title: Centaurea maculosa preliminary study

city: Fort Collins

first: Amanda

last: Broz

state: CO

address: 1173 Campus Delivery

department: Horticulture and Lanscape Architecture

institute: Colorado State University

summary:
Competition is a major determinant of plant community structure consisting of both species-specific and general interactions, either of which may influence competitive competency and plant abundance and size.  In certain cases, competitive competency could arise from altered gene expression and plant function when an individual is confronted with new competitors.  We explored competition at the molecular level by hybridizing transcripts from Centaurea maculosa (spotted knapweed), one of North America's most invasive exotic plant species, to an Arabidopsis microarray chip.  Centaurea was grown in competition with Festuca idahoensis (Idaho fescue), a native grass species that generally has weak competitive effects against Centaurea; Gaillardia aristata (Indian blanketflower), a native herbaceous species that tends to be a much stronger competitor against Centaurea; or alone (control).  The expression of some genes was found to be relatively uninfluenced by the type of plant neighbor, whereas other patterns of gene expression appeared to be more neighbor specific.  To our knowledge, these results are the first to identify genes in an invasive plant that are induced or repressed by plant neighbors and provide a new avenue of insight into the molecular aspects of plant competitive ability.
Keywords: treated vs.untreated

overall_design:
First file, Chip 508 (9-9-05) is preliminary hyb data to see if this cross species hybridization is possible using the OAR27K chip
Experiment 1 (chip 508): The Arabidopsis OAR27K gene chip was hybridized with labeled cDNA probes produced from samples of Centaurea RNA at the W.M. Keck Foundation Biotechnology Resource Laboratory, Yale University.  The OAR27K gene chip consists of 70-mer oligos representing approximately 27,000 genes from Arabidopsis.  Leaf and root samples were reverse-transcribed into cDNA and labeled with different fluorescent dyes (Cy3 and Cy5) using the Genisphere Array 900 kit (Cat No.W500180) (Genisphere, Hatfield PA).  Labeled cDNAs were hybridized to the OAR27Kchip using the Advalytix ArrayBooster DNA Microarray Incubator (Advalytix, Boston MA).  Approximately 10ug of each sample was used for hybridization.Experiment 2:  Hybridization and labeling followed the procedure above, except the experimental control was Centaurea plants grown alone, and the test situations were Centaurea grown with Gaillardia or Festuca neighbors.  In each experiment, RNA from Centaurea grown alone was used as the control (labeled with Cy3) and RNA from Centaurea grown with a neighbor was used as the test (labeled with Cy5).  Two replicates were performed for each test situation.


title: Canine CNVs associated with canFam2 segmental duplications

status: Public on Oct 31 2008

city: Seattle

first: Thomas

middle: J

last: Nicholas

state: WA

address: 1705 NE Pacific St. Foege Building

department: Genome Sciences

country: USA

institute: University of Washington

type: Genome variation profiling by genome tiling array

summary: This study centered on using a custom made Nimblegen aCGH chip that targeted all segmental duplications in the canine genome to identify associated CNVs.  A total of 19 hybridizations were performed in a panel of diverse dogs and a single wolf.

sample_organism:
Canis lupus
Canis lupus familiaris

overall_design: Using computational approaches all segmental duplications were identified in the canFam2 genome of the dog.  A custom aCGH chip was then built that densely interrogated these segmental duplications for CNVs in a panel of diverse dog breeds and a single wolf.

first: Ze

last: Cheng

first: Mario

last: Ventura

first: Katrina

last: Mealey

first: Evan

middle: E

last: Eichler

first: Joshua

middle: M


status: Public on Oct 29 2008

city: BEAUCOUZE

first: Sandra

last: Pelletier

address: 42, rue Georges Morel - BP 60057

country: France

institute: INRA

summary:
Transcriptional and translational status of genes responding to drought stress								 - Drought-stressed plants were compared to non-stressed plants, using total, polysomal or non-polysomal RNAs.

Keywords: treated vs untreated comparison

overall_design: 6 dye-swap - CATMA arrays

first: Jean-Pierre

middle: P

last: Renou

first: Jeff

last: Leung

first: Fabien

last: Jammes

first: Marie-Laure

middle: L

last: Martin-Magniette


title: Moderate long-term drought stress in rice

city: Golm

first: Karin

last: Koehl

state: Brandenburg

address: Am Muehlenberg 1

country: Germany

institute: Max-Planck-Institut of Molecular Plant Physiology

summary:
We investigated the physiological and gene expression response of drought-tolerant (IR57311 and LC-93-4; subgroup indica) and drought-sensitive (Nipponbare and Taipai 309; subgroup japonica) rice (Oryza sativa) cultivars to 18 days of drought stress in climate chamber experiments. Rice plants were grown under water sufficient and water limiting conditions in three independent experiments in a controlled climate chamber with 12 h day length at 600 µE m-2 s-1; temperature was 26°C in the light and 22°C at night, with a relative humidity of 75% in the light and 70% at night. Leaf material for expression profiling analysis was harvested five hours after the beginning of the light period after 18 days of stress or control treatment. Normalization and statistical testing was performed using the R package limma (R 2.3.1, limma version 2.7.3; (Smyth, 2005)). The methods Robustspline for within array normalization and Quantile for between array normalization were applied. A linear model with the effects genotype, drought treatment, genotype x drought, dye was fitted to normalized data in limma that models the systemic variation in the data. Afterwards, for the comparisons of interest, moderated t-statistics that use an empirical Bayes method were calculated. Differentially expressed genes were identified using the decideTests function (method global, Benjamini & Hochberg fdr corrected p-value <0.05 in limma)
Keywords: stress reponse

overall_design:
Growth design. Three independent experiments were performed. The design was a split-plot design with five blocks per drought or control treatment in each experiment.  Each treatment and cultivar was represented by five replicate pots with one plant per pot. Pots were randomized within the blocks. Block position was rotated daily.
Hybridisation design: A total of 28 samples, each representing mRNA from four parallel plants, were hybridized to 14 arrays. The hybridization design was optimized for the estimation of the effects of condition and the condition x cultivar interaction taking a variance minimization approach. In a two-step procedure, a smaller design for one sensitive and one tolerant cultivar was enlarged to encompass all four cultivars by integrating eight additional arrays. The optimization was programmed using R [R Development Core Team 2006] and carried out on the 16-node Beowulf Linux-Cluster at the University of Potsdam. R-scripts are available upon request and from the Bioinformatics Team at the MPI of Molecular Plant Physiology (see the website for more information). Allocation of the three biological replicates of each of the combinations of treatment and cultivar was completed such that a balanced distribution with respect to the labeling was achieved. Each combination of condition and cultivar from all experiments has been used at least once.

last: Degenkolbe

first: Phuc

middle: T

last: Do

first: Ellen

last: Zuther

first: Dirk

last: Repsilber

last: Walther

middle: K

last: Hincha

middle: I


title: Microarray analysis of rat mammary carcinogenesis

status: Public on Oct 28 2008

city: sakuragaoka

first: souda

last: masakazu

state: kagoshima/kagoshima

department: tumor pathology, field of oncology

country: Japan

institute: kagoshima university graduate school of medical and dental sciences

summary:
7,12-Dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinoma is a well-recognized model, however, the genetic alterations during its carcinogenesis have yet to be determined. We used laser capture microdissection to specifically isolate cells from terminal end buds (TEBs), the origin of carcinoma, at 2 weeks after sesame oil- treatment (control) or DMBA-treatment (DMBA-TEBs), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (MC). Using an oligonucleotide microarray representing 20600 rat probe sequences, we analyzed gene expression profiles and validated mRNA and protein levels of genes of interest by real-time quantitative PCR, immunohistochemistry and immunofluorescence. The number of differentially expressed genes was smallest in DMBA-TEBs (63), followed by DCIS (798) and MC (981). Only the expression of PEP-19, an anti-apoptic gene, showed significant increases in DMBA-TEBs (4-fold), DCIS (10-fold) and MC (16-fold). MMP-13 expression was increased markedly in DCIS (19-fold) and MC (61-fold) while OPN expression was increased 6-fold in DCIS and 8-fold in MC. MMP-7 expression was increased 4-fold in MC. Nidogen-1; a participant in the assembly of basement membranes, TSP-2; an inhibitor of angiogenesis and COUP-TFI; a transcription repressor showed significant decreases in DCIS (4-, 9-, and 17-fold, respectively) and MC (10-, 37-, and 100-fold). Network analyses with IPA software revealed that the most significant network was centered on Akt groups in DCIS and ERK groups in MC. The present findings provide insights into the molecular changes and suggest the importance of PEP-19 overexpression very early on during mammary carcinogenesis.

Keywords: Comparative experiments of sesame oil treatment（control）and 7,12-Dimethylbenz[a]anthracene（DMBA）treatment group. Or                                      comparative experiments of tissue type

overall_design: Experiment：7,12-Dimethylbenz[a]anthracene (DMBA)-induced rat mammary carcinoma is a well-recognized model, however, the genetic alterations during carcinogenesis have yet to be determined. We used laser capture microdissection to specifically isolate the cells from terminal end buds (TEBs), the origin of MC, at 2 weeks after sesame oil-(control) or DMBA-treatment (DMBA-TEBs), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (MC). At 50 days after birth, inbred Sprague-Dawley female rats were given 1ml sesame oil (control) or 20 mg DMBA (Nacalai tesque, Inc. Kyoto Japan) dissolved in 1ml sesame oil, by gastric intubations. Animals in control group were euthanized after 2 weeks (W) and DMBA-treated group were euthanized after 2 weeks (W), 6W and 8W, respectively. From for 4 sample(control, DMBA-TEBs, DCIS, MC), we performed a hybridization repeatedly, respectively.

first: Masakazu

last: Souda

first: Yoshihisa

last: Umekita

first: Kazuhiro

last: Abeyama

first: Hiroki

last: Yoshida


title: The growth factor environment defines distinct pluripotent ground states in novel blastocyst derived stem cells

city: Boston

first: Niels

last: Geijsen

state: MA

address: 185 Cambridge St. CPZN 4-4256

department: Center for Regenerative Medicine

institute: Massachusetts General Hospital

summary:
Pluripotent stem cell lines can be derived from blastocyst embryos, which yield embryonic stem cell lines (ES cells), as well as the post-implantation epiblast, which gives rise to epiblast stem cell lines (EpiSCs). Remarkably, ES cells and EpiSCs display profound differences in the combination of growth factors that maintain their pluripotent state. Molecular and functional differences between these two stem cell types demonstrate that the tissue of origin and/or the growth factor milieu may be important determinants of the stem cell identity. We explored how developmental stage of the tissue of origin and culture growth factor conditions affect the stem cell pluripotent state. Our findings reveal that novel stem cell lines can be generated from blastocyst embryos with unique functional and molecular properties. We demonstrate that the culture growth factor environment and cell-cell interaction play a critical role in defining several unique and stable stem cell ground states.

