title: Efficacy of anti-TNF biologic agent, infliximab, for RA patients using transcriptome analysis of white blood cells

status: Public on Mar 09 2010

city: Yokohama

first: Motohiko

last: Tanino

state: Kanagawa

address: 1-1-43, Suehirocho, Tsurumi-ku

institute: DNA Chip Research Inc.

summary: Introduction of biologics, such as infliximab, to the therapy of rheumatoid arthritis (RA) patients has revolutionized the treatment of this disease. However, biomarkers for predicting the efficacy of the drug at an early phase of treatment for selecting real responders have not been found. We here present predictive markers based on a thorough transcriptome analysis of white blood cells from RA patients. RNA from whole blood cells of consecutive 42 patients before the first infusion was analyzed with microarrays for training studies. Samples from the subsequent 26 consecutive patients were used for a prospective study. We categorized the results into no inflammation and residual inflammation groups using the serum C-reactive protein (CRP) level at 14 weeks after the first infusion. The accuracy of prediction in our study was 65.4%.

overall_design: Forty-two and 26 consecutive RA patients followed at Saitama Medical Center, for retrospective and verification studies, respectively, who were resistant to standard MTX treatment, were enrolled. The serum CRP level was used for the definition of the level of inflammation of each patient, 0.3 mg/dl or less as “no inflammation” (NI), and more than 0.3 mg/dl as “residual inflammation” (RI) at 14 weeks after the first treatment with infliximab infusion. Differentially expressed genes between the NI and RI patients in the retrospective/training set were extracted, and these genes were verified using the verification set.

first: Ryo

last: Matoba

first: Seiji

last: Nakamura

first: Hideto

last: Kameda

first: Koichi

last: Amano

first: Toshitsugu

last: Okayama

first: Hayato

last: Nagasawa

first: Katsuya

last: Suzuki

first: Kenichi

last: Matsubara

first: Tsutomu

last: Takeuchi


title: MRC1-Dependent Scaling of the Budding Yeast DNA Replication Timing Program

status: Public on Mar 04 2010

city: Boston

first: Amnon

last: Koren

state: MA

address: 77 Avenue Louis Pasteur

department: Genetics

country: USA

institute: Harvard Medical School

type: Genome binding/occupancy profiling by genome tiling array

summary: Fourteen yeast mutants with an extended S-phase were identified by a novel genome-wide screen. These mutants are associated with the DNA replication machinery, cell-cycle control and dNTP synthesis. We determined the genome-wide DNA replication timing profile of all these mutants as well as wild type, by FACS-sorting G1- and S-phase cells and co-hybridizing their DNA to Agilent genomic tiling arrays, in four repeats each. We find that MRC1 is required for scaling of the DNA replication timing program upon replication perturbation.

overall_design: Four repeats for each strain, S vs G1 phase-cell DNA with different Cy dyes for each


title: Expression data from mouse embryonic stem cell differentiation experiment

status: Public on Feb 25 2010

city: Champaign

first: Feng

last: Hong

state: IL

address: 101 Illini Hall, MC-374, 725 South Wright Street

department: Statistics

institute: University of Illinois at Urbana Champaign

summary:
We differentiated mouse embryonic stem (mES) cells spontaneously into embryoid bodies (EBs). Gene expression of biological replicates of undifferentiated ES cells (0-day), 4-day, 8-day and 14-day EBs were measured by Affymetrix microarrays.

Keywords: time course

overall_design: Mouse embroynic stem cells were spontaneously into EBs. The gene expression was measured on undifferentiated mES cells on gelatin (0-day), undifferentiated mES cells sorted by FACS on Oct4 GFP-day (0-day), 4-day, 8-day and 14-day EBs.

first: Hong

last: Feng


title: Adrenal gland gene expression from chromogranin A knockout mice (Mahapatra et al. 2005) vs. wild-type mice

status: Public on Mar 03 2010

city: La Jolla

first: Ryan

last: Friese

state: CA

institute: University of California San Diego

summary: The objective of the experiment is to determine the genes differentially expressed in the adrenal gland of the chromogranin A knockout mouse (Mahapatra et al., 2005).

overall_design: Adrenal glands from chromogranin A knockout mice (n=3) and wild-type control mice (n=3).

middle: S


title: Saccharomyces cerevisiae GINS complex progression (Psf2) determined ChIP-chip time course through the cell cycle

status: Public on Mar 01 2010

city: Seattle

first: John

last: Aitchison

state: WA

institute: Institute for Systems Biology

summary:
Time points from two separate experiments – a) every 15 min after release from the G1 block from 0 min – 105 min and b) every 5 min beginning 20 min after block (through S-phase) from 20 -50 min.
Data include composite of normalized high and low scan intensities

overall_design: IP samples versus genomic DNA on 4x44k whole genome microarrays (Agilent) with average spatial resolution of ~290 nt

first: Matthew

middle: D

last: Sekdat

first: David

last: Fenyo

first: Richard

last: Rogers

first: Alan

middle: J

last: Tackett

first: Brian

middle: T

last: Chait


title: Human keratinocytes exposed to tungsten carbide and tungsten carbide cobalt nanoparticles and cobalt chloride

status: Public on Feb 01 2010

city: Leipzig

first: Wibke

last: Busch

address: Permoser Str. 15

department: Bioanalytical Ecotoxicology

country: Germany

institute: UFZ Helmholtz Centre for Environmental Research

summary: Comparison of transcriptional profiles after exposure of HaCaT cells to WC or WC-Co nanoparticles and respective amount of free cobalt in form of CoCl2 after 3 hours and 3 days of exposure. Genes responding especially to nanoparticles or leached cobalt ions should be determined.

overall_design: Common refernce: pool of 80 RNA Samples of HaCaT cells (treated and not treated), Cy5 labeled. Samples: RNA of HaCaT cells treated 3 hours and 3 days with 30 µg/ml WC nanoparticles, or 33 µg/ml WC-Co nanoparticles (3 µg/ml cobalt content), or 51 µM CoCl2 (equals 3 µg/ml), or water as control, 5 biological replicates for each treatment, all samples were labeled with Cy3 and hybridised together with the common reference.


title: Expression data from cumulus cells from C57Bl/6 and Ptger2 (EP2) KO mice

status: Public on Feb 20 2010

city: Tokyo

first: Eri

last: Segi-Nishida

state: Tokyo

address: Niijuku 6-3-1

country: Japan

institute: Tokyo University of Science

summary: To understand the role of prostaglandin (PG) receptor EP2 (Ptger2) signaling in ovulation and fertilization, we investigated time-dependent expression profiles in wild-type (WT) and Ptger2-/- cumuli before and after ovulation by using microarrays.

overall_design: We used Affymetrix U74A ver2 microarrays to investigate gonadotropin-induced gene expression profiles in WT and EP2KO cumulus cells before and after ovulation. Immature mice were primed with pregnant mare serum gonadotropin (PMSG) as an FSH-like stimulation. The cumulus-oocyte complexes (COCs) were isolated from mice 48 h after PMSG injection (H0) and from PMSG-primed mice exposed to human chorionic gonadotropin (hCG) as an LH-like stimulation for 3, 9 or 14 h (H3, H9 or H14, respectively).

first: Shigero

last: Tamba

first: Reiko

last: Yodoi

first: Kazushi

last: Morimoto

first: Tomoaki

last: Inazumi

first: Yasushi

last: Okuno

first: Yukihiko

last: Sugimoto


title: Differences in gene expression in islets isolated from pancreata donated after Cardiac Death relative to Brain Death

status: Public on Feb 09 2010

city: Madison

first: Jamie

middle: M

last: Sperger

state: WI

address: 1111 Highland Ave.  RM 5168

department: Department of Surgery

institute: University of Wisconsin - Madison

summary:
The decreasing numbers of Donation after Brain Death (DBD) donors necessitates the comprehensive evaluation of Donation after Cardiac Death Donors (DCD) as a source of pancreata.  The aim of this study was to characterize pancreata and islets from DCD and DBD donors with respect to markers of cellular stress that may indicate compromised islet quality.   Immunohistochemical staining of pre-isolation pancreas biopsies found increased numbers of caspase 3 positive islets in DBD, while markers of oxidative stress (nitrotyrosine, CML, and HNE) were elevated in DCD.  Assessment of islet quality by standard (yield, morphology, fluorescence microscopy, and glucose stimulated insulin secretion) and novel methods (flow cytometry, HPLC quantification of ATP) did not reveal significant differences.  However, the post culture loss of DCD islets was increased compared to DBD, and DCD islets showed delayed functional potency when transplanted into diabetic NOD.scid mice.  Microarray analysis of cultured islets showed increased expression of multiple stress pathway related genes in DCD compared to DBD.  Together these data indicate that the current standard donor management, pancreas recovery and preservation practices are insufficient to quench the oxidative stress injury suffered by DCD islets which leads to loss in culture and may complicate their use in clinical transplant.

Keywords: cell type comparison

overall_design: In the study presented here, 10 islet preparations isolated from Donation after Brain Death pancreata were compared to 9 sets of islet preparations isolated from Donation after Cardiac Death pancreata.  All islet preparations had cold ischemia times less than 11 hours.

first: Jonathan

last: Schug

first: Peter

last: White

first: Klaus

last: Kaestner

first: Luis

middle: A

last: Fernandez


title: Gene Expression Profiling of Airway and Distal Lung Fibroblasts

city: Pittsburgh

first: Kazuhisa

last: Konishi

state: PA

address: 1212 Kaufmann Building  3471 Fifth Ave

department: Pulmonry, Allergy, and Critical Care Medicine

institute: University of Pittsburgh

summary: Fresh lung tissue specimens from  distal lungs (n=3) and  airways (n=3) were obtained through bronchoscopy, and primary fibroblasts were isolated and cultured.  IRB approval was obtained from the University of Pittsburgh Institutional Review Board.

overall_design: Total RNA was isolated from lung tissue using Qiazol (QIAGEN) and purified using the miRNeasy Mini kit (QIAGEN). RNA quantity and quality was assessed using Agilent Nanodrop and Agilent 2100 Bioanalyzer, respectively. 500 ng of total RNA meeting was Cy3 labeled using the Agilent Low RNA Input Linear Amplification Kit PLUS (One-Color). After purification and fragmentation, aliquots of each sample were hybridized to an Agilent Whole Human Genome 4 X 44K microarray, sequentially washed, and scanned on an Agilent DNA microarray scanner.

first: Xiuxia

last: Zhou

first: Jadranka

last: Milosevic

first: Naftali

last: Kaminski

first: Sally

last: Wenzel


title: Circulating endothelial cells in patients with chronic lymphocytic leukemia: clinical and biological characterization

city: Modena

first: Rossana

last: Maffei

state: Modena

address: Via del Pozzo 71

department: Dept of Hematology and Oncology

country: Italy

institute: University of Modena and Reggio Emilia

summary: In cancer patients, circulating endothelial cells (CEC) are increased and correlate with an aggressive course of the disease. However the clinical and biologic significance of CEC in chronic lymphocytic leukemia (CLL) is still uncertain. In this study we show that, in series of 171 CLL patients, CEC are increased in comparison to normal age and sex related controls. A higher level of CEC (> 20 ul) identifies a subset of patients with a more aggressive course of the disease characterized by a shorter time to first treatment both in univariate and multivariate analyses. CEC levels do not correlate with known markers of aggressiveness including stage, CD38 and ZAP70 positivity, and cytogenetics. By FISH analysis, in 7 cases a significant proportion of CEC presented the same cytogenetic lesion of neoplastic lymphocytes and immunophenotypic features of endothelial progenitor cells. The gene expression profile of sorted CEC showed a molecular pattern suggesting a derivation from CLL leukemic cells with increased cell survival and proliferation, diminished cell adhesion to extracellular matrix and enhanced pro-angiogenic function compared to their normal counterpart. These data suggest that in CLL, CEC are tumor-derived and may represent a new biologic marker of aggressiveness playing a role in disease evolution.

