title: DZNep-treated glioblastoma multiforme cancer stem cells

city: Torino

first: Paolo

last: Provero

address: Via Nizza 52

department: Molecular Biotechnology and Health Sciences

institute: University of Turin

summary: Overexpression of the Polycomb group protein Enhancer of Zeste Homolog 2 (EZH2) occurs  in diverse malignancies, including prostate cancer, breast cancer, and glioblastoma  multiforme (GBM) (1). Based on its ability to modulate transcription of key genes implicated  in cell cycle control, DNA repair and cell differentiation, EZH2 is believed to play a crucial  role in tissue-specific stem cell maintenance and tumor development. Here we show that  targeted pharmacologic disruption of EZH2 by the S-adenosylhomocysteine hydrolase  inhibitor 3-Deazaneplanocin A (DZNep), or its specific down-regulation by shRNA, strongly  impairs GBM cancer stem cell self-renewal in vitro and tumor-initiating capacity in vivo.  Using genome-wide expression analysis of DZNep-treated GBM cancer stem cells, we found  the expression of c-myc, recently reported to be essential for GBM cancer stem cells, to be  strongly repressed upon EZH2 depletion. Specific shRNA-mediated down-regulation of  EZH2 in combination with chromatin immunoprecipitation (ChIP) experiments revealed that  c-myc is a direct target of EZH2 in GBM cancer stem cells. Taken together, our observations  provide evidence that direct transcriptional regulation of c-myc by EZH2 may constitute a  novel mechanism underlying GBM cancer stem cell maintenance and suggest that EZH2 may  be a valuable new therapeutic target for GBM management.

overall_design: Three samples of cancer stem-cell enriched gliospheres from primary glioblastoma multiforme cell cultures were treated with DZNep. Untreated gliospheres from the same cultures were used as controls.

first: Mario-Luca

last: Suvà

first: Nicolò

last: Riggi

first: Michalina

last: Janiszewska

first: Ivan

last: Radovanovic

first: Jean-Christophe

last: Stehle

first: Karine

last: Baumer

first: Marie-Aude

last: Le Bitoux

first: Denis

last: Marino

first: Luisa

last: Cironi

first: Victor

last: Marquez

first: Virginie

last: Clément

last: Stamenkovic


title: Transcriptomes in Peripheral Blood Mononuclear Cells of Dementia and Alzheimer Patients

status: Public on Nov 30 2009

city: Kaohsiung

first: Kuang-Den

last: Chen

address: 123, Ta-Pei Rd., Niao-Sung Dist.

department: Translational Research Center in Biomedical Sicences

country: Taiwan

institute: Kaohsiung Chang Gung Memorial Hospital

summary:
With the aging population, there is a growing focus on dementia, especially Alzheimer’s disease (AD). The molecular basis underlying the pathogenesis of AD is gradually being elucidated. Increasing evidence has shown that the immunological function of leukocytes plays a crucial role in the development of neurodegenerative disorders. However, there have been few studies among the Taiwanese population.

The aim of this study was to investigate potential biomarkers for early diagnosis of Alzheimer’s disease from blood leukocytes.

overall_design: The peripheral blood mononuclear cells (PBMC) transcriptomes from 5 patients with mild cognitive impairment (MCI), 4 AD, as well as 4 normal controls (NC), were analyzed by microarray analysis.

first: Pei-Ning

last: Wang

first: Po-Tsung

last: Chang

first: Yueh-Hsin

last: Ping

first: Chin-Wei

last: Yeh

first: Hsin-Chen

last: Lee


title: Conditioned media from SDEC supports human embryonic stem cell proliferation on type I collagen

city: Baltimore

first: Michael

middle: J

last: Betenbaugh

state: MD

address: 3400 N. Charles St.

department: Chemical and Biomolecular Engineering

country: USA

institute: Johns Hopkins University

summary:
Conditioned media from a human embryonic germ cell-derived line called SDEC was found to be supportive of human embryonic stem cell growth in the absence of feeder layers on a simple type I collagen matrix.  We performed gene expression studies comparing this line to non-supportive cell lines (WI-38 and Detroit 551) to try to identify gene targets responsible for this phenomenon.
We used Affymetrix microarrays to identify genes that are differentially regulated in SDEC vs. non-supportive cell lines.  The goal is to determine which genes maybe be contributing to human embryonic stem cell growth in the absence of a mouse fibroblast feeder layer.

overall_design: Total RNA samples were extracted from SDEC, WI-38, and Detroit 551 (D551) cell lines to compare gene expressions.  Three biological replicates of each were analyzed via micorarray.  Gene targets were identified by looking for highly differentially regulated genes in SDEC compared to the non-supportive lines.  We concentrated on secreted proteins (which could potentially be secreted into the conditioned media) which we identified by functional annotations and literature research.

last: Shamblott

first: Meredith

middle: B

last: Jones


title: Expression data following mitogen stimulation from Jurkat Cell

status: Public on Dec 04 2009

city: Bethesda

first: Jung

middle: S

last: Byun

address: 37 convent drive, Bldg 37,  Rm 1042

institute: NCI/NIH

summary:
Gene expression analysis identified 27 of these 744 p300 and pol II associated genes as significantly increased (p≤ 0.05) within the first hour following mitogen stimulation
Keywords: time course

overall_design: Total RNA was purified from Jurkat T-cells treated 0, 30, and 60 min with phorbol ester and ionomycin.  Antisense biotin-labeled RNA (aRNA) suitable for application to the Affymetrix microarray platform was generated using the Ambion MessageAmp 2-Biotin Enhanced kit as suggested by the manufacturer (www.Ambion.com).  Purified biotin-labeled aRNA was fragmented and hybridized to U133 plus 2 GenechipsTM  (Affymetrix), as recommended by the manufacturer.  All samples were hybridized as technical replicates.  Affymetrix U133 plus 2 GenechipsTM contain probe-sets corresponding to over 56,000 targets.

first: Kevin

last: Gardner

first: Audrey

last: Player


title: Generation of induced pluripotent stem cells from cord blood using OCT4 and SOX2

city: Barcelona

first: Stephanie

last: Boue

address: Dr Aiguader 88

country: Spain

institute: CMRB

summary: Induced pluripotent stem (iPS) cells have generated interest for regenerative medicine as they allow for producing patient-specific progenitors in vitro with potential value for cell therapy. In many instances, however, an off-the-shelf approach would be desirable, such as for cell therapy of acute conditions or when the patient's somatic cells are altered as a consequence of chronic disease or aging. Cord blood (CB) stem cells appear ideally suited for this purpose as they are newborn, immunologically immature cells with minimal genetic and epigenetic alterations, and several hundred thousand immunotyped CB units are readily available through a worldwide network of CB banks. Here, we show that CB stem cells can be reprogrammed to pluripotency by retroviral transduction with OCT4, SOX2, KLF4, and c-MYC, in a process that is extremely efficient and fast. The resulting CB-derived iPS (CBiPS) cells are phenotypically and molecularly indistinguishable from human embryonic stem (hES) cells. Furthermore, we show that generation of CBiPS can be efficiently achieved without the use of the c-MYC and KLF4 oncogenes and just by overexpression of OCT4 and SOX2. Our studies set the basis for the creation of a comprehensive bank of HLA-matched CBiPS cells for off-the-shelf applications.

overall_design:
Human cord blood cells enriched for progenitors by sorting with the CD133 antigen were reprogrammed using either 2 factors (Oct4 and Sox2) or 4 factors (Oct4, sox2, Klf4 and c-Myc) to give rise to induced pluripotent stem cells (CBiPS).

Using genome-wide microarray profiling, we compare expression profiles of the starting population (CD133+), the reprogrammed cells either with 2 or 4 factors (CBiPS 2F and 4F) and a population of embryonic stem cells as reference (ES2), each in 2 replicates.

first: Alessandra

last: Giorgetti

first: Nuria

last: Montserrat Pulido

first: Trond

last: Aasen

first: Ignacio

last: Rodriguez

first: Rita

last: Vassena

first: Maria-Jose

last: Barrero

first: Begoña

last: Aran Corbella

first: Marta

last: Torrebadella

first: Anna

last: Veiga

first: Juan-Carlos

last: Izpisua Belmonte


title: Expression data from doxycylin-inducible miR-15a/16-1 and empty vector (EV) expression in a 13q14-\- cell line

status: Public on Dec 01 2009

city: New York

first: Ulf

last: Klein

state: NY

address: 1130 St. Nicholas Avenue

department: Herbert Irving Comprehensive Cancer Center

institute: Columbia University

summary: Reexpression of microRNAs miR-15a/16-1 in a cell line deficient for these miRs (homozygous deletion of chromosomal region 13q14) results in the downregulation of certain mRNAs.

overall_design: A doxycyclin-inducible expression vector containing miR-15a/16-1 was transfected into CLL-I83E95 cells that are deficient for those miRs. miR-expression was induced by addition of doxycyclin to the medium, and cell were harvested 48 hours later. Labeled cRNA was hybridized to microarrays and gene expression data were compared to CLL-I83U95 stably transfected with an empty vector.

first: Riccardo

last: Dalla-Favera


title: Expression data from cytotoxic T cell clone

status: Public on Nov 29 2009

city: Camperdown

first: Jennifer

middle: R

last: Gamble

state: New South Wales

address: Building 93, Royal Prince Alfred Hospital

department: Vascular Biology

country: Australia

institute: CENTENARY INSTITUTE

summary:
T cells contribute to host-tumor interactions in patients with monoclonal gammopathies. Expansions of CD8+CD57+TCRVβ+ restricted cytotoxic T cell (CTL) clones are found in 48% of patients with multiple myeloma and confer a favorable prognosis. We now report the presence of CTL clones with varying TCRVβ repertoire in 70% of patients with Waldenstrom’s Macroglobulinaemia (WM) (n=20). Previous nucleoside analogue (NA) therapy, associated with an increased incidence of transformation to aggressive lymphoma, significantly influenced the presence of TCRVβ expansions (X2=11.6; P<0.001) as 83% of patients without (n=6) and only 7% with TCRVβ expansions (n=14) had received NA. Clonality of CD3+CD8+CD57+TCRVβ+ restricted CTLs were confirmed by TCRVβ CDR3 size analysis and direct sequencing. To characterize CTL clones, samples of CD3+CD8+CD57+TCRVβ+ cells were profiled using DNA microarrays and the results were validated on both gene and protein level. By gene set enrichment analysis, CTL clones not only expressed genes (GZMB, PRF1, FGFBP2) from cytotoxic pathways but also genes which suppress apoptosis, inhibit proliferation, arrest cell cycle G1/S transition and activate T cells (RAS, CSK and TOB pathways). Proliferation tracking confirmed their anergic state. Our studies demonstrate the incidence, NA sensitivity and anergic nature of clonal T cells in a B cell tumor.
We used microarrays to detail the global programme of gene expression underlying T cell clonal expansion and identified distinct classes of up-regulated genes during this process.

overall_design: In this study we have sought T cell clones in the blood of patients with WM and related their presence with nucleoside analog therapy and transformation. We have also sorted CD3+CD4-TCRVβ+CD57+ from CD3+CD4-TCRVβ+CD57- cells and confirmed clonality by TCRVβ CDR3 size determination and sequencing and used carboxy-fluorescein diacetate succinmidyl ester (CFSE) tracking to monitor proliferation of CD3+CD4-TCRVβ+CD57+ clones. Finally we have performed microarray analysis to search for significant differentially expressed genes and pathways in the clonally expanded CD3+CD8+TCRVβ+CD57+ cells, focusing on cytotoxic pathways but also identifying other dysfunctional changes in these cells. Expression of a selection of the most differentially expressed genes detected by microarray was validated at both gene (qPCR) and protein level.

