title: Gene expression in E.coli K-12 MC4100 at 37˚C and 23˚C in M9 glycerol exponential phase growth

status: Public on Jan 22 2008

city: Northampton

first: Christine

middle: Ann

last: White-Ziegler

state: MA

address: Clark Science Center

department: Biological Sciences

institute: Smith College

summary:
Because many virulence genes in E. coli and other pathogens are regulated by temperature, we wanted to determine on a genome-wide scale which genes are modulated in adapting to both human host temperature (37˚C) and ambient room temperature (23˚C).  Overall, 126 genes were found to be more highly expressed at 37˚C (1) whereas 297 genes were more highly expressed at 23˚C (2).  Genes involved in the uptake and utilization of amino acids, carbohydrates, and iron dominated the 37˚C list, supporting a model in which temperature serves as a host cue to increase expression of bacterial genes needed for growth. 122 of the 297 genes more highly expressed at 23˚C are RpoS-controlled, confirming genome-wide the model that low temperature serves as a primary cue to trigger the general stress response.  Several genes expressed at 23˚C overlap with the cold shock response, suggesting that strategies used to adapt to sudden shifts in temperature also mediate long-term growth at 23˚C.  Another category of genes more highly expressed at 23˚C are associated with biofilm development, implicating temperature as an important cue influencing this developmental pathway.

1.) White-Ziegler, C. A., A. J. Malhowski, and S. D. Young.  Human body temperature (37˚C) increases the expression of iron, carbohydrate, and amino acid utilization genes in Escherichia coli K-12.  Journal of Bacteriology 2007.  Vol. 189(15):5429-40. Epub 2007 May 25.

2.)White-Ziegler, C. A., S. Um, N.Peréz, A. L. Berns, A. J. Malhowski, and S. Young. Biofilm, coldshock, and RpoS-dependent genes demonstrate increased expression during low temperature growth (23˚C) in Escherichia coli K-12.  In revision for Microbiology.
Keywords: stress response

overall_design:
Growth and medium conditions were chosen (M9 glycerol medium with aeration) that maximize papBA transcription at 37˚C so we could use this operon as a temperature-regulated positive control in our microarrays.  Parallel cultures grown at 37˚C and 23˚C were harvested in exponential phase (OD600=0.2-0.6) at 9-11 generations growth after inoculation.  Thus, the results presented here reflect the adapted state and signify genes whose expression is differentially maintained over long-term growth at a given temperature.

1.) White-Ziegler, C. A., A. J. Malhowski, and S. D. Young.  Human body temperature (37˚C) increases the expression of iron, carbohydrate, and amino acid utilization genes in Escherichia coli K-12.  Journal of Bacteriology 2007.  Vol. 189(15):5429-40. Epub 2007 May 25.

last: Whi

first: Amy

last: Malhowski

first: Sarah

last: Young


title: Evidence of the role of tick subolesin in gene expression

status: Public on Jan 23 2008

city: Stillwater

first: Patricia

middle: J

last: Ayoubi

state: OK

address: 246 NRC

department: Biochemistry and Molecular Biology

country: USA

institute: Oklahoma State University

summary:
Subolesin is an evolutionary conserved protein that was recently discovered in Ixodes scapularis as a tick protective antigen and has a role in tick blood digestion, reproduction and development.  In other organisms, subolesin orthologs may be involved in the control of developmental processes. Because of the profound effect of subolesin knockdown in ticks and other organisms, we hypothesized that subolesin has a role in gene expression, thus affecting multiple cellular processes. The objective of this study was to provide support for the role of subolesin in gene expression.
Keywords: time course

overall_design: Total RNA was prepared and pooled from subolesin dsRNA- and saline-injected ticks at 6 and 9 dpi (5 and 8 days of feeding).


title: Campylobacter jejuni genes contributing to acid adaptation

status: Public on Jan 21 2008

city: Ottawa

first: Alain

last: Stintzi

state: Ontario

address: 451 Smyth road

department: Biochemistry, Microbiology and Immunology

country: Canada

institute: University of Ottawa

summary:
In order to cause disease, the food- and water-borne pathogen Campylobacter jejuni must face the extreme acidity of the host stomach as well as cope with pH fluctuations in the intestine. In the present study, C. jejuni NCTC 11168 was grown under mild acidic conditions mimicking those encountered in the intestine. The resulting transcriptional profiles revealed how this bacterium fine-tunes gene expression in response to acid stress. This adaptation involves differential expression of respiratory pathways, induction of genes for phosphate transport and repression of energy generation and intermediary metabolism genes.
Keywords: acid shock; dose response; transcriptional response to 3 mild-acidic pH growth conditions (pH6.5, pH 6.0 and pH5.0)

overall_design: Three two-condition experiments: (1) pH7.0 versus pH 6.5; (2) pH7.0 versus pH6.0; and (3) pH 7.0 versus pH 5.5. Two biological replicates per experiment and 3 technical replicates for each biological replicate

first: Anne

last: Reid

first: Reenu

last: Pandey

first: Kiran

last: Palyada


title: Identification of Campylobacter jejuni genes involved in the response to stomach transit

summary:
Campylobacter jejuni causes food- and water-borne gastroenteritis, and as such must survive passage through the stomach in order to reach the gastrointestinal tract. While little is known about how C. jejuni survives transit through the stomach, its low infectious dose suggests it is well equipped to sense and respond to acid shock. In this study, the transcriptional profile of C. jejuni NCTC 11168 was obtained after exposure to in  in vivo (piglet stomach) acid shock.
Keywords: acid shock; in vivo study; transit through the host stomach

overall_design: The in vivo transcriptome profile of C. jejuni was obtained as the bacterium transited through the piglet stomach. The piglets were fed ~10e13 viable bacteria. Total RNA was extracted from the stomach contents of 2 piglets 20 min following oral inoculation with C. jejuni, and the RNA samples were pooled. The extracted RNA was reverse-transcribed, labeled and subjected to microarray analysis by competitive hybridization with cDNA derived from C. jejuni grown to mid-log phase in vitro in MH medium (pH 7.0). The in vivo data comprise 6 technical replicates from a single pool of RNAs (obtained from 2 piglets).


title: The fine-scale and complex architecture of human copy number variation

status: Public on Jan 24 2008

city: Santa Clara

first: Peter

last: Tsang

state: CA

address: 5301 Stevens Creek Blvd.

department: Agilent Labs

institute: Agilent Technologies

type: Genome variation profiling by genome tiling array

summary:
Despite considerable excitement over the potential functional significance of copy number variants (CNVs), we still lack knowledge of the fine-scale architecture of the large majority of CNV regions in the human genome. In this study, we used a high-resolution array-based comparative genomic hybridization (aCGH) platform that targeted known CNV regions of the human genome at ~1 kb resolution to interrogate the genomic DNAs of 30 individuals from four HapMap populations. Our results revealed that 1,020 of 1,153 CNV loci (88%) were actually smaller in size than what is recorded in the Database of Genomic Variants based on previously published studies. A reduction in size of more than 50% was observed for 876 CNV regions (76%). We conclude that the total genomic content of common human CNVs may be smaller than previously thought. In addition, approximately 8% of the CNV regions observed in multiple individuals exhibited genomic architectural complexity in the form of smaller CNVs within larger ones and CNVs with inter-individual variation in breakpoints. Future association studies that aim to capture the potential influences of CNVs on disease phenotypes will need to consider how to best ascertain this previously underappreciated complexity.
Keywords: comparative genomic hybridization

overall_design: The DNA samples are a panel of 30 Hapmap samples, and a female sample NA15510 of undetermined geographic origin, which has been well characterized by fosmid-end sequencing (Tuzun et al. 2005, Nature Genetics 10, p1038). The DNA for this set of 16 female and 15 male samples are all supplied by the Coriell Cell Repository, and is comprised of samples from four populations: 10 unrelated Yoruban, 10 unrelated European-American individuals from Utah (CEPH), 5 unrelated Chinese, and 5 unrelated Japanese. The reference sample, NA10851 is also a male CEPH also from the HapMap Sample set (Corriel Cell Repository). Each of these samples was hybridized in pairs with the reversed labeling polarities. Additionally, 3 self-self control hybridizations were carried out for the reference sample, NA10851, one on each hybridization date. In the Title  for each of the samples, each hybridization session is indicated by the letters A, B or C.

first: George

middle: H

last: Perry

first: Amir

last: Ben-Dor

first: Anya

last: Tsalenko

first: Nick

last: Sampas

first: Laia

last: Rodriguez-Revenga

first: Charles

middle: W

last: Tran

first: Alicia

last: Scheffer

first: Israel

last: Steinfeld

last: Yamada

first: Park

last: Soo

first: Jong

middle: I

last: Kim

first: Jeong

middle: S

last: Seo

first: Zohar

last: Yakhini

first: Stephen

last: Laderman

first: Laurakay

last: Bruhn

last: Lee


title: Gene expression variation in African and European populations of Drosophila melanogaster

city: Planegg-Martinsried

first: Stephan

last: Hutter

state: Bavaria

address: Grosshaderner Strasse 2

department: Department of Biology II

country: Germany

institute: University of Munich

summary:
Background: Differences in levels of gene expression among individuals are an important source of phenotypic variation within populations. Recent microarray studies have revealed that expression variation is abundant in many species, including Drosophila melanogaster. However, previous expression surveys in this species generally focused on a small number of laboratory strains established from derived populations. Thus, these studies were not ideal for population genetic analyses.
Results: We surveyed gene expression variation in adult males of 16 D. melanogaster strains from two natural populations, including an ancestral African population and a derived European population. Levels of expression polymorphism were nearly equal in the two populations, but a higher number of differences was detected when comparing strains between populations. Expression variation was greatest for genes associated with few molecular functions or biological processes, as well as those expressed predominantly in males. Our analysis also identified genes that differed in expression level between the European and African populations, which may be candidates for adaptive regulatory evolution. Genes involved in flight musculature and fatty acid metabolism were over-represented in the list of candidates.
Conclusions: Overall, stabilizing selection appears to be the major force governing gene expression variation within populations. However, positive selection may be responsible for much of the between-population expression divergence. The nature of the genes identified to differ in expression between populations may reveal which traits were important for local adaptation to the European and African environments.
Keywords: Natural variation

overall_design: We used dual channel microarrays to compare genome-wide expression profiles in adult males from 16 inbred strains derived from two natural populations. In total 80 hybidizations were performed including dye-swaps. The hybridization scheme consisted of a balanced loop design, which allowed an unbiased comparison of relative expression levels within and between populations.

first: Sarah

last: Saminadin-Peter

first: Wolfgang

last: Stephan

first: John

last: Parsch


title: Characterization of probiotic Escherichia coli isolates using a novel pangenome microarray

city: Kgs. Lyngby

first: Hanni

last: Willenbrock

address: Kemikalievej, bygning 208

department: Center for Biological Sequence Analysis

country: Denmark

institute: DTU

summary:
Background: Based on 32 Escherichia coli and Shigella genome sequences, we have developed an E. coli pan-genome microarray. Publicly available genomes were annotated in a consistent manor to define all currently known genes potentially present in the species. The chip design was evaluated by hybridization of DNA from two sequenced E. coli strains, K-12 MG1655 (a commensal) and O157:H7 EDL933 (an enterotoxigenic E. coli). A dual channel and single channel analysis approach was compared for the comparative genomic hybridization experiments. Moreover, the microarray was used to characterize four unsequenced probiotic E. coli strains, currently marketed for beneficial effects on the human gut flora.

