title: Antirheumatic Drug Response in Human Chondrocytes: Potential Molecular Targets to Stimulate Cartilage Regeneration

first: Thomas

last: Häupl

address: Charitéplatz 1

department: Rheumatology

institute: Charité

summary:
Rheumatoid arthritis (RA) leads to progressive destruction of articular structures. Despite recent progress in controlling inflammation and pain, little cartilage repair has yet been observed. This in vitro study aims to determine the role of chondrocytes in RA-related cartilage destruction and antirheumatic drug-related regenerative processes. Human chondrocytes were three-dimensionally cultured in alginate beads. To determine the RA-induced gene expression pattern, human chondrocytes were stimulated with supernatant of RA synovial fibroblasts (RASF) and normal donor synovial fibroblasts (NDSF), respectively. To examine antirheumatic drug response signatures, human chondrocytes were stimulated with supernatant of RASF that have been treated with disease-modifying antirheumatic drugs (DMARD; azathioprine, sodium aurothiomalate, chloroquine phosphate, methotrexate), non-steroidal anti-inflammatory drugs (NSAID; piroxicam, diclofenac) or steroidal anti-inflammatory drugs (SAID; methylprednisolone, prednisolone). Genome-wide expression profiling with oligonucleotide microarrays was used to determine differentially expressed genes. Real-time RT-PCR and ELISA were performed for validation of microarray data. Following antirheumatic treatment, microarray analysis disclosed a reverted expression of 94 RA-induced chondrocyte genes involved in inflammation/NF-κB signalling, cytokine/chemokine activity, immune response, proliferation/differentiation and matrix remodelling. Hierarchical clustering analysis showed that treatment of RASF with the DMARD azathioprine, gold sodium thiomalate and methotrexate resulted in chondrocyte gene expression signatures that were closely related to the “healthy” pattern. Treatment with the SAID methylprednisolone and prednisolone strongly reverted the RA-related chondrocyte gene expression, in particular the expression of genes involved in inflammation/NF-κB and cytokine/chemokine activity. The NSAID piroxicam and diclofenac and the DMARD chloroquine phosphate had only moderate to marginal effects. Pathway analysis determined major mechanisms of drug action, for example pathways of cytokine-cytokine receptor interaction, TGF-β/TLR/Jak-STAT signalling and ECM-receptor interaction were targeted. This in vitro study provides a comprehensive molecular insight into the antirheumatic drug response signatures in human chondrocytes, thereby revealing potential molecular targets, pathways and mechanisms of drug action involved in chondrocyte regeneration. Thus, the present study may contribute to the development of novel therapeutic chondro-protective compounds and strategies.

Keywords: drug response

overall_design: Drug-related suppression of gene expression in activated chondrocytes was determined by genome-wide microarray analysis. Chondrocytes were stimulated with supernatant of RASF and NDSF. Effect of treatment with DMARDs, NSAIDs and glucocorticoids was tested by treating RASF prior to collection of supernatant. Two RNA pools were analyzed for each group (RASF-stimulated NDSF stimulated and RASF-treated), each pool consisting of equal amounts of RNA from three different donors.

first: Kristin

last: Andreas

first: Carsten

last: Lübke

first: Jochen

last: Ringe

first: Lars

last: Morawietz

first: Anja

last: Wachtel

first: Michael

last: Sittinger

last: Kaps


title: Expression profiling of the plant cell wall mutants: pmr5, pmr6 and pmr5 pmr6 double mutants

status: Public on Sep 01 2008

city: Berkeley

first: Shauna

last: Somerville

state: CA

address: 130 Calvin Hall, Mail Code 5230

department: Energy Biosciences Institute

country: USA

institute: University of California at Berkeley

summary:
The rosettes from genotype Col-0, pmr5, pmr6 and pmr5 pmr6 double mutant were collected for RNA expression profiling when the plants were 3 weeks old.
Keywords: cell wall mutant

overall_design: The rosettes were collected when the plants were 3 weeks old for RNA extraction and hybridization on Affymetrix microarrays.

first: Sonja

last: Vorwerk

first: Bi-Huei

last: Hou


title: MILDEW-INDUCED LESIONS 4 encodes a novel regulator of the salicylic acid defense response

summary:
The 4 weeks old plant of genotype Col-0, mil4, sid2 and mil4 sid2 double mutants were sprayed with BTH. The rosette tissue were collected for RNA expression profiling.
Keywords: defense response

overall_design: The rosettes were collected when the plants were 4 weeks old for RNA extraction and hybridization on Affymetrix microarrays.

first: Marc

last: Nishimura


title: Suppression of MT1-MMP in Human Fibrosarcoma Impair Tumor Angiogenesis and Enlargement

city: Stony Brook

first: Wei

last: Zeng

state: NY

institute: SUNY at Stony Brook

summary:
To define the role of MT1-MMP in the tumor progression, we suppressed its expression in human fibrosarcoma cell line, HT1080, using RNAi technique. The gene expression pattern was then compared betweent the two experimental cell groups with contrasting MT1-MMP expression level.
Keywords: other

first: Jaclyn

last: Freudenberg

first: Donghai

last: Chen

first: Wen-Tien


title: Arabidopsis thaliana Ler developmental series

status: Public on Sep 17 2008

city: Rehovot

first: Idan

middle: D.

last: Efroni

address: Weizmann

country: Israel

institute: Weizmann Institute

summary: Leaf development has been monitored chiefly by following anatomical markers. Analysis of transcriptome dynamics during leaf maturation revealed multiple expression patterns that rise or fall with age or that display age specific peaks. These were used to formulate a digital differentiation index (DDI), based on a set of selected markers with informative expression during leaf ontogeny. The leaf-based DDI reliably predicted the developmental state of leaf samples from diverse sources and was independent of mitotic cell division transcripts or propensity of the specific cell type. To calibrate and test the DDI a series of Arabidopsis shoot development was used (Efroni et al, 2008)

overall_design: Four samples from different stages of shoot and leaf development taken from plants grown at short days (10 hours light at 20C), starting from 5 days after sowing (DAS) apices to 35 DAS fully expanded leaves. RNA was hybridized to affymatrix ATH1 arrays and done in duplicates.

first: Yuval

last: Eshed

first: Eyal

last: Blum


title: Differential gene expression in primary ovarian tumors from depressed and non-depressed patients

status: Public on Sep 10 2008

city: Los Angeles

first: Steve

last: Cole

address: 11-934 Factor Bldg, UCLA

department: Medicine

institute: UCLA School of Medicine

summary:
Gene expression profiling was carried out on primary ovarian carcinomas from 10 patients.  The primary research question is whether gene expression differs in tissues from individuals with high vs low symptoms of psychological depression.
Keywords: Risk prediction


title: Expression data from melanoma cell lines 224 and BL transfected with siRNA-NRASQ61R

status: Public on Aug 31 2008

city: Princeton

first: Fei

last: Huang

state: NJ

institute: Bristol-Myers Squibb Co

summary: Transcriptional profiling for global characterization of gene expression alterations that resulted from treatment of melanoma cells with siRNA specifically targeting NRASQ61R

overall_design: BL or 224 cells transfected with siRNA against either mutant NRASQ61R (siMut10 and siMut12) or scrambled control (siSC). Untransfected cells (T0) from Day 0 were used as the baseline control for cells transfected with both siMut and siSC

first: M

last: Eskandarpour

first: F

first: K

last: Reeves

first: E

last: Clark

first: J

last: Hansson


title: Periodontitis is associated with a type-1 interferon signature in peripheral blood neutrophils

status: Public on Aug 20 2008

city: Birmingham

first: Helen

middle: Joan

last: Wright

state: West Midlands

address: St Chads Queensway

department: Oral Biology

country: United Kingdom

institute: University of Birmingham

summary: Peripheral blood neutrophils from periodontitis patients exhibit a hyper-reactive and hyper-active phenotype (collectively termed hyper-responsivity) in terms of production of reactive oxygen species (ROS) however the molecular basis for this observation is yet to be determined. Our objectives were to identify genes differentially expressed in hyper-responsive peripheral blood neutrophils from chronic periodontitis patients relative to periodontally healthy controls and use this data to identify potential contributory pathways to the hyper-responsive neutrophil phenotype.

overall_design: Neutrophils taken from 4 chronic periodontitis patients and age/sex matched healthy controls. RNA extracted and subsequently hybridised in dulpicate on to U133A arrays.

first: John

middle: b

last: Matthews

first: Iain

middle: L

last: Chapple

first: Nicola

last: Ling-Mountford

first: Paul

middle: R

last: Cooper


title: Expression data from Arabidopsis seed compartments at the pre-globular stage

status: Public on Aug 12 2008

city: Davis

middle: J

last: Harada

address: One Shields Avenue

department: Plant Biology

institute: University of California, Davis

summary:
We collected pre-globular stage seed compartments from 7-micron paraffin sections using the Leica LMD6000 system in order to identify the mRNAs present in different compartments of seeds containing pre-globular-stage embryos was identified as those seeds containing embryo propers made up of  between 2 and 8 cells. For the purposes of this study we captured 6 compartments: embyro proper, micropylar endosperm, peripheral endosperm, chalazal endosperm, chalazal seed coat and general seed coat. Serial sections of entire seeds were also captured for comparison.

Keywords: cell type comparison

overall_design: Pre-globular stage seed compartments or whole seeds were isolated using the LMD6000 system. Total RNA was amplified and hybridized with Affymetrix ATH1 Arabidopsis array for 14 samples (embryo proper, micropylar endosperm, peripheral endosperm, chalazal endosperm, general seed coat, chalazal seed coat, whole seeds, 2 biological replicates each).

first: Sandra

last: Stone

first: Julie

last: Pelletier

first: Ryan

middle: C

last: Kirkbride

first: Xiao

middle: H

last: Lu

first: Mark

middle: F

last: Belmonte

first: Jiong

last: Fei

first: Chen

last: Cheng

first: Robert

middle: B

last: Golberg


title: Arabidopsis wild-type vs. vip3

status: Public on Aug 30 2008

city: East Lansing

first: Sookyung

last: Oh

state: MI

department: Horticulture

institute: Michigan State University

summary:
Comparison of wild-type and vip3.
The Arabidopsis thaliana VERNALIZATION INDEPENDENCE (VIP) gene class has multiple functions in development, including repression of flowering through activation of MADS box gene FLC. Among VIP genes, VIP genes encode yeast Paf1 complex homolog which is required for histone modification, transcriptional elongation.
To identify genes regulated by VIP3, we performed Affymetrix Arabidopsis gene chip analysis and compared gene transcription profiles from wild-type and vip3 mutant plants.
We found more than 200 genes misregulated in vip3 mutant plants compring to wild-type plant.
Keywords: Comparison of wild -type and vip3 mutants

overall_design: Plants were grown on soil. Total aerial tissues were harvestedfrom 14 day old seedling. Each of two replicates for each genotypes was composed of three independently derived samples. Each sample included more than 100 plants. Total RNA was isolated using Qiagen RNAeasy columns (Qiagen, Valencia, CA). Synthesis of cDNA employed the SuperScript double-stranded cDNA sysnthesis kit (Affymetrix). Synthesis of biotinylated cDNA utilized the BioArray high yield RNA transcript labeling kit (Affymetrix). Hybridization and scanning of microarrays was performed at the Research Techonology Support Facility at Michigan State University. Microarray data were analyzed using the statistical algorithms within the Affymetrix Microarray Suite (MAS) 5.0 software.