first: Jia

last: Li

first: Ross

middle: D

last: Brow


title: Comparing effects of mTR and mTERT deletion on gene expression and DNA damage response: MEF

first: Carol

last: Greider

address: 725 N Wolfe St

department: Molecular Biology and Genetics

institute: Johns Hopkins University School of Medicine

summary: Telomerase, the essential enzyme that maintains telomere length, contains two core components, TERT and TR. While early studies in yeast and mouse both indicated that loss of telomerase leads to phenotypes that arise after an increased number of generations, due to telomere shortening, recent studies claim additional roles for telomerase components in transcription and the response to DNA damage. To test these telomere length maintenance-independent roles of telomerase components, we examined first generation mTR-/- and mTERT-/- mice with long telomeres. We used gene expression profiling and found no genes that were expressed at significantly different levels when independent mTR-/- G1 mice were compared to mTERT-/- G1 mice and to wild-type mice. In addition, we compared the response to DNA damage in mTR-/-G1 and mTERT-/- G1 mouse embryonic fibroblasts, and found no increase in the response to DNA damage in the absence of either telomerase components compared to wild-type. We conclude that in the wild-type physiological telomere length setting, neither mTR nor mTERT act as a transcription factor or have a role in the DNA damage response.

overall_design:
Mouse Embryonic Fibroblasts (MEFs) were generated from age- and sex-matched telomerase wild-type and knock-out C57BL/6J embryos (E12.5-14.5). MEFs were processed for RNA extraction at passage 5.
Telomerase null embryos were first-generation (G1) mTERT KO or mTR KO.
3 independent wild-type + knock-out MEF sets were examined.
In each set, wild-type + knock-out mice were littermates.

first: Sofia

middle: L

last: Vidal-Cardenas

middle: W


title: Complement receptor 2/CD21– human naive B cells contain mostly autoreactive unresponsive clones

first: Isabelle

last: Isnardi

address: 535E 70th Street

institute: Hospital for Special Surgery

summary: Complement receptor 2–negative (CR2/CD21–) B cells have been found enriched in patients with autoimmune diseases and in common variable immunodeficiency (CVID) patients who are prone to autoimmunity. However, the physiology of CD21–/lo  B cells remains poorly characterized. We found that some rheumatoid arthritis (RA) patients also display an increased frequency of CD21–/lo B cells in their blood. A majority of CD21–/lo B cells from RA and CVID patients expressed germline autoreactive antibodies, which recognized nuclear and cytoplasmic structures. In addition, these B cells were unable to induce calcium flux, become activated, or proliferate in response to B-cell receptor and/or CD40 triggering, suggesting that these autoreactive B cells may be anergic. Moreover, gene array analyses of CD21–/lo  B cells revealed molecules specifically expressed in these B  cells and that are likely to induce their unresponsive stage.  Thus, CD21–/lo B cells contain mostly autoreactive unresponsive clones, which express a specific set of molecules that may represent new biomarkers to identify anergic B cells in humans.

overall_design: RNA was extracted from batch sorted CD19+CD21+CD10-CD27- and CD19+CD21-CD10-CD27- naïve B cells isolated from donors using the Absolutely RNA microprep kit (Stratagene). 100-200 ng of RNA was obtained per sample, and the quality of the purified RNA was assessed by the Bioanalyzer from Agilent. Using the Ovation biotin system kit from Nugen, 30-50ng of RNA was amplified and labeled to produce cDNA. Labeled cDNA was hybridized on chips containing the whole human genome (Human Genome U133 2.0 from Affymetrix). Data from CD21+ and CD21- B cell populations were compared in order to determine the gene signature of the newly described CD21- B cells.

first: Eric

last: Meffre


title: Nodular lymphocyte predominant Hodgkin's lymphoma vs T cell/histiocyte rich B cell lymphoma

status: Public on Nov 27 2009

city: Leuven

first: Rekin's

last: Janky

address: Herestraat 49 Box 816

department: Nucleomics Core

country: Belgium

institute: VIB

summary:
T-cell/histiocyte rich B cell lymphoma (THRBL) and nodular lymphocyte predominant Hodgkin's lymphoma (NLPHL) share some morphological characteristics, including a prominent stromal reaction, but display a markedly different prognosis. To investigate the difference between the stromal reactions of these lymphomas at the molecular level, we performed microarray expression profiling on a series of THRBL and NLPHL cases.
Keywords: lymphoma, NLPHL, THRBL, stromal reaction

overall_design: Microarray expression profiling was performed for 10 NLPHL and 10 THRBL cases. A pool of 5 reactive lymph nodes was included as a reference sample.

first: Peter

last: Van Loo

first: Vera

last: Vanhentenrijk

first: Daan

last: Dierickx

last: Vanden Bempt

first: Gregor

last: Verhoef

last: Marynen

first: Patrick

last: Matthys

first: Chris

last: De Wolf-Peeters


title: E. coli MqsR and MqsA whole DNA microarray and nickel DNA enrichment microarray

city: State College

first: Thomas

middle: K.

last: Wood

state: PA

address: Room 161 Fenske Laboratory, University Park

department: Chemical Engineering & Biochemistry and Molecular Biology

institute: Pennsylvania State University

summary: Here evidence is presented that MqsR and B3021 are a novel toxin-antitoxin (TA) system related to biofilm development and quorum sensing.  Using whole-transcriptome studies and nickel enrichment DNA binding microarrays coupled with cell survival studies in which MqsR was overexpressed in isogenic mutants, we identified seven genes involved in MqsR toxicity (clpX, clpP, yfjZ, cspD, relB, relE, and hokA).  Quantitative real-time polymerase chain reaction (qRT-PCR) confirmed consistent induction of the seven genes by the overexpression of MqsR as well as induction of nutrient starvation related genes (cstA, rpoS, and dps).  Taken together, our results indicate that MqsR toxicity is caused via CspD (a DNA replication inhibitor), other TA systems (RelE/RelB and YfjZ), HokA (a small membrane toxin peptide), and nutrient-starvation conditions induced via CstA, RpoS, and Dps.  Additionally, in vivo binding results show antitoxin B3021 binds to the promoter regions of genes encoding essential proteins for stress, growth and normal physiology.

platform_organism:
Escherichia coli
Escherichia coli K-12

overall_design:
Strain: E. coli K-12 BW25113 wt and mqsR deleted mutant
Medium: LB medium
Temperature: 37 oC
Time: 24 h
Cell type: Biofilm grown on glass wool and planktonic culture

first: Younghoon

last: Kim

first: Xue-Song

last: Zhang

middle: K


title: Expression profiling of ATRA treated NB4 cells

status: Public on Nov 26 2009

city: Shanghai

first: Jiantao

last: Shi

state: Shanghai

address: 197 Ruijin Rd II

country: China

institute: Rui-Jin Hospital affiliated to Shanghai Jiao Tong University School of Medicine (RJH-SJTU-SM)

summary: NB4 is an APL derived cell line, carrying the t(15;17) translocation and expressing the PML/RARa fusion protein. Still, an important question that remains to be addressed is whether PML/RARa target genes are transcriptionally suppressed in primary APL cells and re-activated in all-trans retinoic acid (ATRA) treated NB4 cells. Gene expression of NB4 cells treated with ATRA at different time points were analyzed.

overall_design: 6 samples at various times. No replicats.

first: Kankan

last: Wang

first: Ji


title: The Role of Cholesterol Pathways in Norovirus Replication

status: Public on Nov 13 2009

city: Manhattan

first: Kyeong-Ok

last: Chang

address: 1800 Denison Ave

institute: Kansas State Univerisity

summary: Norwalk virus (NV) is a prototype strain of the noroviruses (family Caliciviridae) which have emerged as major causes of acute gastroenteritis worldwide. We have developed NV replicon systems using reporter proteins such as a neomycin resistant protein (NV replicon-bearing cells) and a green fluorescent protein (pNV-GFP), and demonstrated that these systems were excellent tools to study virus replication in cell culture. In this study, we first performed DNA microarray analysis of the replicon-bearing cells to identify cellular factors associated with NV replication. The analysis demonstrated that genes in lipid (cholesterol) or carbohydrate metabolic pathways were significantly (p< 0.001) changed by the gene ontology analysis. Among genes in the cholesterol pathways, we found that mRNA levels of hydroxymethylglutaryl-CoA (HMG-CoA) synthase, squalene epoxidase and acyl-CoA:cholesterol acyltransferase(ACAT) 1, ACAT 2, small heterodimer partner, and low density lipoprotein receptor (LDLR)-related proteins were significantly changed in the cells.

overall_design: To identify host-factors associated with norovirus replication, we have performed DNA microarray analysis of replicon-bearing cells (HG23) and parental Huh-7 cells using a Affymetrix GeneChip. One- or two-day old semi-confluent Huh-7 cells and HG23 cells grown in T-75 flasks were prepared to do DNA microarray analysis. Total RNA which was isolated using the RNeasy kit (Qiagen, Valensia, CA) was used on the DNA array. At least 3 independent cell cultures and RNA preparations were supplied for statistical analysis. The synthesis of cDNA, labeling and hybridization were done using Affymetrix regents according to the instructions from the company. We used Affymetrix GeneChip Human Genome U133 Plus 2.0 (a whole genome array covering 47,000 transcripts) for the DNA array. The entire process was done in the DNA array facility at Kansas State University. The genes either up- or down-regulated in HG23 cells compared to Huh-7 cells were analyzed with the Student’s t test of the three independent samples. The genes showing at least 1.5- or 2-fold difference with p<0.05 were further analyzed.


title: 12 microarrays for cis-DCE mineralization and new stress genes

status: Public on Nov 24 2009

city: College Station

first: Jintae

last: Lee

state: TX

department: Chemical Engineering

institute: Texas A&M University

summary: Metabolically-engineered Escherichia coli has been used previously to degrade the ubiquitous pollutant cis-1,2-dichloroethylene (cis-DCE), and the impact of the metabolic engineering was assessed by investigating the changes in the proteome.  Here, genome-wide transcriptome analysis was performed to confirm that a strong heat shock and/or oxidative stress occurs during enhanced cis-DCE mineralization. Also, seven new stress proteins (YchH, YdeI, YodD, YodC, YgiW, YhcN, and YjaA) that were previously uncharacterized have been identified.

overall_design:
Twelve planktonic RNA samples for the six sets of microarray experiments (set i was repeated) were prepared (Table 1 indicates the rationale of the whole-transcriptome studies): (i) cells of E. coli TG1 with TOM-Green/IsoILR1/GSHI* vs. no cloned genes contacted for 2 h with 1 mM cis-DCE to determine the effect of expressing eight genes (encoding TOM-Green/IsoILR1/GSHI*) on cis-DCE mineralization, (ii) cells of TG1 with TOM-Green/EchA F108L/I219L/C248I vs. TOM-Green contacted for 2 h with 1 mM cis-DCE to determine the effect of the evolved epoxide hydrolase on cis-DCE mineralization, (iii) cells (turbidity of 1.0) of E. coli BW25113 with 2 mM H2O2 vs. no H2O2 contacted for 10 min with shaking to determine gene expression in response to H2O2 stress since cis-DCE mineralization induced many oxidative stress genes (iv) cells (turbidity of 1.0) of the BW25113 ygiW strain with 2 mM H2O2 vs. no H2O2 contacted for 10 min to determine gene expression in response to H2O2 stress in the absence of YgiW, and (v) cells (turbidity of 1.0) of the BW25113 ychH strain with 2 mM H2O2 vs. no H2O2 contacted for 10 min to determine gene expression in response to H2O2 stress in the absence of YchH.