Results: Based on the genomes included in this study, we were able to group together 2,041 genes that were present in all 32 genomes. Furthermore, we predict that the size of the E. coli core genome will approach ~1,560 essential genes, considerably less than previous estimates. Although any individual E. coli genome contains between 4,000 and 5,000 genes, we identified more than twice as many (11,872) distinct gene groups in the total gene pool (“pan-genome”) examined for microarray design. Benchmarking of the design based on sequenced control strain samples demonstrated a high sensitivity and relatively low false positive rate. Moreover, the array was highly sufficient to investigate the gene content of apathogenic isolates, despite the strong bias towards pathogenic E. coli strains that have been sequenced so far. Our analysis of four probiotic E. coli strains demonstrate that they share a gene pool very similar to the E. coli K-12 strains but also show significant similarity with enteropathogenic strains. Nonetheless, virulence genes were largely absent. Strain-specific genes found in probiotic E. coli but absent in E. coli K12 were most frequently phage-related genes, transposases and other genes related to mobile DNA, and metabolic enzymes or factors that may offer colonization fitness, which together with their asymptomatic nature may explain their nature.

Conclusion: This high-density microarray provides an excellent tool for characterizing either DNA content or gene expression from unknown E. coli strains.
Keywords: Comparative genomic hybridizations

overall_design:
Factorial design:
Each of four test samples (G 1/2, G3/10, G 4/9, G5) are co-hybridized with two control strain samples (K-12 MG1655 and O157:H7 EDL933). Additional replicate co-hybridizations are included of the two control strain samples (O157:H7 EDL933 vs. K-12 MG1655).

first: Peter

middle: F

last: Hallin

first: Trudy

last: Wassanar

first: David

last: Ussery


title: In flies, the 5´ ends of both microRNAs and their miRNA* strands are more accurately defined than their 3´ termini

city: Worcester

first: Hervé

last: Seitz

address: 364 Plantation Street

department: Biochemistry and Molecular Pharmacology

institute: University of Massachusetts Medical School

type: Non-coding RNA profiling by high throughput sequencing

summary:
MicroRNAs (miRNAs) are short (∼ 21-23 nt) regulatory RNAs that guide the degradation or translational repression of their RNA targets. The miRNA “seed”—nucleotides 2 through 7—establishes miRNA target specificity, because this region is the primary determinant of RNA binding by both miRNA and small interfering RNAs. Accurate processing of the miRNA 5´ end is thought to be under strong selective pressure, as a shift by just one nucleotide in the 5´ end of a miRNA would alter its seed sequence, redeﬁning its repertoire of targets. Animal miRNAs are produced by the sequential cleavage of partially double-stranded precursor RNAs by the RNase III endonucleases Drosha, which cleaves the primary miRNA transcript in the nucleus to release a pre-miRNA, and Dicer, which cleaves the pre-miRNA to generate a transitory intermediate comprising the mature miRNA paired with its miRNA* strand. Here, we report that in flies, the 5´ end of a miRNA is typically more precisely deﬁned than the 3´ ends of both the miRNA and its miRNA*. Surprisingly, the 5´ end of the miRNA* sequence was also more precisely defined than the adjacent 3´ end of the miRNA. Our data imply either that many miRNA* sequences are under evolutionary pressure to maintain their seed sequences—that is, they have cellular or exogenous RNA targets—or that secondary constraints, such as the sequence requirements for loading small RNAs into functional Argonaute protein complexes narrow the range of miRNA and miRNA* 5´ ends that accumulate in flies.
Keywords: microRNA deep sequencing; pyrosequencing

overall_design:
Four samples were analyzed: fly heads (subjected to beta-elimination, or not), and S2 cells (idem). Beta-elimination prevents RNAs from being ligated on their 3' end, unless they bear a 3' modification (e.g., a 2'-O-methylation).
Small RNAs (18 to 30 nt in length) from each of these sample were submitted to deep sequencing with the 454 technology.
Small RNA sequences were extracted from the 454 reads with 10 different stringencies (tolerating 0, 1, 2 ..., 9 mismatches on the pair of adapters). The proportion of genome-matching inserts is the same when 0 or 1 mismatch is tolerated on the pair of adapters, but it drops when more mismatches are tolerated; hence, in the article (Argonaute Loading Improves the 5' Precision of Both MicroRNAs and Their miRNA* Strands in Flies (2008) Curr. Biol. 18:147; PubMed ID: 18207740), only the inserts identified when 0 or 1 mismatch was tolerated on the pair of adapters, were considered.

Raw data is linked to this submission as supplementary files (.sff files are not available). There are two fasta files, and two corresponding .qual files. Each fasta file contains the sequences of small RNAs from two samples (two samples were analyzed per lane by barcoding with different 3´ adapters).

The file associations are:

IR-NB_S2-NB.fna/IR-NB_S2-NB.qual:
- fly heads, non beta-eliminated
- S2 cells, non beta-eliminated

IR-B_S2-B.fna/IR-B_S2-B.qual:
- fly heads, beta-eliminated
- S2 cells, beta-eliminated

first: Megha

last: Ghildiyal

first: Phillip

middle: D

last: Zamore


title: Identification of Campylobacter jejuni genes involved in the response to acidic pH

first: Alain

last: Stintzi

summary:
Campylobacter jejuni causes food- and water-borne gastroenteritis, and as such must survive passage through the stomach in order to reach the gastrointestinal tract. While little is known about how C. jejuni survives transit through the stomach, its low infectious dose suggests it is well equipped to sense and respond to acid shock. In this study, the transcriptional profile of C. jejuni NCTC 11168 was obtained after exposure to in vitro acid shock.
Keywords: acid shock; in vitro study; time course

overall_design: Time course experiments: C. jejuni gene expression was monitored over a 20 min period following acid shock (pH 4.5). Total RNA was extracted from samples taken 2, 4, 12, 16 and 20 min after the acid challenge. The relative abundance of each transcript was monitored by competitive hybridization to C. jejuni microarray of cDNA obtained from C. jejuni exposed to MH-MES medium at pH 7.0 and cDNA from C. jejuni exposed to MH-MES medium at pH 4.5 for each of the time points. Two biological replicates per experiment and 3 technical replicates for each biological replicate

first: Anne

last: Reid

first: Reenu

last: Pandey

first: Kiran

last: Palyada


title: Similar patterns of additive and non-additive gene expression in maize hybrids with varying levels of heterosis

status: Public on Jan 26 2008

city: Saint Paul

first: Robert

middle: M

last: Stupar

state: MN

address: 1991 Upper Buford Circle

department: Agronomy and Plant Genetics

institute: University of Minnesota

summary:
Expression profiling analyses for 5 maize inbreds and 4 hybrids, chosen to represent diversity in genotypes and heterosis responses, revealed a correlation between genetic diversity and transcriptional variation.  The majority of differentially expressed genes in each of the different hybrids exhibited additive expression patterns, and ~25% exhibited statistically significant non-additive expression profiles.  Among the non-additive profiles, ~80% exhibited hybrid expression levels between the parental levels, ~20% exhibited hybrid expression levels at the parental levels and ~1% exhibited hybrid levels outside the parental range.  These findings indicate that the frequencies of additive and non-additive expression patterns are very similar across a range of hybrid lines.
Keywords: Genotype comparison series

overall_design: Affymetrix expression profiling was used to study gene expression in aerial tissue from 11-day seedlings of maize. Three biological replicates were performed for nine different genotypes; B37, B73, B84, Mo17, Oh43, B37xB73, B84xB73, Oh43xB73 and Oh43xMo17.

first: Nathan

last: Springer


title: Improvement of Lactobacillus plantarum Aerobic Growth by Comprehensive Transcriptome Analysis

status: Public on Jan 18 2008

city: Amsterdam

first: Douwe

last: Molenaar

address: De Boelelaan 1085

department: Systems Bioinformatics

country: Netherlands

institute: Vrije Universiteit Amsterdam

summary:
An aerobic Lactobacillus plantarum culture displayed growth stagnation during early growth. Transcriptome analysis revealed that regain of growth after stagnation correlated with activation of CO2-producing pathways suggesting that limiting CO2-concentration induced stagnation. Analogously providing increased CO2 gas partial pressure during aerobic fermentation prevented the temporal growth stagnation.
Keywords: cell type comparison

overall_design: Two aerobic fermenters (biological replicates Land R) were sampled at two time points, before (1) and after (2) growth stagnation.