overall_design:
Large-scale GEP was performed on 37 samples including 12 CEC, 12 CD19+, 10 CD14+ cells purified from CLL patients as well as 2 CEC and 1 CD19+ samples collected from normal controls. Total RNAs were isolated from all samples using RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). Then, high quality RNAs were amplified and Cy5-labeled using Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Palo alto, CA, USA). Universal Human Reference RNA (UHR) (Stratagene, Cedar Creek, TX), consisted of equal amount of total RNA from 10 human cancer cell lines, was used as reference control in all microarray gene-profiling experiments. Amplified reference cRNA was labelled with cyanine 3-CTP (Agilent Technologies) in each experiment. Agilent RNA Two-Color Spike-In was added in each sample to provide positive controls for monitoring the microarray workflow from sample amplification and labelling to microarray processing. It contains 10 in vitro synthesized, polyadenylated transcripts derived from the Adenovirus E1A transcriptome that are premixed at various ratios. All cRNA products were purified using RNeasy columns (QIAGEN). Samples had to contain at least 10-15 pmole of cyanine dye/ug of cRNA to be considered suitable for subsequent hybridization. 825 ng of Cy5-labelled cRNA were mixed with the same amount of Cy3-labeled reference cRNA and then cRNA mixtures were fragmented to an average size of 50-100 nt by incubation at 60°C for 30 min using in situ Hybridization kit-plus (Agilent Technologies). Samples were hybridized for 17 h at 65°C on 4X44K Whole Human Genome Microarray (Agilent Technologies), comprising over 33’000 (60-mer) experimentally validated oligonucleotide probes and then scanned using a confocal laser scanner (Agilent Technologies).

Gene expression analyses
Fluorescence data were analyzed with Feature Extraction Software v.9.1 (Agilent Technologies). Result data from each scan (Log10 Cy5/Cy3) were imported into the gene expression analysis software Luminator (Rosetta Bio software, Seattle, WA, USA). Two-dimensional clustering analysis was performed using an agglomerative algorithm with an average link heuristics and a correlation with mean subtraction.The identification of genes differentially expressed between subgroups was performed using both Significant Analysis of Microarray (SAM) algorithm with false discovery ratio (FDR)<5% and enhanced Analysis of Variance (ANOVA) with p-value <0.001. To increase the statistical power, the enhanced ANOVA uses as input data both expression level and the estimated technology error associated with the expression level. As a result, the false-positive rate is reduced when the number of replicates is small and, then, sensitivity detection is increased. A complete description of statistical methods used is available in the technology section of Rosetta Bio software website (http://www.rosettabio.com/tech/default.htm). PANTHER (Protein ANalysis THrough Evolutionary Relationships, http://www.pantherdb.org/) Classification System (Applied Biosystems, Foster City, CA, USA) was used to determine cellular pathways of genes identified.

first: GianMatteo

last: Rigolin

first: Lara

last: Rizzotto

first: Maria

last: Ciccone

first: Olga

last: Sofritti

first: Giulia

last: Daghia

first: Francesca

last: Cibien

last: Viglione

first: Cristina

last: Ambrosio

first: Francesco

last: Cavazzini

first: Roberto

last: Marasca

first: Gianluigi

last: Castoldi

first: Antonio

last: Cuneo


title: Gene expression profile of fresh human acute myeloid leukemia cells exposed ex vivo to AS602868

status: Public on Jan 13 2010

city: Lyon

first: Lars

middle: Petter

last: Jordheim

address: 8 Av Rockefeller

country: France

institute: INSERM U590

summary: To study modifications in gene expression profiles induced by the cytotoxic compound AS602868, we employed genome microarray expression profiling on treated and untreated samples from patients with acute myeloid leukemia.

overall_design: Freshly isolated leukemic blasts from AML patients were incubated for 6 hours with 10 µM AS602868. As AS602868 was initially diluted in DMSO, untreated samples were exposed to equivalent volume of DMSO for 6 hours.

first: Céline

last: Keime

first: Charles

last: Dumontet

middle: P


title: Kidney Stone Induces Developmental Stage-specific Alterations in Gene Expression

city: Piscataway

first: Jianmin

last: Chen

state: NJ

address: 604 Allison Rd

department: Cell Biology and Neuroscience

institute: Rutgers University

summary:
OBJECTIVES: Kidney stone diseases are common in premature infants, but the underlying molecular and cellular mechanisms are not fully defined. We carried out a prospective observational study using microarray analysis to identify factors that may be crucial for the initiation and progression of stone-induced injury in the developing mouse kidney.
METHODS: Mice with adenine phosphoribosyltransferase (Aprt) deficiency develop 2,8-dihydroxyadenine (DHA) nephrolithiasis. Gene expression changes between Aprt-/- and Aprt+/+ kidneys from newborn and adult mice were compared using Affymetrix gene chips.
RESULTS: We observed that: (i) gene expression changes induced by Aprt deficiency are developmental stage-specific; (ii) maturation-related gene expression changes are delayed in developing Aprt-/- kidneys; and (iii) immature Aprt-deficient kidneys contain large numbers of intercalated cells blocked from terminal differentiation.
CONCLUSIONS: This study presents a comprehensive picture of the transcriptional changes induced by stone injury in the developing mouse kidney. Our findings help explain growth impairment in kidneys subject to injury during the early stages of development.

overall_design: Total RNA were extracted from kidneys of 12 newly born and 6 adult mice (half Aprt-/- and half control). Gene expression changes between Aprt-/- and Aprt+/+ kidneys from newborn and adult mice were compared using Affymetrix gene chips.

first: Amrik

last: Sahota

first: Jay

last: Tischfield


title: Identification of miR-145 or miR-133a target genes in squamous cell carcinoma

status: Public on Jan 26 2010

city: Chiba

first: Naohiko

last: Seki

state: Chiba

address: 1-8-1, Inohana

department: Functional Genomics

institute: Chiba University

summary: We recently identified a subset of down-regulated miRNAs such as miR-145 and miR-133a in squamous cell carcinoma. Cell growth inhibitions occurred in miR-145 and miR-133a transfectants compared with the controls, suggesting that both miRNAs function as tumor suppressors. The aims of our expression studies were identification of these miRNAs target genes.

overall_design: miR-145 transfected cancer cells vs control cells, miR-133a transfected cancer cells vs control cells


title: Effect of exercise on gene expression profile in unfractionated peripheral blood leukocytes

status: Public on Jan 08 2010

city: Minato-ku

first: Seiji

last: Nakamura

address: 1-15-1, Kaigan, Suzuebaydium 5F

summary: A 4-hr bout of exercise induces immunomodulatory effects. Peripheral blood was withdrawn before, and at 4, 8 and 24hr after the start of exercise. RNA from the unfractionated white blood cells was analyzed using Agilent human 44K microarrays. The expression profiles were sorted into seven clusters based on their unique time-dependent kinetics. In a separate experiment, cell-specific markers were collected and compared among the members in each cluster. Two clusters were assigned as representing neutrophils, one as NK cells, and another mostly as T cells. Three clusters seemed to be mixtures of several cell types.

overall_design: Peripheral blood was withdrawn before, and at 4, 8 and 24hr after a bout of exercise. RNA from the unfractionated white blood cells was analyzed using Agilent human 44K microarrays.

first: Michie

last: Kobayashi

first: Tomohiro

last: Sugino

first: Osami

last: Kajimoto

first: Ryo

last: Matoba

first: Kenichi

last: Matsubara


title: Identification of LY6K target genes in bladder cancer

status: Public on Jan 05 2010

summary: Five bladder cancer (BC) cell lines were subjected to high-resolution array comparative genomic hybridization (CGH), which contained about 244,000 probes. The focus was on the typical gain locus of 8q24.3, which has not been studied in detail for BC. Integrating array-CGH data with the results of gene-expression profiling using oligo-microarray analysis revealed that 34 genes on 8q24.3 were generally up-regulated in the cell lines. Among these 34 genes, lymphocyte antigen 6 complex locus K (LY6K), which is a cancer/testis antigen, was the most up-regulated one in the BC (13.76-fold) as well as in clinical BC samples (2.84-fold). Investigation molecular network of LY6K performed using another oligo-microarray of the LY6K transfectant demonstrated that 269 genes were generally up-regulated more than five-fold in the transfectant. Twenty-one percent of the up-regulated genes were annotated into ‘cell cycle’ category, suggesting that LY6K is a promising molecular target for BC.

overall_design: LY6K transfected cancer cells vs control cells


title: NOD2 and human ileal gene expression

status: Public on Jan 01 2010

city: St Louis

first: Rekha

last: Meyer

state: MO

address: 4320 Forest Park Av

institute: Washington University School of Medicine

summary: Profiling of ileal mucosal gene expression in Chron's disease patients with and without NOD2 risk alleles; through microarray analyses it was discovered that 18 genes related to lymphocyte and phagocyte activation recuitment were upregulated in affected ileum in NOD2R patients as compared to ileum in NOD2s (no risk alleles) patients, thus supporting the concept that NOD2 risk alleles contribute to impaired regulation of inflammation in the ileum.

overall_design: Mucosal biopsies of resected ileum were collected from 8 Crohn's disease patients at risk for NOD2R alleles. The patients were genotyped for the three major NOD2 risk alleles (Arg702Trp, Gly908Arg, Leu1007fs).  Microarray analysis was performed in samples from NOD2R (at least one risk allele) Crohn’s disease patients, NOD2S (no risk alleles) Crohn’s disease patients and NOD2S controls.

first: Ellen

last: Li

first: Steven

last: Hunt


title: RAW264.7 macrophages infected with Brucella abortus, B. melitensis, B. neotomae, and B. ovis

first: Gary

last: Splitter

address: 1656 Linden Dr

department: Pathobiological Sciences

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

overall_design: Refer to individual Series

first: Angela

last: Mathison

first: Linda

last: Eskra


title: Expression data from P7 and adult wt and Tenascin-R (Tnr) deficient mice brain

status: Public on Dec 15 2009

city: Chapel Hill

first: ingo

last: meier

state: NC

address: 416 Fordham Hall

institute: UNC chapel hill

summary:
We used the microarray to investigate the lack of Tenascin-R in brain of adult and P7 mice. The aim was to find differential expressed genes which could explain the behaviour differences between the tenascin wt and ko mice. The data show that not many genes are dysregulated in the Tnr deficient mouse in comparison to the wt mouse brain.
Gas5 was one of the dysregulated genes. Gas5 was dysregulated in the P7 and 2 month old mice.

overall_design: 14:  (P7 4wt vs 4ko), (adult 3wt vs 3ko)

first: Ingo

last: Meier

first: Thomas

last: Streichert


title: Chronic Active B Cell Receptor Signaling in Diffuse Large B Cell Lymphoma

status: Public on Jan 11 2010

city: Bethesda

first: Louis

middle: M.

last: Staudt

state: MD

address: 9000 Rockville Pike, Bldg 10, Rm 4N114

department: Lymphoid Malignancies Branch

institute: National Cancer Institute

summary: To determine gene expression of DLBCL upon NF-kB inhibition or knockdown of BCR signaling components.

