title: Irradiated mouse: Irradiated mouse vs. non-irradiated mouse

city: sendai-city

first: Taisuke

last: Baba

state: Miyagi

address: seiryo-machi 4-1

institute: Tohoku Univ. IDAC

summary:
Adopting boron neutron capture, we separately irradiated Kupffer cells and endothelial cells in mouse liver in vivo and the changes in gene transcriptions were analyzed by an oligonucleotide microarray.
Keywords: Gene expression

overall_design: control(non-irradiated mouse liver) vs. Endothelial(alpha-particle), Kupffer(alpha-particle), neutron or Gamma irradiated mouse liver

first: Mehryar

middle: H

last: Roudkenar

first: Li

last: Li

first: Yoshikazy

last: Kuwahara

first: Hironobu

last: Nakagawa

first: Lu

last: Wang

first: Satoshi

last: Kasaoka

first: Yasuhito

last: Ohkubo

first: Koji

last: Ono

first: Manabu

last: Fukumoto


title: Differentially Expressed Genes in the Peripheral Blood of Patients in Cervical Cancer

status: Public on Oct 20 2007

city: TauYau

first: Yun-Shien

last: Lee

state: Taiwan

address: TauYau

department: Dept. Biotechnology

country: Taiwan

institute: Ming Chuan Univ

summary:
We prospectively investigated gene expression profiles of cervical cancer patients undergoing concurrent chemoradiation. Upregulated genes in the circumstance of anemia were analyzed. Peripheral blood of 20 patients (bulky stage IB –IVA cervical squamous cell carcinomas) undergoing concurrent chemoradiation at four time-points was collected. Total RNA extracted by PaxGene Blood RNA System was analyzed with microarrays and MetaCoreTM functional network analyses. Fifty-three genes were significantly differentially expressed during concurrent chemoradiation. Fetal and embryonic hemoglobin genes were upregulated when patients had been severely myelosuppressed. Twenty-eight genes significantly correlated with the hemoglobin genes are involved in responses to hypoxia and oxygenation, TGF-β signaling, cell cycle suppression, G-protein signaling, and transcriptional regulation. C-Myc has the highest rank in transcriptional co-regulation. Besides, IGKV1D-13 was significantly downregulated in patients with severe hematologic toxicity. These approaches identified biological processes in peripheral blood modulated by concurrent chemoradiation and subsequent anemia.
Keywords: chemoradiation, microarray, network analysis

overall_design: peripheral blood of 54 patients with common reference, cDNA microarray, replication by dye-swapping

first: Angel

last: Chao

first: Tzu-Hao

first: Chyong-Huey

last: Lai


title: 46,XY sex reversed females with complete androgen insensitivity syndrome

city: Kiel

first: Paul-Martin

last: Holterhus

address: Campus Kiel, Schwanenweg 20

department: Pädiatrische Endokrinologie und Diabetologie

country: Germany

institute: Universitätsklinikum Schleswig-Holstein

summary:
This gene set contains skin fibroblasts from either labia majora of 46,XY sex reversed females having complete androgen insensitivity syndrome due to inactivation mutations of the androgen receptor gene and from the scrotum of normal males. Both, labia majora and scrotum origin from the same embryological anlagen, the labioscrotal swellings. The phenotypic difference is due to androgen dependent virilization in males. This is not possible in 46,XY patients with complete androgen insensitivity syndrome because the androgen receptor pathway is knocked out.
A cell type comparison design experiment design type compares cells of different type for example different cell lines.
Cell Line: genital skin fibroblasts from different locations
mutant line: normal 46,XY male and 46,XY sex reversed female due to inactivating mutations of the androgen receptor gene
Keywords: cell_type_comparison_design

overall_design: Computed


title: CGH mapping of Genetic Deficiencies balanced by sDp3 on LGIII

status: Public on Oct 23 2007

city: Vancouver

first: Martin

middle: Richard

last: Jones

state: BC

address: Room 419 NCE Building 2125 East Mall

department: Medical Genetics

country: Canada

institute: University of British Columbia

type: Genome variation profiling by genome tiling array

summary:
Background: A collection of genetic deficiencies covering over 70% of the Caenorhabditis elegans genome exists, however the application of these valuable biological tools has been limited due to the incomplete correlation between their genetic and physical characterization.
Results: We have applied oligonucleotide array Comparative Genomic Hybridization (oaCGH) to the high resolution, molecular characterization of several genetic deficiency and duplication strains in a 5Mb region of Chromosome III. We incorporate this data into a physical deficiency map which is subsequently used to direct the positional cloning of essential genes within the region. From this analysis we are able to quickly determine the molecular identity of several previously unidentified mutations.
Conclusion: We have applied accurate, high resolution molecular analysis to the characterization of genetic mapping tools in Caenorhabditis elegans. Consequently we have generated a valuable physical mapping resource, which we have demonstrated can aid in the rapid molecular identification of mutations of interest.
Keywords: C.elegans Deficiencies CGH

overall_design: 3 test samples from sDp3 balanced strains individually hybridized to reference DNA (N2 Bristol)

middle: R

first: David

middle: L

last: Baillie

first: Donald

middle: G

last: Moerman

first: Stephane

last: Flibotte

first: Jason

middle: S

last: Maydan


title: Fibroblasts from genital and extragenital regions of 46,XY individuals

summary:
This dataset contains fibroblasts from different genital and extragenital regions, mostly from 46,XY individuals. Due to proven androgen receptor gene mutations, several individuals have varying degrees of undervirilization of the external genitalia ranging from complete androgen insensitivity syndrome (CAIS) to partial androgen insensitivity syndrome (PAIS). CAIS is a completely female person despite a 46,XY karyotype. AIS-3 individuals have ambigous external genitalia. AIS-4 means predominantly female (clitoral enlargement), AIS-2 means predominantly male (hypospadias).  It is important for this experiment set, that it contains data from 3 differet independent datasets that are not directly cmparable with each other. This is because of different reference RNAs and different glass slides. The particular normalization procedure is described in the paper accompanying this dataset. Only then, all arrays are allowed to be clustered together. Dataset 2 and 3 have "DS-2" and "DS-3" in their experiment names. All other experiments belong to dataset 1.
A cell type comparison design experiment design type compares cells of different type for example different cell lines.
Disease State: normal male, normal female or ambiguous external genitalia according to grading scheme by Sinnecker et al.
Cell Line: fibroblast cell lines from different regions of the genital skin, some from extragenital skin
Sex/Mating type: 46,XY or 46,XX karyotype
Keywords: cell_type_comparison_design


title: Establishment of a predictive clinicogenomic model for FEC100 regimen in node-positive breast cancer patients

status: Public on Oct 18 2007

city: Saint Herblain

first: Wilfried

last: Gouraud

address: bd Jacques Monod

department: Integrated Center of Oncology René Gauducheau

country: France

institute: ICO (Integrated Center of Oncology Nantes)

summary:
Breast cancer is a hugely heterogeneous disease, and markers for disease subtypes and therapy response remain poorly defined. For that reason, we employed a retrospective study in node-positive breast cancer to identify molecular signatures of gene expression correlating with metastatic free survival. Patients were primarily included in FEC100 (fluorouracil, epirubicin and cyclophosphamide) arms of two multicentric phase III clinical trials (PACS01 and PEGASE01 - FNCLCC). Data from nylon microarrays containing 8.032 cDNA unique sequences, representing 5.776 distinct genes, have been used to develop a predictive model for treatment outcome. We obtained the gene expression profiles of 150 population-based patients, and used stringent univariate selection technique based on Cox regression combined with principal component analysis to identify signature associated with prognosis and impact of FEC100 chemotherapy. Our work identified a gene-signature of metastatic relapse. Most of the 14 selected genes have a clear role in breast cancer, neoplasia or chemotherapy resistance. Furthermore, we showed the interest of combining transcriptomic data with clinical data into a clinicogenomic model for patients subtyping. The described model adds predictive accuracy to that provided by the well established Nottingham prognostic index or by the genomic predictor alone.
Keywords: Gene-expression profiling

overall_design: 150 breast cancer patients at diagnosis examined with spotted cDNA nylon membrane

first: Mario

last: Campone

first: Loïc

last: Campion

first: Henry

last: Roché

first: Catherine

last: Charbonnel

first: Florence

last: Magrangeas

first: Stéphane

last: Minvielle

first: Jean

last: Genève

first: Anne-Laure

last: Martin

first: Régis

last: Bataille

first: Pascal

last: Jézéquel


title: 0.2 mM As(III) stressed yap1 deletion mutant vs 0.2 mM As(III) stressed wild-type after 1 hour

city: Göteborg

first: Erik

last: Kristiansson

address: Chalmers Tvärgata 3

department: Department of Mathematical Sciences

country: Sweden

institute: Chalmers University of Technology

summary:
Arsenic is ubiquitously present in nature and various mechanisms have
evolved enabling cells to evade toxicity and acquire tolerance. Herein, we
explored how Saccharomyces cerevisiae (budding yeast) respond to trivalent
arsenic (arsenite) by quantitative and kinetic transcriptome, proteome and
sulfur metabolite profiling. Arsenite exposure affected transcription of
genes encoding functions related to protein biosynthesis, arsenic
detoxification, oxidative stress defense, redox maintenance and
proteolytic activity. Importantly, enzymes involved in sulfate assimilation
and glutathione biosynthesis were induced at both gene and protein levels.
Kinetic metabolic profiling evidenced a significant increase in the pools of
sulfur metabolites as well as elevated glutathione levels. Moreover, the
flux in the sulfur assimilation pathway as well as the glutathione synthesis
rate strongly increased with a concomitant reduction of sulfur incorporation
into proteins. By combining comparative genomics and molecular analyses, we
pin-pointed transcription factors that mediate thecore of the
transcriptional response to arsenite. Taken together, our data reveals that
arsenite-exposed cells channel a large part of assimilated sulfur into
glutathione biosynthesis and we provide evidence that the transcriptional
regulators Yap1p and Met4p control this response in concert.
Keywords: stress