For the cis-DCE degradation system, the exponentially-grown TG1 cells (Table 1) were contacted with 1 mM cis-DCE together with 0.5 mM IPTG and 5 mM succinate (as a carbon/energy source during the cis-DCE degradation) in 60 mL vials for 2 hr as performed previously in the proteomics study J. Proteome Res. 5: 1388 - 1397 (2006). After 2 hr contact with cis-DCE, samples were centrifuged, and the cell pellets were frozen immediately with dry ice and stored at -80°C.  For the H2O2 stress system, overnight cultures of BW25113, BW25113 ygiW, and BW25113 ychH were re-grown to mid-log phase in LB (turbidity at 600 nm of 1), then the cultures were split into two cultures and 2 mM H2O2 and water (negative control) were added.   After contacting for 10 min, samples were centrifuged and the cell pellets frozen immediately with dry ice and stored at -80°C. RNA was isolated from the biofilm cells as described previously using sonication and a bead beater Appl. Microbiol. Biotechnol. 64: 515-524 (2004)..

For DNA microarray analysis, the E. coli Genechip antisense genome array (Affymetrix, P/N 900381, Santa Clara, CA) and the E. coli GeneChip Genome 2.0 Array (Affymetrix, P/N 900551) were used to study the differential gene expression profile of the K12 cells; the E. coli Genechip antisense genome array contains probe sets for all 4290 open reading frames (ORF), rRNA, tRNA, and 1350 intergenic regions, and the E. coli GeneChip Genome 2.0 Array contains 10,208 probe sets for open reading frames, rRNA, tRNA, and intergenic regions for four E. coli strains: MG1655, CFT073, O157:H7-Sakai, and O157:H7-EDL933. The same type of microarray was used for each binary comparison.  cDNA synthesis, fragmentation, and hybridizations were as described previously J. Bacteriol. 188: 587-598 (2006). Hybridization was performed for 16 h, and the total cell intensity was scaled to an average value of 500. The probe array images were inspected for any image artifact.  Background values, noise values, and scaling factors of both arrays were examined and were comparable. The intensities of polyadenosine RNA control were used to monitor the labeling process. For each binary microarray comparison of differential genes expression, if the gene with the larger transcription rate did not have a consistent transcription rate based on the 11-15 probe pairs (p-value less than 0.05), these genes were discarded.

Extract protocol: To lyse the cells, 1.0 mL RLT buffer (Qiagen, Inc., Valencia, CA) and 0.2 mL 0.1 mm zirconia/silica beads (Biospec) were added to the frozen bead beater tubes containing the cell pellets. The tubes were closed tightly and beat for 50 seconds at the maximum speed in a mini bead beater (cat. no. 3110BX, Biospec). The total RNA was isolated by following the protocol of the RNeasy Mini Kit (Qiagen) including an on-column DNase digestion with RNase-free DNase I (Qiagen).

Label protocol: The total RNA samples were first converted into cDNA through a reverse transcription reaction with poly-A RNA controls spiked into the same reaction mixture to monitor the entire target labeling process.  The cDNA was then digested with DNase I (Amersham Biosciences) to produce 50-200 bp fragments, which was checked by running the fragmented cDNA on a 2% agarose gel.  The fragmented cDNA was labeled at the 3' termini by the Enzo BioArray Terminal Labeling Kit with Biotin-ddUTP (Affymetrix, P/N 900181).

Hybridization protocol: The biotin-labeled target was hybridized to the Affymetrix GeneChip E. coli array at 45°C for 16 hour at 60 rpm using the Hybridization Oven 640 (Affymetrix), then a three-step fluorescent staining was conducted using the Fluidics Station 450 (Affymetrix) during the washing and staining procedure.

Scanning protocol: The microarray was scanned at 570 nm to get an image file by the GeneChip Scanner 3000 (Affymetrix).  Using GeneChip Operating Software, total cell intensity was scaled automatically in the software to an average value of 500.


title: siRNA-mediated Egr-3 knockdown in VEGF-treated HUVEC

city: Mitaka

first: Jun-ichi

last: Suehiro

state: Tokyo

address: 6-20-2, Shinkawa

department: Department of Pharmacology and Toxicology

country: Japan

institute: Kyorin University School of Medicine

summary: Analysis of umbilical vein endothelial cells (HUVEC) treated with Egr-3 siRNA under the VEGF treatment for 0,1, and 4 h. Egr-3, a member of early growth response family, is  immediately and dramatically induced by VEGF in HUVEC, which regulates expression of many genes related to endothelial activation.

overall_design: Total 21 samples were derived from triplicate arrays of VEGF-treated si-Control-transfected cells and duplicate arrays of each of the VEGF-treated si-Egr-3-transfected cells.

first: Takao

last: Hamakubo

first: William

middle: C

last: Aird

first: Tatsuhiko

last: Kodama

first: Takashi

last: Minami


title: Transcriptional profiling of testicular biopsies with Sertoli-cell-only and spermatogonial presence

city: 20246 Hamburg

first: Andrej-Nikolai

last: Spiess

address: Martinistr. 52

department: Andrology

country: Germany

institute: University Hospital Hamburg-Eppendorf

summary:
The aim of the study was to identify in vivo spermatogonial gene expression within the context of their biological niche.
Identification of spermatogonial genes was done by t-testing on the replicates of Score 3 (Spermatogonia) and Score 2 (SCO) testicular biopsies.

overall_design: 3 biological replicates with spermatogonial presence in the tubuli seminiferi, 5 biological replicates with Sertoli-cell-only syndrome.

first: Kathrein

last: von Kopylow

first: Christiane

last: Kirchhoff

first: Davor

last: Jezek

first: Wolfgang

last: Schulze

first: Caroline

last: Feig

first: Michael

last: Primig

first: Volker

last: Steinkraus


title: The purine scaffold  HSP90 inhibitor PU-H71 induces specific changes in gene expression in DLBCL xenografts.

status: Public on Nov 15 2009

first: Ana

middle: I

last: Robles

address: 37 Convent Dr 37/3060D

institute: National Cancer Institute/NIH

summary: Heat shock protein 90 (Hsp90) is an emerging therapeutic target in cancer.  We report that Hsp90 inhibitors selectively kill DLBCLs that are biologically dependent on the BCL6 transcriptional repressor.  We examined the pharmacokinetics, toxicity and efficacy of PUH71, a recently developed purine scaffold Hsp90 inhibitor.  PUH71 preferentially accumulated in tumors vs. normal tissues, and unlike the widely used benzoquinone Hsp90 inhibitors, displayed no signs of organ toxicity.  PUH71 selectively and potently induced the regression of BCL6-dependent DLBCLs in vivo, through reactivation of key BCL6 target genes and apoptosis.

overall_design: Six SCID mice were subcutaneously injected with human Farage DLBCL cells. When tumors reached 1 cm in diameter, four mice were administered PU-H71 (75mg/kg) by intra-peritoneal injection and two mice served as no-treatment controls. Animals were sacrificed by cervical dislocation under anesthesia at 6 and 12 h after the administration of PU-H71.

first: Leandro

last: Cerchietti

first: Jennifer

last: Walling

first: Ari

middle: M

last: Melnick


title: Expression data from two pair of osteosarcoma sublines with difeerental pulmonary

status: Public on Nov 10 2009

city: Xi'an

first: xiang

last: chen

address: Xinsi Rd

institute: Tangdu Hospital

summary: Metastasis of tumor consists of a complex serias of cascades . Molecular mechanisms needs to be elucidated. We used microarrays to explore the global change of gene expression betwwen two pairs of osteosarcoma sublines screened by different method.

overall_design: The assay was performed among three pairs of cublines, the first two pairs of sublines comes from the different passage of sublines established with orthotopic transplantation under the established cell line named Sosp-9607 ,the other pair was screened by the tail-vein injection method of commercial avaliable cell line-Saos-2.

first: Xiang

last: Chen

first: Tongtao

last: Yang

first: Qingyu

last: Fan

first: Baoan

last: Ma

first: Yong

last: Zhou

first: Xiuchun

last: Qiu


title: Regulation of Inflammatory Gene Expression by Immunostimulatory Botanicals

status: Public on Nov 21 2009

city: Tempe

first: Robert

last: Waters

state: AZ

address: 1001 S. McAllister

department: Biodesign Institute

institute: Arizona State University

summary: Using herbogenomics, this study provides novel insight into putative genes which may be involved in physiological responses.

overall_design: PBMCs were treated with 5uL herbal extract for indicated times, followed by RNA isolation with Rneasy kit (Qiagen). Microarray analysis was performed using Affymetrix Human Genome U133 Plus 2.0 Array.

first: Jeffrey

last: Langland

first: Karen

last: Denzler


title: Detection call p-values for Affymetrix low level spike-in experiment

city: Columbus

first: Kellie

middle: J.

last: Archer

state: OH

address: 1841 Neil Ave

department: Division of Biostatistics

institute: Ohio State University

summary:
Four different concentrations (0, 0.75, 1.5, 3 pM) of genes were spiked in groups (group size 6-7 transcripts)
Each concentration combination were spiked into a common backgroung sample (HeLa cell line) and hybridized to three HG-U133A Plus 2.0 Arrays.

Keywords: Latin square design for HG-U133A Plus 2.0 GeneChips

first: Sarah

middle: E

last: Reese


title: Rapamycin treatment of MDA-MB-468 breast cancer cell line and MDA-MB-468 xenografts

status: Public on Nov 20 2009

city: Houston

first: Funda

last: Meric-Bernstam

address: 1515 Holcombe Boulevard

department: Surgical Oncology

institute: University of Texas M.D. Anderson Cancer Center

summary: Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in multiple intracellular signaling pathways promoting tumor growth. mTOR is aberrantly activated in a significant portion of breast cancers and is a promising target for treatment. Rapamycin and its analogues are in clinical trials for breast cancer treatment. Patterns of gene expression (metagenes) may also be used to simulate a biologic process of effects of a drug treatment. In this study, we tested the hypothesis that the gene-expression signature regulated by rapamycin could predict disease outcome for patients with breast cancer. Results: Colony formation and sulforhodamine B (IC50 < 1nM) assays, and xenograft animals showed that MDA-MB-468 cells were sensitive to treatment with rapamycin. The comparison of in vitro and in vivo gene expression data identified a signature, termed rapamycin metagene index (RMI), of 31 genes upregulated by rapamycin treatment in vitro as well as in vivo (false discovery rate of 10%). In the Miller dataset, RMI was significantly associated with tumor size or lymph node status. High (>75) percentile) RMI was significantly associated with longer survival (P = 0.015). On multivariate analysis, RMI (P = 0.029), tumor size (P = 0.015) and lymph node status (P = 0.01) were prognostic. In van 't Veer study, RMI was not associated with the time to develop distant metastasis (P = 0.41). In Wang dataset, RMI predicted time to disease relapse (P = 0.09). Conclusions: Rapamycin-regulated gene expression signature predicts clinical outcome in breast cancer. This supports the central role of mTOR signaling in breast cancer biology and provides further impetus to pursue mTOR-targeted therapies for breast cancer treatment. Mol Cancer. 2009 Sep 24;8(1):75.

overall_design:
Rapamycin treatment of MDA-MB-468 breast cancer cell line:
MDA-MB-468 cell line was treated by DMSO (vehicle) and 100 nM rapamycin for 24 hours. We sought to identify differentially expressed genes.