overall_design: Total RNA (Trizol reagent; Invitrogen, Carlsbad, CA) was prepared from 8 HBL-1 samples following incubation with 25 µmol/L MLN120B (Millennium Pharmaceuticals, Cambridge, MA) for 2, 3, 4, 6, 8, 12, 16, and 24 hours. In addition, 16 HBL-1 samples were infected with retroviral vectors expressing various shRNAs (CARD11, SYK, BTK, CD79A) in a doxycycline-inducible fashion, selected with puromycin, treated with doxycycline for 24 or 48 hr and then harvested for total RNA. Uninduced cultures were prepared in parallel as control samples. Biological replicates were performed for the CARD11, BTK, and CD79A samples.


title: Gene expression profiles of bladder cancer cell lines

first: Naohiko

last: Seki

summary: Bladder cancer is among the five most common malignancies worldwide, and it is the second most common tumor of the genitourinary tract and the second most common cause of death in patients with genitourinary tract malignancies. To identify target genes of human bladder cancer, five cell lines were subject to Agilent whole genome microarray.

overall_design: Five bladder cancer cell lines (BOY, J82, KK47, T24, and UMUC) were compared to human normal bladder RNA


title: Systematic identification of fragile sites via genome-wide location analysis of gamma-H2AX

status: Public on Dec 01 2009

city: Montréal

first: François

last: Robert

state: Québec

address: 110, avenue des Pins Ouest

department: Chromatin and Genomic Expression

country: Canada

institute: Institut de recherches cliniques de Montréal (IRCM)

summary: Phosphorylation of histone H2AX is an early response to DNA damage in eukaryotes. In Saccharomyces cerevisiae, DNA damage or replication fork stalling results in histone H2A phosphorylation to yield gamma-H2A (yeast gamma-H2AX) in a Mec1 (ATR)- and Tel1 (ATM)- dependent manner. Here, we describe the genome-wide location analysis of gamma-H2A as a strategy to identify loci prone to engage the Mec1 and Tel1 pathways. Remarkably, gamma-H2A enrichment overlaps with loci prone to replication fork stalling and is caused by the action of Mec1 and Tel1, indicating that these loci are prone to breakage. Moreover, about half the sites enriched for gamma-H2A map to repressed protein-coding genes, and histone deacetylases are necessary for formation of gamma-H2A at these loci. Finally, our work indicates that high resolution mapping of gamma-H2AX is a fruitful route to map fragile sites in eukaryotic genomes.

overall_design: To identify loci enriched in gamma-H2A, we carried out chromatin immunoprecipitations with a phospho-specific antibody that recognizes gamam-H2A in yeast and hybridized to high-density tiling arrays surveying the genome at an average density of one probe per 275 bp. In a typical experiment, we performed competitive hybridization of DNA precipitated from HTA1 HTA2 cells with DNA precipitated from the gamma-H2A-deficient hta1-S129A hta2-S129A cells (referred to hereafter as S129A). All experiments were done at least in duplicate and combined using a weighted average method.

first: Rachel

middle: K

last: Szilard*

first: Pierre-Étienne

middle: E

last: Jacques*

first: Louise

last: Laramée

first: Benjamin

last: Cheng

first: Sarah

last: Galicia

first: Alain

middle: R

last: Bataille

first: ManTek

last: Yeung

first: Megan

last: Mendez

first: Maxime

last: Bergeron

first: Daniel

last: Durocher


title: PrEC response to Alanine or Sarcosine at 6 hours

status: Public on Dec 31 2009

city: Detroit

first: Laila

last: Poisson

state: MI

address: 1 Ford Place

department: Biostatistics and Research Epidemiology

institute: Henry Ford Health Systems

summary:
PrEC cells treated with either 50uM alanine or 50uM sarcosine (N-methylglycine). Gene expression is measured after 6 hours.
We wish to look at the effects of sarcosine addition in relation to alanine addition.
Keywords: Amino acid addition response

overall_design: A technical replicate is analyzed for both the alanine and the sarcosine treated cells (4 arrays total). Untreated PrEC cells are used as the reference.

first: Arun

last: Sreekumar

first: Arul

last: Chinnaiyan


title: Time-series toxicogenomics analysis of N-nitroso compound exposure in Caco-2 cells

status: Public on Apr 29 2010

city: Maastricht

first: Dennie

last: Hebels

address: Universiteitssingel 50

department: Health Risk Analysis and Toxicology

country: Netherlands

institute: Maastricht University

summary:
N-nitroso compounds (NOC) may be implicated in human colon carcinogenesis, but the toxicological mechanisms involved have not been elucidated. Since it was previously demonstrated that nitrosamines and nitrosamides, representing two classes of NOC, induce distinct gene expression effects in colon cells that are particularly related to oxidative stress, we hypothesized that different radical mechanisms are involved. Using ESR spectroscopy, we investigated radical generating properties of genotoxic NOC concentrations in human colon adenocarcinoma cells (Caco-2). Cells were exposed to nitrosamides (N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosurea) or nitrosamines (N-nitrosodiethylamine, N-nitrosodimethylamine, N-nitrosopiperidine, N-nitrosopyrrolidine). Nitrosamines caused formation of reactive oxygen species (ROS) and carbon centered radicals which was further stimulated in presence of Caco-2 cells. N-methyl-N-nitrosurea exposure resulted in a small ROS signal, and formation of nitrogen centered radicals (NCR), also stimulated by Caco-2 cells. N-methyl-N'-nitro-N-nitrosoguanidine did not cause radical formation at genotoxic concentrations, but at increased exposure levels, both ROS and NCR formation was observed. By associating gene expression patterns with ROS formation, several cellular processes responding to nitrosamine exposure were identified, including apoptosis, cell cycle blockage, DNA repair and oxidative stress. These findings suggest that following NOC exposure in Caco-2 cells, ROS formation plays an important role in deregulation of gene expression patterns which may be relevant for the process of chemical carcinogenesis in the human colon, in addition to the role of DNA alkylation.

Keywords: Nitrosamines, nitrosamides, N-nitroso compounds, free radicals, toxicogenomics, colon carcinogenesis

overall_design: The study investigated differential gene expression in Caco-2 cell line mRNA following 1, 6 or 24 hours of exposure to six different N-nitroso compounds. Two biological replicates per sample compound. One compound per array, hybridized against vehicle control. Dye-swap between biological replicates.

middle: G

first: Jacob

last: Briedé

first: Jos

middle: C

last: Kleinjans

first: Theo

last: de Kok


title: Activation of Host Wound Responses in Breast Cancer Microenvironment

status: Public on Jan 07 2010

first: Melissa

last: Troester

address: 135 Dauer Drive, CB 7435

department: Epidemiology

institute: University of North Carolina at Chapel Hill

summary: Cancer progression is mediated by processes that are also important in wound repair. As a result, cancers have been conceptualized as overhealing wounds or "wounds that do not heal," and gene expression signatures reflective of wound repair have shown value as predictors of breast cancer survival. Despite the widespread acknowledgment of commonalities between host responses to wounds and host responses to cancer, the gene expression responses of normal tissue adjacent to cancers have not been well characterized.

overall_design: Using RNA extracted from histologically normal breast tissue from 105 patients, 121 arrays were performed, including 72 arrays representing 58 reduction mammoplasty patients and 49 arrays representing 47 cancer patients, we measured whole genome expression profiles and identified a gene expression signature that is induced in response to breast cancer.


title: Nutlin-3 treatment of B-CLL

status: Public on Nov 11 2009

city: Aviano

first: daniela

last: marconi

address: Via franco Gallini 2

institute: CRO AVIANO

summary: Background: p53 plays a key role in determining the clinical features of B cell chronic lymphocytic leukemia (CLL). Disruption of p53 by point mutations, deletion at 17p13, or both, occurs in a fraction of cases at diagnosis and predicts poor survival and chemorefractoriness. In cells with functional p53, p53 activity is inhibited through interaction with MDM2. In fact, p53 can be activated upon exposure of cells to inhibitors of p53/MDM2 interaction, like Nutlins. Exposure of CLL cells to Nutlin-3 is effective in raising the levels of p53 protein with subsequent induction of cell cycle arrest and/or apoptosis, independently of the most relevant prognostic markers. Specific gene-sets and GEP were documented to be associated with response or resistance to Nutlin-3 exposure in p53(wt) or p53(del/mut) CLL. These findings may help to identify novel molecular targets for CLL therapy.  Aim: to analyze the gene expression profile (GEP) induced by Nutlin-3 exposure in primary CLL cells from p53(wt) and p53(del/mut) cases.  Methods: purified cells from 24 PB CLL samples, all characterized for IGHV mutational status, CD38 and ZAP-70 and p53 mutations (16 p53(wt) CLL, 8 p53(del/mut) CLL of which 6 with del17p13 and p53 mutations, 1 with del17p13 alone, and 1 with p53 mutations alone), were exposed to 10 uM Nutlin-3 for 24 hours. GEP was performed using a dual labelling strategy; the differential expression of the below reported genes were validated by quantitative real-time PCR.   specific gene-sets and GEP were documented to be associated with response or resistance to Nutlin-3 exposure in p53(wt) or p53(del/mut) CLL. These findings may help to identify novel molecular targets for CLL therapy.

overall_design: Two-condition experiment, Nutlin-3 Treated B-CLL vs. Untreated B-CLL. Biological replicates: 24 control replicates, 24  Treated replicates. Reference design

first: Michele

last: Dal Bo

first: Paola

last: Secchiero

first: Daniela

last: Marconi

first: Riccardo

last: Bomben

first: Dania

last: Benedetti

first: Antonella

last: Zucchetto

first: Gabriele

last: Pozzato

first: Gianluca

last: Gaidano

first: Giovanni

last: Del Poeta

first: Francesco

last: Forconi

first: Giorgio

last: Zauli

first: Valter

last: Gattei


title: Microarray analyses define the scope of the gut derived microbial metabolite butyrate on peripheral and central neuronal targets

status: Public on Nov 10 2009

city: Valhalla

first: Bistra

last: Nankova

state: NY

address: 95 Grassland Rd

institute: New York Medical College

summary: Genome-wide expression profiling showed significant overlap of the genetic networks regulated by butyrate in vivo (rat adrenal medulla) and in vitro (PC12 cells) including TH and other neurotransmitter-related genes (DBH, AADC, GTPCH, ppEnk, NPY).

overall_design:
Studies in PC12 cells were performed to delineate which genetic networks are regulated by SCFA depending on the dose. Experiments in vivo were performed to test wheter similar phenomenon operates in the sympathoadrenal system.

