title: Indole cell signaling occurs primarily at low temperatures in Escherichia coli

status: Public on Jun 05 2008

city: State College

first: Thomas

last: Wood

state: PA

address: Room 161 Fenske Laboratory, University Park

institute: Pennsylvania State University

summary: We have shown the quorum-sensing signals acylhomoserine lactones (AHLs), autoinducer-2 (AI-2), and indole influence the biofilm formation of Escherichia coli.  Here, we investigate how the environment, i.e., temperature, affects indole and AI-2 signaling in E. coli. We show in biofilms that indole addition leads to more extensive differential gene expression at 30°C (186 genes) than at 37°C (59 genes), that indole reduces biofilm formation (without affecting growth) more significantly at 25°C and 30°C than at 37°C, and that the effect is associated with the quorum-sensing protein SdiA. The addition of indole at 30°C compared to 37°C most significantly repressed genes involved in uridine monophosphate (UMP) biosynthesis (carAB, pyrLBI, pyrC, pyrD pyrF, and upp) and uracil transport (uraA). These uracil-related genes are also repressed at 30°C by SdiA, which confirms SdiA is involved in indole signaling. Also, compared to 37°C, indole more significantly decreased flagella-related qseB, flhD, and fliA promoter activity, enhanced antibiotic resistance, and inhibited cell division at 30°C.  In contrast to indole and SdiA, the addition of (S)-4,5-dihydroxy-2,3-pentanedione (the AI-2 precursor) leads to more extensive differential gene expression at 37°C (63 genes) than at 30°C (11 genes), and, rather than repressing UMP synthesis genes, AI-2 induces them at 37°C (but not at 30°C). Also, the addition of AI-2 induces the transcription of virulence genes in enterohemorrhagic E. coli O157:H7 at 37°C but not at 30°C. Hence, cell signals cause diverse responses at different temperatures, and indole- and AI-2-based signaling are intertwined.

overall_design: We performed seven sets of microarray experiments in LB medium (Table 2): (i) biofilm cells of the BW25113 wild-type strain grown for 7 h at 30°C vs. 37°C to determine the effect of temperature on E. coli biofilm formation (initial absorbance of 0.05), (ii) biofilm cells of the tnaA mutant grown for 7 h with 1 mM indole vs. no indole at 30°C to determine the effect of temperature on indole signaling in E. coli (initial absorbance of 0.05), (iii) biofilm cells of the tnaA mutant grown for 7 h with 1 mM indole vs. no indole at 37°C to determine the effect of temperature on indole signaling in E. coli, (iv) suspension cells of the wild-type strain vs. the sdiA mutant at an absorbance of 4.0 at 600 nm  at 30°C (since production of indole takes place primarily in the stationary phase) to discern the genes that SdiA controls (initial absorbance of 0.05), (v) biofilm cells of the sdiA mutant grown for 7 h with 1 mM indole vs. no indole at 30°C to determine gene expression in response to indole in the absence of SdiA, (vi) suspension cells of the luxS mutant with 100 micro M AI-2 vs. no AI-2 at 30°C for 3 h to determine the effect of temperature on AI-2 signaling in E. coli (initial absorbance of 0.5), and (vii) suspension cells of the luxS mutant with 100 micro M AI-2 vs. no AI-2 at 37°C for 3 h to determine the effect of temperature on AI-2 signaling in E. coli.  Ten g of glass wool (Corning Glass Works, Corning, N.Y.) was used to form large amounts of biofilms (Ren et al., 2004a) in 250 mL LB in 1 L Erlenmeyer shake flasks. RNA was isolated from the suspension and biofilm cells as described previously (Ren et al., 2004b).

first: Xue-Song

last: Zhang

first: Jintae

last: Lee


title: Novel Estrogen Receptor-{alpha} Binding Sites and Estradiol Target Genes Identified by ChIP Cloning in Breast Cancer.

status: Public on May 20 2008

city: Chicago

first: Zhihong

last: Lin

state: IL

address: 303 East Superior Street

department: Obstetrics and Gynecology

country: USA

institute: Northwestern University

summary:
Estrogen receptor-{alpha} (ER{alpha}) and its ligand estradiol play critical roles in breast cancer growth and are important therapeutic targets for this disease. Using chromatin immunoprecipitation (ChIP)-on-chip, ligand-bound ER{alpha} was recently found to function as a master transcriptional regulator via binding to many cis-acting sites genome-wide. Here, we used an alternative technology (ChIP cloning) and identified 94 ER{alpha} target loci in breast cancer cells. The ER{alpha}-binding sites contained both classic estrogen response elements and nonclassic binding sequences, showed specific transcriptional activity in reporter gene assay, and interacted with the key transcriptional regulators, including RNA polymerase II and nuclear receptor coactivator-3. The great majority of the binding sites were located in either introns or far distant to coding regions of genes. Forty-three percent of the genes that lie within 50 kb to an ER{alpha}-binding site were regulated by estradiol. Most of these genes are novel estradiol targets encoding receptors, signaling messengers, and ion binders/transporters. mRNA profiling in estradiol-treated breast cancer cell lines and tissues revealed that these genes are highly ER{alpha} responsive both in vitro and in vivo. Among estradiol-induced genes, Wnt11 was found to increase cell survival by significantly reducing apoptosis in breast cancer cells. Taken together, we showed novel genomic binding sites of ER{alpha} that regulate a novel set of genes in response to estradiol in breast cancer. Our findings suggest that at least a subset of these genes, including Wnt11, may play important in vivo and in vitro biological roles in breast cancer.

Keywords: time course

overall_design: This Series currently contains the gene expression data accompanying Zhihong Lin et al. Cancer Research 67,5017-5024(2007).  MCF7 cells were treated with vehicle or E2 at a concentration of 10E-9 mol/L for 3 and 6 h.  All experiments were performed in triplicate.

first: Scott

last: Reierstad

first: Chiang-Ching

last: Huang

first: Serdar

middle: E

last: Bulun


title: Gene expression profile analysis for the Fascin knockdown esophageal squamous carcinoma cells

status: Public on May 08 2008

city: shantou

first: Enmin

last: Li

state: guangdong

address: 22 Xinling Road

country: China

institute: Shantou university, medical college

summary:
Fascin, an actin-binding protein, is upregulated in a variety of cancers, including esophageal squamous cell carcinoma (ESCC) and is proposed to function in cellular growth, mobility and invasiveness. Nonetheless, the molecular mechanisms through which fascin expression contributes to the proliferation and invasiveness of ESCC are unknown.
To address this issue and to identify genes and biological pathways associated with fascin expression, a genome-wide gene expression analysis was performed on a fascin RNAi-depleted cell line and a corresponding ESCC cell line previously determined to express high levels of fascin.
Keywords: cell type comparison

overall_design: We used cDNA microarrays to identify genes that were differentially expressed upon fascin knockdown. For this purpose, we selected PSF8 (Fascin siRNA-depleted cell) and PSC(blank vector control cell ), two ESCC cell lines recently established in our laboratory. Total RNA from PSF8 cells and PSC cells was prepared for use with Affymetrix U133 cDNA microarrays.

first: Jianjun

last: Xie

first: Liyan

last: Xu


title: Comparison of gene expression data between undifferentiated hES cells and MSC-derived hES cells

city: EVRY

first: Christelle

last: ROCHON-BEAUCOURT

address: 5 rue H. Desbruères

country: France

institute: INSERM/UEVE UMR 861/ISTEM,AFM

summary:
Analysis of genes that were differentially expressed in MSC-derived hES cells (VUB01 and SA01) as compared to VUB01 and SA01 undifferentiated hES cells
Keywords: differentiation state analysis

overall_design: Two cell lines (VUB01 and SA01) with three biological replicates for each


title: Gene expression profiles during in vivo human rhinovirus infection: insights into the host response.

status: Public on May 06 2008

city: MASON

first: Jay

middle: Patrick

last: Tiesman

state: OH

address: PO BOX 8006

department: Global Biotechnology

institute: Procter & Gamble

summary: RATIONALE: Human rhinovirus infections cause colds and trigger exacerbations of lower airway diseases. OBJECTIVES: To define changes in gene expression profiles during in vivo rhinovirus infections. METHODS: Nasal epithelial scrapings were obtained before and during experimental rhinovirus infection, and gene expression was evaluated by microarray. Naturally acquired rhinovirus infections, cultured human epithelial cells, and short interfering RNA knockdown were used to further evaluate the role of viperin in rhinovirus infections. MEASUREMENTS AND MAIN RESULTS: Symptom scores and viral titers were measured in subjects inoculated with rhinovirus or sham control, and changes in gene expression were assessed 8 and 48 hours after inoculation. Real-time reverse transcription-polymerase chain reaction for viperin and rhinoviruses was used in naturally acquired infections, and viperin mRNA levels and viral titers were measured in cultured cells. Rhinovirus-induced changes in gene expression were not observed 8 hours after viral infection, but 11,887 gene transcripts were significantly altered in scrapings obtained 2 days postinoculation. Major groups of up-regulated genes included chemokines, signaling molecules, interferon-responsive genes, and antivirals. Viperin expression was further examined and also was increased in naturally acquired rhinovirus infections, as well as in cultured human epithelial cells infected with intact, but not replication-deficient, rhinovirus. Knockdown of viperin with short interfering RNA increased rhinovirus replication in infected epithelial cells. CONCLUSIONS: Rhinovirus infection significantly alters the expression of many genes associated with the immune response, including chemokines and antivirals. The data obtained provide insights into the host response to rhinovirus infection and identify potential novel targets for further evaluation.

overall_design: Randomized, parallel group study.  A total of 31 subjects was analyzed:  15 infected and 16 controls.  Nasal scapings were taken 14 days prior to infection (d14) and 8 hours (d0) and 2 days (d2) after infection.  For each collection, alternating nostrils were used (e.g. -14d = left, 8hr = right, 2d=left) and subjects were randomized for collection (e.g. LRL, RLR).

first: David

last: Proud

first: Ronald

middle: B

last: Turner

first: Birgit

last: Winther

first: Shahina

last: Wiehler

middle: P

first: Tim

middle: D

last: Reichling

first: Kenton

last: Juhlin

first: Andy

middle: W

last: Fulmer

first: Begonia

middle: Y

last: Ho

first: Amy

middle: A

last: Walanski

first: Cathy

middle: L

last: Poore

first: Haruko

last: Mizoguchi

first: Lynn

last: Jump

first: Marsha

last: Moore

first: Claudine

middle: K

last: Zukowski

first: Jeffrey

last: Clymer


title: Hs 294T Cells Treated with Elesclomol in Combination with Paclitaxel or NAC

status: Public on May 29 2008

city: Lexington

first: Jane

middle: Frances

last: Kepros

state: MA

address: 45 Hartwell Ave

department: Biology

institute: Synta Pharmaceuticals Corp.

