was one of the novel molecules that was 10-fold upregulated in tumors. Using tissue microarrays, we validated the expression of testican-1 by immunohistochemical staining. It was overexpressed in 56% (160/282) of the cases tested. Pathway analysis led to the identification of several networks in which SPOCK1 was among the topmost networks of interacting genes. By gene enrichment analysis, we identified several genes involved in cell adhesion and cell proliferation to be significantly upregulated while those corresponding to metabolic pathways were significantly downregulated. The differentially expressed genes identified in this study are candidate biomarkers for gastric adenoacarcinoma. PMID:27030788

WholePathwayScope: a comprehensive pathway-based analysis tool for high-throughput data

PubMed Central

Yi, Ming; Horton, Jay D; Cohen, Jonathan C; Hobbs, Helen H; Stephens, Robert M

2006-01-01

Background Analysis of High Throughput (HTP) Data such as microarray and proteomics data has provided a powerful methodology to study patterns of gene regulation at genome scale. A major unresolved problem in the post-genomic era is to assemble the large amounts of data generated into a meaningful biological context. We have developed a comprehensive software tool, WholePathwayScope (WPS), for deriving biological insights from analysis of HTP data. Result WPS extracts gene lists with shared biological themes through color cue templates. WPS statistically evaluates global functional category enrichment of gene lists and pathway-level pattern enrichment of data. WPS incorporates well-known biological pathways from KEGG (Kyoto Encyclopedia of Genes and Genomes) and Biocarta, GO (Gene Ontology) terms as well as user-defined pathways or relevant gene clusters or groups, and explores gene-term relationships within the derived gene-term association networks (GTANs). WPS simultaneously compares multiple datasets within biological contexts either as pathways or as association networks. WPS also integrates Genetic Association Database and Partial MedGene Database for disease-association information. We have used this program to analyze and compare microarray and proteomics datasets derived from a variety of biological systems. Application examples demonstrated the capacity of WPS to significantly facilitate the analysis of HTP data for integrative discovery. Conclusion This tool represents a pathway-based platform for discovery integration to maximize analysis power. The tool is freely available at . PMID:16423281

Stannous Fluoride Effects on Gene Expression of Streptococcus mutans and Actinomyces viscosus.

PubMed

Shi, Y; Li, R; White, D J; Biesbrock, A R

2018-02-01

A genome-wide transcriptional analysis was performed to elucidate the bacterial cellular response of Streptococcus mutans and Actinomyces viscosus to NaF and SnF 2 . The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of SnF 2 were predetermined before microarray study. Gene expression profiling microarray experiments were carried out in the absence (control) and presence (experimental) of 10 ppm and 100 ppm Sn 2+ (in the form of SnF 2 ) and fluoride controls for 10-min exposures (4 biological replicates/treatment). These Sn 2+ levels and treatment time were chosen because they have been shown to slow bacterial growth of S. mutans (10 ppm) and A. viscosus (100 ppm) without affecting cell viability. All data generated by microarray experiments were analyzed with bioinformatics tools by applying the following criteria: 1) a q value should be ≤0.05, and 2) an absolute fold change in transcript level should be ≥1.5. Microarray results showed SnF 2 significantly inhibited several genes encoding enzymes of the galactose pathway upon a 10-min exposure versus a negative control: lacA and lacB (A and B subunits of the galactose-6-P isomerase), lacC (tagatose-6-P kinase), lacD (tagatose-1,6-bP adolase), galK (galactokinase), galT (galactose-1-phosphate uridylyltransferase), and galE (UDP-glucose 4-epimerase). A gene fruK encoding fructose-1-phosphate kinase in the fructose pathway was also significantly inhibited. Several genes encoding fructose/mannose-specific enzyme IIABC components in the phosphotransferase system (PTS) were also downregulated, as was ldh encoding lactate dehydrogenase, a key enzyme involved in lactic acid synthesis. SnF 2 downregulated the transcription of most key enzyme genes involved in the galactose pathway and also suppressed several key genes involved in the PTS, which transports sugars into the cell in the first step of glycolysis.

Characterizing biomarkers in osteosarcoma metastasis based on an ego-network.

PubMed

Liu, Zhen; Song, Yan

2017-06-01

To characterize biomarkers that underlie osteosarcoma (OS) metastasis based on an ego-network. From the microarray data, we obtained 13,326 genes. By combining PPI data and microarray data, 10,520 shared genes were found and constructed into ego-networks. 17 significant ego-networks were identified with p < 0.05. In the pathway enrichment analysis, seven ego-networks were identified with the most significant pathway. These significant ego-modules were potential biomarkers that reveal the potential mechanisms in OS metastasis, which may contribute to understanding cancer prognoses and providing new perspectives in the treatment of cancer.

Differentiating Human Multipotent Mesenchymal Stromal Cells Regulate microRNAs: Prediction of microRNA Regulation by PDGF During Osteogenesis

PubMed Central

Goff, Loyal A.; Boucher, Shayne; Ricupero, Christopher L.; Fenstermacher, Sara; Swerdel, Mavis; Chase, Lucas; Adams, Christopher; Chesnut, Jonathan; Lakshmipathy, Uma; Hart, Ronald P.

2009-01-01

Objective Human multipotent mesenchymal stromal cells (MSC) have the potential to differentiate into multiple cell types, although little is known about factors that control their fate. Differentiation-specific microRNAs may play a key role in stem cell self renewal and differentiation. We propose that specific intracellular signalling pathways modulate gene expression during differentiation by regulating microRNA expression. Methods Illumina mRNA and NCode microRNA expression analyses were performed on MSC and their differentiated progeny. A combination of bioinformatic prediction and pathway inhibition was used to identify microRNAs associated with PDGF signalling. Results The pattern of microRNA expression in MSC is distinct from that in pluripotent stem cells such as human embryonic stem cells. Specific populations of microRNAs are regulated in MSC during differentiation targeted towards specific cell types. Complementary mRNA expression analysis increases the pool of markers characteristic of MSC or differentiated progeny. To identify microRNA expression patterns affected by signalling pathways, we examined the PDGF pathway found to be regulated during osteogenesis by microarray studies. A set of microRNAs bioinformatically predicted to respond to PDGF signalling was experimentally confirmed by direct PDGF inhibition. Conclusion Our results demonstrate that a subset of microRNAs regulated during osteogenic differentiation of MSCs is responsive to perturbation of the PDGF pathway. This approach not only identifies characteristic classes of differentiation-specific mRNAs and microRNAs, but begins to link regulated molecules with specific cellular pathways. PMID:18657893

Microarray expression profiling in adhesion and normal peritoneal tissues.

PubMed

Ambler, Dana R; Golden, Alicia M; Gell, Jennifer S; Saed, Ghassan M; Carey, David J; Diamond, Michael P

2012-05-01

To identify molecular markers associated with adhesion and normal peritoneal tissue using microarray expression profiling. Comparative study. University hospital. Five premenopausal women. Adhesion and normal peritoneal tissue samples were obtained from premenopausal women. Ribonucleic acid was extracted using standard protocols and processed for hybridization to Affymetrix Whole Transcript Human Gene Expression Chips. Microarray data were obtained from five different patients, each with adhesion tissue and normal peritoneal samples. Real-time polymerase chain reaction was performed for confirmation using standard protocols. Gene expression in postoperative adhesion and normal peritoneal tissues. A total of 1,263 genes were differentially expressed between adhesion and normal tissues. One hundred seventy-three genes were found to be up-regulated and 56 genes were down-regulated in the adhesion tissues compared with normal peritoneal tissues. The genes were sorted into functional categories according to Gene Ontology annotations. Twenty-six up-regulated genes and 11 down-regulated genes were identified with functions potentially relevant to the pathophysiology of postoperative adhesions. We evaluated and confirmed expression of 12 of these specific genes via polymerase chain reaction. The pathogenesis, natural history, and optimal treatment of postoperative adhesive disease remains unanswered. Microarray analysis of adhesions identified specific genes with increased and decreased expression when compared with normal peritoneum. Knowledge of these genes and ontologic pathways with altered expression provide targets for new therapies to treat patients who have or are at risk for postoperative adhesions. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Using scale and feather traits for module construction provides a functional approach to chicken epidermal development.

PubMed

Bao, Weier; Greenwold, Matthew J; Sawyer, Roger H

2017-11-01

Gene co-expression network analysis has been a research method widely used in systematically exploring gene function and interaction. Using the Weighted Gene Co-expression Network Analysis (WGCNA) approach to construct a gene co-expression network using data from a customized 44K microarray transcriptome of chicken epidermal embryogenesis, we have identified two distinct modules that are highly correlated with scale or feather development traits. Signaling pathways related to feather development were enriched in the traditional KEGG pathway analysis and functional terms relating specifically to embryonic epidermal development were also enriched in the Gene Ontology analysis. Significant enrichment annotations were discovered from customized enrichment tools such as Modular Single-Set Enrichment Test (MSET) and Medical Subject Headings (MeSH). Hub genes in both trait-correlated modules showed strong specific functional enrichment toward epidermal development. Also, regulatory elements, such as transcription factors and miRNAs, were targeted in the significant enrichment result. This work highlights the advantage of this methodology for functional prediction of genes not previously associated with scale- and feather trait-related modules.

Additional targets of the Arabidopsis autonomous pathway members, FCA and FY.

PubMed

Marquardt, S; Boss, P K; Hadfield, J; Dean, C

2006-01-01

A central player in the Arabidopsis floral transition is the floral repressor FLC, the MADS-box transcriptional regulator that inhibits the activity of genes required to switch the meristem from vegetative to floral development. One of the many pathways that regulate FLC expression is the autonomous promotion pathway composed of FCA, FY, FLD, FPA, FVE, LD, and FLK. Rather than a hierarchical set of activities the autonomous promotion pathway comprises sub-pathways of genes with different biochemical functions that all share FLC as a target. One sub-pathway involves FCA and FY, which interact to regulate RNA processing of FLC. Several of the identified components (FY, FVE, and FLD) are homologous to yeast and mammalian proteins with rather generic roles in gene regulation. So why do mutations in these genes specifically show a late-flowering phenotype in Arabidopsis? One reason, found during the analysis of fy alleles, is that the mutant alleles identified in flowering screens can be hypomorphic, they still have partial function. A broader role for the autonomous promotion pathway is supported by a microarray analysis which has identified genes mis-regulated in fca mutants, and whose expression is also altered in fy mutants.

Expression Profile of Long Noncoding RNAs in Human Earlobe Keloids: A Microarray Analysis

PubMed Central

Guo, Liang; Xu, Kai; Yan, Hongbo; Feng, Haifeng

2016-01-01

Background. Long noncoding RNAs (lncRNAs) play key roles in a wide range of biological processes and their deregulation results in human disease, including keloids. Earlobe keloid is a type of pathological skin scar, and the molecular pathogenesis of this disease remains largely unknown. Methods. In this study, microarray analysis was used to determine the expression profiles of lncRNAs and mRNAs between 3 pairs of earlobe keloid and normal specimens. Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to identify the main functions of the differentially expressed genes and earlobe keloid-related pathways. Results. A total of 2068 lncRNAs and 1511 mRNAs were differentially expressed between earlobe keloid and normal tissues. Among them, 1290 lncRNAs and 1092 mRNAs were upregulated, and 778 lncRNAs and 419 mRNAs were downregulated. Pathway analysis revealed that 24 pathways were correlated to the upregulated transcripts, while 11 pathways were associated with the downregulated transcripts. Conclusion. We characterized the expression profiles of lncRNA and mRNA in earlobe keloids and suggest that lncRNAs may serve as diagnostic biomarkers for the therapy of earlobe keloid. PMID:28101509

Transcriptome Analysis of Mycobacteria-Specific CD4+ T Cells Identified by Activation-Induced Expression of CD154.

PubMed

Kunnath-Velayudhan, Shajo; Goldberg, Michael F; Saini, Neeraj K; Johndrow, Christopher T; Ng, Tony W; Johnson, Alison J; Xu, Jiayong; Chan, John; Jacobs, William R; Porcelli, Steven A

2017-10-01

Analysis of Ag-specific CD4 + T cells in mycobacterial infections at the transcriptome level is informative but technically challenging. Although several methods exist for identifying Ag-specific T cells, including intracellular cytokine staining, cell surface cytokine-capture assays, and staining with peptide:MHC class II multimers, all of these have significant technical constraints that limit their usefulness. Measurement of activation-induced expression of CD154 has been reported to detect live Ag-specific CD4 + T cells, but this approach remains underexplored and, to our knowledge, has not previously been applied in mycobacteria-infected animals. In this article, we show that CD154 expression identifies adoptively transferred or endogenous Ag-specific CD4 + T cells induced by Mycobacterium bovis bacillus Calmette-Guérin vaccination. We confirmed that Ag-specific cytokine production was positively correlated with CD154 expression by CD4 + T cells from bacillus Calmette-Guérin-vaccinated mice and show that high-quality microarrays can be performed from RNA isolated from CD154 + cells purified by cell sorting. Analysis of microarray data demonstrated that the transcriptome of CD4 + CD154 + cells was distinct from that of CD154 - cells and showed major enrichment of transcripts encoding multiple cytokines and pathways of cellular activation. One notable finding was the identification of a previously unrecognized subset of mycobacteria-specific CD4 + T cells that is characterized by the production of IL-3. Our results support the use of CD154 expression as a practical and reliable method to isolate live Ag-specific CD4 + T cells for transcriptomic analysis and potentially for a range of other studies in infected or previously immunized hosts. Copyright © 2017 by The American Association of Immunologists, Inc.

Augmenting Microarray Data with Literature-Based Knowledge to Enhance Gene Regulatory Network Inference

PubMed Central

Kilicoglu, Halil; Shin, Dongwook; Rindflesch, Thomas C.

2014-01-01

Gene regulatory networks are a crucial aspect of systems biology in describing molecular mechanisms of the cell. Various computational models rely on random gene selection to infer such networks from microarray data. While incorporation of prior knowledge into data analysis has been deemed important, in practice, it has generally been limited to referencing genes in probe sets and using curated knowledge bases. We investigate the impact of augmenting microarray data with semantic relations automatically extracted from the literature, with the view that relations encoding gene/protein interactions eliminate the need for random selection of components in non-exhaustive approaches, producing a more accurate model of cellular behavior. A genetic algorithm is then used to optimize the strength of interactions using microarray data and an artificial neural network fitness function. The result is a directed and weighted network providing the individual contribution of each gene to its target. For testing, we used invasive ductile carcinoma of the breast to query the literature and a microarray set containing gene expression changes in these cells over several time points. Our model demonstrates significantly better fitness than the state-of-the-art model, which relies on an initial random selection of genes. Comparison to the component pathways of the KEGG Pathways in Cancer map reveals that the resulting networks contain both known and novel relationships. The p53 pathway results were manually validated in the literature. 60% of non-KEGG relationships were supported (74% for highly weighted interactions). The method was then applied to yeast data and our model again outperformed the comparison model. Our results demonstrate the advantage of combining gene interactions extracted from the literature in the form of semantic relations with microarray analysis in generating contribution-weighted gene regulatory networks. This methodology can make a significant contribution to understanding the complex interactions involved in cellular behavior and molecular physiology. PMID:24921649

Augmenting microarray data with literature-based knowledge to enhance gene regulatory network inference.

PubMed

Chen, Guocai; Cairelli, Michael J; Kilicoglu, Halil; Shin, Dongwook; Rindflesch, Thomas C

2014-06-01

Gene regulatory networks are a crucial aspect of systems biology in describing molecular mechanisms of the cell. Various computational models rely on random gene selection to infer such networks from microarray data. While incorporation of prior knowledge into data analysis has been deemed important, in practice, it has generally been limited to referencing genes in probe sets and using curated knowledge bases. We investigate the impact of augmenting microarray data with semantic relations automatically extracted from the literature, with the view that relations encoding gene/protein interactions eliminate the need for random selection of components in non-exhaustive approaches, producing a more accurate model of cellular behavior. A genetic algorithm is then used to optimize the strength of interactions using microarray data and an artificial neural network fitness function. The result is a directed and weighted network providing the individual contribution of each gene to its target. For testing, we used invasive ductile carcinoma of the breast to query the literature and a microarray set containing gene expression changes in these cells over several time points. Our model demonstrates significantly better fitness than the state-of-the-art model, which relies on an initial random selection of genes. Comparison to the component pathways of the KEGG Pathways in Cancer map reveals that the resulting networks contain both known and novel relationships. The p53 pathway results were manually validated in the literature. 60% of non-KEGG relationships were supported (74% for highly weighted interactions). The method was then applied to yeast data and our model again outperformed the comparison model. Our results demonstrate the advantage of combining gene interactions extracted from the literature in the form of semantic relations with microarray analysis in generating contribution-weighted gene regulatory networks. This methodology can make a significant contribution to understanding the complex interactions involved in cellular behavior and molecular physiology.

Bisphenol A exposure leads to specific microRNA alterations in placental cells.

PubMed

Avissar-Whiting, Michele; Veiga, Keila R; Uhl, Kristen M; Maccani, Matthew A; Gagne, Luc A; Moen, Erika L; Marsit, Carmen J

2010-07-01

Exposure to bisphenol A (BPA) has been observed to alter developmental pathways and cell processes, at least in part, through epigenetic mechanisms. This study sought to investigate the effect of BPA on microRNAs (miRNAs) in human placental cells. miRNA microarray was performed following BPA treatment in three immortalized cytotrophoblast cell lines and the results validated using quantitative real-time PCR. For functional analysis, overexpression constructs were stably transfected into cells that were then assayed for changes in proliferation and response to toxicants. Microarray analysis revealed several miRNAs to be significantly altered in response to BPA treatment in two cell lines. Real-time PCR results confirmed that miR-146a was particularly strongly induced and its overexpression in cells led to slower proliferation as well as higher sensitivity to the DNA damaging agent, bleomycin. Overall, these results suggest that BPA can alter miRNA expression in placental cells, a potentially novel mode of BPA toxicity.

Bisphenol A Exposure Leads to Specific MicroRNA Alterations in Placental Cells

PubMed Central

Avissar-Whiting, Michele; Veiga, Keila; Uhl, Kristen; Maccani, Matthew; Gagne, Luc; Moen, Erika; Marsit, Carmen J.

2010-01-01

Exposure to bisphenol-A (BPA) has been observed to alter developmental pathways and cell processes, at least in part, through epigenetic mechanisms. This study sought to investigate the effect of BPA on microRNAs (miRNAs) in human placental cells. miRNA microarray was performed following BPA treatment in three immortalized cytotrophoblast cell lines and the results validated using quantitative real-time PCR. For functional analysis, overexpression constructs were stably transfected into cells that were then assayed for changes in proliferation and response to toxicants. Microarray analysis revealed several miRNAs to be significantly altered in response to BPA treatment in two cell lines. Real-time PCR results confirmed that miR-146a was particularly strongly induced and its overexpression in cells led to slower proliferation as well as higher sensitivity to the DNA damaging agent, bleomycin. Overall, these results suggest that BPA can alter miRNA expression in placental cells, a potentially novel mode of BPA toxicity. PMID:20417706

RNA-Seq Profiling Reveals Novel Hepatic Gene Expression Pattern in Aflatoxin B1 Treated Rats

PubMed Central

Merrick, B. Alex; Phadke, Dhiral P.; Auerbach, Scott S.; Mav, Deepak; Stiegelmeyer, Suzy M.; Shah, Ruchir R.; Tice, Raymond R.

2013-01-01

Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1), a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG) than microarray analysis, including low copy and novel transcripts related to AFB1’s carcinogenic activity compared to feed controls (CTRL). Paired-end reads were mapped to the rat genome (Rn4) with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005) compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT’s) on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c. We find the rat transcriptome contains many previously unidentified, AFB1-responsive exons and transcripts supporting RNA-Seq’s capabilities to provide new insights into AFB1-mediated gene expression leading to hepatocellular carcinoma. PMID:23630614

RNA-Seq profiling reveals novel hepatic gene expression pattern in aflatoxin B1 treated rats.

PubMed

Merrick, B Alex; Phadke, Dhiral P; Auerbach, Scott S; Mav, Deepak; Stiegelmeyer, Suzy M; Shah, Ruchir R; Tice, Raymond R

2013-01-01

Deep sequencing was used to investigate the subchronic effects of 1 ppm aflatoxin B1 (AFB1), a potent hepatocarcinogen, on the male rat liver transcriptome prior to onset of histopathological lesions or tumors. We hypothesized RNA-Seq would reveal more differentially expressed genes (DEG) than microarray analysis, including low copy and novel transcripts related to AFB1's carcinogenic activity compared to feed controls (CTRL). Paired-end reads were mapped to the rat genome (Rn4) with TopHat and further analyzed by DESeq and Cufflinks-Cuffdiff pipelines to identify differentially expressed transcripts, new exons and unannotated transcripts. PCA and cluster analysis of DEGs showed clear separation between AFB1 and CTRL treatments and concordance among group replicates. qPCR of eight high and medium DEGs and three low DEGs showed good comparability among RNA-Seq and microarray transcripts. DESeq analysis identified 1,026 differentially expressed transcripts at greater than two-fold change (p<0.005) compared to 626 transcripts by microarray due to base pair resolution of transcripts by RNA-Seq, probe placement within transcripts or an absence of probes to detect novel transcripts, splice variants and exons. Pathway analysis among DEGs revealed signaling of Ahr, Nrf2, GSH, xenobiotic, cell cycle, extracellular matrix, and cell differentiation networks consistent with pathways leading to AFB1 carcinogenesis, including almost 200 upregulated transcripts controlled by E2f1-related pathways related to kinetochore structure, mitotic spindle assembly and tissue remodeling. We report 49 novel, differentially-expressed transcripts including confirmation by PCR-cloning of two unique, unannotated, hepatic AFB1-responsive transcripts (HAfT's) on chromosomes 1.q55 and 15.q11, overexpressed by 10 to 25-fold. Several potentially novel exons were found and exon refinements were made including AFB1 exon-specific induction of homologous family members, Ugt1a6 and Ugt1a7c. We find the rat transcriptome contains many previously unidentified, AFB1-responsive exons and transcripts supporting RNA-Seq's capabilities to provide new insights into AFB1-mediated gene expression leading to hepatocellular carcinoma.

Differentially expressed genes and pathways induced by organophosphates in human neuroblastoma cells.

PubMed

Li, Tianwei; Zhao, Hongtao; Hung, Guo-Chiuan; Han, Jing; Tsai, Shien; Li, Bingjie; Zhang, Jing; Puri, Raj K; Lo, Shyh-Ching

2012-12-01

Organophosphates (OPs) are toxic chemicals commonly used as pesticides and herbicides. Some OPs are highly toxic to humans and have been used in warfare and terrorist attacks. In order to elucidate the molecular mechanisms of injury caused by OPs, the differentially expressed genes were analyzed in human SK-N-SH neuroblastoma cells induced by three OPs. The SK-N-SH cells were treated with one of the three OPs, chlorpyrifos, dichlorvos or methamidophos at LC20 (high-dose), the concentration causing 20% cell death, as well as 1/20 of LC20 (low-dose), a sub-lethal concentration with no detectable cell death, for 24 h. The genome-wide gene changes were identified by Agilent Microarray System, and analyzed by microarray analysis tools. The analysis revealed neuroblastoma cells treated with the high doses of all three OPs markedly activated cell apoptosis and inhibited cell growth and proliferation genes, which would most likely lead to the process of cell death. Interestingly, the analysis also revealed significant decrease in expressions of many genes in a specific spliceosome pathway in cells treated with the low doses of all three different OPs. The change of spliceosome pathway may represent an important mechanism of injury in neuronal cells exposed to low doses of various OPs. In addition to unraveling a potentially different form of OP pathogenesis, this finding could provide a new diagnostic marker in assessing OP-associated injury in cells or tissues. In addition, these results could also contribute to the development of new prevention and/or therapeutic regimens against OP toxicity.

Plasma long noncoding RNA expression profile identified by microarray in patients with Crohn's disease.

PubMed

Chen, Dong; Liu, Jiang; Zhao, Hui-Ying; Chen, Yi-Peng; Xiang, Zun; Jin, Xi

2016-05-21

To investigate the expression pattern of plasma long noncoding RNAs (lncRNAs) in Chrohn's disease (CD) patients. Microarray screening and qRT-PCR verification of lncRNAs and mRNAs were performed in CD and control subjects, followed by hierarchy clustering, GO and KEGG pathway analyses. Significantly dysregulated lncRNAs were categorized into subgroups of antisense lncRNAs, enhancer lncRNAs and lincRNAs. To predict the regulatory effect of lncRNAs on mRNAs, a CNC network analysis was performed and cross linked with significantly changed lncRNAs. The overlapping lncRNAs were randomly selected and verified by qRT-PCR in a larger cohort. Initially, there were 1211 up-regulated and 777 down-regulated lncRNAs as well as 1020 up-regulated and 953 down-regulated mRNAs after microarray analysis; a heat map based on these results showed good categorization into the CD and control groups. GUSBP2 and AF113016 had the highest fold change of the up- and down-regulated lncRNAs, whereas TBC1D17 and CCL3L3 had the highest fold change of the up- and down-regulated mRNAs. Six (SNX1, CYFIP2, CD6, CMTM8, STAT4 and IGFBP7) of 10 mRNAs and 8 (NR_033913, NR_038218, NR_036512, NR_049759, NR_033951, NR_045408, NR_038377 and NR_039976) of 14 lncRNAs showed the same change trends on the microarray and qRT-PCR results with statistical significance. Based on the qRT-PCR verified mRNAs, 1358 potential lncRNAs with 2697 positive correlations and 2287 negative correlations were predicted by the CNC network. The plasma lncRNAs profiles provide preliminary data for the non-invasive diagnosis of CD and a resource for further specific lncRNA-mRNA pathway exploration.

SLEPR: A Sample-Level Enrichment-Based Pathway Ranking Method — Seeking Biological Themes through Pathway-Level Consistency

PubMed Central

Yi, Ming; Stephens, Robert M.

2008-01-01

Analysis of microarray and other high throughput data often involves identification of genes consistently up or down-regulated across samples as the first step in extraction of biological meaning. This gene-level paradigm can be limited as a result of valid sample fluctuations and biological complexities. In this report, we describe a novel method, SLEPR, which eliminates this limitation by relying on pathway-level consistencies. Our method first selects the sample-level differentiated genes from each individual sample, capturing genes missed by other analysis methods, ascertains the enrichment levels of associated pathways from each of those lists, and then ranks annotated pathways based on the consistency of enrichment levels of individual samples from both sample classes. As a proof of concept, we have used this method to analyze three public microarray datasets with a direct comparison with the GSEA method, one of the most popular pathway-level analysis methods in the field. We found that our method was able to reproduce the earlier observations with significant improvements in depth of coverage for validated or expected biological themes, but also produced additional insights that make biological sense. This new method extends existing analyses approaches and facilitates integration of different types of HTP data. PMID:18818771

cDNA microarrays as a tool for identification of biomineralization proteins in the coccolithophorid Emiliania huxleyi (Haptophyta).

PubMed

Quinn, Patrick; Bowers, Robert M; Zhang, Xiaoyu; Wahlund, Thomas M; Fanelli, Michael A; Olszova, Daniela; Read, Betsy A

2006-08-01

Marine unicellular coccolithophore algae produce species-specific calcite scales otherwise known as coccoliths. While the coccoliths and their elaborate architecture have attracted the attention of investigators from various scientific disciplines, our knowledge of the underpinnings of the process of biomineralization in this alga is still in its infancy. The processes of calcification and coccolithogenesis are highly regulated and likely to be complex, requiring coordinated expression of many genes and pathways. In this study, we have employed cDNA microarrays to investigate changes in gene expression associated with biomineralization in the most abundant coccolithophorid, Emiliania huxleyi. Expression profiling of cultures grown under calcifying and noncalcifying conditions has been carried out using cDNA microarrays corresponding to approximately 2,300 expressed sequence tags. A total of 127 significantly up- or down-regulated transcripts were identified using a P value of 0.01 and a change of >2.0-fold. Real-time reverse transcriptase PCR was used to test the overall validity of the microarray data, as well as the relevance of many of the proteins predicted to be associated with biomineralization, including a novel gamma-class carbonic anhydrase (A. R. Soto, H. Zheng, D. Shoemaker, J. Rodriguez, B. A. Read, and T. M. Wahlund, Appl. Environ. Microbiol. 72:5500-5511, 2006). Differentially regulated genes include those related to cellular metabolism, ion channels, transport proteins, vesicular trafficking, and cell signaling. The putative function of the vast majority of candidate transcripts could not be defined. Nonetheless, the data described herein represent profiles of the transcription changes associated with biomineralization-related pathways in E. huxleyi and have identified novel and potentially useful targets for more detailed analysis.

cDNA Microarrays as a Tool for Identification of Biomineralization Proteins in the Coccolithophorid Emiliania huxleyi (Haptophyta)

PubMed Central

Quinn, Patrick; Bowers, Robert M.; Zhang, Xiaoyu; Wahlund, Thomas M.; Fanelli, Michael A.; Olszova, Daniela; Read, Betsy A.

2006-01-01

Marine unicellular coccolithophore algae produce species-specific calcite scales otherwise known as coccoliths. While the coccoliths and their elaborate architecture have attracted the attention of investigators from various scientific disciplines, our knowledge of the underpinnings of the process of biomineralization in this alga is still in its infancy. The processes of calcification and coccolithogenesis are highly regulated and likely to be complex, requiring coordinated expression of many genes and pathways. In this study, we have employed cDNA microarrays to investigate changes in gene expression associated with biomineralization in the most abundant coccolithophorid, Emiliania huxleyi. Expression profiling of cultures grown under calcifying and noncalcifying conditions has been carried out using cDNA microarrays corresponding to approximately 2,300 expressed sequence tags. A total of 127 significantly up- or down-regulated transcripts were identified using a P value of 0.01 and a change of >2.0-fold. Real-time reverse transcriptase PCR was used to test the overall validity of the microarray data, as well as the relevance of many of the proteins predicted to be associated with biomineralization, including a novel gamma-class carbonic anhydrase (A. R. Soto, H. Zheng, D. Shoemaker, J. Rodriguez, B. A. Read, and T. M. Wahlund, Appl. Environ. Microbiol. 72:5500-5511, 2006). Differentially regulated genes include those related to cellular metabolism, ion channels, transport proteins, vesicular trafficking, and cell signaling. The putative function of the vast majority of candidate transcripts could not be defined. Nonetheless, the data described herein represent profiles of the transcription changes associated with biomineralization-related pathways in E. huxleyi and have identified novel and potentially useful targets for more detailed analysis. PMID:16885305

Biologically relevant effects of mRNA amplification on gene expression profiles.

PubMed

van Haaften, Rachel I M; Schroen, Blanche; Janssen, Ben J A; van Erk, Arie; Debets, Jacques J M; Smeets, Hubert J M; Smits, Jos F M; van den Wijngaard, Arthur; Pinto, Yigal M; Evelo, Chris T A

2006-04-11

Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other.Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways.

Biologically relevant effects of mRNA amplification on gene expression profiles

PubMed Central

van Haaften, Rachel IM; Schroen, Blanche; Janssen, Ben JA; van Erk, Arie; Debets, Jacques JM; Smeets, Hubert JM; Smits, Jos FM; van den Wijngaard, Arthur; Pinto, Yigal M; Evelo, Chris TA

2006-01-01

Background Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. Results Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other. Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. Conclusion This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways. PMID:16608515

High throughput gene expression profiling: a molecular approach to integrative physiology

PubMed Central

Liang, Mingyu; Cowley, Allen W; Greene, Andrew S

2004-01-01

Integrative physiology emphasizes the importance of understanding multiple pathways with overlapping, complementary, or opposing effects and their interactions in the context of intact organisms. The DNA microarray technology, the most commonly used method for high-throughput gene expression profiling, has been touted as an integrative tool that provides insights into regulatory pathways. However, the physiology community has been slow in acceptance of these techniques because of early failure in generating useful data and the lack of a cohesive theoretical framework in which experiments can be analysed. With recent advances in both technology and analysis, we propose a concept of multidimensional integration of physiology that incorporates data generated by DNA microarray and other functional, genomic, and proteomic approaches to achieve a truly integrative understanding of physiology. Analysis of several studies performed in simpler organisms or in mammalian model animals supports the feasibility of such multidimensional integration and demonstrates the power of DNA microarray as an indispensable molecular tool for such integration. Evaluation of DNA microarray techniques indicates that these techniques, despite limitations, have advanced to a point where the question-driven profiling research has become a feasible complement to the conventional, hypothesis-driven research. With a keen sense of homeostasis, global regulation, and quantitative analysis, integrative physiologists are uniquely positioned to apply these techniques to enhance the understanding of complex physiological functions. PMID:14678487

Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure.

PubMed

Chater-Diehl, Eric J; Laufer, Benjamin I; Castellani, Christina A; Alberry, Bonnie L; Singh, Shiva M

2016-01-01

The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse's lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as "Free radical scavenging". We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was "Peroxisome biogenesis"; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD.

Non-Small-Cell Lung Cancer Molecular Signatures Recapitulate Lung Developmental Pathways

PubMed Central

Borczuk, Alain C.; Gorenstein, Lyall; Walter, Kristin L.; Assaad, Adel A.; Wang, Liqun; Powell, Charles A.

2003-01-01

Current paradigms hold that lung carcinomas arise from pleuripotent stem cells capable of differentiation into one or several histological types. These paradigms suggest lung tumor cell ontogeny is determined by consequences of gene expression that recapitulate events important in embryonic lung development. Using oligonucleotide microarrays, we acquired gene profiles from 32 microdissected non-small-cell lung tumors. We determined the 100 top-ranked marker genes for adenocarcinoma, squamous cell, large cell, and carcinoid using nearest neighbor analysis. Results were validated by immunostaining for 11 selected proteins using a tissue microarray representing 80 tumors. Gene expression data of lung development were accessed from a publicly available dataset generated with the murine Mu11k genome microarray. Self-organized mapping identified two temporally distinct clusters of murine orthologues. Supervised clustering of lung development data showed large-cell carcinoma gene orthologues were in a cluster expressed in pseudoglandular and canalicular stages whereas adenocarcinoma homologues were predominantly in a cluster expressed later in the terminal sac and alveolar stages of murine lung development. Representative large-cell genes (E2F3, MYBL2, HDAC2, CDK4, PCNA) are expressed in the nucleus and are associated with cell cycle and proliferation. In contrast, adenocarcinoma genes are associated with lung-specific transcription pathways (SFTPB, TTF-1), cell adhesion, and signal transduction. In sum, non-small-cell lung tumors histology gene profiles suggest mechanisms relevant to ontogeny and clinical course. Adenocarcinoma genes are associated with differentiation and glandular formation whereas large-cell genes are associated with proliferation and differentiation arrest. The identification of developmentally regulated pathways active in tumorigenesis provides insights into lung carcinogenesis and suggests early steps may differ according to the eventual tumor morphology. PMID:14578194

Unconventional microarray design reveals the response to obesity is largely tissue specific: analysis of common and divergent responses to diet-induced obesity in insulin-sensitive tissues.

PubMed

Lee, Robyn K; Hittel, Dustin S; Nyamandi, Vongai Z; Kang, Li; Soh, Jung; Sensen, Christoph W; Shearer, Jane

2012-04-01

Obesity is a chronic condition involving the excessive accumulation of adipose tissue that adversely affects all systems in the body. The aim of the present study was to employ an unbiased, genome-wide assessment of transcript abundance in order to identify common gene expression pathways within insulin-sensitive tissues in response to dietary-induced diabetes. Following 20 weeks of chow or high-fat feeding (60% kcal), age-matched mice underwent a euglycemic-hyperinsulinemic clamp to assess insulin sensitivity. High-fat-fed animals were obese and highly insulin resistant, disposing of ∼75% less glucose compared with their chow-fed counterparts. Tissues were collected, and gene expression was examined by microarray in 4 tissues known to exhibit obesity-related metabolic disturbances: white adipose tissue, skeletal muscle, liver, and heart. A total of 463 genes were differentially expressed between diets. Analysis of individual tissues showed skeletal muscle to exhibit the largest number of differentially expressed genes (191) in response to high-fat feeding, followed by adipose tissue (169), liver (115), and heart (65). Analyses revealed that the response of individual genes to obesity is distinct and largely tissue specific, with less than 10% of transcripts being shared among tissues. Although transcripts are largely tissue specific, a systems approach shows numerous commonly activated pathways, including those involved in signal transduction, inflammation, oxidative stress, substrate transport, and metabolism. This suggests a coordinated attempt by tissues to limit metabolic perturbations occurring in early-stage obesity. Many identified genes were associated with a variety of disorders, thereby serving as potential links between obesity and its related health risks.

Gene network and pathway analysis of mice with conditional ablation of Dicer in post-mitotic neurons.

PubMed

Dorval, Véronique; Smith, Pascal Y; Delay, Charlotte; Calvo, Ezequiel; Planel, Emmanuel; Zommer, Nadège; Buée, Luc; Hébert, Sébastien S

2012-01-01

The small non-protein-coding microRNAs (miRNAs) have emerged as critical regulators of neuronal differentiation, identity and survival. To date, however, little is known about the genes and molecular networks regulated by neuronal miRNAs in vivo, particularly in the adult mammalian brain. We analyzed whole genome microarrays from mice lacking Dicer, the enzyme responsible for miRNA production, specifically in postnatal forebrain neurons. A total of 755 mRNA transcripts were significantly (P<0.05, FDR<0.25) misregulated in the conditional Dicer knockout mice. Ten genes, including Tnrc6c, Dnmt3a, and Limk1, were validated by real time quantitative RT-PCR. Upregulated transcripts were enriched in nonneuronal genes, which is consistent with previous studies in vitro. Microarray data mining showed that upregulated genes were enriched in biological processes related to gene expression regulation, while downregulated genes were associated with neuronal functions. Molecular pathways associated with neurological disorders, cellular organization and cellular maintenance were altered in the Dicer mutant mice. Numerous miRNA target sites were enriched in the 3'untranslated region (3'UTR) of upregulated genes, the most significant corresponding to the miR-124 seed sequence. Interestingly, our results suggest that, in addition to miR-124, a large fraction of the neuronal miRNome participates, by order of abundance, in coordinated gene expression regulation and neuronal maintenance. Taken together, these results provide new clues into the role of specific miRNA pathways in the regulation of brain identity and maintenance in adult mice.

Graphite Web: web tool for gene set analysis exploiting pathway topology

PubMed Central

Sales, Gabriele; Calura, Enrica; Martini, Paolo; Romualdi, Chiara

2013-01-01

Graphite web is a novel web tool for pathway analyses and network visualization for gene expression data of both microarray and RNA-seq experiments. Several pathway analyses have been proposed either in the univariate or in the global and multivariate context to tackle the complexity and the interpretation of expression results. These methods can be further divided into ‘topological’ and ‘non-topological’ methods according to their ability to gain power from pathway topology. Biological pathways are, in fact, not only gene lists but can be represented through a network where genes and connections are, respectively, nodes and edges. To this day, the most used approaches are non-topological and univariate although they miss the relationship among genes. On the contrary, topological and multivariate approaches are more powerful, but difficult to be used by researchers without bioinformatic skills. Here we present Graphite web, the first public web server for pathway analysis on gene expression data that combines topological and multivariate pathway analyses with an efficient system of interactive network visualizations for easy results interpretation. Specifically, Graphite web implements five different gene set analyses on three model organisms and two pathway databases. Graphite Web is freely available at http://graphiteweb.bio.unipd.it/. PMID:23666626

Assessing differential expression in two-color microarrays: a resampling-based empirical Bayes approach.

PubMed

Li, Dongmei; Le Pape, Marc A; Parikh, Nisha I; Chen, Will X; Dye, Timothy D

2013-01-01

Microarrays are widely used for examining differential gene expression, identifying single nucleotide polymorphisms, and detecting methylation loci. Multiple testing methods in microarray data analysis aim at controlling both Type I and Type II error rates; however, real microarray data do not always fit their distribution assumptions. Smyth's ubiquitous parametric method, for example, inadequately accommodates violations of normality assumptions, resulting in inflated Type I error rates. The Significance Analysis of Microarrays, another widely used microarray data analysis method, is based on a permutation test and is robust to non-normally distributed data; however, the Significance Analysis of Microarrays method fold change criteria are problematic, and can critically alter the conclusion of a study, as a result of compositional changes of the control data set in the analysis. We propose a novel approach, combining resampling with empirical Bayes methods: the Resampling-based empirical Bayes Methods. This approach not only reduces false discovery rates for non-normally distributed microarray data, but it is also impervious to fold change threshold since no control data set selection is needed. Through simulation studies, sensitivities, specificities, total rejections, and false discovery rates are compared across the Smyth's parametric method, the Significance Analysis of Microarrays, and the Resampling-based empirical Bayes Methods. Differences in false discovery rates controls between each approach are illustrated through a preterm delivery methylation study. The results show that the Resampling-based empirical Bayes Methods offer significantly higher specificity and lower false discovery rates compared to Smyth's parametric method when data are not normally distributed. The Resampling-based empirical Bayes Methods also offers higher statistical power than the Significance Analysis of Microarrays method when the proportion of significantly differentially expressed genes is large for both normally and non-normally distributed data. Finally, the Resampling-based empirical Bayes Methods are generalizable to next generation sequencing RNA-seq data analysis.

Altered gene regulation and synaptic morphology in Drosophila learning and memory mutants

PubMed Central

Guan, Zhuo; Buhl, Lauren K.; Quinn, William G.; Littleton, J. Troy

2011-01-01

Genetic studies in Drosophila have revealed two separable long-term memory pathways defined as anesthesia-resistant memory (ARM) and long-lasting long-term memory (LLTM). ARM is disrupted in radish (rsh) mutants, whereas LLTM requires CREB-dependent protein synthesis. Although the downstream effectors of ARM and LLTM are distinct, pathways leading to these forms of memory may share the cAMP cascade critical for associative learning. Dunce, which encodes a cAMP-specific phosphodiesterase, and rutabaga, which encodes an adenylyl cyclase, both disrupt short-term memory. Amnesiac encodes a pituitary adenylyl cyclase-activating peptide homolog and is required for middle-term memory. Here, we demonstrate that the Radish protein localizes to the cytoplasm and nucleus and is a PKA phosphorylation target in vitro. To characterize how these plasticity pathways may manifest at the synaptic level, we assayed synaptic connectivity and performed an expression analysis to detect altered transcriptional networks in rutabaga, dunce, amnesiac, and radish mutants. All four mutants disrupt specific aspects of synaptic connectivity at larval neuromuscular junctions (NMJs). Genome-wide DNA microarray analysis revealed ∼375 transcripts that are altered in these mutants, suggesting defects in multiple neuronal signaling pathways. In particular, the transcriptional target Lapsyn, which encodes a leucine-rich repeat cell adhesion protein, localizes to synapses and regulates synaptic growth. This analysis provides insights into the Radish-dependent ARM pathway and novel transcriptional targets that may contribute to memory processing in Drosophila. PMID:21422168

Gene expression profiling of whole blood: Comparison of target preparation methods for accurate and reproducible microarray analysis

PubMed Central

Vartanian, Kristina; Slottke, Rachel; Johnstone, Timothy; Casale, Amanda; Planck, Stephen R; Choi, Dongseok; Smith, Justine R; Rosenbaum, James T; Harrington, Christina A

2009-01-01

Background Peripheral blood is an accessible and informative source of transcriptomal information for many human disease and pharmacogenomic studies. While there can be significant advantages to analyzing RNA isolated from whole blood, particularly in clinical studies, the preparation of samples for microarray analysis is complicated by the need to minimize artifacts associated with highly abundant globin RNA transcripts. The impact of globin RNA transcripts on expression profiling data can potentially be reduced by using RNA preparation and labeling methods that remove or block globin RNA during the microarray assay. We compared four different methods for preparing microarray hybridization targets from human whole blood collected in PAXGene tubes. Three of the methods utilized the Affymetrix one-cycle cDNA synthesis/in vitro transcription protocol but varied treatment of input RNA as follows: i. no treatment; ii. treatment with GLOBINclear; or iii. treatment with globin PNA oligos. In the fourth method cDNA targets were prepared with the Ovation amplification and labeling system. Results We find that microarray targets generated with labeling methods that reduce globin mRNA levels or minimize the impact of globin transcripts during hybridization detect more transcripts in the microarray assay compared with the standard Affymetrix method. Comparison of microarray results with quantitative PCR analysis of a panel of genes from the NF-kappa B pathway shows good correlation of transcript measurements produced with all four target preparation methods, although method-specific differences in overall correlation were observed. The impact of freezing blood collected in PAXGene tubes on data reproducibility was also examined. Expression profiles show little or no difference when RNA is extracted from either fresh or frozen blood samples. Conclusion RNA preparation and labeling methods designed to reduce the impact of globin mRNA transcripts can significantly improve the sensitivity of the DNA microarray expression profiling assay for whole blood samples. While blockage of globin transcripts during first strand cDNA synthesis with globin PNAs resulted in the best overall performance in this study, we conclude that selection of a protocol for expression profiling studies in blood should depend on several factors, including implementation requirements of the method and study design. RNA isolated from either freshly collected or frozen blood samples stored in PAXGene tubes can be used without altering gene expression profiles. PMID:19123946

A microarray whole-genome gene expression dataset in a rat model of inflammatory corneal angiogenesis.

PubMed

Mukwaya, Anthony; Lindvall, Jessica M; Xeroudaki, Maria; Peebo, Beatrice; Ali, Zaheer; Lennikov, Anton; Jensen, Lasse Dahl Ejby; Lagali, Neil

2016-11-22

In angiogenesis with concurrent inflammation, many pathways are activated, some linked to VEGF and others largely VEGF-independent. Pathways involving inflammatory mediators, chemokines, and micro-RNAs may play important roles in maintaining a pro-angiogenic environment or mediating angiogenic regression. Here, we describe a gene expression dataset to facilitate exploration of pro-angiogenic, pro-inflammatory, and remodelling/normalization-associated genes during both an active capillary sprouting phase, and in the restoration of an avascular phenotype. The dataset was generated by microarray analysis of the whole transcriptome in a rat model of suture-induced inflammatory corneal neovascularisation. Regions of active capillary sprout growth or regression in the cornea were harvested and total RNA extracted from four biological replicates per group. High quality RNA was obtained for gene expression analysis using microarrays. Fold change of selected genes was validated by qPCR, and protein expression was evaluated by immunohistochemistry. We provide a gene expression dataset that may be re-used to investigate corneal neovascularisation, and may also have implications in other contexts of inflammation-mediated angiogenesis.

Applying Multivariate Adaptive Splines to Identify Genes With Expressions Varying After Diagnosis in Microarray Experiments.

PubMed

Duan, Fenghai; Xu, Ye

2017-01-01

To analyze a microarray experiment to identify the genes with expressions varying after the diagnosis of breast cancer. A total of 44 928 probe sets in an Affymetrix microarray data publicly available on Gene Expression Omnibus from 249 patients with breast cancer were analyzed by the nonparametric multivariate adaptive splines. Then, the identified genes with turning points were grouped by K-means clustering, and their network relationship was subsequently analyzed by the Ingenuity Pathway Analysis. In total, 1640 probe sets (genes) were reliably identified to have turning points along with the age at diagnosis in their expression profiling, of which 927 expressed lower after turning points and 713 expressed higher after the turning points. K-means clustered them into 3 groups with turning points centering at 54, 62.5, and 72, respectively. The pathway analysis showed that the identified genes were actively involved in various cancer-related functions or networks. In this article, we applied the nonparametric multivariate adaptive splines method to a publicly available gene expression data and successfully identified genes with expressions varying before and after breast cancer diagnosis.

DNA Microarray Highlights Nrf2-Mediated Neuron Protection Targeted by Wasabi-Derived Isothiocyanates in IMR-32 Cells

PubMed Central

Trio, Phoebe Zapanta; Fujisaki, Satoru; Tanigawa, Shunsuke; Hisanaga, Ayami; Sakao, Kozue; Hou, De-Xing

2016-01-01

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), 6-(methylthio)hexyl isothiocyanate (6-MTITC), and 4-(methylsulfinyl)butyl isothiocyanate (4-MSITC) are isothiocyanate (ITC) bioactive compounds from Japanese Wasabi. Previous in vivo studies highlighted the neuroprotective potential of ITCs since ITCs enhance the production of antioxidant-related enzymes. Thus, in this present study, a genome-wide DNA microarray analysis was designed to profile gene expression changes in a neuron cell line, IMR-32, stimulated by these ITCs. Among these ITCs, 6-MSITC caused the expression changes of most genes (263), of which 100 genes were upregulated and 163 genes were downregulated. Gene categorization showed that most of the differentially expressed genes are involved in oxidative stress response, and pathway analysis further revealed that Nrf2-mediated oxidative stress pathway is the top of the ITC-modulated signaling pathway. Finally, real-time polymerase chain reaction (PCR) and Western blotting confirmed the gene expression and protein products of the major targets by ITCs. Taken together, Wasabi-derived ITCs might target the Nrf2-mediated oxidative stress pathway to exert neuroprotective effects. PMID:27547033

DNA Microarray Highlights Nrf2-Mediated Neuron Protection Targeted by Wasabi-Derived Isothiocyanates in IMR-32 Cells.

PubMed

Trio, Phoebe Zapanta; Fujisaki, Satoru; Tanigawa, Shunsuke; Hisanaga, Ayami; Sakao, Kozue; Hou, De-Xing

2016-01-01

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), 6-(methylthio)hexyl isothiocyanate (6-MTITC), and 4-(methylsulfinyl)butyl isothiocyanate (4-MSITC) are isothiocyanate (ITC) bioactive compounds from Japanese Wasabi. Previous in vivo studies highlighted the neuroprotective potential of ITCs since ITCs enhance the production of antioxidant-related enzymes. Thus, in this present study, a genome-wide DNA microarray analysis was designed to profile gene expression changes in a neuron cell line, IMR-32, stimulated by these ITCs. Among these ITCs, 6-MSITC caused the expression changes of most genes (263), of which 100 genes were upregulated and 163 genes were downregulated. Gene categorization showed that most of the differentially expressed genes are involved in oxidative stress response, and pathway analysis further revealed that Nrf2-mediated oxidative stress pathway is the top of the ITC-modulated signaling pathway. Finally, real-time polymerase chain reaction (PCR) and Western blotting confirmed the gene expression and protein products of the major targets by ITCs. Taken together, Wasabi-derived ITCs might target the Nrf2-mediated oxidative stress pathway to exert neuroprotective effects.

Seeking unique and common biological themes in multiple gene lists or datasets: pathway pattern extraction pipeline for pathway-level comparative analysis.

PubMed

Yi, Ming; Mudunuri, Uma; Che, Anney; Stephens, Robert M

2009-06-29

One of the challenges in the analysis of microarray data is to integrate and compare the selected (e.g., differential) gene lists from multiple experiments for common or unique underlying biological themes. A common way to approach this problem is to extract common genes from these gene lists and then subject these genes to enrichment analysis to reveal the underlying biology. However, the capacity of this approach is largely restricted by the limited number of common genes shared by datasets from multiple experiments, which could be caused by the complexity of the biological system itself. We now introduce a new Pathway Pattern Extraction Pipeline (PPEP), which extends the existing WPS application by providing a new pathway-level comparative analysis scheme. To facilitate comparing and correlating results from different studies and sources, PPEP contains new interfaces that allow evaluation of the pathway-level enrichment patterns across multiple gene lists. As an exploratory tool, this analysis pipeline may help reveal the underlying biological themes at both the pathway and gene levels. The analysis scheme provided by PPEP begins with multiple gene lists, which may be derived from different studies in terms of the biological contexts, applied technologies, or methodologies. These lists are then subjected to pathway-level comparative analysis for extraction of pathway-level patterns. This analysis pipeline helps to explore the commonality or uniqueness of these lists at the level of pathways or biological processes from different but relevant biological systems using a combination of statistical enrichment measurements, pathway-level pattern extraction, and graphical display of the relationships of genes and their associated pathways as Gene-Term Association Networks (GTANs) within the WPS platform. As a proof of concept, we have used the new method to analyze many datasets from our collaborators as well as some public microarray datasets. This tool provides a new pathway-level analysis scheme for integrative and comparative analysis of data derived from different but relevant systems. The tool is freely available as a Pathway Pattern Extraction Pipeline implemented in our existing software package WPS, which can be obtained at http://www.abcc.ncifcrf.gov/wps/wps_index.php.

Identification of personalized dysregulated pathways in hepatocellular carcinoma.

PubMed

Li, Hong; Jiang, Xiumei; Zhu, Shengjie; Sui, Lihong

2017-04-01

Hepatocellular carcinoma (HCC) is the most common liver malignancy, and ranks the fifth most prevalent malignant tumors worldwide. In general, HCC are detected until the disease is at an advanced stage and may miss the best chance for treatment. Thus, elucidating the molecular mechanisms is critical to clinical diagnosis and treatment for HCC. The purpose of this study was to identify dysregulated pathways of great potential functional relevance in the progression of HCC. Microarray data of 72 pairs of tumor and matched non-tumor surrounding tissues of HCC were transformed to gene expression data. Differentially expressed genes (DEG) between patients and normal controls were identified using Linear Models for Microarray Analysis. Personalized dysregulated pathways were identified using individualized pathway aberrance score module. 169 differentially expressed genes (DEG) were obtained with |logFC|≥1.5 and P≤0.01. 749 dysregulated pathways were obtained with P≤0.01 in pathway statistics, and there were 93 DEG overlapped in the dysregulated pathways. After performing normal distribution analysis, 302 pathways with the aberrance probability≥0.5 were identified. By ranking pathway with aberrance probability, the top 20 pathways were obtained. Only three DEGs (TUBA1C, TPR, CDC20) were involved in the top 20 pathways. These personalized dysregulated pathways and overlapped genes may give new insights into the underlying biological mechanisms in the progression of HCC. Particular attention can be focused on them for further research. Copyright © 2017 Elsevier GmbH. All rights reserved.

Identifying pathogenic processes by integrating microarray data with prior knowledge

PubMed Central

2014-01-01

Background It is of great importance to identify molecular processes and pathways that are involved in disease etiology. Although there has been an extensive use of various high-throughput methods for this task, pathogenic pathways are still not completely understood. Often the set of genes or proteins identified as altered in genome-wide screens show a poor overlap with canonical disease pathways. These findings are difficult to interpret, yet crucial in order to improve the understanding of the molecular processes underlying the disease progression. We present a novel method for identifying groups of connected molecules from a set of differentially expressed genes. These groups represent functional modules sharing common cellular function and involve signaling and regulatory events. Specifically, our method makes use of Bayesian statistics to identify groups of co-regulated genes based on the microarray data, where external information about molecular interactions and connections are used as priors in the group assignments. Markov chain Monte Carlo sampling is used to search for the most reliable grouping. Results Simulation results showed that the method improved the ability of identifying correct groups compared to traditional clustering, especially for small sample sizes. Applied to a microarray heart failure dataset the method found one large cluster with several genes important for the structure of the extracellular matrix and a smaller group with many genes involved in carbohydrate metabolism. The method was also applied to a microarray dataset on melanoma cancer patients with or without metastasis, where the main cluster was dominated by genes related to keratinocyte differentiation. Conclusion Our method found clusters overlapping with known pathogenic processes, but also pointed to new connections extending beyond the classical pathways. PMID:24758699

Multiomics in Grape Berry Skin Revealed Specific Induction of the Stilbene Synthetic Pathway by Ultraviolet-C Irradiation1

PubMed Central

Suzuki, Mami; Nakabayashi, Ryo; Ogata, Yoshiyuki; Sakurai, Nozomu; Tokimatsu, Toshiaki; Goto, Susumu; Suzuki, Makoto; Jasinski, Michal; Martinoia, Enrico; Otagaki, Shungo; Matsumoto, Shogo; Saito, Kazuki; Shiratake, Katsuhiro

2015-01-01

Grape (Vitis vinifera) accumulates various polyphenolic compounds, which protect against environmental stresses, including ultraviolet-C (UV-C) light and pathogens. In this study, we looked at the transcriptome and metabolome in grape berry skin after UV-C irradiation, which demonstrated the effectiveness of omics approaches to clarify important traits of grape. We performed transcriptome analysis using a genome-wide microarray, which revealed 238 genes up-regulated more than 5-fold by UV-C light. Enrichment analysis of Gene Ontology terms showed that genes encoding stilbene synthase, a key enzyme for resveratrol synthesis, were enriched in the up-regulated genes. We performed metabolome analysis using liquid chromatography-quadrupole time-of-flight mass spectrometry, and 2,012 metabolite peaks, including unidentified peaks, were detected. Principal component analysis using the peaks showed that only one metabolite peak, identified as resveratrol, was highly induced by UV-C light. We updated the metabolic pathway map of grape in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and in the KaPPA-View 4 KEGG system, then projected the transcriptome and metabolome data on a metabolic pathway map. The map showed specific induction of the resveratrol synthetic pathway by UV-C light. Our results showed that multiomics is a powerful tool to elucidate the accumulation mechanisms of secondary metabolites, and updated systems, such as KEGG and KaPPA-View 4 KEGG for grape, can support such studies. PMID:25761715

Multiomics in grape berry skin revealed specific induction of the stilbene synthetic pathway by ultraviolet-C irradiation.

PubMed

Suzuki, Mami; Nakabayashi, Ryo; Ogata, Yoshiyuki; Sakurai, Nozomu; Tokimatsu, Toshiaki; Goto, Susumu; Suzuki, Makoto; Jasinski, Michal; Martinoia, Enrico; Otagaki, Shungo; Matsumoto, Shogo; Saito, Kazuki; Shiratake, Katsuhiro

2015-05-01

Grape (Vitis vinifera) accumulates various polyphenolic compounds, which protect against environmental stresses, including ultraviolet-C (UV-C) light and pathogens. In this study, we looked at the transcriptome and metabolome in grape berry skin after UV-C irradiation, which demonstrated the effectiveness of omics approaches to clarify important traits of grape. We performed transcriptome analysis using a genome-wide microarray, which revealed 238 genes up-regulated more than 5-fold by UV-C light. Enrichment analysis of Gene Ontology terms showed that genes encoding stilbene synthase, a key enzyme for resveratrol synthesis, were enriched in the up-regulated genes. We performed metabolome analysis using liquid chromatography-quadrupole time-of-flight mass spectrometry, and 2,012 metabolite peaks, including unidentified peaks, were detected. Principal component analysis using the peaks showed that only one metabolite peak, identified as resveratrol, was highly induced by UV-C light. We updated the metabolic pathway map of grape in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and in the KaPPA-View 4 KEGG system, then projected the transcriptome and metabolome data on a metabolic pathway map. The map showed specific induction of the resveratrol synthetic pathway by UV-C light. Our results showed that multiomics is a powerful tool to elucidate the accumulation mechanisms of secondary metabolites, and updated systems, such as KEGG and KaPPA-View 4 KEGG for grape, can support such studies. © 2015 American Society of Plant Biologists. All Rights Reserved.

Dynamic changes in global microRNAome and transcriptome reveal complex miRNA-mRNA regulated host response to Japanese Encephalitis Virus in microglial cells

PubMed Central

Kumari, Bharti; Jain, Pratistha; Das, Shaoli; Ghosal, Suman; Hazra, Bibhabasu; Trivedi, Ashish Chandra; Basu, Anirban; Chakrabarti, Jayprokas; Vrati, Sudhanshu; Banerjee, Arup

2016-01-01

Microglia cells in the brain play essential role during Japanese Encephalitis Virus (JEV) infection and may lead to change in microRNA (miRNA) and mRNA profile. These changes may together control disease outcome. Using Affymetrix microarray platform, we profiled cellular miRNA and mRNA expression at multiple time points during viral infection in human microglial (CHME3) cells. In silico analysis of microarray data revealed a phased pattern of miRNAs expression, associated with JEV replication and provided unique signatures of infection. Target prediction and pathway enrichment analysis identified anti correlation between differentially expressed miRNA and the gene expression at multiple time point which ultimately affected diverse signaling pathways including Notch signaling pathways in microglia. Activation of Notch pathway during JEV infection was demonstrated in vitro and in vivo. The expression of a subset of miRNAs that target multiple genes in Notch signaling pathways were suppressed and their overexpression could affect JEV induced immune response. Further analysis provided evidence for the possible presence of cellular competing endogenous RNA (ceRNA) associated with innate immune response. Collectively, our data provide a uniquely comprehensive view of the changes in the host miRNAs induced by JEV during cellular infection and identify Notch pathway in modulating microglia mediated inflammation. PMID:26838068

Dynamic changes in global microRNAome and transcriptome reveal complex miRNA-mRNA regulated host response to Japanese Encephalitis Virus in microglial cells.

PubMed

Kumari, Bharti; Jain, Pratistha; Das, Shaoli; Ghosal, Suman; Hazra, Bibhabasu; Trivedi, Ashish Chandra; Basu, Anirban; Chakrabarti, Jayprokas; Vrati, Sudhanshu; Banerjee, Arup

2016-02-03

Microglia cells in the brain play essential role during Japanese Encephalitis Virus (JEV) infection and may lead to change in microRNA (miRNA) and mRNA profile. These changes may together control disease outcome. Using Affymetrix microarray platform, we profiled cellular miRNA and mRNA expression at multiple time points during viral infection in human microglial (CHME3) cells. In silico analysis of microarray data revealed a phased pattern of miRNAs expression, associated with JEV replication and provided unique signatures of infection. Target prediction and pathway enrichment analysis identified anti correlation between differentially expressed miRNA and the gene expression at multiple time point which ultimately affected diverse signaling pathways including Notch signaling pathways in microglia. Activation of Notch pathway during JEV infection was demonstrated in vitro and in vivo. The expression of a subset of miRNAs that target multiple genes in Notch signaling pathways were suppressed and their overexpression could affect JEV induced immune response. Further analysis provided evidence for the possible presence of cellular competing endogenous RNA (ceRNA) associated with innate immune response. Collectively, our data provide a uniquely comprehensive view of the changes in the host miRNAs induced by JEV during cellular infection and identify Notch pathway in modulating microglia mediated inflammation.

A systems biology approach to the pathogenesis of obesity-related nonalcoholic fatty liver disease using reverse phase protein microarrays for multiplexed cell signaling analysis.

PubMed

Calvert, Valerie S; Collantes, Rochelle; Elariny, Hazem; Afendy, Arian; Baranova, Ancha; Mendoza, Michael; Goodman, Zachary; Liotta, Lance A; Petricoin, Emanuel F; Younossi, Zobair M

2007-07-01

Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease. Omental adipose tissue, a biologically active organ secreting adipokines and cytokines, may play a role in the development of NAFLD. We tested this hypothesis with reverse-phase protein microarrays (RPA) for multiplexed cell signaling analysis of adipose tissue from patients with NAFLD. Omental adipose tissue was obtained from 99 obese patients. Liver biopsies obtained at the time of surgery were all read by the same hepatopathologist. Adipose tissue was exposed to rapid pressure cycles to extract protein lysates. RPA was used to investigate intracellular signaling. Analysis of 54 different kinase substrates and cell signaling endpoints showed that an insulin signaling pathway is deranged in different locations in NAFLD patients. Furthermore, components of insulin receptor-mediated signaling differentiate most of the conditions on the NAFLD spectrum. For example, PKA (protein kinase A) and AKT/mTOR (protein kinase B/mammalian target of rapamycin) pathway derangement accurately discriminates patients with NASH from those with the non-progressive forms of NAFLD. PKC (protein kinase C) delta, AKT, and SHC phosphorylation changes occur in patients with simple steatosis. Amounts of the FKHR (forkhead factor Foxo1)phosphorylated at S256 residue were significantly correlated with AST/ALT ratio in all morbidly obese patients. Furthermore, amounts of cleaved caspase 9 and pp90RSK S380 were positively correlated in patients with NASH. Specific insulin pathway signaling events are altered in the adipose tissue of patients with NASH compared with patients with nonprogressive forms of NAFLD. These findings provide evidence for the role of omental fat in the pathogenesis, and potentially, the progression of NAFLD.

A limited innate immune response is induced by a replication-defective herpes simplex virus vector following delivery to the murine central nervous system

PubMed Central

Zeier, Zane; Aguilar, J Santiago; Lopez, Cecilia M; Devi-Rao, G B; Watson, Zachary L; Baker, Henry V; Wagner, Edward K; Bloom, David C

2010-01-01

Herpes simplex virus type 1 (HSV-1)–based vectors readily transduce neurons and have a large payload capacity, making them particularly amenable to gene therapy applications within the central nervous system (CNS). Because aspects of the host responses to HSV-1 vectors in the CNS are largely unknown, we compared the host response of a nonreplicating HSV-1 vector to that of a replication-competent HSV-1 virus using microarray analysis. In parallel, HSV-1 gene expression was tracked using HSV-specific oligonucleotide-based arrays in order to correlate viral gene expression with observed changes in host response. Microarray analysis was performed following stereotactic injection into the right hippocampal formation of mice with either a replication-competent HSV-1 or a nonreplicating recombinant of HSV-1, lacking the ICP4 gene (ICP4−). Genes that demonstrated a significant change (P < .001) in expression in response to the replicating HSV-1 outnumbered those that changed in response to mock or nonreplicating vector by approximately 3-fold. Pathway analysis revealed that both the replicating and nonreplicating vectors induced robust antigen presentation but only mild interferon, chemokine, and cytokine signaling responses. The ICP4− vector was restricted in several of the Toll-like receptor-signaling pathways, indicating reduced stimulation of the innate immune response. These array analyses suggest that although the nonreplicating vector induces detectable activation of immune response pathways, the number and magnitude of the induced response is dramatically restricted compared to the replicating vector, and with the exception of antigen presentation, host gene expression induced by the non-replicating vector largely resembles mock infection. PMID:20095947

Transcriptome analysis supports viral infection and fluoride toxicity as contributors to chronic kidney disease of unknown etiology (CKDu) in Sri Lanka.

PubMed

Sayanthooran, Saravanabavan; Gunerathne, Lishanthe; Abeysekera, Tilak D J; Magana-Arachchi, Dhammika N

2018-05-28

Chronic kidney disease of unknown etiology (CKDu), having epidemic characteristics, is being diagnosed increasingly in certain tropical regions of the world, mainly Latin America and Sri Lanka. They have been observed primarily in farming communities and current hypotheses point toward many environmental and occupational triggers. CKDu does not have common etiologies of chronic kidney disease (CKD) such as hypertension, diabetes, or autoimmune disease. We aimed to understand the molecular processes underlying CKDu in Sri Lanka using transcriptome analysis. RNA extracted from whole blood was reverse transcribed and used for microarray analysis using the Human HT-12 v.4 array (Illumina). Pathway analysis was carried out using ingenuity pathway analysis (IPA-Qiagen). Microarray results were validated using real-time PCR of five selected genes. Pathways related to innate immune response, including interferon signaling, inflammasome signaling and TREM1 signaling had the most significant positive activation z scores, where as EIF2 signaling and mTOR signaling had the most significant negative activation z scores. Pathways previously linked to fluoride toxicity; G-protein activation, Cdc42 signaling, Rac signaling and RhoA signaling were activated in CKDu patients. The most significantly activated biological functions were cell death, cell movement and antimicrobial response. Significant toxicological functions were mitochondrial dysfunction, oxidative stress and apoptosis. Based on the molecular pathway analysis in CKDu patients and review of literature, viral infections and fluoride toxicity appear to be contributing to the molecular mechanisms underlying CKDu.

Differences in gene expression profiles and signaling pathways in rhabdomyolysis-induced acute kidney injury

PubMed Central

Geng, Xiaodong; Wang, Yuanda; Hong, Quan; Yang, Jurong; Zheng, Wei; Zhang, Gang; Cai, Guangyan; Chen, Xiangmei; Wu, Di

2015-01-01

Purpose: Rhabdomyolysis is a threatening syndrome because it causes the breakdown of skeletal muscle. Muscle destruction leads to the release of myoglobin, intracellular proteins, and electrolytes into the circulation. The aim of this study was to investigate the differences in gene expression profiles and signaling pathways upon rhabdomyolysis-induced acute kidney injury (AKI). Methods: In this study, we used glycerol-induced renal injury as a model of rhabdomyolysis-induced AKI. We analyzed data and relevant information from the Gene Expression Omnibus database (No: GSE44925). The gene expression data for three untreated mice were compared to data for five mice with rhabdomyolysis-induced AKI. The expression profiling of the three untreated mice and the five rhabdomyolysis-induced AKI mice was performed using microarray analysis. We examined the levels of Cyp3a13, Rela, Aldh7a1, Jun, CD14. And Cdkn1a using RT-PCR to determine the accuracy of the microarray results. Results: The microarray analysis showed that there were 1050 downregulated and 659 upregulated genes in the rhabdomyolysis-induced AKI mice compared to the control group. The interactions of all differentially expressed genes in the Signal-Net were analyzed. Cyp3a13 and Rela had the most interactions with other genes. The data showed that Rela and Aldh7a1 were the key nodes and had important positions in the Signal-Net. The genes Jun, CD14, and Cdkn1a were also significantly upregulated. The pathway analysis classified the differentially expressed genes into 71 downregulated and 48 upregulated pathways including the PI3K/Akt, MAPK, and NF-κB signaling pathways. Conclusion: The results of this study indicate that the NF-κB, MAPK, PI3K/Akt, and apoptotic pathways are regulated in rhabdomyolysis-induced AKI. PMID:26823722

Analysis of the Metabolic Pathways Affected by Poly(γ-glutamic Acid) in Arabidopsis thaliana Based on GeneChip Microarray.

PubMed

Xu, Zongqi; Lei, Peng; Feng, Xiaohai; Li, Sha; Xu, Hong

2016-08-17

Plant growth is promoted by poly(γ-glutamic acid) (γ-PGA). However, the molecular mechanism underlying such promotion is not yet well understood. Therefore, we used GeneChip microarrays to explore the effects of γ-PGA on gene transcription in Arabidopsis thaliana. Our results revealed 299 genes significantly regulated by γ-PGA. These differently expressed genes participate mainly in metabolic and cellular processes and in stimuli responses. The metabolic pathways linked to these differently expressed genes were also investigated. A total of 64 of the 299 differently expressed genes were shown to be directly involved in 24 pathways such as brassinosteroid biosynthesis, α-linolenic acid metabolism, phenylpropanoid biosynthesis, and nitrogen metabolism, all of which were influenced by γ-PGA. The analysis demonstrated that γ-PGA promoted nitrogen assimilation and biosynthesis of brassinosteroids, jasmonic acid, and lignins, providing a better explanation for why γ-PGA promotes growth and enhances stress tolerance in plants.

Microarray analysis of gene expression profiles in ripening pineapple fruits.

PubMed

Koia, Jonni H; Moyle, Richard L; Botella, Jose R

2012-12-18

Pineapple (Ananas comosus) is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening. Microarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC) analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested. This is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study extends our knowledge of the molecular basis of pineapple fruit ripening and non-climacteric fruit ripening in general.

Microarray analysis of gene expression profiles in ripening pineapple fruits

PubMed Central

2012-01-01

Background Pineapple (Ananas comosus) is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening. Results Microarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC) analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested. Conclusions This is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study extends our knowledge of the molecular basis of pineapple fruit ripening and non-climacteric fruit ripening in general. PMID:23245313

Identification of genes modulated in rheumatoid arthritis using complementary DNA microarray analysis of lymphoblastoid B cell lines from disease-discordant monozygotic twins.

PubMed

Haas, Christian S; Creighton, Chad J; Pi, Xiujun; Maine, Ira; Koch, Alisa E; Haines, G Kenneth; Ling, Song; Chinnaiyan, Arul M; Holoshitz, Joseph

2006-07-01

To identify disease-specific gene expression profiles in patients with rheumatoid arthritis (RA), using complementary DNA (cDNA) microarray analyses on lymphoblastoid B cell lines (LCLs) derived from RA-discordant monozygotic (MZ) twins. The cDNA was prepared from LCLs derived from the peripheral blood of 11 pairs of RA-discordant MZ twins. The RA twin cDNA was labeled with cy5 fluorescent dye, and the cDNA of the healthy co-twin was labeled with cy3. To determine relative expression profiles, cDNA from each twin pair was combined and hybridized on 20,000-element microarray chips. Immunohistochemistry and real-time polymerase chain reaction were used to detect the expression of selected gene products in synovial tissue from patients with RA compared with patients with osteoarthritis and normal healthy controls. In RA twin LCLs compared with healthy co-twin LCLs, 1,163 transcripts were significantly differentially expressed. Of these, 747 were overexpressed and 416 were underexpressed. Gene ontology analysis revealed many genes known to play a role in apoptosis, angiogenesis, proteolysis, and signaling. The 3 most significantly overexpressed genes were laeverin (a novel enzyme with sequence homology to CD13), 11beta-hydroxysteroid dehydrogenase type 2 (a steroid pathway enzyme), and cysteine-rich, angiogenic inducer 61 (a known angiogenic factor). The products of these genes, heretofore uncharacterized in RA, were all abundantly expressed in RA synovial tissues. Microarray cDNA analysis of peripheral blood-derived LCLs from well-controlled patient populations is a useful tool to detect RA-relevant genes and could help in identifying novel therapeutic targets.

Microarray analysis of differentially expressed genes engaged in fruit development between Prunus mume and Prunus armeniaca.

PubMed

Li, Xiaoying; Korir, Nicholas Kibet; Liu, Lili; Shangguan, Lingfei; Wang, Yuzhu; Han, Jian; Chen, Ming; Fang, Jinggui

2012-11-15

Microarray analysis is a technique that can be employed to provide expression profiles of single genes and new insights to elucidate the biological mechanisms responsible for fruit development. To evaluate expression of genes mostly engaged in fruit development between Prunus mume and Prunus armeniaca, we first identified differentially expressed transcripts along the entire fruit life cycle by using microarrays spotted with 10,641 ESTs collected from P. mume and other Prunus EST sequences. A total of 1418 ESTs were selected after quality control of microarray spots and analysis for differential gene expression patterns during fruit development of P. mume and P. Armeniaca. From these, 707 up-regulated and 711 down-regulated genes showing more than two-fold differences in expression level were annotated by GO based on biological processes, molecular functions and cellular components. These differentially expressed genes were found to be involved in several important pathways of carbohydrate, galactose, and starch and sucrose metabolism as well as in biosynthesis of other secondary metabolites via KEGG. This could provide detailed information on the fruit quality differences during development and ripening of these two species. With the results obtained, we provide a practical database for comprehensive understanding of molecular events during fruit development and also lay a theoretical foundation for the cloning of genes regulating in a series of important rate-limiting enzymes involved in vital metabolic pathways during fruit development. Copyright © 2012 Elsevier GmbH. All rights reserved.

Separate enrichment analysis of pathways for up- and downregulated genes.

PubMed

Hong, Guini; Zhang, Wenjing; Li, Hongdong; Shen, Xiaopei; Guo, Zheng

2014-03-06

Two strategies are often adopted for enrichment analysis of pathways: the analysis of all differentially expressed (DE) genes together or the analysis of up- and downregulated genes separately. However, few studies have examined the rationales of these enrichment analysis strategies. Using both microarray and RNA-seq data, we show that gene pairs with functional links in pathways tended to have positively correlated expression levels, which could result in an imbalance between the up- and downregulated genes in particular pathways. We then show that the imbalance could greatly reduce the statistical power for finding disease-associated pathways through the analysis of all-DE genes. Further, using gene expression profiles from five types of tumours, we illustrate that the separate analysis of up- and downregulated genes could identify more pathways that are really pertinent to phenotypic difference. In conclusion, analysing up- and downregulated genes separately is more powerful than analysing all of the DE genes together.

Differential gene expression and a functional analysis of PCB-exposed children: Understanding disease and disorder development

PubMed Central

Dutta, Sisir K; Mitra, Partha S; Ghosh, Somiranjan; Zang, Shizhu; Sonneborn, Dean; Hertz-Picciotto, Irva; Trnovec, Tomas; Palkovicova, Lubica; Sovcikova, Eva; Ghimbovschi, Svetlana; Hoffman, Eric P.

2011-01-01

The goal of the present study is to understand the probable molecular mechanism of toxicities and the associated pathways related to observed pathophysiology in high PCB-exposed populations. We have performed a microarray-based differential gene expression analysis of children (mean age 46.1 months) of Central European descent from Slovak Republic in a well-defined study cohort. The subset of children having high blood PCB concentrations (>75 percentile) were compared against their low PCB counterparts (<25 percentile), with mean lipid-adjusted PCB values of 3.02±1.3 and 0.06±0.03 ng/mg of serum lipid, for the two groups, respectively (18.1±4.4 and 0.3±0.1 ng/ml of serum). The microarray was conducted with the total RNA from the peripheral blood mononuclear cells of the children using an Affymetrix platform (GeneChip Human genome U133 Plus 2.0 Array) and was analyzed by Gene Spring (GX 10.0). A highly significant set of 162 differentially expressed genes between high and low PCB groups (p value <0.00001) were identified and subsequently analyzed using the Ingenuity Pathway Analysis tool. The results indicate that Cell-To-Cell Signaling and Interaction, Cellular Movement, Cell Signaling, Molecular Transport, and Vitamin and Mineral Metabolism were the major molecular and cellular functions associated with the differentially altered gene set in high PCB-exposed children. The differential gene expressions appeared to play a pivotal role in the development of probable diseases and disorders, including cardiovascular disease and cancer, in the PCB-exposed population. The analyses also pointed out possible organ-specific effects, e.g., cardiotoxicity, hepatotoxicity and nephrotoxicity, in high PCB-exposed subjects. A few notable genes, such as BCL2, PON1, and ITGB1, were significantly altered in our study, and the related pathway analysis explained their plausible involvement in the respective disease processes, as mentioned. Our results provided insight into understanding the associated molecular mechanisms of complex gene-environment interactions in a PCB-exposed population. Future endeavors of supervised genotyping of pathway-specific molecular epidemiological studies and population biomarker validations are already underway to reveal individual risk factors in these PCB-exposed populations. PMID:21855147

Insights into significant pathways and gene interaction networks underlying breast cancer cell line MCF-7 treated with 17β-estradiol (E2).