first: Marc

last: Stevens

first: Oscar

last: Kuipers

first: Willem

last: De Vos

first: Eddy

last: Smid

last: Wiersma

first: Michiel

last: Kleerebezem


title: Cell response to Au nanoparticles

status: Public on Jan 20 2008

city: Dallas

first: Lianghao

last: Ding

state: TX

address: 5323 Harry Hines Blvd

institute: UT Southwestern Medical Center

summary:
Human skin fibroblast treated with Au nanoparticles induced gene expression changes
Keywords: Time course and dose response

overall_design: Illumina arrays, one array per sample

first: Liang-Hao

first: Michael

last: Story

first: Shane

last: Scoggin

first: Fanqing

last: Chen


title: In vitro expression profiles of Lactobacillus johnsonii NCC533 in MRS

status: Public on Jan 17 2008

city: Lausanne

first: Emmanuel

last: Denou

state: vaud

address: vers chez les blancs

department: Nutrition & Health

country: Switzerland

institute: Nestlé Research Center

summary:
Work with pathogens like Vibrio cholerae has shown major differences between gene expression in bacteria grown in vitro and in vivo. To explore this subject for commensals, we investigated the transcription of the Lactobacillus johnsonii NCC533 genome during in vitro using the microarray technology. In broth growth, 537, 626 and 277 of the 1756 tested genes were expressed during exponential, “adaptation” (early stationary phase) and stationary phase, respectively. One hundred-one, 150 and 33 genes, respectively, were specifically transcribed in these three phases.  Forty-four per cent of the NCC533 genome were not detectably transcribed under any of the investigated conditions. Non-transcribed genes were clustered on the genome and enriched in the variable genome part.
For gene expression profiling experiments, cells were grown in a Sixfors fermentor system, composed of four individual 500 ml vessels (Infors, Bottmingen, Switzerland). Growth curves were performed at least in triplicate using the four separate fermentation vessels (fermentor 1 to 4), which were inoculated at 0.4% (v/v) with four individual overnight cultures in order to reach a starting optical density (OD600nm) of 0.05. Samples were taken at regular intervals from the four vessels to measure the OD600 nm and determine the CFU/ml until 36 h of fermentation. Aliquots of 15 ml (for early (T1-T2) and mid-exponential phases (T3-T4)) and 10 ml (for adaptation (T5), mid-stationary (T6) and late stationary phases (T7)) were centrifuged for 5 min at 10,000 x g at 4°C. Cell pellets were snap frozen in liquid nitrogen and stored at –80°C until further use.L. johnsonii NCC533 was grown in MRS broth supplemented with 2 % glucose as carbon source and 0.05% cysteine as redox buffer. For mRNA isolation L. johnsonii NCC533 cells were harvested from the broth culture several time points after inoculation (Fig. 1). In a total of 27 hybridization experiments. Genes were scored as expressed when their signal was detected in all hybridization experiments (with two technical replicates per slide) with mRNA preparations obtained from three independent growth experiments.
Keywords: Growth phases of Lactobacillus johnsonii NCC533 in MRS

overall_design:
total of 24 hybridization experiments. Genes were scored as expressed when their signal was detected in all hybridization experiments (with two technical replicates per slide) with mRNA preparations obtained from three independent growth experiments.

A supplementary file is attached with the scored genes for the 18 Samples. A score of 1 represents the gene expressed, 0 represents non detectable.

first: Bernard

last: Berger


title: Yeast (S. cerevisiae SK1) Sporulation/Meiosis

status: Public on Jan 16 2008

city: La Jolla

last: Baitaluk

address: 9500 Gilman dr

department: SDSC

institute: UCSD

summary:
Transription profile of Saccharomyces cerevisiae SK1 cultures undergoing synchronous sporulation.
We have measured mRNA levels in synchronized SK1 cells immediately upon transfer to the sporulation medium and every 30 minutes after that for 6 hours.  mRNA extracted from these cultures were converted to cDNA and hybridized to microarrays and log2 ratios of hybridization signal of each time point was compared to that of time zero (immediately prior to transfer to the sporulation medium).
Keywords: Time course

overall_design: RNA from time zero is control, other time samples are experimental, two replicates per array, dye reverse of each time point on separate arrays, each time sample represents pooled cells from six independent synchronized cultures cultivated on the same day.

first: Animesh

last: Ray

first: Mayya

last: Sedova

first: Kirilynn

last: Svay


title: Transcriptional Analysis of 1M Regions in Glycine max Using Tiling Microarrays

status: Public on Jan 15 2008

city: New Haven

first: Hang

last: He

state: CT

address: 165 Prospect St

department: MCDB

institute: Yale University

type: Expression profiling by genome tiling array

summary:
We report here a transcriptonal analysis in six different organ types of a approximately 1 Mb region in soybean (Glycine max) which is sytenic with legume (Medicago truncatula). We used oligonucleotide tiling microarrays to detecte transcription of over 80% of the predicted genes in both species. We detected differential gene expression in the six examined organ types.
Keywords: RNA

overall_design:
Both the barrel medic and soybean tiling arrays were produced on the Maskless Array Synthesizer platform. Briefly, tiling-paths consisting of 36-mer oligonucleotides offset by five nucleotides were designed to represent both DNA strands of the selected barrel medic and soybean genome sequence. Probes were synthesized at a feature-density of 390,000 probes per array in a “chess board” design. Microarray production and storage were carried out.
Total RNA and mRNA were sequentially isolated using the RNeasy Plant Mini kit (Qiagen, Valencia, CA) and the Oligotex mRNA kit (Qiagen) according to the manufacturer’s recommendations, respectively. mRNA from different organ types was reverse transcribed using a mixture of oligo(dT)18 and random nonamer primers, during which amino-allyl-modified dUTP (aa-dUTP) was incoporated. The aa-dUTP decorated cDNA was fluorescent labeled by conjugating the monofunctional Cy3 dye (GE Healthcare, Piscataway, NJ) to the amino-allyl functional groups in the cDNA. Two μg dye-labeled targets were used for hybridization.

first: Lei

last: Li

first: Juan

last: Zhang

first: Xiangfeng

last: Wang

first: Viktor

last: Stolc

first: Waraporn

last: Tongpraist

first: Oliver

last: Yu

first: XingWang

last: Deng


title: Genomic copy number alterations as predictive markers of systemic recurrence in breast cancer

city: Seoul

first: Wonshik

last: Han

address: 28 Yongon-dong, Chongno-gu

department: Dept. of Surgery

country: South Korea

institute: Seoul National University College of Medicine

summary:
Array CGH containing 4,044 human bacterial artificial chromosome clones was used to assess copy number changes in 31 pairs of clinicopathologically well matched recurred / nonrecurred breast cancer tissues.
Keywords: array CGH

first: Ki-Tae

last: Hwang

first: Dong-Young

last: Noh

first: Song-Ju

last: Yang


title: Microarray expression profiling of C. elegans N2 L4 control(RNAi) vs. mdt-15(RNAi)

city: San Francisco

first: Stefan

last: Taubert

department: CMP

institute: UCSF

summary:
We previously showed that the C. elegans Mediator subunit MDT-15 impacts expression of select genes involved in fatty acid (FA) metabolism (Taubert et al, Genes & Dev, 2006). To comprehensively identify processes downstream of MDT-15 in an unbiased manner, we set out to globally discover new MDT-15-dependent genes.
Keywords: Expression profiling, RNAi depletion

overall_design: We used expression microarrays to compare the transcriptional profiles in wild-type (N2) worms that were fed for 40 hr (i.e. allowed to synchronously develop to larval stage L4) with bacteria harboring vectors for either control or mdt-15 RNA-interference (RNAi). Note that array #4 is a dye-swap. To identify statistically significant changes in gene expression following MDT-15 depletion we analyzed the raw data using “linear models for microarray data” (limma). In total we found 187 genes that were downregulated, and 120 genes that were upregulated (P-value cutoff 0.05). An alternative approach, “statistical analysis of microarrays” (SAM), revealed similar numbers and similar sets of affected genes (data not shown).

first: Marc

middle: R

last: Van Gilst

first: Samantha

middle: B

last: Cooper

first: Keith

last: Yamamoto


title: CTL-OA-OP human IT PF Bone - CompuGen H19K microarray study

status: Public on Jan 14 2008

city: Adelaide

first: Blair

last: Hopwood

state: South Australia

address: Frome Road

department: Tissue Pathology

country: Australia

institute: Institute of Medical and Veterinary Science

summary:
Aims of study: (1) To identify systemic differences in osteoarthritic (OA) bone that contribute to OA pathogenesis. (2) Identify novel osteoporotic (OP) bone-related disease genes.
Study involved comparison of trabecular bone extracted from the intertrochanteric (IT) region of the proximal femur (PF) from OA, OP and normal/control (CTL) individuals.
Bone was obtained from OA and OP individuals at surgery for total hip replacement and from CTL individuals at autopsy.
Keywords: Bone tissue comparison, diseased versus non-diseased, factorial design, linear modelling

overall_design:
Four sets of sample comparisons (39 comparisons in total) were made in this study. These comprised 10 OA-CTL female, 10 OA-CTL male, 10 OA-OP female and 9 OP-CTL female comparisons.
A Compugen Human 19K-oligo library spotted onto glass slides by the Adelaide Microarray facility (AMF) was used in this study. The slides were interrogated by competitive hybridisation of Cy3 and Cy5 labelled pairs of OA-CTL, OA-OP or OP-CTL amplified RNA samples. Sample pairs were age-matched as closely as possible. A biological dye-swap strategy was employed. After hybridisation and washing of the slides they were scanned using a GenePix 4000B Scanner driven by GenePix Pro 4.0. All analyses were performed using the statistical programming and graphics environment R. The “SPOT” software package was used to identify spots by adaptive segmentation method and subtract backgrounds utilising morphological opening approach.  Data analysis was performed in R using Bioconductor. Loess print tip method was used to correct for dye-bias and intensity within each group of adjacent spots printed by one pin.  Linear modelling was performed with the Limma package of Bioconductor. Linear models were used to incorporate all available data into a single analysis. This allowed the use of both direct and indirect comparisons in calculation of expression ratios as well as improved accuracy in estimation of variance for each gene.
Factorial design utilised the following factors: medical condition (OA or OP or CTL); sex (male or female) for OA patients only.