overall_design: Direct comparisons

first: Michael

last: Thorsen

first: Markus

last: Tamas

first: Olle

last: Nerman


title: 0.2 mM As(III) stressed met4 deletion mutant vs 0.2 mM As(III) stressed wild-type after 1 hour

overall_design: Direct comparison


title: The small RNA repertoire of Dictyostelium discoideum

status: Public on Oct 17 2007

city: Uppsala

first: Johan

last: Reimegård

address: Biomedical Center

department: Department of cell and molecular biology

institute: Uppsala University

summary:
Small RNAs play crucial roles in regulation of gene expression in many eukaryotes. Here, we report the cloning and characterization of 18-26 nt RNAs in the social amoeba Dictyostelium discoideum. This survey uncovered developmentally regulated microRNA candidates whose biogenesis, at least in one case, is dependent on a Dicer homolog, DrnB. Furthermore, we identified a large number of 21 nt RNAs originating from the DIRS-1 retrotransposon, clusters of which have been suggested to constitute centromeres. Small RNAs from another retrotransposon, Skipper, were significantly up-regulated in strains depleted of the second Dicer-like protein, DrnA, and a putative RNA-dependent RNA polymerase, RrpC. In contrast, the expression of DIRS-1 small RNAs was not altered in any of the analyzed strains. This suggests the presence of multiple RNAi pathways in D. discoideum. In addition, we isolated several small RNAs with antisense complementarity to mRNAs. Three of these mRNAs are developmentally regulated. Interestingly, all three corresponding genes express longer antisense RNAs from which the small RNAs may originate. In at least one case, the longer antisense RNA is complementary to the spliced but not the unspliced pre-mRNA, indicating synthesis by an RNA-dependent RNA polymerase.
Keywords: cDNA library; small RNA sequencing

overall_design:
The aim of this study was to investigate the small RNA (18-26 nt) profile of Dictyostelium discoideum during growth and development. For this reason, we cloned and sequenced pooled small RNAs from growing single cells and from two different multicellular stages (16 and 24 hours of development).

cDNA libraries of 18-26 nt D. discoideum RNAs were constructed according to two different protocols (Lee, R.C. and Ambros, V. (2001)  Science, 294; Lau, N.C. et al (2001) Science, 294). Briefly, total RNA was isolated from growing D. discoideum AX4 strain cells as well as from cells developed for 16 hours and 24 hours, and the fractions were subsequently pooled. After size fractionation and ligation of a 3’ linker, the RNA was divided into two fractions, one of which was directly ligated to a 5’ linker, thus selecting for small RNAs with 5’ monophosphates. Following RT-PCR, the PCR fragments were cloned and sequenced. The second fraction was subjected to reverse transcription directly after 3’ ligation. Apart from synthesizing a complementary DNA strand, the reverse transcriptase adds a few non-templated C residues at the 3’ end. These C:s were then hybridized to a DNA oligo with three 3’ G:s followed by RT-PCR, cloning and sequencing. This approach is insensitive of the nature of the 5’ end of the small RNA.

first: Andrea

last: Hinas

first: Gerhart

last: Wagner

first: Wolfgang

last: Nellen

first: Victor

last: Ambros

first: Fredrik

last: Söderbom


title: 0.2 mM As(III) stressed wild-type vs (non-stressed) wild-type after 1 hour

overall_design: Stressed wild-type 0.2 mM 1h versus untreated wild-type using direct comparisons.


title: Rsd timecourse

status: Public on Oct 22 2007

city: Ikoma

first: Naotake

last: Ogasawara

state: Nara

country: Japan

institute: Nara Institute of Science and Technology

summary:
The gene expression profile of MC4100 delta rsd with pACYC Rsd and pACYC delta BS plasmids has been analysed using whole genome oligonucleotide microarrays. Control RNA was isolated from 50ml LB cultures of MC4100 delta rsd strain with pACYC delta BS. Experimental RNA was isolated from 50ml LB cultures of MC4100 delta rsd with pACYC Rsd, for each growth condition (OD600 = 0.4: log phase, 0.9: late log phase, 1.1: transition phase, ST(OD600 = >2.0): stationary phase). The experiments performed replicate.
Keywords: timecourse (overproduction of Rsd)

first: Taku

last: Oshima

first: Stephen

middle: J

last: Busby

first: Jon

last: Hobman


title: Association of cohesin and Nipped-B with transcriptionally active regions of the Drosophila melanogaster genome

city: Saint Louis

first: Dale

last: Dorsett

state: MO

address: 1100 South Grand Boulevard

department: Biochemistry and Molecular Biology

country: USA

institute: Saint Louis University School of Medicine

type: Genome binding/occupancy profiling by genome tiling array

summary:
The cohesin complex is a chromosomal component required for sister chromatid cohesion that is conserved from yeast to man. The similarly conserved Nipped-B protein is needed for cohesin to bind to chromosomes. In higher organisms, Nipped-B and cohesin regulate gene expression and development by unknown mechanisms. Using chromatin immunoprecipitation, we find that Nipped-B and cohesin bind to the same sites throughout the entire non-repetitive Drosophila genome. They preferentially bind transcribed regions and overlap with RNA polymerase II. This contrasts sharply with yeast, where cohesin binds almost exclusively between genes. Differences in cohesin and Nipped-B binding between Drosophila cell lines often correlate with differences in gene expression. For example, cohesin and Nipped-B bind the Abd-B homeobox gene in cells in which it is transcribed, but not in cells in which it is silenced. They bind to the Abd-B transcription unit and downstream regulatory region, and thus could regulate both transcriptional elongation and activation. We posit that transcription facilitates cohesin binding, perhaps by unfolding chromatin, and that Nipped-B then regulates gene expression by controlling cohesin dynamics. These mechanisms are likely involved in the etiology of Cornelia de Lange syndrome, in which mutation of one copy of the NIPBL gene encoding the human Nipped B ortholog causes diverse structural and mental birth defects.
Keywords: ChIP-chip, cell type comparison, protein comparison

overall_design: Binding of the Smc1 subunit to the Drosophila melanogaster genome was determined and compared in Kc, Sg4 and ML-DmBG3 cells by ChIP-chip. The Nipped-B cohesin loader and RNA polymerase II binding were mapped and compared in Sg4 and ML-DmBG3 cells, and the SA cohesin subunit was mapped in Sg4 cells. Each ChIP-chip experiment used at least two independent chromatin preparations, two antibody immunoprecipitation samples, and two control (mock immunoprecipitation and/or input chromatin) samples. Binding of Nipped-B to the cohesin subunits was compared in Sg4 and ML-DmBG3 cells. Nipped-B and cohesin binding were compared to RNA polymerase II binding in Sg4 and BG3 cells. Nipped-B binding was compared between Sg4 and ML-DmBG3 cells.


title: Time Course 1.0 mM As(III)

summary:
Arsenic is ubiquitously present in nature and various mechanisms have
evolved enabling cells to evade toxicity and acquire tolerance. Herein, we
explored how Saccharomyces cerevisiae (budding yeast) respond to trivalent
arsenic (arsenite) by quantitative and kinetic transcriptome, proteome and
sulfur metabolite profiling. Arsenite exposure affected transcription of
genes encoding functions related to protein biosynthesis, arsenic
detoxification, oxidative stress defense, redox maintenance and
proteolytic activity. Importantly, enzymes involved in sulfate assimilation
and glutathione biosynthesis were induced at both gene and protein levels.
Kinetic metabolic profiling evidenced a significant increase in the pools of
sulfur metabolites as well as elevated glutathione levels. Moreover, the
flux in the sulfur assimilation pathway as well as the glutathione synthesis
rate strongly increased with a concomitant reduction of sulfur incorporation
into proteins. By combining comparative genomics and molecular analyses, we
pin-pointed transcription factors that mediate thecore of the
transcriptional response to arsenite. Taken together, our data reveals that
arsenite-exposed cells channel a large part of assimilated sulfur into
glutathione biosynthesis and we provide evidence that the transcriptional
regulators Yap1p and Met4p control this response in concert.
Keywords: stress, time course

overall_design: Four time points, each array hybridized to untreated wild-type.