PubMed

Huan, Jinliang; Wang, Lishan; Xing, Li; Qin, Xianju; Feng, Lingbin; Pan, Xiaofeng; Zhu, Ling

2014-01-01

Estrogens are known to regulate the proliferation of breast cancer cells and to alter their cytoarchitectural and phenotypic properties, but the gene networks and pathways by which estrogenic hormones regulate these events are only partially understood. We used global gene expression profiling by Affymetrix GeneChip microarray analysis, with KEGG pathway enrichment, PPI network construction, module analysis and text mining methods to identify patterns and time courses of genes that are either stimulated or inhibited by estradiol (E2) in estrogen receptor (ER)-positive MCF-7 human breast cancer cells. Of the genes queried on the Affymetrix Human Genome U133 plus 2.0 microarray, we identified 628 (12h), 852 (24h) and 880 (48 h) differentially expressed genes (DEGs) that showed a robust pattern of regulation by E2. From pathway enrichment analysis, we found out the changes of metabolic pathways of E2 treated samples at each time point. At 12h time point, the changes of metabolic pathways were mainly focused on pathways in cancer, focal adhesion, and chemokine signaling pathway. At 24h time point, the changes were mainly enriched in neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction and calcium signaling pathway. At 48 h time point, the significant pathways were pathways in cancer, regulation of actin cytoskeleton, cell adhesion molecules (CAMs), axon guidance and ErbB signaling pathway. Of interest, our PPI network analysis and module analysis found that E2 treatment induced enhancement of PRSS23 at the three time points and PRSS23 was in the central position of each module. Text mining results showed that the important genes of DEGs have relationship with signal pathways, such as ERbB pathway (AREG), Wnt pathway (NDP), MAPK pathway (NTRK3, TH), IP3 pathway (TRA@) and some transcript factors (TCF4, MAF). Our studies highlight the diverse gene networks and metabolic and cell regulatory pathways through which E2 operates to achieve its widespread effects on breast cancer cells. © 2013 Elsevier B.V. All rights reserved.

Meta-Analysis of Placental Transcriptome Data Identifies a Novel Molecular Pathway Related to Preeclampsia.

PubMed

van Uitert, Miranda; Moerland, Perry D; Enquobahrie, Daniel A; Laivuori, Hannele; van der Post, Joris A M; Ris-Stalpers, Carrie; Afink, Gijs B

2015-01-01

Studies using the placental transcriptome to identify key molecules relevant for preeclampsia are hampered by a relatively small sample size. In addition, they use a variety of bioinformatics and statistical methods, making comparison of findings challenging. To generate a more robust preeclampsia gene expression signature, we performed a meta-analysis on the original data of 11 placenta RNA microarray experiments, representing 139 normotensive and 116 preeclamptic pregnancies. Microarray data were pre-processed and analyzed using standardized bioinformatics and statistical procedures and the effect sizes were combined using an inverse-variance random-effects model. Interactions between genes in the resulting gene expression signature were identified by pathway analysis (Ingenuity Pathway Analysis, Gene Set Enrichment Analysis, Graphite) and protein-protein associations (STRING). This approach has resulted in a comprehensive list of differentially expressed genes that led to a 388-gene meta-signature of preeclamptic placenta. Pathway analysis highlights the involvement of the previously identified hypoxia/HIF1A pathway in the establishment of the preeclamptic gene expression profile, while analysis of protein interaction networks indicates CREBBP/EP300 as a novel element central to the preeclamptic placental transcriptome. In addition, there is an apparent high incidence of preeclampsia in women carrying a child with a mutation in CREBBP/EP300 (Rubinstein-Taybi Syndrome). The 388-gene preeclampsia meta-signature offers a vital starting point for further studies into the relevance of these genes (in particular CREBBP/EP300) and their concomitant pathways as biomarkers or functional molecules in preeclampsia. This will result in a better understanding of the molecular basis of this disease and opens up the opportunity to develop rational therapies targeting the placental dysfunction causal to preeclampsia.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perkins, Timothy N.; Dentener, Mieke A.

Growth and development of the mature lung is a complex process orchestrated by a number of intricate developmental signaling pathways. Wingless-type MMTV-integration site (WNT) signaling plays critical roles in controlling branching morphogenesis cell differentiation, and formation of the conducting and respiratory airways. In addition, WNT pathways are often re-activated in mature lungs during repair and regeneration. WNT- signaling has been elucidated as a crucial contributor to the development of idiopathic pulmonary fibrosis as well as other hyper-proliferative lung diseases. Silicosis, a detrimental occupational lung disease caused by excessive inhalation of crystalline silica dust, is hallmarked by repeated cycles of damagingmore » inflammation, epithelial hyperplasia, and formation of dense, hyalinized nodules of whorled collagen. However, mechanisms of epithelial cell hyperplasia and matrix deposition are not well understood, as most research efforts have focused on the pronounced inflammatory response. Microarray data from our previous studies has revealed a number of WNT-signaling and WNT-target genes altered by crystalline silica in human lung epithelial cells. In the present study, we utilize pathway analysis to designate connections between genes altered by silica in WNT-signaling networks. Furthermore, we confirm microarray findings by QRT-PCR and demonstrate both activation of canonical (β-catenin) and down-regulation of non-canonical (WNT5A) signaling in immortalized (BEAS-2B) and primary (PBEC) human bronchial epithelial cells. These findings suggest that WNT-signaling and cross-talk with other pathways (e.g. Notch), may contribute to proliferative, fibrogenic and inflammatory responses to silica in lung epithelial cells. - Highlights: • Pathway analysis reveals silica-induced WNT-signaling in lung epithelial cells. • Silica-induced canonical WNT-signaling is mediated by autocrine/paracrine signals. • Crystalline silica decreases non-canonical WNT5A signaling. • Microarray reveals WNT as a novel complex signaling network in silica-mediated injury.« less

Pattern Triggered Immunity (PTI) in Tobacco: Isolation of Activated Genes Suggests Role of the Phenylpropanoid Pathway in Inhibition of Bacterial Pathogens

PubMed Central

Szatmári, Ágnes; Zvara, Ágnes; Móricz, Ágnes M.; Besenyei, Eszter; Szabó, Erika; Ott, Péter G.; Puskás, László G.; Bozsó, Zoltán

2014-01-01

Background Pattern Triggered Immunity (PTI) or Basal Resistance (BR) is a potent, symptomless form of plant resistance. Upon inoculation of a plant with non-pathogens or pathogenicity-mutant bacteria, the induced PTI will prevent bacterial proliferation. Developed PTI is also able to protect the plant from disease or HR (Hypersensitive Response) after a challenging infection with pathogenic bacteria. Our aim was to reveal those PTI-related genes of tobacco (Nicotiana tabacum) that could possibly play a role in the protection of the plant from disease. Methodology/Principal Findings Leaves were infiltrated with Pseudomonas syringae pv. syringae hrcC- mutant bacteria to induce PTI, and samples were taken 6 and 48 hours later. Subtraction Suppressive Hybridization (SSH) resulted in 156 PTI-activated genes. A cDNA microarray was generated from the SSH clone library. Analysis of hybridization data showed that in the early (6 hpi) phase of PTI, among others, genes of peroxidases, signalling elements, heat shock proteins and secondary metabolites were upregulated, while at the late phase (48 hpi) the group of proteolysis genes was newly activated. Microarray data were verified by real time RT-PCR analysis. Almost all members of the phenyl-propanoid pathway (PPP) possibly leading to lignin biosynthesis were activated. Specific inhibition of cinnamic-acid-4-hydroxylase (C4H), rate limiting enzyme of the PPP, decreased the strength of PTI - as shown by the HR-inhibition and electrolyte leakage tests. Quantification of cinnamate and p-coumarate by thin-layer chromatography (TLC)-densitometry supported specific changes in the levels of these metabolites upon elicitation of PTI. Conclusions/Significance We believe to provide first report on PTI-related changes in the levels of these PPP metabolites. Results implicated an actual role of the upregulation of the phenylpropanoid pathway in the inhibition of bacterial pathogenic activity during PTI. PMID:25101956

NRF2-regulated metabolic gene signature as a prognostic biomarker in non-small cell lung cancer

PubMed Central

Namani, Akhileshwar; Cui, Qin Qin; Wu, Yihe; Wang, Hongyan; Wang, Xiu Jun; Tang, Xiuwen

2017-01-01

Mutations in Kelch-like ECH-associated protein 1 (KEAP1) cause the aberrant activation of nuclear factor erythroid-derived 2-like 2 (NRF2), which leads to oncogenesis and drug resistance in lung cancer cells. Our study was designed to identify the genes involved in lung cancer progression targeted by NRF2. A series of microarray experiments in normal and cancer cells, as well as in animal models, have revealed regulatory genes downstream of NRF2 that are involved in wide variety of pathways. Specifically, we carried out individual and combinatorial microarray analysis of KEAP1 overexpression and NRF2 siRNA-knockdown in a KEAP1 mutant-A549 non-small cell lung cancer (NSCLC) cell line. As a result, we identified a list of genes which were mainly involved in metabolic functions in NSCLC by using functional annotation analysis. In addition, we carried out in silico analysis to characterize the antioxidant responsive element sequences in the promoter regions of known and putative NRF2-regulated metabolic genes. We further identified an NRF2-regulated metabolic gene signature (NRMGS) by correlating the microarray data with lung adenocarcinoma RNA-Seq gene expression data from The Cancer Genome Atlas followed by qRT-PCR validation, and finally showed that higher expression of the signature conferred a poor prognosis in 8 independent NSCLC cohorts. Our findings provide novel prognostic biomarkers for NSCLC. PMID:29050246

MAAMD: a workflow to standardize meta-analyses and comparison of affymetrix microarray data

PubMed Central

2014-01-01

Background Mandatory deposit of raw microarray data files for public access, prior to study publication, provides significant opportunities to conduct new bioinformatics analyses within and across multiple datasets. Analysis of raw microarray data files (e.g. Affymetrix CEL files) can be time consuming, complex, and requires fundamental computational and bioinformatics skills. The development of analytical workflows to automate these tasks simplifies the processing of, improves the efficiency of, and serves to standardize multiple and sequential analyses. Once installed, workflows facilitate the tedious steps required to run rapid intra- and inter-dataset comparisons. Results We developed a workflow to facilitate and standardize Meta-Analysis of Affymetrix Microarray Data analysis (MAAMD) in Kepler. Two freely available stand-alone software tools, R and AltAnalyze were embedded in MAAMD. The inputs of MAAMD are user-editable csv files, which contain sample information and parameters describing the locations of input files and required tools. MAAMD was tested by analyzing 4 different GEO datasets from mice and drosophila. MAAMD automates data downloading, data organization, data quality control assesment, differential gene expression analysis, clustering analysis, pathway visualization, gene-set enrichment analysis, and cross-species orthologous-gene comparisons. MAAMD was utilized to identify gene orthologues responding to hypoxia or hyperoxia in both mice and drosophila. The entire set of analyses for 4 datasets (34 total microarrays) finished in ~ one hour. Conclusions MAAMD saves time, minimizes the required computer skills, and offers a standardized procedure for users to analyze microarray datasets and make new intra- and inter-dataset comparisons. PMID:24621103

Macrophage Gene Expression Associated with Remodeling of the Prepartum Rat Cervix: Microarray and Pathway Analyses

PubMed Central

Dobyns, Abigail E.; Goyal, Ravi; Carpenter, Lauren Grisham; Freeman, Tom C.; Longo, Lawrence D.; Yellon, Steven M.

2015-01-01

As the critical gatekeeper for birth, prepartum remodeling of the cervix is associated with increased resident macrophages (Mφ), proinflammatory processes, and extracellular matrix degradation. This study tested the hypothesis that expression of genes unique to Mφs characterizes the prepartum from unremodeled nonpregnant cervix. Perfused cervix from prepartum day 21 postbreeding (D21) or nonpregnant (NP) rats, with or without Mφs, had RNA extracted and whole genome microarray analysis performed. By subtractive analyses, expression of 194 and 120 genes related to Mφs in the cervix from D21 rats were increased and decreased, respectively. In both D21 and NP groups, 158 and 57 Mφ genes were also more or less up- or down-regulated, respectively. Mφ gene expression patterns were most strongly correlated within groups and in 5 major clustering patterns. In the cervix from D21 rats, functional categories and canonical pathways of increased expression by Mφ gene related to extracellular matrix, cell proliferation, differentiation, as well as cell signaling. Pathways were characteristic of inflammation and wound healing, e.g., CD163, CD206, and CCR2. Signatures of only inflammation pathways, e.g., CSF1R, EMR1, and MMP12 were common to both D21 and NP groups. Thus, a novel and complex balance of Mφ genes and clusters differentiated the degraded extracellular matrix and cellular genomic activities in the cervix before birth from the unremodeled state. Predicted Mφ activities, pathways, and networks raise the possibility that expression patterns of specific genes characterize and promote prepartum remodeling of the cervix for parturition at term and with preterm labor. PMID:25811906

Development and Use of Integrated Microarray-Based Genomic Technologies for Assessing Microbial Community Composition and Dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, J.; Wu, L.; Gentry, T.

2006-04-05

To effectively monitor microbial populations involved in various important processes, a 50-mer-based oligonucleotide microarray was developed based on known genes and pathways involved in: biodegradation, metal resistance and reduction, denitrification, nitrification, nitrogen fixation, methane oxidation, methanogenesis, carbon polymer decomposition, and sulfate reduction. This array contains approximately 2000 unique and group-specific probes with <85% similarity to their non-target sequences. Based on artificial probes, our results showed that at hybridization conditions of 50 C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appearedmore » to be specific to their corresponding target genes. Detection limits were about 5-10ng genomic DNA in the absence of background DNA, and 50-100ng ({approx}1.3{sup o} 10{sup 7} cells) in the presence background DNA. Strong linear relationships between signal intensity and target DNA and RNA concentration were observed (r{sup 2} = 0.95-0.99). Application of this microarray to naphthalene-amended enrichments and soil microcosms demonstrated that composition of the microflora varied depending on incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in enrichments, the genes involved in naphthalene degradation from Gram-negative microorganisms such as Ralstonia, Comamonas, and Burkholderia were most abundant in the soil microcosms (as well as those for polyaromatic hydrocarbon and nitrotoluene degradation). Although naphthalene degradation is widely known and studied in Pseudomonas, Pseudomonas genes were not detected in either system. Real-time PCR analysis of 4 representative genes was consistent with microarray-based quantification (r{sup 2} = 0.95). Currently, we are also applying this microarray to the study of several different microbial communities and processes at the NABIR-FRC in Oak Ridge, TN. One project involves the monitoring of the development and dynamics of the microbial community of a fluidized bed reactor (FBR) used for reducing nitrate and the other project monitors microbial community responses to stimulation of uranium reducing populations via ethanol donor additions in situ and in a model system. Additionally, we are developing novel strategies for increasing microarray hybridization sensitivity. Finally, great improvements to our methods of probe design were made by the development of a new computer program, CommOligo. CommOligo designs unique and group-specific oligo probes for whole-genomes, metagenomes, and groups of environmental sequences and uses a new global alignment algorithm to design single or multiple probes for each gene or group. We are now using this program to design a more comprehensive functional gene array for environmental studies. Overall, our results indicate that the 50mer-based microarray technology has potential as a specific and quantitative tool to reveal the composition of microbial communities and their dynamics important to processes within contaminated environments.« less

FH535, a β-catenin pathway inhibitor, represses pancreatic cancer xenograft growth and angiogenesis

PubMed Central

Gong, Fei-Ran; Zhou, Binhua P.; Lian, Lian; Shen, Bairong; Chen, Kai; Duan, Weiming; Wu, Meng-Yao; Tao, Min; Li, Wei

2016-01-01

The WNT/β-catenin pathway plays an important role in pancreatic cancer carcinogenesis. We evaluated the correlation between aberrant β-catenin pathway activation and the prognosis pancreatic cancer, and the potential of applying the β-catenin pathway inhibitor FH535 to pancreatic cancer treatment. Meta-analysis and immunohistochemistry showed that abnormal β-catenin pathway activation was associated with unfavorable outcome. FH535 repressed pancreatic cancer xenograft growth in vivo. Gene Ontology (GO) analysis of microarray data indicated that target genes responding to FH535 participated in stemness maintenance. Real-time PCR and flow cytometry confirmed that FH535 downregulated CD24 and CD44, pancreatic cancer stem cell (CSC) markers, suggesting FH535 impairs pancreatic CSC stemness. GO analysis of β-catenin chromatin immunoprecipitation sequencing data identified angiogenesis-related gene regulation. Immunohistochemistry showed that higher microvessel density correlated with elevated nuclear β-catenin expression and unfavorable outcome. FH535 repressed the secretion of the proangiogenic cytokines vascular endothelial growth factor (VEGF), interleukin (IL)-6, IL-8, and tumor necrosis factor-α, and also inhibited angiogenesis in vitro and in vivo. Protein and mRNA microarrays revealed that FH535 downregulated the proangiogenic genes ANGPT2, VEGFR3, IFN-γ, PLAUR, THPO, TIMP1, and VEGF. FH535 not only represses pancreatic CSC stemness in vitro, but also remodels the tumor microenvironment by repressing angiogenesis, warranting further clinical investigation. PMID:27323403

Transcriptomic profile of host response in mouse brain after exposure to plant toxin abrin.

PubMed

Bhaskar, A S Bala; Gupta, Nimesh; Rao, P V Lakshmana

2012-09-04

Abrin toxin is a plant glycoprotein, which is similar in structure and properties to ricin and is obtained from the seeds of Abrus precatorius (jequirity bean). Abrin is highly toxic, with an estimated human fatal dose of 0.1-1 μg/kg, and has caused death after accidental and intentional poisoning. Abrin is a potent biological toxin warfare agent. There are no chemical antidotes available against the toxin. Neurological symptoms like delirium, hallucinations, reduced consciousness and generalized seizures were reported in human poisoning cases. Death of a patient with symptoms of acute demyelinating encephalopathy with gastrointestinal bleeding due to ingestion of abrin seeds was reported in India. The aim of this study was to examine both dose and time-dependent transcriptional responses induced by abrin in the adult mouse brain. Mice (n=6) were exposed to 1 and 2 LD50 (2.83 and 5.66 μg/kg respectively) dose of abrin by intraperitoneal route and observed over 3 days. A subset of animals (n=3) were sacrificed at 1 and 2 day intervals for microarray and histopathology analysis. None of the 2 LD50 exposed animals survived till 3 days. The histopathological analysis showed the severe damage in brain and the infiltration of inflammatory cells in a dose and time dependent manner. The abrin exposure resulted in the induction of rapid immune and inflammatory response in brain. Clinical biochemistry parameters like lactate dehydrogenase, aspartate aminotransferase, urea and creatinine showed significant increase at 2-day 2 LD50 exposure. The whole genome microarray data revealed the significant regulation of various pathways like MAPK pathway, cytokine-cytokine receptor interaction, calcium signaling pathway, Jak-STAT signaling pathway and natural killer cell mediated toxicity. The comparison of differential gene expression at both the doses showed dose dependent effects of abrin toxicity. The real-time qRT-PCR analysis of selected genes supported the microarray data. This is the first report on host-gene response using whole genome microarray in an animal model after abrin exposure. The data generated provides leads for developing suitable medical counter measures against abrin poisoning. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Transcriptome profiling of a Saccharomyces cerevisiae mutant with a constitutively activated Ras/cAMP pathway.

PubMed

Jones, D L; Petty, J; Hoyle, D C; Hayes, A; Ragni, E; Popolo, L; Oliver, S G; Stateva, L I

2003-12-16

Often changes in gene expression levels have been considered significant only when above/below some arbitrarily chosen threshold. We investigated the effect of applying a purely statistical approach to microarray analysis and demonstrated that small changes in gene expression have biological significance. Whole genome microarray analysis of a pde2Delta mutant, constructed in the Saccharomyces cerevisiae reference strain FY23, revealed altered expression of approximately 11% of protein encoding genes. The mutant, characterized by constitutive activation of the Ras/cAMP pathway, has increased sensitivity to stress, reduced ability to assimilate nonfermentable carbon sources, and some cell wall integrity defects. Applying the Munich Information Centre for Protein Sequences (MIPS) functional categories revealed increased expression of genes related to ribosome biogenesis and downregulation of genes in the cell rescue, defense, cell death and aging category, suggesting a decreased response to stress conditions. A reduced level of gene expression in the unfolded protein response pathway (UPR) was observed. Cell wall genes whose expression was affected by this mutation were also identified. Several of the cAMP-responsive orphan genes, upon further investigation, revealed cell wall functions; others had previously unidentified phenotypes assigned to them. This investigation provides a statistical global transcriptome analysis of the cellular response to constitutive activation of the Ras/cAMP pathway.

Tumor Necrosis Factor-α Regulates Distinct Molecular Pathways and Gene Networks in Cultured Skeletal Muscle Cells

PubMed Central

Gupta, Sanjay K.; Dahiya, Saurabh; Lundy, Robert F.; Kumar, Ashok

2010-01-01

Background Skeletal muscle wasting is a debilitating consequence of large number of disease states and conditions. Tumor necrosis factor-α (TNF-α) is one of the most important muscle-wasting cytokine, elevated levels of which cause significant muscular abnormalities. However, the underpinning molecular mechanisms by which TNF-α causes skeletal muscle wasting are less well-understood. Methodology/Principal Findings We have used microarray, quantitative real-time PCR (QRT-PCR), Western blot, and bioinformatics tools to study the effects of TNF-α on various molecular pathways and gene networks in C2C12 cells (a mouse myoblastic cell line). Microarray analyses of C2C12 myotubes treated with TNF-α (10 ng/ml) for 18h showed differential expression of a number of genes involved in distinct molecular pathways. The genes involved in nuclear factor-kappa B (NF-kappaB) signaling, 26s proteasome pathway, Notch1 signaling, and chemokine networks are the most important ones affected by TNF-α. The expression of some of the genes in microarray dataset showed good correlation in independent QRT-PCR and Western blot assays. Analysis of TNF-treated myotubes showed that TNF-α augments the activity of both canonical and alternative NF-κB signaling pathways in myotubes. Bioinformatics analyses of microarray dataset revealed that TNF-α affects the activity of several important pathways including those involved in oxidative stress, hepatic fibrosis, mitochondrial dysfunction, cholesterol biosynthesis, and TGF-β signaling. Furthermore, TNF-α was found to affect the gene networks related to drug metabolism, cell cycle, cancer, neurological disease, organismal injury, and abnormalities in myotubes. Conclusions TNF-α regulates the expression of multiple genes involved in various toxic pathways which may be responsible for TNF-induced muscle loss in catabolic conditions. Our study suggests that TNF-α activates both canonical and alternative NF-κB signaling pathways in a time-dependent manner in skeletal muscle cells. The study provides novel insight into the mechanisms of action of TNF-α in skeletal muscle cells. PMID:20967264

Time course of gene expression during mouse skeletal muscle hypertrophy

PubMed Central

Lee, Jonah D.; England, Jonathan H.; Esser, Karyn A.; McCarthy, John J.

2013-01-01

The purpose of this study was to perform a comprehensive transcriptome analysis during skeletal muscle hypertrophy to identify signaling pathways that are operative throughout the hypertrophic response. Global gene expression patterns were determined from microarray results on days 1, 3, 5, 7, 10, and 14 during plantaris muscle hypertrophy induced by synergist ablation in adult mice. Principal component analysis and the number of differentially expressed genes (cutoffs ≥2-fold increase or ≥50% decrease compared with control muscle) revealed three gene expression patterns during overload-induced hypertrophy: early (1 day), intermediate (3, 5, and 7 days), and late (10 and 14 days) patterns. Based on the robust changes in total RNA content and in the number of differentially expressed genes, we focused our attention on the intermediate gene expression pattern. Ingenuity Pathway Analysis revealed a downregulation of genes encoding components of the branched-chain amino acid degradation pathway during hypertrophy. Among these genes, five were predicted by Ingenuity Pathway Analysis or previously shown to be regulated by the transcription factor Kruppel-like factor-15, which was also downregulated during hypertrophy. Moreover, the integrin-linked kinase signaling pathway was activated during hypertrophy, and the downregulation of muscle-specific micro-RNA-1 correlated with the upregulation of five predicted targets associated with the integrin-linked kinase pathway. In conclusion, we identified two novel pathways that may be involved in muscle hypertrophy, as well as two upstream regulators (Kruppel-like factor-15 and micro-RNA-1) that provide targets for future studies investigating the importance of these pathways in muscle hypertrophy. PMID:23869057

Time course of gene expression during mouse skeletal muscle hypertrophy.

PubMed

Chaillou, Thomas; Lee, Jonah D; England, Jonathan H; Esser, Karyn A; McCarthy, John J

2013-10-01

The purpose of this study was to perform a comprehensive transcriptome analysis during skeletal muscle hypertrophy to identify signaling pathways that are operative throughout the hypertrophic response. Global gene expression patterns were determined from microarray results on days 1, 3, 5, 7, 10, and 14 during plantaris muscle hypertrophy induced by synergist ablation in adult mice. Principal component analysis and the number of differentially expressed genes (cutoffs ≥2-fold increase or ≥50% decrease compared with control muscle) revealed three gene expression patterns during overload-induced hypertrophy: early (1 day), intermediate (3, 5, and 7 days), and late (10 and 14 days) patterns. Based on the robust changes in total RNA content and in the number of differentially expressed genes, we focused our attention on the intermediate gene expression pattern. Ingenuity Pathway Analysis revealed a downregulation of genes encoding components of the branched-chain amino acid degradation pathway during hypertrophy. Among these genes, five were predicted by Ingenuity Pathway Analysis or previously shown to be regulated by the transcription factor Kruppel-like factor-15, which was also downregulated during hypertrophy. Moreover, the integrin-linked kinase signaling pathway was activated during hypertrophy, and the downregulation of muscle-specific micro-RNA-1 correlated with the upregulation of five predicted targets associated with the integrin-linked kinase pathway. In conclusion, we identified two novel pathways that may be involved in muscle hypertrophy, as well as two upstream regulators (Kruppel-like factor-15 and micro-RNA-1) that provide targets for future studies investigating the importance of these pathways in muscle hypertrophy.

Global gene expression and Ingenuity biological functions analysis on PCBs 153 and 138 induced human PBMC in vitro reveals differential mode(s) of action in developing toxicities.

PubMed

Ghosh, Somiranjan; Zang, Shizhu; Mitra, Partha S; Ghimbovschi, Svetlana; Hoffman, Eric P; Dutta, Sisir K

2011-07-01

Several reports have indicated that low level of polychlorinated biphenyl (PCB) exposure can adversely affect a multitude of physiological disorders and diseases in in vitro, in vivo, and as reported in epidemiological studies. This investigation is focused on the possible contribution of two most prevalent PCB congeners in vitro in developing toxicities. We used PCBs 138 and 153 at the human equivalence level as model agents to test their specificity in developing toxicities. We chose a global approach using oligonucleotide microarray technology to investigate modulated gene expression for biological effects, upon exposure of PCBs, followed by Ingenuity Pathway Analysis (IPA), to understand the underlying consequence in developing disease and disorders. We performed in vitro studies with human peripheral blood mononuclear cells (PBMC), where PBMC cells were exposed to respective PCBs for 48 h. Overall, our observation on gene expression indicated that PCB produces a unique signature affecting different pathways, specific for each congener. While analyzing these data through IPA, the prominent and interesting disease and disorders were neurological disease, cancer, cardiovascular disease, respiratory disease, as well as endocrine system disorders, genetic disorders, and reproductive system disease. They showed strong resemblances with in vitro, in vivo, and in the epidemiological studies. A distinct difference was observed in renal and urological diseases, organisimal injury and abnormalities, dental disease, ophthalmic disease, and psychological disorders, which are only revealed by PCB 138 exposure, but not in PCB 153. The present study emphasizes the challenges of global gene expression in vitro and was correlated with the results of exposed human population. The microarray results give a molecular mechanistic insight and functional effects, following PCB exposure. The extent of changes in genes related to several possible mode(s) of action highlights the changes in cellular functions and signaling pathways that play major roles. In addition to understanding the pathways related to mode of action for chemicals, these data could lead to the identification of genomic signatures that could be used for screening of chemicals for their potential to cause disease and developmental disorders. Copyright © 2011 Elsevier Ltd. All rights reserved.

Exploring Regulatory Mechanisms of Atrial Myocyte Hypertrophy of Mitral Regurgitation through Gene Expression Profiling Analysis: Role of NFAT in Cardiac Hypertrophy

PubMed Central

Chang, Tzu-Hao; Chen, Mien-Cheng; Chang, Jen-Ping; Huang, Hsien-Da; Ho, Wan-Chun; Lin, Yu-Sheng; Pan, Kuo-Li; Huang, Yao-Kuang; Liu, Wen-Hao; Wu, Chia-Chen

2016-01-01

Background Left atrial enlargement in mitral regurgitation (MR) predicts a poor prognosis. The regulatory mechanisms of atrial myocyte hypertrophy of MR patients remain unknown. Methods and Results This study comprised 14 patients with MR, 7 patients with aortic valve disease (AVD), and 6 purchased samples from normal subjects (NC). We used microarrays, enrichment analysis and quantitative RT-PCR to study the gene expression profiles in the left atria. Microarray results showed that 112 genes were differentially up-regulated and 132 genes were differentially down-regulated in the left atria between MR patients and NC. Enrichment analysis of differentially expressed genes demonstrated that “NFAT in cardiac hypertrophy” pathway was not only one of the significant associated canonical pathways, but also the only one predicted with a non-zero score of 1.34 (i.e. activated) through Ingenuity Pathway Analysis molecule activity predictor. Ingenuity Pathway Analysis Global Molecular Network analysis exhibited that the highest score network also showed high association with cardiac related pathways and functions. Therefore, 5 NFAT associated genes (PPP3R1, PPP3CB, CAMK1, MEF2C, PLCE1) were studies for validation. The mRNA expressions of PPP3CB and MEF2C were significantly up-regulated, and CAMK1 and PPP3R1 were significantly down-regulated in MR patients compared to NC. Moreover, MR patients had significantly increased mRNA levels of PPP3CB, MEF2C and PLCE1 compared to AVD patients. The atrial myocyte size of MR patients significantly exceeded that of the AVD patients and NC. Conclusions Differentially expressed genes in the “NFAT in cardiac hypertrophy” pathway may play a critical role in the atrial myocyte hypertrophy of MR patients. PMID:27907007

NeAT: a toolbox for the analysis of biological networks, clusters, classes and pathways.

PubMed

Brohée, Sylvain; Faust, Karoline; Lima-Mendez, Gipsi; Sand, Olivier; Janky, Rekin's; Vanderstocken, Gilles; Deville, Yves; van Helden, Jacques

2008-07-01

The network analysis tools (NeAT) (http://rsat.ulb.ac.be/neat/) provide a user-friendly web access to a collection of modular tools for the analysis of networks (graphs) and clusters (e.g. microarray clusters, functional classes, etc.). A first set of tools supports basic operations on graphs (comparison between two graphs, neighborhood of a set of input nodes, path finding and graph randomization). Another set of programs makes the connection between networks and clusters (graph-based clustering, cliques discovery and mapping of clusters onto a network). The toolbox also includes programs for detecting significant intersections between clusters/classes (e.g. clusters of co-expression versus functional classes of genes). NeAT are designed to cope with large datasets and provide a flexible toolbox for analyzing biological networks stored in various databases (protein interactions, regulation and metabolism) or obtained from high-throughput experiments (two-hybrid, mass-spectrometry and microarrays). The web interface interconnects the programs in predefined analysis flows, enabling to address a series of questions about networks of interest. Each tool can also be used separately by entering custom data for a specific analysis. NeAT can also be used as web services (SOAP/WSDL interface), in order to design programmatic workflows and integrate them with other available resources.

Altered Molecular Expression of the TLR4/NF-κB Signaling Pathway in Mammary Tissue of Chinese Holstein Cattle with Mastitis

PubMed Central

Wu, Jie; Li, Lian; Sun, Yu; Huang, Shuai; Tang, Juan; Yu, Pan; Wang, Genlin

2015-01-01

Toll-like receptor 4 (TLR4) mediated activation of the nuclear transcription factor κB (NF-κB) signaling pathway by mastitis initiates expression of genes associated with inflammation and the innate immune response. In this study, the profile of mastitis-induced differential gene expression in the mammary tissue of Chinese Holstein cattle was investigated by Gene-Chip microarray and bioinformatics. The microarray results revealed that 79 genes associated with the TLR4/NF-κB signaling pathway were differentially expressed. Of these genes, 19 were up-regulated and 29 were down-regulated in mastitis tissue compared to normal, healthy tissue. Statistical analysis of transcript and protein level expression changes indicated that 10 genes, namely TLR4, MyD88, IL-6, and IL-10, were up-regulated, while, CD14, TNF-α, MD-2, IL-β, NF-κB, and IL-12 were significantly down-regulated in mastitis tissue in comparison with normal tissue. Analyses using bioinformatics database resources, such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and the Gene Ontology Consortium (GO) for term enrichment analysis, suggested that these differently expressed genes implicate different regulatory pathways for immune function in the mammary gland. In conclusion, our study provides new evidence for better understanding the differential expression and mechanisms of the TLR4 /NF-κB signaling pathway in Chinese Holstein cattle with mastitis. PMID:25706977

Altered molecular expression of the TLR4/NF-κB signaling pathway in mammary tissue of Chinese Holstein cattle with mastitis.

PubMed

Wu, Jie; Li, Lian; Sun, Yu; Huang, Shuai; Tang, Juan; Yu, Pan; Wang, Genlin

2015-01-01

Toll-like receptor 4 (TLR4) mediated activation of the nuclear transcription factor κB (NF-κB) signaling pathway by mastitis initiates expression of genes associated with inflammation and the innate immune response. In this study, the profile of mastitis-induced differential gene expression in the mammary tissue of Chinese Holstein cattle was investigated by Gene-Chip microarray and bioinformatics. The microarray results revealed that 79 genes associated with the TLR4/NF-κB signaling pathway were differentially expressed. Of these genes, 19 were up-regulated and 29 were down-regulated in mastitis tissue compared to normal, healthy tissue. Statistical analysis of transcript and protein level expression changes indicated that 10 genes, namely TLR4, MyD88, IL-6, and IL-10, were up-regulated, while, CD14, TNF-α, MD-2, IL-β, NF-κB, and IL-12 were significantly down-regulated in mastitis tissue in comparison with normal tissue. Analyses using bioinformatics database resources, such as the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and the Gene Ontology Consortium (GO) for term enrichment analysis, suggested that these differently expressed genes implicate different regulatory pathways for immune function in the mammary gland. In conclusion, our study provides new evidence for better understanding the differential expression and mechanisms of the TLR4 /NF-κB signaling pathway in Chinese Holstein cattle with mastitis.

Identification of Genes in the Phenylalanine Metabolic Pathway by Ectopic Expression of a MYB Transcription Factor in Tomato Fruit[W

PubMed Central

Dal Cin, Valeriano; Tieman, Denise M.; Tohge, Takayuki; McQuinn, Ryan; de Vos, Ric C.H.; Osorio, Sonia; Schmelz, Eric A.; Taylor, Mark G.; Smits-Kroon, Miriam T.; Schuurink, Robert C.; Haring, Michel A.; Giovannoni, James; Fernie, Alisdair R.; Klee, Harry J.

2011-01-01

Altering expression of transcription factors can be an effective means to coordinately modulate entire metabolic pathways in plants. It can also provide useful information concerning the identities of genes that constitute metabolic networks. Here, we used ectopic expression of a MYB transcription factor, Petunia hybrida ODORANT1, to alter Phe and phenylpropanoid metabolism in tomato (Solanum lycopersicum) fruits. Despite the importance of Phe and phenylpropanoids to plant and human health, the pathway for Phe synthesis has not been unambiguously determined. Microarray analysis of ripening fruits from transgenic and control plants permitted identification of a suite of coregulated genes involved in synthesis and further metabolism of Phe. The pattern of coregulated gene expression facilitated discovery of the tomato gene encoding prephenate aminotransferase, which converts prephenate to arogenate. The expression and biochemical data establish an arogenate pathway for Phe synthesis in tomato fruits. Metabolic profiling and 13C flux analysis of ripe fruits further revealed large increases in the levels of a specific subset of phenylpropanoid compounds. However, while increased levels of these human nutrition-related phenylpropanoids may be desirable, there were no increases in levels of Phe-derived flavor volatiles. PMID:21750236

Identification of Wnt Pathway Target Genes Regulating the Division and Differentiation of Larval Seam Cells and Vulval Precursor Cells in Caenorhabditis elegans

PubMed Central

Gorrepati, Lakshmi; Krause, Michael W.; Chen, Weiping; Brodigan, Thomas M.; Correa-Mendez, Margarita; Eisenmann, David M.

2015-01-01

The evolutionarily conserved Wnt/β-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector β-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/β-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type–specific "mRNA tagging" to enrich for VPC and seam cell–specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type–specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells. PMID:26048561

Identification of Wnt Pathway Target Genes Regulating the Division and Differentiation of Larval Seam Cells and Vulval Precursor Cells in Caenorhabditis elegans.

PubMed

Gorrepati, Lakshmi; Krause, Michael W; Chen, Weiping; Brodigan, Thomas M; Correa-Mendez, Margarita; Eisenmann, David M

2015-06-05

The evolutionarily conserved Wnt/β-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector β-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/β-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type-specific "mRNA tagging" to enrich for VPC and seam cell-specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type-specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells. Copyright © 2015 Gorrepati et al.

Bi-directional gene set enrichment and canonical correlation analysis identify key diet-sensitive pathways and biomarkers of metabolic syndrome.

PubMed

Morine, Melissa J; McMonagle, Jolene; Toomey, Sinead; Reynolds, Clare M; Moloney, Aidan P; Gormley, Isobel C; Gaora, Peadar O; Roche, Helen M

2010-10-07

Currently, a number of bioinformatics methods are available to generate appropriate lists of genes from a microarray experiment. While these lists represent an accurate primary analysis of the data, fewer options exist to contextualise those lists. The development and validation of such methods is crucial to the wider application of microarray technology in the clinical setting. Two key challenges in clinical bioinformatics involve appropriate statistical modelling of dynamic transcriptomic changes, and extraction of clinically relevant meaning from very large datasets. Here, we apply an approach to gene set enrichment analysis that allows for detection of bi-directional enrichment within a gene set. Furthermore, we apply canonical correlation analysis and Fisher's exact test, using plasma marker data with known clinical relevance to aid identification of the most important gene and pathway changes in our transcriptomic dataset. After a 28-day dietary intervention with high-CLA beef, a range of plasma markers indicated a marked improvement in the metabolic health of genetically obese mice. Tissue transcriptomic profiles indicated that the effects were most dramatic in liver (1270 genes significantly changed; p < 0.05), followed by muscle (601 genes) and adipose (16 genes). Results from modified GSEA showed that the high-CLA beef diet affected diverse biological processes across the three tissues, and that the majority of pathway changes reached significance only with the bi-directional test. Combining the liver tissue microarray results with plasma marker data revealed 110 CLA-sensitive genes showing strong canonical correlation with one or more plasma markers of metabolic health, and 9 significantly overrepresented pathways among this set; each of these pathways was also significantly changed by the high-CLA diet. Closer inspection of two of these pathways--selenoamino acid metabolism and steroid biosynthesis--illustrated clear diet-sensitive changes in constituent genes, as well as strong correlations between gene expression and plasma markers of metabolic syndrome independent of the dietary effect. Bi-directional gene set enrichment analysis more accurately reflects dynamic regulatory behaviour in biochemical pathways, and as such highlighted biologically relevant changes that were not detected using a traditional approach. In such cases where transcriptomic response to treatment is exceptionally large, canonical correlation analysis in conjunction with Fisher's exact test highlights the subset of pathways showing strongest correlation with the clinical markers of interest. In this case, we have identified selenoamino acid metabolism and steroid biosynthesis as key pathways mediating the observed relationship between metabolic health and high-CLA beef. These results indicate that this type of analysis has the potential to generate novel transcriptome-based biomarkers of disease.

Bi-directional gene set enrichment and canonical correlation analysis identify key diet-sensitive pathways and biomarkers of metabolic syndrome

PubMed Central

2010-01-01

Background Currently, a number of bioinformatics methods are available to generate appropriate lists of genes from a microarray experiment. While these lists represent an accurate primary analysis of the data, fewer options exist to contextualise those lists. The development and validation of such methods is crucial to the wider application of microarray technology in the clinical setting. Two key challenges in clinical bioinformatics involve appropriate statistical modelling of dynamic transcriptomic changes, and extraction of clinically relevant meaning from very large datasets. Results Here, we apply an approach to gene set enrichment analysis that allows for detection of bi-directional enrichment within a gene set. Furthermore, we apply canonical correlation analysis and Fisher's exact test, using plasma marker data with known clinical relevance to aid identification of the most important gene and pathway changes in our transcriptomic dataset. After a 28-day dietary intervention with high-CLA beef, a range of plasma markers indicated a marked improvement in the metabolic health of genetically obese mice. Tissue transcriptomic profiles indicated that the effects were most dramatic in liver (1270 genes significantly changed; p < 0.05), followed by muscle (601 genes) and adipose (16 genes). Results from modified GSEA showed that the high-CLA beef diet affected diverse biological processes across the three tissues, and that the majority of pathway changes reached significance only with the bi-directional test. Combining the liver tissue microarray results with plasma marker data revealed 110 CLA-sensitive genes showing strong canonical correlation with one or more plasma markers of metabolic health, and 9 significantly overrepresented pathways among this set; each of these pathways was also significantly changed by the high-CLA diet. Closer inspection of two of these pathways - selenoamino acid metabolism and steroid biosynthesis - illustrated clear diet-sensitive changes in constituent genes, as well as strong correlations between gene expression and plasma markers of metabolic syndrome independent of the dietary effect. Conclusion Bi-directional gene set enrichment analysis more accurately reflects dynamic regulatory behaviour in biochemical pathways, and as such highlighted biologically relevant changes that were not detected using a traditional approach. In such cases where transcriptomic response to treatment is exceptionally large, canonical correlation analysis in conjunction with Fisher's exact test highlights the subset of pathways showing strongest correlation with the clinical markers of interest. In this case, we have identified selenoamino acid metabolism and steroid biosynthesis as key pathways mediating the observed relationship between metabolic health and high-CLA beef. These results indicate that this type of analysis has the potential to generate novel transcriptome-based biomarkers of disease. PMID:20929581

Querying Co-regulated Genes on Diverse Gene Expression Datasets Via Biclustering.

PubMed

Deveci, Mehmet; Küçüktunç, Onur; Eren, Kemal; Bozdağ, Doruk; Kaya, Kamer; Çatalyürek, Ümit V

2016-01-01

Rapid development and increasing popularity of gene expression microarrays have resulted in a number of studies on the discovery of co-regulated genes. One important way of discovering such co-regulations is the query-based search since gene co-expressions may indicate a shared role in a biological process. Although there exist promising query-driven search methods adapting clustering, they fail to capture many genes that function in the same biological pathway because microarray datasets are fraught with spurious samples or samples of diverse origin, or the pathways might be regulated under only a subset of samples. On the other hand, a class of clustering algorithms known as biclustering algorithms which simultaneously cluster both the items and their features are useful while analyzing gene expression data, or any data in which items are related in only a subset of their samples. This means that genes need not be related in all samples to be clustered together. Because many genes only interact under specific circumstances, biclustering may recover the relationships that traditional clustering algorithms can easily miss. In this chapter, we briefly summarize the literature using biclustering for querying co-regulated genes. Then we present a novel biclustering approach and evaluate its performance by a thorough experimental analysis.

Microarray and Pathway Analysis Reveal Distinct Mechanisms Underlying Cannabinoid-Mediated Modulation of LPS-Induced Activation of BV-2 Microglial Cells

PubMed Central

Juknat, Ana; Kozela, Ewa; Rimmerman, Neta; Levy, Rivka; Gao, Fuying; Coppola, Giovanni; Geschwind, Daniel; Vogel, Zvi

2013-01-01

Cannabinoids are known to exert immunosuppressive activities. However, the mechanisms which contribute to these effects are unknown. Using lipopolysaccharide (LPS) to activate BV-2 microglial cells, we examined how Δ9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, and cannabidiol (CBD) the non-psychoactive component, modulate the inflammatory response. Microarray analysis of genome-wide mRNA levels was performed using Illumina platform and the resulting expression patterns analyzed using the Ingenuity Pathway Analysis to identify functional subsets of genes, and the Ingenuity System Database to denote the gene networks regulated by CBD and THC. From the 5338 transcripts that were differentially expressed across treatments, 400 transcripts were found to be upregulated by LPS, 502 by CBD+LPS and 424 by THC+LPS, while 145 were downregulated by LPS, 297 by CBD+LPS and 149 by THC+LPS, by 2-fold or more (p≤0.005). Results clearly link the effects of CBD and THC to inflammatory signaling pathways and identify new cannabinoid targets in the MAPK pathway (Dusp1, Dusp8, Dusp2), cell cycle related (Cdkn2b, Gadd45a) as well as JAK/STAT regulatory molecules (Socs3, Cish, Stat1). The impact of CBD on LPS-stimulated gene expression was greater than that of THC. We attribute this difference to the fact that CBD highly upregulated several genes encoding negative regulators of both NFκB and AP-1 transcriptional activities, such as Trib3 and Dusp1 known to be modulated through Nrf2 activation. The CBD-specific expression profile reflected changes associated with oxidative stress and glutathione depletion via Trib3 and expression of ATF4 target genes. Furthermore, the CBD affected genes were shown to be controlled by nuclear factors usually involved in regulation of stress response and inflammation, mainly via Nrf2/Hmox1 axis and the Nrf2/ATF4-Trib3 pathway. These observations indicate that CBD, and less so THC, induce a cellular stress response and that this response underlies their high immunosuppressant activities. PMID:23637839

Differential Adipose Tissue Gene Expression Profiles in Abacavir Treated Patients That May Contribute to the Understanding of Cardiovascular Risk: A Microarray Study

PubMed Central

Shahmanesh, Mohsen; Phillips, Kenneth; Boothby, Meg; Tomlinson, Jeremy W.

2015-01-01

Objective To compare changes in gene expression by microarray from subcutaneous adipose tissue from HIV treatment naïve patients treated with efavirenz based regimens containing abacavir (ABC), tenofovir (TDF) or zidovidine (AZT). Design Subcutaneous fat biopsies were obtained before, at 6- and 18–24-months after treatment, and from HIV negative controls. Groups were age, ethnicity, weight, biochemical profile, and pre-treatment CD4 count matched. Microarray data was generated using the Agilent Whole Human Genome Microarray. Identification of differentially expressed genes and genomic response pathways was performed using limma and gene set enrichment analysis. Results There were significant divergences between ABC and the other two groups 6 months after treatment in genes controlling cell adhesion and environmental information processing, with some convergence at 18–24 months. Compared to controls the ABC group, but not AZT or TDF showed enrichment of genes controlling adherence junction, at 6 months and 18–24 months (adjusted p<0.05) and focal adhesions and tight junction at 6 months (p<0.5). Genes controlling leukocyte transendothelial migration (p<0.05) and ECM-receptor interactions (p = 0.04) were over-expressed in ABC compared to TDF and AZT at 6 months but not at 18–24 months. Enrichment of pathways and individual genes controlling cell adhesion and environmental information processing were specifically dysregulated in the ABC group in comparison with other treatments. There was little difference between AZT and TDF. Conclusion After initiating treatment, there is divergence in the expression of genes controlling cell adhesion and environmental information processing between ABC and both TDF and AZT in subcutaneous adipose tissue. If similar changes are also taking place in other tissues including the coronary vasculature they may contribute to the increased risk of cardiovascular events reported in patients recently started on abacavir-containing regimens. PMID:25617630

Francisella tularensis alters human neutrophil gene expression: insights into the molecular basis of delayed neutrophil apoptosis

PubMed Central

Schwartz, Justin T.; Bandyopadhyay, Sarmistha; Kobayashi, Scott D.; McCracken, Jenna; Whitney, Adeline R.; DeLeo, Frank R.; Allen, Lee-Ann H.

2013-01-01

We demonstrated recently that Francisella tularensis profoundly impairs human neutrophil apoptosis, but how this is achieved is largely unknown. Herein we used human oligonucleotide microarrays to test the hypothesis that changes in neutrophil gene expression contribute to this phenotype, and now demonstrate that F. tularensis live vaccine strain (LVS) caused significant changes in neutrophil gene expression over a 24 h time period relative to the uninfected controls. Of ~47,000 genes analyzed, 3,435 were significantly up- or down-regulated by LVS, including 365 unique genes associated with apoptosis and cell survival. Specific targets in this category included genes associated with the intrinsic and extrinsic apoptotic pathways (CFLAR, TNFAIP3, TNFRSF10D, SOD2, BCL2A1, BIRC4, PIM2, TNFSF10, TNFRSF10C, CASP2, and CASP8) and genes that act via the NF B pathway and other mechanisms to prolong cell viability (NFKB1, NFKB2, and RELA, IL1B, CAST, CDK2, GADD45B, BCL3, BIRC3, CDK2, IL1A, PBEF1, IL6, CXCL1, CCL4 and VEGF). The microarray data were confirmed by qPCR and pathway analysis. Moreover, we demonstrate that X-linked inhibitor of apoptosis (XIAP) protein remained abundant in PMNs over 48 h of LVS infection, whereas BAX mRNA and protein were progressively down-regulated. These data strongly suggest that antiapoptotic and pro-survival mechanisms collaborate to sustain the viability of F. tularensis infected neutrophils. PMID:22986450

Global alteration in gene expression profiles of deciduas from women with idiopathic recurrent pregnancy loss.

PubMed

Krieg, S A; Fan, X; Hong, Y; Sang, Q-X; Giaccia, A; Westphal, L M; Lathi, R B; Krieg, A J; Nayak, N R

2012-09-01

Recurrent pregnancy loss (RPL) occurs in ∼5% of women. However, the etiology is still poorly understood. Defects in decidualization of the endometrium during early pregnancy contribute to several pregnancy complications, such as pre-eclampsia and intrauterine growth restriction (IUGR), and are believed to be important in the pathogenesis of idiopathic RPL. We performed microarray analysis to identify gene expression alterations in the deciduas of idiopathic RPL patients. Control patients had one antecedent term delivery, but were undergoing dilation and curettage for current aneuploid miscarriage. Gene expression differences were evaluated using both pathway and gene ontology (GO) analysis. Selected genes were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). A total of 155 genes were found to be significantly dysregulated in the deciduas of RPL patients (>2-fold change, P < 0.05), with 22 genes up-regulated and 133 genes down-regulated. GO analysis linked a large percentage of genes to discrete biological functions, including immune response (23%), cell signaling (18%) and cell invasion (17.1%), and pathway analysis revealed consistent changes in both the interleukin 1 (IL-1) and IL-8 pathways. All genes in the IL-8 pathway were up-regulated while genes in the IL-1 pathway were down-regulated. Although both pathways can promote inflammation, IL-1 pathway activity is important for normal implantation. Additionally, genes known to be critical for degradation of the extracellular matrix, including matrix metalloproteinase 26 and serine peptidase inhibitor Kazal-type 1, were also highly up-regulated. In this first microarray approach to decidual gene expression in RPL patients, our data suggest that dysregulation of genes associated with cell invasion and immunity may contribute significantly to idiopathic recurrent miscarriage.

Identification of novel and known oocyte-specific genes using complementary DNA subtraction and microarray analysis in three different species.

PubMed

Vallée, Maud; Gravel, Catherine; Palin, Marie-France; Reghenas, Hélène; Stothard, Paul; Wishart, David S; Sirard, Marc-André

2005-07-01

The main objective of the present study was to identify novel oocyte-specific genes in three different species: bovine, mouse, and Xenopus laevis. To achieve this goal, two powerful technologies were combined: a polymerase chain reaction (PCR)-based cDNA subtraction, and cDNA microarrays. Three subtractive libraries consisting of 3456 clones were established and enriched for oocyte-specific transcripts. Sequencing analysis of the positive insert-containing clones resulted in the following classification: 53% of the clones corresponded to known cDNAs, 26% were classified as uncharacterized cDNAs, and a final 9% were classified as novel sequences. All these clones were used for cDNA microarray preparation. Results from these microarray analyses revealed that in addition to already known oocyte-specific genes, such as GDF9, BMP15, and ZP, known genes with unknown function in the oocyte were identified, such as a MLF1-interacting protein (MLF1IP), B-cell translocation gene 4 (BTG4), and phosphotyrosine-binding protein (xPTB). Furthermore, 15 novel oocyte-specific genes were validated by reverse transcription-PCR to confirm their preferential expression in the oocyte compared to somatic tissues. The results obtained in the present study confirmed that microarray analysis is a robust technique to identify true positives from the suppressive subtractive hybridization experiment. Furthermore, obtaining oocyte-specific genes from three species simultaneously allowed us to look at important genes that are conserved across species. Further characterization of these novel oocyte-specific genes will lead to a better understanding of the molecular mechanisms related to the unique functions found in the oocyte.

Whole-Genome Analysis of the SHORT-ROOT Developmental Pathway in Arabidopsis

PubMed Central

Busch, Wolfgang; Cui, Hongchang; Wang, Jean Y; Blilou, Ikram; Hassan, Hala; Nakajima, Keiji; Matsumoto, Noritaka; Lohmann, Jan U; Scheres, Ben

2006-01-01

Stem cell function during organogenesis is a key issue in developmental biology. The transcription factor SHORT-ROOT (SHR) is a critical component in a developmental pathway regulating both the specification of the root stem cell niche and the differentiation potential of a subset of stem cells in the Arabidopsis root. To obtain a comprehensive view of the SHR pathway, we used a statistical method called meta-analysis to combine the results of several microarray experiments measuring the changes in global expression profiles after modulating SHR activity. Meta-analysis was first used to identify the direct targets of SHR by combining results from an inducible form of SHR driven by its endogenous promoter, ectopic expression, followed by cell sorting and comparisons of mutant to wild-type roots. Eight putative direct targets of SHR were identified, all with expression patterns encompassing subsets of the native SHR expression domain. Further evidence for direct regulation by SHR came from binding of SHR in vivo to the promoter regions of four of the eight putative targets. A new role for SHR in the vascular cylinder was predicted from the expression pattern of several direct targets and confirmed with independent markers. The meta-analysis approach was then used to perform a global survey of the SHR indirect targets. Our analysis suggests that the SHR pathway regulates root development not only through a large transcription regulatory network but also through hormonal pathways and signaling pathways using receptor-like kinases. Taken together, our results not only identify the first nodes in the SHR pathway and a new function for SHR in the development of the vascular tissue but also reveal the global architecture of this developmental pathway. PMID:16640459

Sex-Related Differences in Rat Choroid Plexus and Cerebrospinal Fluid: A cDNA Microarray and Proteomic Analysis.

PubMed

Quintela, T; Marcelino, H; Deery, M J; Feret, R; Howard, J; Lilley, K S; Albuquerque, T; Gonçalves, I; Duarte, A C; Santos, C R A

2016-01-01

The choroid plexus (CP) epithelium is a unique structure in the brain that forms an interface between the peripheral blood and the cerebrospinal fluid (CSF), which is mostly produced by the CP itself. Because the CP transcriptome is regulated by the sex hormone background, the present study compared gene/protein expression profiles in the CP and CSF from male and female rats aiming to better understand sex-related differences in CP functions and brain physiology. We used data previously obtained by cDNA microarrays to compare the CP transcriptome between male and female rats, and complemented these data with the proteomic analysis of the CSF of castrated and sham-operated males and females. Microarray analysis showed that 17 128 and 17 002 genes are expressed in the male and female CP, which allowed the functional annotation of 141 and 134 pathways, respectively. Among the most expressed genes, canonical pathways associated with mitochondrial dysfunctions and oxidative phosphorylation were the most prominent, whereas the most relevant molecular and cellular functions annotated were protein synthesis, cellular growth and proliferation, cell death and survival, molecular transport, and protein trafficking. No significant differences were found between males and females regarding these pathways. Seminal functions of the CP differentially regulated between sexes were circadian rhythm signalling, as well as several canonical pathways related to stem cell differentiation, metabolism and the barrier function of the CP. The proteomic analysis identified five down-regulated proteins in the CSF samples from male rats compared to females and seven proteins exhibiting marked variation in the CSF of gonadectomised males compared to sham animals, whereas no differences were found between sham and ovariectomised females. These data clearly show sex-related differences in CP gene expression and CSF protein composition that may impact upon neurological diseases. © 2015 British Society for Neuroendocrinology.

Multiplex giant magnetoresistive biosensor microarrays identify interferon-associated autoantibodies in systemic lupus erythematosus

NASA Astrophysics Data System (ADS)

Lee, Jung-Rok; Haddon, D. James; Wand, Hannah E.; Price, Jordan V.; Diep, Vivian K.; Hall, Drew A.; Petri, Michelle; Baechler, Emily C.; Balboni, Imelda M.; Utz, Paul J.; Wang, Shan X.

2016-06-01

High titer, class-switched autoantibodies are a hallmark of systemic lupus erythematosus (SLE). Dysregulation of the interferon (IFN) pathway is observed in individuals with active SLE, although the association of specific autoantibodies with chemokine score, a combined measurement of three IFN-regulated chemokines, is not known. To identify autoantibodies associated with chemokine score, we developed giant magnetoresistive (GMR) biosensor microarrays, which allow the parallel measurement of multiple serum antibodies to autoantigens and peptides. We used the microarrays to analyze serum samples from SLE patients and found individuals with high chemokine scores had significantly greater reactivity to 13 autoantigens than individuals with low chemokine scores. Our findings demonstrate that multiple autoantibodies, including antibodies to U1-70K and modified histone H2B tails, are associated with IFN dysregulation in SLE. Further, they show the microarrays are capable of identifying autoantibodies associated with relevant clinical manifestations of SLE, with potential for use as biomarkers in clinical practice.

Inferring Molecular Processes Heterogeneity from Transcriptional Data.

PubMed

Gogolewski, Krzysztof; Wronowska, Weronika; Lech, Agnieszka; Lesyng, Bogdan; Gambin, Anna

2017-01-01

RNA microarrays and RNA-seq are nowadays standard technologies to study the transcriptional activity of cells. Most studies focus on tracking transcriptional changes caused by specific experimental conditions. Information referring to genes up- and downregulation is evaluated analyzing the behaviour of relatively large population of cells by averaging its properties. However, even assuming perfect sample homogeneity, different subpopulations of cells can exhibit diverse transcriptomic profiles, as they may follow different regulatory/signaling pathways. The purpose of this study is to provide a novel methodological scheme to account for possible internal, functional heterogeneity in homogeneous cell lines, including cancer ones. We propose a novel computational method to infer the proportion between subpopulations of cells that manifest various functional behaviour in a given sample. Our method was validated using two datasets from RNA microarray experiments. Both experiments aimed to examine cell viability in specific experimental conditions. The presented methodology can be easily extended to RNA-seq data as well as other molecular processes. Moreover, it complements standard tools to indicate most important networks from transcriptomic data and in particular could be useful in the analysis of cancer cell lines affected by biologically active compounds or drugs.

Inferring Molecular Processes Heterogeneity from Transcriptional Data

PubMed Central

Wronowska, Weronika; Lesyng, Bogdan; Gambin, Anna

2017-01-01

RNA microarrays and RNA-seq are nowadays standard technologies to study the transcriptional activity of cells. Most studies focus on tracking transcriptional changes caused by specific experimental conditions. Information referring to genes up- and downregulation is evaluated analyzing the behaviour of relatively large population of cells by averaging its properties. However, even assuming perfect sample homogeneity, different subpopulations of cells can exhibit diverse transcriptomic profiles, as they may follow different regulatory/signaling pathways. The purpose of this study is to provide a novel methodological scheme to account for possible internal, functional heterogeneity in homogeneous cell lines, including cancer ones. We propose a novel computational method to infer the proportion between subpopulations of cells that manifest various functional behaviour in a given sample. Our method was validated using two datasets from RNA microarray experiments. Both experiments aimed to examine cell viability in specific experimental conditions. The presented methodology can be easily extended to RNA-seq data as well as other molecular processes. Moreover, it complements standard tools to indicate most important networks from transcriptomic data and in particular could be useful in the analysis of cancer cell lines affected by biologically active compounds or drugs. PMID:29362714

Transcriptomic meta-analysis identifies gene expression characteristics in various samples of HIV-infected patients with nonprogressive disease.

PubMed

Zhang, Le-Le; Zhang, Zi-Ning; Wu, Xian; Jiang, Yong-Jun; Fu, Ya-Jing; Shang, Hong

2017-09-12

A small proportion of HIV-infected patients remain clinically and/or immunologically stable for years, including elite controllers (ECs) who have undetectable viremia (<50 copies/ml) and long-term nonprogressors (LTNPs) who maintain normal CD4 + T cell counts for prolonged periods (>10 years). However, the mechanism of nonprogression needs to be further resolved. In this study, a transcriptome meta-analysis was performed on nonprogressor and progressor microarray data to identify differential transcriptome pathways and potential biomarkers. Using the INMEX (integrative meta-analysis of expression data) program, we performed the meta-analysis to identify consistently differentially expressed genes (DEGs) in nonprogressors and further performed functional interpretation (gene ontology analysis and pathway analysis) of the DEGs identified in the meta-analysis. Five microarray datasets (81 cases and 98 controls in total), including whole blood, CD4 + and CD8 + T cells, were collected for meta-analysis. We determined that nonprogressors have reduced expression of important interferon-stimulated genes (ISGs), CD38, lymphocyte activation gene 3 (LAG-3) in whole blood, CD4 + and CD8 + T cells. Gene ontology (GO) analysis showed a significant enrichment in DEGs that function in the type I interferon signaling pathway. Upregulated pathways, including the PI3K-Akt signaling pathway in whole blood, cytokine-cytokine receptor interaction in CD4 + T cells and the MAPK signaling pathway in CD8 + T cells, were identified in nonprogressors compared with progressors. In each metabolic functional category, the number of downregulated DEGs was more than the upregulated DEGs, and almost all genes were downregulated DEGs in the oxidative phosphorylation (OXPHOS) and tricarboxylic acid (TCA) cycle in the three types of samples. Our transcriptomic meta-analysis provides a comprehensive evaluation of the gene expression profiles in major blood types of nonprogressors, providing new insights in the understanding of HIV pathogenesis and developing strategies to delay HIV disease progression.

Profiling conserved biological pathways in Autosomal Dominant Polycystic Kidney Disorder (ADPKD) to elucidate key transcriptomic alterations regulating cystogenesis: A cross-species meta-analysis approach.

PubMed

Chatterjee, Shatakshee; Verma, Srikant Prasad; Pandey, Priyanka

2017-09-05

Initiation and progression of fluid filled cysts mark Autosomal Dominant Polycystic Kidney Disease (ADPKD). Thus, improved therapeutics targeting cystogenesis remains a constant challenge. Microarray studies in single ADPKD animal models species with limited sample sizes tend to provide scattered views on underlying ADPKD pathogenesis. Thus we aim to perform a cross species meta-analysis to profile conserved biological pathways that might be key targets for therapy. Nine ADPKD microarray datasets on rat, mice and human fulfilled our study criteria and were chosen. Intra-species combined analysis was performed after considering removal of batch effect. Significantly enriched GO biological processes and KEGG pathways were computed and their overlap was observed. For the conserved pathways, biological modules and gene regulatory networks were observed. Additionally, Gene Set Enrichment Analysis (GSEA) using Molecular Signature Database (MSigDB) was performed for genes found in conserved pathways. We obtained 28 modules of significantly enriched GO processes and 5 major functional categories from significantly enriched KEGG pathways conserved in human, mice and rats that in turn suggest a global transcriptomic perturbation affecting cyst - formation, growth and progression. Significantly enriched pathways obtained from up-regulated genes such as Genomic instability, Protein localization in ER and Insulin Resistance were found to regulate cyst formation and growth whereas cyst progression due to increased cell adhesion and inflammation was suggested by perturbations in Angiogenesis, TGF-beta, CAMs, and Infection related pathways. Additionally, networks revealed shared genes among pathways e.g. SMAD2 and SMAD7 in Endocytosis and TGF-beta. Our study suggests cyst formation and progression to be an outcome of interplay between a set of several key deregulated pathways. Thus, further translational research is warranted focusing on developing a combinatorial therapeutic approach for ADPKD redressal. Copyright © 2017 Elsevier B.V. All rights reserved.

Microarray pathway analysis indicated that mitogen-activated protein kinase/extracellular signal-regulated kinase and insulin growth factor 1 signaling pathways were inhibited by small interfering RNA against AT-rich interactive domain 1A in endometrial cancer

PubMed Central

Yang, Ye; Bao, Wei; Sang, Zhengyu; Yang, Yongbing; Lu, Meng; Xi, Xiaowei

2018-01-01

Mutations in the gene encoding AT-rich interactive domain 1A (ARID1A) are frequently observed in endometrial cancer (EC) but the molecular mechanisms linking the genetic changes remain to be fully understood. The present study aimed to elucidate the influence of ARID1A mutations on signaling pathways. Missense, synonymous and nonsense heterozygous ARID1A mutations in the EC HEC-1-A cell line were verified by Sanger sequencing. Mutated ARID1A small interfering RNA was transfected into HEC-1-A cells. Biochemical microarray analysis revealed 13 upregulated pathways, 17 downregulated pathways, 14 significantly affected disease states and functions, 662 upstream and 512 downstream genes in mutated ARID1A-depleted HEC-1-A cells, among which the mitogen-activated protein kinase/extracellular signal-regulated kinase and insulin-like growth factor-1 (IGF1) signaling pathways were the 2 most downregulated pathways. Furthermore, the forkhead box protein O1 pathway was upregulated, while the IGF1 receptor, insulin receptor substrate 1 and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit b pathways were downregulated. Carcinoma tumorigenesis, tumor cell mitosis and tumor cell death were significantly upregulated disease states and functions, while cell proliferation and tumor growth were significantly downregulated. The results of the present study suggested that ARID1A may be a potential prognostic and therapeutic molecular drug target for the prevention of EC progression. PMID:29399196

Mechanical instability and titanium particles induce similar transcriptomic changes in a rat model for periprosthetic osteolysis and aseptic loosening.

PubMed

Amirhosseini, Mehdi; Andersson, Göran; Aspenberg, Per; Fahlgren, Anna

2017-12-01

Wear debris particles released from prosthetic bearing surfaces and mechanical instability of implants are two main causes of periprosthetic osteolysis. While particle-induced loosening has been studied extensively, mechanisms through which mechanical factors lead to implant loosening have been less investigated. This study compares the transcriptional profiles associated with osteolysis in a rat model for aseptic loosening, induced by either mechanical instability or titanium particles. Rats were exposed to mechanical instability or titanium particles. After 15 min, 3, 48 or 120 h from start of the stimulation, gene expression changes in periprosthetic bone tissue was determined by microarray analysis. Microarray data were analyzed by PANTHER Gene List Analysis tool and Ingenuity Pathway Analysis (IPA). Both types of osteolytic stimulation led to gene regulation in comparison to unstimulated controls after 3, 48 or 120 h. However, when mechanical instability was compared to titanium particles, no gene showed a statistically significant difference (fold change ≥ ± 1.5 and adjusted p-value ≤ 0.05) at any time point. There was a remarkable similarity in numbers and functional classification of regulated genes. Pathway analysis showed several inflammatory pathways activated by both stimuli, including Acute Phase Response signaling, IL-6 signaling and Oncostatin M signaling. Quantitative PCR confirmed the changes in expression of key genes involved in osteolysis observed by global transcriptomics. Inflammatory mediators including interleukin (IL)-6, IL-1β, chemokine (C-C motif) ligand (CCL)2, prostaglandin-endoperoxide synthase (Ptgs)2 and leukemia inhibitory factor (LIF) showed strong upregulation, as assessed by both microarray and qPCR. By investigating genome-wide expression changes we show that, despite the different nature of mechanical implant instability and titanium particles, osteolysis seems to be induced through similar biological and signaling pathways in this rat model for aseptic loosening. Pathways associated to the innate inflammatory response appear to be a major driver for osteolysis. Our findings implicate early restriction of inflammation to be critical to prevent or mitigate osteolysis and aseptic loosening of orthopedic implants.

A Systems Biology Methodology Combining Transcriptome and Interactome Datasets to Assess the Implications of Cytokinin Signaling for Plant Immune Networks.

PubMed

Kunz, Meik; Dandekar, Thomas; Naseem, Muhammad

2017-01-01

Cytokinins (CKs) play an important role in plant growth and development. Also, several studies highlight the modulatory implications of CKs for plant-pathogen interaction. However, the underlying mechanisms of CK mediating immune networks in plants are still not fully understood. A detailed analysis of high-throughput transcriptome (RNA-Seq and microarrays) datasets under modulated conditions of plant CKs and its mergence with cellular interactome (large-scale protein-protein interaction data) has the potential to unlock the contribution of CKs to plant defense. Here, we specifically describe a detailed systems biology methodology pertinent to the acquisition and analysis of various omics datasets that delineate the role of plant CKs in impacting immune pathways in Arabidopsis.

CHIR99021 promotes self-renewal of mouse embryonic stem cells by modulation of protein-encoding gene and long intergenic non-coding RNA expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Yongyan; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A and F University, Yangling 712100, Shaanxi; Ai, Zhiying

2013-10-15

Embryonic stem cells (ESCs) can proliferate indefinitely in vitro and differentiate into cells of all three germ layers. These unique properties make them exceptionally valuable for drug discovery and regenerative medicine. However, the practical application of ESCs is limited because it is difficult to derive and culture ESCs. It has been demonstrated that CHIR99021 (CHIR) promotes self-renewal and enhances the derivation efficiency of mouse (m)ESCs. However, the downstream targets of CHIR are not fully understood. In this study, we identified CHIR-regulated genes in mESCs using microarray analysis. Our microarray data demonstrated that CHIR not only influenced the Wnt/β-catenin pathway bymore » stabilizing β-catenin, but also modulated several other pluripotency-related signaling pathways such as TGF-β, Notch and MAPK signaling pathways. More detailed analysis demonstrated that CHIR inhibited Nodal signaling, while activating bone morphogenetic protein signaling in mESCs. In addition, we found that pluripotency-maintaining transcription factors were up-regulated by CHIR, while several developmental-related genes were down-regulated. Furthermore, we found that CHIR altered the expression of epigenetic regulatory genes and long intergenic non-coding RNAs. Quantitative real-time PCR results were consistent with microarray data, suggesting that CHIR alters the expression pattern of protein-encoding genes (especially transcription factors), epigenetic regulatory genes and non-coding RNAs to establish a relatively stable pluripotency-maintaining network. - Highlights: • Combined use of CHIR with LIF promotes self-renewal of J1 mESCs. • CHIR-regulated genes are involved in multiple pathways. • CHIR inhibits Nodal signaling and promotes Bmp4 expression to activate BMP signaling. • Expression of epigenetic regulatory genes and lincRNAs is altered by CHIR.« less

Automatic Identification and Quantification of Extra-Well Fluorescence in Microarray Images.

PubMed

Rivera, Robert; Wang, Jie; Yu, Xiaobo; Demirkan, Gokhan; Hopper, Marika; Bian, Xiaofang; Tahsin, Tasnia; Magee, D Mitchell; Qiu, Ji; LaBaer, Joshua; Wallstrom, Garrick

2017-11-03

In recent studies involving NAPPA microarrays, extra-well fluorescence is used as a key measure for identifying disease biomarkers because there is evidence to support that it is better correlated with strong antibody responses than statistical analysis involving intraspot intensity. Because this feature is not well quantified by traditional image analysis software, identification and quantification of extra-well fluorescence is performed manually, which is both time-consuming and highly susceptible to variation between raters. A system that could automate this task efficiently and effectively would greatly improve the process of data acquisition in microarray studies, thereby accelerating the discovery of disease biomarkers. In this study, we experimented with different machine learning methods, as well as novel heuristics, for identifying spots exhibiting extra-well fluorescence (rings) in microarray images and assigning each ring a grade of 1-5 based on its intensity and morphology. The sensitivity of our final system for identifying rings was found to be 72% at 99% specificity and 98% at 92% specificity. Our system performs this task significantly faster than a human, while maintaining high performance, and therefore represents a valuable tool for microarray image analysis.

Detection of cryptic pathogenic copy number variations and constitutional loss of heterozygosity using high resolution SNP microarray analysis in 117 patients referred for cytogenetic analysis and impact on clinical practice.

PubMed

Bruno, D L; Ganesamoorthy, D; Schoumans, J; Bankier, A; Coman, D; Delatycki, M; Gardner, R J M; Hunter, M; James, P A; Kannu, P; McGillivray, G; Pachter, N; Peters, H; Rieubland, C; Savarirayan, R; Scheffer, I E; Sheffield, L; Tan, T; White, S M; Yeung, A; Bowman, Z; Ngo, C; Choy, K W; Cacheux, V; Wong, L; Amor, D J; Slater, H R

2009-02-01

Microarray genome analysis is realising its promise for improving detection of genetic abnormalities in individuals with mental retardation and congenital abnormality. Copy number variations (CNVs) are now readily detectable using a variety of platforms and a major challenge is the distinction of pathogenic from ubiquitous, benign polymorphic CNVs. The aim of this study was to investigate replacement of time consuming, locus specific testing for specific microdeletion and microduplication syndromes with microarray analysis, which theoretically should detect all known syndromes with CNV aetiologies as well as new ones. Genome wide copy number analysis was performed on 117 patients using Affymetrix 250K microarrays. 434 CNVs (195 losses and 239 gains) were found, including 18 pathogenic CNVs and 9 identified as "potentially pathogenic". Almost all pathogenic CNVs were larger than 500 kb, significantly larger than the median size of all CNVs detected. Segmental regions of loss of heterozygosity larger than 5 Mb were found in 5 patients. Genome microarray analysis has improved diagnostic success in this group of patients. Several examples of recently discovered "new syndromes" were found suggesting they are more common than previously suspected and collectively are likely to be a major cause of mental retardation. The findings have several implications for clinical practice. The study revealed the potential to make genetic diagnoses that were not evident in the clinical presentation, with implications for pretest counselling and the consent process. The importance of contributing novel CNVs to high quality databases for genotype-phenotype analysis and review of guidelines for selection of individuals for microarray analysis is emphasised.

Integrated microarray and ChIP analysis identifies multiple Foxa2 dependent target genes in the notochord.

PubMed

Tamplin, Owen J; Cox, Brian J; Rossant, Janet

2011-12-15

The node and notochord are key tissues required for patterning of the vertebrate body plan. Understanding the gene regulatory network that drives their formation and function is therefore important. Foxa2 is a key transcription factor at the top of this genetic hierarchy and finding its targets will help us to better understand node and notochord development. We performed an extensive microarray-based gene expression screen using sorted embryonic notochord cells to identify early notochord-enriched genes. We validated their specificity to the node and notochord by whole mount in situ hybridization. This provides the largest available resource of notochord-expressed genes, and therefore candidate Foxa2 target genes in the notochord. Using existing Foxa2 ChIP-seq data from adult liver, we were able to identify a set of genes expressed in the notochord that had associated regions of Foxa2-bound chromatin. Given that Foxa2 is a pioneer transcription factor, we reasoned that these sites might represent notochord-specific enhancers. Candidate Foxa2-bound regions were tested for notochord specific enhancer function in a zebrafish reporter assay and 7 novel notochord enhancers were identified. Importantly, sequence conservation or predictive models could not have readily identified these regions. Mutation of putative Foxa2 binding elements in two of these novel enhancers abrogated reporter expression and confirmed their Foxa2 dependence. The combination of highly specific gene expression profiling and genome-wide ChIP analysis is a powerful means of understanding developmental pathways, even for small cell populations such as the notochord. Copyright © 2011 Elsevier Inc. All rights reserved.

Unraveling the Specific Ischemic Core and Penumbra Transcriptome in the Permanent Middle Cerebral Artery Occlusion Mouse Model Brain Treated with the Neuropeptide PACAP38

PubMed Central

Hori, Motohide; Nakamachi, Tomoya; Shibato, Junko; Rakwal, Randeep; Shioda, Seiji; Numazawa, Satoshi

2015-01-01

Our group has been systematically investigating the effects of the neuropeptide pituitary adenylate-cyclase activating polypeptide (PACAP) on the ischemic brain. To do so, we have established and utilized the permanent middle cerebral artery occlusion (PMCAO) mouse model, in which PACAP38 (1 pmol) injection is given intracerebroventrically and compared to a control saline (0.9% sodium chloride, NaCl) injection, to unravel genome-wide gene expression changes using a high-throughput DNA microarray analysis approach. In our previous studies, we have accumulated a large volume of data (gene inventory) from the whole brain (ipsilateral and contralateral hemispheres) after both PMCAO and post-PACAP38 injection. In our latest research, we have targeted specifically infarct or ischemic core (hereafter abbreviated IC) and penumbra (hereafter abbreviated P) post-PACAP38 injections in order to re-examine the transcriptome at 6 and 24 h post injection. The current study aims to delineate the specificity of expression and localization of differentially expressed molecular factors influenced by PACAP38 in the IC and P regions. Utilizing the mouse 4 × 44 K whole genome DNA chip we show numerous changes (≧/≦ 1.5/0.75-fold) at both 6 h (654 and 456, and 522 and 449 up- and down-regulated genes for IC and P, respectively) and 24 h (2568 and 2684, and 1947 and 1592 up- and down-regulated genes for IC and P, respectively) after PACAP38 treatment. Among the gene inventories obtained here, two genes, brain-derived neurotrophic factor (Bdnf) and transthyretin (Ttr) were found to be induced by PACAP38 treatment, which we had not been able to identify previously using the whole hemisphere transcriptome analysis. Using bioinformatics analysis by pathway- or specific-disease-state focused gene classifications and Ingenuity Pathway Analysis (IPA) the differentially expressed genes are functionally classified and discussed. Among these, we specifically discuss some novel and previously identified genes, such as alpha hemoglobin stabilizing protein (Ahsp), cathelicidin antimicrobial peptide (Camp), chemokines, interferon beta 1 (Ifnb1), and interleukin 6 (Il6) in context of PACAP38-mediated neuroprotection in the ischemic brain. Taken together, the DNA microarray analysis provides not only a great resource for further study, but also reinforces the importance of region-specific analyses in genome-wide identification of target molecular factors that might play a role in the neuroprotective function of PACAP38. PMID:27600210

Estimating differential expression from multiple indicators

PubMed Central

Ilmjärv, Sten; Hundahl, Christian Ansgar; Reimets, Riin; Niitsoo, Margus; Kolde, Raivo; Vilo, Jaak; Vasar, Eero; Luuk, Hendrik

2014-01-01

Regardless of the advent of high-throughput sequencing, microarrays remain central in current biomedical research. Conventional microarray analysis pipelines apply data reduction before the estimation of differential expression, which is likely to render the estimates susceptible to noise from signal summarization and reduce statistical power. We present a probe-level framework, which capitalizes on the high number of concurrent measurements to provide more robust differential expression estimates. The framework naturally extends to various experimental designs and target categories (e.g. transcripts, genes, genomic regions) as well as small sample sizes. Benchmarking in relation to popular microarray and RNA-sequencing data-analysis pipelines indicated high and stable performance on the Microarray Quality Control dataset and in a cell-culture model of hypoxia. Experimental-data-exhibiting long-range epigenetic silencing of gene expression was used to demonstrate the efficacy of detecting differential expression of genomic regions, a level of analysis not embraced by conventional workflows. Finally, we designed and conducted an experiment to identify hypothermia-responsive genes in terms of monotonic time-response. As a novel insight, hypothermia-dependent up-regulation of multiple genes of two major antioxidant pathways was identified and verified by quantitative real-time PCR. PMID:24586062

Comprehensive analysis of differentially expressed profiles of lncRNAs and construction of miR-133b mediated ceRNA network in colorectal cancer.