first: Anna

last: Tsykin

first: David

last: Findlay

first: Nicola

middle: L

last: Fazzalari


title: Gene expression profiling of dMyc null Drosophila larvae

city: Seattle

first: Robert

last: Eisenman

state: WA

address: 1100 Fairview Ave N

department: Basic Sciences

institute: Fred Hutchinson Cancer Research Center

summary:
Myc oncoproteins are essential regulators of the growth and proliferation of mammalian cells. In Drosophila the single ortholog of Myc (dMyc), encoded by the dm gene, influences organismal size and the growth of both mitotic and endoreplicating cells. A null mutation in dm (dm4) results in attenuated endoreplication and growth arrest early in larval development. Gene expression analysis indicates that loss of dMyc leads to decreased expression of genes required for ribosome biogenesis and protein synthesis.
Keywords: cDNA expression microarray, mutant analysis

overall_design: Drosophila larvae were collected 24 hours after egg deposition. Sample comparisons were performed using 3 biological independent experimental replicates of dm4 that were each compared to a common reference (wild type larvae of the same genetic background as dm4).  For each comparison, a dye-swapped technical replicate was also performed and the paired results were averaged and used as a single observation.

first: Sarah

last: Pierce

first: Cynthia

last: Yost

middle: A

last: Anderson

first: Erin

last: Flynn

first: Jeffrey

last: Delrow

middle: N


title: The regenerative response in the pig liver remnant varies with the degree of resection and rise in portal pressure.

city: Tjele

first: Jakob

last: Hedegaard

address: PO-box 50

department: Department of Genetics and Biotechnology

institute: Aarhus University, Faculty of Agricultural Sciences

summary:
Following parenchymal loss, the liver regenerates restoring normal mass and metabolic function. Prevailing theories on triggering events leading to regeneration include humoral, metabolic and flow-mediated mechanisms, the latter emphasizing the importance of shear stress mediated nitric oxide (NO) regulation. We aimed to investigate whether the grade of resection and hence the portal venous pressure and sinusoidal shear stress increase, would be reflected in the gene expression profiles in the liver remnant by employing a global porcine cDNA microarray chip with approximately 23 000 genes represented. Six pig livers were resected with 62% (Low Portal Pressure Resection, LPPR) and 75% (High Portal Pressure Resection) resulting in a portal venous pressure increase from a baseline of 6.1 mmHg to 8.2 and 12 mmHg respectively. By sampling consecutive biopsies from the liver remnants we found differentially expressed genes in the HPPR group to have functions related primarily to apoptosis, nitric oxide metabolism and oxidative stress, whereas differentially expressed genes in the LPPR group potentially regulate the cell cycle. Common to both groups was the upregulation of genes regulating inflammation, transport, cell proliferation and development and protein metabolism. Also common to both groups was both up- and downregulation of genes regulating cell-cell signaling, signal transduction, cell adhesion and translation. Genes regulating the metabolism of lipids, hormones, amines, and alcohol were downregulated in both groups. Conclusions: The genetic regenerative response in the liver remnant to varies according to the level of resection.
Keywords: time course, treatment comparison

overall_design: Three pigs underwent a 62% liver resection (low-pressure resection, LPR) and three underwent a 75% resection (high-pressure resection, HPR). Biopsies from the liver remnant were taken from all animals at time points 1, 30, 90, minutes and 3, 4 and 6 hours after resection. Expression profiling was conducted by hybridising each sample against a common reference, consisting of liver RNA from an unrelated animal.

first: Kim

middle: E

last: Mortensen

first: Lene

last: Conley

first: Trine

last: Kalstad

first: Peter

last: Sørensen

first: Christian

last: Bendixen

first: Arthur

last: Revhaug


title: Rat liver: Dexamethasone treatment vs. Control

status: Public on Jan 13 2008

city: Angers

first: Matthieu

last: Arvier

address: 4 rue larrey

department: UMR 694

country: France

institute: INSERM

summary:
Sixteen male Sprague-Dawley rats  were randomly allocated into 2 groups (8 rats per group) as follows: the control group (CON) and the dexamethasone-treated group (DEXA). Dexamethasone-treated rats received a daily intraperitoneal injection of 1.5 mg/kg of dexamethasone for 5 days. All rats were fasted during the night following the fifth day. On the sixth day, the animals were killed by decapitation.

In order to focus our investigation on metabolism-related genes, we developed a metabolism dedicated microarray tool: the Mitoligo. Using this microarray tool, we were able to determine that energy metabolism was deeply modified by dexamethasone treatment. Dexamethasone treatment to rats induces a complete switch of the metabolism toward a maximal rate of ATP synthesis. In this study, we show that substrate supplying for oxidative phosphorylation is greatly enhanced. We also confirm that oxidative phosphorylation capacity is increased by dexamethasone treatment.
Keywords: hormonal treatment

platform_organism:
Homo sapiens
Rattus norvegicus

overall_design:
	The present investigation was performed according to the guiding principles of the French department of Animal and Environmental protection for the care and use of animals and in accordance with the American Physiological Society “Guiding Principles for Research Involving Animals and Human Beings”. Sixteen male Sprague-Dawley rats, born and bred in our animal facilities, were housed in individual cages from the age of 13 weeks (330-400g). Animals were provided with water ad libitum and a standard diet (U-A-R A04) composed of (% total weight) 16% protein, 3% fat, 60% carbohydrate and 21% water, fiber, vitamins and minerals. The metabolisable energy content was 2.9 kcal/day. Rats were randomly allocated into 2 groups (8 rats per group) as follows: the control group (CON) and the dexamethasone-treated group (DEXA). Dexamethasone-treated rats received a daily intraperitoneal injection of 1.5 mg/kg of dexamethasone for 5 days. All rats were fasted during the night following the fifth day. On the sixth day, the animals were killed by decapitation.

Total RNA was isolated from tissue samples using a standard guanidium isothiocyanate protocol (TRIzol reagent, Invitrogen). RNA integrity was determined using a BioAnalyzer 2100 (Agilent Technologies, Waldbronn, Germany).
	Cy3- and Cy5-labelled (amplified) aRNA was prepared using the Amino Allyl MessageAmpII aRNA Amplification Kit (Ambion, 1753). The reference sample consisted of a pool of an equal amount of aRNA obtained after the amplification of tRNA from each non-restricted control rat and was labelled with Cy5 (Amersham). For each of different rat within each group (including control group), two replicate tRNA extractions were prepared using the Amino Allyl MessageAmpII aRNA Amplification Kit (Ambion, 1753) and  labeled individually with Cy3 (Amersham). Each Cy3-labeled sample was mixed with an equal amount of the Cy5- labeled reference sample, pre-incubated with yeast tRNA and polyA RNA, and hybridized to the microarrays.

Microarrays were prepared in-house (Plateforme Puces à ADN-Ouest Génopole, Nantes, France) using 50-mer oligonucléotides probes. The probes were diluted in spotting buffer A (25 μM in buffer A 1x, Ocimum) and spotted onto epoxysilane-coated glass slides (Slide E, Schott nexterion) using the Lucidea Array Spotter from Amersham. Immediately after spotting, the slides were incubated at 42°C and 55% humidity for 12 h. Slides were washed once in 0.2% SDS and twice in H2O and then incubated for 20 minutes in H2O at 50°C, dried by centrifugation (1000 rpm, 3 minutes, 20°C), and stored at 20°C.
	The 982 genes that were represented on the microarray were selected for involvement in mitochondrial metabolism. 350 genes are specific for rat while 600 genes are specific of human mRNA. Each gene was spotted in triplicate on the microarrays. Hybridized arrays were scanned at 10 μm/pixel resolution by fluorescence confocal microscopy (Scanarray 3000, GSI-Lumonics). Signal intensities were extracted with Genepix Pro 5.1.0.19 image analysis software (Axon Instruments, USA). Data were retained based on concordance between the measurements of different types of replicates: biological replicates, technical replicates and combined biological and technical replicates.

Microarray raw data were analyzed using Genespring GX 7.0 (Agilent technologies). Data were normalized using Lowess normalization. Normalized data were compared using ANOVA followed by tukey or student t-test.

first: Jean-François

last: Dumas

first: Yves

last: Malthièry

first: Gilles

last: Simard

first: Patrick

last: Ritz

first: Catherine

last: Chevallier

first: Jean

last: Leger


title: Spironolactone effect on renal RNA expression in 36 wk old NZB/W F1 female mice

status: Public on Jan 12 2008

city: Ann Arbor

first: Seetha

middle: U

last: Monrad

state: MI

address: 1500 E Medical Center Dr, SPC 5358

department: Internal Medicine

institute: University of Michigan

summary:
The purpose of this study was to examine the effect of aldosterone receptor blockade on the immunopathogenesis and progression of nephritis in the (NZBxNZW) F1 murine lupus model. Female NZB/W F1 mice (11 weeks old) were treated daily with 25 or 50 mg/kg of oral spironolactone or vehicle. Proteinuria, renal function and serum autoantibody levels were monitored. Renal histopathology, immune complex deposition, and immunohistochemistry were analyzed at various time points. Targeted microarray analysis was performed on renal tissue, with subsequent real time PCR analysis of several differentially expressed genes.