first: Sunchung

last: Park

first: Steve

last: van Nocker


title: LEAFY COTYLEDON1 is a key regulator of fatty acid biosynthesis in Arabidopsis thaliana

city: Beijing

first: Jinye

last: Mu

address: Datun Road, Chaoyang District,Beijing,China

country: China

institute: Institute of Genetics and Developmental Biology,CAS

summary: In plants, fatty acids are de novo synthesized predominantly in plastids fromacetyl-CoA. Although fatty acid biosynthesis has been biochemically well-studied, little isknown about the regulatory mechanisms of the pathway. Here, we show that overexpressionof the Arabidopsis (Arabidopsis thaliana) LEAFY COTYLEDON1 (LEC1) gene causesglobally increased expression of fatty acid biosynthetic genes, which are involved in keyreactions of condensation, chain elongation and desaturation of fatty acid biosynthesis. Inthe plastidial fatty acid synthetic pathway, over 58% of known enzyme-coding genes areupregulated in LEC1-overexpressing transgenic plants, including those encoding threesubunits of acetyl-CoA carboxylase, a key enzyme controlling the fatty acid biosynthesisflux. Moreover, genes involved in glycolysis and lipid accumulation are also upregulated.Consistent with these results, levels of major fatty acid species and lipids were substantiallyincreased in the transgenic plants. Genetic analysis indicates that the LEC1 function ispartially dependent on ABSCISIC ACID INSENSITIVE3, FUSCA3 and WRINKLED1 in theregulation of fatty acid biosynthesis. Moreover, a similar phenotype was observed intransgenic Arabidopsis plants overexpressing two LEC1-like genes of Brassica napus.These results suggest that LEC1 and LEC1-like genes act as key regulators to coordinate theexpression of fatty acid biosynthetic genes, thereby representing a promising target forgenetic improvement of oil-production plants.

overall_design: The pER8-LEC1 transgenic seedlings were germinated and grown in the presence of 10 µM estradiol for 4 days. The control sample was germinated and grown under the identical conditions without estradiol but containing 0.1% DMSO. Total RNA was prepared from fresh or frozen plant materials using the RNAeasy Plant Mini Kit (Qiagen China, Beijing). The first strand cDNA was synthesized, and then hybridized with the ATH1 oligonucleotide chips as described by the manufacture’s instructions (Affymetrix). The microarray hybridization data were collected and analyzed using related R (http://www.r-project.org/) packages provided by Bioconductor (http://www.biocoductor.org/). In brief, genes differentially expressed between wild type and mutant plants were selected by first removing “absent” genes which were never detected to be expressed in the experiments, then a two-side t-test was applied to remaining genes in order to test the expression difference between wide type and mutant plants. To avoid multiple testing problems, raw p-values were then adjusted into False Discovery Rate (FDR) using Benjamini and Hochberg approach. Finally, differentially expressed genes were defined as those FDR less than 0.2. Functional analysis of differentially expressed genes was carried out using the biological process category of Arabidopsis Gene Ontology. The hierarchical map of GO annotation was constructed according to the ontology tree provided by the Gene Ontology website (http://www.geneontology.com) as described previously (Zheng and Wang, 2008). Ontology categories that are significantly enriched among differentially expressed genes (hypergeometric test and FDR less than 0.1) were displayed as boxes in the map.

first: Qi

last: Zheng

first: Xiujie

last: Wang

first: Jianru

last: Zuo


title: transcript profiling of the adaptive response to decreases in oxygen concentration in the roots of Arabidopsis plants

city: Potsdam

first: Joost

middle: T.

last: van Dongen

address: Am Muehlenberg 1

country: Germany

institute: Max Planck Institute of Molecular Plant Physiology

summary:
-	Background and Aims: Oxygen can fall to low concentrations within plant tissues, either because of environmental factors that decrease the external oxygen concentration or because the movement of oxygen through the plant tissues cannot keep pace with the rate of oxygen consumption. Recent studies document that plants can decrease their oxygen consumption in response to relative small changes in oxygen concentrations to avoid internal anoxia. The molecular mechanisms underlying this response have not been identified yet. The aim of this study was to use transcript and metabolite profiling to investigate the genomic response of Arabidopsis roots to a mild decrease in oxygen concentrations.
-	Methods: Arabidopsis seedlings were grown on vertical agar plates at 21, 8, 4 and 1% (v/v) external oxygen for 0.5, 2 and 48h. Roots were analyzed for changes in transcript levels using Affymetrix whole genome DNA microarrays, and for changes in metabolite levels using routine GC-MS based metabolite profiling. Root extension rates were monitored in parallel to investigate adaptive changes in growth.
-	Key results: Results show that root growth was inhibited and transcript and metabolite profiles were significantly altered in response to a moderate decrease in oxygen concentrations. Low oxygen leads to a preferential up-regulation of genes that might be important to trigger adaptive responses in the plant. A small but highly specific set of genes is induced very early in response to a moderate decrease in oxygen concentrations. Genes that were down-regulated mainly encoded proteins involved in energy-consuming processes. In line with this, root extension growth was significantly decreased which will ultimately save ATP and decrease oxygen consumption. This was accompanied by a differential regulation of metabolite levels at short and long term incubation at low oxygen.
-	Conclusions: Results show that there are adaptive changes in root extension involving large-scale reprogramming of gene expression and metabolism when oxygen concentration is decreased in a very narrow range.

overall_design: Arabidopsis seedlings were grown on vertical agar plates and treated with various oxygen concentrations (1%, 4%, 8%, and 21% as a control), 350ppm CO2 and N2 (rest) for different time periods (0.5 hours, 2 hours and 2 days. At the end of the experiment, the roots of the seedlings were immediately frozen in liquid nitrogen and used for gene expression analysis.

middle: T

first: Anja

last: Froehlich

first: Peter

last: Geigenberger


title: Expression data from Arabidopsis seed compartments at the linear-cotyledon stage

first: John

last: Harada

summary: We collected linear-cotyledon stage seed compartments from 5 to 7 micron paraffin sections using the Leica LMD6000 system in order to identify the mRNAs present in different compartments from seeds containing linear-coyledon-stage embryos. For the purposes of this study we captured 7 compartments: embyro proper, cellularized endosperm, chalazal endosperm, chalazal seed coat, general seed coat, whole seeds and micropylar endosperm.

overall_design: linear-cotyledon stage seed compartments were isolated using the LMD6000 system. Total RNA was amplified and hybridized with Affymetrix ATH1 Arabidopsis array for 14 samples (embryo proper, micropylar endosperm, cellularized endosperm, general seed coat, chalazal seed coat, whole seeds and micropylar endosperm, 2 biological replicates each).

first: Sandra

last: Stone

first: Julie

last: Pelletier


title: Hereditary Inclusion Body Myopathy (HIBM)

status: Public on Sep 04 2008

city: Jerusalem

first: Noa

last: Novershtern

address: Admond Safra Campus

department: School of Computer Science and Engineering

institute: The Hebrew University

summary:
HIBM is a neuromuscular disorder characterized by adult-onset, slowly progressive distal and proximal muscle weakness. Here, gene expression was measured in muscle specimens from 10 HIBM patients carrying the M712T Persian Jewish founder mutation in GNE and presenting with mild histological changes, and from 10 healthy matched control individuals.

Keywords: Muscle specimen

overall_design: Samples were taken from muscle specimens (deltoid, biceps, quadriceps, tibialis), from 10 HIBM patients carrying the M712T Persian Jewish founder mutation in GNE and presenting with mild histological changes. Ages of patients range between 20 to 59. Additional 10 matched samples were taken from healthy control individuals (deltoid, biceps, quadriceps, gluteus, paraspinally and triceps muscles), with age range 18 to 74.

first: Iris

last: Eisenberg

first: Zohar

last: Itzhaki

first: Michal

last: Becker-Cohen

first: Menachem

last: Sadeh

last: Willems

first: Nir

last: Friedman

first: Werner

last: Koopman

first: Stella

last: Mitrani-Rosenbaum


title: Construction of a modular analysis framework for blood Genomics Studies

status: Public on Aug 11 2008

city: Dallas

first: Damien

last: Chaussabel

state: TX

address: 3434 Live Oak

institute: Baylor Institute for Immunology Research

summary: We designed a strategy for microarray analysis that is based on the identification of transcriptional modules formed by genes coordinately expressed in multiple disease data sets. Mapping changes in gene expression at the module level generated disease-specific transcriptional fingerprints that provide a stable framework for the visualization and functional interpretation of microarray data.

overall_design:
The first step of the module-construction process analyzes expression patterns of transcripts across samples for individual diseases: sets of coordinately expressed transcripts were identified with an unsupervised clustering algorithm; in this case, the GeneSpring Version 7.1 (Agilent) implementation of the K-Means algorithm (k = 30). All transcripts detected in at least one sample were used as input; no screening for differential expression was performed. The second step of the module-construction process analyzed the “clustering behavior” of transcripts across diseases, taking into account the possibility that genes may cocluster in some diseases but not in others. Also, in our example, the transcripts that clustered together across all eight diseases were grouped to form a set of modules (round 1 of selection), and the stringency of the analysis was then decreased gradually to identify transcripts that belong to a similar K-means cluster in only a subset of diseases (round 2: seven out of eight diseases; round 3: six out of eight diseases). It is important to note that the module-selection process is “data-driven” and does not involve manual selection of genes by the investigator.

We implemented the module-construction strategy described above, using as input a total of 239 peripheral-blood mononuclear cell (PBMC) samples obtained from individuals with one of the following conditions: systemic juvenile idiopathic arthritis (n = 47), systemic lupus erythematosus (n = 40), type I diabetes (n = 20), metastatic melanoma (n = 39), acute infections (Escherichia coli [n = 22], Staphylococcus aureus [n = 18], Influenza A [n = 16]) or liver-transplant recipients undergoing immunosuppressive therapy (n = 37). Transcriptional profiles were generated with Affymetrix U133A and U133B GeneChips (> 44,000 probe sets). A total of 4742 transcripts, distributed among 28 sets, were selected after running of the module-construction algorithm described above. Each module is assigned a unique identifier indicating the round and order of selection (i.e., M3.1 is the first module identified in the third round of selection).