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

overall_design: Refer to individual Series


title: Genome-wide analysis of estrogen receptor binding sites

status: Public on May 02 2008

city: Houston

first: Scott

middle: Andrew

last: Ochsner

state: TX

address: One Baylor Plaza

department: Molecular and Cellular Biology

institute: Baylor College of Medicine

summary:
The estrogen receptor is the master transcriptional regulator of breast cancer phenotype and the archetype of a molecular therapeutic target. We mapped all estrogen receptor and RNA polymerase II binding sites on a genome-wide scale, identifying the authentic cis binding sites and target genes, in breast cancer cells.  Combining this unique resource with gene expression data demonstrates distinct temporal mechanisms of estrogen-mediated gene regulation,particularly in the case of estrogen-suppressed genes.  Furthermore, this resource has allowed the identification of cis-regulatory sites in previously unexplored regions of the genome and the cooperating transcription factors underlying estrogen signaling in breast cancer.
Keywords: estrogen effect, time course

overall_design: This Series currently contains the gene expression data accompaning Carroll JS et al. Nature Genetics 38,1289-1297(2006).  MCF7 cells were stimulated with 100 nM estrogen for 0, 3, 6, or 12h.  All experiments were performed in triplicate.

first: Jason

middle: S

last: Carroll

first: Clifford

last: Meyer

first: Jun

last: Song

first: Wei

last: Li

first: Timothy

middle: R

last: Geistlinger

first: Jérôme

last: Eeckhoute

first: Alexander

last: Brodsky

first: Erika

last: Keeton

first: Kirsten

middle: C

last: Fertuck

first: Giles

middle: F

last: Hall

first: Qianben

last: Wang

first: Stefan

last: Bekiranov

first: Victor

last: Sementchenko

first: Edward

last: Fox

first: Pamela

last: Silver

first: Thomas

last: Gingeras

first: X

last: Liu

first: Myles

last: Brown


title: Characterization of Plexiform Leiomyomata Provide Further Evidence for Genetic Heterogeneity Underlying Uterine Fibroids

status: Public on May 01 2008

city: Boston

first: Bradley

middle: J

last: Quade

address: 75 Francis Street

department: Pathology

institute: Brigham and Women's Hospital

summary:
Plexiform leiomyomata are a histologically defined subgroup of benign uterine smooth muscle tumors based on their epitheliod cytology and abundant extracellular matrix.
We used microarrays to compare plexiform leiomyomata to normal myometrium (smooth muscle of the uterine wall), typical leiomyomata, cellular or atypical leiomyomata and malignant leiomyosarcoma of the uterus.
Keywords: tumor analysis

overall_design:
Samples analyzed on U133 Plus 2.0 and HuFL (GPL570 and GPL80).
Data from the microarrays was merged by selection data for probe sets in which  Entrez gene ID code on the GPL570 table equaled the Entrez gene ID on the GPL80 table.
If more than one row on the GPL570 matched the value of the Entrez gene ID for a probe set on the GPL80 table, the values from the GPL570 were averaged.  Thus, the GPL570 data was condensed to conform to the GPL80 format.
The reformated raw data from both microarrays was then normalized such that the sum of the expression values was 3 million, and that values less than 20 were subsequently set to 20 (to permit log transformation in some statistical analysis).

first: Cynthia

last: Morton

first: Jennelle

last: Hodge


title: Human Chronic Obstructive Pulmonary Disorder (COPD) Biomarker

status: Public on May 31 2008

city: Rochester

first: Soumyaroop

last: Bhattacharya

state: NY

address: 601 Elmwood Avenue Box 850

department: Pediatrics

institute: University of Rochester Medical Center

summary:
Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease with complex pathological features and largely unknown etiologies. Identification and validation of biomarkers for this disease could facilitate earlier diagnosis, appreciation of disease subtypes and/or determination of response to therapeutic intervention.  To identify gene expression markers for COPD, we performed genome-wide expression profiling of lung tissue from 56 subjects using the Affymetrix U133 Plus 2.0 array. Lung function measurements from these subjects ranged from normal, un-obstructed to severely obstructed. Analysis of differential expression between cases (FEV1<70%, FEV1/FVC<0.7) and controls (FEV1>80%, FEV1/FVC>0.7) identified a set of 65 probe sets representing discrete markers associated with COPD. Correlation of gene expression with quantitative measures of airflow obstruction (FEV1 or FEV1/FVC) identified a set of 220 probe sets. A total of 31 probe sets were identified that showed evidence of significant correlation with quantitative traits and differential expression between cases and controls.
Keywords: Disease state marker

overall_design: We assessed genome-wide expression patterns in lung tissue specimens derived from 56 subjects. These subjects were undergoing lobectomy for removal of a suspected tumor, and tissue for our studies was derived from histologically normal tissue distant from the tumor margin.  Subjects underwent routine spirometry prior to surgery.  Low values for both FEV1 and FEV1/FVC are characteristic features of COPD and associated with the severity of disease.  For our studies, Cases (n=15) were defined as subjects with FEV1<70% and FEV1/FVC<0.7 and Controls (n=18) as subjects with FEV1>80% and FEV1/FVC>0.7. A majority of the subjects were diagnosed with adenocarcinoma (n=26) or squamous cell carcinoma (n=19), while other tumor types or benign lesions were found in the remaining subjects (n=11).

first: Sorachai

last: Srisuma

last: Mariani


title: cross-hybridization of woodchuck HCCs using human, rat and mouse arrays

city: Cleveland

first: Fangjing

address: Bishop building, Room s731, UH

department: Biomedical Engineering

institute: case Western Reserve University

summary:
Woodchucks that were infected with WHC virus developed HCCs. HCCs and surrounding tissues were removed and RNA was extracted from microarray study. Human, rat and mouse arrays were used due to the lack of a woodchuck array. Same procedures and parameters were used to conduct this experiment.
Keywords: comparative study

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: 3 woodchucks with HCCs were used. HCCs and their surrouding normal tissues were harvested. These 3 pairs of tissues were cross-species hybridized into a human, rat and mouse chip under same conditions. Data analysis were performed to compare the results.

first: Zhenghong

last: Lee

first: Paul

last: Anderson


title: Predicting prognosis using molecular profiling in estrogen receptor-positive breast cancer treated with tamoxifen

status: Public on May 07 2008

city: Toronto

first: Benjamin

last: Haibe-Kains

state: Ontario

address: 610 University Avenue

department: Princess Margaret Research

country: Canada

institute: Princess Margaret Cancer Centre

summary:
Background: Estrogen receptor positive (ER+) breast cancers (BC) are heterogeneous with regard to their clinical behavior and response to therapies. The ER is currently the best predictor of response to the anti-estrogen agent tamoxifen, yet up to 30-40% of ER+BC will relapse despite tamoxifen treatment. New prognostic biomarkers and further biological understanding of tamoxifen resistance are required. We used gene expression profiling to develop an outcome-based predictor using a training set of 255 ER+ BC samples from women treated with adjuvant tamoxifen monotherapy. We used clusters of highly correlated genes to develop our predictor to facilitate both signature stability and biological interpretation. Independent validation was performed using 362 tamoxifen-treated ER+ BC samples obtained from multiple institutions and treated with tamoxifen only in the adjuvant and metastatic settings.

Results: We developed a gene classifier consisting of 181 genes belonging to 13 biological clusters. In the independent set of adjuvantly-treated samples, it was able to define two distinct prognostic groups (HR 2.01 95%CI: 1.29-3.13; p=0.002). Six of the 13 gene clusters represented pathways involved in cell cycle and proliferation. In 112 metastatic breast cancer patients treated with tamoxifen, one of the classifier components suggesting a cellular inflammatory mechanism was significantly predictive of response.

Conclusions: We have developed a gene classifier that can predict clinical outcome in tamoxifen-treated ER+ BC patients. Whilst our study emphasizes the important role of proliferation genes in prognosis, our approach proposes other genes and pathways that may elucidate further mechanisms that influence clinical outcome and prediction of response to tamoxifen.
Keywords: disease state analysis

overall_design: dataset of microarray experiments from primary breast tumors of patients treated by Tamoxifen in adjuvant setting. No replicate, no reference sample.

first: S

last: Loi

first: B

first: C

last: Desmedt

first: P

last: Wirapati

first: F

last: Lallemand

first: A

middle: M

last: Tutt

last: Gilett

last: Ellis

first: K

last: Ryder

first: J

last: Reid

first: M

middle: G

last: Daidone

last: Pierotti

first: E

last: Berns

last: Jansen

last: Foekens

last: Delorenzi

first: G

last: Bontempi

last: Piccart

last: Sotiriou


title: Human Herpesvirus-8 infection of pulmonary microvascular entdothelial cells

status: Public on Apr 30 2008

city: Aurora

first: Christopher

last: Coldren

state: CO

address: 12700 East 17th Place

department: Medicine

institute: University of Colorado School of Medicine

summary:
Human herpesvirus-8 (HHV-8) is the causative agent of Kaposi’s sarcoma and is associated with the angioproliferative disorders primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD).  We have previously described evidence of HHV-8 infection within the pulmonary vasculature of patients with idiopathic pulmonary arterial hypertension (IPAH).  We speculated that viral infection of the pulmonary microvascular endothelial cells could cause the angioproliferative phenotype characteristic of severe pulmonary arterial hypertension (PAH).  We now demonstrate the ability of HHV-8 to infect human pulmonary microvascular endothelial cells (HPMVECs) in vitro, confirming both latent and lytic infection.  HHV-8 infection of HPMVECs resulted in significant changes of gene expression including alterations of pathways integral to both cellular apoptosis and angiogenesis.  This infection also results in alterations of genes integral to the bone morphogenic protein (BMP) pathway, including down regulation of bone morphogenic protein receptor 1a (BMPR1a) and bone morphogenic protein 4 (BMP4).  Other genes previously implicated in the development of PAH are also altered in expression by HHV-8 infection.  These include increased expression of Interleukin-6 (IL-6) and the matrix metalloproteinases (MMP)-1, MMP-2 and MMP-10.  Lastly, cells infected with HHV-8 apoptosis resistant.   Infection of pulmonary microvascular endothelial cells with human herepesvirus-8 results in alteration of the BMP pathway as well as an anti-apoptotic phenotype, consistent with the development of plexiform lesions characteristic of pulmonary arterial hypertension.
Keywords: Viral infection of endothelial cells in culture

overall_design:
•	Direct comparison of HHV8-infected and mock-infected human pulmonary microvascular endothelial cells.
•	Triplicate infection and mock infection samples were prepared. One hybridization per sample, 6 total hybridizations
•	Single channel hybridization (no reference).

first: Todd

last: Bull

first: Christine

last: Meadows

first: Mark

last: Moore

first: Serge

last: Nana-Sinkam

first: Thomas

middle: B

last: Campbell

last: Geraci


title: Timecourse of estradiol (10nM) exposure in MCF7 breast cancer cells.