Rapamycin treatment of breast tumor xenografts:
MDA-MB-468 cells were inoculated in the mammary fat pad of female nude mice. When resulting tumor volumes had reached 75-150 mm3, the mice were divided in four groups. Groups 1 and 2 received a single injection of DMSO (vehicle) or rapamycin (15 mg/kg) intraperitoneally and sacrificied 24 h later (1-day groups). Groups 3 and 4 received weekly injections of DMSO or rapamycin for 3 weeks and sacrificied 24 h after the last injection (22-day groups).

first: Argun

last: Akcakanat

first: Li

last: Zhang

first: Spiridon

last: Tsavachidis


title: Expression profile of human T-ALL cell lines treated with DMSO or SAHM1

status: Public on Nov 12 2009

city: Cambridge

first: Raymond

last: Moellering

state: MA

address: 12 Oxford Street

department: Chemistry and Chemical Biology

institute: Harvard University

summary:
NOTCH proteins regulate signaling pathways involved in cellular differentiation, proliferation and death. Overactive Notch signaling as been observed in numerous cancers and has been extensively studied in the context of T-cell acute lymphoblastic leukemia (T-ALL) where more than 50% of pateints harbour mutant NOTCH1. Small molecule modulators of these proteins would be important for understanding the role of NOTCH proteins in malignant and normal biological processes.
We were interested to measure the global changes in gene expression upon treatment of the  human T-ALL cell lines HPB-ALL and KOPT-K1 with either vehicle alone (DMSO) or SAHM1, an alpha-helical hydrocarbon stapled peptide derived from the MAML1 co-activator protein.

overall_design: Triplicate cultures of KOPT-K1 or HPB-ALL cells were treated with either DMSO alone or SAHM1 (20 uM) for 24 hours. Total RNA was extracted and hybridized to Affymetrix human U133 plus 2.0 microarrays (three arrays per treatment per cell line for a total of 12 arrays).


title: Ontogeny of CD24 expression in the human fetal kidney

status: Public on Nov 11 2009

city: Vancouver

first: Douglas

last: Matsell

state: BC

address: 4480 Oak Street, Rm K4-150

country: Canada

institute: BC Children's Hospital

summary:
CD24, or heat stable antigen, is a cell surface sialoglycoprotein expressed on immature cells that disappears after the cells have reached their final differentiation stage. CD24 may be important in human embryonic kidney epithelial cell differentiation. In mice, CD24 expression is up-regulated in the early metanephros and localized to developing epithelial structures but the role and expression of CD24 in the developing human kidney has not been well described. In normal human fetal kidneys from 8 to 38 weeks gestation, CD24 expression was up-regulated and restricted to the early epithelial aggregates of the metanephric blastema and to the committed proliferating tubular epithelia of the S-shape nephron; however individual CD24+ cells were identified in the interstitium of later gestation and postnatal kidneys. In freshly isolated cells, FACS analysis demonstrated distinct CD24+ and CD24+133+ cell populations, constituting up to 16% and 14% respectively of the total cells analyzed. Isolated and expanded CD24+ clones displayed features of an epithelial progenitor cell line. Early fetal urinary tract obstruction resulted in an upregulation of CD24 expression, both in developing epithelial structures of early gestation kidneys and in the cells of the injured tubular epithelium of the later gestation kidneys. These results highlight the cell specific expression of CD24 in the developing human kidney and dysregulation in fetal urinary tract obstruction.
We used microarrays to define the differences in global gene expression between obstructed and normal human fetal kidneys.

overall_design: We analyzed RNA extracted from human fetal kdeys bewteen the gestational ages 8 and 18 weeks gestation.

middle: G

first: Alice

middle: F

last: Tarantal


title: DNMT1 Maintains Progenitor Function in Self-Renewing Somatic Tissue

city: Stanford

first: Paul

middle: A

last: Khavari

state: CA

address: 259 Campus Dr

department: Dermatology

institute: Stanford University

type: Methylation profiling by genome tiling array

summary: Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation. DNA methylation provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1) maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance, a clear role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is uncharacterized. Here we show that DNMT1 is essential for supporting epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. These effects correlated with DNA methylation as genome-wide analysis revealed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation.

overall_design:
Gene expression analysis: To establish a differentiation signature for primary human keratinocytes, total RNA was isolated in biologic duplicate from cells cultured in growth conditions and high calcium differentiation conditions and hybridized to Affymetrix HG-U133 2.0 Plus arrays. This gene signature was also compared to DNMT1 deficient cells cultured in growth conditions.

Methylated DNA profiling: To globally characterize DNA methylation in primary human keratinocytes, genomic DNA was immunoprecipitated using a 5-methylcytidine antibody, amplified, and hybridized to NimbleGen HG18 promoter tiling arrays. Profiling was done using DNA isolated in growth conditions as well as differentiation conditions.

first: George

last: Sen

first: Jason

last: Reuter

first: Daniel

last: Webster

first: Lilly

last: Zhu


title: Comparison of neuroepithelial cells and regionally specified astrocytes from human embryonic stem cells

city: Madison

first: Robert

last: Krencik

state: WI

address: 1500 Highland Ave #641B

department: Waisman Center

institute: University of Wisconsin-Madison

summary: These arrays were conducted in order to confirm the generation of astrocytes from human embryonic stem cells and to determine expression differences between astrocyte subtypes.

overall_design: Human embryonic stem cells were differentated in vitro to neuroepithelial cells (day-17), or astrocytes (day-210) after specification with the morphogens retinoic acid (RA) or FGF8 from days 10-21.

first: Su-Chun


title: PML-RARa binding sites and their correlation with the gene expression in ATRA treated NB4 cells

first: Jiantao

last: Shi

type: Genome binding/occupancy profiling by genome tiling array

summary:
PML-RARa contributes to the development of APL through repression of genes important in myeloid development. Through a global approach, we have identified 2,979 high quality PML-RARa binding sites in ZnSO4 induced PR9 cells. By integration the gene expression data, we found that PML/RARa target genes are transcriptionally suppressed in primary APL cells and re-activated in ATRA treated NB4 cells.

This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Analysis of Meniscal Degeneration and Meniscal Gene Expression

status: Public on Nov 18 2009

city: Charlotte

first: Yubo

last: Sun

state: NC

address: 1542 Garden Terrace

department: Orthopaedic Surgery

institute: Carolinas HealthCare System

summary:
Menisci play a vital role in load transmission, shock absorption and joint stability.  The current dogma is that the menisci simply protects the cartilage and play no role in osteoarthritis (OA) unless they are injured. However, there is increasing evidence suggesting that OA menisci may not merely be bystanders in the disease process of OA. This study sought: 1) to determine the prevalence of meniscal   degeneration in OA patients, 2) to examine gene expression in OA meniscal cells compared to normal control meniscal cells, and 3) to test the hypothesis that OA meniscal cells are different from normal meniscal cells.
The grades of meniscal degeneration correlated in a positive fashion with the grades of articular cartilage degeneration (r = 0.672; P < 0.0001). Many genes classified in the biological processes of immune response, inflammatory response,  biomineral formation and cell proliferation, including major histocompatibility complex, class II, DP alpha 1 (HLA-DPA1), integrin, beta 2 (ITGB2), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), ankylosis, progressive homolog (ANKH) and fibroblast growth factor 7 (FGF7), were expressed at higher levels compared to normal control meniscal cells. In addition, many genes that were previously implicated in OA were also expressed at higher levels in OA meniscal cells, including ADAM metallopeptidase with thrombospondin type 1 motif, 5 (ADAMTS5). Our findings suggest that OA is a whole joint disease. Meniscal cells may   play an active role in the development of OA. Investigation of the gene expression   profiles of OA meniscal cells may reveal new therapeutic targets for OA therapy and   also may uncover novel disease markers for early diagnosis of OA.

overall_design: Studies were approved by our human subjects Institutional Review Board. Menisci and articular cartilage were collected during joint replacement surgery for OA patients (OA specimens) and lower limb amputation surgery for osteosarcoma patients   (normal control specimens), and graded. Meniscal cells were prepared from the meniscal tissues and expanded in monolayer culture. Differential gene expression in OA meniscal cells and normal control meniscal cells was examined using Affymetrix   microarray and real time RT-PCR.

first: David

last: Mauerhan

first: Patrick

last: Honeycutt

first: Jeffrey

last: Kneisl

first: H

middle: J

last: Norton

first: Edward

middle: N

last: Hanley Jr

first: Helen

last: Gruber


title: Differentially expressed genes between MCF-7 and MCF-7-14 cells

status: Public on Nov 06 2009

city: Nagareyama-shi

first: Masahiro

last: Uchino

state: Chiba

address: 105 Higashifukai

institute: Bio Matrix Research, Inc

summary: We investigated the differentially expressed genes between MCF-7 and MCF-7-14, and estimated the similarities of expression profiles between MCF-7-14 and MDA-MB-231. We identified genes differentially expressed between non-invasive/non-metastatic and invasive/metastatic breast cancer cells.

overall_design: We performed microarray analysis as following design: MCF-7 versus MCF-7-14 versus MDA-MB-231 cells.

first: Kenta

last: Wada


title: E. coli BW25113 K-12 hfq vs. wild-type persister

status: Public on Nov 08 2009

first: Thomas

last: Wood

institute: Pennsylvania State University

summary: Persisters are cells which evade stresses like antibiotics and which are characterized by reduced metabolism and a lack of genetic alterations required to achieve this state.  We showed previously that MqsR and MqsA of Escherichia coli are a toxin-antitoxin pair that influence cell physiology (e.g., biofilm formation and motility) via RNase activity as well as through regulation of toxin CspD.  Here, we show that deletion of the mqsRA locus decreases persister cell formation and, consistent with this result, overexpression of MqsR increases persister cell formation.  Furthermore, toxins Hha, CspD, and HokA increase persister cell formation.  In addition, by overproducing MqsR in a series of isogenic mutants, we show that Hha and CspD are necessary for persister cell formation via MqsR overexpression.  Surprisingly, Hfq, a small RNA chaperone, decreases persistence.  A whole-transcriptome study shows that Hfq induces transport-related genes (oppA, oppB, oppC, oppD, oppF, and dppA), outer membrane protein-related genes (ybfM and ybfN), toxins (hha), and proteases (clpX, clpP, and lon).  Taken together, these results indicate that toxins CspD and Hha influence persister cell formation via MqsR and that Hfq plays an important role in the regulation of persister cell formation via regulation of transport or outer membrane proteins.

overall_design:
Strains: E. coli BW25113 K-12 hfq deleted mutant vs. wild-type
Medium: LB
Culture: Planktonic cell grown OD=0.5, adjusted OD=1.0, and then exposed to 100 ug/mL ampicillin for 2 h.

first: Younghoon

last: Kim


title: Tumor expression data from neoadjuvant trial of cisplatin monotherapy in triple negative breast cancer patients

status: Public on Nov 04 2009

city: Lyngby

first: Aron

last: Eklund

address: Kemitorvet, Building 208

department: Center for Biological Sequence Analysis

country: Denmark

institute: Technical University of Denmark

summary:
Evidence suggests that BRCA1 mutation associated tumors have increased sensitivity to DNA damaging agents like cisplatin.  Sporadic triple negative breast cancers (TNBC) have many phenotypic similarities to BRCA1 tumors and may have a similar sensitivity to cisplatin.  We tested the efficacy of cisplatin monotherapy in 28 TNBC patients in a single arm neoadjuvant trial with outcome measured by pathologic treatment response quantified using the Miller-Payne scale.
We used microarrays gene expression profiles to determine tumor subtype of each trial tumor sample and to test various expression signatures for association with pathologic response to cisplatin.

