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Sample records for single-cell gene expression

  1. Determining Physical Mechanisms of Gene Expression Regulation from Single Cell Gene Expression Data

    OpenAIRE

    Ezer, Daphne; Moignard, Victoria; G?ttgens, Berthold; Adryan, Boris

    2016-01-01

    Many genes are expressed in bursts, which can contribute to cell-to-cell heterogeneity. It is now possible to measure this heterogeneity with high throughput single cell gene expression assays (single cell qPCR and RNA-seq). These experimental approaches generate gene expression distributions which can be used to estimate the kinetic parameters of gene expression bursting, namely the rate that genes turn on, the rate that genes turn off, and the rate of transcription. We construct a complete ...

  2. Spatial reconstruction of single-cell gene expression data.

    Science.gov (United States)

    Satija, Rahul; Farrell, Jeffrey A; Gennert, David; Schier, Alexander F; Regev, Aviv

    2015-05-01

    Spatial localization is a key determinant of cellular fate and behavior, but methods for spatially resolved, transcriptome-wide gene expression profiling across complex tissues are lacking. RNA staining methods assay only a small number of transcripts, whereas single-cell RNA-seq, which measures global gene expression, separates cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos and generated a transcriptome-wide map of spatial patterning. We confirmed Seurat's accuracy using several experimental approaches, then used the strategy to identify a set of archetypal expression patterns and spatial markers. Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems.

  3. Spatial reconstruction of single-cell gene expression

    Science.gov (United States)

    Satija, Rahul; Farrell, Jeffrey A.; Gennert, David; Schier, Alexander F.; Regev, Aviv

    2015-01-01

    Spatial localization is a key determinant of cellular fate and behavior, but spatial RNA assays traditionally rely on staining for a limited number of RNA species. In contrast, single-cell RNA-seq allows for deep profiling of cellular gene expression, but established methods separate cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos, inferring a transcriptome-wide map of spatial patterning. We confirmed Seurat’s accuracy using several experimental approaches, and used it to identify a set of archetypal expression patterns and spatial markers. Additionally, Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems. PMID:25867923

  4. A Microchip for Integrated Single-Cell Gene Expression Profiling and Genotoxicity Detection

    Directory of Open Access Journals (Sweden)

    Hui Dong

    2016-09-01

    Full Text Available Microfluidics-based single-cell study is an emerging approach in personalized treatment or precision medicine studies. Single-cell gene expression holds a potential to provide treatment selections with maximized efficacy to help cancer patients based on a genetic understanding of their disease. This work presents a multi-layer microchip for single-cell multiplexed gene expression profiling and genotoxicity detection. Treated by three drug reagents (i.e., methyl methanesulfonate, docetaxel and colchicine with varied concentrations and time lengths, individual human cancer cells (MDA-MB-231 are lysed on-chip, and the released mRNA templates are captured and reversely transcribed into single strand DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, cyclin-dependent kinase inhibitor 1A (CDKN1A, and aurora kinase A (AURKA genes from single cells are amplified and real-time quantified through multiplex polymerase chain reaction. The microchip is capable of integrating all steps of single-cell multiplexed gene expression profiling, and providing precision detection of drug induced genotoxic stress. Throughput has been set to be 18, and can be further increased following the same approach. Numerical simulation of on-chip single cell trapping and heat transfer has been employed to evaluate the chip design and operation.

  5. Single-cell gene-expression profiling and its potential diagnostic applications

    Czech Academy of Sciences Publication Activity Database

    Stahlberg, A.; Kubista, Mikael; Aman, P.

    2011-01-01

    Roč. 11, č. 7 (2011), s. 735-740 ISSN 1473-7159 R&D Projects: GA ČR(CZ) GAP303/10/1338; GA ČR(CZ) GA301/09/1752 Institutional research plan: CEZ:AV0Z50520701 Keywords : gene-expression profiling * RT-qPCR * single-cell gene-expression profiling Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.859, year: 2011

  6. Single-Cell Gene Expression Analysis of Cholinergic Neurons in the Arcuate Nucleus of the Hypothalamus.

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    Jae Hoon Jeong

    Full Text Available The cholinoceptive system in the hypothalamus, in particular in the arcuate nucleus (ARC, plays a role in regulating food intake. Neurons in the ARC contain multiple neuropeptides, amines, and neurotransmitters. To study molecular and neurochemical heterogeneity of ARC neurons, we combine single-cell qRT-PCR and single-cell whole transcriptome amplification methods to analyze expression patterns of our hand-picked 60 genes in individual neurons in the ARC. Immunohistochemical and single-cell qRT-PCR analyses show choline acetyltransferase (ChAT-expressing neurons in the ARC. Gene expression patterns are remarkably distinct in each individual cholinergic neuron. Two-thirds of cholinergic neurons express tyrosine hydroxylase (Th mRNA. A large subset of these Th-positive cholinergic neurons is GABAergic as they express the GABA synthesizing enzyme glutamate decarboxylase and vesicular GABA transporter transcripts. Some cholinergic neurons also express the vesicular glutamate transporter transcript gene. POMC and POMC-processing enzyme transcripts are found in a subpopulation of cholinergic neurons. Despite this heterogeneity, gene expression patterns in individual cholinergic cells appear to be highly regulated in a cell-specific manner. In fact, membrane receptor transcripts are clustered with their respective intracellular signaling and downstream targets. This novel population of cholinergic neurons may be part of the neural circuitries that detect homeostatic need for food and control the drive to eat.

  7. Temporal dynamics and transcriptional control using single-cell gene expression analysis.

    Science.gov (United States)

    Kouno, Tsukasa; de Hoon, Michiel; Mar, Jessica C; Tomaru, Yasuhiro; Kawano, Mitsuoki; Carninci, Piero; Suzuki, Harukazu; Hayashizaki, Yoshihide; Shin, Jay W

    2013-01-01

    Changes in environmental conditions lead to expression variation that manifest at the level of gene regulatory networks. Despite a strong understanding of the role noise plays in synthetic biological systems, it remains unclear how propagation of expression heterogeneity in an endogenous regulatory network is distributed and utilized by cells transitioning through a key developmental event. Here we investigate the temporal dynamics of a single-cell transcriptional network of 45 transcription factors in THP-1 human myeloid monocytic leukemia cells undergoing differentiation to macrophages. We systematically measure temporal regulation of expression and variation by profiling 120 single cells at eight distinct time points, and infer highly controlled regulatory modules through which signaling operates with stochastic effects. This reveals dynamic and specific rewiring as a cellular strategy for differentiation. The integration of both positive and negative co-expression networks further identifies the proto-oncogene MYB as a network hinge to modulate both the pro- and anti-differentiation pathways. Compared to averaged cell populations, temporal single-cell expression profiling provides a much more powerful technique to probe for mechanistic insights underlying cellular differentiation. We believe that our approach will form the basis of novel strategies to study the regulation of transcription at a single-cell level.

  8. Estimating intrinsic and extrinsic noise from single-cell gene expression measurements

    Science.gov (United States)

    Fu, Audrey Qiuyan; Pachter, Lior

    2017-01-01

    Gene expression is stochastic and displays variation (“noise”) both within and between cells. Intracellular (intrinsic) variance can be distinguished from extracellular (extrinsic) variance by applying the law of total variance to data from two-reporter assays that probe expression of identically regulated gene pairs in single cells. We examine established formulas [Elowitz, M. B., A. J. Levine, E. D. Siggia and P. S. Swain (2002): “Stochastic gene expression in a single cell,” Science, 297, 1183–1186.] for the estimation of intrinsic and extrinsic noise and provide interpretations of them in terms of a hierarchical model. This allows us to derive alternative estimators that minimize bias or mean squared error. We provide a geometric interpretation of these results that clarifies the interpretation in [Elowitz, M. B., A. J. Levine, E. D. Siggia and P. S. Swain (2002): “Stochastic gene expression in a single cell,” Science, 297, 1183–1186.]. We also demonstrate through simulation and re-analysis of published data that the distribution assumptions underlying the hierarchical model have to be satisfied for the estimators to produce sensible results, which highlights the importance of normalization. PMID:27875323

  9. Heterogeneity of astrocytes: from development to injury - single cell gene expression.

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    Vendula Rusnakova

    Full Text Available Astrocytes perform control and regulatory functions in the central nervous system; heterogeneity among them is still a matter of debate due to limited knowledge of their gene expression profiles and functional diversity. To unravel astrocyte heterogeneity during postnatal development and after focal cerebral ischemia, we employed single-cell gene expression profiling in acutely isolated cortical GFAP/EGFP-positive cells. Using a microfluidic qPCR platform, we profiled 47 genes encoding glial markers and ion channels/transporters/receptors participating in maintaining K(+ and glutamate homeostasis per cell. Self-organizing maps and principal component analyses revealed three subpopulations within 10-50 days of postnatal development (P10-P50. The first subpopulation, mainly immature glia from P10, was characterized by high transcriptional activity of all studied genes, including polydendrocytic markers. The second subpopulation (mostly from P20 was characterized by low gene transcript levels, while the third subpopulation encompassed mature astrocytes (mainly from P30, P50. Within 14 days after ischemia (D3, D7, D14, additional astrocytic subpopulations were identified: resting glia (mostly from P50 and D3, transcriptionally active early reactive glia (mainly from D7 and permanent reactive glia (solely from D14. Following focal cerebral ischemia, reactive astrocytes underwent pronounced changes in the expression of aquaporins, nonspecific cationic and potassium channels, glutamate receptors and reactive astrocyte markers.

  10. Comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis.

    Science.gov (United States)

    Fox, Bridget C; Devonshire, Alison S; Baradez, Marc-Olivier; Marshall, Damian; Foy, Carole A

    2012-08-15

    Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT-qPCR approach using dilutions of cells and RNA standards to the single cell level. Single cell-level messenger RNA (mRNA) analysis was possible using all three methods, although the precision, linearity, and effect of lysis buffer and cell background differed depending on the approach used. The impact of using a microfluidic qPCR platform versus a standard instrument was investigated for potential variability introduced by preamplification of template or scaling down of the qPCR to nanoliter volumes using laser-dissected single cell samples. The two approaches were found to be comparable. These studies show that accurate gene expression analysis is achievable at the single cell level and highlight the importance of well-validated experimental procedures for low-level mRNA analysis. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. What Population Reveals about Individual Cell Identity: Single-Cell Parameter Estimation of Models of Gene Expression in Yeast.

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    Artémis Llamosi

    2016-02-01

    Full Text Available Significant cell-to-cell heterogeneity is ubiquitously observed in isogenic cell populations. Consequently, parameters of models of intracellular processes, usually fitted to population-averaged data, should rather be fitted to individual cells to obtain a population of models of similar but non-identical individuals. Here, we propose a quantitative modeling framework that attributes specific parameter values to single cells for a standard model of gene expression. We combine high quality single-cell measurements of the response of yeast cells to repeated hyperosmotic shocks and state-of-the-art statistical inference approaches for mixed-effects models to infer multidimensional parameter distributions describing the population, and then derive specific parameters for individual cells. The analysis of single-cell parameters shows that single-cell identity (e.g. gene expression dynamics, cell size, growth rate, mother-daughter relationships is, at least partially, captured by the parameter values of gene expression models (e.g. rates of transcription, translation and degradation. Our approach shows how to use the rich information contained into longitudinal single-cell data to infer parameters that can faithfully represent single-cell identity.

  12. Distinct gene expression signatures in human embryonic stem cells differentiated towards definitive endoderm at single-cell level

    DEFF Research Database (Denmark)

    Norrman, Karin; Strömbeck, Anna; Semb, Henrik

    2013-01-01

    for the three activin A based protocols applied. Our data provide novel insights in DE gene expression at the cellular level of in vitro differentiated human embryonic stem cells, and illustrate the power of using single-cell gene expression profiling to study differentiation heterogeneity and to characterize...... of anterior definitive endoderm (DE). Here, we differentiated human embryonic stem cells towards DE using three different activin A based treatments. Differentiation efficiencies were evaluated by gene expression profiling over time at cell population level. A panel of key markers was used to study DE...... formation. Final DE differentiation was also analyzed with immunocytochemistry and single-cell gene expression profiling. We found that cells treated with activin A in combination with sodium butyrate and B27 serum-free supplement medium generated the most mature DE cells. Cell population studies were...

  13. Heterogeneity of Astrocytes: From Development to Injury - Single Cell Gene Expression

    Czech Academy of Sciences Publication Activity Database

    Rusňáková, Vendula; Honsa, Pavel; Džamba, Dávid; Stahlberg, A.; Kubista, Mikael; Anděrová, Miroslava

    2013-01-01

    Roč. 8, č. 8 (2013), e69734 E-ISSN 1932-6203 R&D Projects: GA ČR GA13-02154S Institutional research plan: CEZ:AV0Z50520701; CEZ:AV0Z50390703 Keywords : Single cell expression profiling * astrocytes * GenEx Subject RIV: EB - Genetics ; Molecular Biology; FH - Neurology (UEM-P) Impact factor: 3.534, year: 2013

  14. Single-cell multiple gene expression analysis based on single-molecule-detection microarray assay for multi-DNA determination

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Xianwei [School of Life Sciences, Shandong University, Jinan 250100 (China); Zhang, Xiaoli [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin, Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2015-01-07

    Highlights: • A single-molecule-detection (SMD) microarray for 10 samples is fabricated. • The based-SMD microarray assay (SMA) can determine 8 DNAs for each sample. • The limit of detection of SMA is as low as 1.3 × 10{sup −16} mol L{sup −1}. • The SMA can be applied in single-cell multiple gene expression analysis. - Abstract: We report a novel ultra-sensitive and high-selective single-molecule-detection microarray assay (SMA) for multiple DNA determination. In the SMA, a capture DNA (DNAc) microarray consisting of 10 subarrays with 9 spots for each subarray is fabricated on a silanized glass coverslip as the substrate. On the subarrays, the spot-to-spot spacing is 500 μm and each spot has a diameter of ∼300 μm. The sequence of the DNAcs on the 9 spots of a subarray is different, to determine 8 types of target DNAs (DNAts). Thus, 8 types of DNAts are captured to their complementary DNAcs at 8 spots of a subarray, respectively, and then labeled with quantum dots (QDs) attached to 8 types of detection DNAs (DNAds) with different sequences. The ninth spot is used to detect the blank value. In order to determine the same 8 types of DNAts in 10 samples, the 10 DNAc-modified subarrays on the microarray are identical. Fluorescence single-molecule images of the QD-labeled DNAts on each spot of the subarray are acquired using a home-made single-molecule microarray reader. The amounts of the DNAts are quantified by counting the bright dots from the QDs. For a microarray, 8 types of DNAts in 10 samples can be quantified in parallel. The limit of detection of the SMA for DNA determination is as low as 1.3 × 10{sup −16} mol L{sup −1}. The SMA for multi-DNA determination can also be applied in single-cell multiple gene expression analysis through quantification of complementary DNAs (cDNAs) corresponding to multiple messenger RNAs (mRNAs) in single cells. To do so, total RNA in single cells is extracted and reversely transcribed into their cDNAs. Three

  15. SigEMD: A powerful method for differential gene expression analysis in single-cell RNA sequencing data.

    Science.gov (United States)

    Wang, Tianyu; Nabavi, Sheida

    2018-04-24

    Differential gene expression analysis is one of the significant efforts in single cell RNA sequencing (scRNAseq) analysis to discover the specific changes in expression levels of individual cell types. Since scRNAseq exhibits multimodality, large amounts of zero counts, and sparsity, it is different from the traditional bulk RNA sequencing (RNAseq) data. The new challenges of scRNAseq data promote the development of new methods for identifying differentially expressed (DE) genes. In this study, we proposed a new method, SigEMD, that combines a data imputation approach, a logistic regression model and a nonparametric method based on the Earth Mover's Distance, to precisely and efficiently identify DE genes in scRNAseq data. The regression model and data imputation are used to reduce the impact of large amounts of zero counts, and the nonparametric method is used to improve the sensitivity of detecting DE genes from multimodal scRNAseq data. By additionally employing gene interaction network information to adjust the final states of DE genes, we further reduce the false positives of calling DE genes. We used simulated datasets and real datasets to evaluate the detection accuracy of the proposed method and to compare its performance with those of other differential expression analysis methods. Results indicate that the proposed method has an overall powerful performance in terms of precision in detection, sensitivity, and specificity. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Single cell analysis of gene expression patterns of competence development and initiation of sporulation in Bacillus subtilis grown on chemically defined media

    NARCIS (Netherlands)

    Veening, J. -W.; Smits, W. K.; Hamoen, L. W.; Kuipers, O. P.

    Aim: Understanding the basis for the heterogeneous (or bistable) expression patterns of competence development and sporulation in Bacillus subtilis. Methods and Results: Using flow cytometric analyses of various promoter-GFP fusions, we have determined the single-cell gene expression patterns of

  17. Single cell subtractive transcriptomics for identification of cell-specifically expressed candidate genes of pyrrolizidine alkaloid biosynthesis.

    Science.gov (United States)

    Sievert, Christian; Beuerle, Till; Hollmann, Julien; Ober, Dietrich

    2015-09-01

    Progress has recently been made in the elucidation of pathways of secondary metabolism. However, because of its diversity, genetic information concerning biosynthetic details is still missing for many natural products. This is also the case for the biosynthesis of pyrrolizidine alkaloids. To close this gap, we tested strategies using tissues that express this pathway in comparison to tissues in which this pathway is not expressed. As many pathways of secondary metabolism are known to be induced by jasmonates, the pyrrolizidine alkaloid-producing species Heliotropium indicum, Symphytum officinale, and Cynoglossum officinale of the Boraginales order were treated with methyl jasmonate. An effect on pyrrolizidine alkaloid levels and on transcript levels of homospermidine synthase, the first specific enzyme of pyrrolizidine alkaloid biosynthesis, was not detectable. Therefore, a method was developed by making use of the often observed cell-specific production of secondary compounds. H. indicum produces pyrrolizidine alkaloids exclusively in the shoot. Homospermidine synthase is expressed only in the cells of the lower leaf epidermis and the epidermis of the stem. Suggesting that the whole pathway of pyrrolizidine alkaloid biosynthesis might be localized in these cells, we have isolated single cells of the upper and lower epidermis by laser-capture microdissection. The resulting cDNA preparations have been used in a subtractive transcriptomic approach. Quantitative real-time polymerase chain reaction has shown that the resulting library is significantly enriched for homospermidine-synthase-coding transcripts providing a valuable source for the identification of further genes involved in pyrrolizidine alkaloid biosynthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Developmental switching in Physarum polycephalum : Petri net analysis of single cell trajectories of gene expression indicates responsiveness and genetic plasticity of the Waddington quasipotential landscape

    International Nuclear Information System (INIS)

    Werthmann, Britta; Marwan, Wolfgang

    2017-01-01

    The developmental switch to sporulation in Physarum polycephalum is a phytochrome-mediated far-red light-induced cell fate decision that synchronously encompasses the entire multinucleate plasmodial cell and is associated with extensive reprogramming of the transcriptome. By repeatedly taking samples of single cells after delivery of a light stimulus pulse, we analysed differential gene expression in two mutant strains and in a heterokaryon of the two strains all of which display a different propensity for making the cell fate decision. Multidimensional scaling of the gene expression data revealed individually different single cell trajectories eventually leading to sporulation. Characterization of the trajectories as walks through states of gene expression discretized by hierarchical clustering allowed the reconstruction of Petri nets that model and predict the observed behavior. Structural analyses of the Petri nets indicated stimulus- and genotype-dependence of both, single cell trajectories and of the quasipotential landscape through which these trajectories are taken. The Petri net-based approach to the analysis and decomposition of complex cellular responses and of complex mutant phenotypes may provide a scaffold for the data-driven reconstruction of causal molecular mechanisms that shape the topology of the quasipotential landscape. (paper)

  19. Developmental switching in Physarum polycephalum: Petri net analysis of single cell trajectories of gene expression indicates responsiveness and genetic plasticity of the Waddington quasipotential landscape

    Science.gov (United States)

    Werthmann, Britta; Marwan, Wolfgang

    2017-11-01

    The developmental switch to sporulation in Physarum polycephalum is a phytochrome-mediated far-red light-induced cell fate decision that synchronously encompasses the entire multinucleate plasmodial cell and is associated with extensive reprogramming of the transcriptome. By repeatedly taking samples of single cells after delivery of a light stimulus pulse, we analysed differential gene expression in two mutant strains and in a heterokaryon of the two strains all of which display a different propensity for making the cell fate decision. Multidimensional scaling of the gene expression data revealed individually different single cell trajectories eventually leading to sporulation. Characterization of the trajectories as walks through states of gene expression discretized by hierarchical clustering allowed the reconstruction of Petri nets that model and predict the observed behavior. Structural analyses of the Petri nets indicated stimulus- and genotype-dependence of both, single cell trajectories and of the quasipotential landscape through which these trajectories are taken. The Petri net-based approach to the analysis and decomposition of complex cellular responses and of complex mutant phenotypes may provide a scaffold for the data-driven reconstruction of causal molecular mechanisms that shape the topology of the quasipotential landscape.

  20. Probabilistic modeling of bifurcations in single-cell gene expression data using a Bayesian mixture of factor analyzers [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Kieran R Campbell

    2017-03-01

    Full Text Available Modeling bifurcations in single-cell transcriptomics data has become an increasingly popular field of research. Several methods have been proposed to infer bifurcation structure from such data, but all rely on heuristic non-probabilistic inference. Here we propose the first generative, fully probabilistic model for such inference based on a Bayesian hierarchical mixture of factor analyzers. Our model exhibits competitive performance on large datasets despite implementing full Markov-Chain Monte Carlo sampling, and its unique hierarchical prior structure enables automatic determination of genes driving the bifurcation process. We additionally propose an Empirical-Bayes like extension that deals with the high levels of zero-inflation in single-cell RNA-seq data and quantify when such models are useful. We apply or model to both real and simulated single-cell gene expression data and compare the results to existing pseudotime methods. Finally, we discuss both the merits and weaknesses of such a unified, probabilistic approach in the context practical bioinformatics analyses.

  1. Dissecting stem cell differentiation using single cell expression profiling

    OpenAIRE

    Moignard, Victoria Rachel; Göttgens, Berthold

    2016-01-01

    Many assumptions about the way cells behave are based on analyses of populations. However, it is now widely recognized that even apparently pure populations can display a remarkable level of heterogeneity. This is particularly true in stem cell biology where it hinders our understanding of normal development and the development of strategies for regenerative medicine. Over the past decade technologies facilitating gene expression analysis at the single cell level have become widespread, provi...

  2. Approaches to systems biology. Four methods to study single-cell gene expression, cell motility, antibody reactivity, and respiratory metabolism

    DEFF Research Database (Denmark)

    Hagedorn, Peter

    To understand how complex systems, such as cells, function, comprehensive Measurements of their constituent parts must be made. This can be achieved by combining methods that are each optimized to measure specific parts of the system. Four such methods,each covering a different area, are presented...... from such measurements allows models of the system to be developed and tested. For each of the methods, such analysis and modelling approaches have beenapplied and are presented: Differentially regulated genes are identified and classified according to function; cell-specfic motility models...... are developed that can distinguish between different surfaces; a method for selecting repertoires of antigens thatseparate mice based on their response to treatment is developed; and the observed concentrations of free and bound NADH is used to build and test a basic model of respiratory metabolism...

  3. Co-expression of Exo-inulinase and Endo-inulinase Genes in the Oleaginous Yeast Yarrowia lipolytica for Efficient Single Cell Oil Production from Inulin.

    Science.gov (United States)

    Shi, Nianci; Mao, Weian; He, Xiaoxia; Chi, Zhe; Chi, Zhenming; Liu, Guanglei

    2018-05-01

    Yarrowia lipolytica is a promising platform for the single cell oil (SCO) production. In this study, a transformant X+N8 in which exo- and endo-inulinase genes were co-expressed could produce an inulinase activity of 124.33 U/mL within 72 h. However, the inulinase activity of a transformant X2 carrying a single exo-inulinase gene was only 47.33 U/mL within 72 h. Moreover, the transformant X+N8 could accumulate 48.13% (w/w) SCO from inulin and the cell dry weight reached 13.63 g/L within 78 h, which were significantly higher than those of the transformant X2 (41.87% (w/w) and 11.23 g/L) under the same conditions. In addition, inulin hydrolysis and utilization of the transformant X+N8 were also more efficient than those of the transformant X2 during the fermentation process. These results demonstrated that the co-expression of the exo- and endo-inulinase genes significantly enhanced the SCO production from inulin due to the improvement of the inulinase activity and the synergistic action of exo- and endo-inulinase. Besides, over 95.01% of the fatty acids from the transformant X+N8 were C16-C18, especially C18:1 (53.10%), suggesting that the fatty acids could be used as feedstock for biodiesel production.

  4. Protein Expression Analyses at the Single Cell Level

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    Masae Ohno

    2014-09-01

    Full Text Available The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level.

  5. Correlating Anatomy and Function with Gene Expression in Individual Neurons by Combining in Vivo Labeling, Patch Clamp, and Single Cell RNA-seq

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    Carsten K. Pfeffer

    2017-11-01

    Full Text Available The classification of neurons into distinct types is an ongoing effort aimed at revealing and understanding the diversity of the components of the nervous system. Recently available methods allow us to determine the gene expression pattern of individual neurons in the mammalian cerebral cortex to generate powerful categorization schemes. For a thorough understanding of neuronal diversity such genetic categorization schemes need to be combined with traditional classification parameters like position, axonal projection or response properties to sensory stimulation. Here we describe a method to link the gene expression of individual neurons with their position, axonal projection, or sensory response properties. Neurons are labeled in vivo based on their anatomical or functional properties and, using patch clamp pipettes, their RNA individually harvested in vitro for RNAseq. We validate the methodology using multiple established molecularly and anatomically distinct cell populations and explore molecular differences between uncharacterized neurons in mouse visual cortex. Gene expression patterns between L5 neurons projecting to frontal or contralateral cortex are distinct while L2 neurons differing in position, projection, or function are molecularly similar. With this method we can determine the genetic expression pattern of functionally and anatomically identified individual neurons.

  6. Characterization of Distinct Astrocytic Populations Responding with Different Volume Changes to Oxygen-Glucose Deprivation: 3d Confocal Morphometry And Single-Cell Gene Expression Profilig

    Czech Academy of Sciences Publication Activity Database

    Benešová, Jana; Rusňáková, Vendula; Honsa, Pavel; Pivoňková, Helena; Kubista, Mikael; Anděrová, Miroslava

    2011-01-01

    Roč. 59, Supplement 1 (2011), S126-S126 ISSN 0894-1491. [European meeting on Glia l Cells in Health and Disease /10./. 13.09.2011-13.09.2011, Prague] Institutional research plan: CEZ:AV0Z50390703; CEZ:AV0Z50520701 Keywords : cell volume regulation * single-cell PCR profiling * ischemia Subject RIV: FH - Neurology

  7. Using machine learning to speed up manual image annotation: application to a 3D imaging protocol for measuring single cell gene expression in the developing C. elegans embryo

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    Waterston Robert H

    2010-02-01

    Full Text Available Abstract Background Image analysis is an essential component in many biological experiments that study gene expression, cell cycle progression, and protein localization. A protocol for tracking the expression of individual C. elegans genes was developed that collects image samples of a developing embryo by 3-D time lapse microscopy. In this protocol, a program called StarryNite performs the automatic recognition of fluorescently labeled cells and traces their lineage. However, due to the amount of noise present in the data and due to the challenges introduced by increasing number of cells in later stages of development, this program is not error free. In the current version, the error correction (i.e., editing is performed manually using a graphical interface tool named AceTree, which is specifically developed for this task. For a single experiment, this manual annotation task takes several hours. Results In this paper, we reduce the time required to correct errors made by StarryNite. We target one of the most frequent error types (movements annotated as divisions and train a support vector machine (SVM classifier to decide whether a division call made by StarryNite is correct or not. We show, via cross-validation experiments on several benchmark data sets, that the SVM successfully identifies this type of error significantly. A new version of StarryNite that includes the trained SVM classifier is available at http://starrynite.sourceforge.net. Conclusions We demonstrate the utility of a machine learning approach to error annotation for StarryNite. In the process, we also provide some general methodologies for developing and validating a classifier with respect to a given pattern recognition task.

  8. Single-Cell Gene Expression Analysis of a Human ESC Model of Pancreatic Endocrine Development Reveals Different Paths to β-Cell Differentiation.

    Science.gov (United States)

    Petersen, Maja Borup Kjær; Azad, Ajuna; Ingvorsen, Camilla; Hess, Katja; Hansson, Mattias; Grapin-Botton, Anne; Honoré, Christian

    2017-10-10

    The production of insulin-producing β cells from human embryonic stem cells (hESCs) in vitro represents a promising strategy for a cell-based therapy for type 1 diabetes mellitus. To explore the cellular heterogeneity and temporal progression of endocrine progenitors and their progeny, we performed single-cell qPCR on more than 500 cells across several stages of in vitro differentiation of hESCs and compared them with human islets. We reveal distinct subpopulations along the endocrine differentiation path and an early lineage bifurcation toward either polyhormonal cells or β-like cells. We uncover several similarities and differences with mouse development and reveal that cells can take multiple paths to the same differentiation state, a principle that could be relevant to other systems. Notably, activation of the key β-cell transcription factor NKX6.1 can be initiated before or after endocrine commitment. The single-cell temporal resolution we provide can be used to improve the production of functional β cells. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  9. The workflow of single-cell expression profiling using quantitative real-time PCR

    Czech Academy of Sciences Publication Activity Database

    Stahlberg, A.; Kubista, Mikael

    2014-01-01

    Roč. 14, č. 3 (2014), s. 323-331 ISSN 1473-7159 R&D Projects: GA ČR GA13-02154S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : single-cell workflow * gene expression profiling * RT-qPCR Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.516, year: 2014

  10. Accounting for technical noise in differential expression analysis of single-cell RNA sequencing data.

    Science.gov (United States)

    Jia, Cheng; Hu, Yu; Kelly, Derek; Kim, Junhyong; Li, Mingyao; Zhang, Nancy R

    2017-11-02

    Recent technological breakthroughs have made it possible to measure RNA expression at the single-cell level, thus paving the way for exploring expression heterogeneity among individual cells. Current single-cell RNA sequencing (scRNA-seq) protocols are complex and introduce technical biases that vary across cells, which can bias downstream analysis without proper adjustment. To account for cell-to-cell technical differences, we propose a statistical framework, TASC (Toolkit for Analysis of Single Cell RNA-seq), an empirical Bayes approach to reliably model the cell-specific dropout rates and amplification bias by use of external RNA spike-ins. TASC incorporates the technical parameters, which reflect cell-to-cell batch effects, into a hierarchical mixture model to estimate the biological variance of a gene and detect differentially expressed genes. More importantly, TASC is able to adjust for covariates to further eliminate confounding that may originate from cell size and cell cycle differences. In simulation and real scRNA-seq data, TASC achieves accurate Type I error control and displays competitive sensitivity and improved robustness to batch effects in differential expression analysis, compared to existing methods. TASC is programmed to be computationally efficient, taking advantage of multi-threaded parallelization. We believe that TASC will provide a robust platform for researchers to leverage the power of scRNA-seq. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Reverse-engineering of gene networks for regulating early blood development from single-cell measurements.

    Science.gov (United States)

    Wei, Jiangyong; Hu, Xiaohua; Zou, Xiufen; Tian, Tianhai

    2017-12-28

    Recent advances in omics technologies have raised great opportunities to study large-scale regulatory networks inside the cell. In addition, single-cell experiments have measured the gene and protein activities in a large number of cells under the same experimental conditions. However, a significant challenge in computational biology and bioinformatics is how to derive quantitative information from the single-cell observations and how to develop sophisticated mathematical models to describe the dynamic properties of regulatory networks using the derived quantitative information. This work designs an integrated approach to reverse-engineer gene networks for regulating early blood development based on singel-cell experimental observations. The wanderlust algorithm is initially used to develop the pseudo-trajectory for the activities of a number of genes. Since the gene expression data in the developed pseudo-trajectory show large fluctuations, we then use Gaussian process regression methods to smooth the gene express data in order to obtain pseudo-trajectories with much less fluctuations. The proposed integrated framework consists of both bioinformatics algorithms to reconstruct the regulatory network and mathematical models using differential equations to describe the dynamics of gene expression. The developed approach is applied to study the network regulating early blood cell development. A graphic model is constructed for a regulatory network with forty genes and a dynamic model using differential equations is developed for a network of nine genes. Numerical results suggests that the proposed model is able to match experimental data very well. We also examine the networks with more regulatory relations and numerical results show that more regulations may exist. We test the possibility of auto-regulation but numerical simulations do not support the positive auto-regulation. In addition, robustness is used as an importantly additional criterion to select candidate

  12. Development of a Method to Monitor Gene Expression in Single Bacterial Cells During the Interaction With Plants and Use to Study the Expression of the Type III Secretion System in Single Cells of Dickeya dadantii in Potato

    Directory of Open Access Journals (Sweden)

    Zhouqi Cui

    2018-06-01

    Full Text Available Dickeya dadantii is a bacterial plant pathogen that causes soft rot disease on a wide range of host plants. The type III secretion system (T3SS is an important virulence factor in D. dadantii. Expression of the T3SS is induced in the plant apoplast or in hrp-inducing minimal medium (hrp-MM, and is repressed in nutrient-rich media. Despite the understanding of induction conditions, how individual cells in a clonal bacterial population respond to these conditions and modulate T3SS expression is not well understood. In our previous study, we reported that in a clonal population, only a small proportion of bacteria highly expressed T3SS genes while the majority of the population did not express T3SS genes under hrp-MM condition. In this study, we developed a method that enabled in situ observation and quantification of gene expression in single bacterial cells in planta. Using this technique, we observed that the expression of the T3SS genes hrpA and hrpN is restricted to a small proportion of D. dadantii cells during the infection of potato. We also report that the expression of T3SS genes is higher at early stages of infection compared to later stages. This expression modulation is achieved through adjusting the ratio of T3SS ON and T3SS OFF cells and the expression intensity of T3SS ON cells. Our findings not only shed light into how bacteria use a bi-stable gene expression manner to modulate an important virulence factor, but also provide a useful tool to study gene expression in individual bacterial cells in planta.

  13. Clustering Single-Cell Expression Data Using Random Forest Graphs.

    Science.gov (United States)

    Pouyan, Maziyar Baran; Nourani, Mehrdad

    2017-07-01

    Complex tissues such as brain and bone marrow are made up of multiple cell types. As the study of biological tissue structure progresses, the role of cell-type-specific research becomes increasingly important. Novel sequencing technology such as single-cell cytometry provides researchers access to valuable biological data. Applying machine-learning techniques to these high-throughput datasets provides deep insights into the cellular landscape of the tissue where those cells are a part of. In this paper, we propose the use of random-forest-based single-cell profiling, a new machine-learning-based technique, to profile different cell types of intricate tissues using single-cell cytometry data. Our technique utilizes random forests to capture cell marker dependences and model the cellular populations using the cell network concept. This cellular network helps us discover what cell types are in the tissue. Our experimental results on public-domain datasets indicate promising performance and accuracy of our technique in extracting cell populations of complex tissues.

  14. Analysis of allelic expression patterns in clonal somatic cells by single-cell RNA-seq.

    Science.gov (United States)

    Reinius, Björn; Mold, Jeff E; Ramsköld, Daniel; Deng, Qiaolin; Johnsson, Per; Michaëlsson, Jakob; Frisén, Jonas; Sandberg, Rickard

    2016-11-01

    Cellular heterogeneity can emerge from the expression of only one parental allele. However, it has remained controversial whether, or to what degree, random monoallelic expression of autosomal genes (aRME) is mitotically inherited (clonal) or stochastic (dynamic) in somatic cells, particularly in vivo. Here we used allele-sensitive single-cell RNA-seq on clonal primary mouse fibroblasts and freshly isolated human CD8 + T cells to dissect clonal and dynamic monoallelic expression patterns. Dynamic aRME affected a considerable portion of the cells' transcriptomes, with levels dependent on the cells' transcriptional activity. Notably, clonal aRME was detected, but it was surprisingly scarce (aRME occurs transiently within individual cells, and patterns of aRME are thus primarily scattered throughout somatic cell populations rather than, as previously hypothesized, confined to patches of clonally related cells.

  15. Evaluation of tools for highly variable gene discovery from single-cell RNA-seq data.

    Science.gov (United States)

    Yip, Shun H; Sham, Pak Chung; Wang, Junwen

    2018-02-21

    Traditional RNA sequencing (RNA-seq) allows the detection of gene expression variations between two or more cell populations through differentially expressed gene (DEG) analysis. However, genes that contribute to cell-to-cell differences are not discoverable with RNA-seq because RNA-seq samples are obtained from a mixture of cells. Single-cell RNA-seq (scRNA-seq) allows the detection of gene expression in each cell. With scRNA-seq, highly variable gene (HVG) discovery allows the detection of genes that contribute strongly to cell-to-cell variation within a homogeneous cell population, such as a population of embryonic stem cells. This analysis is implemented in many software packages. In this study, we compare seven HVG methods from six software packages, including BASiCS, Brennecke, scLVM, scran, scVEGs and Seurat. Our results demonstrate that reproducibility in HVG analysis requires a larger sample size than DEG analysis. Discrepancies between methods and potential issues in these tools are discussed and recommendations are made.

  16. Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system

    Science.gov (United States)

    Lemus-Diaz, Nicolas; Böker, Kai O.; Rodriguez-Polo, Ignacio; Mitter, Michael; Preis, Jasmin; Arlt, Maximilian; Gruber, Jens

    2017-01-01

    Despite major advances on miRNA profiling and target predictions, functional readouts for endogenous miRNAs are limited and frequently lead to contradicting conclusions. Numerous approaches including functional high-throughput and miRISC complex evaluations suggest that the functional miRNAome differs from the predictions based on quantitative sRNA profiling. To resolve the apparent contradiction of expression versus function, we generated and applied a fluorescence reporter gene assay enabling single cell analysis. This approach integrates and adapts a mathematical model for miRNA-driven gene repression. This model predicts three distinct miRNA-groups with unique repression activities (low, mid and high) governed not just by expression levels but also by miRNA/target-binding capability. Here, we demonstrate the feasibility of the system by applying controlled concentrations of synthetic siRNAs and in parallel, altering target-binding capability on corresponding reporter-constructs. Furthermore, we compared miRNA-profiles with the modeled predictions of 29 individual candidates. We demonstrate that expression levels only partially reflect the miRNA function, fitting to the model-projected groups of different activities. Furthermore, we demonstrate that subcellular localization of miRNAs impacts functionality. Our results imply that miRNA profiling alone cannot define their repression activity. The gene regulatory function is a dynamic and complex process beyond a minimalistic conception of “highly expressed equals high repression”. PMID:28338079

  17. Monitoring single-cell gene regulation under dynamically controllable conditions with integrated microfluidics and software

    NARCIS (Netherlands)

    Kaiser, Matthias; Jug, Florian; Julou, Thomas; Deshpande, S.R.; Pfohl, Thomas; Silander, Olin K.; Myers, Gene; Van Nimwegen, Erik

    2018-01-01

    Much is still not understood about how gene regulatory interactions control cell fate decisions in single cells, in part due to the difficulty of directly observing gene regulatory processes in vivo. We introduce here a novel integrated setup consisting of a microfluidic chip and accompanying

  18. Gene expression

    International Nuclear Information System (INIS)

    Hildebrand, C.E.; Crawford, B.D.; Walters, R.A.; Enger, M.D.

    1983-01-01

    We prepared probes for isolating functional pieces of the metallothionein locus. The probes enabled a variety of experiments, eventually revealing two mechanisms for metallothionein gene expression, the order of the DNA coding units at the locus, and the location of the gene site in its chromosome. Once the switch regulating metallothionein synthesis was located, it could be joined by recombinant DNA methods to other, unrelated genes, then reintroduced into cells by gene-transfer techniques. The expression of these recombinant genes could then be induced by exposing the cells to Zn 2+ or Cd 2+ . We would thus take advantage of the clearly defined switching properties of the metallothionein gene to manipulate the expression of other, perhaps normally constitutive, genes. Already, despite an incomplete understanding of how the regulatory switch of the metallothionein locus operates, such experiments have been performed successfully

  19. Dynamic expression of the translational machinery during Bacillus subtilis life cycle at a single cell level.

    Directory of Open Access Journals (Sweden)

    Alex Rosenberg

    Full Text Available The ability of bacteria to responsively regulate the expression of translation components is crucial for rapid adaptation to fluctuating environments. Utilizing Bacillus subtilis (B. subtilis as a model organism, we followed the dynamics of the translational machinery at a single cell resolution during growth and differentiation. By comprehensive monitoring the activity of the major rrn promoters and ribosomal protein production, we revealed diverse dynamics between cells grown in rich and poor medium, with the most prominent dissimilarities exhibited during deep stationary phase. Further, the variability pattern of translational activity varied among the cells, being affected by nutrient availability. We have monitored for the first time translational dynamics during the developmental process of sporulation within the two distinct cellular compartments of forespore and mother-cell. Our study uncovers a transient forespore specific increase in expression of translational components. Finally, the contribution of each rrn promoter throughout the bacterium life cycle was found to be relatively constant, implying that differential expression is not the main purpose for the existence of multiple rrn genes. Instead, we propose that coordination of the rrn operons serves as a strategy to rapidly fine tune translational activities in a synchronized fashion to achieve an optimal translation level for a given condition.

  20. Single Cell Analysis of Dystrophin and SRY Gene by Using Whole Genome Amplification

    Institute of Scientific and Technical Information of China (English)

    徐晨明; 金帆; 黄荷凤; 陶冶; 叶英辉

    2001-01-01

    Objective To develop a reliable and sensitive method for detection of sex and multiloci of Duchenne muscular dystrophy (DMD) gene in single cell Materials & methods Whole genome of single cell were amplified by using 15-base random primers (primer extension preamplification, PEP), then a small aliquot of PEP product were analyzed by using locus-specific nest PCR amplification. The procedure was evaluated by detection dystrophin exons 8, 17, 19, 44, 45, 48 and human testis-determining gene (SRY)in single lymphocytes from known sources and single blastomeres from the couples with no family history of DMD.Results The amplification efficiency rate of six dystrophin exons from single lymphocytes and single blastomeres were 97. 2% (175/180) and 100% (60/60) respectively.Results of SRY showed that 100% (15/15) amplification in single male-derived lymphocytes and 0% (0/15) amplification in single female-derived lymphocytes. Conclusion The technique of single cell PEP-nest PCR for dystrophin exons 8, 17,19, 44, 45, 48 and SRY is highly specifc. PEP-nest PCR is suitable for Preimplantation genetic diagnosis (PGD) of DMD at single cell level.

  1. Single cell genomics indicates horizontal gene transfer and viral infections in a deep subsurface Firmicutes population

    Directory of Open Access Journals (Sweden)

    Jessica eLabonté

    2015-04-01

    Full Text Available A major fraction of Earth's prokaryotic biomass dwells in the deep subsurface, where cellular abundances per volume of sample are lower, metabolism is slower, and generation times are longer than those in surface terrestrial and marine environments. How these conditions impact biotic interactions and evolutionary processes is largely unknown. Here we employed single cell genomics to analyze cell-to-cell genome content variability and signatures of horizontal gene transfer (HGT and viral infections in five cells of Candidatus Desulforudis audaxviator, which were collected from a three km-deep fracture water in the 2.9 Ga-old Witwatersrand Basin of South Africa. Between 0 and 32 % of genes recovered from single cells were not present in the original, metagenomic assembly of Desulforudis, which was obtained from a neighboring subsurface fracture. We found a transposable prophage, a retron, multiple clustered regularly interspaced short palindromic repeats (CRISPRs and restriction-modification systems, and an unusually high frequency of transposases in the analyzed single cell genomes. This indicates that recombination, HGT and viral infections are prevalent evolutionary events in the studied population of microorganisms inhabiting a highly stable deep subsurface environment.

  2. RNA preparation and characterization for gene expression studies

    DEFF Research Database (Denmark)

    Stangegaard, Michael

    2009-01-01

    Much information can be obtained from knowledge of the relative expression level of each gene in the transcriptome. With the current advances in technology as little as a single cell is required as starting material for gene expression experiments. The mRNA from a single cell may be linearly...

  3. Comparison of transcriptional heterogeneity of eight genes between batch Desulfovibrio vulgaris biofilm and planktonic culture at a single-cell level

    Directory of Open Access Journals (Sweden)

    Zhenhua eQi

    2016-04-01

    Full Text Available Sulfate-reducing bacteria (SRB biofilm formed on metal surfaces can change the physicochemical properties of metals and cause metal corrosion. To enhance understanding of differential gene expression in Desulfovibrio vulgaris under planktonic and biofilm growth modes, a single-cell based RT-qPCR approach was applied to determine gene expression levels of 8 selected target genes in four sets of the 31 individual cells isolated from each growth condition (i.e., biofilm formed on a stainless steel (SS) and planktonic cultures, exponential and stationary phases. The results showed obvious gene-expression heterogeneity for the target genes among D. vulgaris single cells of both biofilm and planktonic cultures. In addition, an increased gene-expression heterogeneity in the D. vulgaris biofilm when compared with the planktonic culture was also observed for seven out of eight selected genes, which may be contributing to the increased complexity in terms of structures and morphology in the biofilm. Moreover, the results showed up-regulation of DVU0281 gene encoding exopolysaccharide biosynthesis protein, and down-regulation of genes involved in energy metabolism (i.e., DVU0434 and DVU0588, stress responses (i.e., DVU2410 and response regulator (i.e., DVU3062 in the D. vulgaris biofilm cells. Finally, the gene (DVU2571 involved in iron transportation was found down-regulated, and two genes (DVU1340 and DVU1397 involved in ferric uptake repressor and iron storage were up-regulated in D. vulgaris biofilm, suggesting their possible roles in maintaining normal metabolism of the D. vulgaris biofilm under environments of high concentration of iron. This study showed that the single-cell based analysis could be a useful approach in deciphering metabolism of microbial biofilms.

  4. Single-cell real-time imaging of transgene expression upon lipofection

    Energy Technology Data Exchange (ETDEWEB)

    Fiume, Giuseppe [Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa (Italy); Di Rienzo, Carmine [Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa (Italy); NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12, 56127, Pisa (Italy); Marchetti, Laura [Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa (Italy); Pozzi, Daniela; Caracciolo, Giulio [Department of Molecular Medicine, “Sapienza” University of Rome, Viale Regina Elena 291, 00161, Rome (Italy); Cardarelli, Francesco, E-mail: francesco.cardarelli@iit.it [Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa (Italy)

    2016-05-20

    Here we address the process of lipofection by quantifying the expression of a genetically-encoded fluorescent reporter at the single-cell level, and in real-time, by confocal imaging in live cells. The Lipofectamine gold-standard formulation is compared to the alternative promising DC-Chol/DOPE formulation. In both cases, we report that only dividing cells are able to produce a detectable amount of the fluorescent reporter protein. Notably, by measuring fluorescence over time in each pair of daughter cells, we find that Lipofectamine-based transfection statistically yields a remarkably higher degree of “symmetry” in protein expression between daughter cells as compared to DC-Chol/DOPE. A model is envisioned in which the degree of symmetry of protein expression is linked to the number of bioavailable DNA copies within the cell before nuclear breakdown. Reported results open new perspectives for the understanding of the lipofection mechanism and define a new experimental platform for the quantitative comparison of transfection reagents. -- Highlights: •The process of lipofection is followed by quantifying the transgene expression in real time. •The Lipofectamine gold-standard is compared to the promising DC-Chol/DOPE formulation. •We report that only dividing cells are able to produce the fluorescent reporter protein. •The degree of symmetry of protein expression in daughter cells is linked to DNA bioavailability. •A new experimental platform for the quantitative comparison of transfection reagents is proposed.

  5. Single-cell real-time imaging of transgene expression upon lipofection

    International Nuclear Information System (INIS)

    Fiume, Giuseppe; Di Rienzo, Carmine; Marchetti, Laura; Pozzi, Daniela; Caracciolo, Giulio; Cardarelli, Francesco

    2016-01-01

    Here we address the process of lipofection by quantifying the expression of a genetically-encoded fluorescent reporter at the single-cell level, and in real-time, by confocal imaging in live cells. The Lipofectamine gold-standard formulation is compared to the alternative promising DC-Chol/DOPE formulation. In both cases, we report that only dividing cells are able to produce a detectable amount of the fluorescent reporter protein. Notably, by measuring fluorescence over time in each pair of daughter cells, we find that Lipofectamine-based transfection statistically yields a remarkably higher degree of “symmetry” in protein expression between daughter cells as compared to DC-Chol/DOPE. A model is envisioned in which the degree of symmetry of protein expression is linked to the number of bioavailable DNA copies within the cell before nuclear breakdown. Reported results open new perspectives for the understanding of the lipofection mechanism and define a new experimental platform for the quantitative comparison of transfection reagents. -- Highlights: •The process of lipofection is followed by quantifying the transgene expression in real time. •The Lipofectamine gold-standard is compared to the promising DC-Chol/DOPE formulation. •We report that only dividing cells are able to produce the fluorescent reporter protein. •The degree of symmetry of protein expression in daughter cells is linked to DNA bioavailability. •A new experimental platform for the quantitative comparison of transfection reagents is proposed.

  6. Single-cell real-time imaging of transgene expression upon lipofection.

    Science.gov (United States)

    Fiume, Giuseppe; Di Rienzo, Carmine; Marchetti, Laura; Pozzi, Daniela; Caracciolo, Giulio; Cardarelli, Francesco

    2016-05-20

    Here we address the process of lipofection by quantifying the expression of a genetically-encoded fluorescent reporter at the single-cell level, and in real-time, by confocal imaging in live cells. The Lipofectamine gold-standard formulation is compared to the alternative promising DC-Chol/DOPE formulation. In both cases, we report that only dividing cells are able to produce a detectable amount of the fluorescent reporter protein. Notably, by measuring fluorescence over time in each pair of daughter cells, we find that Lipofectamine-based transfection statistically yields a remarkably higher degree of "symmetry" in protein expression between daughter cells as compared to DC-Chol/DOPE. A model is envisioned in which the degree of symmetry of protein expression is linked to the number of bioavailable DNA copies within the cell before nuclear breakdown. Reported results open new perspectives for the understanding of the lipofection mechanism and define a new experimental platform for the quantitative comparison of transfection reagents. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

    DEFF Research Database (Denmark)

    Ronander, Elena; Bengtsson, Dominique C; Joergensen, Louise

    2012-01-01

    Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes. The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has...... been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors...... fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE(1). Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human...

  8. Full-length single-cell RNA-seq applied to a viral human cancer: applications to HPV expression and splicing analysis in HeLa S3 cells.

    Science.gov (United States)

    Wu, Liang; Zhang, Xiaolong; Zhao, Zhikun; Wang, Ling; Li, Bo; Li, Guibo; Dean, Michael; Yu, Qichao; Wang, Yanhui; Lin, Xinxin; Rao, Weijian; Mei, Zhanlong; Li, Yang; Jiang, Runze; Yang, Huan; Li, Fuqiang; Xie, Guoyun; Xu, Liqin; Wu, Kui; Zhang, Jie; Chen, Jianghao; Wang, Ting; Kristiansen, Karsten; Zhang, Xiuqing; Li, Yingrui; Yang, Huanming; Wang, Jian; Hou, Yong; Xu, Xun

    2015-01-01

    Viral infection causes multiple forms of human cancer, and HPV infection is the primary factor in cervical carcinomas. Recent single-cell RNA-seq studies highlight the tumor heterogeneity present in most cancers, but virally induced tumors have not been studied. HeLa is a well characterized HPV+ cervical cancer cell line. We developed a new high throughput platform to prepare single-cell RNA on a nanoliter scale based on a customized microwell chip. Using this method, we successfully amplified full-length transcripts of 669 single HeLa S3 cells and 40 of them were randomly selected to perform single-cell RNA sequencing. Based on these data, we obtained a comprehensive understanding of the heterogeneity of HeLa S3 cells in gene expression, alternative splicing and fusions. Furthermore, we identified a high diversity of HPV-18 expression and splicing at the single-cell level. By co-expression analysis we identified 283 E6, E7 co-regulated genes, including CDC25, PCNA, PLK4, BUB1B and IRF1 known to interact with HPV viral proteins. Our results reveal the heterogeneity of a virus-infected cell line. It not only provides a transcriptome characterization of HeLa S3 cells at the single cell level, but is a demonstration of the power of single cell RNA-seq analysis of virally infected cells and cancers.

  9. Identification of a core set of rhizobial infection genes using data from single cell-types

    Directory of Open Access Journals (Sweden)

    Da-Song eChen

    2015-07-01

    Full Text Available Genome-wide expression studies on nodulation have varied in their scale from entire root systems to dissected nodules or root sections containing nodule primordia. More recently efforts have focused on developing methods for isolation of root hairs from infected plants and the application of laser-capture microdissection technology to nodules. Here we analyze two published data sets to identify a core set of infection genes that are expressed in the nodule and in root hairs during infection. Among the genes identified were those encoding phenylpropanoid biosynthesis enzymes including Chalcone-O-Methyltransferase which is required for the production of the potent Nod gene inducer 4’,4-dihydroxy-2-methoxychalcone. A promoter-GUS analysis in transgenic hairy roots for two genes encoding Chalcone-O-Methyltransferase isoforms revealed their expression in rhizobially infected root hairs and the nodule infection zone but not in the nitrogen fixation zone. We also describe a group of Rhizobially Induced Peroxidases whose expression overlaps with the production of superoxide in rhizobially infected root hairs and in nodules and roots. Finally, we identify a cohort of co-regulated transcription factors as candidate regulators of these processes.

  10. The correlation between expression profiles measured in single cells and in traditional bulk samples

    Czech Academy of Sciences Publication Activity Database

    Džamba, Dávid; Valihrach, Lukáš; Kubista, Mikael; Anděrová, Miroslava

    2016-01-01

    Roč. 6, nov (2016), s. 37022 ISSN 2045-2322 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) EE2.3.30.0045; GA ČR(CZ) GAP303/12/0855 Institutional support: RVO:68378041 ; RVO:86652036 Keywords : real-time PCR * gene-expression * messenger-rna Subject RIV: FH - Neurology; EI - Biotechnology ; Bionics (BTO-N) Impact factor: 4.259, year: 2016

  11. Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip

    Science.gov (United States)

    Li, Ren; Zhou, Mingxing; Li, Jine; Wang, Zihua; Zhang, Weikai; Yue, Chunyan; Ma, Yan; Peng, Hailin; Wei, Zewen; Hu, Zhiyuan

    2018-03-01

    EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.[Figure not available: see fulltext.

  12. Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system.

    Science.gov (United States)

    Nakamura, Tsuyoshi; Omasa, Takeshi

    2015-09-01

    Therapeutic antibodies are commonly produced by high-expressing, clonal and recombinant Chinese hamster ovary (CHO) cell lines. Currently, CHO cells dominate as a commercial production host because of their ease of use, established regulatory track record, and safety profile. CHO-K1SV is a suspension, protein-free-adapted CHO-K1-derived cell line employing the glutamine synthetase (GS) gene expression system (GS-CHO expression system). The selection of high-producing mammalian cell lines is a crucial step in process development for the production of therapeutic antibodies. In general, cloning by the limiting dilution method is used to isolate high-producing monoclonal CHO cells. However, the limiting dilution method is time consuming and has a low probability of monoclonality. To minimize the duration and increase the probability of obtaining high-producing clones with high monoclonality, an automated single cell-based clone selector, the ClonePix FL system, is available. In this study, we applied the high-throughput ClonePix FL system for cell line development using CHO-K1SV cells and investigated efficient conditions for single cell-based clone selection. CHO-K1SV cell growth at the pre-picking stage was improved by optimizing the formulation of semi-solid medium. The efficiency of picking and cell growth at the post-picking stage was improved by optimization of the plating time without decreasing the diversity of clones. The conditions for selection, including the medium formulation, were the most important factors for the single cell-based clone selection system to construct a high-producing CHO cell line. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  13. Linnorm: improved statistical analysis for single cell RNA-seq expression data.

    Science.gov (United States)

    Yip, Shun H; Wang, Panwen; Kocher, Jean-Pierre A; Sham, Pak Chung; Wang, Junwen

    2017-12-15

    Linnorm is a novel normalization and transformation method for the analysis of single cell RNA sequencing (scRNA-seq) data. Linnorm is developed to remove technical noises and simultaneously preserve biological variations in scRNA-seq data, such that existing statistical methods can be improved. Using real scRNA-seq data, we compared Linnorm with existing normalization methods, including NODES, SAMstrt, SCnorm, scran, DESeq and TMM. Linnorm shows advantages in speed, technical noise removal and preservation of cell heterogeneity, which can improve existing methods in the discovery of novel subtypes, pseudo-temporal ordering of cells, clustering analysis, etc. Linnorm also performs better than existing DEG analysis methods, including BASiCS, NODES, SAMstrt, Seurat and DESeq2, in false positive rate control and accuracy. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Charged residues in the H-NS linker drive DNA binding and gene silencing in single cells.

    Science.gov (United States)

    Gao, Yunfeng; Foo, Yong Hwee; Winardhi, Ricksen S; Tang, Qingnan; Yan, Jie; Kenney, Linda J

    2017-11-21

    Nucleoid-associated proteins (NAPs) facilitate chromosome organization in bacteria, but the precise mechanism remains elusive. H-NS is a NAP that also plays a major role in silencing pathogen genes. We used genetics, single-particle tracking in live cells, superresolution microscopy, atomic force microscopy, and molecular dynamics simulations to examine H-NS/DNA interactions in single cells. We discovered a role for the unstructured linker region connecting the N-terminal oligomerization and C-terminal DNA binding domains. In the present work we demonstrate that linker amino acids promote engagement with DNA. In the absence of linker contacts, H-NS binding is significantly reduced, although no change in chromosome compaction is observed. H-NS is not localized to two distinct foci; rather, it is scattered all around the nucleoid. The linker makes DNA contacts that are required for gene silencing, while chromosome compaction does not appear to be an important H-NS function.

  15. Freedom of expression: cell-type-specific gene profiling.

    Science.gov (United States)

    Otsuki, Leo; Cheetham, Seth W; Brand, Andrea H

    2014-01-01

    Cell fate and behavior are results of differential gene regulation, making techniques to profile gene expression in specific cell types highly desirable. Many methods now enable investigation at the DNA, RNA and protein level. This review introduces the most recent and popular techniques, and discusses key issues influencing the choice between these such as ease, cost and applicability of information gained. Interdisciplinary collaborations will no doubt contribute further advances, including not just in single cell type but single-cell expression profiling. © 2014 Wiley Periodicals, Inc.

  16. Geometry of the Gene Expression Space of Individual Cells.

    Directory of Open Access Journals (Sweden)

    Yael Korem

    2015-07-01

    Full Text Available There is a revolution in the ability to analyze gene expression of single cells in a tissue. To understand this data we must comprehend how cells are distributed in a high-dimensional gene expression space. One open question is whether cell types form discrete clusters or whether gene expression forms a continuum of states. If such a continuum exists, what is its geometry? Recent theory on evolutionary trade-offs suggests that cells that need to perform multiple tasks are arranged in a polygon or polyhedron (line, triangle, tetrahedron and so on, generally called polytopes in gene expression space, whose vertices are the expression profiles optimal for each task. Here, we analyze single-cell data from human and mouse tissues profiled using a variety of single-cell technologies. We fit the data to shapes with different numbers of vertices, compute their statistical significance, and infer their tasks. We find cases in which single cells fill out a continuum of expression states within a polyhedron. This occurs in intestinal progenitor cells, which fill out a tetrahedron in gene expression space. The four vertices of this tetrahedron are each enriched with genes for a specific task related to stemness and early differentiation. A polyhedral continuum of states is also found in spleen dendritic cells, known to perform multiple immune tasks: cells fill out a tetrahedron whose vertices correspond to key tasks related to maturation, pathogen sensing and communication with lymphocytes. A mixture of continuum-like distributions and discrete clusters is found in other cell types, including bone marrow and differentiated intestinal crypt cells. This approach can be used to understand the geometry and biological tasks of a wide range of single-cell datasets. The present results suggest that the concept of cell type may be expanded. In addition to discreet clusters in gene-expression space, we suggest a new possibility: a continuum of states within a

  17. Gene Expression Omnibus (GEO)

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene Expression Omnibus is a public functional genomics data repository supporting MIAME-compliant submissions of array- and sequence-based data. Tools are provided...