city: Singapore

first: Lance

middle: David

last: Miller

address: 60 Bioplois Street, #02-01 Genome

department: Microarray and Expression Genomics

country: Singapore

institute: Genome Institute of Singapore

summary:
Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor alpha (ERalpha) binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERalpha binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5' and 3' ends of adjacent transcript), suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs), 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERalpha binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERalpha-positive from ERalpha-negative breast tumors. The expression dynamics of the genes adjacent to ERalpha binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERalpha appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERalpha target genes. Unexpectedly, we found that only 22%-24% of the bona fide human ERalpha binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERalpha binding and gene regulation.
Keywords: time course

overall_design: We used oligonucleotide expression microarrays (Affymetrix GeneChip U133 Plus 2.0) to identify estradiol (E2)-responsive genes in the estrogen-receptor positive breast cancer cell line, MCF7. MCF7 cells were grown to 30-50% confluency and exposed to 10 nM E2 (or vehicle only) at 12, 24, and 48 hours. Each timepoint was performed in triplicate (ie, biological replicates). Total RNA was isolated from cells using the Qiagen RNeasy kit, and 5 micrograms of total RNA was amplified, labeled and hybridized to the array according to the manufacturer’s protocols.

last: Lin

first: V

last: Vega

last: Thomsen

first: T

last: Zhang

first: S

last: Kong

last: Xie

middle: P

last: Chiu

first: L

last: Lipovich

first: D

middle: H

last: Barnett

last: Stossi

last: Yeo

last: George

last: Kuznetsov

first: Y

last: Lee

last: Charn

first: N

last: Palanisamy

last: Cheung

last: Katzenellenbogen

last: Ruan

last: Bourque

last: Wei

middle: T

last: Liu


title: Human skeletal muscle biopsies from a 3h IL-6 infusion

status: Public on May 28 2008

city: Copenhagen N

first: Ole

middle: Hartvig

last: Mortensen

address: Panum 6.5.28, Blegdamsvej 3

department: Department of Biomedical Sciences

country: Denmark

institute: University of Copenhagen

summary:
Skeletal muscle has been identified as a secretory organ. We hypothesized that IL-6, a cytokine secreted from skeletal muscle during exercise, could induce production of other secreted factors in skeletal muscle.
Keywords: Human skeletal muscle time course response to IL-6 infusion

overall_design: IL-6 was infused for 3 h into healthy young males (n=7) and muscle biopsies obtained at time points 0, 3 and 6 h in these individuals. Affymetrix microarray analysis of gene expression profiling in muscle were carried out on 3 randomly chosen subjects.

first: Kasper

last: Andersen

first: Anders

last: Nielsen

first: Rehannah

last: Borup

first: Bente

last: Pedersen


title: Significant differential expression genes from HUVECs induced by bg-CAT

city: kunming

first: bai

middle: shu

last: liu

state: yunnan

address: jiaochang east road 32#

institute: kunming institute of zoology

summary: In vertebrates, non-lens bg-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive.A naturally existing 72-kDa complex of non-lens bg-crystallin (a-subunit) and trefoil factor (b-subunit), named bg-CAT, was identified from frog Bombina maxima skin secretions. Its a-subunit and b-subunit (containing three trefoil factor domains), with a non-covalently linked form of ab2, show significant sequence homology to ep37 proteins, a group of non-lens bg-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. The bg-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of bg-CAT (25-50 pM) were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC50 10 nM) and apoptosis. The bg-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membrane. However, what exactly target of bg-CAT act on HUVECs nuclear is still unknown. Primary cultured HUVECs treated with bg-CAT (25 nM, 2 h) were selected for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain the genome wide level of significant differential gene expression induced by bg-CAT on HUVECs in order to get clues about bg-CAT action mechanisms. These findings illustrate novel cellular functions of non-lens bg-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals.

overall_design: We used microarrays to analysis genome wide level of significant differential gene expression induced by bg-CAT on HUVECs.Four independent biological replicates of HUVECs treated with bg-CAT (25 nM, 2 h) were compared with normal control by SAM. The significant differential expression of 123 genes (fold change≥3, q value =0, FDR (false discovery rate)=0).  121 genes were up-regulated, among which members of nuclear receptor subfamily 4 (NR4A) were the most prominent. Only 2 genes were down-regulated (including collagen type I) (Supporting information Fig. S1 and Table S1 in accompanying publication). Members of NR4A exhibit differential roles in determining cell growth or cell death depending on its location in the nucleus or mitochondria. NR4A1 (also called Nur77, TR3) mediates cell apoptosis through translocation from nucleus to mitochondria and induction of cytochrome c releasing . Significant activation of NR4A members suggests these orphan nuclear receptors may be involved in the mediation of bg-CAT biological functions, like proapoptotic action of the protein at high concentrations. Up-regulated expression of a number of matrix metalloproteinases by bg-CAT could facilitate cell migration and detachment. The bg-CAT also induced the expression of a number of cytokines, including interleukin 1, interleukin 8 and interleukin 6, as well as various chemokines in HUVECs. These data provide clues for study in detail of molecular pathways through which bg-CAT exerts its biological functions.

first: Shu-Bai

first: Ying-Ying

last: He

first: Yun

last: Zhang


title: Ezrin knockdown in SW480

status: Public on Apr 29 2008

city: Taipei

first: Chan-Yen

last: Tsai

address: No.155, Sec.2, Linong Street

country: Taiwan

institute: Yang-Ming University

summary:
Analysis of SW480 cells following knockdown of Ezrin using RNAi.  Ezrin is a protein that regulate the organization of cytoskeleton.  Ezrin KD SW480 was used to study the role of ezrin in colon cancer.
Keywords: ezrin, colon cancer, migration

overall_design: Knockdown ezrin in SW480 cells and investigate the affection on cell migration and the downstream genes.


title: Leukemia fusion-gene transduced human cord blood cells

city: Cincinnati

first: James

last: Mulloy

address: 3333 Burnet Ave

department: Experimental Hematology

institute: Cincinnati Children's Hospital Research Foundation

summary:
MLL-AF9 expression in normal human umbilical cord blood CD34+ cells leads to long-term proliferation of a myeloid progenitor cell with leukemogenic potential. Expression of a Core Binding Factor leukemia fusion (AML1-ETO or CBFbeta-SMMHC) in human CD34+ cells results in self-renewal of primitive progenitor cells with multilineage potential and stem cell ability, but these cells do not induce leukemia in immunodeficient mice. This comparative microarray study was initiated to determine how faithful these cell cultures are to the transcriptome of patient samples expressing each of these different fusion proteins, and to analyze the signaling pathways that are unique to CBF cultures and MLL-fusion cultures, with the hope of determining why the MLL-fusion cells are leukemogenic while the CBF cells are not.
Keywords: Disease state analysis; comparison of leukemia fusion gene expression in normal human hematopoietic progenitor cells

overall_design: We have established a culture system whereby we retrovirally transduce human CD34+ cells, obtained from cord blood, with the leukemia fusion genes MLL-AF9 (MA9), AML1-ETO (AE) or CBFbetaMYH11 (CM). Cells expressing each of these fusion proteins are able to proliferate long-term in vitro in a cytokine dependent manner. These cultures resemble somewhat the blast samples from patients that contain these different fusion proteins, in that MA9 cells are predominantly monocytic (M5-like), AE cells have a large population of primitive CD34+ cells with continued abnormal differentiation (M2-like) and CM cells also contain a population of primitive CD34+ cells with continued abnormal differentiation but also have a significant population of abnormal eosinophils (M4Eo). We grow these cells in the presence of fetal bovine serum (FBS), and also grow them in serum-free conditions using the BIT supplement from Stem Cell Technologies. For the current experiments we used cell cultures that had been proliferating in vitro for 8-12 weeks, in either serum-free conditions or with FBS. Replicate samples were included in some instances, and the same clonal cultures that were grown under either BIT or FBS were also included.

first: JunPing

first: Mark

last: Wunderlich

first: Michael

last: Jansen

first: Cathy

last: Fox

first: Jim

first: Jorge

last: DiMartino

first: Ondrej

last: Krejci

first: Yi

last: Gu

first: Jamie

last: Wilhelm

first: Sara

last: Alvarez

first: Juan

last: Cigudosa


title: Feedback Circuit among INK4 Tumor Suppressors Constrains Human Glioblastoma Development

first: Yonghong

last: Xiao

address: 450 Brookline Ave., HIM218A

institute: Dana-Farber Cancer Institute

type: Genome variation profiling by genome tiling array

summary:
We have developed a nonheuristic genome topography scan (GTS) algorithm to characterize the patterns of genomic alterations in human glioblastoma (GBM), identifying frequent p18INK4C and p16INK4A codeletion. Functional reconstitution of p18INK4C in GBM cells null for both p16INK4A and p18INK4C resulted in impaired cell-cycle progression and tumorigenic potential. Conversely, RNAi-mediated depletion of p18INK4C in p16INK4A-deficient primary astrocytes or established GBM cells enhanced tumorigenicity in vitro and in vivo. Furthermore, acute suppression of p16INK4A in primary astrocytes induced a concomitant increase in p18INK4C. Together, these findings uncover a feedback regulatory circuit in the astrocytic lineage and demonstrate a bona fide tumor suppressor role for p18INK4C in human GBM wherein it functions cooperatively with other INK4 family members to constrain inappropriate proliferation.

This SuperSeries is composed of the SubSeries listed below.

overall_design:
Refer to individual Series.
DNA copy number and mRNA transcriptome of human glioblastoma tumors were profiled using Agilent and Affymetrix microarrays.

first: Ruprecht

last: Wiedemeyer

first: Cameron

last: Brennan

first: Timothy

last: Heffernan

first: John

last: Mahoney

first: Alexei

last: Protopopov

first: Hongwu

last: Zheng

first: Graham

last: Bignell

first: Frank

last: Furnari

first: Webster

middle: K

last: Cavenee

first: William

last: Hahn

first: Koichi

last: Ichimura

first: V.

last: Collins

first: Gerald

last: Chu

last: Stratton

first: Keith

middle: L

last: Ligon

first: P.

middle: A

last: Futreal

first: Lynda

last: Chin


title: Genome-wide analysis of transcriptional termination in E. coli

status: Public on May 16 2008

city: New York

first: Christopher

middle: John

last: Cardinale

address: 550 1st Avenue

department: Biochemistry

institute: New York University School of Medicine

summary:
Transcription termination factor Rho is essential in enterobacteria. We inhibited Rho activity with bicyclomycin and used microarray experiments to assess Rho function on a genome-wide scale. Rho is a global regulator of gene expression that matches E. coli transcription to translational needs. Remarkably, genes that are most repressed by Rho are prophages and other horizontally-acquired portions of the genome. Elimination of these foreign DNA elements increases resistance to bicyclomycin. Although rho remains essential, such reduced-genome bacteria no longer require Rho cofactors NusA and NusG. Thus, Rho termination, supported by NusA and NusG, is required to suppress the toxic activity of foreign DNA.
Global regulation of transcription termination by Rho, NusA, and NusG.
Keywords: Antibiotic treatment

overall_design: Two sets of experiments are presented. First, treatment of E. coli with Rho inhibitor bicyclomycin was performed in strains MG1655 and O157:H7 EDL933 for twenty minutes at concentrations of 10, 25, or 100 micrograms/milliliter. In the second set of experiments the reduced-genome strain MDS42 was treated with bicyclomycin as well as having deletions of the genes nusA and nusG. Total RNA was extracted and hybridized to the Affymetrix E. coli Genome 2.0 array, which contains complete genome coverage of four strains of E. coli.