PubMed

Wu, Hao; Wu, Runliu; Chen, Miao; Li, Daojiang; Dai, Jing; Zhang, Yi; Gao, Kai; Yu, Jun; Hu, Gui; Guo, Yihang; Lin, Changwei; Li, Xiaorong

2017-03-28

Growing evidence suggests that long non-coding RNAs (lncRNAs) play a key role in tumorigenesis. However, the mechanism remains largely unknown. Thousands of significantly dysregulated lncRNAs and mRNAs were identified by microarray. Furthermore, a miR-133b-meditated lncRNA-mRNA ceRNA network was revealed, a subset of which was validated in 14 paired CRC patient tumor/non-tumor samples. Gene set enrichment analysis (GSEA) results demonstrated that lncRNAs ENST00000520055 and ENST00000535511 shared KEGG pathways with miR-133b target genes. We used microarrays to survey the lncRNA and mRNA expression profiles of colorectal cancer and para-cancer tissues. Gene Ontology (GO) and KEGG pathway enrichment analyses were performed to explore the functions of the significantly dysregulated genes. An innovate method was employed that combined analyses of two microarray data sets to construct a miR-133b-mediated lncRNA-mRNA competing endogenous RNAs (ceRNA) network. Quantitative RT-PCR analysis was used to validate part of this network. GSEA was used to predict the potential functions of these lncRNAs. This study identifies and validates a new method to investigate the miR-133b-mediated lncRNA-mRNA ceRNA network and lays the foundation for future investigation into the role of lncRNAs in colorectal cancer.

Microarray Analysis of Gene Expression Alteration in Human Middle Ear Epithelial Cells Induced by Asian Sand Dust.

PubMed

Go, Yoon Young; Park, Moo Kyun; Kwon, Jee Young; Seo, Young Rok; Chae, Sung-Won; Song, Jae-Jun

2015-12-01

The primary aim of this study is to evaluate the gene expression profile of Asian sand dust (ASD)-treated human middle ear epithelial cell (HMEEC) using microarray analysis. The HMEEC was treated with ASD (400 µg/mL) and total RNA was extracted for microarray analysis. Molecular pathways among differentially expressed genes were further analyzed. For selected genes, the changes in gene expression were confirmed by real-time polymerase chain reaction. A total of 1,274 genes were differentially expressed by ASD. Among them, 1,138 genes were 2 folds up-regulated, whereas 136 genes were 2 folds down-regulated. Up-regulated genes were mainly involved in cellular processes, including apoptosis, cell differentiation, and cell proliferation. Down-regulated genes affected cellular processes, including apoptosis, cell cycle, cell differentiation, and cell proliferation. The 10 genes including ADM, CCL5, EDN1, EGR1, FOS, GHRL, JUN, SOCS3, TNF, and TNFSF10 were identified as main modulators in up-regulated genes. A total of 11 genes including CSF3, DKK1, FOSL1, FST, TERT, MMP13, PTHLH, SPRY2, TGFBR2, THBS1, and TIMP1 acted as main components of pathway associated with 2-fold down regulated genes. We identified the differentially expressed genes in ASD-treated HMEEC. Our work indicates that air pollutant like ASD, may play an important role in the pathogenesis of otitis media.

Cross-species transcriptomic approach reveals genes in hamster implantation sites.

PubMed

Lei, Wei; Herington, Jennifer; Galindo, Cristi L; Ding, Tianbing; Brown, Naoko; Reese, Jeff; Paria, Bibhash C

2014-12-01

The mouse model has greatly contributed to understanding molecular mechanisms involved in the regulation of progesterone (P4) plus estrogen (E)-dependent blastocyst implantation process. However, little is known about contributory molecular mechanisms of the P4-only-dependent blastocyst implantation process that occurs in species such as hamsters, guineapigs, rabbits, pigs, rhesus monkeys, and perhaps humans. We used the hamster as a model of P4-only-dependent blastocyst implantation and carried out cross-species microarray (CSM) analyses to reveal differentially expressed genes at the blastocyst implantation site (BIS), in order to advance the understanding of molecular mechanisms of implantation. Upregulation of 112 genes and downregulation of 77 genes at the BIS were identified using a mouse microarray platform, while use of the human microarray revealed 62 up- and 38 down-regulated genes at the BIS. Excitingly, a sizable number of genes (30 up- and 11 down-regulated genes) were identified as a shared pool by both CSMs. Real-time RT-PCR and in situ hybridization validated the expression patterns of several up- and down-regulated genes identified by both CSMs at the hamster and mouse BIS to demonstrate the merit of CSM findings across species, in addition to revealing genes specific to hamsters. Functional annotation analysis found that genes involved in the spliceosome, proteasome, and ubiquination pathways are enriched at the hamster BIS, while genes associated with tight junction, SAPK/JNK signaling, and PPARα/RXRα signalings are repressed at the BIS. Overall, this study provides a pool of genes and evidence of their participation in up- and down-regulated cellular functions/pathways at the hamster BIS. © 2014 Society for Reproduction and Fertility.

Discovering functional modules by topic modeling RNA-Seq based toxicogenomic data.

PubMed

Yu, Ke; Gong, Binsheng; Lee, Mikyung; Liu, Zhichao; Xu, Joshua; Perkins, Roger; Tong, Weida

2014-09-15

Toxicogenomics (TGx) endeavors to elucidate the underlying molecular mechanisms through exploring gene expression profiles in response to toxic substances. Recently, RNA-Seq is increasingly regarded as a more powerful alternative to microarrays in TGx studies. However, realizing RNA-Seq's full potential requires novel approaches to extracting information from the complex TGx data. Considering read counts as the number of times a word occurs in a document, gene expression profiles from RNA-Seq are analogous to a word by document matrix used in text mining. Topic modeling aiming at to discover the latent structures in text corpora would be helpful to explore RNA-Seq based TGx data. In this study, topic modeling was applied on a typical RNA-Seq based TGx data set to discover hidden functional modules. The RNA-Seq based gene expression profiles were transformed into "documents", on which latent Dirichlet allocation (LDA) was used to build a topic model. We found samples treated by the compounds with the same modes of actions (MoAs) could be clustered based on topic similarities. The topic most relevant to each cluster was identified as a "marker" topic, which was interpreted by gene enrichment analysis with MoAs then confirmed by compound and pathways associations mined from literature. To further validate the "marker" topics, we tested topic transferability from RNA-Seq to microarrays. The RNA-Seq based gene expression profile of a topic specifically associated with peroxisome proliferator-activated receptors (PPAR) signaling pathway was used to query samples with similar expression profiles in two different microarray data sets, yielding accuracy of about 85%. This proof-of-concept study demonstrates the applicability of topic modeling to discover functional modules in RNA-Seq data and suggests a valuable computational tool for leveraging information within TGx data in RNA-Seq era.

Gene profile in the spleen under massive partial hepatectomy using complementary DNA microarray and pathway analysis.

PubMed

Arakawa, Yusuke; Shimada, Mitsuo; Utsunomiya, Tohru; Imura, Satoru; Morine, Yuji; Ikemoto, Tetsuya; Mori, Hiroki; Kanamoto, Mami; Iwahashi, Shuichi; Saito, Yu; Takasu, Chie

2014-08-01

In general, the spleen is one of the abdominal organs connected by the portal system, and a splenectomy improves hepatic functions in the settings of partial hepatectomy (Hx) for portal hypertensive cases or living donor liver transplantation with excessive portal vein flow. Those precise mechanisms remain still unclear; therefore, we investigated the DNA expression profile in the spleen after 90% Hx in rats using complementary DNA microarray and pathway analysis. Messenger RNAs (mRNAs) were prepared from three rat spleens at each time point (0, 3, and 6 h after 90% Hx). Using the gene chip, mRNA was hybridized to Affymetrix GeneChip Rat Genome 230 2.0 Array (Affymetrix®) and pathway analysis was done with Ingenuity Pathway Analysis (IPA®). We determined the 3-h or 6-h/0-h ratio to assess the influence of Hx, and cut-off values were set at more than 2.0-fold or less than 1/2 (0.5)-fold. Chemokine activity-related genes including Cxcl1 (GRO1) and Cxcl2 (MIP-2) related pathway were upregulated in the spleen. Also, immediate early response genes including early growth response-1 (EGR1), FBJ murine osteosarcoma (FOS) and activating transcription factor 3 (ATF3) related pathway were upregulated in the spleen. We concluded that in the spleen the expression of numerous inflammatory-related genes would occur after 90% Hx. The spleen could take a harmful role and provide a negative impact during post Hx phase due to the induction of chemokine and transcription factors including GRO1 and EGR1. © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gentry, T.; Schadt, C.; Zhou, J.

Microarray technology has the unparalleled potential tosimultaneously determine the dynamics and/or activities of most, if notall, of the microbial populations in complex environments such as soilsand sediments. Researchers have developed several types of arrays thatcharacterize the microbial populations in these samples based on theirphylogenetic relatedness or functional genomic content. Several recentstudies have used these microarrays to investigate ecological issues;however, most have only analyzed a limited number of samples withrelatively few experiments utilizing the full high-throughput potentialof microarray analysis. This is due in part to the unique analyticalchallenges that these samples present with regard to sensitivity,specificity, quantitation, and data analysis. Thismore » review discussesspecific applications of microarrays to microbial ecology research alongwith some of the latest studies addressing the difficulties encounteredduring analysis of complex microbial communities within environmentalsamples. With continued development, microarray technology may ultimatelyachieve its potential for comprehensive, high-throughput characterizationof microbial populations in near real-time.« less

Specific Discrimination of Three Pathogenic Salmonella enterica subsp. enterica Serotypes by carB-Based Oligonucleotide Microarray

PubMed Central

Shin, Hwa Hui; Hwang, Byeong Hee; Seo, Jeong Hyun

2014-01-01

It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes. PMID:24185846

Specific discrimination of three pathogenic Salmonella enterica subsp. enterica serotypes by carB-based oligonucleotide microarray.

PubMed

Shin, Hwa Hui; Hwang, Byeong Hee; Seo, Jeong Hyun; Cha, Hyung Joon

2014-01-01

It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes.

Analysis of the Transcriptomes Downstream of Eyeless and the Hedgehog, Decapentaplegic and Notch Signaling Pathways in Drosophila melanogaster

PubMed Central

Nfonsam, Landry E.; Cano, Carlos; Mudge, Joann; Schilkey, Faye D.; Curtiss, Jennifer

2012-01-01

Tissue-specific transcription factors are thought to cooperate with signaling pathways to promote patterned tissue specification, in part by co-regulating transcription. The Drosophila melanogaster Pax6 homolog Eyeless forms a complex, incompletely understood regulatory network with the Hedgehog, Decapentaplegic and Notch signaling pathways to control eye-specific gene expression. We report a combinatorial approach, including mRNAseq and microarray analyses, to identify targets co-regulated by Eyeless and Hedgehog, Decapentaplegic or Notch. Multiple analyses suggest that the transcriptomes resulting from co-misexpression of Eyeless+signaling factors provide a more complete picture of eye development compared to previous efforts involving Eyeless alone: (1) Principal components analysis and two-way hierarchical clustering revealed that the Eyeless+signaling factor transcriptomes are closer to the eye control transcriptome than when Eyeless is misexpressed alone; (2) more genes are upregulated at least three-fold in response to Eyeless+signaling factors compared to Eyeless alone; (3) based on gene ontology analysis, the genes upregulated in response to Eyeless+signaling factors had a greater diversity of functions compared to Eyeless alone. Through a secondary screen that utilized RNA interference, we show that the predicted gene CG4721 has a role in eye development. CG4721 encodes a neprilysin family metalloprotease that is highly up-regulated in response to Eyeless+Notch, confirming the validity of our approach. Given the similarity between D. melanogaster and vertebrate eye development, the large number of novel genes identified as potential targets of Ey+signaling factors will provide novel insights to our understanding of eye development in D. melanogaster and humans. PMID:22952997

Detecting discordance enrichment among a series of two-sample genome-wide expression data sets.

PubMed

Lai, Yinglei; Zhang, Fanni; Nayak, Tapan K; Modarres, Reza; Lee, Norman H; McCaffrey, Timothy A

2017-01-25

With the current microarray and RNA-seq technologies, two-sample genome-wide expression data have been widely collected in biological and medical studies. The related differential expression analysis and gene set enrichment analysis have been frequently conducted. Integrative analysis can be conducted when multiple data sets are available. In practice, discordant molecular behaviors among a series of data sets can be of biological and clinical interest. In this study, a statistical method is proposed for detecting discordance gene set enrichment. Our method is based on a two-level multivariate normal mixture model. It is statistically efficient with linearly increased parameter space when the number of data sets is increased. The model-based probability of discordance enrichment can be calculated for gene set detection. We apply our method to a microarray expression data set collected from forty-five matched tumor/non-tumor pairs of tissues for studying pancreatic cancer. We divided the data set into a series of non-overlapping subsets according to the tumor/non-tumor paired expression ratio of gene PNLIP (pancreatic lipase, recently shown it association with pancreatic cancer). The log-ratio ranges from a negative value (e.g. more expressed in non-tumor tissue) to a positive value (e.g. more expressed in tumor tissue). Our purpose is to understand whether any gene sets are enriched in discordant behaviors among these subsets (when the log-ratio is increased from negative to positive). We focus on KEGG pathways. The detected pathways will be useful for our further understanding of the role of gene PNLIP in pancreatic cancer research. Among the top list of detected pathways, the neuroactive ligand receptor interaction and olfactory transduction pathways are the most significant two. Then, we consider gene TP53 that is well-known for its role as tumor suppressor in cancer research. The log-ratio also ranges from a negative value (e.g. more expressed in non-tumor tissue) to a positive value (e.g. more expressed in tumor tissue). We divided the microarray data set again according to the expression ratio of gene TP53. After the discordance enrichment analysis, we observed overall similar results and the above two pathways are still the most significant detections. More interestingly, only these two pathways have been identified for their association with pancreatic cancer in a pathway analysis of genome-wide association study (GWAS) data. This study illustrates that some disease-related pathways can be enriched in discordant molecular behaviors when an important disease-related gene changes its expression. Our proposed statistical method is useful in the detection of these pathways. Furthermore, our method can also be applied to genome-wide expression data collected by the recent RNA-seq technology.

Microarray data mining using Bioconductor packages.

PubMed

Nie, Haisheng; Neerincx, Pieter B T; van der Poel, Jan; Ferrari, Francesco; Bicciato, Silvio; Leunissen, Jack A M; Groenen, Martien A M

2009-07-16

This paper describes the results of a Gene Ontology (GO) term enrichment analysis of chicken microarray data using the Bioconductor packages. By checking the enriched GO terms in three contrasts, MM8-PM8, MM8-MA8, and MM8-MM24, of the provided microarray data during this workshop, this analysis aimed to investigate the host reactions in chickens occurring shortly after a secondary challenge with either a homologous or heterologous species of Eimeria. The results of GO enrichment analysis using GO terms annotated to chicken genes and GO terms annotated to chicken-human orthologous genes were also compared. Furthermore, a locally adaptive statistical procedure (LAP) was performed to test differentially expressed chromosomal regions, rather than individual genes, in the chicken genome after Eimeria challenge. GO enrichment analysis identified significant (raw p-value < 0.05) GO terms for all three contrasts included in the analysis. Some of the GO terms linked to, generally, primary immune responses or secondary immune responses indicating the GO enrichment analysis is a useful approach to analyze microarray data. The comparisons of GO enrichment results using chicken gene information and chicken-human orthologous gene information showed more refined GO terms related to immune responses when using chicken-human orthologous gene information, this suggests that using chicken-human orthologous gene information has higher power to detect significant GO terms with more refined functionality. Furthermore, three chromosome regions were identified to be significantly up-regulated in contrast MM8-PM8 (q-value < 0.01). Overall, this paper describes a practical approach to analyze microarray data in farm animals where the genome information is still incomplete. For farm animals, such as chicken, with currently limited gene annotation, borrowing gene annotation information from orthologous genes in well-annotated species, such as human, will help improve the pathway analysis results substantially. Furthermore, LAP analysis approach is a relatively new and very useful way to be applied in microarray analysis.

Alteration of the gene expression profile of T-cell receptor αβ-modified T-cells with diffuse large B-cell lymphoma specificity.

PubMed

Zha, Xianfeng; Yin, Qingsong; Tan, Huo; Wang, Chunyan; Chen, Shaohua; Yang, Lijian; Li, Bo; Wu, Xiuli; Li, Yangqiu

2013-05-01

Antigen-specific, T-cell receptor (TCR)-modified cytotoxic T lymphocytes (CTLs) that target tumors are an attractive strategy for specific adoptive immunotherapy. Little is known about whether there are any alterations in the gene expression profile after TCR gene transduction in T cells. We constructed TCR gene-redirected CTLs with specificity for diffuse large B-cell lymphoma (DLBCL)-associated antigens to elucidate the gene expression profiles of TCR gene-redirected T-cells, and we further analyzed the gene expression profile pattern of these redirected T-cells by Affymetrix microarrays. The resulting data were analyzed using Bioconductor software, a two-fold cut-off expression change was applied together with anti-correlation of the profile ratios to render the microarray analysis set. The fold change of all genes was calculated by comparing the three TCR gene-modified T-cells and a negative control counterpart. The gene pathways were analyzed using Bioconductor and Kyoto Encyclopedia of Genes and Genomes. Identical genes whose fold change was greater than or equal to 2.0 in all three TCR gene-redirected T-cell groups in comparison with the negative control were identified as the differentially expressed genes. The differentially expressed genes were comprised of 33 up-regulated genes and 1 down-regulated gene including JUNB, FOS, TNF, INF-γ, DUSP2, IL-1B, CXCL1, CXCL2, CXCL9, CCL2, CCL4, and CCL8. These genes are mainly involved in the TCR signaling, mitogen-activated protein kinase signaling, and cytokine-cytokine receptor interaction pathways. In conclusion, we characterized the gene expression profile of DLBCL-specific TCR gene-redirected T-cells. The changes corresponded to an up-regulation in the differentiation and proliferation of the T-cells. These data may help to explain some of the characteristics of the redirected T-cells.

Maintenance of Self-Renewal and Pluripotency in J1 Mouse Embryonic Stem Cells through Regulating Transcription Factor and MicroRNA Expression Induced by PD0325901.

PubMed

Ai, Zhiying; Shao, Jingjing; Shi, Xinglong; Yu, Mengying; Wu, Yongyan; Du, Juan; Zhang, Yong; Guo, Zekun

2016-01-01

Embryonic stem cells (ESCs) have the ability to grow indefinitely and retain their pluripotency in culture, and this self-renewal capacity is governed by several crucial molecular pathways controlled by specific regulatory genes and epigenetic modifications. It is reported that multiple epigenetic regulators, such as miRNA and pluripotency factors, can be tightly integrated into molecular pathways and cooperate to maintain self-renewal of ESCs. However, mouse ESCs in serum-containing medium seem to be heterogeneous due to the self-activating differentiation signal of MEK/ERK. Thus, to seek for the crucial miRNA and key regulatory genes that establish ESC properties in MEK/ERK pathway, we performed microarray analysis and small RNA deep-sequencing of J1 mESCs treated with or without PD0325901 (PD), a well-known inhibitor of MEK/ERK signal pathway, followed by verification of western blot analysis and quantitative real-time PCR verification; we found that PD regulated the transcript expressions related to self-renewal and differentiation and antagonized the action of retinoic acid- (RA-) induced differentiation. Moreover, PD can significantly modulate the expressions of multiple miRNAs that have crucial functions in ESC development. Overall, our results demonstrate that PD could enhance ESC self-renewal capacity both by key regulatory genes and ES cell-specific miRNA, which in turn influences ESC self-renewal and cellular differentiation.

Initiation of follicular atresia: gene networks during early atresia in pig ovaries.

PubMed

Zhang, Jinbi; Liu, Yang; Yao, Wang; Li, Qifa; Liu, Hong-Lin; Pan, Zengxiang

2018-05-09

In mammals, more than 99% of ovarian follicles undergo a degenerative process known as atresia. The molecular events involve in atresia initiation remain incompletely understood. The objective of this study was to analyze differential gene expression profiles of medium antral ovarian follicles during early atresia in pig. The transcriptome evaluation was performed on cDNA microarrays using healthy and early atretic follicle samples and was validated by quantitative PCR. Annotation analysis applying current database (sus scrofa 11.1) revealed 450 significantly differential expressed genes between healthy and early atretic follicles. Among them, 142 were significantly up-regulated in early atretic with respect to healthy group and 308 were down-regulated. Similar expression trends were observed between microarray data and qRT-PCR confirmation, which indicated the reliability of the microarray analysis. Further analysis of the differential expressed genes revealed the most significantly affected biological functions during early atresia including blood vessel development, regulation of DNA-templated transcription in response to stress and negative regulation of cell adhesion. The pathway and interaction analysis suggested that atresia initiation associates with 1) a crosstalk of cell apoptosis, autophagy, and ferroptosis rather than change of typical apoptosis markers, 2) dramatic shift of steroidogenic enzymes, 3) deficient glutathione metabolism, and 4) vascular degeneration. The novel gene candidates and pathways identified in the current study will lead to a comprehensive view of the molecular regulation of ovarian follicular atresia and a new understanding of atresia initiation.

Identification of Novel Tissue-Specific Genes by Analysis of Microarray Databases: A Human and Mouse Model

PubMed Central

Suh, Yeunsu; Davis, Michael E.; Lee, Kichoon

2013-01-01

Understanding the tissue-specific pattern of gene expression is critical in elucidating the molecular mechanisms of tissue development, gene function, and transcriptional regulations of biological processes. Although tissue-specific gene expression information is available in several databases, follow-up strategies to integrate and use these data are limited. The objective of the current study was to identify and evaluate novel tissue-specific genes in human and mouse tissues by performing comparative microarray database analysis and semi-quantitative PCR analysis. We developed a powerful approach to predict tissue-specific genes by analyzing existing microarray data from the NCBI′s Gene Expression Omnibus (GEO) public repository. We investigated and confirmed tissue-specific gene expression in the human and mouse kidney, liver, lung, heart, muscle, and adipose tissue. Applying our novel comparative microarray approach, we confirmed 10 kidney, 11 liver, 11 lung, 11 heart, 8 muscle, and 8 adipose specific genes. The accuracy of this approach was further verified by employing semi-quantitative PCR reaction and by searching for gene function information in existing publications. Three novel tissue-specific genes were discovered by this approach including AMDHD1 (amidohydrolase domain containing 1) in the liver, PRUNE2 (prune homolog 2) in the heart, and ACVR1C (activin A receptor, type IC) in adipose tissue. We further confirmed the tissue-specific expression of these 3 novel genes by real-time PCR. Among them, ACVR1C is adipose tissue-specific and adipocyte-specific in adipose tissue, and can be used as an adipocyte developmental marker. From GEO profiles, we predicted the processes in which AMDHD1 and PRUNE2 may participate. Our approach provides a novel way to identify new sets of tissue-specific genes and to predict functions in which they may be involved. PMID:23741331

Transcriptome Pathway Analysis of Pathological and Physiological Aldosterone-Producing Human Tissues.

PubMed

Zhou, Junhua; Lam, Brian; Neogi, Sudeshna G; Yeo, Giles S H; Azizan, Elena A B; Brown, Morris J

2016-12-01

Primary aldosteronism is present in ≈10% of hypertensives. We previously performed a microarray assay on aldosterone-producing adenomas and their paired zona glomerulosa and fasciculata. Confirmation of top genes validated the study design and functional experiments of zona glomerulosa selective genes established the role of the encoded proteins in aldosterone regulation. In this study, we further analyzed our microarray data using AmiGO 2 for gene ontology enrichment and Ingenuity Pathway Analysis to identify potential biological processes and canonical pathways involved in pathological and physiological aldosterone regulation. Genes differentially regulated in aldosterone-producing adenoma and zona glomerulosa were associated with steroid metabolic processes gene ontology terms. Terms related to the Wnt signaling pathway were enriched in zona glomerulosa only. Ingenuity Pathway Analysis showed "NRF2-mediated oxidative stress response pathway" and "LPS (lipopolysaccharide)/IL-1 (interleukin-1)-mediated inhibition of RXR (retinoid X receptor) function" were affected in both aldosterone-producing adenoma and zona glomerulosa with associated genes having up to 21- and 8-fold differences, respectively. Comparing KCNJ5-mutant aldosterone-producing adenoma, zona glomerulosa, and zona fasciculata samples with wild-type samples, 138, 56, and 59 genes were differentially expressed, respectively (fold-change >2; P<0.05). ACSS3, encoding the enzyme that synthesizes acetyl-CoA, was the top gene upregulated in KCNJ5-mutant aldosterone-producing adenoma compared with wild-type. NEFM, a gene highly upregulated in zona glomerulosa, was upregulated in KCNJ5 wild-type aldosterone-producing adenomas. NR4A2, the transcription factor for aldosterone synthase, was highly expressed in zona fasciculata adjacent to a KCNJ5-mutant aldosterone-producing adenoma. Further interrogation of these genes and pathways could potentially provide further insights into the pathology of primary aldosteronism. © 2016 The Authors.

High mobility group box associated with cell proliferation appears to play an important role in hepatocellular carcinogenesis in rats and humans.

PubMed

Suzuki, Shugo; Takeshita, Kentaro; Asamoto, Makoto; Takahashi, Satoru; Kandori, Hitoshi; Tsujimura, Kazunari; Saito, Fumiyo; Masuko, Kazuo; Shirai, Tomoyuki

2009-01-31

To identify genes important in hepatocellular carcinogenesis, especially processes involved in malignant transformation, we focused on differences in gene expression between adenomas and carcinomas by DNA microarray. Eighty-one genes for which expression was specific in carcinomas were analyzed using Ingenuity Pathway Analysis software and Gene Ontology, and found to be associated with TP53 and regulators of cell proliferation. In the genes associated with TP53, we selected high mobility group box (HMGB) for detailed analysis. Immunohistochemistry revealed expression of HMGBs in carcinomas to be significantly higher than in other lesions among both human and rat liver, and a positive correlation between HMGBs and TP53 was detected in rat carcinomas. Knock-down of HMGB 2 expression in a rat hepatocellular carcinoma cell line by RNAi resulted in inhibition of cell growth, although no effects on invasion were evident in vitro. These results suggest that acquisition of malignant potential in the liver requires specific signaling pathways related to high cell proliferation associated with TP53. In particular, HMGBs appear to have an important role for progression and cell proliferation associated with loss of TP53 function in rat and in human hepatocarcinogenesis.

Quantitative Proteomic and Microarray Analysis of the Archaeon Methanosarcina Acetivorans Grown with Acetate Versus Methanol*

PubMed Central

Li, Lingyun; Li, Qingbo; Rohlin, Lars; Kim, UnMi; Salmon, Kirsty; Rejtar, Tomas; Gunsalus, Robert P.; Karger, Barry L.; Ferry, James G.

2008-01-01

Summary Methanosarcina acetivorans strain C2A is an acetate- and methanol-utilizing methane-producing organism for which the genome, the largest yet sequenced among the Archaea, reveals extensive physiological diversity. LC linear ion trap-FTICR mass spectrometry was employed to analyze acetate- vs. methanol-grown cells metabolically labeled with 14N vs. 15N, respectively, to obtain quantitative protein abundance ratios. DNA microarray analyses of acetate- vs. methanol-grown cells was also performed to determine gene expression ratios. The combined approaches were highly complementary, extending the physiological understanding of growth and methanogenesis. Of the 1081 proteins detected, 255 were ≥ 3-fold differentially abundant. DNA microarray analysis revealed 410 genes that were ≥ 2.5-fold differentially expressed of 1972 genes with detected expression. The ratios of differentially abundant proteins were in good agreement with expression ratios of the encoding genes. Taken together, the results suggest several novel roles for electron transport components specific to acetate-grown cells, including two flavodoxins each specific for growth on acetate or methanol. Protein abundance ratios indicated that duplicate CO dehydrogenase/acetyl-CoA complexes function in the conversion of acetate to methane. Surprisingly, the protein abundance and gene expression ratios indicated a general stress response in acetate- vs. methanol-grown cells that included enzymes specific for polyphosphate accumulation and oxidative stress. The microarray analysis identified transcripts of several genes encoding regulatory proteins with identity to the PhoU, MarR, GlnK, and TetR families commonly found in the Bacteria domain. An analysis of neighboring genes suggested roles in controlling phosphate metabolism (PhoU), ammonia assimilation (GlnK), and molybdopterin cofactor biosynthesis (TetR). Finally, the proteomic and microarray results suggested roles for two-component regulatory systems specific for each growth substrate. PMID:17269732

Quantitative proteomic analysis of cultured skin fibroblast cells derived from patients with triglyceride deposit cardiomyovasculopathy

PubMed Central

2013-01-01

Background Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare disease, characterized by the massive accumulation of triglyceride (TG) in multiple tissues, especially skeletal muscle, heart muscle and the coronary artery. TGCV is caused by mutation of adipose triglyceride lipase, which is an essential molecule for the hydrolysis of TG. TGCV is at high risk for skeletal myopathy and heart dysfunction, and therefore premature death. Development of therapeutic methods for TGCV is highly desirable. This study aims to discover specific molecules responsible for TGCV pathogenesis. Methods To identify differentially expressed proteins in TGCV patient cells, the stable isotope labeling with amino acids in cell culture (SILAC) method coupled with LC-MS/MS was performed using skin fibroblast cells derived from two TGCV patients and three healthy volunteers. Altered protein expression in TGCV cells was confirmed using the selected reaction monitoring (SRM) method. Microarray-based transcriptome analysis was simultaneously performed to identify changes in gene expression in TGCV cells. Results Using SILAC proteomics, 4033 proteins were quantified, 53 of which showed significantly altered expression in both TGCV patient cells. Twenty altered proteins were chosen and confirmed using SRM. SRM analysis successfully quantified 14 proteins, 13 of which showed the same trend as SILAC proteomics. The altered protein expression data set was used in Ingenuity Pathway Analysis (IPA), and significant networks were identified. Several of these proteins have been previously implicated in lipid metabolism, while others represent new therapeutic targets or markers for TGCV. Microarray analysis quantified 20743 transcripts, and 252 genes showed significantly altered expression in both TGCV patient cells. Ten altered genes were chosen, 9 of which were successfully confirmed using quantitative RT-PCR. Biological networks of altered genes were analyzed using an IPA search. Conclusions We performed the SILAC- and SRM-based identification-through-confirmation study using skin fibroblast cells derived from TGCV patients, and first identified altered proteins specific for TGCV. Microarray analysis also identified changes in gene expression. The functional networks of the altered proteins and genes are discussed. Our findings will be exploited to elucidate the pathogenesis of TGCV and discover clinically relevant molecules for TGCV in the near future. PMID:24360150

Regional and temporal differences in gene expression of LH(BETA)T(AG) retinoblastoma tumors.

PubMed

Houston, Samuel K; Pina, Yolanda; Clarke, Jennifer; Koru-Sengul, Tulay; Scott, William K; Nathanson, Lubov; Schefler, Amy C; Murray, Timothy G

2011-07-23

The purpose of this study was to evaluate by microarray the hypothesis that LH(BETA)T(AG) retinoblastoma tumors exhibit regional and temporal variations in gene expression. LH(BETA)T(AG) mice aged 12, 16, and 20 weeks were euthanatized (n = 9). Specimens were taken from five tumor areas (apex, anterior lateral, center, base, and posterior lateral). Samples were hybridized to gene microarrays. The data were preprocessed and analyzed, and genes with a P < 0.01, according to the ANOVA models, and a log(2)-fold change >2.5 were considered to be differentially expressed. Differentially expressed genes were analyzed for overlap with known networks by using pathway analysis tools. There were significant temporal (P < 10(-8)) and regional differences in gene expression for LH(BETA)T(AG) retinoblastoma tumors. At P < 0.01 and log(2)-fold change >2.5, there were significant changes in gene expression of 190 genes apically, 84 genes anterolaterally, 126 genes posteriorly, 56 genes centrally, and 134 genes at the base. Differentially expressed genes overlapped with known networks, with significant involvement in regulation of cellular proliferation and growth, response to oxygen levels and hypoxia, regulation of cellular processes, cellular signaling cascades, and angiogenesis. There are significant temporal and regional variations in the LH(BETA)T(AG) retinoblastoma model. Differentially expressed genes overlap with key pathways that may play pivotal roles in murine retinoblastoma development. These findings suggest the mechanisms involved in tumor growth and progression in murine retinoblastoma tumors and identify pathways for analysis at a functional level, to determine significance in human retinoblastoma. Microarray analysis of LH(BETA)T(AG) retinal tumors showed significant regional and temporal variations in gene expression, including dysregulation of genes involved in hypoxic responses and angiogenesis.

Gene expression analysis in MCF-7 breast cancer cells treated with recombinant bromelain.

PubMed

Fouz, Nour; Amid, Azura; Hashim, Yumi Zuhanis Has-Yun

2014-08-01

The contributing molecular pathways underlying the pathogenesis of breast cancer need to be better characterized. The principle of our study was to better understand the genetic mechanism of oncogenesis for human breast cancer and to discover new possible tumor markers for use in clinical practice. We used complimentary DNA (cDNA) microarrays to compare gene expression profiles of treated Michigan Cancer Foundation-7 (MCF-7) with recombinant bromelain and untreated MCF-7. SpringGene analysis was carried out of differential expression followed by Ingenuity Pathway Analysis (IPA), to understand the underlying consequence in developing disease and disorders. We identified 1,102 known genes differentially expressed to a significant degree (p<0.001) changed between the treatment. Within this gene set, 20 genes were significantly changed between treated cells and the control cells with cutoff fold change of more than 1.5. These genes are RNA-binding motif, single-stranded interacting protein 1 (RBMS1), ribosomal protein L29 (RPL29), glutathione S-transferase mu 2 (GSTM2), C15orf32, Akt3, B cell translocation gene 1 (BTG1), C6orf62, C7orf60, kinesin-associated protein 3 (KIFAP3), FBXO11, AT-rich interactive domain 4A (ARID4A), COPS2, TBPL1|SLC2A12, TMEM59, SNORD46, glioma tumor suppressor candidate region gene 2 (GLTSCR2), and LRRFIP. Our observation on gene expression indicated that recombinant bromelain produces a unique signature affecting different pathways, specific for each congener. The microarray results give a molecular mechanistic insight and functional effects, following recombinant bromelain treatment. The extent of changes in genes is related to and involved significantly in gap junction signaling, amyloid processing, cell cycle regulation by BTG family proteins, and breast cancer regulation by stathmin1 that play major roles.

Use of principal components analysis and protein microarray to explore the association of HIV-1-specific IgG responses with disease progression.

PubMed

Gerns Storey, Helen L; Richardson, Barbra A; Singa, Benson; Naulikha, Jackie; Prindle, Vivian C; Diaz-Ochoa, Vladimir E; Felgner, Phil L; Camerini, David; Horton, Helen; John-Stewart, Grace; Walson, Judd L

2014-01-01

The role of HIV-1-specific antibody responses in HIV disease progression is complex and would benefit from analysis techniques that examine clusterings of responses. Protein microarray platforms facilitate the simultaneous evaluation of numerous protein-specific antibody responses, though excessive data are cumbersome in analyses. Principal components analysis (PCA) reduces data dimensionality by generating fewer composite variables that maximally account for variance in a dataset. To identify clusters of antibody responses involved in disease control, we investigated the association of HIV-1-specific antibody responses by protein microarray, and assessed their association with disease progression using PCA in a nested cohort design. Associations observed among collections of antibody responses paralleled protein-specific responses. At baseline, greater antibody responses to the transmembrane glycoprotein (TM) and reverse transcriptase (RT) were associated with higher viral loads, while responses to the surface glycoprotein (SU), capsid (CA), matrix (MA), and integrase (IN) proteins were associated with lower viral loads. Over 12 months greater antibody responses were associated with smaller decreases in CD4 count (CA, MA, IN), and reduced likelihood of disease progression (CA, IN). PCA and protein microarray analyses highlighted a collection of HIV-specific antibody responses that together were associated with reduced disease progression, and may not have been identified by examining individual antibody responses. This technique may be useful to explore multifaceted host-disease interactions, such as HIV coinfections.

A Unique Procedure to Identify Cell Surface Markers Through a Spherical Self-Organizing Map Applied to DNA Microarray Analysis.

PubMed

Sugii, Yuh; Kasai, Tomonari; Ikeda, Masashi; Vaidyanath, Arun; Kumon, Kazuki; Mizutani, Akifumi; Seno, Akimasa; Tokutaka, Heizo; Kudoh, Takayuki; Seno, Masaharu

2016-01-01

To identify cell-specific markers, we designed a DNA microarray platform with oligonucleotide probes for human membrane-anchored proteins. Human glioma cell lines were analyzed using microarray and compared with normal and fetal brain tissues. For the microarray analysis, we employed a spherical self-organizing map, which is a clustering method suitable for the conversion of multidimensional data into two-dimensional data and displays the relationship on a spherical surface. Based on the gene expression profile, the cell surface characteristics were successfully mirrored onto the spherical surface, thereby distinguishing normal brain tissue from the disease model based on the strength of gene expression. The clustered glioma-specific genes were further analyzed by polymerase chain reaction procedure and immunocytochemical staining of glioma cells. Our platform and the following procedure were successfully demonstrated to categorize the genes coding for cell surface proteins that are specific to glioma cells. Our assessment demonstrates that a spherical self-organizing map is a valuable tool for distinguishing cell surface markers and can be employed in marker discovery studies for the treatment of cancer.

Thermodynamically optimal whole-genome tiling microarray design and validation.

PubMed

Cho, Hyejin; Chou, Hui-Hsien

2016-06-13

Microarray is an efficient apparatus to interrogate the whole transcriptome of species. Microarray can be designed according to annotated gene sets, but the resulted microarrays cannot be used to identify novel transcripts and this design method is not applicable to unannotated species. Alternatively, a whole-genome tiling microarray can be designed using only genomic sequences without gene annotations, and it can be used to detect novel RNA transcripts as well as known genes. The difficulty with tiling microarray design lies in the tradeoff between probe-specificity and coverage of the genome. Sequence comparison methods based on BLAST or similar software are commonly employed in microarray design, but they cannot precisely determine the subtle thermodynamic competition between probe targets and partially matched probe nontargets during hybridizations. Using the whole-genome thermodynamic analysis software PICKY to design tiling microarrays, we can achieve maximum whole-genome coverage allowable under the thermodynamic constraints of each target genome. The resulted tiling microarrays are thermodynamically optimal in the sense that all selected probes share the same melting temperature separation range between their targets and closest nontargets, and no additional probes can be added without violating the specificity of the microarray to the target genome. This new design method was used to create two whole-genome tiling microarrays for Escherichia coli MG1655 and Agrobacterium tumefaciens C58 and the experiment results validated the design.

Diagnostic Yield of Chromosomal Microarray Analysis in an Autism Primary Care Practice: Which Guidelines to Implement?

ERIC Educational Resources Information Center

McGrew, Susan G.; Peters, Brittany R.; Crittendon, Julie A.; Veenstra-VanderWeele, Jeremy

2012-01-01

Genetic testing is recommended for patients with ASD; however specific recommendations vary by specialty. American Academy of Pediatrics and American Academy of Neurology guidelines recommend G-banded karyotype and Fragile X DNA. The American College of Medical Genetics recommends Chromosomal Microarray Analysis (CMA). We determined the yield of…

Multiplexed Analysis of Serum Breast and Ovarian Cancer Markers by Means of Suspension Bead-quantum Dot Microarrays

NASA Astrophysics Data System (ADS)

Brazhnik, Kristina; Sokolova, Zinaida; Baryshnikova, Maria; Bilan, Regina; Nabiev, Igor; Sukhanova, Alyona

Multiplexed analysis of cancer markers is crucial for early tumor diagnosis and screening. We have designed lab-on-a-bead microarray for quantitative detection of three breast cancer markers in human serum. Quantum dots were used as bead-bound fluorescent tags for identifying each marker by means of flow cytometry. Antigen-specific beads reliably detected CA 15-3, CEA, and CA 125 in serum samples, providing clear discrimination between the samples with respect to the antigen levels. The novel microarray is advantageous over the routine single-analyte ones due to the simultaneous detection of various markers. Therefore the developed microarray is a promising tool for serum tumor marker profiling.

USE OF TRANSCRIPTIONAL COUPLING AND KEGG PATHWAY ANALYSIS OF GLOBAL GENE EXPRESSION TO REVEAL TRANSCRIPTIONAL CHANGES BETWEEN STATIONARY- AND LOG-PHASE SALMONELLA TYPHIMURIUM LT2

EPA Science Inventory

DNA microarray analysis is plagued by a lack of data reproducibility and by limits to the detectability of transcripts by hybridization. To mitigate these limitations, we employed transcriptional coupling within the S. typhimurium genome. This genome has 2664 transcriptionally co...

Analysis of Altered Micro RNA Expression Profiles in Focal Cortical Dysplasia IIB.

PubMed

Li, Lin; Liu, Chang-Qing; Li, Tian-Fu; Guan, Yu-Guang; Zhou, Jian; Qi, Xue-Ling; Yang, Yu-Tao; Deng, Jia-Hui; Xu, Zhi-Qing David; Luan, Guo-Ming

2016-04-01

Focal cortical dysplasia type IIB is a commonly encountered subtype of developmental malformation of the cerebral cortex and is often associated with pharmacoresistant epilepsy. In this study, to investigate the molecular etiology of focal cortical dysplasia type IIB, the authors performed micro ribonucleic acid (RNA) microarray on surgical specimens from 5 children (2 female and 3 male, mean age was 73.4 months, range 50-112 months) diagnosed of focal cortical dysplasia type IIB and matched normal tissue adjacent to the lesion. In all, 24 micro RNAs were differentially expressed in focal cortical dysplasia type IIB, and the microarray results were validated using quantitative real-time polymerase chain reaction (PCR). Then the putative target genes of the differentially expressed micro RNAs were identified by bioinformatics analysis. Moreover, biological significance of the target genes was evaluated by investigating the pathways in which the genes were enriched, and the Hippo signaling pathway was proposed to be highly related with the pathogenesis of focal cortical dysplasia type IIB. © The Author(s) 2015.

Molecular Profiling of Glatiramer Acetate Early Treatment Effects in Multiple Sclerosis

PubMed Central

Achiron, Anat; Feldman, Anna; Gurevich, Michael

2009-01-01

Background: Glatiramer acetate (GA, Copaxone®) has beneficial effects on the clinical course of relapsing-remitting multiple sclerosis (RRMS). However, the exact molecular mechanisms of GA effects are only partially understood. Objective: To characterized GA molecular effects in RRMS patients within 3 months of treatment by microarray profiling of peripheral blood mononuclear cells (PBMC). Methods: Gene-expression profiles were determined in RRMS patients before and at 3 months after initiation of GA treatment using Affimetrix (U133A-2) microarrays containing 14,500 well-characterized human genes. Most informative genes (MIGs) of GA-induced biological convergent pathways operating in RRMS were constructed using gene functional annotation, enrichment analysis and pathway reconstruction bioinformatic softwares. Verification at the mRNA and protein level was performed by qRT-PCR and FACS. Results: GA induced a specific gene expression molecular signature that included altered expression of 480 genes within 3 months of treatment; 262 genes were up-regulated, and 218 genes were down-regulated. The main convergent mechanisms of GA effects were related to antigen-activated apoptosis, inflammation, adhesion, and MHC class-I antigen presentation. Conclusions: Our findings demonstrate that GA treatment induces alternations of immunomodulatory gene expression patterns that are important for suppression of disease activity already at three months of treatment and can be used as molecular markers of GA activity. PMID:19893201

Mining featured biomarkers associated with prostatic carcinoma based on bioinformatics.

PubMed

Piao, Guanying; Wu, Jiarui

2013-11-01

To analyze the differentially expressed genes and identify featured biomarkers from prostatic carcinoma. The software "Significance Analysis of Microarray" (SAM) was used to identify the differentially coexpressed genes (DCGs). The DCGs existed in two datasets were analyzed by GO (Gene Ontology) functional annotation. A total of 389 DCGs were obtained. By GO analysis, we found these DCGs were closely related with the acinus development, TGF-β receptor and signal transduction pathways. Furthermore, five featured biomarkers were discovered by interaction analysis. These important signal pathways and oncogenes may provide potential therapeutic targets for prostatic carcinoma.

Line differences in Cor/Lea and fructan biosynthesis-related gene transcript accumulation are related to distinct freezing tolerance levels in synthetic wheat hexaploids.

PubMed

Yokota, Hirokazu; Iehisa, Julio C M; Shimosaka, Etsuo; Takumi, Shigeo

2015-03-15

In common wheat, cultivar differences in freezing tolerance are considered to be mainly due to allelic differences at two major loci controlling freezing tolerance. One of the two loci, Fr-2, is coincident with a cluster of genes encoding C-repeat binding factors (CBFs), which induce downstream Cor/Lea genes during cold acclimation. Here, we conducted microarray analysis to study comprehensive changes in gene expression profile under long-term low-temperature (LT) treatment and to identify other LT-responsive genes related to cold acclimation in leaves of seedlings and crown tissues of a synthetic hexaploid wheat line. The microarray analysis revealed marked up-regulation of a number of Cor/Lea genes and fructan biosynthesis-related genes under the long-term LT treatment. For validation of the microarray data, we selected four synthetic wheat lines that contain the A and B genomes from the tetraploid wheat cultivar Langdon and the diverse D genomes originating from different Aegilops tauschii accessions with distinct levels of freezing tolerance after cold acclimation. Quantitative RT-PCR showed increased transcript levels of the Cor/Lea, CBF, and fructan biosynthesis-related genes in more freezing-tolerant lines than in sensitive lines. After a 14-day LT treatment, a significant difference in fructan accumulation was observed among the four lines. Therefore, the fructan biosynthetic pathway is associated with cold acclimation in development of wheat freezing tolerance and is another pathway related to diversity in freezing tolerance, in addition to the CBF-mediated Cor/Lea expression pathway. Copyright © 2014 Elsevier GmbH. All rights reserved.

DNA Microarray Profiling Highlights Nrf2-Mediated Chemoprevention Targeted by Wasabi-Derived Isothiocyanates in HepG2 Cells.

PubMed

Trio, Phoebe Zapanta; Kawahara, Atsuyoshi; Tanigawa, Shunsuke; Sakao, Kozue; Hou, De-Xing

2017-01-01

6-MSITC and 6-MTITC are sulforaphane (SFN) analogs found in Japanese Wasabi. As we reported previously, Wasabi isothiocyanates (ITCs) are activators of Nrf2-antioxidant response element pathway, and also inhibitors of pro-inflammatory cyclooxygenase-2. This study is the first to assess the global changes in transcript levels by Wasabi ITCs, comparing with SFN, in HepG2 cells. We performed comparative gene expression profiling by treating HepG2 cells with ITCs, followed by DNA microarray analyses using HG-U133 plus 2.0 oligonucleotide array. Partial array data on selected gene products were confirmed by RT-PCR and Western blotting. Ingenuity Pathway Analysis (IPA) was used to identify functional subsets of genes and biologically significant network pathways. 6-MTITC showed the highest number of differentially altered (≥2 folds) gene expression, of which 114 genes were upregulated and 75 were downregulated. IPA revealed that Nrf2-mediated pathway, together with glutamate metabolism, is the common significantly modulated pathway across treatments. Interestingly, 6-MSITC exhibited the most potent effect toward Nrf2-mediated pathway. Our data suggest that 6-MSITC could exert chemopreventive role against cancer through its underlying antioxidant activity via the activation of Nrf2-mediated subsequent induction of cytoprotective genes.

The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

PubMed Central

Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

2009-01-01

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

Clustering approaches to identifying gene expression patterns from DNA microarray data.

PubMed

Do, Jin Hwan; Choi, Dong-Kug

2008-04-30

The analysis of microarray data is essential for large amounts of gene expression data. In this review we focus on clustering techniques. The biological rationale for this approach is the fact that many co-expressed genes are co-regulated, and identifying co-expressed genes could aid in functional annotation of novel genes, de novo identification of transcription factor binding sites and elucidation of complex biological pathways. Co-expressed genes are usually identified in microarray experiments by clustering techniques. There are many such methods, and the results obtained even for the same datasets may vary considerably depending on the algorithms and metrics for dissimilarity measures used, as well as on user-selectable parameters such as desired number of clusters and initial values. Therefore, biologists who want to interpret microarray data should be aware of the weakness and strengths of the clustering methods used. In this review, we survey the basic principles of clustering of DNA microarray data from crisp clustering algorithms such as hierarchical clustering, K-means and self-organizing maps, to complex clustering algorithms like fuzzy clustering.

Transcriptome profiling in engrailed-2 mutant mice reveals common molecular pathways associated with autism spectrum disorders.

PubMed

Sgadò, Paola; Provenzano, Giovanni; Dassi, Erik; Adami, Valentina; Zunino, Giulia; Genovesi, Sacha; Casarosa, Simona; Bozzi, Yuri

2013-12-19

Transcriptome analysis has been used in autism spectrum disorder (ASD) to unravel common pathogenic pathways based on the assumption that distinct rare genetic variants or epigenetic modifications affect common biological pathways. To unravel recurrent ASD-related neuropathological mechanisms, we took advantage of the En2-/- mouse model and performed transcriptome profiling on cerebellar and hippocampal adult tissues. Cerebellar and hippocampal tissue samples from three En2-/- and wild type (WT) littermate mice were assessed for differential gene expression using microarray hybridization followed by RankProd analysis. To identify functional categories overrepresented in the differentially expressed genes, we used integrated gene-network analysis, gene ontology enrichment and mouse phenotype ontology analysis. Furthermore, we performed direct enrichment analysis of ASD-associated genes from the SFARI repository in our differentially expressed genes. Given the limited number of animals used in the study, we used permissive criteria and identified 842 differentially expressed genes in En2-/- cerebellum and 862 in the En2-/- hippocampus. Our functional analysis revealed that the molecular signature of En2-/- cerebellum and hippocampus shares convergent pathological pathways with ASD, including abnormal synaptic transmission, altered developmental processes and increased immune response. Furthermore, when directly compared to the repository of the SFARI database, our differentially expressed genes in the hippocampus showed enrichment of ASD-associated genes significantly higher than previously reported. qPCR was performed for representative genes to confirm relative transcript levels compared to those detected in microarrays. Despite the limited number of animals used in the study, our bioinformatic analysis indicates the En2-/- mouse is a valuable tool for investigating molecular alterations related to ASD.

Systematic analysis of gene expression pattern in has-miR-197 over-expressed human uterine leiomyoma cells.

PubMed

Ling, Jing; Wu, Xiaoli; Fu, Ziyi; Tan, Jie; Xu, Qing

2015-10-01

Our previous study showed that the expression of miR-197 in leiomyoma was down-regulated compared with myometrium. Further, miR-197 has been identified to affect uterine leiomyoma cell proliferation, apoptosis, and metastasis ability, though the responsible molecular mechanism has not been well elucidated. In this study, we sought to determine the expression patterns of miR-197 targeted genes and to explore their potential functions, participating Pathways and the networks that are involved in the biological behavior of human uterine leiomyoma. After transfection of human uterine leiomyoma cells with miR-197, we confirmed the expression level of miR-197 using quantitative real-time PCR (qRT-PCR), and we detected the gene expression profiles after miR-197 over-expression through DNA microarray analysis. Further, we performed GO and Pathway analysis. The dominantly dys-regulated genes, which were up- or down-regulated by more than 10-fold, compared with parental cells, were confirmed using qRT-PCR technology. Compared with the control group, miR-197 was up-regulated by 30-fold after miR-197 lentiviral transfection. The microarray data showed that 872 genes were dys-regulated by more than 2-fold in human uterine leiomyoma cells after miR-197 overexpression, including 537 up-regulated and 335 down-regulated genes. The GO analysis indicated that the dys-regulated genes were primarily involved in response to stimuli, multicellular organ processes, and the signaling of biological progression. Further, Pathway analysis data showed that these genes participated in regulating several signaling Pathways, including the JAK/STAT signaling Pathway, the Toll-like receptor signaling Pathway, and cytokine-cytokine receptor interaction. The qRT-PCR results confirmed that 17 of the 66 selected genes, which were up- or down-regulated more than 10-fold by miR-197, were consistent with the microarray results, including tumorigenesis-related genes, such as DRT7, SLC549, SFMBT2, FLJ37956, FBLN2, C10orf35, HOXD12, CACNG7, and LOC100134279. Our study explored gene expression patterns after miR-197 overexpression and confirmed 17 dominantly dys-regulated genes, which could expand the insights into the function of miR-197 and the molecular mechanisms during the development and progression of uterine leiomyomas. This study might afford new clues for understanding the pathogenesis of uterine leiomyomas, and it could likely provide a unique method for diagnosing or predicting prognosis in the clinical treatment of leiomyoma. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

ARACNe-based inference, using curated microarray data, of Arabidopsis thaliana root transcriptional regulatory networks

PubMed Central

2014-01-01

Background Uncovering the complex transcriptional regulatory networks (TRNs) that underlie plant and animal development remains a challenge. However, a vast amount of data from public microarray experiments is available, which can be subject to inference algorithms in order to recover reliable TRN architectures. Results In this study we present a simple bioinformatics methodology that uses public, carefully curated microarray data and the mutual information algorithm ARACNe in order to obtain a database of transcriptional interactions. We used data from Arabidopsis thaliana root samples to show that the transcriptional regulatory networks derived from this database successfully recover previously identified root transcriptional modules and to propose new transcription factors for the SHORT ROOT/SCARECROW and PLETHORA pathways. We further show that these networks are a powerful tool to integrate and analyze high-throughput expression data, as exemplified by our analysis of a SHORT ROOT induction time-course microarray dataset, and are a reliable source for the prediction of novel root gene functions. In particular, we used our database to predict novel genes involved in root secondary cell-wall synthesis and identified the MADS-box TF XAL1/AGL12 as an unexpected participant in this process. Conclusions This study demonstrates that network inference using carefully curated microarray data yields reliable TRN architectures. In contrast to previous efforts to obtain root TRNs, that have focused on particular functional modules or tissues, our root transcriptional interactions provide an overview of the transcriptional pathways present in Arabidopsis thaliana roots and will likely yield a plethora of novel hypotheses to be tested experimentally. PMID:24739361

Revealing impaired pathways in the an11 mutant by high-throughput characterization of Petunia axillaris and Petunia inflata transcriptomes.

PubMed

Zenoni, Sara; D'Agostino, Nunzio; Tornielli, Giovanni B; Quattrocchio, Francesca; Chiusano, Maria L; Koes, Ronald; Zethof, Jan; Guzzo, Flavia; Delledonne, Massimo; Frusciante, Luigi; Gerats, Tom; Pezzotti, Mario

2011-10-01

Petunia is an excellent model system, especially for genetic, physiological and molecular studies. Thus far, however, genome-wide expression analysis has been applied rarely because of the lack of sequence information. We applied next-generation sequencing to generate, through de novo read assembly, a large catalogue of transcripts for Petunia axillaris and Petunia inflata. On the basis of both transcriptomes, comprehensive microarray chips for gene expression analysis were established and used for the analysis of global- and organ-specific gene expression in Petunia axillaris and Petunia inflata and to explore the molecular basis of the seed coat defects in a Petunia hybrida mutant, anthocyanin 11 (an11), lacking a WD40-repeat (WDR) transcription regulator. Among the transcripts differentially expressed in an11 seeds compared with wild type, many expected targets of AN11 were found but also several interesting new candidates that might play a role in morphogenesis of the seed coat. Our results validate the combination of next-generation sequencing with microarray analyses strategies to identify the transcriptome of two petunia species without previous knowledge of their genome, and to develop comprehensive chips as useful tools for the analysis of gene expression in P. axillaris, P. inflata and P. hybrida. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

High-Throughput Quantification of SH2 Domain-Phosphopeptide Interactions with Cellulose-Peptide Conjugate Microarrays.

PubMed

Engelmann, Brett W

2017-01-01

The Src Homology 2 (SH2) domain family primarily recognizes phosphorylated tyrosine (pY) containing peptide motifs. The relative affinity preferences among competing SH2 domains for phosphopeptide ligands define "specificity space," and underpins many functional pY mediated interactions within signaling networks. The degree of promiscuity exhibited and the dynamic range of affinities supported by individual domains or phosphopeptides is best resolved by a carefully executed and controlled quantitative high-throughput experiment. Here, I describe the fabrication and application of a cellulose-peptide conjugate microarray (CPCMA) platform to the quantitative analysis of SH2 domain specificity space. Included herein are instructions for optimal experimental design with special attention paid to common sources of systematic error, phosphopeptide SPOT synthesis, microarray fabrication, analyte titrations, data capture, and analysis.

Microbial Diagnostic Microarrays for the Detection and Typing of Food- and Water-Borne (Bacterial) Pathogens

PubMed Central

Kostić, Tanja; Sessitsch, Angela

2011-01-01

Reliable and sensitive pathogen detection in clinical and environmental (including food and water) samples is of greatest importance for public health. Standard microbiological methods have several limitations and improved alternatives are needed. Most important requirements for reliable analysis include: (i) specificity; (ii) sensitivity; (iii) multiplexing potential; (iv) robustness; (v) speed; (vi) automation potential; and (vii) low cost. Microarray technology can, through its very nature, fulfill many of these requirements directly and the remaining challenges have been tackled. In this review, we attempt to compare performance characteristics of the microbial diagnostic microarrays developed for the detection and typing of food and water pathogens, and discuss limitations, points still to be addressed and issues specific for the analysis of food, water and environmental samples. PMID:27605332

Pathway Distiller - multisource biological pathway consolidation

PubMed Central

2012-01-01

Background One method to understand and evaluate an experiment that produces a large set of genes, such as a gene expression microarray analysis, is to identify overrepresentation or enrichment for biological pathways. Because pathways are able to functionally describe the set of genes, much effort has been made to collect curated biological pathways into publicly accessible databases. When combining disparate databases, highly related or redundant pathways exist, making their consolidation into pathway concepts essential. This will facilitate unbiased, comprehensive yet streamlined analysis of experiments that result in large gene sets. Methods After gene set enrichment finds representative pathways for large gene sets, pathways are consolidated into representative pathway concepts. Three complementary, but different methods of pathway consolidation are explored. Enrichment Consolidation combines the set of the pathways enriched for the signature gene list through iterative combining of enriched pathways with other pathways with similar signature gene sets; Weighted Consolidation utilizes a Protein-Protein Interaction network based gene-weighting approach that finds clusters of both enriched and non-enriched pathways limited to the experiments' resultant gene list; and finally the de novo Consolidation method uses several measurements of pathway similarity, that finds static pathway clusters independent of any given experiment. Results We demonstrate that the three consolidation methods provide unified yet different functional insights of a resultant gene set derived from a genome-wide profiling experiment. Results from the methods are presented, demonstrating their applications in biological studies and comparing with a pathway web-based framework that also combines several pathway databases. Additionally a web-based consolidation framework that encompasses all three methods discussed in this paper, Pathway Distiller (http://cbbiweb.uthscsa.edu/PathwayDistiller), is established to allow researchers access to the methods and example microarray data described in this manuscript, and the ability to analyze their own gene list by using our unique consolidation methods. Conclusions By combining several pathway systems, implementing different, but complementary pathway consolidation methods, and providing a user-friendly web-accessible tool, we have enabled users the ability to extract functional explanations of their genome wide experiments. PMID:23134636

Pathway Distiller - multisource biological pathway consolidation.

PubMed

Doderer, Mark S; Anguiano, Zachry; Suresh, Uthra; Dashnamoorthy, Ravi; Bishop, Alexander J R; Chen, Yidong

2012-01-01

One method to understand and evaluate an experiment that produces a large set of genes, such as a gene expression microarray analysis, is to identify overrepresentation or enrichment for biological pathways. Because pathways are able to functionally describe the set of genes, much effort has been made to collect curated biological pathways into publicly accessible databases. When combining disparate databases, highly related or redundant pathways exist, making their consolidation into pathway concepts essential. This will facilitate unbiased, comprehensive yet streamlined analysis of experiments that result in large gene sets. After gene set enrichment finds representative pathways for large gene sets, pathways are consolidated into representative pathway concepts. Three complementary, but different methods of pathway consolidation are explored. Enrichment Consolidation combines the set of the pathways enriched for the signature gene list through iterative combining of enriched pathways with other pathways with similar signature gene sets; Weighted Consolidation utilizes a Protein-Protein Interaction network based gene-weighting approach that finds clusters of both enriched and non-enriched pathways limited to the experiments' resultant gene list; and finally the de novo Consolidation method uses several measurements of pathway similarity, that finds static pathway clusters independent of any given experiment. We demonstrate that the three consolidation methods provide unified yet different functional insights of a resultant gene set derived from a genome-wide profiling experiment. Results from the methods are presented, demonstrating their applications in biological studies and comparing with a pathway web-based framework that also combines several pathway databases. Additionally a web-based consolidation framework that encompasses all three methods discussed in this paper, Pathway Distiller (http://cbbiweb.uthscsa.edu/PathwayDistiller), is established to allow researchers access to the methods and example microarray data described in this manuscript, and the ability to analyze their own gene list by using our unique consolidation methods. By combining several pathway systems, implementing different, but complementary pathway consolidation methods, and providing a user-friendly web-accessible tool, we have enabled users the ability to extract functional explanations of their genome wide experiments.

MiR-141-3p is upregulated in esophageal squamous cell carcinoma and targets pleckstrin homology domain leucine-rich repeat protein phosphatase-2, a negative regulator of the PI3K/AKT pathway.

PubMed

Ishibashi, Osamu; Akagi, Ichiro; Ogawa, Yota; Inui, Takashi

2018-05-11

The phosphatidylinositol-3-kinase (PI3K)/AKT pathway is frequently activated in various human cancers and plays essential roles in their development and progression. Accumulating evidence suggests that dysregulated expression of microRNAs (miRNAs) is closely associated with cancer progression and metastasis. Here, we focused on miRNAs that could regulate genes related to the PI3K/AKT pathway in esophageal squamous cell carcinoma (ESCC). To identify upregulated miRNAs and their possible target genes in ESCC, we performed microarray-based integrative analyses of miRNA and mRNA expression levels in three human ESCC cell lines and a normal esophageal epithelial cell line. The miRNA microarray analysis revealed that miR-31-5p, miR-141-3p, miR-200b-3p, miR-200c-3p, and miR-205-5p were expressed at higher levels in the ESCC cell lines than the normal esophageal epithelial cell line. Bioinformatical analyses of mRNA microarray data identified several AKT/PI3K pathway-related genes as candidate targets of these miRNAs, which include tumor suppressors such as DNA-damage-inducible transcript 4 and pleckstrin homology domain leucine-rich repeat protein phosphatase-2 (PHLPP2). To validate the targets of relevant miRNAs experimentally, synthetic mimics of the miRNAs were transfected into the esophageal epithelial cell line. Here, we report that miR-141-3p suppress the expression of PHLPP2, a negative regulators of the AKT/PI3K pathway, as a target in ESCC. Copyright © 2018 Elsevier Inc. All rights reserved.

The MSX1 homeobox transcription factor is a downstream target of PHOX2B and activates the Delta-Notch pathway in neuroblastoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Revet, Ingrid; Huizenga, Gerda; Chan, Alvin

Neuroblastoma is an embryonal tumour of the peripheral sympathetic nervous system (SNS). One of the master regulator genes for peripheral SNS differentiation, the homeobox transcription factor PHOX2B, is mutated in familiar and sporadic neuroblastomas. Here we report that inducible expression of PHOX2B in the neuroblastoma cell line SJNB-8 down-regulates MSX1, a homeobox gene important for embryonic neural crest development. Inducible expression of MSX1 in SJNB-8 caused inhibition of both cell proliferation and colony formation in soft agar. Affymetrix micro-array and Northern blot analysis demonstrated that MSX1 strongly up-regulated the Delta-Notch pathway genes DLK1, NOTCH3, and HEY1. In addition, the proneuralmore » gene NEUROD1 was down-regulated. Western blot analysis showed that MSX1 induction caused cleavage of the NOTCH3 protein to its activated form, further confirming activation of the Delta-Notch pathway. These experiments describe for the first time regulation of the Delta-Notch pathway by MSX1, and connect these genes to the PHOX2B oncogene, indicative of a role in neuroblastoma biology. Affymetrix micro-array analysis of a neuroblastic tumour series consisting of neuroblastomas and the more benign ganglioneuromas showed that MSX1, NOTCH3 and HEY1 are more highly expressed in ganglioneuromas. This suggests a block in differentiation of these tumours at distinct developmental stages or lineages.« less

Polyadenylation state microarray (PASTA) analysis.

PubMed

Beilharz, Traude H; Preiss, Thomas

2011-01-01

Nearly all eukaryotic mRNAs terminate in a poly(A) tail that serves important roles in mRNA utilization. In the cytoplasm, the poly(A) tail promotes both mRNA stability and translation, and these functions are frequently regulated through changes in tail length. To identify the scope of poly(A) tail length control in a transcriptome, we developed the polyadenylation state microarray (PASTA) method. It involves the purification of mRNA based on poly(A) tail length using thermal elution from poly(U) sepharose, followed by microarray analysis of the resulting fractions. In this chapter we detail our PASTA approach and describe some methods for bulk and mRNA-specific poly(A) tail length measurements of use to monitor the procedure and independently verify the microarray data.

Transcriptomic responses to wounding: meta-analysis of gene expression microarray data.

PubMed

Sass, Piotr Andrzej; Dąbrowski, Michał; Charzyńska, Agata; Sachadyn, Paweł

2017-11-07

A vast amount of microarray data on transcriptomic response to injury has been collected so far. We designed the analysis in order to identify the genes displaying significant changes in expression after wounding in different organisms and tissues. This meta-analysis is the first study to compare gene expression profiles in response to wounding in as different tissues as heart, liver, skin, bones, and spinal cord, and species, including rat, mouse and human. We collected available microarray transcriptomic profiles obtained from different tissue injury experiments and selected the genes showing a minimum twofold change in expression in response to wounding in prevailing number of experiments for each of five wound healing stages we distinguished: haemostasis & early inflammation, inflammation, early repair, late repair and remodelling. During the initial phases after wounding, haemostasis & early inflammation and inflammation, the transcriptomic responses showed little consistency between different tissues and experiments. For the later phases, wound repair and remodelling, we identified a number of genes displaying similar transcriptional responses in all examined tissues. As revealed by ontological analyses, activation of certain pathways was rather specific for selected phases of wound healing, such as e.g. responses to vitamin D pronounced during inflammation. Conversely, we observed induction of genes encoding inflammatory agents and extracellular matrix proteins in all wound healing phases. Further, we selected several genes differentially upregulated throughout different stages of wound response, including established factors of wound healing in addition to those previously unreported  in this context such as PTPRC and AQP4. We found that transcriptomic responses to wounding showed similar traits in a diverse selection of tissues including skin, muscles, internal organs and nervous system. Notably, we distinguished transcriptional induction of inflammatory genes not only in the initial response to wounding, but also later, during wound repair and tissue remodelling.

Type I interferon and pattern recognition receptor signaling following particulate matter inhalation

PubMed Central

2012-01-01

Background Welding, a process that generates an aerosol containing gases and metal-rich particulates, induces adverse physiological effects including inflammation, immunosuppression and cardiovascular dysfunction. This study utilized microarray technology and subsequent pathway analysis as an exploratory search for markers/mechanisms of in vivo systemic effects following inhalation. Mice were exposed by inhalation to gas metal arc – stainless steel (GMA-SS) welding fume at 40 mg/m3 for 3 hr/d for 10 d and sacrificed 4 hr, 14 d and 28 d post-exposure. Whole blood cells, aorta and lung were harvested for global gene expression analysis with subsequent Ingenuity Pathway Analysis and confirmatory qRT-PCR. Serum was collected for protein profiling. Results The novel finding was a dominant type I interferon signaling network with the transcription factor Irf7 as a central component maintained through 28 d. Remarkably, these effects showed consistency across all tissues indicating a systemic type I interferon response that was complemented by changes in serum proteins (decreased MMP-9, CRP and increased VCAM1, oncostatin M, IP-10). In addition, pulmonary expression of interferon α and β and Irf7 specific pattern recognition receptors (PRR) and signaling molecules (Ddx58, Ifih1, Dhx58, ISGF3) were induced, an effect that showed specificity when compared to other inflammatory exposures. Also, a canonical pathway indicated a coordinated response of multiple PRR and associated signaling molecules (Tlr7, Tlr2, Clec7a, Nlrp3, Myd88) to inhalation of GMA-SS. Conclusion This methodological approach has the potential to identify consistent, prominent and/or novel pathways and provides insight into mechanisms that contribute to pulmonary and systemic effects following toxicant exposure. PMID:22776377

Type I interferon and pattern recognition receptor signaling following particulate matter inhalation.

PubMed

Erdely, Aaron; Antonini, James M; Salmen-Muniz, Rebecca; Liston, Angie; Hulderman, Tracy; Simeonova, Petia P; Kashon, Michael L; Li, Shengqiao; Gu, Ja K; Stone, Samuel; Chen, Bean T; Frazer, David G; Zeidler-Erdely, Patti C

2012-07-09

Welding, a process that generates an aerosol containing gases and metal-rich particulates, induces adverse physiological effects including inflammation, immunosuppression and cardiovascular dysfunction. This study utilized microarray technology and subsequent pathway analysis as an exploratory search for markers/mechanisms of in vivo systemic effects following inhalation. Mice were exposed by inhalation to gas metal arc - stainless steel (GMA-SS) welding fume at 40 mg/m3 for 3 hr/d for 10 d and sacrificed 4 hr, 14 d and 28 d post-exposure. Whole blood cells, aorta and lung were harvested for global gene expression analysis with subsequent Ingenuity Pathway Analysis and confirmatory qRT-PCR. Serum was collected for protein profiling. The novel finding was a dominant type I interferon signaling network with the transcription factor Irf7 as a central component maintained through 28 d. Remarkably, these effects showed consistency across all tissues indicating a systemic type I interferon response that was complemented by changes in serum proteins (decreased MMP-9, CRP and increased VCAM1, oncostatin M, IP-10). In addition, pulmonary expression of interferon α and β and Irf7 specific pattern recognition receptors (PRR) and signaling molecules (Ddx58, Ifih1, Dhx58, ISGF3) were induced, an effect that showed specificity when compared to other inflammatory exposures. Also, a canonical pathway indicated a coordinated response of multiple PRR and associated signaling molecules (Tlr7, Tlr2, Clec7a, Nlrp3, Myd88) to inhalation of GMA-SS. This methodological approach has the potential to identify consistent, prominent and/or novel pathways and provides insight into mechanisms that contribute to pulmonary and systemic effects following toxicant exposure.

Identification of rice genes associated with cosmic-ray response via co-expression gene network analysis.

PubMed

Hwang, Sun-Goo; Kim, Dong Sub; Hwang, Jung Eun; Han, A-Reum; Jang, Cheol Seong

2014-05-15

In order to better understand the biological systems that are affected in response to cosmic ray (CR), we conducted weighted gene co-expression network analysis using the module detection method. By using the Pearson's correlation coefficient (PCC) value, we evaluated complex gene-gene functional interactions between 680 CR-responsive probes from integrated microarray data sets, which included large-scale transcriptional profiling of 1000 microarray samples. These probes were divided into 6 distinct modules that contained 20 enriched gene ontology (GO) functions, such as oxidoreductase activity, hydrolase activity, and response to stimulus and stress. In particular, modules 1 and 2 commonly showed enriched annotation categories such as oxidoreductase activity, including enriched cis-regulatory elements known as ROS-specific regulators. These results suggest that the ROS-mediated irradiation response pathway is affected by CR in modules 1 and 2. We found 243 ionizing radiation (IR)-responsive probes that exhibited similarities in expression patterns in various irradiation microarray data sets. The expression patterns of 6 randomly selected IR-responsive genes were evaluated by quantitative reverse transcription polymerase chain reaction following treatment with CR, gamma rays (GR), and ion beam (IB); similar patterns were observed among these genes under these 3 treatments. Moreover, we constructed subnetworks of IR-responsive genes and evaluated the expression levels of their neighboring genes following GR treatment; similar patterns were observed among them. These results of network-based analyses might provide a clue to understanding the complex biological system related to the CR response in plants. Copyright © 2014 Elsevier B.V. All rights reserved.

Gene expression profile of mouse prostate tumors reveals dysregulations in major biological processes and identifies potential murine targets for preclinical development of human prostate cancer therapy.

PubMed

Haram, Kerstyn M; Peltier, Heidi J; Lu, Bin; Bhasin, Manoj; Otu, Hasan H; Choy, Bob; Regan, Meredith; Libermann, Towia A; Latham, Gary J; Sanda, Martin G; Arredouani, Mohamed S

2008-10-01

Translation of preclinical studies into effective human cancer therapy is hampered by the lack of defined molecular expression patterns in mouse models that correspond to the human counterpart. We sought to generate an open source TRAMP mouse microarray dataset and to use this array to identify differentially expressed genes from human prostate cancer (PCa) that have concordant expression in TRAMP tumors, and thereby represent lead targets for preclinical therapy development. We performed microarrays on total RNA extracted and amplified from eight TRAMP tumors and nine normal prostates. A subset of differentially expressed genes was validated by QRT-PCR. Differentially expressed TRAMP genes were analyzed for concordant expression in publicly available human prostate array datasets and a subset of resulting genes was analyzed by QRT-PCR. Cross-referencing differentially expressed TRAMP genes to public human prostate array datasets revealed 66 genes with concordant expression in mouse and human PCa; 56 between metastases and normal and 10 between primary tumor and normal tissues. Of these 10 genes, two, Sox4 and Tubb2a, were validated by QRT-PCR. Our analysis also revealed various dysregulations in major biologic pathways in the TRAMP prostates. We report a TRAMP microarray dataset of which a gene subset was validated by QRT-PCR with expression patterns consistent with previous gene-specific TRAMP studies. Concordance analysis between TRAMP and human PCa associated genes supports the utility of the model and suggests several novel molecular targets for preclinical therapy.

Artificial intelligence in hematology.

PubMed

Zini, Gina

2005-10-01

Artificial intelligence (AI) is a computer based science which aims to simulate human brain faculties using a computational system. A brief history of this new science goes from the creation of the first artificial neuron in 1943 to the first artificial neural network application to genetic algorithms. The potential for a similar technology in medicine has immediately been identified by scientists and researchers. The possibility to store and process all medical knowledge has made this technology very attractive to assist or even surpass clinicians in reaching a diagnosis. Applications of AI in medicine include devices applied to clinical diagnosis in neurology and cardiopulmonary diseases, as well as the use of expert or knowledge-based systems in routine clinical use for diagnosis, therapeutic management and for prognostic evaluation. Biological applications include genome sequencing or DNA gene expression microarrays, modeling gene networks, analysis and clustering of gene expression data, pattern recognition in DNA and proteins, protein structure prediction. In the field of hematology the first devices based on AI have been applied to the routine laboratory data management. New tools concern the differential diagnosis in specific diseases such as anemias, thalassemias and leukemias, based on neural networks trained with data from peripheral blood analysis. A revolution in cancer diagnosis, including the diagnosis of hematological malignancies, has been the introduction of the first microarray based and bioinformatic approach for molecular diagnosis: a systematic approach based on the monitoring of simultaneous expression of thousands of genes using DNA microarray, independently of previous biological knowledge, analysed using AI devices. Using gene profiling, the traditional diagnostic pathways move from clinical to molecular based diagnostic systems.

Transcriptional profiling of host gene expression in chicken embryo lung cells infected with laryngotracheitis virus

PubMed Central

2010-01-01

Background Infection by infectious laryngotracheitis virus (ILTV; gallid herpesvirus 1) causes acute respiratory diseases in chickens often with high mortality. To better understand host-ILTV interactions at the host transcriptional level, a microarray analysis was performed using 4 × 44 K Agilent chicken custom oligo microarrays. Results Microarrays were hybridized using the two color hybridization method with total RNA extracted from ILTV infected chicken embryo lung cells at 0, 1, 3, 5, and 7 days post infection (dpi). Results showed that 789 genes were differentially expressed in response to ILTV infection that include genes involved in the immune system (cytokines, chemokines, MHC, and NF-κB), cell cycle regulation (cyclin B2, CDK1, and CKI3), matrix metalloproteinases (MMPs) and cellular metabolism. Differential expression for 20 out of 789 genes were confirmed by quantitative reverse transcription-PCR (qRT-PCR). A bioinformatics tool (Ingenuity Pathway Analysis) used to analyze biological functions and pathways on the group of 789 differentially expressed genes revealed that 21 possible gene networks with intermolecular connections among 275 functionally identified genes. These 275 genes were classified into a number of functional groups that included cancer, genetic disorder, cellular growth and proliferation, and cell death. Conclusion The results of this study provide comprehensive knowledge on global gene expression, and biological functionalities of differentially expressed genes in chicken embryo lung cells in response to ILTV infections. PMID:20663125

Alteration of gene expression in the colon of colorectal cancer model rat by dietary sodium gluconate.