The stringency of this algorithm was tested statistically by implementation of the same module-construction procedure after randomization of the original data set. This process was repeated 200 times, without a single module identified. Therefore, the analysis of gene-cluster membership across multiple diseases provided a stringent means to identify PBMC transcriptional modules.


title: A modular analysis framework for the discovery of biomarkers of Systemic Lupus Erythematosus

summary: Transcriptional modules were used as a basis for the selection of biomarkers and the development of a multivariate transcriptional indicator of disease progression in patients with systemic lupus erythematosus.

overall_design: The proposed biomarker-selection strategy relies on modules for reducing highly dimensional microarray data sets in a stepwise manner. Starting from the full set of 28 modules, only those for which a set minimum proportion of transcripts are significantly changed between the study groups are selected (e.g., minimum proportion of differentially expressed transcripts at p < 0.05 = 15% overexpressed or underexpressed transcripts; in the example given, 11 SLE modules meet this criterion). This eliminates from the selection pool the modules registering fewer consistent changes that could be attributed to noise. Transcriptional vectors were derived for the entire cohort of 22 untreated pediatric SLE patients with the use of this set of 11 SLE modules. Patient profiles were also generated for an independent set of 31 children with SLE treated with steroids and/or cytotoxic drugs and/or hydroxychloroquine. A nonparametric method for analyzing multivariate ordinal data was used to score the patients. Lupus disease flares can lead to irreversible worsening of the patient's status. We tested the relevance of this multivariate transcriptional score for longitudinal monitoring of the disease activity in a cohort of 20 pediatric SLE patients (two to four time points/patient, intervals between each time point varied from one month to 18 months). Half of the patients had been included in our cross-sectional analysis before they were enrolled in this longitudinal study. Parallel trends were observed between multivariate transcriptional scores and a clinical severity score. The positive association  was verified statistically with the use of a linear-regression model.


title: Expression data from Arabidopsis Seed Compartments at 5 discrete stages of development

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: A Modular Analysis Framework for Blood Genomics Studies: Application to Systemic Lupus Erythematosus

summary:
The analysis of patient blood transcriptional profiles offers a means to investigate the immunological mechanisms relevant to human diseases on a genome-wide scale. In addition, such studies provide a basis for the discovery of clinically relevant biomarker signatures. We designed a strategy for microarray analysis that is based on the identification of transcriptional modules formed by genes coordinately expressed in multiple disease data sets. Mapping changes in gene expression at the module level generated disease-specific transcriptional fingerprints that provide a stable framework for the visualization and functional interpretation of microarray data. These transcriptional modules were used as a basis for the selection of biomarkers and the development of a multivariate transcriptional indicator of disease progression in patients with systemic lupus erythematosus. Thus, this work describes the implementation and application of a methodology designed to support systems-scale analysis of the human immune system in translational research settings.

This SuperSeries is composed of the SubSeries listed below.

overall_design:
Experiment subseries GSE11908 regroups the profiles that have been used to construct the modular transcriptional framework:
The first step of the module-construction process analyzes expression patterns of transcripts across samples for individual diseases: sets of coordinately expressed transcripts were identified with an unsupervised clustering algorithm; in this case, the GeneSpring Version 7.1 (Agilent) implementation of the K-Means algorithm (k = 30). All transcripts detected in at least one sample were used as input; no screening for differential expression was performed. The second step of the module-construction process analyzed the “clustering behavior” of transcripts across diseases, taking into account the possibility that genes may cocluster in some diseases but not in others. Also, in our example, the transcripts that clustered together across all eight diseases were grouped to form a set of modules (round 1 of selection), and the stringency of the analysis was then decreased gradually to identify transcripts that belong to a similar K-means cluster in only a subset of diseases (round 2: seven out of eight diseases; round 3: six out of eight diseases). It is important to note that the module-selection process is “data-driven” and does not involve manual selection of genes by the investigator.
We implemented the module-construction strategy described above, using as input a total of 239 peripheral-blood mononuclear cell (PBMC) samples obtained from individuals with one of the following conditions: systemic juvenile idiopathic arthritis (n = 47), systemic lupus erythematosus (n = 40), type I diabetes (n = 20), metastatic melanoma (n = 39), acute infections (Escherichia coli [n = 22], Staphylococcus aureus [n = 18], Influenza A [n = 16]) or liver-transplant recipients undergoing immunosuppressive therapy (n = 37). Transcriptional profiles were generated with Affymetrix U133A and U133B GeneChips (> 44,000 probe sets). A total of 4742 transcripts, distributed among 28 sets, were selected after running of the module-construction algorithm described above. Each module is assigned a unique identifier indicating the round and order of selection (i.e., M3.1 is the first module identified in the third round of selection).
The stringency of this algorithm was tested statistically by implementation of the same module-construction procedure after randomization of the original data set. This process was repeated 200 times, without a single module identified. Therefore, the analysis of gene-cluster membership across multiple diseases provided a stringent means to identify PBMC transcriptional modules.

Experiment subseries GSE11909 regroups the profiles that have been used to identify and validate biomarkers of SLE disease activity:
The proposed biomarker-selection strategy relies on modules for reducing highly dimensional microarray data sets in a stepwise manner. Starting from the full set of 28 modules, only those for which a set minimum proportion of transcripts are significantly changed between the study groups are selected (e.g., minimum proportion of differentially expressed transcripts at p < 0.05 = 15% overexpressed or underexpressed transcripts; in the example given, 11 SLE modules meet this criterion). This eliminates from the selection pool the modules registering fewer consistent changes that could be attributed to noise. Transcriptional vectors were derived for the entire cohort of 22 untreated pediatric SLE patients with the use of this set of 11 SLE modules. Patient profiles were also generated for an independent set of 31 children with SLE treated with steroids and/or cytotoxic drugs and/or hydroxychloroquine. A nonparametric method for analyzing multivariate ordinal data was used to score the patients. Lupus disease flares can lead to irreversible worsening of the patient's status. We tested the relevance of this multivariate transcriptional score for longitudinal monitoring of the disease activity in a cohort of 20 pediatric SLE patients (two to four time points/patient, intervals between each time point varied from one month to 18 months). Half of the patients had been included in our cross-sectional analysis before they were enrolled in this longitudinal study. Parallel trends were observed between multivariate transcriptional scores and a clinical severity score. The positive association was verified statistically with the use of a linear-regression model.

first: Charles

last: Quinn

first: Jing

last: Shen

first: Pinakeen

last: Patel

first: Casey

last: Glaser

first: Nicole

last: Baldwin

first: Dorothee

last: Stichweh

first: Derek

last: Blankenship

first: Lei

last: Li

first: Indira

last: Munagala

first: Lynda

last: Bennett

first: Florence

last: Allantaz

first: Asuncion

last: Mejias

first: Monica

last: Ardura

first: Ellen

last: Kaizer

first: Laurence

last: Monnet

first: Windy

last: Allman

first: Henry

last: Randall

first: Diane

last: Johnson

first: Aimee

last: Lanier

first: Marilynn

last: Punaro

first: Knut

middle: M

last: Wittkowski

first: Perrin

last: White

first: Joseph

last: Fay

first: Goran

last: Klintmalm

first: Octavio

last: Ramilo

first: Karolina

last: Palucka

first: Jacques

last: Banchereau

first: Virginia

last: Pascual


title: Cross-talk between Estrogen Receptor-related Receptor alpha (ERRalpha) and Estrogen Receptor (ERalpha) pathways.

status: Public on Aug 25 2008

city: Palo Alto

first: Dmitri

middle: A

last: Kazmin

address: 240 Pasteur Dr

department: Institute for Immunity, Transplantation and Infection

institute: Stanford University

summary:
Orphan nuclear receptor estrogen-related receptor alpha (ERRα) has recently been shown to carry negative prognostic significance in breast and ovarian cancers.  The specific role of ERRα in tumor growth and progression, however, is yet to be fully understood. The significant homology between estrogen receptor alpha (ERα) and ERRα initially suggested that these receptors may share similar transcriptional targets.  Using the well-characterized ERα-positive MCF-7 breast cancer cell line, we sought to gain a genome-wide picture of ERα-ERRα cross-talk using an unbiased microarray approach.
Since a small molecule ligand has not been identified for ERRα, its transcriptional activity in these studies was induced using its known coactivator PGC-1α (peroxisome proliferator-activated receptor-γ coactivator-1α) as a protein ligand.  Both wild-type PGC1, as well as ERRa-specific variant (PGC1 2x9) were used to activate ERRa. Non-NR-dependent activities of PGC-1α were controlled for using a variant PGC-1α in which the leucines within the NR-interacting domain had been mutated to alanines (L2L3M). Beta-galactosidase (BGAL) overexpression was used as a non-specific background control. Activation of endogenous ER was achieved by treatment with 10 nM estadiol.
Keywords: Response to stimulus

overall_design: MCF7 cells were infected with adenovirus overexpressing one of the four proteins (PGC1 WT, PGC1 2x9, PGC1 L2L3 and BGAL); Twelve hours post infection cells were then treated with either 10 nM estradiol (E2)or ethanol vehicle for 12 hours. Each combination of infection and treatment was was repeated three times in three independent experiments.  A total of 23 samples are presented. Two replicates instead of three are shown for PGC1 WT E2 for technical reasons.

first: Rebecca

last: Stein

first: Ching

middle: Y

last: Chang

first: James

last: Way

first: Donald

middle: P

last: McDonnell


title: Effect of Seprase/FAP alpha on the metastasis of human melanoma LOX cell

first: Wei

last: Zeng

summary:
In order to detect the molecular mechanism of seprase involved in tumor metastasis, a comparison of gene expression pattern was made between two groups of human melanoma LOX cell sublines, one is transfected with mock vector expressing GFP only (GUS-1<GSM30738>, GUS-2<GSM30729> and GUS-3<GSM30730>) and the other with pGUS expressing GFP and siRNA targeting mRNA of seprase/FAP alpha (sep-1<GSM30731>, sep-2<GSM30732> and sep-3<GSM30740>).
Keywords: other

first: Donghai

last: Chen

first: Carlo

last: Bergamini

first: Jackyn

last: Freudenberg

first: Wen-Tien


title: Two PBAP chromatin remodeling complex-specific subunits have distinct, redundant functions during Drosophila development

status: Public on Jul 23 2008

city: New York

first: Jessica

middle: E

last: Treisman

address: 540 First Avenue

department: Cell Biology (Skirball)

institute: NYUMC

summary:
Chromatin remodeling complexes control the availability of DNA binding sites to transcriptional regulators. Two distinct forms of the major SWI/SNF-related complex that have different activities in vitro can be distinguished by the presence of specific accessory subunits. In Drosophila, the core Brahma complex associates either with Osa to form the BAP complex, or with Bap170 and Bap180 to form the PBAP complex. Mutations affecting the core subunits have stronger developmental phenotypes than osa mutations; these differences could be due to PBAP complex activity . We have generated mutations in the genes encoding the PBAP-specific subunits Bap170 and Bap180 in order to study their functions in vivo. Bap180 is not essential for viability, but is required in ovarian follicle cells for normal eggshell development. Bap170 is necessary to stabilize the Bap180 protein; however, a mutant form that retains this function is sufficient for survival and fertility. The two subunits act redundantly to allow metamorphosis; using gene expression profiling of double mutants, we have found that the PBAP complex regulates genes involved in tissue remodeling and immune system function. Finally, we have generated mutants that lack Bap170, Bap180 and Osa in the germline to demonstrate that the function of the core Brahma complex in oogenesis does not require any of these accessory subunits.
To study PBAP specific subunits Bap170 and Bap180 role in Drosophila melanogaster development