  18. Potentials of single-cell biology in identification and validation of disease biomarkers.

    Science.gov (United States)

    Niu, Furong; Wang, Diane C; Lu, Jiapei; Wu, Wei; Wang, Xiangdong

    2016-09-01

    Single-cell biology is considered a new approach to identify and validate disease-specific biomarkers. However, the concern raised by clinicians is how to apply single-cell measurements for clinical practice, translate the message of single-cell systems biology into clinical phenotype or explain alterations of single-cell gene sequencing and function in patient response to therapies. This study is to address the importance and necessity of single-cell gene sequencing in the identification and development of disease-specific biomarkers, the definition and significance of single-cell biology and single-cell systems biology in the understanding of single-cell full picture, the development and establishment of whole-cell models in the validation of targeted biological function and the figure and meaning of single-molecule imaging in single cell to trace intra-single-cell molecule expression, signal, interaction and location. We headline the important role of single-cell biology in the discovery and development of disease-specific biomarkers with a special emphasis on understanding single-cell biological functions, e.g. mechanical phenotypes, single-cell biology, heterogeneity and organization of genome function. We have reason to believe that such multi-dimensional, multi-layer, multi-crossing and stereoscopic single-cell biology definitely benefits the discovery and development of disease-specific biomarkers. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  19. Single-Cell RNA-Seq of Mouse Dopaminergic Neurons Informs Candidate Gene Selection for Sporadic Parkinson Disease.

    Science.gov (United States)

    Hook, Paul W; McClymont, Sarah A; Cannon, Gabrielle H; Law, William D; Morton, A Jennifer; Goff, Loyal A; McCallion, Andrew S

    2018-03-01

    Genetic variation modulating risk of sporadic Parkinson disease (PD) has been primarily explored through genome-wide association studies (GWASs). However, like many other common genetic diseases, the impacted genes remain largely unknown. Here, we used single-cell RNA-seq to characterize dopaminergic (DA) neuron populations in the mouse brain at embryonic and early postnatal time points. These data facilitated unbiased identification of DA neuron subpopulations through their unique transcriptional profiles, including a postnatal neuroblast population and substantia nigra (SN) DA neurons. We use these population-specific data to develop a scoring system to prioritize candidate genes in all 49 GWAS intervals implicated in PD risk, including genes with known PD associations and many with extensive supporting literature. As proof of principle, we confirm that the nigrostriatal pathway is compromised in Cplx1-null mice. Ultimately, this systematic approach establishes biologically pertinent candidates and testable hypotheses for sporadic PD, informing a new era of PD genetic research. Copyright © 2018 American Society of Human Genetics. All rights reserved.

  20. Single Cell Oncogenesis

    Science.gov (United States)

    Lu, Xin

    It is believed that cancer originates from a single cell that has gone through generations of evolution of genetic and epigenetic changes that associate with the hallmarks of cancer. In some cancers such as various types of leukemia, cancer is clonal. Yet in other cancers like glioblastoma (GBM), there is tremendous tumor heterogeneity that is likely to be caused by simultaneous evolution of multiple subclones within the same tissue. It is obvious that understanding how a single cell develops into a clonal tumor upon genetic alterations, at molecular and cellular levels, holds the key to the real appreciation of tumor etiology and ultimate solution for therapeutics. Surprisingly very little is known about the process of spontaneous tumorigenesis from single cells in human or vertebrate animal models. The main reason is the lack of technology to track the natural process of single cell changes from a homeostatic state to a progressively cancerous state. Recently, we developed a patented compound, photoactivatable (''caged'') tamoxifen analogue 4-OHC and associated technique called optochemogenetic switch (OCG switch), which we believe opens the opportunity to address this urgent biological as well as clinical question about cancer. We propose to combine OCG switch with genetically engineered mouse models of head and neck squamous cell carcinoma and high grade astrocytoma (including GBM) to study how single cells, when transformed through acute loss of tumor suppressor genes PTEN and TP53 and gain of oncogenic KRAS, can develop into tumor colonies with cellular and molecular heterogeneity in these tissues. The abstract is for my invited talk in session ``Beyond Darwin: Evolution in Single Cells'' 3/18/2016 11:15 AM.

  1. Preconceptional paternal glycidamide exposure affects embryonic gene expression: Single embryo gene expression study following in vitro fertilization

    Czech Academy of Sciences Publication Activity Database

    Brevik, A.; Rusňáková, Vendula; Duale, N.; Slagsvold, H.H.; Olsen, A.-K.; Storeng, R.; Kubista, Mikael; Brunborg, G.; Lindeman, B.

    2011-01-01

    Roč. 32, č. 4 (2011), s. 463-471 ISSN 0890-6238 R&D Projects: GA AV ČR(CZ) IAA500520809 Institutional research plan: CEZ:AV0Z50520701 Keywords : Single-cell gene expression * Glycidamide * Acrylamide Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.226, year: 2011

  2. Evaluation of the Cow Rumen Metagenome: Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies (Metagenomics Informatics Challenges Workshop: 10K Genomes at a Time)

    Energy Technology Data Exchange (ETDEWEB)

    Sczyrba, Alex

    2011-10-13

    DOE JGI's Alex Sczyrba on "Evaluation of the Cow Rumen Metagenome" and "Assembly by Single Copy Gene Analysis and Single Cell Genome Assemblies" at the Metagenomics Informatics Challenges Workshop held at the DOE JGI on October 12-13, 2011.

  3. Gene expression and gene therapy imaging

    International Nuclear Information System (INIS)

    Rome, Claire; Couillaud, Franck; Moonen, Chrit T.W.

    2007-01-01

    The fast growing field of molecular imaging has achieved major advances in imaging gene expression, an important element of gene therapy. Gene expression imaging is based on specific probes or contrast agents that allow either direct or indirect spatio-temporal evaluation of gene expression. Direct evaluation is possible with, for example, contrast agents that bind directly to a specific target (e.g., receptor). Indirect evaluation may be achieved by using specific substrate probes for a target enzyme. The use of marker genes, also called reporter genes, is an essential element of MI approaches for gene expression in gene therapy. The marker gene may not have a therapeutic role itself, but by coupling the marker gene to a therapeutic gene, expression of the marker gene reports on the expression of the therapeutic gene. Nuclear medicine and optical approaches are highly sensitive (detection of probes in the picomolar range), whereas MRI and ultrasound imaging are less sensitive and require amplification techniques and/or accumulation of contrast agents in enlarged contrast particles. Recently developed MI techniques are particularly relevant for gene therapy. Amongst these are the possibility to track gene therapy vectors such as stem cells, and the techniques that allow spatiotemporal control of gene expression by non-invasive heating (with MRI guided focused ultrasound) and the use of temperature sensitive promoters. (orig.)

  4. The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.

    Science.gov (United States)

    Wills, Quin F; Mellado-Gomez, Esther; Nolan, Rory; Warner, Damien; Sharma, Eshita; Broxholme, John; Wright, Benjamin; Lockstone, Helen; James, William; Lynch, Mark; Gonzales, Michael; West, Jay; Leyrat, Anne; Padilla-Parra, Sergi; Filippi, Sarah; Holmes, Chris; Moore, Michael D; Bowden, Rory

    2017-01-07

    Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation. As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.

  5. Single-Cell Mass Spectrometry Reveals Changes in Lipid and Metabolite Expression in RAW 264.7 Cells upon Lipopolysaccharide Stimulation

    Science.gov (United States)

    Yang, Bo; Patterson, Nathan Heath; Tsui, Tina; Caprioli, Richard M.; Norris, Jeremy L.

    2018-05-01

    It has been widely recognized that individual cells that exist within a large population of cells, even if they are genetically identical, can have divergent molecular makeups resulting from a variety of factors, including local environmental factors and stochastic processes within each cell. Presently, numerous approaches have been described that permit the resolution of these single-cell expression differences for RNA and protein; however, relatively few techniques exist for the study of lipids and metabolites in this manner. This study presents a methodology for the analysis of metabolite and lipid expression at the level of a single cell through the use of imaging mass spectrometry on a high-performance Fourier transform ion cyclotron resonance mass spectrometer. This report provides a detailed description of the overall experimental approach, including sample preparation as well as the data acquisition and analysis strategy for single cells. Applying this approach to the study of cultured RAW264.7 cells, we demonstrate that this method can be used to study the variation in molecular expression with cell populations and is sensitive to alterations in that expression that occurs upon lipopolysaccharide stimulation. [Figure not available: see fulltext.

  6. Single-Cell Mass Spectrometry Reveals Changes in Lipid and Metabolite Expression in RAW 264.7 Cells upon Lipopolysaccharide Stimulation

    Science.gov (United States)

    Yang, Bo; Patterson, Nathan Heath; Tsui, Tina; Caprioli, Richard M.; Norris, Jeremy L.

    2018-03-01

    It has been widely recognized that individual cells that exist within a large population of cells, even if they are genetically identical, can have divergent molecular makeups resulting from a variety of factors, including local environmental factors and stochastic processes within each cell. Presently, numerous approaches have been described that permit the resolution of these single-cell expression differences for RNA and protein; however, relatively few techniques exist for the study of lipids and metabolites in this manner. This study presents a methodology for the analysis of metabolite and lipid expression at the level of a single cell through the use of imaging mass spectrometry on a high-performance Fourier transform ion cyclotron resonance mass spectrometer. This report provides a detailed description of the overall experimental approach, including sample preparation as well as the data acquisition and analysis strategy for single cells. Applying this approach to the study of cultured RAW264.7 cells, we demonstrate that this method can be used to study the variation in molecular expression with cell populations and is sensitive to alterations in that expression that occurs upon lipopolysaccharide stimulation. [Figure not available: see fulltext.

  7. Self-Organization of Genome Expression from Embryo to Terminal Cell Fate: Single-Cell Statistical Mechanics of Biological Regulation

    Directory of Open Access Journals (Sweden)

    Alessandro Giuliani

    2017-12-01

    Full Text Available A statistical mechanical mean-field approach to the temporal development of biological regulation provides a phenomenological, but basic description of the dynamical behavior of genome expression in terms of autonomous self-organization with a critical transition (Self-Organized Criticality: SOC. This approach reveals the basis of self-regulation/organization of genome expression, where the extreme complexity of living matter precludes any strict mechanistic approach. The self-organization in SOC involves two critical behaviors: scaling-divergent behavior (genome avalanche and sandpile-type critical behavior. Genome avalanche patterns—competition between order (scaling and disorder (divergence reflect the opposite sequence of events characterizing the self-organization process in embryo development and helper T17 terminal cell differentiation, respectively. On the other hand, the temporal development of sandpile-type criticality (the degree of SOC control in mouse embryo suggests the existence of an SOC control landscape with a critical transition state (i.e., the erasure of zygote-state criticality. This indicates that a phase transition of the mouse genome before and after reprogramming (immediately after the late 2-cell state occurs through a dynamical change in a control parameter. This result provides a quantitative open-thermodynamic appreciation of the still largely qualitative notion of the epigenetic landscape. Our results suggest: (i the existence of coherent waves of condensation/de-condensation in chromatin, which are transmitted across regions of different gene-expression levels along the genome; and (ii essentially the same critical dynamics we observed for cell-differentiation processes exist in overall RNA expression during embryo development, which is particularly relevant because it gives further proof of SOC control of overall expression as a universal feature.

  8. Imaging gene expression in gene therapy

    International Nuclear Information System (INIS)

    Wiebe, Leonard I.

    1997-01-01

    Full text. Gene therapy can be used to introduce new genes, or to supplement the function of indigenous genes. At the present time, however, there is non-invasive test to demonstrate efficacy of the gene transfer and expression processes. It has been postulated that scintigraphic imaging can offer unique information on both the site at which the transferred gene is expressed, and the degree of expression, both of which are critical issue for safety and clinical efficacy. Many current studies are based on 'suicide gene therapy' of cancer. Cells modified to express these genes commit metabolic suicide in the presence of an enzyme encoded by the transferred gene and a specifically-convertible pro drug. Pro drug metabolism can lead to selective metabolic trapping, required for scintigraphy. Herpes simplex virus type-1 thymidine kinase (H S V-1 t k + ) has been use for 'suicide' in vivo tumor gene therapy. It has been proposed that radiolabelled nucleosides can be used as radiopharmaceuticals to detect H S V-1 t k + gene expression where the H S V-1 t k + gene serves a reporter or therapeutic function. Animal gene therapy models have been studied using purine-([ 18 F]F H P G; [ 18 F]-A C V), and pyrimidine- ([ 123 / 131 I]I V R F U; [ 124 / 131I ]) antiviral nucleosides. Principles of gene therapy and gene therapy imaging will be reviewed and experimental data for [ 123 / 131I ]I V R F U imaging with the H S V-1 t k + reporter gene will be presented

  9. Imaging gene expression in gene therapy

    Energy Technology Data Exchange (ETDEWEB)

    Wiebe, Leonard I. [Alberta Univ., Edmonton (Canada). Noujaim Institute for Pharmaceutical Oncology Research

    1997-12-31

    Full text. Gene therapy can be used to introduce new genes, or to supplement the function of indigenous genes. At the present time, however, there is non-invasive test to demonstrate efficacy of the gene transfer and expression processes. It has been postulated that scintigraphic imaging can offer unique information on both the site at which the transferred gene is expressed, and the degree of expression, both of which are critical issue for safety and clinical efficacy. Many current studies are based on `suicide gene therapy` of cancer. Cells modified to express these genes commit metabolic suicide in the presence of an enzyme encoded by the transferred gene and a specifically-convertible pro drug. Pro drug metabolism can lead to selective metabolic trapping, required for scintigraphy. Herpes simplex virus type-1 thymidine kinase (H S V-1 t k{sup +}) has been use for `suicide` in vivo tumor gene therapy. It has been proposed that radiolabelled nucleosides can be used as radiopharmaceuticals to detect H S V-1 t k{sup +} gene expression where the H S V-1 t k{sup +} gene serves a reporter or therapeutic function. Animal gene therapy models have been studied using purine-([{sup 18} F]F H P G; [{sup 18} F]-A C V), and pyrimidine- ([{sup 123}/{sup 131} I]I V R F U; [{sup 124}/{sup 131I}]) antiviral nucleosides. Principles of gene therapy and gene therapy imaging will be reviewed and experimental data for [{sup 123}/{sup 131I}]I V R F U imaging with the H S V-1 t k{sup +} reporter gene will be presented

  10. Regulation of eucaryotic gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Brent, R.; Ptashne, M.S

    1989-05-23

    This patent describes a method of regulating the expression of a gene in a eucaryotic cell. The method consists of: providing in the eucaryotic cell, a peptide, derived from or substantially similar to a peptide of a procaryotic cell able to bind to DNA upstream from or within the gene, the amount of the peptide being sufficient to bind to the gene and thereby control expression of the gene.

  11. Differential Gene Expression and Aging

    Directory of Open Access Journals (Sweden)

    Laurent Seroude

    2002-01-01

    Full Text Available It has been established that an intricate program of gene expression controls progression through the different stages in development. The equally complex biological phenomenon known as aging is genetically determined and environmentally modulated. This review focuses on the genetic component of aging, with a special emphasis on differential gene expression. At least two genetic pathways regulating organism longevity act by modifying gene expression. Many genes are also subjected to age-dependent transcriptional regulation. Some age-related gene expression changes are prevented by caloric restriction, the most robust intervention that slows down the aging process. Manipulating the expression of some age-regulated genes can extend an organism's life span. Remarkably, the activity of many transcription regulatory elements is linked to physiological age as opposed to chronological age, indicating that orderly and tightly controlled regulatory pathways are active during aging.

  12. Going single but not solo with podocytes: potentials, limitations, and pitfalls of single-cell analysis.

    Science.gov (United States)

    Schiffer, Mario

    2017-11-01

    Single-cell RNA-sequence (RNA-seq) is a widely used tool to study biological questions in single cells. The discussed study identified 92 genes being predominantly expressed in podocytes based on a 5-fold higher expression compared with endothelial and mesangial cells. In addition to technical pitfalls, the question that is discussed in this commentary is whether results of a single-cell RNAseq study are able to deliver expression data that truly characterize a podocyte. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  13. Single-cell analysis of targeted transcriptome predicts drug sensitivity of single cells within human myeloma tumors.

    Science.gov (United States)

    Mitra, A K; Mukherjee, U K; Harding, T; Jang, J S; Stessman, H; Li, Y; Abyzov, A; Jen, J; Kumar, S; Rajkumar, V; Van Ness, B

    2016-05-01

    Multiple myeloma (MM) is characterized by significant genetic diversity at subclonal levels that have a defining role in the heterogeneity of tumor progression, clinical aggressiveness and drug sensitivity. Although genome profiling studies have demonstrated heterogeneity in subclonal architecture that may ultimately lead to relapse, a gene expression-based prediction program that can identify, distinguish and quantify drug response in sub-populations within a bulk population of myeloma cells is lacking. In this study, we performed targeted transcriptome analysis on 528 pre-treatment single cells from 11 myeloma cell lines and 418 single cells from 8 drug-naïve MM patients, followed by intensive bioinformatics and statistical analysis for prediction of proteasome inhibitor sensitivity in individual cells. Using our previously reported drug response gene expression profile signature at the single-cell level, we developed an R Statistical analysis package available at https://github.com/bvnlabSCATTome, SCATTome (single-cell analysis of targeted transcriptome), that restructures the data obtained from Fluidigm single-cell quantitative real-time-PCR analysis run, filters missing data, performs scaling of filtered data, builds classification models and predicts drug response of individual cells based on targeted transcriptome using an assortment of machine learning methods. Application of SCATT should contribute to clinically relevant analysis of intratumor heterogeneity, and better inform drug choices based on subclonal cellular responses.

  14. Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells

    NARCIS (Netherlands)

    Semrau, Stefan; Goldmann, Johanna E; Soumillon, Magali; Mikkelsen, Tarjei S; Jaenisch, Rudolf; van Oudenaarden, Alexander

    2017-01-01

    Gene expression heterogeneity in the pluripotent state of mouse embryonic stem cells (mESCs) has been increasingly well-characterized. In contrast, exit from pluripotency and lineage commitment have not been studied systematically at the single-cell level. Here we measure the gene expression

  15. Large-scale image-based profiling of single-cell phenotypes in arrayed CRISPR-Cas9 gene perturbation screens.

    Science.gov (United States)

    de Groot, Reinoud; Lüthi, Joel; Lindsay, Helen; Holtackers, René; Pelkmans, Lucas

    2018-01-23

    High-content imaging using automated microscopy and computer vision allows multivariate profiling of single-cell phenotypes. Here, we present methods for the application of the CISPR-Cas9 system in large-scale, image-based, gene perturbation experiments. We show that CRISPR-Cas9-mediated gene perturbation can be achieved in human tissue culture cells in a timeframe that is compatible with image-based phenotyping. We developed a pipeline to construct a large-scale arrayed library of 2,281 sequence-verified CRISPR-Cas9 targeting plasmids and profiled this library for genes affecting cellular morphology and the subcellular localization of components of the nuclear pore complex (NPC). We conceived a machine-learning method that harnesses genetic heterogeneity to score gene perturbations and identify phenotypically perturbed cells for in-depth characterization of gene perturbation effects. This approach enables genome-scale image-based multivariate gene perturbation profiling using CRISPR-Cas9. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

  16. Defining cell populations with single-cell gene expression profiling: correlations and identification of astrocyte subpopulations

    Czech Academy of Sciences Publication Activity Database

    Stahlberg, A.; Andersson, D.; Aurelius, J.; Faiz, M.; Pekna, M.; Kubista, Mikael; Pekny, M.

    2011-01-01

    Roč. 39, č. 4 (2011) ISSN 0305-1048 R&D Projects: GA AV ČR IAA500970904; GA ČR GAP303/10/1338 Institutional research plan: CEZ:AV0Z50520701 Keywords : EMBRYONIC STEM-CELLS * REAL-TIME PCR * MESSENGER-RNA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.026, year: 2011

  17. Gene expression in colorectal cancer

    DEFF Research Database (Denmark)

    Birkenkamp-Demtroder, Karin; Christensen, Lise Lotte; Olesen, Sanne Harder

    2002-01-01

    Understanding molecular alterations in colorectal cancer (CRC) is needed to define new biomarkers and treatment targets. We used oligonucleotide microarrays to monitor gene expression of about 6,800 known genes and 35,000 expressed sequence tags (ESTs) on five pools (four to six samples in each...... pool) of total RNA from left-sided sporadic colorectal carcinomas. We compared normal tissue to carcinoma tissue from Dukes' stages A-D (noninvasive to distant metastasis) and identified 908 known genes and 4,155 ESTs that changed remarkably from normal to tumor tissue. Based on intensive filtering 226...

  18. Correction of gene expression data

    DEFF Research Database (Denmark)

    Darbani Shirvanehdeh, Behrooz; Stewart, C. Neal, Jr.; Noeparvar, Shahin

    2014-01-01

    This report investigates for the first time the potential inter-treatment bias source of cell number for gene expression studies. Cell-number bias can affect gene expression analysis when comparing samples with unequal total cellular RNA content or with different RNA extraction efficiencies....... For maximal reliability of analysis, therefore, comparisons should be performed at the cellular level. This could be accomplished using an appropriate correction method that can detect and remove the inter-treatment bias for cell-number. Based on inter-treatment variations of reference genes, we introduce...

  19. Relaxation rates of gene expression kinetics reveal the feedback signs of autoregulatory gene networks

    Science.gov (United States)

    Jia, Chen; Qian, Hong; Chen, Min; Zhang, Michael Q.

    2018-03-01

    The transient response to a stimulus and subsequent recovery to a steady state are the fundamental characteristics of a living organism. Here we study the relaxation kinetics of autoregulatory gene networks based on the chemical master equation model of single-cell stochastic gene expression with nonlinear feedback regulation. We report a novel relation between the rate of relaxation, characterized by the spectral gap of the Markov model, and the feedback sign of the underlying gene circuit. When a network has no feedback, the relaxation rate is exactly the decaying rate of the protein. We further show that positive feedback always slows down the relaxation kinetics while negative feedback always speeds it up. Numerical simulations demonstrate that this relation provides a possible method to infer the feedback topology of autoregulatory gene networks by using time-series data of gene expression.

  20. Bioinformatics approaches to single-cell analysis in developmental biology.

    Science.gov (United States)

    Yalcin, Dicle; Hakguder, Zeynep M; Otu, Hasan H

    2016-03-01

    Individual cells within the same population show various degrees of heterogeneity, which may be better handled with single-cell analysis to address biological and clinical questions. Single-cell analysis is especially important in developmental biology as subtle spatial and temporal differences in cells have significant associations with cell fate decisions during differentiation and with the description of a particular state of a cell exhibiting an aberrant phenotype. Biotechnological advances, especially in the area of microfluidics, have led to a robust, massively parallel and multi-dimensional capturing, sorting, and lysis of single-cells and amplification of related macromolecules, which have enabled the use of imaging and omics techniques on single cells. There have been improvements in computational single-cell image analysis in developmental biology regarding feature extraction, segmentation, image enhancement and machine learning, handling limitations of optical resolution to gain new perspectives from the raw microscopy images. Omics approaches, such as transcriptomics, genomics and epigenomics, targeting gene and small RNA expression, single nucleotide and structural variations and methylation and histone modifications, rely heavily on high-throughput sequencing technologies. Although there are well-established bioinformatics methods for analysis of sequence data, there are limited bioinformatics approaches which address experimental design, sample size considerations, amplification bias, normalization, differential expression, coverage, clustering and classification issues, specifically applied at the single-cell level. In this review, we summarize biological and technological advancements, discuss challenges faced in the aforementioned data acquisition and analysis issues and present future prospects for application of single-cell analyses to developmental biology. © The Author 2015. Published by Oxford University Press on behalf of the European

  1. Gateway-assisted vector construction to facilitate expression of foreign proteins in the chloroplast of single celled algae.

    Directory of Open Access Journals (Sweden)

    Melanie Oey

    Full Text Available With a rising world population, demand will increase for food, energy and high value products. Renewable production systems, including photosynthetic microalgal biotechnologies, can produce biomass for foods, fuels and chemical feedstocks and in parallel allow the production of high value protein products, including recombinant proteins. Such high value recombinant proteins offer important economic benefits during startup of industrial scale algal biomass and biofuel production systems, but the limited markets for individual recombinant proteins will require a high throughput pipeline for cloning and expression in microalgae, which is currently lacking, since genetic engineering of microalgae is currently complex and laborious. We have introduced the recombination based Gateway® system into the construction process of chloroplast transformation vectors for microalgae. This simplifies the vector construction and allows easy, fast and flexible vector design for the high efficiency protein production in microalgae, a key step in developing such expression pipelines.

  2. Transgenic Arabidopsis Gene Expression System

    Science.gov (United States)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  3. Human papillomavirus gene expression

    International Nuclear Information System (INIS)

    Chow, L.T.; Hirochika, H.; Nasseri, M.; Stoler, M.H.; Wolinsky, S.M.; Chin, M.T.; Hirochika, R.; Arvan, D.S.; Broker, T.R.

    1987-01-01

    To determine the role of tissue differentiation on expression of each of the papillomavirus mRNA species identified by electron microscopy, the authors prepared exon-specific RNA probes that could distinguish the alternatively spliced mRNA species. Radioactively labeled single-stranded RNA probes were generated from a dual promoter vector system and individually hybridized to adjacent serial sections of formalin-fixed, paraffin-embedded biopsies of condylomata. Autoradiography showed that each of the message species had a characteristic tissue distribution and relative abundance. The authors have characterized a portion of the regulatory network of the HPVs by showing that the E2 ORF encodes a trans-acting enhancer-stimulating protein, as it does in BPV-1 (Spalholz et al. 1985). The HPV-11 enhancer was mapped to a 150-bp tract near the 3' end of the URR. Portions of this region are duplicated in some aggressive strains of HPV-6 (Boshart and zur Hausen 1986; Rando et al. 1986). To test the possible biological relevance of these duplications, they cloned tandem arrays of the enhancer and demonstrated, using a chloramphenicol acetyltransferase (CAT) assay, that they led to dramatically increased transcription proportional to copy number. Using the CAT assays, the authors found that the E2 proteins of several papillomavirus types can cross-stimulate the enhancers of most other types. This suggests that prior infection of a tissue with one papillomavirus type may provide a helper effect for superinfection and might account fo the HPV-6/HPV-16 coinfections in condylomata that they have observed

  4. Single Cell Isolation and Analysis

    Directory of Open Access Journals (Sweden)

    Ping Hu

    2016-10-01

    Full Text Available Increasing evidence shows that the heterogeneity of individual cells within a genetically identical population can be critical to their peculiar function and fate. Conventional cell based assays mainly analysis the average responses from a population cells, while the difference within individual cells may often be masked. The cell size, RNA transcripts and protein expression level are quite different within individual cells and these variations are key point to answer the problems in cancer, neurobiology, stem cell biology, immunology and developmental biology. To better understand the cell-to-cell variations, the single cell analysis can provide much more detailed information which may be helpful for therapeutic decisions in an increasingly personalized medicine. In this review, we will focus on the recent development in single cell analysis, including methods used in single cell isolation, analysis and some application examples. The review provides the historical background to single cell analysis, discusses limitations, and current and future possibilities in this exciting field of research.

  5. An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level [v1; ref status: indexed, http://f1000r.es/2tt

    Directory of Open Access Journals (Sweden)

    Shin-ichiro Kojima

    2014-02-01

    Full Text Available RNA interference (RNAi is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA together with an enhanced green fluorescent protein (EGFP; the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry. Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3 and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker (ccdB method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.

  6. An image-based, dual fluorescence reporter assay to evaluate the efficacy of shRNA for gene silencing at the single-cell level [v2; ref status: indexed, http://f1000r.es/39j

    Directory of Open Access Journals (Sweden)

    Shin-ichiro Kojima

    2014-05-01

    Full Text Available RNA interference (RNAi is widely used to suppress gene expression in a specific manner. The efficacy of RNAi is mainly dependent on the sequence of small interfering RNA (siRNA in relation to the target mRNA. Although several algorithms have been developed for the design of siRNA, it is still difficult to choose a really effective siRNA from among multiple candidates. In this article, we report the development of an image-based, quantitative, ratiometric fluorescence reporter assay to evaluate the efficacy of RNAi at the single-cell level. Two fluorescence reporter constructs are used. One expresses the candidate small hairpin RNA (shRNA together with an enhanced green fluorescent protein (EGFP; the other expresses a 19-nt target sequence inserted into a cassette expressing a red fluorescent protein (either DsRed or mCherry. Effectiveness of the candidate shRNA is evaluated as the extent to which it knocks down expression of the red fluorescent protein. Thus, the red-to-green fluorescence intensity ratio (appropriately normalized to controls is used as the read-out for quantifying the siRNA efficacy at the individual cell level. We tested this dual fluorescence assay and compared predictions to actual endogenous knockdown levels for three different genes (vimentin, lamin A/C and Arp3 and twenty different shRNAs. For each of the genes, our assay successfully predicted the target sequences for effective RNAi. To further facilitate testing of RNAi efficacy, we developed a negative selection marker (ccdB method for construction of shRNA and red fluorescent reporter plasmids that allowed us to purify these plasmids directly from transformed bacteria without the need for colony selection and DNA sequencing verification.

  7. Homeobox gene expression in Brachiopoda

    DEFF Research Database (Denmark)

    Altenburger, Andreas; Martinez, Pedro; Wanninger, Andreas

    2011-01-01

    (ectoderm) specification with co-opted functions in notochord formation in chordates and left/right determination in ambulacrarians and vertebrates. The caudal ortholog, TtrCdx, is first expressed in the ectoderm of the gastrulating embryo in the posterior region of the blastopore. Its expression stays......The molecular control that underlies brachiopod ontogeny is largely unknown. In order to contribute to this issue we analyzed the expression pattern of two homeobox containing genes, Not and Cdx, during development of the rhynchonelliform (i.e., articulate) brachiopod Terebratalia transversa...... completion of larval development, which is marked by a three-lobed body with larval setae. Expression starts at gastrulation in two areas lateral to the blastopore and subsequently extends over the animal pole of the gastrula. With elongation of the gastrula, expression at the animal pole narrows to a small...

  8. Vascular Gene Expression: A Hypothesis

    Directory of Open Access Journals (Sweden)

    Angélica Concepción eMartínez-Navarro

    2013-07-01

    Full Text Available The phloem is the conduit through which photoassimilates are distributed from autotrophic to heterotrophic tissues and is involved in the distribution of signaling molecules that coordinate plant growth and responses to the environment. Phloem function depends on the coordinate expression of a large array of genes. We have previously identified conserved motifs in upstream regions of the Arabidopsis genes, encoding the homologs of pumpkin phloem sap mRNAs, displaying expression in vascular tissues. This tissue-specific expression in Arabidopsis is predicted by the overrepresentation of GA/CT-rich motifs in gene promoters. In this work we have searched for common motifs in upstream regions of the homologous genes from plants considered to possess a primitive vascular tissue (a lycophyte, as well as from others that lack a true vascular tissue (a bryophyte, and finally from chlorophytes. Both lycophyte and bryophyte display motifs similar to those found in Arabidopsis with a significantly low E-value, while the chlorophytes showed either a different conserved motif or no conserved motif at all. These results suggest that these same genes are expressed coordinately in non- vascular plants; this coordinate expression may have been one of the prerequisites for the development of conducting tissues in plants. We have also analyzed the phylogeny of conserved proteins that may be involved in phloem function and development. The presence of CmPP16, APL, FT and YDA in chlorophytes suggests the recruitment of ancient regulatory networks for the development of the vascular tissue during evolution while OPS is a novel protein specific to vascular plants.

  9. Single-cell analysis of peptide expression and electrophysiology of right parietal neurons involved in male copulation behavior of a simultaneous hermaphrodite.

    Science.gov (United States)

    El Filali, Z; de Boer, P A C M; Pieneman, A W; de Lange, R P J; Jansen, R F; Ter Maat, A; van der Schors, R C; Li, K W; van Straalen, N M; Koene, J M

    2015-12-01

    Male copulation is a complex behavior that requires coordinated communication between the nervous system and the peripheral reproductive organs involved in mating. In hermaphroditic animals, such as the freshwater snail Lymnaea stagnalis, this complexity increases since the animal can behave both as male and female. The performance of the sexual role as a male is coordinated via a neuronal communication regulated by many peptidergic neurons, clustered in the cerebral and pedal ganglia and dispersed in the pleural and parietal ganglia. By combining single-cell matrix-assisted laser mass spectrometry with retrograde staining and electrophysiology, we analyzed neuropeptide expression of single neurons of the right parietal ganglion and their axonal projections into the penial nerve. Based on the neuropeptide profile of these neurons, we were able to reconstruct a chemical map of the right parietal ganglion revealing a striking correlation with the earlier electrophysiological and neuroanatomical studies. Neurons can be divided into two main groups: (i) neurons that express heptapeptides and (ii) neurons that do not. The neuronal projection of the different neurons into the penial nerve reveals a pattern where (spontaneous) activity is related to branching pattern. This heterogeneity in both neurochemical anatomy and branching pattern of the parietal neurons reflects the complexity of the peptidergic neurotransmission involved in the regulation of male mating behavior in this simultaneous hermaphrodite.

  10. Neighboring Genes Show Correlated Evolution in Gene Expression

    Science.gov (United States)

    Ghanbarian, Avazeh T.; Hurst, Laurence D.

    2015-01-01

    When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

  11. Gene expression profile of pulpitis.

    Science.gov (United States)

    Galicia, J C; Henson, B R; Parker, J S; Khan, A A

    2016-06-01

    The cost, prevalence and pain associated with endodontic disease necessitate an understanding of the fundamental molecular aspects of its pathogenesis. This study was aimed to identify the genetic contributors to pulpal pain and inflammation. Inflamed pulps were collected from patients diagnosed with irreversible pulpitis (n=20). Normal pulps from teeth extracted for various reasons served as controls (n=20). Pain level was assessed using a visual analog scale (VAS). Genome-wide microarray analysis was performed using Affymetrix GeneTitan Multichannel Instrument. The difference in gene expression levels were determined by the significance analysis of microarray program using a false discovery rate (q-value) of 5%. Genes involved in immune response, cytokine-cytokine receptor interaction and signaling, integrin cell surface interactions, and others were expressed at relatively higher levels in the pulpitis group. Moreover, several genes known to modulate pain and inflammation showed differential expression in asymptomatic and mild pain patients (⩾30 mm on VAS) compared with those with moderate to severe pain. This exploratory study provides a molecular basis for the clinical diagnosis of pulpitis. With an enhanced understanding of pulpal inflammation, future studies on treatment and management of pulpitis and on pain associated with it can have a biological reference to bridge treatment strategies with pulpal biology.

  12. Single-cell technologies in environmental omics

    KAUST Repository

    Kodzius, Rimantas; Gojobori, Takashi

    2015-01-01

    Environmental studies are primarily done by culturing isolated microorganisms or by amplifying and sequencing conserved genes. Difficulties understanding the complexity of large numbers of various microorganisms in an environment led to the development of techniques to enrich specific microorganisms for upstream analysis, ultimately leading to single-cell isolation and analyses. We discuss the significance of single-cell technologies in omics studies with focus on metagenomics and metatranscriptomics. We propose that by reducing sample heterogeneity using single-cell genomics, metaomic studies can be simplified.

  13. Single-cell technologies in environmental omics

    KAUST Repository

    Kodzius, Rimantas

    2015-10-22

    Environmental studies are primarily done by culturing isolated microorganisms or by amplifying and sequencing conserved genes. Difficulties understanding the complexity of large numbers of various microorganisms in an environment led to the development of techniques to enrich specific microorganisms for upstream analysis, ultimately leading to single-cell isolation and analyses. We discuss the significance of single-cell technologies in omics studies with focus on metagenomics and metatranscriptomics. We propose that by reducing sample heterogeneity using single-cell genomics, metaomic studies can be simplified.

  14. A Bistable Switch and Anatomical Site Control Vibrio cholerae Virulence Gene Expression in the Intestine

    DEFF Research Database (Denmark)

    Nielsen, Alex Toftgaard; Dolganov, N. A.; Rasmussen, Thomas

    2010-01-01

    A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene...... expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP) and cholera toxin (CT) were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co......, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal...

  15. Using gene expression noise to understand gene regulation

    NARCIS (Netherlands)

    Munsky, B.; Neuert, G.; van Oudenaarden, A.

    2012-01-01

    Phenotypic variation is ubiquitous in biology and is often traceable to underlying genetic and environmental variation. However, even genetically identical cells in identical environments display variable phenotypes. Stochastic gene expression, or gene expression "noise," has been suggested as a

  16. Lysophospholipid Receptors Are Differentially Expressed in Rat Terminal Schwann Cells, As Revealed by a Single Cell RT-PCR and In Situ Hybridization

    International Nuclear Information System (INIS)

    Kobashi, Hiroaki; Yaoi, Takeshi; Oda, Ryo; Okajima, Seiichiro; Fujiwara, Hiroyoshi; Kubo, Toshikazu; Fushiki, Shinji

    2006-01-01

    Terminal Schwann cells (TSCs) that cover motor neuron terminals, are known to play an important role in maintaining neuromuscular junctions, as well as in the repair process after nerve injury. However, the molecular characteristics of TSCs remain unknown, because of the difficulties in analyzing them due to their paucity. By using our previously reported method of selectively and efficiently collecting TSCs, we have analyzed the difference in expression patterns of lysophospholipid (LPL) receptor genes (LPA 1 , LPA 2 , LPA 3 , S1P 1 , S1P 2 , S1P 3 , S1P 4 , and S1P 5 ) between TSCs and myelinating Schwann cells (MSCs). LPL, which includes lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), is the bioactive lipid that induces a myriad of cellular responses through specific members of G-protein coupled receptors for LPA. It turned out that LPA 3 was expressed only in TSCs, whereas S1P 1 was expressed in TSCs and skeletal muscle, but not in MSCs. Other types of LPL receptor genes, including LPA 1 , S1P 2 , S1P 3 , S1P 4 , were expressed in both types of Schwann cells. None of the LPL receptor gene family showed MSCs-specific expression

  17. A constructive approach to gene expression dynamics

    International Nuclear Information System (INIS)

    Ochiai, T.; Nacher, J.C.; Akutsu, T.

    2004-01-01

    Recently, experiments on mRNA abundance (gene expression) have revealed that gene expression shows a stationary organization described by a scale-free distribution. Here we propose a constructive approach to gene expression dynamics which restores the scale-free exponent and describes the intermediate state dynamics. This approach requires only one assumption: Markov property

  18. Modulation of gene expression made easy

    DEFF Research Database (Denmark)

    Solem, Christian; Jensen, Peter Ruhdal

    2002-01-01

    A new approach for modulating gene expression, based on randomization of promoter (spacer) sequences, was developed. The method was applied to chromosomal genes in Lactococcus lactis and shown to generate libraries of clones with broad ranges of expression levels of target genes. In one example...... that the method can be applied to modulating the expression of native genes on the chromosome. We constructed a series of strains in which the expression of the las operon, containing the genes pfk, pyk, and ldh, was modulated by integrating a truncated copy of the pfk gene. Importantly, the modulation affected...

  19. Synthetic promoter libraries- tuning of gene expression

    DEFF Research Database (Denmark)

    Hammer, Karin; Mijakovic, Ivan; Jensen, Peter Ruhdal

    2006-01-01

    knockout and strong overexpression. However, applications such as metabolic optimization and control analysis necessitate a continuous set of expression levels with only slight increments in strength to cover a specific window around the wildtype expression level of the studied gene; this requirement can......The study of gene function often requires changing the expression of a gene and evaluating the consequences. In principle, the expression of any given gene can be modulated in a quasi-continuum of discrete expression levels but the traditional approaches are usually limited to two extremes: gene...

  20. Beta-Poisson model for single-cell RNA-seq data analyses.

    Science.gov (United States)

    Vu, Trung Nghia; Wills, Quin F; Kalari, Krishna R; Niu, Nifang; Wang, Liewei; Rantalainen, Mattias; Pawitan, Yudi

    2016-07-15

    Single-cell RNA-sequencing technology allows detection of gene expression at the single-cell level. One typical feature of the data is a bimodality in the cellular distribution even for highly expressed genes, primarily caused by a proportion of non-expressing cells. The standard and the over-dispersed gamma-Poisson models that are commonly used in bulk-cell RNA-sequencing are not able to capture this property. We introduce a beta-Poisson mixture model that can capture the bimodality of the single-cell gene expression distribution. We further integrate the model into the generalized linear model framework in order to perform differential expression analyses. The whole analytical procedure is called BPSC. The results from several real single-cell RNA-seq datasets indicate that ∼90% of the transcripts are well characterized by the beta-Poisson model; the model-fit from BPSC is better than the fit of the standard gamma-Poisson model in > 80% of the transcripts. Moreover, in differential expression analyses of simulated and real datasets, BPSC performs well against edgeR, a conventional method widely used in bulk-cell RNA-sequencing data, and against scde and MAST, two recent methods specifically designed for single-cell RNA-seq data. An R package BPSC for model fitting and differential expression analyses of single-cell RNA-seq data is available under GPL-3 license at https://github.com/nghiavtr/BPSC CONTACT: yudi.pawitan@ki.se or mattias.rantalainen@ki.se Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. Adaptive Evolution of Gene Expression in Drosophila

    Directory of Open Access Journals (Sweden)

    Armita Nourmohammad

    2017-08-01

    Full Text Available Gene expression levels are important quantitative traits that link genotypes to molecular functions and fitness. In Drosophila, population-genetic studies have revealed substantial adaptive evolution at the genomic level, but the evolutionary modes of gene expression remain controversial. Here, we present evidence that adaptation dominates the evolution of gene expression levels in flies. We show that 64% of the observed expression divergence across seven Drosophila species are adaptive changes driven by directional selection. Our results are derived from time-resolved data of gene expression divergence across a family of related species, using a probabilistic inference method for gene-specific selection. Adaptive gene expression is stronger in specific functional classes, including regulation, sensory perception, sexual behavior, and morphology. Moreover, we identify a large group of genes with sex-specific adaptation of expression, which predominantly occurs in males. Our analysis opens an avenue to map system-wide selection on molecular quantitative traits independently of their genetic basis.

  2. Lessons from single-cell transcriptome analysis of oxygen-sensing cells.

    Science.gov (United States)

    Zhou, Ting; Matsunami, Hiroaki

    2018-05-01

    The advent of single-cell RNA-sequencing (RNA-Seq) technology has enabled transcriptome profiling of individual cells. Comprehensive gene expression analysis at the single-cell level has proven to be effective in characterizing the most fundamental aspects of cellular function and identity. This unbiased approach is revolutionary for small and/or heterogeneous tissues like oxygen-sensing cells in identifying key molecules. Here, we review the major methods of current single-cell RNA-Seq technology. We discuss how this technology has advanced the understanding of oxygen-sensing glomus cells in the carotid body and helped uncover novel oxygen-sensing cells and mechanisms in the mice olfactory system. We conclude by providing our perspective on future single-cell RNA-Seq research directed at oxygen-sensing cells.

  3. Single cell analysis: the new frontier in 'Omics'

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Daojing; Bodovitz, Steven

    2010-01-14

    Cellular heterogeneity arising from stochastic expression of genes, proteins, and metabolites is a fundamental principle of cell biology, but single cell analysis has been beyond the capabilities of 'Omics' technologies. This is rapidly changing with the recent examples of single cell genomics, transcriptomics, proteomics, and metabolomics. The rate of change is expected to accelerate owing to emerging technologies that range from micro/nanofluidics to microfabricated interfaces for mass spectrometry to third- and fourth-generation automated DNA sequencers. As described in this review, single cell analysis is the new frontier in Omics, and single cell Omics has the potential to transform systems biology through new discoveries derived from cellular heterogeneity.

  4. The evolution of gene expression in primates

    OpenAIRE

    Tashakkori Ghanbarian, Avazeh

    2015-01-01

    The evolution of a gene’s expression profile is commonly assumed to be independent of its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between expression of neighboring genes in extant taxa. Indeed, in all eukaryotic genomes, genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their e...

  5. Computational Investigation of Environment-Noise Interaction in Single-Cell Organisms: The Merit of Expression Stochasticity Depends on the Quality of Environmental Fluctuations.

    Science.gov (United States)

    Lück, Anja; Klimmasch, Lukas; Großmann, Peter; Germerodt, Sebastian; Kaleta, Christoph

    2018-01-10

    Organisms need to adapt to changing environments and they do so by using a broad spectrum of strategies. These strategies include finding the right balance between expressing genes before or when they are needed, and adjusting the degree of noise inherent in gene expression. We investigated the interplay between different nutritional environments and the inhabiting organisms' metabolic and genetic adaptations by applying an evolutionary algorithm to an agent-based model of a concise bacterial metabolism. Our results show that constant environments and rapidly fluctuating environments produce similar adaptations in the organisms, making the predictability of the environment a major factor in determining optimal adaptation. We show that exploitation of expression noise occurs only in some types of fluctuating environment and is strongly dependent on the quality and availability of nutrients: stochasticity is generally detrimental in fluctuating environments and beneficial only at equal periods of nutrient availability and above a threshold environmental richness. Moreover, depending on the availability and nutritional value of nutrients, nutrient-dependent and stochastic expression are both strategies used to deal with environmental changes. Overall, we comprehensively characterize the interplay between the quality and periodicity of an environment and the resulting optimal deterministic and stochastic regulation strategies of nutrient-catabolizing pathways.

  6. Sex chromosomes and germline transcriptomics explored by single-cell sequencing and RNA-tomography

    NARCIS (Netherlands)

    Vértesy, Ábel

    2018-01-01

    In our study of germ cell differentiation, we applied two recently developed technologies on the germline of various model organisms: single-cell mRNA sequencing and RNA-tomography. For the first time we could look at gene expression with such a high resolution, and this led us to discover the

  7. SCNS: a graphical tool for reconstructing executable regulatory networks from single-cell genomic data.

    Science.gov (United States)

    Woodhouse, Steven; Piterman, Nir; Wintersteiger, Christoph M; Göttgens, Berthold; Fisher, Jasmin

    2018-05-25

    Reconstruction of executable mechanistic models from single-cell gene expression data represents a powerful approach to understanding developmental and disease processes. New ambitious efforts like the Human Cell Atlas will soon lead to an explosion of data with potential for uncovering and understanding the regulatory networks which underlie the behaviour of all human cells. In order to take advantage of this data, however, there is a need for general-purpose, user-friendly and efficient computational tools that can be readily used by biologists who do not have specialist computer science knowledge. The Single Cell Network Synthesis toolkit (SCNS) is a general-purpose computational tool for the reconstruction and analysis of executable models from single-cell gene expression data. Through a graphical user interface, SCNS takes single-cell qPCR or RNA-sequencing data taken across a time course, and searches for logical rules that drive transitions from early cell states towards late cell states. Because the resulting reconstructed models are executable, they can be used to make predictions about the effect of specific gene perturbations on the generation of specific lineages. SCNS should be of broad interest to the growing number of researchers working in single-cell genomics and will help further facilitate the generation of valuable mechanistic insights into developmental, homeostatic and disease processes.

  8. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy [Davis, CA; Bachkirova, Elena [Davis, CA; Rey, Michael [Davis, CA

    2012-05-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  9. Methods for monitoring multiple gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Berka, Randy; Bachkirova, Elena; Rey, Michael

    2013-10-01

    The present invention relates to methods for monitoring differential expression of a plurality of genes in a first filamentous fungal cell relative to expression of the same genes in one or more second filamentous fungal cells using microarrays containing Trichoderma reesei ESTs or SSH clones, or a combination thereof. The present invention also relates to computer readable media and substrates containing such array features for monitoring expression of a plurality of genes in filamentous fungal cells.

  10. cis sequence effects on gene expression

    Directory of Open Access Journals (Sweden)

    Jacobs Kevin

    2007-08-01

    Full Text Available Abstract Background Sequence and transcriptional variability within and between individuals are typically studied independently. The joint analysis of sequence and gene expression variation (genetical genomics provides insight into the role of linked sequence variation in the regulation of gene expression. We investigated the role of sequence variation in cis on gene expression (cis sequence effects in a group of genes commonly studied in cancer research in lymphoblastoid cell lines. We estimated the proportion of genes exhibiting cis sequence effects and the proportion of gene expression variation explained by cis sequence effects using three different analytical approaches, and compared our results to the literature. Results We generated gene expression profiling data at N = 697 candidate genes from N = 30 lymphoblastoid cell lines for this study and used available candidate gene resequencing data at N = 552 candidate genes to identify N = 30 candidate genes with sufficient variance in both datasets for the investigation of cis sequence effects. We used two additive models and the haplotype phylogeny scanning approach of Templeton (Tree Scanning to evaluate association between individual SNPs, all SNPs at a gene, and diplotypes, with log-transformed gene expression. SNPs and diplotypes at eight candidate genes exhibited statistically significant (p cis sequence effects in our study, respectively. Conclusion Based on analysis of our results and the extant literature, one in four genes exhibits significant cis sequence effects, and for these genes, about 30% of gene expression variation is accounted for by cis sequence variation. Despite diverse experimental approaches, the presence or absence of significant cis sequence effects is largely supported by previously published studies.

  11. Gene expression inference with deep learning.

    Science.gov (United States)

    Chen, Yifei; Li, Yi; Narayan, Rajiv; Subramanian, Aravind; Xie, Xiaohui

    2016-06-15

    Large-scale gene expression profiling has been widely used to characterize cellular states in response to various disease conditions, genetic perturbations, etc. Although the cost of whole-genome expression profiles has been dropping steadily, generating a compendium of expression profiling over thousands of samples is still very expensive. Recognizing that gene expressions are often highly correlated, researchers from the NIH LINCS program have developed a cost-effective strategy of profiling only ∼1000 carefully selected landmark genes and relying on computational methods to infer the expression of remaining target genes. However, the computational approach adopted by the LINCS program is currently based on linear regression (LR), limiting its accuracy since it does not capture complex nonlinear relationship between expressions of genes. We present a deep learning method (abbreviated as D-GEX) to infer the expression of target genes from the expression of landmark genes. We used the microarray-based Gene Expression Omnibus dataset, consisting of 111K expression profiles, to train our model and compare its performance to those from other methods. In terms of mean absolute error averaged across all genes, deep learning significantly outperforms LR with 15.33% relative improvement. A gene-wise comparative analysis shows that deep learning achieves lower error than LR in 99.97% of the target genes. We also tested the performance of our learned model on an independent RNA-Seq-based GTEx dataset, which consists of 2921 expression profiles. Deep learning still outperforms LR with 6.57% relative improvement, and achieves lower error in 81.31% of the target genes. D-GEX is available at https://github.com/uci-cbcl/D-GEX CONTACT: xhx@ics.uci.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  12. Identification of innate lymphoid cells in single-cell RNA-Seq data.

    Science.gov (United States)

    Suffiotti, Madeleine; Carmona, Santiago J; Jandus, Camilla; Gfeller, David

    2017-07-01

    Innate lymphoid cells (ILCs) consist of natural killer (NK) cells and non-cytotoxic ILCs that are broadly classified into ILC1, ILC2, and ILC3 subtypes. These cells recently emerged as important early effectors of innate immunity for their roles in tissue homeostasis and inflammation. Over the last few years, ILCs have been extensively studied in mouse and human at the functional and molecular level, including gene expression profiling. However, sorting ILCs with flow cytometry for gene expression analysis is a delicate and time-consuming process. Here we propose and validate a novel framework for studying ILCs at the transcriptomic level using single-cell RNA-Seq data. Our approach combines unsupervised clustering and a new cell type classifier trained on mouse ILC gene expression data. We show that this approach can accurately identify different ILCs, especially ILC2 cells, in human lymphocyte single-cell RNA-Seq data. Our new model relies only on genes conserved across vertebrates, thereby making it in principle applicable in any vertebrate species. Considering the rapid increase in throughput of single-cell RNA-Seq technology, our work provides a computational framework for studying ILC2 cells in single-cell transcriptomic data and may help exploring their conservation in distant vertebrate species.

  13. Quantifying intrinsic and extrinsic variability in stochastic gene expression models.

    Science.gov (United States)

    Singh, Abhyudai; Soltani, Mohammad

    2013-01-01

    Genetically identical cell populations exhibit considerable intercellular variation in the level of a given protein or mRNA. Both intrinsic and extrinsic sources of noise drive this variability in gene expression. More specifically, extrinsic noise is the expression variability that arises from cell-to-cell differences in cell-specific factors such as enzyme levels, cell size and cell cycle stage. In contrast, intrinsic noise is the expression variability that is not accounted for by extrinsic noise, and typically arises from the inherent stochastic nature of biochemical processes. Two-color reporter experiments are employed to decompose expression variability into its intrinsic and extrinsic noise components. Analytical formulas for intrinsic and extrinsic noise are derived for a class of stochastic gene expression models, where variations in cell-specific factors cause fluctuations in model parameters, in particular, transcription and/or translation rate fluctuations. Assuming mRNA production occurs in random bursts, transcription rate is represented by either the burst frequency (how often the bursts occur) or the burst size (number of mRNAs produced in each burst). Our analysis shows that fluctuations in the transcription burst frequency enhance extrinsic noise but do not affect the intrinsic noise. On the contrary, fluctuations in the transcription burst size or mRNA translation rate dramatically increase both intrinsic and extrinsic noise components. Interestingly, simultaneous fluctuations in transcription and translation rates arising from randomness in ATP abundance can decrease intrinsic noise measured in a two-color reporter assay. Finally, we discuss how these formulas can be combined with single-cell gene expression data from two-color reporter experiments for estimating model parameters.

  14. Determinants of human adipose tissue gene expression

    DEFF Research Database (Denmark)

    Viguerie, Nathalie; Montastier, Emilie; Maoret, Jean-José

    2012-01-01

    weight maintenance diets. For 175 genes, opposite regulation was observed during calorie restriction and weight maintenance phases, independently of variations in body weight. Metabolism and immunity genes showed inverse profiles. During the dietary intervention, network-based analyses revealed strong...... interconnection between expression of genes involved in de novo lipogenesis and components of the metabolic syndrome. Sex had a marked influence on AT expression of 88 transcripts, which persisted during the entire dietary intervention and after control for fat mass. In women, the influence of body mass index...... on expression of a subset of genes persisted during the dietary intervention. Twenty-two genes revealed a metabolic syndrome signature common to men and women. Genetic control of AT gene expression by cis signals was observed for 46 genes. Dietary intervention, sex, and cis genetic variants independently...

  15. Deriving Trading Rules Using Gene Expression Programming

    Directory of Open Access Journals (Sweden)

    Adrian VISOIU

    2011-01-01

    Full Text Available This paper presents how buy and sell trading rules are generated using gene expression programming with special setup. Market concepts are presented and market analysis is discussed with emphasis on technical analysis and quantitative methods. The use of genetic algorithms in deriving trading rules is presented. Gene expression programming is applied in a form where multiple types of operators and operands are used. This gives birth to multiple gene contexts and references between genes in order to keep the linear structure of the gene expression programming chromosome. The setup of multiple gene contexts is presented. The case study shows how to use the proposed gene setup to derive trading rules encoded by Boolean expressions, using a dataset with the reference exchange rates between the Euro and the Romanian leu. The conclusions highlight the positive results obtained in deriving useful trading rules.

  16. Polycistronic gene expression in Aspergillus niger.

    Science.gov (United States)

    Schuetze, Tabea; Meyer, Vera

    2017-09-25

    Genome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. However, the majority of these gene clusters still remain cryptic because they are not expressed in their natural host. Simultaneous expression of all genes belonging to a biosynthetic pathway in a heterologous host is one approach to activate biosynthetic gene clusters and to screen the metabolites produced for bioactivities. Polycistronic expression of all pathway genes under control of a single and tunable promoter would be the method of choice, as this does not only simplify cloning procedures, but also offers control on timing and strength of expression. However, polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger. In this study, we tested the suitability of the viral P2A peptide for co-expression of three genes in A. niger. Two genes descend from Fusarium oxysporum and are essential to produce the secondary metabolite enniatin (esyn1, ekivR). The third gene (luc) encodes the reporter luciferase which was included to study position effects. Expression of the polycistronic gene cassette was put under control of the Tet-On system to ensure tunable gene expression in A. niger. In total, three polycistronic expression cassettes which differed in the position of luc were constructed and targeted to the pyrG locus in A. niger. This allowed direct comparison of the luciferase activity based on the position of the luciferase gene. Doxycycline-mediated induction of the Tet-On expression cassettes resulted in the production of one long polycistronic mRNA as proven by Northern analyses, and ensured comparable production of enniatin in all three strains. Notably, gene position within the polycistronic expression cassette matters, as, luciferase activity was lowest at position one and had a comparable activity at positions two and three. The P2A peptide can be used to express at

  17. Profiling Gene Expression in Germinating Brassica Roots.

    Science.gov (United States)

    Park, Myoung Ryoul; Wang, Yi-Hong; Hasenstein, Karl H

    2014-01-01

    Based on previously developed solid-phase gene extraction (SPGE) we examined the mRNA profile in primary roots of Brassica rapa seedlings for highly expressed genes like ACT7 (actin7), TUB (tubulin1), UBQ (ubiquitin), and low expressed GLK (glucokinase) during the first day post-germination. The assessment was based on the mRNA load of the SPGE probe of about 2.1 ng. The number of copies of the investigated genes changed spatially along the length of primary roots. The expression level of all genes differed significantly at each sample position. Among the examined genes ACT7 expression was most even along the root. UBQ was highest at the tip and root-shoot junction (RS). TUB and GLK showed a basipetal gradient. The temporal expression of UBQ was highest in the MZ 9 h after primary root emergence and higher than at any other sample position. Expressions of GLK in EZ and RS increased gradually over time. SPGE extraction is the result of oligo-dT and oligo-dA hybridization and the results illustrate that SPGE can be used for gene expression profiling at high spatial and temporal resolution. SPGE needles can be used within two weeks when stored at 4 °C. Our data indicate that gene expression studies that are based on the entire root miss important differences in gene expression that SPGE is able to resolve for example growth adjustments during gravitropism.

  18. Chromatin loops, gene positioning, and gene expression

    NARCIS (Netherlands)

    Holwerda, S.; de Laat, W.

    2012-01-01

    Technological developments and intense research over the last years have led to a better understanding of the 3D structure of the genome and its influence on genome function inside the cell nucleus. We will summarize topological studies performed on four model gene loci: the alpha- and beta-globin

  19. Serial analysis of gene expression (SAGE)

    NARCIS (Netherlands)

    van Ruissen, Fred; Baas, Frank

    2007-01-01

    In 1995, serial analysis of gene expression (SAGE) was developed as a versatile tool for gene expression studies. SAGE technology does not require pre-existing knowledge of the genome that is being examined and therefore SAGE can be applied to many different model systems. In this chapter, the SAGE

  20. Expression of Sox genes in tooth development.

    Science.gov (United States)

    Kawasaki, Katsushige; Kawasaki, Maiko; Watanabe, Momoko; Idrus, Erik; Nagai, Takahiro; Oommen, Shelly; Maeda, Takeyasu; Hagiwara, Nobuko; Que, Jianwen; Sharpe, Paul T; Ohazama, Atsushi

    2015-01-01

    Members of the Sox gene family play roles in many biological processes including organogenesis. We carried out comparative in situ hybridization analysis of seventeen sox genes (Sox1-14, 17, 18, 21) during murine odontogenesis from the epithelial thickening to the cytodifferentiation stages. Localized expression of five Sox genes (Sox6, 9, 13, 14 and 21) was observed in tooth bud epithelium. Sox13 showed restricted expression in the primary enamel knots. At the early bell stage, three Sox genes (Sox8, 11, 17 and 21) were expressed in pre-ameloblasts, whereas two others (Sox5 and 18) showed expression in odontoblasts. Sox genes thus showed a dynamic spatio-temporal expression during tooth development.