PubMed

Kameue, Chiyoko; Tsukahara, Takamitsu; Ushida, Kazunari

2006-03-01

Butyrate induces apoptosis of various cancer cell lines in a p53-independent manner and inhibits the proliferation of cancer cells. In a previous report, we reported a significant reduction in tumor incidence in rat colon as a result of dietary sodium gluconate (GNA). The stimulation of apoptosis through enhanced butyrate production in the large intestine was involved in the antitumorigenic effect of GNA. In the present study, a cDNA microarray analysis was performed to investigate the particular mechanism involved in the antitumorigenic effect of GNA. Some up-regulated genes suggested by microarray analysis were further evaluated using real-time PCR. A microarray revealed that GNA regulates the expression of retinoic acid receptor (RAR) and retinoid X receptor (RXR), and several genes known as the target of retinoids in cancer cells. In other words, the antitumorigenic effect of GNA may involve the regulation of the retinoid signaling pathway by butyrate in a retinoid-independent manner.

c-Kit modifies the inflammatory status of smooth muscle cells

PubMed Central

Song, Lei; Martinez, Laisel; Zigmond, Zachary M.; Hernandez, Diana R.; Lassance-Soares, Roberta M.; Selman, Guillermo

2017-01-01

Background c-Kit is a receptor tyrosine kinase present in multiple cell types, including vascular smooth muscle cells (SMC). However, little is known about how c-Kit influences SMC biology and vascular pathogenesis. Methods High-throughput microarray assays and in silico pathway analysis were used to identify differentially expressed genes between primary c-Kit deficient (KitW/W–v) and control (Kit+/+) SMC. Quantitative real-time RT-PCR and functional assays further confirmed the differences in gene expression and pro-inflammatory pathway regulation between both SMC populations. Results The microarray analysis revealed elevated NF-κB gene expression secondary to the loss of c-Kit that affects both the canonical and alternative NF-κB pathways. Upon stimulation with an oxidized phospholipid as pro-inflammatory agent, c-Kit deficient SMC displayed enhanced NF-κB transcriptional activity, higher phosphorylated/total p65 ratio, and increased protein expression of NF-κB regulated pro-inflammatory mediators with respect to cells from control mice. The pro-inflammatory phenotype of mutant cells was ameliorated after restoring c-Kit activity using lentiviral transduction. Functional assays further demonstrated that c-Kit suppresses NF-κB activity in SMC in a TGFβ-activated kinase 1 (TAK1) and Nemo-like kinase (NLK) dependent manner. Discussion Our study suggests a novel mechanism by which c-Kit suppresses NF-κB regulated pathways in SMC to prevent their pro-inflammatory transformation. PMID:28626608

c-Kit modifies the inflammatory status of smooth muscle cells.

PubMed

Song, Lei; Martinez, Laisel; Zigmond, Zachary M; Hernandez, Diana R; Lassance-Soares, Roberta M; Selman, Guillermo; Vazquez-Padron, Roberto I

2017-01-01

c-Kit is a receptor tyrosine kinase present in multiple cell types, including vascular smooth muscle cells (SMC). However, little is known about how c-Kit influences SMC biology and vascular pathogenesis. High-throughput microarray assays and in silico pathway analysis were used to identify differentially expressed genes between primary c-Kit deficient (Kit W/W-v ) and control (Kit +/+ ) SMC. Quantitative real-time RT-PCR and functional assays further confirmed the differences in gene expression and pro-inflammatory pathway regulation between both SMC populations. The microarray analysis revealed elevated NF-κB gene expression secondary to the loss of c-Kit that affects both the canonical and alternative NF-κB pathways. Upon stimulation with an oxidized phospholipid as pro-inflammatory agent, c-Kit deficient SMC displayed enhanced NF-κB transcriptional activity, higher phosphorylated/total p65 ratio, and increased protein expression of NF-κB regulated pro-inflammatory mediators with respect to cells from control mice. The pro-inflammatory phenotype of mutant cells was ameliorated after restoring c-Kit activity using lentiviral transduction. Functional assays further demonstrated that c-Kit suppresses NF-κB activity in SMC in a TGFβ-activated kinase 1 (TAK1) and Nemo-like kinase (NLK) dependent manner. Our study suggests a novel mechanism by which c-Kit suppresses NF-κB regulated pathways in SMC to prevent their pro-inflammatory transformation.

Identification of Key Transcription Factors Associated with Lung Squamous Cell Carcinoma

PubMed Central

Zhang, Feng; Chen, Xia; Wei, Ke; Liu, Daoming; Xu, Xiaodong; Zhang, Xing; Shi, Hong

2017-01-01

Background Lung squamous cell carcinoma (lung SCC) is a common type of lung cancer, but its mechanism of pathogenesis is unclear. The aim of this study was to identify key transcription factors in lung SCC and elucidate its mechanism. Material/Methods Six published microarray datasets of lung SCC were downloaded from Gene Expression Omnibus (GEO) for integrated bioinformatics analysis. Significance analysis of microarrays was used to identify differentially expressed genes (DEGs) between lung SCC and normal controls. The biological functions and signaling pathways of DEGs were mapped in the Gene Otology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, respectively. A transcription factor gene regulatory network was used to obtain insights into the functions of DEGs. Results A total of 1,011 genes, including 539 upregulated genes and 462 downregulated genes, were filtered as DEGs between lung SCC and normal controls. DEGs were significantly enriched in cell cycle, DNA replication, p53 signaling pathway, pathways in cancer, adherens junction, and cell adhesion molecules signaling pathways. There were 57 transcription factors identified, which were used to construct a regulatory network. The network consisted of 736 interactions between 49 transcription factors and 486 DEGs. NFIC, BRCA1, and NFATC2 were the top 3 transcription factors that had the highest connectivity with DEGs and that regulated 83, 82, and 75 DEGs in the network, respectively. Conclusions NFIC, BRCA1, and NFATC2 might be the key transcription factors in the development of lung SCC by regulating the genes involved in cell cycle and DNA replication pathways. PMID:28081052

Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by algU.

PubMed

Shi, Xiang Yang; Dumenyo, C Korsi; Hernandez-Martinez, Rufina; Azad, Hamid; Cooksey, Donald A

2007-11-01

Many virulence genes in plant bacterial pathogens are coordinately regulated by "global" regulatory genes. Conducting DNA microarray analysis of bacterial mutants of such genes, compared with the wild type, can help to refine the list of genes that may contribute to virulence in bacterial pathogens. The regulatory gene algU, with roles in stress response and regulation of the biosynthesis of the exopolysaccharide alginate in Pseudomonas aeruginosa and many other bacteria, has been extensively studied. The role of algU in Xylella fastidiosa, the cause of Pierce's disease of grapevines, was analyzed by mutation and whole-genome microarray analysis to define its involvement in aggregation, biofilm formation, and virulence. In this study, an algU::nptII mutant had reduced cell-cell aggregation, attachment, and biofilm formation and lower virulence in grapevines. Microarray analysis showed that 42 genes had significantly lower expression in the algU::nptII mutant than in the wild type. Among these are several genes that could contribute to cell aggregation and biofilm formation, as well as other physiological processes such as virulence, competition, and survival.

Differential Coexpression Analysis Reveals Extensive Rewiring of Arabidopsis Gene Coexpression in Response to Pseudomonas syringae Infection

PubMed Central

Jiang, Zhenhong; Dong, Xiaobao; Li, Zhi-Gang; He, Fei; Zhang, Ziding

2016-01-01

Plant defense responses to pathogens involve massive transcriptional reprogramming. Recently, differential coexpression analysis has been developed to study the rewiring of gene networks through microarray data, which is becoming an important complement to traditional differential expression analysis. Using time-series microarray data of Arabidopsis thaliana infected with Pseudomonas syringae, we analyzed Arabidopsis defense responses to P. syringae through differential coexpression analysis. Overall, we found that differential coexpression was a common phenomenon of plant immunity. Genes that were frequently involved in differential coexpression tend to be related to plant immune responses. Importantly, many of those genes have similar average expression levels between normal plant growth and pathogen infection but have different coexpression partners. By integrating the Arabidopsis regulatory network into our analysis, we identified several transcription factors that may be regulators of differential coexpression during plant immune responses. We also observed extensive differential coexpression between genes within the same metabolic pathways. Several metabolic pathways, such as photosynthesis light reactions, exhibited significant changes in expression correlation between normal growth and pathogen infection. Taken together, differential coexpression analysis provides a new strategy for analyzing transcriptional data related to plant defense responses and new insights into the understanding of plant-pathogen interactions. PMID:27721457

Development and Comparison of Two Assay Formats for Parallel Detection of Four Biothreat Pathogens by Using Suspension Microarrays

PubMed Central

Janse, Ingmar; Bok, Jasper M.; Hamidjaja, Raditijo A.; Hodemaekers, Hennie M.; van Rotterdam, Bart J.

2012-01-01

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple pathogens. We developed diagnostic suspension microarrays for sensitive and specific detection of the biothreat pathogens Bacillus anthracis, Yersinia pestis, Francisella tularensis and Coxiella burnetii. Two assay chemistries for amplification and labeling were developed, one method using direct hybridization and the other using target-specific primer extension, combined with hybridization to universal arrays. Asymmetric PCR products for both assay chemistries were produced by using a multiplex asymmetric PCR amplifying 16 DNA signatures (16-plex). The performances of both assay chemistries were compared and their advantages and disadvantages are discussed. The developed microarrays detected multiple signature sequences and an internal control which made it possible to confidently identify the targeted pathogens and assess their virulence potential. The microarrays were highly specific and detected various strains of the targeted pathogens. Detection limits for the different pathogen signatures were similar or slightly higher compared to real-time PCR. Probit analysis showed that even a few genomic copies could be detected with 95% confidence. The microarrays detected DNA from different pathogens mixed in different ratios and from spiked or naturally contaminated samples. The assays that were developed have a potential for application in surveillance and diagnostics. PMID:22355407

Development and comparison of two assay formats for parallel detection of four biothreat pathogens by using suspension microarrays.

PubMed

Janse, Ingmar; Bok, Jasper M; Hamidjaja, Raditijo A; Hodemaekers, Hennie M; van Rotterdam, Bart J

2012-01-01

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple pathogens. We developed diagnostic suspension microarrays for sensitive and specific detection of the biothreat pathogens Bacillus anthracis, Yersinia pestis, Francisella tularensis and Coxiella burnetii. Two assay chemistries for amplification and labeling were developed, one method using direct hybridization and the other using target-specific primer extension, combined with hybridization to universal arrays. Asymmetric PCR products for both assay chemistries were produced by using a multiplex asymmetric PCR amplifying 16 DNA signatures (16-plex). The performances of both assay chemistries were compared and their advantages and disadvantages are discussed. The developed microarrays detected multiple signature sequences and an internal control which made it possible to confidently identify the targeted pathogens and assess their virulence potential. The microarrays were highly specific and detected various strains of the targeted pathogens. Detection limits for the different pathogen signatures were similar or slightly higher compared to real-time PCR. Probit analysis showed that even a few genomic copies could be detected with 95% confidence. The microarrays detected DNA from different pathogens mixed in different ratios and from spiked or naturally contaminated samples. The assays that were developed have a potential for application in surveillance and diagnostics.

The function of BTG3 in colorectal cancer cells and its possible signaling pathway.

PubMed

Lv, Chi; Wang, Heling; Tong, Yuxin; Yin, Hongzhuan; Wang, Dalu; Yan, Zhaopeng; Liang, Yichao; Wu, Di; Su, Qi

2018-02-01

B-cell translocation gene 3 (BTG3) has been identified as a candidate driver gene for various cancers, but its specific role in colorectal cancer (CRC) is poorly understood. We aimed to investigate the relationship between expression of BTG3 and clinicopathological features and prognosis, as well as to explore the effects and the role of a possible BTG3 molecular mechanism on aggressive colorectal cancer behavior. BTG3 expression was assessed by immunohistochemistry (IHC) on specimens from 140 patients with CRC. The association of BTG3 expression with clinicopathological features was examined. To confirm the biological role of BTG3 in CRC, two CRC cell lines expressing BTG3 were used and BTG3 expression was knocked down by shRNA. CCK-8, cell cycle, apoptosis, migration, and invasion assays were performed. The influence of BTG3 knockdown was further investigated by genomic microarray to uncover the potential molecular mechanisms underlying BTG3-mediated CRC development and progression. BTG3 was downregulated in colorectal cancer tissues and positively correlated with pathological classification (p = 0.037), depth of invasion (p = 0.016), distant metastasis (p = 0.024), TNM stage (p = 0.007), and overall survival (OS) and disease-free survival (DFS). BTG3 knockdown promoted cell proliferation, migration, invasion, relieved G2 arrest, and inhibited apoptosis in HCT116 and LoVo cells. A genomic microarray analysis showed that numerous tumor-associated signaling pathways and oncogenes were altered by BTG3 knockdown. At the mRNA level, nine genes referred to the extracellular-regulated kinase/mitogen-activated protein kinase pathway were differentially expressed. Western blotting revealed that BTG3 knockdown upregulated PAK2, RPS6KA5, YWHAB, and signal transducer and activator of transcription (STAT)3 protein levels, but downregulated RAP1A, DUSP6, and STAT1 protein expression, which was consistent with the genomic microarray data. BTG3 expression might contribute to CRC carcinogenesis. BTG3 knockdown might strengthen the aggressive colorectal cancer behavior.

ArrayInitiative - a tool that simplifies creating custom Affymetrix CDFs

PubMed Central

2011-01-01

Background Probes on a microarray represent a frozen view of a genome and are quickly outdated when new sequencing studies extend our knowledge, resulting in significant measurement error when analyzing any microarray experiment. There are several bioinformatics approaches to improve probe assignments, but without in-house programming expertise, standardizing these custom array specifications as a usable file (e.g. as Affymetrix CDFs) is difficult, owing mostly to the complexity of the specification file format. However, without correctly standardized files there is a significant barrier for testing competing analysis approaches since this file is one of the required inputs for many commonly used algorithms. The need to test combinations of probe assignments and analysis algorithms led us to develop ArrayInitiative, a tool for creating and managing custom array specifications. Results ArrayInitiative is a standalone, cross-platform, rich client desktop application for creating correctly formatted, custom versions of manufacturer-provided (default) array specifications, requiring only minimal knowledge of the array specification rules and file formats. Users can import default array specifications, import probe sequences for a default array specification, design and import a custom array specification, export any array specification to multiple output formats, export the probe sequences for any array specification and browse high-level information about the microarray, such as version and number of probes. The initial release of ArrayInitiative supports the Affymetrix 3' IVT expression arrays we currently analyze, but as an open source application, we hope that others will contribute modules for other platforms. Conclusions ArrayInitiative allows researchers to create new array specifications, in a standard format, based upon their own requirements. This makes it easier to test competing design and analysis strategies that depend on probe definitions. Since the custom array specifications are easily exported to the manufacturer's standard format, researchers can analyze these customized microarray experiments using established software tools, such as those available in Bioconductor. PMID:21548938

BioCichlid: central dogma-based 3D visualization system of time-course microarray data on a hierarchical biological network.

PubMed

Ishiwata, Ryosuke R; Morioka, Masaki S; Ogishima, Soichi; Tanaka, Hiroshi

2009-02-15

BioCichlid is a 3D visualization system of time-course microarray data on molecular networks, aiming at interpretation of gene expression data by transcriptional relationships based on the central dogma with physical and genetic interactions. BioCichlid visualizes both physical (protein) and genetic (regulatory) network layers, and provides animation of time-course gene expression data on the genetic network layer. Transcriptional regulations are represented to bridge the physical network (transcription factors) and genetic network (regulated genes) layers, thus integrating promoter analysis into the pathway mapping. BioCichlid enhances the interpretation of microarray data and allows for revealing the underlying mechanisms causing differential gene expressions. BioCichlid is freely available and can be accessed at http://newton.tmd.ac.jp/. Source codes for both biocichlid server and client are also available.

Molecular strategies of the Caenorhabditis elegans dauer larva to survive extreme desiccation.

PubMed

Erkut, Cihan; Vasilj, Andrej; Boland, Sebastian; Habermann, Bianca; Shevchenko, Andrej; Kurzchalia, Teymuras V

2013-01-01

Massive water loss is a serious challenge for terrestrial animals, which usually has fatal consequences. However, some organisms have developed means to survive this stress by entering an ametabolic state called anhydrobiosis. The molecular and cellular mechanisms underlying this phenomenon are poorly understood. We recently showed that Caenorhabditis elegans dauer larva, an arrested stage specialized for survival in adverse conditions, is resistant to severe desiccation. However, this requires a preconditioning step at a mild desiccative environment to prepare the organism for harsher desiccation conditions. A systems approach was used to identify factors that are activated during this preconditioning. Using microarray analysis, proteomics, and bioinformatics, genes, proteins, and biochemical pathways that are upregulated during this process were identified. These pathways were validated via reverse genetics by testing the desiccation tolerances of mutants. These data show that the desiccation response is activated by hygrosensation (sensing the desiccative environment) via head neurons. This leads to elimination of reactive oxygen species and xenobiotics, expression of heat shock and intrinsically disordered proteins, polyamine utilization, and induction of fatty acid desaturation pathway. Remarkably, this response is specific and involves a small number of functional pathways, which represent the generic toolkit for anhydrobiosis in plants and animals.

Molecular Strategies of the Caenorhabditis elegans Dauer Larva to Survive Extreme Desiccation

PubMed Central

Erkut, Cihan; Vasilj, Andrej; Boland, Sebastian; Habermann, Bianca; Shevchenko, Andrej; Kurzchalia, Teymuras V.

2013-01-01

Massive water loss is a serious challenge for terrestrial animals, which usually has fatal consequences. However, some organisms have developed means to survive this stress by entering an ametabolic state called anhydrobiosis. The molecular and cellular mechanisms underlying this phenomenon are poorly understood. We recently showed that Caenorhabditis elegans dauer larva, an arrested stage specialized for survival in adverse conditions, is resistant to severe desiccation. However, this requires a preconditioning step at a mild desiccative environment to prepare the organism for harsher desiccation conditions. A systems approach was used to identify factors that are activated during this preconditioning. Using microarray analysis, proteomics, and bioinformatics, genes, proteins, and biochemical pathways that are upregulated during this process were identified. These pathways were validated via reverse genetics by testing the desiccation tolerances of mutants. These data show that the desiccation response is activated by hygrosensation (sensing the desiccative environment) via head neurons. This leads to elimination of reactive oxygen species and xenobiotics, expression of heat shock and intrinsically disordered proteins, polyamine utilization, and induction of fatty acid desaturation pathway. Remarkably, this response is specific and involves a small number of functional pathways, which represent the generic toolkit for anhydrobiosis in plants and animals. PMID:24324795

Microarray-based analysis of gene expression in lycopersicon esculentum seedling roots in response to cadmium, chromium, mercury, and lead.

PubMed

Hou, Jing; Liu, Xinhui; Wang, Juan; Zhao, Shengnan; Cui, Baoshan

2015-02-03

The effects of heavy metals in agricultural soils have received special attention due to their potential for accumulation in crops, which can affect species at all trophic levels. Therefore, there is a critical need for reliable bioassays for assessing risk levels due to heavy metals in agricultural soil. In the present study, we used microarrays to investigate changes in gene expression of Lycopersicon esculentum in response to Cd-, Cr-, Hg-, or Pb-spiked soil. Exposure to (1)/10 median lethal concentrations (LC50) of Cd, Cr, Hg, or Pb for 7 days resulted in expression changes in 29 Cd-specific, 58 Cr-specific, 192 Hg-specific and 864 Pb-specific genes as determined by microarray analysis, whereas conventional morphological and physiological bioassays did not reveal any toxicant stresses. Hierarchical clustering analysis showed that the characteristic gene expression profiles induced by Cd, Cr, Hg, and Pb were distinct from not only the control but also one another. Furthermore, a total of three genes related to "ion transport" for Cd, 14 genes related to "external encapsulating structure organization", "reproductive developmental process", "lipid metabolic process" and "response to stimulus" for Cr, 11 genes related to "cellular metabolic process" and "cellular response to stimulus" for Hg, 78 genes related to 20 biological processes (e.g., DNA metabolic process, monosaccharide catabolic process, cell division) for Pb were identified and selected as their potential biomarkers. These findings demonstrated that microarray-based analysis of Lycopersicon esculentum was a sensitive tool for the early detection of potential toxicity of heavy metals in agricultural soil, as well as an effective tool for identifying the heavy metal-specific genes, which should be useful for assessing risk levels due to heavy metals in agricultural soil.

The pleiotropic effects of fisetin and hesperetin on human acute promyelocytic leukemia cells are mediated through apoptosis, cell cycle arrest, and alterations in signaling networks.

PubMed

Adan, Aysun; Baran, Yusuf

2015-11-01

Fisetin and hesperetin, flavonoids from various plants, have several pharmaceutical activities including antioxidative, anti-inflammatory, and anticancer effects. However, studies elucidating the role and the mechanism(s) of action of fisetin and hesperetin in acute promyelocytic leukemia are absent. In this study, we investigated the mechanism of the antiproliferative and apoptotic actions exerted by fisetin and hesperetin on human HL60 acute promyelocytic leukemia cells. The viability of HL60 cells was evaluated using the MTT assay, apoptosis by annexin V/propidium iodide (PI) staining and cell cycle distribution using flow cytometry, and changes in caspase-3 enzyme activity and mitochondrial transmembrane potential. Moreover, we performed whole-genome microarray gene expression analysis to reveal genes affected by fisetin and hesperetin that can be important for developing of future targeted therapy. Based on data obtained from microarray analysis, we also described biological networks modulated after fisetin and hesperetin treatment by KEGG and IPA analysis. Fisetin and hesperetin treatment showed a concentration- and time-dependent inhibition of proliferation and induced G2/M arrest for both agents and G0/G1 arrest for hesperetin at only the highest concentrations. There was a disruption of mitochondrial membrane potential together with increased caspase-3 activity. Furthermore, fisetin- and hesperetin-triggered apoptosis was confirmed by annexin V/PI analysis. The microarray gene profiling analysis revealed some important biological pathways including mitogen-activated protein kinases (MAPK) and inhibitor of DNA binding (ID) signaling pathways altered by fisetin and hesperetin treatment as well as gave a list of genes modulated ≥2-fold involved in cell proliferation, cell division, and apoptosis. Altogether, data suggested that fisetin and hesperetin have anticancer properties and deserve further investigation.

Impairment of organ-specific T cell negative selection by diabetes susceptibility genes: genomic analysis by mRNA profiling.

PubMed

Liston, Adrian; Hardy, Kristine; Pittelkow, Yvonne; Wilson, Susan R; Makaroff, Lydia E; Fahrer, Aude M; Goodnow, Christopher C

2007-01-01

T cells in the thymus undergo opposing positive and negative selection processes so that the only T cells entering circulation are those bearing a T cell receptor (TCR) with a low affinity for self. The mechanism differentiating negative from positive selection is poorly understood, despite the fact that inherited defects in negative selection underlie organ-specific autoimmune disease in AIRE-deficient people and the non-obese diabetic (NOD) mouse strain Here we use homogeneous populations of T cells undergoing either positive or negative selection in vivo together with genome-wide transcription profiling on microarrays to identify the gene expression differences underlying negative selection to an Aire-dependent organ-specific antigen, including the upregulation of a genomic cluster in the cytogenetic band 2F. Analysis of defective negative selection in the autoimmune-prone NOD strain demonstrates a global impairment in the induction of the negative selection response gene set, but little difference in positive selection response genes. Combining expression differences with genetic linkage data, we identify differentially expressed candidate genes, including Bim, Bnip3, Smox, Pdrg1, Id1, Pdcd1, Ly6c, Pdia3, Trim30 and Trim12. The data provide a molecular map of the negative selection response in vivo and, by analysis of deviations from this pathway in the autoimmune susceptible NOD strain, suggest that susceptibility arises from small expression differences in genes acting at multiple points in the pathway between the TCR and cell death.

Impairment of organ-specific T cell negative selection by diabetes susceptibility genes: genomic analysis by mRNA profiling

PubMed Central

Liston, Adrian; Hardy, Kristine; Pittelkow, Yvonne; Wilson, Susan R; Makaroff, Lydia E; Fahrer, Aude M; Goodnow, Christopher C

2007-01-01

Background T cells in the thymus undergo opposing positive and negative selection processes so that the only T cells entering circulation are those bearing a T cell receptor (TCR) with a low affinity for self. The mechanism differentiating negative from positive selection is poorly understood, despite the fact that inherited defects in negative selection underlie organ-specific autoimmune disease in AIRE-deficient people and the non-obese diabetic (NOD) mouse strain Results Here we use homogeneous populations of T cells undergoing either positive or negative selection in vivo together with genome-wide transcription profiling on microarrays to identify the gene expression differences underlying negative selection to an Aire-dependent organ-specific antigen, including the upregulation of a genomic cluster in the cytogenetic band 2F. Analysis of defective negative selection in the autoimmune-prone NOD strain demonstrates a global impairment in the induction of the negative selection response gene set, but little difference in positive selection response genes. Combining expression differences with genetic linkage data, we identify differentially expressed candidate genes, including Bim, Bnip3, Smox, Pdrg1, Id1, Pdcd1, Ly6c, Pdia3, Trim30 and Trim12. Conclusion The data provide a molecular map of the negative selection response in vivo and, by analysis of deviations from this pathway in the autoimmune susceptible NOD strain, suggest that susceptibility arises from small expression differences in genes acting at multiple points in the pathway between the TCR and cell death. PMID:17239257

Differential expression of thymic DNA repair genes in low-dose-rate irradiated AKR/J mice

PubMed Central

Bong, Jin Jong; Kang, Yu Mi; Shin, Suk Chul; Choi, Seung Jin

2013-01-01

We previously determined that AKR/J mice housed in a low-dose-rate (LDR) (137Cs, 0.7 mGy/h, 2.1 Gy) γ-irradiation facility developed less spontaneous thymic lymphoma and survived longer than those receiving sham or high-dose-rate (HDR) (137Cs, 0.8 Gy/min, 4.5 Gy) radiation. Interestingly, histopathological analysis showed a mild lymphomagenesis in the thymus of LDR-irradiated mice. Therefore, in this study, we investigated whether LDR irradiation could trigger the expression of thymic genes involved in the DNA repair process of AKR/J mice. The enrichment analysis of Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways showed immune response, nucleosome organization, and the peroxisome proliferator-activated receptors signaling pathway in LDR-irradiated mice. Our microarray analysis and quantitative polymerase chain reaction data demonstrated that mRNA levels of Lig4 and RRM2 were specifically elevated in AKR/J mice at 130 days after the start of LDR irradiation. Furthermore, transcriptional levels of H2AX and ATM, proteins known to recruit DNA repair factors, were also shown to be upregulated. These data suggest that LDR irradiation could trigger specific induction of DNA repair-associated genes in an attempt to repair damaged DNA during tumor progression, which in turn contributed to the decreased incidence of lymphoma and increased survival. Overall, we identified specific DNA repair genes in LDR-irradiated AKR/J mice. PMID:23820165

Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

NASA Technical Reports Server (NTRS)

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology

PubMed Central

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Saïd; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis. PMID:12088997

Protein turnover in atrophying muscle: from nutritional intervention to microarray expression analysis

NASA Technical Reports Server (NTRS)

Stein, T. Peter; Wade, Charles E.

2003-01-01

PURPOSE OF REVIEW: In response to decreased usage, skeletal muscle undergoes adaptive reductive remodeling due to the decrease in tension on the weight bearing components of the musculo-skeletal system. This response occurs with uncomplicated disuse (e.g. bed rest, space flight), as a secondary consequence of several widely prevalent chronic diseases for which activity is reduced (e.g. chronic obstructive pulmonary disease and chronic heart failure) and is part of the aging process. The problem is therefore one of considerable clinical importance. RECENT FINDINGS: The impaired function and exercise intolerance is related more to the associated muscle wasting rather than to the specific organ system primarily impacted by the disease. Progress has continued in describing the use of anabolic drugs and dietary manipulation. The major advance in the field has been: (i) the discovery of the atrogin-1 gene and (ii) the application of microarray expression analysis and proteomics with the objectives of obtaining comprehensive understanding of the pathways changed with disuse atrophy. SUMMARY: Disuse atrophy is a common clinical problem. There is a need for therapeutic interventions that do not involve exercise. A better understanding of the changes, particularly at the molecular level, could indicate hitherto unsuspected sites for nutritional and pharmacological intervention.

De Novo Deep Transcriptome Analysis of Medicinal Plants for Gene Discovery in Biosynthesis of Plant Natural Products.

PubMed

Han, R; Rai, A; Nakamura, M; Suzuki, H; Takahashi, H; Yamazaki, M; Saito, K

2016-01-01

Study on transcriptome, the entire pool of transcripts in an organism or single cells at certain physiological or pathological stage, is indispensable in unraveling the connection and regulation between DNA and protein. Before the advent of deep sequencing, microarray was the main approach to handle transcripts. Despite obvious shortcomings, including limited dynamic range and difficulties to compare the results from distinct experiments, microarray was widely applied. During the past decade, next-generation sequencing (NGS) has revolutionized our understanding of genomics in a fast, high-throughput, cost-effective, and tractable manner. By adopting NGS, efficiency and fruitful outcomes concerning the efforts to elucidate genes responsible for producing active compounds in medicinal plants were profoundly enhanced. The whole process involves steps, from the plant material sampling, to cDNA library preparation, to deep sequencing, and then bioinformatics takes over to assemble enormous-yet fragmentary-data from which to comb and extract information. The unprecedentedly rapid development of such technologies provides so many choices to facilitate the task, which can cause confusion when choosing the suitable methodology for specific purposes. Here, we review the general approaches for deep transcriptome analysis and then focus on their application in discovering biosynthetic pathways of medicinal plants that produce important secondary metabolites. © 2016 Elsevier Inc. All rights reserved.

The Effect of Gestational Age on Angiogenic Gene Expression in the Rat Placenta

PubMed Central

Vaswani, Kanchan; Hum, Melissa Wen-Ching; Chan, Hsiu-Wen; Ryan, Jennifer; Wood-Bradley, Ryan J.; Nitert, Marloes Dekker; Mitchell, Murray D.; Armitage, James A.; Rice, Gregory E.

2013-01-01

The placenta plays a central role in determining the outcome of pregnancy. It undergoes changes during gestation as the fetus develops and as demands for energy substrate transfer and gas exchange increase. The molecular mechanisms that coordinate these changes have yet to be fully elucidated. The study performed a large scale screen of the transcriptome of the rat placenta throughout mid-late gestation (E14.25–E20) with emphasis on characterizing gestational age associated changes in the expression of genes invoved in angiogenic pathways. Sprague Dawley dams were sacrificed at E14.25, E15.25, E17.25 and E20 (n = 6 per group) and RNA was isolated from one placenta per dam. Changes in placental gene expression were identifed using Illumina Rat Ref-12 Expression BeadChip Microarrays. Differentially expressed genes (>2-fold change, <1% false discovery rate, FDR) were functionally categorised by gene ontology pathway analysis. A subset of differentially expressed genes identified by microarrays were confirmed using Real-Time qPCR. The expression of thirty one genes involved in the angiogenic pathway was shown to change over time, using microarray analysis (22 genes displayed increased and 9 gene decreased expression). Five genes (4 up regulated: Cd36, Mmp14, Rhob and Angpt4 and 1 down regulated: Foxm1) involved in angiogenesis and blood vessel morphogenesis were subjected to further validation. qPCR confirmed late gestational increased expression of Cd36, Mmp14, Rhob and Angpt4 and a decrease in expression of Foxm1 before labour onset (P<0.0001). The observed acute, pre-labour changes in the expression of the 31 genes during gestation warrant further investigation to elucidate their role in pregnancy. PMID:24391823

Tissue microarrays and quantitative tissue-based image analysis as a tool for oncology biomarker and diagnostic development.

PubMed

Dolled-Filhart, Marisa P; Gustavson, Mark D

2012-11-01

Translational oncology has been improved by using tissue microarrays (TMAs), which facilitate biomarker analysis of large cohorts on a single slide. This has allowed for rapid analysis and validation of potential biomarkers for prognostic and predictive value, as well as for evaluation of biomarker prevalence. Coupled with quantitative analysis of immunohistochemical (IHC) staining, objective and standardized biomarker data from tumor samples can further advance companion diagnostic approaches for the identification of drug-responsive or resistant patient subpopulations. This review covers the advantages, disadvantages and applications of TMAs for biomarker research. Research literature and reviews of TMAs and quantitative image analysis methodology have been surveyed for this review (with an AQUA® analysis focus). Applications such as multi-marker diagnostic development and pathway-based biomarker subpopulation analyses are described. Tissue microarrays are a useful tool for biomarker analyses including prevalence surveys, disease progression assessment and addressing potential prognostic or predictive value. By combining quantitative image analysis with TMAs, analyses will be more objective and reproducible, allowing for more robust IHC-based diagnostic test development. Quantitative multi-biomarker IHC diagnostic tests that can predict drug response will allow for greater success of clinical trials for targeted therapies and provide more personalized clinical decision making.

Identification of rat lung-specific microRNAs by micoRNA microarray: valuable discoveries for the facilitation of lung research.

PubMed

Wang, Yang; Weng, Tingting; Gou, Deming; Chen, Zhongming; Chintagari, Narendranath Reddy; Liu, Lin

2007-01-24

An important mechanism for gene regulation utilizes small non-coding RNAs called microRNAs (miRNAs). These small RNAs play important roles in tissue development, cell differentiation and proliferation, lipid and fat metabolism, stem cells, exocytosis, diseases and cancers. To date, relatively little is known about functions of miRNAs in the lung except lung cancer. In this study, we utilized a rat miRNA microarray containing 216 miRNA probes, printed in-house, to detect the expression of miRNAs in the rat lung compared to the rat heart, brain, liver, kidney and spleen. Statistical analysis using Significant Analysis of Microarray (SAM) and Tukey Honestly Significant Difference (HSD) revealed 2 miRNAs (miR-195 and miR-200c) expressed specifically in the lung and 9 miRNAs co-expressed in the lung and another organ. 12 selected miRNAs were verified by Northern blot analysis. The identified lung-specific miRNAs from this work will facilitate functional studies of miRNAs during normal physiological and pathophysiological processes of the lung.

Integrative analysis of RUNX1 downstream pathways and target genes

PubMed Central

Michaud, Joëlle; Simpson, Ken M; Escher, Robert; Buchet-Poyau, Karine; Beissbarth, Tim; Carmichael, Catherine; Ritchie, Matthew E; Schütz, Frédéric; Cannon, Ping; Liu, Marjorie; Shen, Xiaofeng; Ito, Yoshiaki; Raskind, Wendy H; Horwitz, Marshall S; Osato, Motomi; Turner, David R; Speed, Terence P; Kavallaris, Maria; Smyth, Gordon K; Scott, Hamish S

2008-01-01

Background The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. Results Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFβ, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFβ. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. Conclusion This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications. PMID:18671852

Methoxychlor affects multiple hormone signaling pathways in the largemouth bass (Micropterus salmoides) liver

PubMed Central

Martyniuk, Christopher J.; Spade, Daniel J.; Blum, Jason L.; Kroll, Kevin J.; Denslow, Nancy D.

2011-01-01

Methoxychlor (MXC) is an organochlorine pesticide that has been shown to have estrogenic activity by activating estrogen receptors and inducing vitellogenin production in male fish. Previous studies report that exposure to MXC induces changes in mRNA abundance of reproductive genes in the liver and testes of largemouth bass (Micropterus salmoides). The objective of the present study was to better characterize the mode of action of MXC by measuring the global transcriptomic response in the male largemouth liver using an oligonucleotide microarray. Microarray analysis identified highly significant changes in the expression of 37 transcripts (p<0.001) (20 induced and 17 decreased) in the liver after MXC injection and a total of 900 expression changes (p<0.05) in transcripts with high homology to known genes. Largemouth bass estrogen receptor alpha (esr1) and androgen receptor (ar) were among the transcripts that were increased in the liver after MXC treatment. Functional enrichment analysis identified the molecular functions of steroid binding and androgen receptor activity as well as steroid hormone receptor activity as being significantly over-represented gene ontology terms. Pathway analysis identified c-fos signaling as being putatively affected through both estrogen and androgen signaling. This study provides evidence that MXC elicits transcriptional effects through the estrogen receptor as well as androgen receptor-mediated pathways in the liver. PMID:21276474

Gene profiling, biomarkers and pathways characterizing HCV-related hepatocellular carcinoma

PubMed Central

De Giorgi, Valeria; Monaco, Alessandro; Worchech, Andrea; Tornesello, MariaLina; Izzo, Francesco; Buonaguro, Luigi; Marincola, Francesco M; Wang, Ena; Buonaguro, Franco M

2009-01-01

Background Hepatitis C virus (HCV) infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The molecular mechanisms of HCV-induced hepatocarcinogenesis are not yet fully elucidated. Besides indirect effects as tissue inflammation and regeneration, a more direct oncogenic activity of HCV can be postulated leading to an altered expression of cellular genes by early HCV viral proteins. In the present study, a comparison of gene expression patterns has been performed by microarray analysis on liver biopsies from HCV-positive HCC patients and HCV-negative controls. Methods Gene expression profiling of liver tissues has been performed using a high-density microarray containing 36'000 oligos, representing 90% of the human genes. Samples were obtained from 14 patients affected by HCV-related HCC and 7 HCV-negative non-liver-cancer patients, enrolled at INT in Naples. Transcriptional profiles identified in liver biopsies from HCC nodules and paired non-adjacent non-HCC liver tissue of the same HCV-positive patients were compared to those from HCV-negative controls by the Cluster program. The pathway analysis was performed using the BRB-Array- Tools based on the "Ingenuity System Database". Significance threshold of t-test was set at 0.001. Results Significant differences were found between the expression patterns of several genes falling into different metabolic and inflammation/immunity pathways in HCV-related HCC tissues as well as the non-HCC counterpart compared to normal liver tissues. Only few genes were found differentially expressed between HCV-related HCC tissues and paired non-HCC counterpart. Conclusion In this study, informative data on the global gene expression pattern of HCV-related HCC and non-HCC counterpart, as well as on their difference with the one observed in normal liver tissues have been obtained. These results may lead to the identification of specific biomarkers relevant to develop tools for detection, diagnosis, and classification of HCV-related HCC. PMID:19821982

Development of endosperm transfer cells in barley.

PubMed

Thiel, Johannes

2014-01-01

Endosperm transfer cells (ETCs) are positioned at the intersection of maternal and filial tissues in seeds of cereals and represent a bottleneck for apoplasmic transport of assimilates into the endosperm. Endosperm cellularization starts at the maternal-filial boundary and generates the highly specialized ETCs. During differentiation barley ETCs develop characteristic flange-like wall ingrowths to facilitate effective nutrient transfer. A comprehensive morphological analysis depicted distinct developmental time points in establishment of transfer cell (TC) morphology and revealed intracellular changes possibly associated with cell wall metabolism. Embedded inside the grain, ETCs are barely accessible by manual preparation. To get tissue-specific information about ETC specification and differentiation, laser microdissection (LM)-based methods were used for transcript and metabolite profiling. Transcriptome analysis of ETCs at different developmental stages by microarrays indicated activated gene expression programs related to control of cell proliferation and cell shape, cell wall and carbohydrate metabolism reflecting the morphological changes during early ETC development. Transporter genes reveal distinct expression patterns suggesting a switch from active to passive modes of nutrient uptake with the onset of grain filling. Tissue-specific RNA-seq of the differentiating ETC region from the syncytial stage until functionality in nutrient transfer identified a high number of novel transcripts putatively involved in ETC differentiation. An essential role for two-component signaling (TCS) pathways in ETC development of barley emerged from this analysis. Correlative data provide evidence for abscisic acid and ethylene influences on ETC differentiation and hint at a crosstalk between hormone signal transduction and TCS phosphorelays. Collectively, the data expose a comprehensive view on ETC development, associated pathways and identified candidate genes for ETC specification.

Development of endosperm transfer cells in barley

PubMed Central

Thiel, Johannes

2014-01-01

Endosperm transfer cells (ETCs) are positioned at the intersection of maternal and filial tissues in seeds of cereals and represent a bottleneck for apoplasmic transport of assimilates into the endosperm. Endosperm cellularization starts at the maternal-filial boundary and generates the highly specialized ETCs. During differentiation barley ETCs develop characteristic flange-like wall ingrowths to facilitate effective nutrient transfer. A comprehensive morphological analysis depicted distinct developmental time points in establishment of transfer cell (TC) morphology and revealed intracellular changes possibly associated with cell wall metabolism. Embedded inside the grain, ETCs are barely accessible by manual preparation. To get tissue-specific information about ETC specification and differentiation, laser microdissection (LM)-based methods were used for transcript and metabolite profiling. Transcriptome analysis of ETCs at different developmental stages by microarrays indicated activated gene expression programs related to control of cell proliferation and cell shape, cell wall and carbohydrate metabolism reflecting the morphological changes during early ETC development. Transporter genes reveal distinct expression patterns suggesting a switch from active to passive modes of nutrient uptake with the onset of grain filling. Tissue-specific RNA-seq of the differentiating ETC region from the syncytial stage until functionality in nutrient transfer identified a high number of novel transcripts putatively involved in ETC differentiation. An essential role for two-component signaling (TCS) pathways in ETC development of barley emerged from this analysis. Correlative data provide evidence for abscisic acid and ethylene influences on ETC differentiation and hint at a crosstalk between hormone signal transduction and TCS phosphorelays. Collectively, the data expose a comprehensive view on ETC development, associated pathways and identified candidate genes for ETC specification. PMID:24723929

Cell cycle arrest and induction of apoptosis by cajanin stilbene acid from Cajanus cajan in breast cancer cells.

PubMed

Fu, Yujie; Kadioglu, Onat; Wiench, Benjamin; Wei, Zuofu; Gao, Chang; Luo, Meng; Gu, Chengbo; Zu, Yuangang; Efferth, Thomas

2015-04-15

The low abundant cajanin stilbene acid (CSA) from Pigeon Pea (Cajanus cajan) has been shown to kill estrogen receptor α positive cancer cells in vitro and in vivo. Downstream effects such as cell cycle and apoptosis-related mechanisms have not been analyzed yet. We analyzed the activity of CSA by means of flow cytometry (cell cycle distribution, mitochondrial membrane potential, MMP), confocal laser scanning microscopy (MMP), DNA fragmentation assay (apoptosis), Western blotting (Bax and Bcl-2 expression, caspase-3 activation) as well as mRNA microarray hybridization and Ingenuity pathway analysis. CSA induced G2/M arrest and apoptosis in a concentration-dependent manner from 8.88 to 14.79 µM. The MMP broke down, Bax was upregulated, Bcl-2 downregulated and caspase-3 activated. Microarray profiling revealed that CSA affected BRCA-related DNA damage response and cell cycle-regulated chromosomal replication pathways. CSA inhibited breast cancer cells by DNA damage and cell cycle-related signaling pathways leading to cell cycle arrest and apoptosis. Copyright © 2015 Elsevier GmbH. All rights reserved.

Molecular pathway activation in cancer and tissue following space radiation exposure

NASA Astrophysics Data System (ADS)

Kovyrshina, Tatiana A.

Space radiation exposure is an important safety concern for astronauts, especially since one of the risks is carcinogenesis. This thesis explores the link between lung, colorectal, and breast cancer and iron particles and gamma radiation on a molecular level. We obtained DNA microarrays for each condition from the Gene Expression Omnibus (GEO), a public functional genomics data repository, cleaned up the data, and analysed overexpression and underexpression of pathway analysis. Our results show that pathways which participate in DNA replication appear to be overexpressed in cancer cells and cells exposed to ionizing radiation.

Microarray analysis reveals key genes and pathways in Tetralogy of Fallot

PubMed Central

He, Yue-E; Qiu, Hui-Xian; Jiang, Jian-Bing; Wu, Rong-Zhou; Xiang, Ru-Lian; Zhang, Yuan-Hai

2017-01-01

The aim of the present study was to identify key genes that may be involved in the pathogenesis of Tetralogy of Fallot (TOF) using bioinformatics methods. The GSE26125 microarray dataset, which includes cardiovascular tissue samples derived from 16 children with TOF and five healthy age-matched control infants, was downloaded from the Gene Expression Omnibus database. Differential expression analysis was performed between TOF and control samples to identify differentially expressed genes (DEGs) using Student's t-test, and the R/limma package, with a log2 fold-change of >2 and a false discovery rate of <0.01 set as thresholds. The biological functions of DEGs were analyzed using the ToppGene database. The ReactomeFIViz application was used to construct functional interaction (FI) networks, and the genes in each module were subjected to pathway enrichment analysis. The iRegulon plugin was used to identify transcription factors predicted to regulate the DEGs in the FI network, and the gene-transcription factor pairs were then visualized using Cytoscape software. A total of 878 DEGs were identified, including 848 upregulated genes and 30 downregulated genes. The gene FI network contained seven function modules, which were all comprised of upregulated genes. Genes enriched in Module 1 were enriched in the following three neurological disorder-associated signaling pathways: Parkinson's disease, Alzheimer's disease and Huntington's disease. Genes in Modules 0, 3 and 5 were dominantly enriched in pathways associated with ribosomes and protein translation. The Xbox binding protein 1 transcription factor was demonstrated to be involved in the regulation of genes encoding the subunits of cytoplasmic and mitochondrial ribosomes, as well as genes involved in neurodegenerative disorders. Therefore, dysfunction of genes involved in signaling pathways associated with neurodegenerative disorders, ribosome function and protein translation may contribute to the pathogenesis of TOF. PMID:28713939

A Canonical Correlation Analysis of AIDS Restriction Genes and Metabolic Pathways Identifies Purine Metabolism as a Key Cooperator.

PubMed

Ye, Hanhui; Yuan, Jinjin; Wang, Zhengwu; Huang, Aiqiong; Liu, Xiaolong; Han, Xiao; Chen, Yahong

2016-01-01

Human immunodeficiency virus causes a severe disease in humans, referred to as immune deficiency syndrome. Studies on the interaction between host genetic factors and the virus have revealed dozens of genes that impact diverse processes in the AIDS disease. To resolve more genetic factors related to AIDS, a canonical correlation analysis was used to determine the correlation between AIDS restriction and metabolic pathway gene expression. The results show that HIV-1 postentry cellular viral cofactors from AIDS restriction genes are coexpressed in human transcriptome microarray datasets. Further, the purine metabolism pathway comprises novel host factors that are coexpressed with AIDS restriction genes. Using a canonical correlation analysis for expression is a reliable approach to exploring the mechanism underlying AIDS.

Analysis of the effects of sex hormone background on the rat choroid plexus transcriptome by cDNA microarrays.

PubMed

Quintela, Telma; Gonçalves, Isabel; Carreto, Laura C; Santos, Manuel A S; Marcelino, Helena; Patriarca, Filipa M; Santos, Cecília R A

2013-01-01

The choroid plexus (CP) are highly vascularized branched structures that protrude into the ventricles of the brain, and form a unique interface between the blood and the cerebrospinal fluid (CSF), the blood-CSF barrier, that are the main site of production and secretion of CSF. Sex hormones are widely recognized as neuroprotective agents against several neurodegenerative diseases, and the presence of sex hormones cognate receptors suggest that it may be a target for these hormones. In an effort to provide further insight into the neuroprotective mechanisms triggered by sex hormones we analyzed gene expression differences in the CP of female and male rats subjected to gonadectomy, using microarray technology. In gonadectomized female and male animals, 3045 genes were differentially expressed by 1.5-fold change, compared to sham controls. Analysis of the CP transcriptome showed that the top-five pathways significantly regulated by the sex hormone background are olfactory transduction, taste transduction, metabolism, steroid hormone biosynthesis and circadian rhythm pathways. These results represent the first overview of global expression changes in CP of female and male rats induced by gonadectomy and suggest that sex hormones are implicated in pathways with central roles in CP functions and CSF homeostasis.

Analysis of the Effects of Sex Hormone Background on the Rat Choroid Plexus Transcriptome by cDNA Microarrays

PubMed Central

Quintela, Telma; Gonçalves, Isabel; Carreto, Laura C.; Santos, Manuel A. S.; Marcelino, Helena; Patriarca, Filipa M.; Santos, Cecília R. A.

2013-01-01

The choroid plexus (CP) are highly vascularized branched structures that protrude into the ventricles of the brain, and form a unique interface between the blood and the cerebrospinal fluid (CSF), the blood-CSF barrier, that are the main site of production and secretion of CSF. Sex hormones are widely recognized as neuroprotective agents against several neurodegenerative diseases, and the presence of sex hormones cognate receptors suggest that it may be a target for these hormones. In an effort to provide further insight into the neuroprotective mechanisms triggered by sex hormones we analyzed gene expression differences in the CP of female and male rats subjected to gonadectomy, using microarray technology. In gonadectomized female and male animals, 3045 genes were differentially expressed by 1.5-fold change, compared to sham controls. Analysis of the CP transcriptome showed that the top-five pathways significantly regulated by the sex hormone background are olfactory transduction, taste transduction, metabolism, steroid hormone biosynthesis and circadian rhythm pathways. These results represent the first overview of global expression changes in CP of female and male rats induced by gonadectomy and suggest that sex hormones are implicated in pathways with central roles in CP functions and CSF homeostasis. PMID:23585832

Characterization of a novel RING-type ubiquitin E3 ligase GhRING2 differentially expressed in cotton fiber

USDA-ARS?s Scientific Manuscript database

The ubiquitin-proteasome proteolysis pathway is responsible for the degradation of abnormal and short-lived proteins to regulate many important biochemical activities in eukaryotes. By employing affymetrix microarray analysis, we have identified a novel ubiquitin ligase E3 gene GhRING2 that is diffe...

Cloning and Characterization of a Bursicon-Regulated Gene Su(H) in the House Fly Musca domestica

USDA-ARS?s Scientific Manuscript database

Bursicon is a neuropeptide that regulates cuticle sclerotization (hardening and tanning) in insect via G-protein coupled receptor. However, the signal transduction pathway down stream of the G-protein coupled receptor is currently not well known. In our recent microarray analysis, we identified a pa...

Implementation of mutual information and bayes theorem for classification microarray data

NASA Astrophysics Data System (ADS)

Dwifebri Purbolaksono, Mahendra; Widiastuti, Kurnia C.; Syahrul Mubarok, Mohamad; Adiwijaya; Aminy Ma’ruf, Firda

2018-03-01

Microarray Technology is one of technology which able to read the structure of gen. The analysis is important for this technology. It is for deciding which attribute is more important than the others. Microarray technology is able to get cancer information to diagnose a person’s gen. Preparation of microarray data is a huge problem and takes a long time. That is because microarray data contains high number of insignificant and irrelevant attributes. So, it needs a method to reduce the dimension of microarray data without eliminating important information in every attribute. This research uses Mutual Information to reduce dimension. System is built with Machine Learning approach specifically Bayes Theorem. This theorem uses a statistical and probability approach. By combining both methods, it will be powerful for Microarray Data Classification. The experiment results show that system is good to classify Microarray data with highest F1-score using Bayesian Network by 91.06%, and Naïve Bayes by 88.85%.

Temporal profiling of gene networks associated with the late phase of long-term potentiation in vivo.

PubMed

Ryan, Margaret M; Ryan, Brigid; Kyrke-Smith, Madeleine; Logan, Barbara; Tate, Warren P; Abraham, Wickliffe C; Williams, Joanna M

2012-01-01

Long-term potentiation (LTP) is widely accepted as a cellular mechanism underlying memory processes. It is well established that LTP persistence is strongly dependent on activation of constitutive and inducible transcription factors, but there is limited information regarding the downstream gene networks and controlling elements that coalesce to stabilise LTP. To identify these gene networks, we used Affymetrix RAT230.2 microarrays to detect genes regulated 5 h and 24 h (n = 5) after LTP induction at perforant path synapses in the dentate gyrus of awake adult rats. The functional relationships of the differentially expressed genes were examined using DAVID and Ingenuity Pathway Analysis, and compared with our previous data derived 20 min post-LTP induction in vivo. This analysis showed that LTP-related genes are predominantly upregulated at 5 h but that there is pronounced downregulation of gene expression at 24 h after LTP induction. Analysis of the structure of the networks and canonical pathways predicted a regulation of calcium dynamics via G-protein coupled receptors, dendritogenesis and neurogenesis at the 5 h time-point. By 24 h neurotrophin-NFKB driven pathways of neuronal growth were identified. The temporal shift in gene expression appears to be mediated by regulation of protein synthesis, ubiquitination and time-dependent regulation of specific microRNA and histone deacetylase expression. Together this programme of genomic responses, marked by both homeostatic and growth pathways, is likely to be critical for the consolidation of LTP in vivo.

A Microarray Tool Provides Pathway and GO Term Analysis.

PubMed

Koch, Martin; Royer, Hans-Dieter; Wiese, Michael

2011-12-01

Analysis of gene expression profiles is no longer exclusively a task for bioinformatic experts. However, gaining statistically significant results is challenging and requires both biological knowledge and computational know-how. Here we present a novel, user-friendly microarray reporting tool called maRt. The software provides access to bioinformatic resources, like gene ontology terms and biological pathways by use of the DAVID and the BioMart web-service. Results are summarized in structured HTML reports, each presenting a different layer of information. In these report, contents of diverse sources are integrated and interlinked. To speed up processing, maRt takes advantage of the multi-core technology of modern desktop computers by using parallel processing. Since the software is built upon a RCP infrastructure it might be an outset for developers aiming to integrate novel R based applications. Installer, documentation and various kinds of tutorials are available under LGPL license at the website of our institute http://www.pharma.uni-bonn.de/www/mart. This software is free for academic use. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

CLUSFAVOR 5.0: hierarchical cluster and principal-component analysis of microarray-based transcriptional profiles

PubMed Central

Peterson, Leif E

2002-01-01

CLUSFAVOR (CLUSter and Factor Analysis with Varimax Orthogonal Rotation) 5.0 is a Windows-based computer program for hierarchical cluster and principal-component analysis of microarray-based transcriptional profiles. CLUSFAVOR 5.0 standardizes input data; sorts data according to gene-specific coefficient of variation, standard deviation, average and total expression, and Shannon entropy; performs hierarchical cluster analysis using nearest-neighbor, unweighted pair-group method using arithmetic averages (UPGMA), or furthest-neighbor joining methods, and Euclidean, correlation, or jack-knife distances; and performs principal-component analysis. PMID:12184816

Identification of interactive gene networks: a novel approach in gene array profiling of myometrial events during guinea pig pregnancy.

PubMed

Mason, Clifford W; Swaan, Peter W; Weiner, Carl P

2006-06-01

The transition from myometrial quiescence to activation is poorly understood, and the analysis of array data is limited by the available data mining tools. We applied functional analysis and logical operations along regulatory gene networks to identify molecular processes and pathways underlying quiescence and activation. We analyzed some 18,400 transcripts and variants in guinea pig myometrium at stages corresponding to quiescence and activation, and compared them to the nonpregnant (control) counterpart using a functional mapping tool, MetaCore (GeneGo, St Joseph, MI) to identify novel gene networks composed of biological pathways during mid (MP) and late (LP) pregnancy. Genes altered during quiescence and or activation were identified following gene specific comparisons with myometrium from nonpregnant animals, and then linked to curated pathways and formulated networks. The MP and LP networks were subtracted from each other to identify unique genomic events during those periods. For example, changes 2-fold or greater in genes mediating protein biosynthesis, programmed cell death, microtubule polymerization, and microtubule based movement were noted during the transition to LP. We describe a novel approach combining microarrays and genetic data to identify networks associated with normal myometrial events. The resulting insights help identify potential biomarkers and permit future targeted investigations of these pathways or networks to confirm or refute their importance.

Transcriptomic data analysis and differential gene expression of antioxidant pathways in king penguin juveniles (Aptenodytes patagonicus) before and after acclimatization to marine life.

PubMed

Rey, Benjamin; Dégletagne, Cyril; Duchamp, Claude

2016-12-01

In this article, we present differentially expressed gene profiles in the pectoralis muscle of wild juvenile king penguins that were either naturally acclimated to cold marine environment or experimentally immersed in cold water as compared with penguin juveniles that never experienced cold water immersion. Transcriptomic data were obtained by hybridizing penguins total cDNA on Affymetrix GeneChip Chicken Genome arrays and analyzed using maxRS algorithm , " Transcriptome analysis in non-model species: a new method for the analysis of heterologous hybridization on microarrays " (Dégletagne et al., 2010) [1] . We focused on genes involved in multiple antioxidant pathways. For better clarity, these differentially expressed genes were clustered into six functional groups according to their role in controlling redox homeostasis. The data are related to a comprehensive research study on the ontogeny of antioxidant functions in king penguins, "Hormetic response triggers multifaceted anti-oxidant strategies in immature king penguins (Aptenodytes patagonicus)" (Rey et al., 2016) [2] . The raw microarray dataset supporting the present analyses has been deposited at the Gene Expression Omnibus (GEO) repository under accessions GEO: GSE17725 and GEO: GSE82344.

The genome-wide expression profile of Curcuma longa-treated cisplatin-stimulated HEK293 cells

PubMed Central

Sohn, Sung-Hwa; Ko, Eunjung; Chung, Hwan-Suck; Lee, Eun-Young; Kim, Sung-Hoon; Shin, Minkyu; Hong, Moochang; Bae, Hyunsu

2010-01-01

AIM The rhizome of turmeric, Curcuma longa (CL), is a herbal medicine used in many traditional prescriptions. It has previously been shown that CL treatment showed greater than 47% recovery from cisplatin-induced cell damage in human kidney HEK 293 cells. This study was conducted to evaluate the recovery mechanisms of CL that occur during cisplatin induced nephrotoxicity by examining the genome wide mRNA expression profiles of HEK 293 -cells. METHOD Recovery mechanisms of CL that occur during cisplatin-induced nephrotoxicity were determined by microarray, real-time PCR, immunofluorescent confocal microscopy and Western blot analysis. RESULTS The results of microarray analysis and real-time PCR revealed that NFκB pathway-related genes and apoptosis-related genes were down-regulated in CL-treated HEK 293 cells. In addition, immunofluorescent confocal microscopy and Western blot analysis revealed that NFκB p65 nuclear translocation was inhibited in CL-treated HEK 293 cells. Therefore, the mechanism responsible for the effects of CL on HEK 293 cells is closely associated with regulation of the NFκB pathway. CONCLUSION CL possesses novel therapeutic agents that can be used for the prevention or treatment of cisplatin-induced renal disorders. PMID:20840446

Possible molecular mechanism underlying cadmium-induced circadian rhythms disruption in zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiao, Bo; Chen, Tian-Ming; Zhong, Yingbin

This study was aimed to explore the mechanisms underlying cadmium-induced circadian rhythms disruption. Two groups of zebrafish larvae treated with or without 5 ppm CdCl{sub 2} were incubated in a photoperiod of 14-h light/10-h dark conditions. The mRNA levels of clock1a, bmal1b, per2 and per1b in two groups were determined. Microarray data were generated in two group of samples. Differential expression of genes were identified and the changes in expression level for some genes were validated by RT-PCR. Finally, Gene Ontology functional and KEGG pathway enrichment analysis of differentially expressed genes (DEGs) were performed. In comparison with normal group, the mRNAmore » levels of clock1a, bmal1b, and per2 were significantly changed and varied over the circadian cycle in CdCl2-treated group. DEGs were obtained from the light (84 h, ZT12) and dark (88 h, ZT16) phase. In addition, G-protein coupled receptor protein signaling pathway and immune response were both enriched by DEGs in both groups. While, proteolysis and amino acid metabolism were found associated with DEGs in light phase, and Neuroactive ligand-receptor interaction and oxidation-reduction process were significantly enriched by DEGs in dark phase. Besides, the expression pattern of genes including hsp70l and or115-11 obtained by RT-PCR were consistent with those obtained by microarray analysis. As a consequence, cadmium could make significant effects on circadian rhythms through immune response and G protein-coupled receptor signaling pathway. Besides, between the dark and the light phase, the mechanism by which cadmium inducing disruption of circadian rhythms were different to some extent. - Highlights: • Cadmium could affect the expression levels of circadian rhythm-related genes. • Genes expression in microarray data were consistent with those in RT-PCR analysis. • Immune response and G protein-coupled receptor signaling pathway were identified. • Cadmium induces circadian rhythm disruption by different mechanism in day and night.« less

Workflows for microarray data processing in the Kepler environment.

PubMed

Stropp, Thomas; McPhillips, Timothy; Ludäscher, Bertram; Bieda, Mark

2012-05-17

Microarray data analysis has been the subject of extensive and ongoing pipeline development due to its complexity, the availability of several options at each analysis step, and the development of new analysis demands, including integration with new data sources. Bioinformatics pipelines are usually custom built for different applications, making them typically difficult to modify, extend and repurpose. Scientific workflow systems are intended to address these issues by providing general-purpose frameworks in which to develop and execute such pipelines. The Kepler workflow environment is a well-established system under continual development that is employed in several areas of scientific research. Kepler provides a flexible graphical interface, featuring clear display of parameter values, for design and modification of workflows. It has capabilities for developing novel computational components in the R, Python, and Java programming languages, all of which are widely used for bioinformatics algorithm development, along with capabilities for invoking external applications and using web services. We developed a series of fully functional bioinformatics pipelines addressing common tasks in microarray processing in the Kepler workflow environment. These pipelines consist of a set of tools for GFF file processing of NimbleGen chromatin immunoprecipitation on microarray (ChIP-chip) datasets and more comprehensive workflows for Affymetrix gene expression microarray bioinformatics and basic primer design for PCR experiments, which are often used to validate microarray results. Although functional in themselves, these workflows can be easily customized, extended, or repurposed to match the needs of specific projects and are designed to be a toolkit and starting point for specific applications. These workflows illustrate a workflow programming paradigm focusing on local resources (programs and data) and therefore are close to traditional shell scripting or R/BioConductor scripting approaches to pipeline design. Finally, we suggest that microarray data processing task workflows may provide a basis for future example-based comparison of different workflow systems. We provide a set of tools and complete workflows for microarray data analysis in the Kepler environment, which has the advantages of offering graphical, clear display of conceptual steps and parameters and the ability to easily integrate other resources such as remote data and web services.

RNA-seq based transcriptomic map reveals new insights into mouse salivary gland development and maturation.

PubMed

Gluck, Christian; Min, Sangwon; Oyelakin, Akinsola; Smalley, Kirsten; Sinha, Satrajit; Romano, Rose-Anne

2016-11-16

Mouse models have served a valuable role in deciphering various facets of Salivary Gland (SG) biology, from normal developmental programs to diseased states. To facilitate such studies, gene expression profiling maps have been generated for various stages of SG organogenesis. However these prior studies fall short of capturing the transcriptional complexity due to the limited scope of gene-centric microarray-based technology. Compared to microarray, RNA-sequencing (RNA-seq) offers unbiased detection of novel transcripts, broader dynamic range and high specificity and sensitivity for detection of genes, transcripts, and differential gene expression. Although RNA-seq data, particularly under the auspices of the ENCODE project, have covered a large number of biological specimens, studies on the SG have been lacking. To better appreciate the wide spectrum of gene expression profiles, we isolated RNA from mouse submandibular salivary glands at different embryonic and adult stages. In parallel, we processed RNA-seq data for 24 organs and tissues obtained from the mouse ENCODE consortium and calculated the average gene expression values. To identify molecular players and pathways likely to be relevant for SG biology, we performed functional gene enrichment analysis, network construction and hierarchal clustering of the RNA-seq datasets obtained from different stages of SG development and maturation, and other mouse organs and tissues. Our bioinformatics-based data analysis not only reaffirmed known modulators of SG morphogenesis but revealed novel transcription factors and signaling pathways unique to mouse SG biology and function. Finally we demonstrated that the unique SG gene signature obtained from our mouse studies is also well conserved and can demarcate features of the human SG transcriptome that is different from other tissues. Our RNA-seq based Atlas has revealed a high-resolution cartographic view of the dynamic transcriptomic landscape of the mouse SG at various stages. These RNA-seq datasets will complement pre-existing microarray based datasets, including the Salivary Gland Molecular Anatomy Project by offering a broader systems-biology based perspective rather than the classical gene-centric view. Ultimately such resources will be valuable in providing a useful toolkit to better understand how the diverse cell population of the SG are organized and controlled during development and differentiation.

Specific Transcriptome Changes Associated with Blood Pressure Reduction in Hypertensive Patients After Relaxation Response Training.

PubMed

Bhasin, Manoj K; Denninger, John W; Huffman, Jeff C; Joseph, Marie G; Niles, Halsey; Chad-Friedman, Emma; Goldman, Roberta; Buczynski-Kelley, Beverly; Mahoney, Barbara A; Fricchione, Gregory L; Dusek, Jeffery A; Benson, Herbert; Zusman, Randall M; Libermann, Towia A

2018-05-01

Mind-body practices that elicit the relaxation response (RR) have been demonstrated to reduce blood pressure (BP) in essential hypertension (HTN) and may be an adjunct to antihypertensive drug therapy. However, the molecular mechanisms by which the RR reduces BP remain undefined. Genomic determinants associated with responsiveness to an 8-week RR-based mind-body intervention for lowering HTN in 13 stage 1 hypertensive patients classified as BP responders and 11 as nonresponders were identified. Transcriptome analysis in peripheral blood mononuclear cells identified 1771 genes regulated by the RR in responders. Biological process- and pathway-based analysis of transcriptome data demonstrated enrichment in the following gene categories: immune regulatory pathways and metabolism (among downregulated genes); glucose metabolism, cardiovascular system development, and circadian rhythm (among upregulated genes). Further in silico estimation of cell abundance from the microarray data showed enrichment of the anti-inflammatory M2 subtype of macrophages in BP responders. Nuclear factor-κB, vascular endothelial growth factor, and insulin were critical molecules emerging from interactive network analysis. These findings provide the first insights into the molecular mechanisms that are associated with the beneficial effects of the RR on HTN.

Specific Transcriptome Changes Associated with Blood Pressure Reduction in Hypertensive Patients After Relaxation Response Training

PubMed Central

Bhasin, Manoj K.; Denninger, John W.; Huffman, Jeff C.; Joseph, Marie G.; Niles, Halsey; Chad-Friedman, Emma; Goldman, Roberta; Buczynski-Kelley, Beverly; Mahoney, Barbara A.; Fricchione, Gregory L.; Dusek, Jeffery A.; Benson, Herbert; Zusman, Randall M.

2018-01-01

Abstract Objective: Mind–body practices that elicit the relaxation response (RR) have been demonstrated to reduce blood pressure (BP) in essential hypertension (HTN) and may be an adjunct to antihypertensive drug therapy. However, the molecular mechanisms by which the RR reduces BP remain undefined. Design: Genomic determinants associated with responsiveness to an 8-week RR-based mind–body intervention for lowering HTN in 13 stage 1 hypertensive patients classified as BP responders and 11 as nonresponders were identified. Results: Transcriptome analysis in peripheral blood mononuclear cells identified 1771 genes regulated by the RR in responders. Biological process- and pathway-based analysis of transcriptome data demonstrated enrichment in the following gene categories: immune regulatory pathways and metabolism (among downregulated genes); glucose metabolism, cardiovascular system development, and circadian rhythm (among upregulated genes). Further in silico estimation of cell abundance from the microarray data showed enrichment of the anti-inflammatory M2 subtype of macrophages in BP responders. Nuclear factor-κB, vascular endothelial growth factor, and insulin were critical molecules emerging from interactive network analysis. Conclusions: These findings provide the first insights into the molecular mechanisms that are associated with the beneficial effects of the RR on HTN. PMID:29616846

C. elegans GATA factors EGL-18 and ELT-6 function downstream of Wnt signaling to maintain the progenitor fate during larval asymmetric divisions of the seam cells.

PubMed

Gorrepati, Lakshmi; Thompson, Kenneth W; Eisenmann, David M

2013-05-01

The C. elegans seam cells are lateral epithelial cells arrayed in a single line from anterior to posterior that divide in an asymmetric, stem cell-like manner during larval development. These asymmetric divisions are regulated by Wnt signaling; in most divisions, the posterior daughter in which the Wnt pathway is activated maintains the progenitor seam fate, while the anterior daughter in which the Wnt pathway is not activated adopts a differentiated hypodermal fate. Using mRNA tagging and microarray analysis, we identified the functionally redundant GATA factor genes egl-18 and elt-6 as Wnt pathway targets in the larval seam cells. EGL-18 and ELT-6 have previously been shown to be required for initial seam cell specification in the embryo. We show that in larval seam cell asymmetric divisions, EGL-18 is expressed strongly in the posterior seam-fated daughter. egl-18 and elt-6 are necessary for larval seam cell specification, and for hypodermal to seam cell fate transformations induced by ectopic Wnt pathway overactivation. The TCF homolog POP-1 binds a site in the egl-18 promoter in vitro, and this site is necessary for robust seam cell expression in vivo. Finally, larval overexpression of EGL-18 is sufficient to drive expression of a seam marker in other hypodermal cells in wild-type animals, and in anterior hypodermal-fated daughters in a Wnt pathway-sensitized background. These data suggest that two GATA factors that are required for seam cell specification in the embryo independently of Wnt signaling are reused downstream of Wnt signaling to maintain the progenitor fate during stem cell-like divisions in larval development.

C. elegans GATA factors EGL-18 and ELT-6 function downstream of Wnt signaling to maintain the progenitor fate during larval asymmetric divisions of the seam cells

PubMed Central

Gorrepati, Lakshmi; Thompson, Kenneth W.; Eisenmann, David M.

2013-01-01

The C. elegans seam cells are lateral epithelial cells arrayed in a single line from anterior to posterior that divide in an asymmetric, stem cell-like manner during larval development. These asymmetric divisions are regulated by Wnt signaling; in most divisions, the posterior daughter in which the Wnt pathway is activated maintains the progenitor seam fate, while the anterior daughter in which the Wnt pathway is not activated adopts a differentiated hypodermal fate. Using mRNA tagging and microarray analysis, we identified the functionally redundant GATA factor genes egl-18 and elt-6 as Wnt pathway targets in the larval seam cells. EGL-18 and ELT-6 have previously been shown to be required for initial seam cell specification in the embryo. We show that in larval seam cell asymmetric divisions, EGL-18 is expressed strongly in the posterior seam-fated daughter. egl-18 and elt-6 are necessary for larval seam cell specification, and for hypodermal to seam cell fate transformations induced by ectopic Wnt pathway overactivation. The TCF homolog POP-1 binds a site in the egl-18 promoter in vitro, and this site is necessary for robust seam cell expression in vivo. Finally, larval overexpression of EGL-18 is sufficient to drive expression of a seam marker in other hypodermal cells in wild-type animals, and in anterior hypodermal-fated daughters in a Wnt pathway-sensitized background. These data suggest that two GATA factors that are required for seam cell specification in the embryo independently of Wnt signaling are reused downstream of Wnt signaling to maintain the progenitor fate during stem cell-like divisions in larval development. PMID:23633508

In silico pathway analysis in cervical carcinoma reveals potential new targets for treatment

PubMed Central

van Dam, Peter A.; van Dam, Pieter-Jan H. H.; Rolfo, Christian; Giallombardo, Marco; van Berckelaer, Christophe; Trinh, Xuan Bich; Altintas, Sevilay; Huizing, Manon; Papadimitriou, Kostas; Tjalma, Wiebren A. A.; van Laere, Steven

2016-01-01

An in silico pathway analysis was performed in order to improve current knowledge on the molecular drivers of cervical cancer and detect potential targets for treatment. Three publicly available Affymetrix gene expression data-sets (GSE5787, GSE7803, GSE9750) were retrieved, vouching for a total of 9 cervical cancer cell lines (CCCLs), 39 normal cervical samples, 7 CIN3 samples and 111 cervical cancer samples (CCSs). Predication analysis of microarrays was performed in the Affymetrix sets to identify cervical cancer biomarkers. To select cancer cell-specific genes the CCSs were compared to the CCCLs. Validated genes were submitted to a gene set enrichment analysis (GSEA) and Expression2Kinases (E2K). In the CCSs a total of 1,547 probe sets were identified that were overexpressed (FDR < 0.1). Comparing to CCCLs 560 probe sets (481 unique genes) had a cancer cell-specific expression profile, and 315 of these genes (65%) were validated. GSEA identified 5 cancer hallmarks enriched in CCSs (P < 0.01 and FDR < 0.25) showing that deregulation of the cell cycle is a major component of cervical cancer biology. E2K identified a protein-protein interaction (PPI) network of 162 nodes (including 20 drugable kinases) and 1626 edges. This PPI-network consists of 5 signaling modules associated with MYC signaling (Module 1), cell cycle deregulation (Module 2), TGFβ-signaling (Module 3), MAPK signaling (Module 4) and chromatin modeling (Module 5). Potential targets for treatment which could be identified were CDK1, CDK2, ABL1, ATM, AKT1, MAPK1, MAPK3 among others. The present study identified important driver pathways in cervical carcinogenesis which should be assessed for their potential therapeutic drugability. PMID:26701206

Transcriptomic Analysis and Meta-Analysis of Human Granulosa and Cumulus Cells

PubMed Central

Burnik Papler, Tanja; Vrtacnik Bokal, Eda; Maver, Ales; Kopitar, Andreja Natasa; Lovrečić, Luca

2015-01-01

Specific gene expression in oocytes and its surrounding cumulus (CC) and granulosa (GC) cells is needed for successful folliculogenesis and oocyte maturation. The aim of the present study was to compare genome-wide gene expression and biological functions of human GC and CC. Individual GC and CC were derived from 37 women undergoing IVF procedures. Gene expression analysis was performed using microarrays, followed by a meta-analysis. Results were validated using quantitative real-time PCR. There were 6029 differentially expressed genes (q < 10−4); of which 650 genes had a log2 FC ≥ 2. After the meta-analysis there were 3156 genes differentially expressed. Among these there were genes that have previously not been reported in human somatic follicular cells, like prokineticin 2 (PROK2), higher expressed in GC, and pregnancy up-regulated nonubiquitous CaM kinase (PNCK), higher expressed in CC. Pathways like inflammatory response and angiogenesis were enriched in GC, whereas in CC, cell differentiation and multicellular organismal development were among enriched pathways. In conclusion, transcriptomes of GC and CC as well as biological functions, are distinctive for each cell subpopulation. By describing novel genes like PROK2 and PNCK, expressed in GC and CC, we upgraded the existing data on human follicular biology. PMID:26313571

A chain reaction approach to modelling gene pathways.

PubMed

Cheng, Gary C; Chen, Dung-Tsa; Chen, James J; Soong, Seng-Jaw; Lamartiniere, Coral; Barnes, Stephen

2012-08-01

BACKGROUND: Of great interest in cancer prevention is how nutrient components affect gene pathways associated with the physiological events of puberty. Nutrient-gene interactions may cause changes in breast or prostate cells and, therefore, may result in cancer risk later in life. Analysis of gene pathways can lead to insights about nutrient-gene interactions and the development of more effective prevention approaches to reduce cancer risk. To date, researchers have relied heavily upon experimental assays (such as microarray analysis, etc.) to identify genes and their associated pathways that are affected by nutrient and diets. However, the vast number of genes and combinations of gene pathways, coupled with the expense of the experimental analyses, has delayed the progress of gene-pathway research. The development of an analytical approach based on available test data could greatly benefit the evaluation of gene pathways, and thus advance the study of nutrient-gene interactions in cancer prevention. In the present study, we have proposed a chain reaction model to simulate gene pathways, in which the gene expression changes through the pathway are represented by the species undergoing a set of chemical reactions. We have also developed a numerical tool to solve for the species changes due to the chain reactions over time. Through this approach we can examine the impact of nutrient-containing diets on the gene pathway; moreover, transformation of genes over time with a nutrient treatment can be observed numerically, which is very difficult to achieve experimentally. We apply this approach to microarray analysis data from an experiment which involved the effects of three polyphenols (nutrient treatments), epigallo-catechin-3-O-gallate (EGCG), genistein, and resveratrol, in a study of nutrient-gene interaction in the estrogen synthesis pathway during puberty. RESULTS: In this preliminary study, the estrogen synthesis pathway was simulated by a chain reaction model. By applying it to microarray data, the chain reaction model computed a set of reaction rates to examine the effects of three polyphenols (EGCG, genistein, and resveratrol) on gene expression in this pathway during puberty. We first performed statistical analysis to test the time factor on the estrogen synthesis pathway. Global tests were used to evaluate an overall gene expression change during puberty for each experimental group. Then, a chain reaction model was employed to simulate the estrogen synthesis pathway. Specifically, the model computed the reaction rates in a set of ordinary differential equations to describe interactions between genes in the pathway (A reaction rate K of A to B represents gene A will induce gene B per unit at a rate of K; we give details in the "method" section). Since disparate changes of gene expression may cause numerical error problems in solving these differential equations, we used an implicit scheme to address this issue. We first applied the chain reaction model to obtain the reaction rates for the control group. A sensitivity study was conducted to evaluate how well the model fits to the control group data at Day 50. Results showed a small bias and mean square error. These observations indicated the model is robust to low random noises and has a good fit for the control group. Then the chain reaction model derived from the control group data was used to predict gene expression at Day 50 for the three polyphenol groups. If these nutrients affect the estrogen synthesis pathways during puberty, we expect discrepancy between observed and expected expressions. Results indicated some genes had large differences in the EGCG (e.g., Hsd3b and Sts) and the resveratrol (e.g., Hsd3b and Hrmt12) groups. CONCLUSIONS: In the present study, we have presented (I) experimental studies of the effect of nutrient diets on the gene expression changes in a selected estrogen synthesis pathway. This experiment is valuable because it allows us to examine how the nutrient-containing diets regulate gene expression in the estrogen synthesis pathway during puberty; (II) global tests to assess an overall association of this particular pathway with time factor by utilizing generalized linear models to analyze microarray data; and (III) a chain reaction model to simulate the pathway. This is a novel application because we are able to translate the gene pathway into the chemical reactions in which each reaction channel describes gene-gene relationship in the pathway. In the chain reaction model, the implicit scheme is employed to efficiently solve the differential equations. Data analysis results show the proposed model is capable of predicting gene expression changes and demonstrating the effect of nutrient-containing diets on gene expression changes in the pathway. One of the objectives of this study is to explore and develop a numerical approach for simulating the gene expression change so that it can be applied and calibrated when the data of more time slices are available, and thus can be used to interpolate the expression change at a desired time point without conducting expensive experiments for a large amount of time points. Hence, we are not claiming this is either essential or the most efficient way for simulating this problem, rather a mathematical/numerical approach that can model the expression change of a large set of genes of a complex pathway. In addition, we understand the limitation of this experiment and realize that it is still far from being a complete model of predicting nutrient-gene interactions. The reason is that in the present model, the reaction rates were estimated based on available data at two time points; hence, the gene expression change is dependent upon the reaction rates and a linear function of the gene expressions. More data sets containing gene expression at various time slices are needed in order to improve the present model so that a non-linear variation of gene expression changes at different time can be predicted.

Circular RNA In Invasive and Recurrent Clinical Nonfunctioning Pituitary Adenomas: Expression Profiles and Bioinformatic Analysis.