ISBPC: 1mMSB PC12
GSBPC: 6mMSB PC12
CPCR: CPC12
SBAM: rat SBAdrenal Medulla
CAM: rat Control Adrenal Medulla

first: Priyadarshani

last: Giri

first: Edmund

last: LaGamma


title: ELK1 and NFYA knockdown in MBA-MD-231 cell line

status: Public on Nov 04 2009

city: San Francisco

first: Hani

last: Goodarzi

department: Biochemistry and Biophysics

institute: UCSF

summary: Dual-color transcriptional profiling of siRNA-transfected cells, targeting ELK1 and NFYA, versus mock-transfected cells.

overall_design: Dual-color experiment, siRNA-transfected vs. control. Biological replicates: 2 for ELK1 and 2 for NFYA

first: Olivier

last: Elemento


title: miR-17 seed family specific targets in two Hodgkin lymphoma cell lines

status: Public on Oct 13 2009

city: Groningen

first: Lu Ping

last: Tan

address: Hanzeplein 1

department: Department of Pathology

institute: University Medical Center Groningen

summary: The aim of the study is to identify miRNA specific targets in Hodgkin lymphoma cell lines. By immunoprecipitation (IP) of wild type Ago2, Ago2 associated gene transcripts (ie miRNA targets) are coimmunoprecipitated. In cells transfected with anti-miR-17/20/93/106, the miR-17 seed family specific targets are not coimmunoprecipitated with Ago2. With microarray analysis, signal intensities of probes associated with Ago2 from untransfected and anti-miRNA transfected cells are compared. Gene transcripts that are depleted from the Ago2-IP fraction upon miRNA inhibition (ie miRNA specific targets) are identified.

overall_design: RNA isolated from the Ago2 immunoprecipitated (IP) fraction is hybridized against the RNA from total cell lysate (T) fraction. The experiment is performed in two independent Hodgkin lymphoma cell lines, L1236 and L428. Samples included anti-miR-220 transfected L428, anti-miR-17/20/93/106 transfected L428 and anti-miR-17/20/93/106 transfected L1236. All hybridizations are performed with a dye swap design.

first: L

first: E

last: Seinen

first: G

last: Duns

first: D

last: de Jong

first: O

last: Sibon

first: K

last: Kok

first: B

last: Kroesen

first: S

last: Poppema

first: A

last: van den Berg


title: Expression consequences of decoy vs scrambled oligonucleotides harboring AAAA[AGT]TT motif in MBA-MD-231 cell line

summary: Dual-color transcriptional profiling of decoy-transfected cells, harboring AAAA[AGT]TT motif, versus scrambled-transfected cells as controls.

overall_design: Dual-color experiment, decoy-transfected vs. scrambled-transfected. Biological replicates: 2 sets of decoys and scrambled ds-oligonucleotides


title: Host RAW264.7 macrophage transcript profile following Brucella melitensis, B. neotomae, and B. ovis infections

city: Madison

first: Gary

last: Splitter

institute: University of Wisconsin - Madison

summary:
Brucella dynamically engage macrophages while trafficking to an intracellular replicative niche as macrophages, the first line of innate host defense, attempt to eliminate organisms.  Brucella melitensis, B. neotomae, and B. ovis are highly homologous, yet exhibit a range of host pathogenicity and specificity.  RAW 264.7 macrophages infected with B. melitensis, and B. ovis exhibit divergent patterns of bacterial persistence and clearance; conversely, B. melitensis and B. neotomae exhibit similar patterns of infection.  Evaluating early macrophage interaction with Brucella spp. allows discovery of host entry and intracellular translocation mechanisms, rather than bacterial replication.  Microarray analysis of macrophage transcript levels following a 4 hr Brucella spp. infection revealed 130 probe sets altered compared to uninfected macrophages; specifically, 72 probe sets were increased and 58 probe sets were decreased with any Brucella spp.  Interestingly, much of the inflammatory response was not regulated by the number of Brucella gaining intracellular entry, as macrophage transcript levels were often equivalent among B. melitensis, B. ovis, and B. neotomae infections.  An additional 33 probe sets were identified with altered macrophage transcript levels among Brucella spp. infections that may correlate with species specific host defenses and intracellular survival.  Gene ontological categorization unveiled genes altered among species are involved in cell growth and maintenance, response to external stimuli, transcription regulation, transporter activity, endopeptidase inhibitor activity and G-protein mediated signaling.  Host transcript profiles provide a foundation to understand variations in Brucella spp. infections, while structure of the macrophage response and intracellular niche of Brucella spp. will be revealed through piecewise consideration of host signaling pathways.
Keywords: Macrophage, intracellular pathogen, Brucella melitensis, Brucella neotomae, Brucella ovis, inflammatory immune response, species specificity

overall_design: Eleven MG_U74Av2 GeneChip® microarrays (Affymetrix) were independently hybridized with cRNA from uninfected macrophage samples (2 independent samples) and each of three Brucella spp. infected macrophage samples (3 independent samples for each Brucella species infection – B. melitensis, B. neotomae, and B. ovis).  All probe sets were subjected to analysis by EBarrays, a statistical analysis package in the comprehensive R archive network (http://cran.r-project.org/).  Data conformity to the statistical assumptions was checked (coefficient of variation and log-normal normal model) and best fit models were utilized.  Six hypotheses of altered transcript levels were tested and probe set specific posterior probabilities (EBarrays Probability) calculated with Empirical Bayesian statistics.  The null hypothesis identified probe sets with no change among conditions and 5 experimental hypotheses identified changes in transcript among the experimental conditions.

first: Angela

last: Mathison

first: Linda

last: Eskra

first: Jill

last: Covert


title: CLOCK knockdown in MCF-7 cells

city: New Haven

first: Aaron

middle: Ezra

last: Hoffman

state: CT

address: 60 College St.

department: LEPH

institute: Yale School of Medicine

summary: The aim of the study was to determine the impact of CLOCK knockdown on global gene expression.

overall_design: MCF7 cells were transfected with either a non-targeting siRNA oligo (negative control) or an siRNA oligo targeting CLOCK. Gene expression was measured relative to negative control, and biological replicates were included.

first: Yong

last: Zhu

first: Chun-Hui

last: Yi


title: Ago2 associated gene transcripts in two Hodgkin lymphoma cell lines

status: Public on Sep 21 2009

first: Lu Ping

summary:
The aim of the study is to identify miRNA targets in Hodgkin lymphoma cell lines. By immunoprecipitation of wild type Ago2, it is expected to pull down the Ago2 associated gene transcripts. Through microarray analysis, the Ago2 associated gene transcripts identified are expected to be miRNA targets.

Keywords: Ribonucleoprotein Immunoprecipitation - Gene Chip (RIP-Chip)

overall_design: RNA isolated from the Ago2 immunoprecipitated (IP) fraction is hybridized against the RNA from total cell lysate (T) fraction. The experiment is performed in two independent Hodgkin lymphoma cell lines, L1236 and L428. In addition, RNA of flow through (FT) fraction of L1236 is hybridized against RNA of total cell lysate (T) fraction of L1236 to demonstrate the specificity/enrichment seen in the IP fraction and not in the FT fraction. All hybridizations is done with a dye swap design.

first: Lu

first: Erwin

first: Gerben

first: Debora

first: Ody

first: Klaas

first: Bart-Jan

first: Sibrand

first: Anke


title: Yeast RNase III triggers polyadenylation independent transcription termination

status: Public on Oct 09 2009

first: François

last: Robert

summary: Transcription termination of messenger RNA (mRNA) is normally achieved by polyadenylation followed by Rat1p dependent 5’-3’ exoribonuleolytic degradation of the downstream transcript. Here we show that the yeast orthologue of the dsRNA-specific ribonuclease III (Rnt1p) may trigger Rat1p dependent termination of RNA transcripts that fail to terminate near polyadenylation signals. Rnt1p cleavage sites were found downstream of several genes and the deletion of RNT1 resulted in transcription read-through. Inactivation of Rat1p impaired Rnt1p dependent termination and resulted in the accumulation of 3’ end cleavage products. These results support a new model for transcription termination in which co-transcriptional cleavage by Rnt1p provides access for exoribonucleases in the absence of polyadenylation signals.

overall_design:
In order to examine the global impact of Rnt1p on transcription termination, we analyzed the overall pattern of RNAP II occupancy in the presence and the absence of RNT1. RNAP II specific chromatin immunoprecipitation was performed as described below (IP vs input) from RNT1 or rnt1Δ cells and the extracted DNA hybridized to a DNA microarray containing an average of 4 probes per kilobase across the whole yeast genome.
The raw data were corrected (foreground-background) then normalized using the limma's loess function (Yang et al., 2002) in BioConductor (from the ArrayPipe Analysis Pipeline (Hokamp et al., 2004)) and replicates were combined using a weighted average method as described previously (Ren et al., 2000, Pokholok et al., 2005).
The occupancy of RNAPII in WT and rnt1Δ was assessed respectively in triplicate and in duplicate. The combined datasets are available in the supplemental files of the related publication.

first: Ghada

last: Ghazal

first: Jules

last: Gagnon

first: Pierre-Étienne

last: Jacques

first: Josette

last: Landry

first: Sherif

last: Abou Elela


title: Gene expression profiling of NCAM-deficient mouse brain

city: Hamburg

first: Thomas

last: Streichert

address: Martinistr. 52, Campus Forschung N27, 2. OG

department: Institute For Clinical Chemistry / Central Laboratories

institute: University Medical Center Hamburg-Eppendorf

summary:
Analysis of brains of mice lacking the neural cell adhesion molecule NCAM (Ncam-/-) in comparison to wild-type mice of same age and genetic background (Ncam+/+). NCAM-deficient mice exhibit deficits in long-term potentiation and spatial learning, as well as increased intermale aggression.
We performed a microarray analysis of adult NCAM-deficient mouse brains to identify molecular targets which might underly the phenotype of NCAM-deficient mice.

overall_design: We analyzed the gene expression profiles in 2-3 months old (young adult) mouse brains, with both the Ncam-/- and the Ncam+/+ group consisting of three individuals each. Wild-type and NCAM-deficient animals had the same genetic background. RNA was prepared from total brain.

first: Yong

middle: W

last: Wong


title: Microarray Analysis of Genes Regulated by Macrophage-Conditioned Media Treatment of Human Adipocytes

city: Sharnbrook

first: Fei Ling

last: Lim

state: Bedfordshire

address: Colworth Science Park

country: United Kingdom

institute: Unilever

summary: A major development in obesity research is the recognition that the condition is characterized by chronic mild inflammation. Within adipose tissue, this involves the infiltration of macrophages as well as a direct inflammatory response of adipocytes. This study has used Agilent whole-genome microarrays to examine the effects of macrophage-conditioned medium on the global inflammatory response of human adipocytes.

overall_design: Human adipocytes (SGBS cells), differentiated in culture, were treated with macrophage (U937 cells) conditioned media for 4 or 24 h. Control SGBS cells were treated with unconditioned media (control) or RPMI media alone to control for differences in media used to culture U937 cells. There were 6 replicates per group.

first: Adrian

last: O’Hara

first: Fei

middle: L

first: Dawn

last: Mazzatti

first: Paul

last: Trayhurn


title: Effect of heteronemin on gene expression profiles in chronic myelogenous K562 leukemia cells

status: Public on Oct 14 2009

city: Seoul

first: Marc

last: Diederich

address: Gwanak-ro, Gwanak-gu, 1

department: College of Pharmacy

country: South Korea

institute: Seoul National University

summary: Heteronemin is a sesterterpene first isolated from Hyrtios Erecta in 1976. In this study we decided to investigate the anti-cancer and anti-inflammatory potential  of this marine natural compound at the transcriptomic level in chronic myelogenous K562 leukemia cells. Our results suggest that this sesterterpene has an impact on cell cycle, apoptosis, the MAP kinase signalling pathway and the NF-kappaB transcription factor.

overall_design: We used four experimental conditions : untreated cells, cells treated with heteronemin, cells treated with TNFalpha and cells co-treated with TNFalpha and heteronemin. Experiments were performed in three biological replicates including two technical replicates. Technical replicates were hybridized as dye-swaps. Dye-swap between replicates 1 and 2, 3 and 4, 5 and 6, respectively.

first: Claudia

last: Cerella

first: Serge

last: Eifes

last: Schumacher

first: Sebastien

last: Chateauvieux

first: Mario

last: Dicato


title: Control vs. HMEC-1 cells treated with 100ng/ml TNF-alpha for 4h and 24h

status: Public on Sep 15 2009

city: Tuebingen

first: Peter

last: Rosenberger

address: Waldhoernlestr. 22

department: Anesthesiology

institute: ZMF Universitaet Tuebingen

summary:
Transcriptional profiling of HMEC-1 cells comparing untreated control cells with TNF-alpha-treated cells. Goal was to include more evidence by adding another research method.