Keywords: Clonal analysis of FAB-SC gene expression

overall_design: Cell type characterization - 3 separate single cell clones

first: Yu-Fen

last: Chou

first: Hsu-Hsin

last: Chen

first: Maureen

last: Eijpe

first: Akiko

last: Yabuuchi

middle: G

last: Chenoweth

first: Jun

last: Lu

first: R

middle: D

last: McKay


title: Streptococcus agalactiae genes controlled by the MtaR transcription factor

city: Shreveport

first: Daniel

middle: Wilson

last: Shelver

state: LA

address: 1501 Kings Highway

department: Microbiology and Immunology

institute: LSU Health Sciences Center in Shreveport

summary: The purpose of this study was to determine what S. agalactiae genes are under the control of the MtaR regulatory protein.  Inactivation of the Streptococcus agalactiae mtaR gene resulted in the downregulation of 11 genes and the upregulation of 1 gene in the mtaR mutant.  Genes involved in the uptake of methionine and a gene involved in the degradation of peptides were downregulated. Also, genes involved in the transport and biosynthesis of arginine were downregulated.  The gene encoding the virulence factor cspA was downregulated, as well as a gene showing similarity to plasminogen activators.  Thus, the expression of genes involved in both metabolic functions and virulence are under the influence of mtaR.

overall_design: Three cultures of an mtaR insertional mutant (DS101) and three cultures of the isogenic wild-type strain (COH1) were grown in a chemically-defined medium.  Cells were disrupted with glass-bead and RNA was isolated by a Trizol method.  A custom Affymetrix chip was utilized for microarray analysis.  Genes were considered differentially-expressed if they showed a >2.0-fold difference in expression between the strains and a Bayesian (Cyber-T) t-test value of <0.001.  The posterior probability of differential expression (PPDE) (< p) value for the genes identified was >0.964.

first: Joshua

last: Bryan

first: Roxanne

last: Liles


title: Expression data from chicken B cell lymphoma (DT40) cell lines.

status: Public on Oct 27 2008

city: Huddinge

first: Hossain

last: Nawaz

state: Stockholm

address: Hälsovägen 7

department: LABMED

country: Sweden

institute: Karolinska Institutet

summary:
Global gene expression profiling of the avian B-lymphoma DT40 cell line was used as a model to differentiate among Btk KO and Btk KO cells reconstituted with human Btk. Differences in the gene expression pattern showed statistically significant changes between parental DT40 and all the Btk KO cell populations irrespective of whether they are reconstituted or not. These results imply that in the process of generating a knockout cell line, subclones are selected, which have multiple changes in their gene expression pattern (p<0.01).

Keywords: cell line

overall_design: DT40 cells along with the mutants were grown at various time points in different batches of fetal calf serum with or without any stimulation. All experiments were repeated ten times and then polled together as one sample. In total 28 samples were used RNA extraction and hybridization on Affymetrix microarrays.

first: Emelie

last: Blomberg


title: The Transcriptome of Photo-Biological Hydrogen Production in the Green Alga Chlamydomonas reinhardtii

city: Bielefeld

first: Anh Vu

last: Nguyen

state: NRW

address: G2-154, CEBITEC, Universitaetsstr. 27

department: Cell and Molecular Biology

institute: Bielefeld University

summary:
Algal photo-bio hydrogen production, a promising method for producing clean and renewable fuel in the form of hydrogen gas, has been studied extensively over the last few decades.  In this study, microarray analyses were used to obtain a global expression profile of mRNA abundance in the green alga Chlamydomonas reinhardtii at five different time points before the onset and during the course of sulphur depleted hydrogen production. The present work confirms previous findings on the impacts of sulphur deprivation but also provides new insights into photosynthesis, sulphur assimilation and carbon metabolism under sulphur starvation towards hydrogen production. For instance, while a general trend towards repression of transcripts encoding photosynthetic genes was observed, the abundance of Lhcbm9 (encoding a major light harvesting polypeptide) and LhcSR1 (encoding a chlorophyll binding protein) was strongly elevated throughout the experiment, suggesting remodeling of the photosystem II light harvesting complex as well as an important function of Lhcbm9 under sulphur starvation. This study presents the first global transcriptional analysis of C. reinhardtii during hydrogen production using five major time points at Peak Oxygen, Mid Oxygen, Zero Oxygen, Mid Hydrogen and Peak Hydrogen.
Keywords: Time course, sulfur deprivation, hydrogen production.

overall_design: Time course microarray analyses were used to analyze the global gene expression in sulfur depleted hydrogen producing C. reinhardtii. When depleted of sulfur, a sealed an illuminated C. reinhardtii culture slowly becomes anaerobic and produces hydrogen gas. Based on the changes in the dissolved O2 level and H2 production rate, the whole course of H2 production from the starting of S deprivation to the end of H2 production can be divided into five phases including an Aerobic Phase (I) in which the dissolved O2 level goes up (I), an O2 Consumption Phase (II), an Anaerobic Phase (III), a H2 Production Phase (IV) and a Termination phase (V). Samples were taken from three different bioreactors (biological replicates) at the following time points after the start of S depletion: 6 h, 16 h, 21 h, 37 h and 52 h corresponding to Peak O2, Mid O2, Zero O2, Mid H2 and Peak H2.

first: Anh

middle: V

first: Jen

last: Rupprecht

first: Skye

last: Thomas-Hall

first: Ben

last: Hankamer

first: Olaf

last: Kruse

first: Peer

last: Schenk


title: Arabidopsis at the stage of Golovinomyces cichoracearum haustorium formation

status: Public on Oct 21 2008

city: Stanford

last: Stanford Microarray Database

state: CA

address: 300 Pasteur Drive

department: Stanford University, School of Medicine

institute: Stanford Microarray Database (SMD)

summary:
Here, we sought to describe qualitative and quantitative changes in the global gene expression profiles of susceptible Arabidopsis plants supporting the development of G. cichoracearum haustoria. We analyzed the features of compatibility at this infection stage, and further evaluated the contribution of the SA- and JA/ET-dependent defense signaling pathways in the pathogen-induced responses by comparing responses in infected wild-type, npr1-1, and jar1-1 plants. Our findings collectively contribute to knowledge regarding early host cell alterations generated in response to attack by this virulent obligate biotrophic fungus.
Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc.
Disease State: plant infected/uninfected with Golovinomyces orontii
Genotype: wt/mutant strains

Keywords: Logical Set

overall_design: Computed

first: Georgina

last: Fabro


title: Integrating chromosomal aberrations and gene expression profiles to dissect rectal cancer

city: Amsterdam

first: Esther

last: Lips

address: Plesmanlaan 121

department: Molecular Pathology

country: Netherlands

institute: NKI-AVL

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

summary:
Accurate staging of rectal tumors is essential for making the correct treatment choice. In a previous study, we found that loss of 17p, 18q and gain of 8q, 13q and 20q could distinguish adenoma from carcinoma tissue and that loss of 1q was related to lymph node metastasis. In order to find markers for tumor staging, we searched for candidate genes on these specific chromosomes. We performed gene expression microarray analysis on 79 rectal tumors and integrated these data with genomic data from the same sample series (Series GSE7946, Samples GSM194994-GSM195028). We performed supervised analysis to find candidate genes on affected chromosomes and validated the results with qRT-PCR and immunohistochemistry. Approximately 8% of the genes were significantly different between adenomas and carcinomas; the most differently expressed genes were involved in cell adhesion and cell cycle processes. Integration of gene expression and chromosomal instability data revealed a significant genome-wide correlation between these two data types. Supervised analysis identified up-regulation of EFNA1 in cases with 1q gain, and EFNA1 expression was correlated with the expression of a target gene (VEGF). The BOP1 gene, involved in ribosome biogenesis and related to chromosomal instability, was over-expressed in cases with 8q gain. SMAD2 was the most down-regulated gene on 18q, and on 20q, STMN3 and TGIF2 were highly up-regulated. Immunohistochemistry for SMAD4 correlated with SMAD2 gene expression and 18q loss. This study showed a good correlation between chromosomal aberrations and gene expression data. In the near future, specific genes identified by such integrative methods could be of additional value for explaining rectal tumorigenesis.

Keywords: disease state analysis

overall_design: Samples GSM307294-GSM307372: 79 Tumor RNAs were hybridised, all against a common reference sample, consisting of a mixture of cell line and normal colon RNA. 9 replicates (primarily duplicates) were performed (tumor samples: 217, 544, 608, 609, TME1344, TME625, TME884, TME947).

middle: H

first: Hans

last: Morreau


title: Cryptic Transcription in a set2∆ in Saccharomyces cerevisiae

city: Chapel Hill

first: Jason

last: Lieb

state: NC

address: 408 Fordham Hal, CB#3280

department: Biology

institute: UNC-Chapel Hill

type: Expression profiling by genome tiling array

summary: In cells lacking the histone methyltransferase Set2, initiation of RNA polymerase II transcription occurs inappropriately within the protein-coding regions of genes, rather than being restricted to the proximal promoter. Here, we mapped the transcripts produced in an S. cerevisiae strain lacking Set2, and applied rigorous statistical methods to identify sites of cryptic transcription at high resolution.

overall_design: Wild type (BY4741) and set2∆ (BY4741) strains were grown at 30°C in YPD (1% yeast extract, 2% peptone, 2% dextrose) to an OD600 of 0.6-0.8. For each of the three replicates, Total RNA was extracted by acid-phenol method (Xiao et al. 2003). Double-stranded cDNA was prepared using an Invitrogen SuperScript™ (Cat No. 11917-010) primed with Oligo(dt) and random hexamers. For each replicate, the wt and set2∆ cDNA were independetly fluorescently labeled and comparatively hybridized to high-resolution 385K Saccharomyces cerevisiae CGH arrays (2005-08-16_SCER_WG_CGH) with Tm-normalized probes. In one of the replicates, assignment of the fluorescent label was reversed.

first: Colin

middle: R

last: Lickwar

first: Bhargavi

last: Rao

first: Andrey

middle: A

last: Shabalin

first: Andrew

middle: B

last: Nobel

first: Brian

last: Strahl


title: Analysis of the physiological status of Geobacter uraniireducens growing in uranium-contaminated subsurface sediments

status: Public on Oct 20 2008

city: Amherst

first: Derek

last: Lovley

address: University of Massachusetts at Amherst, Dept. of Microbiology, Morrill IV north room 202

department: Microbiology

institute: University of Massachusetts Amherst

summary:
Geobacteraceae transfer electrons from a donor such as acetate to an electron acceptor such as Fe(III) or U(VI).  Geobacter uraniireducens is found in uranium-contaminated sites and plays an important role in in situ bioremediation.  In this experiment, gene expression was compared between G. uraniireducens cultures grown in sediments from a uranium contaminated site amended with acetate and cultures grown in acetate/fumarate medium.
Keywords: two-condition comparison

overall_design: This was a dual channel experiment using aRNA directly labeled with either cy5 (experimental) or cy3 (control). Three biological replicates were labeled and hybridized in duplicate (two technical replicates).

first: Dawn

last: Holmes

first: Regina

last: O'Neil

first: Milind

last: Chavan

first: Lorrie

last: Perpetua

first: Juliana

last: Larrahondo

first: Raymond

last: DiDonato

first: Anna

last: Liu


title: Transcriptional profiles of Staphylococcus aureus strains in the presence of the lantibiotic mersacidin.