Keywords: genetic modification, chromatin remodelling, gene regulation, microarray

overall_design: Two males and two females for each genotype (wild type and bap170;bap180 double mutant) were processed at white prepupae stage (0 hour APF)

first: Inés

last: Carrera

first: Jiri

last: Zavadil


title: Dynamics of mRNA abundance and translation in response to short and prolonged hypoxia and reoxygenation

status: Public on Jul 22 2008

city: Riverside

first: Charles

last: Jang

address: 2215 Batchelor Hall

department: Botany and Plant Sciences

institute: UC Riverside

summary:
Gene expression analysis of 7d-old Arabidopsis seedlings exposed to short term (2 h) hypoxia, long term (9 h) hypoxia, and 1 h reoxygenation after long term (9 h) hypoxia to evaluate the regulation of gene expression at the level of translation.
Keywords: Time Course, hypoxia recovery, polysomal mRNA, IP RNA, polysomes, hypoxia stress, reoxygenation, translational control.

overall_design: 30 samples, 5 conditions (2 hr hypoxia stress, 2hr non-stress, 9 hr hypoxia stress , 9 hr non-stress, 9 hr hypoxia with 1 hr recovery), 2 RNA pools (Total mRNA and polysomal mRNA), 3 replicates

first: Cristina

last: Branco-Price

first: Julia

last: Bailey-Serres


title: affy_aba_ath1-Characterization of aba3-1 suppressor mutants

city: Beaucouze

first: sandrine

last: balzergue

address: Rue G. Morel

country: France

institute: INRA

summary:
affy_aba_ath1 - affy_aba_ath1 - In order to identify new genes implicated in ABA signalisation pathways, we have carried out a screen for suppressor mutants of the ABA deficient mutant aba3-1. Infrared thermography was used to identify plants whose cold leaf phenotype was reverted. The transcriptome analysis of four recessive suppressor mutants was undertaken to determine which genes are differentially expressed compared to the wild type or aba3-1. Such genes could represent the mutated gene itself or indicate signalling or response pathways affected by the mutation-comparison of four suppressor mutants (g-radiation induced) to wild type and the ABA deficient mutant aba3-1
Keywords: wt vs mutant comparison

overall_design: 12 arrays - ATH1

first: Sandrine

last: Balzergue

first: Helen

last: North

first: Anne

last: Plessis

first: Marie-Laure

last: Martin-Magniette


title: A Precisely Regulated Gene Expression Cassette Potently Modulates Metastasis and Survival in Multiple Solid Cancers

city: Singapore

first: Kun

last: Yu

department: Cellular & Molecular Research

country: Singapore

institute: National Cancer Centre

summary: Successful carcinogenesis involves the integration of both pro- and anti-oncogenic pathways. We postulated that genes critical for balancing these opposing pathways are likely to be precisely controlled in tumors, since even subtle alterations in their activity might cause substantial alterations in tumor growth and survival. Using a novel genomic approach, we identified a 48-gene “Poised Gene Cassette” (PGC) showing tight regulation specifically in human cancers but not in corresponding nonmalignant tissues. We show, using a wide variety of in vitro and in vivo approaches, that small alterations in PGC expression are consistently associated with significant differences in experimental metastasis and patient survival, and we demonstrate a direct functional role for five PGC genes (p53CSV, MAP3K11, MTCH2, CPSF6 and SKIP) in cancer invasion. Our findings support the existence of a novel class of ultrasensitive genes that may regulate various cancer-associated phenotypes such as metastasis. Such precisely controlled genes could represent appealing drug targets, since even partial alterations in their activity should prove sufficient to induce potent effects on tumors. Besides cancer, our analytical approach is quite generalizable and likely to be applicable to other disease conditions.

overall_design: Primary human tumor and adjacent normal tissues were obtained from the Tissue Repository of the National Cancer Centre of Singapore (NCCS).  Appropriate institutional approvals were obtained from the NCCS Tissue Repository and Ethics Committees. In the operating theater, morphologically visible tumor and adjacent matched normal tissues were removed by surgery and examined by a surgical pathologist to confirm the presence of cancer cells by cryosections. The tissue samples were divided into discrete aliquots, flash frozen and subsequently stored in liquid nitrogen. The detailed information regarding samples is available in Supplementary Information S1. The training set used in this study comprises 341 samples (270 tumors and 71 matched normals) from patients with breast, colon, liver, lung, oesophagal and thyroid cancer (Tissue Type/Tumor/Normal : Lung/18/12, Thyroid/35/16, Liver/9/8, Oesophagus/16/13, Colon/9/9, Breast/183/13.)

first: Patrick

last: Tan


title: Non-supervised hierarchical clustering of gene expression data

city: Chicago

first: yuanyuan

last: zha

state: IL

department: Department of Pathology

institute: University of Chicago

summary:
Despite the frequent detection of circulating tumor antigen-specific T cells, either spontaneously or following active immunization or adoptive transfer, immune-mediated cancer regression occurs only in the minority of patients.  One theoretical rate-limiting step is whether effector T cells successfully migrate into metastatic tumor sites.  Affymetrix gene expression profiling performed on a series of metastatic melanoma biopsies revealed a major segregation of samples based on the presence or absence of T cell-associated transcripts.  The presence of lymphocytes correlated with the expression of defined chemokine genes.  A subset of 6 chemokines (CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10) was confirmed by protein array and/or quantitative RT-PCR to be preferentially expressed in tumors that contained T cells.  Corresponding chemokine receptors were found to be upregulated on human CD8+ effector T cells, and transwell migration assays confirmed the ability of each of these chemokines to promote migration of CD8+ effector cells in vitro.  Screening by chemokine protein array identified a subset of melanoma cell lines produced a similar broad array of chemokines.  These melanoma cells more effectively recruited human CD8+ effector T cells when implanted as xenografts in NOD/scid mice in vivo.  Chemokine blockade with specific antibodies inhibited migration of CD8+ T cells.  Our results suggest that lack of critical chemokines in a subset of melanoma metastases may limit the migration of activated T cells, which in turn could limit the effectiveness of anti-tumor immunity.

Keywords: Tumor sample microarray

overall_design: To examine associations between CD8 expression and a diverse panel of chemokines, affymetrix gene expression profiling performed on a series of metastatic melanoma biopsies. Non-supervised hierarchical clustering analysis revealed a major segregation of samples based on the presence or absence of T-cell-associated transcripts.

first: Helena

last: Harlin

first: Yuru

last: Meng

first: Amy

last: Perterson

first: Yuanyuan

last: Zha

first: Maria

last: Tretiakova

first: Craig

last: Slingluff

first: Mark

last: McKee

first: Thomas

last: Gajewski


title: Expression data from  Congenital disorders of Glycosylation type-1 patients (CDG-I)

status: Public on Jul 11 2008

city: Zurich

first: Thierry

last: Hennet

department: Institute of physiology

country: Switzerland

institute: University of Zurich

summary:
Disruption of N-linked glycosylation has a broad impact on proper glycosylation of nascent glycoproteins in the endoplasmic reticulum, which affect multiple signalling pathways( by changing the stability of membrane proteins or the signalling ability of membrane receptors) and may be responsible of the fibrotic stage associated to CDG type-I.
We used microarrays to characterize the global changes in  gene expression in three distinct groups of CDG-I patients and we identified a common perturbation in the expression of genes encoding for proteins involved in the stress as well as in the fibrotic responses.
Keywords: disease state analysis

overall_design: Primary fibroblasts, obtained from forearm skin biopsies of healthy control subjects and three distinct groups of CDG-I patients,  were cultured in F12 DMEM supplemented with 4.5 g/l glucose and 10% fetal calf serum prior to RNA extraction and Hybridization on  human HG 133 A Affymetrix Microarrays. To avoid bias related to the analysis of a specific CDG-I type or to a specific individual expression pattern, we have  considered three patients for the CDG-Ic and -Ie group  and two patients for the CDG-Ig group. Moreover the total RNA  was extracted from three different independent biological replicates.

last: Lecca

first: Charlotte

last: Maag

first: Eric

last: Berger

first: Ulrich

last: Wagner

first: Andrea

last: Patrignani


title: Gene expression profile of keratinocyte stem cells

status: Public on Jul 31 2008

city: Stony Brook

first: Maja

last: Matic

address: SUNY at Stony Brook

institute: SUNY at Stony Brook

summary:
Background: Human interfollicular epidermis is sustained by the proliferation of stem cells and their progeny, transient amplifying cells. Molecular characterization of these two cell populations is essential for better understanding of self renewal, differentiation and mechanisms of skin pathogenesis. The purpose of this study was to obtain gene expression profiles of alpha 6+/MHCI+, transient amplifying cells and alpha 6+/MHCI- , putative stem cells,  and to compare them with existing data bases of gene expression profiles for mouse hair follicle stem cells. The expression of Major Histocompatibility Complex (MHC) class I, previously shown to be absent in stem cells in several tissues, and alpha 6 integrin were used to isolate MHCI positive basal cells , and MHCI low/negative basal cells.

Results: Transcriptional profiles of the two cell populations were determined and comparisons made with published data for hair follicle stem cell gene expression profiles. We demonstrate that presumptive interfollicular stem cells, alpha 6+/MHCI- cells, are enriched in messenger RNAs encoding surface receptors, cell adhesion molecules, extracellular matrix proteins, transcripts encoding members of IFN-alpha family proteins and components of IFN signaling, but contain lower levels of transcripts encoding proteins which take part in energy metabolism, cell cycle, ribosome biosynthesis, splicing, protein translation, degradation, DNA replication, repair, and chromosome remodeling. Furthermore, our data indicate that the cell signaling pathways Notch1 and NF-κB are downregulated/inhibited in MHC negative basal cells.

Conclusion: This study demonstrates that alpha 6+/MHCI- cells have additional characteristics attributed to stem cells. In addition, the transcription profile of alpha 6+/MHCI- cells shows similarities to transcription profiles of mouse hair follicle bulge cells known to be enriched for stem cells. Collectively, our data suggests that alpha 6+/MHCI- cells may be enriched for stem cells. This study is the first comprehensive gene expression profile of putative human epithelial cells and their progeny that were isolated directly from neonatal foreskin tissue. Our study is important for understanding self renewal and differentiation of epidermal stem cells, and for elucidating signaling pathways involved in those processes. The generated data base may serve those working with other human epithelial tissue progenitors.

Keywords: Comparison of stem and TA cells

overall_design: In the present work we employ microarray technology, to report global transcriptional profiles of two cell populations: the basal cells that express MHCI, α6+/MHCI+ (transient amplifying cells) and the basal cells that have low/negative MHCI expression, α6+/MHCI- cells, (putative stem cells). Cells were isolated using fluorescence-activated cell sorter (FACS) directly from human epidermis. Further comparisons were made with published data of hair follicle stem cell gene expression profiles.

first: Salih

last: Kocer

first: Petar

last: Djuric

first: Monica

last: Bugallo

first: Sanford

last: Simon


title: Transcript abundance data from seedlings of wild-type Ws and ged1 (greening after extended darkness 1) mutant

city: Cambridge

first: Mun-Kit

last: Choy

state: Cambridgeshire

address: Box 110, Level 6, ACCI Building, Addenbrooke’s Hospital, Hills Road

department: Division of Cardiovascular Medicine

institute: University of Cambridge

summary:
An Arabidopsis mutant showing an altered ability to green on illumination after extended periods of darkness has been isolated in a screen for genomes uncoupled (gun) mutants. Following illumination for 24 h, 10-day-old dark-grown mutant seedlings accumulated 5 times more chlorophyll than wild-type seedlings and this was correlated with differences in plastid morphology observed by transmission electron microscopy. The mutant has been named greening after extended darkness 1 (ged1). We used microarrays to detail the global profiles of transcript abundances in the mutant in comparison to the wild type. Microarray analysis showed much lower amounts of transcripts of genes encoding seed storage proteins, oleosins and late embryogenesis abundant (LEA) proteins in 7-day-old seedlings of ged1 compared to wild type. RNA-gel-blot analyses confirmed very low levels of transcripts of seed protein genes in ged1 seedlings grown for 2-10 days in the dark, and showed higher amounts of transcripts of photosynthesis-related genes in illuminated 10-day-old dark-grown ged1 seedlings compared to wild type.