Keywords: Inflammatory stimulation

overall_design: Two-condition experiment, control vs. TNF-alpha-treated HMEC-1 cells. 1 control after 4h, 1 TNF-alpha-treated sample after 4h, 1 control after 24h, 1 TNF-alpha-treated sample after 24h. For each condition, 3 replicates of HMEC-1 cultures were grown and treated independently, then harvested and pooled for microarray.


title: Genes regulated by high calcium medium in parathyroid tissue explants from patients with primary hyperparathyroidism

status: Public on Oct 23 2009

city: Shinjuku-ku

first: Kanji

last: Sato

address: Kawada-cho 8-1

department: Institute of Clinical Endocrinology

institute: Tokyo Women's Medical University

summary: To investigate genes modulated in the parathyroid glands by calcium, expression levels of mRNA for all genes expressed in parathyroid tissue explants (PTEs) obtained from patients with primary hyperparathyroidism (I-HPT) were analyzed by oligo-DNA microarray. PTEs obtained from 4 patients with I-HPT were precultured in normocalcemic medium (Ca++ 1.0-1.1 mM) for 7 days and then cultured in hypocalcemic medium (Ca++ 0.60 mM) or hypercalcemic (Ca++ 1.60 mM) medium for an additional 7 days. As expected, expression levels of mRNA for PTH and chromogranin A were decreased to less than 50%　in hypercalcemic medium. Furthermore, oligo-DNA microarray analyses followed by GeneSpring GX analyses revealed that 7 genes were up-regulated by more than 2-fold and more than 30 genes were down-regulated by more than 1/2 in PTEs obtained from patients with I-HPT.  Interestingly, 9 of these genes (up-regulated genes: chemokine ligand 8[CCL8], multiple C2 domain and transmembrane region protein 1 [MCTP1]; down-regulated genes: matrix metallopeptidase-9 [MMP9], B-box and SPRY domain-containing protein [BSPRY], nitric oxide synthase 2A [NOS2A], parathyroid hormone [PTH], cartilage acidic protein 1 [CRTAC1], chromogranin A [CHGA], and fibrin 1 [FBLN1]) were involved in calcium metabolism or calcium-signaling pathways in the parathyroid tissue. Unexpectedly, however, the expression level of mRNA for alpha-klotho was variable, and it was not constantly decreased in hypercalcemic medium under the present experimental conditions. Although it was not possible to use normal parathyroid tissue, this is the first reported study to have investigated the expression levels of mRNA for all genes in human parathyroid adenomas that are modulated by high calcium concentration in organ culture.

overall_design: Organ culture experiments, cultured in low vs.high calcium medium.

first: Kishiko

last: Nakajima


title: Gene profiling of intermediolateral column, medial, and lateral motoneuron after spinal cord transection

status: Public on Sep 23 2009

city: Atlanta

first: Dapeng

last: Cui

state: GA

address: 24 Peachtree Center Avenue

department: Biology

institute: Georgia State University

summary: To determine spinal cord injury (SCI) induced changes in gene expression, laser capture microscopy (LCM) and Affymetrix microarrays were used to profiles three distinct populations of motor neurons (MNs) in five sets of littermates. In each set, one littermate received a lamenectomy (sham operated), and the other received a complete spinal cord transection. Each MN population was caudal to the transection and not axotomized. Thus, MNs in transected animals were responding to two major insults: deafferentation and changing microenvironments due to spreading immune and inflammatory responses. A total of 30 expression profiles from sham operated control animals have been uploaded to GEO database with accession number GSE2595. The current series contains 32 motoneuron expression profiles from spinalized mice.  All chips (30 sham + 32 transection) were generated together using RMA in 2004.

overall_design: In this study we determine SCI induced changes in gene expression in lateral motoneurons (LMN), medial motoneurons (MMN) and intermediolateral column motoneurons (IML).  Five sets of male littermates were used in these experiments (sets a-e). In each set, one littermate received a lamenectomy (sham operated), and the other received a complete spinal cord transection.  Four of the sets (a-d) consisted of one transect and one littermate sham operated control. The fifth set (e) had an additional transect. All surgeries were performed on postnatal day (P) 24.  Three weeks later (P45), the animals were sacrificed and their spinal cords were removed and sectioned for LCM. Each of the three cell types was captured from every animal. The expression profiles of the three classes of MNs were obtained using Affymetrix microarrays.

first: Deborah

last: Baro

first: Shawn

last: Hochman

first: Kimberly

last: Dougherty

first: David

last: Machacek

first: Michael

last: Sawchuk


title: Runx1 (Aml1) knockdown in AMkL Meg-01 cells

status: Public on Aug 01 2009

first: Yubin

last: Ge

address: 110 East Warren Ave.

institute: Karmanos Cancer Institute

summary: Transcriptional profiling of the effect of shRNA silencing of Runx1 in human AMkL Meg-01 cells.  RNA samples obtained from two independent colonies were compared to RNAs from negative control transductions in a two-color design. A total of four microarrays were completed, with a dye-swapped pair performed for each colony.

overall_design: Two-condition experiment, Runx1 knockdown vs. negative transduction controls. Biological replicates: 2 knockdown replicates, 2 control replicates.


title: Meiotic time course: open chromatin and expression profile

status: Public on Sep 25 2009

city: Chicago

first: Sean

middle: Erik

last: Hanlon

state: IL

address: 920 E. 58th Street – CLSC 515E

department: Department of Human Genetics

institute: University of Chicago

type: Genome binding/occupancy profiling by array

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

overall_design: Refer to individual Series

first: Luke

last: Berchowitz

first: Jason

last: Lieb

first: Gregory

last: Copenhaver


title: Endometrial gene expression analysis at the time of embryo implantation in women with unexplained infertility

status: Public on Oct 17 2009

city: Valencia

first: Jose

middle: Antonio

last: Martinez-Conejero

address: C/ Guadassuar 1, bajo

country: Spain

institute: iGenomix

summary: The aim of this study was to investigate the endometrial gene expression profile in women with unexplained infertility in comparison to fertile controls at the time of embryo implantation in order to find potential informative markers of uterine receptivity, and further, to identify the molecular mechanisms related to infertility.

overall_design: High-density, oligonucleotide gene arrays, comprising 44,000 gene targets, were used to define the endometrial gene expression profile in infertile (n = 4) and fertile (n = 5) women during the mid-secretory phase (LH +7).

first: Signe

last: Altmäe

first: A

last: Salumets

first: Carlos

last: Simón

first: José

last: Horcajadas

first: Anneli

last: Stavreus-Evers


title: Scavenger receptor B2 is a cellular receptor for enterovirus 71

status: Public on Jun 21 2009

city: Tsukuba

first: Taro

last: Takemura

state: Ibaraki

address: 1-2-1 Sengen

department: Nanotechnology Innovation Station

institute: National Institute for Materials Science

summary: Enterovirus 71 (EV71) belongs to human enterovirus species A of the genus Enterovirus within the family Picornaviridae. We established transformant cells by transfection of mouse cells with genomic DNA from human cells and then detected two EV71-susceptible cell lines.  Using microarray with the two cell lines we found that scavenger receptor B2 is a cellular receptor for EV71.

overall_design: We compared EV71-susceptaible cell line Ltr051 or Ltr246 to wild type cell L929 using microarray. Each microarray experiment was repeated three times.

first: Seiya

last: Yamayoshi

first: Yasuko

last: Yamashita

first: Jifen

last: Li

first: Nobutaka

last: Hanagata

first: Takashi

last: Minowa

first: Satoshi

last: Koike


title: Effect of Genetic Variant in miR-196a-2 in MCF-7 cells

status: Public on Jun 08 2009

first: Aaron

last: Hoffman

summary: The impact of a SNP (rs11614913) in miR-196a-2 was examined by introducing expression vectors containing both the common and variant forms of the miRNA precursor.

overall_design: MCF7 cells were transfected with either an empty vector (negative control) or a vector expressing pre-miR-196a-2 with either the common (C allele) or variant (T allele) sequence.  Gene expression was measured relative to negative control, and biological replicates of each allele were included.

first: Yong

last: Zhu


title: The kinase TNIK is an essential activator of Wnt target genes

status: Public on Aug 13 2009

city: London

first: Vivian

address: 1 Midland Road

institute: The Francis Crick Institute

summary: Wnt signalling maintains the undifferentiated state of intestinal crypt/progenitor cells through the TCF4/ß-catenin activating transcriptional complex. In colorectal cancer, activating mutations in Wnt pathway components lead to inappropriate activation of the TCF4/ß-catenin transcriptional program and tumourigenesis in the gut epithelium. The mechanisms by which TCF4/ß-catenin activate key target genes are not well understood. Using a proteomics approach, we identified Tnik, a member of the Germinal centre kinase family, as a Tcf4 interactor in the proliferative crypts of mouse small intestine. Tnik is recruited to promoters of Wnt target genes in mouse crypts and in Ls174T colorectal cancer cells in a ß-catenin dependent manner. Depletion of TNIK and expression of TNIK kinase mutants abrogated TCF-LEF transcription, highlighting the essential role of the kinase activity in Wnt target gene activation. siRNA depletion of TNIK followed by expression array analysis demonstrated that TNIK is an essential and exclusive activator of Wnt induced transcriptional program. As an essential component in the TCF4/ß-catenin activator complex, the kinase TNIK may present an attractive candidate for drug targeting in colorectal cancer.

overall_design: HEK293T cells: Wnt3A vs control medium (CM) induction for 4, 7 and 9 hours; si-TNIK vs si-control after Wnt3A induction at 4 and 7 hours (2 biological replicates for 7 hour time point); dyeswap for each experiment (i.e. 12 arrays in total).

first: Tokameh

last: Mahmoudi

middle: S

first: Ser

last: Ng

first: Nadia

last: Taouatas

first: Robert

last: Vries

first: Shabaz

last: Mohammed

first: Albert

middle: J

last: Heck

first: Hans

last: Clevers


status: Public on Sep 01 2009

city: Salamanca

first: Javier

last: De Las Rivas

address: Campus Miguel de Unamuno

department: CSIC and University of Salamanca

institute: Cancer Research Center (CiC-IBMCC)

summary:
Serum-dependent transcriptional networks identify distinct functional roles for H-ras and N-ras during initial stages of the cell cycle

Using oligonucleotide microarrays, we compared gene expression transcriptional profiles corresponding to the initial cell cycle stages of mouse fibroblasts lacking H-Ras and/or NRas with those of matching, WT controls.
The similarity of transcription profiles among serum-starved fibroblasts of all different WT and ras knockout genotypes tested, indicated that H-Ras and N-Ras do not play significant roles in control of transcriptional responses to serum deprivation stress. In contrast, genomic disruption of H-ras or N-ras, individually or in combination, determined highly specific, differential gene expression profiles in response to post-starvation stimulation with serum for 1 hour (G0/G1 transition) or for 8 hours (mid-G1 progression). The absence of N-Ras caused significantly higher changes than the absence of H-Ras on the wave of transcriptional activation linked to G0/G1 transition. In contrast, the absence of H-Ras affected more potently the profile of the transcriptional wave detected during mid-G1 progression. Functional analysis demonstrated a predominant functional association of H-Ras with growth and proliferation, whereas N-Ras exhibited a closer functional link to development or cell cycle regulation as well as immunomodulation and apoptosis. Mechanistic analysis indicated that ERK-dependent activation of Stat1 mediates the regulatory effect of N-Ras on defense and immunity, whereas the pro-apoptotic effects of N-Ras are mediated through ERK and p38 signaling.  Our observations support previous reports of an absolute requirement for different peaks of Ras activity during the initial stages of the cell cycle and confirm the notion of functional specificity for the H-Ras and N-Ras isoforms.