status: Public on Oct 23 2008

city: Bonn

first: Peter

last: Sass

address: Sigmund-Freud-Straße 25

department: Institute for Microbiology (IMMIP)

institute: University of Bonn

summary:
The lantibiotic mersacidin is an antimicrobial peptide of 20 amino acids that is ribosomally produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin acts by complexing the sugar phosphate head group of the peptidoglycan precursor lipid II, thereby inhibiting the transglycosylation reaction of peptidoglycan biosynthesis.
Here, we studied the transcriptomic response of S. aureus to subinhibitory concentrations of mersacidin using microarray and qRT-PCR techniques. The transcriptomics revealed an extensive induction of the cell wall stress response of S. aureus, which is partly controlled by the two-component regulatory system VraSR. In contrast to other cell wall-active antibiotics such as vancomycin, 0.2 x MIC of mersacidin was sufficient for induction. Interestingly, the cell wall stress response was equally induced in vancomycin intermediate resistant S. aureus (VISA) as well as in a highly susceptible strain. Furthermore, the efficiency of mersacidin was not affected by an increased cell wall thickness, which is part of the VISA-type resistance mechanism. Since the transcription of the VraDE ABC transporter genes was induced up to 1700-fold in our experiments, we analyzed the role of VraDE in the response of S. aureus to mersacidin. Unexpectedly, the deletion of the vraE gene did not result in an increased susceptibility to mersacidin compared to the wild type strain. In conclusion, mersacidin appears to be a strong inducer of the cell wall stress response of S. aureus at very low concentrations, which reflects its general mode of action as a cell wall-active peptide as well as its use of a unique target site on lipid II.
Keywords: antibiotic induced response

platform_organism: Staphylococcus aureus subsp. aureus N315

overall_design:
In this experiment, the transcriptomic response of S. aureus was assayed via microarray analysis.
To this end, S. aureus strains SA137/93A [mersacidin MIC 35 mg/L] and SA137/93G [mersacidin MIC 30 mg/L] were grown without or with mersacidin [16 mg/L] to determine the genes being induced or repressed by the lantibiotic. SA137/93G was additionally grown in the presence of 4 mg/L mersacidin. Furthermore, the highly susceptible S. aureus strain SG511 [mersacidin MIC 1 mg/L] was also grown without or with mersacidin [1 mg/L].
Each experiment was performed 4 times including a dye swap resulting in four chips per competitive comparison to increase reproducibility. Only strain SA137/93G incubated with 0.2 x MIC of mersacidin was reproduced in duplicate. All hybridizations were done with equal amounts of cDNA probes displaying similar picomoles of incorporated dye.


title: Response of L. lactis faced to S. cerevisiae

city: Toulouse

first: Muriel

last: Cocaign-Bousquet

address: 135 avenue de Rangueil

department: UMR5504 INSA/CNRS - UMR792 INSA/INRA

institute: Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés

summary:
The response of L. lactis to the presence of S. cerevisiae was analyzed during the exponential growth phase in fermentors in defined growth conditions. Although no growth kinetic difference was observed between the pure and mixed culture of L. lactis, the mRNA level of genes was significantly modified. More particularly, a strong reorientation of pyrimidine metabolism was observed when L. lactis was grown in the mixed culture.
Keywords: microbial interaction, time course

overall_design: A pure culture of L. lactis was conducted in parallel with a co-culture of L. lactis and S. cerevisiae. For the transcriptomic analysis, the mixed culture (test condition, 2.5 hours of culture, B’) was compared to the pure culture (reference, 2.5 hours of culture, B) on the same slide at the same time. The mRNA level changes between 2 and 3 hours (A and C samples) flanking B sample (2.5 hours) in the pure culture were also determined on another slide. Total RNA was extracted from these samples and labelled cDNA (Cy3/Cy5) were prepared and hybridized on glass slides exhibiting 2004 amplicons specific of Lactococcus lactis IL1403 genes. 3 independent repetitions were performed.

first: Mathieu

last: Maligoy

first: Pascal

last: Loubière


title: Localized Rho GTPase Activation Regulates RNA Dynamics and Compartmentalization in Tumor Cell Protrusions

status: Public on Oct 17 2008

city: Vancouver

first: Ivan

middle: Robert

last: Nabi

state: British columbia

address: LSI, 2350 health sciences Mall

department: CPS

country: Canada

institute: University of British Columbia

summary:
mRNA trafficking and local protein translation are associated with protrusive cellular domains, such as neuronal growth cones, and deregulated control of protein translation is associated with tumor malignancy. We show here that activated RhoA, but not Rac1, is enriched in pseudopodia of MSV-MDCK-INV tumor cells and that Rho, Rho kinase (ROCK) and myosin II regulate the microtubule-independent targeting of RNA to these tumor cell domains. ROCK inhibition does not affect pseudopodial actin turnover but significantly reduces the dynamics of pseudopodial RNA turnover. Gene array analysis shows that 7.3% of the total genes analyzed exhibited a greater than 1.6-fold difference between the pseudopod and cell body fractions. Of these, only 13.2% (261 genes) are enriched in pseudopodia, suggesting that only a limited number of total cellular mRNAs are enriched in tumor cell protrusions. Comparison of the tumor pseudopod mRNA cohort and a cohort of mRNAs enriched in neuronal processes identified tumor pseudopod-specific signaling networks that were defined by expression of M-Ras and the Shp2 protein phosphatase. Pseudopod expression of M-Ras and Shp2 mRNA were diminished by ROCK inhibition linking pseudopodial Rho/ROCK activation to the localized expression of specific mRNAs. Pseudopodial enrichment for mRNAs involved in protein translation and signaling suggests that local mRNA translation regulates pseudopodial expression of less stable signaling molecules as well as the cellular machinery to translate these mRNAs. mRNA trafficking and local protein translation are associated with protrusive cellular domains, such as neuronal growth cones, and deregulated control of protein translation is associated with tumor malignancy. We show here that activated RhoA, but not Rac1, is enriched in pseudopodia of MSV-MDCK-INV tumor cells and that Rho, Rho kinase (ROCK) and myosin II regulate the microtubule-independent targeting of RNA to these tumor cell domains. ROCK inhibition does not affect pseudopodial actin turnover but significantly reduces the dynamics of pseudopodial RNA turnover. Gene array analysis shows that 7.3% of the total genes analyzed exhibited a greater than 1.6-fold difference between the pseudopod and cell body fractions. Of these, only 13.2% (261 genes) are enriched in pseudopodia, suggesting that only a limited number of total cellular mRNAs are enriched in tumor cell protrusions. Comparison of the tumor pseudopod mRNA cohort and a cohort of mRNAs enriched in neuronal processes identified tumor pseudopod-specific signaling networks that were defined by expression of M-Ras and the Shp2 protein phosphatase. Pseudopod expression of M-Ras and Shp2 mRNA were diminished by ROCK inhibition linking pseudopodial Rho/ROCK activation to the localized expression of specific mRNAs. Pseudopodial enrichment for mRNAs involved in protein translation and signaling suggests that local mRNA translation regulates pseudopodial expression of less stable signaling molecules as well as the cellular machinery to translate these mRNAs. Pseudopodial Rho/ROCK activation may impact on tumor cell migration and metastasis by stimulating the pseudopodial translocation of mRNAs and thereby regulating the expression of local signaling cascades.

Keywords: tumor cells, protrusive cellular domains, pseudopodia vs cell body, RNA trafficking

overall_design: Pseudopod purification- Pseudopodia purification was performed as previously described (Jia et al, J. Biol. Chem. 280, 30564-73, 2005). Briefly, 107 MSV-MDCK-INV cells were plated on 100 mm 1um pore filters mounted between two 3-3.5” custom-made washers (Boulons Plus, Anjou, QC) in a 100 mm Falcon Petri dish and the exterior sealed with agarose to prevent cell leakage to the bottom of the filter. After 24 hours culture, the filter was washed 4 times with cold PBS containing 0.1 mM Ca2+ and 1 mM Mg2+ and the top (cell bodies) and bottom (pseudopodia) of the filter scraped with a glass cover slip in ice-cold lysis buffer (20 mM Tris-HCl pH 7.6, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA containing freshly added 2 mM DTT, 0.5 mM PMSF, 1 mM sodium vanadate, 2.5 mM sodium fluoride and 1uM leupeptin).   Affymetrix gene array- GeneChips were processed at the London Regional Genomics Centre (Robarts Research Institute, London, Ontario, Canada; http://www.lrge.ca). Six Affymetrix canine arrays (3 pseudopodia, 3 cell body) were scanned with the Affymetrix GeneChip Scanner 3000 and signal intensities for genes generated with GCOS1.2 (Affymetrix Inc., Santa Clara, CA) using default values for statistical expression algorithm parameters, a target signal of 150 for all probe sets and a normalization value of 1. To determine significant differences in gene expression levels between pseudopodia and cell body samples, 1 way-ANOVA was performed using Partek Pro (St. Louis, MO). Significantly modulated genes were defined as those with absolute log2 fold change greater than 0.7. Genes were summarized together using hierarchical clustering by Z score normalized data crossing all 6 samples.

first: Zongjian

last: Jia

first: T

last: Underhill


title: Molecular response of an invasive weed to different competitive conditions.

first: Amanda

last: Broz

overall_design: Files; chip 618 (12-7-05) and chip 623 (1-20-06) are replicates.  One channel is root cDNA from Centaurea maculosa grown in isolation and the other channel is root cDNA from Centaurea maculosa grown with a strong competitor, Gaillardia aristata. For each chip, RNA extractions on unique biological samples were performed. Files; chip 720 (2-23-06) and chip 723 (3-17-06) are replicates.  One channel is root cDNA from Centaurea maculosa grown in isolation and the other channel is root cDNA from Centaurea maculosa grown with a weak competitor, Festuca idahoensis. For each chip, RNA extractions on unique biological samples were performed.


title: High resolution mapping of DNA replication origins in human genome

status: Public on Oct 22 2008

city: Paris

first: Jean-Charles

last: Cadoret

address: 15 rue Hélène Brion

institute: CNRS-IJM

summary:
Because of the lack of information, regulation of DNA replication initiation in mammals is still poorly understood. In order to identify general rules, we have mapped replication origins along 1% of the human genome in HeLa cells. We found large gene-poor regions lacking origin and G+C rich regions containing clusters of closely spaced origins. Half of the 283 origins mapped are within or near CpG islands. The connection with gene expression is further reinforced by the observation that most origins overlap with DNAseI hypersensitive sites found at transcriptional regulatory elements. We show, however, that this association is independent of chromatin structure and transcriptional activity. Replication timing analyses coupled to our origin mapping demonstrate that origin dense regions and isolated origins are replicated at every moment in S phase. All together, our data suggest that a relatively strict origin-timing programme regulates DNA replication of the human genome.
Keywords: Nascent strands, ENCODE project, HeLAS3 cells, SNS-Chip

overall_design: Four independent preparations of Short Nascent Strands (SNS) were performed. In order to have enough material for microarray hybridisation, we coupled the stringent preparation of SNS with the TLAD method, a technique of linear amplification that can generate several µg of amplified material from 10-20 ng of DNA (Liu et al., 2003).Two were amplified by TLAD (experiments A and B) and hybridized on DNA microarrays, and the other two (experiments C and D) were used for the validation by real-time quantitative PCR (qPCR) of results obtained on micro-arrays. We performed also a gDNA/gDNA hybridization where gDNA are also amplified by TLAD to order to do a control.