  1. Positron emission tomography imaging of gene expression

    International Nuclear Information System (INIS)

    Tang Ganghua

    2001-01-01

    The merging of molecular biology and nuclear medicine is developed into molecular nuclear medicine. Positron emission tomography (PET) of gene expression in molecular nuclear medicine has become an attractive area. Positron emission tomography imaging gene expression includes the antisense PET imaging and the reporter gene PET imaging. It is likely that the antisense PET imaging will lag behind the reporter gene PET imaging because of the numerous issues that have not yet to be resolved with this approach. The reporter gene PET imaging has wide application into animal experimental research and human applications of this approach will likely be reported soon

  2. The functional landscape of mouse gene expression

    Directory of Open Access Journals (Sweden)

    Zhang Wen

    2004-12-01

    Full Text Available Abstract Background Large-scale quantitative analysis of transcriptional co-expression has been used to dissect regulatory networks and to predict the functions of new genes discovered by genome sequencing in model organisms such as yeast. Although the idea that tissue-specific expression is indicative of gene function in mammals is widely accepted, it has not been objectively tested nor compared with the related but distinct strategy of correlating gene co-expression as a means to predict gene function. Results We generated microarray expression data for nearly 40,000 known and predicted mRNAs in 55 mouse tissues, using custom-built oligonucleotide arrays. We show that quantitative transcriptional co-expression is a powerful predictor of gene function. Hundreds of functional categories, as defined by Gene Ontology 'Biological Processes', are associated with characteristic expression patterns across all tissues, including categories that bear no overt relationship to the tissue of origin. In contrast, simple tissue-specific restriction of expression is a poor predictor of which genes are in which functional categories. As an example, the highly conserved mouse gene PWP1 is widely expressed across different tissues but is co-expressed with many RNA-processing genes; we show that the uncharacterized yeast homolog of PWP1 is required for rRNA biogenesis. Conclusions We conclude that 'functional genomics' strategies based on quantitative transcriptional co-expression will be as fruitful in mammals as they have been in simpler organisms, and that transcriptional control of mammalian physiology is more modular than is generally appreciated. Our data and analyses provide a public resource for mammalian functional genomics.

  3. A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.

    Science.gov (United States)

    Nestorowa, Sonia; Hamey, Fiona K; Pijuan Sala, Blanca; Diamanti, Evangelia; Shepherd, Mairi; Laurenti, Elisa; Wilson, Nicola K; Kent, David G; Göttgens, Berthold

    2016-08-25

    Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice. © 2016 by The American Society of Hematology.

  4. Chemical memory reactions induced bursting dynamics in gene expression.

    Science.gov (United States)

    Tian, Tianhai

    2013-01-01

    Memory is a ubiquitous phenomenon in biological systems in which the present system state is not entirely determined by the current conditions but also depends on the time evolutionary path of the system. Specifically, many memorial phenomena are characterized by chemical memory reactions that may fire under particular system conditions. These conditional chemical reactions contradict to the extant stochastic approaches for modeling chemical kinetics and have increasingly posed significant challenges to mathematical modeling and computer simulation. To tackle the challenge, I proposed a novel theory consisting of the memory chemical master equations and memory stochastic simulation algorithm. A stochastic model for single-gene expression was proposed to illustrate the key function of memory reactions in inducing bursting dynamics of gene expression that has been observed in experiments recently. The importance of memory reactions has been further validated by the stochastic model of the p53-MDM2 core module. Simulations showed that memory reactions is a major mechanism for realizing both sustained oscillations of p53 protein numbers in single cells and damped oscillations over a population of cells. These successful applications of the memory modeling framework suggested that this innovative theory is an effective and powerful tool to study memory process and conditional chemical reactions in a wide range of complex biological systems.

  5. Identification of transcription-factor genes expressed in the Arabidopsis female gametophyte

    Directory of Open Access Journals (Sweden)

    Kang Il-Ho

    2010-06-01

    Full Text Available Abstract Background In flowering plants, the female gametophyte is typically a seven-celled structure with four cell types: the egg cell, the central cell, the synergid cells, and the antipodal cells. These cells perform essential functions required for double fertilization and early seed development. Differentiation of these distinct cell types likely involves coordinated changes in gene expression regulated by transcription factors. Therefore, understanding female gametophyte cell differentiation and function will require dissection of the gene regulatory networks operating in each of the cell types. These efforts have been hampered because few transcription factor genes expressed in the female gametophyte have been identified. To identify such genes, we undertook a large-scale differential expression screen followed by promoter-fusion analysis to detect transcription-factor genes transcribed in the Arabidopsis female gametophyte. Results Using quantitative reverse-transcriptase PCR, we analyzed 1,482 Arabidopsis transcription-factor genes and identified 26 genes exhibiting reduced mRNA levels in determinate infertile 1 mutant ovaries, which lack female gametophytes, relative to ovaries containing female gametophytes. Spatial patterns of gene transcription within the mature female gametophyte were identified for 17 transcription-factor genes using promoter-fusion analysis. Of these, ten genes were predominantly expressed in a single cell type of the female gametophyte including the egg cell, central cell and the antipodal cells whereas the remaining seven genes were expressed in two or more cell types. After fertilization, 12 genes were transcriptionally active in the developing embryo and/or endosperm. Conclusions We have shown that our quantitative reverse-transcriptase PCR differential-expression screen is sufficiently sensitive to detect transcription-factor genes transcribed in the female gametophyte. Most of the genes identified in this

  6. A comparative gene expression database for invertebrates

    Directory of Open Access Journals (Sweden)

    Ormestad Mattias

    2011-08-01

    Full Text Available Abstract Background As whole genome and transcriptome sequencing gets cheaper and faster, a great number of 'exotic' animal models are emerging, rapidly adding valuable data to the ever-expanding Evo-Devo field. All these new organisms serve as a fantastic resource for the research community, but the sheer amount of data, some published, some not, makes detailed comparison of gene expression patterns very difficult to summarize - a problem sometimes even noticeable within a single lab. The need to merge existing data with new information in an organized manner that is publicly available to the research community is now more necessary than ever. Description In order to offer a homogenous way of storing and handling gene expression patterns from a variety of organisms, we have developed the first web-based comparative gene expression database for invertebrates that allows species-specific as well as cross-species gene expression comparisons. The database can be queried by gene name, developmental stage and/or expression domains. Conclusions This database provides a unique tool for the Evo-Devo research community that allows the retrieval, analysis and comparison of gene expression patterns within or among species. In addition, this database enables a quick identification of putative syn-expression groups that can be used to initiate, among other things, gene regulatory network (GRN projects.

  7. Adaptive Evolution of Gene Expression in Drosophila.

    Science.gov (United States)

    Nourmohammad, Armita; Rambeau, Joachim; Held, Torsten; Kovacova, Viera; Berg, Johannes; Lässig, Michael

    2017-08-08

    Gene expression levels are important quantitative traits that link genotypes to molecular functions and fitness. In Drosophila, population-genetic studies have revealed substantial adaptive evolution at the genomic level, but the evolutionary modes of gene expression remain controversial. Here, we present evidence that adaptation dominates the evolution of gene expression levels in flies. We show that 64% of the observed expression divergence across seven Drosophila species are adaptive changes driven by directional selection. Our results are derived from time-resolved data of gene expression divergence across a family of related species, using a probabilistic inference method for gene-specific selection. Adaptive gene expression is stronger in specific functional classes, including regulation, sensory perception, sexual behavior, and morphology. Moreover, we identify a large group of genes with sex-specific adaptation of expression, which predominantly occurs in males. Our analysis opens an avenue to map system-wide selection on molecular quantitative traits independently of their genetic basis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Differential gene expression during Trypanosoma cruzi metacyclogenesis

    Directory of Open Access Journals (Sweden)

    Marco Aurelio Krieger

    1999-09-01

    Full Text Available The transformation of epimastigotes into metacyclic trypomastigotes involves changes in the pattern of expressed genes, resulting in important morphological and functional differences between these developmental forms of Trypanosoma cruzi. In order to identify and characterize genes involved in triggering the metacyclogenesis process and in conferring to metacyclic trypomastigotes their stage specific biological properties, we have developed a method allowing the isolation of genes specifically expressed when comparing two close related cell populations (representation of differential expression or RDE. The method is based on the PCR amplification of gene sequences selected by hybridizing and subtracting the populations in such a way that after some cycles of hybridization-amplification genes specific to a given population are highly enriched. The use of this method in the analysis of differential gene expression during T. cruzi metacyclogenesis (6 hr and 24 hr of differentiation and metacyclic trypomastigotes resulted in the isolation of several clones from each time point. Northern blot analysis showed that some genes are transiently expressed (6 hr and 24 hr differentiating cells, while others are present in differentiating cells and in metacyclic trypomastigotes. Nucleotide sequencing of six clones characterized so far showed that they do not display any homology to gene sequences available in the GeneBank.

  9. Stochastic gene expression in Arabidopsis thaliana.

    Science.gov (United States)

    Araújo, Ilka Schultheiß; Pietsch, Jessica Magdalena; Keizer, Emma Mathilde; Greese, Bettina; Balkunde, Rachappa; Fleck, Christian; Hülskamp, Martin

    2017-12-14

    Although plant development is highly reproducible, some stochasticity exists. This developmental stochasticity may be caused by noisy gene expression. Here we analyze the fluctuation of protein expression in Arabidopsis thaliana. Using the photoconvertible KikGR marker, we show that the protein expressions of individual cells fluctuate over time. A dual reporter system was used to study extrinsic and intrinsic noise of marker gene expression. We report that extrinsic noise is higher than intrinsic noise and that extrinsic noise in stomata is clearly lower in comparison to several other tissues/cell types. Finally, we show that cells are coupled with respect to stochastic protein expression in young leaves, hypocotyls and roots but not in mature leaves. Our data indicate that stochasticity of gene expression can vary between tissues/cell types and that it can be coupled in a non-cell-autonomous manner.

  10. Characterizing human stem cell-derived sensory neurons at the single-cell level reveals their ion channel expression and utility in pain research.

    Science.gov (United States)

    Young, Gareth T; Gutteridge, Alex; Fox, Heather DE; Wilbrey, Anna L; Cao, Lishuang; Cho, Lily T; Brown, Adam R; Benn, Caroline L; Kammonen, Laura R; Friedman, Julia H; Bictash, Magda; Whiting, Paul; Bilsland, James G; Stevens, Edward B

    2014-08-01

    The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell-derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders.

  11. Characterizing Human Stem Cell–derived Sensory Neurons at the Single-cell Level Reveals Their Ion Channel Expression and Utility in Pain Research

    Science.gov (United States)

    Young, Gareth T; Gutteridge, Alex; Fox, Heather DE; Wilbrey, Anna L; Cao, Lishuang; Cho, Lily T; Brown, Adam R; Benn, Caroline L; Kammonen, Laura R; Friedman, Julia H; Bictash, Magda; Whiting, Paul; Bilsland, James G; Stevens, Edward B

    2014-01-01

    The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell–derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders. PMID:24832007

  12. Gene expression in periodontal tissues following treatment

    Directory of Open Access Journals (Sweden)

    Eisenacher Martin

    2008-07-01

    Full Text Available Abstract Background In periodontitis, treatment aimed at controlling the periodontal biofilm infection results in a resolution of the clinical and histological signs of inflammation. Although the cell types found in periodontal tissues following treatment have been well described, information on gene expression is limited to few candidate genes. Therefore, the aim of the study was to determine the expression profiles of immune and inflammatory genes in periodontal tissues from sites with severe chronic periodontitis following periodontal therapy in order to identify genes involved in tissue homeostasis. Gingival biopsies from 12 patients with severe chronic periodontitis were taken six to eight weeks following non-surgical periodontal therapy, and from 11 healthy controls. As internal standard, RNA of an immortalized human keratinocyte line (HaCaT was used. Total RNA was subjected to gene expression profiling using a commercially available microarray system focusing on inflammation-related genes. Post-hoc confirmation of selected genes was done by Realtime-PCR. Results Out of the 136 genes analyzed, the 5% most strongly expressed genes compared to healthy controls were Interleukin-12A (IL-12A, Versican (CSPG-2, Matrixmetalloproteinase-1 (MMP-1, Down syndrome critical region protein-1 (DSCR-1, Macrophage inflammatory protein-2β (Cxcl-3, Inhibitor of apoptosis protein-1 (BIRC-1, Cluster of differentiation antigen 38 (CD38, Regulator of G-protein signalling-1 (RGS-1, and Finkel-Biskis-Jinkins murine osteosarcoma virus oncogene (C-FOS; the 5% least strongly expressed genes were Receptor-interacting Serine/Threonine Kinase-2 (RIP-2, Complement component 3 (C3, Prostaglandin-endoperoxide synthase-2 (COX-2, Interleukin-8 (IL-8, Endothelin-1 (EDN-1, Plasminogen activator inhibitor type-2 (PAI-2, Matrix-metalloproteinase-14 (MMP-14, and Interferon regulating factor-7 (IRF-7. Conclusion Gene expression profiles found in periodontal tissues following

  13. Widespread ectopic expression of olfactory receptor genes

    Directory of Open Access Journals (Sweden)

    Yanai Itai

    2006-05-01

    Full Text Available Abstract Background Olfactory receptors (ORs are the largest gene family in the human genome. Although they are expected to be expressed specifically in olfactory tissues, some ectopic expression has been reported, with special emphasis on sperm and testis. The present study systematically explores the expression patterns of OR genes in a large number of tissues and assesses the potential functional implication of such ectopic expression. Results We analyzed the expression of hundreds of human and mouse OR transcripts, via EST and microarray data, in several dozens of human and mouse tissues. Different tissues had specific, relatively small OR gene subsets which had particularly high expression levels. In testis, average expression was not particularly high, and very few highly expressed genes were found, none corresponding to ORs previously implicated in sperm chemotaxis. Higher expression levels were more common for genes with a non-OR genomic neighbor. Importantly, no correlation in expression levels was detected for human-mouse orthologous pairs. Also, no significant difference in expression levels was seen between intact and pseudogenized ORs, except for the pseudogenes of subfamily 7E which has undergone a human-specific expansion. Conclusion The OR superfamily as a whole, show widespread, locus-dependent and heterogeneous expression, in agreement with a neutral or near neutral evolutionary model for transcription control. These results cannot reject the possibility that small OR subsets might play functional roles in different tissues, however considerable care should be exerted when offering a functional interpretation for ectopic OR expression based only on transcription information.

  14. Regulation of Gene Expression in Protozoa Parasites

    Directory of Open Access Journals (Sweden)

    Consuelo Gomez

    2010-01-01

    Full Text Available Infections with protozoa parasites are associated with high burdens of morbidity and mortality across the developing world. Despite extensive efforts to control the transmission of these parasites, the spread of populations resistant to drugs and the lack of effective vaccines against them contribute to their persistence as major public health problems. Parasites should perform a strict control on the expression of genes involved in their pathogenicity, differentiation, immune evasion, or drug resistance, and the comprehension of the mechanisms implicated in that control could help to develop novel therapeutic strategies. However, until now these mechanisms are poorly understood in protozoa. Recent investigations into gene expression in protozoa parasites suggest that they possess many of the canonical machineries employed by higher eukaryotes for the control of gene expression at transcriptional, posttranscriptional, and epigenetic levels, but they also contain exclusive mechanisms. Here, we review the current understanding about the regulation of gene expression in Plasmodium sp., Trypanosomatids, Entamoeba histolytica and Trichomonas vaginalis.

  15. Regulation of gene expression in protozoa parasites.

    Science.gov (United States)

    Gomez, Consuelo; Esther Ramirez, M; Calixto-Galvez, Mercedes; Medel, Olivia; Rodríguez, Mario A

    2010-01-01

    Infections with protozoa parasites are associated with high burdens of morbidity and mortality across the developing world. Despite extensive efforts to control the transmission of these parasites, the spread of populations resistant to drugs and the lack of effective vaccines against them contribute to their persistence as major public health problems. Parasites should perform a strict control on the expression of genes involved in their pathogenicity, differentiation, immune evasion, or drug resistance, and the comprehension of the mechanisms implicated in that control could help to develop novel therapeutic strategies. However, until now these mechanisms are poorly understood in protozoa. Recent investigations into gene expression in protozoa parasites suggest that they possess many of the canonical machineries employed by higher eukaryotes for the control of gene expression at transcriptional, posttranscriptional, and epigenetic levels, but they also contain exclusive mechanisms. Here, we review the current understanding about the regulation of gene expression in Plasmodium sp., Trypanosomatids, Entamoeba histolytica and Trichomonas vaginalis.

  16. Inferring gene networks from discrete expression data

    KAUST Repository

    Zhang, L.; Mallick, B. K.

    2013-01-01

    graphical models applied to continuous data, which give a closedformmarginal likelihood. In this paper,we extend network modeling to discrete data, specifically data from serial analysis of gene expression, and RNA-sequencing experiments, both of which

  17. Gene Expression and Microarray Investigation of Dendrobium ...

    African Journals Online (AJOL)

    blood glucose > 16.7 mmol/L were used as the model group and treated with Dendrobium mixture. (DEN ... Keywords: Diabetes, Gene expression, Dendrobium mixture, Microarray testing ..... homeostasis in airway smooth muscle. Am J.

  18. Identification of genes showing differential expression profile ...

    Indian Academy of Sciences (India)

    3Department of Natural Sciences, International Christian University, Mitaka, Tokyo 181-8585, Japan ... the changes of expression predicted from gene function suggested association ... ate School of Science and Technology, Niigata University.

  19. Drosophila melanogaster gene expression changes after spaceflight.

    Data.gov (United States)

    National Aeronautics and Space Administration — Gene expression levels were determined in 3rd instar and adult Drosophila melanogaster reared during spaceflight to elucidate the genetic and molecular mechanisms...

  20. Exertional Heat Illness and Human Gene Expression

    National Research Council Canada - National Science Library

    Sonna, L.A; Sawka, M. N; Lilly, C. M

    2007-01-01

    Microarray analysis of gene expression at the level of RNA has generated new insights into the relationship between cellular responses to acute heat shock in vitro, exercise, and exertional heat illness...

  1. Expression Profiling of Tyrosine Kinase Genes

    National Research Council Canada - National Science Library

    Weier, Heinz

    2000-01-01

    ... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

  2. Regulation of meiotic gene expression in plants

    Directory of Open Access Journals (Sweden)

    Adele eZhou

    2014-08-01

    Full Text Available With the recent advances in genomics and sequencing technologies, databases of transcriptomes representing many cellular processes have been built. Meiotic transcriptomes in plants have been studied in Arabidopsis thaliana, rice (Oryza sativa, wheat (Triticum aestivum, petunia (Petunia hybrida, sunflower (Helianthus annuus, and maize (Zea mays. Studies in all organisms, but particularly in plants, indicate that a very large number of genes are expressed during meiosis, though relatively few of them seem to be required for the completion of meiosis. In this review, we focus on gene expression at the RNA level and analyze the meiotic transcriptome datasets and explore expression patterns of known meiotic genes to elucidate how gene expression could be regulated during meiosis. We also discuss mechanisms, such as chromatin organization and non-coding RNAs, that might be involved in the regulation of meiotic transcription patterns.

  3. Identification of genes preferentially expressed during

    African Journals Online (AJOL)

    雨林木风

    2012-08-16

    Aug 16, 2012 ... The suppression subtractive hybridization (SSH) method conducted to generate ... which showed the lack of genomic information currently available for lily. ..... characterization of genes expressed during somatic embryo.

  4. Mining gene expression data of multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Pi Guo

    Full Text Available Microarray produces a large amount of gene expression data, containing various biological implications. The challenge is to detect a panel of discriminative genes associated with disease. This study proposed a robust classification model for gene selection using gene expression data, and performed an analysis to identify disease-related genes using multiple sclerosis as an example.Gene expression profiles based on the transcriptome of peripheral blood mononuclear cells from a total of 44 samples from 26 multiple sclerosis patients and 18 individuals with other neurological diseases (control were analyzed. Feature selection algorithms including Support Vector Machine based on Recursive Feature Elimination, Receiver Operating Characteristic Curve, and Boruta algorithms were jointly performed to select candidate genes associating with multiple sclerosis. Multiple classification models categorized samples into two different groups based on the identified genes. Models' performance was evaluated using cross-validation methods, and an optimal classifier for gene selection was determined.An overlapping feature set was identified consisting of 8 genes that were differentially expressed between the two phenotype groups. The genes were significantly associated with the pathways of apoptosis and cytokine-cytokine receptor interaction. TNFSF10 was significantly associated with multiple sclerosis. A Support Vector Machine model was established based on the featured genes and gave a practical accuracy of ∼86%. This binary classification model also outperformed the other models in terms of Sensitivity, Specificity and F1 score.The combined analytical framework integrating feature ranking algorithms and Support Vector Machine model could be used for selecting genes for other diseases.

  5. Optimal Reference Genes for Gene Expression Normalization in Trichomonas vaginalis

    Science.gov (United States)

    dos Santos, Odelta; de Vargas Rigo, Graziela; Frasson, Amanda Piccoli; Macedo, Alexandre José; Tasca, Tiana

    2015-01-01

    Trichomonas vaginalis is the etiologic agent of trichomonosis, the most common non-viral sexually transmitted disease worldwide. This infection is associated with several health consequences, including cervical and prostate cancers and HIV acquisition. Gene expression analysis has been facilitated because of available genome sequences and large-scale transcriptomes in T. vaginalis, particularly using quantitative real-time polymerase chain reaction (qRT-PCR), one of the most used methods for molecular studies. Reference genes for normalization are crucial to ensure the accuracy of this method. However, to the best of our knowledge, a systematic validation of reference genes has not been performed for T. vaginalis. In this study, the transcripts of nine candidate reference genes were quantified using qRT-PCR under different cultivation conditions, and the stability of these genes was compared using the geNorm and NormFinder algorithms. The most stable reference genes were α-tubulin, actin and DNATopII, and, conversely, the widely used T. vaginalis reference genes GAPDH and β-tubulin were less stable. The PFOR gene was used to validate the reliability of the use of these candidate reference genes. As expected, the PFOR gene was upregulated when the trophozoites were cultivated with ferrous ammonium sulfate when the DNATopII, α-tubulin and actin genes were used as normalizing gene. By contrast, the PFOR gene was downregulated when the GAPDH gene was used as an internal control, leading to misinterpretation of the data. These results provide an important starting point for reference gene selection and gene expression analysis with qRT-PCR studies of T. vaginalis. PMID:26393928

  6. Evaluation of suitable reference genes for gene expression studies ...

    Indian Academy of Sciences (India)

    2011-12-14

    Dec 14, 2011 ... MADS family of TFs control floral organ identity within each whorl of the flower by activating downstream genes. Measuring gene expression in different tissue types and developmental stages is of fundamental importance in TFs functional research. In last few years, quantitative real-time. PCR (qRT-PCR) ...

  7. PRAME gene expression profile in medulloblastoma

    Directory of Open Access Journals (Sweden)

    Tânia Maria Vulcani-Freitas

    2011-02-01

    Full Text Available Medulloblastoma is the most common malignant tumors of central nervous system in the childhood. The treatment is severe, harmful and, thus, has a dismal prognosis. As PRAME is present in various cancers, including meduloblastoma, and has limited expression in normal tissues, this antigen can be an ideal vaccine target for tumor immunotherapy. In order to find a potential molecular target, we investigated PRAME expression in medulloblastoma fragments and we compare the results with the clinical features of each patient. Analysis of gene expression was performed by real-time quantitative PCR from 37 tumor samples. The Mann-Whitney test was used to analysis the relationship between gene expression and clinical characteristics. Kaplan-Meier curves were used to evaluate survival. PRAME was overexpressed in 84% samples. But no statistical association was found between clinical features and PRAME overexpression. Despite that PRAME gene could be a strong candidate for immunotherapy since it is highly expressed in medulloblastomas.

  8. Comparative gene expression between two yeast species

    Directory of Open Access Journals (Sweden)

    Guan Yuanfang

    2013-01-01

    Full Text Available Abstract Background Comparative genomics brings insight into sequence evolution, but even more may be learned by coupling sequence analyses with experimental tests of gene function and regulation. However, the reliability of such comparisons is often limited by biased sampling of expression conditions and incomplete knowledge of gene functions across species. To address these challenges, we previously systematically generated expression profiles in Saccharomyces bayanus to maximize functional coverage as compared to an existing Saccharomyces cerevisiae data repository. Results In this paper, we take advantage of these two data repositories to compare patterns of ortholog expression in a wide variety of conditions. First, we developed a scalable metric for expression divergence that enabled us to detect a significant correlation between sequence and expression conservation on the global level, which previous smaller-scale expression studies failed to detect. Despite this global conservation trend, between-species gene expression neighborhoods were less well-conserved than within-species comparisons across different environmental perturbations, and approximately 4% of orthologs exhibited a significant change in co-expression partners. Furthermore, our analysis of matched perturbations collected in both species (such as diauxic shift and cell cycle synchrony demonstrated that approximately a quarter of orthologs exhibit condition-specific expression pattern differences. Conclusions Taken together, these analyses provide a global view of gene expression patterns between two species, both in terms of the conditions and timing of a gene's expression as well as co-expression partners. Our results provide testable hypotheses that will direct future experiments to determine how these changes may be specified in the genome.

  9. Single cell analysis of Vibrio harveyi uncovers functional heterogeneity in response to quorum sensing signals

    Directory of Open Access Journals (Sweden)

    Anetzberger Claudia

    2012-09-01

    Full Text Available Abstract Background Vibrio harveyi and closely related species are important pathogens in aquaculture. A complex quorum sensing cascade involving three autoinducers controls bioluminescence and several genes encoding virulence factors. Single cell analysis of a V. harveyi population has already indicated intercellular heterogeneity in the production of bioluminescence. This study was undertaken to analyze the expression of various autoinducer-dependent genes in individual cells. Results Here we used reporter strains bearing promoter::gfp fusions to monitor the induction/repression of three autoinducer-regulated genes in wild type conjugates at the single cell level. Two genes involved in pathogenesis - vhp and vscP, which code for an exoprotease and a component of the type III secretion system, respectively, and luxC (the first gene in the lux operon were chosen for analysis. The lux operon and the exoprotease gene are induced, while vscP is repressed at high cell density. As controls luxS and recA, whose expression is not dependent on autoinducers, were examined. The responses of the promoter::gfp fusions in individual cells from the same culture ranged from no to high induction. Importantly, simultaneous analysis of two autoinducer induced phenotypes, bioluminescence (light detection and exoproteolytic activity (fluorescence of a promoter::gfp fusion, in single cells provided evidence for functional heterogeneity within a V. harveyi population. Conclusions Autoinducers are not only an indicator for cell density, but play a pivotal role in the coordination of physiological activities within the population.

  10. Single cell analysis of Vibrio harveyi uncovers functional heterogeneity in response to quorum sensing signals.

    Science.gov (United States)

    Anetzberger, Claudia; Schell, Ursula; Jung, Kirsten

    2012-09-18

    Vibrio harveyi and closely related species are important pathogens in aquaculture. A complex quorum sensing cascade involving three autoinducers controls bioluminescence and several genes encoding virulence factors. Single cell analysis of a V. harveyi population has already indicated intercellular heterogeneity in the production of bioluminescence. This study was undertaken to analyze the expression of various autoinducer-dependent genes in individual cells. Here we used reporter strains bearing promoter::gfp fusions to monitor the induction/repression of three autoinducer-regulated genes in wild type conjugates at the single cell level. Two genes involved in pathogenesis - vhp and vscP, which code for an exoprotease and a component of the type III secretion system, respectively, and luxC (the first gene in the lux operon) were chosen for analysis. The lux operon and the exoprotease gene are induced, while vscP is repressed at high cell density. As controls luxS and recA, whose expression is not dependent on autoinducers, were examined. The responses of the promoter::gfp fusions in individual cells from the same culture ranged from no to high induction. Importantly, simultaneous analysis of two autoinducer induced phenotypes, bioluminescence (light detection) and exoproteolytic activity (fluorescence of a promoter::gfp fusion), in single cells provided evidence for functional heterogeneity within a V. harveyi population. Autoinducers are not only an indicator for cell density, but play a pivotal role in the coordination of physiological activities within the population.

  11. Inferring gene networks from discrete expression data

    KAUST Repository

    Zhang, L.

    2013-07-18

    The modeling of gene networks from transcriptional expression data is an important tool in biomedical research to reveal signaling pathways and to identify treatment targets. Current gene network modeling is primarily based on the use of Gaussian graphical models applied to continuous data, which give a closedformmarginal likelihood. In this paper,we extend network modeling to discrete data, specifically data from serial analysis of gene expression, and RNA-sequencing experiments, both of which generate counts of mRNAtranscripts in cell samples.We propose a generalized linear model to fit the discrete gene expression data and assume that the log ratios of the mean expression levels follow a Gaussian distribution.We restrict the gene network structures to decomposable graphs and derive the graphs by selecting the covariance matrix of the Gaussian distribution with the hyper-inverse Wishart priors. Furthermore, we incorporate prior network models based on gene ontology information, which avails existing biological information on the genes of interest. We conduct simulation studies to examine the performance of our discrete graphical model and apply the method to two real datasets for gene network inference. © The Author 2013. Published by Oxford University Press. All rights reserved.

  12. Gene expression profiling in autoimmune diseases

    DEFF Research Database (Denmark)

    Bovin, Lone Frier; Brynskov, Jørn; Hegedüs, Laszlo

    2007-01-01

    A central issue in autoimmune disease is whether the underlying inflammation is a repeated stereotypical process or whether disease specific gene expression is involved. To shed light on this, we analysed whether genes previously found to be differentially regulated in rheumatoid arthritis (RA...

  13. Bayesian assignment of gene ontology terms to gene expression experiments.

    Science.gov (United States)

    Sykacek, P

    2012-09-15

    Gene expression assays allow for genome scale analyses of molecular biological mechanisms. State-of-the-art data analysis provides lists of involved genes, either by calculating significance levels of mRNA abundance or by Bayesian assessments of gene activity. A common problem of such approaches is the difficulty of interpreting the biological implication of the resulting gene lists. This lead to an increased interest in methods for inferring high-level biological information. A common approach for representing high level information is by inferring gene ontology (GO) terms which may be attributed to the expression data experiment. This article proposes a probabilistic model for GO term inference. Modelling assumes that gene annotations to GO terms are available and gene involvement in an experiment is represented by a posterior probabilities over gene-specific indicator variables. Such probability measures result from many Bayesian approaches for expression data analysis. The proposed model combines these indicator probabilities in a probabilistic fashion and provides a probabilistic GO term assignment as a result. Experiments on synthetic and microarray data suggest that advantages of the proposed probabilistic GO term inference over statistical test-based approaches are in particular evident for sparsely annotated GO terms and in situations of large uncertainty about gene activity. Provided that appropriate annotations exist, the proposed approach is easily applied to inferring other high level assignments like pathways. Source code under GPL license is available from the author. peter.sykacek@boku.ac.at.

  14. Bayesian assignment of gene ontology terms to gene expression experiments

    Science.gov (United States)

    Sykacek, P.

    2012-01-01

    Motivation: Gene expression assays allow for genome scale analyses of molecular biological mechanisms. State-of-the-art data analysis provides lists of involved genes, either by calculating significance levels of mRNA abundance or by Bayesian assessments of gene activity. A common problem of such approaches is the difficulty of interpreting the biological implication of the resulting gene lists. This lead to an increased interest in methods for inferring high-level biological information. A common approach for representing high level information is by inferring gene ontology (GO) terms which may be attributed to the expression data experiment. Results: This article proposes a probabilistic model for GO term inference. Modelling assumes that gene annotations to GO terms are available and gene involvement in an experiment is represented by a posterior probabilities over gene-specific indicator variables. Such probability measures result from many Bayesian approaches for expression data analysis. The proposed model combines these indicator probabilities in a probabilistic fashion and provides a probabilistic GO term assignment as a result. Experiments on synthetic and microarray data suggest that advantages of the proposed probabilistic GO term inference over statistical test-based approaches are in particular evident for sparsely annotated GO terms and in situations of large uncertainty about gene activity. Provided that appropriate annotations exist, the proposed approach is easily applied to inferring other high level assignments like pathways. Availability: Source code under GPL license is available from the author. Contact: peter.sykacek@boku.ac.at PMID:22962488

  15. A bistable switch and anatomical site control Vibrio cholerae virulence gene expression in the intestine.

    Directory of Open Access Journals (Sweden)

    Alex T Nielsen

    2010-09-01

    Full Text Available A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP and cholera toxin (CT were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co-localize with virulence gene expression. Significant heterogeneity in the expression of tcpA, the repeating subunit of TCP, was observed late in the infectious process. The expression of tcpA, studied in single cells in a homogeneous medium, demonstrated unimodal induction of tcpA after addition of bicarbonate, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal master regulator of virulence gene expression also exhibited the bifurcation phenotype. The bifurcation phenotype was found to be reversible, epigenetic and to persist after removal of bicarbonate, features consistent with bistable switches. The bistable switch requires the positive-feedback circuit controlling ToxT expression and formation of the CRP-cAMP complex during entry into stationary phase. Key features of this bistable switch also were demonstrated in vivo, where striking heterogeneity in tcpA expression was observed in luminal fluid in later stages of the infection. When this fluid was diluted into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a

  16. Cell individuality: the bistable gene expression of the type III secretion system in Dickeya dadantii 3937.

    Science.gov (United States)

    Zeng, Quan; Laiosa, Michael D; Steeber, Douglas A; Biddle, Eulandria M; Peng, Quan; Yang, Ching-Hong

    2012-01-01

    Dickeya dadantii 3937 is a gram-negative phytopathogenic bacterium that expresses genes encoding a type III secretion system (T3SS) in a bistable pattern when cultured in a homogeneous minimal media. In this work, we further characterized the bistable gene expression of T3SS at the single-cell level. We demonstrated that bistable expression of the HrpL-regulon genes, such as hrpA and hrpN, is controlled by the same regulatory mechanism. We also showed that the expression level of the T3SS master regulatory gene hrpL plays an important role in the development of the bistable expression of hrpA. A high expression level of hrpL is required but unable to guarantee the high-state expression of hrpA in a cell. In addition, bistable expression patterns of T3SS genes in other gram-negative pathogens of the Enterobacteriaceae and Pseudomonadaceae families were also described in this study. This suggests that the T3SS bistability might be a conserved population behavior in several gram-negative bacterial pathogens.

  17. Immobilisation of barley aleurone layers enables parallelisation of assays and analysis of transient gene expression in single cells

    DEFF Research Database (Denmark)

    Zor, Kinga; Mark, Christina; Heiskanen, Arto

    2017-01-01

    at a single time point. By immobilising barley aleurone layer tissue on polydimethylsiloxane pillars in the lid of a multiwell plate, continuous monitoring of living tissue is enabled using multiple non-destructive assays in parallel. Cell viability and menadione reducing capacity were monitored in the same...

  18. Reference Gene Screening for Analyzing Gene Expression Across Goat Tissue

    Directory of Open Access Journals (Sweden)

    Yu Zhang

    2013-12-01

    Full Text Available Real-time quantitative PCR (qRT-PCR is one of the important methods for investigating the changes in mRNA expression levels in cells and tissues. Selection of the proper reference genes is very important when calibrating the results of real-time quantitative PCR. Studies on the selection of reference genes in goat tissues are limited, despite the economic importance of their meat and dairy products. We used real-time quantitative PCR to detect the expression levels of eight reference gene candidates (18S, TBP, HMBS, YWHAZ, ACTB, HPRT1, GAPDH and EEF1A2 in ten tissues types sourced from Boer goats. The optimal reference gene combination was selected according to the results determined by geNorm, NormFinder and Bestkeeper software packages. The analyses showed that tissue is an important variability factor in genes expression stability. When all tissues were considered, 18S, TBP and HMBS is the optimal reference combination for calibrating quantitative PCR analysis of gene expression from goat tissues. Dividing data set by tissues, ACTB was the most stable in stomach, small intestine and ovary, 18S in heart and spleen, HMBS in uterus and lung, TBP in liver, HPRT1 in kidney and GAPDH in muscle. Overall, this study provided valuable information about the goat reference genes that can be used in order to perform a proper normalisation when relative quantification by qRT-PCR studies is undertaken.

  19. Microfluidics for single cell analysis

    DEFF Research Database (Denmark)

    Jensen, Marie Pødenphant

    Isolation and manipulation of single cells have gained an increasing interest from researchers because of the heterogeneity of cells from the same cell culture. Single cell analysis can ensure a better understanding of differences between individual cells and potentially solve a variety of clinical...... problems. In this thesis lab on a chip systems for rare single cell analysis are investigated. The focus was to develop a commercial, disposable device for circulating tumour cell (CTC) analysis. Such a device must be able to separate rare cells from blood samples and subsequently capture the specific...... cells, and simultaneously be fabricated and operated at low costs and be user-friendly. These challenges were addressed through development of two microfluidic devices, one for rare cell isolation based on pinched flow fractionation (PFF) and one for single cell capture based on hydrodynamic trapping...

  20. Noise minimization in eukaryotic gene expression.

    Directory of Open Access Journals (Sweden)

    Hunter B Fraser

    2004-06-01

    Full Text Available All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or "noise." Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.

  1. Noise minimization in eukaryotic gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, Hunter B.; Hirsh, Aaron E.; Giaever, Guri; Kumm, Jochen; Eisen, Michael B.

    2004-01-15

    All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or noise. Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection.

  2. Noise minimization in eukaryotic gene expression

    International Nuclear Information System (INIS)

    Fraser, Hunter B.; Hirsh, Aaron E.; Giaever, Guri; Kumm, Jochen; Eisen, Michael B.

    2004-01-01

    All organisms have elaborate mechanisms to control rates of protein production. However, protein production is also subject to stochastic fluctuations, or noise. Several recent studies in Saccharomyces cerevisiae and Escherichia coli have investigated the relationship between transcription and translation rates and stochastic fluctuations in protein levels, or more generally, how such randomness is a function of intrinsic and extrinsic factors. However, the fundamental question of whether stochasticity in protein expression is generally biologically relevant has not been addressed, and it remains unknown whether random noise in the protein production rate of most genes significantly affects the fitness of any organism. We propose that organisms should be particularly sensitive to variation in the protein levels of two classes of genes: genes whose deletion is lethal to the organism and genes that encode subunits of multiprotein complexes. Using an experimentally verified model of stochastic gene expression in S. cerevisiae, we estimate the noise in protein production for nearly every yeast gene, and confirm our prediction that the production of essential and complex-forming proteins involves lower levels of noise than does the production of most other genes. Our results support the hypothesis that noise in gene expression is a biologically important variable, is generally detrimental to organismal fitness, and is subject to natural selection

  3. visnormsc: A Graphical User Interface to Normalize Single-cell RNA Sequencing Data.

    Science.gov (United States)

    Tang, Lijun; Zhou, Nan

    2017-12-26

    Single-cell RNA sequencing (RNA-seq) allows the analysis of gene expression with high resolution. The intrinsic defects of this promising technology imports technical noise into the single-cell RNA-seq data, increasing the difficulty of accurate downstream inference. Normalization is a crucial step in single-cell RNA-seq data pre-processing. SCnorm is an accurate and efficient method that can be used for this purpose. An R implementation of this method is currently available. On one hand, the R package possesses many excellent features from R. On the other hand, R programming ability is required, which prevents the biologists who lack the skills from learning to use it quickly. To make this method more user-friendly, we developed a graphical user interface, visnormsc, for normalization of single-cell RNA-seq data. It is implemented in Python and is freely available at https://github.com/solo7773/visnormsc . Although visnormsc is based on the existing method, it contributes to this field by offering a user-friendly alternative. The out-of-the-box and cross-platform features make visnormsc easy to learn and to use. It is expected to serve biologists by simplifying single-cell RNA-seq normalization.

  4. Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants

    Energy Technology Data Exchange (ETDEWEB)

    Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta; Haas, Markus; Marwan, Wolfgang, E-mail: wolfgang.marwan@ovgu.de

    2013-05-24

    Highlights: •We investigate reprogramming of gene expression in multinucleate single cells. •Cells of two differentiation control mutants are fused. •Fused cells proceed to alternative gene expression patterns. •The population of nuclei damps stochastic fluctuations in gene expression. •Dynamic processes of cellular reprogramming can be observed by repeated sampling of a cell. -- Abstract: Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states. The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level.

  5. Switch-like reprogramming of gene expression after fusion of multinucleate plasmodial cells of two Physarum polycephalum sporulation mutants

    International Nuclear Information System (INIS)

    Walter, Pauline; Hoffmann, Xenia-Katharina; Ebeling, Britta; Haas, Markus; Marwan, Wolfgang

    2013-01-01

    Highlights: •We investigate reprogramming of gene expression in multinucleate single cells. •Cells of two differentiation control mutants are fused. •Fused cells proceed to alternative gene expression patterns. •The population of nuclei damps stochastic fluctuations in gene expression. •Dynamic processes of cellular reprogramming can be observed by repeated sampling of a cell. -- Abstract: Nonlinear dynamic processes involving the differential regulation of transcription factors are considered to impact the reprogramming of stem cells, germ cells, and somatic cells. Here, we fused two multinucleate plasmodial cells of Physarum polycephalum mutants defective in different sporulation control genes while being in different physiological states. The resulting heterokaryons established one of two significantly different expression patterns of marker genes while the plasmodial halves that were fused to each other synchronized spontaneously. Spontaneous synchronization suggests that switch-like control mechanisms spread over and finally control the entire plasmodium as a result of cytoplasmic mixing. Regulatory molecules due to the large volume of the vigorously streaming cytoplasm will define concentrations in acting on the population of nuclei and in the global setting of switches. Mixing of a large cytoplasmic volume is expected to damp stochasticity when individual nuclei deliver certain RNAs at low copy number into the cytoplasm. We conclude that spontaneous synchronization, the damping of molecular noise in gene expression by the large cytoplasmic volume, and the option to take multiple macroscopic samples from the same plasmodium provide unique options for studying the dynamics of cellular reprogramming at the single cell level

  6. Manipulation of gene expression by infrared laser heat shock and its application to the study of tracheal development in Drosophila.

    Science.gov (United States)

    Miao, Guangxia; Hayashi, Shigeo

    2015-03-01

    Induction of gene expression in a specific cell and a defined time window is desirable to investigate gene function at the cellular level during morphogenesis. To achieve this, we attempted to introduce the infrared laser-evoked gene operator system (IR-LEGO, Kamei et al., 2009) in the Drosophila embryo. In this technique, infrared laser light illumination induces genes to be expressed under the control of heat shock promoters at the single cell level. We applied IR-LEGO to a transgenic fly stock, HS-eGFP, in which the enhanced green fluorescent protein (eGFP) gene is placed under the control of heat shock protein 70 promoter, and showed that eGFP expression can be induced in single cells within 1-2 hr after IR illumination. Furthermore, induction of HS-Branchless transgene encoding the Drosophila fibroblast growth factor (FGF) effectively altered the migration and branching patterns of the tracheal system. Our results indicated that IR-LEGO is a promising choice for the timely control of gene expression in a small group of cells in the Drosophila embryo. By using IR-LEGO, we further demonstrated that the tracheal terminal branching program is sensitive to localized expression of exogenous FGF. © 2014 Wiley Periodicals, Inc.

  7. Expression Study of Banana Pathogenic Resistance Genes

    Directory of Open Access Journals (Sweden)

    Fenny M. Dwivany

    2016-10-01

    Full Text Available Banana is one of the world's most important trade commodities. However, infection of banana pathogenic fungi (Fusarium oxysporum race 4 is one of the major causes of decreasing production in Indonesia. Genetic engineering has become an alternative way to control this problem by isolating genes that involved in plant defense mechanism against pathogens. Two of the important genes are API5 and ChiI1, each gene encodes apoptosis inhibitory protein and chitinase enzymes. The purpose of this study was to study the expression of API5 and ChiI1 genes as candidate pathogenic resistance genes. The amplified fragments were then cloned, sequenced, and confirmed with in silico studies. Based on sequence analysis, it is showed that partial API5 gene has putative transactivation domain and ChiI1 has 9 chitinase family GH19 protein motifs. Data obtained from this study will contribute in banana genetic improvement.

  8. Dlx homeobox gene family expression in osteoclasts.

    Science.gov (United States)

    Lézot, F; Thomas, B L; Blin-Wakkach, C; Castaneda, B; Bolanos, A; Hotton, D; Sharpe, P T; Heymann, D; Carles, G F; Grigoriadis, A E; Berdal, A

    2010-06-01

    Skeletal growth and homeostasis require the finely orchestrated secretion of mineralized tissue matrices by highly specialized cells, balanced with their degradation by osteoclasts. Time- and site-specific expression of Dlx and Msx homeobox genes in the cells secreting these matrices have been identified as important elements in the regulation of skeletal morphology. Such specific expression patterns have also been reported in osteoclasts for Msx genes. The aim of the present study was to establish the expression patterns of Dlx genes in osteoclasts and identify their function in regulating skeletal morphology. The expression patterns of all Dlx genes were examined during the whole osteoclastogenesis using different in vitro models. The results revealed that Dlx1 and Dlx2 are the only Dlx family members with a possible function in osteoclastogenesis as well as in mature osteoclasts. Dlx5 and Dlx6 were detected in the cultures but appear to be markers of monocytes and their derivatives. In vivo, Dlx2 expression in osteoclasts was examined using a Dlx2/LacZ transgenic mouse. Dlx2 is expressed in a subpopulation of osteoclasts in association with tooth, brain, nerve, and bone marrow volumetric growths. Altogether the present data suggest a role for Dlx2 in regulation of skeletal morphogenesis via functions within osteoclasts. (c) 2010 Wiley-Liss, Inc.

  9. Hepatocyte specific expression of human cloned genes

    Energy Technology Data Exchange (ETDEWEB)

    Cortese, R

    1986-01-01

    A large number of proteins are specifically synthesized in the hepatocyte. Only the adult liver expresses the complete repertoire of functions which are required at various stages during development. There is therefore a complex series of regulatory mechanisms responsible for the maintenance of the differentiated state and for the developmental and physiological variations in the pattern of gene expression. Human hepatoma cell lines HepG2 and Hep3B display a pattern of gene expression similar to adult and fetal liver, respectively; in contrast, cultured fibroblasts or HeLa cells do not express most of the liver specific genes. They have used these cell lines for transfection experiments with cloned human liver specific genes. DNA segments coding for alpha1-antitrypsin and retinol binding protein (two proteins synthesized both in fetal and adult liver) are expressed in the hepatoma cell lines HepG2 and Hep3B, but not in HeLa cells or fibroblasts. A DNA segment coding for haptoglobin (a protein synthesized only after birth) is only expressed in the hepatoma cell line HepG2 but not in Hep3B nor in non hepatic cell lines. The information for tissue specific expression is located in the 5' flanking region of all three genes. In vivo competition experiments show that these DNA segments bind to a common, apparently limiting, transacting factor. Conventional techniques (Bal deletions, site directed mutagenesis, etc.) have been used to precisely identify the DNA sequences responsible for these effects. The emerging picture is complex: they have identified multiple, separate transcriptional signals, essential for maximal promoter activation and tissue specific expression. Some of these signals show a negative effect on transcription in fibroblast cell lines.

  10. Gene expression profiles in skeletal muscle after gene electrotransfer

    DEFF Research Database (Denmark)

    Hojman, Pernille; Zibert, John R; Gissel, Hanne

    2007-01-01

    BACKGROUND: Gene transfer by electroporation (DNA electrotransfer) to muscle results in high level long term transgenic expression, showing great promise for treatment of e.g. protein deficiency syndromes. However little is known about the effects of DNA electrotransfer on muscle fibres. We have...... caused down-regulation of structural proteins e.g. sarcospan and catalytic enzymes. Injection of DNA induced down-regulation of intracellular transport proteins e.g. sentrin. The effects on muscle fibres were transient as the expression profiles 3 weeks after treatment were closely related......) followed by a long low voltage pulse (LV, 100 V/cm, 400 ms); a pulse combination optimised for efficient and safe gene transfer. Muscles were transfected with green fluorescent protein (GFP) and excised at 4 hours, 48 hours or 3 weeks after treatment. RESULTS: Differentially expressed genes were...

  11. Gene expression analysis of flax seed development

    Science.gov (United States)

    2011-01-01

    Background Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed. Results We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions LIBEST_026995 to LIBEST_027011) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development. Conclusions We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise

  12. Gene expression analysis of flax seed development

    Directory of Open Access Journals (Sweden)

    Sharpe Andrew

    2011-04-01

    Full Text Available Abstract Background Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed. Results We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages seed coats (globular and torpedo stages and endosperm (pooled globular to torpedo stages and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST (GenBank accessions LIBEST_026995 to LIBEST_027011 were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152 had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development. Conclusions We have developed a foundational database of expressed sequences and collection of plasmid

  13. Lithium ions induce prestalk-associated gene expression and inhibit prespore gene expression in Dictyostelium discoideum

    NARCIS (Netherlands)

    Peters, Dorien J.M.; Lookeren Campagne, Michiel M. van; Haastert, Peter J.M. van; Spek, Wouter; Schaap, Pauline

    1989-01-01

    We investigated the effect of Li+ on two types of cyclic AMP-regulated gene expression and on basal and cyclic AMP-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Li+ effectively inhibits cyclic AMP-induced prespore gene expression, half-maximal inhibition occurring at about 2mM-LiCl.

  14. Scaling of gene expression data allowing the comparison of different gene expression platforms

    NARCIS (Netherlands)

    van Ruissen, Fred; Schaaf, Gerben J.; Kool, Marcel; Baas, Frank; Ruijter, Jan M.

    2008-01-01

    Serial analysis of gene expression (SAGE) and microarrays have found a widespread application, but much ambiguity exists regarding the amalgamation of the data resulting from these technologies. Cross-platform utilization of gene expression data from the SAGE and microarray technology could reduce

  15. Advancing haematopoietic stem and progenitor cell biology through single-cell profiling

    OpenAIRE

    Hamey, Fiona; Nestorowa, Sonia; Wilson, Nicola Kaye; Göttgens, Berthold

    2016-01-01

    Haematopoietic stem and progenitor cells (HSPCs) sit at the top of the haematopoietic hierarchy, and their fate choices need to be carefully controlled to ensure balanced production of all mature blood cell types. As cell fate decisions are made at the level of the individual cells, recent technological advances in measuring gene and protein expression in increasingly large numbers of single cells have been rapidly adopted to study both normal and pathological HSPC function. In this review we...

  16. Using a periclinal chimera to unravel layer-specific gene expression in plants.

    Science.gov (United States)

    Filippis, Ioannis; Lopez-Cobollo, Rosa; Abbott, James; Butcher, Sarah; Bishop, Gerard J

    2013-09-01

    Plant organs are made from multiple cell types, and defining the expression level of a gene in any one cell or group of cells from a complex mixture is difficult. Dicotyledonous plants normally have three distinct layers of cells, L1, L2 and L3. Layer L1 is the single layer of cells making up the epidermis, layer L2 the single cell sub-epidermal layer and layer L3 constitutes the rest of the internal cells. Here we show how it is possible to harvest an organ and characterise the level of layer-specific expression by using a periclinal chimera that has its L1 layer from Solanum pennellii and its L2 and L3 layers from Solanum lycopersicum. This is possible by measuring the level of the frequency of species-specific transcripts. RNA-seq analysis enabled the genome-wide assessment of whether a gene is expressed in the L1 or L2/L3 layers. From 13 277 genes that are expressed in both the chimera and the parental lines and with at least one polymorphism between the parental alleles, we identified 382 genes that are preferentially expressed in L1 in contrast to 1159 genes in L2/L3. Gene ontology analysis shows that many genes preferentially expressed in L1 are involved in cutin and wax biosynthesis, whereas numerous genes that are preferentially expressed in L2/L3 tissue are associated with chloroplastic processes. These data indicate the use of such chimeras and provide detailed information on the level of layer-specific expression of genes. © 2013 East Malling Research The Plant Journal © 2013 John Wiley & Sons Ltd.

  17. Renal Gene Expression Database (RGED): a relational database of gene expression profiles in kidney disease.

    Science.gov (United States)

    Zhang, Qingzhou; Yang, Bo; Chen, Xujiao; Xu, Jing; Mei, Changlin; Mao, Zhiguo

    2014-01-01

    We present a bioinformatics database named Renal Gene Expression Database (RGED), which contains comprehensive gene expression data sets from renal disease research. The web-based interface of RGED allows users to query the gene expression profiles in various kidney-related samples, including renal cell lines, human kidney tissues and murine model kidneys. Researchers can explore certain gene profiles, the relationships between genes of interests and identify biomarkers or even drug targets in kidney diseases. The aim of this work is to provide a user-friendly utility for the renal disease research community to query expression profiles of genes of their own interest without the requirement of advanced computational skills. Website is implemented in PHP, R, MySQL and Nginx and freely available from http://rged.wall-eva.net. http://rged.wall-eva.net. © The Author(s) 2014. Published by Oxford University Press.

  18. Renal Gene Expression Database (RGED): a relational database of gene expression profiles in kidney disease

    Science.gov (United States)

    Zhang, Qingzhou; Yang, Bo; Chen, Xujiao; Xu, Jing; Mei, Changlin; Mao, Zhiguo

    2014-01-01

    We present a bioinformatics database named Renal Gene Expression Database (RGED), which contains comprehensive gene expression data sets from renal disease research. The web-based interface of RGED allows users to query the gene expression profiles in various kidney-related samples, including renal cell lines, human kidney tissues and murine model kidneys. Researchers can explore certain gene profiles, the relationships between genes of interests and identify biomarkers or even drug targets in kidney diseases. The aim of this work is to provide a user-friendly utility for the renal disease research community to query expression profiles of genes of their own interest without the requirement of advanced computational skills. Availability and implementation: Website is implemented in PHP, R, MySQL and Nginx and freely available from http://rged.wall-eva.net. Database URL: http://rged.wall-eva.net PMID:25252782

  19. Co-ordinate single-cell expression of LEE4- and LEE5-encoded proteins of Escherichia coli O157:H7.

    Science.gov (United States)

    Roe, Andrew J; Naylor, Stuart W; Spears, Kevin J; Yull, Helen M; Dransfield, Tracy A; Oxford, Matthew; McKendrick, Iain J; Porter, Megan; Woodward, Martin J; Smith, David G E; Gally, David L

    2004-10-01

    Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.

  20. Single-cell RNA sequencing reveals metallothionein heterogeneity during hESC differentiation to definitive endoderm

    Directory of Open Access Journals (Sweden)

    Junjie Lu

    2018-04-01

    Full Text Available Differentiation of human pluripotent stem cells towards definitive endoderm (DE is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. This study improves our understanding of the cellular heterogeneity during in-vitro directed differentiation and provides a valuable resource to improve DE differentiation efficiency. Keywords: hPSC, Differentiation, Definitive endoderm, Heterogeneity, Single cell, RNA sequencing

  1. Gene expression in developing fibres of Upland cotton (Gossypium hirsutum L.) was massively altered by domestication.

    Science.gov (United States)

    Rapp, Ryan A; Haigler, Candace H; Flagel, Lex; Hovav, Ran H; Udall, Joshua A; Wendel, Jonathan F

    2010-11-15

    Understanding the evolutionary genetics of modern crop phenotypes has a dual relevance to evolutionary biology and crop improvement. Modern upland cotton (Gossypium hirsutum L.) was developed following thousands of years of artificial selection from a wild form, G. hirsutum var. yucatanense, which bears a shorter, sparser, layer of single-celled, ovular trichomes ('fibre'). In order to gain an insight into the nature of the developmental genetic transformations that accompanied domestication and crop improvement, we studied the transcriptomes of cotton fibres from wild and domesticated accessions over a developmental time course. Fibre cells were harvested between 2 and 25 days post-anthesis and encompassed the primary and secondary wall synthesis stages. Using amplified messenger RNA and a custom microarray platform designed to interrogate expression for 40,430 genes, we determined global patterns of expression during fibre development. The fibre transcriptome of domesticated cotton is far more dynamic than that of wild cotton, with over twice as many genes being differentially expressed during development (12,626 versus 5273). Remarkably, a total of 9465 genes were diagnosed as differentially expressed between wild and domesticated fibres when summed across five key developmental time points. Human selection during the initial domestication and subsequent crop improvement has resulted in a biased upregulation of components of the transcriptional network that are important for agronomically advanced fibre, especially in the early stages of development. About 15% of the differentially expressed genes in wild versus domesticated cotton fibre have no homology to the genes in databases. We show that artificial selection during crop domestication can radically alter the transcriptional developmental network of even a single-celled structure, affecting nearly a quarter of the genes in the genome. Gene expression during fibre development within accessions and expression

  2. Gene expression analysis identifies global gene dosage sensitivity in cancer

    DEFF Research Database (Denmark)

    Fehrmann, Rudolf S. N.; Karjalainen, Juha M.; Krajewska, Malgorzata

    2015-01-01

    Many cancer-associated somatic copy number alterations (SCNAs) are known. Currently, one of the challenges is to identify the molecular downstream effects of these variants. Although several SCNAs are known to change gene expression levels, it is not clear whether each individual SCNA affects gen...

  3. Metallothionein gene expression in renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Deeksha Pal

    2014-01-01

    Full Text Available Introduction: Metallothioneins (MTs are a group of low-molecular weight, cysteine-rich proteins. In general, MT is known to modulate three fundamental processes: (1 the release of gaseous mediators such as hydroxyl radical or nitric oxide, (2 apoptosis and (3 the binding and exchange of heavy metals such as zinc, cadmium or copper. Previous studies have shown a positive correlation between the expression of MT with invasion, metastasis and poor prognosis in various cancers. Most of the previous studies primarily used immunohistochemistry to analyze localization of MT in renal cell carcinoma (RCC. No information is available on the gene expression of MT2A isoform in different types and grades of RCC. Materials and Methods: In the present study, total RNA was isolated from 38 histopathologically confirmed cases of RCC of different types and grades. Corresponding adjacent normal renal parenchyma was taken as control. Real-time polymerase chain reaction (RT PCR analysis was done for the MT2A gene expression using b-actin as an internal control. All statistical calculations were performed using SPSS software. Results: The MT2A gene expression was found to be significantly increased (P < 0.01 in clear cell RCC in comparison with the adjacent normal renal parenchyma. The expression of MT2A was two to three-fold higher in sarcomatoid RCC, whereas there was no change in papillary and collecting duct RCC. MT2A gene expression was significantly higher in lower grade (grades I and II, P < 0.05, while no change was observed in high-grade tumor (grade III and IV in comparison to adjacent normal renal tissue. Conclusion: The first report of the expression of MT2A in different types and grades of RCC and also these data further support the role of MT2A in tumorigenesis.

  4. Model-based deconvolution of cell cycle time-series data reveals gene expression details at high resolution.

    Directory of Open Access Journals (Sweden)

    Dan Siegal-Gaskins

    2009-08-01

    Full Text Available In both prokaryotic and eukaryotic cells, gene expression is regulated across the cell cycle to ensure "just-in-time" assembly of select cellular structures and molecular machines. However, present in all time-series gene expression measurements is variability that arises from both systematic error in the cell synchrony process and variance in the timing of cell division at the level of the single cell. Thus, gene or protein expression data collected from a population of synchronized cells is an inaccurate measure of what occurs in the average single-cell across a cell cycle. Here, we present a general computational method to extract "single-cell"-like information from population-level time-series expression data. This method removes the effects of 1 variance in growth rate and 2 variance in the physiological and developmental state of the cell. Moreover, this method represents an advance in the deconvolution of molecular expression data in its flexibility, minimal assumptions, and the use of a cross-validation analysis to determine the appropriate level of regularization. Applying our deconvolution algorithm to cell cycle gene expression data from the dimorphic bacterium Caulobacter crescentus, we recovered critical features of cell cycle regulation in essential genes, including ctrA and ftsZ, that were obscured in population-based measurements. In doing so, we highlight the problem with using population data alone to decipher cellular regulatory mechanisms and demonstrate how our deconvolution algorithm can be applied to produce a more realistic picture of temporal regulation in a cell.