Keywords: different gene KO mice and time course

overall_design: Mus musculus cell lines from the appropriate ras genotype were harvested on Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum (10% FBS), glutamine (2mM), penicillin (100 U/ml) and streptomycin (100 mg/ml). Cultures were grown in a humidified CO2 (5%) atmosphere at 37°C and when subconfluent cells were starved for 24 hours. After starvation cells were either used for RNA isolation, or induced for 1 hour or 8 hours with 20% fetal bovine serum and then RNA extraction and isolation was carried out for hybridization on Affymetrix microarrays.

first: Eugenio

last: Santos

first: Carmen

last: Guerrero

first: Alejandro

last: Nuñez

first: Esther

last: Castellano


title: Identification of LATS transcriptional targets in HeLa cells using whole human genome oligonucleotide micorarray

status: Public on Sep 17 2009

city: Kingston

first: Xiaolong

last: Yang

state: Ontario

address: 88 Stuart Street

department: Pathology and Molecular Medicine

institute: Queen's University

summary: Human LATS1 and LATS2 (LATS1/2) are tumor suppressors that have been shown to be mutated or downregulated in several human cancers including leukemia, lung, prostate and breast cancers.  However, the precise mechanisms and the proteins modulated by LATS1/2 that are responsible for these events remain largely unknown.  To elucidate potential signaling pathways, the current study investigated the expression profile in HeLa cells with reduced expression of LATS1/2.  Using RNA-mediated intereference, both LATS1 and LATS2 were substantially knocked-down, and accordingly, this lead to an increase in multiple phenotypes associated with tumor progression, including enhanced cell proliferation, resistance to drug-induced cell death, and increase cell migration.  Using whole human genome oligo (60-mer) arrays (Agilent), gene modulated by loss of LATS1/2 were identified and functionally grouped into categories including cell proliferation, cell death, cell adhesion and motility, as well as cell communication.  Selected genes, including known tumor suppressor genes and oncogenes such as CDKN1A, WISP2, SLIT2, TP53INP1, BIRC4BP, SPRY2, SPRY4, SPRED1, FAT4,and CYR61 were confirmed by qRT-PCR to be significantly differentially expressed.  Importantly, the collection of genes identified suggests that LATS1/2 functions through diverse mechanisms and multiple signaling pathways including the Hippo signaling pathway, as well as the p53, Ras-ERK, or WNT networks, in inhibit tumor progression.

overall_design: HeLa cells were treated with a negative control siRNA with scrambled sequence (siRNA-Control) or a combination of siRNA targeting LATS1 and siRNA targeting LATS2 (siRNA-LATS1/2).  Total RNA was extracted using TRIzol and RNA was further purified with RNease Mini Kit (Qiagen).  Whole Human Genome Oligo (60-mer) array gene expression analysis was performed with siRNA-LATS1/2 and siRNA-Control HeLa cells a total of 4 times.  Two biological replicates were performed, in which LATS1/2 were knocked-down in separate experiments and two technilogical replicates were performed in which the Cy3 and Cy5 dyes were flipped.  Data were normalized by the linear lowess method.  Genes that were 1.5-fold differentially expressed on 3 of the 4 arrays were scored as significant.  Furthermore, genes must meet a p-value of ≤ 0.05 based on a student t-test to be included in further analysis.  Molecular functions of genes were classified according to Gene Ontology.

first: Stacy

last: Visser


title: Gene expression signature of naïve and in vitro activated CD8 T cells in response to IL-12 and Type I IFN

status: Public on Jul 22 2009

city: Minneapolis

first: PUJYA

last: AGARWAL

state: MN

address: 6-246 NHH, 312 Church Street, SE

department: Laboratory,Medicine and Pathology

institute: University of Minnesota

summary:
Differentiation of naive CD8 T cells into cytotoxic effector cells requires three distinct signals- antigen (signal 1), costimulation -B7-1 (signal 2) and cytokine, either interleukin-12 or interferon-a/b (signal 3). Interaction of naive CD8 T cells with antigen and B7-1 programs cell division and proliferation whereas the presence of cytokines- IL-12 or IFNa/b promote survival, differentiation and memory establishment. In the absence of signal 3, the cells interacting with antigen/B7-1 undergo tolerance induction. The objective of this study was to elucidate the mechanisms how the provision of signal 3 promotes differentiation and averts tolerance induction in CD8 T cells.  Trichostatin A is a pharmacological agent that inhibits histone deacetylase activity, hence regulating chromatin structure and gene expression and differentiation in many cell types. Gene signature profiles of IL-12, IFNa/b and trichostatin A stimulated cells were compared to elucidate the molecular mechanisms of gene regulation.
Oligonucleotide microarray analysis is carried out to determine the extent and molecular nature of the CD8 T cell differentiation program induced by IL-12 or IFNa/b in concert with antigen and B7-1 signal.

overall_design: The programming for development of function and memory in presence of signal 3 occurs over three days of initial stimulation, and antigen-B7 and IL-12 or IFNa/b must be present for most of this period to achieve maximal responses. We analyzed gene expression in highly purified naive CD8 T cells at 0, 24, 48 and 72h of in vitro culture stimulated with antigen-B7 and with or without IL-12 or IFNa/b to tease apart gene expression profiles of naive, 2-signal and 3-signal stimulated cells over the course of 3-days. Gene expression of cells stimulated with trichostatin A for 72hr were compared with IL-12 or IFNa/b stimulated cells. 20 arrays.

first: Pujya

last: Agarwal

first: Matthew

middle: F

last: Mescher


title: Gene expression profiles of in vitro-infected (Leishmania major) and uninfected mouse bone marrow-derived macrophages

status: Public on Sep 04 2009

city: Parkville

first: Gordon

last: Smyth

state: Vic

address: 1G Royal Pde

department: Bioinformatics

country: Australia

institute: Walter and Eliza Hall Institute of Medical Research

summary:
Gene expression profiles of in vitro-infected (Leishmania major) and uninfected bone-marrow-derived macrophages from BALB/c, C57BL/6, C.B6-(lmr1, lmr2) and B6.c-(lmr1, lmr2) mouse strains.
We utilised microarrays to investigate a number of issues. Firstly, we determined which genes were differentially regulated in macrophages in response to infection with Leishmania major. Secondly, we hoped to gain some insight into the differences between C-B6-(lmr1, lmr2) congenic mice and their parental control BALB/c, as well as differences between B6-c-(lmr1, lmr2) and their parental control C57BL/6. This would aid us in the search for the genes underlying our loci, since the only genomic regions differing between the two strains are those of the two congenic intervals on chromosomes 9 and 17. Any genes differentially regulated would be contributing to the experimental infection differences observed between the congenic mice and their parental controls.

overall_design: L. major-infected and uninfected bone marrow-derived macrophages from C57BL/6, BALB/c, B6.c-(lmr1, lmr2) and C.B6-(lmr1, lmr2) mice were analyzed. Macrophages from 6 mice were pooled for each sample.

first: Anuratha

last: Sakthianandeswared

first: Colleen

last: Elso

first: Simon

last: Foote

first: Emanuela

last: Handman


title: NMD inhibition fails to identify tumour suppressor genes in microsatellite stable gastric cancer cell lines

status: Public on Sep 19 2009

city: Amsterdam

first: Daoud

last: Sie

address: De Boelelaan 1117

department: Pathology

institute: Vrije Universiteit Medical Center

type:
Expression profiling by array
Genome variation profiling by array

summary:
Gastric cancers frequently show chromosomal alterations which can cause activation of oncogenes, and/or inactivation of tumour suppressor genes. In gastric cancer several chromosomal regions are described to be frequently lost, but for most of the regions, no tumour suppressor genes have been identified yet. The present study aimed to identify tumour suppressor genes inactivated by nonsense mutation and deletion in gastric cancer by means of GINI (gene identification by nonsense mediated decay inhibition) and whole genome copy number analysis.
Material and Methods: Two non-commercial gastric cancer cell lines, GP202 and IPA220, were transfected with siRNA directed against UPF1, to specifically inhibit the nonsense mediated decay (NMD) pathway, and with siRNA directed against non-specific siRNA duplexes (CVII) as a control. Microarray expression experiments were performed in triplicate on 4x44 K agilent arrays by hybridizing RNA from UPF1-transfected cells against non-specific CVII-transfected cells. In addition, array CGH of the two cell lines was performed on 4x44K agilent arrays to obtain the DNA copy number profiles. Mutation analysis of GINI candidates was performed by sequencing.
Results:  UPF1 expression was reduced for >70% and >80% in the GP202 and IPA220 gastric cancer cell lines, respectively. Integration of array CGH and microarray expression data provided a list of 134 and 50 candidate genes inactivated by nonsense mutation and deletion for GP202 and IPA220, respectively. We selected 12 candidate genes for mutation analysis. Sequence analysis was performed on 11 genes. One gene, PLA2G4A, showed a silent mutation, and in two genes, CTSA and PTPRJ, missense mutations were detected. No nonsense mutations were detected in any of the 11 genes tested.
Conclusions: Although UPF1 was substantially repressed, thus resulting in the inhibition of the NMD system, we did not find genes inactivated by nonsense mutations. Our results show that the GINI strategy leads to a high number of false positives.

first: Tineke

last: Buffart

first: Marianne

last: Tijssen

first: Jamila

last: El-Bchiri

first: Alex

last: Duval

first: Mark

middle: A

last: van der Wal

first: Bauke

last: Ylstra

first: Raquel

last: Seruca

first: Gerrit

last: Meijer

first: Beatriz

last: Carvalho


title: BxPC-3 cells: growth in different nutrients

status: Public on Aug 17 2009

city: Salt Lake City

first: Mohan

middle: Rao

last: Kaadige

state: UT

address: 2000 Circle of Hope

department: Oncological Sciences

institute: Huntsman Cancer Institute

summary: The MondoA transcription factor forms a heterocomplex with its obligate partner Mlx to regulate ~75% of glucose-dependent transcription. By mediating glucose-induced activation of thioredoxin-interacting protein (TXNIP), MondoA directly represses glucose uptake. Given the predominant role of MondoA in controlling glucose-dependent transcription and glucose uptake, we asked whether glutamine regulates MondoA activity. Expression profiles from glucose and glutamine starved BxPC-3 cells (-G-Q) were compared with those from cells grown in glucose only (+G-Q), glutamine only (-G+Q) or glucose plus glutamine (+G+Q). As expected, TXNIP expression was highly induced by glucose. However, the addition of glutamine repressed the glucose-dependent induction of TXNIP. We show that glutamine inhibits MondoA-dependent transcriptional activation of TXNIP by triggering the recruitment of a histone deacetylase-dependent corepressor to the amino terminus of MondoA. Consistent with the repression of TXNIP, glucose uptake is elevated in cells grown in the presence of glucose and glutamine. Finally, alpha-ketoglutarate, a tricarboxylic acid cycle intermediate, also blocks MondoA-dependent activation of TXNIP and stimulates glucose uptake. Together, these results suggest that glutamine-dependent mitochondrial anapleurosis stimulates glucose uptake by restricting TXNIP expression via MondoA:Mlx complexes.