Keywords: Genotype

overall_design: Total RNA samples were extracted from wild-type Ws and ged1 seedlings grown for 5 days in the dark followed by 2 days in the light and hybridised on Affymetrix ATH-121501 arrays. Transcript amounts between wild-type Ws and ged1 seedlings were compared globally.

first: John

last: Gray


title: Gene expression profiles of the intralobular and interlobular fibroblast populations within the human breast

status: Public on Aug 01 2008

city: Bethesda

first: Jodie

last: Fleming

state: MD

address: Bldg 37, 37 Convent drive

department: National Cancer Institute

institute: National Institutes of Health

summary:
     The normal growth and function of mammary epithelial cells depend on interactions with the supportive stroma.  Alterations in this communication can lead to the progression or expansion of malignant growth.  The human mammary gland contains two distinctive types of fibroblasts within the stroma.  The epithelial cells are surrounded by loosely connected intralobular fibroblasts, which are subsequently surrounded by the more compacted interlobular fibroblasts.  The different proximity of these fibroblasts to the epithelial cells suggests distinctive functions for these two subtypes. In this report, we compared the gene expression profiles between the two stromal subtypes.

     No statistically significant difference was detected between the gene expression profiles of the interlobular and intralobular fibroblasts by microarray analysis and RT-PCR.  However, for some of the genes tested, the protein expression patterns between the two subtypes of fibroblasts were significantly different.

     This study is the first to report the gene expression profiles of the two distinct fibroblast populations within the human mammary gland.  While there was no significant difference in the gene expression profiles between the groups, there was an obvious difference in the expression pattern of several proteins tested.  This report also highlights the importance of studying gene regulation at both the transcriptional and post-translational level.

overall_design:
Sample Collection
This study was performed in accordance with the guidelines of the National Cancer Institute Review Board, protocol 02-C-0144.  All patients provided written informed consent.  Fresh human mammary tissue was collected from four (two Caucasian, one African-American, and one Hispanic) female, pre-menopausal, reduction mammoplasty patients, ages ranging from 18 to 40 years old.  The tissue was embedded in Tissue-Tek O.C.T. embedding medium (Sankura Finetek Inc., Torrance CA) and frozen on dry ice immediately after surgery.  Eight - 10 micron sections of tissue were cut using a Leica 2800 Frigocut-E cryostat (Bannockburn, IL).  Every tenth section was subjected to hematoxylin and eosin staining.  For each patient sample, sections with distinctive intralobular and interlobular regions were selected for laser capture and microarray analysis.

Laser capture microdissection and microarrays
Laser capture microdissection (LCM) and microarrays were performed by Cogenics, Inc. (Morrisville, NC).  Briefly, selected intralobular and interlobular stroma sections of frozen tissue were subjected to an AutoPixTM automated LCM system from Arcturus, using static image settings.  RNA was isolated from each specimen, pooled, and then evaluated by spectrophotometry and by using an Agilent Bioanalyzer before proceeding to sample amplification.  For each sample, 50 ng of total RNA was amplified using Affymetrix Two-Cycle Target Labeling kit (Santa Clara, CA).   Ten micrograms of biotinylated cRNA spiked with bioB, bioC, bioD, and cre as a control was hybridized to the Affymetrix Human Genome U133 GeneChip® for 16 h at 45°C.  Following hybridization, arrays were washed and stained with Affymetrix GeneChip Fluidics Station.  Stained arrays were scanned with an Affymetrix GeneChip Scanner 3000.  Quality check and preliminary data analysis were carried out using Affymetrix GeneChip Operating Software and Quality Reporter.

Microarray analysis
Microarray data were analyzed using the Affy package available at the Bioconductor website (www.bioconductor.org). The raw data were first background-corrected by the Robust Multichip Average (RMA) method and then normalized by an invariant set method. Unsupervised hierarchical clustering analysis was performed on 1,115 most variable genes. The difference of gene expression between the inter- and intra-stromal samples was analyzed by the Limma package available at the Bioconductor website.  P-values obtained from the multiple comparison tests were corrected by false discovery rates.

first: Lori

last: Long

first: Erika

last: Ginsburg

first: Daniel

last: Gerscovich

first: Paul

last: Meltzer

first: Barbara

middle: K

last: Vonderhaar


city: Raleigh

first: Mike

last: Magwire

state: NC

address: 3501 Gardner Hall

department: Genetics

institute: NCSU

summary:
ABSTRACT
MAGWIRE, MICHAEL MAHLON. Mutations Increasing Drosophila melanogaster
Life Span. (Under the direction of Trudy F. C. Mackay.)
Better living conditions and advances in medicine have made it possible for
humans to live considerably longer than before. This has uncovered many
health problems associated with age. By identifying genes involved in the
limitation of life span, we may better understand the processes that lead to aging.
In model organisms, we can use mutagenesis to discover mutations that
increase life span, and hence infer that the wild-type allele limits life span by some means.
We have assessed longevity in a collection of 1332 co-isogenic gene-trap
P-element insertion lines and determined changes in life span of each insert line relative to the corresponding control line. Significant lines were determined via two different methods of analysis: A 95% confidence interval was established based on deviations of the mutant life spans from the controls and Dunnett’s two tailed t-tests were used to examine differences between mutant and control in each individual block. Based on the 95% confidence interval, 139 inserts displayed an increase in life span and 194 inserts a decrease. Using Dunnett’s, 70 lines increased in life span, while 270 decreased in life span. An additional 48 increased longevity lines were close enough to meeting Dunnett’s criteria to be considered for additional investigation. Combining these two analyses, we chose 83 inserts associated with increased life span for a secondary screen using an
additional twelve replicates. 58 of these lines remained significant. We have
determined the P-element insertion site for 50 of these lines.
Starvation resistance, chill coma recovery time and climbing activity were
measured on the lines remaining significant after the the secondary screen to identify pleiotropic effects. A positive correlation was found for males between life span and starvation resistance as well as between life span and chill coma recovery. Females only displayed a correlation between life span and chill coma recovery, which was negative. None of the lines indicated increased fitness for all phenotypes, indicating there may be some type of trade-off. There also appears to be a lot of pleiotropic variation depending on background and sex.
Ten of the lines, all with the same parental background, were chosen for a
half-diallel cross to identify epistasis between the mutants. There were
substantial epistatic interactions between all ten lines. Furthermore, males and females displayed vastly different patterns of epistasis, again indicating major differences in life span regulation between the sexes.
Finally, a subset of seven of the lines used in the epistatic study, in
addition to the corresponding parental control, were chosen for microarray
analysis to look for possible pathways and novel genes involved in increasing life span. 1,996 probe sets were significant at a false discovery rate q-value
threshold of q < 0.0001. Tukeys tests were carried out for these probe sets to determine how each of the seven mutant backgrounds differed compared to the control and each other. In addition, each mutant background was compared individually with the control to identify any changes in expression. Gene ontologies were determined for each of the lines to identify over-represented biological functions which would indicate possible longevity pathways. Dozens of pathways were suggested, including several novel pathways.
Keywords: Genetic (P-element insertion) modification and comparison to control line

overall_design: We used seven mutant lines homozygous for a P-element insertion and a control line.  All lines were isogenic other than the P-element insert.  Males and females were done separately with two replicates for a total of 32 arrays.

first: Michael

first: Akihiko

last: Yamamoto

first: MaryAnna

last: Carbone

first: Natalia

middle: V

last: Roshina

first: Elena

middle: G

last: Pasyukova

first: Tatiana

last: Morozova

first: Trudy

last: Mackay


title: Global Analysis of the Drosophila NELF complex

status: Public on Jul 14 2008

city: RTP

first: NIEHS

last: Microarray Core

address: 111 T.W. Alexander Drive

department: DIR

institute: NIEHS

summary:
To determine the physiological targets of the NELF complex, and provide insight into the mechanism of NELF activity in vivo.
Keywords: gene expression, transcription elongation, stress response

overall_design: Drosophila melanogaster S2 cells, obtained from the Drosophila Genomics Resource Center, were untreated, or treated with dsRNA for 72 hours, as described in Armknecht, S. et al. (2005) Methods in Enzymology, 392: pp. 55-73.  Total RNA was then extracted using the RNeasy RNA extraction kit, with on column DNAse digestion (Qiagen), according to manufacturer’s protocol.  Gene expression analysis was conducted using Drosophila Genome 2.0 Genechip® arrays (Affymetrix, Santa Clara, CA).  Starting with 1ug of total RNA, biotin-labeled cRNA was produced using the Affymetrix 3’ Amplification One-Cycle Target labeling kit according to manufacturer’s protocol.  For each array, 10ug of amplified cRNAs were fragmented and hybridized to the array for 16 hours in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol.  Slides were stained and washed as indicated in the Antibody Amplification Stain for Eukaryotic Targets protocol using the Affymetrix Fluidics Station FS450.  Arrays were then scanned with an Affymetrix Scanner 3000 and data was obtained using the Genechip® Operating Software (Version 1.2.0.037) and imported into the Rosetta Resolver system (Version 6.0).

last: Gilchrist

first: Chanhyo

last: Lee

first: Jennifer

last: Collins

first: Saikat

last: Ghosh

first: David

middle: S

last: Gilmour

first: Karen

last: Adelman


title: NFYA5, a CCAAT binding transcription factor important for drought resistance in Arabidopsis

status: Public on Jul 09 2008

first: Jian-Kang

last: Zhu

address: 2150 Batchelor Hall

institute: University of California

summary: NFYA5 is a transcription factor important for drought resistance.  Drought stress up-regulates NFYA5 gene expression not only at the transcriptional level but also at the posttranscriptional level by down-regulating the expression of miR169a that targets NFYA5 transcript for cleavage.