first: Robert

middle: S

last: Washburn

first: Vasisht

last: Tadigotla

first: Jiri

last: Zavadil

first: Lewis

last: Brown

first: Max

middle: E

last: Gottesman

first: Evgeny

last: Nudler


first: Yonghong

last: Xiao

summary:
We have developed a nonheuristic genome topography scan (GTS) algorithm to characterize the patterns of genomic alterations in human glioblastoma (GBM), identifying frequent p18INK4C and p16INK4A codeletion. Functional reconstitution of p18INK4C in GBM cells null for both p16INK4A and p18INK4C resulted in impaired cell-cycle progression and tumorigenic potential. Conversely, RNAi-mediated depletion of p18INK4C in p16INK4A-deficient primary astrocytes or established GBM cells enhanced tumorigenicity in vitro and in vivo. Furthermore, acute suppression of p16INK4A in primary astrocytes induced a concomitant increase in p18INK4C. Together, these findings uncover a feedback regulatory circuit in the astrocytic lineage and demonstrate a bona fide tumor suppressor role for p18INK4C in human GBM wherein it functions cooperatively with other INK4 family members to constrain inappropriate proliferation.
Keywords: static expression

overall_design:
Expression profiles of human glioblastoma frozen tumors and cell lines were obtained to study copy number abberation driven expressin alteration.
Affymetrix human genome U133plus2 arrays were used to obtain the expression profiles of human glioblastoma tumors and cell lines.

first: Ruprecht

last: Wiedemeyer

first: Cameron

last: Brennan

first: Timothy

last: Heffernan

first: John

last: Mahoney

first: Alexei

last: Protopopov

first: Hongwu

last: Zheng

first: Graham

last: Bignell

last: Furnari

middle: C

first: Michael

middle: R


title: Chronic nicotine exposure (kuo-affy-human-232930)

status: Public on Apr 19 2008

city: Phoenix

first: Winnie

last: Liang

state: AZ

address: 445 N. Fifth Street

institute: Translational Genomics

summary:
The long-term goal of this project is to establish whether and how chronic nicotine exposure affects nervous system function.  The biological targets of nicotine action are diverse members of the superfamily of neurotransmitter-gated ion channels called nicotinic acetylcholine receptors (nAChR).  nAChR play multiple, critical roles in chemical signaling throughout the brain and body.  They also must be involved in nicotine dependence, which drives tobacco product use responsible for tremendous economic and personal costs.
To define changes in gene expression induced by nicotine exposure in a model neuronal cell lines expressing at least two nicotinic receptor subtypes.
Nicotine exposure exerst at least some of its effects on nervous system function by altering gene expression.
Cells of the SH-SY5Y human neuroblastoma will be exposed to an efficacious dose of nicotine or to control medium for two different periods.
Keywords: time-course

first: Yen-Ping

last: Kuo


title: Genome wide genotyping and gene expression data of childhood B-cell precursor ALL without known genetic aberrations

status: Public on Apr 25 2008

city: Padova

first: Andrea

last: Zangrando

address: Via Giustiniani, 3

department: Woman and Child's Health

country: Italy

institute: University of Padova

type:
Expression profiling by array
Genome variation profiling by SNP array
SNP genotyping by SNP array

summary:
Acute lymphoblastic pediatric leukemia specimens without known genetic hallmarks are examined for hidden genomic aberrancies and related gene expression profiles
Integration of genomic variation and gene expression profiling identifies hidden genetic lesions and novel pathways involved in BP-ALL pathogenesis
Keywords: expression data

overall_design: To identify variation in expression of genes affected by recurrent genomic alterations in BP-ALL genotype and GEP data of the same samples were integrated

first: Silvia

last: Bungaro

first: Geertruy

last: te Kronnie


title: Vaccinia E3L mutant virus infected HeLa cell lines (langl-affy-human-215499)

status: Public on Apr 23 2008

summary:
Smallpox is a highly communicable, often fatal diseae.  There is currently no licensed treatment for smallpox and vaccinia virus (VV) is currently used for immunization.  While immunization with VV can provide good protection against exposure to the smallpox virus, the current vaccine is far from optimal.  Complications occur in 1/1,000-1/10,000 vaccinees, with at least one death per million vaccinees.  We have constructed recombinant VV strains which are less pathogenic, yet provide a protective immune response.  These viruses contain various mutations in the E3L which is known to block the host antiviral response.  Identifying the host genes involved in producing a strong protective immunological response to these attenuated viruses would not only increase our understanding of the proteins and pathways involved in effective smallpox vaccination, but aid in the development of alternative vaccine strains which enhance these specific immune responses.
We will determine gene expression patterns in HeLa cells at various times following infection with wtVV and several VV constructs containing mutations in the E3L gene.
The VV E3L gene product blocks the host antiviral response by sequestering viral danger signals, including double-stranded RNA and Z-DNA.  VV constructs containing mutations in E3L which allow host cell recognition of either of these danger signals leads to a decrease in viral pathogensis.  In this project we will dissect the cellular inflammatory response to infection with wtVV in comparison to VV containing  mutations in the E3L gene.  By understanding why certain strains of VV are non-pathogenic, yet highly immunogenic, it is possible to gain a better understanding on the mechanisms of poxvirus pathogenesis and the host response.
We will examine three times points following infection with VV:  2 HPI, 6 HPI and 9 HPI.  These times points represent keys points in the virus replication cycle.  Several VV constructs will be used which contain mutations in the E3L gene.  These constructs alter the ability of E3L to sequester double-stranded RNA and/or Z-DNA and therefore have a direct effect on viral pathogenesis.  Fourteen constructs will be used including: mock, wtVV, VVdelE3L, VVE3Ldel83N, VVE3Ldel37N, VVE3Ldel26C, VVE3Ldel7C, VVE3L Y48A, VVE3L P63A, VVE3L K167T, VV-ATV, VV-ADAR/E3L, VVdelK3L, VVdelK3L-E3Ldel37N.  Cells will be infected at an MOI of 5 to allow infection of all cells.  At each time point, cells will be harvested by scraping.  RNA will be isolated using a Trizol RNA extraction protocol (Invitrogen) followed by RNA purification using the RNeasy cleanup kit available from Qiagen.
Keywords: time-course

first: Jeffrey

last: Langland


title: Interferon signaling and treatment outcome in chronic hepatitis C

status: Public on Apr 17 2008

city: Basel

first: Edward

middle: James

last: Oakeley

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

summary:
Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The current standard therapy for chronic hepatitis C (CHC) consists of a combination of pegylated IFN alpha (pegIFN-alpha) and ribavirin. It achieves a sustained viral clearance in only 50–60% of patients. To learn more about molecular mechanisms underlying treatment failure, we investigated IFN-induced signaling in paired liver biopsies collected from CHC patients before and after administration of pegIFN-alpha. In patients with a rapid virological response to treatment, pegIFN-alpha induced a strong up-regulation of IFN-stimulated genes (ISGs). As shown previously, nonresponders had high expression levels of ISGs before therapy. Analysis of posttreatment biopsies of these patients revealed that pegIFN-alpha did not induce expression of ISGs above the pretreatment levels. In accordance with ISG expression data, phosphorylation, DNA binding, and nuclear localization of STAT1 indicated that the IFN signaling pathway in nonresponsive patients is preactivated and refractory to further stimulation. Some features characteristic of nonresponders were more accentuated in patients infected with HCV genotypes 1 and 4 compared with genotypes 2 and 3, providing a possible explanation for the poor response of the former group to therapy. Taken together with previous findings, our data support the concept that activation of the endogenous IFN system in CHC not only is ineffective in clearing the infection but also may impede the response to therapy, most likely by inducing a refractory state of the IFN signaling pathway.
Keywords: Comparison of human patient biopses before and after interferon treatment

overall_design: Total of 78 Samples (before and after interferon treatement) are analyzed using Affymetrix Human U133 Plus 2.0 Array.

first: Filipowicz

last: Magda

first: Oakeley

last: Edward

first: Heim

last: Markus


title: Coordinated over-expression of genes in the EGFR pathway predicts sensitivity to EGFR inhibition in pancreatic cancer

status: Public on Apr 16 2008

city: Baltimore

first: Aik Choon

last: Tan

state: MD

address: 1650 Orleans Street, RM 1M86

department: The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins

institute: Johns Hopkins University

summary:
Tumors from pancreatic cancer specimens obtained at surgery were used for efficacy testing and biologic analysis
Keywords: Pharmacogenetics

overall_design: Eighteen tumor samples were profiled in duplicates.  Ten samples were used in the training set and eight samples were used as the validation set.

first: Aik-Choon

first: Antonio

last: Jimeno

first: Manuel

last: Hidalgo


title: Local regulation and clinical impact of complement gene expression in deceased and living donor kidney allografts

city: Stanford

first: Li

last: Li

state: CA

address: 300 pastuer dr.,

department: Pediatric

institute: Stanford University

summary:
The biopsy samples obtained at implantation segregated in 2 distinct groups according to donor origin, with a cluster of 319 unique identified genes higher expressed in DD compared to LD kidneys, and 329 genes lower expressed (false discovery rate <5%). Using pathway analysis software a significant local renal overrepresentation of complement genes in DD implantation biopsies was identified. Complement gene expression in DD kidneys related both to donor death and cold ischemia duration, and was associated with a slower onset of renal allograft function. In post-transplantation protocol biopsies, there was a continued overexpression of complement genes, regardless of donor source. The local renal complement gene expression variability in post-transplantation biopsies correlated with renal graft function.