PubMed

Wang, Jianpeng; Wang, Dong; Wan, Dehong; Ma, Qingxia; Liu, Qian; Li, Jiye; Li, Zhaojian; Gao, Yang; Jiang, Guohui; Ma, Leina; Liu, Jia; Li, Chuzhong

2018-06-14

The invasion and recurrence of clinical nonfunctioning pituitary adenomas (NFA) often lead to surgical treatment failure. Circular RNAs (circRNAs) are a novel class of RNAs whose 3' and 5' ends are joined together and have been shown to play important roles in cancer development. Up to now, the roles of circRNAs remain unclear in invasive and recurrent NFA. We detected and summarized the circRNA expression pattern in 75 NFA tissues from 10 non-invasive cases and 65 invasive cases and 9 pairs NFA tumor tissues from 9 recurrent cases by circRNA microarrays. Accordingly, functional enrichment analysis and pathway analysis were performed and circRNA-microRNA(miRNA) network were generated by bioinformatic analysis tools. 5 new invasive NFA samples and 5 non-invasive NFA samples were collected to measure the microarray results. 570 dysregulated circRNAs (Invasive Tumor vs. Non-invasive Tumor) and 10 up-regulated circRNAs (Recurrent tumor Tissue vs. First surgery tumor Tissue) were identified based on the situation (FC>2, P<0.05). The parental genes of the dysregulated circRNAs in the comparison between invasion tumor and non-invasion tumor were found to be enriched in some cell adhesion signaling pathways such as Focal adhesion, Hippo signaling pathway, PI3K-Akt signaling pathway, and Adherens junction. The circRNA-miRNA network showed that the dysregulated circRNA may function as miRNA sponges. This is the first study to conduct and comprehensively analyze the circRNA expression profile in invasive and recurrent NFA. Our finding will provide evidence for the significance of circRNAs in NFA diagnosis, prognosis and clinical treatment. Copyright © 2018 Elsevier Inc. All rights reserved.

Gastroesophageal reflux activates the NF-κB pathway and impairs esophageal barrier function in mice

PubMed Central

Fang, Yu; Chen, Hao; Hu, Yuhui; Djukic, Zorka; Tevebaugh, Whitney; Shaheen, Nicholas J.; Orlando, Roy C.; Hu, Jianguo

2013-01-01

The barrier function of the esophageal epithelium is a major defense against gastroesophageal reflux disease. Previous studies have shown that reflux damage is reflected in a decrease in transepithelial electrical resistance associated with tight junction alterations in the esophageal epithelium. To develop novel therapies, it is critical to understand the molecular mechanisms whereby contact with a refluxate impairs esophageal barrier function. In this study, surgical models of duodenal and mixed reflux were developed in mice. Mouse esophageal epithelium was analyzed by gene microarray. Gene set enrichment analysis showed upregulation of inflammation-related gene sets and the NF-κB pathway due to reflux. Significance analysis of microarrays revealed upregulation of NF-κB target genes. Overexpression of NF-κB subunits (p50 and p65) and NF-κB target genes (matrix metalloproteinases-3 and -9, IL-1β, IL-6, and IL-8) confirmed activation of the NF-κB pathway in the esophageal epithelium. In addition, real-time PCR, Western blotting, and immunohistochemical staining also showed downregulation and mislocalization of claudins-1 and -4. In a second animal experiment, treatment with an NF-κB inhibitor, BAY 11-7085 (20 mg·kg−1·day−1 ip for 10 days), counteracted the effects of duodenal and mixed reflux on epithelial resistance and NF-κB-regulated cytokines. We conclude that gastroesophageal reflux activates the NF-κB pathway and impairs esophageal barrier function in mice and that targeting the NF-κB pathway may strengthen esophageal barrier function against reflux. PMID:23639809

Insulin immuno-neutralization in fed chickens: effects on liver and muscle transcriptome.

PubMed

Simon, Jean; Milenkovic, Dragan; Godet, Estelle; Cabau, Cedric; Collin, Anne; Métayer-Coustard, Sonia; Rideau, Nicole; Tesseraud, Sophie; Derouet, Michel; Crochet, Sabine; Cailleau-Audouin, Estelle; Hennequet-Antier, Christelle; Gespach, Christian; Porter, Tom E; Duclos, Michel J; Dupont, Joëlle; Cogburn, Larry A

2012-03-01

Chickens mimic an insulin-resistance state by exhibiting several peculiarities with regard to plasma glucose level and its control by insulin. To gain insight into the role of insulin in the control of chicken transcriptome, liver and leg muscle transcriptomes were compared in fed controls and "diabetic" chickens, at 5 h after insulin immuno-neutralization, using 20.7K-chicken oligo-microarrays. At a level of false discovery rate <0.01, 1,573 and 1,225 signals were significantly modified by insulin privation in liver and muscle, respectively. Microarray data agreed reasonably well with qRT-PCR and some protein level measurements. Differentially expressed mRNAs with human ID were classified using Biorag analysis and Ingenuity Pathway Analysis. Multiple metabolic pathways, structural proteins, transporters and proteins of intracellular trafficking, major signaling pathways, and elements of the transcriptional control machinery were largely represented in both tissues. At least 42 mRNAs have already been associated with diabetes, insulin resistance, obesity, energy expenditure, or identified as sensors of metabolism in mice or humans. The contribution of the pathways presently identified to chicken physiology (particularly those not yet related to insulin) needs to be evaluated in future studies. Other challenges include the characterization of "unknown" mRNAs and the identification of the steps or networks, which disturbed tissue transcriptome so extensively, quickly after the turning off of the insulin signal. In conclusion, pleiotropic effects of insulin in chickens are further evidenced; major pathways controlled by insulin in mammals have been conserved despite the presence of unique features of insulin signaling in chicken muscle.

Bifidobacterium breve B-3 exerts metabolic syndrome-suppressing effects in the liver of diet-induced obese mice: a DNA microarray analysis.

PubMed

Kondo, S; Kamei, A; Xiao, J Z; Iwatsuki, K; Abe, K

2013-09-01

We previously reported that supplementation with Bifidobacterium breve B-3 reduced body weight gain and accumulation of visceral fat in a dose-dependent manner, and improved serum levels of total cholesterol, glucose and insulin in a mouse model of diet-induced obesity. In this study, we investigated the expression of genes in the liver using DNA microarray analysis and q-PCR to reveal the mechanism of these anti-obesity effects in this mouse model. Administration of B. breve B-3 led to regulated gene expression of pathways involved in lipid metabolism and response to stress. The results indicate that these regulations in the liver are related to the anti-metabolic syndrome effects of B. breve B-3.

Construction of diagnosis system and gene regulatory networks based on microarray analysis.

PubMed

Hong, Chun-Fu; Chen, Ying-Chen; Chen, Wei-Chun; Tu, Keng-Chang; Tsai, Meng-Hsiun; Chan, Yung-Kuan; Yu, Shyr Shen

2018-05-01

A microarray analysis generally contains expression data of thousands of genes, but most of them are irrelevant to the disease of interest, making analyzing the genes concerning specific diseases complicated. Therefore, filtering out a few essential genes as well as their regulatory networks is critical, and a disease can be easily diagnosed just depending on the expression profiles of a few critical genes. In this study, a target gene screening (TGS) system, which is a microarray-based information system that integrates F-statistics, pattern recognition matching, a two-layer K-means classifier, a Parameter Detection Genetic Algorithm (PDGA), a genetic-based gene selector (GBG selector) and the association rule, was developed to screen out a small subset of genes that can discriminate malignant stages of cancers. During the first stage, F-statistic, pattern recognition matching, and a two-layer K-means classifier were applied in the system to filter out the 20 critical genes most relevant to ovarian cancer from 9600 genes, and the PDGA was used to decide the fittest values of the parameters for these critical genes. Among the 20 critical genes, 15 are associated with cancer progression. In the second stage, we further employed a GBG selector and the association rule to screen out seven target gene sets, each with only four to six genes, and each of which can precisely identify the malignancy stage of ovarian cancer based on their expression profiles. We further deduced the gene regulatory networks of the 20 critical genes by applying the Pearson correlation coefficient to evaluate the correlationship between the expression of each gene at the same stages and at different stages. Correlationships between gene pairs were calculated, and then, three regulatory networks were deduced. Their correlationships were further confirmed by the Ingenuity pathway analysis. The prognostic significances of the genes identified via regulatory networks were examined using online tools, and most represented biomarker candidates. In summary, our proposed system provides a new strategy to identify critical genes or biomarkers, as well as their regulatory networks, from microarray data. Copyright © 2018. Published by Elsevier Inc.

A High Throughput Protein Microarray Approach to Classify HIV Monoclonal Antibodies and Variant Antigens

PubMed Central

Dotsey, Emmanuel Y.; Gorlani, Andrea; Ingale, Sampat; Achenbach, Chad J.; Forthal, Donald N.; Felgner, Philip L.; Gach, Johannes S.

2015-01-01

In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs) has been applied to greatly advance our understanding of the specificity, and functional activity of antibodies against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip comprised of the HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001) with concentrations from ELISA binding measurements. Polyclonal immune responses in plasma samples from HIV-1 infected subjects exhibited different binding patterns, and reactivity against printed proteins. Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV. PMID:25938510

Meta-analysis of chicken--salmonella infection experiments.

PubMed

Te Pas, Marinus F W; Hulsegge, Ina; Schokker, Dirkjan; Smits, Mari A; Fife, Mark; Zoorob, Rima; Endale, Marie-Laure; Rebel, Johanna M J

2012-04-24

Chicken meat and eggs can be a source of human zoonotic pathogens, especially Salmonella species. These food items contain a potential hazard for humans. Chickens lines differ in susceptibility for Salmonella and can harbor Salmonella pathogens without showing clinical signs of illness. Many investigations including genomic studies have examined the mechanisms how chickens react to infection. Apart from the innate immune response, many physiological mechanisms and pathways are reported to be involved in the chicken host response to Salmonella infection. The objective of this study was to perform a meta-analysis of diverse experiments to identify general and host specific mechanisms to the Salmonella challenge. Diverse chicken lines differing in susceptibility to Salmonella infection were challenged with different Salmonella serovars at several time points. Various tissues were sampled at different time points post-infection, and resulting host transcriptional differences investigated using different microarray platforms. The meta-analysis was performed with the R-package metaMA to create lists of differentially regulated genes. These gene lists showed many similarities for different chicken breeds and tissues, and also for different Salmonella serovars measured at different times post infection. Functional biological analysis of these differentially expressed gene lists revealed several common mechanisms for the chicken host response to Salmonella infection. The meta-analysis-specific genes (i.e. genes found differentially expressed only in the meta-analysis) confirmed and expanded the biological functional mechanisms. The meta-analysis combination of heterogeneous expression profiling data provided useful insights into the common metabolic pathways and functions of different chicken lines infected with different Salmonella serovars.

Meta-analysis of Chicken – Salmonella infection experiments

PubMed Central

2012-01-01

Background Chicken meat and eggs can be a source of human zoonotic pathogens, especially Salmonella species. These food items contain a potential hazard for humans. Chickens lines differ in susceptibility for Salmonella and can harbor Salmonella pathogens without showing clinical signs of illness. Many investigations including genomic studies have examined the mechanisms how chickens react to infection. Apart from the innate immune response, many physiological mechanisms and pathways are reported to be involved in the chicken host response to Salmonella infection. The objective of this study was to perform a meta-analysis of diverse experiments to identify general and host specific mechanisms to the Salmonella challenge. Results Diverse chicken lines differing in susceptibility to Salmonella infection were challenged with different Salmonella serovars at several time points. Various tissues were sampled at different time points post-infection, and resulting host transcriptional differences investigated using different microarray platforms. The meta-analysis was performed with the R-package metaMA to create lists of differentially regulated genes. These gene lists showed many similarities for different chicken breeds and tissues, and also for different Salmonella serovars measured at different times post infection. Functional biological analysis of these differentially expressed gene lists revealed several common mechanisms for the chicken host response to Salmonella infection. The meta-analysis-specific genes (i.e. genes found differentially expressed only in the meta-analysis) confirmed and expanded the biological functional mechanisms. Conclusions The meta-analysis combination of heterogeneous expression profiling data provided useful insights into the common metabolic pathways and functions of different chicken lines infected with different Salmonella serovars. PMID:22531008

A Sorghum bicolor expression atlas reveals dynamic genotype-specific expression profiles for vegetative tissues of grain, sweet and bioenergy sorghums.

PubMed

Shakoor, Nadia; Nair, Ramesh; Crasta, Oswald; Morris, Geoffrey; Feltus, Alex; Kresovich, Stephen

2014-01-23

Effective improvement in sorghum crop development necessitates a genomics-based approach to identify functional genes and QTLs. Sequenced in 2009, a comprehensive annotation of the sorghum genome and the development of functional genomics resources is key to enable the discovery and deployment of regulatory and metabolic genes and gene networks for crop improvement. This study utilizes the first commercially available whole-transcriptome sorghum microarray (Sorgh-WTa520972F) to identify tissue and genotype-specific expression patterns for all identified Sorghum bicolor exons and UTRs. The genechip contains 1,026,373 probes covering 149,182 exons (27,577 genes) across the Sorghum bicolor nuclear, chloroplast, and mitochondrial genomes. Specific probesets were also included for putative non-coding RNAs that may play a role in gene regulation (e.g., microRNAs), and confirmed functional small RNAs in related species (maize and sugarcane) were also included in our array design. We generated expression data for 78 samples with a combination of four different tissue types (shoot, root, leaf and stem), two dissected stem tissues (pith and rind) and six diverse genotypes, which included 6 public sorghum lines (R159, Atlas, Fremont, PI152611, AR2400 and PI455230) representing grain, sweet, forage, and high biomass ideotypes. Here we present a summary of the microarray dataset, including analysis of tissue-specific gene expression profiles and associated expression profiles of relevant metabolic pathways. With an aim to enable identification and functional characterization of genes in sorghum, this expression atlas presents a new and valuable resource to the research community.

A Sorghum bicolor expression atlas reveals dynamic genotype-specific expression profiles for vegetative tissues of grain, sweet and bioenergy sorghums

PubMed Central

2014-01-01

Background Effective improvement in sorghum crop development necessitates a genomics-based approach to identify functional genes and QTLs. Sequenced in 2009, a comprehensive annotation of the sorghum genome and the development of functional genomics resources is key to enable the discovery and deployment of regulatory and metabolic genes and gene networks for crop improvement. Results This study utilizes the first commercially available whole-transcriptome sorghum microarray (Sorgh-WTa520972F) to identify tissue and genotype-specific expression patterns for all identified Sorghum bicolor exons and UTRs. The genechip contains 1,026,373 probes covering 149,182 exons (27,577 genes) across the Sorghum bicolor nuclear, chloroplast, and mitochondrial genomes. Specific probesets were also included for putative non-coding RNAs that may play a role in gene regulation (e.g., microRNAs), and confirmed functional small RNAs in related species (maize and sugarcane) were also included in our array design. We generated expression data for 78 samples with a combination of four different tissue types (shoot, root, leaf and stem), two dissected stem tissues (pith and rind) and six diverse genotypes, which included 6 public sorghum lines (R159, Atlas, Fremont, PI152611, AR2400 and PI455230) representing grain, sweet, forage, and high biomass ideotypes. Conclusions Here we present a summary of the microarray dataset, including analysis of tissue-specific gene expression profiles and associated expression profiles of relevant metabolic pathways. With an aim to enable identification and functional characterization of genes in sorghum, this expression atlas presents a new and valuable resource to the research community. PMID:24456189

Fisetin and hesperetin induced apoptosis and cell cycle arrest in chronic myeloid leukemia cells accompanied by modulation of cellular signaling.

PubMed

Adan, Aysun; Baran, Yusuf

2016-05-01

Fisetin and hesperetin, naturally occurring flavonoids, have been reported as novel antioxidants with chemopreventive/chemotherapeutic potential against various types of cancer. However, their mechanism of action in CML is still unknown. This particular study aims to evaluate the therapeutic potentials of fisetin and hesperetin and their effects on cell proliferation, apoptosis, and cell cycle progression in human K562 CML cells. The results indicated that fisetin and hesperetin inhibited cell proliferation and triggered programmed cell death in these cells. The latter was confırmed by mitochondrial membrane depolarization and an increase in caspase-3 activation. In addition to that, we have detected S and G2/M cell cycle arrests and G0/G1 arrest upon fisetin and hesperetin treatment, respectively. To identify the altered genes and genetic networks in response to fisetin and hesperetin, whole-genome microarray analysis was performed. The microarray gene profiling analysis revealed some important signaling pathways including JAK/STAT pathway, KIT receptor signaling, and growth hormone receptor signaling that were altered upon fisetin and hesperetin treatment. Moreover, microarray data suggested potential candidate genes for targeted CML therapy. Fisetin and hesperetin significantly modulated the expression of genes involved in cell proliferation and division, apoptosis, cell cycle regulation, and other significant cellular processes such as replication, transcription, and translation. In conclusion, our results suggest that fisetin and hesperetin as potential natural agents for CML therapy.

Integrative ChIP-seq/Microarray Analysis Identifies a CTNNB1 Target Signature Enriched in Intestinal Stem Cells and Colon Cancer

PubMed Central

Watanabe, Kazuhide; Biesinger, Jacob; Salmans, Michael L.; Roberts, Brian S.; Arthur, William T.; Cleary, Michele; Andersen, Bogi; Xie, Xiaohui; Dai, Xing

2014-01-01

Background Deregulation of canonical Wnt/CTNNB1 (beta-catenin) pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 are highly frequent in colon cancer and cause aberrant stabilization of CTNNB1, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of CTNNB1 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of CTNNB1 in colon cancer cells. Results We observed 3629 CTNNB1 binding peaks across the genome and a significant correlation between CTNNB1 binding and knockdown-induced gene expression change. Our integrative analysis led to the discovery of a direct Wnt target signature composed of 162 genes. Gene ontology analysis of this signature revealed a significant enrichment of Wnt pathway genes, suggesting multiple feedback regulations of the pathway. We provide evidence that this gene signature partially overlaps with the Lgr5+ intestinal stem cell signature, and is significantly enriched in normal intestinal stem cells as well as in clinical colorectal cancer samples. Interestingly, while the expression of the CTNNB1 target gene set does not correlate with survival, elevated expression of negative feedback regulators within the signature predicts better prognosis. Conclusion Our data provide a genome-wide view of chromatin occupancy and gene regulation of Wnt/CTNNB1 signaling in colon cancer cells. PMID:24651522

Integrative ChIP-seq/microarray analysis identifies a CTNNB1 target signature enriched in intestinal stem cells and colon cancer.

PubMed

Watanabe, Kazuhide; Biesinger, Jacob; Salmans, Michael L; Roberts, Brian S; Arthur, William T; Cleary, Michele; Andersen, Bogi; Xie, Xiaohui; Dai, Xing

2014-01-01

Deregulation of canonical Wnt/CTNNB1 (beta-catenin) pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 are highly frequent in colon cancer and cause aberrant stabilization of CTNNB1, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of CTNNB1 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of CTNNB1 in colon cancer cells. We observed 3629 CTNNB1 binding peaks across the genome and a significant correlation between CTNNB1 binding and knockdown-induced gene expression change. Our integrative analysis led to the discovery of a direct Wnt target signature composed of 162 genes. Gene ontology analysis of this signature revealed a significant enrichment of Wnt pathway genes, suggesting multiple feedback regulations of the pathway. We provide evidence that this gene signature partially overlaps with the Lgr5+ intestinal stem cell signature, and is significantly enriched in normal intestinal stem cells as well as in clinical colorectal cancer samples. Interestingly, while the expression of the CTNNB1 target gene set does not correlate with survival, elevated expression of negative feedback regulators within the signature predicts better prognosis. Our data provide a genome-wide view of chromatin occupancy and gene regulation of Wnt/CTNNB1 signaling in colon cancer cells.

A novel dysregulated pathway-identification analysis based on global influence of within-pathway effects and crosstalk between pathways

PubMed Central

Han, Junwei; Li, Chunquan; Yang, Haixiu; Xu, Yanjun; Zhang, Chunlong; Ma, Jiquan; Shi, Xinrui; Liu, Wei; Shang, Desi; Yao, Qianlan; Zhang, Yunpeng; Su, Fei; Feng, Li; Li, Xia

2015-01-01

Identifying dysregulated pathways from high-throughput experimental data in order to infer underlying biological insights is an important task. Current pathway-identification methods focus on single pathways in isolation; however, consideration of crosstalk between pathways could improve our understanding of alterations in biological states. We propose a novel method of pathway analysis based on global influence (PAGI) to identify dysregulated pathways, by considering both within-pathway effects and crosstalk between pathways. We constructed a global gene–gene network based on the relationships among genes extracted from a pathway database. We then evaluated the extent of differential expression for each gene, and mapped them to the global network. The random walk with restart algorithm was used to calculate the extent of genes affected by global influence. Finally, we used cumulative distribution functions to determine the significance values of the dysregulated pathways. We applied the PAGI method to five cancer microarray datasets, and compared our results with gene set enrichment analysis and five other methods. Based on these analyses, we demonstrated that PAGI can effectively identify dysregulated pathways associated with cancer, with strong reproducibility and robustness. We implemented PAGI using the freely available R-based and Web-based tools (http://bioinfo.hrbmu.edu.cn/PAGI). PMID:25551156

Assessing probe-specific dye and slide biases in two-color microarray data

USDA-ARS?s Scientific Manuscript database

A primary reason for using two-color microarrays is that the use of two samples labeled with different dyes on the same slide and that bind to probes on the same spot is supposed to adjust for many factors that introduce noise and errors into the analysis. Most users assume that any differences bet...

A Discovery Resource of Rare Copy Number Variations in Individuals with Autism Spectrum Disorder

PubMed Central

Prasad, Aparna; Merico, Daniele; Thiruvahindrapuram, Bhooma; Wei, John; Lionel, Anath C.; Sato, Daisuke; Rickaby, Jessica; Lu, Chao; Szatmari, Peter; Roberts, Wendy; Fernandez, Bridget A.; Marshall, Christian R.; Hatchwell, Eli; Eis, Peggy S.; Scherer, Stephen W.

2012-01-01

The identification of rare inherited and de novo copy number variations (CNVs) in human subjects has proven a productive approach to highlight risk genes for autism spectrum disorder (ASD). A variety of microarrays are available to detect CNVs, including single-nucleotide polymorphism (SNP) arrays and comparative genomic hybridization (CGH) arrays. Here, we examine a cohort of 696 unrelated ASD cases using a high-resolution one-million feature CGH microarray, the majority of which were previously genotyped with SNP arrays. Our objective was to discover new CNVs in ASD cases that were not detected by SNP microarray analysis and to delineate novel ASD risk loci via combined analysis of CGH and SNP array data sets on the ASD cohort and CGH data on an additional 1000 control samples. Of the 615 ASD cases analyzed on both SNP and CGH arrays, we found that 13,572 of 21,346 (64%) of the CNVs were exclusively detected by the CGH array. Several of the CGH-specific CNVs are rare in population frequency and impact previously reported ASD genes (e.g., NRXN1, GRM8, DPYD), as well as novel ASD candidate genes (e.g., CIB2, DAPP1, SAE1), and all were inherited except for a de novo CNV in the GPHN gene. A functional enrichment test of gene-sets in ASD cases over controls revealed nucleotide metabolism as a potential novel pathway involved in ASD, which includes several candidate genes for follow-up (e.g., DPYD, UPB1, UPP1, TYMP). Finally, this extensively phenotyped and genotyped ASD clinical cohort serves as an invaluable resource for the next step of genome sequencing for complete genetic variation detection. PMID:23275889

Microarray Analysis of Differential Gene Expression Profile Between Human Fetal and Adult Heart.

PubMed

Geng, Zhimin; Wang, Jue; Pan, Lulu; Li, Ming; Zhang, Jitai; Cai, Xueli; Chu, Maoping

2017-04-01

Although many changes have been discovered during heart maturation, the genetic mechanisms involved in the changes between immature and mature myocardium have only been partially elucidated. Here, gene expression profile changed between the human fetal and adult heart was characterized. A human microarray was applied to define the gene expression signatures of the fetal (13-17 weeks of gestation, n = 4) and adult hearts (30-40 years old, n = 4). Gene ontology analyses, pathway analyses, gene set enrichment analyses, and signal transduction network were performed to predict the function of the differentially expressed genes. Ten mRNAs were confirmed by quantificational real-time polymerase chain reaction. 5547 mRNAs were found to be significantly differentially expressed. "Cell cycle" was the most enriched pathway in the down-regulated genes. EFGR, IGF1R, and ITGB1 play a central role in the regulation of heart development. EGFR, IGF1R, and FGFR2 were the core genes regulating cardiac cell proliferation. The quantificational real-time polymerase chain reaction results were concordant with the microarray data. Our data identified the transcriptional regulation of heart development in the second trimester and the potential regulators that play a prominent role in the regulation of heart development and cardiac cells proliferation.

Evaluation of Different Normalization and Analysis Procedures for Illumina Gene Expression Microarray Data Involving Small Changes

PubMed Central

Johnstone, Daniel M.; Riveros, Carlos; Heidari, Moones; Graham, Ross M.; Trinder, Debbie; Berretta, Regina; Olynyk, John K.; Scott, Rodney J.; Moscato, Pablo; Milward, Elizabeth A.

2013-01-01

While Illumina microarrays can be used successfully for detecting small gene expression changes due to their high degree of technical replicability, there is little information on how different normalization and differential expression analysis strategies affect outcomes. To evaluate this, we assessed concordance across gene lists generated by applying different combinations of normalization strategy and analytical approach to two Illumina datasets with modest expression changes. In addition to using traditional statistical approaches, we also tested an approach based on combinatorial optimization. We found that the choice of both normalization strategy and analytical approach considerably affected outcomes, in some cases leading to substantial differences in gene lists and subsequent pathway analysis results. Our findings suggest that important biological phenomena may be overlooked when there is a routine practice of using only one approach to investigate all microarray datasets. Analytical artefacts of this kind are likely to be especially relevant for datasets involving small fold changes, where inherent technical variation—if not adequately minimized by effective normalization—may overshadow true biological variation. This report provides some basic guidelines for optimizing outcomes when working with Illumina datasets involving small expression changes. PMID:27605185

Genome-Wide Analysis of Host Responses to Four Different Types of Microorganisms in Bombyx Mori (Lepidoptera: Bombycidae).

PubMed

Cheng, Tingcai; Lin, Ping; Huang, Lulin; Wu, Yuqian; Jin, Shengkai; Liu, Chun; Xia, Qingyou

2016-01-01

Several pathogenic microorganisms have been used to investigate the genome-wide transcriptional responses of Bombyx mori to infection. However, studies have so far each focused on one microorganism, and systematic genome-wide comparison of transcriptional responses to different pathogenic microorganisms has not been undertaken. Here, we surveyed transcriptional responses of B. mori to its natural bacterial, viral, and fungal pathogens, Bacillus bombyseptieus, B. mori nucleopolyhedrovirus (BmNPV), and Beauveria bassiana, respectively, and to nonpathogenic Escherichia coli, by microarray analysis. In total, the expression of 2,436, 1,804, 1,743, and 912 B. mori genes was modulated by infection with B. bombyseptieus, BmNPV, B. bassiana, and E. coli, respectively. Notably, the expression of 620, 400, 177, or 165 of these genes was only modulated by infection with B. bombyseptieus, BmNPV, B. bassiana, or E. coli, respectively. In contrast to the expression of genes related to juvenile hormone synthesis and metabolism, that of genes encoding juvenile hormone binding proteins was microorganism-specific. Three basal metabolic pathways were modulated by infection with any of the four microorganisms, and 3, 14, 5, and 2 metabolic pathways were specifically modulated by infection with B. bombyseptieus, BmNPV, B. bassiana, and E. coli, respectively. Interestingly, BmNPV infection modulated the JAK/STAT signaling pathway, whereas both the Imd and Toll signaling pathways were modulated by infection with B. bombyseptieus, B. bassiana, or E. coli These results elucidate potential molecular mechanisms of the host response to different microorganisms, and provide a foundation for further work on host-pathogen interaction. © The Author 2016. Published by Oxford University Press on behalf of the Entomological Society of America.

ALDH isozymes downregulation affects cell growth, cell motility and gene expression in lung cancer cells.

PubMed

Moreb, Jan S; Baker, Henry V; Chang, Lung-Ji; Amaya, Maria; Lopez, M Cecilia; Ostmark, Blanca; Chou, Wayne

2008-11-24

Aldehyde dehydrogenase isozymes ALDH1A1 and ALDH3A1 are highly expressed in non small cell lung cancer. Neither the mechanisms nor the biologic significance for such over expression have been studied. We have employed oligonucleotide microarrays to analyze changes in gene profiles in A549 lung cancer cell line in which ALDH activity was reduced by up to 95% using lentiviral mediated expression of siRNA against both isozymes (Lenti 1+3). Stringent analysis methods were used to identify gene expression patterns that are specific to the knock down of ALDH activity and significantly different in comparison to wild type A549 cells (WT) or cells similarly transduced with green fluorescent protein (GFP) siRNA. We confirmed significant and specific down regulation of ALDH1A1 and ALDH3A1 in Lenti 1+3 cells and in comparison to 12 other ALDH genes detected. The results of the microarray analysis were validated by real time RT-PCR on RNA obtained from Lenti 1+3 or WT cells treated with ALDH activity inhibitors. Detailed functional analysis was performed on 101 genes that were significantly different (P < 0.001) and their expression changed by > or = 2 folds in the Lenti 1+3 group versus the control groups. There were 75 down regulated and 26 up regulated genes. Protein binding, organ development, signal transduction, transcription, lipid metabolism, and cell migration and adhesion were among the most affected pathways. These molecular effects of the ALDH knock-down are associated with in vitro functional changes in the proliferation and motility of these cells and demonstrate the significance of ALDH enzymes in cell homeostasis with a potentially significant impact on the treatment of lung cancer.

Advantages of RNA-seq compared to RNA microarrays for transcriptome profiling of anterior cruciate ligament tears.

PubMed

Rai, Muhammad Farooq; Tycksen, Eric D; Sandell, Linda J; Brophy, Robert H

2018-01-01

Microarrays and RNA-seq are at the forefront of high throughput transcriptome analyses. Since these methodologies are based on different principles, there are concerns about the concordance of data between the two techniques. The concordance of RNA-seq and microarrays for genome-wide analysis of differential gene expression has not been rigorously assessed in clinically derived ligament tissues. To demonstrate the concordance between RNA-seq and microarrays and to assess potential benefits of RNA-seq over microarrays, we assessed differences in transcript expression in anterior cruciate ligament (ACL) tissues based on time-from-injury. ACL remnants were collected from patients with an ACL tear at the time of ACL reconstruction. RNA prepared from torn ACL remnants was subjected to Agilent microarrays (N = 24) and RNA-seq (N = 8). The correlation of biological replicates in RNA-seq and microarrays data was similar (0.98 vs. 0.97), demonstrating that each platform has high internal reproducibility. Correlations between the RNA-seq data and the individual microarrays were low, but correlations between the RNA-seq values and the geometric mean of the microarrays values were moderate. The cross-platform concordance for differentially expressed transcripts or enriched pathways was linearly correlated (r = 0.64). RNA-Seq was superior in detecting low abundance transcripts and differentiating biologically critical isoforms. Additional independent validation of transcript expression was undertaken using microfluidic PCR for selected genes. PCR data showed 100% concordance (in expression pattern) with RNA-seq and microarrays data. These findings demonstrate that RNA-seq has advantages over microarrays for transcriptome profiling of ligament tissues when available and affordable. Furthermore, these findings are likely transferable to other musculoskeletal tissues where tissue collection is challenging and cells are in low abundance. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:484-497, 2018. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Genomic expression patterns of cardiac tissues from dogs with dilated cardiomyopathy.

PubMed

Oyama, Mark A; Chittur, Sridar

2005-07-01

To evaluate global genome expression patterns of left ventricular tissues from dogs with dilated cardiomyopathy (DCM). Tissues obtained from the left ventricle of 2 Doberman Pinschers with end-stage DCM and 5 healthy control dogs. Transcriptional activities of 23,851 canine DNA sequences were determined by use of an oligonucleotide microarray. Genome expression patterns of DCM tissue were evaluated by measuring the relative amount of complementary RNA hybridization to the microarray probes and comparing it with gene expression for tissues from 5 healthy control dogs. 478 transcripts were differentially expressed (> or = 2.5-fold change). In DCM tissue, expression of 173 transcripts was upregulated and expression of 305 transcripts was downregulated, compared with expression for control tissues. Of the 478 transcripts, 167 genes could be specifically identified. These genes were grouped into 1 of 8 categories on the basis of their primary physiologic function. Grouping revealed that pathways involving cellular energy production, signaling and communication, and cell structure were generally downregulated, whereas pathways involving cellular defense and stress responses were upregulated. Many previously unreported genes that may contribute to the pathophysiologic aspects of heart disease were identified. Evaluation of global expression patterns provides a molecular portrait of heart failure, yields insights into the pathophysiologic aspects of DCM, and identifies intriguing genes and pathways for further study.

Identification of new autoantigens for primary biliary cirrhosis using human proteome microarrays.

PubMed

Hu, Chao-Jun; Song, Guang; Huang, Wei; Liu, Guo-Zhen; Deng, Chui-Wen; Zeng, Hai-Pan; Wang, Li; Zhang, Feng-Chun; Zhang, Xuan; Jeong, Jun Seop; Blackshaw, Seth; Jiang, Li-Zhi; Zhu, Heng; Wu, Lin; Li, Yong-Zhe

2012-09-01

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology and is considered to be an autoimmune disease. Autoantibodies are important tools for accurate diagnosis of PBC. Here, we employed serum profiling analysis using a human proteome microarray composed of about 17,000 full-length unique proteins and identified 23 proteins that correlated with PBC. To validate these results, we fabricated a PBC-focused microarray with 21 of these newly identified candidates and nine additional known PBC antigens. By screening the PBC microarrays with additional cohorts of 191 PBC patients and 321 controls (43 autoimmune hepatitis, 55 hepatitis B virus, 31 hepatitis C virus, 48 rheumatoid arthritis, 45 systematic lupus erythematosus, 49 systemic sclerosis, and 50 healthy), six proteins were confirmed as novel PBC autoantigens with high sensitivities and specificities, including hexokinase-1 (isoforms I and II), Kelch-like protein 7, Kelch-like protein 12, zinc finger and BTB domain-containing protein 2, and eukaryotic translation initiation factor 2C, subunit 1. To facilitate clinical diagnosis, we developed ELISA for Kelch-like protein 12 and zinc finger and BTB domain-containing protein 2 and tested large cohorts (297 PBC and 637 control sera) to confirm the sensitivities and specificities observed in the microarray-based assays. In conclusion, our research showed that a strategy using high content protein microarray combined with a smaller but more focused protein microarray can effectively identify and validate novel PBC-specific autoantigens and has the capacity to be translated to clinical diagnosis by means of an ELISA-based method.

The efficacy of microarray screening for autosomal recessive retinitis pigmentosa in routine clinical practice

PubMed Central

van Huet, Ramon A. C.; Pierrache, Laurence H.M.; Meester-Smoor, Magda A.; Klaver, Caroline C.W.; van den Born, L. Ingeborgh; Hoyng, Carel B.; de Wijs, Ilse J.; Collin, Rob W. J.; Hoefsloot, Lies H.

2015-01-01

Purpose To determine the efficacy of multiple versions of a commercially available arrayed primer extension (APEX) microarray chip for autosomal recessive retinitis pigmentosa (arRP). Methods We included 250 probands suspected of arRP who were genetically analyzed with the APEX microarray between January 2008 and November 2013. The mode of inheritance had to be autosomal recessive according to the pedigree (including isolated cases). If the microarray identified a heterozygous mutation, we performed Sanger sequencing of exons and exon–intron boundaries of that specific gene. The efficacy of this microarray chip with the additional Sanger sequencing approach was determined by the percentage of patients that received a molecular diagnosis. We also collected data from genetic tests other than the APEX analysis for arRP to provide a detailed description of the molecular diagnoses in our study cohort. Results The APEX microarray chip for arRP identified the molecular diagnosis in 21 (8.5%) of the patients in our cohort. Additional Sanger sequencing yielded a second mutation in 17 patients (6.8%), thereby establishing the molecular diagnosis. In total, 38 patients (15.2%) received a molecular diagnosis after analysis using the microarray and additional Sanger sequencing approach. Further genetic analyses after a negative result of the arRP microarray (n = 107) resulted in a molecular diagnosis of arRP (n = 23), autosomal dominant RP (n = 5), X-linked RP (n = 2), and choroideremia (n = 1). Conclusions The efficacy of the commercially available APEX microarray chips for arRP appears to be low, most likely caused by the limitations of this technique and the genetic and allelic heterogeneity of RP. Diagnostic yields up to 40% have been reported for next-generation sequencing (NGS) techniques that, as expected, thereby outperform targeted APEX analysis. PMID:25999674

Cross species analysis of microarray expression data

PubMed Central

Lu, Yong; Huggins, Peter; Bar-Joseph, Ziv

2009-01-01

Motivation: Many biological systems operate in a similar manner across a large number of species or conditions. Cross-species analysis of sequence and interaction data is often applied to determine the function of new genes. In contrast to these static measurements, microarrays measure the dynamic, condition-specific response of complex biological systems. The recent exponential growth in microarray expression datasets allows researchers to combine expression experiments from multiple species to identify genes that are not only conserved in sequence but also operated in a similar way in the different species studied. Results: In this review we discuss the computational and technical challenges associated with these studies, the approaches that have been developed to address these challenges and the advantages of cross-species analysis of microarray data. We show how successful application of these methods lead to insights that cannot be obtained when analyzing data from a single species. We also highlight current open problems and discuss possible ways to address them. Contact: zivbj@cs.cmu.edu PMID:19357096

Expression profiling in canine osteosarcoma: identification of biomarkers and pathways associated with outcome

PubMed Central

2010-01-01

Background Osteosarcoma (OSA) spontaneously arises in the appendicular skeleton of large breed dogs and shares many physiological and molecular biological characteristics with human OSA. The standard treatment for OSA in both species is amputation or limb-sparing surgery, followed by chemotherapy. Unfortunately, OSA is an aggressive cancer with a high metastatic rate. Characterization of OSA with regard to its metastatic potential and chemotherapeutic resistance will improve both prognostic capabilities and treatment modalities. Methods We analyzed archived primary OSA tissue from dogs treated with limb amputation followed by doxorubicin or platinum-based drug chemotherapy. Samples were selected from two groups: dogs with disease free intervals (DFI) of less than 100 days (n = 8) and greater than 300 days (n = 7). Gene expression was assessed with Affymetrix Canine 2.0 microarrays and analyzed with a two-tailed t-test. A subset of genes was confirmed using qRT-PCR and used in classification analysis to predict prognosis. Systems-based gene ontology analysis was conducted on genes selected using a standard J5 metric. The genes identified using this approach were converted to their human homologues and assigned to functional pathways using the GeneGo MetaCore platform. Results Potential biomarkers were identified using gene expression microarray analysis and 11 differentially expressed (p < 0.05) genes were validated with qRT-PCR (n = 10/group). Statistical classification models using the qRT-PCR profiles predicted patient outcomes with 100% accuracy in the training set and up to 90% accuracy upon stratified cross validation. Pathway analysis revealed alterations in pathways associated with oxidative phosphorylation, hedgehog and parathyroid hormone signaling, cAMP/Protein Kinase A (PKA) signaling, immune responses, cytoskeletal remodeling and focal adhesion. Conclusions This profiling study has identified potential new biomarkers to predict patient outcome in OSA and new pathways that may be targeted for therapeutic intervention. PMID:20860831

The SNP rs6500843 in 16p13.3 is associated with survival specifically among chemotherapy-treated breast cancer patients.

PubMed

Fagerholm, Rainer; Schmidt, Marjanka K; Khan, Sofia; Rafiq, Sajjad; Tapper, William; Aittomäki, Kristiina; Greco, Dario; Heikkinen, Tuomas; Muranen, Taru A; Fasching, Peter A; Janni, Wolfgang; Weinshilboum, Richard; Loehberg, Christian R; Hopper, John L; Southey, Melissa C; Keeman, Renske; Lindblom, Annika; Margolin, Sara; Mannermaa, Arto; Kataja, Vesa; Chenevix-Trench, Georgia; Lambrechts, Diether; Wildiers, Hans; Chang-Claude, Jenny; Seibold, Petra; Couch, Fergus J; Olson, Janet E; Andrulis, Irene L; Knight, Julia A; García-Closas, Montserrat; Figueroa, Jonine; Hooning, Maartje J; Jager, Agnes; Shah, Mitul; Perkins, Barbara J; Luben, Robert; Hamann, Ute; Kabisch, Maria; Czene, Kamila; Hall, Per; Easton, Douglas F; Pharoah, Paul D P; Liu, Jianjun; Eccles, Diana; Blomqvist, Carl; Nevanlinna, Heli

2015-04-10

We have utilized a two-stage study design to search for SNPs associated with the survival of breast cancer patients treated with adjuvant chemotherapy. Our initial GWS data set consisted of 805 Finnish breast cancer cases (360 treated with adjuvant chemotherapy). The top 39 SNPs from this stage were analyzed in three independent data sets: iCOGS (n=6720 chemotherapy-treated cases), SUCCESS-A (n=3596), and POSH (n=518). Two SNPs were successfully validated: rs6500843 (any chemotherapy; per-allele HR 1.16, 95% C.I. 1.08-1.26, p=0.0001, p(adjusted)=0.0091), and rs11155012 (anthracycline therapy; per-allele HR 1.21, 95% C.I. 1.08-1.35, p=0.0010, p(adjusted)=0.0270). The SNP rs6500843 was found to specifically interact with adjuvant chemotherapy, independently of standard prognostic markers (p(interaction)=0.0009), with the rs6500843-GG genotype corresponding to the highest hazard among chemotherapy-treated cases (HR 1.47, 95% C.I. 1.20-1.80). Upon trans-eQTL analysis of public microarray data, the rs6500843 locus was found to associate with the expression of a group of genes involved in cell cycle control, notably AURKA, the expression of which also exhibited differential prognostic value between chemotherapy-treated and untreated cases in our analysis of microarray data. Based on previously published information, we propose that the eQTL genes may be connected to the rs6500843 locus via a RBFOX1-FOXM1 -mediated regulatory pathway.

Microarray and network-based identification of functional modules and pathways of active tuberculosis.

PubMed

Bian, Zhong-Rui; Yin, Juan; Sun, Wen; Lin, Dian-Jie

2017-04-01

Diagnose of active tuberculosis (TB) is challenging and treatment response is also difficult to efficiently monitor. The aim of this study was to use an integrated analysis of microarray and network-based method to the samples from publically available datasets to obtain a diagnostic module set and pathways in active TB. Towards this goal, background protein-protein interactions (PPI) network was generated based on global PPI information and gene expression data, following by identification of differential expression network (DEN) from the background PPI network. Then, ego genes were extracted according to the degree features in DEN. Next, module collection was conducted by ego gene expansion based on EgoNet algorithm. After that, differential expression of modules between active TB and controls was evaluated using random permutation test. Finally, biological significance of differential modules was detected by pathways enrichment analysis based on Reactome database, and Fisher's exact test was implemented to extract differential pathways for active TB. Totally, 47 ego genes and 47 candidate modules were identified from the DEN. By setting the cutoff-criteria of gene size >5 and classification accuracy ≥0.9, 7 ego modules (Module 4, Module 7, Module 9, Module 19, Module 25, Module 38 and Module 43) were extracted, and all of them had the statistical significance between active TB and controls. Then, Fisher's exact test was conducted to capture differential pathways for active TB. Interestingly, genes in Module 4, Module 25, Module 38, and Module 43 were enriched in the same pathway, formation of a pool of free 40S subunits. Significant pathway for Module 7 and Module 9 was eukaryotic translation termination, and for Module 19 was nonsense mediated decay enhanced by the exon junction complex (EJC). Accordingly, differential modules and pathways might be potential biomarkers for treating active TB, and provide valuable clues for better understanding of molecular mechanism of active TB. Copyright © 2017 Elsevier Ltd. All rights reserved.

The statistics of identifying differentially expressed genes in Expresso and TM4: a comparison

PubMed Central

Sioson, Allan A; Mane, Shrinivasrao P; Li, Pinghua; Sha, Wei; Heath, Lenwood S; Bohnert, Hans J; Grene, Ruth

2006-01-01

Background Analysis of DNA microarray data takes as input spot intensity measurements from scanner software and returns differential expression of genes between two conditions, together with a statistical significance assessment. This process typically consists of two steps: data normalization and identification of differentially expressed genes through statistical analysis. The Expresso microarray experiment management system implements these steps with a two-stage, log-linear ANOVA mixed model technique, tailored to individual experimental designs. The complement of tools in TM4, on the other hand, is based on a number of preset design choices that limit its flexibility. In the TM4 microarray analysis suite, normalization, filter, and analysis methods form an analysis pipeline. TM4 computes integrated intensity values (IIV) from the average intensities and spot pixel counts returned by the scanner software as input to its normalization steps. By contrast, Expresso can use either IIV data or median intensity values (MIV). Here, we compare Expresso and TM4 analysis of two experiments and assess the results against qRT-PCR data. Results The Expresso analysis using MIV data consistently identifies more genes as differentially expressed, when compared to Expresso analysis with IIV data. The typical TM4 normalization and filtering pipeline corrects systematic intensity-specific bias on a per microarray basis. Subsequent statistical analysis with Expresso or a TM4 t-test can effectively identify differentially expressed genes. The best agreement with qRT-PCR data is obtained through the use of Expresso analysis and MIV data. Conclusion The results of this research are of practical value to biologists who analyze microarray data sets. The TM4 normalization and filtering pipeline corrects microarray-specific systematic bias and complements the normalization stage in Expresso analysis. The results of Expresso using MIV data have the best agreement with qRT-PCR results. In one experiment, MIV is a better choice than IIV as input to data normalization and statistical analysis methods, as it yields as greater number of statistically significant differentially expressed genes; TM4 does not support the choice of MIV input data. Overall, the more flexible and extensive statistical models of Expresso achieve more accurate analytical results, when judged by the yardstick of qRT-PCR data, in the context of an experimental design of modest complexity. PMID:16626497

PathMAPA: a tool for displaying gene expression and performing statistical tests on metabolic pathways at multiple levels for Arabidopsis.

PubMed

Pan, Deyun; Sun, Ning; Cheung, Kei-Hoi; Guan, Zhong; Ma, Ligeng; Holford, Matthew; Deng, Xingwang; Zhao, Hongyu

2003-11-07

To date, many genomic and pathway-related tools and databases have been developed to analyze microarray data. In published web-based applications to date, however, complex pathways have been displayed with static image files that may not be up-to-date or are time-consuming to rebuild. In addition, gene expression analyses focus on individual probes and genes with little or no consideration of pathways. These approaches reveal little information about pathways that are key to a full understanding of the building blocks of biological systems. Therefore, there is a need to provide useful tools that can generate pathways without manually building images and allow gene expression data to be integrated and analyzed at pathway levels for such experimental organisms as Arabidopsis. We have developed PathMAPA, a web-based application written in Java that can be easily accessed over the Internet. An Oracle database is used to store, query, and manipulate the large amounts of data that are involved. PathMAPA allows its users to (i) upload and populate microarray data into a database; (ii) integrate gene expression with enzymes of the pathways; (iii) generate pathway diagrams without building image files manually; (iv) visualize gene expressions for each pathway at enzyme, locus, and probe levels; and (v) perform statistical tests at pathway, enzyme and gene levels. PathMAPA can be used to examine Arabidopsis thaliana gene expression patterns associated with metabolic pathways. PathMAPA provides two unique features for the gene expression analysis of Arabidopsis thaliana: (i) automatic generation of pathways associated with gene expression and (ii) statistical tests at pathway level. The first feature allows for the periodical updating of genomic data for pathways, while the second feature can provide insight into how treatments affect relevant pathways for the selected experiment(s).

PathMAPA: a tool for displaying gene expression and performing statistical tests on metabolic pathways at multiple levels for Arabidopsis

PubMed Central

Pan, Deyun; Sun, Ning; Cheung, Kei-Hoi; Guan, Zhong; Ma, Ligeng; Holford, Matthew; Deng, Xingwang; Zhao, Hongyu

2003-01-01

Background To date, many genomic and pathway-related tools and databases have been developed to analyze microarray data. In published web-based applications to date, however, complex pathways have been displayed with static image files that may not be up-to-date or are time-consuming to rebuild. In addition, gene expression analyses focus on individual probes and genes with little or no consideration of pathways. These approaches reveal little information about pathways that are key to a full understanding of the building blocks of biological systems. Therefore, there is a need to provide useful tools that can generate pathways without manually building images and allow gene expression data to be integrated and analyzed at pathway levels for such experimental organisms as Arabidopsis. Results We have developed PathMAPA, a web-based application written in Java that can be easily accessed over the Internet. An Oracle database is used to store, query, and manipulate the large amounts of data that are involved. PathMAPA allows its users to (i) upload and populate microarray data into a database; (ii) integrate gene expression with enzymes of the pathways; (iii) generate pathway diagrams without building image files manually; (iv) visualize gene expressions for each pathway at enzyme, locus, and probe levels; and (v) perform statistical tests at pathway, enzyme and gene levels. PathMAPA can be used to examine Arabidopsis thaliana gene expression patterns associated with metabolic pathways. Conclusion PathMAPA provides two unique features for the gene expression analysis of Arabidopsis thaliana: (i) automatic generation of pathways associated with gene expression and (ii) statistical tests at pathway level. The first feature allows for the periodical updating of genomic data for pathways, while the second feature can provide insight into how treatments affect relevant pathways for the selected experiment(s). PMID:14604444

Development and experimental validation of a 20K Atlantic cod (Gadus morhua) oligonucleotide microarray based on a collection of over 150,000 ESTs.

PubMed

Booman, Marije; Borza, Tudor; Feng, Charles Y; Hori, Tiago S; Higgins, Brent; Culf, Adrian; Léger, Daniel; Chute, Ian C; Belkaid, Anissa; Rise, Marlies; Gamperl, A Kurt; Hubert, Sophie; Kimball, Jennifer; Ouellette, Rodney J; Johnson, Stewart C; Bowman, Sharen; Rise, Matthew L

2011-08-01

The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research.

Discrimination of Influenza Infection (A/2009 H1N1) from Prior Exposure by Antibody Protein Microarray Analysis

PubMed Central

te Beest, Dennis; de Bruin, Erwin; Imholz, Sandra; Wallinga, Jacco; Teunis, Peter; Koopmans, Marion; van Boven, Michiel

2014-01-01

Reliable discrimination of recent influenza A infection from previous exposure using hemagglutination inhibition (HI) or virus neutralization tests is currently not feasible. This is due to low sensitivity of the tests and the interference of antibody responses generated by previous infections. Here we investigate the diagnostic characteristics of a newly developed antibody (HA1) protein microarray using data from cross-sectional serological studies carried out before and after the pandemic of 2009. The data are analysed by mixture models, providing a probabilistic classification of sera (susceptible, prior-exposed, recently infected). Estimated sensitivity and specificity for identifying A/2009 infections are low using HI (66% and 51%), and high when using A/2009 microarray data alone or together with A/1918 microarray data (96% and 95%). As a heuristic, a high A/2009 to A/1918 antibody ratio (>1.05) is indicative of recent infection, while a low ratio is indicative of a pre-existing response, even if the A/2009 titer is high. We conclude that highly sensitive and specific classification of individual sera is possible using the protein microarray, thereby enabling precise estimation of age-specific infection attack rates in the population even if sample sizes are small. PMID:25405997

Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis.

PubMed

Negm, Ola H; Hamed, Mohamed R; Dilnot, Elizabeth M; Shone, Clifford C; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E; Edwards, Laura J; Tighe, Patrick J; Wilcox, Mark H; Monaghan, Tanya M

2015-09-01

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis

PubMed Central

Negm, Ola H.; Hamed, Mohamed R.; Dilnot, Elizabeth M.; Shone, Clifford C.; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E.; Edwards, Laura J.; Tighe, Patrick J.; Wilcox, Mark H.

2015-01-01

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. PMID:26178385

Assessing co-regulation of directly linked genes in biological networks using microarray time series analysis.

PubMed

Del Sorbo, Maria Rosaria; Balzano, Walter; Donato, Michele; Draghici, Sorin

2013-11-01

Differential expression of genes detected with the analysis of high throughput genomic experiments is a commonly used intermediate step for the identification of signaling pathways involved in the response to different biological conditions. The impact analysis was the first approach for the analysis of signaling pathways involved in a certain biological process that was able to take into account not only the magnitude of the expression change of the genes but also the topology of signaling pathways including the type of each interactions between the genes. In the impact analysis, signaling pathways are represented as weighted directed graphs with genes as nodes and the interactions between genes as edges. Edges weights are represented by a β factor, the regulatory efficiency, which is assumed to be equal to 1 in inductive interactions between genes and equal to -1 in repressive interactions. This study presents a similarity analysis between gene expression time series aimed to find correspondences with the regulatory efficiency, i.e. the β factor as found in a widely used pathway database. Here, we focused on correlations among genes directly connected in signaling pathways, assuming that the expression variations of upstream genes impact immediately downstream genes in a short time interval and without significant influences by the interactions with other genes. Time series were processed using three different similarity metrics. The first metric is based on the bit string matching; the second one is a specific application of the Dynamic Time Warping to detect similarities even in presence of stretching and delays; the third one is a quantitative comparative analysis resulting by an evaluation of frequency domain representation of time series: the similarity metric is the correlation between dominant spectral components. These three approaches are tested on real data and pathways, and a comparison is performed using Information Retrieval benchmark tools, indicating the frequency approach as the best similarity metric among the three, for its ability to detect the correlation based on the correspondence of the most significant frequency components. Copyright © 2013. Published by Elsevier Ireland Ltd.

Biological Pathways

MedlinePlus

... Sheets A Brief Guide to Genomics About NHGRI Research About the International HapMap Project Biological Pathways Chromosome Abnormalities Chromosomes Cloning Comparative Genomics DNA Microarray Technology DNA Sequencing Deoxyribonucleic Acid ( ...

Cardiac transcriptome profiling of diabetic Akita mice using microarray and next generation sequencing

PubMed Central

Kesherwani, Varun; Shahshahan, Hamid R.

2017-01-01

Although diabetes mellitus (DM) causes cardiomyopathy and exacerbates heart failure, the underlying molecular mechanisms for diabetic cardiomyopathy/heart failure are poorly understood. Insulin2 mutant (Ins2+/-) Akita is a mouse model of T1DM, which manifests cardiac dysfunction. However, molecular changes at cardiac transcriptome level that lead to cardiomyopathy remain unclear. To understand the molecular changes in the heart of diabetic Akita mice, we profiled cardiac transcriptome of Ins2+/- Akita and Ins2+/+ control mice using next generation sequencing (NGS) and microarray, and determined the implications of differentially expressed genes on various heart failure signaling pathways using Ingenuity pathway (IPA) analysis. First, we validated hyperglycemia, increased cardiac fibrosis, and cardiac dysfunction in twelve-week male diabetic Akita. Then, we analyzed the transcriptome levels in the heart. NGS analyses on Akita heart revealed 137 differentially expressed transcripts, where Bone Morphogenic Protein-10 (BMP10) was the most upregulated and hairy and enhancer of split-related (HELT) was the most downregulated gene. Moreover, twelve long non-coding RNAs (lncRNAs) were upregulated. The microarray analyses on Akita heart showed 351 differentially expressed transcripts, where vomeronasal-1 receptor-180 (Vmn1r180) was the most upregulated and WD Repeat Domain 83 Opposite Strand (WDR83OS) was the most downregulated gene. Further, miR-101c and H19 lncRNA were upregulated but Neat1 lncRNA was downregulated in Akita heart. Eleven common genes were upregulated in Akita heart in both NGS and microarray analyses. IPA analyses revealed the role of these differentially expressed genes in key signaling pathways involved in diabetic cardiomyopathy. Our results provide a platform to initiate focused future studies by targeting these genes and/or non-coding RNAs, which are differentially expressed in Akita hearts and are involved in diabetic cardiomyopathy. PMID:28837672

Microarray analysis of the rat lacrimal gland following the loss of parasympathetic control of secretion

PubMed Central

Nguyen, Doan H.; Toshida, Hiroshi; Schurr, Jill; Beuerman, Roger W.

2010-01-01

Previous studies showed that loss of muscarinic parasympathetic input to the lacrimal gland (LG) leads to a dramatic reduction in tear secretion and profound changes to LG structure. In this study, we used DNA microarrays to examine the regulation of the gene expression of the genes for secretory function and organization of the LG. Long-Evans rats anesthetized with a mixture of ketamine/xylazine (80:10 mg/kg) underwent unilateral sectioning of the greater superficial petrosal nerve, the input to the pterygopalatine ganglion. After 7 days, tear secretion was measured, the animals were killed, and structural changes in the LG were examined by light microscopy. Total RNA from control and experimental LGs (n = 5) was used for DNA microarray analysis employing the U34A GeneChip. Three statistical algorithms (detection, change call, and signal log ratio) were used to determine differential gene expression using the Microarray Suite (5.0) and Data Mining Tools (3.0). Tear secretion was significantly reduced and corneal ulcers developed in all experimental eyes. Light microscopy showed breakdown of the acinar structure of the LG. DNA microarray analysis showed downregulation of genes associated with the endoplasmic reticulum and Golgi, including genes involved in protein folding and processing. Conversely, transcripts for cytoskeleton and extracellular matrix components, inflammation, and apoptosis were upregulated. The number of significantly upregulated genes (116) was substantially greater than the number of downregulated genes (49). Removal of the main secretory input to the rat LG resulted in clinical symptoms associated with severe dry eye. Components of the secretory pathway were negatively affected, and the increase in cell proliferation and inflammation may lead to loss of organization in the parasympathectomized lacrimal gland. PMID:15084711

Oxidative stress status accompanying diabetic bladder cystopathy results in the activation of protein degradation pathways

PubMed Central

Kanika, Nirmala; Chang, Jinsook; Tong, Yuehong; Tiplitsky, Scott; Lin, Juan; Yohannes, Elizabeth; Tar, Moses; Chance, Mark; Christ, George J.; Melman, Arnold; Davies, Kelvin

2010-01-01

Objectives To investigate the role that oxidative stress plays in the development of diabetic cystopathy. Materials and methods Comparative gene expression in the bladder of non-diabetic and streptozotocin (STZ)-induced 2-month-old diabetic rats was carried out using microarray analysis. Evidence of oxidative stress was investigated in the bladder by analyzing glutathione S-transferase activity, lipid peroxidation, and carbonylation and nitrosylation of proteins. The activity of protein degradation pathways was assessed using western blot analysis. Results Analysis of global gene expression showed that detrusor smooth muscle tissue of STZ-induced diabetes undergoes significant enrichment in targets involved in the production or regulation of reactive oxygen species (P = 1.27 × 10−10). The microarray analysis was confirmed by showing that markers of oxidative stress were all significantly increased in the diabetic bladder. It was hypothesized that the sequelae to oxidative stress would be increased protein damage and apoptosis. This was confirmed by showing that two key proteins involved in protein degradation (Nedd4 and LC3B) were greatly up-regulated in diabetic bladders compared to controls by 12.2 ± 0.76 and 4.4 ± 1.0-fold, respectively, and the apoptosis inducing protein, BAX, was up-regulated by 6.76 ± 0.76-fold. Conclusions Overall, the findings obtained in the present study add to the growing body of evidence showing that diabetic cystopathy is associated with oxidative damage of smooth muscle cells, and results in protein damage and activation of apoptotic pathways that may contribute to a deterioration in bladder function. PMID:21518418

Identification of miR-194-5p as a potential biomarker for postmenopausal osteoporosis

PubMed Central

Pan, Nanan; Sun, Ning; Wang, Qiujun; Fan, Jingxue; Zhou, Ping

2015-01-01

The incidence of osteoporosis is high in postmenopausal women due to altered estrogen levels and continuous calcium loss that occurs with aging. Recent studies have shown that microRNAs (miRNAs) are involved in the development of osteoporosis. These miRNAs may be used as potential biomarkers to identify women at a high risk for developing the disease. In this study, whole blood samples were collected from 48 postmenopausal Chinese women with osteopenia or osteoporosis and pooled into six groups according to individual T-scores. A miRNA microarray analysis was performed on pooled blood samples to identify potential miRNA biomarkers for postmenopausal osteoporosis. Five miRNAs (miR-130b-3p, -151a-3p, -151b, -194-5p, and -590-5p) were identified in the microarray analysis. These dysregulated miRNAs were subjected to a pathway analysis investigating whether they were involved in regulating osteoporosis-related pathways. Among them, only miR-194-5p was enriched in multiple osteoporosis-related pathways. Enhanced miR-194-5p expression in women with osteoporosis was confirmed by quantitative reverse transcription–polymerase chain reaction analysis. For external validation, a significant correlation between the expression of miR-194-5p and T-scores was found in an independent patient collection comprised of 24 postmenopausal women with normal bone mineral density, 30 postmenopausal women with osteopenia, and 32 postmenopausal women with osteoporosis (p < 0.05). Taken together, the present findings suggest that miR-194-5p may be a viable miRNA biomarker for postmenopausal osteoporosis. PMID:26038726

Identification of miR-194-5p as a potential biomarker for postmenopausal osteoporosis.

PubMed

Meng, Jia; Zhang, Dapeng; Pan, Nanan; Sun, Ning; Wang, Qiujun; Fan, Jingxue; Zhou, Ping; Zhu, Wenliang; Jiang, Lihong

2015-01-01

The incidence of osteoporosis is high in postmenopausal women due to altered estrogen levels and continuous calcium loss that occurs with aging. Recent studies have shown that microRNAs (miRNAs) are involved in the development of osteoporosis. These miRNAs may be used as potential biomarkers to identify women at a high risk for developing the disease. In this study, whole blood samples were collected from 48 postmenopausal Chinese women with osteopenia or osteoporosis and pooled into six groups according to individual T-scores. A miRNA microarray analysis was performed on pooled blood samples to identify potential miRNA biomarkers for postmenopausal osteoporosis. Five miRNAs (miR-130b-3p, -151a-3p, -151b, -194-5p, and -590-5p) were identified in the microarray analysis. These dysregulated miRNAs were subjected to a pathway analysis investigating whether they were involved in regulating osteoporosis-related pathways. Among them, only miR-194-5p was enriched in multiple osteoporosis-related pathways. Enhanced miR-194-5p expression in women with osteoporosis was confirmed by quantitative reverse transcription-polymerase chain reaction analysis. For external validation, a significant correlation between the expression of miR-194-5p and T-scores was found in an independent patient collection comprised of 24 postmenopausal women with normal bone mineral density, 30 postmenopausal women with osteopenia, and 32 postmenopausal women with osteoporosis (p < 0.05). Taken together, the present findings suggest that miR-194-5p may be a viable miRNA biomarker for postmenopausal osteoporosis.

Gene Expression and Pathway Analysis of Effects of the CMAH Deactivation on Mouse Lung, Kidney and Heart

PubMed Central

Kwon, Deug-Nam; Chang, Byung-Soo; Kim, Jin-Hoi

2014-01-01

Background N-glycolylneuraminic acid (Neu5Gc) is generated by hydroxylation of CMP-Neu5Ac to CMP-Neu5Gc, catalyzed by CMP-Neu5Ac hydroxylase (CMAH). However, humans lack this common mammalian cell surface molecule, Neu5Gc, due to inactivation of the CMAH gene during evolution. CMAH is one of several human-specific genes whose function has been lost by disruption or deletion of the coding frame. It has been suggested that CMAH inactivation has resulted in biochemical or physiological characteristics that have resulted in human-specific diseases. Methodology/Principal Findings To identify differential gene expression profiles associated with the loss of Neu5Gc expression, we performed microarray analysis using Illumina MouseRef-8 v2 Expression BeadChip, using the main tissues (lung, kidney, and heart) from control mice and CMP-Neu5Ac hydroxylase (Cmah) gene knock-out mice, respectively. Out of a total of 25,697 genes, 204, 162, and 147 genes were found to be significantly modulated in the lung, kidney, and heart tissues of the Cmah null mouse, respectively. In this study, we examined the gene expression profiles, using three commercial pathway analysis software packages: Ingenuity Pathways Analysis, Kyoto Encyclopedia of Genes and Genomes analysis, and Pathway Studio. The gene ontology analysis revealed that the top 6 biological processes of these genes included protein metabolism and modification, signal transduction, lipid, fatty acid, and steroid metabolism, nucleoside, nucleotide and nucleic acid metabolism, immunity and defense, and carbohydrate metabolism. Gene interaction network analysis showed a common network that was common to the different tissues of the Cmah null mouse. However, the expression of most sialytransferase mRNAs of Hanganutziu-Deicher antigen, sialy-Tn antigen, Forssman antigen, and Tn antigen was significantly down-regulated in the liver tissue of Cmah null mice. Conclusions/Significance Mice bearing a human-like deletion of the Cmah gene serve as an important model for the study of abnormal pathogenesis and/or metabolism caused by the evolutionary loss of Neu5Gc synthesis in humans. PMID:25229777

High resolution Chromatin Immunoprecipitation (ChIP) sequencing reveals novel bindings targets and prognostic role for SOX11 in Mantle cell lymphoma

PubMed Central

Kuo, Pei-Yu; Leshchenko, Violetta V.; Fazzari, Melissa J.; Perumal, Deepak; Gellen, Tobias; He, Tianfang; Iqbal, Javeed; Baumgartner-Wennerholm, Stefanie; Nygren, Lina; Zhang, Fan; Zhang, Weijia; Suh, K. Stephen; Goy, Andre; Yang, David T.; Chan, Wing-Chung; Kahl, Brad S.; Verma, Amit K.; Gascoyne, Randy D.; Kimby, Eva; Sander, Birgitta; Ye, B. Hilda; Melnick, Ari M.; Parekh, Samir

2015-01-01

SOX11 (Sex determining region Y-box 11) expression is specific for MCL as compared to other Non-Hodgkin's lymphomas. However, the function and direct binding targets of SOX11 in MCL are largely unknown. We used high-resolution ChIP-Seq to identify the direct target genes of SOX11 in a genome-wide, unbiased manner and elucidate its functional significance. Pathway analysis identified WNT, PKA and TGF-beta signaling pathways as significantly enriched by SOX11 target genes. qCHIP and promoter reporter assays confirmed that SOX11 directly binds to individual genes and modulates their transcription activities in these pathways in MCL. Functional studies using RNA interference demonstrate that SOX11 directly regulates WNT in MCL. We analyzed SOX11 expression in three independent well-annotated tissue microarrays from the University of Wisconsin (UW), Karolinska Institute and British Columbia Cancer Agency (BCCA). Our findings suggest that high SOX11 expression is associated with improved survival in a subset of MCL patients, particularly those treated with intensive chemotherapy. Transcriptional regulation of WNT and other biological pathways affected by SOX11 target genes may help explain the impact of SOX11 expression on patient outcomes. PMID:24681958

High-Dimensional Sparse Factor Modeling: Applications in Gene Expression Genomics

PubMed Central

Carvalho, Carlos M.; Chang, Jeffrey; Lucas, Joseph E.; Nevins, Joseph R.; Wang, Quanli; West, Mike

2010-01-01

We describe studies in molecular profiling and biological pathway analysis that use sparse latent factor and regression models for microarray gene expression data. We discuss breast cancer applications and key aspects of the modeling and computational methodology. Our case studies aim to investigate and characterize heterogeneity of structure related to specific oncogenic pathways, as well as links between aggregate patterns in gene expression profiles and clinical biomarkers. Based on the metaphor of statistically derived “factors” as representing biological “subpathway” structure, we explore the decomposition of fitted sparse factor models into pathway subcomponents and investigate how these components overlay multiple aspects of known biological activity. Our methodology is based on sparsity modeling of multivariate regression, ANOVA, and latent factor models, as well as a class of models that combines all components. Hierarchical sparsity priors address questions of dimension reduction and multiple comparisons, as well as scalability of the methodology. The models include practically relevant non-Gaussian/nonparametric components for latent structure, underlying often quite complex non-Gaussianity in multivariate expression patterns. Model search and fitting are addressed through stochastic simulation and evolutionary stochastic search methods that are exemplified in the oncogenic pathway studies. Supplementary supporting material provides more details of the applications, as well as examples of the use of freely available software tools for implementing the methodology. PMID:21218139

Data on quantification of signaling pathways activated by KIT and PDGFRA mutants.

PubMed

Bahlawane, Christelle; Schmitz, Martine; Letellier, Elisabeth; Arumugam, Karthik; Nicot, Nathalie; Nazarov, Petr V; Haan, Serge

2016-12-01

The present data are related to the article entitled "Insights into ligand stimulation effects on gastro-intestinal stromal tumors signaling" (C. Bahlawane, M. Schmitz, E. Letellier, K. Arumugam, N. Nicot, P.V. Nazarov, S. Haan, 2016) [1]. Constitutive and ligand-derived signaling pathways mediated by KIT and PDGFRA mutated proteins found in gastrointestinal stromal tumors (GIST) were compared. Expression of mutant proteins was induced by doxycycline in an isogenic background (Hek293 cells). Kit was identified by FACS at the cell surface and found to be quickly degraded or internalized upon SCF stimulation for both Kit Wild type and Kit mutant counterparts. Investigation of the main activated pathways in GIST unraveled a new feature specific for oncogenic KIT mutants, namely their ability to be further activated by Kit ligand, the stem cell factor (scf). We were also able to identify the MAPK pathway as the most prominent target for a common inhibition of PDGFRA and KIT oncogenic signaling. Western blotting and micro-array analysis were applied to analyze the capacities of the mutant to induce an effective STATs response. Among all Kit mutants, only Kit Ex11 deletion mutant was able to elicit an effective STATs response whereas all PDGFRA were able to do so.

Pathway results from the chicken data set using GOTM, Pathway Studio and Ingenuity softwares

PubMed Central

Bonnet, Agnès; Lagarrigue, Sandrine; Liaubet, Laurence; Robert-Granié, Christèle; SanCristobal, Magali; Tosser-Klopp, Gwenola

2009-01-01

Background As presented in the introduction paper, three sets of differentially regulated genes were found after the analysis of the chicken infection data set from EADGENE. Different methods were used to interpret these results. Results GOTM, Pathway Studio and Ingenuity softwares were used to investigate the three lists of genes. The three softwares allowed the analysis of the data and highlighted different networks. However, only one set of genes, showing a differential expression between primary and secondary response gave significant biological interpretation. Conclusion Combining these databases that were developed independently on different annotation sources supplies a useful tool for a global biological interpretation of microarray data, even if they may contain some imperfections (e.g. gene not or not well annotated). PMID:19615111

Ossification of the posterior longitudinal ligament related genes identification using microarray gene expression profiling and bioinformatics analysis.

PubMed

He, Hailong; Mao, Lingzhou; Xu, Peng; Xi, Yanhai; Xu, Ning; Xue, Mingtao; Yu, Jiangming; Ye, Xiaojian

2014-01-10

Ossification of the posterior longitudinal ligament (OPLL) is a kind of disease with physical barriers and neurological disorders. The objective of this study was to explore the differentially expressed genes (DEGs) in OPLL patient ligament cells and identify the target sites for the prevention and treatment of OPLL in clinic. Gene expression data GSE5464 was downloaded from Gene Expression Omnibus; then DEGs were screened by limma package in R language, and changed functions and pathways of OPLL cells compared to normal cells were identified by DAVID (The Database for Annotation, Visualization and Integrated Discovery); finally, an interaction network of DEGs was constructed by string. A total of 1536 DEGs were screened, with 31 down-regulated and 1505 up-regulated genes. Response to wounding function and Toll-like receptor signaling pathway may involve in the development of OPLL. Genes, such as PDGFB, PRDX2 may involve in OPLL through response to wounding function. Toll-like receptor signaling pathway enriched genes such as TLR1, TLR5, and TLR7 may involve in spine cord injury in OPLL. PIK3R1 was the hub gene in the network of DEGs with the highest degree; INSR was one of the most closely related genes of it. OPLL related genes screened by microarray gene expression profiling and bioinformatics analysis may be helpful for elucidating the mechanism of OPLL. © 2013.

Ganoderma lucidum polysaccharides in human monocytic leukemia cells: from gene expression to network construction

PubMed Central

Cheng, Kun-Chieh; Huang, Hsuan-Cheng; Chen, Jenn-Han; Hsu, Jia-Wei; Cheng, Hsu-Chieh; Ou, Chern-Han; Yang, Wen-Bin; Chen, Shui-Tein; Wong, Chi-Huey; Juan, Hsueh-Fen

2007-01-01

Background Ganoderma lucidum has been widely used as a herbal medicine for promoting health and longevity in China and other Asian countries. Polysaccharide extracts from Ganoderma lucidum have been reported to exhibit immuno-modulating and anti-tumor activities. In previous studies, F3, the active component of the polysaccharide extract, was found to activate various cytokines such as IL-1, IL-6, IL-12, and TNF-α. This gave rise to our investigation on how F3 stimulates immuno-modulating or anti-tumor effects in human leukemia THP-1 cells. Results Here, we integrated time-course DNA microarray analysis, quantitative PCR assays, and bioinformatics methods to study the F3-induced effects in THP-1 cells. Significantly disturbed pathways induced by F3 were identified with statistical analysis on microarray data. The apoptosis induction through the DR3 and DR4/5 death receptors was found to be one of the most significant pathways and play a key role in THP-1 cells after F3 treatment. Based on time-course gene expression measurements of the identified pathway, we reconstructed a plausible regulatory network of the involved genes using reverse-engineering computational approach. Conclusion Our results showed that F3 may induce death receptor ligands to initiate signaling via receptor oligomerization, recruitment of specialized adaptor proteins and activation of caspase cascades. PMID:17996095

Ganoderma lucidum polysaccharides in human monocytic leukemia cells: from gene expression to network construction.

PubMed

Cheng, Kun-Chieh; Huang, Hsuan-Cheng; Chen, Jenn-Han; Hsu, Jia-Wei; Cheng, Hsu-Chieh; Ou, Chern-Han; Yang, Wen-Bin; Chen, Shui-Tein; Wong, Chi-Huey; Juan, Hsueh-Fen

2007-11-09

Ganoderma lucidum has been widely used as a herbal medicine for promoting health and longevity in China and other Asian countries. Polysaccharide extracts from Ganoderma lucidum have been reported to exhibit immuno-modulating and anti-tumor activities. In previous studies, F3, the active component of the polysaccharide extract, was found to activate various cytokines such as IL-1, IL-6, IL-12, and TNF-alpha. This gave rise to our investigation on how F3 stimulates immuno-modulating or anti-tumor effects in human leukemia THP-1 cells. Here, we integrated time-course DNA microarray analysis, quantitative PCR assays, and bioinformatics methods to study the F3-induced effects in THP-1 cells. Significantly disturbed pathways induced by F3 were identified with statistical analysis on microarray data. The apoptosis induction through the DR3 and DR4/5 death receptors was found to be one of the most significant pathways and play a key role in THP-1 cells after F3 treatment. Based on time-course gene expression measurements of the identified pathway, we reconstructed a plausible regulatory network of the involved genes using reverse-engineering computational approach. Our results showed that F3 may induce death receptor ligands to initiate signaling via receptor oligomerization, recruitment of specialized adaptor proteins and activation of caspase cascades.

Comparative transcriptome analysis of Methylibium petroleiphilum PM1 exposed to the fuel oxygenates methyl tert-butyl ether and ethanol.

PubMed

Hristova, Krassimira R; Schmidt, Radomir; Chakicherla, Anu Y; Legler, Tina C; Wu, Janice; Chain, Patrick S; Scow, Kate M; Kane, Staci R

2007-11-01

High-density whole-genome cDNA microarrays were used to investigate substrate-dependent gene expression of Methylibium petroleiphilum PM1, one of the best-characterized aerobic methyl tert-butyl ether (MTBE)-degrading bacteria. Differential gene expression profiling was conducted with PM1 grown on MTBE and ethanol as sole carbon sources. Based on microarray high scores and protein similarity analysis, an MTBE regulon located on the megaplasmid was identified for further investigation. Putative functions for enzymes encoded in this regulon are described with relevance to the predicted MTBE degradation pathway. A new unique dioxygenase enzyme system that carries out the hydroxylation of tert-butyl alcohol to 2-methyl-2-hydroxy-1-propanol in M. petroleiphilum PM1 was discovered. Hypotheses regarding the acquisition and evolution of MTBE genes as well as the involvement of IS elements in these complex processes were formulated. The pathways for toluene, phenol, and alkane oxidation via toluene monooxygenase, phenol hydroxylase, and propane monooxygenase, respectively, were upregulated in MTBE-grown cells compared to ethanol-grown cells. Four out of nine putative cyclohexanone monooxygenases were also upregulated in MTBE-grown cells. The expression data allowed prediction of several hitherto-unknown enzymes of the upper MTBE degradation pathway in M. petroleiphilum PM1 and aided our understanding of the regulation of metabolic processes that may occur in response to pollutant mixtures and perturbations in the environment.

Microarray-Based Gene Expression Analysis for Veterinary Pathologists: A Review.

PubMed

Raddatz, Barbara B; Spitzbarth, Ingo; Matheis, Katja A; Kalkuhl, Arno; Deschl, Ulrich; Baumgärtner, Wolfgang; Ulrich, Reiner

2017-09-01

High-throughput, genome-wide transcriptome analysis is now commonly used in all fields of life science research and is on the cusp of medical and veterinary diagnostic application. Transcriptomic methods such as microarrays and next-generation sequencing generate enormous amounts of data. The pathogenetic expertise acquired from understanding of general pathology provides veterinary pathologists with a profound background, which is essential in translating transcriptomic data into meaningful biological knowledge, thereby leading to a better understanding of underlying disease mechanisms. The scientific literature concerning high-throughput data-mining techniques usually addresses mathematicians or computer scientists as the target audience. In contrast, the present review provides the reader with a clear and systematic basis from a veterinary pathologist's perspective. Therefore, the aims are (1) to introduce the reader to the necessary methodological background; (2) to introduce the sequential steps commonly performed in a microarray analysis including quality control, annotation, normalization, selection of differentially expressed genes, clustering, gene ontology and pathway analysis, analysis of manually selected genes, and biomarker discovery; and (3) to provide references to publically available and user-friendly software suites. In summary, the data analysis methods presented within this review will enable veterinary pathologists to analyze high-throughput transcriptome data obtained from their own experiments, supplemental data that accompany scientific publications, or public repositories in order to obtain a more in-depth insight into underlying disease mechanisms.