Keywords: treatment study

overall_design: At 36 weeks of age,  8 mice from each treatment group (vehicle, 25 mg/kg/d spironolactone and 50 mg/kg/d spironolactone) were anesthetized and perfused with cold saline; then one kidney per mouse was removed , homogenized in Tripure, and frozen at -80 until RNA extraction. RNA was extracted using Tripure and a Qiagen RNEasy Minikit. RNA was pooled equally by weight within each treatment group and frozen at -80. Subsequently, the three batches of pooled RNA were processed for microarray analysis.

first: Paul

last: Killen

first: Marc

last: Anderson

first: Amanda

last: Bradke

first: Mariana

last: Kaplan


title: Evaluation of Pseudomonas putida KT2440 gene expression in the Arabidopsis thaliana rhizosphere

status: Public on Jan 11 2008

city: Davis

first: Enid

middle: T

last: Gonzalez

address: One Shields Drive

department: Crops Pathology and Genetics Research Unit

institute: USDA-Agricultural Research Service

summary:
Gene expression patterns of the plant colonizing bacterium,Pseudomonas putida KT2440 were evaluated as a function of growth in the Arabidopsis thaliana rhizosphere.  Gene expression in rhizosphere grown P. putida cells was compared to gene expression in non-rhizosphere grown cells.
Keywords: Gene expression

overall_design: The experiment contained three biological replicates, two conditions (plus plant, minus plant), and two time points (48 and 120 hours).

first: Daniel

last: Kluepfel


title: Expression Profiling of Oral Squamous Cell Carcinoma and Mucosa of healthy non-tumor patients

city: Mannheim

first: Carsten

last: Sticht

address: Theodor-Kutzer-Ufer

department: ZMF

institute: University Heidelberg

summary:
In an attempt to further elucidate the pathomechanisms in oral squamous cell carcinoma (OSCC), gene expression profiling to a set of 35 primary OSCCs compared to 6 oral mucosa from healthy non-tumor patients was performed.
Keywords: expression profiling

overall_design: Gene expression profiling using a whole-transcriptome chip that contains 35,035 gene-specific 70mere oligonucleotides (Human Oligoset 4.0; Operon) to a set of 35 primary OSCCs and 6 mucosa of healthy non-tumor patients.

first: Kolja

last: Freier

first: Karl

last: Knoepfle

first: Christa

last: Flechtenmacher

first: Susanne

last: Pungs

first: Christof

last: Hofele

first: Meinhard

last: Hahn

first: Stefan

last: Joos

first: Peter

last: Lichter


title: Culture of Campylobacter jejuni with Sodium Deoxycholate induces Virulence Gene Expression

status: Public on Jan 10 2008

city: Albany

first: Craig

last: Parker

address: 800 Buchanan St

department: PSM

institute: USDA ARS

summary:
C. jejuni, a spiral-shaped gram-negative bacterium, is a leading bacterial cause of human foodborne illness.  Acute disease is associated with C. jejuni invasion of the intestinal epithelium.  Further, maximal host cell invasion requires the secretion of proteins termed Campylobacter invasion antigens (Cia).  As bile acids are known to alter the pathogenic behavior of other gastrointestinal pathogens, we hypothesized that the virulence potential of Campylobacter may be triggered by the bile acid deoxycholate (DOC).  In support of this hypothesis, culturing C. jejuni with a physiologically relevant concentration of DOC significantly altered the kinetics of cell invasion as evidenced by gentamicin-protection assays.  In contrast to C. jejuni harvested from Mueller-Hinton (MH) agar plates, C. jejuni harvested from MH agar plates supplemented with DOC demonstrated Cia secretion as judged by metabolic labeling experiments.  DOC was also found to induce the expression of the ciaB gene as judged by B-galactosidase reporter assays and real-time RT-PCR.  Microarray analysis revealed that DOC induced the expression of virulence genes (i.e., ciaB, cmeABC, dccR, and tlyA).  In summary, we demonstrate that it is possible to enhance the pathogenic behavior of C. jejuni by modifying the culture conditions.  These results provide a foundation to identify genes expressed by C. jejuni in response to in vivo-like culture conditions.
Keywords: Stress response

overall_design:
For the expression profiling arrays, an indirect comparison of gene expression was performed, where the expression profile of the C. jejuni F38011 cultured in the presence and absence of DOC was measured separately on different slides as described previously (26).  Briefly, Cy5 labeled reference DNA from the C. jejuni F38011 strain was mixed with Cy3 labeled test cDNA (C. jejuni F38011 cultured in the presence or absence of DOC) and hybridized to the Campylobacter cDNA array (26) on separate slides.  DNA microarrays were scanned using an Axon GenePix 4000B microarray laser scanner (Axon Instruments, Union City, CA) and the data for spot and background intensities were processed using the GenePix 4.0 software. To compensate for any effect of the amount of template and uneven Cy-dye incorporation, data normalization was performed as previously described (26).
For the comparison of genes differentially expressed in the presence and absence of DOC,six hybridization measurements were generated per biological experiment (three technical replicate arrays and two replicate features per array).

first: Preeti

last: Malik-Kale

first: Michael

last: Konkel


title: Microarray analysis of the effect of the xenobiotic beta-aminobutyric acid in Arabidopsis ecotype Co

city: Taipei

first: Laurent

last: Zimmerli

address: Roosevelt road

country: Taiwan

institute: National Taiwan University

summary:
Summary: The non-protein amino acid beta-aminobutyric acid (BABA) primes Arabidopsis to respond more quickly and strongly to pathogen and osmotic stress. Here, we report that BABA also significantly enhanced acquired thermotolerance in Arabidopsis. This thermotolerance was dependent on heat shock protein 101, a critical component of the normal heat shock response. BABA did not enhance basal thermotolerance under a severe heat shock treatment. No roles for the hormones ethylene and salicylic acid in BABA-induced acquired thermotolerance were identified by mutant analysis. By global gene expression analysis, transcript levels for several transcription factors and DNA binding proteins regulating responses to the stress hormone abscisic acid (ABA) were found to be elevated in BABA-treated relative to water-treated plants. The role of ABA in BABA-induced thermotolerance was complex. BABA-enhanced thermotolerance was partially compromised in the ABA-insensitive mutant, abi1-1, but was augmented in abi2-1. In an unrelated process, BABA, like ABA, inhibited root growth and the level of inhibition was roughly additive in roots treated with both compounds. Root growth of both abi1-1 and abi2-1 was also inhibited by BABA. Unexpectedly, abi1-1 and abi2-1 root growth was inhibited more strongly by combined ABA and BABA treatments than by BABA alone. Our results together with previously published data suggest that BABA is a general enhancer of plant stress resistance and that cross-talk occurs between BABA and ABA signalling cascades. Specifically, the BABA-mediated accumulation of ABA transcription factors without concomitant activation of a downstream ABA response could represent one component of the BABA-primed state in Arabidopsis. Overall design:
A replicate experimental design type is where a series of replicates are performed to evaluate reproducibility or as a pilot study to determine the appropriate number of replicates for a subsequent experiments.
Keywords: replicate_design

overall_design:
Arabidopsis Col-0 plants were grown in ProMix HP (Premier Horticulture, Red Hill, PA, USA) for 14 days and the soil was drenched with a 0.25 mM BABA solution 1 day prior to harvesting samples for RNA preparation. These plants were grown in a growth chamber at 21?C with a 14-hr photoperiod. All plants were grown at a light intensity of about 100 ? 150 ?E m-2 s-1. BABA (mixed isomers, Sigma, Milwaukee, WI, USA) was diluted in water at indicated concentration. A biological sample represents a comparison between plants that were water-treated (green channel) to plants drenched with BABA (red channel). This study represents 4 independent biological replicates.
Computed


title: Immunity to Grb10, a signal transduction molecule, inhibits the growth of breast cancer in mice.

status: Public on Jan 09 2008

city: Northbrook

last: Falduto

state: IL

address: 1808 Janke, Unit M

institute: GenUs BioSystems, Inc.

summary:
This study describes the application of a unique strategy to identify breast cancer antigens (TAA). In a mouse model, the strategy led to the identification of growth factor receptor-bound protein10 (Grb10) as a newly identified TAA. Grb10 is signal transduction molecule associated with multiple transmembrane tyrosine-kinase receptors. It was discovered by comparing microarrays of cellular breast cancer vaccines highly enriched for cells that induced breast cancer immunity in tumor-bearing mice, with non enriched vaccines.

The vaccines were prepared by transferring a cDNA expression library derived from SB5b cells, a breast cancer cell line (C3H/He origin (H-2k), into LM mouse fibroblasts (H-2k). As the transferred cDNA integrates spontaneously into the genome of the recipient cells, replicates as the cells divide, and is expressed, the vaccine could be prepared from microgram amounts of tumor tissue. Relatively few cells in the transduced cell-population, however, incorporated cDNA-fragments that included genes specifying TAA. (The vast majority specified normal cellular constituents.) A unique strategy was employed, therefore, to enrich the vaccine for immunotherapeutic cells. Twenty genes were over-represented in the enriched vaccines. One, the gene for Grb10, was approximately 100 fold over-represented. To determine if Grb10 in the enriched vaccine was partly responsible for its therapeutic benefits, the gene was transferred into the fibroblast cell line, which was then used as a vaccine. Mice with established breast cancer treated solely by immunization with the modified fibroblasts developed robust immunity to the breast cancer cells, which, in some instances, was sufficient to result in tumor-rejection.
Keywords: cell type comparisons, genetic modifications, transfected cells, fibroblasts, immunity

overall_design:
Experimental animals, tumor cell lines and monoclonal antibodies (mAbs). Pathogen-free C3H/He female mice (H-2k) between 10 to 14 weeks old were from the Jackson Laboratory (Bar Harbor, ME). They were maintained according to NIH Guidelines for the Care and Use of Laboratory Animals. SB5b cells were a breast cancer cell line established from an adenocarcinoma that arose spontaneously in the mammary gland of a C3H/He mouse in our animal colony. LM fibroblasts, of C3H/He mouse origin, were from the American Type Culture Collection (ATCC). B16F1 cells, a melanoma cell line of C57BL/6 mouse origin (H-2b), were from the ATCC. Each of the cell types was maintained at 370 in a humidified 7% CO2/air atmosphere in DMEM (Gibco BRL, Grand Island, NY) supplemented with 10% heat inactivated fetal bovine serum (FBS) and antibiotics (Gibco BRL) (growth medium). mAbs for CD4+, CD8+ and NK1.1 cells and fluoroscein-conjugated mAbs for H-2Kb class I-determinants were from B-D Pharmingen (San Jose, CA).

Modification of LM fibroblasts to secrete IL-2. The fibroblasts were modified to secrete IL-2 before transfection (LM-IL-2 cells) as a means of augmenting their non-specific immunogenic properties, as described previously (30).