This study demonstrates a significant and clinically relevant local overexpression of complement genes in DD kidneys at engraftment and continuous functionally important regulation of complement gene expression after transplantation, regardless of donor source. Targeted therapy interfering with complement activation is an attractive therapeutic target that deserves further investigation in solid organ transplantation.
Keywords: time course, genomics gene expression

overall_design: A total of 95 human renal allograft protocol biopsies were included in this study, 28 biopsies (14 DD, 14 LD) obtained at implantation prior to revascularization and 67 protocol biopsies obtained after transplantation. Whole genome expression profiles were assessed using microarrays.

first: Maarten

last: Naesens

first: Lihua

last: Ying

first: Poonam

last: Sansanwal

first: Tara

last: Sigdel

first: Szu-chuan

last: Hsieh

first: Neeraja

last: Kambham

first: Evelyne

last: Lerut

first: Oscar

last: Salvatierra

first: Atul

middle: J

last: Butte

first: Minnie

middle: M

last: Sarwal


title: Generation of pluripotent stem cells from adult human testis

status: Public on May 09 2008

city: Tuebingen

first: Michael

last: Bonin

state: BW

address: CAlwerstr. 7

department: Medical Genetics

country: Germany

institute: University Tuebingen

summary:
Human primordial germ cells and mouse neonatal and adult germline stem cells are pluripotent and derive embryonic stem cell properties.
Here we report the successful establishment of stable pluripotent human adult germline stem cells (haGSCs) derived from spermatogonial cells of adult human testis. Cellular and molecular characterization of haGSCs revealed many similarities to human embryonic stem (hES) cells and haGSCs produced teratomas after subcutaneous transplantation into immunodeficient mice. The haGSCs differentiated into various types of somatic cells of all three germ layers when grown under conditions used to induce the differentiation of hES cells.
We conclude that the generation of haGSCs from testicular biopsies may provide simple and non-controversial access to individual cell-based therapy without the ethical and immunological problems associated with hES cells.
Keywords: pluripotent stem cells characterisation

overall_design:
•	Spermatogonial cells (hGS) from 3 different normal patients (patient 59, 73 and 81, passage 0) were cultured for 4 days in Knockout medium, 20% Knockout-FBS, 1% L-glutamine and 4ng/ml GDNF. After enrichment with CD49f and matrix selection with collagen and laminin the cells were washed with phosphate buffered saline (PBS) and pelleted at 1000rpm/10min./4°C, lysed with RLT buffer + ß-Mercaptoethanol and stored at -80°C.
•	hES(H1) cells (hES) were cultured for 10 days (passage 48, 57 and 67) in Knockout medium, 20% Knockout-Serum-Replacer, 1% L-glutamine, 1% NEAA, 1% penicillin/streptomycin and 4ng/ml FGF-2, on CF1 inactivated feeder on 0,1% gelatine coated dishes. After the clusters were manually cut, the isolated clusters were washed
with phosphate buffered saline (PBS) and pelleted at 1000rpm/10min./4°C and lysed with RLT buffer + ß-Mercaptoethanol and stored at -80°C.
•	human adult germ stem cells (haGSCs) were derived from hGS cells from 3 different normal patients after the enrichment and further cultivation in LIF supplemented medium. haGSC from patient 52, 54 and 35 (from lower and higher passages) were cultured for 10 days in basic medium (DMEM high glucose, 15% FCS (Biochrom), 1% non-essential amino acids (NEAA), 1% L-glutamine and 0.05 mM ß-mercaptoethanol (Gibco) with 103 units/ml leukemia inhibitory factor (LIF, human, Chemicon) on 0,1% gelatine coated dishes. After  the clusters were manually cut, the isolated clusters were  washed with phosphate buffered saline (PBS) and pelleted at 1000rpm/10min./4°C and lysed with RLT buffer + ß-Mercaptoethanol and stored at -80°C.

first: Sabine

last: Conrad

first: Thomas

last: Skutella

first: Markus

last: Renninger

first: Jörg

last: Hennenlotter

first: Tina

last: Wiesner

first: Lothar

last: Just

first: Wilhelm

last: Aicher

first: Hans-Jörg

last: Bühring

first: Ulrich

last: Mattheus

first: Hans-Joachim

last: Wagner

first: Stephen

last: Minger

first: Matthias

last: Matzkies

last: Reppel

first: Jürgen

last: Hescheler

first: Karl-Dietrich

last: Sievert

first: Arnulf

last: Stenzl


title: Human sclera

status: Public on Apr 10 2008

city: Setagaya-ku

first: Akihiro

last: Umezawa

state: Tokyo

department: Center for Regenerative Medicine

country: Japan

institute: National Center for Child Health and Development

summary:
The sclera maintains and protects the eye ball, which receives visual inputs. The aim of this study is to identify characteristics of the human sclera as one of the connective tissues derived from the neural crest and mesoderm. We have here demonstrated microarray data of cultured human scleral cells.
Keywords: sclera, cartilage

overall_design: Affymetrix human U133 plus 2.0 array was used to transcriptionally profile to compare sclera cells and others.

first: Yuko

last: Seko

first: Noriyuki

last: Azuma

first: Yoriko

last: Takahashi

first: Hatsune

last: Makino

first: Takeshi

last: Muneta

first: Kenji

last: Matsumoto

first: Hirohisa

last: Saito

first: Ichiro

last: Sekiya


title: Nicotine effect on CEM model T cell line (kuo-affy-human-232861)

status: Public on Apr 12 2008

first: Winnie

last: Liang

summary:
The long-term objective of this project is to establish roles played in development and function of the immune system by nicotinic acetylcholine receptors (nAChR) and exposure to the tobacco alkaloid, nicotine.  The planned gene chip studies will allow us to assess effects of nicotine exposure on gene expression in a model T cell line, and the findings are expected to help direct further efforts.  One of the biomedically-relevant hypotheses of this project is that tobacco use and nicotine exposure affect immune system development.

To establish effects of nicotine exposure on gene expression in the CEM model T cell line.
Immune system nAChR acting as ion channels or via novel signaling cascades mediate their effects on T cell development by altering expression of genes involved in T cell receptor rearrangements, cytokine expression, and cell death/survival decisions.
CEM cells will be treated for one hour or one day with an effective dose of nicotine or with the same medium change but in the absence of nicotine.
Keywords: nicotine, T cell line

first: Yen-Ping

last: Kuo


title: E. coli Isoleucine starvation and stringent response network

status: Public on Apr 08 2008

city: Norman

first: Tyrrell

last: Conway

state: OK

address: 101 David L. Boren Blvd.

department: Advanced Center for Genome Technology

institute: University of Oklahoma

summary:
Transcription profiling of wild type, relA-, and relA-spoT-, crp-, dksA-, rpoS-, lrp- mutant strains of E. coli starved for isoleucine
Bacteria comprehensively reorganize their global gene expression when faced with nutrient exhaustion.  In Escherichia coli and other free-living bacteria, the alarmone ppGpp facilitates this massive response by directly or indirectly coordinating the down-regulation of genes of the translation apparatus, and the induction of biosynthetic genes and the general stress response.  Such a large reorientation likely requires the cooperative activities of many different genetic regulators, yet the structure of the transcription network below the level of ppGpp remains poorly defined.  Using isoleucine starvation as an experimental model system for amino acid starvation, we identified genes that required ppGpp, Lrp, and RpoS for their induction.  Surprisingly, despite the fact that the overwhelming majority of genes controlled by Lrp and RpoS required ppGpp for their activation, we found that these two regulons were not induced simultaneously.  The data reported here suggest that metabolic genes, such as those of the Lrp regulon, require only a low basal level of ppGpp for their efficient induction.  In contrast, the RpoS-dependent general stress response is not robustly induced until relatively high levels of ppGpp accumulate.  Here we describe a data-driven conceptual model that explains how bacterial cells allocate transcriptional resources between metabolic and stress survival processes by discretely tuning regulatory activities to a central indicator of cellular physiology.  The regulatory structure that emerges is consistent with a rheostatic model of the stringent response that allows cells to efficiently adapt to a wide range of nutritional environments.
Keywords: genetic modification design; stress response; isoleucine starvation

sample_organism: Escherichia coli str. K-12 substr. MG1655

platform_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2

overall_design:
Two experiments were run.
First experiment: WT and several mutant strains were starved for isoleucine (exhaustion of 60 uM ile). 40 minutes after starvation, RNA was extracted. All samples were compared to RNA from rapidly growing WT cells in identical medium replete with isoleucine (400 uM).  16 samples were hybridized: duplicates of 8 strains/conditions.  Wildtype in log phase (OD = 0.4) with replete ile was control for ile starved samples.
Second experiment: WT strain was starved for isoleucine (exhaustion of 60 uM ile).  RNA was extracted at 12 timepoints as the cells entered stationary phase. All samples were compared to RNA from rapidly growing WT cells in identical medium replete with isoleucine (400 uM).  14 samples were hybridized: duplicates of the control condition and single  timepoints during starvation.  Wildtype in log phase (OD = 0.4) with replete ile was control for ile starved samples.

first: Matthew

last: Traxler

first: Joe

last: Grissom


title: Profiling Gene Expression in Human Placentae of Different Gestational Ages: an OPRU Network and UW SCOR Study

city: Seattle

first: James

middle: William

last: MacDonald

state: WA

address: 4225 Roosevelt Way NE

department: Environmental and Occupational Health Sciences

institute: University of Washington

summary:
We used a whole genome approach to identify major functional gene categories (including xenobiotic transporters and metabolizing enzymes) whose expression depends on gestational age. STUDY DESIGN: We compared gene expression profiles of 1st (45-59 days) and 2nd trimester (109-115 days), and C-section term placentae. RESULTS: In 1st trimester placentae, genes related to cell cycle, DNA, aminoacids and carbohydrate metabolism were significantly overrepresented, while genes related to signal transduction were downregulated. In the organism defense category, we identified genes involved in chemical response, metabolism, and transport.  Analysis of signal transduction pathways suggested, and subsequently confirmed independently, that the Wnt pathway was regulated by gestational age.  CONCLUSIONS: Our study will serve as a reference database to gain insight into the regulation of gene expression in the developing placentae and, thus, allow comparisons with placentae from complicated pregnancies such as those in women experiencing gestational diabetes, pre-eclampsia and teratogenic sequelae.
Keywords: time series

overall_design: Comparison of plaental gene expression profiles of three gestational stages (1st trimester (45-59 days), 2nd trimester (109-115 days), and C-section term placentae), with four replicates each,

first: Richard

last: Beyer


title: Cells silenced for SDHB expression and tumor phenotype

status: Public on Apr 01 2008

city: Valencia

first: Manolo

last: Mata

address: Avda. Blasco Ibanez, 17

department: UCIM

country: Spain

institute: university of valencia

summary:
Effect of SDHB silencing using siRNA methodologies in the tumor phenotype
We used microarrays to detail the global programme of gene expression when SDHB is silenced.
Keywords: siRNA experiment (loss of function)

overall_design: Human hepatocellular carcinoma cell line Hep3B was stably silenced with control or SDHB-specific siRNAs

first: Ana

last: Cervera

first: Manuel

first: Kenneth

last: McCreath


title: ES cells, EBs grown in suspension (d6), PEL (d10) stage and neural rosettes (d17) (zhang-affy-human-346640)

summary:
Human embryonic stem cells not only provide a continuous cell source for potential cell therapy but also offer a system to unveil events of embryonic development in humans.  This proposal will examine how the earliest neural cells, neuroepithelia, are specified from the naïve ES cells, and test the hypothesis that neural specification in humans employs a similar mechanism as in other vertebrates.  We will first re-create in culture the developmental events of the first 2-3 weeks of human embryonic development during which ES cells will be differentiated through the stages of embryoid bodies, primitive ectoderm cells, neural tube-like rosette cells.  The stage-specific events will be defined by DNA microarray analysis along with the characteristic morphologic changes.  This study will lead to an optimized procedure for generating enriched neural precursor cells, which will lay the groundwork for potential use of human ES cells in the treatment of neurological injuries and diseases.
Aim 1:  Establish a stepwise neural differentiation system from human ES cells.
Aim 2.  Determine the mechanism of Neural Specification in humans.
Aim 3:  Define the identity and function of ES cell-derived neural precursor cells.
ES cells offer an alternative approach to study early developmental events in humans.  This however requires re-creating the neural differentiation process in culture.  Cell fate specification is determined by interactions between environmental factors and intrinsic signals.  Our preliminary data suggest that the temporal pattern of neural differentiation and, to some degree, the spatial organization of neural precursors from human ES cells recapitulate in vivo neural development.  We hypothesize that the intrinsic neural specification program may be preserved in culture, which offers a controlled system to examine the effect of extrinsic signals on neural specification.  Distinctive morphological changes along the neural differentiation pathway are presumably accompanied by molecular changes.   DNA microarray analysis will be used to determine the gene expression pattern by cells at each of the given stages.
By analogy with early embryonic development and using morphological and antigenic markers, we can now subdivide the human ES cell neural differentiation process into four identifiable stages: ES, EB, PEL cells, and neural rosette cells.  This definition is based on the assumption that early human development is the same as in other species, and employs the limited known markers from mouse ES cells.  We will systematically investigate the molecular profile of cells at each of the neural differentiation stages using DNA microarray analysis.
Total RNAs will be extracted from cells at the following developmental stages: ES cells, EBs grown in suspension (d6), PEL (d10) stage and neural rosettes (d17).  Since the neural rosette culture contains non-neural lineage cells, we will separate the neural rosette cells from the surrounding non-neural cells through differential enzymatic response and differential adhesion.  Three independent biological replicates consisting of three pooled experiments will be run for each of the four developmental time points, for a total of twelve chips.
Keywords: time-course