AURKA induces EMT by regulating histone modification through Wnt/β-catenin and PI3K/Akt signaling pathway in gastric cancer

PubMed Central

Liu, Xi; Li, Zhaoxia; Song, Yue; Wang, Rui; Han, Lei; Wang, Qixue; Jiang, Kui; Kang, Chunsheng; Zhang, Qingyu

2016-01-01

Gastric cancer, a highly invasive and aggressive malignancy, is the third leading cause of death from cancer worldwide. Genetic association studies have successfully revealed several important genes consistently associated with gastric cancer to date. However, these robust gastric cancer-associated genes do not fully elucidate the mechanisms underlying the development and progression of the disease. In the present study, we performed an alternative approach, a gene expression-based genome-wide association study (eGWAS) across 13 independent microarray experiments (including 251 gastric cancer cases and 428 controls), to identify top candidates (p<0.00001). Additionally, we conducted gene ontology analysis, pathway analysis and network analysis and identified aurora kinase A (AURKA) as our candidate. We observed that MLN8237, which is a specific inhibitor of AURKA, decreased the β-catenin and the phosphorylation of Akt1 and GSK-3β, as well as blocked the Akt and Wnt signaling pathways. Furthermore, MLN8237 arrested the cells in the G2/M phase. The activity of Wnt and Akt signaling pathways affected the level of histone methylation significantly, and we supposed that MLN8237 affected the level of histone methylation through these two signaling pathways. Additionally, the treatment of MLN8237 influenced the level of H3K4 me1/2/3 and H3K27 me1/2/3. Chip data on cell lines suggested that MLN8237 increases the level of H3K27 me3 on the promoter of Twist and inhibits EMT (epithelial-mesenchymal transition). In summary, AURKA is a potential therapeutic target in gastric cancer and induces EMT through histone methylation. PMID:27121204

Microarray analysis of long non-coding RNA expression profiles in monocytic myeloid-derived suppressor cells in Echinococcus granulosus-infected mice.

PubMed

Yu, Aiping; Wang, Ying; Yin, Jianhai; Zhang, Jing; Cao, Shengkui; Cao, Jianping; Shen, Yujuan

2018-05-30

Cystic echinococcosis is a worldwide chronic zoonotic disease caused by infection with the larval stage of Echinococcus granulosus. Previously, we found significant accumulation of myeloid-derived suppressor cells (MDSCs) in E. granulosus infection mouse models and that they play a key role in immunosuppressing T lymphocytes. Here, we compared the long non-coding RNA (lncRNA) and mRNA expression patterns between the splenic monocytic MDSCs (M-MDSCs) of E. granulosus protoscoleces-infected mice and normal mice using microarray analysis. LncRNA functions were predicted using Gene Ontology enrichment and the Kyoto Encyclopedia of Genes and Genomes pathway analysis. Cis- and trans-regulation analyses revealed potential relationships between the lncRNAs and their target genes or related transcription factors. We found that 649 lncRNAs were differentially expressed (fold change ≥ 2, P < 0.05): 582 lncRNAs were upregulated and 67 lncRNAs were downregulated; respectively, 28 upregulated mRNAs and 1043 downregulated mRNAs were differentially expressed. The microarray data was validated by quantitative reverse transcription-PCR. The results indicated that mRNAs co-expressed with the lncRNAs are mainly involved in regulating the actin cytoskeleton, Salmonella infection, leishmaniasis, and the vascular endothelial growth factor (VEGF) signaling pathway. The lncRNA NONMMUT021591 was predicted to cis-regulate the retinoblastoma gene (Rb1), whose expression is associated with abnormal M-MDSCs differentiation. We found that 372 lncRNAs were predicted to interact with 60 transcription factors; among these, C/EBPβ (CCAAT/enhancer binding protein beta) was previously demonstrated to be a transcription factor of MDSCs. Our study identified dysregulated lncRNAs in the M-MDSCs of E. granulosus infection mouse models; they might be involved in M-MDSC-derived immunosuppression in related diseases.

Brain in situ hybridization maps as a source for reverse-engineering transcriptional regulatory networks: Alzheimer's disease insights

DOE Office of Scientific and Technical Information (OSTI.GOV)

Acquaah-Mensah, George K.; Taylor, Ronald C.

Microarray data have been a valuable resource for identifying transcriptional regulatory relationships among genes. As an example, brain region-specific transcriptional regulatory events have the potential of providing etiological insights into Alzheimer Disease (AD). However, there is often a paucity of suitable brain-region specific expression data obtained via microarrays or other high throughput means. The Allen Brain Atlas in situ hybridization (ISH) data sets (Jones et al., 2009) represent a potentially valuable alternative source of high-throughput brain region-specific gene expression data for such purposes. In this study, Allen BrainAtlasmouse ISH data in the hippocampal fields were extracted, focusing on 508 genesmore » relevant to neurodegeneration. Transcriptional regulatory networkswere learned using three high-performing network inference algorithms. Only 17% of regulatory edges from a network reverse-engineered based on brain region-specific ISH data were also found in a network constructed upon gene expression correlations inmousewhole brain microarrays, thus showing the specificity of gene expression within brain sub-regions. Furthermore, the ISH data-based networks were used to identify instructive transcriptional regulatory relationships. Ncor2, Sp3 and Usf2 form a unique three-party regulatory motif, potentially affecting memory formation pathways. Nfe2l1, Egr1 and Usf2 emerge among regulators of genes involved in AD (e.g. Dhcr24, Aplp2, Tia1, Pdrx1, Vdac1, andSyn2). Further, Nfe2l1, Egr1 and Usf2 are sensitive to dietary factors and could be among links between dietary influences and genes in the AD etiology. Thus, this approach of harnessing brain region-specific ISH data represents a rare opportunity for gleaning unique etiological insights for diseases such as AD.« less

Identification of novel target genes involved in Indian Fanconi anemia patients using microarray.

PubMed

Shyamsunder, Pavithra; Ganesh, Kripa S; Vidyasekar, Prasanna; Mohan, Sheila; Verma, Rama Shanker

2013-12-01

Fanconi anemia (FA) is a genetic disorder characterized by progressive bone marrow failure and a predisposition to cancers. Mutations have been documented in 15 FA genes that participate in the FA-BRCA DNA repair pathway, a fundamental pathway in the development of the disease and the presentation of its characteristic symptoms. Certain symptoms such as oxygen sensitivity, hematological abnormalities and impaired immunity suggest that FA proteins could participate in or independently control other pathways as well. In this study, we identified 9 DNA repair genes that were down regulated in a genome wide analysis of 6 Indian Fanconi anemia patients. Functional clustering of a total of 233 dysregulated genes identified key biological processes that included regulation of transcription, DNA repair, cell cycle and chromosomal organization. Microarray data revealed the down regulation of ATXN3, ARID4A and ETS-1, which were validated by RTPCR in a subsequent sample set of 9 Indian FA patients. Here we report for the first time a gene expression profile of Fanconi anemia patients from the Indian population and a pool of genes that might aid in the acquisition and progression of the FA phenotype. © 2013 Elsevier B.V. All rights reserved.

Screening of potential genes contributing to the macrocycle drug resistance of C. albicans via microarray analysis

PubMed Central

Yang, Jing; Zhang, Wei; Sun, Jian; Xi, Zhiqin; Qiao, Zusha; Zhang, Jinyu; Wang, Yan; Ji, Ying; Feng, Wenli

2017-01-01

The aim of the present study was to investigate the potential genes involved in drug resistance of Candida albicans (C. albicans) by performing microarray analysis. The gene expression profile of GSE65396 was downloaded from the Gene Expression Omnibus, including a control, 15-min and 45-min macrocyclic compound RF59-treated group with three repeats for each. Following preprocessing using RAM, the differentially expressed genes (DEGs) were screened using the Limma package. Subsequently, the Kyoto Encyclopedia of Genes and Genomes pathways of these genes were analyzed using the Database for Annotation, Visualization and Integrated Discovery. Based on interactions estimated by the Search Tool for Retrieval of Interacting Gene, the protein-protein interaction (PPI) network was visualized using Cytoscape. Subnetwork analysis was performed using ReactomeFI. A total of 154 upregulated and 27 downregulated DEGs were identified in the 15-min treated group, compared with the control, and 235 upregulated and 233 downregulated DEGs were identified in the 45-min treated group, compared with the control. The upregulated DEGs were significantly enriched in the ribosome pathway. Based on the PPI network, PRP5, RCL1, NOP13, NOP4 and MRT4 were the top five nodes in the 15-min treated comparison. GIS2, URA3, NOP58, ELP3 and PLP7 were the top five nodes in the 45-min treated comparison, and its subnetwork was significantly enriched in the ribosome pathway. The macrocyclic compound RF59 had a notable effect on the ribosome and its associated pathways of C. albicans. RCL1, NOP4, MRT4, GIS2 and NOP58 may be important in RF59-resistance. PMID:28944888

Subcutaneous and gonadal adipose tissue transcriptome differences in lean and obese female dogs.

PubMed

Grant, Ryan W; Vester Boler, Brittany M; Ridge, Tonya K; Graves, Thomas K; Swanson, Kelly S

2013-12-01

Canine obesity leads to shortened life span and increased disease incidence. Adipose tissue depots are known to have unique metabolic and gene expression profiles in rodents and humans, but few comparisons of depot gene expression have been performed in the dog. Using microarray technology, our objective was to identify differentially expressed genes and enriched functional pathways between subcutaneous and gonadal adipose of lean and obese dogs to better understand the pathogenesis of obesity in the dog. Because no depot × body weight status interactions were identified in the microarray data, depot differences were the primary focus. A total of 946 and 703 transcripts were differentially expressed (FDR P < 0.05) between gonadal and subcutaneous adipose tissue in obese and lean dogs respectively. Of the adipose depot-specific differences in gene expression, 162 were present in both lean and obese dogs, with the majority (85%) expressed in the same direction. Both lean and obese dog gene lists had enrichment of the complement and coagulation cascade and systemic lupus erythematosus pathways. Obese dogs had enrichment of lysosome, extracellular matrix-receptor interaction, renin-angiotensin system and hematopoietic cell lineage pathways. Lean dogs had enrichment of glutathione metabolism and synthesis and degradation of ketone bodies. We have identified a core set of genes differentially expressed between subcutaneous and gonadal adipose tissue in dogs regardless of body weight. These genes contribute to depot-specific differences in immune function, extracellular matrix remodeling and lysosomal function and may contribute to the physiological differences noted between depots. © 2013 The Authors, Animal Genetics © 2013 Stichting International Foundation for Animal Genetics.

Microarray expression profiling and co-expression network analysis of circulating LncRNAs and mRNAs associated with neurotoxicity induced by BPA.

PubMed

Pang, Wei; Lian, Fu-Zhi; Leng, Xue; Wang, Shu-Min; Li, Yi-Bo; Wang, Zi-Yu; Li, Kai-Ren; Gao, Zhi-Xian; Jiang, Yu-Gang

2018-05-01

A growing body of evidence has shown bisphenol A (BPA), an estrogen-like industrial chemical, has adverse effects on the nervous system. In this study, we investigated the transcriptional behavior of long non-coding RNAs (lncRNAs) and mRNAs to provide the information to explore neurotoxic effects induced by BPA. By microarray expression profiling, we discovered 151 differentially expressed lncRNAs and 794 differentially expressed mRNAs in the BPA intervention group compared with the control group. Gene ontology analysis indicated the differentially expressed mRNAs were mainly involved in fundamental metabolic processes and physiological and pathological conditions, such as development, synaptic transmission, homeostasis, injury, and neuroinflammation responses. In the expression network of the BPA-induced group, a great number of nodes and connections were found in comparison to the control-derived network. We identified lncRNAs that were aberrantly expressed in the BPA group, among which, growth arrest specific 5 (GAS5) might participate in the BPA-induced neurotoxicity by regulating Jun, RAS, and other pathways indirectly through these differentially expressed genes. This study provides the first investigation of genome-wide lncRNA expression and correlation between lncRNA and mRNA expression in the BPA-induced neurotoxicity. Our results suggest that the elevated expression of lncRNAs is a major biomarker in the neurotoxicity induced by BPA.

Gene expression patterns during the larval development of European sea bass (dicentrarchus labrax) by microarray analysis.

PubMed

Darias, M J; Zambonino-Infante, J L; Hugot, K; Cahu, C L; Mazurais, D

2008-01-01

During the larval period, marine teleosts undergo very fast growth and dramatic changes in morphology, metabolism, and behavior to accomplish their metamorphosis into juvenile fish. Regulation of gene expression is widely thought to be a key mechanism underlying the management of the biological processes required for harmonious development over this phase of life. To provide an overall analysis of gene expression in the whole body during sea bass larval development, we monitored the expression of 6,626 distinct genes at 10 different points in time between 7 and 43 days post-hatching (dph) by using heterologous hybridization of a rainbow trout cDNA microarray. The differentially expressed genes (n = 485) could be grouped into two categories: genes that were generally up-expressed early, between 7 and 23 dph, and genes up-expressed between 25 and 43 dph. Interestingly, among the genes regulated during the larval period, those related to organogenesis, energy pathways, biosynthesis, and digestion were over-represented compared with total set of analyzed genes. We discuss the quantitative regulation of whole-body contents of these specific transcripts with regard to the ontogenesis and maturation of essential functions that take place over larval development. Our study is the first utilization of a transcriptomic approach in sea bass and reveals dynamic changes in gene expression patterns in relation to marine finfish larval development.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andersen, G.L.; He, Z.; DeSantis, T.Z.

Microarrays have proven to be a useful and high-throughput method to provide targeted DNA sequence information for up to many thousands of specific genetic regions in a single test. A microarray consists of multiple DNA oligonucleotide probes that, under high stringency conditions, hybridize only to specific complementary nucleic acid sequences (targets). A fluorescent signal indicates the presence and, in many cases, the abundance of genetic regions of interest. In this chapter we will look at how microarrays are used in microbial ecology, especially with the recent increase in microbial community DNA sequence data. Of particular interest to microbial ecologists, phylogeneticmore » microarrays are used for the analysis of phylotypes in a community and functional gene arrays are used for the analysis of functional genes, and, by inference, phylotypes in environmental samples. A phylogenetic microarray that has been developed by the Andersen laboratory, the PhyloChip, will be discussed as an example of a microarray that targets the known diversity within the 16S rRNA gene to determine microbial community composition. Using multiple, confirmatory probes to increase the confidence of detection and a mismatch probe for every perfect match probe to minimize the effect of cross-hybridization by non-target regions, the PhyloChip is able to simultaneously identify any of thousands of taxa present in an environmental sample. The PhyloChip is shown to reveal greater diversity within a community than rRNA gene sequencing due to the placement of the entire gene product on the microarray compared with the analysis of up to thousands of individual molecules by traditional sequencing methods. A functional gene array that has been developed by the Zhou laboratory, the GeoChip, will be discussed as an example of a microarray that dynamically identifies functional activities of multiple members within a community. The recent version of GeoChip contains more than 24,000 50mer oligonucleotide probes and covers more than 10,000 gene sequences in 150 gene categories involved in carbon, nitrogen, sulfur, and phosphorus cycling, metal resistance and reduction, and organic contaminant degradation. GeoChip can be used as a generic tool for microbial community analysis, and also link microbial community structure to ecosystem functioning. Examples of the application of both arrays in different environmental samples will be described in the two subsequent sections.« less

VitaPad: visualization tools for the analysis of pathway data.

PubMed

Holford, Matthew; Li, Naixin; Nadkarni, Prakash; Zhao, Hongyu

2005-04-15

Packages that support the creation of pathway diagrams are limited by their inability to be readily extended to new classes of pathway-related data. VitaPad is a cross-platform application that enables users to create and modify biological pathway diagrams and incorporate microarray data with them. It improves on existing software in the following areas: (i) It can create diagrams dynamically through graph layout algorithms. (ii) It is open-source and uses an open XML format to store data, allowing for easy extension or integration with other tools. (iii) It features a cutting-edge user interface with intuitive controls, high-resolution graphics and fully customizable appearance. http://bioinformatics.med.yale.edu matthew.holford@yale.edu; hongyu.zhao@yale.edu.

Transcriptomic characterization of temperature stress responses in larval zebrafish.

PubMed

Long, Yong; Li, Linchun; Li, Qing; He, Xiaozhen; Cui, Zongbin

2012-01-01

Temperature influences nearly all biochemical, physiological and life history activities of fish, but the molecular mechanisms underlying the temperature acclimation remains largely unknown. Previous studies have identified many temperature-regulated genes in adult tissues; however, the transcriptional responses of fish larvae to temperature stress are not well understood. In this study, we characterized the transcriptional responses in larval zebrafish exposed to cold or heat stress using microarray analysis. In comparison with genes expressed in the control at 28 °C, a total of 2680 genes were found to be affected in 96 hpf larvae exposed to cold (16 °C) or heat (34 °C) for 2 and 48h and most of these genes were expressed in a temperature-specific and temporally regulated manner. Bioinformatic analysis identified multiple temperature-regulated biological processes and pathways. Biological processes overrepresented among the earliest genes induced by temperature stress include regulation of transcription, nucleosome assembly, chromatin organization and protein folding. However, processes such as RNA processing, cellular metal ion homeostasis and protein transport and were enriched in genes up-regulated under cold exposure for 48 h. Pathways such as mTOR signalling, p53 signalling and circadian rhythm were enriched among cold-induced genes, while adipocytokine signalling, protein export and arginine and praline metabolism were enriched among heat-induced genes. Although most of these biological processes and pathways were specifically regulated by cold or heat, common responses to both cold and heat stresses were also found. Thus, these findings provide new interesting clues for elucidation of mechanisms underlying the temperature acclimation in fish.

Transcriptome of Cultured Lung Fibroblasts in Idiopathic Pulmonary Fibrosis: Meta-Analysis of Publically Available Microarray Datasets Reveals Repression of Inflammation and Immunity Pathways.

PubMed

Plantier, Laurent; Renaud, Hélène; Respaud, Renaud; Marchand-Adam, Sylvain; Crestani, Bruno

2016-12-13

Heritable profibrotic differentiation of lung fibroblasts is a key mechanism of idiopathic pulmonary fibrosis (IPF). Its mechanisms are yet to be fully understood. In this study, individual data from four independent microarray studies comparing the transcriptome of fibroblasts cultured in vitro from normal (total n = 20) and IPF (total n = 20) human lung were compiled for meta-analysis following normalization to z-scores. One hundred and thirteen transcripts were upregulated and 115 were downregulated in IPF fibroblasts using the Significance Analysis of Microrrays algorithm with a false discovery rate of 5%. Downregulated genes were highly enriched for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional classes related to inflammation and immunity such as Defense response to virus, Influenza A, tumor necrosis factor (TNF) mediated signaling pathway, interferon-inducible absent in melanoma2 (AIM2) inflammasome as well as Apoptosis. Although upregulated genes were not enriched for any functional class, select factors known to play key roles in lung fibrogenesis were overexpressed in IPF fibroblasts, most notably connective tissue growth factor ( CTGF ) and serum response factor ( SRF ), supporting their role as drivers of IPF. The full data table is available as a supplement.

Transcriptomic Assessment of Isozymes in the Biphenyl Pathway of Rhodococcus sp. Strain RHA1†

PubMed Central

Gonçalves, Edmilson R.; Hara, Hirofumi; Miyazawa, Daisuke; Davies, Julian E.; Eltis, Lindsay D.; Mohn, William W.

2006-01-01

Rhodococcus sp. RHA1 grows on a broad range of aromatic compounds and vigorously degrades polychlorinated biphenyls (PCBs). Previous work identified RHA1 genes encoding multiple isozymes for most of the seven steps of the biphenyl (BPH) pathway, provided evidence for coexpression of some of these isozymes, and indicated the involvement of some of these enzymes in the degradation of BPH, ethylbenzene (ETB), and PCBs. To investigate the expression of these isozymes and better understand how they contribute to the robust degradative capacity of RHA1, we comprehensively analyzed the 9.7-Mb genome of RHA1 for BPH pathway genes and characterized the transcriptome of RHA1 growing on benzoate (BEN), BPH, and ETB. Sequence analyses revealed 54 potential BPH pathway genes, including 28 not previously reported. Transcriptomic analysis with a DNA microarray containing 70-mer probes for 8,213 RHA1 genes revealed a suite of 320 genes of diverse functions that were upregulated during growth both on BPH and on ETB, relative to growth on the control substrate, pyruvate. By contrast, only 65 genes were upregulated during growth on BEN. Quantitative PCR assays confirmed microarray results for selected genes and indicated that some of the catabolic genes were upregulated over 10,000-fold. Our analysis suggests that up to 22 enzymes, including 8 newly identified ones, may function in the BPH pathway of RHA1. The relative expression levels of catabolic genes did not differ for BPH and ETB, suggesting a common regulatory mechanism. This study delineated a suite of catabolic enzymes for biphenyl and alkyl-benzenes in RHA1, which is larger than previously recognized and which may serve as a model for catabolism in other environmentally important bacteria having large genomes. PMID:16957245

Pulmonary instillation of low doses of titanium dioxide nanoparticles in mice leads to particle retention and gene expression changes in the absence of inflammation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Husain, Mainul, E-mail: mainul.husain@hc-sc.gc.ca; Saber, Anne T., E-mail: ats@nrcwe.dk; Guo, Charles, E-mail: charles.guo@hc-sc.gc.ca

2013-06-15

We investigated gene expression, protein synthesis, and particle retention in mouse lungs following intratracheal instillation of varying doses of nano-sized titanium dioxide (nano-TiO{sub 2}). Female C57BL/6 mice were exposed to rutile nano-TiO{sub 2} via single intratracheal instillations of 18, 54, and 162 μg/mouse. Mice were sampled 1, 3, and 28 days post-exposure. The deposition of nano-TiO{sub 2} in the lungs was assessed using nanoscale hyperspectral microscopy. Biological responses in the pulmonary system were analyzed using DNA microarrays, pathway-specific real-time RT-PCR (qPCR), gene-specific qPCR arrays, and tissue protein ELISA. Hyperspectral mapping showed dose-dependent retention of nano-TiO{sub 2} in the lungs upmore » to 28 days post-instillation. DNA microarray analysis revealed approximately 3000 genes that were altered across all treatment groups (± 1.3 fold; p < 0.1). Several inflammatory mediators changed in a dose- and time-dependent manner at both the mRNA and protein level. Although no influx of neutrophils was detected at the low dose, changes in the expression of several genes and proteins associated with inflammation were observed. Resolving inflammation at the medium dose, and lack of neutrophil influx in the lung fluid at the low dose, were associated with down-regulation of genes involved in ion homeostasis and muscle regulation. Our gene expression results imply that retention of nano-TiO{sub 2} in the absence of inflammation over time may potentially perturb calcium and ion homeostasis, and affect smooth muscle activities. - Highlights: • Pulmonary effects following exposure to low doses of nano-TiO{sub 2} were examined. • Particle retention in lungs was assessed using nanoscale hyperspectral microscopy. • Particles persisted up to 28 days in lungs in all dose groups. • Inflammation was the pathway affected in the high dose group at all time points. • Ion homeostasis and muscle activity pathways were affected in the low dose group.« less

Microarray-based determination of anti-inflammatory genes targeted by 6-(methylsulfinyl)hexyl isothiocyanate in macrophages.

PubMed

Chen, Jihua; Uto, Takuhiro; Tanigawa, Shunsuke; Yamada-Kato, Tomeo; Fujii, Makoto; Hou, DE-Xing

2010-01-01

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a bioactive ingredient of wasabi [Wasabia japonica (Miq.) Matsumura], which is a popular pungent spice of Japan. To evaluate the anti-inflammatory function and underlying genes targeted by 6-MSITC, gene expression profiling through DNA microarray was performed in mouse macrophages. Among 22,050 oligonucleotides, the expression levels of 406 genes were increased by ≥3-fold in lipopolysaccharide (LPS)-activated RAW264 cells, 238 gene signals of which were attenuated by 6-MSITC (≥2-fold). Expression levels of 717 genes were decreased by ≥3-fold in LPS-activated cells, of which 336 gene signals were restored by 6-MSITC (≥2-fold). Utilizing group analysis, 206 genes affected by 6-MSITC with a ≥2-fold change were classified into 35 categories relating to biological processes (81), molecular functions (108) and signaling pathways (17). The genes were further categorized as 'defense, inflammatory response, cytokine activities and receptor activities' and some were confirmed by real-time polymerase chain reaction. Ingenuity pathway analysis further revealed that wasabi 6-MSITC regulated the relevant networks of chemokines, interleukins and interferons to exert its anti-inflammatory function.

Microarray-based determination of anti-inflammatory genes targeted by 6-(methylsulfinyl)hexyl isothiocyanate in macrophages

PubMed Central

CHEN, JIHUA; UTO, TAKUHIRO; TANIGAWA, SHUNSUKE; YAMADA-KATO, TOMEO; FUJII, MAKOTO; HOU, DE-XING

2010-01-01

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a bioactive ingredient of wasabi [Wasabia japonica (Miq.) Matsumura], which is a popular pungent spice of Japan. To evaluate the anti-inflammatory function and underlying genes targeted by 6-MSITC, gene expression profiling through DNA microarray was performed in mouse macrophages. Among 22,050 oligonucleotides, the expression levels of 406 genes were increased by ≥3-fold in lipopolysaccharide (LPS)-activated RAW264 cells, 238 gene signals of which were attenuated by 6-MSITC (≥2-fold). Expression levels of 717 genes were decreased by ≥3-fold in LPS-activated cells, of which 336 gene signals were restored by 6-MSITC (≥2-fold). Utilizing group analysis, 206 genes affected by 6-MSITC with a ≥2-fold change were classified into 35 categories relating to biological processes (81), molecular functions (108) and signaling pathways (17). The genes were further categorized as ‘defense, inflammatory response, cytokine activities and receptor activities’ and some were confirmed by real-time polymerase chain reaction. Ingenuity pathway analysis further revealed that wasabi 6-MSITC regulated the relevant networks of chemokines, interleukins and interferons to exert its anti-inflammatory function. PMID:23136589

Akt/FOXO3a signaling modulates the endothelial stress response through regulation of heat shock protein 70 expression.

PubMed

Kim, Hyo-Soo; Skurk, Carsten; Maatz, Henrike; Shiojima, Ichiro; Ivashchenko, Yuri; Yoon, Suk-Won; Park, Young-Bae; Walsh, Kenneth

2005-06-01

To identify new antiapoptotic targets of the PI3K-Akt signaling pathway in endothelial cells, adenovirus-mediated Akt1 gene transfer and oligonucleotide microarrays were used to examine Akt-regulated transcripts. DNA microarray analysis revealed that HSP70 expression underwent the greatest fold activation of 12,532 transcripts examined in human umbilical vein endothelial cells (HUVEC) transduced with constitutively active Akt1. Akt1 gene transfer increased HSP70 transcript expression by 24.8-fold as determined by quantitative PCR and promoted a dose-dependent up-regulation of HSP70 protein as determined by Western immunoblot analysis. Gene transfer of FOXO3a, a downstream target of Akt in endothelial cells, significantly suppressed both basal and stress-induced HSP70 protein expression. FOXO3a induced caspase-9-dependent apoptosis in HUVEC, and cotransduction with Ad-HSP70 rescued endothelial cells from FOXO3a-induced apoptosis under basal and stress conditions. Our results identify HSP70 as a new antiapoptotic target of Akt-FOXO3a signaling in endothelial cells that controls viability through modulation of the stress-induced intrinsic cell death pathway.

Overview of long non-coding RNA and mRNA expression in response to methamphetamine treatment in vitro.

PubMed

Xiong, Kun; Long, Lingling; Zhang, Xudong; Qu, Hongke; Deng, Haixiao; Ding, Yanjun; Cai, Jifeng; Wang, Shuchao; Wang, Mi; Liao, Lvshuang; Huang, Jufang; Yi, Chun-Xia; Yan, Jie

2017-10-01

Long non-coding RNAs (lncRNAs) display multiple functions including regulation of neuronal injury. However, their impact in methamphetamine (METH)-induced neurotoxicity has rarely been reported. Here, using microarray analysis, we investigated the expression profiling of lncRNAs and mRNAs in primary cultured prefrontal cortical neurons after METH treatment. We observed a difference in lncRNA and mRNA expression between the experimental and sham control groups. Using bioinformatics, we analyzed the highest enriched gene ontology (GO) terms of biological process, cellular component, and molecular function, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and pathway network analysis. Furthermore, an lncRNA-mRNA co-expression sub-network for aberrantly expressed terms revealed possible interactions of lncRNA NR_110713 and NR_027943 with their related genes. Afterwards, three lncRNAs (NR_110713, NR_027943, GAS5) and two mRNAs (Ddit3, Casp12) were targeted to validate the microarray data by qRT-PCR. This presented an overview of lncRNA and mRNA expression profiling and indicated that lncRNA might participate in METH-induced neuronal apoptosis by regulating the coding genes of neurons. Copyright © 2017 Elsevier Ltd. All rights reserved.

Higher Matrix Stiffness Upregulates Osteopontin Expression in Hepatocellular Carcinoma Cells Mediated by Integrin β1/GSK3β/β-Catenin Signaling Pathway.

PubMed

You, Yang; Zheng, Qiongdan; Dong, Yinying; Wang, Yaohui; Zhang, Lan; Xue, Tongchun; Xie, Xiaoying; Hu, Chao; Wang, Zhiming; Chen, Rongxin; Wang, Yanhong; Cui, Jiefeng; Ren, Zhenggang

2015-01-01

Increased stromal stiffness is associated with hepatocellular carcinoma (HCC) development and progression. However, the molecular mechanism by which matrix stiffness stimuli modulate HCC progress is largely unknown. In this study, we explored whether matrix stiffness-mediated effects on osteopontin (OPN) expression occur in HCC cells. We used a previously reported in vitro culture system with tunable matrix stiffness and found that OPN expression was remarkably upregulated in HCC cells with increasing matrix stiffness. Furthermore, the phosphorylation level of GSK3β and the expression of nuclear β-catenin were also elevated, indicating that GSK3β/β-catenin pathway might be involved in OPN regulation. Knock-down analysis of integrin β1 showed that OPN expression and p-GSK3β level were downregulated in HCC cells grown on high stiffness substrate compared with controls. Simultaneously, inhibition of GSK-3β led to accumulation of β-catenin in the cytoplasm and its enhanced nuclear translocation, further triggered the rescue of OPN expression, suggesting that the integrin β1/GSK-3β/β-catenin pathway is specifically activated for matrix stiffness-mediated OPN upregulation in HCC cells. Tissue microarray analysis confirmed that OPN expression was positively correlated with the expression of LOX and COL1. Taken together, high matrix stiffness upregulated OPN expression in HCC cells via the integrin β1/GSK-3β/β-catenin signaling pathway. It highlights a new insight into a pathway involving physical mechanical signal and biochemical signal molecules which contributes to OPN expression in HCC cells.

Identification of intracellular proteins and signaling pathways in human endothelial cells regulated by angiotensin-(1-7).

PubMed

Meinert, Christian; Gembardt, Florian; Böhme, Ilka; Tetzner, Anja; Wieland, Thomas; Greenberg, Barry; Walther, Thomas

2016-01-01

The study aimed to identify proteins regulated by the cardiovascular protective peptide angiotensin-(1-7) and to determine potential intracellular signaling cascades. Human endothelial cells were stimulated with Ang-(1-7) for 1 h, 3 h, 6 h, and 9 h. Peptide effects on intracellular signaling were assessed via antibody microarray, containing antibodies against 725 proteins. Bioinformatics software was used to identify affected intracellular signaling pathways. Microarray data was verified exemplarily by Western blot, Real-Time RT-PCR, and immunohistochemical studies. The microarray identified 110 regulated proteins after 1 h, 119 after 3 h, 31 after 6 h, and 86 after 9 h Ang-(1-7) stimulation. Regulated proteins were associated with high significance to several metabolic pathways like “Molecular Mechanism of Cancer” and “p53 signaling” in a time dependent manner. Exemplarily, Western blots for the E3-type small ubiquitin-like modifier ligase PIAS2 confirmed the microarray data and displayed a decrease by more than 50% after Ang-(1-7) stimulation at 1 h and 3 h without affecting its mRNA. Immunohistochemical studies with PIAS2 in human endothelial cells showed a decrease in cytoplasmic PIAS2 after Ang-(1-7) treatment. The Ang-(1-7) mediated decrease of PIAS2 was reproduced in other endothelial cell types. The results suggest that angiotensin-(1-7) plays a role in metabolic pathways related to cell death and cell survival in human endothelial cells.

Porous Silicon Antibody Microarrays for Quantitative Analysis: Measurement of Free and Total PSA in Clinical Plasma Samples

PubMed Central

Tojo, Axel; Malm, Johan; Marko-Varga, György; Lilja, Hans; Laurell, Thomas

2014-01-01

The antibody microarrays have become widespread, but their use for quantitative analyses in clinical samples has not yet been established. We investigated an immunoassay based on nanoporous silicon antibody microarrays for quantification of total prostate-specific-antigen (PSA) in 80 clinical plasma samples, and provide quantitative data from a duplex microarray assay that simultaneously quantifies free and total PSA in plasma. To further develop the assay the porous silicon chips was placed into a standard 96-well microtiter plate for higher throughput analysis. The samples analyzed by this quantitative microarray were 80 plasma samples obtained from men undergoing clinical PSA testing (dynamic range: 0.14-44ng/ml, LOD: 0.14ng/ml). The second dataset, measuring free PSA (dynamic range: 0.40-74.9ng/ml, LOD: 0.47ng/ml) and total PSA (dynamic range: 0.87-295ng/ml, LOD: 0.76ng/ml), was also obtained from the clinical routine. The reference for the quantification was a commercially available assay, the ProStatus PSA Free/Total DELFIA. In an analysis of 80 plasma samples the microarray platform performs well across the range of total PSA levels. This assay might have the potential to substitute for the large-scale microtiter plate format in diagnostic applications. The duplex assay paves the way for a future quantitative multiplex assay, which analyses several prostate cancer biomarkers simultaneously. PMID:22921878

Neurofibromin Deficiency-Associated Transcriptional Dysregulation Suggests a Novel Therapy for Tibial Pseudoarthrosis in NF1

PubMed Central

Paria, Nandina; Cho, Tae-Joon; Choi, In Ho; Kamiya, Nobuhiro; Kayembe, Kay; Mao, Rong; Margraf, Rebecca L.; Obermosser, Gerlinde; Oxendine, Ila; Sant, David W.; Song, Mi Hyun; Stevenson, David A.; Viskochil, David H.; Wise, Carol A.; Kim, Harry K.W.; Rios, Jonathan J

2014-01-01

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease caused by mutations in NF1. Among the earliest manifestations is tibial pseudoarthrosis and persistent nonunion after fracture. To further understand the pathogenesis of pseudoarthrosis and the underlying bone remodeling defect, pseudoarthrosis tissue and cells cultured from surgically resected pseudoarthrosis tissue from NF1 individuals were analyzed using whole-exome and whole-transcriptome sequencing as well as genomewide microarray analysis. Genomewide analysis identified multiple genetic mechanisms resulting in somatic bi-allelic NF1 inactivation; no other genes with recurring somatic mutations were identified. Gene expression profiling identified dysregulated pathways associated with neurofibromin deficiency, including phosphoinosital-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways. Unlike aggressive NF1-associated malignancies, tibial pseudoarthrosis tissue does not harbor a high frequency of somatic mutations in oncogenes or other tumor-suppressor genes, such as p53. However, gene expression profiling indicates pseudoarthrosis tissue has a tumor-promoting transcriptional pattern, despite lacking tumorigenic somatic mutations. Significant over-expression of specific cancer-associated genes in pseudoarthrosis highlights a potential for receptor tyrosine kinase inhibitors to target neurofibromin-deficient pseudoarthrosis and promote proper bone remodeling and fracture healing. PMID:24932921

A Study of the Differential Effects of Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) on Gene Expression Profiles of Stimulated Thp-1 Macrophages.

PubMed

Allam-Ndoul, Bénédicte; Guénard, Frédéric; Barbier, Olivier; Vohl, Marie-Claude

2017-04-25

Background: An appropriate intake of omega-3 ( n -3) fatty acids (FAs) such as eicosapentaenoic and docosahexaenoic acid (EPA/DHA) from marine sources is known to have anti-inflammatory effects. However, molecular mechanisms underlying their beneficial effects on health are not fully understood. The aim of the present study was to characterize gene expression profiles of THP-1 macrophages, incubated in either EPA or DHA and stimulated with lipopolysaccharide (LPS), a pro-inflammatory agent. Methods: THP-1 macrophages were incubated into 10, 50 and 75 µM of EPA or DHA for 24 h, and 100 nM of LPS was added to the culture media for 18 h. Total mRNA was extracted and gene expression examined by microarray analysis using Illumina Human HT-12 expression beadchips (Illumina). Results: Pathway analysis revealed that EPA and DHA regulate genes involved in cell cycle regulation, apoptosis, immune response and inflammation, oxidative stress and cancer pathways in a differential and dose-dependent manner. Conclusions: EPA and DHA appear to exert differential effects on gene expression in THP-1 macrophages. Specific effects of n -3 FAs on gene expression levels are also dose-dependent.

Use of a bovine genome chip to identify new biological pathways for beef quality in cattle.

PubMed

Guifen, Liu; Xiaomu, Liu; Fachun, Wan; Xiuwen, Tan; Haijian, Cheng; Enliang, Song

2012-12-01

The accumulation of muscle is largely influenced by the genetic background of cattle. Muscle tissue was collected from the longissimus muscle of Lilu beef cattle at 12, 18, 24 and 30 months old. Using meat quality analysis, we found that the Lilu beef cattle have good production and slaughter performance, the performance meets the criterion of beef cattle. Microarray analysis was able to identify a total of 4,219 genes that are differentially expressed (P ≤ 0.01) between the two groups of cattle (12 vs 18; 18 vs 24; 24 vs 30). Bioinformatics analysis results suggested that most of the differentially expressed genes are involved in the metabolic pathways and neuroactive ligand-receptor interaction pathways. In the future study that aims to look for genes relating to growth and meat quality, we will focus on the genes that have been shown to have a significant variation between groups and are involved in the two pathways.

Microarray expression technology: from start to finish.

PubMed

Elvidge, Gareth

2006-01-01

The recent introduction of new microarray expression technologies and the further development of established platforms ensure that the researcher is presented with a range of options for performing an experiment. Whilst this has opened up the possibilities for future applications, such as exon-specific arrays, increased sample throughput and 'chromatin immunoprecipitation (ChIP) on chip' experiments, the initial decision processes and experiment planning are made more difficult. This review will give an overview of the various technologies that are available to perform a microarray expression experiment, from the initial planning stages through to the final data analysis. Both practical aspects and data analysis options will be considered. The relative advantages and disadvantages will be discussed with insights provided for future directions of the technology.

Characterization of microRNA profile in mammary tissue of dairy and beef breed heifers.

PubMed

Wicik, Z; Gajewska, M; Majewska, A; Walkiewicz, D; Osińska, E; Motyl, T

2016-02-01

MicroRNAs (miRNAs) are small non-coding RNAs that participate in the regulation of gene expression. Their role during mammary gland development is still largely unknown. In this study, we performed a microarray analysis to identify miRNAs associated with high mammogenic potential of the bovine mammary gland. We identified 54 significantly differentially expressed miRNAs between the mammary tissue of dairy (Holstein-Friesian, HF) and beef (Limousin, LM) postpubertal heifers. Fifty-two miRNAs had higher expression in the mammary tissue of LM heifers. The expression of the top candidate miRNAs (bta-miR-10b, bta-miR-29b, bta-miR-101, bta-miR-375, bta-miR-2285t, bta-miR-146b, bta-let7b, bta-miR-107, bta-miR-1434-3p) identified in the microarray experiment was additionally evaluated by qPCR. Enrichment analyses for targeted genes revealed that the major differences between miRNA expression in the mammary gland of HF versus LM were associated with the regulation of signalling pathways that are crucial for mammary gland development, such as TGF-beta, insulin, WNT and inflammatory pathways. Moreover, a number of genes potentially targeted by significantly differentially expressed miRNAs were associated with the activity of mammary stem cells. These data indicate that the high developmental potential of the mammary gland in dairy cattle, leading to high milk productivity, depends also on a specific miRNA expression pattern. © 2015 Blackwell Verlag GmbH.

Biological Characteristics and Genetic Heterogeneity between Carcinoma-Associated Fibroblasts and Their Paired Normal Fibroblasts in Human Breast Cancer

PubMed Central

Hou, Yixuan; Sun, Yan; Wang, Liyang; Luo, Haojun; Peng, Huimin; Liu, Manran

2013-01-01

Background The extensional signals in cross-talk between stromal cells and tumor cells generated from extracellular matrix molecules, soluble factor, and cell-cell adhesion complexes cooperate at the extra- and intracellular level in the tumor microenvironment. CAFs are the primary type of stromal cells in the tumor microenvironment and play a pivotal role in tumorigenesis and development. Hitherto, there is hardly any systematic analysis of the intrinsic relationship between CAFs function and its abnormal signaling pathway. The extreme complexity of CAFs’ features and their role in tumor development are needed to be further investigated. Methodology/Principal Findings We primary cultured CAFs and NFs from early stages of breast cancer tissue and identified them using their biomarker by immunohistochemistry for Fibronectin, α-SMA and FAP. Microarray was applied to analyze gene expression profiles of human breast CAFs and the paired NFs. The Up-regulated genes classified by Gene Ontology, signal pathways enriched by DAVID pathway analysis. Abnormal signaling pathways in breast cancer CAFs are involved in cell cycle, cell adhesion, signal transduction and protein transport being reported in CAFs derived from other tumors. Significantly, the altered ATM signaling pathway, a set of cell cycle regulated signaling, and immune associated signaling are identified to be changed in CAFs. Conclusions/Significance CAFs have the vigorous ability of proliferation and potential of invasion and migration comparing with NFs. CAFs could promote breast cancer cell invasion under co-culture conditions through up-regulated CCL18 and CXCL12. Consistently with its biologic behavior, the gene expression profiling analyzed by microarray shows that some of key signaling pathways, such as cell cycle, cell adhesion, and secreting factors play an important role in CAFs. The altered ATM signaling pathway is abnormally active in the early stage of breast cancer. The set of immune associated signaling may be involved in tumor cell immune evasion. PMID:23577100

Regulation of the activity of the promoter of RNA-induced silencing, C3PO.

PubMed

Sahu, Shriya; Williams, Leo; Perez, Alberto; Philip, Finly; Caso, Giuseppe; Zurawsky, Walter; Scarlata, Suzanne

2017-09-01

RNA-induced silencing is a process which allows cells to regulate the synthesis of specific proteins. RNA silencing is promoted by the protein C3PO (component 3 of RISC). We have previously found that phospholipase Cβ, which increases intracellular calcium levels in response to specific G protein signals, inhibits C3PO activity towards certain genes. Understanding the parameters that control C3PO activity and which genes are impacted by G protein activation would help predict which genes are more vulnerable to downregulation. Here, using a library of 10 18 oligonucleotides, we show that C3PO binds oligonucleotides with structural specificity but little sequence specificity. Alternately, C3PO hydrolyzes oligonucleotides with a rate that is sensitive to substrate stability. Importantly, we find that oligonucleotides with higher Tm values are inhibited by bound PLCβ. This finding is supported by microarray analysis in cells over-expressing PLCβ1. Taken together, this study allows predictions of the genes whose post-transcriptional regulation is responsive to the G protein/phospholipase Cβ/calcium signaling pathway. © 2017 The Protein Society.

The Fast and Transient Transcriptional Network of Gravity and Mechanical Stimulation in the Arabidopsis Root Apex1[w

PubMed Central

Kimbrough, Jeffery M.; Salinas-Mondragon, Raul; Boss, Wendy F.; Brown, Christopher S.; Sederoff, Heike Winter

2004-01-01

Plant root growth is affected by both gravity and mechanical stimulation (Massa GD, Gilroy S [2003] Plant J 33: 435–445). A coordinated response to both stimuli requires specific and common elements. To delineate the transcriptional response mechanisms, we carried out whole-genome microarray analysis of Arabidopsis root apices after gravity stimulation (reorientation) and mechanical stimulation and monitored transcript levels of 22,744 genes in a time course during the first hour after either stimulus. Rapid, transient changes in the relative abundance of specific transcripts occurred in response to gravity or mechanical stimulation, and these transcript level changes reveal clusters of coordinated events. Transcriptional regulation occurs in the root apices within less than 2 min after either stimulus. We identified genes responding specifically to each stimulus as well as transcripts regulated in both signal transduction pathways. Several unknown genes were specifically induced only during gravitropic stimulation (gravity induced genes). We also analyzed the network of transcriptional regulation during the early stages of gravitropism and mechanical stimulation. PMID:15347791

Microarray evaluation of gene expression profiles in inflamed and healthy human dental pulp: the role of IL1beta and CD40 in pulp inflammation.

PubMed

Gatta, V; Zizzari, V L; Dd ' Amico, V; Salini, L; D' Aurora, M; Franchi, S; Antonucci, I; Sberna, M T; Gherlone, E; Stuppia, L; Tetè, S

2012-01-01

Dental pulp undergoes a number of changes passing from healthy status to inflammation due to deep decay. These changes are regulated by several genes resulting differently expressed in inflamed and healthy dental pulp, and the knowledge of the processes underlying this differential expression is of great relevance in the identification of the pathogenesis of the disease. In this study, the gene expression profile of inflamed and healthy dental pulps were compared by microarray analysis, and data obtained were analyzed by Ingenuity Pathway Analysis (IPA) software. This analysis allows to focus on a variety of genes, typically expressed in inflamed tissues. The comparison analysis showed an increased expression of several genes in inflamed pulp, among which IL1β and CD40 resulted of particular interest. These results indicate that gene expression profile of human dental pulp in different physiological and pathological conditions may become an useful tool for improving our knowledge about processes regulating pulp inflammation.

A Sol-gel Integrated Dual-readout Microarray Platform for Quantification and Identification of Prostate-specific Antigen.

PubMed

Lee, SangWook; Lee, Jong Hyun; Kwon, Hyuck Gi; Laurell, Thomas; Jeong, Ok Chan; Kim, Soyoun

2018-01-01

Here, we report a sol-gel integrated affinity microarray for on-chip matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) that enables capture and identification of prostate?specific antigen (PSA) in samples. An anti-PSA antibody (H117) was mixed with a sol?gel, and the mixture was spotted onto a porous silicon (pSi) surface without additional surface modifications. The antibody easily penetrates the sol-gel macropore fluidic network structure, making possible high affinities. To assess the capture affinity of the platform, we performed a direct assay using fluorescein isothiocyanate-labeled PSA. Pure PSA was subjected to on-chip MALDI-TOF-MS analysis, yielding three clear mass peptide peaks (m/z = 1272, 1407, and 1872). The sol-gel microarray platform enables dual readout of PSA both fluorometric and MALDI-TOF MS analysis in biological samples. Here we report a useful method for a means for discovery of biomarkers in complex body fluids.

Deregulation of Rab and Rab Effector Genes in Bladder Cancer

PubMed Central

Ho, Joel R.; Chapeaublanc, Elodie; Kirkwood, Lisa; Nicolle, Remy; Benhamou, Simone; Lebret, Thierry; Allory, Yves; Southgate, Jennifer; Radvanyi, François; Goud, Bruno

2012-01-01

Growing evidence indicates that Rab GTPases, key regulators of intracellular transport in eukaryotic cells, play an important role in cancer. We analysed the deregulation at the transcriptional level of the genes encoding Rab proteins and Rab-interacting proteins in bladder cancer pathogenesis, distinguishing between the two main progression pathways so far identified in bladder cancer: the Ta pathway characterized by a high frequency of FGFR3 mutation and the carcinoma in situ pathway where no or infrequent FGFR3 mutations have been identified. A systematic literature search identified 61 genes encoding Rab proteins and 223 genes encoding Rab-interacting proteins. Transcriptomic data were obtained for normal urothelium samples and for two independent bladder cancer data sets corresponding to 152 and 75 tumors. Gene deregulation was analysed with the SAM (significant analysis of microarray) test or the binomial test. Overall, 30 genes were down-regulated, and 13 were up-regulated in the tumor samples. Five of these deregulated genes (LEPRE1, MICAL2, RAB23, STXBP1, SYTL1) were specifically deregulated in FGFR3-non-mutated muscle-invasive tumors. No gene encoding a Rab or Rab-interacting protein was found to be specifically deregulated in FGFR3-mutated tumors. Cluster analysis showed that the RAB27 gene cluster (comprising the genes encoding RAB27 and its interacting partners) was deregulated and that this deregulation was associated with both pathways of bladder cancer pathogenesis. Finally, we found that the expression of KIF20A and ZWINT was associated with that of proliferation markers and that the expression of MLPH, MYO5B, RAB11A, RAB11FIP1, RAB20 and SYTL2 was associated with that of urothelial cell differentiation markers. This systematic analysis of Rab and Rab effector gene deregulation in bladder cancer, taking relevant tumor subgroups into account, provides insight into the possible roles of Rab proteins and their effectors in bladder cancer pathogenesis. This approach is applicable to other group of genes and types of cancer. PMID:22724020

UV-B Irradiation Changes Specifically the Secondary Metabolite Profile in Broccoli Sprouts: Induced Signaling Overlaps with Defense Response to Biotic Stressors

PubMed Central

Mewis, Inga; Schreiner, Monika; Nguyen, Chau Nhi; Krumbein, Angelika; Ulrichs, Christian; Lohse, Marc; Zrenner, Rita

2012-01-01

Only a few environmental factors have such a pronounced effect on plant growth and development as ultraviolet light (UV). Concerns have arisen due to increased UV-B radiation reaching the Earth’s surface as a result of stratospheric ozone depletion. Ecologically relevant low to moderate UV-B doses (0.3–1 kJ m–2 d–1) were applied to sprouts of the important vegetable crop Brassica oleracea var. italica (broccoli), and eco-physiological responses such as accumulation of non-volatile secondary metabolites were related to transcriptional responses with Agilent One-Color Gene Expression Microarray analysis using the 2×204 k format Brassica microarray. UV-B radiation effects have usually been linked to increases in phenolic compounds. As expected, the flavonoids kaempferol and quercetin accumulated in broccoli sprouts (the aerial part of the seedlings) 24 h after UV-B treatment. A new finding is the specific UV-B-mediated induction of glucosinolates (GS), especially of 4-methylsulfinylbutyl GS and 4-methoxy-indol-3-ylmethyl GS, while carotenoids and Chl levels remained unaffected. Accumulation of defensive GS metabolites was accompanied by increased expression of genes associated with salicylate and jasmonic acid signaling defense pathways and up-regulation of genes responsive to fungal and bacterial pathogens. Concomitantly, plant pre-exposure to moderate UV-B doses had negative effects on the performance of the caterpillar Pieris brassicae (L.) and on the population growth of the aphid Myzus persicae (Sulzer). Moreover, insect-specific induction of GS in broccoli sprouts was affected by UV-B pre-treatment. PMID:22773681

Using Ambystoma mexicanum (Mexican Axolotl) Embryos, Chemical Genetics, and Microarray Analysis to Identify Signaling Pathways Associated with Tissue Regeneration

PubMed Central

Ponomareva, Larissa V.; Athippozhy, Antony; Thorson, Jon S.; Voss, S. Randal

2015-01-01

Amphibian vertebrates are important models in regenerative biology because they present exceptional regenerative capabilities throughout life. However, it takes considerable effort to rear amphibians to juvenile and adult stages for regeneration studies and the relatively large sizes that frogs and salamanders achieve during development make them difficult to use in chemical screens. Here we introduce a new tail regeneration model using late stage Mexican axolotl embryos. We show that axolotl embryos completely regenerate amputated tails in 7 days before they exhaust their yolk supply and begin to feed. Further, we show that axolotl embryos can be efficiently reared in microtiter plates to achieve moderate throughput screening of soluble chemicals to investigate toxicity and identify molecules that alter regenerative outcome. As proof of principle, we identified integration 1 / wingless (Wnt), transforming growth factor beta (Tgf-β), and fibroblast growth factor (Fgf) pathway antagonists that completely block tail regeneration and additional chemicals that significantly affected tail outgrowth. Furthermore, we used microarray analysis to show that inhibition of Wnt signaling broadly affects transcription of genes associated with Wnt, Fgf, Tgf-β, epidermal growth factor (Egf), Notch, nerve growth factor (Ngf), homeotic gene (Hox), rat sarcoma/mitogen-activated protein kinase (Ras/Mapk), myelocytomatosis viral oncogene (Myc), tumor protein 53 (p53), and retinoic acid (RA) pathways. Punctuated changes in the expression of genes known to regulate vertebrate development were observed; this suggests the tail regeneration transcriptional program is hierarchically structured and temporally ordered. Our study establishes the axolotl as a chemical screening model to investigate signaling pathways associated with tissue regeneration. PMID:26092703

The Glycan Microarray Story from Construction to Applications.

PubMed

Hyun, Ji Young; Pai, Jaeyoung; Shin, Injae

2017-04-18

Not only are glycan-mediated binding processes in cells and organisms essential for a wide range of physiological processes, but they are also implicated in various pathological processes. As a result, elucidation of glycan-associated biomolecular interactions and their consequences is of great importance in basic biological research and biomedical applications. In 2002, we and others were the first to utilize glycan microarrays in efforts aimed at the rapid analysis of glycan-associated recognition events. Because they contain a number of glycans immobilized in a dense and orderly manner on a solid surface, glycan microarrays enable multiple parallel analyses of glycan-protein binding events while utilizing only small amounts of glycan samples. Therefore, this microarray technology has become a leading edge tool in studies aimed at elucidating roles played by glycans and glycan binding proteins in biological systems. In this Account, we summarize our efforts on the construction of glycan microarrays and their applications in studies of glycan-associated interactions. Immobilization strategies of functionalized and unmodified glycans on derivatized glass surfaces are described. Although others have developed immobilization techniques, our efforts have focused on improving the efficiencies and operational simplicity of microarray construction. The microarray-based technology has been most extensively used for rapid analysis of the glycan binding properties of proteins. In addition, glycan microarrays have been employed to determine glycan-protein interactions quantitatively, detect pathogens, and rapidly assess substrate specificities of carbohydrate-processing enzymes. More recently, the microarrays have been employed to identify functional glycans that elicit cell surface lectin-mediated cellular responses. Owing to these efforts, it is now possible to use glycan microarrays to expand the understanding of roles played by glycans and glycan binding proteins in biological systems.

Gene expression profiling at early organogenesis reveals both common and diverse mechanisms in foregut patterning

PubMed Central

Fagman, Henrik; Amendola, Elena; Parrillo, Luca; Zoppoli, Pietro; Marotta, Pina; Scarfò, Marzia; De Luca, Pasquale; de Carvalho, Denise Pires; Ceccarelli, Michele; De Felice, Mario; Di Lauro, Roberto

2011-01-01

The thyroid and lungs originate as neighboring bud shaped outgrowths from the midline of the embryonic foregut. When and how organ specific programs regulate development into structures of distinct shapes, positions and functions is incompletely understood. To characterize, at least in part, the genetic basis of these events, we have employed laser capture microdissection and microarray analysis to define gene expression in the mouse thyroid and lung primordia at E10.5. By comparing the transcriptome of each bud to that of the whole embryo as well as to each other, we broadly describe the genes that are preferentially expressed in each developing organ as well as those with an enriched expression common to both. The results thus obtained provide a valuable resource for further analysis of genes previously unrecognized to participate in thyroid and lung morphogenesis and to discover organ specific as well as common developmental mechanisms. As an initial step in this direction we describe a regulatory pathway involving the anti-apoptotic gene Bcl2 that controls cell survival in early thyroid development. PMID:21924257

Analysis of Protein-DNA Interaction by Chromatin Immunoprecipitation and DNA Tiling Microarray (ChIP-on-chip).

PubMed

Gao, Hui; Zhao, Chunyan

2018-01-01

Chromatin immunoprecipitation (ChIP) has become the most effective and widely used tool to study the interactions between specific proteins or modified forms of proteins and a genomic DNA region. Combined with genome-wide profiling technologies, such as microarray hybridization (ChIP-on-chip) or massively parallel sequencing (ChIP-seq), ChIP could provide a genome-wide mapping of in vivo protein-DNA interactions in various organisms. Here, we describe a protocol of ChIP-on-chip that uses tiling microarray to obtain a genome-wide profiling of ChIPed DNA.

Identification and comparison of aberrant key regulatory networks in breast, colon, liver, lung, and stomach cancers through methylome database analysis.

PubMed

Kim, Byungtak; Kang, Seongeun; Jeong, Gookjoo; Park, Sung-Bin; Kim, Sun Jung

2014-01-01

Aberrant methylation of specific CpG sites at the promoter is widely responsible for genesis and development of various cancer types. Even though the microarray-based methylome analyzing techniques have contributed to the elucidation of the methylation change at the genome-wide level, the identification of key methylation markers or top regulatory networks appearing common in highly incident cancers through comparison analysis is still limited. In this study, we in silico performed the genome-wide methylation analysis on each 10 sets of normal and cancer pairs of five tissues: breast, colon, liver, lung, and stomach. The methylation array covers 27,578 CpG sites, corresponding to 14,495 genes, and significantly hypermethylated or hypomethylated genes in the cancer were collected (FDR adjusted p-value <0.05; methylation difference >0.3). Analysis of the dataset confirmed the methylation of previously known methylation markers and further identified novel methylation markers, such as GPX2, CLDN15, and KL. Cluster analysis using the methylome dataset resulted in a diagram with a bipartite mode distinguishing cancer cells from normal cells regardless of tissue types. The analysis further revealed that breast cancer was closest with lung cancer, whereas it was farthest from colon cancer. Pathway analysis identified that either the "cancer" related network or the "cancer" related bio-function appeared as the highest confidence in all the five cancers, whereas each cancer type represents its tissue-specific gene sets. Our results contribute toward understanding the essential abnormal epigenetic pathways involved in carcinogenesis. Further, the novel methylation markers could be applied to establish markers for cancer prognosis.

Microarray Data Processing Techniques for Genome-Scale Network Inference from Large Public Repositories.

PubMed

Chockalingam, Sriram; Aluru, Maneesha; Aluru, Srinivas

2016-09-19

Pre-processing of microarray data is a well-studied problem. Furthermore, all popular platforms come with their own recommended best practices for differential analysis of genes. However, for genome-scale network inference using microarray data collected from large public repositories, these methods filter out a considerable number of genes. This is primarily due to the effects of aggregating a diverse array of experiments with different technical and biological scenarios. Here we introduce a pre-processing pipeline suitable for inferring genome-scale gene networks from large microarray datasets. We show that partitioning of the available microarray datasets according to biological relevance into tissue- and process-specific categories significantly extends the limits of downstream network construction. We demonstrate the effectiveness of our pre-processing pipeline by inferring genome-scale networks for the model plant Arabidopsis thaliana using two different construction methods and a collection of 11,760 Affymetrix ATH1 microarray chips. Our pre-processing pipeline and the datasets used in this paper are made available at http://alurulab.cc.gatech.edu/microarray-pp.

Transcriptional Changes in Canine Distemper Virus-Induced Demyelinating Leukoencephalitis Favor a Biphasic Mode of Demyelination

PubMed Central

Ulrich, Reiner; Puff, Christina; Wewetzer, Konstantin; Kalkuhl, Arno; Deschl, Ulrich; Baumgärtner, Wolfgang

2014-01-01

Canine distemper virus (CDV)-induced demyelinating leukoencephalitis in dogs (Canis familiaris) is suggested to represent a naturally occurring translational model for subacute sclerosing panencephalitis and multiple sclerosis in humans. The aim of this study was a hypothesis-free microarray analysis of the transcriptional changes within cerebellar specimens of five cases of acute, six cases of subacute demyelinating, and three cases of chronic demyelinating and inflammatory CDV leukoencephalitis as compared to twelve non-infected control dogs. Frozen cerebellar specimens were used for analysis of histopathological changes including demyelination, transcriptional changes employing microarrays, and presence of CDV nucleoprotein RNA and protein using microarrays, RT-qPCR and immunohistochemistry. Microarray analysis revealed 780 differentially expressed probe sets. The dominating change was an up-regulation of genes related to the innate and the humoral immune response, and less distinct the cytotoxic T-cell-mediated immune response in all subtypes of CDV leukoencephalitis as compared to controls. Multiple myelin genes including myelin basic protein and proteolipid protein displayed a selective down-regulation in subacute CDV leukoencephalitis, suggestive of an oligodendrocyte dystrophy. In contrast, a marked up-regulation of multiple immunoglobulin-like expressed sequence tags and the delta polypeptide of the CD3 antigen was observed in chronic CDV leukoencephalitis, in agreement with the hypothesis of an immune-mediated demyelination in the late inflammatory phase of the disease. Analysis of pathways intimately linked to demyelination as determined by morphometry employing correlation-based Gene Set Enrichment Analysis highlighted the pathomechanistic importance of up-regulated genes comprised by the gene ontology terms “viral replication” and “humoral immune response” as well as down-regulated genes functionally related to “metabolite and energy generation”. PMID:24755553

Transcriptional changes in canine distemper virus-induced demyelinating leukoencephalitis favor a biphasic mode of demyelination.

PubMed

Ulrich, Reiner; Puff, Christina; Wewetzer, Konstantin; Kalkuhl, Arno; Deschl, Ulrich; Baumgärtner, Wolfgang

2014-01-01

Canine distemper virus (CDV)-induced demyelinating leukoencephalitis in dogs (Canis familiaris) is suggested to represent a naturally occurring translational model for subacute sclerosing panencephalitis and multiple sclerosis in humans. The aim of this study was a hypothesis-free microarray analysis of the transcriptional changes within cerebellar specimens of five cases of acute, six cases of subacute demyelinating, and three cases of chronic demyelinating and inflammatory CDV leukoencephalitis as compared to twelve non-infected control dogs. Frozen cerebellar specimens were used for analysis of histopathological changes including demyelination, transcriptional changes employing microarrays, and presence of CDV nucleoprotein RNA and protein using microarrays, RT-qPCR and immunohistochemistry. Microarray analysis revealed 780 differentially expressed probe sets. The dominating change was an up-regulation of genes related to the innate and the humoral immune response, and less distinct the cytotoxic T-cell-mediated immune response in all subtypes of CDV leukoencephalitis as compared to controls. Multiple myelin genes including myelin basic protein and proteolipid protein displayed a selective down-regulation in subacute CDV leukoencephalitis, suggestive of an oligodendrocyte dystrophy. In contrast, a marked up-regulation of multiple immunoglobulin-like expressed sequence tags and the delta polypeptide of the CD3 antigen was observed in chronic CDV leukoencephalitis, in agreement with the hypothesis of an immune-mediated demyelination in the late inflammatory phase of the disease. Analysis of pathways intimately linked to demyelination as determined by morphometry employing correlation-based Gene Set Enrichment Analysis highlighted the pathomechanistic importance of up-regulated genes comprised by the gene ontology terms "viral replication" and "humoral immune response" as well as down-regulated genes functionally related to "metabolite and energy generation".

Systems Biology Analysis of Gene Expression during In Vivo Mycobacterium avium paratuberculosis Enteric Colonization Reveals Role for Immune Tolerance

PubMed Central

Khare, Sangeeta; Lawhon, Sara D.; Drake, Kenneth L.; Nunes, Jairo E. S.; Figueiredo, Josely F.; Rossetti, Carlos A.; Gull, Tamara; Everts, Robin E.; Lewin, Harris A.; Galindo, Cristi L.; Garner, Harold R.; Adams, Leslie Garry

2012-01-01

Survival and persistence of Mycobacterium avium subsp. paratuberculosis (MAP) in the intestinal mucosa is associated with host immune tolerance. However, the initial events during MAP interaction with its host that lead to pathogen survival, granulomatous inflammation, and clinical disease progression are poorly defined. We hypothesize that immune tolerance is initiated upon initial contact of MAP with the intestinal Peyer's patch. To test our hypothesis, ligated ileal loops in neonatal calves were infected with MAP. Intestinal tissue RNAs were collected (0.5, 1, 2, 4, 8 and 12 hrs post-infection), processed, and hybridized to bovine gene expression microarrays. By comparing the gene transcription responses of calves infected with the MAP, informative complex patterns of expression were clearly visible. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis, and genes were grouped into the specific pathways and gene ontology categories to create a holistic model. This model revealed three different phases of responses: i) early (30 min and 1 hr post-infection), ii) intermediate (2, 4 and 8 hrs post-infection), and iii) late (12 hrs post-infection). We describe here the data that include expression profiles for perturbed pathways, as well as, mechanistic genes (genes predicted to have regulatory influence) that are associated with immune tolerance. In the Early Phase of MAP infection, multiple pathways were initiated in response to MAP invasion via receptor mediated endocytosis and changes in intestinal permeability. During the Intermediate Phase, perturbed pathways involved the inflammatory responses, cytokine-cytokine receptor interaction, and cell-cell signaling. During the Late Phase of infection, gene responses associated with immune tolerance were initiated at the level of T-cell signaling. Our study provides evidence that MAP infection resulted in differentially regulated genes, perturbed pathways and specifically modified mechanistic genes contributing to the colonization of Peyer's patch. PMID:22912686

Major carcinogenic pathways identified by gene expression analysis of peritoneal mesotheliomas following chemical treatment in F344 rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Yongbaek; Thai-Vu Ton; De Angelo, Anthony B.

2006-07-15

This study was performed to characterize the gene expression profile and to identify the major carcinogenic pathways involved in rat peritoneal mesothelioma (RPM) formation following treatment of Fischer 344 rats with o-nitrotoluene (o-NT) or bromochloracetic acid (BCA). Oligo arrays, with over 20,000 target genes, were used to evaluate o-NT- and BCA-induced RPMs, when compared to a non-transformed mesothelial cell line (Fred-PE). Analysis using Ingenuity Pathway Analysis software revealed 169 cancer-related genes that were categorized into binding activity, growth and proliferation, cell cycle progression, apoptosis, and invasion and metastasis. The microarray data were validated by positive correlation with quantitative real-time RT-PCRmore » on 16 selected genes including igf1, tgfb3 and nov. Important carcinogenic pathways involved in RPM formation included insulin-like growth factor 1 (IGF-1), p38 MAPkinase, Wnt/{beta}-catenin and integrin signaling pathways. This study demonstrated that mesotheliomas in rats exposed to o-NT- and BCA were similar to mesotheliomas in humans, at least at the cellular and molecular level.« less

Identification of differentially-expressed genes potentially implicated in drought response in pitaya (Hylocereus undatus) by suppression subtractive hybridization and cDNA microarray analysis.

PubMed

Fan, Qing-Jie; Yan, Feng-Xia; Qiao, Guang; Zhang, Bing-Xue; Wen, Xiao-Peng

2014-01-01

Drought is one of the most severe threats to the growth, development and yield of plant. In order to unravel the molecular basis underlying the high tolerance of pitaya (Hylocereus undatus) to drought stress, suppression subtractive hybridization (SSH) and cDNA microarray approaches were firstly combined to identify the potential important or novel genes involved in the plant responses to drought stress. The forward (drought over drought-free) and reverse (drought-free over drought) suppression subtractive cDNA libraries were constructed using in vitro shoots of cultivar 'Zihonglong' exposed to drought stress and drought-free (control). A total of 2112 clones, among which half were from either forward or reverse SSH library, were randomly picked up to construct a pitaya cDNA microarray. Microarray analysis was carried out to verify the expression fluctuations of this set of clones upon drought treatment compared with the controls. A total of 309 expressed sequence tags (ESTs), 153 from forward library and 156 from reverse library, were obtained, and 138 unique ESTs were identified after sequencing by clustering and blast analyses, which included genes that had been previously reported as responsive to water stress as well as some functionally unknown genes. Thirty six genes were mapped to 47 KEGG pathways, including carbohydrate metabolism, lipid metabolism, energy metabolism, nucleotide metabolism, and amino acid metabolism of pitaya. Expression analysis of the selected ESTs by reverse transcriptase polymerase chain reaction (RT-PCR) corroborated the results of differential screening. Moreover, time-course expression patterns of these selected ESTs further confirmed that they were closely responsive to drought treatment. Among the differentially expressed genes (DEGs), many are related to stress tolerances including drought tolerance. Thereby, the mechanism of drought tolerance of this pitaya genotype is a very complex physiological and biochemical process, in which multiple metabolism pathways and many genes were implicated. The data gained herein provide an insight into the mechanism underlying the drought stress tolerance of pitaya, as well as may facilitate the screening of candidate genes for drought tolerance. © 2013 Elsevier B.V. All rights reserved.

Partial Least Squares Based Gene Expression Analysis in EBV- Positive and EBV-Negative Posttransplant Lymphoproliferative Disorders.

PubMed

Wu, Sa; Zhang, Xin; Li, Zhi-Ming; Shi, Yan-Xia; Huang, Jia-Jia; Xia, Yi; Yang, Hang; Jiang, Wen-Qi

2013-01-01

Post-transplant lymphoproliferative disorder (PTLD) is a common complication of therapeutic immunosuppression after organ transplantation. Gene expression profile facilitates the identification of biological difference between Epstein-Barr virus (EBV) positive and negative PTLDs. Previous studies mainly implemented variance/regression analysis without considering unaccounted array specific factors. The aim of this study is to investigate the gene expression difference between EBV positive and negative PTLDs through partial least squares (PLS) based analysis. With a microarray data set from the Gene Expression Omnibus database, we performed PLS based analysis. We acquired 1188 differentially expressed genes. Pathway and Gene Ontology enrichment analysis identified significantly over-representation of dysregulated genes in immune response and cancer related biological processes. Network analysis identified three hub genes with degrees higher than 15, including CREBBP, ATXN1, and PML. Proteins encoded by CREBBP and PML have been reported to be interact with EBV before. Our findings shed light on expression distinction of EBV positive and negative PTLDs with the hope to offer theoretical support for future therapeutic study.

Bioinformatics Analysis Reveals Distinct Molecular Characteristics of Hepatitis B-Related Hepatocellular Carcinomas from Very Early to Advanced Barcelona Clinic Liver Cancer Stages.

PubMed

Kong, Fan-Yun; Wei, Xiao; Zhou, Kai; Hu, Wei; Kou, Yan-Bo; You, Hong-Juan; Liu, Xiao-Mei; Zheng, Kui-Yang; Tang, Ren-Xian

2016-01-01

Hepatocellular carcinoma (HCC)is the fifth most common malignancy associated with high mortality. One of the risk factors for HCC is chronic hepatitis B virus (HBV) infection. The treatment strategy for the disease is dependent on the stage of HCC, and the Barcelona clinic liver cancer (BCLC) staging system is used in most HCC cases. However, the molecular characteristics of HBV-related HCC in different BCLC stages are still unknown. Using GSE14520 microarray data from HBV-related HCC cases with BCLC stages from 0 (very early stage) to C (advanced stage) in the gene expression omnibus (GEO) database, differentially expressed genes (DEGs), including common DEGs and unique DEGs in different BCLC stages, were identified. These DEGs were located on different chromosomes. The molecular functions and biology pathways of DEGs were identified by gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and the interactome networks of DEGs were constructed using the NetVenn online tool. The results revealed that both common DEGs and stage-specific DEGs were associated with various molecular functions and were involved in special biological pathways. In addition, several hub genes were found in the interactome networks of DEGs. The identified DEGs and hub genes promote our understanding of the molecular mechanisms underlying the development of HBV-related HCC through the different BCLC stages, and might be used as staging biomarkers or molecular targets for the treatment of HCC with HBV infection.

Differential Expression and Functional Analysis of High-Throughput -Omics Data Using Open Source Tools.

PubMed

Kebschull, Moritz; Fittler, Melanie Julia; Demmer, Ryan T; Papapanou, Panos N

2017-01-01

Today, -omics analyses, including the systematic cataloging of messenger RNA and microRNA sequences or DNA methylation patterns in a cell population, organ, or tissue sample, allow for an unbiased, comprehensive genome-level analysis of complex diseases, offering a large advantage over earlier "candidate" gene or pathway analyses. A primary goal in the analysis of these high-throughput assays is the detection of those features among several thousand that differ between different groups of samples. In the context of oral biology, our group has successfully utilized -omics technology to identify key molecules and pathways in different diagnostic entities of periodontal disease.A major issue when inferring biological information from high-throughput -omics studies is the fact that the sheer volume of high-dimensional data generated by contemporary technology is not appropriately analyzed using common statistical methods employed in the biomedical sciences.In this chapter, we outline a robust and well-accepted bioinformatics workflow for the initial analysis of -omics data generated using microarrays or next-generation sequencing technology using open-source tools. Starting with quality control measures and necessary preprocessing steps for data originating from different -omics technologies, we next outline a differential expression analysis pipeline that can be used for data from both microarray and sequencing experiments, and offers the possibility to account for random or fixed effects. Finally, we present an overview of the possibilities for a functional analysis of the obtained data.

ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping.

PubMed

Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren; Nielsen, Morten

2017-01-01

Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope.

ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping

PubMed Central

Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren

2017-01-01

Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope. PMID:28095436

Integrative analyses of miRNA and proteomics identify potential biological pathways associated with onset of pulmonary fibrosis in the bleomycin rat model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fukunaga, Satoki; Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 3-1-98 Kasugade-Naka, Konohana-ku, Osaka 554-8558; Kakehashi, Anna

To determine miRNAs and their predicted target proteins regulatory networks which are potentially involved in onset of pulmonary fibrosis in the bleomycin rat model, we conducted integrative miRNA microarray and iTRAQ-coupled LC-MS/MS proteomic analyses, and evaluated the significance of altered biological functions and pathways. We observed that alterations of miRNAs and proteins are associated with the early phase of bleomycin-induced pulmonary fibrosis, and identified potential target pairs by using ingenuity pathway analysis. Using the data set of these alterations, it was demonstrated that those miRNAs, in association with their predicted target proteins, are potentially involved in canonical pathways reflective ofmore » initial epithelial injury and fibrogenic processes, and biofunctions related to induction of cellular development, movement, growth, and proliferation. Prediction of activated functions suggested that lung cells acquire proliferative, migratory, and invasive capabilities, and resistance to cell death especially in the very early phase of bleomycin-induced pulmonary fibrosis. The present study will provide new insights for understanding the molecular pathogenesis of idiopathic pulmonary fibrosis. - Highlights: • We analyzed bleomycin-induced pulmonary fibrosis in the rat. • Integrative analyses of miRNA microarray and proteomics were conducted. • We determined the alterations of miRNAs and their potential target proteins. • The alterations may control biological functions and pathways in pulmonary fibrosis. • Our result may provide new insights of pulmonary fibrosis.« less

Microarray Я US: a user-friendly graphical interface to Bioconductor tools that enables accurate microarray data analysis and expedites comprehensive functional analysis of microarray results.

PubMed

Dai, Yilin; Guo, Ling; Li, Meng; Chen, Yi-Bu

2012-06-08

Microarray data analysis presents a significant challenge to researchers who are unable to use the powerful Bioconductor and its numerous tools due to their lack of knowledge of R language. Among the few existing software programs that offer a graphic user interface to Bioconductor packages, none have implemented a comprehensive strategy to address the accuracy and reliability issue of microarray data analysis due to the well known probe design problems associated with many widely used microarray chips. There is also a lack of tools that would expedite the functional analysis of microarray results. We present Microarray Я US, an R-based graphical user interface that implements over a dozen popular Bioconductor packages to offer researchers a streamlined workflow for routine differential microarray expression data analysis without the need to learn R language. In order to enable a more accurate analysis and interpretation of microarray data, we incorporated the latest custom probe re-definition and re-annotation for Affymetrix and Illumina chips. A versatile microarray results output utility tool was also implemented for easy and fast generation of input files for over 20 of the most widely used functional analysis software programs. Coupled with a well-designed user interface, Microarray Я US leverages cutting edge Bioconductor packages for researchers with no knowledge in R language. It also enables a more reliable and accurate microarray data analysis and expedites downstream functional analysis of microarray results.

A framework for the establishment of a cnidarian gene regulatory network for "endomesoderm" specification: the inputs of ß-catenin/TCF signaling.

PubMed

Röttinger, Eric; Dahlin, Paul; Martindale, Mark Q

2012-01-01

Understanding the functional relationship between intracellular factors and extracellular signals is required for reconstructing gene regulatory networks (GRN) involved in complex biological processes. One of the best-studied bilaterian GRNs describes endomesoderm specification and predicts that both mesoderm and endoderm arose from a common GRN early in animal evolution. Compelling molecular, genomic, developmental, and evolutionary evidence supports the hypothesis that the bifunctional gastrodermis of the cnidarian-bilaterian ancestor is derived from the same evolutionary precursor of both endodermal and mesodermal germ layers in all other triploblastic bilaterian animals. We have begun to establish the framework of a provisional cnidarian "endomesodermal" gene regulatory network in the sea anemone, Nematostella vectensis, by using a genome-wide microarray analysis on embryos in which the canonical Wnt/ß-catenin pathway was ectopically targeted for activation by two distinct pharmaceutical agents (lithium chloride and 1-azakenpaullone) to identify potential targets of endomesoderm specification. We characterized 51 endomesodermally expressed transcription factors and signaling molecule genes (including 18 newly identified) with fine-scale temporal (qPCR) and spatial (in situ) analysis to define distinct co-expression domains within the animal plate of the embryo and clustered genes based on their earliest zygotic expression. Finally, we determined the input of the canonical Wnt/ß-catenin pathway into the cnidarian endomesodermal GRN using morpholino and mRNA overexpression experiments to show that NvTcf/canonical Wnt signaling is required to pattern both the future endomesodermal and ectodermal domains prior to gastrulation, and that both BMP and FGF (but not Notch) pathways play important roles in germ layer specification in this animal. We show both evolutionary conserved as well as profound differences in endomesodermal GRN structure compared to bilaterians that may provide fundamental insight into how GRN subcircuits have been adopted, rewired, or co-opted in various animal lineages that give rise to specialized endomesodermal cell types.

Comprehensive analysis of correlation coefficients estimated from pooling heterogeneous microarray data

PubMed Central

2013-01-01

Background The synthesis of information across microarray studies has been performed by combining statistical results of individual studies (as in a mosaic), or by combining data from multiple studies into a large pool to be analyzed as a single data set (as in a melting pot of data). Specific issues relating to data heterogeneity across microarray studies, such as differences within and between labs or differences among experimental conditions, could lead to equivocal results in a melting pot approach. Results We applied statistical theory to determine the specific effect of different means and heteroskedasticity across 19 groups of microarray data on the sign and magnitude of gene-to-gene Pearson correlation coefficients obtained from the pool of 19 groups. We quantified the biases of the pooled coefficients and compared them to the biases of correlations estimated by an effect-size model. Mean differences across the 19 groups were the main factor determining the magnitude and sign of the pooled coefficients, which showed largest values of bias as they approached ±1. Only heteroskedasticity across the pool of 19 groups resulted in less efficient estimations of correlations than did a classical meta-analysis approach of combining correlation coefficients. These results were corroborated by simulation studies involving either mean differences or heteroskedasticity across a pool of N > 2 groups. Conclusions The combination of statistical results is best suited for synthesizing the correlation between expression profiles of a gene pair across several microarray studies. PMID:23822712

Pathway and network-based analysis of genome-wide association studies and RT-PCR validation in polycystic ovary syndrome

PubMed Central

Shen, Haoran; Liang, Zhou; Zheng, Saihua; Li, Xuelian

2017-01-01

The purpose of this study was to identify promising candidate genes and pathways in polycystic ovary syndrome (PCOS). Microarray dataset GSE345269 obtained from the Gene Expression Omnibus database includes 7 granulosa cell samples from PCOS patients, and 3 normal granulosa cell samples. Differentially expressed genes (DEGs) were screened between PCOS and normal samples. Pathway enrichment analysis was conducted for DEGs using ClueGO and CluePedia plugin of Cytoscape. A Reactome functional interaction (FI) network of the DEGs was built using ReactomeFIViz, and then network modules were extracted, followed by pathway enrichment analysis for the modules. Expression of DEGs in granulosa cell samples was measured using quantitative RT-PCR. A total of 674 DEGs were retained, which were significantly enriched with inflammation and immune-related pathways. Eight modules were extracted from the Reactome FI network. Pathway enrichment analysis revealed significant pathways of each module: module 0, Regulation of RhoA activity and Signaling by Rho GTPases pathways shared ARHGAP4 and ARHGAP9; module 2, GlycoProtein VI-mediated activation cascade pathway was enriched with RHOG; module 3, Thromboxane A2 receptor signaling, Chemokine signaling pathway, CXCR4-mediated signaling events pathways were enriched with LYN, the hub gene of module 3. Results of RT-PCR confirmed the finding of the bioinformatic analysis that ARHGAP4, ARHGAP9, RHOG and LYN were significantly upregulated in PCOS. RhoA-related pathways, GlycoProtein VI-mediated activation cascade pathway, ARHGAP4, ARHGAP9, RHOG and LYN may be involved in the pathogenesis of PCOS. PMID:28949383

Self-directed student research through analysis of microarray datasets: a computer-based functional genomics practical class for masters-level students.