overall_design: 2 replicates for WT (Col-0) were compared to 2 replicates for nfya5 (mutant)

first: Wenxue

last: Li

first: Youko

last: Oono

first: Jianhua

first: Xin-Jian

last: He

first: Jian-Min

last: Wu

first: Kei

last: Iida

first: Xiao-Yan

last: Lu

first: Xinping

last: Cui

first: Hailing

last: Jin


title: Expression data from overexpression and knockdown of HOXC6 in LNCaP cells

status: Public on Jul 01 2008

city: Atlanta

first: Colleen

middle: D

last: McCabe

state: GA

address: 615 Michael St

institute: Emory University

summary:
The transcription factor HOXC6 is upregulated in human prostate cancer. SiRNA knockdown of HOXC6 induces apoptosis in LNCaP cells while upregulation rescued LNCaP cells from siRNA-induced apoptosis.
We used microarrays to identify the genes whose expression underly the anti-apoptotic and proliferative effects of HOXC6 in LNCaP cells.
Keywords: transient overexpression and knockdown

overall_design: LNCaP cells were transiently transfected with a cocktail of 5 siRNAs to HOXC6 to a final concentration of 100nM, with a HOXC6 gene expression vector, or control siRNA and vector.  Cells were collected a 48hours post-transfection

first: Carlos

last: Moreno


title: Human Dendritic Cell Subtype Gene Arrays

city: Albany

first: Stephen

middle: Henry

last: Wrzesinski

address: 317 S. Manning Blvd

department: Medical Oncology

institute: St. Peter's Cancer Care Center

type:
Expression profiling by array
Expression profiling by MPSS

summary: Among the dendritic cell (DC) subsets, plasmacytoid DC’s are thought to be important in both generating antiviral and antitumor responses.  These cells may be useful in developing dendritic cell-based tumor vaccines, however, the rarity of these cells in the peripheral blood have hampered attempts to understand their biology.  To provide better insight into the biology of plasmacytoid DCs, we isolated these cells from the peripheral blood of healthy donors in order to further characterize their gene expression.  Using gene array technology we compared the genetic profiles of these cells to those of CD14+ monocytes isolated from the same donors and found several immune related genes upregulated in this cell population.  Understanding the genetic profiles of this dendritic cell subtype as well as others such as the BDCA-1 expressing myeloid DCs may enable us to manipulate these cells ex-vivo to generate enhanced DC-based tumor vaccines inducing more robust antitumor responses.

overall_design: 4 replicates of BDCA cells and CD14 cells

first: Jill

last: Uhlenhake

first: Jan

middle: L

last: Fisher

first: Marc

last: Ernstoff


title: Vitamin D3/Hoxa10 pathway supports Mafb function during the monocyte differentiation of human CD34++ hematopoietic cells

city: Modena

first: CLAUDIA

last: GEMELLI

state: Modena

address: Via Campi 287

department: Biomedical Science

country: Italy

institute: University of Modena and Reggio Emilia

summary: Although a considerable number of reports indicate an involvement of the Hox-A10 gene in the molecular control of hematopoiesis, the conclusions of such studies are quite controversial since they support, in some cases, a role in the stimulation of stem cell self-renewal and myeloid progenitor expansion while, in others, implicate this transcription factor in the induction of monocyte - macrophage differentiation. To clarify this issue we analyzed the biological effects and the transcriptome changes determined in human primary CD34+ hematopoietic progenitors by retroviral transduction of a full length Hox-A10 cDNA. The results obtained clearly indicated that this homeogene is an inducer of monocyte differentiation, at least partly acting through the up-regulation of MafB gene, recently identified as master regulator of such maturation pathway. By using a combined approach based on computational analysis, EMSA experiments and luciferase assays, we were able to demonstrate the presence of a Hox-A10 binding site in the promoter region of the MafB gene, which suggested the likely molecular mechanism underlying the observed effect. Interestingly, stimulation of the same cells with the Vitamin D3 monocyte differentiation inducer resulted in a clear increase of Hox-A10 and MafB transcripts, indicating the existence of a precise transactivation cascade involving VDR, Hox-A10 and MafB transcription factors. Altogether these data allow to conclude that the Vitamin D3 / Hox-A10 pathway supports MafB function during the induction of monocyte differentiation.

overall_design: RNA pools (100 ng) of LXIDN and LHoxA10IDN transduced CD34+ cells, obtained from three independent experiments, were converted in labelled cRNA according with the “Two cycle” protocol advised by Affymetrix. cRNA has been used to hybridise Affymetrix HG-U133A GeneChip arrays. Images obtained by scanning chips of LXIDN and LHoxA10IDN transduced CD34+ cells were processed using the GeneChip Operating Software. Microarray analysis of Hox-A10 transduced CD34+ cells provided a substantial contribute for a better comprehension of the biological effects driven by this transcription factor in human primary hematopoietic stem / progenitor cells. Results of this analysis confirmed the stimulatory effect exerted by Hox-A10 on monocytopoiesis, disclosing an up-regulated expression of transcription factors and differentiation markers (CD antigens, granule proteins, cytokines / chemokines) that are typically associated with this maturation lineage. In addition, they also evidenced a decreased expression of genes related to erythroid and granulocyte differentiation programs. This last effect was also confirmed by cytochemical and morphological evaluation of Hox-A10 transduced CD34+ cells.

first: Claudia

last: Gemelli

last: Orlandi

first: Tommaso

last: Zanocco marani

first: Andrea

last: Martello

first: Tatiana

last: Vignudelli

first: Francesco

last: Ferrari

first: Monica

last: Montanari

first: Sandra

last: Parenti

first: Anna

last: Testa

first: Alexis

last: Grande

first: Sergio


title: Gene expression index for olfactory behavior in Drosophila melanogaster

status: Public on Jun 26 2008

first: Deepa

last: Sambandan

address: 100, Derieux

institute: North Carolina State University

summary:
Transcriptional analysis for GEI in olfactory behavior
Keywords: whole body, olfactory behavior

overall_design: Larvae were reared on three different media: standard cornmeal-molasses medium, standard medium supplemented with 9% ethanol  and standard medium enriched with tomato paste. After eclosion, we transferred all flies to standard medium at 25°C under a 12:12 hour light/dark cycle prior to characterization of behavioral responses to benzaldehyde and transcription profiling.


title: Intestinal xenotransplants infected with Shigella

status: Public on Jun 23 2008

city: Paris

first: Thierry

last: Pedron

address: 28 rue du Docteur Roux

department: Biologie cellulaire et Infection

institute: Pasteur Institute

summary:
to analyse the transcriptomic response of human intestinal tissue engrafted in SCID mice to Shigella infection
Keywords: infection, Shigella, gene expression, intestinal cell

overall_design:
Four experimental conditions with two biological replicates.
After 2 hours infection, tissues were removed and ARN were extracted before labeling and hybridizations on U-133A genechip.

first: Brice

last: Sperandio

first: Beatrice

last: Regnault

first: Jianhua

last: Guo

first: Zhi

last: Zhang

first: Samuel

last: Stanley

first: Philippe

middle: J

last: Sansonetti


title: Brassinazole treatment of arf2 and wild-type dark-grown seedlings

status: Public on Jun 20 2008

city: Seattle

first: Jennifer

last: Nemhauser

state: WA

institute: Univ. of Washington

summary:
While the close relationship between BRs and auxin has been widely reported, the molecular mechanism for combinatorial control of shared target genes has remained elusive. In this work, we demonstrate that BRs synergistically increase seedling sensitivity to auxin and show that combined treatment with both hormones can increase the magnitude and duration of gene expression. arf2 mutants are less sensitive to changes in endogenous BR levels, while a large number of genes affected in an arf2 background are returned to near wild-type levels by altering BR biosynthesis. Together, these data suggest a model where BIN2 increases expression of auxin-induced genes by directly inactivating repressor ARFs, leading to synergistic increases in transcription.
Keywords: arf2 vs. Col, BRZ vs. mock

overall_design: Total RNA was extracted from 4-day-old, etiolated Arabidopsis seedlings grown on 0.5 µM BRZ or mock treatments and used to probe ATH1 microarrays (Affymetrix), according to manufacturer’s protocols. There are three independent biological replicates for each genotype and treatment, except for arf2 mutants with BRZ where only two arrays passed quality control. We performed standard Affymetrix quality-control procedures using the BioConductor packages simpleaffy. Expression was normalized and estimated using the gcRMA package of BioConductor.

first: Cristina

last: Walcher

first: Grégory

last: Vert

first: Joanne

last: Chory


title: CD34 Overexpression

status: Public on Jun 03 2008

first: Rossella

last: Manfredini

address: Via Gottardi 100

department: Life Sciences

institute: Centre for Regenerative Medicine

summary:
In order to investigate the role of CD34 antigen in haematopoietic commitment, we overexpressed the human CD34 cDNA in human CD34+ cells by retroviral gene transfer.
Keywords: treatment comparison

overall_design: In a set of 10 independent experiments, gene transfer efficiency, assessed by flow cytometry analysis of ΔLNGFR positivity, ranged 15 to 30%. Transduced cells were than purified for NGFR expression at day 3 of liquid culture with the retroviral vector. Phenotypic analysis, performed on transduced/NGFR purified cells evidenced that the CD34 transgene was highly expressed at the protein level (80% of LCD34IΔN vs 30% of controls at 5 days post-infection) up to 14 days of liquid culture.

first: Roberta

last: Zini

first: Simona

last: Salati

first: Elisa

last: Bianchi

first: Fulvio

last: Mavilio

last: Ferrari


title: Molecular profiling of pediatric mature B-cell lymphoma treated in population-based prospective clinical trials

status: Public on Jun 17 2008

city: Regensburg

first: Stefan

last: Bentink

state: Bayern

address: Josef Engertstr. 9

department: Institute of Functional Genomics

institute: University of Regensburg

summary:
The spectrum of entities, the therapeutic strategy and the outcome of mature aggressive B-cell lymphomas (maB-NHL) differs between children and adolescents on the one hand and adult patients on the other. Whereas adult maB-NHL have been studied in detail, data on molecular profiling of pediatric maB-NHL are hitherto lacking. Our aim was to characterize pediatric maB-NHL on the molecular level and to evaluate whether a molecular diagnosis of pediatric maB-NHL reveals clinically relevant groups.
Keywords: pediatric disease state analysis

overall_design: 36 pediatric diffuse large B-cell lymphoma and Burkitt lymphoma samples were hybridized to HGU133A Affymetrix GeneChips

first: Wolfram

last: Klapper

first: Monika

last: Szczepanowski

first: Birgit

last: Burkhardt

first: Hilmar

last: Berger

first: Maciej

last: Rosolowski

first: Carsten

last: Schwaenen

first: Swen

last: Wessendorf

first: Rainer

last: Spang

first: Peter

last: Möller

first: Martin-Leo

last: Hansmann

first: Heinz-Wolfram

last: Bernd

first: German

last: Ott

first: Michael

last: Hummel

first: Harald

last: Stein

first: Markus

last: Loeffler

first: Lorenz

last: Trümper

first: Martin

last: Zimmerman

first: Alfred

last: Reiter

first: Reiner

last: Siebert


title: Global gene expression in Atss3 mutant and WT over short day diurnal cycle

status: Public on Jun 12 2008

city: Ames

first: Ling

last: Li

state: IA

address: 443 Bessey Hall

department: Department of Genetics, Development, and Cell Biology

institute: Iowa State University

summary: The Atss3 mutant and WT plants were arranged according to a Randomized Complete Block Design. The plants were planted in rows with seven rows in each flat; two plants of the same genotype/pot. Plants were grown under a SD photoperiod (8 h light/16 h dark) in a growth chamber as described. Eight randomly selected rows were harvested for each time point from different flats. Plant material was harvested at five time points in the diurnal cycle (1, 4, 8.5, 12, and 16 h; Time 0 is the beginning of the light period); harvesting was conducted under a green safety light. Each sample consisted of rosette leaves (leaves 5 to 8, staged following Bowmann (1994); photosynthetically active (Stessman et al., 2002)) from sixteen six-week-old plants. Leaf samples were frozen in liquid N2 immediately after harvest and stored at -80‚°C for RNA extraction. The experiment was done twice and independent randomizations for plant growth and harvest were used for the two replicates.