  5. Analysis of baseline gene expression levels from ...

    Science.gov (United States)

    The use of gene expression profiling to predict chemical mode of action would be enhanced by better characterization of variance due to individual, environmental, and technical factors. Meta-analysis of microarray data from untreated or vehicle-treated animals within the control arm of toxicogenomics studies has yielded useful information on baseline fluctuations in gene expression. A dataset of control animal microarray expression data was assembled by a working group of the Health and Environmental Sciences Institute's Technical Committee on the Application of Genomics in Mechanism Based Risk Assessment in order to provide a public resource for assessments of variability in baseline gene expression. Data from over 500 Affymetrix microarrays from control rat liver and kidney were collected from 16 different institutions. Thirty-five biological and technical factors were obtained for each animal, describing a wide range of study characteristics, and a subset were evaluated in detail for their contribution to total variability using multivariate statistical and graphical techniques. The study factors that emerged as key sources of variability included gender, organ section, strain, and fasting state. These and other study factors were identified as key descriptors that should be included in the minimal information about a toxicogenomics study needed for interpretation of results by an independent source. Genes that are the most and least variable, gender-selectiv

  6. Gene expression of the endolymphatic sac

    DEFF Research Database (Denmark)

    Friis, Morten; Martin-Bertelsen, Tomas; Friis-Hansen, Lennart

    2011-01-01

    that the endolymphatic sac has multiple and diverse functions in the inner ear. Objectives:The objective of this study was to provide a comprehensive review of the genes expressed in the endolymphatic sac in the rat and perform a functional characterization based on measured mRNA abundance. Methods:Microarray technology...

  7. Gene expression in early stage cervical cancer

    NARCIS (Netherlands)

    Biewenga, Petra; Buist, Marrije R.; Moerland, Perry D.; van Thernaat, Emiel Ver Loren; van Kampen, Antoine H. C.; ten Kate, Fiebo J. W.; Baas, Frank

    2008-01-01

    Objective. Pelvic lymph node metastases are the main prognostic factor for survival in early stage cervical cancer, yet accurate detection methods before surgery are lacking. In this study, we examined whether gene expression profiling can predict the presence of lymph node metastasis in early stage

  8. Shrinkage Approach for Gene Expression Data Analysis

    Czech Academy of Sciences Publication Activity Database

    Haman, Jiří; Valenta, Zdeněk; Kalina, Jan

    2013-01-01

    Roč. 1, č. 1 (2013), s. 65-65 ISSN 1805-8698. [EFMI 2013 Special Topic Conference. 17.04.2013-19.04.2013, Prague] Institutional support: RVO:67985807 Keywords : shrinkage estimation * covariance matrix * high dimensional data * gene expression Subject RIV: IN - Informatics, Computer Science

  9. Regulation of methane genes and genome expression

    Energy Technology Data Exchange (ETDEWEB)

    John N. Reeve

    2009-09-09

    At the start of this project, it was known that methanogens were Archaeabacteria (now Archaea) and were therefore predicted to have gene expression and regulatory systems different from Bacteria, but few of the molecular biology details were established. The goals were then to establish the structures and organizations of genes in methanogens, and to develop the genetic technologies needed to investigate and dissect methanogen gene expression and regulation in vivo. By cloning and sequencing, we established the gene and operon structures of all of the “methane” genes that encode the enzymes that catalyze methane biosynthesis from carbon dioxide and hydrogen. This work identified unique sequences in the methane gene that we designated mcrA, that encodes the largest subunit of methyl-coenzyme M reductase, that could be used to identify methanogen DNA and establish methanogen phylogenetic relationships. McrA sequences are now the accepted standard and used extensively as hybridization probes to identify and quantify methanogens in environmental research. With the methane genes in hand, we used northern blot and then later whole-genome microarray hybridization analyses to establish how growth phase and substrate availability regulated methane gene expression in Methanobacterium thermautotrophicus ΔH (now Methanothermobacter thermautotrophicus). Isoenzymes or pairs of functionally equivalent enzymes catalyze several steps in the hydrogen-dependent reduction of carbon dioxide to methane. We established that hydrogen availability determine which of these pairs of methane genes is expressed and therefore which of the alternative enzymes is employed to catalyze methane biosynthesis under different environmental conditions. As were unable to establish a reliable genetic system for M. thermautotrophicus, we developed in vitro transcription as an alternative system to investigate methanogen gene expression and regulation. This led to the discovery that an archaeal protein

  10. Fluid Mechanics, Arterial Disease, and Gene Expression.

    Science.gov (United States)

    Tarbell, John M; Shi, Zhong-Dong; Dunn, Jessilyn; Jo, Hanjoong

    2014-01-01

    This review places modern research developments in vascular mechanobiology in the context of hemodynamic phenomena in the cardiovascular system and the discrete localization of vascular disease. The modern origins of this field are traced, beginning in the 1960s when associations between flow characteristics, particularly blood flow-induced wall shear stress, and the localization of atherosclerotic plaques were uncovered, and continuing to fluid shear stress effects on the vascular lining endothelial) cells (ECs), including their effects on EC morphology, biochemical production, and gene expression. The earliest single-gene studies and genome-wide analyses are considered. The final section moves from the ECs lining the vessel wall to the smooth muscle cells and fibroblasts within the wall that are fluid me chanically activated by interstitial flow that imposes shear stresses on their surfaces comparable with those of flowing blood on EC surfaces. Interstitial flow stimulates biochemical production and gene expression, much like blood flow on ECs.

  11. Gene Expression Commons: an open platform for absolute gene expression profiling.

    Directory of Open Access Journals (Sweden)

    Jun Seita

    Full Text Available Gene expression profiling using microarrays has been limited to comparisons of gene expression between small numbers of samples within individual experiments. However, the unknown and variable sensitivities of each probeset have rendered the absolute expression of any given gene nearly impossible to estimate. We have overcome this limitation by using a very large number (>10,000 of varied microarray data as a common reference, so that statistical attributes of each probeset, such as the dynamic range and threshold between low and high expression, can be reliably discovered through meta-analysis. This strategy is implemented in a web-based platform named "Gene Expression Commons" (https://gexc.stanford.edu/ which contains data of 39 distinct highly purified mouse hematopoietic stem/progenitor/differentiated cell populations covering almost the entire hematopoietic system. Since the Gene Expression Commons is designed as an open platform, investigators can explore the expression level of any gene, search by expression patterns of interest, submit their own microarray data, and design their own working models representing biological relationship among samples.

  12. Comparative gene expression of intestinal metabolizing enzymes.

    Science.gov (United States)

    Shin, Ho-Chul; Kim, Hye-Ryoung; Cho, Hee-Jung; Yi, Hee; Cho, Soo-Min; Lee, Dong-Goo; Abd El-Aty, A M; Kim, Jin-Suk; Sun, Duxin; Amidon, Gordon L

    2009-11-01

    The purpose of this study was to compare the expression profiles of drug-metabolizing enzymes in the intestine of mouse, rat and human. Total RNA was isolated from the duodenum and the mRNA expression was measured using Affymetrix GeneChip oligonucleotide arrays. Detected genes from the intestine of mouse, rat and human were ca. 60% of 22690 sequences, 40% of 8739 and 47% of 12559, respectively. Total genes of metabolizing enzymes subjected in this study were 95, 33 and 68 genes in mouse, rat and human, respectively. Of phase I enzymes, the mouse exhibited abundant gene expressions for Cyp3a25, Cyp4v3, Cyp2d26, followed by Cyp2b20, Cyp2c65 and Cyp4f14, whereas, the rat showed higher expression profiles of Cyp3a9, Cyp2b19, Cyp4f1, Cyp17a1, Cyp2d18, Cyp27a1 and Cyp4f6. However, the highly expressed P450 enzymes were CYP3A4, CYP3A5, CYP4F3, CYP2C18, CYP2C9, CYP2D6, CYP3A7, CYP11B1 and CYP2B6 in the human. For phase II enzymes, glucuronosyltransferase Ugt1a6, glutathione S-transferases Gstp1, Gstm3 and Gsta2, sulfotransferase Sult1b1 and acyltransferase Dgat1 were highly expressed in the mouse. The rat revealed predominant expression of glucuronosyltransferases Ugt1a1 and Ugt1a7, sulfotransferase Sult1b1, acetyltransferase Dlat and acyltransferase Dgat1. On the other hand, in human, glucuronosyltransferases UGT2B15 and UGT2B17, glutathione S-transferases MGST3, GSTP1, GSTA2 and GSTM4, sulfotransferases ST1A3 and SULT1A2, acetyltransferases SAT1 and CRAT, and acyltransferase AGPAT2 were dominantly detected. Therefore, current data indicated substantial interspecies differences in the pattern of intestinal gene expression both for P450 enzymes and phase II drug-metabolizing enzymes. This genomic database is expected to improve our understanding of interspecies variations in estimating intestinal prehepatic clearance of oral drugs.

  13. Trans-acting GC-rich non-coding RNA at var expression site modulates gene counting in malaria parasite.

    Science.gov (United States)

    Guizetti, Julien; Barcons-Simon, Anna; Scherf, Artur

    2016-11-16

    Monoallelic expression of the var multigene family enables immune evasion of the malaria parasite Plasmodium falciparum in its human host. At a given time only a single member of the 60-member var gene family is expressed at a discrete perinuclear region called the 'var expression site'. However, the mechanism of var gene counting remains ill-defined. We hypothesize that activation factors associating specifically with the expression site play a key role in this process. Here, we investigate the role of a GC-rich non-coding RNA (ncRNA) gene family composed of 15 highly homologous members. GC-rich genes are positioned adjacent to var genes in chromosome-central gene clusters but are absent near subtelomeric var genes. Fluorescence in situ hybridization demonstrates that GC-rich ncRNA localizes to the perinuclear expression site of central and subtelomeric var genes in trans. Importantly, overexpression of distinct GC-rich ncRNA members disrupts the gene counting process at the single cell level and results in activation of a specific subset of var genes in distinct clones. We identify the first trans-acting factor targeted to the elusive perinuclear var expression site and open up new avenues to investigate ncRNA function in antigenic variation of malaria and other protozoan pathogens. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Structure and expression of thyroglobulin gene

    Energy Technology Data Exchange (ETDEWEB)

    Vassart, G; Brocas, H; Christophe, D; de Martynoff, G; Leriche, A; Mercken, L; Pohl, V; van Heuverswyn, B [Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucleaire (IRIBHN), Faculte de Medecine, Universite libre de Bruxelles, Campus Hopital Erasme, Brussels (Belgium)

    1982-01-01

    Thyroglobulin is composed of two 300000 dalton polypeptide chains, translated from an 8000 base mRNA. Preparation of a full length cDNA and its cloning in E. coli have lead to the demonstration that the polypeptides of thyroglobulin protomers were identical. Used as molecular probes, the cloned cDNA allowed the isolation of a fragment of thyroglobulin gene. Electron microscopic studies have demonstrated that this gene contains more than 90 % intronic material separating small size exons (<200 bp). Sequencing of bovine thyroglobulin structural gene is in progress. Preliminary results show evidence for the existence of repetitive segments. Availability of cloned DNA complementary to bovine and human thyroglobulin mRNA allows the study of genetic defects of thyroglobulin gene expression in the human and in various animal models.

  15. Cerebrovascular gene expression in spontaneously hypertensive rats

    DEFF Research Database (Denmark)

    Grell, Anne-Sofie; Frederiksen, Simona Denise; Edvinsson, Lars

    2017-01-01

    Hypertension is a hemodynamic disorder and one of the most important and well-established risk factors for vascular diseases such as stroke. Blood vessels exposed to chronic shear stress develop structural changes and remodeling of the vascular wall through many complex mechanisms. However......, the molecular mechanisms involved are not fully understood. Hypertension-susceptible genes may provide a novel insight into potential molecular mechanisms of hypertension and secondary complications associated with hypertension. The aim of this exploratory study was to identify gene expression differences......, the identified genes in the middle cerebral arteries from spontaneously hypertensive rats could be possible mediators of the vascular changes and secondary complications associated with hypertension. This study supports the selection of key genes to investigate in the future research of hypertension-induced end...

  16. Digital gene expression analysis of gene expression differences within Brassica diploids and allopolyploids.

    Science.gov (United States)

    Jiang, Jinjin; Wang, Yue; Zhu, Bao; Fang, Tingting; Fang, Yujie; Wang, Youping

    2015-01-27

    Brassica includes many successfully cultivated crop species of polyploid origin, either by ancestral genome triplication or by hybridization between two diploid progenitors, displaying complex repetitive sequences and transposons. The U's triangle, which consists of three diploids and three amphidiploids, is optimal for the analysis of complicated genomes after polyploidization. Next-generation sequencing enables the transcriptome profiling of polyploids on a global scale. We examined the gene expression patterns of three diploids (Brassica rapa, B. nigra, and B. oleracea) and three amphidiploids (B. napus, B. juncea, and B. carinata) via digital gene expression analysis. In total, the libraries generated between 5.7 and 6.1 million raw reads, and the clean tags of each library were mapped to 18547-21995 genes of B. rapa genome. The unambiguous tag-mapped genes in the libraries were compared. Moreover, the majority of differentially expressed genes (DEGs) were explored among diploids as well as between diploids and amphidiploids. Gene ontological analysis was performed to functionally categorize these DEGs into different classes. The Kyoto Encyclopedia of Genes and Genomes analysis was performed to assign these DEGs into approximately 120 pathways, among which the metabolic pathway, biosynthesis of secondary metabolites, and peroxisomal pathway were enriched. The non-additive genes in Brassica amphidiploids were analyzed, and the results indicated that orthologous genes in polyploids are frequently expressed in a non-additive pattern. Methyltransferase genes showed differential expression pattern in Brassica species. Our results provided an understanding of the transcriptome complexity of natural Brassica species. The gene expression changes in diploids and allopolyploids may help elucidate the morphological and physiological differences among Brassica species.

  17. Age-Related Gene Expression in the Frontal Cortex Suggests Synaptic Function Changes in Specific Inhibitory Neuron Subtypes

    Directory of Open Access Journals (Sweden)

    Leon French

    2017-05-01

    Full Text Available Genome-wide expression profiling of the human brain has revealed genes that are differentially expressed across the lifespan. Characterizing these genes adds to our understanding of both normal functions and pathological conditions. Additionally, the specific cell-types that contribute to the motor, sensory and cognitive declines during aging are unclear. Here we test if age-related genes show higher expression in specific neural cell types. Our study leverages data from two sources of murine single-cell expression data and two sources of age-associations from large gene expression studies of postmortem human brain. We used nonparametric gene set analysis to test for age-related enrichment of genes associated with specific cell-types; we also restricted our analyses to specific gene ontology groups. Our analyses focused on a primary pair of single-cell expression data from the mouse visual cortex and age-related human post-mortem gene expression information from the orbitofrontal cortex. Additional pairings that used data from the hippocampus, prefrontal cortex, somatosensory cortex and blood were used to validate and test specificity of our findings. We found robust age-related up-regulation of genes that are highly expressed in oligodendrocytes and astrocytes, while genes highly expressed in layer 2/3 glutamatergic neurons were down-regulated across age. Genes not specific to any neural cell type were also down-regulated, possibly due to the bulk tissue source of the age-related genes. A gene ontology-driven dissection of the cell-type enriched genes highlighted the strong down-regulation of genes involved in synaptic transmission and cell-cell signaling in the Somatostatin (Sst neuron subtype that expresses the cyclin dependent kinase 6 (Cdk6 and in the vasoactive intestinal peptide (Vip neuron subtype expressing myosin binding protein C, slow type (Mybpc1. These findings provide new insights into cell specific susceptibility to normal aging

  18. Single cell Hi-C reveals cell-to-cell variability in chromosome structure

    Science.gov (United States)

    Schoenfelder, Stefan; Yaffe, Eitan; Dean, Wendy; Laue, Ernest D.; Tanay, Amos; Fraser, Peter

    2013-01-01

    Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single cell Hi-C, combined with genome-wide statistical analysis and structural modeling of single copy X chromosomes, to show that individual chromosomes maintain domain organisation at the megabase scale, but show variable cell-to-cell chromosome territory structures at larger scales. Despite this structural stochasticity, localisation of active gene domains to boundaries of territories is a hallmark of chromosomal conformation. Single cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organisation underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns. PMID:24067610

  19. Single-Cell RNA Sequencing of Glioblastoma Cells.

    Science.gov (United States)

    Sen, Rajeev; Dolgalev, Igor; Bayin, N Sumru; Heguy, Adriana; Tsirigos, Aris; Placantonakis, Dimitris G

    2018-01-01

    Single-cell RNA sequencing (sc-RNASeq) is a recently developed technique used to evaluate the transcriptome of individual cells. As opposed to conventional RNASeq in which entire populations are sequenced in bulk, sc-RNASeq can be beneficial when trying to better understand gene expression patterns in markedly heterogeneous populations of cells or when trying to identify transcriptional signatures of rare cells that may be underrepresented when using conventional bulk RNASeq. In this method, we describe the generation and analysis of cDNA libraries from single patient-derived glioblastoma cells using the C1 Fluidigm system. The protocol details the use of the C1 integrated fluidics circuit (IFC) for capturing, imaging and lysing cells; performing reverse transcription; and generating cDNA libraries that are ready for sequencing and analysis.

  20. Global gene expression in Escherichia coli biofilms

    DEFF Research Database (Denmark)

    Schembri, Mark; Kjærgaard, K.; Klemm, Per

    2003-01-01

    It is now apparent that microorganisms undergo significant changes during the transition from planktonic to biofilm growth. These changes result in phenotypic adaptations that allow the formation of highly organized and structured sessile communities, which possess enhanced resistance to antimicr......It is now apparent that microorganisms undergo significant changes during the transition from planktonic to biofilm growth. These changes result in phenotypic adaptations that allow the formation of highly organized and structured sessile communities, which possess enhanced resistance...... the transition to biofilm growth, and these included genes expressed under oxygen-limiting conditions, genes encoding (putative) transport proteins, putative oxidoreductases and genes associated with enhanced heavy metal resistance. Of particular interest was the observation that many of the genes altered...... in expression have no current defined function. These genes, as well as those induced by stresses relevant to biofilm growth such as oxygen and nutrient limitation, may be important factors that trigger enhanced resistance mechanisms of sessile communities to antibiotics and hydrodynamic shear forces....

  1. Aberrant Gene Expression in Acute Myeloid Leukaemia

    DEFF Research Database (Denmark)

    Bagger, Frederik Otzen

    model to investigate the role of telomerase in AML, we were able to translate the observed effect into human AML patients and identify specific genes involved, which also predict survival patterns in AML patients. During these studies we have applied methods for investigating differentially expressed......-based gene-lookup webservices, called HemaExplorer and BloodSpot. These web-services support the aim of making data and analysis of haematopoietic cells from mouse and human accessible for researchers without bioinformatics expertise. Finally, in order to aid the analysis of the very limited number...

  2. Gene expression in Pseudomonas aeruginosa swarming motility

    Directory of Open Access Journals (Sweden)

    Déziel Eric

    2010-10-01

    Full Text Available Abstract Background The bacterium Pseudomonas aeruginosa is capable of three types of motilities: swimming, twitching and swarming. The latter is characterized by a fast and coordinated group movement over a semi-solid surface resulting from intercellular interactions and morphological differentiation. A striking feature of swarming motility is the complex fractal-like patterns displayed by migrating bacteria while they move away from their inoculation point. This type of group behaviour is still poorly understood and its characterization provides important information on bacterial structured communities such as biofilms. Using GeneChip® Affymetrix microarrays, we obtained the transcriptomic profiles of both bacterial populations located at the tip of migrating tendrils and swarm center of swarming colonies and compared these profiles to that of a bacterial control population grown on the same media but solidified to not allow swarming motility. Results Microarray raw data were corrected for background noise with the RMA algorithm and quantile normalized. Differentially expressed genes between the three conditions were selected using a threshold of 1.5 log2-fold, which gave a total of 378 selected genes (6.3% of the predicted open reading frames of strain PA14. Major shifts in gene expression patterns are observed in each growth conditions, highlighting the presence of distinct bacterial subpopulations within a swarming colony (tendril tips vs. swarm center. Unexpectedly, microarrays expression data reveal that a minority of genes are up-regulated in tendril tip populations. Among them, we found energy metabolism, ribosomal protein and transport of small molecules related genes. On the other hand, many well-known virulence factors genes were globally repressed in tendril tip cells. Swarm center cells are distinct and appear to be under oxidative and copper stress responses. Conclusions Results reported in this study show that, as opposed to

  3. Decomposition of gene expression state space trajectories.

    Directory of Open Access Journals (Sweden)

    Jessica C Mar

    2009-12-01

    Full Text Available Representing and analyzing complex networks remains a roadblock to creating dynamic network models of biological processes and pathways. The study of cell fate transitions can reveal much about the transcriptional regulatory programs that underlie these phenotypic changes and give rise to the coordinated patterns in expression changes that we observe. The application of gene expression state space trajectories to capture cell fate transitions at the genome-wide level is one approach currently used in the literature. In this paper, we analyze the gene expression dataset of Huang et al. (2005 which follows the differentiation of promyelocytes into neutrophil-like cells in the presence of inducers dimethyl sulfoxide and all-trans retinoic acid. Huang et al. (2005 build on the work of Kauffman (2004 who raised the attractor hypothesis, stating that cells exist in an expression landscape and their expression trajectories converge towards attractive sites in this landscape. We propose an alternative interpretation that explains this convergent behavior by recognizing that there are two types of processes participating in these cell fate transitions-core processes that include the specific differentiation pathways of promyelocytes to neutrophils, and transient processes that capture those pathways and responses specific to the inducer. Using functional enrichment analyses, specific biological examples and an analysis of the trajectories and their core and transient components we provide a validation of our hypothesis using the Huang et al. (2005 dataset.

  4. Global expression differences and tissue specific expression differences in rice evolution result in two contrasting types of differentially expressed genes

    KAUST Repository

    Horiuchi, Youko; Harushima, Yoshiaki; Fujisawa, Hironori; Mochizuki, Takako; Fujita, Masahiro; Ohyanagi, Hajime; Kurata, Nori

    2015-01-01

    Since the development of transcriptome analysis systems, many expression evolution studies characterized evolutionary forces acting on gene expression, without explicit discrimination between global expression differences and tissue

  5. Blood Gene Expression Predicts Bronchiolitis Obliterans Syndrome

    Directory of Open Access Journals (Sweden)

    Richard Danger

    2018-01-01

    Full Text Available Bronchiolitis obliterans syndrome (BOS, the main manifestation of chronic lung allograft dysfunction, leads to poor long-term survival after lung transplantation. Identifying predictors of BOS is essential to prevent the progression of dysfunction before irreversible damage occurs. By using a large set of 107 samples from lung recipients, we performed microarray gene expression profiling of whole blood to identify early biomarkers of BOS, including samples from 49 patients with stable function for at least 3 years, 32 samples collected at least 6 months before BOS diagnosis (prediction group, and 26 samples at or after BOS diagnosis (diagnosis group. An independent set from 25 lung recipients was used for validation by quantitative PCR (13 stables, 11 in the prediction group, and 8 in the diagnosis group. We identified 50 transcripts differentially expressed between stable and BOS recipients. Three genes, namely POU class 2 associating factor 1 (POU2AF1, T-cell leukemia/lymphoma protein 1A (TCL1A, and B cell lymphocyte kinase, were validated as predictive biomarkers of BOS more than 6 months before diagnosis, with areas under the curve of 0.83, 0.77, and 0.78 respectively. These genes allow stratification based on BOS risk (log-rank test p < 0.01 and are not associated with time posttransplantation. This is the first published large-scale gene expression analysis of blood after lung transplantation. The three-gene blood signature could provide clinicians with new tools to improve follow-up and adapt treatment of patients likely to develop BOS.

  6. Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

    International Nuclear Information System (INIS)

    Salem, Tamer Z.; Zhang, Fengrui; Thiem, Suzanne M.

    2013-01-01

    Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

  7. Reduced expression of Autographa californica nucleopolyhedrovirus ORF34, an essential gene, enhances heterologous gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Salem, Tamer Z. [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbial Molecular Biology, AGERI, Agricultural Research Center, Giza 12619 (Egypt); Division of Biomedical Sciences, Zewail University, Zewail City of Science and Technology, Giza 12588 (Egypt); Zhang, Fengrui [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Thiem, Suzanne M., E-mail: smthiem@msu.edu [Department of Entomology, Michigan State University, East Lansing, MI 48824 (United States); Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States)

    2013-01-20

    Autographa californica multiple nucleopolyhedrovirus ORF34 is part of a transcriptional unit that includes ORF32, encoding a viral fibroblast growth factor (FGF) and ORF33. We identified ORF34 as a candidate for deletion to improve protein expression in the baculovirus expression system based on enhanced reporter gene expression in an RNAi screen of virus genes. However, ORF34 was shown to be an essential gene. To explore ORF34 function, deletion (KO34) and rescue bacmids were constructed and characterized. Infection did not spread from primary KO34 transfected cells and supernatants from KO34 transfected cells could not infect fresh Sf21 cells whereas the supernatant from the rescue bacmids transfection could recover the infection. In addition, budded viruses were not observed in KO34 transfected cells by electron microscopy, nor were viral proteins detected from the transfection supernatants by western blots. These demonstrate that ORF34 is an essential gene with a possible role in infectious virus production.

  8. Three gene expression vector sets for concurrently expressing multiple genes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Ishii, Jun; Kondo, Takashi; Makino, Harumi; Ogura, Akira; Matsuda, Fumio; Kondo, Akihiko

    2014-05-01

    Yeast has the potential to be used in bulk-scale fermentative production of fuels and chemicals due to its tolerance for low pH and robustness for autolysis. However, expression of multiple external genes in one host yeast strain is considerably labor-intensive due to the lack of polycistronic transcription. To promote the metabolic engineering of yeast, we generated systematic and convenient genetic engineering tools to express multiple genes in Saccharomyces cerevisiae. We constructed a series of multi-copy and integration vector sets for concurrently expressing two or three genes in S. cerevisiae by embedding three classical promoters. The comparative expression capabilities of the constructed vectors were monitored with green fluorescent protein, and the concurrent expression of genes was monitored with three different fluorescent proteins. Our multiple gene expression tool will be helpful to the advanced construction of genetically engineered yeast strains in a variety of research fields other than metabolic engineering. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  9. Deconstructing stem cell population heterogeneity: Single-cell analysis and modeling approaches

    Science.gov (United States)

    Wu, Jincheng; Tzanakakis, Emmanuel S.

    2014-01-01

    Isogenic stem cell populations display cell-to-cell variations in a multitude of attributes including gene or protein expression, epigenetic state, morphology, proliferation and proclivity for differentiation. The origins of the observed heterogeneity and its roles in the maintenance of pluripotency and the lineage specification of stem cells remain unclear. Addressing pertinent questions will require the employment of single-cell analysis methods as traditional cell biochemical and biomolecular assays yield mostly population-average data. In addition to time-lapse microscopy and flow cytometry, recent advances in single-cell genomic, transcriptomic and proteomic profiling are reviewed. The application of multiple displacement amplification, next generation sequencing, mass cytometry and spectrometry to stem cell systems is expected to provide a wealth of information affording unprecedented levels of multiparametric characterization of cell ensembles under defined conditions promoting pluripotency or commitment. Establishing connections between single-cell analysis information and the observed phenotypes will also require suitable mathematical models. Stem cell self-renewal and differentiation are orchestrated by the coordinated regulation of subcellular, intercellular and niche-wide processes spanning multiple time scales. Here, we discuss different modeling approaches and challenges arising from their application to stem cell populations. Integrating single-cell analysis with computational methods will fill gaps in our knowledge about the functions of heterogeneity in stem cell physiology. This combination will also aid the rational design of efficient differentiation and reprogramming strategies as well as bioprocesses for the production of clinically valuable stem cell derivatives. PMID:24035899

  10. Single cells from human primary colorectal tumors exhibit polyfunctional heterogeneity in secretions of ELR+ CXC chemokines.

    Science.gov (United States)

    Adalsteinsson, Viktor A; Tahirova, Narmin; Tallapragada, Naren; Yao, Xiaosai; Campion, Liam; Angelini, Alessandro; Douce, Thomas B; Huang, Cindy; Bowman, Brittany; Williamson, Christina A; Kwon, Douglas S; Wittrup, K Dane; Love, J Christopher

    2013-10-01

    Cancer is an inflammatory disease of tissue that is largely influenced by the interactions between multiple cell types, secreted factors, and signal transduction pathways. While single-cell sequencing continues to refine our understanding of the clonotypic heterogeneity within tumors, the complex interplay between genetic variations and non-genetic factors ultimately affects therapeutic outcome. Much has been learned through bulk studies of secreted factors in the tumor microenvironment, but the secretory behavior of single cells has been largely uncharacterized. Here we directly profiled the secretions of ELR+ CXC chemokines from thousands of single colorectal tumor and stromal cells, using an array of subnanoliter wells and a technique called microengraving to characterize both the rates of secretion of several factors at once and the numbers of cells secreting each chemokine. The ELR+ CXC chemokines are highly redundant, pro-angiogenic cytokines that signal via the CXCR1 and CXCR2 receptors, influencing tumor growth and progression. We find that human primary colorectal tumor and stromal cells exhibit polyfunctional heterogeneity in the combinations and magnitudes of secretions for these chemokines. In cell lines, we observe similar variance: phenotypes observed in bulk can be largely absent among the majority of single cells, and discordances exist between secretory states measured and gene expression for these chemokines among single cells. Together, these measures suggest secretory states among tumor cells are complex and can evolve dynamically. Most importantly, this study reveals new insight into the intratumoral phenotypic heterogeneity of human primary tumors.

  11. Using measures of single-cell physiology and physiological state to understand organismic aging.

    Science.gov (United States)

    Mendenhall, Alexander; Driscoll, Monica; Brent, Roger

    2016-02-01

    Genetically identical organisms in homogeneous environments have different lifespans and healthspans. These differences are often attributed to stochastic events, such as mutations and 'epimutations', changes in DNA methylation and chromatin that change gene function and expression. But work in the last 10 years has revealed differences in lifespan- and health-related phenotypes that are not caused by lasting changes in DNA or identified by modifications to DNA or chromatin. This work has demonstrated persistent differences in single-cell and whole-organism physiological states operationally defined by values of reporter gene signals in living cells. While some single-cell states, for example, responses to oxygen deprivation, were defined previously, others, such as a generally heightened ability to make proteins, were, revealed by direct experiment only recently, and are not well understood. Here, we review technical progress that promises to greatly increase the number of these measurable single-cell physiological variables and measureable states. We discuss concepts that facilitate use of single-cell measurements to provide insight into physiological states and state transitions. We assert that researchers will use this information to relate cell level physiological readouts to whole-organism outcomes, to stratify aging populations into groups based on different physiologies, to define biomarkers predictive of outcomes, and to shed light on the molecular processes that bring about different individual physiologies. For these reasons, quantitative study of single-cell physiological variables and state transitions should provide a valuable complement to genetic and molecular explanations of how organisms age. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  12. Gene Expression Profiling of Xeroderma Pigmentosum

    Directory of Open Access Journals (Sweden)

    Bowden Nikola A

    2006-05-01

    Full Text Available Abstract Xeroderma pigmentosum (XP is a rare recessive disorder that is characterized by extreme sensitivity to UV light. UV light exposure results in the formation of DNA damage such as cyclobutane dimers and (6-4 photoproducts. Nucleotide excision repair (NER orchestrates the removal of cyclobutane dimers and (6-4 photoproducts as well as some forms of bulky chemical DNA adducts. The disease XP is comprised of 7 complementation groups (XP-A to XP-G, which represent functional deficiencies in seven different genes, all of which are believed to be involved in NER. The main clinical feature of XP is various forms of skin cancers; however, neurological degeneration is present in XPA, XPB, XPD and XPG complementation groups. The relationship between NER and other types of DNA repair processes is now becoming evident but the exact relationships between the different complementation groups remains to be precisely determined. Using gene expression analysis we have identified similarities and differences after UV light exposure between the complementation groups XP-A, XP-C, XP-D, XP-E, XP-F, XP-G and an unaffected control. The results reveal that there is a graded change in gene expression patterns between the mildest, most similar to the control response (XP-E and the severest form (XP-A of the disease, with the exception of XP-D. Distinct differences between the complementation groups with neurological symptoms (XP-A, XP-D and XP-G and without (XP-C, XP-E and XP-F were also identified. Therefore, this analysis has revealed distinct gene expression profiles for the XP complementation groups and the first step towards understanding the neurological symptoms of XP.

  13. Changes in gene expression following androgen receptor blockade ...

    Indian Academy of Sciences (India)

    Madhu urs

    of gene expression in the ventral prostate, it is not clear whether all the gene expression ... These include clusterin, methionine adenosyl transferase IIα, and prostate-specific ..... MAGEE1 melanoma antigen and no similarity was found with the ...

  14. Rubisco activity and gene expression of tropical tree species under ...

    African Journals Online (AJOL)

    Young

    2013-05-15

    May 15, 2013 ... Proteomics analysis associated with gene expression of plants reveal .... Consequently, Rubisco enzyme plays a role in assi- milating into ... technique for examining gene expression encoded at the. mRNA level .... Ammonia.

  15. Gene structure, phylogeny and expression profile of the sucrose ...

    Indian Academy of Sciences (India)

    Gene structure, phylogeny and expression profile of the sucrose synthase gene family in .... 24, 701–713. Bate N. and Twell D. 1998 Functional architecture of a late pollen .... Manzara T. and Gruissem W. 1988 Organization and expression.

  16. Cholinergic regulation of VIP gene expression in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Kristensen, Bo; Georg, Birgitte; Fahrenkrug, Jan

    1997-01-01

    Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing......Vasoactive intestinal polypeptide, muscarinic receptor, neuroblastoma cell, mRNA, gene expression, peptide processing...

  17. Single-cell analysis reveals early manifestation of cancerous phenotype in pre-malignant esophageal cells.

    Directory of Open Access Journals (Sweden)

    Jiangxin Wang

    Full Text Available Cellular heterogeneity plays a pivotal role in a variety of functional processes in vivo including carcinogenesis. However, our knowledge about cell-to-cell diversity and how differences in individual cells manifest in alterations at the population level remains very limited mainly due to the lack of appropriate tools enabling studies at the single-cell level. We present a study on changes in cellular heterogeneity in the context of pre-malignant progression in response to hypoxic stress. Utilizing pre-malignant progression of Barrett's esophagus (BE as a disease model system we studied molecular mechanisms underlying the progression from metaplastic to dysplastic (pre-cancerous stage. We used newly developed methods enabling measurements of cell-to-cell differences in copy numbers of mitochondrial DNA, expression levels of a set of mitochondrial and nuclear genes involved in hypoxia response pathways, and mitochondrial membrane potential. In contrast to bulk cell studies reported earlier, our study shows significant differences between metaplastic and dysplastic BE cells in both average values and single-cell parameter distributions of mtDNA copy numbers, mitochondrial function, and mRNA expression levels of studied genes. Based on single-cell data analysis, we propose that mitochondria may be one of the key factors in pre-malignant progression in BE.

  18. Single-Cell mRNA-Seq Using the Fluidigm C1 System and Integrated Fluidics Circuits.

    Science.gov (United States)

    Gong, Haibiao; Do, Devin; Ramakrishnan, Ramesh

    2018-01-01

    Single-cell mRNA-seq is a valuable tool to dissect expression profiles and to understand the regulatory network of genes. Microfluidics is well suited for single-cell analysis owing both to the small volume of the reaction chambers and easiness of automation. Here we describe the workflow of single-cell mRNA-seq using C1 IFC, which can isolate and process up to 96 cells. Both on-chip procedure (lysis, reverse transcription, and preamplification PCR) and off-chip sequencing library preparation protocols are described. The workflow generates full-length mRNA information, which is more valuable compared to 3' end counting method for many applications.

  19. Nuclear AXIN2 represses MYC gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Rennoll, Sherri A.; Konsavage, Wesley M.; Yochum, Gregory S., E-mail: gsy3@psu.edu

    2014-01-03

    Highlights: •AXIN2 localizes to cytoplasmic and nuclear compartments in colorectal cancer cells. •Nuclear AXIN2 represses the activity of Wnt-responsive luciferase reporters. •β-Catenin bridges AXIN2 to TCF transcription factors. •AXIN2 binds the MYC promoter and represses MYC gene expression. -- Abstract: The β-catenin transcriptional coactivator is the key mediator of the canonical Wnt signaling pathway. In the absence of Wnt, β-catenin associates with a cytosolic and multi-protein destruction complex where it is phosphorylated and targeted for proteasomal degradation. In the presence of Wnt, the destruction complex is inactivated and β-catenin translocates into the nucleus. In the nucleus, β-catenin binds T-cell factor (TCF) transcription factors to activate expression of c-MYC (MYC) and Axis inhibition protein 2 (AXIN2). AXIN2 is a member of the destruction complex and, thus, serves in a negative feedback loop to control Wnt/β-catenin signaling. AXIN2 is also present in the nucleus, but its function within this compartment is unknown. Here, we demonstrate that AXIN2 localizes to the nuclei of epithelial cells within normal and colonic tumor tissues as well as colorectal cancer cell lines. In the nucleus, AXIN2 represses expression of Wnt/β-catenin-responsive luciferase reporters and forms a complex with β-catenin and TCF. We demonstrate that AXIN2 co-occupies β-catenin/TCF complexes at the MYC promoter region. When constitutively localized to the nucleus, AXIN2 alters the chromatin structure at the MYC promoter and directly represses MYC gene expression. These findings suggest that nuclear AXIN2 functions as a rheostat to control MYC expression in response to Wnt/β-catenin signaling.

  20. Nuclear AXIN2 represses MYC gene expression

    International Nuclear Information System (INIS)

    Rennoll, Sherri A.; Konsavage, Wesley M.; Yochum, Gregory S.

    2014-01-01

    Highlights: •AXIN2 localizes to cytoplasmic and nuclear compartments in colorectal cancer cells. •Nuclear AXIN2 represses the activity of Wnt-responsive luciferase reporters. •β-Catenin bridges AXIN2 to TCF transcription factors. •AXIN2 binds the MYC promoter and represses MYC gene expression. -- Abstract: The β-catenin transcriptional coactivator is the key mediator of the canonical Wnt signaling pathway. In the absence of Wnt, β-catenin associates with a cytosolic and multi-protein destruction complex where it is phosphorylated and targeted for proteasomal degradation. In the presence of Wnt, the destruction complex is inactivated and β-catenin translocates into the nucleus. In the nucleus, β-catenin binds T-cell factor (TCF) transcription factors to activate expression of c-MYC (MYC) and Axis inhibition protein 2 (AXIN2). AXIN2 is a member of the destruction complex and, thus, serves in a negative feedback loop to control Wnt/β-catenin signaling. AXIN2 is also present in the nucleus, but its function within this compartment is unknown. Here, we demonstrate that AXIN2 localizes to the nuclei of epithelial cells within normal and colonic tumor tissues as well as colorectal cancer cell lines. In the nucleus, AXIN2 represses expression of Wnt/β-catenin-responsive luciferase reporters and forms a complex with β-catenin and TCF. We demonstrate that AXIN2 co-occupies β-catenin/TCF complexes at the MYC promoter region. When constitutively localized to the nucleus, AXIN2 alters the chromatin structure at the MYC promoter and directly represses MYC gene expression. These findings suggest that nuclear AXIN2 functions as a rheostat to control MYC expression in response to Wnt/β-catenin signaling

  1. Single-Cell RNA-Sequencing Reveals a Continuous Spectrum of Differentiation in Hematopoietic Cells

    Directory of Open Access Journals (Sweden)

    Iain C. Macaulay

    2016-02-01

    Full Text Available The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment.

  2. Molecular mechanisms of curcumin action: gene expression.

    Science.gov (United States)

    Shishodia, Shishir

    2013-01-01

    Curcumin derived from the tropical plant Curcuma longa has a long history of use as a dietary agent, food preservative, and in traditional Asian medicine. It has been used for centuries to treat biliary disorders, anorexia, cough, diabetic wounds, hepatic disorders, rheumatism, and sinusitis. The preventive and therapeutic properties of curcumin are associated with its antioxidant, anti-inflammatory, and anticancer properties. Extensive research over several decades has attempted to identify the molecular mechanisms of curcumin action. Curcumin modulates numerous molecular targets by altering their gene expression, signaling pathways, or through direct interaction. Curcumin regulates the expression of inflammatory cytokines (e.g., TNF, IL-1), growth factors (e.g., VEGF, EGF, FGF), growth factor receptors (e.g., EGFR, HER-2, AR), enzymes (e.g., COX-2, LOX, MMP9, MAPK, mTOR, Akt), adhesion molecules (e.g., ELAM-1, ICAM-1, VCAM-1), apoptosis related proteins (e.g., Bcl-2, caspases, DR, Fas), and cell cycle proteins (e.g., cyclin D1). Curcumin modulates the activity of several transcription factors (e.g., NF-κB, AP-1, STAT) and their signaling pathways. Based on its ability to affect multiple targets, curcumin has the potential for the prevention and treatment of various diseases including cancers, arthritis, allergies, atherosclerosis, aging, neurodegenerative disease, hepatic disorders, obesity, diabetes, psoriasis, and autoimmune diseases. This review summarizes the molecular mechanisms of modulation of gene expression by curcumin. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

  3. Studying the Complex Expression Dependences between Sets of Coexpressed Genes

    Directory of Open Access Journals (Sweden)

    Mario Huerta

    2014-01-01

    Full Text Available Organisms simplify the orchestration of gene expression by coregulating genes whose products function together in the cell. The use of clustering methods to obtain sets of coexpressed genes from expression arrays is very common; nevertheless there are no appropriate tools to study the expression networks among these sets of coexpressed genes. The aim of the developed tools is to allow studying the complex expression dependences that exist between sets of coexpressed genes. For this purpose, we start detecting the nonlinear expression relationships between pairs of genes, plus the coexpressed genes. Next, we form networks among sets of coexpressed genes that maintain nonlinear expression dependences between all of them. The expression relationship between the sets of coexpressed genes is defined by the expression relationship between the skeletons of these sets, where this skeleton represents the coexpressed genes with a well-defined nonlinear expression relationship with the skeleton of the other sets. As a result, we can study the nonlinear expression relationships between a target gene and other sets of coexpressed genes, or start the study from the skeleton of the sets, to study the complex relationships of activation and deactivation between the sets of coexpressed genes that carry out the different cellular processes present in the expression experiments.

  4. Experimental and Modeling Approaches for Understanding the Effect of Gene Expression Noise in Biological Development

    Directory of Open Access Journals (Sweden)

    David M. Holloway

    2018-04-01

    Full Text Available Biological development involves numerous chemical and physical processes which must act in concert to reliably produce a cell, a tissue, or a body. To be successful, the developing organism must be robust to variability at many levels, such as the environment (e.g., temperature, moisture, upstream information (such as long-range positional information gradients, or intrinsic noise due to the stochastic nature of low concentration chemical kinetics. The latter is especially relevant to the regulation of gene expression in cell differentiation. The temporal stochasticity of gene expression has been studied in single celled organisms for nearly two decades, but only recently have techniques become available to gather temporally-resolved data across spatially-distributed gene expression patterns in developing multicellular organisms. These demonstrate temporal noisy “bursting” in the number of gene transcripts per cell, raising the question of how the transcript number defining a particular cell type is produced, such that one cell type can reliably be distinguished from a neighboring cell of different type along a tissue boundary. Stochastic spatio-temporal modeling of tissue-wide expression patterns can identify signatures for specific types of gene regulation, which can be used to extract regulatory mechanism information from experimental time series. This Perspective focuses on using this type of approach to study gene expression noise during the anterior-posterior segmentation of the fruit fly embryo. Advances in experimental and theoretical techniques will lead to an increasing quantification of expression noise that can be used to understand how regulatory mechanisms contribute to embryonic robustness across a range of developmental processes.

  5. The relationship among gene expression, the evolution of gene dosage, and the rate of protein evolution.

    Directory of Open Access Journals (Sweden)

    Jean-François Gout

    2010-05-01

    Full Text Available The understanding of selective constraints affecting genes is a major issue in biology. It is well established that gene expression level is a major determinant of the rate of protein evolution, but the reasons for this relationship remain highly debated. Here we demonstrate that gene expression is also a major determinant of the evolution of gene dosage: the rate of gene losses after whole genome duplications in the Paramecium lineage is negatively correlated to the level of gene expression, and this relationship is not a byproduct of other factors known to affect the fate of gene duplicates. This indicates that changes in gene dosage are generally more deleterious for highly expressed genes. This rule also holds for other taxa: in yeast, we find a clear relationship between gene expression level and the fitness impact of reduction in gene dosage. To explain these observations, we propose a model based on the fact that the optimal expression level of a gene corresponds to a trade-off between the benefit and cost of its expression. This COSTEX model predicts that selective pressure against mutations changing gene expression level or affecting the encoded protein should on average be stronger in highly expressed genes and hence that both the frequency of gene loss and the rate of protein evolution should correlate negatively with gene expression. Thus, the COSTEX model provides a simple and common explanation for the general relationship observed between the level of gene expression and the different facets of gene evolution.

  6. Retrotransposons as regulators of gene expression.

    Science.gov (United States)

    Elbarbary, Reyad A; Lucas, Bronwyn A; Maquat, Lynne E

    2016-02-12

    Transposable elements (TEs) are both a boon and a bane to eukaryotic organisms, depending on where they integrate into the genome and how their sequences function once integrated. We focus on two types of TEs: long interspersed elements (LINEs) and short interspersed elements (SINEs). LINEs and SINEs are retrotransposons; that is, they transpose via an RNA intermediate. We discuss how LINEs and SINEs have expanded in eukaryotic genomes and contribute to genome evolution. An emerging body of evidence indicates that LINEs and SINEs function to regulate gene expression by affecting chromatin structure, gene transcription, pre-mRNA processing, or aspects of mRNA metabolism. We also describe how adenosine-to-inosine editing influences SINE function and how ongoing retrotransposition is countered by the body's defense mechanisms. Copyright © 2016, American Association for the Advancement of Science.

  7. Single-cell Analysis of Lambda Immunity Regulation

    DEFF Research Database (Denmark)

    Bæk, Kristoffer Torbjørn; Svenningsen, Sine Lo; Eisen, Harvey

    2003-01-01

    We have examined expression of the ¿cI operon in single cells via a rexgfp substitution. Although average fluorescence agreed with expectations for expression of ¿-repressor, fluorescence fluctuated greatly from cell-to-cell. Fluctuations in repressor concentration are not predicted by previous m...

  8. Single-cell mRNA cytometry via sequence-specific nanoparticle clustering and trapping

    Science.gov (United States)

    Labib, Mahmoud; Mohamadi, Reza M.; Poudineh, Mahla; Ahmed, Sharif U.; Ivanov, Ivaylo; Huang, Ching-Lung; Moosavi, Maral; Sargent, Edward H.; Kelley, Shana O.

    2018-05-01

    Cell-to-cell variation in gene expression creates a need for techniques that can characterize expression at the level of individual cells. This is particularly true for rare circulating tumour cells, in which subtyping and drug resistance are of intense interest. Here we describe a method for cell analysis—single-cell mRNA cytometry—that enables the isolation of rare cells from whole blood as a function of target mRNA sequences. This approach uses two classes of magnetic particles that are labelled to selectively hybridize with different regions of the target mRNA. Hybridization leads to the formation of large magnetic clusters that remain localized within the cells of interest, thereby enabling the cells to be magnetically separated. Targeting specific intracellular mRNAs enablescirculating tumour cells to be distinguished from normal haematopoietic cells. No polymerase chain reaction amplification is required to determine RNA expression levels and genotype at the single-cell level, and minimal cell manipulation is required. To demonstrate this approach we use single-cell mRNA cytometry to detect clinically important sequences in prostate cancer specimens.

  9. Single-cell photoacoustic thermometry

    Science.gov (United States)

    Gao, Liang; Wang, Lidai; Li, Chiye; Liu, Yan; Ke, Haixin; Zhang, Chi

    2013-01-01

    Abstract. A novel photoacoustic thermometric method is presented for simultaneously imaging cells and sensing their temperature. With three-seconds-per-frame imaging speed, a temperature resolution of 0.2°C was achieved in a photo-thermal cell heating experiment. Compared to other approaches, the photoacoustic thermometric method has the advantage of not requiring custom-developed temperature-sensitive biosensors. This feature should facilitate the conversion of single-cell thermometry into a routine lab tool and make it accessible to a much broader biological research community. PMID:23377004

  10. Gene expression profiling of cutaneous wound healing

    Directory of Open Access Journals (Sweden)

    Wang Ena

    2007-02-01

    Full Text Available Abstract Background Although the sequence of events leading to wound repair has been described at the cellular and, to a limited extent, at the protein level this process has yet to be fully elucidated. Genome wide transcriptional analysis tools promise to further define the global picture of this complex progression of events. Study Design This study was part of a placebo-controlled double-blind clinical trial in which basal cell carcinomas were treated topically with an immunomodifier – toll-like receptor 7 agonist: imiquimod. The fourteen patients with basal cell carcinoma in the placebo arm of the trial received placebo treatment consisting solely of vehicle cream. A skin punch biopsy was obtained immediately before treatment and at the end of the placebo treatment (after 2, 4 or 8 days. 17.5K cDNA microarrays were utilized to profile the biopsy material. Results Four gene signatures whose expression changed relative to baseline (before wound induction by the pre-treatment biopsy were identified. The largest group was comprised predominantly of inflammatory genes whose expression was increased throughout the study. Two additional signatures were observed which included preferentially pro-inflammatory genes in the early post-treatment biopsies (2 days after pre-treatment biopsies and repair and angiogenesis genes in the later (4 to 8 days biopsies. The fourth and smallest set of genes was down-regulated throughout the study. Early in wound healing the expression of markers of both M1 and M2 macrophages were increased, but later M2 markers predominated. Conclusion The initial response to a cutaneous wound induces powerful transcriptional activation of pro-inflammatory stimuli which may alert the host defense. Subsequently and in the absence of infection, inflammation subsides and it is replaced by angiogenesis and remodeling. Understanding this transition which may be driven by a change from a mixed macrophage population to predominately M2

  11. Differential expression of cell adhesion genes

    DEFF Research Database (Denmark)

    Stein, Wilfred D; Litman, Thomas; Fojo, Tito

    2005-01-01

    that compare cells grown in suspension to similar cells grown attached to one another as aggregates have suggested that it is adhesion to the extracellular matrix of the basal membrane that confers resistance to apoptosis and, hence, resistance to cytotoxins. The genes whose expression correlates with poor...... in cell adhesion and the cytoskeleton. If the proteins involved in tethering cells to the extracellular matrix are important in conferring drug resistance, it may be possible to improve chemotherapy by designing drugs that target these proteins....

  12. Network Completion for Static Gene Expression Data

    Directory of Open Access Journals (Sweden)

    Natsu Nakajima

    2014-01-01

    Full Text Available We tackle the problem of completing and inferring genetic networks under stationary conditions from static data, where network completion is to make the minimum amount of modifications to an initial network so that the completed network is most consistent with the expression data in which addition of edges and deletion of edges are basic modification operations. For this problem, we present a new method for network completion using dynamic programming and least-squares fitting. This method can find an optimal solution in polynomial time if the maximum indegree of the network is bounded by a constant. We evaluate the effectiveness of our method through computational experiments using synthetic data. Furthermore, we demonstrate that our proposed method can distinguish the differences between two types of genetic networks under stationary conditions from lung cancer and normal gene expression data.

  13. Quartz-Seq2: a high-throughput single-cell RNA-sequencing method that effectively uses limited sequence reads.

    Science.gov (United States)

    Sasagawa, Yohei; Danno, Hiroki; Takada, Hitomi; Ebisawa, Masashi; Tanaka, Kaori; Hayashi, Tetsutaro; Kurisaki, Akira; Nikaido, Itoshi

    2018-03-09

    High-throughput single-cell RNA-seq methods assign limited unique molecular identifier (UMI) counts as gene expression values to single cells from shallow sequence reads and detect limited gene counts. We thus developed a high-throughput single-cell RNA-seq method, Quartz-Seq2, to overcome these issues. Our improvements in the reaction steps make it possible to effectively convert initial reads to UMI counts, at a rate of 30-50%, and detect more genes. To demonstrate the power of Quartz-Seq2, we analyzed approximately 10,000 transcriptomes from in vitro embryonic stem cells and an in vivo stromal vascular fraction with a limited number of reads.

  14. Inferring gene expression dynamics via functional regression analysis

    Directory of Open Access Journals (Sweden)

    Leng Xiaoyan

    2008-01-01

    Full Text Available Abstract Background Temporal gene expression profiles characterize the time-dynamics of expression of specific genes and are increasingly collected in current gene expression experiments. In the analysis of experiments where gene expression is obtained over the life cycle, it is of interest to relate temporal patterns of gene expression associated with different developmental stages to each other to study patterns of long-term developmental gene regulation. We use tools from functional data analysis to study dynamic changes by relating temporal gene expression profiles of different developmental stages to each other. Results We demonstrate that functional regression methodology can pinpoint relationships that exist between temporary gene expression profiles for different life cycle phases and incorporates dimension reduction as needed for these high-dimensional data. By applying these tools, gene expression profiles for pupa and adult phases are found to be strongly related to the profiles of the same genes obtained during the embryo phase. Moreover, one can distinguish between gene groups that exhibit relationships with positive and others with negative associations between later life and embryonal expression profiles. Specifically, we find a positive relationship in expression for muscle development related genes, and a negative relationship for strictly maternal genes for Drosophila, using temporal gene expression profiles. Conclusion Our findings point to specific reactivation patterns of gene expression during the Drosophila life cycle which differ in characteristic ways between various gene groups. Functional regression emerges as a useful tool for relating gene expression patterns from different developmental stages, and avoids the problems with large numbers of parameters and multiple testing that affect alternative approaches.

  15. Single-Cell RNA-Seq Reveals the Transcriptional Landscape and Heterogeneity of Aortic Macrophages in Murine Atherosclerosis.

    Science.gov (United States)

    Cochain, Clément; Vafadarnejad, Ehsan; Arampatzi, Panagiota; Jaroslav, Pelisek; Winkels, Holger; Ley, Klaus; Wolf, Dennis; Saliba, Antoine-Emmanuel; Zernecke, Alma

    2018-03-15

    Rationale: It is assumed that atherosclerotic arteries contain several macrophage subsets endowed with specific functions. The precise identity of these subsets is poorly characterized as they ha ve been defined by the expression of a restricted number of markers. Objective: We have applied single-cell RNA-seq as an unbiased profiling strategy to interrogate and classify aortic macrophage heterogeneity at the single-cell level in atherosclerosis. Methods and Results: We performed single-cell RNA sequencing of total aortic CD45 + cells extracted from the non-diseased (chow fed) and atherosclerotic (11 weeks of high fat diet) aorta of Ldlr -/- mice. Unsupervised clustering singled out 13 distinct aortic cell clusters. Among the myeloid cell populations, Resident-like macrophages with a gene expression profile similar to aortic resident macrophages were found in healthy and diseased aortae, whereas monocytes, monocyte-derived dendritic cells (MoDC), and two populations of macrophages were almost exclusively detectable in atherosclerotic aortae, comprising Inflammatory macrophages showing enrichment in I l1b , and previously undescribed TREM2 hi macrophages. Differential gene expression and gene ontology enrichment analyses revealed specific gene expression patterns distinguishing these three macrophage subsets and MoDC, and uncovered putative functions of each cell type. Notably, TREM2 hi macrophages appeared to be endowed with specialized functions in lipid metabolism and catabolism, and presented a gene expression signature reminiscent of osteoclasts, suggesting a role in lesion calcification. TREM2 expression was moreover detected in human lesional macrophages. Importantly, these macrophage populations were present also in advanced atherosclerosis and in Apoe -/- aortae, indicating relevance of our findings in different stages of atherosclerosis and mouse models. Conclusions: These data unprecedentedly uncovered the transcriptional landscape and phenotypic

  16. Global expression differences and tissue specific expression differences in rice evolution result in two contrasting types of differentially expressed genes

    KAUST Repository

    Horiuchi, Youko

    2015-12-23

    Background Since the development of transcriptome analysis systems, many expression evolution studies characterized evolutionary forces acting on gene expression, without explicit discrimination between global expression differences and tissue specific expression differences. However, different types of gene expression alteration should have different effects on an organism, the evolutionary forces that act on them might be different, and different types of genes might show different types of differential expression between species. To confirm this, we studied differentially expressed (DE) genes among closely related groups that have extensive gene expression atlases, and clarified characteristics of different types of DE genes including the identification of regulating loci for differential expression using expression quantitative loci (eQTL) analysis data. Results We detected differentially expressed (DE) genes between rice subspecies in five homologous tissues that were verified using japonica and indica transcriptome atlases in public databases. Using the transcriptome atlases, we classified DE genes into two types, global DE genes and changed-tissues DE genes. Global type DE genes were not expressed in any tissues in the atlas of one subspecies, however changed-tissues type DE genes were expressed in both subspecies with different tissue specificity. For the five tissues in the two japonica-indica combinations, 4.6 ± 0.8 and 5.9 ± 1.5 % of highly expressed genes were global and changed-tissues DE genes, respectively. Changed-tissues DE genes varied in number between tissues, increasing linearly with the abundance of tissue specifically expressed genes in the tissue. Molecular evolution of global DE genes was rapid, unlike that of changed-tissues DE genes. Based on gene ontology, global and changed-tissues DE genes were different, having no common GO terms. Expression differences of most global DE genes were regulated by cis-eQTLs. Expression

  17. Epigenetic control of virulence gene expression in Pseudomonas aeruginosa by a LysR-type transcription regulator.

    Directory of Open Access Journals (Sweden)

    Keith H Turner

    2009-12-01

    Full Text Available Phenotypic variation within an isogenic bacterial population is thought to ensure the survival of a subset of cells in adverse conditions. The opportunistic pathogen Pseudomonas aeruginosa variably expresses several phenotypes, including antibiotic resistance, biofilm formation, and the production of CupA fimbriae. Here we describe a previously unidentified bistable switch in P. aeruginosa. This switch controls the expression of a diverse set of genes, including aprA, which encodes the secreted virulence factor alkaline protease. We present evidence that bistable expression of PA2432, herein named bexR (bistable expression regulator, which encodes a LysR-type transcription regulator, controls this switch. In particular, using DNA microarrays, quantitative RT-PCR analysis, chromatin immunoprecipitation, and reporter gene fusions, we identify genes directly under the control of BexR and show that these genes are bistably expressed. Furthermore, we show that bexR is itself bistably expressed and positively autoregulated. Finally, using single-cell analyses of a GFP reporter fusion, we present evidence that positive autoregulation of bexR is necessary for bistable expression of the BexR regulon. Our findings suggest that a positive feedback loop involving a LysR-type transcription regulator serves as the basis for an epigenetic switch that controls virulence gene expression in P. aeruginosa.

  18. Measuring single-cell density.

    Science.gov (United States)

    Grover, William H; Bryan, Andrea K; Diez-Silva, Monica; Suresh, Subra; Higgins, John M; Manalis, Scott R

    2011-07-05

    We have used a microfluidic mass sensor to measure the density of single living cells. By weighing each cell in two fluids of different densities, our technique measures the single-cell mass, volume, and density of approximately 500 cells per hour with a density precision of 0.001 g mL(-1). We observe that the intrinsic cell-to-cell variation in density is nearly 100-fold smaller than the mass or volume variation. As a result, we can measure changes in cell density indicative of cellular processes that would be otherwise undetectable by mass or volume measurements. Here, we demonstrate this with four examples: identifying Plasmodium falciparum malaria-infected erythrocytes in a culture, distinguishing transfused blood cells from a patient's own blood, identifying irreversibly sickled cells in a sickle cell patient, and identifying leukemia cells in the early stages of responding to a drug treatment. These demonstrations suggest that the ability to measure single-cell density will provide valuable insights into cell state for a wide range of biological processes.