PubMed

Grenville-Briggs, Laura J; Stansfield, Ian

2011-01-01

This report describes a linked series of Masters-level computer practical workshops. They comprise an advanced functional genomics investigation, based upon analysis of a microarray dataset probing yeast DNA damage responses. The workshops require the students to analyse highly complex transcriptomics datasets, and were designed to stimulate active learning through experience of current research methods in bioinformatics and functional genomics. They seek to closely mimic a realistic research environment, and require the students first to propose research hypotheses, then test those hypotheses using specific sections of the microarray dataset. The complexity of the microarray data provides students with the freedom to propose their own unique hypotheses, tested using appropriate sections of the microarray data. This research latitude was highly regarded by students and is a strength of this practical. In addition, the focus on DNA damage by radiation and mutagenic chemicals allows them to place their results in a human medical context, and successfully sparks broad interest in the subject material. In evaluation, 79% of students scored the practical workshops on a five-point scale as 4 or 5 (totally effective) for student learning. More broadly, the general use of microarray data as a "student research playground" is also discussed. Copyright © 2011 Wiley Periodicals, Inc.

Identification and handling of artifactual gene expression profiles emerging in microarray hybridization experiments

PubMed Central

Brodsky, Leonid; Leontovich, Andrei; Shtutman, Michael; Feinstein, Elena

2004-01-01

Mathematical methods of analysis of microarray hybridizations deal with gene expression profiles as elementary units. However, some of these profiles do not reflect a biologically relevant transcriptional response, but rather stem from technical artifacts. Here, we describe two technically independent but rationally interconnected methods for identification of such artifactual profiles. Our diagnostics are based on detection of deviations from uniformity, which is assumed as the main underlying principle of microarray design. Method 1 is based on detection of non-uniformity of microarray distribution of printed genes that are clustered based on the similarity of their expression profiles. Method 2 is based on evaluation of the presence of gene-specific microarray spots within the slides’ areas characterized by an abnormal concentration of low/high differential expression values, which we define as ‘patterns of differentials’. Applying two novel algorithms, for nested clustering (method 1) and for pattern detection (method 2), we can make a dual estimation of the profile’s quality for almost every printed gene. Genes with artifactual profiles detected by method 1 may then be removed from further analysis. Suspicious differential expression values detected by method 2 may be either removed or weighted according to the probabilities of patterns that cover them, thus diminishing their input in any further data analysis. PMID:14999086

Novel calibration tools and validation concepts for microarray-based platforms used in molecular diagnostics and food safety control.

PubMed

Brunner, C; Hoffmann, K; Thiele, T; Schedler, U; Jehle, H; Resch-Genger, U

2015-04-01

Commercial platforms consisting of ready-to-use microarrays printed with target-specific DNA probes, a microarray scanner, and software for data analysis are available for different applications in medical diagnostics and food analysis, detecting, e.g., viral and bacteriological DNA sequences. The transfer of these tools from basic research to routine analysis, their broad acceptance in regulated areas, and their use in medical practice requires suitable calibration tools for regular control of instrument performance in addition to internal assay controls. Here, we present the development of a novel assay-adapted calibration slide for a commercialized DNA-based assay platform, consisting of precisely arranged fluorescent areas of various intensities obtained by incorporating different concentrations of a "green" dye and a "red" dye in a polymer matrix. These dyes present "Cy3" and "Cy5" analogues with improved photostability, chosen based upon their spectroscopic properties closely matching those of common labels for the green and red channel of microarray scanners. This simple tool allows to efficiently and regularly assess and control the performance of the microarray scanner provided with the biochip platform and to compare different scanners. It will be eventually used as fluorescence intensity scale for referencing of assays results and to enhance the overall comparability of diagnostic tests.

Global gene expression analysis of peripheral blood mononuclear cells in rhesus monkey infants with CA16 infection-induced HFMD.

PubMed

Song, Jie; Hu, Yajie; Hu, Yunguang; Wang, Jingjing; Zhang, Xiaolong; Wang, Lichun; Guo, Lei; Wang, Yancui; Ning, Ruotong; Liao, Yun; Zhang, Ying; Zheng, Huiwen; Shi, Haijing; He, Zhanlong; Li, Qihan; Liu, Longding

2016-03-02

Coxsackievirus A16 (CA16) is a dominant pathogen that results in hand, foot, and mouth disease and causes outbreaks worldwide, particularly in the Asia-Pacific region. However, the underlying molecular mechanisms remain unclear. Our previous study has demonstrated that the basic CA16 pathogenic process was successfully mimicked in rhesus monkey infant. The present study focused on the global gene expression changes in peripheral blood mononuclear cells of rhesus monkey infants with hand, foot, and mouth disease induced by CA16 infection at different time points. Genome-wide expression analysis was performed with Agilent whole-genome microarrays and established bioinformatics tools. Nine hundred and forty-eight significant differentially expressed genes that were associated with 5 gene ontology categories, including cell communication, cell cycle, immune system process, regulation of transcription and metabolic process were identified. Subsequently, the mapping of genes related to the immune system process by PANTHER pathway analysis revealed the predominance of inflammation mediated by chemokine and cytokine signaling pathways and the interleukin signaling pathway. Ultimately, co-expressed genes and their networks were analyzed. The results revealed the gene expression profile of the immune system in response to CA16 in rhesus monkey infants and suggested that such an immune response was generated as a result of the positive mobilization of the immune system. This initial microarray study will provide insights into the molecular mechanism of CA16 infection and will facilitate the identification of biomarkers for the evaluation of vaccines against this virus. Copyright © 2016 Elsevier B.V. All rights reserved.

Strengthening insights into host responses to mastitis infection in ruminants by combining heterogeneous microarray data sources

PubMed Central

2011-01-01

Background Gene expression profiling studies of mastitis in ruminants have provided key but fragmented knowledge for the understanding of the disease. A systematic combination of different expression profiling studies via meta-analysis techniques has the potential to test the extensibility of conclusions based on single studies. Using the program Pointillist, we performed meta-analysis of transcription-profiling data from six independent studies of infections with mammary gland pathogens, including samples from cattle challenged in vivo with S. aureus, E. coli, and S. uberis, samples from goats challenged in vivo with S. aureus, as well as cattle macrophages and ovine dendritic cells infected in vitro with S. aureus. We combined different time points from those studies, testing different responses to mastitis infection: overall (common signature), early stage, late stage, and cattle-specific. Results Ingenuity Pathway Analysis of affected genes showed that the four meta-analysis combinations share biological functions and pathways (e.g. protein ubiquitination and polyamine regulation) which are intrinsic to the general disease response. In the overall response, pathways related to immune response and inflammation, as well as biological functions related to lipid metabolism were altered. This latter observation is consistent with the milk fat content depression commonly observed during mastitis infection. Complementarities between early and late stage responses were found, with a prominence of metabolic and stress signals in the early stage and of the immune response related to the lipid metabolism in the late stage; both mechanisms apparently modulated by few genes, including XBP1 and SREBF1. The cattle-specific response was characterized by alteration of the immune response and by modification of lipid metabolism. Comparison of E. coli and S. aureus infections in cattle in vivo revealed that affected genes showing opposite regulation had the same altered biological functions and provided evidence that E. coli caused a stronger host response. Conclusions This meta-analysis approach reinforces previous findings but also reveals several novel themes, including the involvement of genes, biological functions, and pathways that were not identified in individual studies. As such, it provides an interesting proof of principle for future studies combining information from diverse heterogeneous sources. PMID:21569310

Identification and Analyses of AUX-IAA target genes controlling multiple pathways in developing fiber cells of Gossypium hirsutum L

PubMed Central

Nigam, Deepti; Sawant, Samir V

2013-01-01

Technological development led to an increased interest in systems biological approaches in plants to characterize developmental mechanism and candidate genes relevant to specific tissue or cell morphology. AUX-IAA proteins are important plant-specific putative transcription factors. There are several reports on physiological response of this family in Arabidopsis but in cotton fiber the transcriptional network through which AUX-IAA regulated its target genes is still unknown. in-silico modelling of cotton fiber development specific gene expression data (108 microarrays and 22,737 genes) using Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) reveals 3690 putative AUX-IAA target genes of which 139 genes were known to be AUX-IAA co-regulated within Arabidopsis. Further AUX-IAA targeted gene regulatory network (GRN) had substantial impact on the transcriptional dynamics of cotton fiber, as showed by, altered TF networks, and Gene Ontology (GO) biological processes and metabolic pathway associated with its target genes. Analysis of the AUX-IAA-correlated gene network reveals multiple functions for AUX-IAA target genes such as unidimensional cell growth, cellular nitrogen compound metabolic process, nucleosome organization, DNA-protein complex and process related to cell wall. These candidate networks/pathways have a variety of profound impacts on such cellular functions as stress response, cell proliferation, and cell differentiation. While these functions are fairly broad, their underlying TF networks may provide a global view of AUX-IAA regulated gene expression and a GRN that guides future studies in understanding role of AUX-IAA box protein and its targets regulating fiber development. PMID:24497725

Development and Validation of Sandwich ELISA Microarrays with Minimal Assay Interference

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gonzalez, Rachel M.; Servoss, Shannon; Crowley, Sheila A.

Sandwich enzyme-linked immunosorbent assay (ELISA) microarrays are emerging as a strong candidate platform for multiplex biomarker analysis because of the ELISA’s ability to quantitatively measure rare proteins in complex biological fluids. Advantages of this platform are high-throughput potential, assay sensitivity and stringency, and the similarity to the standard ELISA test, which facilitates assay transfer from a research setting to a clinical laboratory. However, a major concern with the multiplexing of ELISAs is maintaining high assay specificity. In this study, we systematically determine the amount of assay interference and noise contributed by individual components of the multiplexed 24-assay system. We findmore » that non-specific reagent cross-reactivity problems are relatively rare. We did identify the presence of contaminant antigens in a “purified antigen”. We tested the validated ELISA microarray chip using paired serum samples that had been collected from four women at a 6-month interval. This analysis demonstrated that protein levels typically vary much more between individuals then within an individual over time, a result which suggests that longitudinal studies may be useful in controlling for biomarker variability across a population. Overall, this research demonstrates the importance of a stringent screening protocol and the value of optimizing the antibody and antigen concentrations when designing chips for ELISA microarrays.« less

Epigenetic modification with trichostatin A does not correct specific errors of somatic cell nuclear transfer at the transcriptomic level; highlighting the non-random nature of oocyte-mediated reprogramming errors.

PubMed

Hosseini, Sayyed Morteza; Dufort, Isabelle; Nieminen, Julie; Moulavi, Fariba; Ghanaei, Hamid Reza; Hajian, Mahdi; Jafarpour, Farnoosh; Forouzanfar, Mohsen; Gourbai, Hamid; Shahverdi, Abdol Hossein; Nasr-Esfahani, Mohammad Hossein; Sirard, Marc-André

2016-01-04

The limited duration and compromised efficiency of oocyte-mediated reprogramming, which occurs during the early hours following somatic cell nuclear transfer (SCNT), may significantly interfere with epigenetic reprogramming, contributing to the high incidence of ill/fatal transcriptional phenotypes and physiological anomalies occurring later during pre- and post-implantation events. A potent histone deacetylase inhibitor, trichostatin A (TSA), was used to understand the effects of assisted epigenetic modifications on transcriptional profiles of SCNT blastocysts and to identify specific or categories of genes affected. TSA improved the yield and quality of in vitro embryo development compared to control (CTR-NT). Significance analysis of microarray results revealed that of 37,238 targeted gene transcripts represented on the microarray slide, a relatively small number of genes were differentially expressed in CTR-NT (1592 = 4.3 %) and TSA-NT (1907 = 5.1 %) compared to IVF embryos. For both SCNT groups, the majority of downregulated and more than half of upregulated genes were common and as much as 15 % of all deregulated transcripts were located on chromosome X. Correspondence analysis clustered CTR-NT and IVF transcriptomes close together regardless of the embryo production method, whereas TSA changed SCNT transcriptome to a very clearly separated cluster. Ontological classification of deregulated genes using IPA uncovered a variety of functional categories similarly affected in both SCNT groups with a preponderance of genes required for biological processes. Examination of genes involved in different canonical pathways revealed that the WNT and FGF pathways were similarly affected in both SCNT groups. Although TSA markedly changed epigenetic reprogramming of donor cells (DNA-methylation, H3K9 acetylation), reconstituted oocytes (5mC, 5hmC), and blastocysts (DNA-methylation, H3K9 acetylation), these changes did not recapitulate parallel marked changes in chromatin remodeling, and nascent mRNA and OCT4-EGFP expression of TSA-NT vs. CRT-NT embryos. The results obtained suggest that despite the extensive reprogramming of donor cells that occurred by the blastocyst stage, SCNT-specific errors are of a non-random nature in bovine and are not responsive to epigenetic modifications by TSA.

Profiling of zinc altered gene expression in human prostate normal versus cancer cells: a time course study

PubMed Central

Lin, Shu-fei; Wei, Hua; Maeder, Dennis; Franklin, Renty B.; Feng, Pei

2010-01-01

We have demonstrated that zinc exposure induces apoptosis in human prostate cancer cells (PC-3) and benign hyperplasia cells (BPH), but not in normal prostate cells (HPR-1). However, the mechanisms underlying the effects of zinc on prostate cancer cell growth and zinc homeostasis remain unclear. To explore the zinc effect on gene expression profiles in normal (HPR-1) and malignant prostate cells (PC-3), we conducted a time course study of Zn treatment with microarray analysis. Microarray data were evaluated and profiled using computational approach for the primary and secondary data analyses. Final analyses were focused on the genes: 1. highly sensitive to zinc, 2. associated with zinc homeostasis, i.e. metallothioneins (MTs), solute zinc carriers (ZIPs) and zinc exporters (ZnTs), 3. relevant to several oncogenic pathways. Zinc-mediated mRNA levels of MT isotypes were further validated by semi-quantitative RT-PCR. Results showed that zinc effect on genome-wide expression patterns was cell type specific, and zinc appeared to have mainly down-regulatory effects on thousands of genes (1,953 in HPR-1; 3,534 in PC-3) with a threshold of ±2.5-fold, while fewer genes were up-regulated (872 in HPR-1; 571 in PC-3). The patterns of zinc effect on functional MT genes’ expression provided evidence for the cell-type dependent zinc accumulation and zinc-induced apoptosis in prostate cells. In PC-3 cells, zinc significantly up-regulated the expression of MT-1 isotypes -J and -M, denoted previously as “non-functional” MT genes, and now a depictive molecular structure of MT-1J was proposed. Examination of genes involved in oncogenic pathways indicated that certain genes, e.g. Fos, Akt1, Jak3 and PI3K were highly regulated by zinc with cell type specificity. This work provided an extensive database on zinc related prostate cancer research. The strategy of data analysis was devoted to find genes highly sensitive to Zn, and the genes associated with zinc accumulation and zinc-induced apoptosis. The results indicate that zinc regulation of gene expression is cell-type specific, and MT genes play important roles in prostate malignancy. PMID:19071009

Oxidative stress status accompanying diabetic bladder cystopathy results in the activation of protein degradation pathways.

PubMed

Kanika, Nirmala D; Chang, Jinsook; Tong, Yuehong; Tiplitsky, Scott; Lin, Juan; Yohannes, Elizabeth; Tar, Moses; Chance, Mark; Christ, George J; Melman, Arnold; Davies, Kelvin D

2011-05-01

• To investigate the role that oxidative stress plays in the development of diabetic cystopathy. • Comparative gene expression in the bladder of non-diabetic and streptozotocin (STZ)-induced 2-month- old diabetic rats was carried out using microarray analysis. • Evidence of oxidative stress was investigated in the bladder by analyzing glutathione S-transferase activity, lipid peroxidation, and carbonylation and nitrosylation of proteins. • The activity of protein degradation pathways was assessed using Western blot analysis. • Analysis of global gene expression showed that detrusor smooth muscle tissue of STZ-induced diabetes undergoes significant enrichment in targets involved in the production or regulation of reactive oxygen species (P = 1.27 × 10(-10)). The microarray analysis was confirmed by showing that markers of oxidative stress were all significantly increased in the diabetic bladder. • It was hypothesized that the sequelae to oxidative stress would be increased protein damage and apoptosis. • This was confirmed by showing that two key proteins involved in protein degradation (Nedd4 and LC3B) were greatly up-regulated in diabetic bladders compared to controls by 12.2 ± 0.76 and 4.4 ± 1.0-fold, respectively, and the apoptosis inducing protein, BAX, was up-regulated by 6.76 ± 0.76-fold. • Overall, the findings obtained in the present study add to the growing body of evidence showing that diabetic cystopathy is associated with oxidative damage of smooth muscle cells, and results in protein damage and activation of apoptotic pathways that may contribute to a deterioration in bladder function. © 2010 THE AUTHORS; BJU INTERNATIONAL © 2010 BJU INTERNATIONAL.

Quantitative comparison of microarray experiments with published leukemia related gene expression signatures.

PubMed

Klein, Hans-Ulrich; Ruckert, Christian; Kohlmann, Alexander; Bullinger, Lars; Thiede, Christian; Haferlach, Torsten; Dugas, Martin

2009-12-15

Multiple gene expression signatures derived from microarray experiments have been published in the field of leukemia research. A comparison of these signatures with results from new experiments is useful for verification as well as for interpretation of the results obtained. Currently, the percentage of overlapping genes is frequently used to compare published gene signatures against a signature derived from a new experiment. However, it has been shown that the percentage of overlapping genes is of limited use for comparing two experiments due to the variability of gene signatures caused by different array platforms or assay-specific influencing parameters. Here, we present a robust approach for a systematic and quantitative comparison of published gene expression signatures with an exemplary query dataset. A database storing 138 leukemia-related published gene signatures was designed. Each gene signature was manually annotated with terms according to a leukemia-specific taxonomy. Two analysis steps are implemented to compare a new microarray dataset with the results from previous experiments stored and curated in the database. First, the global test method is applied to assess gene signatures and to constitute a ranking among them. In a subsequent analysis step, the focus is shifted from single gene signatures to chromosomal aberrations or molecular mutations as modeled in the taxonomy. Potentially interesting disease characteristics are detected based on the ranking of gene signatures associated with these aberrations stored in the database. Two example analyses are presented. An implementation of the approach is freely available as web-based application. The presented approach helps researchers to systematically integrate the knowledge derived from numerous microarray experiments into the analysis of a new dataset. By means of example leukemia datasets we demonstrate that this approach detects related experiments as well as related molecular mutations and may help to interpret new microarray data.

DNA methylation regulated microRNAs in HPV-16-induced head and neck squamous cell carcinoma (HNSCC).

PubMed

Sannigrahi, M K; Sharma, Rajni; Singh, Varinder; Panda, Naresh K; Rattan, Vidya; Khullar, Madhu

2018-02-17

Epigenetic modifications have been reported to play an important role in regulating gene expression and these modifications become critical when they have a role in controlling another important layer of epigenetic regulation namely microRNAs. In the present study, we have identified the microRNAs that may be regulated by promoter DNA methylation and histone acetylation in Human papilloma virus-positive head and neck squamous cell carcinoma. HPV-negative cell line (UPCI:SCC-116) and HPV-16 +ve cell line (UPCI:SCC-090) were treated with methylation inhibitor (5-aza-2'-deoxycytidine, AZA) and acetylation inhibitor (Trichostatin-A, TSA), followed by micro-array analysis. The differentially expressed miRNAs were validated in control (n = 10), HPV-16 +ve (n = 30), and HPV -ve (n = 30) HNC, TCGA (n = 529) tissue samples, and two HPV -ve (SCC116 and Hacat) and two HPV +ve (SCC090 and SiHa) cell lines. Methylation-specific PCR (MSP) and chromatin immunoprecipitation assay (CHIP) were performed to validate their regulation. In silico and in vitro analyses of identified miRNAs were done to study putative pathways they target and their possible role in carcinogenesis. Among 10 miRNAs specifically up-regulated in microarray analysis of AZA-treated SCC090 cells, we observed significantly decreased expression of hsa-miR-181c-5p, hsa-miR-132-5p, hsa-miR-658 in HPV +ve HNC cohort, TCGA tissue samples, and cell lines as compared to their HPV -ve counterpart, and their promoter region also possesses CpG islands. MSP and analysis of TCGA data (MethHC) revealed increased frequency of methylation at the promoter of hsa-miR-132-5p that is negatively correlated with its expression. In TSA-treated SCC090 cells, out of 7 miRNAs, two namely Hsa-miR-129-2-3p and Hsa-miR-449a were found to be up-regulated as compared to HPV -ve cells. However, the levels of enrichment by anti-acetyl-H3 and anti-acetyl-H4 were significantly low in cell lines compared to respective controls and both were up-regulated in HPV +ve compared to HPV -ve TCGA tissue samples. In silico analysis revealed hsa-miR-132-5p targeted canonical β-catenin/wnt pathway and modulation of down-stream genes of the pathway was observed on over-expression/inhibition of hsa-miR-132-5p. This study suggests the role of epigenetic modifications in regulating expression of miRNAs in HPV +ve HNSCC.

The Use of Signal-Transduction and Metabolic Pathways to Predict Human Disease Targets from Electric and Magnetic Fields Using in vitro Data in Human Cell Lines

PubMed Central

Parham, Fred; Portier, Christopher J.; Chang, Xiaoqing; Mevissen, Meike

2016-01-01

Using in vitro data in human cell lines, several research groups have investigated changes in gene expression in cellular systems following exposure to extremely low frequency (ELF) and radiofrequency (RF) electromagnetic fields (EMF). For ELF EMF, we obtained five studies with complete microarray data and three studies with only lists of significantly altered genes. Likewise, for RF EMF, we obtained 13 complete microarray datasets and 5 limited datasets. Plausible linkages between exposure to ELF and RF EMF and human diseases were identified using a three-step process: (a) linking genes associated with classes of human diseases to molecular pathways, (b) linking pathways to ELF and RF EMF microarray data, and (c) identifying associations between human disease and EMF exposures where the pathways are significantly similar. A total of 60 pathways were associated with human diseases, mostly focused on basic cellular functions like JAK–STAT signaling or metabolic functions like xenobiotic metabolism by cytochrome P450 enzymes. ELF EMF datasets were sporadically linked to human diseases, but no clear pattern emerged. Individual datasets showed some linkage to cancer, chemical dependency, metabolic disorders, and neurological disorders. RF EMF datasets were not strongly linked to any disorders but strongly linked to changes in several pathways. Based on these analyses, the most promising area for further research would be to focus on EMF and neurological function and disorders. PMID:27656641

Microarray analysis of gene expression in the cyclooxygenase knockout mice - a connection to autism spectrum disorder.

PubMed

Rai-Bhogal, Ravneet; Ahmad, Eizaaz; Li, Hongyan; Crawford, Dorota A

2018-03-01

The cellular and molecular events that take place during brain development play an important role in governing function of the mature brain. Lipid-signalling molecules such as prostaglandin E 2 (PGE 2 ) play an important role in healthy brain development. Abnormalities along the COX-PGE 2 signalling pathway due to genetic or environmental causes have been linked to autism spectrum disorder (ASD). This study aims to evaluate the effect of altered COX-PGE 2 signalling on development and function of the prenatal brain using male mice lacking cyclooxygenase-1 and cyclooxygenase-2 (COX-1 -/- and COX-2 -/- ) as potential model systems of ASD. Microarray analysis was used to determine global changes in gene expression during embryonic days 16 (E16) and 19 (E19). Gene Ontology: Biological Process (GO:BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were implemented to identify affected developmental genes and cellular processes. We found that in both knockouts the brain at E16 had nearly twice as many differentially expressed genes, and affected biological pathways containing various ASD-associated genes important in neuronal function. Interestingly, using GeneMANIA and Cytoscape we also show that the ASD-risk genes identified in both COX-1 -/- and COX-2 -/- models belong to protein-interaction networks important for brain development despite of different cellular localization of these enzymes. Lastly, we identified eight genes that belong to the Wnt signalling pathways exclusively in the COX-2 -/- mice at E16. The level of PKA-phosphorylated β-catenin (S552), a major activator of the Wnt pathway, was increased in this model, suggesting crosstalk between the COX-2-PGE 2 and Wnt pathways during early brain development. Overall, these results provide further molecular insight into the contribution of the COX-PGE 2 pathways to ASD and demonstrate that COX-1 -/- and COX-2 -/- animals might be suitable new model systems for studying the disorders. © 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

The effects of microgravity on gene expression of Arabidopsis

NASA Astrophysics Data System (ADS)

Correll, Melanie; Stimpson, Alexander; Pereira, Rhea; Kiss, John Z.

TROPI (for TROPIsms) consisted of a series of experiments on the International Space Station to study the interaction between phototropism and gravitropism. As part of TROPI, we received frozen Arabidopsis seedlings from the ISS on three shuttle missions (STS-116, STS-117 and STS-120). These seedlings are being used for gene expression studies. Unfortunately, the quality of RNA returned from the first return mission was poor while that from the second and third missions were of high quality. This indicates that some environmental parameters were not maintained during first return mission since all of these samples were stored in the same location at -80° C on the ISS. Therefore, due to the loss during the first sample return, we had to develop new protocols to maximize RNA yields and optimize labeling techniques for microarray analysis. Using these new protocols, RNA was extracted from several sets of seedlings grown in various light treatments and µg levels and microarray analyses performed. Hundreds of genes were shown to be regulated in response to microgravity and include transcription factors (WRKY, MYB, ZF families) and those involved in plant hormone signaling (auxin, ethylene, and ABA responsive genes). The characterization of the regulated pathways and genes specific to gravity and light treatments is underway. (This project is Supported By: NASA NCC2-1200).

Gene expression profiling to characterize sediment toxicity – a pilot study using Caenorhabditis elegans whole genome microarrays

PubMed Central

Menzel, Ralph; Swain, Suresh C; Hoess, Sebastian; Claus, Evelyn; Menzel, Stefanie; Steinberg, Christian EW; Reifferscheid, Georg; Stürzenbaum, Stephen R

2009-01-01

Background Traditionally, toxicity of river sediments is assessed using whole sediment tests with benthic organisms. The challenge, however, is the differentiation between multiple effects caused by complex contaminant mixtures and the unspecific toxicity endpoints such as survival, growth or reproduction. The use of gene expression profiling facilitates the identification of transcriptional changes at the molecular level that are specific to the bio-available fraction of pollutants. Results In this pilot study, we exposed the nematode Caenorhabditis elegans to three sediments of German rivers with varying (low, medium and high) levels of heavy metal and organic contamination. Beside chemical analysis, three standard bioassays were performed: reproduction of C. elegans, genotoxicity (Comet assay) and endocrine disruption (YES test). Gene expression was profiled using a whole genome DNA-microarray approach to identify overrepresented functional gene categories and derived cellular processes. Disaccharide and glycogen metabolism were found to be affected, whereas further functional pathways, such as oxidative phosphorylation, ribosome biogenesis, metabolism of xenobiotics, aging and several developmental processes were found to be differentially regulated only in response to the most contaminated sediment. Conclusion This study demonstrates how ecotoxicogenomics can identify transcriptional responses in complex mixture scenarios to distinguish different samples of river sediments. PMID:19366437

Identifying transcription factor functions and targets by phenotypic activation

PubMed Central

Chua, Gordon; Morris, Quaid D.; Sopko, Richelle; Robinson, Mark D.; Ryan, Owen; Chan, Esther T.; Frey, Brendan J.; Andrews, Brenda J.; Boone, Charles; Hughes, Timothy R.

2006-01-01

Mapping transcriptional regulatory networks is difficult because many transcription factors (TFs) are activated only under specific conditions. We describe a generic strategy for identifying genes and pathways induced by individual TFs that does not require knowledge of their normal activation cues. Microarray analysis of 55 yeast TFs that caused a growth phenotype when overexpressed showed that the majority caused increased transcript levels of genes in specific physiological categories, suggesting a mechanism for growth inhibition. Induced genes typically included established targets and genes with consensus promoter motifs, if known, indicating that these data are useful for identifying potential new target genes and binding sites. We identified the sequence 5′-TCACGCAA as a binding sequence for Hms1p, a TF that positively regulates pseudohyphal growth and previously had no known motif. The general strategy outlined here presents a straightforward approach to discovery of TF activities and mapping targets that could be adapted to any organism with transgenic technology. PMID:16880382

Eye-Specific Gene Expression following Embryonic Ethanol Exposure in Zebrafish: Roles for Heat Shock Factor 1

PubMed Central

Kashyap, Bhavani; Pegorsch, Laurel; Frey, Ruth A.; Sun, Chi; Shelden, Eric A.; Stenkamp, Deborah L.

2014-01-01

The mechanisms through which ethanol exposure results in developmental defects remain unclear. We used the zebrafish model to elucidate eye-specific mechanisms that underlie ethanol-mediated microphthalmia (reduced eye size), through time-series microarray analysis of gene expression within eyes of embryos exposed to 1.5% ethanol. 62 genes were differentially expressed (DE) in ethanol-treated as compared to control eyes sampled during retinal neurogenesis (24-48 hours post-fertilization). The EDGE (extraction of differential gene expression) algorithm identified >3000 genes DE over developmental time in ethanol-exposed eyes as compared to controls. The DE lists included several genes indicating a mis-regulated cellular stress response due to ethanol exposure. Combined treatment with sub-threshold levels of ethanol and a morpholino targeting heat shock factor 1 mRNA resulted in microphthalmia, suggesting convergent molecular pathways. Thermal preconditioning partially prevented ethanol-mediated microphthalmia while maintaining Hsf-1 expression. These data suggest roles for reduced Hsf-1 in mediating microphthalmic effects of embryonic ethanol exposure. PMID:24355176

Changes in Global Transcriptional Profiling of Women Following Obesity Surgery Bypass.

PubMed

Pinhel, Marcela Augusta de Souza; Noronha, Natalia Yumi; Nicoletti, Carolina Ferreira; de Oliveira, Bruno Affonso Parente; Cortes-Oliveira, Cristiana; Pinhanelli, Vitor Caressato; Salgado Junior, Wilson; Machry, Ana Julia; da Silva Junior, Wilson Araújo; Souza, Dorotéia Rossi Silva; Marchini, Júlio Sérgio; Nonino, Carla Barbosa

2018-01-01

Differential gene expression in peripheral blood mononuclear cells (PBMCs) after Roux-en-Y gastric bypass (RYGB) is poorly characterized. Markers of these processes may provide a deeper understanding of the mechanisms that underlie these events. The main goal of this study was to identify changes in PBMC gene expression in women with obesity before and 6 months after RYGB-induced weight loss. The ribonucleic acid (RNA) of PBMCs from 13 obese women was analyzed before and 6 months after RYGB; the RNA of PBMCs from nine healthy women served as control. The gene expression levels were determined by microarray analysis. Significant differences in gene expression were validated by real-time quantitative polymerase chain reaction (RT-qPCR). Microarray analysis for comparison of the pre- and postoperative periods showed that 1366 genes were differentially expressed genes (DEGs). The main pathways were related to gene transcription; lipid, energy, and glycide metabolism; inflammatory and immunological response; cell differentiation; oxidative stress regulation; response to endogenous and exogenous stimuli; substrate oxidation; mTOR signaling pathway; interferon signaling; mitogen-activated protein kinases (MAPK), cAMP response element binding protein (CREB1), heat shock factor 1 (HSF1), and sterol regulatory element binding protein 1c (SREBP-1c) gene expression; adipocyte differentiation; and methylation. Six months after bariatric surgery and significant weight loss, many molecular pathways involved in obesity and metabolic diseases change. These findings are an important tool to identify potential targets for therapeutic intervention and clinical practice of nutritional genomics in obesity.

Role of Insulin-Like Growth Factor-1 Signaling Pathway in Cisplatin-Resistant Lung Cancer Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun Yunguang; Zheng Siyuan; Torossian, Artour

2012-03-01

Purpose: The development of drug-resistant phenotypes has been a major obstacle to cisplatin use in non-small-cell lung cancer. We aimed to identify some of the molecular mechanisms that underlie cisplatin resistance using microarray expression analysis. Methods and Materials: H460 cells were treated with cisplatin. The differences between cisplatin-resistant lung cancer cells and parental H460 cells were studied using Western blot, MTS, and clonogenic assays, in vivo tumor implantation, and microarray analysis. The cisplatin-R cells were treated with human recombinant insulin-like growth factor (IGF) binding protein-3 and siRNA targeting IGF-1 receptor. Results: Cisplatin-R cells illustrated greater expression of the markers CD133more » and aldehyde dehydrogenase, more rapid in vivo tumor growth, more resistance to cisplatin- and etoposide-induced apoptosis, and greater survival after treatment with cisplatin or radiation than the parental H460 cells. Also, cisplatin-R demonstrated decreased expression of insulin-like growth factor binding protein-3 and increased activation of IGF-1 receptor signaling compared with parental H460 cells in the presence of IGF-1. Human recombinant IGF binding protein-3 reversed cisplatin resistance in cisplatin-R cells and targeting of IGF-1 receptor using siRNA resulted in sensitization of cisplatin-R-cells to cisplatin and radiation. Conclusions: The IGF-1 signaling pathway contributes to cisplatin-R to cisplatin and radiation. Thus, this pathway represents a potential target for improved lung cancer response to treatment.« less

Construction and application of EST library from Setaria italica in response to dehydration stress.

PubMed

Zhang, Jinpeng; Liu, Tingsong; Fu, Junjie; Zhu, Yun; Jia, Jinping; Zheng, Jun; Zhao, Yinhe; Zhang, Ying; Wang, Guoying

2007-07-01

Foxtail millet is a gramineous crop with low water requirement. Despite its high water use efficiency, less attention has been paid to the molecular genetics of foxtail millet. This article reports the construction of subtracted cDNA libraries from foxtail millet seedlings under dehydration stress and the expression profile analysis of 1947 UniESTs from the subtracted cDNA libraries by a cDNA microarray. The results showed that 95 and 57 ESTs were upregulated by dehydration stress, respectively, in roots and shoots of seedlings and that 10 and 27 ESTs were downregulated, respectively, in roots and shoots. The expression profile analysis showed that genes induced in foxtail millet roots were different from those in shoots during dehydration stress and that the early response to dehydration stress in foxtail millet roots was the activation of the glycolysis metabolism. Moreover, protein degradation pathway may also play a pivotal role in drought-tolerant responses of foxtail millet. Finally, Northern blot analysis validated well the cDNA microarray data.

CNV-ROC: A cost effective, computer-aided analytical performance evaluator of chromosomal microarrays

PubMed Central

Goodman, Corey W.; Major, Heather J.; Walls, William D.; Sheffield, Val C.; Casavant, Thomas L.; Darbro, Benjamin W.

2016-01-01

Chromosomal microarrays (CMAs) are routinely used in both research and clinical laboratories; yet, little attention has been given to the estimation of genome-wide true and false negatives during the assessment of these assays and how such information could be used to calibrate various algorithmic metrics to improve performance. Low-throughput, locus-specific methods such as fluorescence in situ hybridization (FISH), quantitative PCR (qPCR), or multiplex ligation-dependent probe amplification (MLPA) preclude rigorous calibration of various metrics used by copy number variant (CNV) detection algorithms. To aid this task, we have established a comparative methodology, CNV-ROC, which is capable of performing a high throughput, low cost, analysis of CMAs that takes into consideration genome-wide true and false negatives. CNV-ROC uses a higher resolution microarray to confirm calls from a lower resolution microarray and provides for a true measure of genome-wide performance metrics at the resolution offered by microarray testing. CNV-ROC also provides for a very precise comparison of CNV calls between two microarray platforms without the need to establish an arbitrary degree of overlap. Comparison of CNVs across microarrays is done on a per-probe basis and receiver operator characteristic (ROC) analysis is used to calibrate algorithmic metrics, such as log2 ratio threshold, to enhance CNV calling performance. CNV-ROC addresses a critical and consistently overlooked aspect of analytical assessments of genome-wide techniques like CMAs which is the measurement and use of genome-wide true and false negative data for the calculation of performance metrics and comparison of CNV profiles between different microarray experiments. PMID:25595567

Integration analysis of quantitative proteomics and transcriptomics data identifies potential targets of frizzled-8 protein-related antiproliferative factor in vivo.

PubMed

Yang, Wei; Kim, Yongsoo; Kim, Taek-Kyun; Keay, Susan K; Kim, Kwang Pyo; Steen, Hanno; Freeman, Michael R; Hwang, Daehee; Kim, Jayoung

2012-12-01

What's known on the subject? and What does the study add? Interstitial cystitis (IC) is a prevalent and debilitating pelvic disorder generally accompanied by chronic pain combined with chronic urinating problems. Over one million Americans are affected, especially middle-aged women. However, its aetiology or mechanism remains unclear. No efficient drug has been provided to patients. Several urinary biomarker candidates have been identified for IC; among the most promising is antiproliferative factor (APF), whose biological activity is detectable in urine specimens from >94% of patients with both ulcerative and non-ulcerative IC. The present study identified several important mediators of the effect of APF on bladder cell physiology, suggesting several candidate drug targets against IC. In an attempt to identify potential proteins and genes regulated by APF in vivo, and to possibly expand the APF-regulated network identified by stable isotope labelling by amino acids in cell culture (SILAC), we performed an integration analysis of our own SILAC data and the microarray data of Gamper et al. (2009) BMC Genomics 10: 199. Notably, two of the proteins (i.e. MAPKSP1 and GSPT1) that are down-regulated by APF are involved in the activation of mTORC1, suggesting that the mammalian target of rapamycin (mTOR) pathway is potentially a critical pathway regulated by APF in vivo. Several components of the mTOR pathway are currently being studied as potential therapeutic targets in other diseases. Our analysis suggests that this pathway might also be relevant in the design of diagnostic tools and medications targeting IC. • To enhance our understanding of the interstitial cystitis urine biomarker antiproliferative factor (APF), as well as interstitial cystitis biology more generally at the systems level, we reanalyzed recently published large-scale quantitative proteomics and in vivo transcriptomics data sets using an integration analysis tool that we have developed. • To identify more differentially expressed genes with a lower false discovery rate from a previously published microarray data set, an integrative hypothesis-testing statistical approach was applied. • For validation experiments, expression and phosphorylation levels of select proteins were evaluated by western blotting. • Integration analysis of this transcriptomics data set with our own quantitative proteomics data set identified 10 genes that are potentially regulated by APF in vivo from 4140 differentially expressed genes identified with a false discovery rate of 1%. • Of these, five (i.e. JUP, MAPKSP1, GSPT1, PTGS2/COX-2 and XPOT) were found to be prominent after network modelling of the common genes identified in the proteomics and microarray studies. • This molecular signature reflects the biological processes of cell adhesion, cell proliferation and inflammation, which is consistent with the known physiological effects of APF. • Lastly, we found the mammalian target of rapamycin pathway was down-regulated in response to APF. • This unbiased integration analysis of in vitro quantitative proteomics data with in vivo quantitative transcriptomics data led to the identification of potential downstream mediators of the APF signal transduction pathway. © 2012 THE AUTHORS. BJU INTERNATIONAL © 2012 BJU INTERNATIONAL.

Subpathway-GM: identification of metabolic subpathways via joint power of interesting genes and metabolites and their topologies within pathways.

PubMed

Li, Chunquan; Han, Junwei; Yao, Qianlan; Zou, Chendan; Xu, Yanjun; Zhang, Chunlong; Shang, Desi; Zhou, Lingyun; Zou, Chaoxia; Sun, Zeguo; Li, Jing; Zhang, Yunpeng; Yang, Haixiu; Gao, Xu; Li, Xia

2013-05-01

Various 'omics' technologies, including microarrays and gas chromatography mass spectrometry, can be used to identify hundreds of interesting genes, proteins and metabolites, such as differential genes, proteins and metabolites associated with diseases. Identifying metabolic pathways has become an invaluable aid to understanding the genes and metabolites associated with studying conditions. However, the classical methods used to identify pathways fail to accurately consider joint power of interesting gene/metabolite and the key regions impacted by them within metabolic pathways. In this study, we propose a powerful analytical method referred to as Subpathway-GM for the identification of metabolic subpathways. This provides a more accurate level of pathway analysis by integrating information from genes and metabolites, and their positions and cascade regions within the given pathway. We analyzed two colorectal cancer and one metastatic prostate cancer data sets and demonstrated that Subpathway-GM was able to identify disease-relevant subpathways whose corresponding entire pathways might be ignored using classical entire pathway identification methods. Further analysis indicated that the power of a joint genes/metabolites and subpathway strategy based on their topologies may play a key role in reliably recalling disease-relevant subpathways and finding novel subpathways.

An alternative method to amplify RNA without loss of signal conservation for expression analysis with a proteinase DNA microarray in the ArrayTube format.

PubMed

Schüler, Susann; Wenz, Ingrid; Wiederanders, B; Slickers, P; Ehricht, R

2006-06-12

Recent developments in DNA microarray technology led to a variety of open and closed devices and systems including high and low density microarrays for high-throughput screening applications as well as microarrays of lower density for specific diagnostic purposes. Beside predefined microarrays for specific applications manufacturers offer the production of custom-designed microarrays adapted to customers' wishes. Array based assays demand complex procedures including several steps for sample preparation (RNA extraction, amplification and sample labelling), hybridization and detection, thus leading to a high variability between several approaches and resulting in the necessity of extensive standardization and normalization procedures. In the present work a custom designed human proteinase DNA microarray of lower density in ArrayTube format was established. This highly economic open platform only requires standard laboratory equipment and allows the study of the molecular regulation of cell behaviour by proteinases. We established a procedure for sample preparation and hybridization and verified the array based gene expression profile by quantitative real-time PCR (QRT-PCR). Moreover, we compared the results with the well established Affymetrix microarray. By application of standard labelling procedures with e.g. Klenow fragment exo-, single primer amplification (SPA) or In Vitro Transcription (IVT) we noticed a loss of signal conservation for some genes. To overcome this problem we developed a protocol in accordance with the SPA protocol, in which we included target specific primers designed individually for each spotted oligomer. Here we present a complete array based assay in which only the specific transcripts of interest are amplified in parallel and in a linear manner. The array represents a proof of principle which can be adapted to other species as well. As the designed protocol for amplifying mRNA starts from as little as 100 ng total RNA, it presents an alternative method for detecting even low expressed genes by microarray experiments in a highly reproducible and sensitive manner. Preservation of signal integrity is demonstrated out by QRT-PCR measurements. The little amounts of total RNA necessary for the analyses make this method applicable for investigations with limited material as in clinical samples from, for example, organ or tumour biopsies. Those are arguments in favour of the high potential of our assay compared to established procedures for amplification within the field of diagnostic expression profiling. Nevertheless, the screening character of microarray data must be mentioned, and independent methods should verify the results.

A hierarchical approach employing metabolic and gene expression profiles to identify the pathways that confer cytotoxicity in HepG2 cells

PubMed Central

Li, Zheng; Srivastava, Shireesh; Yang, Xuerui; Mittal, Sheenu; Norton, Paul; Resau, James; Haab, Brian; Chan, Christina

2007-01-01

Background Free fatty acids (FFA) and tumor necrosis factor alpha (TNF-α) have been implicated in the pathogenesis of many obesity-related metabolic disorders. When human hepatoblastoma cells (HepG2) were exposed to different types of FFA and TNF-α, saturated fatty acid was found to be cytotoxic and its toxicity was exacerbated by TNF-α. In order to identify the processes associated with the toxicity of saturated FFA and TNF-α, the metabolic and gene expression profiles were measured to characterize the cellular states. A computational model was developed to integrate these disparate data to reveal the underlying pathways and mechanisms involved in saturated fatty acid toxicity. Results A hierarchical framework consisting of three stages was developed to identify the processes and genes that regulate the toxicity. First, discriminant analysis identified that fatty acid oxidation and intracellular triglyceride accumulation were the most relevant in differentiating the cytotoxic phenotype. Second, gene set enrichment analysis (GSEA) was applied to the cDNA microarray data to identify the transcriptionally altered pathways and processes. Finally, the genes and gene sets that regulate the metabolic responses identified in step 1 were identified by integrating the expression of the enriched gene sets and the metabolic profiles with a multi-block partial least squares (MBPLS) regression model. Conclusion The hierarchical approach suggested potential mechanisms involved in mediating the cytotoxic and cytoprotective pathways, as well as identified novel targets, such as NADH dehydrogenases, aldehyde dehydrogenases 1A1 (ALDH1A1) and endothelial membrane protein 3 (EMP3) as modulator of the toxic phenotypes. These predictions, as well as, some specific targets that were suggested by the analysis were experimentally validated. PMID:17498300

Oligonucleotide microarray for the identification of potential mycotoxigenic fungi

PubMed Central

2010-01-01

Background Mycotoxins are secondary metabolites which are produced by numerous fungi and pose a continuous challenge to the safety and quality of food commodities in South Africa. These toxins have toxicologically relevant effects on humans and animals that eat contaminated foods. In this study, a diagnostic DNA microarray was developed for the identification of the most common food-borne fungi, as well as the genes leading to toxin production. Results A total of 40 potentially mycotoxigenic fungi isolated from different food commodities, as well as the genes that are involved in the mycotoxin synthetic pathways, were analyzed. For fungal identification, oligonucleotide probes were designed by exploiting the sequence variations of the elongation factor 1-alpha (EF-1 α) coding regions and the internal transcribed spacer (ITS) regions of the rRNA gene cassette. For the detection of fungi able to produce mycotoxins, oligonucleotide probes directed towards genes leading to toxin production from different fungal strains were identified in data available in the public domain. The probes selected for fungal identification and the probes specific for toxin producing genes were spotted onto microarray slides. Conclusions The diagnostic microarray developed can be used to identify single pure strains or cultures of potentially mycotoxigenic fungi as well as genes leading to toxin production in both laboratory samples and maize-derived foods offering an interesting potential for microbiological laboratories. PMID:20307326

Identification of genomic aberrations in hemangioblastoma by droplet digital PCR and SNP microarray highlights novel candidate genes and pathways for pathogenesis.

PubMed

Mehrian-Shai, Ruty; Yalon, Michal; Moshe, Itai; Barshack, Iris; Nass, Dvorah; Jacob, Jasmine; Dor, Chen; Reichardt, Juergen K V; Constantini, Shlomi; Toren, Amos

2016-01-14

The genetic mechanisms underlying hemangioblastoma development are still largely unknown. We used high-resolution single nucleotide polymorphism microarrays and droplet digital PCR analysis to detect copy number variations (CNVs) in total of 45 hemangioblastoma tumors. We identified 94 CNVs with a median of 18 CNVs per sample. The most frequently gained regions were on chromosomes 1 (p36.32) and 7 (p11.2). These regions contain the EGFR and PRDM16 genes. Recurrent losses were located at chromosome 12 (q24.13), which includes the gene PTPN11. Our findings provide the first high-resolution genome-wide view of chromosomal changes in hemangioblastoma and identify 23 candidate genes: EGFR, PRDM16, PTPN11, HOXD11, HOXD13, FLT3, PTCH, FGFR1, FOXP1, GPC3, HOXC13, HOXC11, MKL1, CHEK2, IRF4, GPHN, IKZF1, RB1, HOXA9, and micro RNA, such as hsa-mir-196a-2 for hemangioblastoma pathogenesis. Furthermore, our data implicate that cell proliferation and angiogenesis promoting pathways may be involved in the molecular pathogenesis of hemangioblastoma.

Transcriptional profiling identifies differentially expressed genes in developing turkey skeletal muscle

PubMed Central

2011-01-01

Background Skeletal muscle growth and development from embryo to adult consists of a series of carefully regulated changes in gene expression. Understanding these developmental changes in agriculturally important species is essential to the production of high quality meat products. For example, consumer demand for lean, inexpensive meat products has driven the turkey industry to unprecedented production through intensive genetic selection. However, achievements of increased body weight and muscle mass have been countered by an increased incidence of myopathies and meat quality defects. In a previous study, we developed and validated a turkey skeletal muscle-specific microarray as a tool for functional genomics studies. The goals of the current study were to utilize this microarray to elucidate functional pathways of genes responsible for key events in turkey skeletal muscle development and to compare differences in gene expression between two genetic lines of turkeys. To achieve these goals, skeletal muscle samples were collected at three critical stages in muscle development: 18d embryo (hyperplasia), 1d post-hatch (shift from myoblast-mediated growth to satellite cell-modulated growth by hypertrophy), and 16wk (market age) from two genetic lines: a randombred control line (RBC2) maintained without selection pressure, and a line (F) selected from the RBC2 line for increased 16wk body weight. Array hybridizations were performed in two experiments: Experiment 1 directly compared the developmental stages within genetic line, while Experiment 2 directly compared the two lines within each developmental stage. Results A total of 3474 genes were differentially expressed (false discovery rate; FDR < 0.001) by overall effect of development, while 16 genes were differentially expressed (FDR < 0.10) by overall effect of genetic line. Ingenuity Pathways Analysis was used to group annotated genes into networks, functions, and canonical pathways. The expression of 28 genes involved in extracellular matrix regulation, cell death/apoptosis, and calcium signaling/muscle function, as well as genes with miscellaneous function was confirmed by qPCR. Conclusions The current study identified gene pathways and uncovered novel genes important in turkey muscle growth and development. Future experiments will focus further on several of these candidate genes and the expression and mechanism of action of their protein products. PMID:21385442

Superoxide Dismutases, SOD1 and SOD2, Play a Distinct Role in the Fat Body during Pupation in Silkworm Bombyx mori

PubMed Central

Nojima, Yosui; Ito, Katsuhiko; Ono, Hiromasa; Nakazato, Takeru; Bono, Hidemasa; Yokoyama, Takeshi; Sato, Ryoichi; Suetsugu, Yoshitaka; Nakamura, Yuki; Yamamoto, Kimiko; Satoh, Jun-ichi; Tabunoki, Hiroko; Fugo, Hajime

2015-01-01

One way that aerobic biological systems counteract the generation of reactive oxygen species (ROS) is with superoxide dismutase proteins SOD1 and SOD2 that metabolize superoxide radicals to molecular oxygen and hydrogen peroxide or scavenge oxygen radicals produced by the extensive oxidation-reduction and electron-transport reactions that occur in mitochondria. We characterized SOD1 and SOD2 of Bombyx mori isolated from the fat body of larvae. Immunological analysis demonstrated the presence of BmSOD1 and BmSOD2 in the silk gland, midgut, fat body, Malpighian tubules, testis and ovary from larvae to adults. We found that BmSOD2 had a unique expression pattern in the fat body through the fifth instar larval developmental stage. The anti-oxidative functions of BmSOD1 and BmSOD2 were assessed by exposing larvae to insecticide rotenone or vasodilator isosorbide dinitrate, which is an ROS generator in BmN4 cells; however, exposure to these compounds had no effect on the expression levels of either BmSOD protein. Next, we investigated the physiological role of BmSOD1 and BmSOD2 under environmental oxidative stress, applied through whole-body UV irradiation and assayed using quantitative RT-PCR, immunoblotting and microarray analysis. The mRNA expression level of both BmSOD1 and BmSOD2 was markedly increased but protein expression level was increased only slightly. To examine the differences in mRNA and protein level due to UV irradiation intensity, we performed microarray analysis. Gene set enrichment analysis revealed that genes in the insulin signaling pathway and PPAR signaling pathway were significantly up-regulated after 6 and 12 hours of UV irradiation. Taken together, the activities of BmSOD1 and BmSOD2 may be related to the response to UV irradiation stress in B. mori. These results suggest that BmSOD1 and BmSOD2 modulate environmental oxidative stress in the cell and have a specific role in fat body of B. mori during pupation. PMID:25714339

Microarray analysis of pancreatic gene expression during biotin repletion in biotin-deficient rats.

PubMed

Dakshinamurti, Krishnamurti; Bagchi, Rushita A; Abrenica, Bernard; Czubryt, Michael P

2015-12-01

Biotin is a B vitamin involved in multiple metabolic pathways. In humans, biotin deficiency is relatively rare but can cause dermatitis, alopecia, and perosis. Low biotin levels occur in individuals with type-2 diabetes, and supplementation with biotin plus chromium may improve blood sugar control. The acute effect on pancreatic gene expression of biotin repletion following chronic deficiency is unclear, therefore we induced biotin deficiency in adult male rats by feeding them a 20% raw egg white diet for 6 weeks. Animals were then randomized into 2 groups: one group received a single biotin supplement and returned to normal chow lacking egg white, while the second group remained on the depletion diet. After 1 week, pancreata were removed from biotin-deficient (BD) and biotin-repleted (BR) animals and RNA was isolated for microarray analysis. Biotin depletion altered gene expression in a manner indicative of inflammation, fibrosis, and defective pancreatic function. Conversely, biotin repletion activated numerous repair and anti-inflammatory pathways, reduced fibrotic gene expression, and induced multiple genes involved in pancreatic endocrine and exocrine function. A subset of the results was confirmed by quantitative real-time PCR analysis, as well as by treatment of pancreatic AR42J cells with biotin. The results indicate that biotin repletion, even after lengthy deficiency, results in the rapid induction of repair processes in the pancreas.

Functional analysis of PGRP-LA in Drosophila immunity.

PubMed

Gendrin, Mathilde; Zaidman-Rémy, Anna; Broderick, Nichole A; Paredes, Juan; Poidevin, Mickaël; Roussel, Alain; Lemaitre, Bruno

2013-01-01

PeptidoGlycan Recognition Proteins (PGRPs) are key regulators of the insect innate antibacterial response. Even if they have been intensively studied, some of them have yet unknown functions. Here, we present a functional analysis of PGRP-LA, an as yet uncharacterized Drosophila PGRP. The PGRP-LA gene is located in cluster with PGRP-LC and PGRP-LF, which encode a receptor and a negative regulator of the Imd pathway, respectively. Structure predictions indicate that PGRP-LA would not bind to peptidoglycan, pointing to a regulatory role of this PGRP. PGRP-LA expression was enriched in barrier epithelia, but low in the fat body. Use of a newly generated PGRP-LA deficient mutant indicates that PGRP-LA is not required for the production of antimicrobial peptides by the fat body in response to a systemic infection. Focusing on the respiratory tract, where PGRP-LA is strongly expressed, we conducted a genome-wide microarray analysis of the tracheal immune response of wild-type, Relish, and PGRP-LA mutant larvae. Comparing our data to previous microarray studies, we report that a majority of genes regulated in the trachea upon infection differ from those induced in the gut or the fat body. Importantly, antimicrobial peptide gene expression was reduced in the tracheae of larvae and in the adult gut of PGRP-LA-deficient Drosophila upon oral bacterial infection. Together, our results suggest that PGRP-LA positively regulates the Imd pathway in barrier epithelia.

Jagged1 is the pathological link between Wnt and Notch pathways in colorectal cancer

PubMed Central

Rodilla, Verónica; Villanueva, Alberto; Obrador-Hevia, Antonia; Robert-Moreno, Àlex; Fernández-Majada, Vanessa; Grilli, Andrea; López-Bigas, Nuria; Bellora, Nicolás; Albà, M. Mar; Torres, Ferran; Duñach, Mireia; Sanjuan, Xavier; Gonzalez, Sara; Gridley, Thomas; Capella, Gabriel; Bigas, Anna; Espinosa, Lluís

2009-01-01

Notch has been linked to β-catenin-dependent tumorigenesis; however, the mechanisms leading to Notch activation and the contribution of the Notch pathway to colorectal cancer is not yet understood. By microarray analysis, we have identified a group of genes downstream of Wnt/β-catenin (down-regulated when blocking Wnt/β-catenin) that are directly regulated by Notch (repressed by γ-secretase inhibitors and up-regulated by active Notch1 in the absence of β-catenin signaling). We demonstrate that Notch is downstream of Wnt in colorectal cancer cells through β-catenin-mediated transcriptional activation of the Notch-ligand Jagged1. Consistently, expression of activated Notch1 partially reverts the effects of blocking Wnt/β-catenin pathway in tumors implanted s.c. in nude mice. Crossing APCMin/+ with Jagged1+/Δ mice is sufficient to significantly reduce the size of the polyps arising in the APC mutant background indicating that Notch is an essential modulator of tumorigenesis induced by nuclear β-catenin. We show that this mechanism is operating in human tumors from Familial Adenomatous Polyposis patients. We conclude that Notch activation, accomplished by β-catenin-mediated up-regulation of Jagged1, is required for tumorigenesis in the intestine. The Notch-specific genetic signature is sufficient to block differentiation and promote vasculogenesis in tumors whereas proliferation depends on both pathways. PMID:19325125

Jagged1 is the pathological link between Wnt and Notch pathways in colorectal cancer.

PubMed

Rodilla, Verónica; Villanueva, Alberto; Obrador-Hevia, Antonia; Robert-Moreno, Alex; Fernández-Majada, Vanessa; Grilli, Andrea; López-Bigas, Nuria; Bellora, Nicolás; Albà, M Mar; Torres, Ferran; Duñach, Mireia; Sanjuan, Xavier; Gonzalez, Sara; Gridley, Thomas; Capella, Gabriel; Bigas, Anna; Espinosa, Lluís

2009-04-14

Notch has been linked to beta-catenin-dependent tumorigenesis; however, the mechanisms leading to Notch activation and the contribution of the Notch pathway to colorectal cancer is not yet understood. By microarray analysis, we have identified a group of genes downstream of Wnt/beta-catenin (down-regulated when blocking Wnt/beta-catenin) that are directly regulated by Notch (repressed by gamma-secretase inhibitors and up-regulated by active Notch1 in the absence of beta-catenin signaling). We demonstrate that Notch is downstream of Wnt in colorectal cancer cells through beta-catenin-mediated transcriptional activation of the Notch-ligand Jagged1. Consistently, expression of activated Notch1 partially reverts the effects of blocking Wnt/beta-catenin pathway in tumors implanted s.c. in nude mice. Crossing APC(Min/+) with Jagged1(+/Delta) mice is sufficient to significantly reduce the size of the polyps arising in the APC mutant background indicating that Notch is an essential modulator of tumorigenesis induced by nuclear beta-catenin. We show that this mechanism is operating in human tumors from Familial Adenomatous Polyposis patients. We conclude that Notch activation, accomplished by beta-catenin-mediated up-regulation of Jagged1, is required for tumorigenesis in the intestine. The Notch-specific genetic signature is sufficient to block differentiation and promote vasculogenesis in tumors whereas proliferation depends on both pathways.

[Preparation of the cDNA microarray on the differential expressed cDNA of senescence-accelerated mouse's hippocampus].

PubMed

Cheng, Xiao-Rui; Zhou, Wen-Xia; Zhang, Yong-Xiang

2006-05-01

Alzheimer' s disease (AD) is the most common form of dementia in the elderly. AD is an invariably fatal neurodegenerative disorder with no effective treatment. Senescence-accelerated mouse prone 8 (SAMP8) is a model for studying age-related cognitive impairments and also is a good model to study brain aging and one of mouse model of AD. The technique of cDNA microarray can monitor the expression levels of thousands of genes simultaneously and can be used to study AD with the character of multi-mechanism, multi-targets and multi-pathway. In order to disclose the mechanism of AD and find the drug targets of AD, cDNA microarray containing 3136 cDNAs amplified from the suppression subtracted cDNA library of hippocampus of SAMP8 and SAMR1 was prepared with 16 blocks and 14 x 14 pins, the housekeeping gene beta-actin and G3PDH as inner conference. The background of this microarray was low and unanimous, and dots divided evenly. The conditions of hybridization and washing were optimized during the hybridization of probe and target molecule. After the data of hybridization analysis, the differential expressed cDNAs were sequenced and analyzed by the bioinformatics, and some of genes were quantified by the real time RT-PCR and the reliability of this cDNA microarray were validated. This cDNA microarray may be the good means to select the differential expressed genes and disclose the molecular mechanism of SAMP8's brain aging and AD.

In-depth characterization of breast cancer tumor-promoting cell transcriptome by RNA sequencing and microarrays

PubMed Central

Soldà, Giulia; Merlino, Giuseppe; Fina, Emanuela; Brini, Elena; Moles, Anna; Cappelletti, Vera; Daidone, Maria Grazia

2016-01-01

Numerous studies have reported the existence of tumor-promoting cells (TPC) with self-renewal potential and a relevant role in drug resistance. However, pathways and modifications involved in the maintenance of such tumor subpopulations are still only partially understood. Sequencing-based approaches offer the opportunity for a detailed study of TPC including their transcriptome modulation. Using microarrays and RNA sequencing approaches, we compared the transcriptional profiles of parental MCF7 breast cancer cells with MCF7-derived TPC (i.e. MCFS). Data were explored using different bioinformatic approaches, and major findings were experimentally validated. The different analytical pipelines (Lifescope and Cufflinks based) yielded similar although not identical results. RNA sequencing data partially overlapped microarray results and displayed a higher dynamic range, although overall the two approaches concordantly predicted pathway modifications. Several biological functions were altered in TPC, ranging from production of inflammatory cytokines (i.e., IL-8 and MCP-1) to proliferation and response to steroid hormones. More than 300 non-coding RNAs were defined as differentially expressed, and 2,471 potential splicing events were identified. A consensus signature of genes up-regulated in TPC was derived and was found to be significantly associated with insensitivity to fulvestrant in a public breast cancer patient dataset. Overall, we obtained a detailed portrait of the transcriptome of a breast cancer TPC line, highlighted the role of non-coding RNAs and differential splicing, and identified a gene signature with a potential as a context-specific biomarker in patients receiving endocrine treatment. PMID:26556871

Non-Targeted Metabolomics Analysis of Golden Retriever Muscular Dystrophy-Affected Muscles Reveals Alterations in Arginine and Proline Metabolism, and Elevations in Glutamic and Oleic Acid In Vivo.

PubMed

Abdullah, Muhammad; Kornegay, Joe N; Honcoop, Aubree; Parry, Traci L; Balog-Alvarez, Cynthia J; O'Neal, Sara K; Bain, James R; Muehlbauer, Michael J; Newgard, Christopher B; Patterson, Cam; Willis, Monte S

2017-07-29

Like Duchenne muscular dystrophy (DMD), the Golden Retriever Muscular Dystrophy (GRMD) dog model of DMD is characterized by muscle necrosis, progressive paralysis, and pseudohypertrophy in specific skeletal muscles. This severe GRMD phenotype includes moderate atrophy of the biceps femoris (BF) as compared to unaffected normal dogs, while the long digital extensor (LDE), which functions to flex the tibiotarsal joint and serves as a digital extensor, undergoes the most pronounced atrophy. A recent microarray analysis of GRMD identified alterations in genes associated with lipid metabolism and energy production. We, therefore, undertook a non-targeted metabolomics analysis of the milder/earlier stage disease GRMD BF muscle versus the more severe/chronic LDE using GC-MS to identify underlying metabolic defects specific for affected GRMD skeletal muscle. Untargeted metabolomics analysis of moderately-affected GRMD muscle (BF) identified eight significantly altered metabolites, including significantly decreased stearamide (0.23-fold of controls, p = 2.89 × 10 -3 ), carnosine (0.40-fold of controls, p = 1.88 × 10 -2 ), fumaric acid (0.40-fold of controls, p = 7.40 × 10 -4 ), lactamide (0.33-fold of controls, p = 4.84 × 10 -2 ), myoinositol-2-phosphate (0.45-fold of controls, p = 3.66 × 10 -2 ), and significantly increased oleic acid (1.77-fold of controls, p = 9.27 × 10 -2 ), glutamic acid (2.48-fold of controls, p = 2.63 × 10 -2 ), and proline (1.73-fold of controls, p = 3.01 × 10 -2 ). Pathway enrichment analysis identified significant enrichment for arginine/proline metabolism (p = 5.88 × 10 -4 , FDR 4.7 × 10 -2 ), where alterations in L-glutamic acid, proline, and carnosine were found. Additionally, multiple Krebs cycle intermediates were significantly decreased (e.g., malic acid, fumaric acid, citric/isocitric acid, and succinic acid), suggesting that altered energy metabolism may be underlying the observed GRMD BF muscle dysfunction. In contrast, two pathways, inosine-5'-monophosphate (VIP Score 3.91) and 3-phosphoglyceric acid (VIP Score 3.08) mainly contributed to the LDE signature, with two metabolites (phosphoglyceric acid and inosine-5'-monophosphate) being significantly decreased. When the BF and LDE were compared, the most significant metabolite was phosphoric acid, which was significantly less in the GRMD BF compared to control and GRMD LDE groups. The identification of elevated BF oleic acid (a long-chain fatty acid) is consistent with recent microarray studies identifying altered lipid metabolism genes, while alterations in arginine and proline metabolism are consistent with recent studies identifying elevated L-arginine in DMD patient sera as a biomarker of disease. Together, these studies demonstrate muscle-specific alterations in GRMD-affected muscle, which illustrate previously unidentified metabolic changes.

Using Ambystoma mexicanum (Mexican axolotl) embryos, chemical genetics, and microarray analysis to identify signaling pathways associated with tissue regeneration.

PubMed

Ponomareva, Larissa V; Athippozhy, Antony; Thorson, Jon S; Voss, S Randal

2015-12-01

Amphibian vertebrates are important models in regenerative biology because they present exceptional regenerative capabilities throughout life. However, it takes considerable effort to rear amphibians to juvenile and adult stages for regeneration studies, and the relatively large sizes that frogs and salamanders achieve during development make them difficult to use in chemical screens. Here, we introduce a new tail regeneration model using late stage Mexican axolotl embryos. We show that axolotl embryos completely regenerate amputated tails in 7days before they exhaust their yolk supply and begin to feed. Further, we show that axolotl embryos can be efficiently reared in microtiter plates to achieve moderate throughput screening of soluble chemicals to investigate toxicity and identify molecules that alter regenerative outcome. As proof of principle, we identified integration 1 / wingless (Wnt), transforming growth factor beta (Tgf-β), and fibroblast growth factor (Fgf) pathway antagonists that completely block tail regeneration and additional chemicals that significantly affected tail outgrowth. Furthermore, we used microarray analysis to show that inhibition of Wnt signaling broadly affects transcription of genes associated with Wnt, Fgf, Tgf-β, epidermal growth factor (Egf), Notch, nerve growth factor (Ngf), homeotic gene (Hox), rat sarcoma/mitogen-activated protein kinase (Ras/Mapk), myelocytomatosis viral oncogene (Myc), tumor protein 53 (p53), and retinoic acid (RA) pathways. Punctuated changes in the expression of genes known to regulate vertebrate development were observed; this suggests the tail regeneration transcriptional program is hierarchically structured and temporally ordered. Our study establishes the axolotl as a chemical screening model to investigate signaling pathways associated with tissue regeneration. Copyright © 2015 Elsevier Inc. All rights reserved.

Functional genomic analysis of drug sensitivity pathways to guide adjuvant strategies in breast cancer

PubMed Central

Swanton, Charles; Szallasi, Zoltan; Brenton, James D; Downward, Julian

2008-01-01

The widespread introduction of high throughput RNA interference screening technology has revealed tumour drug sensitivity pathways to common cytotoxics such as paclitaxel, doxorubicin and 5-fluorouracil, targeted agents such as trastuzumab and inhibitors of AKT and Poly(ADP-ribose) polymerase (PARP) as well as endocrine therapies such as tamoxifen. Given the limited power of microarray signatures to predict therapeutic response in associative studies of small clinical trial cohorts, the use of functional genomic data combined with expression or sequence analysis of genes and microRNAs implicated in drug response in human tumours may provide a more robust method to guide adjuvant treatment strategies in breast cancer that are transferable across different expression platforms and patient cohorts. PMID:18986507

Modulation of Cholinergic Pathways and Inflammatory Mediators in Blast-Induced Traumatic Brain Injury

DTIC Science & Technology

2013-01-01

matic brain injury (TBI). Centrally acting acetylcholinesterase (AChE) inhibitors are also being considered as potential therapeutic candidates...repeated blast exposures [12]. AChE inhibitors are possible therapeutic candidates against Alzheimer’s disease and TBI [13–15]. In this study, we...esterase inhibitor , as described earlier [12,17–19]. Brain AChE activity was expressed as milliunits/mg protein. 2.3. Microarray analysis Various

Alteration of peripheral blood monocyte gene expression in humans following diesel exhaust inhalation

PubMed Central

Pettit, Ashley P.; Brooks, Andrew; Laumbach, Robert; Fiedler, Nancy; Wang, Qi; Strickland, Pamela Ohman; Madura, Kiran; Zhang, Junfeng; Kipen, Howard M.

2013-01-01

Context Epidemiologic associations between acutely increased cardiorespiratory morbidity and mortality and particulate air pollution are well established, but the effects of acute pollution exposure on human gene expression changes are not well understood. Objective In order to identify potential mechanisms underlying epidemiologic associations between air pollution and morbidity, we explored changes in gene expression in humans following inhalation of fresh diesel exhaust (DE), a model for particulate air pollution. Materials and methods Fourteen ethnically homogeneous (white males), young, healthy subjects underwent 60-min inhalation exposures on 2 separate days with clean filtered air (CA) or freshly generated and diluted DE at a concentration of 300 μg/m3 PM2.5. Prior to and 24 h following each session, whole blood was sampled and fractionated for peripheral blood mononuclear cell (PBMC) isolation, RNA extraction, and generation of cDNA, followed by hybridization with Agilent Whole Human Genome (4X44K) arrays. Results Oxidative stress and the ubiquitin proteasome pathway, as well as the coagulation system, were among hypothesized pathways identified by analysis of differentially expressed genes. Nine genes from these pathways were validated using real-time polymerase chain reaction (PCR) to compare fold change in expression between DE exposed and CA days. Quantitative gene fold changes generated by real-time PCR were directionally consistent with the fold changes from the microarray analysis. Discussion and conclusion Changes in gene expression connected with key oxidative stress, protein degradation, and coagulation pathways are likely to underlie observed physiologic and clinical outcomes and suggest specific avenues and sensitive time points for further physiologic exploration. PMID:22369193

Identification of the Key Genes and Pathways in Esophageal Carcinoma.

PubMed

Su, Peng; Wen, Shiwang; Zhang, Yuefeng; Li, Yong; Xu, Yanzhao; Zhu, Yonggang; Lv, Huilai; Zhang, Fan; Wang, Mingbo; Tian, Ziqiang

2016-01-01

Objective . Esophageal carcinoma (EC) is a frequently common malignancy of gastrointestinal cancer in the world. This study aims to screen key genes and pathways in EC and elucidate the mechanism of it. Methods . 5 microarray datasets of EC were downloaded from Gene Expression Omnibus. Differentially expressed genes (DEGs) were screened by bioinformatics analysis. Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction (PPI) network construction were performed to obtain the biological roles of DEGs in EC. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the expression level of DEGs in EC. Results . A total of 1955 genes were filtered as DEGs in EC. The upregulated genes were significantly enriched in cell cycle and the downregulated genes significantly enriched in Endocytosis. PPI network displayed CDK4 and CCT3 were hub proteins in the network. The expression level of 8 dysregulated DEGs including CDK4, CCT3, THSD4, SIM2, MYBL2, CENPF, CDCA3, and CDKN3 was validated in EC compared to adjacent nontumor tissues and the results were matched with the microarray analysis. Conclusion . The significantly DEGs including CDK4, CCT3, THSD4, and SIM2 may play key roles in tumorigenesis and development of EC involved in cell cycle and Endocytosis.

EDGE3: A web-based solution for management and analysis of Agilent two color microarray experiments

PubMed Central

Vollrath, Aaron L; Smith, Adam A; Craven, Mark; Bradfield, Christopher A

2009-01-01

Background The ability to generate transcriptional data on the scale of entire genomes has been a boon both in the improvement of biological understanding and in the amount of data generated. The latter, the amount of data generated, has implications when it comes to effective storage, analysis and sharing of these data. A number of software tools have been developed to store, analyze, and share microarray data. However, a majority of these tools do not offer all of these features nor do they specifically target the commonly used two color Agilent DNA microarray platform. Thus, the motivating factor for the development of EDGE3 was to incorporate the storage, analysis and sharing of microarray data in a manner that would provide a means for research groups to collaborate on Agilent-based microarray experiments without a large investment in software-related expenditures or extensive training of end-users. Results EDGE3 has been developed with two major functions in mind. The first function is to provide a workflow process for the generation of microarray data by a research laboratory or a microarray facility. The second is to store, analyze, and share microarray data in a manner that doesn't require complicated software. To satisfy the first function, EDGE3 has been developed as a means to establish a well defined experimental workflow and information system for microarray generation. To satisfy the second function, the software application utilized as the user interface of EDGE3 is a web browser. Within the web browser, a user is able to access the entire functionality, including, but not limited to, the ability to perform a number of bioinformatics based analyses, collaborate between research groups through a user-based security model, and access to the raw data files and quality control files generated by the software used to extract the signals from an array image. Conclusion Here, we present EDGE3, an open-source, web-based application that allows for the storage, analysis, and controlled sharing of transcription-based microarray data generated on the Agilent DNA platform. In addition, EDGE3 provides a means for managing RNA samples and arrays during the hybridization process. EDGE3 is freely available for download at . PMID:19732451

EDGE(3): a web-based solution for management and analysis of Agilent two color microarray experiments.

PubMed

Vollrath, Aaron L; Smith, Adam A; Craven, Mark; Bradfield, Christopher A

2009-09-04

The ability to generate transcriptional data on the scale of entire genomes has been a boon both in the improvement of biological understanding and in the amount of data generated. The latter, the amount of data generated, has implications when it comes to effective storage, analysis and sharing of these data. A number of software tools have been developed to store, analyze, and share microarray data. However, a majority of these tools do not offer all of these features nor do they specifically target the commonly used two color Agilent DNA microarray platform. Thus, the motivating factor for the development of EDGE(3) was to incorporate the storage, analysis and sharing of microarray data in a manner that would provide a means for research groups to collaborate on Agilent-based microarray experiments without a large investment in software-related expenditures or extensive training of end-users. EDGE(3) has been developed with two major functions in mind. The first function is to provide a workflow process for the generation of microarray data by a research laboratory or a microarray facility. The second is to store, analyze, and share microarray data in a manner that doesn't require complicated software. To satisfy the first function, EDGE3 has been developed as a means to establish a well defined experimental workflow and information system for microarray generation. To satisfy the second function, the software application utilized as the user interface of EDGE(3) is a web browser. Within the web browser, a user is able to access the entire functionality, including, but not limited to, the ability to perform a number of bioinformatics based analyses, collaborate between research groups through a user-based security model, and access to the raw data files and quality control files generated by the software used to extract the signals from an array image. Here, we present EDGE(3), an open-source, web-based application that allows for the storage, analysis, and controlled sharing of transcription-based microarray data generated on the Agilent DNA platform. In addition, EDGE(3) provides a means for managing RNA samples and arrays during the hybridization process. EDGE(3) is freely available for download at http://edge.oncology.wisc.edu/.

Pathway and network-based analysis of genome-wide association studies and RT-PCR validation in polycystic ovary syndrome.

PubMed

Shen, Haoran; Liang, Zhou; Zheng, Saihua; Li, Xuelian

2017-11-01

The purpose of this study was to identify promising candidate genes and pathways in polycystic ovary syndrome (PCOS). Microarray dataset GSE345269 obtained from the Gene Expression Omnibus database includes 7 granulosa cell samples from PCOS patients, and 3 normal granulosa cell samples. Differentially expressed genes (DEGs) were screened between PCOS and normal samples. Pathway enrichment analysis was conducted for DEGs using ClueGO and CluePedia plugin of Cytoscape. A Reactome functional interaction (FI) network of the DEGs was built using ReactomeFIViz, and then network modules were extracted, followed by pathway enrichment analysis for the modules. Expression of DEGs in granulosa cell samples was measured using quantitative RT-PCR. A total of 674 DEGs were retained, which were significantly enriched with inflammation and immune-related pathways. Eight modules were extracted from the Reactome FI network. Pathway enrichment analysis revealed significant pathways of each module: module 0, Regulation of RhoA activity and Signaling by Rho GTPases pathways shared ARHGAP4 and ARHGAP9; module 2, GlycoProtein VI-mediated activation cascade pathway was enriched with RHOG; module 3, Thromboxane A2 receptor signaling, Chemokine signaling pathway, CXCR4-mediated signaling events pathways were enriched with LYN, the hub gene of module 3. Results of RT-PCR confirmed the finding of the bioinformatic analysis that ARHGAP4, ARHGAP9, RHOG and LYN were significantly upregulated in PCOS. RhoA-related pathways, GlycoProtein VI-mediated activation cascade pathway, ARHGAP4, ARHGAP9, RHOG and LYN may be involved in the pathogenesis of PCOS.

Use of transcriptome sequencing to understand the pistillate flowering in hickory (Carya cathayensis Sarg.).