overall_design: The Atss3 mutant and WT plants were arranged according to a Randomized Complete Block Design. The plants were planted in rows with seven rows in each flat; two plants of the same genotype/pot. Plants were grown under a SD photoperiod (8 h light/16 h dark) in a growth chamber as described. Eight randomly selected rows were harvested for each time point from different flats. Plant material was harvested at five time points in the diurnal cycle (1, 4, 8.5, 12, and 16 h; Time 0 is the beginning of the light period); harvesting was conducted under a green safety light. Each sample consisted of rosette leaves (leaves 5 to 8, staged following Bowmann (1994); photosynthetically active (Stessman et al., 2002)) from sixteen six-week-old plants. Leaf samples were frozen in liquid N2 immediately after harvest and stored at -80°C for RNA extraction. The experiment was done twice and independent randomizations for plant growth and harvest were used for the two replicates.

first: Eve

last: Wurtele


title: Effect of uniconazole on wt and pkl mutant germinating seeds

status: Public on Jun 27 2008

city: West Lafayette

first: Joe

last: Ogas

state: IN

address: 175 S. University St.

department: Biochemistry

institute: Purdue University

summary: Relative expression data from germinating seeds of Columbia (wt), the pkl mutant (pkl), Columbia plus uniconazole-P (Uwt) and the pkl-mutant plus uniconazole-P (Upkl).

overall_design: Each experimental condition (wt, pkl, Uwt and Upkl) has six biological replicates for a total of 24 chips.

first: Heng

last: Zhang

first: Stanley

last: Rider

first: James

last: Henderson

first: Matthew

last: Fountain

first: King

last: Chuang

first: Vasundhara

last: Kandachar

first: Alexis

last: Simons

first: Howard

last: Edenberg

first: Jeanne

last: Romero-Severson

first: William

last: Muir


title: Gene expression profile of cancer cell lines of different origin

status: Public on Jun 24 2008

city: Budapest

first: Balazs

last: Györffy

address: Bókay 53

country: Hungary

institute: Hungarian Academy of Sciences

summary: Gene expression profile of cancer cell lines of breast, lung, pancreatic, gasctric, ovarian, hepatocellular, prostate carcinomas and melanomas.

overall_design: Cells were harvested after 24h of seeding. RNA isolation was performed usign Qiagen RNeasy kit. Hybridisation was performed using Affymetrix protocols. No replicate measurements were performed.

last: Gyorffy

first: Manfred

last: Dietel

first: Reinhold

last: Schaefer

first: Hermann

last: Lage


title: CD34 gene silencing

first: Rossella

last: Manfredini

summary:
In order to investigate the role of CD34 antigen in haematopoietic commitment, we silenced the CD34 gene expression in CD34+ stem/progenitor cells using a siRNA approach.
Keywords: treatment comparison

overall_design: To maximize siRNA transfection efficiency, we utilized the NucleofectorTM technology (Amaxa). CD34+ cells were transfected with a mixture of 4 siRNAs targeting CD34 mRNA and with a non-targeting siRNA as a negative control. The expression level of CD34 antigen on control cells (MOCK and negative control treated cells) and CD34siRNA treated cells was assessed by immunofluorescence analysis at 24 and 48h post-nucleofection.

first: Simona

last: Salati

first: Roberta

last: Zini

first: Elisa

last: Bianchi

first: Anna

last: Testa

first: Fulvio

last: Mavilio

first: Sergio

last: Ferrari


title: Identification of TGA-regulated genes in response to phytoprostane A1 and OPDA

status: Public on Jun 02 2008

city: Wuerzburg

first: Susanne

last: Berger

address: Julius-von-Sachs-Platz 2

department: Pharmaceutical Biology

institute: Julius-von-Sachs-Institute

summary:
12-oxo-phytodienoic acid (OPDA) and phytoprostane A1 (PPA1) are cyclopentenone oxylipins that are formed via the enzymatic
jasmonate pathway and a nonenzymatic, free radical–catalyzed pathway, respectively. Both types of cyclopentenone oxylipins
induce the expression of genes related to detoxification, stress responses, and secondary metabolism, a profile clearly distinct
from that of the cyclopentanone jasmonic acid.
To investigate the role of TGA transcriction factors in oxylipin responses, the regulation of gene expression by OPDA and PPA1 defective in
the expression of TGA2, TGA5, and TGA6 was analyzed using
Affymetrix ATH1 chips.
Keywords: Arabidopsis tga2-5-6 mutant, Oxylipin treatment, ATH1 Chip

overall_design: 18 hybs total


title: Response of Arabidopsis cell culture to cyclopentenone oxylipins

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Gene Expression of Circulating B Lymphocytes for Osteoporosis

status: Public on Jun 01 2008

city: Omaha

first: Peng

last: Xiao

state: NE

address: 601 N 30th ST Suite 6730

department: Biomedical Sciences & Osteoporosis Research Center

institute: Creighton University Medical Center

summary:
B cells produce important cytokines regulate bone metabolism. We comparison gene expression patterns of circulating B cells in blood from 20 postmenopausal females with low or high bone mineral density (BMD): 10 low BMD vs. 10 high BMD.
In total 29 differentially expressed genes were identified including some novel genes to be relevant to bone metabolism. These results provide insight into the role of B cells in pathologic osteoporosis.
Keywords: disease state analysis

overall_design: B cells were isolated from 70 ml of whole blood from each of 20 women, 10 with high BMD and 10 with low BMD, using B cell positive isolation method (Dynabeads CD19,Pan B) from Invitrogen Life Technologies Dynal Biotech Inc,CA. Total RNA was extracted from B cells using Qiagen RNeasy Mini Kit.A total of 4ug totalRNA was used to produced targets for each subject according to standard Affymetrix procedures. Hybridization was made for each subject. Robust Multiarray Algorithm was used to normalize the expression data and comparison was performed between high BMD and low BMD subgroups using t-statistics under mutiple-testing adjustment.

first: Yuan

last: Chen

first: Hui

last: Jiang

first: Feng

last: Pan

first: Tielin

last: Yang

first: Hong-Wen

last: Deng


title: Role of P. gingivalis SerB in Gingival Epithelial Cell Cytoskeletal Remodeling and Cytokine Productions.

city: Gainesville

first: Martin

last: Handfield

state: FL

address: 1600 SW Archer Road, BOX 100424

department: Oral Biology

institute: University of Florida

summary:
Transcriptional profiling of oral keratinocytes was utilized to define the biological role of P. gingivalis SerB.
We used microarrays to detail the global programme of gene expression underlying infection with an isogenic mutant in SerB, and identified distinct classes of up- and down-regulated genes during this process.
Keywords: infection state

overall_design: Gingival epithelial HIGK cells were infected with either the oral pathogen P. gingivalis (Pg) or an isogenic mutant for SerB in the same parental strain in order to gain a better understanding on the role of SerB. These samples were hybridized to Affymetrix microarrays. SerB was found to be a multifunctional invasin and modulin of P. gingivalis and associated with broadly based regulation of the epithelial cell transcriptome.

first: Yoshiaki

last: Hasegawa

first: Gena

last: Tribble

first: Henry

last: Baker

first: Jeffrey

last: Mans

first: Richard

last: Lamont


title: Response of Arabidopsis cell culture to phytoprostane A1

summary:
12-Oxo-phytodienoic acid (OPDA) and several phytoprostanes are structurally related cyclopentenone oxylipins that can be formed via the enzymatic jasmonate pathway and a non-enzymatic, free radical-catalyzed pathway, respectively. To elucidate the biological activities of phytoprostanes in comparison to OPDA as well as the metabolism we performed genome-wide expression analysis.
Keywords: mixotrophic Arabidopsis cell culture, ATH1 Chip

overall_design: In order to get a comprehensive view of the biological activity of non-enzymatically formed reactive oxylipins the regulation of gene expression in Arabidopsis thaliana was analyzed using the ATH1 chip representing 22500 genes. A mixotrophic cell culture of Arabidopsis was treated with 75 µM phytoprostane A1 or with 0.5% methanol in water (Control) and harvested after 4 h. Three independent replicates of each treatment were analyzed and the fold change of normalized signals from PPA1-treated cell culture versus control cell culture was calculated.


title: GAL4-responsive genes in larval salivary glands of late Drosophila prepupae

status: Public on May 29 2008

city: Fayetteville

first: Michael

last: Lehmann

state: AR

address: SCEN 601

department: Biological Sciences

institute: University of Arkansas

summary:
The yeast transcription factor GAL4 has been reported to cause cell death and to have other biological effects when expressed in Drosophila (Kramer and Staveley, 2003: Genet. Mol. Res. 2, 43; Rezaval et al., 2007: Eur. J. Neurosci. 25, 683). Using heat-shock-induced expression of GAL4 to drive expression of a UAS-senseless responder gene in transcriptional profiling experiments, we found that the underlying cause of these effects might be a genomic response to GAL4. To further characterize this response and to account for GAL4-independent changes caused by the transgene integration, GAL4 was expressed from two copies of the transgene in two independent lines, P{GAL4-Hsp70.PB}89-2-1 (short P{hs-GAL4}89) and P{hs-GAL4}X1. In addition, GAL4 was expressed from only one copy of the transgene in P{hs-GAL4}89 prepupae to account for the dosage dependence of observed effects. Prepupae carrying the hs-GAL4 transgenes were subjected to a 30-min heat shock treatment (37 °C) at 9 hours after puparium formation. RNA was isolated from salivary glands dissected from these and similarly treated w1118 control animals at 14 hours after puparium formation and subjected to microarray analysis with Affymetrix GeneChips. The microarray data identified an overlapping set of 1,009 genes that showed an at least 1.5-fold change in expression in both of the GAL4-expressing lines, defining a core set of GAL4-responsive genes in the salivary glands. This set includes genes involved in the control and execution of programmed cell death and in other important regulatory pathways.
Keywords: ectopic expression experiment

overall_design: Experimental RNA samples were obtained from GAL4-expressing salivary glands of the line P{hs-GAL4}X1 in 3 biological replicates (SG_P{hsGAL4}X1_14APF_rep1, SG_P{hsGAL4}X1_14APF_rep2, SG_P{hsGAL4}X1_14APF_rep3); of the line P{hs-GAL4}89 in one replicate (SG_P{hsGAL4}89_14APF_rep1); of animals carrying only one copy of P{hs-GAL4}89 in one replicate (SG_1P{hsGAL4}89_14APF_rep1). The samples were compared to control samples obtained in two biological replicates from the salivary glands of w1118 control animals (SG_w1118(2)_14APF_rep1, SG_w1118(2)_14APF_rep2). All samples were compared using dChip and normalized to the same baseline array (median intensity 145). Statistical analysis of differences between the P{hs-GAL4}X1 and w1118 samples was carried out using GeneSpring (Agilent Technologies) and a false discovery rate (FDR) cutoff of 0.05.

first: Yanling

last: Liu


title: Expression data from dark grown Arabidopsis Wild type (Wt, col-o) and pif1-2 (At2g20810, Salk_072677) mutant seedlings

status: Public on May 30 2008

city: Austin

first: Enamul

last: Huq

address: BIO21B, 205 W 24th St,

department: Molecular Cell & Developmental Biology

institute: The University of Texas at Austin

summary:
Arabidopsis bHLH transcription factor PIF1 has been shown function as a negative regulator of Chlorophyll biosynthesis. Pif1-2 mutant accumulate more protochlorophylide during prolonged dark growth condations, therefore the comparation between Wt and pif1-2 mutant will help us find the PIF1 target genes in Chlorophyll biosynthesis pathway at genome wide level under the dark grown conditions.
We used microarrays to detail the global gene expression under the regulation of PIF1.