Modification of the cytokine-secreting fibroblasts to express H-2Kb-class I-determinants, allogeneic in C3H/He mice.  Allogeneic class I-determinants are strong immune adjuvants (32,33). To stimulate up take of the vaccine by dendritic cells of the tumor-bearing host, and to ensure rejection, the fibroblasts (H-2k) were modified to express H-2Kb-determinants, allogeneic in C3H/He mice (LM-IL-2Kb cells), as described previously (34, 35).

Preparation of cDNA libraries from SB5b breast cancer cells. Guanidine isothiocyanate was used to recover total RNA from SB5b cells or from B16F1 melanoma cells.  The mRNA, derived from approximately 1 x 107 cells, was isolated using an mRNA isolation system (Promega, Madison, WI). The cDNA-expression libraries were constructed with a Lambda Zap vector using a cDNA library kit (Stratgene, La Jolla, CA). cDNAs greater than 0.5 kb were selected by size fractionation via gel filtration and directionally cloned into a pBK-CMV vector with an EcoRI restriction site at the 5’ end and an XhoI site at the 3’ end. The expression libraries yielded approximately 4 x 105 PFU/μg cDNA with an individual cDNA insert. The size distribution of the cDNA transduced into the modified fibroblasts was 0.5-7.0 kb.

Preparation of the cDNA-based cellular vaccines. LM-IL-2Kb cells were transduced with a cDNA library from SB5b cells, or, for use as a specificity control, with a cDNA library from B16F1 cells, using Lipfectamine 2000 (Invitrogen, Calsbad, CA) to aid cDNA uptake. In brief, 30 μg of cDNA from either of the cell types were mixed with 3 μg pcDNA6/Bla (Invitrogen), a plasmid specifying a gene conferring resistance to blasticidin, used for selection. Afterward, the cDNA/pcDNA6/Bla mixture was added to Lipfectamine 2000 and then to 2.0 x 107 LM-IL-2Kb cells divided into four 100 mm plastic cell culture dishes 24 hours previously. After incubation for 18 hours, the cells were divided into sixteen 100 mm dishes, and incubated for 14 days in fresh growth medium containing 5 ug/ml blasticidin and 500 ug/ml G418. The surviving blasticin/G418-resistant cells (at least 2 x 106 colonies) were pooled and maintained as cell lines for use in the experiments (LM-IL-2Kb/cSB5b and LM-IL-2Kb/B16F1 cells respectively). For use as a control, the same procedure was followed except that the fibroblasts were transduced with pcDNA/Bla alone (LM-IL-2Kb cells).

Mouse interferon gamma (IFN-γ) ELISPOT IFN-γ assays. Mouse ELISPOT IFN-γ assays were used to determine the number of responding T cells in mice immunized with the transduced fibroblasts. The spots were counted by computer-assisted image analysis (ImmunoSpot Series 2 analyzer: Cellular Technology Limited, Cleveland, OH).

51Cr-release cytotoxicity assays. Mononuclear cells from the spleens of C3H/He mice immunized with the cDNA-transduced cells, or with cells transduced with a plasmid encoding the gene specifying Grb10, were isolated by Ficoll-Hypaque density gradient centrifugation. After washing, the cells were co cultured at 370 with mitomycin C-treated (45 min, 50 μg/ml) SB5b cells for 5 days (ratio of spleen cells:SB5b cells = 30:1). Afterward, the population that failed to adhere to the plastic cell culture flasks was collected and used as effector cells for the cytotoxicity determinations. Spleen cell mediated cytotoxicity was determined in a standard 51Cr-release assay, using 51Cr-labeled SB5b cells as targets in the reaction.

The percent specific cytolysis was calculated as:

Experimental 51Cr release - Spontaneous 51Cr release
---------------------------------------------------------------X 100
Maximum 51Cr release - Spontaneous 51Cr release

The spontaneous release of 51Cr was less than 15% of the total release in each instance.

Enrichment of the vaccine for cells that induce immunity to (SB5b) mammary carcinoma cells.
The vaccine was prepared by transfer of a cDNA library derived from SB5b cells into the modified mouse fibroblasts. Since only a small proportion of the transduced cell population was expected to have incorporated cDNAs that included genes specifying tumor antigens (TAA), a unique strategy was used to enrich the vaccine for TAA-positive cells, as described previously (30). In brief, aliquots of the suspension of transduced cells were added to each of ten wells of a 96 well plate. Each pool contained a starting inoculum of 1 x 103 cells. Wells containing higher numbers of TAA-positive cells were detected by comparing the response of C3H/He mice to immunization with cells from the individual pools, as determined by both ELISPOT IFN-γ and 51Cr-release cytotoxicity assays. To obtain a sufficient number of cells for immunization, cells from the individual pools were allowed to increase to approximately 5 x 107, through periodic transfers to larger culture plates and eventually cell culture flasks. An aliquot of each of the expanded cell populations was maintained frozen/viable (for later recovery). The remaining portion was used for immunization. Frozen cells derived from the pool that stimulated immunity to the breast cancer cells to the greatest extent (immunohigh), and, for use as a control, from the pool that induced immunity to SB5b cells to the least extent (immunolow), were recovered, reestablished in culture and subjected to additional rounds of positive or negative immune selection (30). (As an additional control, one pool was not subjected to either positive or negative selection (master pool)). After five rounds of selection, microarrays were used to compare the gene expression profiles of cells in the immunohigh and immunolow pools.

Microarrays of cellular vaccines enriched for transduced fibroblasts that induced immunity to SB5b cells.
cRNA microarrays were used to the compare the gene expression profiles of transduced fibroblasts from the immunohigh and immunolow pools, as described previously (36).

RT-PCR of Grb10, a candidate gene specifying a breast cancer antigen, identified by comparing microarrays of the enriched and non enriched vaccines.
Grb10 was highly over represented in cells from immunohigh pools. RT-PCR was used to determine if the gene was expressed. Approximately 6 x 106 cells from the immunohigh pool in monolayer culture were disrupted and homogenized. 1 volume of 70% ethanol was added before the extracts were loaded onto RNeasy mini columns. RT-PCR was performed on RNA eluted from the column with a one-step RT-PCR kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. 1 ug RNA was mixed with buffer containing 1.25 mM MgCl2, 40uM dNTPs, 0.6 μM of each forward and backward primers and 2 μl of a mixture containing reverse transcriptase and Taq polymerase.

first: InSug

last: O-Sullivan

first: Amla

last: Chopra

first: Janai

last: Carr

first: TaeSung

last: Kim

first: Edward

middle: P

last: Cohen


title: Mutant gshF peroxide treatment

city: Wageningen

first: L.

middle: Mariela

last: Serrano

address: Nieuwe Kanaal 9A

institute: TI Food and Nutrition

summary:
The goal of this study is to characterize a gshF::cam L. plantarum strain NZ7608. From phenotype characterization we have observed that the gene gshF plays a role in hydrogen peroxide resistance in L. plantarum and therefore we wanted to analyze the global transcriptome response of strain NZ7608 towards peroxide and compared this to wild type response. In this experiment we compared two L. plantarum strains: WCFS1 (wild type) and NZ7618 (gshF::cam). Both strains were grown -in duplo- on CDM containing 0.5% glucose at 37 degrees celsius. When cell density reached OD600=1.0 a hydrogen peroxide stress was given to the cultures to a final concentration of 10mM. Samples were taken at times (0 10 and 30 minutes) upon hydrogen peroxide. All withdrawn samples were used for this microarray experiment.
Keywords: peroxide, gshF mutant

overall_design: Per array two cDNA labeled targets were hybridized on custom designed L. plantarum WCFS1 11K Agilent oligo microarrays (GEO Acc. Nr. GPL5874) using the Agilent 60-mer oligo microarray processing protocol version 4.1. An oligo-microarray contained an average of 3 probes per transcript. Dried slides were scanned in the Scan Array Express (PerkinElmer Life Sciences; Packard Bioscience) for both Cy3 and Cy5 at 10 microns and a PMT Gain between 40-50%.  Spot intensity data was normalized and quantified (average intensity) using a customized grid in ImaGene (BioDiscovery, Inc.). Signal intensities of all probes were corrected against background in BioArray Software Environment (BASE). The ratio of each probe (M) was defined as log [cy5 intensity/cy3 intensity] where cy3 is the wild type. Fold change (FC) is defined as 2M. For the statistical analysis we used Linear Models for Microarray Analysis (Limma) (R/limma).  Significant transcripts were defined as transcripts with a False Discovery Rate (FDR) adjusted for the pvalues of less than 1% and FC = 1.5

first: L

first: Douwe

last: Molenaar

first: Bas

last: Teusink

first: Willem

last: de Vos

first: Eddy

middle: J

last: Smid


title: The genome of Streptomyces rimosus shows a novel structure compared to other Streptomyces

first: Ralph

last: Kirby

address: Li-Nong Road

institute: National Yang-Ming University

summary:
This study compared the genome of Streptomyces rimosus rimosus against that of Streptomyces coelicolor. It also compared 4 strains with changes in oxytetracycline production and derived from G7, the type strain, against G7.
Keywords: Comparative genomic hybridization

sample_organism:
Streptomyces rimosus
Streptomyces coelicolor A3(2)

overall_design: Duplicate samples, each replicate may be found in the corresponding supplementary file for the Sample

first: Gan

last: Teng-Kuei

first: Emma

last: Tilley

first: Paul

last: Herron

first: Iain

last: Hunter


title: Profiling the transcriptome of Thalassiosira pseudonana under silicon replete and deplete growth.

first: E.

middle: Virginia

last: Armbrust

address: Ben Hall IRB, 616 NE Northlake Pl, Room 306

department: Oceanography

institute: University of Washington

summary:
T. pseudonana tiling arrays were used to validate gene models and to predict new genes in the genome of this diatom. The tiling array data validated transcription of about 41% (4,653) of the 11,390 computationally predicted genes. An additional 1,132 transcripts were identified that did not correspond to modeled genes with few of these transcripts (<17%) predicted to encode proteins with homology (e-value < 10-05) to publicly available proteins. These newly identified transcripts have an average length of 1,549 bp, comparable to the average length of the computationally derived genes. Whole genome tiling arrays were conducted under silicon replete and deplete growth conditions to identify new genes involved in the synthesis of the diatom silica cell wall.
Keywords: silicon, stress response, cell wall

overall_design: We analyzed 2 sets of arrays (8 arrays each): one from growth under silicon replete and another one from growth under silicon deplete conditions.

first: Thomas

last: Mock

first: Manoj

last: Samanta

first: Vaughn

last: Iverson

first: Chris

last: Berthiaume

first: Matthew

last: Robison

last: Rodesch

first: Karie

last: Holtermann

first: Colleen

last: Durkin

first: Sandra

last: SplinterBonDurant

first: Kathryn

last: Richmond

first: Toivo

last: Kallas

last: Huttlin

first: Francesco

last: Cerrina

first: Michael

last: Sussman

first: Elizabeth

middle: V


title: Rat liver: Caloric Restriction vs. Fasting vs. COntrol

first: Matthieu

last: Arvier

summary:
Transcriptional profiling of rat liver after a three days caloric restriction or 3 days starvation.