first: Françoise

last: Meisch

first: Vahideh

last: Hassan Zadeh

first: Marie-Noëlle

last: Prioleau


title: Transcriptome Characterization of Bovine Peripheral Blood Mononuclear Cells Stimulated by LPS

status: Public on Oct 16 2008

city: Beltsville

first: Robert

middle: W

last: Li

state: MD

address: Barc East

department: Animal & Natural Resources Institute

institute: USDA

summary: This study aimed to identify transcription networks and signatures of bovine peripheral blood mononuclear cells in response to LPS stimulation.   A total of 464 genes including at least 17 transcription factors were identified to be significantly induced by   LPS using a high density bovine oligonucleotide microarray.  The network analysis revealed that alternation in gene expression was regulated by transcription factors through potential interaction within the pathway networks in the LPS stimulated cells.  Our results demonstrated that specific pathway networks are responsible for transcription regulation in bovine peripheral blood mononuclear cells in response to pathogen components such as LPS.

overall_design: One class unpaired: Bovine Peripheral Blood Mononuclear Cells from 10 BLV positive cattle are divided into 2 groups: 5 treated with PBS in vitro for 2h serve as controls. The remaining 5 samples are stimulated with LPS in vitro for 2h.

first: Conjun

first: Theodore

last: Elsasser

first: Stanislaw

last: Kahl


title: Small RNA from S2 cell infected with FHV deltaB2 particle

city: Riverside

first: Qingfa

last: Wu

address: 900 University Ave

institute: UC Riverside

type: Non-coding RNA profiling by high throughput sequencing

summary:
S2 cells were infected with FHV deltaB2 particle and the S2 cells were harvested to isolate total RNA at 96 hpi. Small RNAs were separated on a denaturing 15% polyacrylamide gel. Ligation to adapters requires 5' monophosphate and 3' OH.

Note: The base quality (.qual) file corresponding to the fasta format (.fna) file linked to Sample GSM306489 is unavailable.

overall_design: Small RNAs were sequenced from S2 cell after virus infection.


title: Aspergillus CEA10 compared to SRBA mutant under hypoxic conditions

status: Public on Oct 15 2008

city: Bozeman

middle: A.

last: Cramer

state: MT

department: Department of Veterinary Molecular Biology

institute: Montana State University

summary:
Aspergillus fumigatus strains CEA10 and srbA mutant expression patterns were compared after exposure to 24h of hypoxic conditions
Goal of this study was to find genes that are directly and/or indirectly regulated by the transcription factor srbA, that is know to be crucial for growth under hypoxic conditions

Keywords: hypoxic stress response

overall_design: two strains, 2 biological replicates with dye-swaps

first: Sven

last: Willger


title: Activity motifs reveal principles of timing in transcriptional control of the yeast metabolic network

city: Worcester

first: Oliver

last: Rando

address: 364 Plantation St.

institute: UMass Medical School

summary: Significant insight about biological networks arises from the study of network motifs –overly abundant network subgraphs, but such wiring patterns do not specify when and how potential routes within a cellular network are used. To address this limitation, we introduce activity motifs, which capture patterns in the dynamic use of a network. Using this framework to analyze transcription in Saccharomyces cerevisiae metabolism, we find that cells use different timing activity motifs to optimize transcription timing in response to changing conditions: forward activation to produce metabolic compounds efficiently, backward shutoff to rapidly stop production of a detrimental product and synchronized activation for co-production of metabolites required for the same reaction Measuring protein abundance over a time course reveals that mRNA timing motifs also occur at the protein level.  Timing motifs significantly overlap with binding activity motifs, where genes in a linear chain have ordered binding affinity to a transcription factor, suggesting a mechanism for ordered transcription. Finely timed transcriptional regulation is therefore abundant in yeast metabolism, optimizing the organism's adaptation to new environmental conditions.

overall_design:
We generated a set of 13 time courses by measuring gene expression after a metabolic change. Yeast strain KCN118 (MATalpha ade2) was grown at 28 °C in 400 ml of synthetic complete media with 2% dextrose (SCD) to an OD600 of 0.6. Synthetic complete was prepared using the standard recipe, except 75 uM inositol was included. At OD600 of 0.6, 100 ml of cells were collected by centrifugation and frozen as a reference sample, and the remaining cells were rapidly collected by filtration, washed with distilled water and resuspended in 300 ml of one of the following media: SCE (SC + 2% ethanol), SCG (SC + 2% galactose), SM1 (SCD lacking amino acids A, R, N, C, Q, G, K, P, S, F and T), SM2 (SCD lacking amino acids L, I, V, W, H and M), S0 (SCD lacking all amino acids), S0G (no amino acids, 2% galactose) or S0E (no amino acids, 2% ethanol).

The data appears in Figures 2 and 4 of the manuscript, as it relates to the global analysis of all the arrays used in the dataset.

All time courses consist of the following time points (in min):  15, 30, 60, 120, 240, and were hybridized against the t = 0 time point of cells grown in SCD.

Each time course was performed as one single biological replicate and one technological replicate, except where noted below.

Specifically, the 13 time courses break down into the following groups:

Media key:

SCD (synthetic complete, not including inositol)
SCE (SC + 2% ethanol)
SCG (SC + 2% galactose)
SM1 (SCD lacking amino acids A, R, N, C, Q, G, K, P, S, F and T)
SM2 (SCD lacking amino acids L, I, V, W, H and M)
S0 (SCD lacking all amino acids)
S0G (no amino acids, 2% galactose)
S0E (no amino acids, 2% ethanol).
ino = inositol
aa = all amino acids supplemented

The following group descriptions are the media as described above, followed by the description as indicated in the long title of the individual arrays:

1.  S0 (5 arrays) = SD
2.  SCD (6 arrays, t = 240 min has 2 tech replicates) = SD+aa
3.  SM2 (5 arrays) = SD+aa:ARNCQGKPSDEFTY+ino
4.  SM1 + ino (5 arrays) = SD+aa:LIVWHM+ino
5.  S0 + ino (6 arrays, t = 240 min has 2 tech replicates) = SD+ino
6.  S0E (5 arrays) = SEtOH
7.  S0E + aa (7 arrays, t = 240 min and 60 min each have 2 tech replicates) = SEtOH+aa
8.  S0E + aa + ino (5 arrays) = SEtOH+aa+ino
9.  S0E + ino (8 arrays, t = 240 min, 15 min and 30 min each have 2 tech replicates) = SEtOH+ino
10. S0G (5 arrays) = Sgal
11. S0G + aa (5 arrays) = Sgal+aa
12. S0G + aa + ino (5 arrays) = Sgal+aa+ino
13. S0G + ino (6 arrays, t = 60 min has 2 tech replicates) = Sgal+ino

first: Gal

last: Chechik

first: Eugene

last: Oh

first: Jonathan

last: Weissman

first: Aviv

last: Regev

first: Daphne

last: Koller


title: Tolerance of Arabidopsis to thrip feeding

status: Public on Oct 25 2008

city: Kaga-gun, Okayama

first: Yoshihiro

last: Narusaka

address: 7549-1, Kibityuo-town Yoshikawa

institute: Research Institute for Biological Sciences

summary:
We analyzed the interaction between Arabidopsis and western flower thrips (Frankliniella occidentalis), which are one of the most serious insect pests of cultivated plants.
A total of 1.2K potential biotic and abiotic stress-related genes were selected from the genes covered by the Arabidopsis 7K array (RIKEN, Japan) and Arabidopsis oligo microarray (Agilent Technologies, USA) for this study. The 21- day-old plants were feeded by western flower thrips in an acryl cylinder chamber with air ventilation windows covered with a fine mesh. Leaves were harvested several time after the feeding.
Our results indicate that JA plays an important role in Arabidopsis in terms of response to, and tolerance against, thrip feeding.

overall_design:
A total of 1.2K potential stress-related genes that encode known or putative protein were selected for this study from the genes covered by Arabidopsis 7K array (RIKEN, Japan) and Arabidopsis oligo microarray (Agilent technologies, USA), which these genes include biotic and abiotic stress-inducible genes.
Complementary DNA was synthesized and labeled with either Cy5 for the treated samples or Cy3 the untreated plants grown in parallel. No dye swaps were carried out.The microarrays were spotted using the microarray stamping machine (model SpotArray24; PerkinElmer, Wellesley, MA, USA). Hybridization was carried out in our laboratory, and hybridized chips were scanned on a ScanArray Lite scanner (PerkinElmer, Wellesley, MA, USA) and quantified using ScanArray Express ver.3 software. We calculated the median of the pixel intensities for each spot.
time course: 2, 5, 10, 24 hours

first: Mari

first: Hiroshi

last: Abe


title: Genomics-based molecular epidemiology of human clinical and feedlot cattle C. jejuni isolates in Alberta, Canada

city: Lethbridge

first: Eduardo

middle: N

last: Taboada

state: Alberta

address: PO Box 640, TWP Rd. 9-1

institute: Public Health Agency of Canada

summary:
Oligonucleotide DNA microarrays were used as a platform to compare C. jejuni isolates from feedlot cattle and human clinical cases from Alberta. Comparative genomic hybridization (CGH) analysis was performed on 87 isolates (46 bovine, 41 human) obtained within the same geographical regions and time frame.  In addition, We also performed gene association analysis to determine if any genes may be differentially distributed between human and cattle sources or between clusters dominated by either human or cattle isolates (“human enriched” vs “cattle enriched”).

Keywords: Comparative Genomic Hybridization; Genomic epidemiology; Gene-association study

platform_organism: Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819

overall_design: Data from 119 microarrays is included in the dataset, representing the 89 bacterial strains analyzed (87 field isolates, 2 control laboratory strains). Replicate arrays for 20 of the 87 field isolates were included in the dataset, as well as 5 replicates for each of the 2 laboratory controls. An array representing 1546 ORFs from strain NCTC 11168 was used in CGH experiments. CGH was performed by comparing signal from each Tester field isolate analyzed vs. signal from the Control strain NCTC 11168. Values represent mean of triplicate spots.

first: Sherry

middle: J

last: Hannon

first: Margaret

last: Russell

first: Brenda

last: Allan

first: Cheryl

last: Waldner

first: Heather

last: Wilson

first: Andrew

last: Potter

first: Lorne

last: Babiuk

first: Hugh

last: Townsend


title: Epigenomic analysis of neonatal dendritic cells in mice with increased allergic susceptibility

status: Public on Nov 04 2008

city: Boston

first: Alexey

last: Fedulov

address: 75 Francis St.

department: Brigham and Women's Hospital

institute: Harvard Medical School

type: Methylation profiling by genome tiling array

summary:
RATIONALE: In a mouse model of maternal transmission of asthma risk (J. Immunol 170:1683, 2003), baby mice of asthmatic (As), but not normal (Nrm), mothers show increased susceptibility to allergy. We previously showed that adoptive transfer to normal baby mice of dendritic cells (DCs) harvested from asthma-susceptible but allergen-naïve neonates reproduces the increased susceptibility to asthma. Hence, the maternal effect is mediated by altered neonatal DCs, which skew immune responses towards a pro-allergic Th2 phenotype. To identify potential molecular mechanisms, we performed epigenomic profiling of isolated neonatal DCs.
METHODS: BALB/c mice were sensitized by 2 i.p. injections of ovalbumin (OVA) in alum and repeatedly challenged with OVA aerosols (As) prior to mating with normal males. Purified splenic CD11c+ DCs of 14-day old allergen-naïve offspring from these As and control Nrm mothers were isolated using magnetic beads. Methylation profiles of genomic DNA were obtained using Switchgear epigenomic chip arrays. After normalization and background correction analysis using significance analysis for microarrays (SAM) and ANOVA was performed. RESULTS: We identified 300 to 6000 (depending on stringency) chromosomal regions with significantly different methylation status, (2 – 10 fold). Clustering methods and pathway analysis identified several interrelated gene groups that merit further study. CONCLUSION: Maternal asthma causes multiple significant epigenetic changes in neonatal dendritic cells.