  19. Interactive visualization of gene regulatory networks with associated gene expression time series data

    NARCIS (Netherlands)

    Westenberg, M.A.; Hijum, van S.A.F.T.; Lulko, A.T.; Kuipers, O.P.; Roerdink, J.B.T.M.; Linsen, L.; Hagen, H.; Hamann, B.

    2008-01-01

    We present GENeVis, an application to visualize gene expression time series data in a gene regulatory network context. This is a network of regulator proteins that regulate the expression of their respective target genes. The networks are represented as graphs, in which the nodes represent genes,

  20. Single cell transcriptomics of neighboring hyphae of Aspergillus niger

    Science.gov (United States)

    2011-01-01

    Single cell profiling was performed to assess differences in RNA accumulation in neighboring hyphae of the fungus Aspergillus niger. A protocol was developed to isolate and amplify RNA from single hyphae or parts thereof. Microarray analysis resulted in a present call for 4 to 7% of the A. niger genes, of which 12% showed heterogeneous RNA levels. These genes belonged to a wide range of gene categories. PMID:21816052

  1. Positive selection on gene expression in the human brain

    DEFF Research Database (Denmark)

    Khaitovich, Philipp; Tang, Kun; Franz, Henriette

    2006-01-01

    Recent work has shown that the expression levels of genes transcribed in the brains of humans and chimpanzees have changed less than those of genes transcribed in other tissues [1] . However, when gene expression changes are mapped onto the evolutionary lineage in which they occurred, the brain...... shows more changes than other tissues in the human lineage compared to the chimpanzee lineage [1] , [2] and [3] . There are two possible explanations for this: either positive selection drove more gene expression changes to fixation in the human brain than in the chimpanzee brain, or genes expressed...... in the brain experienced less purifying selection in humans than in chimpanzees, i.e. gene expression in the human brain is functionally less constrained. The first scenario would be supported if genes that changed their expression in the brain in the human lineage showed more selective sweeps than other genes...

  2. Identification of Human HK Genes and Gene Expression Regulation Study in Cancer from Transcriptomics Data Analysis

    Science.gov (United States)

    Zhang, Zhang; Liu, Jingxing; Wu, Jiayan; Yu, Jun

    2013-01-01

    The regulation of gene expression is essential for eukaryotes, as it drives the processes of cellular differentiation and morphogenesis, leading to the creation of different cell types in multicellular organisms. RNA-Sequencing (RNA-Seq) provides researchers with a powerful toolbox for characterization and quantification of transcriptome. Many different human tissue/cell transcriptome datasets coming from RNA-Seq technology are available on public data resource. The fundamental issue here is how to develop an effective analysis method to estimate expression pattern similarities between different tumor tissues and their corresponding normal tissues. We define the gene expression pattern from three directions: 1) expression breadth, which reflects gene expression on/off status, and mainly concerns ubiquitously expressed genes; 2) low/high or constant/variable expression genes, based on gene expression level and variation; and 3) the regulation of gene expression at the gene structure level. The cluster analysis indicates that gene expression pattern is higher related to physiological condition rather than tissue spatial distance. Two sets of human housekeeping (HK) genes are defined according to cell/tissue types, respectively. To characterize the gene expression pattern in gene expression level and variation, we firstly apply improved K-means algorithm and a gene expression variance model. We find that cancer-associated HK genes (a HK gene is specific in cancer group, while not in normal group) are expressed higher and more variable in cancer condition than in normal condition. Cancer-associated HK genes prefer to AT-rich genes, and they are enriched in cell cycle regulation related functions and constitute some cancer signatures. The expression of large genes is also avoided in cancer group. These studies will help us understand which cell type-specific patterns of gene expression differ among different cell types, and particularly for cancer. PMID:23382867

  3. Predicting cellular growth from gene expression signatures.

    Directory of Open Access Journals (Sweden)

    Edoardo M Airoldi

    2009-01-01

    Full Text Available Maintaining balanced growth in a changing environment is a fundamental systems-level challenge for cellular physiology, particularly in microorganisms. While the complete set of regulatory and functional pathways supporting growth and cellular proliferation are not yet known, portions of them are well understood. In particular, cellular proliferation is governed by mechanisms that are highly conserved from unicellular to multicellular organisms, and the disruption of these processes in metazoans is a major factor in the development of cancer. In this paper, we develop statistical methodology to identify quantitative aspects of the regulatory mechanisms underlying cellular proliferation in Saccharomyces cerevisiae. We find that the expression levels of a small set of genes can be exploited to predict the instantaneous growth rate of any cellular culture with high accuracy. The predictions obtained in this fashion are robust to changing biological conditions, experimental methods, and technological platforms. The proposed model is also effective in predicting growth rates for the related yeast Saccharomyces bayanus and the highly diverged yeast Schizosaccharomyces pombe, suggesting that the underlying regulatory signature is conserved across a wide range of unicellular evolution. We investigate the biological significance of the gene expression signature that the predictions are based upon from multiple perspectives: by perturbing the regulatory network through the Ras/PKA pathway, observing strong upregulation of growth rate even in the absence of appropriate nutrients, and discovering putative transcription factor binding sites, observing enrichment in growth-correlated genes. More broadly, the proposed methodology enables biological insights about growth at an instantaneous time scale, inaccessible by direct experimental methods. Data and tools enabling others to apply our methods are available at http://function.princeton.edu/growthrate.

  4. Single-cell regulome data analysis by SCRAT.

    Science.gov (United States)

    Ji, Zhicheng; Zhou, Weiqiang; Ji, Hongkai

    2017-09-15

    Emerging single-cell technologies (e.g. single-cell ATAC-seq, DNase-seq or ChIP-seq) have made it possible to assay regulome of individual cells. Single-cell regulome data are highly sparse and discrete. Analyzing such data is challenging. User-friendly software tools are still lacking. We present SCRAT, a Single-Cell Regulome Analysis Toolbox with a graphical user interface, for studying cell heterogeneity using single-cell regulome data. SCRAT can be used to conveniently summarize regulatory activities according to different features (e.g. gene sets, transcription factor binding motif sites, etc.). Using these features, users can identify cell subpopulations in a heterogeneous biological sample, infer cell identities of each subpopulation, and discover distinguishing features such as gene sets and transcription factors that show different activities among subpopulations. SCRAT is freely available at https://zhiji.shinyapps.io/scrat as an online web service and at https://github.com/zji90/SCRAT as an R package. hji@jhu.edu. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

  5. Analysis of multiplex gene expression maps obtained by voxelation.

    Science.gov (United States)

    An, Li; Xie, Hongbo; Chin, Mark H; Obradovic, Zoran; Smith, Desmond J; Megalooikonomou, Vasileios

    2009-04-29

    Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological disease. Researchers have previously used voxelation in combination with microarrays for acquisition of genome-wide atlases of expression patterns in the mouse brain. On the other hand, some work has been performed on studying gene functions, without taking into account the location information of a gene's expression in a mouse brain. In this paper, we present an approach for identifying the relation between gene expression maps obtained by voxelation and gene functions. To analyze the dataset, we chose typical genes as queries and aimed at discovering similar gene groups. Gene similarity was determined by using the wavelet features extracted from the left and right hemispheres averaged gene expression maps, and by the Euclidean distance between each pair of feature vectors. We also performed a multiple clustering approach on the gene expression maps, combined with hierarchical clustering. Among each group of similar genes and clusters, the gene function similarity was measured by calculating the average gene function distances in the gene ontology structure. By applying our methodology to find similar genes to certain target genes we were able to improve our understanding of gene expression patterns and gene functions. By applying the clustering analysis method, we obtained significant clusters, which have both very similar gene expression maps and very similar gene functions respectively to their corresponding gene ontologies. The cellular component ontology resulted in prominent clusters expressed in cortex and corpus callosum. The molecular function ontology gave prominent clusters in cortex, corpus callosum and hypothalamus. The biological process ontology resulted in clusters in cortex, hypothalamus and choroid plexus. Clusters from all three ontologies combined were most prominently expressed in cortex and corpus callosum. The experimental

  6. Analysis of multiplex gene expression maps obtained by voxelation

    Directory of Open Access Journals (Sweden)

    Smith Desmond J

    2009-04-01

    Full Text Available Abstract Background Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological disease. Researchers have previously used voxelation in combination with microarrays for acquisition of genome-wide atlases of expression patterns in the mouse brain. On the other hand, some work has been performed on studying gene functions, without taking into account the location information of a gene's expression in a mouse brain. In this paper, we present an approach for identifying the relation between gene expression maps obtained by voxelation and gene functions. Results To analyze the dataset, we chose typical genes as queries and aimed at discovering similar gene groups. Gene similarity was determined by using the wavelet features extracted from the left and right hemispheres averaged gene expression maps, and by the Euclidean distance between each pair of feature vectors. We also performed a multiple clustering approach on the gene expression maps, combined with hierarchical clustering. Among each group of similar genes and clusters, the gene function similarity was measured by calculating the average gene function distances in the gene ontology structure. By applying our methodology to find similar genes to certain target genes we were able to improve our understanding of gene expression patterns and gene functions. By applying the clustering analysis method, we obtained significant clusters, which have both very similar gene expression maps and very similar gene functions respectively to their corresponding gene ontologies. The cellular component ontology resulted in prominent clusters expressed in cortex and corpus callosum. The molecular function ontology gave prominent clusters in cortex, corpus callosum and hypothalamus. The biological process ontology resulted in clusters in cortex, hypothalamus and choroid plexus. Clusters from all three ontologies combined were most prominently expressed in

  7. Classification across gene expression microarray studies

    Directory of Open Access Journals (Sweden)

    Kuner Ruprecht

    2009-12-01

    Full Text Available Abstract Background The increasing number of gene expression microarray studies represents an important resource in biomedical research. As a result, gene expression based diagnosis has entered clinical practice for patient stratification in breast cancer. However, the integration and combined analysis of microarray studies remains still a challenge. We assessed the potential benefit of data integration on the classification accuracy and systematically evaluated the generalization performance of selected methods on four breast cancer studies comprising almost 1000 independent samples. To this end, we introduced an evaluation framework which aims to establish good statistical practice and a graphical way to monitor differences. The classification goal was to correctly predict estrogen receptor status (negative/positive and histological grade (low/high of each tumor sample in an independent study which was not used for the training. For the classification we chose support vector machines (SVM, predictive analysis of microarrays (PAM, random forest (RF and k-top scoring pairs (kTSP. Guided by considerations relevant for classification across studies we developed a generalization of kTSP which we evaluated in addition. Our derived version (DV aims to improve the robustness of the intrinsic invariance of kTSP with respect to technologies and preprocessing. Results For each individual study the generalization error was benchmarked via complete cross-validation and was found to be similar for all classification methods. The misclassification rates were substantially higher in classification across studies, when each single study was used as an independent test set while all remaining studies were combined for the training of the classifier. However, with increasing number of independent microarray studies used in the training, the overall classification performance improved. DV performed better than the average and showed slightly less variance. In

  8. Intersection of FOXO- and RUNX1-mediated gene expression programs in single breast epithelial cells during morphogenesis and tumor progression.

    Science.gov (United States)

    Wang, Lixin; Brugge, Joan S; Janes, Kevin A

    2011-10-04

    Gene expression networks are complicated by the assortment of regulatory factors that bind DNA and modulate transcription combinatorially. Single-cell measurements can reveal biological mechanisms hidden by population averages, but their value has not been fully explored in the context of mRNA regulation. Here, we adapted a single-cell expression profiling technique to examine the gene expression program downstream of Forkhead box O (FOXO) transcription factors during 3D breast epithelial acinar morphogenesis. By analyzing patterns of mRNA fluctuations among individual matrix-attached epithelial cells, we found that a subset of FOXO target genes was jointly regulated by the transcription factor Runt-related transcription factor 1 (RUNX1). Knockdown of RUNX1 causes hyperproliferation and abnormal morphogenesis, both of which require normal FOXO function. Down-regulating RUNX1 and FOXOs simultaneously causes widespread oxidative stress, which arrests proliferation and restores normal acinar morphology. In hormone-negative breast cancers lacking human epidermal growth factor receptor 2 (HER2) amplification, we find that RUNX1 down-regulation is strongly associated with up-regulation of FOXO1, which may be required to support growth of RUNX1-negative tumors. The coordinate function of these two tumor suppressors may provide a failsafe mechanism that inhibits cancer progression.

  9. Codon usage and amino acid usage influence genes expression level.

    Science.gov (United States)

    Paul, Prosenjit; Malakar, Arup Kumar; Chakraborty, Supriyo

    2018-02-01

    Highly expressed genes in any species differ in the usage frequency of synonymous codons. The relative recurrence of an event of the favored codon pair (amino acid pairs) varies between gene and genomes due to varying gene expression and different base composition. Here we propose a new measure for predicting the gene expression level, i.e., codon plus amino bias index (CABI). Our approach is based on the relative bias of the favored codon pair inclination among the genes, illustrated by analyzing the CABI score of the Medicago truncatula genes. CABI showed strong correlation with all other widely used measures (CAI, RCBS, SCUO) for gene expression analysis. Surprisingly, CABI outperforms all other measures by showing better correlation with the wet-lab data. This emphasizes the importance of the neighboring codons of the favored codon in a synonymous group while estimating the expression level of a gene.

  10. Understanding gene expression in coronary artery disease through ...

    Indian Academy of Sciences (India)

    Understanding gene expression in coronary artery disease through global profiling, network analysis and independent validation of key candidate genes. Prathima ... Table 2. Differentially expressed genes in CAD compared to age and gender matched controls. .... Regulation of nuclear pre-mRNA domain containing 1A.

  11. Improved gene expression signature of testicular carcinoma in situ

    DEFF Research Database (Denmark)

    Almstrup, Kristian; Leffers, Henrik; Lothe, Ragnhild A

    2007-01-01

    on global gene expression in testicular CIS have been previously published. We have merged the two data sets on CIS samples (n = 6) and identified the shared gene expression signature in relation to expression in normal testis. Among the top-20 highest expressed genes, one-third was transcription factors...... development' were significantly altered and could collectively affect cellular pathways like the WNT signalling cascade, which thus may be disrupted in testicular CIS. The merged CIS data from two different microarray platforms, to our knowledge, provide the most precise CIS gene expression signature to date....

  12. Peak flood estimation using gene expression programming

    Science.gov (United States)

    Zorn, Conrad R.; Shamseldin, Asaad Y.

    2015-12-01

    As a case study for the Auckland Region of New Zealand, this paper investigates the potential use of gene-expression programming (GEP) in predicting specific return period events in comparison to the established and widely used Regional Flood Estimation (RFE) method. Initially calibrated to 14 gauged sites, the GEP derived model was further validated to 10 and 100 year flood events with a relative errors of 29% and 18%, respectively. This is compared to the RFE method providing 48% and 44% errors for the same flood events. While the effectiveness of GEP in predicting specific return period events is made apparent, it is argued that the derived equations should be used in conjunction with those existing methodologies rather than as a replacement.

  13. Dissecting Transcriptional Heterogeneity in Pluripotency: Single Cell Analysis of Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Guedes, Ana M V; Henrique, Domingos; Abranches, Elsa

    2016-01-01

    Mouse Embryonic Stem cells (mESCs) show heterogeneous and dynamic expression of important pluripotency regulatory factors. Single-cell analysis has revealed the existence of cell-to-cell variability in the expression of individual genes in mESCs. Understanding how these heterogeneities are regulated and what their functional consequences are is crucial to obtain a more comprehensive view of the pluripotent state.In this chapter we describe how to analyze transcriptional heterogeneity by monitoring gene expression of Nanog, Oct4, and Sox2, using single-molecule RNA FISH in single mESCs grown in different cell culture medium. We describe in detail all the steps involved in the protocol, from RNA detection to image acquisition and processing, as well as exploratory data analysis.

  14. Metagenomics, metatranscriptomics and single cell genomics reveal functional response of active Oceanospirillales to Gulf oil spill

    Energy Technology Data Exchange (ETDEWEB)

    Mason, Olivia U.; Hazen, Terry C.; Borglin, Sharon; Chain, Patrick S. G.; Dubinsky, Eric A.; Fortney, Julian L.; Han, James; Holman, Hoi-Ying N.; Hultman, Jenni; Lamendella, Regina; Mackelprang, Rachel; Malfatti, Stephanie; Tom, Lauren M.; Tringe, Susannah G.; Woyke, Tanja; Zhou, Jizhong; Rubin, Edward M.; Jansson, Janet K.

    2012-06-12

    The Deepwater Horizon oil spill in the Gulf of Mexico resulted in a deep-sea hydrocarbon plume that caused a shift in the indigenous microbial community composition with unknown ecological consequences. Early in the spill history, a bloom of uncultured, thus uncharacterized, members of the Oceanospirillales was previously detected, but their role in oil disposition was unknown. Here our aim was to determine the functional role of the Oceanospirillales and other active members of the indigenous microbial community using deep sequencing of community DNA and RNA, as well as single-cell genomics. Shotgun metagenomic and metatranscriptomic sequencing revealed that genes for motility, chemotaxis and aliphatic hydrocarbon degradation were significantly enriched and expressed in the hydrocarbon plume samples compared with uncontaminated seawater collected from plume depth. In contrast, although genes coding for degradation of more recalcitrant compounds, such as benzene, toluene, ethylbenzene, total xylenes and polycyclic aromatic hydrocarbons, were identified in the metagenomes, they were expressed at low levels, or not at all based on analysis of the metatranscriptomes. Isolation and sequencing of two Oceanospirillales single cells revealed that both cells possessed genes coding for n-alkane and cycloalkane degradation. Specifically, the near-complete pathway for cyclohexane oxidation in the Oceanospirillales single cells was elucidated and supported by both metagenome and metatranscriptome data. The draft genome also included genes for chemotaxis, motility and nutrient acquisition strategies that were also identified in the metagenomes and metatranscriptomes. These data point towards a rapid response of members of the Oceanospirillales to aliphatic hydrocarbons in the deep sea.

  15. Genetic Variants Contribute to Gene Expression Variability in Humans

    Science.gov (United States)

    Hulse, Amanda M.; Cai, James J.

    2013-01-01

    Expression quantitative trait loci (eQTL) studies have established convincing relationships between genetic variants and gene expression. Most of these studies focused on the mean of gene expression level, but not the variance of gene expression level (i.e., gene expression variability). In the present study, we systematically explore genome-wide association between genetic variants and gene expression variability in humans. We adapt the double generalized linear model (dglm) to simultaneously fit the means and the variances of gene expression among the three possible genotypes of a biallelic SNP. The genomic loci showing significant association between the variances of gene expression and the genotypes are termed expression variability QTL (evQTL). Using a data set of gene expression in lymphoblastoid cell lines (LCLs) derived from 210 HapMap individuals, we identify cis-acting evQTL involving 218 distinct genes, among which 8 genes, ADCY1, CTNNA2, DAAM2, FERMT2, IL6, PLOD2, SNX7, and TNFRSF11B, are cross-validated using an extra expression data set of the same LCLs. We also identify ∼300 trans-acting evQTL between >13,000 common SNPs and 500 randomly selected representative genes. We employ two distinct scenarios, emphasizing single-SNP and multiple-SNP effects on expression variability, to explain the formation of evQTL. We argue that detecting evQTL may represent a novel method for effectively screening for genetic interactions, especially when the multiple-SNP influence on expression variability is implied. The implication of our results for revealing genetic mechanisms of gene expression variability is discussed. PMID:23150607

  16. Expression regulation of design process gene in product design

    DEFF Research Database (Denmark)

    Li, Bo; Fang, Lusheng; Li, Bo

    2011-01-01

    To improve the design process efficiency, this paper proposes the principle and methodology that design process gene controls the characteristics of design process under the framework of design process reuse and optimization based on design process gene. First, the concept of design process gene...... is proposed and analyzed, as well as its three categories i.e., the operator gene, the structural gene and the regulator gene. Second, the trigger mechanism that design objectives and constraints trigger the operator gene is constructed. Third, the expression principle of structural gene is analyzed...... with the example of design management gene. Last, the regulation mode that the regulator gene regulates the expression of the structural gene is established and it is illustrated by taking the design process management gene as an example. © (2011) Trans Tech Publications....

  17. Dissecting specific and global transcriptional regulation of bacterial gene expression

    NARCIS (Netherlands)

    Gerosa, Luca; Kochanowski, Karl; Heinemann, Matthias; Sauer, Uwe

    Gene expression is regulated by specific transcriptional circuits but also by the global expression machinery as a function of growth. Simultaneous specific and global regulation thus constitutes an additional-but often neglected-layer of complexity in gene expression. Here, we develop an

  18. Gene expression of the mismatch repair gene MSH2 in primary colorectal cancer

    DEFF Research Database (Denmark)

    Jensen, Lars Henrik; Kuramochi, Hidekazu; Crüger, Dorthe Gylling

    2011-01-01

    promoter was only detected in 14 samples and only at a low level with no correlation to gene expression. MSH2 gene expression was not a prognostic factor for overall survival in univariate or multivariate analysis. The gene expression of MSH2 is a potential quantitative marker ready for further clinical...

  19. Bistable expression of virulence genes in salmonella leads to the formation of an antibiotic-tolerant subpopulation.

    Directory of Open Access Journals (Sweden)

    Markus Arnoldini

    2014-08-01

    Full Text Available Phenotypic heterogeneity can confer clonal groups of organisms with new functionality. A paradigmatic example is the bistable expression of virulence genes in Salmonella typhimurium, which leads to phenotypically virulent and phenotypically avirulent subpopulations. The two subpopulations have been shown to divide labor during S. typhimurium infections. Here, we show that heterogeneous virulence gene expression in this organism also promotes survival against exposure to antibiotics through a bet-hedging mechanism. Using microfluidic devices in combination with fluorescence time-lapse microscopy and quantitative image analysis, we analyzed the expression of virulence genes at the single cell level and related it to survival when exposed to antibiotics. We found that, across different types of antibiotics and under concentrations that are clinically relevant, the subpopulation of bacterial cells that express virulence genes shows increased survival after exposure to antibiotics. Intriguingly, there is an interplay between the two consequences of phenotypic heterogeneity. The bet-hedging effect that arises through heterogeneity in virulence gene expression can protect clonal populations against avirulent mutants that exploit and subvert the division of labor within these populations. We conclude that bet-hedging and the division of labor can arise through variation in a single trait and interact with each other. This reveals a new degree of functional complexity of phenotypic heterogeneity. In addition, our results suggest a general principle of how pathogens can evade antibiotics: Expression of virulence factors often entails metabolic costs and the resulting growth retardation could generally increase tolerance against antibiotics and thus compromise treatment.

  20. Using RNA-Seq data to select refence genes for normalizing gene expression in apple roots

    Science.gov (United States)

    Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for t...

  1. CDX2 gene expression in acute lymphoblastic leukemia

    International Nuclear Information System (INIS)

    Arnaoaut, H.H.; Mokhtar, D.A.; Samy, R.M.; Omar, Sh.A.; Khames, S.A.

    2014-01-01

    CDX genes are classically known as regulators of axial elongation during early embryogenesis. An unsuspected role for CDX genes has been revealed during hematopoietic development. The CDX gene family member CDX2 belongs to the most frequent aberrantly expressed proto-oncogenes in human acute leukemias and is highly leukemogenic in experimental models. We used reversed transcriptase polymerase chain reaction (RT-PCR) to determine the expression level of CDX2 gene in 30 pediatric patients with acute lymphoblastic leukemia (ALL) at diagnosis and 30 healthy volunteers. ALL patients were followed up to detect minimal residual disease (MRD) on days 15 and 42 of induction. We found that CDX2 gene was expressed in 50% of patients and not expressed in controls. Associations between gene expression and different clinical and laboratory data of patients revealed no impact on different findings. With follow up, we could not confirm that CDX2 expression had a prognostic significance.

  2. Developmentally regulated expression of reporter gene in adult ...

    Indian Academy of Sciences (India)

    pression of reporter gene in adult brain specific GAL4 enhancer traps of. Drosophila ... genes based on their expression pattern, thus enabling us to overcome the ... order association and storage centres of olfactory learning and memory, and ...

  3. Expression profiling identifies genes involved in emphysema severity

    Directory of Open Access Journals (Sweden)

    Bowman Rayleen V

    2009-09-01

    Full Text Available Abstract Chronic obstructive pulmonary disease (COPD is a major public health problem. The aim of this study was to identify genes involved in emphysema severity in COPD patients. Gene expression profiling was performed on total RNA extracted from non-tumor lung tissue from 30 smokers with emphysema. Class comparison analysis based on gas transfer measurement was performed to identify differentially expressed genes. Genes were then selected for technical validation by quantitative reverse transcriptase-PCR (qRT-PCR if also represented on microarray platforms used in previously published emphysema studies. Genes technically validated advanced to tests of biological replication by qRT-PCR using an independent test set of 62 lung samples. Class comparison identified 98 differentially expressed genes (p p Gene expression profiling of lung from emphysema patients identified seven candidate genes associated with emphysema severity including COL6A3, SERPINF1, ZNHIT6, NEDD4, CDKN2A, NRN1 and GSTM3.

  4. GSEH: A Novel Approach to Select Prostate Cancer-Associated Genes Using Gene Expression Heterogeneity.

    Science.gov (United States)

    Kim, Hyunjin; Choi, Sang-Min; Park, Sanghyun

    2018-01-01

    When a gene shows varying levels of expression among normal people but similar levels in disease patients or shows similar levels of expression among normal people but different levels in disease patients, we can assume that the gene is associated with the disease. By utilizing this gene expression heterogeneity, we can obtain additional information that abets discovery of disease-associated genes. In this study, we used collaborative filtering to calculate the degree of gene expression heterogeneity between classes and then scored the genes on the basis of the degree of gene expression heterogeneity to find "differentially predicted" genes. Through the proposed method, we discovered more prostate cancer-associated genes than 10 comparable methods. The genes prioritized by the proposed method are potentially significant to biological processes of a disease and can provide insight into them.

  5. Automated discovery of functional generality of human gene expression programs.

    Directory of Open Access Journals (Sweden)

    Georg K Gerber

    2007-08-01

    Full Text Available An important research problem in computational biology is the identification of expression programs, sets of co-expressed genes orchestrating normal or pathological processes, and the characterization of the functional breadth of these programs. The use of human expression data compendia for discovery of such programs presents several challenges including cellular inhomogeneity within samples, genetic and environmental variation across samples, uncertainty in the numbers of programs and sample populations, and temporal behavior. We developed GeneProgram, a new unsupervised computational framework based on Hierarchical Dirichlet Processes that addresses each of the above challenges. GeneProgram uses expression data to simultaneously organize tissues into groups and genes into overlapping programs with consistent temporal behavior, to produce maps of expression programs, which are sorted by generality scores that exploit the automatically learned groupings. Using synthetic and real gene expression data, we showed that GeneProgram outperformed several popular expression analysis methods. We applied GeneProgram to a compendium of 62 short time-series gene expression datasets exploring the responses of human cells to infectious agents and immune-modulating molecules. GeneProgram produced a map of 104 expression programs, a substantial number of which were significantly enriched for genes involved in key signaling pathways and/or bound by NF-kappaB transcription factors in genome-wide experiments. Further, GeneProgram discovered expression programs that appear to implicate surprising signaling pathways or receptor types in the response to infection, including Wnt signaling and neurotransmitter receptors. We believe the discovered map of expression programs involved in the response to infection will be useful for guiding future biological experiments; genes from programs with low generality scores might serve as new drug targets that exhibit minimal

  6. Analysis of multiplex gene expression maps obtained by voxelation

    OpenAIRE

    An, L; Xie, H; Chin, MH; Obradovic, Z; Smith, DJ; Megalooikonomou, V

    2009-01-01

    Abstract Background Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological disease. Researchers have previously used voxelation in combination with microarrays for acquisition of genome-wide atlases of expression patterns in the mouse brain. On the other hand, some work has been performed on studying gene functions, without taking into account the location information of a gene's expression in a mouse brain. In this paper, we presen...

  7. Host Gene Expression Analysis in Sri Lankan Melioidosis Patients

    Science.gov (United States)

    2017-06-19

    CCL5 Chemokine (C-C motif) ligand 5 /RANTES. IFNγ Interferon gamma TNFα Tumor necrosis factor alpha HMGB1 High mobility group box 1 protein /high...aim of this study was to analyze gene expression levels of human host factors in melioidosis patients and establish useful correlation with disease...PBMC’s) of study subjects. Gene expression profiles of 25 gene targets including 19 immune response genes and 6 epigenetic factors were analyzed by

  8. Granatum: a graphical single-cell RNA-Seq analysis pipeline for genomics scientists.

    Science.gov (United States)

    Zhu, Xun; Wolfgruber, Thomas K; Tasato, Austin; Arisdakessian, Cédric; Garmire, David G; Garmire, Lana X

    2017-12-05

    Single-cell RNA sequencing (scRNA-Seq) is an increasingly popular platform to study heterogeneity at the single-cell level. Computational methods to process scRNA-Seq data are not very accessible to bench scientists as they require a significant amount of bioinformatic skills. We have developed Granatum, a web-based scRNA-Seq analysis pipeline to make analysis more broadly accessible to researchers. Without a single line of programming code, users can click through the pipeline, setting parameters and visualizing results via the interactive graphical interface. Granatum conveniently walks users through various steps of scRNA-Seq analysis. It has a comprehensive list of modules, including plate merging and batch-effect removal, outlier-sample removal, gene-expression normalization, imputation, gene filtering, cell clustering, differential gene expression analysis, pathway/ontology enrichment analysis, protein network interaction visualization, and pseudo-time cell series construction. Granatum enables broad adoption of scRNA-Seq technology by empowering bench scientists with an easy-to-use graphical interface for scRNA-Seq data analysis. The package is freely available for research use at http://garmiregroup.org/granatum/app.

  9. Single Cell Assay for Analyzing Single Cell Exosome and Endocrine Secretion and Cancer Markers

    Science.gov (United States)

    Chiu, Yu-Jui

    To understand the inhomogeneity of cells in biological systems, there is a growing demand for the capability to characterize the properties of individual single cells. Since single cell studies require continuous monitoring of the cell behaviors instead of a snapshot test at a single time point, an effective single-cell assay that can support time lapsed studies in a high throughput manner is desired. Most currently available single-cell technologies cannot provide proper environments to sustain cell growth and cannot provide, for appropriate cell types, proliferation of single cells and convenient, non-invasive tests of single cell behaviors from molecular markers. In this dissertation, I present a highly versatile single-cell assay that can accommodate different cellular types, enable easy and efficient single cell loading and culturing, and be suitable for the study of effects of in-vitro environmental factors in combination with drug screening. The salient features of the assay are the non-invasive collection and surveying of single cell secretions at different time points and massively parallel translocation of single cells by user defined criteria, producing very high compatibility to the downstream process such as single cell qPCR and sequencing. Above all, the acquired information is quantitative -- for example, one of the studies is measured by the number of exosomes each single cell secretes for a given time period. Therefore, our single-cell assay provides a convenient, low-cost, and enabling tool for quantitative, time lapsed studies of single cell properties.

  10. Single cell enzyme diagnosis on the chip

    DEFF Research Database (Denmark)

    Jensen, Sissel Juul; Harmsen, Charlotte; Nielsen, Mette Juul

    2013-01-01

    Conventional diagnosis based on ensemble measurements often overlooks the variation among cells. Here, we present a droplet-microfluidics based platform to investigate single cell activities. Adopting a previously developed isothermal rolling circle amplification-based assay, we demonstrate...... detection of enzymatic activities down to the single cell level with small quantities of biological samples, which outcompetes existing techniques. Such a system, capable of resolving single cell activities, will ultimately have clinical applications in diagnosis, prediction of drug response and treatment...

  11. Heterologous gene expression in filamentous fungi.

    Science.gov (United States)

    Su, Xiaoyun; Schmitz, George; Zhang, Meiling; Mackie, Roderick I; Cann, Isaac K O

    2012-01-01

    Filamentous fungi are critical to production of many commercial enzymes and organic compounds. Fungal-based systems have several advantages over bacterial-based systems for protein production because high-level secretion of enzymes is a common trait of their decomposer lifestyle. Furthermore, in the large-scale production of recombinant proteins of eukaryotic origin, the filamentous fungi become the vehicle of choice due to critical processes shared in gene expression with other eukaryotic organisms. The complexity and relative dearth of understanding of the physiology of filamentous fungi, compared to bacteria, have hindered rapid development of these organisms as highly efficient factories for the production of heterologous proteins. In this review, we highlight several of the known benefits and challenges in using filamentous fungi (particularly Aspergillus spp., Trichoderma reesei, and Neurospora crassa) for the production of proteins, especially heterologous, nonfungal enzymes. We review various techniques commonly employed in recombinant protein production in the filamentous fungi, including transformation methods, selection of gene regulatory elements such as promoters, protein secretion factors such as the signal peptide, and optimization of coding sequence. We provide insights into current models of host genomic defenses such as repeat-induced point mutation and quelling. Furthermore, we examine the regulatory effects of transcript sequences, including introns and untranslated regions, pre-mRNA (messenger RNA) processing, transcript transport, and mRNA stability. We anticipate that this review will become a resource for researchers who aim at advancing the use of these fascinating organisms as protein production factories, for both academic and industrial purposes, and also for scientists with general interest in the biology of the filamentous fungi. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Rhythmic diel pattern of gene expression in juvenile maize leaf.

    Directory of Open Access Journals (Sweden)

    Maciej Jończyk

    Full Text Available BACKGROUND: Numerous biochemical and physiological parameters of living organisms follow a circadian rhythm. Although such rhythmic behavior is particularly pronounced in plants, which are strictly dependent on the daily photoperiod, data on the molecular aspects of the diurnal cycle in plants is scarce and mostly concerns the model species Arabidopsis thaliana. Here we studied the leaf transcriptome in seedlings of maize, an important C4 crop only distantly related to A. thaliana, throughout a cycle of 10 h darkness and 14 h light to look for rhythmic patterns of gene expression. RESULTS: Using DNA microarrays comprising ca. 43,000 maize-specific probes we found that ca. 12% of all genes showed clear-cut diel rhythms of expression. Cluster analysis identified 35 groups containing from four to ca. 1,000 genes, each comprising genes of similar expression patterns. Perhaps unexpectedly, the most pronounced and most common (concerning the highest number of genes expression maxima were observed towards and during the dark phase. Using Gene Ontology classification several meaningful functional associations were found among genes showing similar diel expression patterns, including massive induction of expression of genes related to gene expression, translation, protein modification and folding at dusk and night. Additionally, we found a clear-cut tendency among genes belonging to individual clusters to share defined transcription factor-binding sequences. CONCLUSIONS: Co-expressed genes belonging to individual clusters are likely to be regulated by common mechanisms. The nocturnal phase of the diurnal cycle involves gross induction of fundamental biochemical processes and should be studied more thoroughly than was appreciated in most earlier physiological studies. Although some general mechanisms responsible for the diel regulation of gene expression might be shared among plants, details of the diurnal regulation of gene expression seem to differ

  13. FARO server: Meta-analysis of gene expression by matching gene expression signatures to a compendium of public gene expression data

    DEFF Research Database (Denmark)

    Manijak, Mieszko P.; Nielsen, Henrik Bjørn

    2011-01-01

    circumvented by instead matching gene expression signatures to signatures of other experiments. FINDINGS: To facilitate this we present the Functional Association Response by Overlap (FARO) server, that match input signatures to a compendium of 242 gene expression signatures, extracted from more than 1700...... Arabidopsis microarray experiments. CONCLUSIONS: Hereby we present a publicly available tool for robust characterization of Arabidopsis gene expression experiments which can point to similar experimental factors in other experiments. The server is available at http://www.cbs.dtu.dk/services/faro/....

  14. Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells

    Directory of Open Access Journals (Sweden)

    Spyros Darmanis

    2016-01-01

    Full Text Available Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell’s phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment.

  15. Platforms for Single-Cell Collection and Analysis

    Directory of Open Access Journals (Sweden)

    Lukas Valihrach

    2018-03-01

    Full Text Available Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS. In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments.

  16. Platforms for Single-Cell Collection and Analysis.

    Science.gov (United States)

    Valihrach, Lukas; Androvic, Peter; Kubista, Mikael

    2018-03-11

    Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS). In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments.

  17. Scientist, Single Cell Analysis Facility | Center for Cancer Research

    Science.gov (United States)

    The Cancer Research Technology Program (CRTP) develops and implements emerging technology, cancer biology expertise and research capabilities to accomplish NCI research objectives.  The CRTP is an outward-facing, multi-disciplinary hub purposed to enable the external cancer research community and provides dedicated support to NCI’s intramural Center for Cancer Research (CCR).  The dedicated units provide electron microscopy, protein characterization, protein expression, optical microscopy and nextGen sequencing. These research efforts are an integral part of CCR at the Frederick National Laboratory for Cancer Research (FNLCR).  CRTP scientists also work collaboratively with intramural NCI investigators to provide research technologies and expertise. KEY ROLES AND RESPONSIBILITIES We are seeking a highly motivated Scientist II to join the newly established Single Cell Analysis Facility (SCAF) of the Center for Cancer Research (CCR) at NCI. The SCAF will house state of the art single cell sequencing technologies including 10xGenomics Chromium, BD Genomics Rhapsody, DEPPArray, and other emerging single cell technologies. The Scientist: Will interact with close to 200 laboratories within the CCR to design and carry out single cell experiments for cancer research Will work on single cell isolation/preparation from various tissues and cells and related NexGen sequencing library preparation Is expected to author publications in peer reviewed scientific journals

  18. Diversification of gene expression during formation of static submerged biofilms by Escherichia coli

    Directory of Open Access Journals (Sweden)

    Olga Besharova

    2016-10-01

    Full Text Available Many bacteria primarily exist in nature as structured multicellular communities, so called biofilms. Biofilm formation is a highly regulated process that includes the transition from the motile planktonic to sessile biofilm lifestyle. Cellular differentiation within a biofilm is a commonly accepted concept but it remains largely unclear when, where and how exactly such differentiation arises. Here we used fluorescent transcriptional reporters to quantitatively analyze spatio-temporal expression patterns of several groups of genes during the formation of submerged Escherichia coli biofilms in an open static system. We first confirm that formation of such submerged biofilms as well as pellicles at the liquid-air interface requires the major matrix component, curli, and flagella-mediated motility. We further demonstrate that in this system, diversification of gene expression leads to emergence of at least three distinct subpopulations of E. coli, which differ in their levels of curli and flagella expression, and in the activity of the stationary phase sigma factor σS. Our study reveals mutually exclusive expression of curli fibers and flagella at the single cell level, with high curli levels being confined to dense cell aggregates/microcolonies and flagella expression showing an opposite expression pattern. Interestingly, despite the known σS-dependence of curli induction, there was only a partial correlation between the σS activity and curli expression, with subpopulations of cells having high σS activity but low curli expression and vice versa. Finally, consistent with different physiology of the observed subpopulations, we show striking differences between the growth rates of cells within and outside of aggregates.

  19. PRAME Gene Expression in Acute Leukemia and Its Clinical Significance

    International Nuclear Information System (INIS)

    Ding, Kai; Wang, Xiao-ming; Fu, Rong; Ruan, Er-bao; Liu, Hui; Shao, Zong-hong

    2012-01-01

    To investigate the expression of the preferentially expressed antigen of melanoma (PRAME) gene in acute leukemia and its clinical significance. The level of expressed PRAME mRNA in bone marrow mononuclear cells from 34 patients with acute leukemia (AL) and in 12 bone marrow samples from healthy volunteers was measured via RT-PCR. Correlation analyses between PRAME gene expression and the clinical characteristics (gender, age, white blood count, immunophenotype of leukemia, percentage of blast cells, and karyotype) of the patients were performed. The PRAME gene was expressed in 38.2% of all 34 patients, in 40.7% of the patients with acute myelogenous leukemia (AML, n=27), and in 28.6% of the patients with acute lymphoblastic leukemia (ALL, n=7), but was not expressed in the healthy volunteers. The difference in the expression levels between AML and ALL patients was statistically significant. The rate of gene expression was 80% in M 3 , 33.3% in M 2 , and 28.6% in M 5 . Gene expression was also found to be correlated with CD15 and CD33 expression and abnormal karyotype, but not with age, gender, white blood count or percentage of blast cells. The PRAME gene is highly expressed in acute leukemia and could be a useful marker to monitor minimal residual disease. This gene is also a candidate target for the immunotherapy of acute leukemia

  20. Global gene expression analysis for evaluation and design of biomaterials

    Directory of Open Access Journals (Sweden)

    Nobutaka Hanagata, Taro Takemura and Takashi Minowa

    2010-01-01

    Full Text Available Comprehensive gene expression analysis using DNA microarrays has become a widespread technique in molecular biological research. In the biomaterials field, it is used to evaluate the biocompatibility or cellular toxicity of metals, polymers and ceramics. Studies in this field have extracted differentially expressed genes in the context of differences in cellular responses among multiple materials. Based on these genes, the effects of materials on cells at the molecular level have been examined. Expression data ranging from several to tens of thousands of genes can be obtained from DNA microarrays. For this reason, several tens or hundreds of differentially expressed genes are often present in different materials. In this review, we outline the principles of DNA microarrays, and provide an introduction to methods of extracting information which is useful for evaluating and designing biomaterials from comprehensive gene expression data.

  1. Global gene expression analysis for evaluation and design of biomaterials

    International Nuclear Information System (INIS)

    Hanagata, Nobutaka; Takemura, Taro; Minowa, Takashi

    2010-01-01

    Comprehensive gene expression analysis using DNA microarrays has become a widespread technique in molecular biological research. In the biomaterials field, it is used to evaluate the biocompatibility or cellular toxicity of metals, polymers and ceramics. Studies in this field have extracted differentially expressed genes in the context of differences in cellular responses among multiple materials. Based on these genes, the effects of materials on cells at the molecular level have been examined. Expression data ranging from several to tens of thousands of genes can be obtained from DNA microarrays. For this reason, several tens or hundreds of differentially expressed genes are often present in different materials. In this review, we outline the principles of DNA microarrays, and provide an introduction to methods of extracting information which is useful for evaluating and designing biomaterials from comprehensive gene expression data. (topical review)

  2. Comprehensive Identification and Spatial Mapping of Habenular Neuronal Types Using Single-Cell RNA-Seq.

    Science.gov (United States)

    Pandey, Shristi; Shekhar, Karthik; Regev, Aviv; Schier, Alexander F

    2018-04-02

    The identification of cell types and marker genes is critical for dissecting neural development and function, but the size and complexity of the brain has hindered the comprehensive discovery of cell types. We combined single-cell RNA-seq (scRNA-seq) with anatomical brain registration to create a comprehensive map of the zebrafish habenula, a conserved forebrain hub involved in pain processing and learning. Single-cell transcriptomes of ∼13,000 habenular cells with 4× cellular coverage identified 18 neuronal types and dozens of marker genes. Registration of marker genes onto a reference atlas created a resource for anatomical and functional studies and enabled the mapping of active neurons onto neuronal types following aversive stimuli. Strikingly, despite brain growth and functional maturation, cell types were retained between the larval and adult habenula. This study provides a gene expression atlas to dissect habenular development and function and offers a general framework for the comprehensive characterization of other brain regions. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. Redox regulation of photosynthetic gene expression.

    Science.gov (United States)

    Queval, Guillaume; Foyer, Christine H

    2012-12-19

    Redox chemistry and redox regulation are central to the operation of photosynthesis and respiration. However, the roles of different oxidants and antioxidants in the regulation of photosynthetic or respiratory gene expression remain poorly understood. Leaf transcriptome profiles of a range of Arabidopsis thaliana genotypes that are deficient in either hydrogen peroxide processing enzymes or in low molecular weight antioxidant were therefore compared to determine how different antioxidant systems that process hydrogen peroxide influence transcripts encoding proteins targeted to the chloroplasts or mitochondria. Less than 10 per cent overlap was observed in the transcriptome patterns of leaves that are deficient in either photorespiratory (catalase (cat)2) or chloroplastic (thylakoid ascorbate peroxidase (tapx)) hydrogen peroxide processing. Transcripts encoding photosystem II (PSII) repair cycle components were lower in glutathione-deficient leaves, as were the thylakoid NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) dehydrogenases (NDH) mRNAs. Some thylakoid NDH mRNAs were also less abundant in tAPX-deficient and ascorbate-deficient leaves. Transcripts encoding the external and internal respiratory NDHs were increased by low glutathione and low ascorbate. Regulation of transcripts encoding specific components of the photosynthetic and respiratory electron transport chains by hydrogen peroxide, ascorbate and glutathione may serve to balance non-cyclic and cyclic electron flow pathways in relation to oxidant production and reductant availability.

  4. Cell cycle gene expression under clinorotation

    Science.gov (United States)

    Artemenko, Olga

    2016-07-01

    Cyclins and cyclin-dependent kinase (CDK) are main regulators of the cell cycle of eukaryotes. It's assumes a significant change of their level in cells under microgravity conditions and by other physical factors actions. The clinorotation use enables to determine the influence of gravity on simulated events in the cell during the cell cycle - exit from the state of quiet stage and promotion presynthetic phase (G1) and DNA synthesis phase (S) of the cell cycle. For the clinorotation effect study on cell proliferation activity is the necessary studies of molecular mechanisms of cell cycle regulation and development of plants under altered gravity condition. The activity of cyclin D, which is responsible for the events of the cell cycle in presynthetic phase can be controlled by the action of endogenous as well as exogenous factors, but clinorotation is one of the factors that influence on genes expression that regulate the cell cycle.These data can be used as a model for further research of cyclin - CDK complex for study of molecular mechanisms regulation of growth and proliferation. In this investigation we tried to summarize and analyze known literature and own data we obtained relatively the main regulators of the cell cycle in altered gravity condition.

  5. Image segmentation and dynamic lineage analysis in single-cell fluorescence microscopy.

    Science.gov (United States)

    Wang, Quanli; Niemi, Jarad; Tan, Chee-Meng; You, Lingchong; West, Mike

    2010-01-01

    An increasingly common component of studies in synthetic and systems biology is analysis of dynamics of gene expression at the single-cell level, a context that is heavily dependent on the use of time-lapse movies. Extracting quantitative data on the single-cell temporal dynamics from such movies remains a major challenge. Here, we describe novel methods for automating key steps in the analysis of single-cell, fluorescent images-segmentation and lineage reconstruction-to recognize and track individual cells over time. The automated analysis iteratively combines a set of extended morphological methods for segmentation, and uses a neighborhood-based scoring method for frame-to-frame lineage linking. Our studies with bacteria, budding yeast and human cells, demonstrate the portability and usability of these methods, whether using phase, bright field or fluorescent images. These examples also demonstrate the utility of our integrated approach in facilitating analyses of engineered and natural cellular networks in diverse settings. The automated methods are implemented in freely available, open-source software.

  6. Social Regulation of Gene Expression in Threespine Sticklebacks.

    Directory of Open Access Journals (Sweden)

    Anna K Greenwood

    Full Text Available Identifying genes that are differentially expressed in response to social interactions is informative for understanding the molecular basis of social behavior. To address this question, we described changes in gene expression as a result of differences in the extent of social interactions. We housed threespine stickleback (Gasterosteus aculeatus females in either group conditions or individually for one week, then measured levels of gene expression in three brain regions using RNA-sequencing. We found that numerous genes in the hindbrain/cerebellum had altered expression in response to group or individual housing. However, relatively few genes were differentially expressed in either the diencephalon or telencephalon. The list of genes upregulated in fish from social groups included many genes related to neural development and cell adhesion as well as genes with functions in sensory signaling, stress, and social and reproductive behavior. The list of genes expressed at higher levels in individually-housed fish included several genes previously identified as regulated by social interactions in other animals. The identified genes are interesting targets for future research on the molecular mechanisms of normal social interactions.

  7. Large scale gene expression meta-analysis reveals tissue-specific, sex-biased gene expression in humans

    Directory of Open Access Journals (Sweden)

    Benjamin Mayne

    2016-10-01

    Full Text Available The severity and prevalence of many diseases are known to differ between the sexes. Organ specific sex-biased gene expression may underpin these and other sexually dimorphic traits. To further our understanding of sex differences in transcriptional regulation, we performed meta-analyses of sex biased gene expression in multiple human tissues. We analysed 22 publicly available human gene expression microarray data sets including over 2500 samples from 15 different tissues and 9 different organs. Briefly, by using an inverse-variance method we determined the effect size difference of gene expression between males and females. We found the greatest sex differences in gene expression in the brain, specifically in the anterior cingulate cortex, (1818 genes, followed by the heart (375 genes, kidney (224 genes, colon (218 genes and thyroid (163 genes. More interestingly, we found different parts of the brain with varying numbers and identity of sex-biased genes, indicating that specific cortical regions may influence sexually dimorphic traits. The majority of sex-biased genes in other tissues such as the bladder, liver, lungs and pancreas were on the sex chromosomes or involved in sex hormone production. On average in each tissue, 32% of autosomal genes that were expressed in a sex-biased fashion contained androgen or estrogen hormone response elements. Interestingly, across all tissues, we found approximately two-thirds of autosomal genes that were sex-biased were not under direct influence of sex hormones. To our knowledge this is the largest analysis of sex-biased gene expression in human tissues to date. We identified many sex-biased genes that were not under the direct influence of sex chromosome genes or sex hormones. These may provide targets for future development of sex-specific treatments for diseases.

  8. Microarray gene expression profiling and analysis in renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Sadhukhan Provash

    2004-06-01

    Full Text Available Abstract Background Renal cell carcinoma (RCC is the most common cancer in adult kidney. The accuracy of current diagnosis and prognosis of the disease and the effectiveness of the treatment for the disease are limited by the poor understanding of the disease at the molecular level. To better understand the genetics and biology of RCC, we profiled the expression of 7,129 genes in both clear cell RCC tissue and cell lines using oligonucleotide arrays. Methods Total RNAs isolated from renal cell tumors, adjacent normal tissue and metastatic RCC cell lines were hybridized to affymatrix HuFL oligonucleotide arrays. Genes were categorized into different functional groups based on the description of the Gene Ontology Consortium and analyzed based on the gene expression levels. Gene expression profiles of the tissue and cell line samples were visualized and classified by singular value decomposition. Reverse transcription polymerase chain reaction was performed to confirm the expression alterations of selected genes in RCC. Results Selected genes were annotated based on biological processes and clustered into functional groups. The expression levels of genes in each group were also analyzed. Seventy-four commonly differentially expressed genes with more than five-fold changes in RCC tissues were identified. The expression alterations of selected genes from these seventy-four genes were further verified using reverse transcription polymerase chain reaction (RT-PCR. Detailed comparison of gene expression patterns in RCC tissue and RCC cell lines shows significant differences between the two types of samples, but many important expression patterns were preserved. Conclusions This is one of the initial studies that examine the functional ontology of a large number of genes in RCC. Extensive annotation, clustering and analysis of a large number of genes based on the gene functional ontology revealed many interesting gene expression patterns in RCC. Most

  9. A stochastic approach to multi-gene expression dynamics

    International Nuclear Information System (INIS)

    Ochiai, T.; Nacher, J.C.; Akutsu, T.

    2005-01-01

    In the last years, tens of thousands gene expression profiles for cells of several organisms have been monitored. Gene expression is a complex transcriptional process where mRNA molecules are translated into proteins, which control most of the cell functions. In this process, the correlation among genes is crucial to determine the specific functions of genes. Here, we propose a novel multi-dimensional stochastic approach to deal with the gene correlation phenomena. Interestingly, our stochastic framework suggests that the study of the gene correlation requires only one theoretical assumption-Markov property-and the experimental transition probability, which characterizes the gene correlation system. Finally, a gene expression experiment is proposed for future applications of the model

  10. Clinicopathologic and gene expression parameters predict liver cancer prognosis

    International Nuclear Information System (INIS)

    Hao, Ke; Zhong, Hua; Greenawalt, Danielle; Ferguson, Mark D; Ng, Irene O; Sham, Pak C; Poon, Ronnie T; Molony, Cliona; Schadt, Eric E; Dai, Hongyue; Luk, John M; Lamb, John; Zhang, Chunsheng; Xie, Tao; Wang, Kai; Zhang, Bin; Chudin, Eugene; Lee, Nikki P; Mao, Mao

    2011-01-01

    The prognosis of hepatocellular carcinoma (HCC) varies following surgical resection and the large variation remains largely unexplained. Studies have revealed the ability of clinicopathologic parameters and gene expression to predict HCC prognosis. However, there has been little systematic effort to compare the performance of these two types of predictors or combine them in a comprehensive model. Tumor and adjacent non-tumor liver tissues were collected from 272 ethnic Chinese HCC patients who received curative surgery. We combined clinicopathologic parameters and gene expression data (from both tissue types) in predicting HCC prognosis. Cross-validation and independent studies were employed to assess prediction. HCC prognosis was significantly associated with six clinicopathologic parameters, which can partition the patients into good- and poor-prognosis groups. Within each group, gene expression data further divide patients into distinct prognostic subgroups. Our predictive genes significantly overlap with previously published gene sets predictive of prognosis. Moreover, the predictive genes were enriched for genes that underwent normal-to-tumor gene network transformation. Previously documented liver eSNPs underlying the HCC predictive gene signatures were enriched for SNPs that associated with HCC prognosis, providing support that these genes are involved in key processes of tumorigenesis. When applied individually, clinicopathologic parameters and gene expression offered similar predictive power for HCC prognosis. In contrast, a combination of the two types of data dramatically improved the power to predict HCC prognosis. Our results also provided a framework for understanding the impact of gene expression on the processes of tumorigenesis and clinical outcome

  11. Confocal quantification of cis-regulatory reporter gene expression in living sea urchin.

    Science.gov (United States)

    Damle, Sagar; Hanser, Bridget; Davidson, Eric H; Fraser, Scott E

    2006-11-15

    Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.

  12. Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.

    Directory of Open Access Journals (Sweden)

    Lucie Kosinová

    Full Text Available The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3 in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information

  13. Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood

    Directory of Open Access Journals (Sweden)

    Turner Renee J

    2009-08-01

    Full Text Available Abstract Background Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. Methods Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT, 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. Results Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder. Conclusion The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.

  14. Characterization of differentially expressed genes using high-dimensional co-expression networks

    DEFF Research Database (Denmark)

    Coelho Goncalves de Abreu, Gabriel; Labouriau, Rodrigo S.

    2010-01-01

    We present a technique to characterize differentially expressed genes in terms of their position in a high-dimensional co-expression network. The set-up of Gaussian graphical models is used to construct representations of the co-expression network in such a way that redundancy and the propagation...... that allow to make effective inference in problems with high degree of complexity (e.g. several thousands of genes) and small number of observations (e.g. 10-100) as typically occurs in high throughput gene expression studies. Taking advantage of the internal structure of decomposable graphical models, we...... construct a compact representation of the co-expression network that allows to identify the regions with high concentration of differentially expressed genes. It is argued that differentially expressed genes located in highly interconnected regions of the co-expression network are less informative than...

  15. Gene Expression Measurement Module (GEMM) - a fully automated, miniaturized instrument for measuring gene expression in space

    Science.gov (United States)

    Karouia, Fathi; Ricco, Antonio; Pohorille, Andrew; Peyvan, Kianoosh

    2012-07-01

    The capability to measure gene expression on board spacecrafts opens the doors to a large number of experiments on the influence of space environment on biological systems that will profoundly impact our ability to conduct safe and effective space travel, and might also shed light on terrestrial physiology or biological function and human disease and aging processes. Measurements of gene expression will help us to understand adaptation of terrestrial life to conditions beyond the planet of origin, identify deleterious effects of the space environment on a wide range of organisms from microbes to humans, develop effective countermeasures against these effects, determine metabolic basis of microbial pathogenicity and drug resistance, test our ability to sustain and grow in space organisms that can be used for life support and in situ resource utilization during long-duration space exploration, and monitor both the spacecraft environment and crew health. These and other applications hold significant potential for discoveries in space biology, biotechnology and medicine. Accordingly, supported by funding from the NASA Astrobiology Science and Technology Instrument Development Program, we are developing a fully automated, miniaturized, integrated fluidic system for small spacecraft capable of in-situ measuring microbial expression of thousands of genes from multiple samples. The instrument will be capable of (1) lysing bacterial cell walls, (2) extracting and purifying RNA released from cells, (3) hybridizing it on a microarray and (4) providing electrochemical readout, all in a microfluidics cartridge. The prototype under development is suitable for deployment on nanosatellite platforms developed by the NASA Small Spacecraft Office. The first target application is to cultivate and measure gene expression of the photosynthetic bacterium Synechococcus elongatus, i.e. a cyanobacterium known to exhibit remarkable metabolic diversity and resilience to adverse conditions

  16. Differentially expressed genes in iron-induced prion protein conversion

    International Nuclear Information System (INIS)

    Kim, Minsun; Kim, Eun-hee; Choi, Bo-Ran; Woo, Hee-Jong

    2016-01-01

    The conversion of the cellular prion protein (PrP C ) to the protease-resistant isoform is the key event in chronic neurodegenerative diseases, including transmissible spongiform encephalopathies (TSEs). Increased iron in prion-related disease has been observed due to the prion protein-ferritin complex. Additionally, the accumulation and conversion of recombinant PrP (rPrP) is specifically derived from Fe(III) but not Fe(II). Fe(III)-mediated PK-resistant PrP (PrP res ) conversion occurs within a complex cellular environment rather than via direct contact between rPrP and Fe(III). In this study, differentially expressed genes correlated with prion degeneration by Fe(III) were identified using Affymetrix microarrays. Following Fe(III) treatment, 97 genes were differentially expressed, including 85 upregulated genes and 12 downregulated genes (≥1.5-fold change in expression). However, Fe(II) treatment produced moderate alterations in gene expression without inducing dramatic alterations in gene expression profiles. Moreover, functional grouping of identified genes indicated that the differentially regulated genes were highly associated with cell growth, cell maintenance, and intra- and extracellular transport. These findings showed that Fe(III) may influence the expression of genes involved in PrP folding by redox mechanisms. The identification of genes with altered expression patterns in neural cells may provide insights into PrP conversion mechanisms during the development and progression of prion-related diseases. - Highlights: • Differential genes correlated with prion degeneration by Fe(III) were identified. • Genes were identified in cell proliferation and intra- and extracellular transport. • In PrP degeneration, redox related genes were suggested. • Cbr2, Rsad2, Slc40a1, Amph and Mvd were expressed significantly.

  17. Gene expression profile data for mouse facial development

    Directory of Open Access Journals (Sweden)

    Sonia M. Leach

    2017-08-01

    Full Text Available This article contains data related to the research articles "Spatial and Temporal Analysis of Gene Expression during Growth and Fusion of the Mouse Facial Prominences" (Feng et al., 2009 [1] and “Systems Biology of facial development: contributions of ectoderm and mesenchyme” (Hooper et al., 2017 In press [2]. Embryonic mammalian craniofacial development is a complex process involving the growth, morphogenesis, and fusion of distinct facial prominences into a functional whole. Aberrant gene regulation during this process can lead to severe craniofacial birth defects, including orofacial clefting. As a means to understand the genes involved in facial development, we had previously dissected the embryonic mouse face into distinct prominences: the mandibular, maxillary or nasal between E10.5 and E12.5. The prominences were then processed intact, or separated into ectoderm and mesenchyme layers, prior analysis of RNA expression using microarrays (Feng et al., 2009, Hooper et al., 2017 in press [1,2]. Here, individual gene expression profiles have been built from these datasets that illustrate the timing of gene expression in whole prominences or in the separated tissue layers. The data profiles are presented as an indexed and clickable list of the genes each linked to a graphical image of that gene׳s expression profile in the ectoderm, mesenchyme, or intact prominence. These data files will enable investigators to obtain a rapid assessment of the relative expression level of any gene on the array with respect to time, tissue, prominence, and expression trajectory.