overall_design: Pretreatment tumor samples from the clinical trial (N=24 with adequate tissue) were used for RNA extraction, linear amplification, biotin labeling and hybridization to Affymetrix U133 plus 2.0 arrays.  A reference set of 51 primary breast tumors representing all subtypes of breast cancer were processed in a similar manner to include linear amplification, and hybridized to Affymetrix arrays.

first: Andrea

last: Richardson


title: Placentas delivered by term spontaneous labour compared to those delivered by elective term Cesarean section

status: Public on Nov 02 2009

city: Shatin

first: Stephen

middle: S

last: Chim

address: Dept of Obstetrics & Gynaecology, The Chinese University of Hong Kong

department: Obstetrics & Gynaecology

country: Hong Kong

institute: The Chinese University of Hong Kong

summary:
Differentially expressed mRNA transcripts in the placenta delivered by term spontaneous labour compared to those delivered by elective term cesarean section.
We hypothesized that the labour process involves changes in mRNA expression in the placenta. To test this hypothesis, we interrogated the mRNA levels of >50,000 genes and transcript variants using gene expression microarray (Human Genome U133 Plus 2.0 Array, Affymetrix) on 5 placentas collected from term spontaneous delivery and another 5 placentas collected from elective term cesarean delivery. To minimize the effect of gestational age on gene expression, these two groups of placentas were matched for their gestational ages at delivery.
We have identified 134 and 128 genes that were up- or down-regulated, respectively, for more than 3-fold in the term (spontaneous) labour placentas compared to the term (elective) cesarean placentas (Mann-Whitney Rank Sum Test, p-value <= 0.05 after Benjamini and Hochberg adjustment for multiple testing).

overall_design: Placentas collected from (i) term spontaeous labour and (ii) elective term cesarean section were subjected to RNA extraction and hybridization on Affymetrix microarrays. To identify gene expression patterns that are commonly involved in term spontaneous labour, we analyzed 5 placentas from each of these 2 groups and tested for any differentially expressed genes by non-parametric statistical methods.

first: Wing

last: Lee

first: Rossa

last: Chiu

first: Tak

middle: Y

last: Leung

first: Tze

last: Lau

first: Yuk

last: Lo


title: Expression data from human triploid and diploid fibroblast cell cultures

city: Seattle

first: Xinxian

last: Deng

state: WA

address: 1959 NE Pacific St.

department: Laboratory Medicine and Pathology

institute: University of Washington

summary:
Dosage compensation restores a balanced network of gene expression between autosomes and sex chromosomes in males (XY) and females (XX). In mammals, this is achieved by doubling the expression of X-linked genes in both sexes, together with X inactivation in females. X up-regulation may be controlled by DNA sequence based and/or epigenetic mechanisms that double the X output potentially in response to an autosomal counting factor. Human triploids with either one or two active X chromosomes (Xa) provide a mean to test X chromosome expression in the presence of three sets of autosomes, which will help understand the underlying mechanisms of X up-regulation.
We measured whole genome gene expression in human triploid cell cultures with either one or two active X. We found that overall X-linked gene expression is not tripled in the presence of three sets of autosomes. However, in triploid cells with a single active X chromosome, its expression is adjusted upward, presumably by an epigenetic mechanism that senses the active X-autosome ratio.

overall_design: Six human XXX triploid fibroblast clones with either one or two active X, three XYY triploid fibroblast cultures, and two male (XY) and two female (XaXi) control diploid fibroblast cultures were selected for RNA extraction and hybridization on Affymetrix whole genome expression arrays (HG-U133 2.0 plus chip). Probe labeling, array hybridization and scanning were done by the University of Washington Microarray Center.

first: Di

last: Nguyen

first: Scott

middle: R

last: Hansen

first: Daniel

last: Van Dyke

first: Stanley

last: Gartler

first: Christine

last: Disteche


title: Effects on small RNA MgrR in Escherichia coli

city: BETHESDA

first: Deborah

last: Hinton

address: 8 center Dr., Building 8, Rm 2A21

institute: NIH

summary: MgrR is a newly characterized Hfq dependent small RNA RNA.  The expression of MgrR is regulated by Two component system, PhoPQ regulon, which senses low Mg2+ in environment. It has been reported that  Hfq-binding sRNAs base pair with target RNAs, frequently leading to rapid degradation of target messages or, less frequently, to stabilization, both of which can be assayed by using microarrays.  In order to search for the target genes of MgrR, we therefore examined the consequences of MgrR expression on mRNA abundance under two conditions.  In condition 1, the chromosomal copy of mgrR was deleted and MgrR was expressed for 15’ from an induced plac-mgrR plasmid and compared to cells carrying a vector induced for the same period.  In condition 2, the expression of mRNAs was compared in wild-type cells (mgrR+) and the mgrR deletion strain, both grown in LB; because MgrR levels are fairly high under our normal growth conditions, this allowed analysis of both the direct and indirect (long-term) effects of MgrR.

overall_design: The strains used are either wild type which is derivatives of MG1655 or mgrR::kn knockout to restrict MgrR expression to the inducible pBR Plac-mgrR vector.  The mgrR knockout mutants carried  either pBR Plac-mgrR or the control vector pBR Plac.  A Fresh colony of bacteria was inoculated in LB media with ampicilline at final concentration of 100ug/ml at 37°C with agitation.  To induce MgrR expression, cultures carrying the pBR Plac-mgrR construct were grown to an O.D.600 of 0.5 and IPTG was added to the culture at a final concentration of 100uM.  Both cultures for wild type and mgrR deletion mutant were grown to O.D. 600 of 0.5 and cells were collected.   Total RNA was extracted from cells at the indicated time using the hot phenol procedure.

first: Kyung

last: Moon

first: Susan

last: Gottesman


title: Gene Expression in Inflammatory Diseases

city: Portland

first: James

last: Rosenbaum

state: OR

address: 3181 SW Sam Jackson Park Road

department: Casey Eye Institute

institute: Oregon Health & Science University

summary:
We hypothesized that patients with sarcoidosis have characteristic mRNA profiles. Microarray analysis of gene expression was done on peripheral blood. Comparing peripheral blood from patients with sarcoidosis to controls, 872 transcripts were upregulated and 1039 were downregulated at >1.5-fold change and a significant q value. Several transcripts associated with interferon and STAT1 were upregulated. Lung and lymph node analyses also showed dramatic increases in STAT1 and STAT1-regulated chemokines. Granulomas in lymph nodes of patients with sarcoidosis expressed abundant STAT1 and phosphorylated STAT1. STAT1 might play an important role in sarcoidosis. This novel hypothesis unites seemingly disparate observations with regard to sarcoidosis including implication of a casual role for interferons, a suspected infectious trigger, TH1 predominating lymphocytes in bronchoalveolar lavage, and the association with hypercalcemia.
This study tested the hypothesis that SpA is characterized by a distinct pattern of gene expression in peripheral blood of affected individuals compared with healthy controls.  High-density, human GeneChip® probe arrays were used to profile mRNA of peripheral blood cells from 18 subjects with SpA and 25 normal individuals. Samples were processed as two separate sets at different times. Blood samples were taken at a time when patients were not receiving systemic immunomodulatory therapy. Differential expression was defined as a 1.5-fold change with a q value <5%. Gene ontology and pathway information were also studied. Signals from 134 probe sets (representing 95 known and 12 unknown gene transcripts) were consistently different from controls in both Sets 1 and 2.  Included among these were transcripts for a group of 20 genes, such as interleukin-1 receptors 1 and 2, NLRP2, SLPI, SPARC, and TREM-1 that are clearly related to the immune or inflammatory response and a group of 4 transcripts that have a strong role in bone remodeling. Our observations are the first to implicate SPARC, SLPI, and NLRP2, a component of the innate immune system, in the pathogenesis of SpA. Our results also indicate a possible role for interleukin-1 and its receptors in SpA. In accord with the bone pathology component of SpA, we also found that expression levels of transcripts reflecting bone remodeling factors are also distinguishable in peripheral blood from patients with SpA versus controls.  These results confirm some previously identified biomarkers implicated in the pathogenesis of SpA and also point to novel mediators in this disease.

overall_design: Peripheral blood was collected from diseased and control subjects.  The sarcoidosis portion of the study included 12 patients and 12 control subjects.  The spondyloarthropathy study was done in two independent sets. The first set included 11 patients and the same 12 control subjects used for the sarcoidosis study.  The second set included 7 patients and 13 control subjects.  For each sample, total RNA was isolated and analyzed by hybridization on Affymetrix arrays.

middle: T

last: Planck

first: Justine

last: Smith

first: Christina

last: Harrington

first: Dongseok

last: Choi

first: Sirichai

last: Pasadhika

first: Srilakshmi

last: Sharma


status: Public on Nov 07 2009

city: Philadelphia

first: xin

last: wu

address: 3850 baltimore Ave,

department: Animal Biology

institute: School of Veterinary Medicine, University of Pennsylvania

summary:
Full title: Prepubertal Human Spermatogonia and Mouse Gonocytes Share Conserved Gene Expression of Germline Stem Cell Regulatory Molecules

Oligonucleotide Microarray Analysis Reveals Novel Genes Expressed in Prepubertal Human Spermatogonia and Mouse Gonocytes

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design:
Four hundred cells were selected for each group of germ cells or testis somatic cells from three independent testis cell preparations from both human (age 2, 8 and 10 yr) and mouse (age all 3 days)
Selected germ and somatic cells were placed in 25 µl Trizol reagent (Invitrogen) immediately after isolation
Oligonucleotide microarray was utilized to identify genes enriched in human and mouse selected germ cell populations and testis somatic cells.

first: Xin

last: Wu

first: Ralph

middle: L

last: Brinster


title: Expression profiling of breast cancer cell lines MCF-7 and MCF-7R4

city: Jacksonville

first: Yan

middle: W.

last: Asmann

state: FL

address: 4500 San Pablo Road South

institute: Mayo Clinic

summary: Human breast cancer cell line MCF-7 is usually sensitive to chemotherapy drug BMS-554417, an insulin receptor (IR) and insulin-like growth factor receptor (IGFR) inhibitor. However, through step-wise increase in BMS-554417 doses in culture media, we were able able to screen and select a single MCF-7 clone that is BMS-554417 resistant.  It is cross resistant to BMS-536924. This new line of MCF-7 cells was named as MCF-7R4. The transcriptome profiling of both MCF-7 and MCF-7R4 was performed using Affymetrix HG-U133 plus2.0 GeneChip arrays.

overall_design: Five replicates of MCF-7 and five replicates of MCF-7R4 were profiled.

first: Paul

last: Haluska

first: Xiaonan

last: Hou

first: Joan

last: Carboni

first: Karen

last: Flatten

middle: W

first: Cynthia

last: Eyck

first: Takeo

last: Nakanishi

first: Jennifer

middle: D

last: Tibodeau

first: Douglas

last: Ross

middle: H

last: Kaufmann

first: Marco

last: Gottardis

first: Charles

last: Erlichman


title: Broad defects in epidermal cornification in atopic dermatitis (AD) identified through genomic analysis

status: Public on Nov 01 2009

city: New York

first: Mayte

last: Suarez-Farinas

state: NY

address: 1425 Madison Ave, L2-70C, Box 1077,

institute: Mount SinaiSchool of Medicine

summary:
In this study we used genomic profiling to characterize differences in expression of genes related to epidermal growth/differentiation and inflammatory circuits in skin lesions of psoriasis and atopic dermatitis (AD), comparing expression values to normal skin. Skin biopsies were collected from 9 patients with chronic atopic dermatitis, 15 psoriasis patients, and 9 healthy volunteers.