Keywords: Dendritic cells, genomic DNA, DNA methylation, allergy, asthma

overall_design: The analysis includes 9 samples of genomic DNA from isolated splenic CD11c+ dendritic cells (>95% pure) per group. The two groups are neonates born to mothers with induced allergy to ovalbumin, and normal control neonates. All neonates are genetically and environmentally identical, and allergen-naive.

first: Lester

last: Kobzik


title: Identification of genes regulated by gadE in E. coli O157:H7

status: Public on Oct 11 2008

city: East Lansing

first: Thomas

middle: S

last: Whittam

state: MI

address: 165 Food Safety & Tox

institute: Michigan State University

summary: Integrating laterally acquired virulence genes into the backbone regulatory network is important for the pathogenesis of Escherichia coli O157:H7, which has captured many virulence genes through horizontal transfer during evolution. GadE is an essential transcriptional activator of glutamate decarboxylase (GAD) system, the most efficient acid resistance mechanism in E. coli. The full contribution of GadE to the acid resistance and virulence of pathogenic E. coli O157:H7 remains largely unknown. We inactivated gadE in E. coli O157:H7 Sakai and compared global transcription profiles with that of wild type in exponential and stationary phases of growth using microarrays containing 6088 ORFs from three E. coli genomes. gadE inactivation significantly altered the expression of 60 genes independent of growth phase and 122 genes in a growth phase-dependent manner. Inactivation of gadE markedly down-regulated the expression of gadA, gadB, gadC and many acid fitness island genes in a growth phase-dependent manner. Nineteen genes encoded on the locus of enterocyte effacement (LEE), including ler, showed a significant increase in expression upon gadE inactivation. Altogether, our data indicate that GadE is critical for acid resistance of E. coli O157:H7 and plays an important role in virulence by down-regulating expression of LEE.

overall_design: The results are based on O157:H7 Sakai wild type and gadE mutant exponential and stationary phase cultures grown in MOPS minimal medium. Differences in transcript levels were determined using a mixed model ANOVA in R/MAANOVA which tested for significant differences due to growth phase (exponential or stationary), strain (wild type or mutant) and the interaction of these two factors using the following linear model: array+dye+sample (biological replicate)+ phase+strain+phase*strain. We incorporated the dye-swaps among the biological replicates.

first: Sivapriya

last: Kailasan Vanaja

first: Teresa

middle: M

last: Bergholz


title: Artemisinic Acid Production Stress in Yeast

city: Emeryville

first: Mario

last: Ouellet

address: 5885 Hollis St., 4th Floor

institute: Joint BioEnergy Institute

summary: We have enginereed S. cerevisiae to produce high titers of Artemisinic acid, an anti-malarial drug precursor. Here we compare the gene expression profiles of the producer strain (EPY330) and a strain in which the last enzyme of the pathway (CYP71AV1, AMO) was inactivated by a point mutation (EPY338).

overall_design: 5 independent clones of each strain were precultured and inoculated in galactose-inducing medium for production. Each clone was harvested after 24, 48 and 72h of induction and total RNA was extracted. RNA from replicate clones was pooled in equal proportions and used for labeling and hybridization of 4 or 5 replicate slides for each time point.

first: Dae-Kyun

last: Ro

first: Jay

last: Keasling


title: Secretome of Bone Marrow Stroma-Derived Neural Progenitor Cells: Proteomic Analysis

status: Public on Oct 18 2008

city: Louisville

last: Li

state: KY

address: 580 S. Preston st.,

institute: Gheens Center on Aging

summary:
We found the bone marrow stromal-derived neural progenitor cells secretome have the neural protection effect. Proteomic analysis was performed nn order to analyze the protection factor in the secretome.

Keywords: Neural protection, secretome

overall_design: Secretome samples were run first by HPLC. After that, 20 fractions were selected for MS/MS.

first: Harshini

last: Sarojini

first: Jin

last: An

first: Eugenia

last: Wang


title: Zymomonas mobilis oxygen stress responses

status: Public on Oct 10 2008

city: Raleigh

first: Stan

last: Martin

address: 1404 Crete Dr.

institute: Science Apps, L3C

summary:
Zymomonas mobilis ZM4 produces near theoretical yields of ethanol with high specific productivity and recombinant strains are able to ferment both C-5 and C-6 sugars. However, the genetic and physiological basis of the ZM4 response to various industrially-relevant stresses is poorly understood. In this study, the dynamics of ZM4 oxygen stress responses were elucidated by characterizing the transcriptomic and metabolomic profiles of aerobic and anaerobic fermentations using whole-genome microarray analysis and gas chromatography-mass spectrometry. In the absence of oxygen, ZM4 consumed glucose more rapidly, had a higher growth rate, and ethanol was the major end-product. Under aerobic conditions, much less ethanol was observed with other end-products produced, including acetate, lactate, and acetoin. In the early exponential phase, no genes were detected as being significantly differentially expressed between aerobic and anaerobic conditions via microarray analysis. However, microarray analysis of the stationary phase cultures revealed that 166 genes were significantly differentially expressed by more than two-fold. Quantitative-PCR validated the expression values for seventeen genes from different categories of stationary phase microarray data. Transcripts for Entner-Doudoroff  pathway genes  and gene pdc, encoding a key enzyme leading to the ethanol production, were at least 30-fold more abundant under anaerobic conditions at stationary phase based on quantitative-PCR results. Expression of stress response genes was found to be greater under oxygen stress conditions. GC-MS analysis of stationary phase intracellular metabolites indicated that ZM4 under anaerobic conditions contained lower levels of amino acids, glucose and ED pathway intermediates, whereas metabolites such as ribitol, trehalose, myristic acid, glyceric acid, glucose 6-phosphate, mannose 6-phosphate, and 4-hydroxybutanoic acid were more abundant than under aerobic conditions. Transcriptomic and metabolomic data were consistent with faster glucose consumption and greater ethanol production under anaerobic conditions and suggested gene targets for deletion and improved fermentation.
Keywords: Time course/ Stress Response

overall_design: A total of 24 samples were analyzed. These consisted of two times points, (3 hours and 26 hours) under two conditions (anaerobic and aerobic).  There were 3 biological replicates and two dye swaps.  Each microarray containted one to two probes per predicted coding sequence.


title: miRNA profiling in male and female chicken embryonic gonads during sexual differentiation

city: Geelong

first: Stephanie

middle: C

last: Bannister

state: Victoria

address: 5 Portarlington Road

department: Livestock Industries

country: Australia

institute: CSIRO

summary:
MicroRNAs (miRNAs) are a highly conserved class of small RNAs which function in a sequence-specific manner to post-transcriptionally regulate expression of target genes.  Tissue-specific miRNA expression studies have discovered numerous functions for miRNAs in various aspects of embryonic development, but a role for miRNAs in gonadal development and sex differentiation has not yet been reported.  Using the chicken embryo as a vertebrate model, differential miRNA expression between male and female embryonic gonads, was analysed at three developmental stages (embryonic days (E) 5.5, E6.5 and E9.5), using custom-designed 4x2K CombiMatrix miRNA microarray.  The aims of this study were to: 1-identify miRNAs differentialy expressed by sex; 2-identify sex-specific miRNAs; 3-analyse global changes in miRNA up-regulation in male versus female gonads before, during and after the histological onset of sexual differentiation. This study provides a basis for establishing whetehr miRNAs are involved in either initiating or regulating vertebrate gonadal sex differentiation.

Keywords: miRNA, sex comparison, developmental stage comparison.

platform_organism:
Danio rerio
Gallus gallus
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: miRNA samples from male and female embryonic chicken gonads from three developmental stages: embryonic day (E) 5.5 (Hamilton & Hamburger (HH) stage 27-28), E6.5 (HH stage 29-30) & E9.5 (HH stage 35-36).  Samples are listed with biological replicates used for analysis in brackets following: 1 - Male E5.5 (5); 2 - Female E5.5 (4); 3 - Male E6.5 (5); 4 - Female E6.5 (3); 5 - Male E9.5 (4); 6 - Female E9.5 (4).

first: Craig

last: Smith

first: Mark

middle: L

last: Tizard

last: Sinclair

first: Timothy

last: Doran


title: Integrated genomic and transcriptional profiling yields putative marker genes for cervical cancer

city: Amsterdam

first: Daoud

last: Sie

address: De Boelelaan 1117

department: Pathology

institute: Vrije Universiteit Medical Center

summary:
A better understanding of the consequences of recurrent (epi)genetic alterations that occur during cervical carcinogenesis is essential in the search for novel biomarkers. In this study we determined genome-wide expression profiles of 10 squamous cell carcinomas (SCCs), 5 adenocarcinomas (AdCAs) and 6 normal epithelial samples. Expression patterns were subsequently combined with previously determined chromosomal profiles in the same carcinomas.
Differential gene expression analysis identified 76 genes with altered expression in carcinomas compared to normal epithelium. Microarray results for a subset of these genes were validated by real-time RT-PCR. Among the differentially expressed genes a relative overrepresentation of genes located at chromosome 3q, one of the most frequently gained areas in SCCs, was observed (false discovery rate (FDR)<0.005). To further investigate the relationship between gene expression and chromosomal alterations 2 statistical approaches were used, i.e. differential gene locus mapping (DIGMAP) and the array CGH expression integration tool (ACE-it). Using these methods we found that increased gene expression was linked to increased gene copy numbers at 1q32.1, 3q13.32-22.3, 3q26.32-27.3, and 20q11.21-13.33, whereas a loss at 11q22.3-25 correlated with recurrent decreased gene expression.
Integrated genome-wide chromosomal and transcriptional analysis of cervical carcinomas highlighted 7 genes (i.e. FLJ21291, DTX3L, CCDC14, MCM2, PIK3R4, ATP2C1 and SLC25A36), which were identified by differential gene expression analysis and were located within the chromosomal regions identified by DIGMAP and/or ACE-it as well. Further investigations of these promising marker genes in warranted.
Keywords: microarray analysis, array CGH, cervical cancer

overall_design:
For microarray mRNA expression analysis 10 SCCs, 5 AdCAs and 6 normal epithelial controls were used. Normal cervical controls consisted of 1 pool of 3 normal cervices distant from tumour, 1 pool of 4 normal ectocervical smears and 1 pool of 5 normal endocervical smears. To expand our number of normal squamous epithelial controls, we included expression profiles of 3 uvulas of non-cancer patients who underwent uvulopalatopharyngoplasty. In addition, we hybridised RNA isolated from 2 different biopsies of the same SCC (SCC12 and SCC13) as a biological replicate. The pool of 4 normal ectocervical smears was hybridised twice as a technical replicate to determine technical variation.
Genomic profiling of the same 10 SCCs and 5 AdCAs was done using array CGH (Wilting et al, J Pathol 2006, volume 209, p 220-30).