title: Comparison of Mycobacterium bovis-infected cattle versus control non-infected cattle using the BOTL-5 microarray

status: Public on Oct 16 2007

city: Dublin

first: David

middle: E

last: MacHugh

address: University College Dublin, Belfield

department: College of Agriculture, Food Science and Veterinary Medicine

country: Ireland

institute: University College Dublin

summary:
Background: Bovine tuberculosis is an enduring disease of cattle that has significant repercussions for human health. The advent of high-throughput functional genomics technologies has facilitated large-scale analyses of the immune response to this disease that will ultimately lead to novel diagnostics and therapeutic targets. Analysis of mRNA abundance in peripheral blood mononuclear cells (PBMC) from six Mycobacterium bovis infected cattle and six non-infected controls was performed. A targeted immunospecific bovine cDNA microarray with duplicated spot features representing 1,391 genes was used to test the hypothesis that a novel gene expression profile may exist in M. bovis infected animals in vivo.
Results: In total, 378 gene features were differentially expressed at the P ≤ 0.05 level in bovine tuberculosis (BTB)-infected and control animals, of which 244 were expressed at lower levels (65%) in the infected group. Lower relative expression of key innate immune genes such as the Toll-like receptor 2 (TLR2) and TLR4 genes, lack of differential expression of indicator adaptive immune gene transcripts (IFNG, IL2, IL4), and lower BOLA major histocompatability complex – class I (BOLA) and class II (BOLA-DRA) gene expression was consistent with innate immune gene repression in the BTB-infected animals. Supervised hierarchical cluster analysis and class prediction validation identified a panel of 15 genes predictive of disease status and selected gene transcripts were validated (n = 8 per group) by real time quantitative reverse transcription PCR.
Conclusion: These results suggest that large-scale expression profiling can identify gene signatures of disease in peripheral blood that can be used to classify animals on the basis of in vivo infection, in the absence of exogenous antigenic stimulation.
Keywords: Disease state analysis

overall_design:
Experimental animals
Sixteen cattle were used for this study. The eight infected animals were chosen from herds with a recent history of chronic infection with M. bovis. The animals were selected on the basis of the skin-fold thickness response to bovine and avian tuberculin in the single intradermal comparative tuberculin test (SICTT).  The SICTT reactor animals were selected where the skin-fold thickness response to PPD-bovine exceeded that of PPD-avian by at least 12 mm. All of these animals were also measured positive in a whole blood IFN-g assay. The cattle were confirmed positive for tuberculosis following detailed post-mortem pathological examination and/or culture. Bronchial, mediastinal, submandibular, retropharyngeal, mesenteric and hepatic lymph nodes and lungs were examined macroscopically for tuberculosis lesions. Suspected lesions were cultured on Stonebrinks and Lowenstein-Jensen media at 37°C for eight weeks to detect M. bovis. The eight non-infected control animals were selected from a herd without a recent history of tuberculosis and were SICTT and IFN-g test negative.
Blood sampling and analysis
400 ml of blood was collected from each animal in sterile heparinised bottles. Five ml of blood was used for haematological analysis using an Abbott CELL-DYN 3500R automated haematology analyzer (Abbott Laboratories). Leukocyte cell population subsets were compared between infected and control groups (n = 8) using Student’s t-test.
PBMC separation, RNA extraction and quality control
PBMC were isolated using the Percoll™ gradient method with a standard protocol [48]. PBMC were seeded at 10^7 per culture plate and cultured in RPMI 1640 medium supplemented with 5% FBS, 0.1% mercaptoethanol and 0.1% gentamicin. All PBMC samples were cultured overnight at 37°C in 5% CO2. Overnight culture was carried out to minimise noise in gene expression measurements potentially introduced by the mechanical disruption of cells associated with PBMC isolation. Residual cells not seeded for culture were immediately suspended in 3 ml TriReagent® (Molecular Research Centre Inc.) and frozen in 1.5 ml cryotubes at -80°C for use later as a common reference RNA (CRR) pool. Total RNA was extracted using a combined TriReagent®, DNase treatment and Qiagen RNeasy® method (Qiagen Ltd.) according to the manufacturers’ instructions. The integrity and stability of RNA samples is crucial for gene expression analyses using microarray technology; therefore, RNA yield and quality were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). The two-step method for RNA extraction described above was found to produce RNA of high yield and quality (ratios of 18S to 28S ribosomal RNA averaged > 1.6).
Microarray experimental design
The 3,888 feature BOTL-5 immunogenetic cDNA microarray system used has been described previously [49]. Technical information with gene content and sequence information for the BOTL-5 array can be downloaded from the ‘Links’ section on the MSU Center for Animal Functional Genomics website (http://www.nbfgc.msu.edu). The immunobiology-targeted BOTL-5 array contains 1,391 genes or ESTs spotted in duplicate with multiple additional control features (blank spots, negative spots, housekeeping genes) and is an expanded version of the BOTL-4 array described previously. A reference design was used for microarray hybridizations, such that all RNA samples were labelled using Cy3 and co-hybridized with Cy5 labelled CRR pool. It was hypothesized that the CRR pool would display similar mRNA expression levels and gene coverage as the target samples, therefore allowing accurate and consistent comparison of gene expression data without arbitrarily pairing animals from the two groups. The CRR pool contained equal amounts of total RNA from the treated and control animal groups. Twelve arrays were hybridized in total, representing six individual animals from each treatment group.

first: Kieran

last: Meade

first: Eamonn

last: Gormley

first: Mairéad

middle: B

last: Doyle

first: Tara

last: Fitzsimons

first: Cliona

last: O’Farrelly

first: Eamon

last: Costello

first: Joseph

last: Keane

first: Yingdong

last: Zhao


title: Comparative genomic hybridization of human embryonic stem cell lines deposited in the National Stem Cell Bank

city: Madison

first: Melodee

last: Patterson

state: WI

address: 510 Charmany Dr Ste 58 & 59

institute: WiCell

summary:
The Purpose of this series of experiments is to identify copy number variations, duplications, and deletions in human embryonic stem (hES) cell lines deposited to the National Stem Cell bank and reveal the difference between different hES cell lines. CGH can achieve these aims at higher resolution.
Keywords: comparative genomic hybridization

overall_design: Genomic DNA of different human embryonic stem cell lines deposited to the National Stem Cell Bank are extracted and hybridized to NimbleGen CGH microarray, using genomic DNA of hESC H1 or H9 as common reference. Difference between different stem cell lines will be revealed.

first: Karen

last: Mongomery

first: Chuhu

last: Yang


title: Array-CGH of human hepatocellular carcinoma

status: Public on Oct 15 2007

city: Heidelberg

first: Christof

middle: Simon

last: Schlaeger

state: BW

address: INF 580

department: Molecular Genetics (B060)

institute: DKFZ

summary:
Comparison of normal versus tumor DNA (hepatocellular carcinoma). Whole-genome screening of DNA-copy number changes by array-based or matrix comparative genomic hybridization (aCGH). Tumor DNA labeled in Cy3 and pooled DNA of lymphocytes from healthy donors labeled in Cy5.
Keywords: Genetic modification

overall_design: DNA Copy number profiling of 67 human hepatocellular carcinomas of various etiologies

first: Bernhard

last: Radlwimmer


title: Profiling differentially expressed earthworm genes in response to TNT exposure.

city: Vicksburg

first: Ping

last: Gong

state: MS

address: 3909 Halls Ferry Road

department: Environmental Laboratory

institute: US Army ERDC

summary:
To understand molecular mechanisms of the chronic, sublethal toxicity of 2,4,6-trinitrotoluene (TNT), a widely used ordnance compound of public concerns, we constructed a microarray consisting of 4,032 cDNA isolated from the earthworm Eisenia fetida using the suppressive subtractive hybridization technique. Worms were exposed to a gradient of TNT-spiked soil for 28 days. Based on the reproduction response to TNT, four treatments, i.e., control, 7, 35 and 139 ppm, were selected for gene expression studies.
Keywords: Sublethal toxicity of TNT (dose-response) to earthworm (Eisenia fetida)

overall_design: We analyzed 40 arrays for 4 treatments (control, 7ppm, 35ppm, 139ppm) with 5 biological replicates per treatment using an interwoven loop designed hybridization scheme.

first: Xin

last: Guan

first: Edward

last: Perkins


title: CtsR Regulon in Listeria monocytogenes

status: Public on Oct 14 2007

city: Ithaca

first: Sarita

last: Raengpradub

state: NY

address: 412 Stocking Hall

department: Food Science

institute: Cornell University

summary:
The foodborne pathogen Listeria monocytogenes uses a number of transcriptional regulators, including the negative regulator CtsR, to control gene expression under different environmental conditions and in response to stress. Gene expression patterns of DctsR log phase cells were compared to both wt and ictsR-mcsA log phase cells grown with 0.5mM IPTG to identify CtsR-dependent genes.We identified 62 CtsR-dependent genes that showed significant expression ratios (adj. P < 0.05), with ≥ 1.5-fold differential expression either between ΔctsR and wt or between ΔctsR and ictsR-mcsA.
Keywords: Listeria monocytogenes, CtsR regulon, log phase

platform_organism:
Listeria monocytogenes
Listeria innocua

overall_design: Independent RNA isolations were performed for each growth experiment (log phase cells). For wt vs. ctsR null mutant comparisons, three biological replicates were used in competitive whole-genome microarray experiments. For ictsR-mcsA vs ctsR null mutant comparisons, two biological replicates were used in competitive whole-genome microarray experiments. For each set of hybridizations, RNA from the parent strain (10403S) or the ictsR-mcsA strain were hybridized to RNA from the ctsR null mutant.

first: Yuewei

last: Hu

first: Martin

last: Wiedmann

first: Kathryn

last: Boor


title: CGH mapping of Genetic Deficiencies balanced by hT2 and eT1 on LGIII

status: Public on Oct 11 2007

last: Jones

summary:
Background: A collection of genetic deficiencies covering over 70% of the Caenorhabditis elegans genome exists, however the application of these valuable biological tools has been limited due to the incomplete correlation between their genetic and physical characterization.    !Series_summary = Results: We have applied oligonucleotide array Comparative Genomic Hybridization (oaCGH) to the high resolution, molecular characterization of several genetic deficiency and duplication strains in a 5Mb region of Chromosome III. We incorporate this data into a physical deficiency map which is subsequently used to direct the positional cloning of essential genes within the region. From this analysis we are able to quickly determine the molecular identity of several previously unidentified mutations.
Conclusion: We have applied accurate, high resolution molecular analysis to the characterization of genetic mapping tools in Caenorhabditis elegans. Consequently we have generated a valuable physical mapping resource, which we have demonstrated can aid in the rapid molecular identification of mutations of interest.
Keywords: C.elegans Deficiencies CGH

overall_design: 7 test samples from heterozygous deficiency containing strains individually hybridized to reference DNA from strain JK2689 (5 hT2 balanced strains) or BC2842 (2 eT1 balanced strains)

first: David

last: Baillie

first: Donald

last: Moerman

first: Stephane

last: Flibotte

first: Jason

last: Maydan


title: Examination of the function of NtMYBGR1 and NtDC1B in tobacco BY-2 cells.