  18. Stably Expressed Genes Involved in Basic Cellular Functions.

    Directory of Open Access Journals (Sweden)

    Kejian Wang

    Full Text Available Stably Expressed Genes (SEGs whose expression varies within a narrow range may be involved in core cellular processes necessary for basic functions. To identify such genes, we re-analyzed existing RNA-Seq gene expression profiles across 11 organs at 4 developmental stages (from immature to old age in both sexes of F344 rats (n = 4/group; 320 samples. Expression changes (calculated as the maximum expression / minimum expression for each gene of >19000 genes across organs, ages, and sexes ranged from 2.35 to >109-fold, with a median of 165-fold. The expression of 278 SEGs was found to vary ≤4-fold and these genes were significantly involved in protein catabolism (proteasome and ubiquitination, RNA transport, protein processing, and the spliceosome. Such stability of expression was further validated in human samples where the expression variability of the homologous human SEGs was significantly lower than that of other genes in the human genome. It was also found that the homologous human SEGs were generally less subject to non-synonymous mutation than other genes, as would be expected of stably expressed genes. We also found that knockout of SEG homologs in mouse models was more likely to cause complete preweaning lethality than non-SEG homologs, corroborating the fundamental roles played by SEGs in biological development. Such stably expressed genes and pathways across life-stages suggest that tight control of these processes is important in basic cellular functions and that perturbation by endogenous (e.g., genetics or exogenous agents (e.g., drugs, environmental factors may cause serious adverse effects.

  19. ANALYSES ON DIFFERENTIALLY EXPRESSED GENES ASSOCIATED WITH HUMAN BREAST CANCER

    Institute of Scientific and Technical Information of China (English)

    MENG Xu-li; DING Xiao-wen; XU Xiao-hong

    2006-01-01

    Objective: To investigate the molecular etiology of breast cancer by way of studying the differential expression and initial function of the related genes in the occurrence and development of breast cancer. Methods: Two hundred and eighty-eight human tumor related genes were chosen for preparation of the oligochips probe. mRNA was extracted from 16 breast cancer tissues and the corresponding normal breast tissues, and cDNA probe was prepared through reverse-transcription and hybridized with the gene chip. A laser focused fluorescent scanner was used to scan the chip. The different gene expressions were thereafter automatically compared and analyzed between the two sample groups. Cy3/Cy5>3.5 meant significant up-regulation. Cy3/Cy5<0.25 meant significant down-regulation. Results: The comparison between the breast cancer tissues and their corresponding normal tissues showed that 84 genes had differential expression in the Chip. Among the differently expressed genes, there were 4 genes with significant down-regulation and 6 with significant up-regulation. Compared with normal breast tissues, differentially expressed genes did partially exist in the breast cancer tissues. Conclusion: Changes in multi-gene expression regulations take place during the occurrence and development of breast cancer; and the research on related genes can help understanding the mechanism of tumor occurrence.

  20. Regulation of mitochondrial gene expression, the epigenetic enigma

    NARCIS (Netherlands)

    Mposhi, Archibold; van der Wijst, Monique G. P.; Faber, Klaas Nico; Rots, Marianne G.

    2017-01-01

    Epigenetics provides an important layer of information on top of the DNA sequence and is essential for establishing gene expression profiles. Extensive studies have shown that nuclear DNA methylation and histone modifications influence nuclear gene expression. However, it remains unclear whether

  1. Expression of KLK2 gene in prostate cancer

    Directory of Open Access Journals (Sweden)

    Sajad Shafai

    2018-01-01

    Conclusion: The expression of KLK2 gene in people with prostate cancer is the higher than the healthy person; finally, according to the results, it could be mentioned that the KLK2 gene considered as a useful factor in prostate cancer, whose expression is associated with progression and development of the prostate cancer.

  2. Comparative genomics of the relationship between gene structure and expression

    NARCIS (Netherlands)

    Ren, X.

    2006-01-01

    The relationship between the structure of genes and their expression is a relatively new aspect of genome organization and regulation. With more genome sequences and expression data becoming available, bioinformatics approaches can help the further elucidation of the relationships between gene

  3. The gene expressions of DNA methylation/demethylation enzymes ...

    African Journals Online (AJOL)

    user

    2011-01-31

    Jan 31, 2011 ... A decrease in mRNA levels for cytochrome c oxidase (COX) subunits was observed in skeletal muscle of hypothyroid rats. However, the precise expression mechanisms of the related genes in hypothyroid state still remain unclear. This study investigated gene expressions of DNA methyltransferases.

  4. Genome polymorphism markers and stress genes expression for ...

    African Journals Online (AJOL)

    SAM

    2014-06-11

    Jun 11, 2014 ... RNA extraction and purification for SOD and PAL gene expression. Fresh leaf tissues (100 mg), from ... Data analysis. Gelquant program for quantification of protein, DNA and RNA gel. (version 1.8.2) was used for .... by reprogramming the expression of endogenous genes. Higher level of these antioxidant ...

  5. Genome organization and expression of the rat ACBP gene family

    DEFF Research Database (Denmark)

    Mandrup, S; Andreasen, P H; Knudsen, J

    1993-01-01

    pool former. We have molecularly cloned and characterized the rat ACBP gene family which comprises one expressed and four processed pseudogenes. One of these was shown to exist in two allelic forms. A comprehensive computer-aided analysis of the promoter region of the expressed ACBP gene revealed...

  6. Effects of heat stress on gene expression in eggplant ( Solanum ...

    African Journals Online (AJOL)

    In order to identify differentially expressed genes involved in heat shock response, cDNA amplified fragment length polymorphism (cDNA-AFLP) and quantitative real-time polymerase chain reaction (QPCR) were used to study gene expression of eggplant seedlings subjected to 0, 6 and 12 h at 43°C. A total of 53 of over ...

  7. The gene expressions of DNA methylation/demethylation enzymes ...

    African Journals Online (AJOL)

    A decrease in mRNA levels for cytochrome c oxidase (COX) subunits was observed in skeletal muscle of hypothyroid rats. However, the precise expression mechanisms of the related genes in hypothyroid state still remain unclear. This study investigated gene expressions of DNA methyltransferases (Dnmts), DNA ...

  8. Microarray analysis of the gene expression profile in triethylene ...

    African Journals Online (AJOL)

    Microarray analysis of the gene expression profile in triethylene glycol dimethacrylate-treated human dental pulp cells. ... Conclusions: Our results suggest that TEGDMA can change the many functions of hDPCs through large changes in gene expression levels and complex interactions with different signaling pathways.

  9. Amniotic Fluid Cells Show Higher Pluripotency-Related Gene Expression Than Allantoic Fluid Cells.

    Science.gov (United States)

    Kehl, Debora; Generali, Melanie; Görtz, Sabrina; Geering, Diego; Slamecka, Jaroslav; Hoerstrup, Simon P; Bleul, Ulrich; Weber, Benedikt

    2017-10-01

    Amniotic fluid represents an abundant source of multipotent stem cells, referred as broadly multipotent given their differentiation potential and expression of pluripotency-related genes. However, the origin of this broadly multipotent cellular fraction is not fully understood. Several sources have been proposed so far, including embryonic and extraembryonic tissues. In this regard, the ovine developmental model uniquely allows for direct comparison of fetal fluid-derived cells from two separate fetal fluid cavities, the allantois and the amnion, over the entire duration of gestation. As allantoic fluid mainly collects fetal urine, cells originating from the efferent urinary tract can directly be compared with cells deriving from the extraembryonic amniotic tissues and the fetus. This study shows isolation of cells from the amniotic [ovine amniotic fluid cells (oAFCs)] and allantoic fluid [ovine allantoic fluid cells (oALCs)] in a strictly paired fashion with oAFCs and oALCs derived from the same fetus. Both cell types showed cellular phenotypes comparable to standard mesenchymal stem cells (MSCs), with trilineage differentiation potential, and expression of common ovine MSC markers. However, the expression of MSC markers per single cell was higher in oAFCs as measured by flow cytometry. oAFCs exhibited higher proliferative capacities and showed significantly higher expression of pluripotency-related genes OCT4, STAT3, NANOG, and REX1 by quantitative real-time polymerase chain reaction compared with paired oALCs. No significant decrease of pluripotency-related gene expression was noted over gestation, implying that cells with high differentiation potential may be isolated at the end of pregnancy. In conclusion, this study suggests that cells with highest stem cell characteristics may originate from the fetus itself or the amniotic fetal adnexa rather than from the efferent urinary tract or the allantoic fetal adnexa.

  10. Fungal and plant gene expression in arbuscular mycorrhizal symbiosis.

    Science.gov (United States)

    Balestrini, Raffaella; Lanfranco, Luisa

    2006-11-01

    Arbuscular mycorrhizas (AMs) are a unique example of symbiosis between two eukaryotes, soil fungi and plants. This association induces important physiological changes in each partner that lead to reciprocal benefits, mainly in nutrient supply. The symbiosis results from modifications in plant and fungal cell organization caused by specific changes in gene expression. Recently, much effort has gone into studying these gene expression patterns to identify a wider spectrum of genes involved. We aim in this review to describe AM symbiosis in terms of current knowledge on plant and fungal gene expression profiles.

  11. Expression and clinical significance of Pax6 gene in retinoblastoma

    Directory of Open Access Journals (Sweden)

    Hai-Dong Huang

    2013-07-01

    Full Text Available AIM: To discuss the expression and clinical significance of Pax6 gene in retinoblastoma(Rb. METHODS: Totally 15 cases of fresh Rb organizations were selected as observation group and 15 normal retinal organizations as control group. Western-Blot and reverse transcriptase polymerase chain reaction(RT-PCRmethods were used to detect Pax6 protein and Pax6 mRNA expressions of the normal retina organizations and Rb organizations. At the same time, Western Blot method was used to detect the Pax6 gene downstream MATH5 and BRN3b differentiation gene protein level expression. After the comparison between two groups, the expression and clinical significance of Pax6 gene in Rb were discussed. RESULTS: In the observation group, average value of mRNA expression of Pax6 gene was 0.99±0.03; average value of Pax6 gene protein expression was 2.07±0.15; average value of BRN3b protein expression was 0.195±0.016; average value of MATH5 protein expression was 0.190±0.031. They were significantly higher than the control group, and the differences were statistically significant(PCONCLUSION: Abnormal expression of Pax6 gene is likely to accelerate the occurrence of Rb.

  12. Gene expression in cerebral ischemia: a new approach for neuroprotection.

    Science.gov (United States)

    Millán, Mónica; Arenillas, Juan

    2006-01-01

    Cerebral ischemia is one of the strongest stimuli for gene induction in the brain. Hundreds of genes have been found to be induced by brain ischemia. Many genes are involved in neurodestructive functions such as excitotoxicity, inflammatory response and neuronal apoptosis. However, cerebral ischemia is also a powerful reformatting and reprogramming stimulus for the brain through neuroprotective gene expression. Several genes may participate in both cellular responses. Thus, isolation of candidate genes for neuroprotection strategies and interpretation of expression changes have been proven difficult. Nevertheless, many studies are being carried out to improve the knowledge of the gene activation and protein expression following ischemic stroke, as well as in the development of new therapies that modify biochemical, molecular and genetic changes underlying cerebral ischemia. Owing to the complexity of the process involving numerous critical genes expressed differentially in time, space and concentration, ongoing therapeutic efforts should be based on multiple interventions at different levels. By modification of the acute gene expression induced by ischemia or the apoptotic gene program, gene therapy is a promising treatment but is still in a very experimental phase. Some hurdles will have to be overcome before these therapies can be introduced into human clinical stroke trials. Copyright 2006 S. Karger AG, Basel.

  13. Genetic architecture of gene expression in the chicken

    Directory of Open Access Journals (Sweden)

    Stanley Dragana

    2013-01-01

    Full Text Available Abstract Background The annotation of many genomes is limited, with a large proportion of identified genes lacking functional assignments. The construction of gene co-expression networks is a powerful approach that presents a way of integrating information from diverse gene expression datasets into a unified analysis which allows inferences to be drawn about the role of previously uncharacterised genes. Using this approach, we generated a condition-free gene co-expression network for the chicken using data from 1,043 publically available Affymetrix GeneChip Chicken Genome Arrays. This data was generated from a diverse range of experiments, including different tissues and experimental conditions. Our aim was to identify gene co-expression modules and generate a tool to facilitate exploration of the functional chicken genome. Results Fifteen modules, containing between 24 and 473 genes, were identified in the condition-free network. Most of the modules showed strong functional enrichment for particular Gene Ontology categories. However, a few showed no enrichment. Transcription factor binding site enrichment was also noted. Conclusions We have demonstrated that this chicken gene co-expression network is a useful tool in gene function prediction and the identification of putative novel transcription factors and binding sites. This work highlights the relevance of this methodology for functional prediction in poorly annotated genomes such as the chicken.

  14. Decoupling Linear and Nonlinear Associations of Gene Expression

    KAUST Repository

    Itakura, Alan

    2013-01-01

    The FANTOM consortium has generated a large gene expression dataset of different cell lines and tissue cultures using the single-molecule sequencing technology of HeliscopeCAGE. This provides a unique opportunity to investigate novel associations between gene expression over time and different cell types. Here, we create a MatLab wrapper for a powerful and computationally intensive set of statistics known as Maximal Information Coefficient, and then calculate this statistic for a large, comprehensive dataset containing gene expression of a variety of differentiating tissues. We then distinguish between linear and nonlinear associations, and then create gene association networks. Following this analysis, we are then able to identify clusters of linear gene associations that then associate nonlinearly with other clusters of linearity, providing insight to much more complex connections between gene expression patterns than previously anticipated.

  15. Decoupling Linear and Nonlinear Associations of Gene Expression

    KAUST Repository

    Itakura, Alan

    2013-05-01

    The FANTOM consortium has generated a large gene expression dataset of different cell lines and tissue cultures using the single-molecule sequencing technology of HeliscopeCAGE. This provides a unique opportunity to investigate novel associations between gene expression over time and different cell types. Here, we create a MatLab wrapper for a powerful and computationally intensive set of statistics known as Maximal Information Coefficient, and then calculate this statistic for a large, comprehensive dataset containing gene expression of a variety of differentiating tissues. We then distinguish between linear and nonlinear associations, and then create gene association networks. Following this analysis, we are then able to identify clusters of linear gene associations that then associate nonlinearly with other clusters of linearity, providing insight to much more complex connections between gene expression patterns than previously anticipated.

  16. Gene expression profiling of placentas affected by pre-eclampsia

    DEFF Research Database (Denmark)

    Hoegh, Anne Mette; Borup, Rehannah; Nielsen, Finn Cilius

    2010-01-01

    Several studies point to the placenta as the primary cause of pre-eclampsia. Our objective was to identify placental genes that may contribute to the development of pre-eclampsia. RNA was purified from tissue biopsies from eleven pre-eclamptic placentas and eighteen normal controls. Messenger RNA...... expression from pooled samples was analysed by microarrays. Verification of the expression of selected genes was performed using real-time PCR. A surprisingly low number of genes (21 out of 15,000) were identified as differentially expressed. Among these were genes not previously associated with pre-eclampsia...... as bradykinin B1 receptor and a 14-3-3 protein, but also genes that have already been connected with pre-eclampsia, for example, inhibin beta A subunit and leptin. A low number of genes were repeatedly identified as differentially expressed, because they may represent the endpoint of a cascade of events...

  17. Laser capture microdissection of enriched populations of neurons or single neurons for gene expression analysis after traumatic brain injury.

    Science.gov (United States)

    Boone, Deborah R; Sell, Stacy L; Hellmich, Helen Lee

    2013-04-10

    Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function. Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al., has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types. We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 °C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving

  18. Evolution of New cis-Regulatory Motifs Required for Cell-Specific Gene Expression in Caenorhabditis.

    Directory of Open Access Journals (Sweden)

    Michalis Barkoulas

    2016-09-01

    Full Text Available Patterning of C. elegans vulval cell fates relies on inductive signaling. In this induction event, a single cell, the gonadal anchor cell, secretes LIN-3/EGF and induces three out of six competent precursor cells to acquire a vulval fate. We previously showed that this developmental system is robust to a four-fold variation in lin-3/EGF genetic dose. Here using single-molecule FISH, we find that the mean level of expression of lin-3 in the anchor cell is remarkably conserved. No change in lin-3 expression level could be detected among C. elegans wild isolates and only a low level of change-less than 30%-in the Caenorhabditis genus and in Oscheius tipulae. In C. elegans, lin-3 expression in the anchor cell is known to require three transcription factor binding sites, specifically two E-boxes and a nuclear-hormone-receptor (NHR binding site. Mutation of any of these three elements in C. elegans results in a dramatic decrease in lin-3 expression. Yet only a single E-box is found in the Drosophilae supergroup of Caenorhabditis species, including C. angaria, while the NHR-binding site likely only evolved at the base of the Elegans group. We find that a transgene from C. angaria bearing a single E-box is sufficient for normal expression in C. elegans. Even a short 58 bp cis-regulatory fragment from C. angaria with this single E-box is able to replace the three transcription factor binding sites at the endogenous C. elegans lin-3 locus, resulting in the wild-type expression level. Thus, regulatory evolution occurring in cis within a 58 bp lin-3 fragment, results in a strict requirement for the NHR binding site and a second E-box in C. elegans. This single-cell, single-molecule, quantitative and functional evo-devo study demonstrates that conserved expression levels can hide extensive change in cis-regulatory site requirements and highlights the evolution of new cis-regulatory elements required for cell-specific gene expression.

  19. Isolation and characterization of LHY homolog gene expressed in ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-05-02

    May 2, 2008 ... responsible in negative feedback loop reaction of central oscillator in plant circadian clock system. The level of gene expression was found to be high four hours after dawn in flowering shoots and flower. This paper reported the isolation and characterization of the gene. Key words: LHY gene, circadian ...

  20. Gene mining a marama bean expressed sequence tags (ESTs ...

    African Journals Online (AJOL)

    The authors reported the identification of genes associated with embryonic development and microsatellite sequences. The future direction will entail characterization of these genes using gene over-expression and mutant assays. Key words: Namibia, simple sequence repeats (SSR), data mining, homology searches, ...

  1. Expression profiles of genes involved in tanshinone biosynthesis of ...

    Indian Academy of Sciences (India)

    Expression profiles of genes involved in tanshinone biosynthesis of two. Salvia miltiorrhiza genotypes with different tanshinone contents. Zhenqiao Song, Jianhua Wang and Xingfeng Li. J. Genet. 95, 433–439. Table 1. S. miltiorrhiza genes and primer pairs used for qRT-PCR. Gene. GenBank accession. Primer name.

  2. Differentially expressed genes in the midgut of Silkworm infected ...

    African Journals Online (AJOL)

    In this report, we employed suppression subtractive hybridization to compare differentially expressed genes in the midguts of CPV-infected and normal silkworm larvae. 36 genes and 20 novel ESTs were obtained from 2 reciprocal subtractive libraries. Three up-regulated genes (ferritin, rpL11 and alkaline nuclease) and 3 ...

  3. Adaptive differences in gene expression in European flounder ( Platichthys flesus )

    DEFF Research Database (Denmark)

    Larsen, Peter Foged; Eg Nielsen, Einar; Williams, T.D.

    2007-01-01

    levels of neutral genetic divergence, a high number of genes were significantly differentially expressed between North Sea and Baltic Sea flounders maintained in a long-term reciprocal transplantation experiment mimicking natural salinities. Several of the differentially regulated genes could be directly...... linked to fitness traits. These findings demonstrate that flounders, despite little neutral genetic divergence between populations, are differently adapted to local environmental conditions and imply that adaptation in gene expression could be common in other marine organisms with similar low levels...

  4. Gene Expression and the Diversity of Identified Neurons

    OpenAIRE

    Buck, L.; Stein, R.; Palazzolo, M.; Anderson, D. J.; Axel, R.

    1983-01-01

    Nervous systems consist of diverse populations of neurons that are anatomically and functionally distinct. The diversity of neurons and the precision with which they are interconnected suggest that specific genes or sets of genes are activated in some neurons but not expressed in others. Experimentally, this problem may be considered at two levels. First, what is the total number of genes expressed in the brain, and how are they distributed among the different populations of neurons? Second, ...

  5. Evaluating the consistency of gene sets used in the analysis of bacterial gene expression data

    Directory of Open Access Journals (Sweden)

    Tintle Nathan L

    2012-08-01

    Full Text Available Abstract Background Statistical analyses of whole genome expression data require functional information about genes in order to yield meaningful biological conclusions. The Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG are common sources of functionally grouped gene sets. For bacteria, the SEED and MicrobesOnline provide alternative, complementary sources of gene sets. To date, no comprehensive evaluation of the data obtained from these resources has been performed. Results We define a series of gene set consistency metrics directly related to the most common classes of statistical analyses for gene expression data, and then perform a comprehensive analysis of 3581 Affymetrix® gene expression arrays across 17 diverse bacteria. We find that gene sets obtained from GO and KEGG demonstrate lower consistency than those obtained from the SEED and MicrobesOnline, regardless of gene set size. Conclusions Despite the widespread use of GO and KEGG gene sets in bacterial gene expression data analysis, the SEED and MicrobesOnline provide more consistent sets for a wide variety of statistical analyses. Increased use of the SEED and MicrobesOnline gene sets in the analysis of bacterial gene expression data may improve statistical power and utility of expression data.

  6. Rethinking cell-cycle-dependent gene expression in Schizosaccharomyces pombe.

    Science.gov (United States)

    Cooper, Stephen

    2017-11-01

    Three studies of gene expression during the division cycle of Schizosaccharomyces pombe led to the proposal that a large number of genes are expressed at particular times during the S. pombe cell cycle. Yet only a small fraction of genes proposed to be expressed in a cell-cycle-dependent manner are reproducible in all three published studies. In addition to reproducibility problems, questions about expression amplitudes, cell-cycle timing of expression, synchronization artifacts, and the problem with methods for synchronizing cells must be considered. These problems and complications prompt the idea that caution should be used before accepting the conclusion that there are a large number of genes expressed in a cell-cycle-dependent manner in S. pombe.

  7. HIV Cell-to-Cell Spread Results in Earlier Onset of Viral Gene Expression by Multiple Infections per Cell.

    Directory of Open Access Journals (Sweden)

    Mikaël Boullé

    2016-11-01

    Full Text Available Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses.

  8. Validation of reference genes for quantifying changes in gene expression in virus-infected tobacco.

    Science.gov (United States)

    Baek, Eseul; Yoon, Ju-Yeon; Palukaitis, Peter

    2017-10-01

    To facilitate quantification of gene expression changes in virus-infected tobacco plants, eight housekeeping genes were evaluated for their stability of expression during infection by one of three systemically-infecting viruses (cucumber mosaic virus, potato virus X, potato virus Y) or a hypersensitive-response-inducing virus (tobacco mosaic virus; TMV) limited to the inoculated leaf. Five reference-gene validation programs were used to establish the order of the most stable genes for the systemically-infecting viruses as ribosomal protein L25 > β-Tubulin > Actin, and the least stable genes Ubiquitin-conjugating enzyme (UCE) genes were EF1α > Cysteine protease > Actin, and the least stable genes were GAPDH genes, three defense responsive genes were examined to compare their relative changes in gene expression caused by each virus. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Olig2 and Hes regulatory dynamics during motor neuron differentiation revealed by single cell transcriptomics.

    Directory of Open Access Journals (Sweden)

    Andreas Sagner

    2018-02-01

    Full Text Available During tissue development, multipotent progenitors differentiate into specific cell types in characteristic spatial and temporal patterns. We addressed the mechanism linking progenitor identity and differentiation rate in the neural tube, where motor neuron (MN progenitors differentiate more rapidly than other progenitors. Using single cell transcriptomics, we defined the transcriptional changes associated with the transition of neural progenitors into MNs. Reconstruction of gene expression dynamics from these data indicate a pivotal role for the MN determinant Olig2 just prior to MN differentiation. Olig2 represses expression of the Notch signaling pathway effectors Hes1 and Hes5. Olig2 repression of Hes5 appears to be direct, via a conserved regulatory element within the Hes5 locus that restricts expression from MN progenitors. These findings reveal a tight coupling between the regulatory networks that control patterning and neuronal differentiation and demonstrate how Olig2 acts as the developmental pacemaker coordinating the spatial and temporal pattern of MN generation.

  10. A Gene Expression Classifier of Node-Positive Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Paul F. Meeh

    2009-10-01

    Full Text Available We used digital long serial analysis of gene expression to discover gene expression differences between node-negative and node-positive colorectal tumors and developed a multigene classifier able to discriminate between these two tumor types. We prepared and sequenced long serial analysis of gene expression libraries from one node-negative and one node-positive colorectal tumor, sequenced to a depth of 26,060 unique tags, and identified 262 tags significantly differentially expressed between these two tumors (P < 2 x 10-6. We confirmed the tag-to-gene assignments and differential expression of 31 genes by quantitative real-time polymerase chain reaction, 12 of which were elevated in the node-positive tumor. We analyzed the expression levels of these 12 upregulated genes in a validation panel of 23 additional tumors and developed an optimized seven-gene logistic regression classifier. The classifier discriminated between node-negative and node-positive tumors with 86% sensitivity and 80% specificity. Receiver operating characteristic analysis of the classifier revealed an area under the curve of 0.86. Experimental manipulation of the function of one classification gene, Fibronectin, caused profound effects on invasion and migration of colorectal cancer cells in vitro. These results suggest that the development of node-positive colorectal cancer occurs in part through elevated epithelial FN1 expression and suggest novel strategies for the diagnosis and treatment of advanced disease.

  11. With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies.

    Science.gov (United States)

    Chapman, Joanne R; Waldenström, Jonas

    2015-01-01

    The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.

  12. Gene expression profiling of resting and activated vascular smooth muscle cells by serial analysis of gene expression and clustering analysis

    NARCIS (Netherlands)

    Beauchamp, Nicholas J.; van Achterberg, Tanja A. E.; Engelse, Marten A.; Pannekoek, Hans; de Vries, Carlie J. M.

    2003-01-01

    Migration and proliferation of vascular smooth muscle cells (SMCs) are key events in atherosclerosis. However, little is known about alterations in gene expression upon transition of the quiescent, contractile SMC to the proliferative SMC. We performed serial analysis of gene expression (SAGE) of

  13. Using PCR to Target Misconceptions about Gene Expression

    Directory of Open Access Journals (Sweden)

    Leslie K. Wright

    2013-02-01

    Full Text Available We present a PCR-based laboratory exercise that can be used with first- or second-year biology students to help overcome common misconceptions about gene expression. Biology students typically do not have a clear understanding of the difference between genes (DNA and gene expression (mRNA/protein and often believe that genes exist in an organism or cell only when they are expressed. This laboratory exercise allows students to carry out a PCR-based experiment designed to challenge their misunderstanding of the difference between genes and gene expression. Students first transform E. coli with an inducible GFP gene containing plasmid and observe induced and un-induced colonies. The following exercise creates cognitive dissonance when actual PCR results contradict their initial (incorrect predictions of the presence of the GFP gene in transformed cells. Field testing of this laboratory exercise resulted in learning gains on both knowledge and application questions on concepts related to genes and gene expression.

  14. Expression of Separate Proteins in the Same Plant Leaves and Cells Using Two Independent Virus-Based Gene Vectors

    Directory of Open Access Journals (Sweden)

    Maria R. Mendoza

    2017-11-01

    Full Text Available Plant viral vectors enable the expression of proteins at high levels in a relatively short time. For many purposes (e.g., cell biological interaction studies it may be desirable to express more than one protein in a single cell but that is often not feasible when using a single virus vector. Such a co-expression strategy requires the simultaneous delivery by two compatible and non-competitive viruses that can co-exist to each express a separate protein. Here, we report on the use of two agro-launchable coat-protein gene substitution GFP-expressing virus vector systems based on Tomato bushy stunt virus (TBSV referred to as TG, and Tobacco mosaic virus (TMV annotated as TRBO-G. TG expressed GFP in Nicotiana benthamiana, tomato, lettuce and cowpea, whereas expression from TRBO-G was detected only in the first two species. Upon co-infiltration of the two vectors co-expression was monitored by: molecular detection of the two slightly differently sized GFPs, suppressor-complementation assays, and using TG in combination with TRBO-RFP. All the results revealed that in N. benthamiana and tomato the TBSV and TMV vectors accumulated and expressed proteins in the same plants, the same leaves, and in the same cells. Therefore, co-expression by these two vectors provides a platform for fast and high level expression of proteins to study their cell biology or other properties.

  15. Dynamic association rules for gene expression data analysis.

    Science.gov (United States)

    Chen, Shu-Chuan; Tsai, Tsung-Hsien; Chung, Cheng-Han; Li, Wen-Hsiung

    2015-10-14

    The purpose of gene expression analysis is to look for the association between regulation of gene expression levels and phenotypic variations. This association based on gene expression profile has been used to determine whether the induction/repression of genes correspond to phenotypic variations including cell regulations, clinical diagnoses and drug development. Statistical analyses on microarray data have been developed to resolve gene selection issue. However, these methods do not inform us of causality between genes and phenotypes. In this paper, we propose the dynamic association rule algorithm (DAR algorithm) which helps ones to efficiently select a subset of significant genes for subsequent analysis. The DAR algorithm is based on association rules from market basket analysis in marketing. We first propose a statistical way, based on constructing a one-sided confidence interval and hypothesis testing, to determine if an association rule is meaningful. Based on the proposed statistical method, we then developed the DAR algorithm for gene expression data analysis. The method was applied to analyze four microarray datasets and one Next Generation Sequencing (NGS) dataset: the Mice Apo A1 dataset, the whole genome expression dataset of mouse embryonic stem cells, expression profiling of the bone marrow of Leukemia patients, Microarray Quality Control (MAQC) data set and the RNA-seq dataset of a mouse genomic imprinting study. A comparison of the proposed method with the t-test on the expression profiling of the bone marrow of Leukemia patients was conducted. We developed a statistical way, based on the concept of confidence interval, to determine the minimum support and minimum confidence for mining association relationships among items. With the minimum support and minimum confidence, one can find significant rules in one single step. The DAR algorithm was then developed for gene expression data analysis. Four gene expression datasets showed that the proposed

  16. Epigenetic regulation on the gene expression signature in esophagus adenocarcinoma.

    Science.gov (United States)

    Xi, Ting; Zhang, Guizhi

    2017-02-01

    Understanding the molecular mechanisms represents an important step in the development of diagnostic and therapeutic measures of esophagus adenocarcinoma (NOS). The objective of this study is to identify the epigenetic regulation on gene expression in NOS, shedding light on the molecular mechanisms of NOS. In this study, 78 patients with NOS were included and the data of mRNA, miRNA and DNA methylation of were downloaded from The Cancer Genome Atlas (TCGA). Differential analysis between NOS and controls was performed in terms of gene expression, miRNA expression, and DNA methylation. Bioinformatic analysis was followed to explore the regulation mechanisms of miRNA and DNA methylationon gene expression. Totally, up to 1320 differentially expressed genes (DEGs) and 32 differentially expressed miRNAs were identified. 240 DEGs that were not only the target genes but also negatively correlated with the screened differentially expressed miRNAs. 101 DEGs were found to be highlymethylated in CpG islands. Then, 8 differentially methylated genes (DMGs) were selected, which showed down-regulated expression in NOS. Among of these genes, 6 genes including ADHFE1, DPP6, GRIA4, CNKSR2, RPS6KA6 and ZNF135 were target genes of differentially expressed miRNAs (hsa-mir-335, hsa-mir-18a, hsa-mir-93, hsa-mir-106b and hsa-mir-21). The identified altered miRNA, genes and DNA methylation site may be applied as biomarkers for diagnosis and prognosis of NOS. Copyright © 2016 Elsevier GmbH. All rights reserved.

  17. Identifying potential maternal genes of Bombyx mori using digital gene expression profiling

    Science.gov (United States)

    Xu, Pingzhen

    2018-01-01

    Maternal genes present in mature oocytes play a crucial role in the early development of silkworm. Although maternal genes have been widely studied in many other species, there has been limited research in Bombyx mori. High-throughput next generation sequencing provides a practical method for gene discovery on a genome-wide level. Herein, a transcriptome study was used to identify maternal-related genes from silkworm eggs. Unfertilized eggs from five different stages of early development were used to detect the changing situation of gene expression. The expressed genes showed different patterns over time. Seventy-six maternal genes were annotated according to homology analysis with Drosophila melanogaster. More than half of the differentially expressed maternal genes fell into four expression patterns, while the expression patterns showed a downward trend over time. The functional annotation of these material genes was mainly related to transcription factor activity, growth factor activity, nucleic acid binding, RNA binding, ATP binding, and ion binding. Additionally, twenty-two gene clusters including maternal genes were identified from 18 scaffolds. Altogether, we plotted a profile for the maternal genes of Bombyx mori using a digital gene expression profiling method. This will provide the basis for maternal-specific signature research and improve the understanding of the early development of silkworm. PMID:29462160

  18. Extracting gene expression patterns and identifying co-expressed genes from microarray data reveals biologically responsive processes

    Directory of Open Access Journals (Sweden)

    Paules Richard S

    2007-11-01

    Full Text Available Abstract Background A common observation in the analysis of gene expression data is that many genes display similarity in their expression patterns and therefore appear to be co-regulated. However, the variation associated with microarray data and the complexity of the experimental designs make the acquisition of co-expressed genes a challenge. We developed a novel method for Extracting microarray gene expression Patterns and Identifying co-expressed Genes, designated as EPIG. The approach utilizes the underlying structure of gene expression data to extract patterns and identify co-expressed genes that are responsive to experimental conditions. Results Through evaluation of the correlations among profiles, the magnitude of variation in gene expression profiles, and profile signal-to-noise ratio's, EPIG extracts a set of patterns representing co-expressed genes. The method is shown to work well with a simulated data set and microarray data obtained from time-series studies of dauer recovery and L1 starvation in C. elegans and after ultraviolet (UV or ionizing radiation (IR-induced DNA damage in diploid human fibroblasts. With the simulated data set, EPIG extracted the appropriate number of patterns which were more stable and homogeneous than the set of patterns that were determined using the CLICK or CAST clustering algorithms. However, CLICK performed better than EPIG and CAST with respect to the average correlation between clusters/patterns of the simulated data. With real biological data, EPIG extracted more dauer-specific patterns than CLICK. Furthermore, analysis of the IR/UV data revealed 18 unique patterns and 2661 genes out of approximately 17,000 that were identified as significantly expressed and categorized to the patterns by EPIG. The time-dependent patterns displayed similar and dissimilar responses between IR and UV treatments. Gene Ontology analysis applied to each pattern-related subset of co-expressed genes revealed underlying

  19. Gene expression results in lipopolysaccharide-stimulated monocytes depend significantly on the choice of reference genes

    Directory of Open Access Journals (Sweden)

    Øvstebø Reidun

    2010-05-01

    Full Text Available Abstract Background Gene expression in lipopolysaccharide (LPS-stimulated monocytes is mainly studied by quantitative real-time reverse transcription PCR (RT-qPCR using GAPDH (glyceraldehyde 3-phosphate dehydrogenase or ACTB (beta-actin as reference gene for normalization. Expression of traditional reference genes has been shown to vary substantially under certain conditions leading to invalid results. To investigate whether traditional reference genes are stably expressed in LPS-stimulated monocytes or if RT-qPCR results are dependent on the choice of reference genes, we have assessed and evaluated gene expression stability of twelve candidate reference genes in this model system. Results Twelve candidate reference genes were quantified by RT-qPCR in LPS-stimulated, human monocytes and evaluated using the programs geNorm, Normfinder and BestKeeper. geNorm ranked PPIB (cyclophilin B, B2M (beta-2-microglobulin and PPIA (cyclophilin A as the best combination for gene expression normalization in LPS-stimulated monocytes. Normfinder suggested TBP (TATA-box binding protein and B2M as the best combination. Compared to these combinations, normalization using GAPDH alone resulted in significantly higher changes of TNF-α (tumor necrosis factor-alpha and IL10 (interleukin 10 expression. Moreover, a significant difference in TNF-α expression between monocytes stimulated with equimolar concentrations of LPS from N. meningitides and E. coli, respectively, was identified when using the suggested combinations of reference genes for normalization, but stayed unrecognized when employing a single reference gene, ACTB or GAPDH. Conclusions Gene expression levels in LPS-stimulated monocytes based on RT-qPCR results differ significantly when normalized to a single gene or a combination of stably expressed reference genes. Proper evaluation of reference gene stabiliy is therefore mandatory before reporting RT-qPCR results in LPS-stimulated monocytes.

  20. Expression profiles for six zebrafish genes during gonadal sex differentiation

    DEFF Research Database (Denmark)

    Jørgensen, Anne; Morthorst, Jane Ebsen; Andersen, Ole

    2008-01-01

    BACKGROUND: The mechanism of sex determination in zebrafish is largely unknown and neither sex chromosomes nor a sex-determining gene have been identified. This indicates that sex determination in zebrafish is mediated by genetic signals from autosomal genes. The aim of this study was to determine...... the precise timing of expression of six genes previously suggested to be associated with sex differentiation in zebrafish. The current study investigates the expression of all six genes in the same individual fish with extensive sampling dates during sex determination and -differentiation. RESULTS......: In the present study, we have used quantitative real-time PCR to investigate the expression of ar, sox9a, dmrt1, fig alpha, cyp19a1a and cyp19a1b during the expected sex determination and gonadal sex differentiation period. The expression of the genes expected to be high in males (ar, sox9a and dmrt1a) and high...

  1. Interplay of bistable kinetics of gene expression during cellular growth

    International Nuclear Information System (INIS)

    Zhdanov, Vladimir P

    2009-01-01

    In cells, the bistable kinetics of gene expression can be observed on the level of (i) one gene with positive feedback between protein and mRNA production, (ii) two genes with negative mutual feedback between protein and mRNA production, or (iii) in more complex cases. We analyse the interplay of two genes of type (ii) governed by a gene of type (i) during cellular growth. In particular, using kinetic Monte Carlo simulations, we show that in the case where gene 1, operating in the bistable regime, regulates mutually inhibiting genes 2 and 3, also operating in the bistable regime, the latter genes may eventually be trapped either to the state with high transcriptional activity of gene 2 and low activity of gene 3 or to the state with high transcriptional activity of gene 3 and low activity of gene 2. The probability to get to one of these states depends on the values of the model parameters. If genes 2 and 3 are kinetically equivalent, the probability is equal to 0.5. Thus, our model illustrates how different intracellular states can be chosen at random with predetermined probabilities. This type of kinetics of gene expression may be behind complex processes occurring in cells, e.g., behind the choice of the fate by stem cells

  2. Detecting microRNA activity from gene expression data

    LENUS (Irish Health Repository)

    Madden, Stephen F

    2010-05-18

    Abstract Background MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by binding to the messenger RNA (mRNA) of protein coding genes. They control gene expression by either inhibiting translation or inducing mRNA degradation. A number of computational techniques have been developed to identify the targets of miRNAs. In this study we used predicted miRNA-gene interactions to analyse mRNA gene expression microarray data to predict miRNAs associated with particular diseases or conditions. Results Here we combine correspondence analysis, between group analysis and co-inertia analysis (CIA) to determine which miRNAs are associated with differences in gene expression levels in microarray data sets. Using a database of miRNA target predictions from TargetScan, TargetScanS, PicTar4way PicTar5way, and miRanda and combining these data with gene expression levels from sets of microarrays, this method produces a ranked list of miRNAs associated with a specified split in samples. We applied this to three different microarray datasets, a papillary thyroid carcinoma dataset, an in-house dataset of lipopolysaccharide treated mouse macrophages, and a multi-tissue dataset. In each case we were able to identified miRNAs of biological importance. Conclusions We describe a technique to integrate gene expression data and miRNA target predictions from multiple sources.

  3. Detecting microRNA activity from gene expression data.

    LENUS (Irish Health Repository)

    Madden, Stephen F

    2010-01-01

    BACKGROUND: MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by binding to the messenger RNA (mRNA) of protein coding genes. They control gene expression by either inhibiting translation or inducing mRNA degradation. A number of computational techniques have been developed to identify the targets of miRNAs. In this study we used predicted miRNA-gene interactions to analyse mRNA gene expression microarray data to predict miRNAs associated with particular diseases or conditions. RESULTS: Here we combine correspondence analysis, between group analysis and co-inertia analysis (CIA) to determine which miRNAs are associated with differences in gene expression levels in microarray data sets. Using a database of miRNA target predictions from TargetScan, TargetScanS, PicTar4way PicTar5way, and miRanda and combining these data with gene expression levels from sets of microarrays, this method produces a ranked list of miRNAs associated with a specified split in samples. We applied this to three different microarray datasets, a papillary thyroid carcinoma dataset, an in-house dataset of lipopolysaccharide treated mouse macrophages, and a multi-tissue dataset. In each case we were able to identified miRNAs of biological importance. CONCLUSIONS: We describe a technique to integrate gene expression data and miRNA target predictions from multiple sources.

  4. An Interactive Database of Cocaine-Responsive Gene Expression

    Directory of Open Access Journals (Sweden)

    Willard M. Freeman

    2002-01-01

    Full Text Available The postgenomic era of large-scale gene expression studies is inundating drug abuse researchers and many other scientists with findings related to gene expression. This information is distributed across many different journals, and requires laborious literature searches. Here, we present an interactive database that combines existing information related to cocaine-mediated changes in gene expression in an easy-to-use format. The database is limited to statistically significant changes in mRNA or protein expression after cocaine administration. The Flash-based program is integrated into a Web page, and organizes changes in gene expression based on neuroanatomical region, general function, and gene name. Accompanying each gene is a description of the gene, links to the original publications, and a link to the appropriate OMIM (Online Mendelian Inheritance in Man entry. The nature of this review allows for timely modifications and rapid inclusion of new publications, and should help researchers build second-generation hypotheses on the role of gene expression changes in the physiology and behavior of cocaine abuse. Furthermore, this method of organizing large volumes of scientific information can easily be adapted to assist researchers in fields outside of drug abuse.

  5. A Marfan syndrome gene expression phenotype in cultured skin fibroblasts

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    Emond Mary

    2007-09-01

    Full Text Available Abstract Background Marfan syndrome (MFS is a heritable connective tissue disorder caused by mutations in the fibrillin-1 gene. This syndrome constitutes a significant identifiable subtype of aortic aneurysmal disease, accounting for over 5% of ascending and thoracic aortic aneurysms. Results We used spotted membrane DNA macroarrays to identify genes whose altered expression levels may contribute to the phenotype of the disease. Our analysis of 4132 genes identified a subset with significant expression differences between skin fibroblast cultures from unaffected controls versus cultures from affected individuals with known fibrillin-1 mutations. Subsequently, 10 genes were chosen for validation by quantitative RT-PCR. Conclusion Differential expression of many of the validated genes was associated with MFS samples when an additional group of unaffected and MFS affected subjects were analyzed (p-value -6 under the null hypothesis that expression levels in cultured fibroblasts are unaffected by MFS status. An unexpected observation was the range of individual gene expression. In unaffected control subjects, expression ranges exceeding 10 fold were seen in many of the genes selected for qRT-PCR validation. The variation in expression in the MFS affected subjects was even greater.

  6. Single-cell mRNA transfection studies: delivery, kinetics and statistics by numbers.

    Science.gov (United States)

    Leonhardt, Carolin; Schwake, Gerlinde; Stögbauer, Tobias R; Rappl, Susanne; Kuhr, Jan-Timm; Ligon, Thomas S; Rädler, Joachim O

    2014-05-01

    In artificial gene delivery, messenger RNA (mRNA) is an attractive alternative to plasmid DNA (pDNA) since it does not require transfer into the cell nucleus. Here we show that, unlike for pDNA transfection, the delivery statistics and dynamics of mRNA-mediated expression are generic and predictable in terms of mathematical modeling. We measured the single-cell expression time-courses and levels of enhanced green fluorescent protein (eGFP) using time-lapse microscopy and flow cytometry (FC). The single-cell analysis provides direct access to the distribution of onset times, life times and expression rates of mRNA and eGFP. We introduce a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby the dose-response relation. Our results establish a statistical framework for mRNA transfection and as such should advance the development of RNA carriers and small interfering/micro RNA-based drugs. This team of authors established a statistical framework for mRNA transfection by using a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby their dose-response relation. This study establishes a nice connection between theory and experimental planning and will aid the cellular delivery of mRNA molecules. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Automated Single Cell Data Decontamination Pipeline

    Energy Technology Data Exchange (ETDEWEB)

    Tennessen, Kristin [Lawrence Berkeley National Lab. (LBNL), Walnut Creek, CA (United States). Dept. of Energy Joint Genome Inst.; Pati, Amrita [Lawrence Berkeley National Lab. (LBNL), Walnut Creek, CA (United States). Dept. of Energy Joint Genome Inst.

    2014-03-21

    Recent technological advancements in single-cell genomics have encouraged the classification and functional assessment of microorganisms from a wide span of the biospheres phylogeny.1,2 Environmental processes of interest to the DOE, such as bioremediation and carbon cycling, can be elucidated through the genomic lens of these unculturable microbes. However, contamination can occur at various stages of the single-cell sequencing process. Contaminated data can lead to wasted time and effort on meaningless analyses, inaccurate or erroneous conclusions, and pollution of public databases. A fully automated decontamination tool is necessary to prevent these instances and increase the throughput of the single-cell sequencing process

  8. Novel redox nanomedicine improves gene expression of polyion complex vector

    Directory of Open Access Journals (Sweden)

    Kazuko Toh, Toru Yoshitomi, Yutaka Ikeda and Yukio Nagasaki

    2011-01-01

    Full Text Available Gene therapy has generated worldwide attention as a new medical technology. While non-viral gene vectors are promising candidates as gene carriers, they have several issues such as toxicity and low transfection efficiency. We have hypothesized that the generation of reactive oxygen species (ROS affects gene expression in polyplex supported gene delivery systems. The effect of ROS on the gene expression of polyplex was evaluated using a nitroxide radical-containing nanoparticle (RNP as an ROS scavenger. When polyethyleneimine (PEI/pGL3 or PEI alone was added to the HeLa cells, ROS levels increased significantly. In contrast, when (PEI/pGL3 or PEI was added with RNP, the ROS levels were suppressed. The luciferase expression was increased by the treatment with RNP in a dose-dependent manner and the cellular uptake of pDNA was also increased. Inflammatory cytokines play an important role in ROS generation in vivo. In particular, tumor necrosis factor (TNF-α caused intracellular ROS generation in HeLa cells and decreased gene expression. RNP treatment suppressed ROS production even in the presence of TNF-α and increased gene expression. This anti-inflammatory property of RNP suggests that it may be used as an effective adjuvant for non-viral gene delivery systems.

  9. Biasogram: visualization of confounding technical bias in gene expression data

    DEFF Research Database (Denmark)

    Krzystanek, Marcin; Szallasi, Zoltan Imre; Eklund, Aron Charles

    2013-01-01

    Gene expression profiles of clinical cohorts can be used to identify genes that are correlated with a clinical variable of interest such as patient outcome or response to a particular drug. However, expression measurements are susceptible to technical bias caused by variation in extraneous factors...... such as RNA quality and array hybridization conditions. If such technical bias is correlated with the clinical variable of interest, the likelihood of identifying false positive genes is increased. Here we describe a method to visualize an expression matrix as a projection of all genes onto a plane defined...... by a clinical variable and a technical nuisance variable. The resulting plot indicates the extent to which each gene is correlated with the clinical variable or the technical variable. We demonstrate this method by applying it to three clinical trial microarray data sets, one of which identified genes that may...

  10. Identification of reference genes in human myelomonocytic cells for gene expression studies in altered gravity.

    Science.gov (United States)

    Thiel, Cora S; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Unverdorben, Felix; Buttron, Isabell; Lauber, Beatrice; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E; Ullrich, Oliver

    2015-01-01

    Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes ("housekeeping genes") are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

  11. Simple Comparative Analyses of Differentially Expressed Gene Lists May Overestimate Gene Overlap.

    Science.gov (United States)

    Lawhorn, Chelsea M; Schomaker, Rachel; Rowell, Jonathan T; Rueppell, Olav

    2018-04-16

    Comparing the overlap between sets of differentially expressed genes (DEGs) within or between transcriptome studies is regularly used to infer similarities between biological processes. Significant overlap between two sets of DEGs is usually determined by a simple test. The number of potentially overlapping genes is compared to the number of genes that actually occur in both lists, treating every gene as equal. However, gene expression is controlled by transcription factors that bind to a variable number of transcription factor binding sites, leading to variation among genes in general variability of their expression. Neglecting this variability could therefore lead to inflated estimates of significant overlap between DEG lists. With computer simulations, we demonstrate that such biases arise from variation in the control of gene expression. Significant overlap commonly arises between two lists of DEGs that are randomly generated, assuming that the control of gene expression is variable among genes but consistent between corresponding experiments. More overlap is observed when transcription factors are specific to their binding sites and when the number of genes is considerably higher than the number of different transcription factors. In contrast, overlap between two DEG lists is always lower than expected when the genetic architecture of expression is independent between the two experiments. Thus, the current methods for determining significant overlap between DEGs are potentially confounding biologically meaningful overlap with overlap that arises due to variability in control of expression among genes, and more sophisticated approaches are needed.

  12. The gsdf gene locus harbors evolutionary conserved and clustered genes preferentially expressed in fish previtellogenic oocytes.

    Science.gov (United States)

    Gautier, Aude; Le Gac, Florence; Lareyre, Jean-Jacques

    2011-02-01

    The gonadal soma-derived factor (GSDF) belongs to the transforming growth factor-β superfamily and is conserved in teleostean fish species. Gsdf is specifically expressed in the gonads, and gene expression is restricted to the granulosa and Sertoli cells in trout and medaka. The gsdf gene expression is correlated to early testis differentiation in medaka and was shown to stimulate primordial germ cell and spermatogonia proliferation in trout. In the present study, we show that the gsdf gene localizes to a syntenic chromosomal fragment conserved among vertebrates although no gsdf-related gene is detected on the corresponding genomic region in tetrapods. We demonstrate using quantitative RT-PCR that most of the genes localized in the synteny are specifically expressed in medaka gonads. Gsdf is the only gene of the synteny with a much higher expression in the testis compared to the ovary. In contrast, gene expression pattern analysis of the gsdf surrounding genes (nup54, aff1, klhl8, sdad1, and ptpn13) indicates that these genes are preferentially expressed in the female gonads. The tissue distribution of these genes is highly similar in medaka and zebrafish, two teleostean species that have diverged more than 110 million years ago. The cellular localization of these genes was determined in medaka gonads using the whole-mount in situ hybridization technique. We confirm that gsdf gene expression is restricted to Sertoli and granulosa cells in contact with the premeiotic and meiotic cells. The nup54 gene is expressed in spermatocytes and previtellogenic oocytes. Transcripts corresponding to the ovary-specific genes (aff1, klhl8, and sdad1) are detected only in previtellogenic oocytes. No expression was detected in the gonocytes in 10 dpf embryos. In conclusion, we show that the gsdf gene localizes to a syntenic chromosomal fragment harboring evolutionary conserved genes in vertebrates. These genes are preferentially expressed in previtelloogenic oocytes, and thus, they

  13. Blood cell gene expression profiling in rheumatoid arthritis. Discriminative genes and effect of rheumatoid factor

    DEFF Research Database (Denmark)

    Bovin, Lone Frier; Rieneck, Klaus; Workman, Christopher

    2004-01-01

    To study the pathogenic importance of the rheumatoid factor (RF) in rheumatoid arthritis (RA) and to identify genes differentially expressed in patients and healthy individuals, total RNA was isolated from peripheral blood mononuclear cells (PBMC) from eight RF-positive and six RF-negative RA...... patients, and seven healthy controls. Gene expression of about 10,000 genes were examined using oligonucleotide-based DNA chip microarrays. The analyses showed no significant differences in PBMC expression patterns from RF-positive and RF-negative patients. However, comparisons of gene expression patterns...

  14. Plasticity-Related Gene Expression During Eszopiclone-Induced Sleep.

    Science.gov (United States)

    Gerashchenko, Dmitry; Pasumarthi, Ravi K; Kilduff, Thomas S

    2017-07-01

    Experimental evidence suggests that restorative processes depend on synaptic plasticity changes in the brain during sleep. We used the expression of plasticity-related genes to assess synaptic plasticity changes during drug-induced sleep. We first characterized sleep induced by eszopiclone in mice during baseline conditions and during the recovery from sleep deprivation. We then compared the expression of 18 genes and two miRNAs critically involved in synaptic plasticity in these mice. Gene expression was assessed in the cerebral cortex and hippocampus by the TaqMan reverse transcription polymerase chain reaction and correlated with sleep parameters. Eszopiclone reduced the latency to nonrapid eye movement (NREM) sleep and increased NREM sleep amounts. Eszopiclone had no effect on slow wave activity (SWA) during baseline conditions but reduced the SWA increase during recovery sleep (RS) after sleep deprivation. Gene expression analyses revealed three distinct patterns: (1) four genes had higher expression either in the cortex or hippocampus in the group of mice with increased amounts of wakefulness; (2) a large proportion of plasticity-related genes (7 out of 18 genes) had higher expression during RS in the cortex but not in the hippocampus; and (3) six genes and the two miRNAs showed no significant changes across conditions. Even at a relatively high dose (20 mg/kg), eszopiclone did not reduce the expression of plasticity-related genes during RS period in the cortex. These results indicate that gene expression associated with synaptic plasticity occurs in the cortex in the presence of a hypnotic medication. © Sleep Research Society 2017. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

  15. Molecular subsets in the gene expression signatures of scleroderma skin.

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    Ausra Milano

    2008-07-01

    Full Text Available Scleroderma is a clinically heterogeneous disease with a complex phenotype. The disease is characterized by vascular dysfunction, tissue fibrosis, internal organ dysfunction, and immune dysfunction resulting in autoantibody production.We analyzed the genome-wide patterns of gene expression with DNA microarrays in skin biopsies from distinct scleroderma subsets including 17 patients with systemic sclerosis (SSc with diffuse scleroderma (dSSc, 7 patients with SSc with limited scleroderma (lSSc, 3 patients with morphea, and 6 healthy controls. 61 skin biopsies were analyzed in a total of 75 microarray hybridizations. Analysis by hierarchical clustering demonstrates nearly identical patterns of gene expression in 17 out of 22 of the forearm and back skin pairs of SSc patients. Using this property of the gene expression, we selected a set of 'intrinsic' genes and analyzed the inherent data-driven groupings. Distinct patterns of gene expression separate patients with dSSc from those with lSSc and both are easily distinguished from normal controls. Our data show three distinct patient groups among the patients with dSSc and two groups among patients with lSSc. Each group can be distinguished by unique gene expression signatures indicative of proliferating cells, immune infiltrates and a fibrotic program. The intrinsic groups are statistically significant (p<0.001 and each has been mapped to clinical covariates of modified Rodnan skin score, interstitial lung disease, gastrointestinal involvement, digital ulcers, Raynaud's phenomenon and disease duration. We report a 177-gene signature that is associated with severity of skin disease in dSSc.Genome-wide gene expression profiling of skin biopsies demonstrates that the heterogeneity in scleroderma can be measured quantitatively with DNA microarrays. The diversity in gene expression demonstrates multiple distinct gene expression programs in the skin of patients with scleroderma.

  16. Gene expression profiling reveals multiple toxicity endpoints induced by hepatotoxicants

    Energy Technology Data Exchange (ETDEWEB)

    Huang Qihong; Jin Xidong; Gaillard, Elias T.; Knight, Brian L.; Pack, Franklin D.; Stoltz, James H.; Jayadev, Supriya; Blanchard, Kerry T

    2004-05-18

    Microarray technology continues to gain increased acceptance in the drug development process, particularly at the stage of toxicology and safety assessment. In the current study, microarrays were used to investigate gene expression changes associated with hepatotoxicity, the most commonly reported clinical liability with pharmaceutical agents. Acetaminophen, methotrexate, methapyrilene, furan and phenytoin were used as benchmark compounds capable of inducing specific but different types of hepatotoxicity. The goal of the work was to define gene expression profiles capable of distinguishing the different subtypes of hepatotoxicity. Sprague-Dawley rats were orally dosed with acetaminophen (single dose, 4500 mg/kg for 6, 24 and 72 h), methotrexate (1 mg/kg per day for 1, 7 and 14 days), methapyrilene (100 mg/kg per day for 3 and 7 days), furan (40 mg/kg per day for 1, 3, 7 and 14 days) or phenytoin (300 mg/kg per day for 14 days). Hepatic gene expression was assessed using toxicology-specific gene arrays containing 684 target genes or expressed sequence tags (ESTs). Principal component analysis (PCA) of gene expression data was able to provide a clear distinction of each compound, suggesting that gene expression data can be used to discern different hepatotoxic agents and toxicity endpoints. Gene expression data were applied to the multiplicity-adjusted permutation test and significantly changed genes were categorized and correlated to hepatotoxic endpoints. Repression of enzymes involved in lipid oxidation (acyl-CoA dehydrogenase, medium chain, enoyl CoA hydratase, very long-chain acyl-CoA synthetase) were associated with microvesicular lipidosis. Likewise, subsets of genes associated with hepatotocellular necrosis, inflammation, hepatitis, bile duct hyperplasia and fibrosis have been identified. The current study illustrates that expression profiling can be used to: (1) distinguish different hepatotoxic endpoints; (2) predict the development of toxic endpoints; and

  17. Bovine mammary gene expression profiling during the onset of lactation.

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    Yuanyuan Gao

    Full Text Available BACKGROUND: Lactogenesis includes two stages. Stage I begins a few weeks before parturition. Stage II is initiated around the time of parturition and extends for several days afterwards. METHODOLOGY/PRINCIPAL FINDINGS: To better understand the molecular events underlying these changes, genome-wide gene expression profiling was conducted using digital gene expression (DGE on bovine mammary tissue at three time points (on approximately day 35 before parturition (-35 d, day 7 before parturition (-7 d and day 3 after parturition (+3 d. Approximately 6.2 million (M, 5.8 million (M and 6.1 million (M 21-nt cDNA tags were sequenced in the three cDNA libraries (-35 d, -7 d and +3 d, respectively. After aligning to the reference sequences, the three cDNA libraries included 8,662, 8,363 and 8,359 genes, respectively. With a fold change cutoff criteria of ≥ 2 or ≤-2 and a false discovery rate (FDR of ≤ 0.001, a total of 812 genes were significantly differentially expressed at -7 d compared with -35 d (stage I. Gene ontology analysis showed that those significantly differentially expressed genes were mainly associated with cell cycle, lipid metabolism, immune response and biological adhesion. A total of 1,189 genes were significantly differentially expressed at +3 d compared with -7 d (stage II, and these genes were mainly associated with the immune response and cell cycle. Moreover, there were 1,672 genes significantly differentially expressed at +3 d compared with -35 d. Gene ontology analysis showed that the main differentially expressed genes were those associated with metabolic processes. CONCLUSIONS: The results suggest that the mammary gland begins to lactate not only by a gain of function but also by a broad suppression of function to effectively push most of the cell's resources towards lactation.

  18. Divergent and nonuniform gene expression patterns in mouse brain

    Science.gov (United States)

    Morris, John A.; Royall, Joshua J.; Bertagnolli, Darren; Boe, Andrew F.; Burnell, Josh J.; Byrnes, Emi J.; Copeland, Cathy; Desta, Tsega; Fischer, Shanna R.; Goldy, Jeff; Glattfelder, Katie J.; Kidney, Jolene M.; Lemon, Tracy; Orta, Geralyn J.; Parry, Sheana E.; Pathak, Sayan D.; Pearson, Owen C.; Reding, Melissa; Shapouri, Sheila; Smith, Kimberly A.; Soden, Chad; Solan, Beth M.; Weller, John; Takahashi, Joseph S.; Overly, Caroline C.; Lein, Ed S.; Hawrylycz, Michael J.; Hohmann, John G.; Jones, Allan R.