first: Saskia

last: Wilting

first: Jillian

last: de Wilde

first: Chris

last: Meijer

first: Johannes

last: Berkhof

first: Yajun

last: Yi

first: Wessel

last: van Wieringen

first: Boudewijn

last: Braakhuis

first: Bauke

last: Ylstra

first: Peter

last: Snijders

first: Renske

last: Steenbergen


title: Protective response to Brucella infection

city: Ciudad Real

first: Ruth

middle: Cecilia

last: Galindo

state: Ciudad Real

address: Ronda de Toledo s/n

department: Animal Health

country: Spain

institute: Instituto de Investigacion en Recursos Cinegéticos

summary: Brucella ovis causes an important disease characterized by decreased fertility in rams, sporadic abortions in ewes and increased lamb mortality.  The live attenuated B. melitensis Rev.1 vaccine is considered the best vaccine available against this infection. However, this vaccine shows variable protective efficacy ranging from 40% to 100%.The objective of this study was to identify possible correlates of protective response to B. ovis infection through the characterization by microarray hybridization and real-time RT-PCR of inflammatory and immune response genes upregulated in rams previously immunized with the B. melitensis Rev 1 vaccine strain and experimentally challenged with B. ovis. Gene expression profiles were compared before and after challenge with B. ovis between rams protected and those vaccinated but found infected after challenge. The genes upregulated in vaccinated and protected rams provide possible correlates of protective response to B. ovis infection in rams immunized with the B. melitensis Rev 1 vaccine.

overall_design: Gene expression profiles were compared before and after vaccination and challenge with  B. ovis between rams protected with the B. melitensis Rev 1 vaccine strain and those vaccinated but infected after challenge. Total RNA was isolated from buffy coat samples before vaccination (T0),  after vaccination and before challenge (T1) and 60 days post challenge (T2) using TriReagent (Sigma, St. Louis, MO, USA)

first: Pilar

last: Munoz

first: Maria

last: de Miguel

first: Clara

last: Marin

first: Jose

last: Blasco

first: Christian

last: Gortazar

first: Katherine

last: Kocan

last: de la Fuente


title: Transcriptome Analysis of the Zebrafish Mind Bomb Mutant

status: Public on Oct 08 2008

city: Daegu

first: Sanggyu

last: Lee

address: 1370 Sankyuk-dong, Bukgu

country: South Korea

institute: Kyungpook National University

summary: To identify the genes differentially expressed in the zebrafish mibta52b mutant, genome-wide transcriptome analysis was performed using Serial Analysis of Gene Expression (SAGE). 335 transcripts were identified whose expressions were significantly altered in the mibta52b mutant as compared with the wild-type.

overall_design: After mRNA isolation using oligo(dT)25 beads (Dynal Biotech ASA, Oslo, Norway), cDNA synthesis was carried out using anchored oligo(dT) primers to reduce truncated cDNA through internal poly(A) priming (Nam et al., 2002). SAGE analysis was carried out as described (Velculescu et al., 1995) but was modified slightly according to Lee et al (Lee et al., 2007).  We performed a low-cycle PCR (14 cycles) using the GeneAmp PCR System 2400 (Applied Biosystem, Foster City, CA) to amplify 3’cDNA in order to generate a sufficient amount of templates for SAGE analysis. The sense primer used was a SAGE primer 1 (5’-GGA TTT GCT GGT GCA GTA CA-3’) or SAGE primer 2 (5’-CTG CTC GAA TTC AAG CTT CT-3’); the antisense primer used was 5’-biotin- ACT ATC TAG AGC GGC CGC TT-3’, which was located in the 3’ end of all cDNAs generated from the anchored oligo(dT) primers. BsmFI-released fragments containing SAGE tags, were gel purified before being used for ditag formation and concatenation to provide high-quality tags for SAGE analysis. SAGE tag sequences were collected with Big-Dye sequencing and an ABI3730 sequencer, and tag sequences were extracted with SAGE 300 software. A total of 92,970 SAGE tags were collected from the two libraries.

first: Junmo

last: Hwang

first: Hyung-Soo

last: Kim

first: Ji-Woong

last: Seok

first: Soonil

last: Koun

first: Yong-Chul

last: Jung

first: San-Ming

last: Wang

first: Tae-Lin

last: Huh


title: Chromosome 11q and its association with CCND1 gene amplification and tamoxifen resistance in premenopausal breast cancer

city: Lund

first: Johan

last: Staaf

address: Medicon Village

institute: SCIBLU - Swegene Centre for Integrative Biology at Lund University

summary:
Background: The amplification event occurring at chromosome locus 11q13, reported in several different cancers, includes a number of potential oncogenes. We have previously reported amplification of one such oncogene, CCND1, to be correlated with an adverse effect of tamoxifen in premenopausal breast cancer patients. Overexpression of cyclin D1 protein however, confers tamoxifen resistance but not a tamoxifen induced adverse effect. Potentially, co-amplification of an additional 11q13 gene, with a resulting protein overexpression, is required to cause an agonistic effect. Moreover, during 11q13 amplification a deletion of the distal 11q region has been described. In order to assess the potential impact of the deletion we have examined a selected marker for this event. Methods: Array CGH analysis was employed to identify and confirm changes in the gene expression of a number of different genes mapping to the 11q chromosomal region, associated with CCND1 amplification. The subsequent protein expression of these candidate genes was then examined in a clinical material of 500 primary breast cancers from premenopausal patients randomized to either tamoxifen or no adjuvant treatment. The protein expression was also compared to the CGH data in a subset of 56 breast cancer samples. Results: Cortactin and FADD overexpression was linked to CCND1 amplification, determined by FISH, but was not associated with a diminished effect of tamoxifen. However, deletion of distal chromosome 11q, defined as downregulation of the marker Chk1, was associated with an impaired tamoxifen response, and interestingly, also with low proliferative breast cancer of low grade. For Pak1 and cyclin D1 the protein expression corresponded to the gene expression data from the CGH analysis. Conclusions: The results indicate that many 11q13 associated gene products are overexpressed in conjunction with cyclin D1 but not linked to an agonistic effect of tamoxifen. Finally, the deletion of distal 11q, linked to 11q13 amplification, might be an important event affecting breast cancer outcome and tamoxifen response.

Keywords: comparative genomic hybridisation

overall_design: 56 samples with 11q13 amplification have been analysed and compared with protein expression results from immunohistochemical analyses.

first: Katja

last: Lundgren

first: Karolina

last: Holm

first: Bo

last: Nordenskjold

first: Ake

last: Borg

first: Goran

last: Landberg


title: Identification of Resistance-related Defense Responses in Soybean against Aphis glycines

status: Public on Oct 13 2008

city: Urbana

first: min

last: li

state: IL

address: Rm230 1101 W peabody Dr

institute: University of Illinois at Urbana-Champaign

summary:
Gene expression profiles in resistant (cv. Dowling) and susceptible (Williams 82) soybean genotypes [Glycine max (L.) Merrill] were compared at 6 and 12 h with and without aphid (Aphis glycines Matsumura) infestation using cDNA microarrays consisting of approximately 18,000 soybean-expressed sequence tags. More genes were induced in Dowling than Williams 82 at 6 h after infestation. Genes that were differentially expressed between aphid and control treatments were selected as aphid-response genes. Eighty-four genes showed specific responses in Dowling and included genes related to defense and other processes. Expression of three defense-related genes was examined at 6, 12, 24, 48, and 72 h after infestation in both genotypes by quantitative real-time PCR. The increases in the transcripts of three defense-related genes were earlier and stronger at 6, 12 and 24 h after infestation in Dowling compared to Williams 82. The differential gene expression between the two genotypes without aphids was determined, and five genes with constitutively higher expression levels were found in Dowling.
Keywords = genomic
Keywords = Defense Responses
Keywords = plant
Keywords = DNA-binding protein
Keywords = PR proteins
Keywords = plant resistance
Keywords = signal transduction
keywords = insect
Keywords: susceptible vs resistant

overall_design: loop design (6 hpi and 12 hpi) and Dye swap (0 hpi).  6 biological samples, 2 microarray slide sets and 2 biological repeats

first: Yan

last: Li

first: Jijun

last: Zou

first: Min

first: Lila

middle: O

last: Vodkin

first: Glen

last: Hartman

first: Steven

last: Clough


title: Meiotic Consequences of Aneuploidy:  Mouse Models of Down Syndrome

city: Bar Harbor

first: Laura

middle: G

last: Reinholdt

state: ME

address: 600 Main Street

institute: The Jackson Laboratory

summary: Aneuploidy, and especially the presence of unpaired chromosomal axes during meiosis, can cause infertility.  However, it is not known if extra, unpaired autosomal chromosome segments acquire characteristic protein modifications and undergo transcriptional silencing (meiotic silencing of unpaired chromatin, or MSUC) similar to the unpaired regions of the X and Y chromosomes during spermatogenesis.  We used three mouse models of Down syndrome, involving either an extra chromosome or translocation trisomy, to test requirements and consequences of meiotic protein modification and gene silencing in spermatocytes.  These models reveal that copy number alone is not sufficient for up-regulation of genes in the trisomic interval, and that MSUC-promoting modifications are not sufficient for down-regulation of genes that reside in unpaired chromatin during meiosis.  The position of a trisomic region relative to a centromere, the pairing status of the centromere, and the physical extent of the unpaired chromosomal region all affected assembly of meiotic protein modifications typical of MSUC.  One key determinant to modification of unpaired chromatin and infertility in trisomy male mice appears to be proximity of unpaired chromatin to a centromere.  Furthermore, the presence of an extra, unpaired centromere, but not translocation trisomy, causes global misregulation of transcription in spermatocytes.  Thus, neither trisomy per se, nor chromatin modifications of unpaired chromosomal segments, have major effects on gene expression or meiotic success, but an intact unpaired chromosome has profoundly negative effects on meiotic gene expression.

overall_design: Three biological replicate RNA samples from enriched male germ cells from two mouse Down Syndrome mouse models, Ts1Cje and Ts65Dn, and diploid wild-type littermate controls, WT, from two time points, 16-17 and 19-20 days post partum(dpp), were assayed for gene expression using Illumina MouseWG-6 v1.1 BeadArrays.

first: Anne

last: Czechanski

first: Sonya

last: Kamdar

first: Benjamin

last: King

first: Fengyun

last: Sun

first: MaryAnn

last: Handel


title: Natural antisense transcripts accumulate for over 70% of maize genes and exhibit genotypic-specific expression patterns

status: Public on Oct 09 2008

city: Ames

first: Patrick

middle: S.