city: Yokohama

first: Ken

last: Matsuoka

state: Kanagawa

address: 1-7-22 Suehirocho, Tsurumi-ku,

department: Plant Science Center

institute: RIKEN

summary:
Overexpression lines for each of NtMYBGR1 (Genbank accession No. AB279612)  and NtDC1B (Genbank accession No. AB279614) were used to characterize the function of NtMYBGR1 and NtDC1B in tobacco BY-2 cells. We then analyzed the effect of overexpression of these genes on the expression of other genes, using the tobacco microarray. As control sample, tobacco cells transformed with empty vector pMAT137 vector were used. Two control and two transgenic cell lines were analyzed at day 4 after subculture into fresh media.
Keywords: Genetic modification

overall_design: 1. pMAT vector control, 2. NtMYBGR1 overexpressor, 3. NtDC1B overexpressor.

first: Tomonori

last: Shinya

first: Ivan

last: Gális

first: Tomoko

last: Narisawa

first: Mam

last: Sasaki

first: Hiroo

last: Fukuda

first: Hideaki

first: Mikako

last: Saito


title: Response of tobacco cells to Glucan elicitor

summary:
We have previously reported that the responses of tobacco tissues treated with AaGlucan are similar to those for laminarin [1]. To better understand the overall responses and signaling pathways in tobacco, we have identified here AaGlucan and laminarin early response genes, using a custom 16K BY-2 microarray [2,3]. BY-2 cells were treated with laminarin or an AaGlucan-enriched fraction prepared from A. alternata 102 culture-medium and subjected to microarray analysis.
[1] Shinya, T., Menard, R., Kozone, I., Matsuoka, H., Shibuya, N., Kauffmann, S., Matsuoka, K. and Saito, M. (2006) Novel b-1,3-, 1,6-oligoglucan elicitor from Alternaria alternata 102 for defense responses in tobacco. FEBS J. 273: 2421-2431.
[2] Matsuoka, K., Demura, T., Galis, I., Horiguchi, T., Sasaki, M., Tashiro, G. and Fukuda, H. (2004) A comprehensive gene expression analysis toward the understanding of growth and differentiation of tobacco BY-2 cells. Plant Cell Physiol. 45: 1280-1289.
[3] Galis, I., Simek, P., Narisawa, T., Sasaki, M., Horiguchi, T., Fukuda, H. and Matsuoka, K. (2006) A novel R2R3 MYB transcription factor NtMYBJS1 is a methyl jasmonate-dependent regulator of phenylpropanoid-conjugate biosynthesis in tobacco. Plant J. 46: 573-592.
Keywords: Stress response

overall_design:
AaGlucan was purified from Alternaria alternata 102 as a potent elicitor for tobacco [1]
Four-day-old tobacco BY-2 cells after subculture were treated with 10 microg/ml AaGlucan enriched fraction for 1h. Total RNA was prepared from the control and treated samples and used for microarray experiment after reverse transcription and labeling with Cy5 fluorescent dye.
[1] Shinya, T., Menard, R., Kozone, I., Matsuoka, H., Shibuya, N., Kauffmann, S., Matsuoka, K. and Saito, M. (2006) Novel b-1,3-, 1,6-oligoglucan elicitor from Alternaria alternata 102 for defense responses in tobacco. FEBS J. 273: 2421-2431.

first: Mami


title: 1.0 mM As(III) stressed yap1 deletion mutant vs 1.0 mM As(III) stressed wild-type after 1 hour

first: Erik

last: Kristiansson

summary:
volved enabling cells to evade toxicity and acquire tolerance. Herein, we
explored how Saccharomyces cerevisiae (budding yeast) respond to trivalent
arsenic (arsenite) by quantitative and kinetic transcriptome, proteome and
sulfur metabolite profiling. Arsenite exposure affected transcription of
genes encoding functions related to protein biosynthesis, arsenic
detoxification, oxidative stress defense, redox maintenance and
proteolytic activity. Importantly, enzymes involved in sulfate assimilation
and glutathione biosynthesis were induced at both gene and protein levels.
Kinetic metabolic profiling evidenced a significant increase in the pools of
sulfur metabolites as well as elevated glutathione levels. Moreover, the
flux in the sulfur assimilation pathway as well as the glutathione synthesis
rate strongly increased with a concomitant reduction of sulfur incorporation
into proteins. By combining comparative genomics and molecular analyses, we
pin-pointed transcription factors that mediate thecore of the
transcriptional response to arsenite. Taken together, our data reveals that
arsenite-exposed cells channel a large part of assimilated sulfur into
glutathione biosynthesis and we provide evidence that the transcriptional
regulators Yap1p and Met4p control this response in concert.
Keywords: stress

first: Michael

last: Thorsen

first: Markus

last: Tamas

first: Olle

last: Nerman


title: Inflammatory and immune responses in lymph nodes of Iberian red deer infected with Mycobacterium bovis.

city: Stillwater

first: Patricia

last: Ayoubi

state: OK

address: 246 NRC

institute: Oklahoma State University

summary:
Bovine tuberculosis (bTB), caused by Mycobacterium bovis (Mycobacterium tuberculosis complex), is a zoonotic disease that affects cattle and wildlife worldwide. In some regions of Spain, Iberian red deer (Cervus elaphus hispanicus) can serve as reservoir of infection, thus increasing the risk of human and cattle exposure and infection. Mesenteric lymph nodes are naturally infected with M. bovis in Iberian red deer, in which the digestive route of infection is particularly important in Mediterranean Spain. In this study we characterized the differential expression of inflammatory and immune response genes in mesenteric lymph nodes of Iberian red deer naturally infected with M. bovis using a Ruminant Immuno-inflammatory Gene Universal Array (RIGUA) and real-time RT-PCR. Of the 600 genes that were analyzed in the microarray, 157 showed ≥ 1.2 fold changes in expression in infected or uninfected deer and 17 genes displayed an expression fold change greater than 1.7 with a P-value ≤ 0.05 and were selected for further analysis. These genes included tight junction proteins (Z02 and occluding), IL-11R, bactenecin, CD62L, CD74, desmoglein, IgA and IgM that constitute new findings and suggest new mechanisms by which M. bovis may modulate host inflammatory and immune responses. Identification of genes differentially expressed in animals and tissues naturally infected with M. bovis contributes to our basic understanding of the mechanisms of pathogenesis and protective immunity to mycobacterial infections and may have important implications for future functional genomic and vaccine studies to aid in the control of bTB in deer and other wildlife reservoir species.
Keywords: disease state analysis

platform_organism:
Sus scrofa
Bos taurus
Ovis aries

overall_design: Mesenteric lymph node RNA from four different uninfected Iberian red deer stags and two Iberian red deer stags infected with Mycobacterium bovis.  Infected animals were naturally infected with M. bovis.  All animals were hunter-harvested and the tissues retrieved 2-6 hrs after animal hunting.


title: 1.0 mM As(III) stressed wild-type vs (non-stressed) wild-type after 1 hour

summary:
Arsenic is ubiquitously present in nature and various mechanisms have evolved enabling cells to evade toxicity and acquire tolerance. Herein, we explored how Saccharomyces cerevisiae (budding yeast) respond to trivalent arsenic (arsenite) by quantitative and kinetic transcriptome, proteome and sulfur metabolite profiling. Arsenite exposure affected transcription of genes encoding functions related to protein biosynthesis, arsenic detoxification, oxidative stress defense, redox maintenance and
proteolytic activity. Importantly, enzymes involved in sulfate assimilation and glutathione biosynthesis were induced at both gene and protein levels. Kinetic metabolic profiling evidenced a significant increase in the pools of sulfur metabolites as well as elevated glutathione levels. Moreover, the flux in the sulfur assimilation pathway as well as the glutathione synthesis rate strongly increased with a concomitant reduction of sulfur incorporation into proteins. By combining comparative genomics and molecular analyses, we pin-pointed transcription factors that mediate thecore of the transcriptional response to arsenite. Taken together, our data reveals that arsenite-exposed cells channel a large part of assimilated sulfur into glutathione biosynthesis and we provide evidence that the transcriptional regulators Yap1p and Met4p control this response in concert.
Keywords: stress

overall_design: Stressed wild-type 1mM 1h versus untreated wild-type using direct comparisons.