overall_design: Four growth conditons; four biological replicates

first: Don

last: Ayer


title: Convergence of Mutation and Epigenetic Alterations Identifies Common Target Genes in Breast and Colon Cancer

city: Ghent

first: Leander

last: Van Neste

address: Coupure Links 653

department: Molecular Biotechnology

country: Belgium

institute: Ghent University

summary:
The identification and characterization of tumor suppressor genes has enhanced our understanding of the biology of cancer and enabled the development of new diagnostic and therapeutic modalities.  Whereas in past decades, a handful of tumor suppressors have been slowly identified using techniques such as linkage analysis, large-scale sequencing of the cancer genome has enabled the rapid identification of a large number of genes that are mutated in cancer.  However, determining which of these many genes play key roles in cancer development has proven challenging.  Specifically, recent sequencing of human breast and colon cancers has revealed a large number of somatic gene mutations, but virtually all are heterozygous, occur at low frequency, and are tumor-type specific.
Keywords: Microarray, Hypermethylome, DNA-hypermethylation, DAC, TSA, Epigenetic, Mutation, Colorectal Cancer, Breast Cancer

overall_design:
The candidate hypermethylome genes were identified as described below. These genes were compared to the CAN genes identified by Sjöblom T et al (Science, 2006).
Cell culture and treatment. For drug treatments, log phase MCF7, T-47D, MDA-MB231 and MDA-MB-468 cells were cultured in McCoys 5A media (Invitrogen) containing 10% BCS and 1x penicillin/streptomycin with 5M 5aza-deoxycytidine (DAC) (Sigma; stock solution: 1mM in PBS) for 96 hours, replacing media and DAC every 24 hours. Cell treatment with 300nM Trichostatin A (Sigma; stock solution: 1.5mM dissolved in ethanol) was performed for 18 hours. Control cells underwent mock treatment in parallel with addition of equal volume of PBS or ethanol without drugs.
Microarray analysis. Total RNA was harvested from log phase cells using the Qiagen kit according to the manufacturers instructions, including a DNAase step. RNA was quantified using the NanoDrop ND-100 followed by quality assessment with 2100 Bioanalyzer (Agilent Technologies). RNA concentrations for individual samples were greater than 200ng/ul, with 28s/18s ratios greater than 2.2 and RNA integrity numbers of 10 (highest). Sample amplification and labeling procedures were carried out using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies) according to the manufacturers instructions. The labeled cRNA was purified using the RNeasy mini kit (Qiagen) and quantified. RNA spike-in controls (Agilent Technologies) were added to RNA samples before amplification. 0.75 microgram of samples labeled with Cy3 or Cy5 were mixed with control targets (Agilent Technologies), assembled on Oligo Microarray, hybridized, and processed according to the Agilent microarray protocol. Scanning was performed with the Agilent G2565BA microarray scanner under default settings recommended by Agilent Technologies.
Data analysis. All arrays were subject to quality checks recommended by the manufacturer. Images were visually inspected for artifacts and distributions of signal and background intensity of both red and green channels were examined to identify anomalous arrays. No irregularities were observed, and all arrays were retained and used. All calculations were performed using the R statistical computing platform and packages from Bioconductor bioinformatics software project. The log ratio of red signal to green signal was calculated and Loess normalization as implemented in the limma package from Bioconductor.

first: Sabine

last: Glöckner

first: Timothy

last: Chan

first: Kornel

last: Schuebel

first: Nita

last: Ahuja

first: Stephen

middle: B

last: Baylin


title: GENE PROFILING AND PATHWAY ANALYSIS OF NASP EXPRESSION IN HELA CELLS

status: Public on Aug 24 2009

city: Chapel Hill

first: Oleg

middle: M

last: Alekseev

address: 210 Taylor Hall

department: Cell and Developmental Biology

institute: University of North Carolina at Chapel Hill

summary:
Background
NASP (Nuclear Autoantigenic Sperm Protein) is a linker histone chaperone required for normal cell division. Changes in NASP expression significantly affect cell growth and development; loss of gene function results in embryonic lethality. However, the mechanism by which NASP exerts its effects in the cell cycle is not understood. To understand the pathways and networks that may involve NASP function, we evaluated gene expression in HeLa cells in which NASP was either overexpressed or inhibited by siRNA.
Methods
Total RNA was converted to cRNA with incorporation of Cy5-CTP (experimental samples), or Cy3-CTP (control samples). The labeled cRNA samples were hybridized to oligonucleotide microarrays.
Results
From approximately 36 thousand genes present in a total human genome microarray, we identified a set of 57 up-regulated and 11 down-regulated genes as a result of NASP overexpression. Similarly we identified a set of 68 up-regulated and 92 down-regulated genes as a result of NASP siRNA treatment. Gene ontology, molecular network and canonical pathway analysis of NASP overexpression demonstrated that the most significant changes were in proteins participating in organismal injury, immune response and cellular growth and cancer pathways (major “hubs”: TNF, FOS, EGR1, NFkB, IRF7, STAT1, IL6). Inhibition of NASP elicited the changed expression of proteins involved in DNA replication, repair and development, followed by reproductive system disease, and cancer and cell cycle pathways (major “hubs”: E2F8, TP53, FGF, FSH, FST, hCG, NFkB, TRAF6).
Conclusions
This study has demonstrated that NASP belongs to a network of genes and gene functions that are critical for cell survival. Most of these genes have not been previously reported to be connected to expression levels of NASP. Gene ontology and protein network analysis identified general biological processes as well as individual genes/gene products and possible interaction networks. Networks with cancer-related functions had the highest significance. We found signaling pathways which were affected as a result of changed NASP expression. This analysis revealed that, despite some overlap, each reactive response was associated with a unique gene signature. These results place NASP in important cell regulatory networks and suggest a number of directions for further research.

overall_design:
Total cellular RNA was purified from HeLa cells using RNeasy ® Mini Kit (Qiagen, Valencia, CA) according to manufacturers’ instructions. RNA samples representing four separate experiments from cells overexpressing tNASP and four experiments from cells treated with NASP siRNA, along with appropriate controls, were submitted for analysis. After the RNA Quality check was performed the samples of RNA from treated cells were amplified with incorporation of Cy5-CTP (fluorescent in the red region), while control samples were labeled by Cy3-CTP (fluorescent in the green region) and purified. The labeled cRNA samples were then fragmented in fragmentation buffer at 60°C for 30 min before the microarray hybridization. Each sample was hybridized to a whole separate Human Genome (4×44K) microarray (Agilent Technologies, Wilmington, DE) overnight at 65°C in a hybridization oven. The hybridization slides were washed, stabilized, dried, and immediately scanned by Agilent Technologies Microarray Scanner (Agilent Technologies, Wilmington, DE). RNA hybridization was performed in the Genomics Core and Microarray Facility (Lineberger Comprehensive Cancer Center, UNC-CH) according to the protocol suggested by Agilent (Agilent Technologies, Wilmington, DE).
Statistical analysis – During the initial analysis at UNC Microarray Database, all genes were retrieved, appropriately annotated, and filtered. Eventually, only genes with an absolute value of a Log2 Red/Green Lowess Normalized Ratio of at least 1 (doubled in intensity) for all 4 arrays were selected.

first: Richard

middle: T

last: Richardson

first: Michael

last: O'Rand


title: Iodide effect on mRNA expression levels of genes related to immunological function in cultured human thyroid follicles

status: Public on Jul 14 2009

first: Kanji

last: Sato

state: Tokyo

summary: We are going to submit a paper entitled “Iodide-induced Chemokines and Genes   Related to Immunological Function in Cultured Human Thyroid Follicles” to THYROID

overall_design: One conditioned experiments, control NaI 10-8M vs. NaI 10-5M , cultured for 3 hours or 48 hours.

first: Kazuko

last: Yamazaki


title: Polycomb CBX7 represses expression of tumor suppressor genes

summary:
Polycomb CBX7 was stably expressed in embryonal carcinoma cells in order to identify target genes.  The study was specifically geared toward unveiling gene targets that are susceptible to DNA hypermethylation in adult cancer.  A number of CBX7 target tumor suppressor genes were thereby identified and used in further studies to characterize the mechanism surrounding CBX7 mediated gene suppression.
Keywords: CBX7 target gene identification

overall_design: A cDNA encoding the ORF of polycomb CBX7 under the control of EF1alpha promoter was transfected into the Tera-2 embryonal carcinoma cell line.  Empty vector was used to transfect control cells.  The cells were subject to stable selection using puromycin and a pooled population of CBX7 overexpressing cells was established.  Gene expression analysis was performed by comparing the gene expression profile of CBX7 cells to empty vector control cells.

first: Helai

middle: P

last: Mohammad


title: Lenses expression profile of DBL oncogene transgenic mice

status: Public on Jun 01 2009

city: Genoa

first: Paolo

last: Fardin

address: Largo G. Gaslini 5

department: Molecular Biology

institute: Istiuto Giannina Gaslini

summary: The Dbl family of proteins represents a large group of proto-oncogenes involved in cell growth regulation. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders and neoplastic transformation. We have generated transgenic mice introducing the onco-Dbl cDNA sequences linked to the metallothionein promoter into the germ line of FVB mice and found that onco-Dbl expression affected proliferation, migration and differentiation of lens epithelial cells.  We used high density oligonucleotide microarray to define the transcriptional profile induced by Dbl in the lenses of transgenic mice and observed modulation of genes encoding proteins promoting epithelial-mesenchymal transition (EMT). Moreover, genes encoding proteins involved in the positive regulation of apoptosis were markedly down regulated while anti-apoptotic genes were strongly up-regulated. Finally, several genes encoding proteins involved in the process of angiogenesis were up-regulated. These observations were validated by histological and immunohistochemical examination of the transgenic lenses, where vascularization can be readily observed. Thus, onco-Dbl expression in mouse lenses induces disruption of the lens architecture, epithelial cell proliferation, EMT, evasion from cell death, and aberrant angiogenesis.

overall_design: Whole lenses were removed from 2, 14, and 42 day old Dbl transgenic and wild type mice. 4 to 8 individual lenses were pooled . Three independent pools from each time and condition were used as replicates.

first: Marzia

last: Ognibene

first: Cristina

last: Vanni

first: Amleto

last: Desantanna

first: Luigi

last: Varesio

first: Alessandra

last: Eva


title: Gene expression profiles of reactive stroma in prostate cancer

city: Houston

first: Chad

last: Creighton

state: TX

address: One Baylor Plaza, Mail Stop: BCM305

department: Biostatistics, Ducan Cancer Center

institute: Baylor College of Medicine

summary:
This is an analysis of stroma from 17 Grade 3 reactive stroma prostate cancer tissues and matched normal peripheral zone tissues.