    2010-01-01

    Considerable progress has been made in understanding variations in gene sequence and expression level associated with phenotype, yet how genetic diversity translates into complex phenotypic differences remains poorly understood. Here, we examine the relationship between genetic background and spatial patterns of gene expression across seven strains of mice, providing the most extensive cellular-resolution comparative analysis of gene expression in the mammalian brain to date. Using comprehensive brainwide anatomic coverage (more than 200 brain regions), we applied in situ hybridization to analyze the spatial expression patterns of 49 genes encoding well-known pharmaceutical drug targets. Remarkably, over 50% of the genes examined showed interstrain expression variation. In addition, the variability was nonuniformly distributed across strain and neuroanatomic region, suggesting certain organizing principles. First, the degree of expression variance among strains mirrors genealogic relationships. Second, expression pattern differences were concentrated in higher-order brain regions such as the cortex and hippocampus. Divergence in gene expression patterns across the brain could contribute significantly to variations in behavior and responses to neuroactive drugs in laboratory mouse strains and may help to explain individual differences in human responsiveness to neuroactive drugs. PMID:20956311

  19. Anterior-posterior regionalized gene expression in the Ciona notochord.

    Science.gov (United States)

    Reeves, Wendy; Thayer, Rachel; Veeman, Michael

    2014-04-01

    In the simple ascidian chordate Ciona, the signaling pathways and gene regulatory networks giving rise to initial notochord induction are largely understood and the mechanisms of notochord morphogenesis are being systematically elucidated. The notochord has generally been thought of as a non-compartmentalized or regionalized organ that is not finely patterned at the level of gene expression. Quantitative imaging methods have recently shown, however, that notochord cell size, shape, and behavior vary consistently along the anterior-posterior (AP) axis. Here we screen candidate genes by whole mount in situ hybridization for potential AP asymmetry. We identify 4 genes that show non-uniform expression in the notochord. Ezrin/radixin/moesin (ERM) is expressed more strongly in the secondary notochord lineage than the primary. CTGF is expressed stochastically in a subset of notochord cells. A novel calmodulin-like gene (BCamL) is expressed more strongly at both the anterior and posterior tips of the notochord. A TGF-β ortholog is expressed in a gradient from posterior to anterior. The asymmetries in ERM, BCamL, and TGF-β expression are evident even before the notochord cells have intercalated into a single-file column. We conclude that the Ciona notochord is not a homogeneous tissue but instead shows distinct patterns of regionalized gene expression. Copyright © 2013 Wiley Periodicals, Inc.

  20. A deep auto-encoder model for gene expression prediction.

    Science.gov (United States)

    Xie, Rui; Wen, Jia; Quitadamo, Andrew; Cheng, Jianlin; Shi, Xinghua

    2017-11-17

    Gene expression is a key intermediate level that genotypes lead to a particular trait. Gene expression is affected by various factors including genotypes of genetic variants. With an aim of delineating the genetic impact on gene expression, we build a deep auto-encoder model to assess how good genetic variants will contribute to gene expression changes. This new deep learning model is a regression-based predictive model based on the MultiLayer Perceptron and Stacked Denoising Auto-encoder (MLP-SAE). The model is trained using a stacked denoising auto-encoder for feature selection and a multilayer perceptron framework for backpropagation. We further improve the model by introducing dropout to prevent overfitting and improve performance. To demonstrate the usage of this model, we apply MLP-SAE to a real genomic datasets with genotypes and gene expression profiles measured in yeast. Our results show that the MLP-SAE model with dropout outperforms other models including Lasso, Random Forests and the MLP-SAE model without dropout. Using the MLP-SAE model with dropout, we show that gene expression quantifications predicted by the model solely based on genotypes, align well with true gene expression patterns. We provide a deep auto-encoder model for predicting gene expression from SNP genotypes. This study demonstrates that deep learning is appropriate for tackling another genomic problem, i.e., building predictive models to understand genotypes' contribution to gene expression. With the emerging availability of richer genomic data, we anticipate that deep learning models play a bigger role in modeling and interpreting genomics.

  1. Micro-PIXE for single cell analysis

    International Nuclear Information System (INIS)

    Ortega, Richard

    2012-01-01

    The knowledge of the intracellular distribution of biological relevant metals is important to understand their mechanisms of action in cells, either for physiological, toxicological or pathological processes. However, the direct detection of trace metals in single cells is a challenging task that requires sophisticated analytical developments. The combination of micro-PIXE with RBS and STIM (Scanning Transmission Ion Microscopy) allows the quantitative determination of trace metal content within sub-cellular compartments. The application of STIM analysis provides high spatial resolution imaging (< 200 nm) and excellent mass sensitivity (< 0.1 ng). Application of the STIM-PIXE-RBS methodology is absolutely needed when organic mass loss appears during PIXE-RBS irradiation. This combination of STIM-PIXE-RBS provides fully quantitative determination of trace element content, expressed in μg/g, which is a quite unique capability for micro-PIXE compared to other micro-analytical methods such as the electron and synchrotron x-ray fluorescence. Examples of micro-PIXE studies for sub-cellular imaging of trace elements in various fields of interest will be presented: in patho-physiology of trace elements involved in neurodegenerative diseases such as Parkinson's disease, and in toxicology of metals such as cobalt. (author)

  2. Assays for noninvasive imaging of reporter gene expression

    International Nuclear Information System (INIS)

    Gambhir, S.S.; Barrio, J.R.; Herschman, H.R.; Phelps, M.E.

    1999-01-01

    Repeated, noninvasive imaging of reporter gene expression is emerging as a valuable tool for monitoring the expression of genes in animals and humans. Monitoring of organ/cell transplantation in living animals and humans, and the assessment of environmental, behavioral, and pharmacologic modulation of gene expression in transgenic animals should soon be possible. The earliest clinical application is likely to be monitoring human gene therapy in tumors transduced with the herpes simplex virus type 1 thymidine kinase (HSV1-tk) suicide gene. Several candidate assays for imaging reporter gene expression have been studied, utilizing cytosine deaminase (CD), HSV1-tk, and dopamine 2 receptor (D2R) as reporter genes. For the HSV1-tk reporter gene, both uracil nucleoside derivatives (e.g., 5-iodo-2'-fluoro-2'-deoxy-1-β-D-arabinofuranosyl-5-iodouracil [FIAU] labeled with 124 I, 131 I ) and acycloguanosine derivatives {e.g., 8-[ 18 F]fluoro-9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine (8-[ 18 F]-fluoroganciclovir) ([ 18 F]FGCV), 9-[(3-[ 18 F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([ 18 F]FHPG)} have been investigated as reporter probes. For the D2R reporter gene, a derivative of spiperone {3-(2'-[ 18 F]-Fluoroethyl)spiperone ([ 18 F]FESP)} has been used with positron emission tomography (PET) imaging. In this review, the principles and specific assays for imaging reporter gene expression are presented and discussed. Specific examples utilizing adenoviral-mediated delivery of a reporter gene as well as tumors expressing reporter genes are discussed

  3. Electrophysiological and gene expression characterization of the ontogeny of nestin-expressing cells in the adult mouse midbrain

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    Anupama Dey

    2017-08-01

    Full Text Available The birth of new neurons, or neurogenesis, in the adult midbrain is important for progressing dopamine cell-replacement therapies for Parkinson's disease. Most studies suggest newborn cells remain undifferentiated or differentiate into glia within the adult midbrain. However, some studies suggest nestin + neural precursor cells (NPCs have a propensity to generate new neurons here. We sought to confirm this by administering tamoxifen to adult NesCreERT2/R26eYFP transgenic mice, which permanently labelled adult nestin-expressing cells and their progeny with enhanced yellow fluorescent protein (eYFP. eYFP+ midbrain cells were then characterized 1–32 weeks later in acutely prepared brain slices using whole-cell patch clamp electrophysiology combined with single-cell RT-qPCR. Most eYFP+ cells exhibited a mature neuronal phenotype with large amplitude fast action potentials (APs, spontaneous post-synaptic currents (sPSCs, and expression of ‘mature’ neuronal genes (NeuN, Gad1, Gad2 and/or VGLUT2. This was the case even at the earliest time-point following tamoxifen (i.e. 1 week. In comparison to neighboring eYFP− (control cells, eYFP+ cells discharged more APs per unit current injection, and had faster AP time-to-peak, hyperpolarized resting membrane potential, smaller membrane capacitance and shorter duration sPSCs. eYFP+ cells were also differentiated from eYFP− cells by increased expression of ‘immature’ pro-neuronal genes (Pax6, Ngn2 and/or Msx1. However, further analyses failed to reveal evidence of a place of birth, neuronal differentiation, maturation and integration indicative of classical neurogenesis. Thus our findings do not support the notion that nestin + NPCs in the adult SNc and midbrain generate new neurons via classical neurogenesis. Rather, they raise the possibility that mature neurons express nestin under unknown circumstances, and that this is associated with altered physiology and gene expression.

  4. Systematic identification of human housekeeping genes possibly useful as references in gene expression studies.

    Science.gov (United States)

    Caracausi, Maria; Piovesan, Allison; Antonaros, Francesca; Strippoli, Pierluigi; Vitale, Lorenza; Pelleri, Maria Chiara

    2017-09-01

    The ideal reference, or control, gene for the study of gene expression in a given organism should be expressed at a medium‑high level for easy detection, should be expressed at a constant/stable level throughout different cell types and within the same cell type undergoing different treatments, and should maintain these features through as many different tissues of the organism. From a biological point of view, these theoretical requirements of an ideal reference gene appear to be best suited to housekeeping (HK) genes. Recent advancements in the quality and completeness of human expression microarray data and in their statistical analysis may provide new clues toward the quantitative standardization of human gene expression studies in biology and medicine, both cross‑ and within‑tissue. The systematic approach used by the present study is based on the Transcriptome Mapper tool and exploits the automated reassignment of probes to corresponding genes, intra‑ and inter‑sample normalization, elaboration and representation of gene expression values in linear form within an indexed and searchable database with a graphical interface recording quantitative levels of expression, expression variability and cross‑tissue width of expression for more than 31,000 transcripts. The present study conducted a meta‑analysis of a pool of 646 expression profile data sets from 54 different human tissues and identified actin γ 1 as the HK gene that best fits the combination of all the traditional criteria to be used as a reference gene for general use; two ribosomal protein genes, RPS18 and RPS27, and one aquaporin gene, POM121 transmembrane nucleporin C, were also identified. The present study provided a list of tissue‑ and organ‑specific genes that may be most suited for the following individual tissues/organs: Adipose tissue, bone marrow, brain, heart, kidney, liver, lung, ovary, skeletal muscle and testis; and also provides in these cases a representative

  5. A longitudinal study of gene expression in healthy individuals

    Directory of Open Access Journals (Sweden)

    Tessier Michel

    2009-06-01

    Full Text Available Abstract Background The use of gene expression in venous blood either as a pharmacodynamic marker in clinical trials of drugs or as a diagnostic test requires knowledge of the variability in expression over time in healthy volunteers. Here we defined a normal range of gene expression over 6 months in the blood of four cohorts of healthy men and women who were stratified by age (22–55 years and > 55 years and gender. Methods Eleven immunomodulatory genes likely to play important roles in inflammatory conditions such as rheumatoid arthritis and infection in addition to four genes typically used as reference genes were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR, as well as the full genome as represented by Affymetrix HG U133 Plus 2.0 microarrays. Results Gene expression levels as assessed by qRT-PCR and microarray were relatively stable over time with ~2% of genes as measured by microarray showing intra-subject differences over time periods longer than one month. Fifteen genes varied by gender. The eleven genes examined by qRT-PCR remained within a limited dynamic range for all individuals. Specifically, for the seven most stably expressed genes (CXCL1, HMOX1, IL1RN, IL1B, IL6R, PTGS2, and TNF, 95% of all samples profiled fell within 1.5–2.5 Ct, the equivalent of a 4- to 6-fold dynamic range. Two subjects who experienced severe adverse events of cancer and anemia, had microarray gene expression profiles that were distinct from normal while subjects who experienced an infection had only slightly elevated levels of inflammatory markers. Conclusion This study defines the range and variability of gene expression in healthy men and women over a six-month period. These parameters can be used to estimate the number of subjects needed to observe significant differences from normal gene expression in clinical studies. A set of genes that varied by gender was also identified as were a set of genes with elevated

  6. Gene expression during testis development in Duroc boars

    DEFF Research Database (Denmark)

    Lervik, Siri; Kristoffersen, Anja Bråthen; Conley, Lene

    2015-01-01

    . Nine clusters of genes with significant differential expression over time and 49 functional charts were found in the analysed testis samples. Prominent pathways in the prepubertal testis were associated with tissue renewal, cell respiration and increased endocytocis. E-cadherines may be associated...... with the onset of pubertal development. With elevated steroidogenesis (weeks 16 to 27), there was an increase in the expression of genes in the MAPK pathway, STAR and its analogue STARD6. A pubertal shift in genes coding for cellular cholesterol transport was observed. Increased expression of meiotic pathways...

  7. Cloning and selection of reference genes for gene expression ...

    African Journals Online (AJOL)

    Full length mRNA sequences of Ac-β-actin and Ac-gapdh, and partial mRNA sequences of Ac-18SrRNA and Ac-ubiquitin were cloned from pineapple in this study. The four genes were tested as housekeeping genes in three experimental sets. GeNorm and NormFinder analysis revealed that β-actin was the most ...

  8. Integration of biological networks and gene expression data using Cytoscape

    DEFF Research Database (Denmark)

    Cline, M.S.; Smoot, M.; Cerami, E.

    2007-01-01

    of an interaction network obtained for genes of interest. Five major steps are described: (i) obtaining a gene or protein network, (ii) displaying the network using layout algorithms, (iii) integrating with gene expression and other functional attributes, (iv) identifying putative complexes and functional modules......Cytoscape is a free software package for visualizing, modeling and analyzing molecular and genetic interaction networks. This protocol explains how to use Cytoscape to analyze the results of mRNA expression profiling, and other functional genomics and proteomics experiments, in the context...... and (v) identifying enriched Gene Ontology annotations in the network. These steps provide a broad sample of the types of analyses performed by Cytoscape....

  9. The Role of Nuclear Bodies in Gene Expression and Disease

    Science.gov (United States)

    Morimoto, Marie; Boerkoel, Cornelius F.

    2013-01-01

    This review summarizes the current understanding of the role of nuclear bodies in regulating gene expression. The compartmentalization of cellular processes, such as ribosome biogenesis, RNA processing, cellular response to stress, transcription, modification and assembly of spliceosomal snRNPs, histone gene synthesis and nuclear RNA retention, has significant implications for gene regulation. These functional nuclear domains include the nucleolus, nuclear speckle, nuclear stress body, transcription factory, Cajal body, Gemini of Cajal body, histone locus body and paraspeckle. We herein review the roles of nuclear bodies in regulating gene expression and their relation to human health and disease. PMID:24040563

  10. Cell-Type-Specific Transcriptome Analysis in the Drosophila Mushroom Body Reveals Memory-Related Changes in Gene Expression.

    Science.gov (United States)

    Crocker, Amanda; Guan, Xiao-Juan; Murphy, Coleen T; Murthy, Mala

    2016-05-17

    Learning and memory formation in Drosophila rely on a network of neurons in the mushroom bodies (MBs). Whereas numerous studies have delineated roles for individual cell types within this network in aspects of learning or memory, whether or not these cells can also be distinguished by the genes they express remains unresolved. In addition, the changes in gene expression that accompany long-term memory formation within the MBs have not yet been studied by neuron type. Here, we address both issues by performing RNA sequencing on single cell types (harvested via patch pipets) within the MB. We discover that the expression of genes that encode cell surface receptors is sufficient to identify cell types and that a subset of these genes, required for sensory transduction in peripheral sensory neurons, is not only expressed within individual neurons of the MB in the central brain, but is also critical for memory formation. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  11. Cloning of Plasmodium falciparum by single-cell sorting.

    Science.gov (United States)

    Miao, Jun; Li, Xiaolian; Cui, Liwang

    2010-10-01

    Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. Copyright 2010 Elsevier Inc. All rights reserved.

  12. Cloning of Plasmodium falciparum by single-cell sorting

    Science.gov (United States)

    Miao, Jun; Li, Xiaolian; Cui, Liwang

    2010-01-01

    Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. PMID:20435038

  13. In Vivo Imaging of mdrla Gene Expression

    National Research Council Canada - National Science Library

    Synold, Timothy W

    2005-01-01

    .... With the advent of new bioimaging technology and the advancement of efficient gene targeting strategies, they found an opportunity to apply these state-of-the-art molecular tools to their problem...

  14. Dihydrotestostenone increase the gene expression of androgen ...

    African Journals Online (AJOL)

    HNTEP cells were grown in basal medium and treated with DHT in different conditions. HNTEP cells under treatment with DHT (10-13 M) induced an increase in FHL-2 expression. In turn, high DHT concentrations (10-8 M) induced an increase in the expression SHP-1. The present data suggest that the SHP-1 and FHL-2 ...

  15. Epigenetics reloaded: the single-cell revolution.

    Science.gov (United States)

    Bheda, Poonam; Schneider, Robert

    2014-11-01

    Mechanistically, how epigenetic states are inherited through cellular divisions remains an important open question in the chromatin field and beyond. Defining the heritability of epigenetic states and the underlying chromatin-based mechanisms within a population of cells is complicated due to cell heterogeneity combined with varying levels of stability of these states; thus, efforts must be focused toward single-cell analyses. The approaches presented here constitute the forefront of epigenetics research at the single-cell level using classic and innovative methods to dissect epigenetics mechanisms from the limited material available in a single cell. This review further outlines exciting future avenues of research to address the significance of epigenetic heterogeneity and the contributions of microfluidics technologies to single-cell isolation and analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Gene duplication, silencing and expression alteration govern the molecular evolution of PRC2 genes in plants.

    Science.gov (United States)

    Furihata, Hazuka Y; Suenaga, Kazuya; Kawanabe, Takahiro; Yoshida, Takanori; Kawabe, Akira

    2016-10-13

    PRC2 genes were analyzed for their number of gene duplications, d N /d S ratios and expression patterns among Brassicaceae and Gramineae species. Although both amino acid sequences and copy number of the PRC2 genes were generally well conserved in both Brassicaceae and Gramineae species, we observed that some rapidly evolving genes experienced duplications and expression pattern changes. After multiple duplication events, all but one or two of the duplicated copies tend to be silenced. Silenced copies were reactivated in the endosperm and showed ectopic expression in developing seeds. The results indicated that rapid evolution of some PRC2 genes is initially caused by a relaxation of selective constraint following the gene duplication events. Several loci could become maternally expressed imprinted genes and acquired functional roles in the endosperm.

  17. Gene expression studies of reference genes for quantitative real-time PCR: an overview in insects.

    Science.gov (United States)

    Shakeel, Muhammad; Rodriguez, Alicia; Tahir, Urfa Bin; Jin, Fengliang

    2018-02-01

    Whenever gene expression is being examined, it is essential that a normalization process is carried out to eliminate non-biological variations. The use of reference genes, such as glyceraldehyde-3-phosphate dehydrogenase, actin, and ribosomal protein genes, is the usual method of choice for normalizing gene expression. Although reference genes are used to normalize target gene expression, a major problem is that the stability of these genes differs among tissues, developmental stages, species, and responses to abiotic factors. Therefore, the use and validation of multiple reference genes are required. This review discusses the reasons that why RT-qPCR has become the preferred method for validating results of gene expression profiles, the use of specific and non-specific dyes and the importance of use of primers and probes for qPCR as well as to discuss several statistical algorithms developed to help the validation of potential reference genes. The conflicts arising in the use of classical reference genes in gene normalization and their replacement with novel references are also discussed by citing the high stability and low stability of classical and novel reference genes under various biotic and abiotic experimental conditions by employing various methods applied for the reference genes amplification.

  18. Integrated olfactory receptor and microarray gene expression databases

    Directory of Open Access Journals (Sweden)

    Crasto Chiquito J

    2007-06-01

    Full Text Available Abstract Background Gene expression patterns of olfactory receptors (ORs are an important component of the signal encoding mechanism in the olfactory system since they determine the interactions between odorant ligands and sensory neurons. We have developed the Olfactory Receptor Microarray Database (ORMD to house OR gene expression data. ORMD is integrated with the Olfactory Receptor Database (ORDB, which is a key repository of OR gene information. Both databases aim to aid experimental research related to olfaction. Description ORMD is a Web-accessible database that provides a secure data repository for OR microarray experiments. It contains both publicly available and private data; accessing the latter requires authenticated login. The ORMD is designed to allow users to not only deposit gene expression data but also manage their projects/experiments. For example, contributors can choose whether to make their datasets public. For each experiment, users can download the raw data files and view and export the gene expression data. For each OR gene being probed in a microarray experiment, a hyperlink to that gene in ORDB provides access to genomic and proteomic information related to the corresponding olfactory receptor. Individual ORs archived in ORDB are also linked to ORMD, allowing users access to the related microarray gene expression data. Conclusion ORMD serves as a data repository and project management system. It facilitates the study of microarray experiments of gene expression in the olfactory system. In conjunction with ORDB, ORMD integrates gene expression data with the genomic and functional data of ORs, and is thus a useful resource for both olfactory researchers and the public.

  19. Gene Expression Measurement Module (GEMM) - A Fully Automated, Miniaturized Instrument for Measuring Gene Expression in Space

    Science.gov (United States)

    Pohorille, Andrew; Peyvan, Kia; Karouia, Fathi; Ricco, Antonio

    2012-01-01

    The capability to measure gene expression on board spacecraft opens the door to a large number of high-value experiments on the influence of the space environment on biological systems. For example, measurements of gene expression will help us to understand adaptation of terrestrial life to conditions beyond the planet of origin, identify deleterious effects of the space environment on a wide range of organisms from microbes to humans, develop effective countermeasures against these effects, and determine the metabolic bases of microbial pathogenicity and drug resistance. These and other applications hold significant potential for discoveries in space biology, biotechnology, and medicine. Supported by funding from the NASA Astrobiology Science and Technology Instrument Development Program, we are developing a fully automated, miniaturized, integrated fluidic system for small spacecraft capable of in-situ measurement of expression of several hundreds of microbial genes from multiple samples. The instrument will be capable of (1) lysing cell walls of bacteria sampled from cultures grown in space, (2) extracting and purifying RNA released from cells, (3) hybridizing the RNA on a microarray and (4) providing readout of the microarray signal, all in a single microfluidics cartridge. The device is suitable for deployment on nanosatellite platforms developed by NASA Ames' Small Spacecraft Division. To meet space and other technical constraints imposed by these platforms, a number of technical innovations are being implemented. The integration and end-to-end technological and biological validation of the instrument are carried out using as a model the photosynthetic bacterium Synechococcus elongatus, known for its remarkable metabolic diversity and resilience to adverse conditions. Each step in the measurement process-lysis, nucleic acid extraction, purification, and hybridization to an array-is assessed through comparison of the results obtained using the instrument with

  20. Gene expression patterns in pancreatic tumors, cells and tissues.

    Directory of Open Access Journals (Sweden)

    Anson W Lowe

    2007-03-01

    Full Text Available Cancers of the pancreas originate from both the endocrine and exocrine elements of the organ, and represent a major cause of cancer-related death. This study provides a comprehensive assessment of gene expression for pancreatic tumors, the normal pancreas, and nonneoplastic pancreatic disease.DNA microarrays were used to assess the gene expression for surgically derived pancreatic adenocarcinomas, islet cell tumors, and mesenchymal tumors. The addition of normal pancreata, isolated islets, isolated pancreatic ducts, and pancreatic adenocarcinoma cell lines enhanced subsequent analysis by increasing the diversity in gene expression profiles obtained. Exocrine, endocrine, and mesenchymal tumors displayed unique gene expression profiles. Similarities in gene expression support the pancreatic duct as the origin of adenocarcinomas. In addition, genes highly expressed in other cancers and associated with specific signal transduction pathways were also found in pancreatic tumors.The scope of the present work was enhanced by the inclusion of publicly available datasets that encompass a wide spectrum of human tissues and enabled the identification of candidate genes that may serve diagnostic and therapeutic goals.

  1. SIGNATURE: A workbench for gene expression signature analysis

    Directory of Open Access Journals (Sweden)

    Chang Jeffrey T

    2011-11-01

    Full Text Available Abstract Background The biological phenotype of a cell, such as a characteristic visual image or behavior, reflects activities derived from the expression of collections of genes. As such, an ability to measure the expression of these genes provides an opportunity to develop more precise and varied sets of phenotypes. However, to use this approach requires computational methods that are difficult to implement and apply, and thus there is a critical need for intelligent software tools that can reduce the technical burden of the analysis. Tools for gene expression analyses are unusually difficult to implement in a user-friendly way because their application requires a combination of biological data curation, statistical computational methods, and database expertise. Results We have developed SIGNATURE, a web-based resource that simplifies gene expression signature analysis by providing software, data, and protocols to perform the analysis successfully. This resource uses Bayesian methods for processing gene expression data coupled with a curated database of gene expression signatures, all carried out within a GenePattern web interface for easy use and access. Conclusions SIGNATURE is available for public use at http://genepattern.genome.duke.edu/signature/.

  2. Population and sex differences in Drosophila melanogaster brain gene expression

    Directory of Open Access Journals (Sweden)

    Catalán Ana

    2012-11-01

    Full Text Available Abstract Background Changes in gene regulation are thought to be crucial for the adaptation of organisms to their environment. Transcriptome analyses can be used to identify candidate genes for ecological adaptation, but can be complicated by variation in gene expression between tissues, sexes, or individuals. Here we use high-throughput RNA sequencing of a single Drosophila melanogaster tissue to detect brain-specific differences in gene expression between the sexes and between two populations, one from the ancestral species range in sub-Saharan Africa and one from the recently colonized species range in Europe. Results Relatively few genes (Cyp6g1 and CHKov1. Conclusions Analysis of the brain transcriptome revealed many genes differing in expression between populations that were not detected in previous studies using whole flies. There was little evidence for sex-specific regulatory adaptation in the brain, as most expression differences between populations were observed in both males and females. The enrichment of genes with sexually dimorphic expression on the X chromosome is consistent with dosage compensation mechanisms affecting sex-biased expression in somatic tissues.

  3. A comparative study of three different gene expression analysis methods.

    Science.gov (United States)

    Choe, Jae Young; Han, Hyung Soo; Lee, Seon Duk; Lee, Hanna; Lee, Dong Eun; Ahn, Jae Yun; Ryoo, Hyun Wook; Seo, Kang Suk; Kim, Jong Kun

    2017-12-04

    TNF-α regulates immune cells and acts as an endogenous pyrogen. Reverse transcription polymerase chain reaction (RT-PCR) is one of the most commonly used methods for gene expression analysis. Among the alternatives to PCR, loop-mediated isothermal amplification (LAMP) shows good potential in terms of specificity and sensitivity. However, few studies have compared RT-PCR and LAMP for human gene expression analysis. Therefore, in the present study, we compared one-step RT-PCR, two-step RT-LAMP and one-step RT-LAMP for human gene expression analysis. We compared three gene expression analysis methods using the human TNF-α gene as a biomarker from peripheral blood cells. Total RNA from the three selected febrile patients were subjected to the three different methods of gene expression analysis. In the comparison of three gene expression analysis methods, the detection limit of both one-step RT-PCR and one-step RT-LAMP were the same, while that of two-step RT-LAMP was inferior. One-step RT-LAMP takes less time, and the experimental result is easy to determine. One-step RT-LAMP is a potentially useful and complementary tool that is fast and reasonably sensitive. In addition, one-step RT-LAMP could be useful in environments lacking specialized equipment or expertise.

  4. Changes in gene expression during male meiosis in Petunia hybrida.

    Science.gov (United States)

    Cnudde, Filip; Hedatale, Veena; de Jong, Hans; Pierson, Elisabeth S; Rainey, Daphne Y; Zabeau, Marc; Weterings, Koen; Gerats, Tom; Peters, Janny L

    2006-01-01

    We analyzed changes in gene expression during male meiosis in Petunia by combining the meiotic staging of pollen mother cells from a single anther with cDNA-AFLP transcript profiling of mRNA from the synchronously developing sister anthers. The transcript profiling experiments focused on the identification of genes with a modulated expression profile during meiosis, while premeiotic archesporial cells and postmeiotic microspores served as a reference. About 8000 transcript tags, estimated at 30% of the total transcriptome, were generated, of which around 6% exhibited a modulated gene expression pattern at meiosis. Cluster analysis revealed a transcriptional cascade that coincides with the initiation and progression through all stages of the two meiotic divisions. Fragments that exhibited high expression specifically during meiosis I were characterized further by sequencing; 90 out of the 293 sequenced fragments showed homology with known genes, belonging to a wide range of gene classes, including previously characterized meiotic genes. In-situ hybridization experiments were performed to determine the spatial expression pattern for five selected transcript tags. Its concurrence with cDNA-AFLP transcript profiles indicates that this is an excellent approach to study genes involved in specialized processes such as meiosis. Our data set provides the potential to unravel unique meiotic genes that are as yet elusive to reverse genetics approaches.

  5. GESearch: An Interactive GUI Tool for Identifying Gene Expression Signature

    Directory of Open Access Journals (Sweden)

    Ning Ye

    2015-01-01

    Full Text Available The huge amount of gene expression data generated by microarray and next-generation sequencing technologies present challenges to exploit their biological meanings. When searching for the coexpression genes, the data mining process is largely affected by selection of algorithms. Thus, it is highly desirable to provide multiple options of algorithms in the user-friendly analytical toolkit to explore the gene expression signatures. For this purpose, we developed GESearch, an interactive graphical user interface (GUI toolkit, which is written in MATLAB and supports a variety of gene expression data files. This analytical toolkit provides four models, including the mean, the regression, the delegate, and the ensemble models, to identify the coexpression genes, and enables the users to filter data and to select gene expression patterns by browsing the display window or by importing knowledge-based genes. Subsequently, the utility of this analytical toolkit is demonstrated by analyzing two sets of real-life microarray datasets from cell-cycle experiments. Overall, we have developed an interactive GUI toolkit that allows for choosing multiple algorithms for analyzing the gene expression signatures.

  6. VH gene expression and regulation in the mutant Alicia rabbit. Rescue of VHa2 allotype expression.

    Science.gov (United States)

    Chen, H T; Alexander, C B; Young-Cooper, G O; Mage, R G

    1993-04-01

    Rabbits of the Alicia strain, derived from rabbits expressing the VHa2 allotype, have a mutation in the H chain locus that has a cis effect upon the expression of VHa2 and VHa- genes. A small deletion at the most J-proximal (3') end of the VH locus leads to low expression of all the genes on the entire chromosome in heterozygous ali mutants and altered relative expression of VH genes in homozygotes. To study VH gene expression and regulation, we used the polymerase chain reaction to amplify the VH genes expressed in spleens of young and adult wild-type and mutant Alicia rabbits. The cDNA from reverse transcription of splenic mRNA was amplified and polymerase chain reaction libraries were constructed and screened with oligonucleotides from framework regions 1 and 3, as well as JH. Thirty-three VH-positive clones were sequenced and analyzed. We found that in mutant Alicia rabbits, products of the first functional VH gene (VH4a2), (or VH4a2-like genes) were expressed in 2- to 8-wk-olds. Expression of both the VHx and VHy types of VHa- genes was also elevated but the relative proportions of VHx and VHy, especially VHx, decreased whereas the relative levels of expression of VH4a2 or VH4a2-like genes increased with age. Our results suggest that the appearance of sequences resembling that of the VH1a2, which is deleted in the mutant ali rabbits, could be caused by alterations of the sequences of the rearranged VH4a2 genes by gene conversions and/or rearrangement of upstream VH1a2-like genes later in development.

  7. Estradiol-induced gene expression in largemouth bass (Micropterus salmoides)

    Science.gov (United States)

    Bowman, C.J.; Kroll, K.J.; Gross, T.G.; Denslow, N.D.

    2002-01-01

    Vitellogenin (Vtg) and estrogen receptor (ER) gene expression levels were measured in largemouth bass to evaluate the activation of the ER-mediated pathway by estradiol (E2). Single injections of E2 ranging from 0.0005 to 5 mg/kg up-regulated plasma Vtg in a dose-dependent manner. Vtg and ER mRNAs were measured using partial cDNA sequences corresponding to the C-terminal domain for Vtg and the ligand-binding domain of ER?? sequences. After acute E2-exposures (2 mg/kg), Vtg and ER mRNAs and plasma Vtg levels peaked after 2 days. The rate of ER mRNA accumulation peaked 36-42 h earlier than Vtg mRNA. The expression window for ER defines the primary response to E2 in largemouth bass and that for Vtg a delayed primary response. The specific effect of E2 on other estrogen-regulated genes was tested during these same time windows using differential display RT-PCR. Specific up-regulated genes that are expressed in the same time window as Vtg were ERp72 (a membrane-bound disulfide isomerase) and a gene with homology to an expressed gene identified in zebrafish. Genes that were expressed in a pattern that mimics the ER include the gene for zona radiata protein ZP2, and a gene with homology to an expressed gene found in winter flounder. One gene for fibrinogen ?? was down-regulated and an unidentified gene was transiently up-regulated after 12 h of exposure and returned to basal levels by 48 h. Taken together these studies indicate that the acute molecular response to E2 involves a complex network of responses over time. ?? 2002 Elsevier Science Ireland Ltd. All rights reserved.

  8. Domestication rewired gene expression and nucleotide diversity patterns in tomato.

    Science.gov (United States)

    Sauvage, Christopher; Rau, Andrea; Aichholz, Charlotte; Chadoeuf, Joël; Sarah, Gautier; Ruiz, Manuel; Santoni, Sylvain; Causse, Mathilde; David, Jacques; Glémin, Sylvain

    2017-08-01

    Plant domestication has led to considerable phenotypic modifications from wild species to modern varieties. However, although changes in key traits have been well documented, less is known about the underlying molecular mechanisms, such as the reduction of molecular diversity or global gene co-expression patterns. In this study, we used a combination of gene expression and population genetics in wild and crop tomato to decipher the footprints of domestication. We found a set of 1729 differentially expressed genes (DEG) between the two genetic groups, belonging to 17 clusters of co-expressed DEG, suggesting that domestication affected not only individual genes but also regulatory networks. Five co-expression clusters were enriched in functional terms involving carbohydrate metabolism or epigenetic regulation of gene expression. We detected differences in nucleotide diversity between the crop and wild groups specific to DEG. Our study provides an extensive profiling of the rewiring of gene co-expression induced by the domestication syndrome in one of the main crop species. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  9. The human cumulus--oocyte complex gene-expression profile

    Science.gov (United States)

    Assou, Said; Anahory, Tal; Pantesco, Véronique; Le Carrour, Tanguy; Pellestor, Franck; Klein, Bernard; Reyftmann, Lionel; Dechaud, Hervé; De Vos, John; Hamamah, Samir

    2006-01-01

    BACKGROUND The understanding of the mechanisms regulating human oocyte maturation is still rudimentary. We have identified transcripts differentially expressed between immature and mature oocytes, and cumulus cells. METHODS Using oligonucleotides microarrays, genome wide gene expression was studied in pooled immature and mature oocytes or cumulus cells from patients who underwent IVF. RESULTS In addition to known genes such as DAZL, BMP15 or GDF9, oocytes upregulated 1514 genes. We show that PTTG3 and AURKC are respectively the securin and the Aurora kinase preferentially expressed during oocyte meiosis. Strikingly, oocytes overexpressed previously unreported growth factors such as TNFSF13/APRIL, FGF9, FGF14, and IL4, and transcription factors including OTX2, SOX15 and SOX30. Conversely, cumulus cells, in addition to known genes such as LHCGR or BMPR2, overexpressed cell-tocell signaling genes including TNFSF11/RANKL, numerous complement components, semaphorins (SEMA3A, SEMA6A, SEMA6D) and CD genes such as CD200. We also identified 52 genes progressively increasing during oocyte maturation, comprising CDC25A and SOCS7. CONCLUSION The identification of genes up and down regulated during oocyte maturation greatly improves our understanding of oocyte biology and will provide new markers that signal viable and competent oocytes. Furthermore, genes found expressed in cumulus cells are potential markers of granulosa cell tumors. PMID:16571642

  10. AffyMiner: mining differentially expressed genes and biological knowledge in GeneChip microarray data

    Directory of Open Access Journals (Sweden)

    Xia Yuannan

    2006-12-01

    Full Text Available Abstract Background DNA microarrays are a powerful tool for monitoring the expression of tens of thousands of genes simultaneously. With the advance of microarray technology, the challenge issue becomes how to analyze a large amount of microarray data and make biological sense of them. Affymetrix GeneChips are widely used microarrays, where a variety of statistical algorithms have been explored and used for detecting significant genes in the experiment. These methods rely solely on the quantitative data, i.e., signal intensity; however, qualitative data are also important parameters in detecting differentially expressed genes. Results AffyMiner is a tool developed for detecting differentially expressed genes in Affymetrix GeneChip microarray data and for associating gene annotation and gene ontology information with the genes detected. AffyMiner consists of the functional modules, GeneFinder for detecting significant genes in a treatment versus control experiment and GOTree for mapping genes of interest onto the Gene Ontology (GO space; and interfaces to run Cluster, a program for clustering analysis, and GenMAPP, a program for pathway analysis. AffyMiner has been used for analyzing the GeneChip data and the results were presented in several publications. Conclusion AffyMiner fills an important gap in finding differentially expressed genes in Affymetrix GeneChip microarray data. AffyMiner effectively deals with multiple replicates in the experiment and takes into account both quantitative and qualitative data in identifying significant genes. AffyMiner reduces the time and effort needed to compare data from multiple arrays and to interpret the possible biological implications associated with significant changes in a gene's expression.

  11. Single-cell magnetic imaging using a quantum diamond microscope.

    Science.gov (United States)

    Glenn, D R; Lee, K; Park, H; Weissleder, R; Yacoby, A; Lukin, M D; Lee, H; Walsworth, R L; Connolly, C B

    2015-08-01

    We apply a quantum diamond microscope for detection and imaging of immunomagnetically labeled cells. This instrument uses nitrogen-vacancy (NV) centers in diamond for correlated magnetic and fluorescence imaging. Our device provides single-cell resolution and a field of view (∼1 mm(2)) two orders of magnitude larger than that of previous NV imaging technologies, enabling practical applications. To illustrate, we quantified cancer biomarkers expressed by rare tumor cells in a large population of healthy cells.

  12. Pervasive Effects of Aging on Gene Expression in Wild Wolves

    Science.gov (United States)

    Charruau, Pauline; Johnston, Rachel A.; Stahler, Daniel R.; Lea, Amanda; Snyder-Mackler, Noah; Smith, Douglas W.; vonHoldt, Bridgett M.; Cole, Steven W.; Tung, Jenny; Wayne, Robert K.

    2016-01-01

    Abstract Gene expression levels change as an individual ages and responds to environmental conditions. With the exception of humans, such patterns have principally been studied under controlled conditions, overlooking the array of developmental and environmental influences that organisms encounter under conditions in which natural selection operates. We used high-throughput RNA sequencing (RNA-Seq) of whole blood to assess the relative impacts of social status, age, disease, and sex on gene expression levels in a natural population of gray wolves (Canis lupus). Our findings suggest that age is broadly associated with gene expression levels, whereas other examined factors have minimal effects on gene expression patterns. Further, our results reveal evolutionarily conserved signatures of senescence, such as immunosenescence and metabolic aging, between wolves and humans despite major differences in life history and environment. The effects of aging on gene expression levels in wolves exhibit conservation with humans, but the more rapid expression differences observed in aging wolves is evolutionarily appropriate given the species’ high level of extrinsic mortality due to intraspecific aggression. Some expression changes that occur with age can facilitate physical age-related changes that may enhance fitness in older wolves. However, the expression of these ancestral patterns of aging in descendant modern dogs living in highly modified domestic environments may be maladaptive and cause disease. This work provides evolutionary insight into aging patterns observed in domestic dogs and demonstrates the applicability of studying natural populations to investigate the mechanisms of aging. PMID:27189566

  13. Expression of streptavidin gene in bacteria and plants

    International Nuclear Information System (INIS)

    Guan, Xueni; Wurtele, E.S.; Nikolau, B.J.

    1990-01-01

    Six biotin-containing proteins are present in plants, representing at least four different biotin enzymes. The physiological function of these biotin enzymes is not understood. Streptavidin, a protein from Streptomyces avidinii, binds tightly and specifically to biotin causing inactivation of biotin enzymes. One approach to elucidating the physiological function of biotin enzymes in plant metabolism is to create transgenic plants expressing the streptavidin gene. A plasmid containing a fused streptavidin-beta-galactosidase gene has been expressed in E. coli. We also have constructed various fusion genes that include an altered CaMV 35S promoter, signal peptides to target the streptavidin protein to specific organelles, and the streptavidin coding gene. We are examining the expression of these genes in cells of carrot

  14. The evolution of gene expression levels in mammalian organs

    DEFF Research Database (Denmark)

    Brawand, David; Soumillon, Magali; Necsulea, Anamaria

    2011-01-01

    and chromosomes, owing to differences in selective pressures: transcriptome change was slow in nervous tissues and rapid in testes, slower in rodents than in apes and monotremes, and rapid for the X chromosome right after its formation. Although gene expression evolution in mammals was strongly shaped......Changes in gene expression are thought to underlie many of the phenotypic differences between species. However, large-scale analyses of gene expression evolution were until recently prevented by technological limitations. Here we report the sequencing of polyadenylated RNA from six organs across...... ten species that represent all major mammalian lineages (placentals, marsupials and monotremes) and birds (the evolutionary outgroup), with the goal of understanding the dynamics of mammalian transcriptome evolution. We show that the rate of gene expression evolution varies among organs, lineages...

  15. Gene expression programming for power system static security ...

    African Journals Online (AJOL)

    user

    Keywords: static security, gene expression programming, probabilistic neural network ... Hence digital computers are usually installed in operations control centers to gather ...... power system protection, and applications of AI in power systems.

  16. GAL4 enhancer trap strains with reporter gene expression during ...

    Indian Academy of Sciences (India)

    the development of adult brain in Drosophila melanogaster. C. R. VENKATESH ... vous system (CNS), at different time points during the pupal stage—a critical .... in frontal view, with further reduced reporter gene expression. Orthodenticle and ...

  17. Research Article Gene expression profiling for coronary artery ...

    Indian Academy of Sciences (India)

    Shiridhar Kashyap

    stored at -80˚C in nuclease free water for gene expression experiments. ..... So, identification of a unique signature for CAD globally as treatment target and early diagnostic biomarker needs ..... The colour of bar, blue, brown, grey and yellow.

  18. Global analysis of differential expressed genes in ECV304 ...

    African Journals Online (AJOL)

    EB

    Abstract. Background: Human cytomegalovirus (HCMV) is a virus which has the potential to alter cellular gene expression through .... and (reverse: 5'-CAG CAC CAT CCT CCT CTT. CCT CT ..... acute respiratory syndrome (SARS) coronavirus.

  19. Enhancement of plasmid-mediated stable gene expression by ...

    African Journals Online (AJOL)

    ARL

    2012-06-12

    Jun 12, 2012 ... production and faithful translation and processing of proteins (Baldi et al., ..... deeper understanding of the interaction of cellular factors and regulatory DNA .... mediated transgene expression in the rat brain. Gene Ther., 7: ...

  20. Long SAGE analysis of genes differentially expressed in the midgut ...

    African Journals Online (AJOL)

    Long SAGE analysis of genes differentially expressed in the midgut and silk gland between the sexes of the silkwormBombyx mori. Liping Gan, Ying Wang, Jian Xi, Yanshan Niu, Hongyou Qin, Yanghu Sima, Shiqing Xu ...

  1. State-related alterations of gene expression in bipolar disorder

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Vinberg, Maj; Berk, Michael

    2012-01-01

    Munkholm K, Vinberg M, Berk M, Kessing LV. State-related alterations of gene expression in bipolar disorder: a systematic review. Bipolar Disord 2012: 14: 684-696. © 2012 The Authors. Journal compilation © 2012 John Wiley & Sons A/S. Objective:  Alterations in gene expression in bipolar disorder...... have been found in numerous studies. It is unclear whether such alterations are related to specific mood states. As a biphasic disorder, mood state-related alterations in gene expression have the potential to point to markers of disease activity, and trait-related alterations might indicate...... vulnerability pathways. This review therefore evaluated the evidence for whether gene expression in bipolar disorder is state or trait related. Methods:  A systematic review, using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guideline for reporting systematic reviews, based...

  2. Tyrosine Kinase Gene Expression Profiling in Prostate Cancer

    National Research Council Canada - National Science Library

    Weier, Heinz-Ulrich

    2001-01-01

    ... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

  3. Tyrosine Kinase Gene Expression Profiling in Prostate Cancer

    National Research Council Canada - National Science Library

    Weier, Heinz-Ulrich

    2002-01-01

    ... of these genes parallels the progression of tumors to a more malignant phenotype. We developed a DNA micro-array based screening system to monitor the level of expression of tyrosine kinase (tk...

  4. Visually Relating Gene Expression and in vivo DNA Binding Data

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Min-Yu; Mackey, Lester; Ker?,; nen, Soile V. E.; Weber, Gunther H.; Jordan, Michael I.; Knowles, David W.; Biggin, Mark D.; Hamann, Bernd

    2011-09-20

    Gene expression and in vivo DNA binding data provide important information for understanding gene regulatory networks: in vivo DNA binding data indicate genomic regions where transcription factors are bound, and expression data show the output resulting from this binding. Thus, there must be functional relationships between these two types of data. While visualization and data analysis tools exist for each data type alone, there is a lack of tools that can easily explore the relationship between them. We propose an approach that uses the average expression driven by multiple of ciscontrol regions to visually relate gene expression and in vivo DNA binding data. We demonstrate the utility of this tool with examples from the network controlling early Drosophila development. The results obtained support the idea that the level of occupancy of a transcription factor on DNA strongly determines the degree to which the factor regulates a target gene, and in some cases also controls whether the regulation is positive or negative.

  5. A role for gene duplication and natural variation of gene expression in the evolution of metabolism.

    Directory of Open Access Journals (Sweden)

    Daniel J Kliebenstein

    Full Text Available BACKGROUND: Most eukaryotic genomes have undergone whole genome duplications during their evolutionary history. Recent studies have shown that the function of these duplicated genes can diverge from the ancestral gene via neo- or sub-functionalization within single genotypes. An additional possibility is that gene duplicates may also undergo partitioning of function among different genotypes of a species leading to genetic differentiation. Finally, the ability of gene duplicates to diverge may be limited by their biological function. METHODOLOGY/PRINCIPAL FINDINGS: To test these hypotheses, I estimated the impact of gene duplication and metabolic function upon intraspecific gene expression variation of segmental and tandem duplicated genes within Arabidopsis thaliana. In all instances, the younger tandem duplicated genes showed higher intraspecific gene expression variation than the average Arabidopsis gene. Surprisingly, the older segmental duplicates also showed evidence of elevated intraspecific gene expression variation albeit typically lower than for the tandem duplicates. The specific biological function of the gene as defined by metabolic pathway also modulated the level of intraspecific gene expression variation. The major energy metabolism and biosynthetic pathways showed decreased variation, suggesting that they are constrained in their ability to accumulate gene expression variation. In contrast, a major herbivory defense pathway showed significantly elevated intraspecific variation suggesting that it may be under pressure to maintain and/or generate diversity in response to fluctuating insect herbivory pressures. CONCLUSION: These data show that intraspecific variation in gene expression is facilitated by an interaction of gene duplication and biological activity. Further, this plays a role in controlling diversity of plant metabolism.

  6. Multiscale Embedded Gene Co-expression Network Analysis.

    Directory of Open Access Journals (Sweden)

    Won-Min Song

    2015-11-01

    Full Text Available Gene co-expression network analysis has been shown effective in identifying functional co-expressed gene modules associated with complex human diseases. However, existing techniques to construct co-expression networks require some critical prior information such as predefined number of clusters, numerical thresholds for defining co-expression/interaction, or do not naturally reproduce the hallmarks of complex systems such as the scale-free degree distribution of small-worldness. Previously, a graph filtering technique called Planar Maximally Filtered Graph (PMFG has been applied to many real-world data sets such as financial stock prices and gene expression to extract meaningful and relevant interactions. However, PMFG is not suitable for large-scale genomic data due to several drawbacks, such as the high computation complexity O(|V|3, the presence of false-positives due to the maximal planarity constraint, and the inadequacy of the clustering framework. Here, we developed a new co-expression network analysis framework called Multiscale Embedded Gene Co-expression Network Analysis (MEGENA by: i introducing quality control of co-expression similarities, ii parallelizing embedded network construction, and iii developing a novel clustering technique to identify multi-scale clustering structures in Planar Filtered Networks (PFNs. We applied MEGENA to a series of simulated data and the gene expression data in breast carcinoma and lung adenocarcinoma from The Cancer Genome Atlas (TCGA. MEGENA showed improved performance over well-established clustering methods and co-expression network construction approaches. MEGENA revealed not only meaningful multi-scale organizations of co-expressed gene clusters but also novel targets in breast carcinoma and lung adenocarcinoma.

  7. Multiscale Embedded Gene Co-expression Network Analysis.

    Science.gov (United States)

    Song, Won-Min; Zhang, Bin

    2015-11-01

    Gene co-expression network analysis has been shown effective in identifying functional co-expressed gene modules associated with complex human diseases. However, existing techniques to construct co-expression networks require some critical prior information such as predefined number of clusters, numerical thresholds for defining co-expression/interaction, or do not naturally reproduce the hallmarks of complex systems such as the scale-free degree distribution of small-worldness. Previously, a graph filtering technique called Planar Maximally Filtered Graph (PMFG) has been applied to many real-world data sets such as financial stock prices and gene expression to extract meaningful and relevant interactions. However, PMFG is not suitable for large-scale genomic data due to several drawbacks, such as the high computation complexity O(|V|3), the presence of false-positives due to the maximal planarity constraint, and the inadequacy of the clustering framework. Here, we developed a new co-expression network analysis framework called Multiscale Embedded Gene Co-expression Network Analysis (MEGENA) by: i) introducing quality control of co-expression similarities, ii) parallelizing embedded network construction, and iii) developing a novel clustering technique to identify multi-scale clustering structures in Planar Filtered Networks (PFNs). We applied MEGENA to a series of simulated data and the gene expression data in breast carcinoma and lung adenocarcinoma from The Cancer Genome Atlas (TCGA). MEGENA showed improved performance over well-established clustering methods and co-expression network construction approaches. MEGENA revealed not only meaningful multi-scale organizations of co-expressed gene clusters but also novel targets in breast carcinoma and lung adenocarcinoma.

  8. Ranking candidate disease genes from gene expression and protein interaction: a Katz-centrality based approach.

    Directory of Open Access Journals (Sweden)

    Jing Zhao

    Full Text Available Many diseases have complex genetic causes, where a set of alleles can affect the propensity of getting the disease. The identification of such disease genes is important to understand the mechanistic and evolutionary aspects of pathogenesis, improve diagnosis and treatment of the disease, and aid in drug discovery. Current genetic studies typically identify chromosomal regions associated specific diseases. But picking out an unknown disease gene from hundreds of candidates located on the same genomic interval is still challenging. In this study, we propose an approach to prioritize candidate genes by integrating data of gene expression level, protein-protein interaction strength and known disease genes. Our method is based only on two, simple, biologically motivated assumptions--that a gene is a good disease-gene candidate if it is differentially expressed in cases and controls, or that it is close to other disease-gene candidates in its protein interaction network. We tested our method on 40 diseases in 58 gene expression datasets of the NCBI Gene Expression Omnibus database. On these datasets our method is able to predict unknown disease genes as well as identifying pleiotropic genes involved in the physiological cellular processes of many diseases. Our study not only provides an effective algorithm for prioritizing candidate disease genes but is also a way to discover phenotypic interdependency, cooccurrence and shared pathophysiology between different disorders.

  9. Equivalent Gene Expression Profiles between Glatopa™ and Copaxone®.

    Directory of Open Access Journals (Sweden)

    Josephine S D'Alessandro

    Full Text Available Glatopa™ is a generic glatiramer acetate recently approved for the treatment of patients with relapsing forms of multiple sclerosis. Gene expression profiling was performed as a means to evaluate equivalence of Glatopa and Copaxone®. Microarray analysis containing 39,429 unique probes across the entire genome was performed in murine glatiramer acetate--responsive Th2-polarized T cells, a test system highly relevant to the biology of glatiramer acetate. A closely related but nonequivalent glatiramoid molecule was used as a control to establish assay sensitivity. Multiple probe-level (Student's t-test and sample-level (principal component analysis, multidimensional scaling, and hierarchical clustering statistical analyses were utilized to look for differences in gene expression induced by the test articles. The analyses were conducted across all genes measured, as well as across a subset of genes that were shown to be modulated by Copaxone. The following observations were made across multiple statistical analyses: the expression of numerous genes was significantly changed by treatment with Copaxone when compared against media-only control; gene expression profiles induced by Copaxone and Glatopa were not significantly different; and gene expression profiles induced by Copaxone and the nonequivalent glatiramoid were significantly different, underscoring the sensitivity of the test system and the multiple analysis methods. Comparative analysis was also performed on sets of transcripts relevant to T-cell biology and antigen presentation, among others that are known to be modulated by glatiramer acetate. No statistically significant differences were observed between Copaxone and Glatopa in the expression levels (magnitude and direction of these glatiramer acetate-regulated genes. In conclusion, multiple methods consistently supported equivalent gene expression profiles between Copaxone and Glatopa.

  10. Caffeine exposure alters cardiac gene expression in embryonic cardiomyocytes

    Science.gov (United States)

    Fang, Xiefan; Mei, Wenbin; Barbazuk, William B.; Rivkees, Scott A.

    2014-01-01

    Previous studies demonstrated that in utero caffeine treatment at embryonic day (E) 8.5 alters DNA methylation patterns, gene expression, and cardiac function in adult mice. To provide insight into the mechanisms, we examined cardiac gene and microRNA (miRNA) expression in cardiomyocytes shortly after exposure to physiologically relevant doses of caffeine. In HL-1 and primary embryonic cardiomyocytes, caffeine treatment for 48 h significantly altered the expression of cardiac structural genes (Myh6, Myh7, Myh7b, Tnni3), hormonal genes (Anp and BnP), cardiac transcription factors (Gata4, Mef2c, Mef2d, Nfatc1), and microRNAs (miRNAs; miR208a, miR208b, miR499). In addition, expressions of these genes were significantly altered in embryonic hearts exposed to in utero caffeine. For in utero experiments, pregnant CD-1 dams were treated with 20–60 mg/kg of caffeine, which resulted in maternal circulation levels of 37.3–65.3 μM 2 h after treatment. RNA sequencing was performed on embryonic ventricles treated with vehicle or 20 mg/kg of caffeine daily from E6.5-9.5. Differential expression (DE) analysis revealed that 124 genes and 849 transcripts were significantly altered, and differential exon usage (DEU) analysis identified 597 exons that were changed in response to prenatal caffeine exposure. Among the DE genes identified by RNA sequencing were several cardiac structural genes and genes that control DNA methylation and histone modification. Pathway analysis revealed that pathways related to cardiovascular development and diseases were significantly affected by caffeine. In addition, global cardiac DNA methylation was reduced in caffeine-treated cardiomyocytes. Collectively, these data demonstrate that caffeine exposure alters gene expression and DNA methylation in embryonic cardiomyocytes. PMID:25354728

  11. Gene Expression Analysis of Four Radiation-resistant Bacteria

    OpenAIRE

    Gao, Na; Ma, Bin-Guang; Zhang, Yu-Sheng; Song, Qin; Chen, Ling-Ling; Zhang, Hong-Yu

    2009-01-01

    To investigate the general radiation-resistant mechanisms of bacteria, bioinformatic method was employed to predict highly expressed genes for four radiation-resistant bacteria, i.e. Deinococcus geothermalis (D. geo), Deinococcus radiodurans (D. rad), Kineococcus radiotolerans (K. rad) and Rubrobacter xylanophilus (R. xyl). It is revealed that most of the three reference gene sets, i.e. ribosomal proteins, transcription factors and major chaperones, are generally highly expressed in the four ...

  12. Multiobjective optimization in Gene Expression Programming for Dew Point

    OpenAIRE

    Shroff, Siddharth; Dabhi, Vipul

    2013-01-01

    The processes occurring in climatic change evolution and their variations play a major role in environmental engineering. Different techniques are used to model the relationship between temperatures, dew point and relative humidity. Gene expression programming is capable of modelling complex realities with great accuracy, allowing, at the same time, the extraction of knowledge from the evolved models compared to other learning algorithms. This research aims to use Gene Expression Programming ...

  13. Semi-supervised consensus clustering for gene expression data analysis

    OpenAIRE

    Wang, Yunli; Pan, Youlian

    2014-01-01

    Background Simple clustering methods such as hierarchical clustering and k-means are widely used for gene expression data analysis; but they are unable to deal with noise and high dimensionality associated with the microarray gene expression data. Consensus clustering appears to improve the robustness and quality of clustering results. Incorporating prior knowledge in clustering process (semi-supervised clustering) has been shown to improve the consistency between the data partitioning and do...

  14. Bioconductor workflow for single-cell RNA sequencing: Normalization, dimensionality reduction, clustering, and lineage inference [version 1; referees: 1 approved, 2 approved with reservations

    Directory of Open Access Journals (Sweden)

    Fanny Perraudeau

    2017-07-01

    Full Text Available Novel single-cell transcriptome sequencing assays allow researchers to measure gene expression levels at the resolution of single cells and offer the unprecendented opportunity to investigate at the molecular level fundamental biological questions, such as stem cell differentiation or the discovery and characterization of rare cell types. However, such assays raise challenging statistical and computational questions and require the development of novel methodology and software. Using stem cell differentiation in the mouse olfactory epithelium as a case study, this integrated workflow provides a step-by-step tutorial to the methodology and associated software for the following four main tasks: (1 dimensionality reduction accounting for zero inflation and over dispersion and adjusting for gene and cell-level covariates; (2 cell clustering using resampling-based sequential ensemble clustering; (3 inference of cell lineages and pseudotimes; and (4 differential expression analysis along lineages.

  15. Identifying key genes in rheumatoid arthritis by weighted gene co-expression network analysis.

    Science.gov (United States)

    Ma, Chunhui; Lv, Qi; Teng, Songsong; Yu, Yinxian; Niu, Kerun; Yi, Chengqin

    2017-08-01

    This study aimed to identify rheumatoid arthritis (RA) related genes based on microarray data using the WGCNA (weighted gene co-expression network analysis) method. Two gene expression profile datasets GSE55235 (10 RA samples and 10 healthy controls) and GSE77298 (16 RA samples and seven healthy controls) were downloaded from Gene Expression Omnibus database. Characteristic genes were identified using metaDE package. WGCNA was used to find disease-related networks based on gene expression correlation coefficients, and module significance was defined as the average gene significance of all genes used to assess the correlation between the module and RA status. Genes in the disease-related gene co-expression network were subject to functional annotation and pathway enrichment analysis using Database for Annotation Visualization and Integrated Discovery. Characteristic genes were also mapped to the Connectivity Map to screen small molecules. A total of 599 characteristic genes were identified. For each dataset, characteristic genes in the green, red and turquoise modules were most closely associated with RA, with gene numbers of 54, 43 and 79, respectively. These genes were enriched in totally enriched in 17 Gene Ontology terms, mainly related to immune response (CD97, FYB, CXCL1, IKBKE, CCR1, etc.), inflammatory response (CD97, CXCL1, C3AR1, CCR1, LYZ, etc.) and homeostasis (C3AR1, CCR1, PLN, CCL19, PPT1, etc.). Two small-molecule drugs sanguinarine and papaverine were predicted to have a therapeutic effect against RA. Genes related to immune response, inflammatory response and homeostasis presumably have critical roles in RA pathogenesis. Sanguinarine and papaverine have a potential therapeutic effect against RA. © 2017 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.

  16. Dissecting human cerebral organoids and fetal neocortex using single-cell RNAseq

    Science.gov (United States)

    Treutlein, Barbara

    Cerebral organoids - three-dimensional cultures of human cerebral tissue derived from pluripotent stem cells - have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and novel interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages, and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue in order to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.