Keywords: Genetic-pathology

overall_design: Psoriasis and AD are common inflammatory skin diseases which share important features, including: 1) large infiltrates of T-cells and inflammatory dendritic cells in skin lesions, 2) immune activation with up-regulated expression of many cytokines, chemokines, and inflammatory molecules 3) marked epidermal hyperplasia in chronic diseased skin and 4) defective barrier function with increased transepidermal water loss (TEWL), which reflects underlying alterations in keratinocyte differentiation. Using genomic profiling we provide a comprehensive comparison of chronic psoriasis and AD skin lesions as compared with normal skin.

last: Suárez-Fariñas

first: Emma

last: Guttman-Yassky

first: James

last: Krueger


title: Mucosal expression profiling in patients with inflammatory bowel disease before and after first infliximab treatment

status: Public on Nov 03 2009

city: Leuven

first: Ingrid

last: Arijs

address: Herestraat 49

department: Clinical and Experimental Medicine

institute: KULeuven

summary:
We used microarrays to identify mucosal gene signatures predictive of response to infliximab (IFX) in patients with inflammatory bowel disease (IBD) and to gain more insight into the pathogenesis of IBD.

Keywords: drug response and treatment effect

overall_design: Mucosal biopsies were obtained at endoscopy in actively inflamed mucosa from 61 IBD patients (24 ulcerative colitis (UC), 19 Crohn’s colitis (CDc) and 18 Crohn’s ileitis (CDi)), refractory to corticosteroids and/or immunosuppression, before and 4-6 weeks after (except for 1 CDc patient) their first infliximab infusion and in normal mucosa from 12 control patients (6 colon and 6 ileum). The patients were classified for response to infliximab based on endoscopic and histologic findings at 4-6 weeks after first infliximab treatment. Total RNA was isolated from intestinal mucosal biopsies, labelled and hybridized to Affymetrix Human Genome U133 Plus 2.0 Arrays.

first: Leentje

last: Van Lommel

first: Kristel

last: Van Steen

first: Gert

last: De Hertogh

first: Karel

last: Geboes

first: Frans

last: Schuit

last: Rutgeerts


title: Human iPS cells and fibroblasts

city: Baltimore

first: Akiko

last: Doi

state: MD

address: 855 N Wolfe St, Rangos 580

department: Medicine

institute: Johns Hopkins University School of Medicine

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

first: In-Hyun

last: Park

first: Bo

last: Wen

first: Peter

last: Murakami

first: George

middle: Q

last: Daley

first: Andrew

middle: P

last: Feinberg


title: Expression data from human iPS cells and fibroblasts

summary:
DNA methylation is often inversely correlated with gene expression.
We used expression microarrays to determine the relationship between DNA methylation and gene expression in 6 iPS cell lines and the fibroblasts from which they were derived.

overall_design: We isolated RNA from iPS cells and fibroblasts for which we have genome-wide DNA methylation data and hybridized to Affymetrix gene expression microarrays.


title: Therapeutic efficacy of human umbilical cord blood-derived mesenchymal stem cells in myocardial repair after infarction

status: Public on Oct 31 2009

city: Seoul

first: Hyo-Soo

last: Kim

address: 28 Yongon-dong, Chongno-gu

country: South Korea

institute: Seoul National University Hospital

summary:
Human UCB-MSCs showed donor-specific variation of therapeutic efficacy in improving LV systolic function, reducing infarct area, and preserving wall thickness after MI, even though there were no significant differences in MSC phenotypes. UCB-MSCs (M02) which showed better efficacy had better paracrine activity and unique gene expression profile than others.
In DNA microarray, in contrast to M01, M02 showed unique gene expression profiles; up-regulated anti-apoptotic and down-regulated apoptotic gene expression.

overall_design: To evaluate the correlation of gene expression profile and therapeutic efficacy of UCB-MSCs, we selected two UCB-MSCs (M01 and M02) which showed worst and best efficacy, respectively, in improving post-infarction LV remodeling. Under 3 days differentiation condition as described previously, we compared four groups of UCB-MSCs (naïve M01, differentiated M01, naïve M02 and differentiated M02). And, we examined the transcriptome profiles of undifferentiated and differentiated cells using Affymetrix Human Genome U133 Plus 2.0 Array covering more than 47,000 human transcripts.

first: Woong-Yang


title: A Genomic Approach to Improve Prognosis and Predict Therapeutic Response in Chronic Lymphocytic Leukemia

city: Durham

first: Daphne

last: Friedman

address: 101 Science Drive, Room 2121, CIEMAS Bldg

institute: Duke University Medical Center


title: A Genomic Approach to Improve Prognosis and Predict Therapeutic Response in Chronic Lymphocytic Leukemia (Duke_VA)

summary:
Chronic lymphocytic leukemia (CLL) is a heterogeneous malignancy, characterized by a variable clinical course. While clinical and laboratory parameters are increasingly being used to refine prognosis, they do not accurately predict response to commonly used therapy.  We used gene expression profiling to generate and further refine prognostic and predictive markers.  Genomic signatures that reflect progressive disease and responses to chemotherapy or chemo-immunotherapy were created using cancer cell lines and patient leukemia samples.  We validated these signatures using independent clinical data from four separate cohorts representing a total of 301 CLL patients.  A prognostic genomic signature created from patient leukemic cell gene expression data coupled with clinical parameters could statistically differentiate patients with stable or progressive disease in the training dataset.  The progression signature was then validated in two independent datasets, demonstrating a capacity to accurately identify patients at risk for progressive disease.  In addition, two distinct genomic signatures that predict response to chlorambucil or pentostatin, cyclophosphamide, and rituximab were also generated and were shown to accurately distinguish responding and non-responding CLL patients.  Microarray analysis of CLL patients’ lymphocytes can be used to refine prognosis and predict response to different therapies.  These results have direct implications for standard and investigational therapeutics in CLL patients.
Keywords: Gene Expression Profiling of two phenotypic states

overall_design: For the prognostic genomic signature, 68 CLL leukemia samples were used (36 from patients with stable disease and 32 from patients with progressive disease).

first: J-Brice

last: Weinberg

first: Neil

middle: E

last: Kay

first: Diane

last: Jelinek

first: Renee

last: Tschumper

first: John

last: Byrd

first: Alicia

last: Volkheimer

first: Karen

middle: M

last: Bond

first: Youwei

last: Chen

first: Ning

last: Jiang

first: Joseph

middle: O

last: Moore

first: Jon

last: Gockerman

first: Louis

last: Diehl

first: Carlos

last: Decastro

first: Anil

last: Potti

last: Nevins


title: Differentially expressed mRNA transcripts in the placenta delivered by preterm versus term spontaneous labour

first: Stephen

last: Chim

summary:
We hypothesized that preterm spontaneous labor involves aberrant changes in mRNA expression in the placenta. To test this hypothesis, we interrogated the mRNA levels of >50,000 genes and transcript variants using gene expression microarray (Human Genome U133 Plus 2.0 Array, Affymetrix) on 5 placentas collected from preterm spontaneous delivery (<34 weeks of gestation) and another 5 placentas collected from term spontaneous delivery (38-39 weeks).
We have identified 229 and 162 genes that were up- or down-regulated, respectively, for more than 3-fold in the preterm placentas compared to the term placentas (Mann-Whitney Rank Sum Test, with multiple testing correction by the Benjamini-Hochberg method, adjusted p-value <= 0.05).

overall_design: Placentas collected from (i) preterm spontaneous delivery (<34 weeks of gestation) and (ii) term spontaneous delivery (38-39 weeks of gestation) were subjected to RNA extraction and hybridization on Affymetrix microarrays. To identify gene expression patterns that are commonly involved in preterm spontaneous labour, we analyzed 5 placentas from each of these 2 groups and tested for any differentially expressed genes by Mann-Whitney Rank Sum Test.

first: Wing

last: Lee

first: Rossa

last: Chiu

first: Tak

last: Leung

first: Tze

middle: K

last: Lau

first: Yuk

last: Lo


title: The biological and molecular heterogeneity of breast cancers correlate with their cancer stem cell content

city: Milano

first: Stefano

last: Confalonieri

address: Via Adamello 16

country: Italy

institute: IFOM - The FIRC Institute for Molecular Oncology Foundation

summary: Pathways that govern normal stem cell (SC) function are often subverted in cancer. Here, we report the isolation to near purity of human normal mammary SC (hNMSCs), from cultured mammospheres, based on their ability to retain the lipophilic dye PKH26 as a consequence of their quiescent nature. We demonstrated that PKH26-positive cells possess all the characteristics of hNMSCs. The transcriptional profile of PKH26-positive cells (hNMSC signature) was able to predict biological and molecular features of breast cancers. By using markers of the hNMSC signature, we could prospectively isolate SCs from the normal gland and from breast tumors. Poorly-differentiated aggressive (G3) cancers displayed higher content of prospectively isolated cancer SCs, than well-differentiated less aggressive (G1) cancers. By comparing G3 and G1 tumors in xenotransplantation experiments, we directly demonstrated that G3s are enriched in cancer SCs. Our data support the notion that the heterogeneous phenotypical and molecular traits of human breast cancers are a function of their SC content.

overall_design: Epithelial cells, from reductive mammoplasties, labeled with PKH26 (Sigma, 10-7 M, 5 min) and subjected to FACS analysis using a FACS Vantage SE flow cytometer (Becton&Dickinson) to yield PKH-POS and PKH-NEG cells, were prepared for RNA extraction and hybridization on Affymetrix microarrays. Three independent pools of cells were used, and six Gene Expression Arrays were obtained.  No replicates are available.


title: Establishment of a novel model to assess the function of proteins under in vivo conditions

status: Public on Oct 27 2009

city: Zurich

first: Ursula

last: Quitterer

address: Winterthurerstrasse 190

department: Molecular Pharmacology

country: Switzerland

institute: ETH Zurich

summary: We established a novel model to assess the function of proteins under in vivo conditions. The model relies on the expansion of HEK293 cells in immunodeficient NOD.Scid mice. To validate the novel model, we performed microarray gene expression profiling of NOD.Scid-expanded HEK293 cells relative to conventionally cultivated cells. Microarray analysis revealed that cell expansion in NOD.Scid mice restored an imbalanced chaperone system without inducing a major upregulation of the entire protein folding machinery.

overall_design: Human embryonic kidney (HEK293) cells were injected subcutaneously into immunodeficient NOD.Scid mice. After three weeks, the expanded cell pellet was isolated, and total RNA was extracted. In parallel, total RNA was prepared from HEK293 cells cultivated in vitro under standard cell culture conditions in a commercially available medium containing 450 mg/dl glucose (DMEM). Microarray gene expression profiling was performed to determine differential gene expression between in vivo expanded and in vitro cultivated HEK293 cells. Two biological replicates for each condition were made (in vitro cultivated HEK293 cells: Cells-1 and Cells-2;  and NOD.Scid-expanded HEK293 cells: Scid-1 and Scid-2).