Keywords: two group comparison

overall_design: Laser capture microdissection and expression array analysis of stroma from 17 Grade 3 reactive stroma prostate cancer tissues and matched normal peripheral zone tissues.

last: Ittmann

first: Olga

last: Dakhova


title: Analysis of the Heat Shock Response in Mouse Liver Reveals Transcriptional Dependence on the Nuclear Receptor PPARα

status: Public on May 01 2009

city: Research Triangle Park

first: beena

last: vallanat

address: 109 Tw Alexanser Dr

institute: EPA

summary:
The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance.  Many of these genes are also transcriptionally regulated by heat shock (HS) through activation by HS factor-1 (HSF1).  We hypothesized that there are interactions on a genetic level between PPARα and the HS response mediated by HSF1.  Wild-type and PPARα-null mice were exposed to HS, the PPARα agonist WY-14,643 (WY), or both; gene and protein expression was examined in the livers of the mice 4 or 24 hrs after HS.  Gene expression profiling identified a number of Hsp family members that were altered similarly in both mouse strains.  However, most of the targets of HS did not overlap between strains.  A subset of genes was shown by microarray and RT-PCR to be regulated by HS in a PPARα-dependent manner.  HS also down-regulated a large set of mitochondrial genes specifically in PPARα-null mice that are known targets of PPARg co-activator 1 (PGC-1) family members.  Pretreatment of PPARα-null mice with WY increased expression of PGC-1b and target genes and prevented the down-regulation of the mitochondrial genes by HS.  A comparison of HS genes regulated in our dataset with those identified in wild-type and HSF1-null mouse embryonic fibroblasts indicated that although many HS genes are regulated independently of both PPARα and HSF1, a number require both factors for HS responsiveness.  These findings demonstrate that the PPARα genotype has a dramatic effect on the transcriptional targets of HS and support an expanded role for PPARα in the regulation of proteome maintenance genes after exposure to diverse forms of environmental stress including HS.

Keywords: Gene Expresssion-Heat shock

overall_design: total 24 samples, 3 per group, for controls and treated.

first: Beena

last: Vallanat

first: Steven

last: Anderson

first: Holly

last: Brown-Borg

first: Hongzu

last: Ren

first: Sander

last: Kersten

first: Sudhakar

last: Jonnalagadda

first: Rajagopalan

last: Srinivasan

first: J

last: Corton


title: Long intronic non-coding RNA from the KLHL29 locus regulates proliferation of human tumorigenic cell lines

status: Public on Jul 30 2009

city: São Paulo

first: Eduardo

last: Reis

address: Av. professor Lineu Prestes, 748

department: Departamento de Bioquímica

country: Brazil

institute: Instituto de Química da Universidade de São Paulo

summary: Many classes of noncoding RNAs (ncRNAs) are known to be involved in gene expression regulation; however, long intronic ncRNAs have received little attention.  In previous works, our group has provided evidence that intronic regions, transcribed from 80% of all RefSeq gene loci, are key sources of potentially regulatory ncRNAs. Intronic transcripts were shown to be correlated to the degree of prostate cancer differentiation, regulated by physiological stimuli, and highly expressed in genomic loci related to transcriptional regulation. In the present work we aimed at functional characterization of KLHL29 intronic transcript, one of the most highly expressed in human tissues. Although the protein-coding KLHL29 transcripts are still poorly studied, genes from this family are involved in a wide variety of biological processes, including cell cycle regulation mechanisms. We have observed that the long intronic ncRNA is predominantly transcribed from the opposite strand at the KLHL29 locus, and that the ncRNA overexpression does not affect the levels of protein-coding mRNAs from the same locus. Intriguingly, we found 2,002 genes with significant (q< 5.3%) differential expression between HEK293 cells overexpressing KLHL29 sense (S) or antisense (AS) intronic transcript. Within these genes, GO categories such as ‘Regulation of cell cycle’ and ‘Transcription’ were significantly enriched. Furthermore, human tumorigenic cell lines overexpressing KLHL29 AS intronic transcript showed increased proliferation rates in vitro and increased tumorigenic capacity in nude mice. In conclusion, we suggest that KLHL29 long intronic ncRNA controls the cell cycle mechanisms, probably through a fine-tuning regulation of gene expression.

overall_design: Genes were defined as expressed when their intensity signals were above the average negative control value plus three standard deviations; a set of 153 negative controls (Agilent Technologies) was present on the array. Only genes that were detected as expressed in all four replicates of the same HEK293 transfection (or not expressed in the four replicates) were used in further analysis. The intensity values between experiments were normalized by the 40% trimmed mean intensity.  For each transfection, one entire replicate experiment was excluded from the analyses, based on evaluating the coefficient of variance among the samples (for S KLHL29 intronic transcript, exclusion of the outlier replicate decreased the coefficient of variance from 0.21-0.24 (obtained by excluding any other replicate) to 0.19; for AS transcript, the coefficient of variance decreased from 0.17-0.18 to 0.12). Differentially expressed genes were identified using three transfection replicates for each construction, employing the Significance Analysis of Microarrays (SAM) tool (Tusher et al., 2001) with the following parameters: two-class response, 100 permutations and K-Nearest Neighbors Imputer. We applied 5.4 or 10.3% FDR (False Discovery Rate) cutoffs, as described in the results. A fold-change filter (1.4-fold cutoff) was applied after identification of significant differentially expressed genes.

first: Anna

last: Smirnova

first: Rodrigo

last: Louro

first: Debora

last: Loturco

first: Endrigo

middle: I

last: Orsini-Palma

first: Camila

last: Brito-Leite

first: Nadja

last: Soares-Andrade

first: Luiz

last: Dufner-Almeida

first: Sergio

last: Verjovski-Almeida


title: miR101 overexpression vs. EZH2 RNAi

status: Public on Apr 28 2009

city: Ann Arbor

first: Christopher

last: Maher

address: 1500 E. Medical Center Drive

institute: Michigan Center for Translational Pathology

summary:
Transcriptional analysis of genes in SKBr3 cells.

Enhancer of zeste homolog 2 (EZH2) is a mammalian histone methyltransferase that contributes to the epigenetic silencing of target genes and regulates the survival and metastasis of cancer cells. EZH2 is overexpressed in aggressive solid tumors by mechanisms that remain unclear. Here we show that the expression and function of EZH2 in cancer cell lines are inhibited by microRNA-101 (miR-101).

overall_design: Two-condition experiment: miR101 overexpression vs. EZH2 RNAi in SKBr3 breast cancer cells.


title: Genome-Wide Discovery of STAT3 Functional Binding Sites

status: Public on May 05 2009

city: Torino

first: Raffaele

last: Calogero

state: To

address: Via Nizza 52

department: Molecular Biotechnology Center

institute: University of Torino

summary:
STAT3 is a transcription factor playing a crucial role in inflammation, immunity and oncogenesis, able to induce distinct subsets of target genes in different cell types or under different conditions. Identification of direct transcriptional targets however has only defined a relatively limited set of genes, not sufficient to explain its variegated functions. In order to improve our understanding of the STAT3 transcriptional network we decided to develop a computational approach for the discovery of STAT3 functional binding sites. Upon generating a Positional Weight Matrix to define STAT3 binding sites, we applied a loglikelihood ratio scoring function and were able to assign affinity scores with very high specificity (93.5%) as measured by EMSA. STAT3 binding sites scoring above a stringent threshold have been identified genome-wide in Homo sapiens and Mus musculus and selected for phylogenetic conservation by genomic sequence alignment using eight vertebrate species. Validation was carried out on a subset of predicted
sites within genes previously identified as STAT3-responsive by microarray analysis. The high percentage of sites able to bind STAT3 in vivo, as assessed by Chromatin Immunoprecipitation (ChIP) analysis, revealed the high predictive power of our method.

overall_design: Three prototypic situation were investigated using two replications for each experimental point: STAT3+/+ versus STAT3-/- MEFs , STAT3+/+ versus STAT3+/+ treated with OSM and STAT3-/- versus STAT3-/- treated with OSM.

first: Francesco

last: Vallania

first: Davide

last: Schiavone

first: Sarah

last: Dewilde

first: Emanuela

last: Pupo

first: Serge

last: Garbay

first: Marco

last: Pontoglio

first: Paolo

last: Provero

first: Valeria

last: Poli


title: A Novel Androgen Receptor Splice Variant Is Upregulated during Prostate Cancer Progression

status: Public on Mar 17 2009

city: Baltimore

first: Hegang

last: Chen

address: 660 West Redwood Street

institute: University of Maryland School of Medicine

summary: The androgen receptor (AR) plays a key role in progression to incurable androgen-ablation resistant prostate cancer (PCA).  We have identified three novel AR splice variants lacking the ligand binding domain (designated as AR3, AR4 and AR5) in hormone insensitive PCA cells. AR3, one of the major splice variants expressed in human prostate tissues, is constitutively active and its transcriptional activity is not regulated by androgens or antiandrogens. Immunohistochemistry analysis on tissue microarrays containing 429 human prostate tissue samples shows that AR3 is significantly upregulated during PCA progression and AR3 expression level is correlated with the risk of tumor recurrence after radical prostatectomy. Overexpression of AR3 confers ablation-independent growth of PCA cells while specific knock-down of AR3 expression (without altering AR level) in hormone resistant PCA cells attenuates their growth under androgen-depleted conditions in both cell culture and xenograft models, suggesting an indispensable role of AR3 in ablation-independent growth of PCA cells.  Furthermore, AR3 may play a distinct yet essential role in ablation-independent growth through regulating a unique set of genes including AKT1, which are not regulated by the prototype AR. Our data suggest that aberrant expression of AR splice variants may be a novel mechanism underlying ablation-independence during PCA progression and AR3 may serve as a prognostic marker to predict patient outcome in response to hormonal therapy. Given that these novel AR splice variants are not inhibited by currently available anti-androgen drugs, development of new drugs targeting these AR isoforms may potentially be effective for treatment of ablation-resistant PCA.

overall_design: Total RNA was extracted from CWR-R1 and 22Rv1 cells treated with shAR3-1, shARa and the scrambled shRNA control, respectively. Each of CWR-R1 and 22Rv1 cells treated with shAR3-1 was compared with the scrambled shRNA control. The same experiments were performed for the cells treated with shARa.


title: Strain-specific Variation in Mouse Gene Expression and the Onset of Gap Junction Mutation-induced Cataracts

city: Chicago

first: Nalin

last: Kumar

address: 1855 West Taylor Street

department: Ophthalmology and Visual Sciences

institute: University of Illinois at Chicago

summary:
Disruption of the mouse gene encoding the gap junction subunit alpha3 connexin 46 (Cx46) results in the formation of lens cataracts. The timing of the onset of this lens opacity is affected by the genetic background, i.e. the mouse strain. To elucidate the mechanism by which cataracts form in the 129Sv/Jae strain earlier than in the C57BL/6J strain, global gene expression was quantitated in the lenses of these strains. Lens cDNAs were analyzed by hybridization to DNA microarrays and with real time-PCR. Theories are proposed based on the observed higher level of expression of the stress-response genes in the C57BL/6J strain and variations in the expression levels of genes involved in protein synthesis, metabolism, catabolism and cell proliferation. How these variations in gene expression might affect the response of lens fiber cells to the increased calcium, caused by lack of alpha3Cx46, is considered. The possibility that the proteins coded by the strain-variable genes might influence the cataract-associated proteolysis of gamma-crystallin is also addressed.
Keywords: Lens, cataract, microarray, connexin 46, gene knock out

overall_design: To determine differences in transcript expression between the lenses of two mouse wild type strains (129 SvJae and C57BL/6J), as well as between alpha3Cx46 KO and wild-type mice. The former comparison may lead to identification of potential candidate(s) genes that prevent (or promote) cataract formation, whereas the latter comparison may provide insights into the mechanism by which cataract formation occurs in the alpha3Cx46 KO mice. Total 8 samples were used ( two separate samples for each of the following 4 types of mice: 129SvJae wild type, 129SvJae alpha3Cx46 KO, C57BL/6J wild type and C57BL/6J alpha3Cx46 KO)

first: Yajun

last: Tang

first: Thomas

middle: E

last: Crowley