last: Schnable

state: IA

address: 2035B Roy J Carver Co-Lab

institute: Iowa State University

summary:
Natural Antisense Transcripts (NATs) can regulate gene expression by virtue of their ability to form double-stranded RNA duplexes. To investigate NATs in the maize transcriptome, cDNAs from seedling of two inbred lines (B73 and Mo17) were hybridized to an oligonucleotide microarray designed to validate the expression of in silico detected NATs and to screen for NATs that can anneal to a random set of 3’ UTRs and selected repeats found in 3’ UTRs regions. Quantitative Real-Time PCR experiments were conducted to determine the minimum detection threshold of microarray experiment and to thereby identify genes for which both sense and antisense transcripts accumulate to detectable levels. Two independent approaches, strand-specific RT-PCR and S1 nuclease assays were conducted to validate the results of the microarray experiment. Based on these conservative assays, NATs accumulate in seedlings that can anneal to over 70% of a random set of maize genes. In addition, both sense and antisense transcripts anneal to more than 80% of a set of maize repeats. Significantly, sense and antisense transcripts exhibit significant different expression patterns between the two genotypes. Based on these findings we hypothesize that interactions between sense and antisense transcripts may contribute to the differential patterns of gene expression in maize hybrids and to heterosis.
Keywords: Global antisense transcripts profiling between two maize inbreds

overall_design: To systematically identify NATs in maize, we employed multiple strategies to computationally identify putative antisense transcripts from our partial genome assembly (MAGIs) and a collection of ESTs we sequenced with known sequence orientation. Additionally, we randomly sampled and surveyed maize UTRs which often harbor transposons.  Strand-specific oligonucleotides which can hybridize to the antisense strand were designed and spotted with together with the oligonucleotides hybridizing sense strands on a custom, strand-specific oligoarray, providing the first global expression profiling study of NATs in maize UTRs. In total, 10 biological replication were conducted to compare the global gene expression profiles between two maize genotypes (B73 and Mo17).


title: Epstein-Barr virus growth/latency III program alters cellular microRNA expression

status: Public on Oct 07 2008

city: New Orleans

first: Erik

middle: K

last: Flemington

address: 1430 Tulane Ave., SL79

institute: Tulane Health Sciences Center

summary: The Epstein-Barr virus (EBV) is associated with lymphoid and epithelial cancers.  Initial EBV infection alters lymphocyte gene expression, inducing cellular proliferation and differentiation as the virus transitions through consecutive latency transcription programs.  Cellular microRNAs (miRNAs) are important regulators of signaling pathways and are implicated in carcinogenesis.  The extent to which EBV exploits cellular miRNAs is unknown.  Using micro-array analysis and quantitative PCR, we demonstrate differential expression of cellular miRNAs in type III versus type I EBV latency including elevated expression of miR-21, miR-23a, miR-24, miR-27a, miR-34a, miR-146a and b, and miR-155.  In contrast, miR-28 expression was found to be lower in type III latency.   The EBV-mediated regulation of cellular miRNAs may contribute to EBV signaling and associated cancers.

overall_design:
There were two major objectives for this study.  The first objective was to determine whether EBV infected cells exhibiting type I latency influence cellular microRNA expression.  For this study, four EBV negative derivatives of the type I latency cell line, Mutu I, were derived by retroviral infection with a dominant negative from of the EBV episomal replication factor, EBNA1.  RNA from these four clones were compared to parental EBV positive Mutu I cells.  Four dual labeling experiments were carried out for this comparison with dye reversal for every second pair of RNAs.

The second objective was to determine whether EBV type III latency cells exhibit altered cellular microRNA gene expression compared to type I latency cells or EBV negative B cells.  Four dual labeling experiments were carried out for this analysis with dye reversal for every second pair of RNAs.

first: Jennifer

middle: E

last: Cameron

first: Claire

last: Fewell

first: Qinyan

last: Yin

first: Jane

last: McBride

first: Xia

last: Wang

first: Zhen

last: Lin


title: Targets of the E. histolytica transcription factor URE3-BP

status: Public on Oct 14 2008

city: Charlottesville

first: Carol

middle: A

last: Gilchrist

state: VA

address: Dept. of Medicine/Division of Infectious Diseases, Building MR4, Rm 2126,PO Box 801365

department: Medicine

institute: University of Virginia

summary: The E. histolytica transcription factor URE3-BP is a calcium-responsive regulator of two E. histolytica virulence genes, hgl5 and fdx1. URE3-BP was previously identified by a yeast one-hybrid screen of E. histolytica proteins capable of binding to the sequence TATTCTATT (URE3) in the promoter regions of hgl5 and fdx1.  In this work precise definition of the consensus URE3 element was performed by electrophoretic mobility shift assays (EMSA) using base-substituted oligonucleotides, and the consensus motif validated using episomal reporter constructs. Transcriptome profiling of a strain induced to produce a dominant-positive URE3-BP was then used to identify additional genes regulated by URE3-BP. Fifty modulated transcripts were identified, and of these the EMSA defined motif T[atg]T[tc][cg]T[at][tgc][tg] was found in over half of the promoters (54% p<0.0001). Fifteen of the URE3-BP regulated genes were potential membrane proteins suggesting that one function of URE3-BP is to remodel the surface of E. histolytica in response to a calcium signal.  Induction of URE3-BP lead to an increase in tranwell migration suggesting a possible role in the regulation of cellular motility.

overall_design: 11 samples are used for two strains:EF and STOP at 0 and 9 hour time points

first: Duza

last: Baba

first: Yan

last: Zhang

first: Oswald

last: Crasta

first: Clive

last: Evans

first: Elisabet

last: Caler

first: Bruno

last: Sobral

first: Christina

last: Bousquet

first: Megan

last: Leo

first: Ameilia

last: Hochreiter

first: Sarah

last: Connell

first: Barbara

last: Mann

first: William

last: Petri. Jr


title: Widespread transcriptional changes preempt the critical pelagic-benthic transition in the vetigastropod Haliotis asinina

status: Public on Oct 04 2008

city: Brisbane

first: Elizabeth

last: Williams

state: QLD

address: St Lucia campus

department: School of Integrative Biology

institute: University of Queensland

summary:
Larval settlement and metamorphosis is a vital transition period for marine invertebrates and can have far-reaching effects on the ecology and evolution of a species. To explore the molecular mechanisms of this critical process in a non-model organism, the tropical abalone Haliotis asinina, we employed cDNA microarray methods. By comparing gene expression profiles through mid to late larval development and metamorphosis, we identified 144 genes as likely candidates for a role in competence and/or metamorphosis. Gene characterization showed that ~60% of these were significantly similar to previously described genes from other taxa, while ~40% had no significant similarities to any known genes. A high 49.3% of genes were gastropod- or abalone-specific, but none appear to be Lophotrochozoan-specific, despite the fact that metamorphosis is thought to have had a separate origin in this group. Based on temporal expression profiles, the differentially expressed larval and postlarval genes can be clustered into 5 categories that reveal there are strikingly different transcriptional patterns occurring during this phase of development. Some classes of gene activation are contingent upon exogenous cues and correlate with the initiation of settlement and metamorphosis. Importantly, there is also extensive gene activity associated with the endogenous attainment of competence, which occurs prior to, and independent of, the exogenous induction of settlement. Our results show that as the haliotid veliger larva matures, it requires the coordinated regulation of temporally different batteries of genes involved in a wide range of physiological and developmental processes associated with colonisation of the benthos. Although the signalling pathways operating at metamorphosis may be conserved across the animal kingdom, it appears they are regulating the expression of novel genes specific to abalone, gastropods and molluscs during H. asinina metamorphosis.

Keywords: timecourse; metamorphosis; marine ecology

overall_design: Each microarray slide contained a different combination of 2 of the 9 developmental stages used in the experiment (66 hpf, 78 hpf, 90 hpf, 108 hpf, 120 hpf, 144 hpf, 12 hpi, 24 hpi, 48 hpi). Each developmental stage was subjected to 4 hybridisations – amounting to 4 technical replicates per stage - in a loop design (Churchill 2002; Oleksiak et al. 2002). This design led to raw data consisting of 72 measurements - 9 stages with 8 replicates (including 2 replicates per chip) - for each of 5541 spots.

first: Bernard

last: Degnan

first: Helen

last: Gunter

first: Daniel

last: Jackson

first: Ben

last: Woodcroft

first: Sandie


title: Signaling pathways after 30 minutes and 4 hours treatment with 500 μM Na2S in intestinal epithelial cells

first: H

middle: Rex

last: Gaskins

address: 1207 W Gregory dr

department: Institute for Genomic Biology

institute: University of Illinois

summary:
Hydrogen sulfide (H2S), a metabolic endproduct of sulfate-reducing bacteria in the large intestine, represents a genotoxic insult to the colonic epithelium, which may also be linked with chronic disorders such as ulcerative colitis and colorectal cancer. The present study defined the early (30 minutes) and late (4 hours) response of non-transformed human intestinal epithelial cells (FHs 74 Int) to sulfide.
We measured the genotoxicity of Na2S using the single cell gel electrophoresis (comet) assay. Changes in gene expression were analyzed after exposure to a genotoxic–but not cytotoxic–concentration of sulfide (500 μM Na2S) using pathway specific quantitative RT-PCR gene arrays

overall_design: 12 Samples were analysed using the Superarray Signal Transduction PathwayFinder APHS-014b QRT-PCR array. The cells were treated for either 30 minutes or 4 hours with 500 μM Na2S (6 samples for each time). For each time we had 3 samples as a control (no Na2S) 3 samples treated (500 μM Na2S)

first: Matias

last: Attene-Ramos

first: Gerardo

last: Nava

first: Mark

last: Muellner

middle: D

last: Wagner

first: Michjael

last: Plewa

middle: R


title: miRNA analysis of HBMVECs exposed or unexposed to U87 glioma cells

city: Charlestown

first: Thomas

last: Wurdinger

state: MA

address: 149 13th Street

institute: MGH

summary:
A key step in angiogenesis is the upregulation of growth factor receptors on endothelial cells. Here we demonstrate that a small regulatory microRNA, miR-296 has a major role in this process. Glioma cells and angiogenic growth factors elevate the level of miR-296 in primary human brain microvascular endothelial cells in culture. The miR-296 level is also elevated in primary tumor endothelial cells isolated from human brain tumors compared to normal brain endothelial cells. Growth factor-induced miR-296 contributes significantly to angiogenesis by directly targeting the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) mRNA, leading to decreased levels of HGS and thereby reducing HGS-mediated degradation of the growth factor receptors VEGFR2 and PDGFR-β. Furthermore, inhibition of miR-296 with antagomirs reduces angiogenesis in tumor xenografts in vivo.