title: lonB mutant

city: manhattan

first: Xiaoyan

last: Tang

email: xtang@ksu.edu, llan@ksu.edu

state: KS

address: 4702 Throckmorton

department: Plant Pathology

institute: Kansas State University

summary:
The bacterial Lon protease participates in a variety of biological processes. In Pseudomonas syringae, mutation of lon is known to activate hrpL and a few hrpL-regulated genes in the rich medium. The elevated expression of hrpL and hrpL-regulated genes results from of increased stability of HrpR, the transcriptional activator of hrpL, in the lon mutant. Here we conducted a microarray analysis to determine genes that are differently expressed in the lon- mutant of P. s. pv. tomato DC3000 grown in the rich medium KB. Most genes induced in the lon- mutant belong to the HrpL-regulon or are related to the transcription, protein synthesis, and energy metabolism. The major group of genes reduced in the lon- mutant is related to cell wall biogenesis. The HrpL-regulated genes exhibit different induction patterns in the lon- mutant, suggesting the involvement of regulators additional to HrpL in regulating these genes. Compared with the wild type bacteria, the lon- mutants exhibit elevated hrpL expression in KB medium but reduced hrpL expression in the minimal medium (MM). The reduced hrpL RNA is correlated with the reduced hrpR and hrpS RNAs, suggesting that the Lon-mediated regulation of hrpL involves different mechanisms in KB and MM. Consistent with the reduced expression of the hrpL in MM, the lon- mutants are less pathogenic on host plants.
Keywords: Expression profiles of lonB mutant in KB

sample_organism: Pseudomonas syringae pv. tomato str. DC3000

overall_design: three samples ( biological replicates) of mutation were analyzed

first: Lefu

last: Lan


title: Expression data from planktonic and biofilm cultures of Mycobacterium smegmatis

city: Pittsburgh

first: Graham

last: Hatfull

state: PA

address: 4249 Fifth Avenue

department: Biological Sciences

institute: University of Pittsburgh

summary:
Transition of microbial growth from planktonic to biofilm is associated with programmed changes in the global patterns of gene expression. These changes are likely to faciliate the appropriate physiological and metabolic adjustments that bacteria need to make during the development of biofilms.
Using microarrays we have examined the changes in pattern of gene expression associated with growth of Mycobacterium smegmatis in various stages of planktonic and biofilm cultures.
Keywords: developmental time course

overall_design: M.smegmatis cells were harvested from logarithmic and stationary phase planktonic as well as 3day and 4day biofilm cultures. RNA extracted from these cells were subjected to labelling and hybridization on TIGR microarray chips according to the protocol described by the manufacturer.

first: Anil

middle: K

last: Ojha

middle: F


title: Molecular targets of  bortezomib on adult T-cell leukemia cells

status: Public on Oct 09 2007

city: Tokyo

first: Junko

last: Ohyashiki

address: 6-7-1, Nishishinjuku, Shinjuku-ku

department: Intractable Immune System Diseases Research Center

institute: Tokyo Medical University

summary:
BACKGROUND: The molecular mechanism of proteasome inhibitor-mediated anti-cancer activity has been logically hypothesized that the major pathway is inhibition of nuclear factor kappa-B (NF-kB) cascades, however, the precise mechanism is still unclear. Adult T-cell leukemia (ATL) is a fatal neoplasia derived from HTLV-1 infected T lymphocytes exhibiting constitutive activation of NF-kB. AIM: To elucidate the complex molecular mechanism of anti-tumor effect of the proteasome inhibitor, bortezomib in ATL cells, we attempted to perform network-based gene expression profiling. METHODS and RESULTS: Assessment of gene regulation by microarray analysis revealed that down-regulation of genes involved in anti-apoptosis (i.e., BCL2, and IAP5), up-regulation of genes related with apoptosis (i.e., FAF1), heat shock proteins (i.e., HSPA, HSPCA), and oxygen stress (i.e., heme oxygenase-1: HMOX-1). Gene network analysis employing the Bayesian statistical framework also suggests that HMOX-1 which is known as an inducer of reactive oxygen species (ROS), as well as SPARC play an important role in bortezomib-treated ATL cells. The HMOX-1 inhibitor, ZnPP, in addition to bortezomib partially inhibited the apoptotic effect of bortezomib on TaY cells. CONCLUSION: Our results suggest that in addition to the inhibition of NF-kB, bortezomib activated ROS pathway through HMOX-1, and SPARC may participate bortezomib-induced apoptosis, providing novel insight into the bortezomib mediated anti-tumor activity in ATL cells.
Keywords: dose response and time course

overall_design: For dual color analysis, cDNA obtained from uninfected TaY cells was labeled with Cy3 as a reference, and cDNA obtained from the HHV-6B infected cells was labeled with Cy5.

first: Ryoko

last: Hamamura


title: Long Serial Analysis of Gene Expression (LongSAGE) of Chinook salmon (Oncorhynchus tshawytscha) brain tissue.

status: Public on Oct 10 2007

city: Newport

first: Jeremiah

last: Bernier

state: OR

address: 2030 SE Marine Science Drive

department: Fisheries and Wildlife

institute: Hatfield Marine Science Center, Oregon State University

summary:
Chinook salmon (Oncorhynchus tshawytscha) display the greatest variability of return times to freshwater of all Pacific salmon. Populations return to freshwater for spawning at many different times of year, resulting in segregated populations that may use differing molecular pathways for these large behavioral and physiological differences. Using a population of Chinook from California’s Central Valley, we sought to generate novel expressed sequences using Long Serial Analysis of Gene Expression (LongSAGE). We constructed three LongSAGE libraries from brains of samples caught in the spring and fall in freshwater and from the ocean. Using cDNA libraries from Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss), we were able to assign 59% of putatively differentially expressed tags to genes. Additionally, we tested the expression levels of seven genes, indicated by LongSAGE to be putatively differentially expressed between the fall and spring, and found none significantly differentially expressed. This study is the first to apply LongSAGE to salmon and provides a framework for conducting future research on gene expression differences between Chinook salmon of different populations, as well as underlying mechanisms of differing physiology and behavior.
Keywords: seasonal difference

overall_design: Single individuals were used to construct each LongSAGE library.  The fall, spring and ocean samples were then compared between each other and examined for differences in the number of tags observed.

middle: C

first: Shanda

last: Birkeland

last: Cipriano

first: Andrew

last: McArthur

middle: A

last: Banks


title: The cardiac calsequestrin gene (CASQ2) is up-regulated in the thyroid in patients with Graves’ ophthalmopathy

city: Sydney

first: Leon

middle: Edward

last: Wescombe

state: New South Wales

address: Nepean Hospital, Derby Street, Penrith

department: Medicine

country: Australia

institute: University of Sydney

summary:
Thyroid-associated ophthalmopathy (TAO) is a complex eye and orbital disorder that is uniquely linked to Graves’ hyperthyroidism (GH). A popular explanation for the development of orbital inflammation in these patients is autoimmunity against the TSH-receptor expressed in orbital preadipocytes.  However, this has not been proven and alternative hypotheses should be entertained. We have shown recently that antibodies against highly purified rabbit skeletal muscle calsequestrin are sensitive and specific markers of eye muscle inflammation in patients with thyroid autoimmunity.
Keywords: disease state comparison

overall_design: In the study presented here, RNA from thyroid tissue of hyperthyroid patients with (n=10) and without (n=8) symptoms of ophthalmopathy was compared across 20,589 genes.

first: Bamini

last: Gopinath

first: Jack

last: Wall


title: Nutrition-induced ketosis alters metabolic and signaling gene networks in liver of periparturient dairy cows

city: Urbana

first: Juan

middle: J.

last: Loor

state: IL

address: 1207 West Gregory Drive

department: Animal Sciences

institute: University of Illinois

summary:
Hepatic molecular adaptations underlying periparturient metabolic diseases such as ketosis in dairy cows are largely unknown.  We used a simple model for induction of ketosis to examine liver gene expression profiles using a microarray consisting of 13,257 annotated cattle oligonucleotides.  At 4 days post-partum, 7 cows classified as healthy after a physical examination were fed at 50% of intake at day 4 from day 5 to signs of ketosis or until 14 days post-partum. Another group of 7 healthy cows served as controls. Liver was biopsied at 9-14 (ketosis) or 14 days post-partum (controls).  More than 9,000 sequences represented on the microarray were expressed in liver.
Keywords: disease state analysis

overall_design: Fourteen multiparous Holstein cows classified as healthy after a thorough physical examination on day 4 postpartum were randomly assigned to a healthy control group or to a ketosis induction group.  Ketosis induction was by feed restriction.  At 5 days postpartum cows in the ketosis induction group were fed at 50% of their intake at 4 days post-partum until signs of clinical ketosis (anorexia, ataxia, or abnormal behavior) or until 14 days postpartum, and then were returned to ad libitum intake and treated, if necessary.  Total RNA from liver and a reference standard (derived from cattle tissues) was extracted and labelled with Cy3 and Cy5 in a dye-swap design.  Samples were co-hybridize for 48 hours to microarrays containing 13,257 elements.

first: Robin

last: Everts

first: Massimo

last: Bionaz

first: Heather

middle: M

last: Dann

first: Dawn

last: Morin

first: Rosane

last: Oliveira

first: Sandra

last: Rodriguez-Zas

first: James

last: Drackley

first: Harris

last: Lewin


title: Genome position and gene amplification - higher resolution mapping of amplicons

status: Public on Oct 08 2007

city: San Francisco

first: Donna

last: Albertson

state: CA

department: Comprihensive Cancer Center

institute: University of California San Francisco

summary:
We investigated the influence of genome position on propensity to amplify. First, we integrated a mutant form of DHFR into different positions in the human genome, challenged cells with methotrexate and then studied the genomic alterations arising in drug resistant cells.
We observed site specific differences in methotrexate sensitivity, organization of amplicons and amplification frequency. One site was uniquely associated with a significantly enhanced propensity to amplify and recurrent amplicon boundaries, possibly implicating a rare folate sensitive fragile site in initiating amplification. Hierarchical clustering of gene expression patterns and subsequent gene enrichment analysis revealed two clusters differing significantly in expression of MYC target genes independent of integration site.
Keywords: Gene amplification, array CGH, chromosomal fragile sites

overall_design: We introduced a mutant form of DHFR (L22F), which confers greater resistance to methotrexate than the wild type (endogenous) gene into HCT116+chr3 cells and isolated independent clones containing DHFR* at different positions in the genome and identified genome sequences flanking the integration site of DHFR* using inverse PCR. For further analysis, we selected only clones, which were considered to have a single insertion of DHFR* by inverse PCR (13 independent insertion sites). The individual insertion site clones were further characterized with respect to genome copy number profiles. To select methotrexate resistant colonies, we exposed cells to a concentration of methotrexate that was three to four times the IC-50 for each integration site. Genomic copy number profiles were obtained for isolated resistant colonies (GSE6262) bu using UCSF HumArray platform (GPL4421). This dataset contains higher resolution mapping of six selected amplicons on the 32K BAC genome tiling path array (GPL4422).