In the present study, we wanted to investigate the expression of multiple genes linked to pathways of energy metabolism. We developed a metabolism dedicated microarray tool: the Mitoligo. This microarray gathers genes linked to the main functions of energy metabolism, such as glycolysis or fatty acid oxidation. This biochip has also been designed to study the expression of genes encoding for all the subunits of proteins constituting the ETC. Eventually, genes coding for protein linked to ROS scavenging systems can also be studied using the Mitoligo.
Keywords: Nutritional intervention

overall_design:
The present investigation was performed according to the guiding principles of the French department of Animal and Environmental protection for the care and use of animals and in accordance with the American Physiological Society “Guiding Principles for Research Involving Animals and Human Beings”. Twenty-four male Sprague-Dawley rats, born and bred in our animal facilities, were housed in individual cages at 12 weeks of age (370-420g). Animals were provided with water ad libitum and a standard diet (U.A.R A04) consisting (by weight) of 16% protein, 3% fat, 60% carbohydrate and 21% water, fiber, vitamins and minerals. The metabolizable energy content was 2.9 kcal/g. Rats were randomly separated into 3 groups as follows: control fed (CON) group (n=6), the calorie restricted (CR) group and the 100% calorie restricted (fasted) group. The rats in the CON group consumed the standard diet ad libitum while the other rats were food-restricted and received 25 and 50% (CR group) of the food given to the control group. The rats in the fested group were totally deprivated of feeding. The experimental design was conducted for 3 days. On the 4th day, the animals were killed by decapitation. Liver was removed rapidly and weighed. Some tissue samples were immediately used for respiration and potential membrane measurements and the remainder were frozen in liquid nitrogen and stored at -80°C in order to measure enzyme activity levels and gene expression levels.

Total RNA was isolated from tissue samples using a standard guanidium isothiocyanate protocol (TRIzol reagent, Invitrogen). RNA integrity was determined using a BioAnalyzer 2100 (Agilent Technologies, Waldbronn, Germany).
	Cy3- and Cy5-labelled (amplified) aRNA was prepared using the Amino Allyl MessageAmpII aRNA Amplification Kit (Ambion, 1753). The reference sample consisted of a pool of an equal amount of aRNA obtained after the amplification of tRNA from each non-restricted control rat and was labelled with Cy5 (Amersham). For each of different rat within each group (including control group), two replicate tRNA extractions were prepared using the Amino Allyl MessageAmpII aRNA Amplification Kit (Ambion, 1753) and  labeled individually with Cy3 (Amersham). Each Cy3-labeled sample was mixed with an equal amount of the Cy5- labeled reference sample, pre-incubated with yeast tRNA and polyA RNA, and hybridized to the microarrays.

Microarrays were prepared in-house (Plateforme Puces à ADN-Ouest Génopole, Nantes, France) using 50-mer oligonucléotides probes. The probes were diluted in spotting buffer A (25 μM in buffer A 1x, Ocimum) and spotted onto epoxysilane-coated glass slides (Slide E, Schott nexterion) using the Lucidea Array Spotter from Amersham. Immediately after spotting, the slides were incubated at 42°C and 55% humidity for 12 h. Slides were washed once in 0.2% SDS and twice in H2O and then incubated for 20 minutes in H2O at 50°C, dried by centrifugation (1000 rpm, 3 minutes, 20°C), and stored at 20°C.
	The 982 genes that were represented on the microarray were selected for involvement in mitochondrial metabolism. 350 genes are specific for rat while 600 genes are specific of human mRNA. Each gene was spotted in triplicate on the microarrays. Hybridized arrays were scanned at 10 μm/pixel resolution by fluorescence confocal microscopy (Scanarray 3000, GSI-Lumonics). Signal intensities were extracted with Genepix Pro 5.1.0.19 image analysis software (Axon Instruments, USA). Data were retained based on concordance between the measurements of different types of replicates: biological replicates, technical replicates and combined biological and technical replicates.

Microarray raw data were analyzed using Genespring GX 7.0 (Agilent technologies). Data were normalized using Lowess normalization. Normalized data were compared using ANOVA followed by tukey or student t-test.

first: Jean-François

last: Dumas

first: Yves

last: Malthièry

first: Gilles

last: Simard

first: Patrick

last: Ritz

first: Catherine

last: Chevallier

first: Jean

last: Leger


title: DH06 Adrenal ACTH Sus scrofa

status: Public on Jan 07 2008

city: Bordeaux

first: Dominique

last: Hazard

address: Institut Francois Magendie 146 rue Léo Saignat

department: Animal Genetic

institute: INRA

summary:
Large White and Meishan pigs were either non-treated or injected with mammalian 1-24 ACTH (Immediate Synachten, Novartis France) at the dose of 250 µg per animal. Pigs were sacrificed either immediately after capture from their home cage (non-treated animals) or 1 hour following ACTH injection. Adrenal glands were immediately collected from pigs and frozen on dry ice and then stored at -80°C until RNA isolation.
Keywords: stress response, adrenal, gene expression, pig

overall_design: 47 samples


title: Maintenance of Long-Range DNA Interactions after Inhibition of Ongoing RNA Polymerase II Transcription.

city: Rotterdam

first: Wouter

last: de Laat

address: Dr. Molewaterplein 50

department: Cell Biology

institute: Erasmus MC

summary:
A relationship exists between nuclear architecture and gene activity and it has been proposed that the activity of ongoing RNA polymerase II transcription determines genome organization in the mammalian cell nucleus. Recently developed 3C and 4C technology allowed us to test the importance of transcription for nuclear architecture. We demonstrate that upon transcription inhibition binding of RNA polymerase II to gene regulatory elements is severely reduced. However, contacts between regulatory DNA elements and genes in the β-globin locus are unaffected and the locus still interacts with the same genomic regions elsewhere on the chromosome. This is a general phenomenon since the great majority of intra- and interchromosomal interactions with the ubiquitously expressed Rad23a gene are also not affected. Our data demonstrate that without transcription the organization and modification of nucleosomes at active loci and the local binding of specific trans-acting factors is unaltered. We propose that these parameters, more than transcription or RNA polymerase II binding, determine the maintenance of long-range DNA interactions.
Keywords: transcription inhibition 4C

overall_design: 4C experiments were performed in duplo, dye swap was included. Hybridisation was performed on dedicated micro-arrays. Probes (60-mers) were selected from the sequences 100 bp up –and downstream of HindIII sites. To prevent cross-hybridization, probes that had any similarity with highly abundant repeats (RepBase 10.09) were removed from the probeset. In addition, probes that gave more than two BLAST hits in the genome were also removed from the probeset. Sequence alignments were performed using MegaBLAST using the standard settings. A hit was defined as an alignment of 30 nt or longer.

first: Robert-Jan

last: Palstra

first: Marieke

last: Simonis

first: Petra

last: Klous

first: Emilie

last: Brasset

first: Bart

last: Eijkelkamp


title: Analysis of CNVs in the rhesus macaque genome identifies candidate loci for evolutionary and human disease studies

city: Boston

first: Arthur

middle: S

last: Lee

address: 221 Longwood Ave.

institute: Harvard Medical School

summary:
Copy number variants (CNVs) are heritable gains and losses of genomic DNA in normal individuals. While copy number variation is widely studied in humans, our knowledge of CNVs in other mammalian species is more limited. We have designed a custom array-based comparative genomic hybridization (aCGH) platform with 385,000 oligonucleotide probes based on the reference genome sequence of the rhesus macaque (Macaca mulatta), the most widely studied non-human primate in biomedical research. We used this platform to identify 123 CNVs among 10 unrelated macaque individuals, with 24% of the CNVs observed in multiple individuals. We found that segmental duplications were significantly enriched at macaque CNV loci. We also observed significant overlap between rhesus macaque and human CNVs, suggesting that certain genomic regions are prone to recurrent CNV formation and instability, even across a total of ~50 million years of primate evolution (~25 million years in each lineage). Furthermore, for 8 of the CNVs that were observed in both humans and macaques, previous human studies have reported a relationship between copy number and gene expression or disease susceptibility. Therefore, the rhesus macaque offers an intriguing, non-human primate model organism for which hypotheses concerning the specific functions of phenotypically-relevant human CNVs can be tested.
Keywords: array-based comparative genomic hybridization, oligonucleotide probes

overall_design: We performed array-based comparative genomic hybridization using a 385,000 probe custom oligonucleotide platform based on the rhesus macaque reference genome (rheMac2) on 10 unrelated rhesus macaques (9 test individuals vs. 1 reference). As a control, we performed 2 self-self hybridization experiments using the single female reference sample as well as a single male sample from the 9 test individuals.


title: Polymorphism and Regulation of the spxB (Pyruvate Oxidase) Virulence Factor Gene in Streptococcus pneumoniae

status: Public on Jan 04 2008

city: Bloomington

first: Ho-Ching

middle: Tiffany

last: Tsui

state: IN

address: 1001 E. Third St.