first: Su-chun

last: Zhang


title: Ductal Carcinoma Progression

status: Public on Apr 05 2008

city: São Paulo

first: Nádia

middle: Pereira

last: Castro

state: SP

address: R: Prof Antônio Prudente, 211

country: Brazil

institute: Hospital AC Camargo

summary:
To obtain insight into the molecular basis of ductal carcinoma in situ (DCIS) and its progression by infiltrating surrounding tissue, we have performed cellular-based gene expression analysis of pure DCIS and DCIS with co-existing invasive ductal carcinoma (IDC) and compared the histological and molecular aspects between these morphologically identical lesions seeking to find key genes involved in DCIS progression. For that, 30 samples were evaluated, 4 non-neoplastic (N), 5 pure DCIS, 11 DCIS with co-existing IDC (DCIS-IDC) and 10 IDC. All samples were laser capture microdissected and RNAs were amplified using T7-based methodology. Microarray technology was performed using a customized cDNA platform containing 4,608 human genes. To classify the 4 sample groups according to molecular similarity we performed comparisons of their general expression pattern using ANOVA test (pFDR<0,01) followed by Tukey´s test (fold>l2l). Among the 4 sample groups, as expected, non-neoplastic cells reported the most distinct gene expression pattern. However, among the 3 groups of neoplastic cells, in contrast to morphological aspects, pure DCIS had the most distinct expression profile when compared to the other lesions: DCIS-IDC and IDC. Additionally, by comparison among pure DCIS, DCIS-IDC and N, we identified 147 genes potentially involved in DCIS progression. Unsupervised hierarchical cluster based on expression profile of this gene-set could discriminate DCIS-IDC from 60% of pure DCIS samples.
Keywords: disease state analysis

overall_design:
Fresh-frozen human breast samples were retrieved from the Tumor Tissue Biobank of the Medical and Research Center - Hospital A. C. Camargo, São Paulo. This research was approved by Ethic Committee of the Medical and Research Center - Hospital A. C. Camargo under number 587/04 and a written informed consent signed by all participants. All patients had presented at least 5 years of follow-up. Thirty samples were evaluated, 4 non-neoplastic breast samples (MN), 5 pure DCIS (stage 0; DCIS-0), 11 DCIS with co-existing IDC (DCIS) and 10 IDC. The non-neoplastic samples were obtained from perilesional mammary specimens from patients submitted to resection of benign lesions. All cells types were laser capture microdissected using PixCell II LCM system (Arcturus Engineering, Mountain View, CA). The total RNA was extracted by using the PicoPureä RNA Isolation kit (Arcturus Engineering # KT0204). A two-round linear amplification procedure based on T7-driven amplification was carried out. Amplified RNA was then used in a transcriptase reverse reaction into cDNA in the presence of Cy3- or Cy5-labeled dCTP. HB4a normal luminal epithelial mammary cell line (O’Hare et al 1991) was amplified following the same protocol and used as reference for microarray hybridizations. Dye swap was performed for each sample analyzed and used as replicate samples.
For the raw_data (lowess_data), see Web Link below.

middle: P

first: Cynthia

last: Osório

first: César

last: Torres

first: Elen

last: Bastos

first: Fernando

last: Soares

first: Helena

last: Brentani

first: Dirce

last: Carraro


title: mRNA expression profiles in human cell lines

city: Sakyo-ku

first: Yoshinao

last: Ruike

state: Kyoto

address: 46-29 Yoshida-Shimo-Adachi-cho

department: Pharmaceutical Sciences

institute: Kyoto University

summary:
We performed a global analysis of both miRNAs and mRNAs expression across sixteen human cell lines and extracted negatively correlated pairs of miRNA and mRNA which indicate miRNA-target relationship. The many of known-target of miR-124a showed negative correlation, suggesting our analysis were valid. We further extracted physically relevant miRNA-target gene pairs, applying computational target prediction algorism with inverse correlations of miRNA and mRNA expression. Furthermore, Gene Ontology-based annotation and functional enrichment analysis of the extracted miRNA-target gene pairs indicated putative functions of miRNAs.
Keywords: Cell type comparison

overall_design: 16 samples were analyzed. Replicate were not included.

first: Atsuhiko

last: Ichimura

first: Soken

last: Tsuchiya

first: Kazuharu

last: Shimizu

first: Ryo

last: Kunimoto

first: Yasushi

last: Okuno

first: Gozoh

last: Tsujimoto


title: Argyrin A is a p27 stabilizing drug with potent antiproliferative activity in vivo

city: Braunschweig

first: Robert

last: Geffers

address: Inhoffenstr. 7

department: Dep. Molecular Bacteriology

institute: HCI - Helmholtz Centre for Infection Research

summary:
Reduction in the cellular levels of the cyclin kinase inhibitor p27kip1 are frequently found in many human cancers and correlate directly with patient prognosis. Specifically ubiquitin dependent proteasomal turnover has been shown to cause reduced p27 expression in many human cancers. We recently demonstated that expression of a stabilized version of p27kip1 (p27kip1T187A) in a genetically modified mouse significantly reduced the number of intestinal adenomatous polyps which progressed to invasive carcinomas. Based on this work we set out to identify compounds which lead to a re-expression of p27 in cancer tissues. In this work we identify Argyrin A a compound derived from myxobacterium archangium gephyra as a potent inducer of p27kip1 expression. Argyrin A induces apoptosis in human colon cancer xenografts and tumor vasculature in vivo leading to a profound reduction in tumor size at well tolerated levels. Argyrin A functions are strictly dependent on the expression of p27kip1 as neither tumor cells nor endothelial cells which do not express p27kip1 respond to this compound. Surprisingly the molecular mechanism by which Argyrin A exerts its p27 dependent biological function is through a potent inhibition of the 20S proteasome.
Keywords: protasome inhibitor, time course, MCF7 cells, genome wide  gene expression response, siRNA

overall_design: MCF7 cells were incubated with proteasome inhibitors bortezomib or Argyrin A or proteasome activity was reduced by treatment with siRNA against proteasomal subunits and gene expression profiles were determined at different timepoints thereafter.

first: R

first: N

last: Malek


title: Acute graft-versus-host disease (gene expression profiling)

status: Public on Apr 09 2008

city: Tampa

first: Matthew

last: Buzzeo

state: FL

address: 12901 Bruce B. Downs Blvd

institute: University of South Florida College of Medicine

summary: Allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice for high-risk hematological malignancies, yet a major complication associated with this therapy is acute graft-versus-host disease (GVHD). Despite a well-defined pathophysiological mechanism, there are no definitive markers for predicting acute GVHD development or progression to advanced stages. In the current study, we enrolled four acute GVHD and four acute GVHD-free recipients of allogeneic HSCT and collected peripheral blood just prior to onset of clinical acute GVHD for analysis on Affymetrix GeneChip Human Genome U133 Plus 2.0 microarrays. We noted significant differences in expression of 1,658 genes between control and acute GVHD patients, based on an analysis of covariance (ANCOVA) by type of transplant, a pooled error estimate, and a false discovery rate (FDR) of 10%. In conclusion, we offer the first report of a preliminary molecular signature of acute GVHD in allogeneic HSCT patients.

overall_design: Samples 1-4 are control patients and samples 5-8 are acute GVHD patients.  Peripheral blood was collected at engraftment and used for microarray analysis. The type of transplant (unrelated/related donor) was used as a covariate to assess if there were significant changes in gene expression between the control (1-4) and acute GVHD (5-8) groups, based on a 10% FDR with a pooled error estimate.

first: Vijay

last: Reddy


title: Gene expression microarray profiles of cumulus cells in lean and overweight-obese polycystic ovary syndrome patients

status: Public on Mar 27 2008

city: Toronto

first: Shlomit

last: Kenigsberg

address: 790 Bay st.

department: Research

institute: Create Fertility Centre

summary:
Objective: The etiology of PCOS is mostly unknown. Existing data support both genetic and environmental factors in its pathogenesis.  Design: Prospective case - control study. Setting: University Hospital. Patients:  25 patients undergoing IVF-ICSI treatment.  Intervention: Genome-wide oligonucleotide microarray technology was used to study differential gene-expression patterns of cultured human cumulus cells from IVF patients divided into 4 groups according to disease state (PCOS vs. Control) and BMI (Obese vs. Lean).  Results: Two differential PCOS gene expression profiles were established: Lean-Type was formed by comparing PCOS lean (PL) vs. non-PCOS lean (NL) individuals; Obese-Type was formed by comparing PCOS obese (PO) vs. non-PCOS (NO) obese patients.   Conclusions:  Different molecular pathways are associated with PCOS in Lean and Obese individuals, as demonstrated by gene expression profiling of cumulus cells. Our findings provide insights into the molecular pathogenesis of PCOS.
We used microarrays to study the gene expression of human cultured cumulus cells. We  compared the genes expression of lean PCOS,  Obese PCOS, lean controls and obese controls. Different molecular pathways are associated with PCOS in Lean and Obese patients.
Keywords: disease state analysis

overall_design: Cumulus cells obtained from woman undergoing IVF/ICSI.  Following oocyte retrieval, cumulus cells were stripped from the oocyte, in preparation for the ICSI process, with a micropipette. After 48h in culture the cumulus cells were collected for RNA extraction and hybridization on Affymetrix microarrays. We compered the expression profile of 4 groups - lean PCOS, obese PCOS, lean controls and obese controls.

first: Yaakov

last: Bentov

first: Vered

last: Chalifa-Caspi

first: Gad

last: Potashnik

first: Rivka

last: Ofir

first: Ohad

last: Birk


title: Strand-specific 5'-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity.