first: Joshua

last: Abd Alla

first: Kristin

last: Reeck

first: Thomas

last: Streichert

first: Said

last: AbdAlla


title: Comparison of human lower and upper entorhinal cortex layers gene expression

status: Public on Oct 23 2009

city: Campus de la Universidad Autónoma de Madrid

first: Ismael

last: Santa-Maria

state: Madrid

address: Nicolás Cabrera 1

department: Biologia Molecular

institute: Centro de Biologia Molecular Severo Ochoa

summary:
Specific vulnerability of neurons in the human entorhinal cortex has been associated with the onset of disease.
Gene expression is analyzed to define the molecular characteristic of those neurons.

overall_design: Human tissue collection and dissection.   Brain samples were collected from four individuals with no clinical evidence of neurological disease and no neuropathological evidence of neurodegeneration. Tissue samples were obtained from the neurological tissue bank (UIPA) and from the Neurological Research Tissue Bank (BTIN, Madrid). The mean postmortem interval (PMI) of the tissue was 6 h and each subject died in hospital due to either cardiac or infectious diseases. The tissue was obtained according to local ethical and legal regulations concerning the use of human post-mortem tissue for biomedical research.  Frozen tissue samples were collected from the entorhinal cortex [EC; Brodmann area (BA) 28], at coronal level 27 of the Atlas of Paxinos.   Tissue samples corresponded to either upper (CES), lower (CEI) or the entire EC (CET). Two adjacent vertical columns comprising the full thickness of the EC were dissected under magnification with a Leica M50 stereomicroscope. One of them was then divided into two blocks, corresponding to CES and CEI, while the remaining column was processed as CET.  RNA sample preparation.   Cerebral tissue was homogenised in liquid nitrogen with a pestle and mortar, and the total RNA was isolated using the RNeasy Mini Kit and QIAshredder (Qiagen). The total RNA concentration and purity were determined using an Agilent2100 Bioanalyzer (Agilent Biotechnologies, Palo, Alto, CA) and by agarose gel electrophoresis.   Subsequently, cDNA was synthesized using the One-Cycle cDNA Synthesis kit (Affymetrix), according to the protocol described in the Expression Analysis Technical Manual. Biotinylated cRNA probes were generated from each cDNA sample following the IVT Labeling kit instructions (Affymetrix), and the cRNA synthesized was purified with the GeneChip Sample Cleanup Module (Affymetrix). The concentration and purity of the biotinylated cRNA was determined using an Agilent2100 Bioanalyzer (Agilent Biotechnologies, Palo, Alto, CA) and by agarose gel electrophoresis.

first: I

first: J

last: Avila

first: A

last: Rabano


title: UV- or oxidized lipid treated dermal skin fibroblasts

status: Public on Oct 22 2009

city: Vienna

first: Martin

last: Bilban

country: Austria

institute: Medical University of Vienna

summary:
Long wavelength Ultraviolet (UVA-1) radiation causes oxidative stress that leads to the formation of noxious substances within the skin. As a defensive mechanism skin cells produce detoxifying enzymes and antioxidants when they detect modified molecules. We have recently shown that UVA-1 irradiation oxidizes the abundant membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), which then induced the synthesis of the stress response protein heme oxygenase 1 (HO-1) in dermal fibroblasts. Here we examined the effects of UVA-1 and (UV-) oxidized phospholipids on the global gene expression in human dermal fibroblasts. We identified a cluster of genes that were co-induced by UVA-1-oxidized PAPC and UVA-1 radiation. The cluster included HO-1, glutamate-cysteine ligase modifier subunit (GCLM), aldo-keto reductases-1-C1 and -C2 (AKR1C1, AKR1C2), and interleukin 8 (IL8). These genes are members of the cellular stress response system termed “antioxidant response” or “Phase II detoxification”. Accordingly, the regulatory regions of all these genes contain binding sites for NF-E2-related factor 2 (Nrf2), a major regulator of the antioxidant response.
 Both UVA-1 irradiation and treatment with oxidized lipids led to increased nuclear accumulation of Nrf2. Silencing expression of Nrf2 using siRNA or using cells and tissue from Nrf2-deficient mice, we show that the induction of the co-regulated genes was suppressed. Expression of other canonical UVA-1-induced genes, including cyclooxygenase 2 (Cox2) and interleukin 6 (IL6) was unaltered in the absence of Nrf2. Together, our data show that UVA-1-mediated lipid oxidation induces induction of antioxidant response genes, which is dependent on the redox-regulated transcription factor Nrf2. To activate Nrf2 is a major strategy for novel antioxidant drugs, the skin photo-adaptation (SPA) inducers. Our finding that specific uv-oxidized lipids act similar sheds a new (ultraviolet) light on the usually detrimental “image” of UV generated lipid mediators.

overall_design: we profiled global mRNA expression levels in human dermal fibroblasts that had been treated with either UVA-1 or oxidized lipids. To investigate the effect of oxidized phospholipids on gene regulation, we used two  preparations, which differed in their degree of oxidation; the minimally oxidized UV-PAPC resulting from UVA-1 irradiation of PAPC, and air-oxidized PAPC (OxPAPC), which represents the full spectrum of oxidation products (Gruber 07) (Reis et al., 2005). We irradiated dermal fibroblasts with UVA-1 (40J/cm²) or treated them with UV-PAPC, OxPAPC or native PAPC (100µg/ml each). We analyzed global gene expression four hours after stimulation with gene arrays (Affymetrix U133A Plus 2.0 Gene Chips).

first: Florian

last: Gruber

first: Erwin

last: Tschachler


title: Effect of mir-210 overexpression or down-modulation on human umbilical vein cells

city: rotterdam

first: Mario

last: Pescatori

address: Westplein 11

country: Netherlands

institute: Health e-genomics

summary: MicroRNAs (miRNAs) are small non-protein-coding RNAs that are incorporated into the RNA-induced silencing complex (RISC) and inhibit gene expression by regulating the stability and/or the translational efficiency of target mRNAs. Previously, we demonstrated that miR-210 is a key player of endothelial cell (EC) response to hypoxia, modulating EC survival, migration and ability to form capillary like-structures. Moreover, the receptor tyrosine kinase ligand Ephrin-A3 was identified as one functionally relevant target. Since each miRNA regulates hundreds of mRNAs, different approaches were combined to identify new miR-210 targets: a Using target prediction software, 32 new miR-210 potential targets were identified. b The proteomic profiling of miR-210 over-expressing ECs identified 11 proteins that were specifically inhibited by miR-210, either directly or indirectly. c Affymetrix based gene expression profiles identified 51 genes that were both down-modulated by miR-210 over-expression and de-repressed when miR-210 was blocked. Surprisingly, only few genes identified either by proteomics or transcriptomics were recognized as miR-210 targets by target prediction algorithms. However, a low-stringency pairing research revealed enrichment for miR-210 putative binding sites, raising the possibility that these genes were targeted via non-canonical recognition sequences. To clarify this issue, miR-210-loaded RISC was purified by immuno-precipitation along with its mRNA targets. The presence of Ephrin-A3 mRNA in the complex validated this approach. We found that 32 potential targets were indeed enriched in miR-210-loaded RISC, and thus can be considered as genuine miR-210 targets. In keeping with this conclusion, we were able to further validate a sub-set of them by 3’UTR-reporter assays. Gene ontology analysis of the targets confirmed the known miR-210 activity in differentiation and cell cycle regulation, highlighting new functions such as involvement in RNA processing, DNA binding, development, membrane trafficking and amino acid catabolism.  In conclusion, we validated a multidisciplinary approach for miRNAs target identification and indicated novel molecular mechanisms underpinning miR-210 role in EC response to hypoxia.

overall_design: Gene expression modifications induced by both miR-210 over-expression and blockade were evaluated. In order to identify new direct and indirect miR-210 targets, transcripts repressed by miR-210 over-expression and up-regulated by miR-210 inhibition (and vice versa) were selected.

first: Pasquale

last: Fasanaro

first: Maurizio

middle: C

last: Capogrossi

first: Fabio

last: Martelli


title: Comparison of lens gene expression between the control and DicerCN mouse embryos at E13.5

status: Public on Oct 28 2009

city: Bethesda

first: Yan

last: Li

address: 5625 Fishers Lane, Room 1S-02

institute: NIH\NEI

summary: In order to investigate the mechanism for the progressive lens degeneration caused by targeted deletion of Dicer, we compared expression profiles of protein-coding genes in wild type and DicerCN lenses at E13.5, at a time before gross morphological changes had occurred.  We identified distinct classes of differentially expressed genes in the conditional knockout lenses.

overall_design: Intact lenses were dissected from five control and five DicerCN mice at E13.5 for RNA extraction and hybridization on Affymetrix microarrays.

first: Joram

last: Piatigorsky


title: Molecular profiling of the of conjunctival epithelial side population cells

status: Public on Oct 30 2009

first: J. Mario

last: Wolosin

address: One Gustave levy Pl

department: Ophthalmology

institute: Mount Siani School of Medicine

summary:
Side population (SP) cells isolated from limbal and conjunctival epithelia derive from cells that are slow cycling in vivo, a known feature of tissue stem cells. The purpose of this study was to define the molecular signature of the conjunctival side population cells by global differential gene expression to identify markers and signaling pathways associated with this cell phenotype.
Four overnight cultures of freshly isolated human conjunctival epithelial cells stained with Hoechst 3342 were cytometrically sorted into SP and non-SP cohorts. RNA was isolated and processed for microarray analysis using a commercial oligonucleotide array representing more than 55,000 transcripts derived from about 30,000 different genes.  Selected microarray results were validated at the gene and protein levels by quantitative PCR, and immunological methods. Data mining methods were used to identify cellular processes relevant for stem cell function.
Comparative analysis of transcripts expression based on expression levels and present/absent software calls across 4 replicate experiments identified 16993 expressed conjunctival epithelial transcripts including 10,266 unique known genes. Of those genes, 1254 and 363 were over expressed (> 2-fold ) or under expressed (< 0.5-fold), respectively, in the SP.  The overexpressed set included genes coding for non-epithelial genes (e.g.,  CD62E/E-selectin and CD93), genes that have been associated with stem cell function in other cellular systems, including several homeodomain genes and genes whose over- or under-expression may underpin the stem cell slow cycling phenotype  ( e.g., dual specificity phosphatases and cyclin kinases).

overall_design:
Whole human conjunctivae from unidentifiable cadaver donors, aged 55 to 65, were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA) within 48 hours of collection. No donor details apart from age, sex, and cause of death were released.  The use of human tissue in the study was in accordance with the provisions of the Declaration of Helsinki and sanctioned by the Institutional Review Board.  Fresh rabbit tissue was obtained from local abattoirs within one hour of sacrifice. Unless stated otherwise all reagents were procured from Sigma (St. Louis, Mo).