PubMed

Huang, You-Jun; Liu, Li-Li; Huang, Jian-Qin; Wang, Zheng-Jia; Chen, Fang-Fang; Zhang, Qi-Xiang; Zheng, Bing-Song; Chen, Ming

2013-10-10

Different from herbaceous plants, the woody plants undergo a long-period vegetative stage to achieve floral transition. They then turn into seasonal plants, flowering annually. In this study, a preliminary model of gene regulations for seasonal pistillate flowering in hickory (Carya cathayensis) was proposed. The genome-wide dynamic transcriptome was characterized via the joint-approach of RNA sequencing and microarray analysis. Differential transcript abundance analysis uncovered the dynamic transcript abundance patterns of flowering correlated genes and their major functions based on Gene Ontology (GO) analysis. To explore pistillate flowering mechanism in hickory, a comprehensive flowering gene regulatory network based on Arabidopsis thaliana was constructed by additional literature mining. A total of 114 putative flowering or floral genes including 31 with differential transcript abundance were identified in hickory. The locations, functions and dynamic transcript abundances were analyzed in the gene regulatory networks. A genome-wide co-expression network for the putative flowering or floral genes shows three flowering regulatory modules corresponding to response to light abiotic stimulus, cold stress, and reproductive development process, respectively. Totally 27 potential flowering or floral genes were recruited which are meaningful to understand the hickory specific seasonal flowering mechanism better. Flowering event of pistillate flower bud in hickory is triggered by several pathways synchronously including the photoperiod, autonomous, vernalization, gibberellin, and sucrose pathway. Totally 27 potential flowering or floral genes were recruited from the genome-wide co-expression network function module analysis. Moreover, the analysis provides a potential FLC-like gene based vernalization pathway and an 'AC' model for pistillate flower development in hickory. This work provides an available framework for pistillate flower development in hickory, which is significant for insight into regulation of flowering and floral development of woody plants.

Use of transcriptome sequencing to understand the pistillate flowering in hickory (Carya cathayensis Sarg.)

PubMed Central

2013-01-01

Background Different from herbaceous plants, the woody plants undergo a long-period vegetative stage to achieve floral transition. They then turn into seasonal plants, flowering annually. In this study, a preliminary model of gene regulations for seasonal pistillate flowering in hickory (Carya cathayensis) was proposed. The genome-wide dynamic transcriptome was characterized via the joint-approach of RNA sequencing and microarray analysis. Results Differential transcript abundance analysis uncovered the dynamic transcript abundance patterns of flowering correlated genes and their major functions based on Gene Ontology (GO) analysis. To explore pistillate flowering mechanism in hickory, a comprehensive flowering gene regulatory network based on Arabidopsis thaliana was constructed by additional literature mining. A total of 114 putative flowering or floral genes including 31 with differential transcript abundance were identified in hickory. The locations, functions and dynamic transcript abundances were analyzed in the gene regulatory networks. A genome-wide co-expression network for the putative flowering or floral genes shows three flowering regulatory modules corresponding to response to light abiotic stimulus, cold stress, and reproductive development process, respectively. Totally 27 potential flowering or floral genes were recruited which are meaningful to understand the hickory specific seasonal flowering mechanism better. Conclusions Flowering event of pistillate flower bud in hickory is triggered by several pathways synchronously including the photoperiod, autonomous, vernalization, gibberellin, and sucrose pathway. Totally 27 potential flowering or floral genes were recruited from the genome-wide co-expression network function module analysis. Moreover, the analysis provides a potential FLC-like gene based vernalization pathway and an 'AC’ model for pistillate flower development in hickory. This work provides an available framework for pistillate flower development in hickory, which is significant for insight into regulation of flowering and floral development of woody plants. PMID:24106755

Exposure to Cobalt Causes Transcriptomic and Proteomic Changes in Two Rat Liver Derived Cell Lines

PubMed Central

Permenter, Matthew G.; Dennis, William E.; Sutto, Thomas E.; Jackson, David A.; Lewis, John A.; Stallings, Jonathan D.

2013-01-01

Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies. PMID:24386269

Novel in silico technology in combination with microarrays: a state-of-the-art technology for allergy diagnosis and management?

PubMed

Melioli, Giovanni; Passalacqua, Giovanni; Canonica, Giorgio W

2014-12-01

'Allergen microarrays, in poly-sensitized allergic patients, represent a real value added in the accurate IgE profiling and in the identification of allergen(s) to administer for an effective allergen immunotherapy.' Allergen microarrays (AMA) were developed in the early 2000s to improve the diagnostic pathway of patients with allergic reactions. Nowadays, AMA are constituted by more than 100 different components (either purified or recombinant), representing genuine and cross-reacting molecules from plants and animals. The cost of the procedure had suggested its use as third-level diagnostics (following in vivo- and in vitro-specific IgE tests) in poly-sensitized patients. The complexity of the interpretation had inspired the development of in silico technologies to help clinicians in their work. Both machine learning techniques and expert systems are now available. In particular, an expert system that has been recently developed not only identifies positive and negative components but also lists dangerous components and classifies patients based on their potential responsiveness to allergen immunotherapy, on the basis of published algorithms. For these characteristics, AMA represents the state-of-the-art technology for allergy diagnosis in poly-sensitized patients.

Pathway Analysis Hints Towards Beneficial Effects of Long-Term Vibration on Human Chondrocytes.

PubMed

Lützenberg, Ronald; Solano, Kendrick; Buken, Christoph; Sahana, Jayashree; Riwaldt, Stefan; Kopp, Sascha; Krüger, Marcus; Schulz, Herbert; Saar, Kathrin; Huebner, Norbert; Hemmersbach, Ruth; Bauer, Johann; Infanger, Manfred; Grimm, Daniela; Wehland, Markus

2018-06-27

Spaceflight negatively influences the function of cartilage tissue in vivo. In vitro human chondrocytes exhibit an altered gene expression of inflammation markers after a two-hour exposure to vibration. Little is known about the impact of long-term vibration on chondrocytes. Human cartilage cells were exposed for up to 24 h (VIB) on a specialised vibration platform (Vibraplex) simulating the vibration profile which occurs during parabolic flights and compared to static control conditions (CON). Afterwards, they were investigated by phase-contrast microscopy, rhodamine phalloidin staining, microarray analysis, qPCR and western blot analysis. Morphological investigations revealed no changes between CON and VIB chondrocytes. F-Actin staining showed no alterations of the cytoskeleton in VIB compared with CON cells. DAPI and TUNEL staining did not identify apoptotic cells. ICAM-1 was elevated and vimentin, beta-tubulin and osteopontin proteins were significantly reduced in VIB compared to CON cells. qPCR of cytoskeletal genes, ITGB1, SOX3, SOX5, SOX9 did not reveal differential regulations. Microarray analysis detected 13 differentially expressed genes, mostly indicating unspecific stimulations. Pathway analyses demonstrated interactions of PSMD4 and CNOT7 with ICAM. Long-term vibration did not damage human chondrocytes in vitro. The reduction of osteopontin protein and the down-regulation of PSMD4 and TBX15 gene expression suggest that in vitro long-term vibration might even positively influence cultured chondrocytes. © 2018 The Author(s). Published by S. Karger AG, Basel.

Inflammatory Pathways in Parkinson's Disease; A BNE Microarray Study

PubMed Central

Durrenberger, Pascal. F.; Grünblatt, Edna; Fernando, Francesca S.; Monoranu, Camelia Maria; Evans, Jordan; Riederer, Peter; Reynolds, Richard; Dexter, David T.

2012-01-01

The aetiology of Parkinson's disease (PD) is yet to be fully understood but it is becoming more and more evident that neuronal cell death may be multifactorial in essence. The main focus of PD research is to better understand substantia nigra homeostasis disruption, particularly in relation to the wide-spread deposition of the aberrant protein α-synuclein. Microarray technology contributed towards PD research with several studies to date and one gene, ALDH1A1 (Aldehyde dehydrogenase 1 family, member A1), consistently reappeared across studies including the present study, highlighting dopamine (DA) metabolism dysfunction resulting in oxidative stress and most probably leading to neuronal cell death. Neuronal cell death leads to increased inflammation through the activation of astrocytes and microglia. Using our dataset, we aimed to isolate some of these pathways so to offer potential novel neuroprotective therapeutic avenues. To that effect our study has focused on the upregulation of P2X7 (purinergic receptor P2X, ligand-gated ion channel, 7) receptor pathway (microglial activation) and on the NOS3 (nitric oxide synthase 3) pathway (angiogenesis). In summary, although the exact initiator of striatal DA neuronal cell death remains to be determined, based on our analysis, this event does not remain without consequence. Extracellular ATP and reactive astrocytes appear to be responsible for the activation of microglia which in turn release proinflammatory cytokines contributing further to the parkinsonian condition. In addition to tackling oxidative stress pathways we also suggest to reduce microglial and endothelial activation to support neuronal outgrowth. PMID:22548201

Identification of key genes associated with the effect of estrogen on ovarian cancer using microarray analysis.

PubMed

Zhang, Shi-tao; Zuo, Chao; Li, Wan-nan; Fu, Xue-qi; Xing, Shu; Zhang, Xiao-ping

2016-02-01

To identify key genes related to the effect of estrogen on ovarian cancer. Microarray data (GSE22600) were downloaded from Gene Expression Omnibus. Eight estrogen and seven placebo treatment samples were obtained using a 2 × 2 factorial designs, which contained 2 cell lines (PEO4 and 2008) and 2 treatments (estrogen and placebo). Differentially expressed genes were identified by Bayesian methods, and the genes with P < 0.05 and |log2FC (fold change)| ≥0.5 were chosen as cut-off criterion. Differentially co-expressed genes (DCGs) and differentially regulated genes (DRGs) were, respectively, identified by DCe function and DRsort function in DCGL package. Topological structure analysis was performed on the important transcriptional factors (TFs) and genes in transcriptional regulatory network using tYNA. Functional enrichment analysis was, respectively, performed for DEGs and the important genes using Gene Ontology and KEGG databases. In total, 465 DEGs were identified. Functional enrichment analysis of DEGs indicated that ACVR2B, LTBP1, BMP7 and MYC involved in TGF-beta signaling pathway. The 2285 DCG pairs and 357 DRGs were identified. Topological structure analysis showed that 52 important TFs and 65 important genes were identified. Functional enrichment analysis of the important genes showed that TP53 and MLH1 participated in DNA damage response and the genes (ACVR2B, LTBP1, BMP7 and MYC) involved in TGF-beta signaling pathway. TP53, MLH1, ACVR2B, LTBP1 and BMP7 might participate in the pathogenesis of ovarian cancer.

A Sorghum bicolor expression atlas reveals dynamic genotype-specific expression profiles for vegetative tissues of grain, sweet and bioenergy sorghums

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shakoor, N; Nair, R; Crasta, O

2014-01-23

Background: Effective improvement in sorghum crop development necessitates a genomics-based approach to identify functional genes and QTLs. Sequenced in 2009, a comprehensive annotation of the sorghum genome and the development of functional genomics resources is key to enable the discovery and deployment of regulatory and metabolic genes and gene networks for crop improvement. Results: This study utilizes the first commercially available whole-transcriptome sorghum microarray (Sorgh-WTa520972F) to identify tissue and genotype-specific expression patterns for all identified Sorghum bicolor exons and UTRs. The genechip contains 1,026,373 probes covering 149,182 exons (27,577 genes) across the Sorghum bicolor nuclear, chloroplast, and mitochondrial genomes. Specificmore » probesets were also included for putative non-coding RNAs that may play a role in gene regulation (e. g., microRNAs), and confirmed functional small RNAs in related species (maize and sugarcane) were also included in our array design. We generated expression data for 78 samples with a combination of four different tissue types (shoot, root, leaf and stem), two dissected stem tissues (pith and rind) and six diverse genotypes, which included 6 public sorghum lines (R159, Atlas, Fremont, PI152611, AR2400 and PI455230) representing grain, sweet, forage, and high biomass ideotypes. Conclusions: Here we present a summary of the microarray dataset, including analysis of tissue-specific gene expression profiles and associated expression profiles of relevant metabolic pathways. With an aim to enable identification and functional characterization of genes in sorghum, this expression atlas presents a new and valuable resource to the research community.« less

Distinctive genomic signature of neural and intestinal organoids from familial Parkinson's disease patient-derived induced pluripotent stem cells.

PubMed

Son, M-Y; Sim, H; Son, Y S; Jung, K B; Lee, M-O; Oh, J-H; Chung, S-K; Jung, C-R; Kim, J

2017-12-01

The leucine-rich repeat kinase 2 (LRRK2) G2019S mutation is the most common genetic cause of Parkinson's disease (PD). There is compelling evidence that PD is not only a brain disease but also a gastrointestinal disorder; nonetheless, its pathogenesis remains unclear. We aimed to develop human neural and intestinal tissue models of PD patients harbouring an LRRK2 mutation to understand the link between LRRK2 and PD pathology by investigating the gene expression signature. We generated PD patient-specific induced pluripotent stem cells (iPSCs) carrying an LRRK2 G2019S mutation (LK2GS) and then differentiated into three-dimensional (3D) human neuroectodermal spheres (hNESs) and human intestinal organoids (hIOs). To unravel the gene and signalling networks associated with LK2GS, we analysed differentially expressed genes in the microarray data by functional clustering, gene ontology (GO) and pathway analyses. The expression profiles of LK2GS were distinct from those of wild-type controls in hNESs and hIOs. The most represented GO biological process in hNESs and hIOs was synaptic transmission, specifically synaptic vesicle trafficking, some defects of which are known to be related to PD. The results were further validated in four independent PD-specific hNESs and hIOs by microarray and qRT-PCR analysis. We provide the first evidence that LK2GS also causes significant changes in gene expression in the intestinal cells. These hNES and hIO models from the same genetic background of PD patients could be invaluable resources for understanding PD pathophysiology and for advancing the complexity of in vitro models with 3D expandable organoids. © 2017 British Neuropathological Society.

Microarray analysis reveals overlapping and specific transcriptional responses to different plant hormones in rice

PubMed Central

Garg, Rohini; Tyagi, Akhilesh K.; Jain, Mukesh

2012-01-01

Hormones exert pleiotropic effects on plant growth and development throughout the life cycle. Many of these effects are mediated at molecular level via altering gene expression. In this study, we investigated the exogenous effect of plant hormones, including auxin, cytokinin, abscisic acid, ethylene, salicylic acid and jasmonic acid, on the transcription of rice genes at whole genome level using microarray. Our analysis identified a total of 4171 genes involved in several biological processes, whose expression was altered significantly in the presence of different hormones. Further, 28% of these genes exhibited overlapping transcriptional responses in the presence of any two hormones, indicating crosstalk among plant hormones. In addition, we identified genes showing only a particular hormone-specific response, which can be used as hormone-specific markers. The results of this study will facilitate further studies in hormone biology in rice. PMID:22827941

A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria.

PubMed

Strauss, Christian; Endimiani, Andrea; Perreten, Vincent

2015-01-01

A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplification were then performed by classic PCR amplification using biotinylated primers specific for the linkers. The microarray developed by Perreten et al. (Perreten, V., Vorlet-Fawer, L., Slickers, P., Ehricht, R., Kuhnert, P., Frey, J., 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J.Clin.Microbiol. 43, 2291-2302.) was improved by additional oligonucleotides. A total of 244 oligonucleotides (26 to 37 nucleotide length and with similar melting temperatures) were spotted on the microarray, including genes conferring resistance to clinically important antibiotic classes like β-lactams, macrolides, aminoglycosides, glycopeptides and tetracyclines. Each antibiotic resistance gene is represented by at least 2 oligonucleotides designed from consensus sequences of gene families. The specificity of the oligonucleotides and the quality of the amplification and labeling were verified by analysis of a collection of 65 strains belonging to 24 species. Association between genotype and phenotype was verified for 6 antibiotics using 77 Staphylococcus strains belonging to different species and revealed 95% test specificity and a 93% predictive value of a positive test. The DNA labeling and amplification is independent of the species and of the target genes and could be used for different types of microarrays. This system has also the advantage to detect several genes within one bacterium at once, like in Staphylococcus aureus strain BM3318, in which up to 15 genes were detected. This new microarray-based detection system offers a large potential for applications in clinical diagnostic, basic research, food safety and surveillance programs for antimicrobial resistance. Copyright © 2014 Elsevier B.V. All rights reserved.

A High Phosphorus Diet Affects Lipid Metabolism in Rat Liver: A DNA Microarray Analysis

PubMed Central

Chun, Sunwoo; Bamba, Takeshi; Suyama, Tatsuya; Ishijima, Tomoko; Fukusaki, Eiichiro; Abe, Keiko; Nakai, Yuji

2016-01-01

A high phosphorus (HP) diet causes disorders of renal function, bone metabolism, and vascular function. We previously demonstrated that DNA microarray analysis is an appropriate method to comprehensively evaluate the effects of a HP diet on kidney dysfunction such as calcification, fibrillization, and inflammation. We reported that type IIb sodium-dependent phosphate transporter is significantly up-regulated in this context. In the present study, we performed DNA microarray analysis to investigate the effects of a HP diet on the liver, which plays a pivotal role in energy metabolism. DNA microarray analysis was performed with total RNA isolated from the livers of rats fed a control diet (containing 0.3% phosphorus) or a HP diet (containing 1.2% phosphorus). Gene Ontology analysis of differentially expressed genes (DEGs) revealed that the HP diet induced down-regulation of genes involved in hepatic amino acid catabolism and lipogenesis, while genes related to fatty acid β-oxidation process were up-regulated. Although genes related to fatty acid biosynthesis were down-regulated in HP diet-fed rats, genes important for the elongation and desaturation reactions of omega-3 and -6 fatty acids were up-regulated. Concentrations of hepatic arachidonic acid and eicosapentaenoic acid were increased in HP diet-fed rats. These essential fatty acids activate peroxisome proliferator-activated receptor alpha (PPARα), a transcription factor for fatty acid β-oxidation. Evaluation of the upstream regulators of DEGs using Ingenuity Pathway Analysis indicated that PPARα was activated in the livers of HP diet-fed rats. Furthermore, the serum concentration of fibroblast growth factor 21, a hormone secreted from the liver that promotes fatty acid utilization in adipose tissue as a PPARα target gene, was higher (p = 0.054) in HP diet-fed rats than in control diet-fed rats. These data suggest that a HP diet enhances energy expenditure through the utilization of free fatty acids released via lipolysis of white adipose tissue. PMID:27187182

New technology and resources for cryptococcal research

PubMed Central

Zhang, Nannan; Park, Yoon-Dong; Williamson, Peter R.

2014-01-01

Rapid advances in molecular biology and genome sequencing have enabled the generation of new technology and resources for cryptococcal research. RNAi-mediated specific gene knock down has become routine and more efficient by utilizing modified shRNA plasmids and convergent promoter RNAi constructs. This system was recently applied in a high-throughput screen to identify genes involved in host-pathogen interactions. Gene deletion efficiencies have also been improved by increasing rates of homologous recombination through a number of approaches, including a combination of double-joint PCR with split-marker transformation, the use of dominant selectable markers and the introduction of Cre-Loxp systems into Cryptococcus. Moreover, visualization of cryptococcal proteins has become more facile using fusions with codon-optimized fluorescent tags, such as green or red fluorescent proteins or, mCherry. Using recent genome-wide analytical tools, new transcriptional factors and regulatory proteins have been identified in novel virulence-related signaling pathways by employing microarray analysis, RNA-sequencing and proteomic analysis. PMID:25460849

Somatic ACE regulates self-renewal of mouse spermatogonial stem cells via the MAPK signaling pathway.

PubMed

Gao, Tingting; Zhao, Xin; Liu, Chenchen; Shao, Binbin; Zhang, Xi; Li, Kai; Cai, Jinyang; Wang, Su; Huang, Xiaoyan

2018-05-24

Spermatogonial stem cell (SSC) self-renewal is an indispensable part of spermatogenesis. Angiotensin I-converting enzyme (ACE) is a zinc dipeptidyl carboxypeptidase that plays a critical role in regulation of the renin-angiotensin system. Here, we used RT-PCR and Western blot analysis to confirm that somatic ACE (sACE) but not testicular ACE (tACE) is highly expressed in mouse testis before postpartum day 7 and in cultured SSCs. Our results revealed that sACE is located on the membrane of SSCs. Treating cultured SSCs with the ACE competitive inhibitor captopril was found to inhibit sACE activity, and significantly reduced the proliferation rate of SSCs. Microarray analysis identified 651 genes with significant differential expression. KEGG pathway analysis showed that these differentially expressed genes are mainly involved in the mitogen-activated protein kinase (MAPK) signaling pathway and cell cycle. sACE was found to play an important role in SSC self-renewal via the regulation of MAPK-dependent cell proliferation.

Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)

PubMed Central

Deutsch, Eric W; Ball, Catherine A; Berman, Jules J; Bova, G Steven; Brazma, Alvis; Bumgarner, Roger E; Campbell, David; Causton, Helen C; Christiansen, Jeffrey H; Daian, Fabrice; Dauga, Delphine; Davidson, Duncan R; Gimenez, Gregory; Goo, Young Ah; Grimmond, Sean; Henrich, Thorsten; Herrmann, Bernhard G; Johnson, Michael H; Korb, Martin; Mills, Jason C; Oudes, Asa J; Parkinson, Helen E; Pascal, Laura E; Pollet, Nicolas; Quackenbush, John; Ramialison, Mirana; Ringwald, Martin; Salgado, David; Sansone, Susanna-Assunta; Sherlock, Gavin; Stoeckert, Christian J; Swedlow, Jason; Taylor, Ronald C; Walashek, Laura; Warford, Anthony; Wilkinson, David G; Zhou, Yi; Zon, Leonard I; Liu, Alvin Y; True, Lawrence D

2015-01-01

One purpose of the biomedical literature is to report results in sufficient detail so that the methods of data collection and analysis can be independently replicated and verified. Here we present for consideration a minimum information specification for gene expression localization experiments, called the “Minimum Information Specification For In Situ Hybridization and Immunohistochemistry Experiments (MISFISHIE)”. It is modelled after the MIAME (Minimum Information About a Microarray Experiment) specification for microarray experiments. Data specifications like MIAME and MISFISHIE specify the information content without dictating a format for encoding that information. The MISFISHIE specification describes six types of information that should be provided for each experiment: Experimental Design, Biomaterials and Treatments, Reporters, Staining, Imaging Data, and Image Characterizations. This specification has benefited the consortium within which it was initially developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal. PMID:18327244

Identification of Differentially Expressed IGFBP5-Related Genes in Breast Cancer Tumor Tissues Using cDNA Microarray Experiments.

PubMed

Akkiprik, Mustafa; Peker, İrem; Özmen, Tolga; Amuran, Gökçe Güllü; Güllüoğlu, Bahadır M; Kaya, Handan; Özer, Ayşe

2015-11-10

IGFBP5 is an important regulatory protein in breast cancer progression. We tried to identify differentially expressed genes (DEGs) between breast tumor tissues with IGFBP5 overexpression and their adjacent normal tissues. In this study, thirty-eight breast cancer and adjacent normal breast tissue samples were used to determine IGFBP5 expression by qPCR. cDNA microarrays were applied to the highest IGFBP5 overexpressed tumor samples compared to their adjacent normal breast tissue. Microarray analysis revealed that a total of 186 genes were differentially expressed in breast cancer compared with normal breast tissues. Of the 186 genes, 169 genes were downregulated and 17 genes were upregulated in the tumor samples. KEGG pathway analyses showed that protein digestion and absorption, focal adhesion, salivary secretion, drug metabolism-cytochrome P450, and phenylalanine metabolism pathways are involved. Among these DEGs, the prominent top two genes (MMP11 and COL1A1) which potentially correlated with IGFBP5 were selected for validation using real time RT-qPCR. Only COL1A1 expression showed a consistent upregulation with IGFBP5 expression and COL1A1 and MMP11 were significantly positively correlated. We concluded that the discovery of coordinately expressed genes related with IGFBP5 might contribute to understanding of the molecular mechanism of the function of IGFBP5 in breast cancer. Further functional studies on DEGs and association with IGFBP5 may identify novel biomarkers for clinical applications in breast cancer.

Analysis of High-Throughput ELISA Microarray Data

DOE Office of Scientific and Technical Information (OSTI.GOV)

White, Amanda M.; Daly, Don S.; Zangar, Richard C.

Our research group develops analytical methods and software for the high-throughput analysis of quantitative enzyme-linked immunosorbent assay (ELISA) microarrays. ELISA microarrays differ from DNA microarrays in several fundamental aspects and most algorithms for analysis of DNA microarray data are not applicable to ELISA microarrays. In this review, we provide an overview of the steps involved in ELISA microarray data analysis and how the statistically sound algorithms we have developed provide an integrated software suite to address the needs of each data-processing step. The algorithms discussed are available in a set of open-source software tools (http://www.pnl.gov/statistics/ProMAT).

EzArray: A web-based highly automated Affymetrix expression array data management and analysis system

PubMed Central

Zhu, Yuerong; Zhu, Yuelin; Xu, Wei

2008-01-01

Background Though microarray experiments are very popular in life science research, managing and analyzing microarray data are still challenging tasks for many biologists. Most microarray programs require users to have sophisticated knowledge of mathematics, statistics and computer skills for usage. With accumulating microarray data deposited in public databases, easy-to-use programs to re-analyze previously published microarray data are in high demand. Results EzArray is a web-based Affymetrix expression array data management and analysis system for researchers who need to organize microarray data efficiently and get data analyzed instantly. EzArray organizes microarray data into projects that can be analyzed online with predefined or custom procedures. EzArray performs data preprocessing and detection of differentially expressed genes with statistical methods. All analysis procedures are optimized and highly automated so that even novice users with limited pre-knowledge of microarray data analysis can complete initial analysis quickly. Since all input files, analysis parameters, and executed scripts can be downloaded, EzArray provides maximum reproducibility for each analysis. In addition, EzArray integrates with Gene Expression Omnibus (GEO) and allows instantaneous re-analysis of published array data. Conclusion EzArray is a novel Affymetrix expression array data analysis and sharing system. EzArray provides easy-to-use tools for re-analyzing published microarray data and will help both novice and experienced users perform initial analysis of their microarray data from the location of data storage. We believe EzArray will be a useful system for facilities with microarray services and laboratories with multiple members involved in microarray data analysis. EzArray is freely available from . PMID:18218103

CNV-ROC: A cost effective, computer-aided analytical performance evaluator of chromosomal microarrays.

PubMed

Goodman, Corey W; Major, Heather J; Walls, William D; Sheffield, Val C; Casavant, Thomas L; Darbro, Benjamin W

2015-04-01

Chromosomal microarrays (CMAs) are routinely used in both research and clinical laboratories; yet, little attention has been given to the estimation of genome-wide true and false negatives during the assessment of these assays and how such information could be used to calibrate various algorithmic metrics to improve performance. Low-throughput, locus-specific methods such as fluorescence in situ hybridization (FISH), quantitative PCR (qPCR), or multiplex ligation-dependent probe amplification (MLPA) preclude rigorous calibration of various metrics used by copy number variant (CNV) detection algorithms. To aid this task, we have established a comparative methodology, CNV-ROC, which is capable of performing a high throughput, low cost, analysis of CMAs that takes into consideration genome-wide true and false negatives. CNV-ROC uses a higher resolution microarray to confirm calls from a lower resolution microarray and provides for a true measure of genome-wide performance metrics at the resolution offered by microarray testing. CNV-ROC also provides for a very precise comparison of CNV calls between two microarray platforms without the need to establish an arbitrary degree of overlap. Comparison of CNVs across microarrays is done on a per-probe basis and receiver operator characteristic (ROC) analysis is used to calibrate algorithmic metrics, such as log2 ratio threshold, to enhance CNV calling performance. CNV-ROC addresses a critical and consistently overlooked aspect of analytical assessments of genome-wide techniques like CMAs which is the measurement and use of genome-wide true and false negative data for the calculation of performance metrics and comparison of CNV profiles between different microarray experiments. Copyright © 2015 Elsevier Inc. All rights reserved.

An object model and database for functional genomics.

PubMed

Jones, Andrew; Hunt, Ela; Wastling, Jonathan M; Pizarro, Angel; Stoeckert, Christian J

2004-07-10

Large-scale functional genomics analysis is now feasible and presents significant challenges in data analysis, storage and querying. Data standards are required to enable the development of public data repositories and to improve data sharing. There is an established data format for microarrays (microarray gene expression markup language, MAGE-ML) and a draft standard for proteomics (PEDRo). We believe that all types of functional genomics experiments should be annotated in a consistent manner, and we hope to open up new ways of comparing multiple datasets used in functional genomics. We have created a functional genomics experiment object model (FGE-OM), developed from the microarray model, MAGE-OM and two models for proteomics, PEDRo and our own model (Gla-PSI-Glasgow Proposal for the Proteomics Standards Initiative). FGE-OM comprises three namespaces representing (i) the parts of the model common to all functional genomics experiments; (ii) microarray-specific components; and (iii) proteomics-specific components. We believe that FGE-OM should initiate discussion about the contents and structure of the next version of MAGE and the future of proteomics standards. A prototype database called RNA And Protein Abundance Database (RAPAD), based on FGE-OM, has been implemented and populated with data from microbial pathogenesis. FGE-OM and the RAPAD schema are available from http://www.gusdb.org/fge.html, along with a set of more detailed diagrams. RAPAD can be accessed by registration at the site.

A Study of the Differential Effects of Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) on Gene Expression Profiles of Stimulated Thp-1 Macrophages

PubMed Central

Allam-Ndoul, Bénédicte; Guénard, Frédéric; Barbier, Olivier; Vohl, Marie-Claude

2017-01-01

Background: An appropriate intake of omega-3 (n-3) fatty acids (FAs) such as eicosapentaenoic and docosahexaenoic acid (EPA/DHA) from marine sources is known to have anti-inflammatory effects. However, molecular mechanisms underlying their beneficial effects on health are not fully understood. The aim of the present study was to characterize gene expression profiles of THP-1 macrophages, incubated in either EPA or DHA and stimulated with lipopolysaccharide (LPS), a pro-inflammatory agent. Methods: THP-1 macrophages were incubated into 10, 50 and 75 µM of EPA or DHA for 24 h, and 100 nM of LPS was added to the culture media for 18 h. Total mRNA was extracted and gene expression examined by microarray analysis using Illumina Human HT-12 expression beadchips (Illumina). Results: Pathway analysis revealed that EPA and DHA regulate genes involved in cell cycle regulation, apoptosis, immune response and inflammation, oxidative stress and cancer pathways in a differential and dose-dependent manner. Conclusions: EPA and DHA appear to exert differential effects on gene expression in THP-1 macrophages. Specific effects of n-3 FAs on gene expression levels are also dose-dependent. PMID:28441337

Reverse engineering biological networks :applications in immune responses to bio-toxins.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martino, Anthony A.; Sinclair, Michael B.; Davidson, George S.

Our aim is to determine the network of events, or the regulatory network, that defines an immune response to a bio-toxin. As a model system, we are studying T cell regulatory network triggered through tyrosine kinase receptor activation using a combination of pathway stimulation and time-series microarray experiments. Our approach is composed of five steps (1) microarray experiments and data error analysis, (2) data clustering, (3) data smoothing and discretization, (4) network reverse engineering, and (5) network dynamics analysis and fingerprint identification. The technological outcome of this study is a suite of experimental protocols and computational tools that reverse engineermore » regulatory networks provided gene expression data. The practical biological outcome of this work is an immune response fingerprint in terms of gene expression levels. Inferring regulatory networks from microarray data is a new field of investigation that is no more than five years old. To the best of our knowledge, this work is the first attempt that integrates experiments, error analyses, data clustering, inference, and network analysis to solve a practical problem. Our systematic approach of counting, enumeration, and sampling networks matching experimental data is new to the field of network reverse engineering. The resulting mathematical analyses and computational tools lead to new results on their own and should be useful to others who analyze and infer networks.« less

Detecting novel genes with sparse arrays

PubMed Central

Haiminen, Niina; Smit, Bart; Rautio, Jari; Vitikainen, Marika; Wiebe, Marilyn; Martinez, Diego; Chee, Christine; Kunkel, Joe; Sanchez, Charles; Nelson, Mary Anne; Pakula, Tiina; Saloheimo, Markku; Penttilä, Merja; Kivioja, Teemu

2014-01-01

Species-specific genes play an important role in defining the phenotype of an organism. However, current gene prediction methods can only efficiently find genes that share features such as sequence similarity or general sequence characteristics with previously known genes. Novel sequencing methods and tiling arrays can be used to find genes without prior information and they have demonstrated that novel genes can still be found from extensively studied model organisms. Unfortunately, these methods are expensive and thus are not easily applicable, e.g., to finding genes that are expressed only in very specific conditions. We demonstrate a method for finding novel genes with sparse arrays, applying it on the 33.9 Mb genome of the filamentous fungus Trichoderma reesei. Our computational method does not require normalisations between arrays and it takes into account the multiple-testing problem typical for analysis of microarray data. In contrast to tiling arrays, that use overlapping probes, only one 25mer microarray oligonucleotide probe was used for every 100 b. Thus, only relatively little space on a microarray slide was required to cover the intergenic regions of a genome. The analysis was done as a by-product of a conventional microarray experiment with no additional costs. We found at least 23 good candidates for novel transcripts that could code for proteins and all of which were expressed at high levels. Candidate genes were found to neighbour ire1 and cre1 and many other regulatory genes. Our simple, low-cost method can easily be applied to finding novel species-specific genes without prior knowledge of their sequence properties. PMID:20691772

Transcriptional signature associated with early rheumatoid arthritis and healthy individuals at high risk to develop the disease

PubMed Central

Macías-Segura, N.; Bastian, Y.; Santiago-Algarra, D.; Castillo-Ortiz, J. D.; Alemán-Navarro, A. L.; Jaime-Sánchez, E.; Gomez-Moreno, M.; Saucedo-Toral, C. A.; Lara-Ramírez, Edgar E.; Zapata-Zuñiga, M.; Enciso-Moreno, L.; González-Amaro, R.; Ramos-Remus, C.; Enciso-Moreno, J. A.

2018-01-01

Background Little is known regarding the mechanisms underlying the loss of tolerance in the early and preclinical stages of autoimmune diseases. The aim of this work was to identify the transcriptional profile and signaling pathways associated to non-treated early rheumatoid arthritis (RA) and subjects at high risk. Several biomarker candidates for early RA are proposed. Methods Whole blood total RNA was obtained from non-treated early RA patients with <1 year of evolution as well as from healthy first-degree relatives of patients with RA (FDR) classified as ACCP+ and ACCP- according to their antibodies serum levels against cyclic citrullinated peptides. Complementary RNA (cRNA) was synthetized and hybridized to high-density microarrays. Data was analyzed in Genespring Software and functional categories were assigned to a specific transcriptome identified in subjects with RA and FDR ACCP positive. Specific signaling pathways for genes associated to RA were identified. Gene expression was evaluated by qPCR. Receiver operating characteristic (ROC) analysis was used to evaluate these genes as biomarkers. Results A characteristic transcriptome of 551 induced genes and 4,402 repressed genes were identified in early RA patients. Bioinformatics analysis of the data identified a specific transcriptome in RA patients. Moreover, some overlapped transcriptional profiles between patients with RA and ACCP+ were identified, suggesting an up-regulated distinctive transcriptome from the preclinical stages up to progression to an early RA state. A total of 203 pathways have up-regulated genes that are shared between RA and ACCP+. Some of these genes show potential to be used as progression biomarkers for early RA with area under the curve of ROC > 0.92. These genes come from several functional categories associated to inflammation, Wnt signaling and type I interferon pathways. Conclusion The presence of a specific transcriptome in whole blood of RA patients suggests the activation of a specific inflammatory transcriptional signature in early RA development. The set of overexpressed genes in early RA patients that are shared with ACCP+ subjects but not with ACCP- subjects, can represent a transcriptional signature involved with the transition of a preclinical to a clinical RA stage. Some of these particular up-regulated and down-regulated genes are related to inflammatory processes and could be considered as biomarker candidates for disease progression in subjects at risk to develop RA. PMID:29584756

DNA Microarray Gene Expression Profile of Marginal Zone versus Follicular B cells and Idiotype Positive Marginal Zone B cells Before and After Immunization with Streptococcus pneumoniae 1

PubMed Central

Liu, Jiabin; Behrens, Timothy W.; Kearney, John F.

2014-01-01

Marginal Zone (MZ) B cells play an important role in the clearance of blood-borne bacterial infections via rapid T-independent IgM responses. We have previously demonstrated that MZ B cells respond rapidly and robustly to bacterial particulates. To determine the MZ-specific genes that are expressed to allow for this response, MZ and Follicular (FO) B cells were sort-purified and analyzed via DNA microarray analysis. We identified 181 genes that were significantly different between the two B cell populations. 99 genes were more highly expressed in MZ B cells while 82 genes were more highly expressed in FO B cells. To further understand the molecular mechanisms by which MZ B cells respond so rapidly to bacterial challenge, idiotype positive and negative MZ B cells were sort-purified before (0 hour) or after (1 hour) i.v. immunization with heat killed Streptococcus pneumoniae, R36A, and analyzed via DNA microarray analysis. We identified genes specifically up regulated or down regulated at 1 hour following immunization in the idiotype positive MZ B cells. These results give insight into the gene expression pattern in resting MZ vs. FO B cells and the specific regulation of gene expression in antigen-specific MZ B cells following interaction with antigen. PMID:18453586

sigReannot: an oligo-set re-annotation pipeline based on similarities with the Ensembl transcripts and Unigene clusters.

PubMed

Casel, Pierrot; Moreews, François; Lagarrigue, Sandrine; Klopp, Christophe

2009-07-16

Microarray is a powerful technology enabling to monitor tens of thousands of genes in a single experiment. Most microarrays are now using oligo-sets. The design of the oligo-nucleotides is time consuming and error prone. Genome wide microarray oligo-sets are designed using as large a set of transcripts as possible in order to monitor as many genes as possible. Depending on the genome sequencing state and on the assembly state the knowledge of the existing transcripts can be very different. This knowledge evolves with the different genome builds and gene builds. Once the design is done the microarrays are often used for several years. The biologists working in EADGENE expressed the need of up-to-dated annotation files for the oligo-sets they share including information about the orthologous genes of model species, the Gene Ontology, the corresponding pathways and the chromosomal location. The results of SigReannot on a chicken micro-array used in the EADGENE project compared to the initial annotations show that 23% of the oligo-nucleotide gene annotations were not confirmed, 2% were modified and 1% were added. The interest of this up-to-date annotation procedure is demonstrated through the analysis of real data previously published. SigReannot uses the oligo-nucleotide design procedure criteria to validate the probe-gene link and the Ensembl transcripts as reference for annotation. It therefore produces a high quality annotation based on reference gene sets.

Altered Global Gene Expression in First Trimester Placentas of Women Destined to Develop Preeclampsia

PubMed Central

Founds, Sandra A.; Conley, Yvette P.; Lyons-Weiler, James F.; Jeyabalan, Arun; Hogge, W. Allen; Conrad, Kirk P.

2009-01-01

Background Preeclampsia is a pregnancy-specific disorder that remains a leading cause of maternal, fetal and neonatal morbidity and mortality, and is associated with risk for future cardiovascular disease. There are no reliable predictors, specific preventative measures or treatments other than delivery. A widely-held view is that the antecedents of preeclampsia lie with impaired placentation in early pregnancy. Accordingly, we hypothesized dysregulation of global gene expression in first trimester placentas of women who later manifested preeclampsia. Methods Surplus chorionic villus sampling (CVS) tissues were collected at 10–12 weeks gestation in 160 patients with singleton fetuses. Four patients developed preeclampsia, and their banked CVS specimens were matched to 8 control samples from patients with unaffected pregnancies. Affymetrix HG-U133 Plus 2.0 GeneChips were utilized for microarray analysis. Naïve Bayes prediction modeling and pathway analysis were conducted. qRT-PCR examined three of the dysregulated genes. Results Thirty-six differentially expressed genes were identified in the preeclampsia placentas. qRT-PCR verified the microarray analysis. Thirty-one genes were down-regulated. Many were related to inflammation/immunoregulation and cell motility. Decidual gene dysregulation was prominent. No evidence was found for alterations in hypoxia and oxidative stress regulated genes. Conclusions To our knowledge, this is the first study to show dysregulation of gene expression in the early placentas of women ~6 months before developing preeclampsia, thereby reinforcing a placental origin of the disorder. We hypothesize that placentation in preeclampsia is compromised in the first trimester by maternal and fetal immune dysregulation, abnormal decidualization, or both, thereby impairing trophoblast invasion. Several of the genes provide potential targets for the development of clinical biomarkers in maternal blood during the first trimester. Supplementary materials are available for this article via the publisher’s online edition. PMID:19027158

Phthalimide neovascular factor 1 (PNF1) modulates MT1-MMP activity in human microvascular endothelial cells

PubMed Central

Wieghaus, Kristen A.; Gianchandani, Erwin P.; Neal, Rebekah A.; Paige, Mikell A.; Brown, Milton L.; Papin, Jason A.; Botchwey, Edward A.

2009-01-01

We are creating synthetic pharmaceuticals with angiogenic activity and potential to promote vascular invasion. We previously demonstrated that one of these molecules, phthalimide neovascular factor 1 (PNF1), significantly expands microvascular networks in vivo following sustained release from poly(lactic-co-glycolic acid) (PLAGA) films. In addition, to probe PNF1 mode-of-action, we recently applied a novel pathway-based compendium analysis to a multi-timepoint, controlled microarray dataset of PNF1-treated (versus control) human microvascular endothelial cells (HMVECs), and we identified induction of tumor necrosis factor-alpha (TNF-α) and, subsequently, transforming growth factor-beta (TGF-β) signaling networks by PNF1. Here we validate this microarray data-set with quantitative real-time polymerase chain reaction (RT-PCR) analysis. Subsequently, we probe this dataset and identify three specific TGF-β-induced genes with regulation by PNF1 conserved over multiple timepoints—amyloid beta (A4) precursor protein (APP), early growth response 1 (EGR-1), and matrix metalloproteinase 14 (MMP14 or MT1-MMP)—that are also implicated in angiogenesis. We further focus on MMP14 given its unique role in angiogenesis, and we validate MT1-MMP modulation by PNF1 with an in vitro fluorescence assay that demonstrates the direct effects that PNF1 exerts on functional metalloproteinase activity. We also utilize endothelial cord formation in collagen gels to show that PNF1-induced stimulation of endothelial cord network formation in vitro is in some way MT1-MMP-dependent. Ultimately, this new network analysis of our transcriptional footprint characterizing PNF1 activity 1–48 h post-supplementation in HMVECs coupled with corresponding validating experiments suggests a key set of a few specific targets that are involved in PNF1 mode-of-action and important for successful promotion of the neovascularization that we have observed by the drug in vivo. PMID:19326468

Phthalimide neovascular factor 1 (PNF1) modulates MT1-MMP activity in human microvascular endothelial cells.

PubMed

Wieghaus, Kristen A; Gianchandani, Erwin P; Neal, Rebekah A; Paige, Mikell A; Brown, Milton L; Papin, Jason A; Botchwey, Edward A

2009-07-01

We are creating synthetic pharmaceuticals with angiogenic activity and potential to promote vascular invasion. We previously demonstrated that one of these molecules, phthalimide neovascular factor 1 (PNF1), significantly expands microvascular networks in vivo following sustained release from poly(lactic-co-glycolic acid) (PLAGA) films. In addition, to probe PNF1 mode of action, we recently applied a novel pathway-based compendium analysis to a multi-timepoint, controlled microarray data set of PNF1-treated (vs. control) human microvascular endothelial cells (HMVECs), and we identified induction of tumor necrosis factor-alpha (TNF-alpha) and, subsequently, transforming growth factor-beta (TGF-beta) signaling networks by PNF1. Here we validate this microarray data set with quantitative real-time polymerase chain reaction (RT-PCR) analysis. Subsequently, we probe this data set and identify three specific TGF-beta-induced genes with regulation by PNF1 conserved over multiple timepoints-amyloid beta (A4) precursor protein (APP), early growth response 1 (EGR-1), and matrix metalloproteinase 14 (MMP14 or MT1-MMP)-that are also implicated in angiogenesis. We further focus on MMP14 given its unique role in angiogenesis, and we validate MT1-MMP modulation by PNF1 with an in vitro fluorescence assay that demonstrates the direct effects that PNF1 exerts on functional metalloproteinase activity. We also utilize endothelial cord formation in collagen gels to show that PNF1-induced stimulation of endothelial cord network formation in vitro is in some way MT1-MMP-dependent. Ultimately, this new network analysis of our transcriptional footprint characterizing PNF1 activity 1-48 h post-supplementation in HMVECs coupled with corresponding validating experiments suggests a key set of a few specific targets that are involved in PNF1 mode of action and important for successful promotion of the neovascularization that we have observed by the drug in vivo.

Alteration of gene expression by alcohol exposure at early neurulation.

PubMed

Zhou, Feng C; Zhao, Qianqian; Liu, Yunlong; Goodlett, Charles R; Liang, Tiebing; McClintick, Jeanette N; Edenberg, Howard J; Li, Lang

2011-02-21

We have previously demonstrated that alcohol exposure at early neurulation induces growth retardation, neural tube abnormalities, and alteration of DNA methylation. To explore the global gene expression changes which may underline these developmental defects, microarray analyses were performed in a whole embryo mouse culture model that allows control over alcohol and embryonic variables. Alcohol caused teratogenesis in brain, heart, forelimb, and optic vesicle; a subset of the embryos also showed cranial neural tube defects. In microarray analysis (accession number GSM9545), adopting hypothesis-driven Gene Set Enrichment Analysis (GSEA) informatics and intersection analysis of two independent experiments, we found that there was a collective reduction in expression of neural specification genes (neurogenin, Sox5, Bhlhe22), neural growth factor genes [Igf1, Efemp1, Klf10 (Tieg), and Edil3], and alteration of genes involved in cell growth, apoptosis, histone variants, eye and heart development. There was also a reduction of retinol binding protein 1 (Rbp1), and de novo expression of aldehyde dehydrogenase 1B1 (Aldh1B1). Remarkably, four key hematopoiesis genes (glycophorin A, adducin 2, beta-2 microglobulin, and ceruloplasmin) were absent after alcohol treatment, and histone variant genes were reduced. The down-regulation of the neurospecification and the neurotrophic genes were further confirmed by quantitative RT-PCR. Furthermore, the gene expression profile demonstrated distinct subgroups which corresponded with two distinct alcohol-related neural tube phenotypes: an open (ALC-NTO) and a closed neural tube (ALC-NTC). Further, the epidermal growth factor signaling pathway and histone variants were specifically altered in ALC-NTO, and a greater number of neurotrophic/growth factor genes were down-regulated in the ALC-NTO than in the ALC-NTC embryos. This study revealed a set of genes vulnerable to alcohol exposure and genes that were associated with neural tube defects during early neurulation.

Evaluating between-pathway models with expression data.

PubMed

Hescott, B J; Leiserson, M D M; Cowen, L J; Slonim, D K

2010-03-01

Between-pathway models (BPMs) are network motifs consisting of pairs of putative redundant pathways. In this article, we show how adding another source of high-throughput data--microarray gene expression data from knockout experiments--allows us to identify a compensatory functional relationship between genes from the two BPM pathways. We evaluate the quality of the BPMs from four different studies, and we describe how our methods might be extended to refine pathways.

Microarray analysis of gene expression in West Nile virus–infected human retinal pigment epithelium

PubMed Central

Munoz-Erazo, Luis; Natoli, Ricardo; Provis, Jan Marie; Madigan, Michelle Catherine

2012-01-01

Purpose To identify key genes differentially expressed in the human retinal pigment epithelium (hRPE) following low-level West Nile virus (WNV) infection. Methods Primary hRPE and retinal pigment epithelium cell line (ARPE-19) cells were infected with WNV (multiplicity of infection 1). RNA extracted from mock-infected and WNV-infected cells was assessed for differential expression of genes using Affymetrix microarray. Quantitative real-time PCR analysis of 23 genes was used to validate the microarray results. Results Functional annotation clustering of the microarray data showed that gene clusters involved in immune and antiviral responses ranked highly, involving genes such as chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 5 (CCL5), chemokine (C-X-C motif) ligand 10 (CXCL10), and toll like receptor 3 (TLR3). In conjunction with the quantitative real-time PCR analysis, other novel genes regulated by WNV infection included indoleamine 2,3-dioxygenase (IDO1), genes involved in the transforming growth factor–β pathway (bone morphogenetic protein and activin membrane-bound inhibitor homolog [BAMBI] and activating transcription factor 3 [ATF3]), and genes involved in apoptosis (tumor necrosis factor receptor superfamily, member 10d [TNFRSF10D]). WNV-infected RPE did not produce any interferon-γ, suggesting that IDO1 is induced by other soluble factors, by the virus alone, or both. Conclusions Low-level WNV infection of hRPE cells induced expression of genes that are typically associated with the host cell response to virus infection. We also identified other genes, including IDO1 and BAMBI, that may influence the RPE and therefore outer blood-retinal barrier integrity during ocular infection and inflammation, or are associated with degeneration, as seen for example in aging. PMID:22509103

Chemiluminescence microarrays in analytical chemistry: a critical review.

PubMed

Seidel, Michael; Niessner, Reinhard

2014-09-01

Multi-analyte immunoassays on microarrays and on multiplex DNA microarrays have been described for quantitative analysis of small organic molecules (e.g., antibiotics, drugs of abuse, small molecule toxins), proteins (e.g., antibodies or protein toxins), and microorganisms, viruses, and eukaryotic cells. In analytical chemistry, multi-analyte detection by use of analytical microarrays has become an innovative research topic because of the possibility of generating several sets of quantitative data for different analyte classes in a short time. Chemiluminescence (CL) microarrays are powerful tools for rapid multiplex analysis of complex matrices. A wide range of applications for CL microarrays is described in the literature dealing with analytical microarrays. The motivation for this review is to summarize the current state of CL-based analytical microarrays. Combining analysis of different compound classes on CL microarrays reduces analysis time, cost of reagents, and use of laboratory space. Applications are discussed, with examples from food safety, water safety, environmental monitoring, diagnostics, forensics, toxicology, and biosecurity. The potential and limitations of research on multiplex analysis by use of CL microarrays are discussed in this review.

DNA microarray-based experimental strategy for trustworthy expression profiling of the hippocampal genes by astaxanthin supplementation in adult mouse

PubMed Central

Yook, Jang Soo; Shibato, Junko; Rakwal, Randeep; Soya, Hideaki

2015-01-01

Naturally occurring astaxantin (ASX) is one of the noticeable carotenoid and dietary supplement, which has strong antioxidant and anti-inflammatory properties, and neuroprotective effects in the brain through crossing the blood–brain barrier. Specially, we are interested in the role of ASX as a brain food. Although ASX has been suggested to have potential benefit to the brain function, the underlying molecular mechanisms and events mediating such effect remain unknown. Here we examined molecular factors in the hippocampus of adult mouse fed ASX diets (0.1% and 0.5% doses) using DNA microarray (Agilent 4 × 44 K whole mouse genome chip) analysis. In this study, we described in detail our experimental workflow and protocol, and validated quality controls with the housekeeping gene expression (Gapdh and Beta-actin) on the dye-swap based approach to advocate our microarray data, which have been uploaded to Gene Expression Omnibus (accession number GSE62197) as a gene resource for the scientific community. This data will also form an important basis for further detailed experiments and bioinformatics analysis with an aim to unravel the potential molecular pathways or mechanisms underlying the positive effects of ASX supplementation on the brain, in particular the hippocampus. PMID:26981356

Sho-saiko-to, a traditional herbal medicine, regulates gene expression and biological function by way of microRNAs in primary mouse hepatocytes

PubMed Central

2014-01-01

Background Sho-saiko-to (SST) (also known as so-shi-ho-tang or xiao-chai-hu-tang) has been widely prescribed for chronic liver diseases in traditional Oriental medicine. Despite the substantial amount of clinical evidence for SST, its molecular mechanism has not been clearly identified at a genome-wide level. Methods By using a microarray, we analyzed the temporal changes of messenger RNA (mRNA) and microRNA expression in primary mouse hepatocytes after SST treatment. The pattern of genes regulated by SST was identified by using time-series microarray analysis. The biological function of genes was measured by pathway analysis. For the identification of the exact targets of the microRNAs, a permutation-based correlation method was implemented in which the temporal expression of mRNAs and microRNAs were integrated. The similarity of the promoter structure between temporally regulated genes was measured by analyzing the transcription factor binding sites in the promoter region. Results The SST-regulated gene expression had two major patterns: (1) a temporally up-regulated pattern (463 genes) and (2) a temporally down-regulated pattern (177 genes). The integration of the genes and microRNA demonstrated that 155 genes could be the targets of microRNAs from the temporally up-regulated pattern and 19 genes could be the targets of microRNAs from the temporally down-regulated pattern. The temporally up-regulated pattern by SST was associated with signaling pathways such as the cell cycle pathway, whereas the temporally down-regulated pattern included drug metabolism-related pathways and immune-related pathways. All these pathways could be possibly associated with liver regenerative activity of SST. Genes targeted by microRNA were moreover associated with different biological pathways from the genes not targeted by microRNA. An analysis of promoter similarity indicated that co-expressed genes after SST treatment were clustered into subgroups, depending on the temporal expression patterns. Conclusions We are the first to identify that SST regulates temporal gene expression by way of microRNA. MicroRNA targets and non-microRNA targets moreover have different biological roles. This functional segregation by microRNA would be critical for the elucidation of the molecular activities of SST. PMID:24410935

Sho-saiko-to, a traditional herbal medicine, regulates gene expression and biological function by way of microRNAs in primary mouse hepatocytes.

PubMed

Song, Kwang Hoon; Kim, Yun Hee; Kim, Bu-Yeo

2014-01-11

Sho-saiko-to (SST) (also known as so-shi-ho-tang or xiao-chai-hu-tang) has been widely prescribed for chronic liver diseases in traditional Oriental medicine. Despite the substantial amount of clinical evidence for SST, its molecular mechanism has not been clearly identified at a genome-wide level. By using a microarray, we analyzed the temporal changes of messenger RNA (mRNA) and microRNA expression in primary mouse hepatocytes after SST treatment. The pattern of genes regulated by SST was identified by using time-series microarray analysis. The biological function of genes was measured by pathway analysis. For the identification of the exact targets of the microRNAs, a permutation-based correlation method was implemented in which the temporal expression of mRNAs and microRNAs were integrated. The similarity of the promoter structure between temporally regulated genes was measured by analyzing the transcription factor binding sites in the promoter region. The SST-regulated gene expression had two major patterns: (1) a temporally up-regulated pattern (463 genes) and (2) a temporally down-regulated pattern (177 genes). The integration of the genes and microRNA demonstrated that 155 genes could be the targets of microRNAs from the temporally up-regulated pattern and 19 genes could be the targets of microRNAs from the temporally down-regulated pattern. The temporally up-regulated pattern by SST was associated with signaling pathways such as the cell cycle pathway, whereas the temporally down-regulated pattern included drug metabolism-related pathways and immune-related pathways. All these pathways could be possibly associated with liver regenerative activity of SST. Genes targeted by microRNA were moreover associated with different biological pathways from the genes not targeted by microRNA. An analysis of promoter similarity indicated that co-expressed genes after SST treatment were clustered into subgroups, depending on the temporal expression patterns. We are the first to identify that SST regulates temporal gene expression by way of microRNA. MicroRNA targets and non-microRNA targets moreover have different biological roles. This functional segregation by microRNA would be critical for the elucidation of the molecular activities of SST.

Effect of storage time on gene expression data acquired from unfrozen archived newborn blood spots.

PubMed

Ho, Nhan T; Busik, Julia V; Resau, James H; Paneth, Nigel; Khoo, Sok Kean

2016-11-01

Unfrozen archived newborn blood spots (NBS) have been shown to retain sufficient messenger RNA (mRNA) for gene expression profiling. However, the effect of storage time at ambient temperature for NBS samples in relation to the quality of gene expression data is relatively unknown. Here, we evaluated mRNA expression from quantitative real-time PCR (qRT-PCR) and microarray data obtained from NBS samples stored at ambient temperature to determine the effect of storage time on the quality of gene expression. These data were generated in a previous case-control study examining NBS in 53 children with cerebral palsy (CP) and 53 matched controls. NBS sample storage period ranged from 3 to 16years at ambient temperature. We found persistently low RNA integrity numbers (RIN=2.3±0.71) and 28S/18S rRNA ratios (~0) across NBS samples for all storage periods. In both qRT-PCR and microarray data, the expression of three common housekeeping genes-beta cytoskeletal actin (ACTB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and peptidylprolyl isomerase A (PPIA)-decreased with increased storage time. Median values of each microarray probe intensity at log 2 scale also decreased over time. After eight years of storage, probe intensity values were largely reduced to background intensity levels. Of 21,500 genes tested, 89% significantly decreased in signal intensity, with 13,551, 10,730, and 9925 genes detected within 5years, > 5 to <10years, and >10years of storage, respectively. We also examined the expression of two gender-specific genes (X inactivation-specific transcript, XIST and lysine-specific demethylase 5D, KDM5D) and seven gene sets representing the inflammatory, hypoxic, coagulative, and thyroidal pathways hypothesized to be related to CP risk to determine the effect of storage time on the detection of these biologically relevant genes. We found the gender-specific genes and CP-related gene sets detectable in all storage periods, but exhibited differential expression (between male vs. female or CP vs. control) only within the first six years of storage. We concluded that gene expression data quality deteriorates in unfrozen archived NBS over time and that differential gene expression profiling and analysis is recommended for those NBS samples collected and stored within six years at ambient temperature. Copyright © 2016 Elsevier Inc. All rights reserved.

Fluorescence-based bioassays for the detection and evaluation of food materials.

PubMed

Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

2015-10-13

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials.

Fluorescence-Based Bioassays for the Detection and Evaluation of Food Materials

PubMed Central

Nishi, Kentaro; Isobe, Shin-Ichiro; Zhu, Yun; Kiyama, Ryoiti

2015-01-01

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials. PMID:26473869

Down-weighting overlapping genes improves gene set analysis

PubMed Central

2012-01-01

Background The identification of gene sets that are significantly impacted in a given condition based on microarray data is a crucial step in current life science research. Most gene set analysis methods treat genes equally, regardless how specific they are to a given gene set. Results In this work we propose a new gene set analysis method that computes a gene set score as the mean of absolute values of weighted moderated gene t-scores. The gene weights are designed to emphasize the genes appearing in few gene sets, versus genes that appear in many gene sets. We demonstrate the usefulness of the method when analyzing gene sets that correspond to the KEGG pathways, and hence we called our method Pathway Analysis with Down-weighting of Overlapping Genes (PADOG). Unlike most gene set analysis methods which are validated through the analysis of 2-3 data sets followed by a human interpretation of the results, the validation employed here uses 24 different data sets and a completely objective assessment scheme that makes minimal assumptions and eliminates the need for possibly biased human assessments of the analysis results. Conclusions PADOG significantly improves gene set ranking and boosts sensitivity of analysis using information already available in the gene expression profiles and the collection of gene sets to be analyzed. The advantages of PADOG over other existing approaches are shown to be stable to changes in the database of gene sets to be analyzed. PADOG was implemented as an R package available at: http://bioinformaticsprb.med.wayne.edu/PADOG/or http://www.bioconductor.org. PMID:22713124

Mig-6 regulates endometrial genes involved in cell cycle and progesterone signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoo, Jung-Yoon; Kim, Tae Hoon; Lee, Jae Hee

2015-07-10

Mitogen inducible gene 6 (Mig-6) is an important mediator of progesterone (P4) signaling to inhibit estrogen (E2) signaling in the uterus. Ablation of Mig-6 in the murine uterus leads to the development of endometrial hyperplasia and E2-induced endometrial cancer. To identify the molecular pathways regulated by Mig-6, we performed microarray analysis on the uterus of ovariectomized Mig-6{sup f/f} and PGR{sup cre/+}Mig-6{sup f/f} (Mig-6{sup d/d}) mice treated with vehicle or P4 for 6 h. The results revealed that 772 transcripts were significantly regulated in the Mig-6{sup d/d} uterus treated with vehicle as compared with Mig-6{sup f/f} mice. The pathway analysis showed thatmore » Mig-6 suppressed the expression of gene-related cell cycle regulation in the absence of ovarian steroid hormone. The epithelium of Mig-6{sup d/d} mice showed a significant increase in the number of proliferative cells compared to Mig-6{sup f/f} mice. This microarray analysis also revealed that 324 genes are regulated by P4 as well as Mig-6. Cited2, the developmentally important transcription factor, was identified as being regulated by the P4-Mig-6 axis. To determine the role of Cited2 in the uterus, we used the mice with Cited2 that were conditionally ablated in progesterone receptor-positive cells (PGR{sup cre/+}Cited2{sup f/f}; Cited2{sup d/d}). Ablation of Cited2 in the uterus resulted in a significant reduction in the ability of the uterus to undergo a hormonally induced decidual reaction. Identification and analysis of these responsive genes will help define the role of P4 as well as Mig-6 in regulating uterine biology. - Highlights: • We identify Mig-6- and P4-regulated uterine genes by microarray analysis. • Mig-6 suppresses cell cycle progression and epithelial cell proliferation in uterus. • We identify the Mig-6 dependent induced genes by P4. • Cited2 plays an important role for decidualization as a P4 and Mig-6 target gene.« less

Knowledge-based analysis of microarrays for the discovery of transcriptional regulation relationships

PubMed Central

2010-01-01

Background The large amount of high-throughput genomic data has facilitated the discovery of the regulatory relationships between transcription factors and their target genes. While early methods for discovery of transcriptional regulation relationships from microarray data often focused on the high-throughput experimental data alone, more recent approaches have explored the integration of external knowledge bases of gene interactions. Results In this work, we develop an algorithm that provides improved performance in the prediction of transcriptional regulatory relationships by supplementing the analysis of microarray data with a new method of integrating information from an existing knowledge base. Using a well-known dataset of yeast microarrays and the Yeast Proteome Database, a comprehensive collection of known information of yeast genes, we show that knowledge-based predictions demonstrate better sensitivity and specificity in inferring new transcriptional interactions than predictions from microarray data alone. We also show that comprehensive, direct and high-quality knowledge bases provide better prediction performance. Comparison of our results with ChIP-chip data and growth fitness data suggests that our predicted genome-wide regulatory pairs in yeast are reasonable candidates for follow-up biological verification. Conclusion High quality, comprehensive, and direct knowledge bases, when combined with appropriate bioinformatic algorithms, can significantly improve the discovery of gene regulatory relationships from high throughput gene expression data. PMID:20122245

Knowledge-based analysis of microarrays for the discovery of transcriptional regulation relationships.

PubMed

Seok, Junhee; Kaushal, Amit; Davis, Ronald W; Xiao, Wenzhong

2010-01-18

The large amount of high-throughput genomic data has facilitated the discovery of the regulatory relationships between transcription factors and their target genes. While early methods for discovery of transcriptional regulation relationships from microarray data often focused on the high-throughput experimental data alone, more recent approaches have explored the integration of external knowledge bases of gene interactions. In this work, we develop an algorithm that provides improved performance in the prediction of transcriptional regulatory relationships by supplementing the analysis of microarray data with a new method of integrating information from an existing knowledge base. Using a well-known dataset of yeast microarrays and the Yeast Proteome Database, a comprehensive collection of known information of yeast genes, we show that knowledge-based predictions demonstrate better sensitivity and specificity in inferring new transcriptional interactions than predictions from microarray data alone. We also show that comprehensive, direct and high-quality knowledge bases provide better prediction performance. Comparison of our results with ChIP-chip data and growth fitness data suggests that our predicted genome-wide regulatory pairs in yeast are reasonable candidates for follow-up biological verification. High quality, comprehensive, and direct knowledge bases, when combined with appropriate bioinformatic algorithms, can significantly improve the discovery of gene regulatory relationships from high throughput gene expression data.

Targeted exploration and analysis of large cross-platform human transcriptomic compendia

PubMed Central

Zhu, Qian; Wong, Aaron K; Krishnan, Arjun; Aure, Miriam R; Tadych, Alicja; Zhang, Ran; Corney, David C; Greene, Casey S; Bongo, Lars A; Kristensen, Vessela N; Charikar, Moses; Li, Kai; Troyanskaya, Olga G.

2016-01-01

We present SEEK (http://seek.princeton.edu), a query-based search engine across very large transcriptomic data collections, including thousands of human data sets from almost 50 microarray and next-generation sequencing platforms. SEEK uses a novel query-level cross-validation-based algorithm to automatically prioritize data sets relevant to the query and a robust search approach to identify query-coregulated genes, pathways, and processes. SEEK provides cross-platform handling, multi-gene query search, iterative metadata-based search refinement, and extensive visualization-based analysis options. PMID:25581801

ASK1-dependent recruitment and activation of macrophages induce hair growth in skin wounds

PubMed Central

Osaka, Nao; Takahashi, Takumi; Murakami, Shiori; Matsuzawa, Atsushi; Noguchi, Takuya; Fujiwara, Takeshi; Aburatani, Hiroyuki; Moriyama, Keiji; Takeda, Kohsuke; Ichijo, Hidenori

2007-01-01

Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein 3-kinase family that activates both c-Jun NH2-terminal kinase and p38 pathways in response to inflammatory cytokines and physicochemical stress. We report that ASK1 deficiency in mice results in dramatic retardation of wounding-induced hair regrowth in skin. Oligonucleotide microarray analysis revealed that expression of several chemotactic and activating factors for macrophages, as well as several macrophage-specific marker genes, was reduced in the skin wound area of ASK1-deficient mice. Intracutaneous transplantation of cytokine-activated bone marrow-derived macrophages strongly induced hair growth in both wild-type and ASK1-deficient mice. These findings indicate that ASK1 is required for wounding-induced infiltration and activation of macrophages, which play central roles in inflammation-dependent hair regrowth in skin. PMID:17389227

Microarray Analysis Reveals Increased Expression of Matrix Metalloproteases and Cytokines of Interleukin-20 Subfamily in the Kidneys of Neonate Rats Underwent Unilateral Ureteral Obstruction: A Potential Role of IL-24 in the Regulation of Inflammation and Tissue Remodeling.

PubMed

Pap, Domonkos; Sziksz, Erna; Kiss, Zoltán; Rokonay, Réka; Veres-Székely, Apor; Lippai, Rita; Takács, István Márton; Kis, Éva; Fekete, Andrea; Reusz, György; Szabó, Attila J; Vannay, Adam

2017-01-01

Congenital obstructive nephropathy (CON) is the main cause of pediatric chronic kidney diseases leading to renal fibrosis. High morbidity and limited treatment opportunities of CON urge the better understanding of the underlying molecular mechanisms. To identify the differentially expressed genes, microarray analysis was performed on the kidney samples of neonatal rats underwent unilateral ureteral obstruction (UUO). Microarray results were then validated by real-time RT-PCR and bioinformatics analysis was carried out to identify the relevant genes, functional groups and pathways involved in the pathomechanism of CON. Renal expression of matrix metalloproteinase (MMP)-12 and interleukin (IL)-24 were evaluated by real-time RT-PCR, flow cytometry and immunohistochemical analysis. Effect of the main profibrotic factors on the expression of MMP-12 and IL-24 was investigated on HK-2 and HEK-293 cell lines. Finally, the effect of IL-24 treatment on the expression of pro-inflammatory cytokines and MMPs were tested in vitro. Microarray analysis revealed 880 transcripts showing >2.0-fold change following UUO, enriched mainly in immune response related processes. The most up-regulated genes were MMPs and members of IL-20 cytokine subfamily, including MMP-3, MMP-7, MMP-12, IL-19 and IL-24. We found that while TGF-β treatment inhibits the expression of MMP-12 and IL-24, H2O2 or PDGF-B treatment induce the epithelial expression of MMP-12. We demonstrated that IL-24 treatment decreases the expression of IL-6 and MMP-3 in the renal epithelial cells. This study provides an extensive view of UUO induced changes in the gene expression profile of the developing kidney and describes novel molecules, which may play significant role in the pathomechanism of CON. © 2017 The Author(s)Published by S. Karger AG, Basel.

FREQUENT SUBGRAPH MINING OF PERSONALIZED SIGNALING PATHWAY NETWORKS GROUPS PATIENTS WITH FREQUENTLY DYSREGULATED DISEASE PATHWAYS AND PREDICTS PROGNOSIS.

PubMed

Durmaz, Arda; Henderson, Tim A D; Brubaker, Douglas; Bebek, Gurkan

2017-01-01

Large scale genomics studies have generated comprehensive molecular characterization of numerous cancer types. Subtypes for many tumor types have been established; however, these classifications are based on molecular characteristics of a small gene sets with limited power to detect dysregulation at the patient level. We hypothesize that frequent graph mining of pathways to gather pathways functionally relevant to tumors can characterize tumor types and provide opportunities for personalized therapies. In this study we present an integrative omics approach to group patients based on their altered pathway characteristics and show prognostic differences within breast cancer (p < 9:57E - 10) and glioblastoma multiforme (p < 0:05) patients. We were able validate this approach in secondary RNA-Seq datasets with p < 0:05 and p < 0:01 respectively. We also performed pathway enrichment analysis to further investigate the biological relevance of dysregulated pathways. We compared our approach with network-based classifier algorithms and showed that our unsupervised approach generates more robust and biologically relevant clustering whereas previous approaches failed to report specific functions for similar patient groups or classify patients into prognostic groups. These results could serve as a means to improve prognosis for future cancer patients, and to provide opportunities for improved treatment options and personalized interventions. The proposed novel graph mining approach is able to integrate PPI networks with gene expression in a biologically sound approach and cluster patients in to clinically distinct groups. We have utilized breast cancer and glioblastoma multiforme datasets from microarray and RNA-Seq platforms and identified disease mechanisms differentiating samples. Supplementary methods, figures, tables and code are available at https://github.com/bebeklab/dysprog.

Use of an activated beta-catenin to identify Wnt pathway target genes in caenorhabditis elegans, including a subset of collagen genes expressed in late larval development.

PubMed

Jackson, Belinda M; Abete-Luzi, Patricia; Krause, Michael W; Eisenmann, David M

2014-04-16

The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin-dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1 col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.

Synergistic Effect of Combinatorial Treatment with Curcumin and Mitomycin C on the Induction of Apoptosis of Breast Cancer Cells: A cDNA Microarray Analysis

PubMed Central

Zhou, Qian-Mei; Chen, Qi-Long; Du, Jia; Wang, Xiu-Feng; Lu, Yi-Yu; Zhang, Hui; Su, Shi-Bing

2014-01-01

In order to explore the synergistic mechanisms of combinatorial treatment using curcumin and mitomycin C (MMC) for breast cancer, MCF-7 breast cancer xenografts were conducted to observe the synergistic effect of combinatorial treatment using curcumin and MMC at various dosages. The synergistic mechanisms of combinatorial treatment using curcumin and MMC on the inhibition of tumor growth were explored by differential gene expression profile, gene ontology (GO), ingenuity pathway analysis (IPA) and Signal–Net network analysis. The expression levels of selected genes identified by cDNA microarray expression profiling were validated by quantitative RT-PCR (qRT-PCR) and Western blot analysis. Effect of combinatorial treatment on the inhibition of cell growth was observed by MTT assay. Apoptosis was detected by flow cytometric analysis and Hoechst 33258 staining. The combinatorial treatment of 100 mg/kg curcumin and 1.5 mg/kg MMC revealed synergistic inhibition on tumor growth. Among 1501 differentially expressed genes, the expression of 25 genes exhibited an obvious change and a significant difference in 27 signal pathways was observed (p < 0.05). In addition, Mapk1 (ERK) and Mapk14 (MAPK p38) had more cross-interactions with other genes and revealed an increase in expression by 8.14- and 11.84-fold, respectively during the combinatorial treatment by curcumin and MMC when compared with the control. Moreover, curcumin can synergistically improve tumoricidal effect of MMC in another human breast cancer MDA-MB-231 cells. Apoptosis was significantly induced by the combinatorial treatment (p < 0.05) and significantly inhibited by ERK inhibitor (PD98059) in MCF-7 cells (p < 0.05). The synergistic effect of combinatorial treatment by curcumin and MMC on the induction of apoptosis in breast cancer cells may be via the ERK pathway. PMID:25226537

Digital microarray analysis for digital artifact genomics

NASA Astrophysics Data System (ADS)

Jaenisch, Holger; Handley, James; Williams, Deborah

2013-06-01

We implement a Spatial Voting (SV) based analogy of microarray analysis for digital gene marker identification in malware code sections. We examine a famous set of malware formally analyzed by Mandiant and code named Advanced Persistent Threat (APT1). APT1 is a Chinese organization formed with specific intent to infiltrate and exploit US resources. Manidant provided a detailed behavior and sting analysis report for the 288 malware samples available. We performed an independent analysis using a new alternative to the traditional dynamic analysis and static analysis we call Spatial Analysis (SA). We perform unsupervised SA on the APT1 originating malware code sections and report our findings. We also show the results of SA performed on some members of the families associated by Manidant. We conclude that SV based SA is a practical fast alternative to dynamics analysis and static analysis.

Promzea: a pipeline for discovery of co-regulatory motifs in maize and other plant species and its application to the anthocyanin and phlobaphene biosynthetic pathways and the Maize Development Atlas.

PubMed

Liseron-Monfils, Christophe; Lewis, Tim; Ashlock, Daniel; McNicholas, Paul D; Fauteux, François; Strömvik, Martina; Raizada, Manish N

2013-03-15

The discovery of genetic networks and cis-acting DNA motifs underlying their regulation is a major objective of transcriptome studies. The recent release of the maize genome (Zea mays L.) has facilitated in silico searches for regulatory motifs. Several algorithms exist to predict cis-acting elements, but none have been adapted for maize. A benchmark data set was used to evaluate the accuracy of three motif discovery programs: BioProspector, Weeder and MEME. Analysis showed that each motif discovery tool had limited accuracy and appeared to retrieve a distinct set of motifs. Therefore, using the benchmark, statistical filters were optimized to reduce the false discovery ratio, and then remaining motifs from all programs were combined to improve motif prediction. These principles were integrated into a user-friendly pipeline for motif discovery in maize called Promzea, available at http://www.promzea.org and on the Discovery Environment of the iPlant Collaborative website. Promzea was subsequently expanded to include rice and Arabidopsis. Within Promzea, a user enters cDNA sequences or gene IDs; corresponding upstream sequences are retrieved from the maize genome. Predicted motifs are filtered, combined and ranked. Promzea searches the chosen plant genome for genes containing each candidate motif, providing the user with the gene list and corresponding gene annotations. Promzea was validated in silico using a benchmark data set: the Promzea pipeline showed a 22% increase in nucleotide sensitivity compared to the best standalone program tool, Weeder, with equivalent nucleotide specificity. Promzea was also validated by its ability to retrieve the experimentally defined binding sites of transcription factors that regulate the maize anthocyanin and phlobaphene biosynthetic pathways. Promzea predicted additional promoter motifs, and genome-wide motif searches by Promzea identified 127 non-anthocyanin/phlobaphene genes that each contained all five predicted promoter motifs in their promoters, perhaps uncovering a broader co-regulated gene network. Promzea was also tested against tissue-specific microarray data from maize. An online tool customized for promoter motif discovery in plants has been generated called Promzea. Promzea was validated in silico by its ability to retrieve benchmark motifs and experimentally defined motifs and was tested using tissue-specific microarray data. Promzea predicted broader networks of gene regulation associated with the historic anthocyanin and phlobaphene biosynthetic pathways. Promzea is a new bioinformatics tool for understanding transcriptional gene regulation in maize and has been expanded to include rice and Arabidopsis.

Subpathway-GM: identification of metabolic subpathways via joint power of interesting genes and metabolites and their topologies within pathways

PubMed Central

Li, Chunquan; Han, Junwei; Yao, Qianlan; Zou, Chendan; Xu, Yanjun; Zhang, Chunlong; Shang, Desi; Zhou, Lingyun; Zou, Chaoxia; Sun, Zeguo; Li, Jing; Zhang, Yunpeng; Yang, Haixiu; Gao, Xu; Li, Xia

2013-01-01

Various ‘omics’ technologies, including microarrays and gas chromatography mass spectrometry, can be used to identify hundreds of interesting genes, proteins and metabolites, such as differential genes, proteins and metabolites associated with diseases. Identifying metabolic pathways has become an invaluable aid to understanding the genes and metabolites associated with studying conditions. However, the classical methods used to identify pathways fail to accurately consider joint power of interesting gene/metabolite and the key regions impacted by them within metabolic pathways. In this study, we propose a powerful analytical method referred to as Subpathway-GM for the identification of metabolic subpathways. This provides a more accurate level of pathway analysis by integrating information from genes and metabolites, and their positions and cascade regions within the given pathway. We analyzed two colorectal cancer and one metastatic prostate cancer data sets and demonstrated that Subpathway-GM was able to identify disease-relevant subpathways whose corresponding entire pathways might be ignored using classical entire pathway identification methods. Further analysis indicated that the power of a joint genes/metabolites and subpathway strategy based on their topologies may play a key role in reliably recalling disease-relevant subpathways and finding novel subpathways. PMID:23482392

Frontotemporal dementia: insights into the biological underpinnings of disease through gene co-expression network analysis.

PubMed

Ferrari, Raffaele; Forabosco, Paola; Vandrovcova, Jana; Botía, Juan A; Guelfi, Sebastian; Warren, Jason D; Momeni, Parastoo; Weale, Michael E; Ryten, Mina; Hardy, John

2016-02-24

In frontotemporal dementia (FTD) there is a critical lack in the understanding of biological and molecular mechanisms involved in disease pathogenesis. The heterogeneous genetic features associated with FTD suggest that multiple disease-mechanisms are likely to contribute to the development of this neurodegenerative condition. We here present a systems biology approach with the scope of i) shedding light on the biological processes potentially implicated in the pathogenesis of FTD and ii) identifying novel potential risk factors for FTD. We performed a gene co-expression network analysis of microarray expression data from 101 individuals without neurodegenerative diseases to explore regional-specific co-expression patterns in the frontal and temporal cortices for 12 genes (MAPT, GRN, CHMP2B, CTSC, HLA-DRA, TMEM106B, C9orf72, VCP, UBQLN2, OPTN, TARDBP and FUS) associated with FTD and we then carried out gene set enrichment and pathway analyses, and investigated known protein-protein interactors (PPIs) of FTD-genes products. Gene co-expression networks revealed that several FTD-genes (such as MAPT and GRN, CTSC and HLA-DRA, TMEM106B, and C9orf72, VCP, UBQLN2 and OPTN) were clustering in modules of relevance in the frontal and temporal cortices. Functional annotation and pathway analyses of such modules indicated enrichment for: i) DNA metabolism, i.e. transcription regulation, DNA protection and chromatin remodelling (MAPT and GRN modules); ii) immune and lysosomal processes (CTSC and HLA-DRA modules), and; iii) protein meta/catabolism (C9orf72, VCP, UBQLN2 and OPTN, and TMEM106B modules). PPI analysis supported the results of the functional annotation and pathway analyses. This work further characterizes known FTD-genes and elaborates on their biological relevance to disease: not only do we indicate likely impacted regional-specific biological processes driven by FTD-genes containing modules, but also do we suggest novel potential risk factors among the FTD-genes interactors as targets for further mechanistic characterization in hypothesis driven cell biology work.