first: Pavla

last: Gajduskova

first: Antoine

last: Snijders

first: Serena

last: Kwek

first: Ritu

last: Roydasgupta

first: Jane

last: Fridlyand

first: Taku

last: Tokuyasu

first: Daniel

last: Pinkel


title: Transcriptional Profiling of high pigment-2dg Tomato Mutant Fruit

city: Rishon Le-Zion

first: Igor

middle: Michael

last: Kolotilin

address: Dereh Ha-Macabim

department: Plant sciences

country: Israel

institute: Volcani inst.

summary:
Phenotypes of the tomato (Solanum lycopersicum L.) high pigment-2dg (hp-2dg) mutant are caused by a mutation in the gene encoding DEETIOLATED1, a negative regulator of light signaling. Homozygous hp-2dg plants display a plethora of distinctive developmental and metabolic phenotypes in comparison to their normal isogenic counterparts. This mutant is however best known for the increased levels of lycopene and other plastid-accumulating functional metabolites. In this study we analyzed the transcriptional alterations in mature-green, breaker and early-red fruits of hp-2dg/hp-2dg plants in relation to their normal counterparts using microarray technology. Results show that a large portion of the genes that are affected by hp-2dg mutation, display a tendency for up- rather than down-regulation. Ontology assignment of these differentially regulated transcripts, revealed a consistent upregulation of those related to chloroplast biogenesis and photosynthesis in hp-2dg mutants throughout fruit ripening. A tendency of upregulation was also observed in structural genes involved in phytonutrient biosynthesis. However, this upregulation was not as consistent, positioning plastid biogenesis as an important determinant of phytonutrient overproduction in hp-2dg mutant fruits. Microscopic observations revealed a highly significant increase in chloroplasts size and number in pericarp cells of mature-green hp-2dg/hp-2dg fruits in comparison to their normal counterparts. This increase could be observed from early stages of fruit development. Therefore, the molecular trigger that drives phytonutrient overproduction in hp-2dg mutant fruits should be initially traced at early stages of fruit development.
Keywords: Comparative Transcriptional Profiling of tomato fruit pericarp tissue

overall_design:
Representative plants of hp-2dg tomato mutants and their normal (WT) counterparts were planted in a randomized-block design (3 blocks, 5 plants in each block) in an environmentally controlled greenhouse at the Volcani Center, Bet Dagan, Israel, during the winter seasons of 2004 and 2005 (minimal temperature kept at 15˚C with no supplemental light). At each of 3 developmental stages (mature green, breaker and pink-red) a single fruit was harvested from a randomly chosen plant of each genotype within each block. In each harvest different plants in each block were taken to represent each genotype. Following harvest, equal pericarp samples were cut from the equator region of each fruit. These samples were pooled according to genotype prior to RNA extraction, allowing equal representation of each block in the final sample. A total of 3 harvests taken from each genotype were eventually analyzed– each harvest representing a biological repeat.
Transcriptional profiling of tomato pericarp tissue of hp-2dg/hp-2dg plants was obtained using direct comparisons with their normal counterparts in each developmental stage separately. Two-color hybridizations were carried out to the TOM1 microarray slide (CGEP website at Cornell).
Three microgram of amplified mRNA products were subjected to reverse transcription and then labeled with Cy3 and Cy5 (Amersham Biosciences, Uppsala, Sweden) by the indirect amino-allyl method (Invitrogen, Carlsbad, CA, USA). Hybridization of labeled targets to the tomato microarray slide TOM1 was carried out at 42°C for 14 to 16 h using a covered hybridization oven (Amersham Pharmacia, Buckinghamshire, England). Microarray slides were washed seven times for 10 min each: at room temperature in 2XSSC/0.1% SDS- twice, room temperature in 0.2XSSC/0.1% SDS- twice, room temperature with 0.2XSSC- twice, and once in 0.2XSSC at 42°C. Excess fluids were drained from the arrays by centrifugation (5 min at 450g).
For each developmental stage (mature green, breaker and early-red), 4 hybridizations were carried out, representing 3 independent biological repeats and one technical repeat of swapped dyes. Separate images for each fluorescence dye were acquired using ScanArray 4000 genetic analyzer [GSI Lumonics (Packard / Perkin Elmer), Ramsey MN, USA]. The analyzer scan was carried out at 10 μm resolution per pixel, adjusting the photomultiplier and laser power to achieve an optimal distribution of signals with minimal saturation. Quantification was carried out using QuantArray version 3 software, applying histogram method (Billerica, Packard BioScience, MA, USA). Data analysis was performed using Genespring® 7.2 software (Silicon Genetics, Redwood City, CA, USA), applying per-spot and per-chip normalizations: For each hybridization Lowess curve was fit to the log-intensity versus log-ratio plot. 40% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the value of the normal genotype for each measurement (control channel). If the control channel was lower than 10 then 10 was used instead. Low-quality data were identified and discarded when their signal to noise ratio was <2.

first: Carmiya

last: Bar-Or

first: Hinanit

last: Koltai

first: Ya'akov

last: Tadmor

first: Moshe

last: Reuveni

first: Ilan

last: Levin


title: Genome Position and Gene Amplification

summary:
Amplifications, regions of focal high level copy number change, lead to overexpression of oncogenes or drug resistance genes in tumors.  Their presence is often associated with poor prognosis; however the use of amplification as a mechanism for overexpression of a particular gene in tumors varies. To investigate the influence of genome position on
propensity to amplify, we integrated a mutant form of DHFR into different positions in the human genome, challenged cells with methotrexate and then studied the genomic alterations arising in drug resistant cells.
We observed site specific differences in methotrexate sensitivity, organization of amplicons and amplification frequency. One site was uniquely associated with a significantly enhanced propensity to amplify and recurrent amplicon boundaries, possibly implicating a rare folate sensitive fragile site in initiating amplification.  Hierarchical clustering of gene expression patterns and subsequent gene enrichment analysis revealed two clusters differing significantly in expression of MYC target genes independent of integration site.
These studies suggest that genome context together with the particular challenges to genome stability experienced during the progression to cancer contribute to the propensity to amplify a specific oncogene or drug resistance gene, whereas the overall functional response to drug (or other) challenge may be independent of the genomic location of an oncogene.
Keywords: Gene amplification, array CGH, chromosomal fragile sites

overall_design:
We introduced a mutant form of DHFR (L22F), which confers greater resistance to methotrexate than the wild type (endogenous) gene into
HCT116+chr3 cells. We isolated independent clones containing DHFR* at different positions in the genome and identified genome sequences flanking the integration site of DHFR* using inverse PCR. For further analysis, we selected only clones, which were considered to have a single insertion of DHFR* by inverse PCR (13 independent insertion sites). The individual insertion site clones were further characterized with respect to genome copy number profiles. To select methotrexate resistant colonies, we exposed cells to a concentration of
methotrexate that was three to four times the IC-50 for each integration site. Genomic copy number profiles were obtained for isolated resistant colonies (82 methotrexate resistant colonies).


title: Vasopressin (AVP) and the V2R specific agonist dDAVP 7 day infusion into Aquaporin 1 knockout animals

status: Public on Oct 07 2007

city: Tucson

first: Kevin

last: Greer

state: AZ

department: Biomedical Engineering

institute: University of Arizona

summary:
Vasopressin, the antidiuretic hormone, acts on the renal collecting duct. In this experiment both vasopressin (AVP) and the V2R specific agonist dDAVP were infused into Aquaporin 1 knockout animals for 7 days. The aim of the experiment was to identify genes increased by vasopressin receptors in the renal medullary collecting ducts, in the absence of an increase in renal medullary osmolarity (the AQP1 knockouts are concentrating mechanism knockouts). All experiments used inner medulla tissue for the RNA isolation.
Keywords: Vasopressin treatment study

overall_design: Hybridizations were performed that compared kidney inner medulla total RNA from three control mice against kidney medulla total RNA from 3 mice infused with either arginine vasopressin (AVP) or des-amino-D-arginine vasopressin (dDAVP).