Eight microarray experiments using side population (Hoechst 33342-transporting cells; SP) or non side population (Hoechst 33342-stained cells; nSP) cells from four cadavers.

first: M

middle: A

last: Akinci

first: Helen

last: Turner

first: Maria

last: Taveras


title: SKPs derive from hair follicle precursors and exhibit properties of adult dermal stem cells

city: Toronto

first: Freda

last: Miller

state: ON

address: 555 University Avenue

institute: Hospital for Sick Children

summary:
Global expression analysis of neural crest-like skin-derived precursors (SKPs) and Sox2-positive follicle dermal cells that SKPs originate from.
In spite of the remarkable regenerative capacity of mammalian skin, an adult dermal stem cell has not yet been identified. Here, we provide evidence that SKPs, multipotent neural crest-like skin-derived precursors, represent an adult dermal stem cell. When transplanted into adult skin, SKPs can reconstitute the adult dermis, contribute to dermal wound-healing, home to a hair follicle niche, and instruct epidermal cells to make hair follicles. Hair follicle-derived SKPs self-renew, maintain their multipotency, and serially reconstitute hair follicles. The endogenous origin of SKPs are Sox2-positive follicle dermal cells that share a similar global gene expression profile with SKPs. These endogenous cells home back to their follicle niche, induce hair follicle morphogenesis, and differentiate into neural and dermal progeny. Hair follicle-associated dermal cells will move out of their niche to contribute to dermal maintenance and wound-healing. These studies therefore identify a dermal stem cell, and provide a biological rationale for the presence of a multipotent precursor within adult dermis, findings with important therapeutic implications.

overall_design: RNA was isolated from 4 independent neonatal backskin primary passage SKP preparations and from 5 independent neonatal Sox2:EGFP-positive, CD34-negative backskin cell isolates prepared in this way. These RNA samples were analyzed on Affymetrix GeneChip Mouse Gene 1.0 ST Arrays. No technical replicates were performed.

first: Jeffrey

last: Biernaskie

first: Maryline

last: Paris

first: Olena

last: Morozova

first: B

last: Fagan

first: Marco

last: Marra

first: Larysa

last: Pevny


title: Knockdown of transactive response DNA-binding protein TDP-43 downregulates histone deacetylase 6

status: Public on Oct 20 2009

city: Tuebingen

first: Michael

middle: H.

last: Walter

address: Calwer Str. 7

department: Department of Medical Genetics

institute: University of Tuebingen

summary: TDP-43 is an RNA/DNA-binding protein implicated in transcriptional repression and  mRNA processing. Inclusions of TDP-43 are hallmarks of frontotemporal dementias  and amyotrophic lateral sclerosis. Besides aggregation of TDP-43, loss of nuclear  localization is observed in disease. To identify relevant targets of TDP-43, we performed  an expression profiling study. Thereby, histone deacetylase 6 (HDAC6) downregulation  was discovered upon TDP-43 silencing on mRNA and protein level in human embryonic  kidney HEK293E and neuronal SH-SY5Y cells. This was accompanied by accumulation  of the major HDAC6 substrate, acetyl-tubulin. Expression of wild-type but neither  RNA-binding- nor nuclear-localization-deficient TDP-43 restored HDAC6 expression.  Moreover, TDP-43 bound specifically to HDAC6 mRNA arguing for a direct functional  interaction. Importantly, in vivo validation in TDP-43 knockout Drosophila melanogaster  also showed HDAC6 mRNA decrease. HDAC6 is necessary for protein aggregate  formation and degradation. Indeed, downregulation of HDAC6 reduced aggregate  formation and increased cytotoxicity of expanded poly-glutamine ataxin-3 in TDP-43  silenced cells. This was completely restored by co-transfection with HDAC6. In  conclusion, loss of functional TDP-43 causes HDAC6 downregulation and might thereby  contribute to pathogenesis.

overall_design: Comparison of TDP-43 knockdown in HEK293E cells against cells transfected with scrambled siRNA. 4 independend transfections per siRNA were used.

first: Fabienne

last: Fiesel

first: Marlene

last: Anderson

first: Chris

last: Van den Haute

first: Aaron

last: Voigt

first: Thorsten

last: Schmidt

first: Manuela

last: Neumann

first: Susann

last: Klein

first: Wolfdieter

last: Springer

first: Jörg

middle: B

last: Schulz

last: Bonin

first: Veerle

last: Baekelandt

first: Philipp

middle: J

last: Kahle


title: E. coli gene expression following CHIR-090 treatment

city: Memphis

first: Matthew

middle: Wilmer

last: Frank

state: TN

address: 262 Danny Thomas Place

department: Infectious Diseases

institute: St Jude Children's Research Hospital

summary:
CHIR-090 is an inhibitor of lipid A biosytnesis. CHIR-090 treatment on E. coli sinificantly increased the expression of fatty acid biosythesis gene fabA and fabB.

Keywords: Inhibition response

overall_design: The lipid A biosynthesis in E. coli was inhibited by CHIR-090, and the RNA was extracted after 10 minutes treatment to capture the effect of increased saturated fatty acid on global gene expression.

first: Kun

last: Zhu

first: Yong-Mei

last: Zhang

first: Karen

last: Miller

first: Charles

last: Rock


title: DEHP activation of PPAR(alpha) and CAR regulartory pathway in mouse liver

city: RTP

first: Hongzu

last: Ren

address: 109 TW Alexander Dr

institute: US Environmental Protection Agency

summary:
Characterization of Peroxisome Proliferator-Activated Receptor alpha (PPAR(alpha)) - Independent Effects of PPAR(alpha) Activators in the Rodent Liver: Di-(2-ethylhexyl) phthalate Activates the Constitutive Activated Receptor
data files in this series indicate the involvement of PPAR(alpha) and CAR regulatory pathway after DEHP treatment.

Keywords: gene expression/microarray

overall_design: Wild-type and PPAR(alpha)-null mice were treated with DEHP for 4 days and liver tissues were collected for microarray assay.

first: Lauren

last: Aleksunes

first: Carmen

last: Wood

first: Beena

last: Vallanat

first: Michael

last: George

first: Curtis

last: Klaassen

first: J

last: Corton


title: Expression data from ependymal layer of wild type and FoxJ1 null postnatal male mice

status: Public on Oct 21 2009

city: Raleigh

first: Troy

last: Ghashghaei

address: 4700 Hillsborough Street

department: Molecular Biomedical Sciences

institute: North Carolina State University

summary:
FoxJ1 dependent gene expression is required for establishment of ependymal cells in the postnatal brain.  This data set compares gene expression profiles of wildtype and FoxJ1 null microdissected dissected tissues at multiple postnatal time points.
We used microarrays to detail the FoxJ1-dependent programme of gene expression underlying ependymal cell differentiation in the mouse brain.

overall_design: Using FoxJ1-EGFP brains, EGFP(+) domains were microdissected using laser capture microscopy from P1, P7 and P14 wildtype and FoxJ1 null brains.

first: H

middle: T


title: A gene signature predictive for outcome in advanced ovarian cancer identifies a novel survival factor: MAGP2

status: Public on Oct 17 2009

city: Boston

last: Birrer

address: 70 Blossom Street

institute: MGH

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

overall_design: Refer to individual Series


title: Whole-genome oligonucleotide expression analysis of papillary serous ovarian adenocarcinomas

summary:
To demonstrate the use of a whole-genome oligonucleotide array to perform expression profiling on a series of microdissected late-stage, high-grade papillary serous ovarian adenocarcinomas to establish a prognostic gene signature correlating with survival and to identify novel survival factors in ovarian cancer.
Advanced stage papillary serous tumors of the ovary are responsible for the majority of ovarian cancer deaths, yet the molecular determinants modulating patient survival are poorly characterized. We identify and validate a prognostic gene expression signature correlating with survival in a series of microdissected serous ovarian tumors.

overall_design: We identified 53 advanced stage, high-grade primary tumor specimens and 10 normal ovarian surface epithelium (OSE) brushings.


status: Public on Oct 15 2009

first: Celia

last: Longhurst

address: 956 Court Avenue Room H300

institute: UTHSC

summary: Expression of the TM4SF member CD9 on the human Burkitts lymphoma cell line Raji induced increased cell proliferation, motilty and adhesion to fibronectin. CD9 promoted increases in Raji cell proliferation was dependent upon histone deacetyalase (HDAC) activity as treatment with HDAC inhibitors trichostain A or cucurmin attenuated CD9 mediated increases in Raji cell proliferation. Gene expression of Raji cells stably expressing human CD9 via transfection with expression vector PRVCMVCD9 was compared with corresponding Mock transfected cell by microarray analysis using the Affymetrix U133 2.0 platform.

overall_design: Transfected Raji cells that had been in cultured in RPMI, 10% FBS in the presence of 1mg/ml G418 were harvested during exponential growth and RNA was extracted using TRiReagent according to manufacturer's protocol (Sigma).

first: Lisa

last: Jennings


title: Natural compound screening

status: Public on Oct 05 2009

city: Uppsala

first: Mårten

last: Fryknäs

address: Akademiska sjh ingång 61

department: Medical sciencies

country: Sweden

institute: Uppsala university

summary:
Transcriptional expression data for bioactive small molecules for mechanism identification.

Keywords: Expression profiling by array

overall_design: MCF7 cells were treated with either NSC76022 (thaspine) or DMSO control for six hours, to identify compound specific gene regulation.

first: Marten

last: Fryknas


title: Hi-C Expression Data

status: Public on Oct 01 2009

city: Cambridge

first: Erez

last: Lieberman-Aiden

address: 7 Cambridge Center

institute: Broad Institute

summary:
We describe Hi-C, a method that probes the three-dimensional architecture of whole genomes by coupling proximity-based ligation with massively parallel sequencing. We constructed spatial proximity maps of the human genome with Hi-C at a resolution of 1Mb. These maps confirm the presence of chromosome territories and the spatial proximity of small, gene-rich chromosomes.  We identified an additional level of genome organization that is characterized by the spatial segregation of open and closed chromatin to form two genome-wide compartments.  At the megabase scale, the chromatin conformation is consistent with a fractal globule, a knot-free conformation that enables maximally dense packing while preserving the ability to easily fold and unfold any genomic locus. The fractal globule is distinct from the more commonly used globular equilibrium model. Our results demonstrate the power of Hi-C to map the dynamic conformations of whole genomes.

These expression data are used in the paper to show that there are marked differences in mRNA expression between loci in the open and closed chromatin compartments.

overall_design: Affy Expression array for GM06690 cells

first: Nynke

last: Van Berkum


title: Expression data from Human MicroVascular Endothelial Cells (HMVECS)

status: Public on Oct 13 2009

city: Zürich

first: Lubor

last: Borsig

department: Institute of Physiology

institute: University of Zürich

summary:
The activation of endothelium by tumor cells is one of the main steps by tumor metastasis. The role of the blood components (platelets and leukocytes) in this process remain unclear.
We used microarrays to detail the repsonse of endothelial cells to tumor cells in the presence or absence of blood components.

overall_design: HMVECs were cultured to 90% confluency. Tumor cells (LS180 or HT-29) in the presence or in the absence of blood components were co-cultured for 8 h with HMVECs. After the purfication of HMVECS by MACS sorting RNA was isolated.

first: Heinz

last: Läubli


title: Expression profiles of  beta-hematin (BH)- or 4-hydroxy-2-nonenal (HNE)-treated RAW 264.7 cells

status: Public on Oct 16 2009

city: Nashville

first: David

last: Wright

address: SC Stevenson Center 7, Station B 351822

department: Chemistry

institute: Vanderbilt University

summary:
Hemozoin phagocytosis results in immunomodulation. In the present study, gene expression profiles were analyzed to identify transcriptional changes associated with two of the individual components of Hz in a model macrophage cell line. LPS-stimulated RAW 264.7 cells were exposed to the synthetic form of Hz, β-hematin (BH, 0.1 mg/mL) and HNE (35 μM) for 6 or 24 h.

Keywords: variable treatment

overall_design: Three biological replicates were analyzed for each 3 treatment types (HNE, BH, bead) in LPS stimulated cells in addition to 3 replicates of untreated LPS stimulated cells (control). Each replicate was composed of 6 pooled wells from a 6-well plate. In order to identify transcriptional changes dependent on interactions of BH rather than those due to phagocytosis, differentially expressed mRNAs were controlled by a corresponding particulate latex bead challenge.

first: Alexandra

last: Schrimpe