Keywords: comparitive miRNA analysis

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: 3 biological replicates (HBMVECs) are compared to 3 biological replicates (HBMVECs exposed to U87 glioma cells)

first: Anna

last: Krichevsky


title: Mrc1 and DNA polymerase ε function together in linking DNA replication and the S phase checkpoint

city: Bunkyo-ku

first: Katsuhiko

last: Shirahige

state: Tokyo

address: 1-1-1 Yayoi

department: Research Center for Epigenetic Disease

institute: The University of Tokyo

type: Genome binding/occupancy profiling by genome tiling array

summary: Yeast Mrc1, ortholog of metazoan Claspin, is both a central component of normal DNA replication forks and a mediator of the S phase checkpoint.  We report that Mrc1 interacts with Pol2, the catalytic subunit of DNA polymerase ε, essential for leading strand DNA replication and for the checkpoint.  In unperturbed cells, Mrc1 interacts independently with both the N-terminal and C-terminal halves of Pol2 (Pol2N and Pol2C).  Strikingly, phosphorylation of Mrc1 during the S phase checkpoint abolishes Pol2N binding but not Pol2C interaction.  Mrc1 is required to stabilize Pol2 at replication forks stalled in HU.  The bimodal Mrc1/Pol2 interaction may identify a novel step in regulating the S phase checkpoint response to DNA damage on the leading strand.  We propose that Mrc1, which also interacts with the MCMs,  may modulate coupling of polymerization and unwinding at the replication fork.

overall_design:
Type of experiment: ChIP-chip (Chromatin immunoprecipitation on DNA chip) analyses of replication related, checkpoint related proteins and BrdU incorporated regions at 300bp resolution. S.cerevisiae chromosome VI tiling array produced by affymetrix was used.

Experimental factors:Distribution of DNA Pol2 (epsilon) on chromosome VI during S-phase (with hydroxy urea). Wild type, tof1 deletion mutant, and mrc1 deletion mutant were investigated.

The type of reference used for the hybridizations, if any: For the analysis of HA tagged Pol2 protein, POL1-3XFLAG was analyzed simultaneously as a reference.

Hybridization design: Comparison of ChIP fraction with SUP fraction for protein binding profile. No locus was scored as significantly enriched in the control ChIP experiment using HU treated untagged cells.

Quality control steps taken: Confirmation of ChIP fraction by Western blotting. Confirmation by conventional ChIP PCR methods for selected locus.

Manipulation of biological samples and protocols used:For the preparation of HU-arrested cells, we synchronized yeast cells with alpha-factor (2mM) and then released cells into 200mM HU for 60 min. at 23°C. Cells were fixed by 1% Formaldehyde and then used for ChIP of protein of interest.

Protocol for preparing the hybridization extract:We disrupted 1.5X10^8 cells by MULTI-BEADS SHOCKER (MB400U, YASUI KIKAI, Osaka), which was able to keep cells precisely at lower than 6°C during disruption by glass beads. This enabled us to retrieve more than 60% of tagged proteins to soluble fraction routinely. Chromosomal DNA was shared into the average size of 400-600bp by sonication. Anti-HA monoclonal antibody HA.11 (16B12) (CRP Inc., Denver, PA) and anti-FLAG monoclonal antibody M2 (Sigma-Aldrich Co., St Louis, MO) were used for chromatin immuno-precipitation. Immunoprecipitated chromosomal DNA was subsequently purified and amplified by PCR. 2ug of amplified DNA was digested with DNaseI to a mean size of 100bp and used for labeling as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998).

Labeling protocol:DNA was end-labelled with Biotin-N6ddATP by Terminal Transferase as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998)

The protocol and conditions used during hybridization, blocking and washing: Hybridization, blocking and washing steps were carried out as previously described by Winzeler et al. (Science. 281, 1194-1197, 1998). Each sample was hybridized to the array in 150ul containing 6XSSPE, 0.005% TritonX-100, 15ug fragmented denatured salmon sperm DNA (Gibco-BRL), and 1nmole of a 3’-biotin control oligonucleotide (oligo B2 probes) that hybridized to the border features on the array. Samples were heated to 100°C for 10 min., and then cooled on ice. Samples were hybridized for 16h at 42°C in a hybridization oven (GeneChip hybri oven 320, Affymetrix, CA). Washing and staining protocol (Mini_euk1 ver2) provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 400, Affymetrix, CA).

Measurement data and specifications:Each array was scanned by HP GeneArray Scanner (Affymetrix, CA) at an emission wavelength of 560nm at 7.5uM resolution. Grids were aligned to the scanned images using the know feature of the array. Primary data analyses were carried out using the Affymetrix Microarray Suite Ver.5.0 software to obtain hybridization intensity, fold change value, change p-value, and detection p-value for each locus. For the discrimination of positive and negative signals for the binding, we compared ChIP fraction with Sup (supernatant) fraction by using three criteria as follows. First, the reliability of strength of signal was judged by detection p-value of each locus (p-value lower than 0.025 was considered as significant). Secondly, reliability of binding ratio was judged by change p-value (p-value lower than 0.025 was considered as significant). Thirdly, clusters consisted of at least three contiguous loci which filled above two criteria were selected as significantly enriched locus, because it is apparent that a single site of protein-DNA interaction will result in immuno-precipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbors. This third criterion is very unique, make the data highly reliable and can be only applicable to the chip data obtained by our high-resolution tiling array.

first: Huiqiang

last: Lou

first: Makiko

last: Komata

first: Yuki

last: Katou

first: Zhiyun

last: Guan

first: Clara

last: Reis

first: Martin

last: Budd

first: Judith

last: Campbell


title: Normalization of Illumina Infinium whole-genome SNP data improves copy number estimates and allelic intensity ratios

first: Johan

last: Staaf

summary:
Illumina Infinium whole genome genotyping (WGG) arrays are increasingly being applied in cancer genomics to study gene copy number alterations and allele-specific aberrations such as loss-of-heterozygosity (LOH). Methods developed for normalization of WGG arrays have mostly focused on diploid, normal samples. However, for cancer samples genomic aberrations may confound normalization and data interpretation. Therefore, we examined the effects of the conventionally used normalization method for Illumina Infinium arrays when applied to cancer samples.

We demonstrate an asymmetry in the detection of the two alleles for each SNP, which deleteriously influences both allelic proportions and copy number estimates. The asymmetry is caused by a remaining bias between the two dyes used in the Infinium II assay after using the normalization method in Illumina’s proprietary software (BeadStudio). We propose a quantile normalization strategy for correction of this dye bias. We tested the normalization strategy using 535 individual hybridizations from 10 data sets from the analysis of cancer genomes and normal blood samples generated on Illumina Infinium II 300k version 1 and 2, 370k and 550k BeadChips. We show that the proposed normalization strategy successfully removes asymmetry in estimates of both allelic proportions and copy numbers. Additionally, the normalization strategy reduces the technical variation for copy number estimates while retaining the response to copy number alterations.

The proposed normalization strategy represents a valuable low-level analysis tool that improves the quality of data obtained from Illumina Infinium arrays, in particular when used for LOH and copy number variation studies.

overall_design: To investigate the effects of a quantile normalization of Illumina Infinium data, compared to conventional normalization using BeadStudio (www.illumina.com), we renormalized 535 individual hybridizations conducted on Illumina 300K, 370K and 550K BeadChips. Sample types included breast cancer, colon cancer, urothelial carcinoma, leukemia as well as normal blood and HapMap samples. This series includes the 6 breast cancers hybridized on Illumina HumanHap 550K BeadChips.

last: Vallon-Christersson

first: David

last: Lindgren

first: Gunnar

last: Juliusson

first: Richard

last: Rosenquist

first: Mattias

last: Höglund

first: Ake

last: Borg

first: Markus

last: Ringner


title: Identification of the calcium-responsive genes dependent on CrzA in the filamentous fungus Aspergillus nidulans

status: Public on Oct 03 2008

city: Sendai

first: Daisuke

last: Hagiwara

state: Miyagi

address: 6-6-10 Aoba Aramaki

department: New Industry Creation Hatchery Center

institute: Tohoku university

summary:
Microarray analysis was used to identify the calcium-responsive genes dependent on CrzA in the filamentous fungus Aspergillus nidulans.
In order to identify such genes, we conducted the two types of experiment. One was a comparison between  wild type with calcium treatment and wild type without calcium treatment. Another was a comparison between wild type with calcium treatment and crzA mutant with calcium treatment.
From a comparison between the results of these experiments, we could identify the genes whose expression was induced or repressed in response to calcium in a manner dependent on CrzA.
KEY WORD; Aspergillus nidulans, calcium response, crzA

overall_design:
Conidia of wild type or crzA mutant strains were incubated at 37C in CD medium for 18h and treated with or without calcium (final concentration; 50 mM)  for 15 min. The mycelia were harvested and frozen in liquid nitrogen, ground to a powder, and used for RNA preparation. mRNA was purified and used for hybridization experiments.

A total of 2 hybridizations were performed for each microarray experiment described in the summary. The following replicates were carried out:
1. In-slide replicates were carried out for each analyses.
2. Dye swap replicates were carried out for each analyses.

The slides were scanned with an Axon GenePix 4000B scanner (Molecular Devices). The resulting TIFF images were imported into GenePix Pro and fluorescent intensity of spots were calculated for each of the Cy3 and Cy5 channels. Global normalization was applied to all analyses. Following normalization, spots whose Cy3 or Cy5 intensity was less than 0 were removed from the data set (the exceptional case was that intensity of the other channel was more than 100). The dye-swap replicates and in-slide replicates were subjected to all analyses. Finally, gene expression ratios (channel 2/channel 1) were calculated for each replicates. Gene expression was considered to be significantly higher or lower whenever the spot intensity changed by at least 2-fold in all four replicates.

first: Atsushi

last: Kondo

first: Tomonori

last: Fujioka

first: Keietsu

last: Abe


title: Early and long-standing rheumatoid arthritis: molecular signatures identified by gene expression profiling in synovium

city: ROUEN

first: Jean

middle: Philippe

last: SALIER

address: Bvd Gambetta

department: Inserm Unit 519

institute: Faculty of Medicine and Pharmacy

summary: At a given time, rheumatoid arthritis (RA) is a heterogeneous disease and its underlying molecular mechanisms is still poorly understood. Because previous microarrays studies have only focused on late RA stages, we aimed to compare the biological and molecular profiles in early and long-standing (LS) RA.  Synovial biopsies obtained by arthroscopy were performed in 4 early untreated RA patients, 4 LS treated RA patients and 7 control patients. Extracted mRNA were linearly amplified and used for large-scale gene expression profiling by cDNA arrays. By gene ontology analysis, the different gene combinations obtained by comparison of early, LS RA and normal synovium were used to identify the biological processes, molecular functions and pathways involved at each stage. Three combinations of 719, 116 and 52 transcripts dissociated respectively early from LS RA patients, early or LS RA from normal synovium. We identified several gene clusters and distinct molecular signatures specifically related to early or LS RA suggesting the involvement of different pathological mechanisms at each stage. Early and LS RA have distinct molecular signatures with the participation of different biological processes during the course of the disease. These results suggest that a better knowledge of the main biological processes involved at a given RA stage might help the rheumatologist to choose the more appropriate treatment.

overall_design: PBMC then RNA were extracted from synovial biopsies of  4 eraly RA patients, 4 long-standing RA patients and from 7 normal synovium used as control. Each sample was hybridized three times in different nylon membrane.

first: T

last: Lequerre

first: C

last: Bansard

first: O

last: Vittecoq

last: Derambure

first: M

last: Hiron

last: Daveau

first: F

last: Tron

last: Dougados

first: N

last: Biga

first: I

last: Auquit-Auckbur

first: P

middle: Y

last: Milliez

first: G

last: Chiocchia

first: X

last: Le

first: J

middle: P

last: Salier