first: Heddwen

last: Brooks


title: mRNA expression profile in peripheral blood cells of RA patients following treatment with an anti-TNFa mAb, infliximab

city: Kawagoe-shi, Saitama

first: Tsutomu

last: Takeuchi

address: 1981 Tsujido-machi, Kamoda

department: Department of Internal Medicine

institute: Saitama Medical University

summary:
Objectives: We monitored the mRNA expression profiles of peripheral blood cells during treatment with a tumor necrosis factor-a inhibitor, infliximab, in patients with rheumatoid arthritis (RA). Using a DNA microarray analysis, we demonstrated a unique set of genes, with distinct baseline and post-treatment changes in expression between responders and non-responders to infliximab treatment.Methods: Using a customized low-density cDNA microarray and a reliable data collection system, we monitored the mRNA expression profiles of whole blood cells from 18 RA patients before and after the infusion of infliximab for up to 22 weeks.  The clinical response to treatment with infliximab was determined using the American College of Rheumatology (ACR) response criteria, the disease activity score of 28 joints (DAS28), and individual clinical parameters. The patients were classified as responders or non-responders based on their ACR50% response at 22 weeks. Results: Approximately 15% of the total genes were found to exhibit a greater than 1.5-fold change, compared to their reference values, at one or more time points during the 22 weeks of infliximab therapy. The expression of inflammatory genes, such as interferon-related genes, was strongly correlated with the serum level of C-reactive protein and the DAS28.  The increased expression of inflammatory genes in responders was normalized within 2 weeks and then remained at a normal level during the treatment period.  In contrast, in the non-responders, the elevated expression at baseline, although it was significantly decreased at 2 weeks, returned to the baseline level after 14 weeks. In addition to inflammatory genes, we identified several groups of genes with distinct differences in expression between the responders and non-responders.Conclusions: Our results suggest that a customized low-density microarray is useful for monitoring mRNA expression profiles in peripheral blood cells, enabling us to identify a unique set of genes with differentially regulated expressions in responders and non-responders to a TNF inhibitor among patients with RA.
Keywords: disease state analysis

overall_design: 18 RA Patients, We have collected blood samples into PAXgene tube systems for microarray measurement from patients as follows, just before first iv-injection of infliximab, 2 weeks, 14 weeks, and 22 weeks after first infliximab injection.

first: Naoya

last: Sekiguchi

first: Satoshi

last: Kawauchi

first: Takako

last: Furuya

first: Niro

last: Inaba

first: Koichiro

last: Matsuda

last: Ando

first: Masayoshi

last: Ogasawara

first: Hiroyuki

last: Aburatani

first: Hideto

last: Kameda

first: Kouichi

last: Amano

first: Tohru

last: Abe

first: Satoru

last: Ito


title: Effect of ethosuximide on gene expression in Drosophila head

status: Public on Oct 05 2007

city: Delhi

first: Abhay

last: Sharma

state: Delhi

address: Delhi University campus, Mall Road

department: Functional Genomics Unit

country: India

institute: CSIR-Institute of Genomics and Integrative Biology (IGIB)

summary:
We recently developed a fly model of pentylenetetrazol (PTZ)-induced long-term behavioral plasticity. Pharmacological validation suggests this model to be kindling-like. Hence, our model is of relevance in understanding the molecular pathogenesis of epileptogenesis as well as in screening potential antiepileptogenic agents. Ethosuximide (ETH) is an antiepileptic drug, and in the process of developing our fly model we generated gene expression profile of flies’ head secondary to treatment of the insects with ETH. Our results provide novel insights in to the drug’s mode of action.
Keywords: Dose response

overall_design: Oregon-R wild type D. melanogaster was grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin. Flies were cultured at 24+/-1ºC 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Ten to eleven days old unmated adult males were grown in either normal food (NF) or food containing 3.48 mg/ml of ETH for three days. Each culture vials contained 30 flies. Flies frozen in liquid nitrogen were agitated and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four ETH treated individuals, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche), and the cDNA purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of NF control and ETH treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and ETH treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The 16 bit TIFF images were preprocessed and quantified using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 2.21, Excel Add-In, Stanford) under the conditions of one class response and 100 permutations.

first: Priyanka

last: Singh

first: Mohd

last: Farhan


title: RNA quality and the influence on gene expression

status: Public on Oct 04 2007

city: Lund

first: Carina

last: Strand

address: Barngatan 2B

department: IKVL, Oncology

institute: Lund University

summary:
We created RNA with different degree of degradation and investigated if the RNA quality influenced gene expression
We used oligo-cDNA-microarrays to compare gene expressions
Keywords: RNA quality, breast cancer, gene expression

overall_design:
Six breast cancer samples were used, each sample were divided in four and the pieces were left in room temperature for four different time periods (50 sec, 2-3 min, 10 min, 30 min)
Stratagene Universal Human RNA was used as reference


title: Effect of levetiracetam on gene expression in Drosophila head

summary:
We recently developed a fly model of pentylenetetrazol (PTZ)-induced long-term behavioral plasticity. Pharmacological validation suggests this model to be kindling-like. Hence, our model is of relevance in understanding the molecular pathogenesis of epileptogenesis as well as in screening potential antiepileptogenic agents. Levetiracetam (LEV) is considered to have antiepileptogenic activity. Our fly model also predicted similar activity in LEV. Since the mechanism of action of LEV is not clearly known, we have examined the gene expression profiles of fly heads secondary to three days long chronic LEV treatment in Drosophila. Our results provide novel insights in to the drug’s mode of action.
Keywords: Drug Response

overall_design: Oregon-R wild type D. melanogaster was grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin.  Flies were cultured at 24 +/- 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Seven to ten days old unmated adult males were grown in either normal food (NF) or food containing 5 mg/ml of LEV for three days. Each culture vials contained 30 flies. Flies frozen in liquid nitrogen were agitated and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four LEV treated individuals, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche), and the cDNA purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of NF control and LEV treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and LEV treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The 16 bit TIFF images were preprocessed and quantified using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), and were detectable above background (SNR >= 2). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 3.0, Excel Add-In, Stanford) under the conditions of one class response and 100 permutations

first: Mohammad


title: Effect of vigabatrin on gene expression in Drosophila head

summary:
We recently developed a fly model of pentylenetetrazol (PTZ)-induced long-term behavioral plasticity. Pharmacological validation suggests this model to be kindling-like. Hence, our model is of relevance in understanding the molecular pathogenesis of epileptogenesis as well as in screening potential antiepileptogenic agents. Vigabatrin (VGB) is an antiepileptic drug, and in the process of developing our fly model we generated gene expression profile of flies’ head secondary to treatment of the insects with VGB. Our results provide novel insights in to the drug’s mode of action.
Keywords: Dose response

overall_design: Oregon-R wild type D. melanogaster was grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin.  Flies were cultured at 24 +/- 1ºC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Ten to eleven days old unmated adult males were grown in either normal food (NF) or food containing 24 mg/ml of VGB for three days. Each culture vials contained 30 flies. Flies frozen in liquid nitrogen were agitated and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four VGB treated individuals, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche), and the cDNA purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of NF control and VGB treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and VGB treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The 16 bit TIFF images were preprocessed and quantified using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 2.21, Excel Add-In, Stanford) under the conditions of one class response and 100 permutations.


title: Mapping drought-responsive genes in cotton

status: Public on Oct 03 2007

city: Lubbock

first: Paxton

last: Payton

state: TX

address: 3810 4th Street

institute: USDA-ARS

summary:
Structural and functional approaches were used to study cotton (Gossypium) genes implicated in water-deficit stress. A genetic map representing the hypothetical ancestral diploid genome (Consensus Map) was used to map 1,907 of 15,784 tentative consensus sequences (TCs). These TCs represent 25,119 cotton ESTs derived from various tissues under irrigated and water-limited condition. The correspondence of mapped TCs and 42 stress-related quantitative trait loci (QTLs) revealed that 391 of the initial 1907 TCs co-localized within a QTL interval. About 31% of these TCs were annotated as genes involved in plant responses to abiotic stress. By comparison, only 18% of the total annotated TCs mapped on the Consensus map were classified as abiotic stress genes.  The enrichment of stress-related TCs that map to stress-related QTLs could not be explained by chance (P = 1.5 x 10-7). Gene expression profiling experiments were carried out using a microarray composed of 12,006 oligonucleotides. Transcriptional responses to imposed water-deficit stress in root and leaf tissue of 8-week old cotton plants revealed 1401 transcripts identified as drought responsive. A total of 158 (84 drought-induced and 74 drought-repressed) genes were mapped, of which 22 (8 induced and 14 repressed) genes co-localized with a QTL. A total of 539 unique genes were identified in the drought-stressed libraries. However, only 91 of these genes are contained on the array.  Of these genes, 12 showed significant changes in transcript abundance between stressed and irrigated leaf and root. Forty-five candidate genes implicated in drought-stress response at some level of characterization were identified.
Keywords: stress response

platform_organism:
Gossypium hirsutum
Gossypium arboreum
Gossypium raimondii

overall_design:
Cotton plants (FiberMax 989) were grown under greenhouse conditions (30ºC/25ºC, day/night) for 8 weeks.  Plants were fertilized twice weekly with 50% Hoagland’s solution.  Water-deficit stress was imposed by withholding irrigation and monitored based on leaf water potential measured with a pressure bomb.  Additionally, leaf level gas-exchange was measured as water-deficit increased using a portable infrared gas analyzer (Li-COR, Model LI-6400, Lincoln, NE, USA).  Leaf-to-air vapor pressure deficit (VPD), air temperature, and CO2 concentration (400 μmol mol−1) of the cuvette were set to ambient environmental values for each measurement period and maintained constant for all measurements across plots.  Irradiance was set to saturating light conditions (2000 μmol m−2 s−1) using a light-emitting diode (Licor LI-6400-002).  Data were logged three times for each leaf and then averaged for each plant to be used as a statistical unit.  At the time of leaf and root tissue harvest, leaf water potentials averaged -8.7 Bars (+ 0.37 SE) for fully irrigated control plants and -23.1 Bars (+ 0.28 SE) for stressed plants, and leaf photosynthetic rates had decreased to approximately 60% of the rates seen in the irrigated controls.  The youngest, nearly fully expanded leaf (4th leaf from the apical meristem) was sampled from each plant and immediately frozen in liquid nitrogen.  Root tissue was harvested by sampling lateral roots in two sequential steps of flash freezing in liquid nitrogen.
Gene expression profiling experiments were carried out using the second-generation cotton oligonucleotide microarray.  The array is composed of 12,006 oligonucleotides derived from an assembly of more than 180,000 Gossypium ESTs sequenced from 30 cDNA libraries (www.cottonevolution.info/microarray).  Expression data were subjected to LOWESS normalization and a Benjamini and Hochberg multiple testing correction.  Differential expression was defined as >2 fold change in expression level at p <0.05 in at least 4 of 6 replicate hybridizations of 3 biological replicates in dye-swap comparisons.