status: Public on Apr 02 2008

first: Dimos

last: Gaidatzis

institute: Friedrich Miescher Institute

summary:
Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide catalytic sequence-specific cleavage of fully or nearly fully complementary target mRNAs or control translation and/or stability of many mRNAs that share 6-8 nucleotides (nt) of complementarity to the siRNA and miRNA 5' end. siRNA- and miRNA-containing ribonucleoprotein silencing complexes are assembled from double-stranded 21- to 23-nt RNase III processing intermediates that carry 5' phosphates and 2-nt overhangs with free 3' hydroxyl groups. Despite the structural symmetry of a duplex siRNA, the nucleotide sequence asymmetry can generate a bias for preferred loading of one of the two duplex-forming strands into the RNA-induced silencing complex (RISC). Here we show that the 5'-phosphorylation status of the siRNA strands also acts as an important determinant for strand selection. 5'-O-methylated siRNA duplexes refractory to 5' phosphorylation were examined for their biases in siRNA strand selection. Asymmetric, single methylation of siRNA duplexes reduced the occupancy of the silencing complex by the methylated strand with concomitant elimination of its off-targeting signature and enhanced off-targeting signature of the phosphorylated strand. Methylation of both siRNA strands reduced but did not completely abolish RNA silencing, without affecting strand selection relative to that of the unmodified siRNA. We conclude that asymmetric 5' modification of siRNA duplexes can be useful for controlling targeting specificity.
Keywords: siRNA, transfection, chemical modification, off-targets

overall_design: For details please see: Chen PY, Weinmann L, Gaidatzis D, Pei Y, Zavolan M, Tuschl T, Meister G. RNA (2008) 14(2):263-74.

first: Po

middle: Y

last: Chen

first: Lasse

last: Weinmann

first: Yi

last: Pei

first: Mihaela

last: Zavolan

first: Thomas

last: Tuschl

first: Gunter

last: Meister


title: VS94 SAPI AI-2 Temporal study

status: Public on Mar 21 2008

city: College Station

first: Tarun

last: Bansal

address: 3122 TAMU

department: Chemical Engineering

institute: Texas A & M University

summary:
VS94 gene expression at different time-points in SAPI medium in absence and presence of AI-2 was studied.

Autoinducer-2 (AI-2) is produced by many species of bacteria, including various commensal bacteria and is involved in inter-species communication. Since, pathogens encounter AI-2 once they enter the human gastro-intestinal tract; we studied the effects of presence of AI-2 on various phenotypes associated with infection and colonization of enterohemorrhagic Escherichia coli (EHEC) namely, chemotaxis, motility and attachment to HeLa cells. AI-2 attracted EHEC when observed in agarose plug assays and also increased EHEC motility by 1.44-fold. AI-2 also increased EHEC attachment to HeLa cells by 1.6-fold; hence, suggesting that exposure to AI-2 inside the gastro-intestinal tract can play an important role in EHEC colonization. We then investigated the global effects of AI-2 on EHEC gene expression using DNA microarrays at various time-points. We found that AI-2 controls virulence gene expression and several other groups of genes (flagellar genes, iron related genes, biofilm genes etc.) associated with virulence in a time-dependent manner. Hence, through these studies we have shown that AI-2 may be a key component in EHEC infection of human gastro-intestinal tract.
Keywords: Time course

overall_design:
Strain: VS94 (EHEC luxS mutant)
Medium: SAPI
Temperature: 37C
Cell type: Suspension
Chemical: 100 uM AI-2

Overnight cultures of VS94 were diluted in SAPI medium to a turbidity of 0.1. The cells were allowed to grow to a turbidity of 0.5 at 37 oC and 100 µM AI-2 was added to the culture. In the control flasks, no AI-2 was added. The cultures were then allowed to grow for further 3.5 hours, 4 hours, 4.5 hours and 5.5 hours before cell pellets were collected by centrifugation at the above mentioned time-points and stored at -80oC.
Total RNA was isolated from isolated cell pellets and RNA quality was assessed using gel electrophoresis. E. coli Genome 2.0 arrays (Affymetrix, California) containing 10,208 probe sets for all 20,366 genes present in four strains of E. coli, including E. coli O157:H7, were used to profile changes in gene expression using RNA samples for each treatment. Hybridization was performed for 16 hours, and the total cell intensity was scaled automatically in the software to an average value of 500. The data were inspected for quality and analyzed according to the procedures described by the manufacturer (Affymetrix: Data Analysis Fundamentals) which include using premixed polyadenylated transcripts of the B. subtilis genes (lys, phe, thr, and dap) at different concentrations. Genes were identified as differentially expressed if the expression ratio (between AI-2 and control cells at different time-points) was greater than 1.5 (based on the standard deviation between values measuring relative changes in expression) (Ren et al. 2004) and the change in the P value was less than 0.05. The differentially expressed genes were annotated using gene ontology definitions available in the Affymetrix NetAffx Analysis Center (http://www.affymetrix.com/analysis/index.affx).

last: Wood

first: Arul

last: Jayaraman


title: The role of RIP140 in retinoid mediated signaling

status: Public on Mar 24 2008

city: Urbana

first: Michael

last: Spinella

state: IL

address: 2001 South Lincoln Ave

department: Comparative Biosciences

institute: University of Illinois

summary:
Cell-and context-specific activities of nuclear receptors may in part be due to distinct coregulator complexes recruited to distinct subsets of target genes.   RIP140 (also called NRIP1) is a ligand-dependent corepressor that is inducible with retinoic acid (RA).  We have shown previously that silencing of RIP140 enhances RA-induced differentiation and enhances the induction of model RA target genes in human embryonal carcinoma cells (EC).  Through use of microarray technology we sought to elucidate in a de novo fashion the global role of RIP140 in RA-dependent signaling. RIP140-dependent gene expression was largely consistent with RIP140 functioning to limit RAR signaling. Few if any genes were regulated in a manner to support a role for RIP140 in “active repression”.   Interestingly, approximately half of the RA-dependent genes were unaffected by RIP140, suggesting that RIP140 may discriminate between different classes of RA target genes.  RIP140 silencing also accelerated RA target gene activation and sensitized EC cells to low doses of RA.   Together the data suggests that the RIP140-dependent RA target genes identified here may be particularly important in mediating RA-induced tumor cell differentiation. RIP140 may be an attractive target to sensitize tumor cells to retinoid-based differentiation therapy.
Keywords: Drug treatment with siRNA

overall_design:
The current investigation sought to determine the global effects of RIP140 on RA-dependent and RA–independent gene expression. A dose of siRNA of 90 nM was chosen. Twelve independent hybridizations were performed from mRNA isolated from 12 independent samples. One scrambled and two independent RISC-free siRNA transfections and transfections with three distinct RIP140 siRNAs were performed in NT2/D1 cells.  Each siRNA treatment was further treated with either 10 μM RA or DMSO control for 24 hours.  Hence, the design consisted of four groups, the three siRNA controls treated with vehicle, Group 1; the siRNA controls treated with RA, Group 2; siRNA to RIP140 treated with vehicle, Group 3; and siRNA to RIP140 treated with RA, Group 4 .
The treatments were done in the presence of normal sera, since charcoal absorption of sera removes many small hydrophobic ligands to the nuclear receptor family in addition to retinoids.  This measure was taken with the aim to perhaps identify target genes of other nuclear receptors including orphan receptors that could be regulated by RIP140 in the presence of low levels of their cognate ligands.   RA treatment of 10 μM for 24 hours was chosen since previous experience with NT2/D1 cells indicated that a reasonably sized set of altered genes would be obtained.  In addition, NT2/D1 cells commit to RA-induced differentiation by 48 hours and RIP140 repression enhances and accelerates the effects of RA in these cells.  Therefore, the 24-hour time point is well-within the range of the RA commitment “window” and a larger percentage of direct RA-target genes could be expect to be uncovered compared to later time points.
	Based on the current understanding of the properties of RIP140, a number of potential outcomes were anticipated: 1) RIP140 may limit RA target gene activation by negative-feedback inhibition upon RA addition, 2) RIP140 may participate in “active repression” of gene expression upon RA addition, 3) an unexpected outcome that RIP140 may participate in the activation of a subset of target genes, 4) RIP140 may regulate the transcription of target genes of other nuclear receptors or even non-nuclear receptor transcription factors.

Total RNA was purified using RNeasy columns (Qiagen).  Hybridizations were performed according to Affymetrix guidelines at the Dartmouth College Microarray Shared Resource using an Affymetrix GeneChip Workstation.  Biotin-labeled cRNA was generated from 5 μg of total RNA and hybridized to the Human Genome (HG) U133 Plus 2.0 chip   A total of 12 hybridizations were performed comprising 12-independent biologic samples organized into the four groups of three.

first: Kelly

last: Heim

first: Kristina

last: White-Branco


title: Monocytes of patients with familial hypercholesterolemia show alterations in cholesterol metabolism

city: Jena

first: Sandy

last: Mosig

address: Bachstrasse 18

department: Molecular Hemostaseology - Institute of Vascular Medicine

institute: University Hospital Jena

summary:
Background: Elevated plasma cholesterol promotes the formation of atherosclerotic lesions in which monocyte-derived lipid-laden macrophages are frequently found. To analyze, if circulating monocytes already show increased lipid content and differences in lipoprotein metabolism, we compared monocytes from patients with Familial Hypercholesterolemia (FH) with those from healthy individuals.
Methods: Cholesterol and oxidized cholesterol metabolite serum levels of FH and of healthy, gender/age matched control subjects were measured by combined gas chromatography ? mass spectroscopy. Monocytes from patients with FH and from healthy subjects were isolated by antibody-assisted density centrifugation. Gene expression profiles of isolated monocytes were measured using Affymetrix HG-U 133 Plus 2.0 microarrays. We compared monocyte gene expression profiles from FH patients with healthy controls using a Welch T-test with correction for multiple testing (p < 0.05; Benjamini Hochberg correction, False Discovery Rate = 0.05). The differential expression of FH associated genes was validated at the mRNA level by qRT-PCR and/or at the protein level by Western Blot or flow cytometry. Functional validation of monocyte scavenger receptor activities were done by binding assays and dose/time dependent uptake analysis using native and oxidized LDL.
Results: Using microarray analysis we found in FH patients a significant up-regulation of 1,617 genes and a down-regulation of 701 genes compared to monocytes from healthy individuals. These include genes of proteins that are involved in the uptake, biosynthesis, disposition, and cellular efflux of cholesterol. In addition, plasma from FH patients contains elevated amounts of sterols and oxysterols. An increased uptake of oxidized as well as of native LDL by FH monocytes combined with a down-regulation of NPC1 and ABCA1 explains the lipid accumulation observed in these cells.
Conclusion: Our data demonstrate that circulating FH monocytes show differences in cell physiology that may contribute to the early onset of atherosclerosis in this disease.