Keywords: Genotype comparison

overall_design: 4 days dark grown Wt and pif1-2 seedlings were collected for isolation RNA. Standard Affymetrix protocal and 25K Affymetrix chip (ATH1) were used for Microarry hybridition.

last: Shen


title: The ß-Catenin Pathway is Overexpressed in Focal Nodular Hyperplasia but not in Cirrhotic FNH-like Nodules

status: Public on Jun 18 2008

first: Sandrine

last: Imbeaud

address: 15 rue de l'Ecole de Médecine

department: INSERM UMRS1138-EQUIPE 28, Sorbonne Université-Inserm-Université de Paris

institute: Centre de Recherche des Cordeliers

summary:
Focal nodular hyperplasias (FNHs) are benign liver lesions considered to be a hyperplastic response to increased blood flow in otherwise normal liver. In contrast, FNH-like nodules occur in cirrhotic liver but share similar histopathological features. To better understand the pathophysiology of FNH, we performed a transcriptomic analysis. Methods: Affymetrix and cDNA microarrays were used to compare gene expression in eight FNHs with that in tissue from six normal livers. Selected genes were validated with quantitative RT-PCR in 70 benign liver tumors including adenomas and cirrhotic and FNH-like lesions.  Results: Among the deregulated genes in FNHs, 19 were physiologically zonated in the normal liver lobule. All six periveinous genes were up-regulated in FNH, whereas 13 genes normally expressed in the periportal area were down-regulated. Immunohistochemistry revealed that glutamine synthetase was markedly overexpressed, forming anastomosed areas usually centered on visible veins. ß-catenin mRNA was slightly but significantly overexpressed, as were several known ß-catenin target genes. Moreover, activated hypophosphorylated ß-catenin protein accumulated in FNH in the absence of activating mutations. These results suggest zonated activation of the ß-catenin pathway specifically in FNH, whereas the other benign hepatocellular tumors, including FNH-like lesions, demonstrated an entirely different pattern of ß-catenin expression. Conclusions: In FNH, increased expression of the ß-catenin pathway was restricted to enlarged periveinous areas, which may explain the slight polyclonal over-proliferation of hepatocytes at the origin of the lesion. FNH-like nodules may have a different pathogenetic origin.
Keywords: Disease state analysis

overall_design:
In a first experiment, microarray analyses were applied to 7 Focal nodular hyperplasias (FNHs) and 8 non related non-tumor livers using cDNA in-house manufactured arrays following a randomized and blinded unbalanced design. RNA labelling, hybridization and analysis of fluorescence was carried out, as described in [Graudens et al., Genome Biol 2006, 7: R19; PMID: 16542501], considering that uneven numbers of samples were randomly allocated to each of the engineers who were not aware of sample phenotypes. To assess data reproducibility and minimize dye bias effects, each of the samples was measured four times, twice with Cy3 and twice with Cy5. To ensure robustness and flexibility in data analysis, a reference design was used with a universal reference sample (Stratagene, USA) serving as a baseline for the comparisons of tumor samples. Hybridizations were performed onto an 11K human array (11K_VJF-ARRAY, GPL3282), which provides a genome-wide coverage of functional pathways. Raw data were obtained using the ArrayVision™ 7.0 software (Imaging Research Inc., USA); the resulting hybridization data points collected from 30 arrays were stored in a MIAME-compliant database and pre-processed for normalization and filtering as described in [Graudens et al., Genome Biol 2006, 7: R19; PMID: 16542501].

In a second experiment, HG-U133A Affymetrix GeneChipTM arrays were used to compare the expression profiles of 4 Focal nodular hyperplasias (FNHs) and 4 non related non-tumor livers. RNA labelling, hybridization and analysis were carried out following the manufacturer’s instructions (Affymetrix, Santa Clara, CA). Raw data were obtained by using Microarray Suite 5.0 (MAS5) software, embedded in the Affymetrix GeneChip Operating Software (Santa Clara, USA); the resulting raw numerical data (CEL files) - available as supplementary files - collected from 8 Affymetrix GeneChips were pre-processed for normalization and filtering as described in [Rebouissou et al., J Biol Chem 2007, 282(19):14437-46, PMID: 17379603; and Rebuissou et al., in preparation].

A platform-dependant statistical comparison was performed considering Focal nodular hyperplasias (FNHs) versus non tumor liver tissues, using multiple testing procedures to evaluate statistical significance for differentially expressed genes, as described in [Graudens et al., Genome Biol 2006, 7: R19; PMID: 16542501] and in the supplemental experimental procedures in [Rebouissou et al., J Biol Chem 2007, 282(19):14437-46, PMID: 17379603; and Rebuissou et al., in preparation]. Thus, a combined cross-platform analysis was done considering the UGCluster Identifiants as a common primary key (Unigene Build184-June05).

Additional information on the procedures is provided in the supplemental experimental procedures in [Rebouissou et al., J Biol Chem 2007, 282(19):14437-46, PMID: 17379603; and Rebuissou et al., in preparation].

first: Sandra

last: Rebuissou

first: Gabrielle

last: Couchy

first: Louis

last: Libbrecht

first: Charles

last: Balabaud

last: Auffray

first: Tania

last: Roskams

first: Paulette

last: Bioulac-Sage

first: Jessica

last: Zucman-Rossi


title: Genetic Analysis of Human Traits In-Vitro: Drug Response and Gene Expression in Lymphoblastoid Cell Lines

city: Boston

first: Roman

last: Yelensky

state: MA

address: Massachusetts General Hospital Department of Molecular Biology 185 Cambridge St., Simches Ctr., CPZN-6818

institute: MGH

summary:
Lymphoblastoid cell lines (LCLs), originally collected as renewable sources of DNA, are now being used as a model system to study genotype-phenotype relationships in human cells.  These cell lines have been used to search for genetic variants that are associated with drug response as well as with more basic cellular traits such as RNA levels.  In setting out to extend such studies by searching for genetic variants contributing to drug response, we observed that phenotypes in LCLs were, in our lab and others, significantly affected by experimental confounders (i.e. in vitro growth rate, metabolic state, and relative levels of the Epstein-Barr virus used to transform the cells).  As we did not find any SNPs associated with genome-wide significance to drug response, we evaluated whether incorporating RNA expression levels (and eQTLs) in the analysis could increase power to detect such effects.  As previously shown, cis-acting eQTLs were detectable for a sizeable fraction of RNAs and baseline levels of many RNAs predicted response to several drugs.  However, we found only limited evidence that SNPs influenced drug response through their effect on expression of RNA.  Efforts to use LCLs to map genes underlying cellular traits will require great care to control experimental confounders, unbiased methods for integrating and interpreting such multi-dimensional data, and much larger sample sizes than have been applied to date.

Keywords: baseline RNA expression

sample_organism:
Pan troglodytes
Homo sapiens

overall_design:
We studied 269 cell lines densely genotyped by the International HapMap Project [31]. Cell lines were cultured and characterized at baseline for a variety of cellular phenotypes including growth rate, ATP levels, mitochondrial DNA copy number, EBV copy number, and measures of B-cell relevant cell surface receptors and cytokine levels.  Each cell line was exposed in 384-well plates to a range of doses for each of seven drugs selected based on their divergent mechanisms of action and importance in clinical use for treatment of B-cell diseases, focusing on anti-cancer agents: 5-fluorouracil (5FU), methotrexate (MTX), simvastatin, SAHA, 6-mercaptopurine (6MP), rapamycin, and bortezomib.  Drug response was measured using Celltiter Glo, an ATP-activated intracellular luminescent marker that, when compared to mock-treated control wells, can represent relative levels of cellular viability and metabolic activity.  RNA was collected at baseline and RNA transcript levels were measured genome-wide on the Affymetrix platform.    Baseline characterization and plating for drug response experiments was performed using batches of 90 cell lines from each HapMap analysis panel (CEU, JPT / CHB, and YRI) on each of three experiment days.  The order of cell lines within each panel was randomized to avoid inducing artificial intra-familial correlation.  Each drug was tested at a range of doses around the expected IC50 as reported for the drug by the NCI DTP; each dose of drug was tested in two wells per plate and on two separate plates.  These replicate measurements for each cell line allowed assessment of intra-experimental variation.      To evaluate day-to-day (i.e. inter-experimental) variation in all traits, a subset of 90 cell lines (30 from each of the three HapMap panels) was grown from a fresh aliquot and the entire experiment was repeated.  To evaluate the effect of technical error on measured RNA levels, a set of 22 RNAs previously expression profiled (using Illumina HumanChip) at Wellcome Trust Sanger Institute (WTSI) (generously provided by Emmanouil T. Dermatsakis) was included in expression profiling at the Broad on Affymetrix arrays.     Data can be downloaded from the Broad Institute web site: (http://www.broad.mit.edu/~yelensky/cell_lines_paper/). Please see Materials and Methods for details of QC, normalization, etc.

Note - Roman Yelensky (12/22/2009): A recent application of novel micro-array experiment QC methods by the scientific community (Lude Franke) has revealed the possibility of a mixup of a handful of RNA samples in the CHB/JPT population subset. Specifically, it has been suggested that RNA data for sample pairs:

- NA18609 and NA18971
- NA18592 and NA18605
- NA18637 and NA18966
- NA18550 and NA18635
- NA18994 and NA18524

may have been accidentally swapped. While we cannot verify whether a swap indeed occurred, users of the dataset are encouraged to also consider this alternate sample mapping (e.g. the array for NA18609 is actually NA18971 and vice versa)  in their analyses.

first: Edwin

last: Choy

first: David

last: Altshuler

first: Mark

last: Daly


title: Pediatric malignant germ cell tumors show characteristic transcriptome profiles

status: Public on May 31 2008

city: Cambridge

first: Roger

middle: David

last: Palmer

address: Box 197 Hills Road

department: MRC Cancer Cell Unit

institute: Hutchison/MRC Research Centre

summary:
To compare the transcriptome profiles of the two principal histological variants of malignant germ cell tumor that occur in childhood
Keywords: Disease state analysis

overall_design: Here, we analyzed the global gene expression profiles of 27 pediatric MGCTs from the two principal histologies (18 yolk sac tumors [YSTs] and 9 seminomas).

first: Nuno

last: Barbosa-Morais


title: CD34 antigen role in haematopoietic commitment

first: Rossella

last: Manfredini

first: Simona

last: Salati

first: Roberta

last: Zini

first: Elisa

last: Bianchi

first: Anna

last: Testa

first: Fulvio

last: Mavilio

first: Sergio

last: Ferrari