first: Joseph

last: Quilantan

first: Hirut

last: Kebede

first: Randy

middle: D

last: Allen

first: Robert

last: Wright


title: Gene Expression Analysis of Human Endometrial Endothelial Cells Exposed to o,p'-DDT

city: Uppsala

first: Carolina

last: Bredhult

address: Uppsala University Hospital

department: Women's and Children's Health

institute: Uppsala University

summary:
The endocrine disrupting chemical o,p'-DDT can affect reproductive organs, tissues and cells in several species. Treatment of human endometrial endothelial cells (HEEC) with 50 µM o,p'-DDT decreased the cell proliferation compared to the control. Microarray analyses revealed that o,p'-DDT affected biological processes such as the cell cycle, cell division, defence response and lipid and steroid metabolism, in cellular components such as the plasma membrane and chromosomes, with molecular functions involved in signalling, receptor and cytokine activity, confirming the results of the proliferation assay. The present study supports our previous findings of decreased proliferation and increased cell death in response to o,p'-DDT, and may offer important clues in the deduction of the mechanisms of action of o,p'-DDT.
Keywords: Exposure analysis

overall_design:
Two-condition experiments, o,p'-DDT-exposed vs. control HEEC, and individual control vs control pool. Biological replicates: 5 HEEC cultures, independently grown, exposed and harvested. One replicate per array. Dye-swaps for exposed and reference samples between replicates.

variable = Exposure: o,p'-DDT-treated: Indiv1 s1 op-DDT Cy3, Indiv2 s19 op-DDT Cy3, Indiv3 s37 op-DDT Cy3, Indiv4 s55 op-DDT Cy5, Indiv5 s73 op-DDT Cy5
variable = Exposure: control: Indiv1 s2 control Cy5, Indiv2 s20 control Cy5, Indiv3 s38 control Cy5, Indiv4 s56 control Cy3, Indiv5 s74 control Cy3
variable = Exposure: individual control: Indiv1 s17 control Cy3, Indiv2 s35 control Cy5, Indiv3 s53 control Cy3, Indiv4 s71 control Cy5, Indiv5 s89 control Cy3
variable = Exposure: control pool: Pooled control s18/36/54/72/90 Cy5, Pooled control s18/36/54/72/90 Cy3, Pooled control s18/36/54/72/90 Cy5, Pooled control s18/36/54/72/90 Cy3, Pooled control s18/36/54/72/90 Cy5
repeat = Biological replicate: Indiv1 s1 op-DDT Cy3, Indiv1 s2 control Cy5, Indiv1 s17 control Cy3, sample 18 in Pooled control
repeat = Biological replicate: Indiv2 s19 op-DDT Cy3, Indiv2 s20 control Cy5, Indiv2 s35 control Cy5, sample 36 in Pooled control
repeat = Biological replicate: Indiv3 s37 op-DDT Cy3, Indiv3 s38 control Cy5, Indiv3 s53 control Cy3, sample 54 in Pooled control
repeat = Biological replicate: Indiv4 s55 op-DDT Cy5, Indiv4 s56 control Cy3, Indiv4 s71 control Cy5, sample 72 in Pooled control
repeat = Biological replicate: Indiv5 s73 op-DDT Cy5, Indiv5 s74 control Cy3, Indiv5 s89 control Cy3, sample 90 in Pooled control

first: Lena

last: Sahlin

first: Matts

last: Olovsson


title: Translation initiation factor 4E confers primary human cells with neoplastic properties

city: Stockholm

first: Ola

last: Larsson

department: Department of oncology-pathology

institute: Karolinska Institutet

summary:
Deregulation of translational control is an obligatory step in oncogenesis; however, this step has not been addressed by prior genomic and transcriptional profiling studies of cancer biology. Here we simulate the translational deregulation found in cancer by ectopically over expressing translation initiation factor eIF4E in primary human mammary epithelial cells; and examine its impact on cell biology and the pattern of ribosomal recruitment to mRNA genome wide. Over expression of eIF4E allows cells to bypass M0 premature growth arrest, but does not confer other malignant properties. However, in concert with hTERT, eIF4E imparts cells with growth and survival autonomy - and profoundly alters the pattern of polyribosome-associated mRNA encoding cell cycle and apoptosis regulators. The translational response to increased eIF4E is not only a unidirectional activation of oncogenic drivers, but also consists of complex intrinsic translational mechanisms that mitigate the acquisition of neoplastic properties.
Keywords: Cell line comparison using both total and polyribosomal RNA

overall_design: A comparison of HMEC/hTERT and HMEC/hTERT/eIF4E cells using both total and polyribosoaml associated RNA.


title: Effect of sodium valproate on gene expression in Drosophila head

summary:
We recently developed a fly model of pentylenetetrazol (PTZ)-induced long-term behavioral plasticity. Pharmacological validation suggests this model to be kindling-like. Hence, our model is of relevance in understanding the molecular pathogenesis of epileptogenesis as well as in screening potential antiepileptogenic agents. Sodium valproate (NaVP) is an antiepileptic drug, and in the process of developing our fly model we generated gene expression profile of flies’ head secondary to treatment of the insects with NaVP. Our results provide novel insights in to the drug’s mode of action.
Keywords: Dose response

overall_design: Oregon-R wild type D. melanogaster was grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin.  Flies were cultured at 24 +/- 1ºC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Ten to eleven days old unmated adult males were grown in either normal food (NF) or food containing 0.33 mg/ml of NaVP for three days. Each culture vials contained 30 flies. Flies frozen in liquid nitrogen were agitated and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four NaVP treated individuals, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche), and the cDNA purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of NF control and NaVP treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and NaVP treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The 16 bit TIFF images were preprocessed and quantified using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 2.21, Excel Add-In, Stanford) under the conditions of one class response and 100 permutations.


title: Genome-Wide Expression Profiling of the Arabidopsis Female Gametophyte Identifies Families of Small, Secreted Proteins

city: Chicago

first: Matthew

last: Jones-Rhoades

address: W519, 929 E. 57th Street

institute: University of Chicago

type: Expression profiling by genome tiling array

summary:
Comparison of expression in wild type ovules to that in dif1 mutant (no embro sac) and myb98 mutant (impaired pollen tube guidance)
Keywords: gene expression analysis

overall_design: 12 samples were analyzed, with replicates per genotpye (wild type, dif1, myb98).  Ovules were dissected out of muture ovaries.  Total RNA was isloated by spin column purification, 2 ug were  used as template for reverse transcription, seceond strand synthesis and biotin labeling.

first: Borevitz

last: Justin

first: Daphne

last: Preuss


title: CGH result of Neisseria meningitidis ST-7 isolates

city: Beijing

first: Junping

last: Peng

address: No 6, Rongjing Eastern Street, BDA

country: China

institute: Chinese center for disease control and prevention

summary:
Neisseria meningitidis is a major cause of bacterial meningitis and septicemia worldwide. Seven new serogroup C meningococci were isolated from two provinces of China in January, 2006. Their PorA VR types were P1.20, 9. Multilocus sequence typing results indicated that they all belonged to ST-7. It is a new serogroup C N. meningitidis sequence type clone identified in China. Here we also present the results of a genomic comparison of these isolates with other 15 N. meningitidis serogroup A and B isolates, which belonged to ST-7, based on comparative genomic hybridization analysis. The data described here would be helpful to monitor the spread of this new serogroup C meningococci sequence type clone in China and worldwide.
Keywords: comparative genomic hybridization

overall_design: To compare the genome compositions of these menC ST-7 isolates with those of menC ST-4821 isolates, menA ST-7 isolates and menB ST-7 isolates, we performed comparative genomic hybridization (CGH) analysis among 17 N. meningitidis isolates (including two newly identified menC ST-7 isolates) using an updated version of the whole-genome microarray of N. meningitidis serogroup C isolate 053442 .

first: Xiaobing

last: Zhang

first: Qi

last: Jin


title: Chromatin IP of POF, HP1 and H3K9ac in S2 cells and salivary glands.

city: Umea

first: Jan

address: Umea University

department: Molecular Biology

institute: Umea University

summary:
Two chromosome-targeting and gene regulatory systems are present in Drosophila melanogaster. The male X-chromosome is targeted by the male-specific lethal complex believed to mediate the two-fold up-regulation of the X-linked genes. The highly heterochromatic 4th chromosome is specifically targeted by the Painting of Fourth (POF) protein which together with heterochromatin protein 1 (HP1) modulate the expression level of genes on the 4th chromosome. Here we report high-resolution mapping of POF and HP1 on the 4th chromosome using chromatin immunoprecipitation followed by tiling microarrays (ChIP-chip) in S2 cells and salivary glands. The enrichments are compared to transcript profiles of the two cell types used. POF specifically binds to genes with a strong preference for exons. The POF binding profile correlates to the binding profile of HP1 which in addition displays a typical “peak” in the promoter regions of bound genes. POF and HP1 binds to active genes and the binding correlates with levels of transcription and changes in binding levels are paralleled by a comparable change in transcription. Our results provide a high resolution description of the chromosome 4 specific gene regulatory system provided by the combinatorial effects of POF and HP1.
Keywords: ChIP-chip

overall_design: Chromatin IP of POF, HP1 and H3K9ac in S2 cells. Three biological replicates, one replicate per array.

first: Per

last: Stenberg

first: Anna-Mia

last: Johansson