Keywords: Micro-array analysis, cell type comparison,

overall_design: 23 monocytes samples: 4 homozygous FH, 6 heterozygous FH, 13 control participants

first: Knut

last: Rennert

first: Petra

last: Buettner

first: Muhidien

last: Soufi

first: Siegfried

last: Krause

first: Julia

last: Kzhyshkowska

first: Sergej

last: Goerdt

first: Regine

last: Heller

first: Juergen

last: Schaefer

first: Harald

last: Funke

first: Barbara

last: Kuehn

first: Kerstin

last: Neunuebel

last: Schreiner


title: Germline NRAS mutation causes a novel human autoimmune lymphoproliferative syndrome

city: Sao Paulo

first: Joao

middle: Bosco

last: Oliveira

state: Sao Paulo

address: Av. Dr. Eneas de carvalho Aguiar, 500 IMT Bldg. II 3rd Floor

department: Dermatology

institute: University os São Paulo

summary:
The p21 RAS subfamily of small GTPases, including KRAS, HRAS, and NRAS, regulates cell proliferation, cytoskeletal organization and other signaling networks, and is the most frequent target of activating mutations in cancer.  Activating germline mutations of KRAS and HRAS cause severe developmental abnormalities leading to Noonan, cardio-facial-cutaneous and Costello syndrome, but activating germline mutations of NRAS have not been reported.  Autoimmune lymphoproliferative syndrome (ALPS) is the most common genetic disease of lymphocyte apoptosis and causes autoimmunity as well as excessive lymphocyte accumulation, particularly of CD4-, CD8- ab T cells.  Mutations in ALPS typically affect CD95 (Fas/APO-1)-mediated apoptosis, one of the extrinsic death pathways involving tumor necrosis factor receptor (TNFR) superfamily proteins, but certain ALPS individuals have no such mutations.  We show here that the salient features of ALPS as well as a predisposition to hematological malignancies can be caused by a heterozygous germline Gly13Asp activating mutation of the NRAS oncogene that does not impair CD95-mediated apoptosis.  The increase in active, GTP-bound NRAS augments RAF/MEK/ERK signaling which markedly decreases the pro-apoptotic protein BIM and attenuates intrinsic, nonreceptor-mediated mitochondrial apoptosis.  Thus, germline activating mutations in NRAS differ from other p21 Ras oncoproteins by causing selective immune abnormalities without general developmental defects.  Our observations on the effects of NRAS activation indicate that RAS-inactivating drugs, such as farnesyl-transferase inhibitors (FTIs) should be examined in human autoimmune and lymphocyte homeostasis disorders.
Keywords: NRAS

overall_design: Describes the discovery of a new gene underlying a novel type of autoimmune lymphoproliferative syndrome, and characterizes the mechanisms involved in the pathogenesis of the disease.

middle: B

first: Nicolas

last: Bidere

first: Julie

last: Niemela

first: Lixin

last: Zheng

first: Keiko

last: Sakai

first: Cynthia

last: Nix

first: Robert

last: Danner

first: Jennifer

last: Barb

first: Peter

last: Munson

last: Puck

first: Janet

last: Dale

first: Stephen

last: Straus

first: Thomas

last: Fleisher

last: Lenardo


title: Gene expression profile in HUVECs before and after Angiopoietin stimulation

city: Osaka

first: NAOKI

last: MOCHIZUKI

address: 5-7-1 Fujishirodai, Suita

department: Structural Analysis

institute: Natl. Cardiovasc. Ctr. Res. Inst

summary:
Angiopoietin-Tie2 sytem has been inplicated in both vascular quiescence and angiogenesis. It is unclear how these two opposing signals are regulated from the same receptor-mediated intracellular signal transduction. We have noticed that Tie2 localization upon Angiopoietin stimulation depends upon the presence or absence of cell-cell contacts.
Therefore, to identify the downstream signaling of Tie2 upon Angiopoietin stimulation, we performed DNA microarray analyais using RNAs obtained from confluent human umbirical vein endothelial cells (HUVECs) or sparse HUVECs stimulated with after Angiopoietin-1. There is striking difference on gene expression profile between confluent and sparse HUVECs.
Keywords: endothelial cell, angiopoietin

overall_design:
To identify the genes in HUVECs which are upregulated or downregulated by the stimulation with Angiopoietin-1 in the presence (confluent) or absence (sparse) of cell-cell contacts, mRNAs were prepared from either confluent HUVECs or sparse HUVECs stimulated or unstimulated with 100 ng/ml COMP-Ang1 (more potent Angiopoietin-1). RNAs were obtained from HUVECs three times cultured independently three times and mixed as one RNA sample for further DNA microarray analyses.
   Microarray (Human Genome U133 Plus 2.0 array, Affymetrix) analyses have been performed in duplicate. Biotin labeled probes were used for the series of analyses.  Hybridization and Scan were performed according to the recommendation by Affymetric. Data have been processed following the MAS 5.0 with target intensity = 100.

first: Shigetomo

last: Fukuhara

first: Takashi

last: Minami

first: Tatsuhiko

last: Kodama

first: Naoki

last: Mochizuki


title: Imprints of atherosclerosis are present in circulating T cells of patients with Familial Hypercholesterolemia

first: Sandy

last: Mosig

summary:
Atherosclerosis is the major cause of death in industrialized countries. This disease has initially been characterized as a lipid disorder, but current concepts argue for an inflammatory disease, which develops in the background of hypercholesterolemia and other risk factors. In response to initial events of atherosclerosis formation, such as LDL-deposition in the subendothelial space, monocytes and T cells interact with the vessel wall. However, little is known about the properties and the behavior of these cells in this context. Using familial hypercholesterolemia (FH) as a model we demonstrate substantial differences in the gene expression of freshly isolated human monocytes and T lymphocytes. In FH monocytes we found an increased uptake of oxidized LDL, elevated amounts of scavenger receptors and adhesion molecules, and differences in the regulation of intracellular lipoprotein metabolism compared to monocytes from healthy individuals. Furthermore, the monocyte subpopulation of CD14+/CD16+ cells is less frequent in FH but exhibits significantly higher levels of CD11c and CD29 which increases the likelihood for their transmigration through the endothelial layer. The presence of increased amounts of CD69 in T lymphocytes from FH patients suggests that these cells are more activated than control cells. Our results indicate that some important steps of atherosclerosis formation already take place in circulating blood cells which extends current atherosclerosis models to the plasma compartment.
Keywords: atherosclerosis, T cells, Familial Hypercholesterolemia

overall_design: 23 T cells samples: 3 homozygous FH, 7 heterozygous FH, 13 control participants

first: Knut

last: Rennert

first: Petra

last: Buettner

first: Muhidien

last: Soufi

first: Siegfried

last: Krause

first: Julia

last: Kzhyshkowska

first: Sergej

last: Goerdt

first: Regine

last: Heller

first: Juergen

last: Schaefer

first: Harald

last: Funke

first: Barbara

last: Kuehn

first: Kerstin

last: Neunuebel

last: Schreiner


title: Expression profiles of immortal lung fibroblasts

city: Hiroshima

first: Keiko

last: Hiyama

state: Hiroshima

address: 1-2-3 Kasumi, Minami-ku

department: Research Institute for Radiation Biology and Medicine

institute: Hiroshima University

summary:
Activation of telomerase often endows cancer cells, but rarely normal somatic cells, with immortality. Especially, fetal lung fibroblasts are known to be hardly immortalized by TERT overexpression. We here established an immortal non-transformed lung fibroblast cell line only by TERT transfection, as well as an immortal transformed cell line by transfection of TERT and SV40 early antigens. Comparing the expression profiles of these cell lines with those of mortal cell strains with elongated lifespan after TERT transfection, 51 genes, including 19 upregulated and 32 downregulated, were explored to be the candidates responsible for regulation of cellular proliferation of lung fibroblasts. These included the genes previously reported to be involved in cellular proliferation, transformation, or self-renewal capacity, and those highly expressed in lung tissues obtained from patients with idiopathic pulmonary fibrosis or hypersensitivity pneumonitis. This set of lung fibrobrast cell lines/strains of identical genetic background with different proliferative capacity, mortal and immortal non-transformed fibroblasts may become useful model cells for research on lung fibroblast growth regulation and the candidate genes explored in this study may provide promising biomarkers or molecular targets of pulmonary fibrosis.
Keywords: Cell type comparison

overall_design: We analyzed 6 arrays with Affymetrix, 4 arrays with Agilent, and 7 arrays with CodeLink oligonucleotide microarrays for lung fibroblasts with various lifespan with or without transfection of TERT and/or SV40 early antigens.

first: Eiso

first: Keiji

last: Tanimoto

first: Masahiko

last: Nishiyama


title: Impact of curcumin on human monocytes (U937 cells) exposed to oxidative stress

status: Public on Mar 20 2008

city: Horsham

first: Paul

last: Kirkham

state: west Sussex

address: Wimblehurst Road

country: United Kingdom

institute: Novartis Institutes for Biomedical Research

summary:
Oxidative stress as a result of cigarette smoking is an important etiological factor in the pathogenesis of chronic obstructive pulmonary disease (COPD), a chronic steroid-insensitive inflammatory disease of the airways. The activity of the transcriptional co-repressor Histone deacetylase-2 (HDAC2) is dramatically reduced in COPD and cells exposed to oxidative stress or cigarette smoke. Moreover, curcumin (diferuloylmethane), a dietary polyphenol, at concentrations upto 1uM specifically restores cigarette smoke extract (CSE)- or oxidative stress- impaired HDAC2 activity. The aim of this study was to therefore identify any links through those gene sets that are affected by oxidative stress and subsequent treatment with curcumin in order to determine whether or not this could explain the impact of curcumin on restoration of oxidant impaired HDAC2 transcriptional co-repressor activity.
Keywords: time course

overall_design: Human PMA-differentiated U937 cells, unexposed or exposed to oxidative stress (100mM H2O2)  were either left untreated or treated with curcumin (1 mM) for either 4 or 18 hr. Performed in triplicate


title: The effect of the HDAC inhibitor OSU-HDAC42 on gene expression in esophageal tissues and adenocarcinoma cells

city: Columbus

first: Laura

last: Kresty

state: OH

address: CCC Building, Rm 302B

department: Internal Medicine

institute: The Ohio State University

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

sample_organism:
Homo sapiens
Rattus norvegicus

platform_organism:
Homo sapiens
Rattus norvegicus

overall_design: Refer to individual Series


title: The effect of the HDAC inhibitor OSU-HDAC42 on gene expression in esophageal tissues and adenocarcinoma cells I

summary:
Title: In vitro and in vivo effects of the orally bioavailable phenylbutyrate-derived histone deacetylase inhibitor OSU-HDAC42 on gene expression in esophageal tissues and in esophageal adenocarcinoma cells
Histone deacetylases (HDACs) modulate nucleosomal packaging of DNA, thereby influencing gene transcription and multiple cancer-associated processes.
Thus, we conducted microarray analysis at multiple time-points to assess the ability of OSU-HDAC42, the S enantiomer of the previously published compound HDAC-42,  to modulate key acid-induced changes as well as to impact other genes altered as the normal esophageal epithelium progresses along the metaplasia-dysplasia-esophageal adenocarcinoma continuum.
Keywords: acid-pulsed cells pretreated with OSU-HDAC42 or vehicle

overall_design: SEG-1 cells were pretreated for 24 hours with vehicle or OSU-HDAC42, pulsed with acidified media (pH 3.5; 20 mins at 37C), replenished with media or OSU-HDAC42, and harvested 3, 6 and 24 hours later.  Global gene expression analysis was conducted using the human genome chip U133 2.0 Plus.