  17. Evaluation of the similarity of gene expression data estimated with SAGE and Affymetrix GeneChips

    NARCIS (Netherlands)

    van Ruissen, Fred; Ruijter, Jan M.; Schaaf, Gerben J.; Asgharnegad, Lida; Zwijnenburg, Danny A.; Kool, Marcel; Baas, Frank

    2005-01-01

    Background: Serial Analysis of Gene Expression ( SAGE) and microarrays have found awidespread application, but much ambiguity exists regarding the evaluation of these technologies. Cross-platform utilization of gene expression data from the SAGE and microarray technology could reduce the need for

  18. Design parameters to control synthetic gene expression in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Mark Welch

    Full Text Available BACKGROUND: Production of proteins as therapeutic agents, research reagents and molecular tools frequently depends on expression in heterologous hosts. Synthetic genes are increasingly used for protein production because sequence information is easier to obtain than the corresponding physical DNA. Protein-coding sequences are commonly re-designed to enhance expression, but there are no experimentally supported design principles. PRINCIPAL FINDINGS: To identify sequence features that affect protein expression we synthesized and expressed in E. coli two sets of 40 genes encoding two commercially valuable proteins, a DNA polymerase and a single chain antibody. Genes differing only in synonymous codon usage expressed protein at levels ranging from undetectable to 30% of cellular protein. Using partial least squares regression we tested the correlation of protein production levels with parameters that have been reported to affect expression. We found that the amount of protein produced in E. coli was strongly dependent on the codons used to encode a subset of amino acids. Favorable codons were predominantly those read by tRNAs that are most highly charged during amino acid starvation, not codons that are most abundant in highly expressed E. coli proteins. Finally we confirmed the validity of our models by designing, synthesizing and testing new genes using codon biases predicted to perform well. CONCLUSION: The systematic analysis of gene design parameters shown in this study has allowed us to identify codon usage within a gene as a critical determinant of achievable protein expression levels in E. coli. We propose a biochemical basis for this, as well as design algorithms to ensure high protein production from synthetic genes. Replication of this methodology should allow similar design algorithms to be empirically derived for any expression system.

  19. Evaluation of suitable reference genes for gene expression studies in bovine muscular tissue

    Directory of Open Access Journals (Sweden)

    Dunner Susana

    2008-09-01

    Full Text Available Abstract Background Real-time reverse transcriptase quantitative polymerase chain reaction (real-time RTqPCR is a technique used to measure mRNA species copy number as a way to determine key genes involved in different biological processes. However, the expression level of these key genes may vary among tissues or cells not only as a consequence of differential expression but also due to different factors, including choice of reference genes to normalize the expression levels of the target genes; thus the selection of reference genes is critical for expression studies. For this purpose, ten candidate reference genes were investigated in bovine muscular tissue. Results The value of stability of ten candidate reference genes included in three groups was estimated: the so called 'classical housekeeping' genes (18S, GAPDH and ACTB, a second set of genes used in expression studies conducted on other tissues (B2M, RPII, UBC and HMBS and a third set of novel genes (SF3A1, EEF1A2 and CASC3. Three different statistical algorithms were used to rank the genes by their stability measures as produced by geNorm, NormFinder and Bestkeeper. The three methods tend to agree on the most stably expressed genes and the least in muscular tissue. EEF1A2 and HMBS followed by SF3A1, ACTB, and CASC3 can be considered as stable reference genes, and B2M, RPII, UBC and GAPDH would not be appropriate. Although the rRNA-18S stability measure seems to be within the range of acceptance, its use is not recommended because its synthesis regulation is not representative of mRNA levels. Conclusion Based on geNorm algorithm, we propose the use of three genes SF3A1, EEF1A2 and HMBS as references for normalization of real-time RTqPCR in muscle expression studies.

  20. Gene expression profiles in stages II and III colon cancers

    DEFF Research Database (Denmark)

    Thorsteinsson, Morten; Kirkeby, Lene T; Hansen, Raino

    2012-01-01

    PURPOSE: A 128-gene signature has been proposed to predict outcome in patients with stages II and III colorectal cancers. In the present study, we aimed to reproduce and validate the 128-gene signature in external and independent material. METHODS: Gene expression data from the original material...... were retrieved from the Gene Expression Omnibus (GEO) (n¿=¿111) in addition to a Danish data set (n¿=¿37). All patients had stages II and III colon cancers. A Prediction Analysis of Microarray classifier, based on the 128-gene signature and the original training set of stage I (n¿=¿65) and stage IV (n...... correctly predicted as stage IV-like, and the remaining patients were predicted as stage I-like and unclassifiable, respectively. Stage II patients could not be stratified. CONCLUSIONS: The 128-gene signature showed reproducibility in stage III colon cancer, but could not predict recurrence in stage II...

  1. Serial analysis of gene expression (SAGE) in rat liver regeneration

    International Nuclear Information System (INIS)

    Cimica, Velasco; Batusic, Danko; Haralanova-Ilieva, Borislava; Chen, Yonglong; Hollemann, Thomas; Pieler, Tomas; Ramadori, Giuliano

    2007-01-01

    We have applied serial analysis of gene expression for studying the molecular mechanism of the rat liver regeneration in the model of 70% partial hepatectomy. We generated three SAGE libraries from a normal control liver (NL library: 52,343 tags), from a sham control operated liver (Sham library: 51,028 tags), and from a regenerating liver (PH library: 53,061 tags). By SAGE bioinformatics analysis we identified 40 induced genes and 20 repressed genes during the liver regeneration. We verified temporal expression of such genes by real time PCR during the regeneration process and we characterized 13 induced genes and 3 repressed genes. We found connective tissue growth factor transcript and protein induced very early at 4 h after PH operation before hepatocytes proliferation is triggered. Our study suggests CTGF as a growth factor signaling mediator that could be involved directly in the mechanism of liver regeneration induction

  2. Ebola virus infection induces irregular dendritic cell gene expression.

    Science.gov (United States)

    Melanson, Vanessa R; Kalina, Warren V; Williams, Priscilla

    2015-02-01

    Filoviruses subvert the human immune system in part by infecting and replicating in dendritic cells (DCs). Using gene arrays, a phenotypic profile of filovirus infection in human monocyte-derived DCs was assessed. Monocytes from human donors were cultured in GM-CSF and IL-4 and were infected with Ebola virus Kikwit variant for up to 48 h. Extracted DC RNA was analyzed on SuperArray's Dendritic and Antigen Presenting Cell Oligo GEArray and compared to uninfected controls. Infected DCs exhibited increased expression of cytokine, chemokine, antiviral, and anti-apoptotic genes not seen in uninfected controls. Significant increases of intracellular antiviral and MHC I and II genes were also noted in EBOV-infected DCs. However, infected DCs failed to show any significant difference in co-stimulatory T-cell gene expression from uninfected DCs. Moreover, several chemokine genes were activated, but there was sparse expression of chemokine receptors that enabled activated DCs to home to lymph nodes. Overall, statistically significant expression of several intracellular antiviral genes was noted, which may limit viral load but fails to stop replication. EBOV gene expression profiling is of vital importance in understanding pathogenesis and devising novel therapeutic treatments such as small-molecule inhibitors.

  3. Random Subspace Aggregation for Cancer Prediction with Gene Expression Profiles

    Directory of Open Access Journals (Sweden)

    Liying Yang

    2016-01-01

    Full Text Available Background. Precisely predicting cancer is crucial for cancer treatment. Gene expression profiles make it possible to analyze patterns between genes and cancers on the genome-wide scale. Gene expression data analysis, however, is confronted with enormous challenges for its characteristics, such as high dimensionality, small sample size, and low Signal-to-Noise Ratio. Results. This paper proposes a method, termed RS_SVM, to predict gene expression profiles via aggregating SVM trained on random subspaces. After choosing gene features through statistical analysis, RS_SVM randomly selects feature subsets to yield random subspaces and training SVM classifiers accordingly and then aggregates SVM classifiers to capture the advantage of ensemble learning. Experiments on eight real gene expression datasets are performed to validate the RS_SVM method. Experimental results show that RS_SVM achieved better classification accuracy and generalization performance in contrast with single SVM, K-nearest neighbor, decision tree, Bagging, AdaBoost, and the state-of-the-art methods. Experiments also explored the effect of subspace size on prediction performance. Conclusions. The proposed RS_SVM method yielded superior performance in analyzing gene expression profiles, which demonstrates that RS_SVM provides a good channel for such biological data.

  4. Drosophila Myc is required for normal DREF gene expression

    International Nuclear Information System (INIS)

    Dang Thi Phuong Thao; Seto, Hirokazu; Yamaguchi, Masamitsu

    2008-01-01

    The Drosophila DNA replication-related element-binding factor (dDREF) is required for the expression of many proliferation-related genes carrying the DRE sequence, 5'-TATCGATA. Finding a canonical E-box, 5'-CACGTG, in the dDREF gene promoter prompted us to explore the possibility that the dDREF gene is a target of Drosophila Myc (dMyc). Luciferase transient expression assays combined with RNA interference in Drosophila S2 cells revealed that knockdown of dmyc reduced dDREF gene promoter activity by 35% to 82%, an effect at least partly mediated by the E-box in the promoter. dm 4 /Y hemizygous mutant larvae demonstrated no maternal dMyc and severe impairment of dDREF mRNA transcription. dMyc loss of function in dm 2 /dm 2 homozygous mutant follicle cell clones also resulted in loss of anti-dDREF immunostaining in nuclei. In contrast, co-expression of dMyc-dMax up-regulated dDREF promoter activity in S2 cells. Furthermore, dMyc over-expressing clones exhibited a high level of dDREF gene expression in wing and eye discs. These results taken together indicate that dMyc is indeed required for dDREF gene expression

  5. Screening for interaction effects in gene expression data.

    Directory of Open Access Journals (Sweden)

    Peter J Castaldi

    Full Text Available Expression quantitative trait (eQTL studies are a powerful tool for identifying genetic variants that affect levels of messenger RNA. Since gene expression is controlled by a complex network of gene-regulating factors, one way to identify these factors is to search for interaction effects between genetic variants and mRNA levels of transcription factors (TFs and their respective target genes. However, identification of interaction effects in gene expression data pose a variety of methodological challenges, and it has become clear that such analyses should be conducted and interpreted with caution. Investigating the validity and interpretability of several interaction tests when screening for eQTL SNPs whose effect on the target gene expression is modified by the expression level of a transcription factor, we characterized two important methodological issues. First, we stress the scale-dependency of interaction effects and highlight that commonly applied transformation of gene expression data can induce or remove interactions, making interpretation of results more challenging. We then demonstrate that, in the setting of moderate to strong interaction effects on the order of what may be reasonably expected for eQTL studies, standard interaction screening can be biased due to heteroscedasticity induced by true interactions. Using simulation and real data analysis, we outline a set of reasonable minimum conditions and sample size requirements for reliable detection of variant-by-environment and variant-by-TF interactions using the heteroscedasticity consistent covariance-based approach.

  6. Vascular Gene Expression in Nonneoplastic and Malignant Brain

    Science.gov (United States)

    Madden, Stephen L.; Cook, Brian P.; Nacht, Mariana; Weber, William D.; Callahan, Michelle R.; Jiang, Yide; Dufault, Michael R.; Zhang, Xiaoming; Zhang, Wen; Walter-Yohrling, Jennifer; Rouleau, Cecile; Akmaev, Viatcheslav R.; Wang, Clarence J.; Cao, Xiaohong; St. Martin, Thia B.; Roberts, Bruce L.; Teicher, Beverly A.; Klinger, Katherine W.; Stan, Radu-Virgil; Lucey, Brenden; Carson-Walter, Eleanor B.; Laterra, John; Walter, Kevin A.

    2004-01-01

    Malignant gliomas are uniformly lethal tumors whose morbidity is mediated in large part by the angiogenic response of the brain to the invading tumor. This profound angiogenic response leads to aggressive tumor invasion and destruction of surrounding brain tissue as well as blood-brain barrier breakdown and life-threatening cerebral edema. To investigate the molecular mechanisms governing the proliferation of abnormal microvasculature in malignant brain tumor patients, we have undertaken a cell-specific transcriptome analysis from surgically harvested nonneoplastic and tumor-associated endothelial cells. SAGE-derived endothelial cell gene expression patterns from glioma and nonneoplastic brain tissue reveal distinct gene expression patterns and consistent up-regulation of certain glioma endothelial marker genes across patient samples. We define the G-protein-coupled receptor RDC1 as a tumor endothelial marker whose expression is distinctly induced in tumor endothelial cells of both brain and peripheral vasculature. Further, we demonstrate that the glioma-induced gene, PV1, shows expression both restricted to endothelial cells and coincident with endothelial cell tube formation. As PV1 provides a framework for endothelial cell caveolar diaphragms, this protein may serve to enhance glioma-induced disruption of the blood-brain barrier and transendothelial exchange. Additional characterization of this extensive brain endothelial cell gene expression database will provide unique molecular insights into vascular gene expression. PMID:15277233

  7. Paternal irradiation perturbs the expression of circadian genes in offspring

    International Nuclear Information System (INIS)

    Gomes, Andre M.G.F.; Barber, Ruth C.; Dubrova, Yuri E.

    2015-01-01

    Highlights: • We have analysed gene expression in the offspring of irradiated male mice. • CBA/Ca and BALB/c male mice were used in our study. • The pattern of gene expression was established in four tissues. • Expression of genes in involved in rhythmic process/circadian rhythm is compromised. • Our data may explain the phenomenon of transgenerational genomic instability. - Abstract: The circadian system represents a complex network which influences the timing of many biological processes. Recent studies have established that circadian alterations play an important role in the susceptibility to many human diseases, including cancer. Here we report that paternal irradiation in mice significantly affects the expression of genes involved in rhythmic processes in their first-generation offspring. Using microarrays, the patterns of gene expression were established for brain, kidney, liver and spleen samples from the non-exposed offspring of irradiated CBA/Ca and BALB/c male mice. The most over-represented categories among the genes differentially expressed in the offspring of control and irradiated males were those involved in rhythmic process, circadian rhythm and DNA-dependent regulation of transcription. The results of our study therefore provide a plausible explanation for the transgenerational effects of paternal irradiation, including increased transgenerational carcinogenesis described in other studies

  8. Paternal irradiation perturbs the expression of circadian genes in offspring

    Energy Technology Data Exchange (ETDEWEB)

    Gomes, Andre M.G.F.; Barber, Ruth C.; Dubrova, Yuri E., E-mail: yed2@le.ac.uk

    2015-05-15

    Highlights: • We have analysed gene expression in the offspring of irradiated male mice. • CBA/Ca and BALB/c male mice were used in our study. • The pattern of gene expression was established in four tissues. • Expression of genes in involved in rhythmic process/circadian rhythm is compromised. • Our data may explain the phenomenon of transgenerational genomic instability. - Abstract: The circadian system represents a complex network which influences the timing of many biological processes. Recent studies have established that circadian alterations play an important role in the susceptibility to many human diseases, including cancer. Here we report that paternal irradiation in mice significantly affects the expression of genes involved in rhythmic processes in their first-generation offspring. Using microarrays, the patterns of gene expression were established for brain, kidney, liver and spleen samples from the non-exposed offspring of irradiated CBA/Ca and BALB/c male mice. The most over-represented categories among the genes differentially expressed in the offspring of control and irradiated males were those involved in rhythmic process, circadian rhythm and DNA-dependent regulation of transcription. The results of our study therefore provide a plausible explanation for the transgenerational effects of paternal irradiation, including increased transgenerational carcinogenesis described in other studies.

  9. Evaluation of Appropriate Reference Genes for Gene Expression Normalization during Watermelon Fruit Development.

    Directory of Open Access Journals (Sweden)

    Qiusheng Kong

    Full Text Available Gene expression analysis in watermelon (Citrullus lanatus fruit has drawn considerable attention with the availability of genome sequences to understand the regulatory mechanism of fruit development and to improve its quality. Real-time quantitative reverse-transcription PCR (qRT-PCR is a routine technique for gene expression analysis. However, appropriate reference genes for transcript normalization in watermelon fruits have not been well characterized. The aim of this study was to evaluate the appropriateness of 12 genes for their potential use as reference genes in watermelon fruits. Expression variations of these genes were measured in 48 samples obtained from 12 successive developmental stages of parthenocarpic and fertilized fruits of two watermelon genotypes by using qRT-PCR analysis. Considering the effects of genotype, fruit setting method, and developmental stage, geNorm determined clathrin adaptor complex subunit (ClCAC, β-actin (ClACT, and alpha tubulin 5 (ClTUA5 as the multiple reference genes in watermelon fruit. Furthermore, ClCAC alone or together with SAND family protein (ClSAND was ranked as the single or two best reference genes by NormFinder. By using the top-ranked reference genes to normalize the transcript abundance of phytoene synthase (ClPSY1, a good correlation between lycopene accumulation and ClPSY1 expression pattern was observed in ripening watermelon fruit. These validated reference genes will facilitate the accurate measurement of gene expression in the studies on watermelon fruit biology.

  10. Evaluation of Appropriate Reference Genes for Gene Expression Normalization during Watermelon Fruit Development.

    Science.gov (United States)

    Kong, Qiusheng; Yuan, Jingxian; Gao, Lingyun; Zhao, Liqiang; Cheng, Fei; Huang, Yuan; Bie, Zhilong

    2015-01-01

    Gene expression analysis in watermelon (Citrullus lanatus) fruit has drawn considerable attention with the availability of genome sequences to understand the regulatory mechanism of fruit development and to improve its quality. Real-time quantitative reverse-transcription PCR (qRT-PCR) is a routine technique for gene expression analysis. However, appropriate reference genes for transcript normalization in watermelon fruits have not been well characterized. The aim of this study was to evaluate the appropriateness of 12 genes for their potential use as reference genes in watermelon fruits. Expression variations of these genes were measured in 48 samples obtained from 12 successive developmental stages of parthenocarpic and fertilized fruits of two watermelon genotypes by using qRT-PCR analysis. Considering the effects of genotype, fruit setting method, and developmental stage, geNorm determined clathrin adaptor complex subunit (ClCAC), β-actin (ClACT), and alpha tubulin 5 (ClTUA5) as the multiple reference genes in watermelon fruit. Furthermore, ClCAC alone or together with SAND family protein (ClSAND) was ranked as the single or two best reference genes by NormFinder. By using the top-ranked reference genes to normalize the transcript abundance of phytoene synthase (ClPSY1), a good correlation between lycopene accumulation and ClPSY1 expression pattern was observed in ripening watermelon fruit. These validated reference genes will facilitate the accurate measurement of gene expression in the studies on watermelon fruit biology.

  11. Importance of globin gene order for correct developmental expression.

    NARCIS (Netherlands)

    O. Hanscombe (Olivia); D. Whyatt (David); P.J. Fraser (Peter); N. Yannoutsos (Nikos); D.R. Greaves (David); N.O. Dillon (Niall); F.G. Grosveld (Frank)

    1991-01-01

    textabstractWe have used transgenic mice to study the influence of position of the human globin genes relative to the locus control region (LCR) on their expression pattern during development. The LCR, which is located 5' of the globin gene cluster, is normally required for the activation of all the

  12. NORMAL NASAL GENE EXPRESSION LEVELS USING CDNA ARRAY TECHNOLOGY

    Science.gov (United States)

    Normal Nasal Gene Expression Levels Using cDNA Array Technology. The nasal epithelium is a target site for chemically-induced toxicity and carcinogenicity. To detect and analyze genetic events which contribute to nasal tumor development, we first defined the gene expressi...

  13. Comparison of gene expression profiles in Bacillus megaterium ...

    African Journals Online (AJOL)

    Abstract. The MP agent, prepared from Bacillus megaterium isolated from the soil near tobacco fields, can improve metabolic products, and hence the aroma, of tobacco (Nicotiana tabacum) leaf. To explore genes regulating metabolic responses in tobacco leaf, we used microarrays to analyze differentially expressed genes ...

  14. Flies selected for longevity retain a young gene expression profile

    DEFF Research Database (Denmark)

    Sarup, Pernille Merete; Sørensen, Peter; Loeschcke, Volker

    2011-01-01

      We investigated correlated responses in the transcriptomes of longevity-selected lines of Drosophila melanogaster to identify pathways that affect life span in metazoan systems. We evaluated the gene expression profile in young, middle-aged, and old male flies, finding that 530 genes were...

  15. Gene expression profiling of chicken intestinal host responses

    NARCIS (Netherlands)

    Hemert, van S.

    2007-01-01

    Chicken lines differ in genetic disease susceptibility. The scope of the research described in this thesis was to identify genes involved in genetic disease resistance in the chicken intestine. Therefore gene expression in the jejunum was investigated using a microarray approach. An intestine

  16. GOBO: gene expression-based outcome for breast cancer online.

    Directory of Open Access Journals (Sweden)

    Markus Ringnér

    Full Text Available Microarray-based gene expression analysis holds promise of improving prognostication and treatment decisions for breast cancer patients. However, the heterogeneity of breast cancer emphasizes the need for validation of prognostic gene signatures in larger sample sets stratified into relevant subgroups. Here, we describe a multifunctional user-friendly online tool, GOBO (http://co.bmc.lu.se/gobo, allowing a range of different analyses to be performed in an 1881-sample breast tumor data set, and a 51-sample breast cancer cell line set, both generated on Affymetrix U133A microarrays. GOBO supports a wide range of applications including: 1 rapid assessment of gene expression levels in subgroups of breast tumors and cell lines, 2 identification of co-expressed genes for creation of potential metagenes, 3 association with outcome for gene expression levels of single genes, sets of genes, or gene signatures in multiple subgroups of the 1881-sample breast cancer data set. The design and implementation of GOBO facilitate easy incorporation of additional query functions and applications, as well as additional data sets irrespective of tumor type and array platform.

  17. Gene Expression Signature in Endemic Osteoarthritis by Microarray Analysis

    Directory of Open Access Journals (Sweden)

    Xi Wang

    2015-05-01

    Full Text Available Kashin-Beck Disease (KBD is an endemic osteochondropathy with an unknown pathogenesis. Diagnosis of KBD is effective only in advanced cases, which eliminates the possibility of early treatment and leads to an inevitable exacerbation of symptoms. Therefore, we aim to identify an accurate blood-based gene signature for the detection of KBD. Previously published gene expression profile data on cartilage and peripheral blood mononuclear cells (PBMCs from adults with KBD were compared to select potential target genes. Microarray analysis was conducted to evaluate the expression of the target genes in a cohort of 100 KBD patients and 100 healthy controls. A gene expression signature was identified using a training set, which was subsequently validated using an independent test set with a minimum redundancy maximum relevance (mRMR algorithm and support vector machine (SVM algorithm. Fifty unique genes were differentially expressed between KBD patients and healthy controls. A 20-gene signature was identified that distinguished between KBD patients and controls with 90% accuracy, 85% sensitivity, and 95% specificity. This study identified a 20-gene signature that accurately distinguishes between patients with KBD and controls using peripheral blood samples. These results promote the further development of blood-based genetic biomarkers for detection of KBD.

  18. Identification of salt-stress induced differentially expressed genes in ...

    African Journals Online (AJOL)

    Identification of salt-stress induced differentially expressed genes in barley leaves using the annealingcontrol- primer-based GeneFishing technique. S Lee, K Lee, K Kim, GJ Choi, SH Yoon, HC Ji, S Seo, YC Lim, N Ahsan ...

  19. A Critical Perspective On Microarray Breast Cancer Gene Expression Profiling

    NARCIS (Netherlands)

    Sontrop, H.M.J.

    2015-01-01

    Microarrays offer biologists an exciting tool that allows the simultaneous assessment of gene expression levels for thousands of genes at once. At the time of their inception, microarrays were hailed as the new dawn in cancer biology and oncology practice with the hope that within a decade diseases

  20. Analysis and visualization of gene expression data using ...

    African Journals Online (AJOL)

    Several clustering and biclustering methods have been introduced to analyze the gene expression data by identifying the similar patterns and grouping genes into subsets that share biological significance. However, it is not clear how the different methods compare with each other with respect to the biological relevance of ...

  1. Differential expressions of putative genes in various floral organs of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-06-03

    Jun 3, 2009 ... Full Length Research Paper. Differential expressions of putative genes in various floral organs of the Pigeon orchid (Dendrobium crumenatum) using GeneFishing. Faridah, Q. Z.1, 2, Ng, B. Z.3, Raha, A. R.4, Umi, K. A. B.5 and Khosravi, A. R.2*. 1Department of Biology, Faculty Science, University Putra ...

  2. Genes differentially expressed in medulloblastoma and fetal brain

    NARCIS (Netherlands)

    Michiels, E. M.; Oussoren, E.; van Groenigen, M.; Pauws, E.; Bossuyt, P. M.; Voûte, P. A.; Baas, F.

    1999-01-01

    Serial analysis of gene expression (SAGE) was used to identify genes that might be involved in the development or growth of medulloblastoma, a childhood brain tumor. Sequence tags from medulloblastoma (10229) and fetal brain (10692) were determined. The distributions of sequence tags in each

  3. GSNFS: Gene subnetwork biomarker identification of lung cancer expression data.

    Science.gov (United States)

    Doungpan, Narumol; Engchuan, Worrawat; Chan, Jonathan H; Meechai, Asawin

    2016-12-05

    Gene expression has been used to identify disease gene biomarkers, but there are ongoing challenges. Single gene or gene-set biomarkers are inadequate to provide sufficient understanding of complex disease mechanisms and the relationship among those genes. Network-based methods have thus been considered for inferring the interaction within a group of genes to further study the disease mechanism. Recently, the Gene-Network-based Feature Set (GNFS), which is capable of handling case-control and multiclass expression for gene biomarker identification, has been proposed, partly taking into account of network topology. However, its performance relies on a greedy search for building subnetworks and thus requires further improvement. In this work, we establish a new approach named Gene Sub-Network-based Feature Selection (GSNFS) by implementing the GNFS framework with two proposed searching and scoring algorithms, namely gene-set-based (GS) search and parent-node-based (PN) search, to identify subnetworks. An additional dataset is used to validate the results. The two proposed searching algorithms of the GSNFS method for subnetwork expansion are concerned with the degree of connectivity and the scoring scheme for building subnetworks and their topology. For each iteration of expansion, the neighbour genes of a current subnetwork, whose expression data improved the overall subnetwork score, is recruited. While the GS search calculated the subnetwork score using an activity score of a current subnetwork and the gene expression values of its neighbours, the PN search uses the expression value of the corresponding parent of each neighbour gene. Four lung cancer expression datasets were used for subnetwork identification. In addition, using pathway data and protein-protein interaction as network data in order to consider the interaction among significant genes were discussed. Classification was performed to compare the performance of the identified gene subnetworks with three

  4. Alteration of gene expression by alcohol exposure at early neurulation.

    Science.gov (United States)

    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

  5. Single-cell analyses identify bioengineered niches for enhanced maintenance of hematopoietic stem cells.

    Science.gov (United States)

    Roch, Aline; Giger, Sonja; Girotra, Mukul; Campos, Vasco; Vannini, Nicola; Naveiras, Olaia; Gobaa, Samy; Lutolf, Matthias P

    2017-08-09

    The in vitro expansion of long-term hematopoietic stem cells (HSCs) remains a substantial challenge, largely because of our limited understanding of the mechanisms that control HSC fate choices. Using single-cell multigene expression analysis and time-lapse microscopy, here we define gene expression signatures and cell cycle hallmarks of murine HSCs and the earliest multipotent progenitors (MPPs), and analyze systematically single HSC fate choices in culture. Our analysis revealed twelve differentially expressed genes marking the quiescent HSC state, including four genes encoding cell-cell interaction signals in the niche. Under basal culture conditions, most HSCs rapidly commit to become early MPPs. In contrast, when we present ligands of the identified niche components such as JamC or Esam within artificial niches, HSC cycling is reduced and long-term multipotency in vivo is maintained. Our approach to bioengineer artificial niches should be useful in other stem cell systems.Haematopoietic stem cell (HSC) self-renewal is not sufficiently understood to recapitulate in vitro. Here, the authors generate gene signature and cell cycle hallmarks of single murine HSCs, and use identified endothelial receptors Esam and JamC as substrates to enhance HSC growth in engineered niches.

  6. CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification

    Directory of Open Access Journals (Sweden)

    Tamar Hashimshony

    2012-09-01

    Full Text Available High-throughput sequencing has allowed for unprecedented detail in gene expression analyses, yet its efficient application to single cells is challenged by the small starting amounts of RNA. We have developed CEL-Seq, a method for overcoming this limitation by barcoding and pooling samples before linearly amplifying mRNA with the use of one round of in vitro transcription. We show that CEL-Seq gives more reproducible, linear, and sensitive results than a PCR-based amplification method. We demonstrate the power of this method by studying early C. elegans embryonic development at single-cell resolution. Differential distribution of transcripts between sister cells is seen as early as the two-cell stage embryo, and zygotic expression in the somatic cell lineages is enriched for transcription factors. The robust transcriptome quantifications enabled by CEL-Seq will be useful for transcriptomic analyses of complex tissues containing populations of diverse cell types.

  7. Combined histochemical staining, RNA amplification, regional, and single cell cDNA analysis within the hippocampus.

    Science.gov (United States)

    Ginsberg, Stephen D; Che, Shaoli

    2004-08-01

    The use of five histochemical stains (cresyl violet, thionin, hematoxylin & eosin, silver stain, and acridine orange) was evaluated in combination with an expression profiling paradigm that included regional and single cell analyses within the hippocampus of post-mortem human brains and adult mice. Adjacent serial sections of human and mouse hippocampus were labeled by histochemistry or neurofilament immunocytochemistry. These tissue sections were used as starting material for regional and single cell microdissection followed by a newly developed RNA amplification procedure (terminal continuation (TC) RNA amplification) and subsequent hybridization to custom-designed cDNA arrays. Results indicated equivalent levels of global hybridization signal intensity and relative expression levels for individual genes for hippocampi stained by cresyl violet, thionin, and hematoxylin & eosin, and neurofilament immunocytochemistry. Moreover, no significant differences existed between the Nissl stains and neurofilament immunocytochemistry for individual CA1 neurons obtained via laser capture microdissection. In contrast, a marked decrement was observed in adjacent hippocampal sections stained for silver stain and acridine orange, both at the level of the regional dissection and at the CA1 neuron population level. Observations made on the cDNA array platform were validated by real-time qPCR using primers directed against beta-actin and glyceraldehyde-3 phosphate dehydrogenase. Thus, this report demonstrated the utility of using specific Nissl stains, but not stains that bind RNA species directly, in both human and mouse brain tissues at the regional and cellular level for state-of-the-art molecular fingerprinting studies.

  8. The Medicago truncatula gene expression atlas web server

    Directory of Open Access Journals (Sweden)

    Tang Yuhong

    2009-12-01

    Full Text Available Abstract Background Legumes (Leguminosae or Fabaceae play a major role in agriculture. Transcriptomics studies in the model legume species, Medicago truncatula, are instrumental in helping to formulate hypotheses about the role of legume genes. With the rapid growth of publically available Affymetrix GeneChip Medicago Genome Array GeneChip data from a great range of tissues, cell types, growth conditions, and stress treatments, the legume research community desires an effective bioinformatics system to aid efforts to interpret the Medicago genome through functional genomics. We developed the Medicago truncatula Gene Expression Atlas (MtGEA web server for this purpose. Description The Medicago truncatula Gene Expression Atlas (MtGEA web server is a centralized platform for analyzing the Medicago transcriptome. Currently, the web server hosts gene expression data from 156 Affymetrix GeneChip® Medicago genome arrays in 64 different experiments, covering a broad range of developmental and environmental conditions. The server enables flexible, multifaceted analyses of transcript data and provides a range of additional information about genes, including different types of annotation and links to the genome sequence, which help users formulate hypotheses about gene function. Transcript data can be accessed using Affymetrix probe identification number, DNA sequence, gene name, functional description in natural language, GO and KEGG annotation terms, and InterPro domain number. Transcripts can also be discovered through co-expression or differential expression analysis. Flexible tools to select a subset of experiments and to visualize and compare expression profiles of multiple genes have been implemented. Data can be downloaded, in part or full, in a tabular form compatible with common analytical and visualization software. The web server will be updated on a regular basis to incorporate new gene expression data and genome annotation, and is accessible

  9. Expression of T4 Lysozyme Gene (gene e) in Streptococcus ...

    African Journals Online (AJOL)

    pL2 plasmid isolated from E. coli was introduced into S. salivarius subsp. thermophilus and Lactococcus lactis cells by electro-transformation. The lysozyme enzymes expressing by these bacteria were found to be active on Micrococcus luteus cells and thereby preventing their growth on assay plates. Thermostability of ...

  10. Mapping photothermally induced gene expression in living cells and tissues by nanorod-locked nucleic acid complexes.

    Science.gov (United States)

    Riahi, Reza; Wang, Shue; Long, Min; Li, Na; Chiou, Pei-Yu; Zhang, Donna D; Wong, Pak Kin

    2014-04-22

    The photothermal effect of plasmonic nanostructures has numerous applications, such as cancer therapy, photonic gene circuit, large cargo delivery, and nanostructure-enhanced laser tweezers. The photothermal operation can also induce unwanted physical and biochemical effects, which potentially alter the cell behaviors. However, there is a lack of techniques for characterizing the dynamic cell responses near the site of photothermal operation with high spatiotemporal resolution. In this work, we show that the incorporation of locked nucleic acid probes with gold nanorods allows photothermal manipulation and real-time monitoring of gene expression near the area of irradiation in living cells and animal tissues. The multimodal gold nanorod serves as an endocytic delivery reagent to transport the probes into the cells, a fluorescence quencher and a binding competitor to detect intracellular mRNA, and a plasmonic photothermal transducer to induce cell ablation. We demonstrate the ability of the gold nanorod-locked nucleic acid complex for detecting the spatiotemporal gene expression in viable cells and tissues and inducing photothermal ablation of single cells. Using the gold nanorod-locked nucleic acid complex, we systematically characterize the dynamic cellular heat shock responses near the site of photothermal operation. The gold nanorod-locked nucleic acid complex enables mapping of intracellular gene expressions and analyzes the photothermal effects of nanostructures toward various biomedical applications.

  11. Acute effects of heavy metals on the expression of glutathione-related antioxidant genes in the marine ciliate Euplotes crassus

    International Nuclear Information System (INIS)

    Kim, Se-Hun; Kim, Se-Joo; Lee, Jae-Seong; Lee, Young-Mi

    2014-01-01

    Highlights: • Significant higher increases in the relative ROS and total GSH levels were observed after exposure to heavy metals. • Real-time PCR data showed expression levels of GPx and GR mRNA were sensitively modulated within 8 h of exposure to heavy metals. • E. crassus GPx and GR genes may be involved in cellular defense mechanisms against heavy metal-induced oxidative stress. • E. crassus GPx and GR genes will be useful as potential molecular markers for monitoring heavy metal contamination. - Abstract: Euplotes crassus, a single-celled eukaryote, is directly affected by environmental contaminants. Here, exponentially cultured E. crassus were exposed to cadmium, copper, lead, and zinc and then the reactive oxygen species (ROS) and total glutathione (GSH) levels were measured. Subsequently, the transcriptional modulation of glutathione peroxidase (GPx) and glutathione reductase (GR) were estimated by quantitative RT-PCR. After an 8-h exposure, significantly higher increases in the relative ROS and total GSH levels were observed in exposed group, compared to the controls. Real-time PCR data revealed that the expression levels of GPx and GR mRNA were sensitively modulated within 8 h of exposure to all heavy metals. These findings suggest that these genes may be involved in cellular defense mechanisms by modulating their gene expression against heavy metal-induced oxidative stress. Thus, they may be useful as potential molecular biomarkers to assess sediment environments for contaminants

  12. Vaginal Gene Expression During Treatment With Aromatase Inhibitors.

    Science.gov (United States)

    Kallak, Theodora Kunovac; Baumgart, Juliane; Nilsson, Kerstin; Åkerud, Helena; Poromaa, Inger Sundström; Stavreus-Evers, Anneli

    2015-12-01

    Aromatase inhibitor (AI) treatment suppresses estrogen biosynthesis and causes genitourinary symptoms of menopause such as vaginal symptoms, ultimately affecting the quality of life for many postmenopausal women with breast cancer. Thus, the aim of this study was to examine vaginal gene expression in women during treatment with AIs compared with estrogen-treated women. The secondary aim was to study the presence and localization of vaginal aromatase. Vaginal biopsies were collected from postmenopausal women treated with AIs and from age-matched control women treated with vaginal estrogen therapy. Differential gene expression was studied with the Affymetrix Gene Chip Gene 1.0 ST Array (Affymetrix Inc, Santa Clara, CA) system, Ingenuity pathway analysis, quantitative real-time polymerase chain reaction, and immunohistochemistry. The expression of 279 genes differed between the 2 groups; AI-treated women had low expression of genes involved in cell differentiation, proliferation, and cell adhesion. Some differentially expressed genes were found to interact indirectly with the estrogen receptor alpha. In addition, aromatase protein staining was evident in the basal and the intermediate vaginal epithelium layers, and also in stromal cells with a slightly stronger staining intensity found in AI-treated women. In this study, we demonstrated that genes involved in cell differentiation, proliferation, and cell adhesion are differentially expressed in AI-treated women. The expression of vaginal aromatase suggests that this could be the result of local and systemic inhibition of aromatase. Our results emphasize the role of estrogen for vaginal cell differentiation and proliferation and future drug candidates should be aimed at improving cell differentiation and proliferation. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon

    Directory of Open Access Journals (Sweden)

    Hackett Perry B

    2006-06-01

    Full Text Available Abstract Background Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern. Results Here we report the systematic development and testing of a transposon-based gene-trap system incorporating the doxycycline-repressible Tet-Off (tTA system that is capable of activating the expression of genes under control of a Tet response element (TRE promoter. We demonstrate that the gene trap system is fully functional in vitro by introducing the "gene-trap tTA" vector into human cells by transposition and identifying clones that activate expression of a TRE-luciferase transgene in a doxycycline-dependent manner. In transgenic mice, we mobilize gene-trap tTA vectors, discover parameters that can affect germline mobilization rates, and identify candidate gene insertions to demonstrate the in vivo functionality of the vector system. We further demonstrate that the gene-trap can act as a reporter of endogenous gene expression and it can be coupled with bioluminescent imaging to identify genes with tissue-specific expression patterns. Conclusion Akin to the GAL4/UAS system used in the fly, we have made progress developing a tool for mutating and revealing the expression of mouse genes by generating the tTA transactivator in the presence of a secondary TRE-regulated reporter molecule. A vector like the gene

  14. Patterns of expression of cell wall related genes in sugarcane

    Directory of Open Access Journals (Sweden)

    Lima D.U.

    2001-01-01

    Full Text Available Our search for genes related to cell wall metabolism in the sugarcane expressed sequence tag (SUCEST database (http://sucest.lbi.dcc.unicamp.br resulted in 3,283 reads (1% of the total reads which were grouped into 459 clusters (potential genes with an average of 7.1 reads per cluster. To more clearly display our correlation coefficients, we constructed surface maps which we used to investigate the relationship between cell wall genes and the sugarcane tissues libraries from which they came. The only significant correlations that we found between cell wall genes and/or their expression within particular libraries were neutral or synergetic. Genes related to cellulose biosynthesis were from the CesA family, and were found to be the most abundant cell wall related genes in the SUCEST database. We found that the highest number of CesA reads came from the root and stem libraries. The genes with the greatest number of reads were those involved in cell wall hydrolases (e.g. beta-1,3-glucanases, xyloglucan endo-beta-transglycosylase, beta-glucosidase and endo-beta-mannanase. Correlation analyses by surface mapping revealed that the expression of genes related to biosynthesis seems to be associated with the hydrolysis of hemicelluloses, pectin hydrolases being mainly associated with xyloglucan hydrolases. The patterns of cell wall related gene expression in sugarcane based on the number of reads per cluster reflected quite well the expected physiological characteristics of the tissues. This is the first work to provide a general view on plant cell wall metabolism through the expression of related genes in almost all the tissues of a plant at the same time. For example, developing flowers behaved similarly to both meristematic tissues and leaf-root transition zone tissues. Besides providing a basis for future research on the mechanisms of plant development which involve the cell wall, our findings will provide valuable tools for plant engineering in the

  15. Quick and sensitive determination of gene expression of fatty acid ...

    African Journals Online (AJOL)

    User

    2011-05-16

    May 16, 2011 ... from fatty acid synthase (FAS) with a different glucose level in ... By using the following formula, this study was able to quantify the mRNA expression of ... hypertension, heart disease and diabetes. ... regulation of gene expression has emerged in recent ... stages of adipocyte meta-bolism are relatively well.

  16. Genetic effects on gene expression across human tissues

    NARCIS (Netherlands)

    Battle, Alexis; Brown, Christopher D.; Engelhardt, Barbara E.; Montgomery, Stephen B.; Aguet, François; Ardlie, Kristin G.; Cummings, Beryl B.; Gelfand, Ellen T.; Getz, Gad; Hadley, Kane; Handsaker, Robert E.; Huang, Katherine H.; Kashin, Seva; Karczewski, Konrad J.; Lek, Monkol; Li, Xiao; MacArthur, Daniel G.; Nedzel, Jared L.; Nguyen, Duyen T.; Noble, Michael S.; Segrè, Ayellet V.; Trowbridge, Casandra A.; Tukiainen, Taru; Abell, Nathan S.; Balliu, Brunilda; Barshir, Ruth; Basha, Omer; Bogu, Gireesh K.; Brown, Andrew; Castel, Stephane E.; Chen, Lin S.; Chiang, Colby; Conrad, Donald F.; Cox, Nancy J.; Damani, Farhan N.; Davis, Joe R.; Delaneau, Olivier; Dermitzakis, Emmanouil T.; Eskin, Eleazar; Ferreira, Pedro G.; Frésard, Laure; Gamazon, Eric R.; Garrido-Martín, Diego; Gewirtz, Ariel D. H.; Gliner, Genna; Gloudemans, Michael J.; Guigo, Roderic; Hall, Ira M.; Han, Buhm; He, Yuan

    2017-01-01

    Characterization of the molecular function of the human genome and its variation across individuals is essential for identifying the cellular mechanisms that underlie human genetic traits and diseases. The Genotype-Tissue Expression (GTEx) project aims to characterize variation in gene expression

  17. Comparison of gene expression patterns between porcine cumulus ...

    African Journals Online (AJOL)

    These results suggest that the aberrant of gene expression patterns detected in the oocytes of NOs compared with COCs explains their reduced quality in terms of development and maturation. In conclusion, these differentially expressed mRNAs may be involved in cellular interactions between oocytes and cumulus cells ...

  18. Expression of bgt gene in transgenic birch (Betula platyphylla Suk ...

    African Journals Online (AJOL)

    Study on the characteristics of integration and expression is the basis of genetic stability of foreign genes in transgenic trees. To obtain insight into the relationship of transgene copy number and expression level, we screened 22 transgenic birch lines. Southern blot analysis of the transgenic birch plants indicated that the ...

  19. Gene-expression Classifier in Papillary Thyroid Carcinoma

    DEFF Research Database (Denmark)

    Londero, Stefano Christian; Jespersen, Marie Louise; Krogdahl, Annelise

    2016-01-01

    BACKGROUND: No reliable biomarker for metastatic potential in the risk stratification of papillary thyroid carcinoma exists. We aimed to develop a gene-expression classifier for metastatic potential. MATERIALS AND METHODS: Genome-wide expression analyses were used. Development cohort: freshly...

  20. Transient expression of β-glucuronidase gene in indica and ...

    African Journals Online (AJOL)

    Owner

    co-transfer of DNAs to cells was monitored by analyzing transient gus expression 24 h after .... induction frequency was determined by measuring the ... Effect of age on transient expression of gus gene and production of hygromycin .... japonica varieties via electric discharge particle acceleration of .... yellow stem borer.

  1. Relative expression of genes related with cold tolerance in ...

    African Journals Online (AJOL)

    Low temperature is one of the main abiotic stresses affecting rice yield in Chile. Alterations in phenology and physiology of the crop are observed after a cold event. The objective of this work was to study the relative expression of genes related with cold stress in Chilean cultivars of rice. For this, we analyzed the expression ...

  2. Expression of bgt gene in transgenic birch (Betula platyphylla Suk.)

    African Journals Online (AJOL)

    STORAGESEVER

    2009-08-04

    Aug 4, 2009 ... Study on the characteristics of integration and expression is the basis of genetic stability of foreign genes in transgenic trees. To obtain insight into the relationship of transgene copy number and expression level, we screened 22 transgenic birch lines. Southern blot analysis of the transgenic birch.

  3. VESPUCCI: exploring patterns of gene expression in grapevine

    Directory of Open Access Journals (Sweden)

    Marco eMoretto

    2016-05-01

    Full Text Available Large-scale transcriptional studies aim to decipher the dynamic cellular responses to a stimulus, like different environmental conditions. In the era of high-throughput omics biology, the most used technologies for these purposes are microarray and RNA-Seq, whose data are usually required to be deposited in public repositories upon publication. Such repositories have the enormous potential to provide a comprehensive view of how different experimental conditions lead to expression changes, by comparing gene expression across all possible measured conditions. Unfortunately, this task is greatly impaired by differences among experimental platforms that make direct comparisons difficult.In this paper we present the Vitis Expression Studies Platform Using COLOMBOS Compendia Instances (VESPUCCI, a gene expression compendium for grapevine which was built by adapting an approach originally developed for bacteria, and show how it can be used to investigate complex gene expression patterns. We integrated nearly all publicly available microarray and RNA-Seq expression data: 1608 gene expression samples from 10 different technological platforms. Each sample has been manually annotated using a controlled vocabulary developed ad hoc to ensure both human readability and computational tractability. Expression data in the compendium can be visually explored using several tools provided by the web interface or can be programmatically accessed using the REST interface. VESPUCCI is freely accessible at http://vespucci.colombos.fmach.it.

  4. The rules of gene expression in plants: Organ identity and gene body methylation are key factors for regulation of gene expression in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Gutiérrez Rodrigo A

    2008-09-01

    Full Text Available Abstract Background Microarray technology is a widely used approach for monitoring genome-wide gene expression. For Arabidopsis, there are over 1,800 microarray hybridizations representing many different experimental conditions on Affymetrix™ ATH1 gene chips alone. This huge amount of data offers a unique opportunity to infer the principles that govern the regulation of gene expression in plants. Results We used bioinformatics methods to analyze publicly available data obtained using the ATH1 chip from Affymetrix. A total of 1887 ATH1 hybridizations were normalized and filtered to eliminate low-quality hybridizations. We classified and compared control and treatment hybridizations and determined differential gene expression. The largest differences in gene expression were observed when comparing samples obtained from different organs. On average, ten-fold more genes were differentially expressed between organs as compared to any other experimental variable. We defined "gene responsiveness" as the number of comparisons in which a gene changed its expression significantly. We defined genes with the highest and lowest responsiveness levels as hypervariable and housekeeping genes, respectively. Remarkably, housekeeping genes were best distinguished from hypervariable genes by differences in methylation status in their transcribed regions. Moreover, methylation in the transcribed region was inversely correlated (R2 = 0.8 with gene responsiveness on a genome-wide scale. We provide an example of this negative relationship using genes encoding TCA cycle enzymes, by contrasting their regulatory responsiveness to nitrate and methylation status in their transcribed regions. Conclusion Our results indicate that the Arabidopsis transcriptome is largely established during development and is comparatively stable when faced with external perturbations. We suggest a novel functional role for DNA methylation in the transcribed region as a key determinant

  5. Novel gene expression tools for rice biotechnology

    Science.gov (United States)

    Biotechnology is an effective and important method of improving both quality and agronomic traits in rice. We are developing novel molecular tools for genetic engineering, with a focus on developing novel transgene expression control elements (i.e. promoters) for rice. A suite of monocot grass promo...

  6. Real-time PCR gene expression profiling

    Czech Academy of Sciences Publication Activity Database

    Kubista, Mikael; Sjögreen, B.; Forootan, A.; Šindelka, Radek; Jonák, Jiří; Andrade, J.M.

    2007-01-01

    Roč. 1, - (2007), s. 56-60 ISSN 1360-8606 R&D Projects: GA AV ČR KJB500520601 Institutional research plan: CEZ:AV0Z50520514 Keywords : real - time PCR, * expression profiling * statistical analysis Subject RIV: EB - Genetics ; Molecular Biology

  7. Cytokine gene expression of peripheral blood lymphocytes ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-03-20

    Mar 20, 2009 ... Key words: Lipopolysaccharide, lymphocytes, TLRs, cytokines. INTRODUCTION. Lipopolysaccharide (LPS), a predominant glycolipid in the outer membranes of Gam-negative bacteria, stimulates monocyte, macrophages, and neutrophils and increase expression of cell adhesion molecules (Trent et al., ...

  8. Gene Expression in the Human Endolymphatic Sac

    DEFF Research Database (Denmark)

    Møller, Martin Nue; Kirkeby, Svend; Vikeså, Jonas

    2015-01-01

    a1 sodium-bicarbonate transporter, SLC9a2 sodium-hydrogen transporter, SLC12a3 thiazide-sensitive Na-Cl transporter, and SLC34a2 sodium-phosphate transporter. CONCLUSIONS: Several important ion transporters of the SLC family are expressed in the human endolymphatic sac, including Pendrin...

  9. Selection of reference genes for gene expression studies in heart failure for left and right ventricles.

    Science.gov (United States)

    Li, Mengmeng; Rao, Man; Chen, Kai; Zhou, Jianye; Song, Jiangping

    2017-07-15

    Real-time quantitative reverse transcriptase-PCR (qRT-PCR) is a feasible tool for determining gene expression profiles, but the accuracy and reliability of the results depends on the stable expression of selected housekeeping genes in different samples. By far, researches on stable housekeeping genes in human heart failure samples are rare. Moreover the effect of heart failure on the expression of housekeeping genes in right and left ventricles is yet to be studied. Therefore we aim to provide stable housekeeping genes for both ventricles in heart failure and normal heart samples. In this study, we selected seven commonly used housekeeping genes as candidates. By using the qRT-PCR, the expression levels of ACTB, RAB7A, GAPDH, REEP5, RPL5, PSMB4 and VCP in eight heart failure and four normal heart samples were assessed. The stability of candidate housekeeping genes was evaluated by geNorm and Normfinder softwares. GAPDH showed the least variation in all heart samples. Results also indicated the difference of gene expression existed in heart failure left and right ventricles. GAPDH had the highest expression stability in both heart failure and normal heart samples. We also propose using different sets of housekeeping genes for left and right ventricles respectively. The combination of RPL5, GAPDH and PSMB4 is suitable for the right ventricle and the combination of GAPDH, REEP5 and RAB7A is suitable for the left ventricle. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. In situ gene expression and ecophysiology of thermophilic Cyanobacteria

    DEFF Research Database (Denmark)

    Jensen, Sheila Ingemann

    -378), the expression patterns of various functional genes (with an emphasis on nif genes involved in N2-fixation), the protein levels of nitrogenase (NifH), the N2-fixation activity, as well as microsensor based measurements on O2 availability, production and consumption were investigated in situ over the entire diel...... cycle. Interestingly, it was found that while the nif genes are expressed, and nitrogenase is synthesized once the mat gets anoxic in the early evening, the largest N2-fixation activity occurs as a burst during dim light in the early morning, albeit protein levels remained high over the entire course...

  11. Repressor-mediated tissue-specific gene expression in plants

    Science.gov (United States)

    Meagher, Richard B [Athens, GA; Balish, Rebecca S [Oxford, OH; Tehryung, Kim [Athens, GA; McKinney, Elizabeth C [Athens, GA

    2009-02-17

    Plant tissue specific gene expression by way of repressor-operator complexes, has enabled outcomes including, without limitation, male sterility and engineered plants having root-specific gene expression of relevant proteins to clean environmental pollutants from soil and water. A mercury hyperaccumulation strategy requires that mercuric ion reductase coding sequence is strongly expressed. The actin promoter vector, A2pot, engineered to contain bacterial lac operator sequences, directed strong expression in all plant vegetative organs and tissues. In contrast, the expression from the A2pot construct was restricted primarily to root tissues when a modified bacterial repressor (LacIn) was coexpressed from the light-regulated rubisco small subunit promoter in above-ground tissues. Also provided are analogous repressor operator complexes for selective expression in other plant tissues, for example, to produce male sterile plants.

  12. Novel gene sets improve set-level classification of prokaryotic gene expression data.

    Science.gov (United States)

    Holec, Matěj; Kuželka, Ondřej; Železný, Filip

    2015-10-28

    Set-level classification of gene expression data has received significant attention recently. In this setting, high-dimensional vectors of features corresponding to genes are converted into lower-dimensional vectors of features corresponding to biologically interpretable gene sets. The dimensionality reduction brings the promise of a decreased risk of overfitting, potentially resulting in improved accuracy of the learned classifiers. However, recent empirical research has not confirmed this expectation. Here we hypothesize that the reported unfavorable classification results in the set-level framework were due to the adoption of unsuitable gene sets defined typically on the basis of the Gene ontology and the KEGG database of metabolic networks. We explore an alternative approach to defining gene sets, based on regulatory interactions, which we expect to collect genes with more correlated expression. We hypothesize that such more correlated gene sets will enable to learn more accurate classifiers. We define two families of gene sets using information on regulatory interactions, and evaluate them on phenotype-classification tasks using public prokaryotic gene expression data sets. From each of the two gene-set families, we first select the best-performing subtype. The two selected subtypes are then evaluated on independent (testing) data sets against state-of-the-art gene sets and against the conventional gene-level approach. The novel gene sets are indeed more correlated than the conventional ones, and lead to significantly more accurate classifiers. The novel gene sets are indeed more correlated than the conventional ones, and lead to significantly more accurate classifiers. Novel gene sets defined on the basis of regulatory interactions improve set-level classification of gene expression data. The experimental scripts and other material needed to reproduce the experiments are available at http://ida.felk.cvut.cz/novelgenesets.tar.gz.

  13. Differential neutrophil gene expression in early bovine pregnancy

    Directory of Open Access Journals (Sweden)

    Kizaki Keiichiro

    2013-02-01

    Full Text Available Abstract Background In food production animals, especially cattle, the diagnosis of gestation is important because the timing of gestation directly affects the running of farms. Various methods have been used to detect gestation, but none of them are ideal because of problems with the timing of detection or the accuracy, simplicity, or cost of the method. A new method for detecting gestation, which involves assessing interferon-tau (IFNT-stimulated gene expression in peripheral blood leukocytes (PBL, was recently proposed. PBL fractionation methods were used to examine whether the expression profiles of various PBL populations could be used as reliable diagnostic markers of bovine gestation. Methods PBL were collected on days 0 (just before artificial insemination, 7, 14, 17, 21, and 28 of gestation. The gene expression levels of the PBL were assessed with microarray analysis and/or quantitative real-time reverse transcription (q PCR. PBL fractions were collected by flow cytometry or density gradient cell separation using Histopaque 1083 or Ficoll-Conray solutions. The expression levels of four IFNT-stimulated genes, interferon-stimulated protein 15 kDa (ISG15, myxovirus-resistance (MX 1 and 2, and 2′-5′-oligoadenylate synthetase (OAS1, were then analyzed in each fraction through day 28 of gestation using qPCR. Results Microarray analysis detected 72 and 28 genes in whole PBL that were significantly higher on days 14 and 21 of gestation, respectively, than on day 0. The upregulated genes included IFNT-stimulated genes. The expression levels of these genes increased with the progression of gestation until day 21. In flow cytometry experiments, on day 14 the expression levels of all of the genes were significantly higher in the granulocyte fraction than in the other fractions. Their expression gradually decreased through day 28 of gestation. Strong correlations were observed between the expression levels of the four genes in the granulocyte

  14. Whole-body gene expression pattern registration in Platynereis larvae.

    Science.gov (United States)

    Asadulina, Albina; Panzera, Aurora; Verasztó, Csaba; Liebig, Christian; Jékely, Gáspár

    2012-12-03

    Digital anatomical atlases are increasingly used in order to depict different gene expression patterns and neuronal morphologies within a standardized reference template. In evo-devo, a discipline in which the comparison of gene expression patterns is a widely used approach, such standardized anatomical atlases would allow a more rigorous assessment of the conservation of and changes in gene expression patterns during micro- and macroevolutionary time scales. Due to its small size and invariant early development, the annelid Platynereis dumerilii is particularly well suited for such studies. Recently a reference template with registered gene expression patterns has been generated for the anterior part (episphere) of the Platynereis trochophore larva and used for the detailed study of neuronal development. Here we introduce and evaluate a method for whole-body gene expression pattern registration for Platynereis trochophore and nectochaete larvae based on whole-mount in situ hybridization, confocal microscopy, and image registration. We achieved high-resolution whole-body scanning using the mounting medium 2,2'-thiodiethanol (TDE), which allows the matching of the refractive index of the sample to that of glass and immersion oil thereby reducing spherical aberration and improving depth penetration. This approach allowed us to scan entire whole-mount larvae stained with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) in situ hybridization and counterstained fluorescently with an acetylated-tubulin antibody and the nuclear stain 4'6-diamidino-2-phenylindole (DAPI). Due to the submicron isotropic voxel size whole-mount larvae could be scanned in any orientation. Based on the whole-body scans, we generated four different reference templates by the iterative registration and averaging of 40 individual image stacks using either the acetylated-tubulin or the nuclear-stain signal for each developmental stage. We then registered to these templates the

  15. Whole-body gene expression pattern registration in Platynereis larvae

    Directory of Open Access Journals (Sweden)

    Asadulina Albina

    2012-12-01

    Full Text Available Abstract Background Digital anatomical atlases are increasingly used in order to depict different gene expression patterns and neuronal morphologies within a standardized reference template. In evo-devo, a discipline in which the comparison of gene expression patterns is a widely used approach, such standardized anatomical atlases would allow a more rigorous assessment of the conservation of and changes in gene expression patterns during micro- and macroevolutionary time scales. Due to its small size and invariant early development, the annelid Platynereis dumerilii is particularly well suited for such studies. Recently a reference template with registered gene expression patterns has been generated for the anterior part (episphere of the Platynereis trochophore larva and used for the detailed study of neuronal development. Results Here we introduce and evaluate a method for whole-body gene expression pattern registration for Platynereis trochophore and nectochaete larvae based on whole-mount in situ hybridization, confocal microscopy, and image registration. We achieved high-resolution whole-body scanning using the mounting medium 2,2’-thiodiethanol (TDE, which allows the matching of the refractive index of the sample to that of glass and immersion oil thereby reducing spherical aberration and improving depth penetration. This approach allowed us to scan entire whole-mount larvae stained with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP in situ hybridization and counterstained fluorescently with an acetylated-tubulin antibody and the nuclear stain 4’6-diamidino-2-phenylindole (DAPI. Due to the submicron isotropic voxel size whole-mount larvae could be scanned in any orientation. Based on the whole-body scans, we generated four different reference templates by the iterative registration and averaging of 40 individual image stacks using either the acetylated-tubulin or the nuclear-stain signal for each developmental

  16. Identification of differentially expressed genes in cutaneous squamous cell carcinoma by microarray expression profiling

    Directory of Open Access Journals (Sweden)

    Sterry Wolfram

    2006-08-01

    Full Text Available Abstract Background Carcinogenesis is a multi-step process indicated by several genes up- or down-regulated during tumor progression. This study examined and identified differentially expressed genes in cutaneous squamous cell carcinoma (SCC. Results Three different biopsies of 5 immunosuppressed organ-transplanted recipients each normal skin (all were pooled, actinic keratosis (AK (two were pooled, and invasive SCC and additionally 5 normal skin tissues from immunocompetent patients were analyzed. Thus, total RNA of 15 specimens were used for hybridization with Affymetrix HG-U133A microarray technology containing 22,283 genes. Data analyses were performed by prediction analysis of microarrays using nearest shrunken centroids with the threshold 3.5 and ANOVA analysis was independently performed in order to identify differentially expressed genes (p vs. AK and SCC were observed for 118 genes. Conclusion The majority of identified differentially expressed genes in cutaneous SCC were previously not described.

  17. AGEMAP: a gene expression database for aging in mice.

    Directory of Open Access Journals (Sweden)

    Jacob M Zahn

    2007-11-01

    Full Text Available We present the AGEMAP (Atlas of Gene Expression in Mouse Aging Project gene expression database, which is a resource that catalogs changes in gene expression as a function of age in mice. The AGEMAP database includes expression changes for 8,932 genes in 16 tissues as a function of age. We found great heterogeneity in the amount of transcriptional changes with age in