department: Biology

institute: Indiana University

summary:
spxB-encoded pyruvate oxidase is a major virulence factor of Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes H2O2 and acetyl phosphate, which play roles in metabolism, signaling, and oxidative stress. We report here the first cis- and trans-acting regulatory elements for spxB transcription. These elements were identified in a genetic screen for spontaneous mutations that caused colonies of strain D39 to change from a semi-transparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency 3x10-5) were impaired for SpxB function or expression. Two mutations changed amino acids in SpxB likely required for cofactor or subunit binding. One mutation defined a cis-acting adjacent direct repeat required for optimal spxB transcription. The other three spontaneous mutations created the same frameshift near the start of the trans-acting spxR regulatory gene. The SpxR protein contains helix-turn-helix, CBS, and HotDog domains implicated in binding DNA, adenosyl compounds, and CoA-containing compounds, respectively, and suggest that SpxR positively regulates spxB transcription in response to energy and metabolic state. Finally, microarray analyses of a spxR mutant demonstrated that SpxR positively regulates the strH exoglycosidase gene, which like spxB, has been implicated in colonization.
Keywords: bacterial genetic modification

overall_design: Microarray analyses were performed comparing relative transcript amounts for strains D39 (IU1690) with D39(IU1690) spxR6 (Table 2). Total RNA was prepared from bacteria grown exponentially in CDM broth to OD620 = 0.2 as described previously {Lanie, 2007 #58; Ng, 2005 #35}. S. pneumoniae microarrays were purchased from Ocimum Biosolutions). Samples were collected from three independent biological replicates and included one dye swap. Synthesis, labeling, hybridization, scanning, and analysis using the Cyber-T web interface were performed as described previously {Lanie, 2007 #58; Ng, 2005 #35} with the exception of normalization software used. Data were normalized with web-based BASE (BioArray Software Environment, iubase.cgb.indiana.edu/index/phtml) without background subtraction, excluding empty wells and Arabidopsis controls with the plug-in Lowess Normalization

first: Smirla

last: Ramos-Montanez

first: Kyle

last: Wayne

first: Jordan

last: Morris

first: Lindsey

last: Peters

first: Faming

last: Zhang

first: Krystyna

last: Kazmierczak

first: Lok-To

last: Sham

first: Malcolm

last: Winkler


title: Hypothalamic transcriptional profiling during the development of adiposity

status: Public on Jan 03 2008

city: College Park

first: Tom

last: Porter

state: MD

address: Building #142

department: Animal and Avian Sciences

institute: University of Maryland

summary:
Hypothalami from Fat (FL) and Lean lines (LL) of juvenile broiler chickens were obtained by divergent selection for high or low levels of abdominal fat at SRA-INRA, France. Gene expression patterns were measured during the development of adiposity at 1 to 7 weeks of age. Differentially expressed genes associated with line, age, or line-by-age were identified. Various phenotypic and metabolic alterations are present between the lines (i.e., abdominal fat, T3 levels and glycemia), however there are no alterations in ad libitum food intake between the lines. A balanced block hybrdization design and the Del-Mar 14K chicken integrated systems microarrays were used to measure gene expression during development. 561 genes were differentially expressed between line, and 442 genes were significant for line-by-age interactions. The greatest number of genes were differentially expressed at week 1 of age, prior to any divergence in adiposity between the two lines.
Keywords: Chickens divergently selected for fatness or leanness, transcriptional profiling, differentially expressed genes

overall_design: Four biological replicates were used for each genotype (FL and LL) at four different ages (weeks 1, 3, 5 and 7). 20 micrograms of total RNA from each sample were analyzed using DEL-MAR 14K integrated systems microarrays in a reference design. Each experimental sample was labeled with Cy3 and then hybridized with an aliquot of the reference pool (Cy5). The reference pool of hypothalamic total RNA was generated from an equal aliquot of all the RNA samples (fat and lean lines, week 1, 3, 5, and 7).

first: M

last: Byerly

first: T


title: Stress induced gene expression profiling in Penaeus monodon

city: Charleston

first: Enrique

last: de la Vega

state: SC

address: 331 Fort Johnson Road

department: Biochemistry

institute: Medical University of South Carolina

summary:
Cultured shrimp are continuously exposed to variable environmental conditions which are associated with stress and subsequent outbreaks of disease. To investigate the effect of environmental stress on Penaeus monodon gene expression, a 3853 random cDNA microarray chip was generated with clones originating from 6 stress-enriched hemocyte libraries generated by suppression subtractive hybridization and a normal hemocyte cDNA library. Changes in temporal gene expression were analyzed from shrimp exposed to hypoxic, hyperthermic and hypoosmotic conditions. 3.1% of the cDNAs were differentially expressed in response to at least one of the environmental stressors. 70% of the differentially expressed clones had no significant sequence similarity to previously known genes. Among those genes with high identity to known sequences, the most common functional groups were immune related genes and non-LTR retrotransposons. Hierarchical clustering revealed a set of cDNAs with temporal and stressspecific gene expression profiles as well as a set of cDNAs indicating a common stress response between stressors. Hypoxic and hyperthermic stressors induced the most severe short-term response in terms of gene regulation, and the osmotic stress had the least variation in expression profiles relative to the control. These expression data agree with observed differences in shrimp physical appearance and behavior following exposure to stress conditions.

Keywords: stress response, shrimp, time series

overall_design: An even loop hybridization design with 2 independent (replicate) loops and 12 slides per loop was used to perform a time series comparison of the gene expression profiles in juvenile shrimp subject to different types of stress. Both loops contained different pooled (3 separate shrimp) amplified RNA sample sets to provide biological replication and estimation of population variability. Being a balanced loop design, all samples were labelled with both Cy3 and Cy5. Further information on the design, refer to: (Kerr MK.  Biometrics 59: 822-828, 2003).

first: Michael

last: Hall

first: Kate

last: Wilson

first: Antonio

last: Reverter

first: Rick

middle: G

last: Woods

first: Bernard

last: Degnan


title: Alteration of microRNA gene expression by EBV encoded LMP1 oncogene

city: New Orleans

first: Erik

middle: K

last: Flemington

state: LA

address: 1430 Tulane Ave., SL79

department: Pathology

institute: Tulane Health Sciences Center

summary:
The Epstein Barr virus (EBV) encoded latent membrane protein-1 (LMP1) is a functional homologue of the tumor necrosis factor receptor family and contributes substantially to the oncogenic potential of EBV through activation of Nuclear Factor-kappaB (NF-kappaB). MicroRNAs (miRNAs) are a class of small RNA molecules that are involved in the regulation of cellular processes such as growth, development, and apoptosis, and have recently been linked to cancer phenotypes. Through miRNA microarray analysis, we demonstrate that LMP1 dysregulates the expression of several cellular miRNAs, including the most highly regulated of these, miR-146a. Quantitative RT-PCR analysis confirmed induced expression of miR-146a by LMP1. Analysis of miR-146a expression in EBV latency type III and type I cell lines revealed substantial expression of miR-146a in type III (which express LMP1) but not in type I cell lines. Reporter studies demonstrated that LMP1 induces miR-146a predominantly through two NF-kappaB binding sites in the miR-146a promoter and identified a role for an OCT-1 site in conferring basal and induced expression. Array analysis of cellular mRNAs expressed in Akata cells transduced with an miR-146a expressing retrovirus identified genes that are directly or indirectly regulated by miR-146a, including a group of interferon responsive genes that are inhibited by miR-146a. Since miR-146a is known to be induced by agents that activate the interferon response pathway (including LMP1), these results suggest that miR-146a functions in a negative feedback loop to modulate the intensity and/or duration of the interferon response.
Keywords: microRNA expression modified by EBV encoded oncogene, LMP1

overall_design: EBV negative Mutu E1dn Cl. 3 cells were transduced with either a control retrovirus (pEhyg) or an LMP1 containing retrovirus (pEhyg-FLAG-LMP1).  Cells were selected with hygromycin for approximately 2 weeks afterwhich 5 pairs of RNA preps were carried out on 5 successive days.  Arrays GSM255656 and GSM255659 are dye swaps in which LMP1 samples were labeled with Cy3 and control samples were labeled with Cy5.

first: Jennifer

last: Cameron

first: Qinyan

last: Yin

first: Claire

last: Fewell

first: Michelle

last: Lacey

first: Jane

last: McBride

first: Xia

last: Wang

first: Zhen

last: Lin

first: Brian

middle: C

last: Schaefer


title: Allele-Specific Expression 2

status: Public on Jan 05 2008

city: Baltimore

first: Aik Choon

last: Tan

address: 1650 Orleans Street, RM 1M86

department: The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins

institute: Johns Hopkins University

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

summary:
Physiologic allele-specific expression (ASE) in germline tissues occurs during random X-chromosome inactivation and in genomic imprinting, wherein the two alleles of a gene in a heterozygous individual are not expressed equally. Recent studies have confirmed the existence of ASE in apparently non-imprinted autosomal genes; however, the extent of ASE in the human genome is unknown.  We explored ASE in lymphoblastoid cell lines of 145 individuals using an oligonucleotide array based assay.  ASE of autosomal genes was found to be a very common phenomenon in ~20% of heterozygotes at 78% of SNPs at 84% of the genes examined.  Comparison of 100 affected individuals from familial pancreatic cancer kindreds and 45 controls revealed three types of changes in the germline: (a) loss of ASE, (b) gain of ASE, and, (c) rare instances of "extreme" (near monoallelic) ASE. The latter changes identified heterozygous deleterious mutations in a subset of these genes.  Consequently, an ASE assay efficiently identifies candidate disease genes with altered germline expression properties as compared to controls, and provides insights into mechanisms that confer disease risk as for pancreatic cancer.
Keywords: allele specific expression

overall_design:
The goal of this study is to identify genes showing allele-specific expression (ASE) in familial pancreatic cancer vs normal germline.

Please see readme.pdf for more details.

first: Aik


title: Profiling the transcriptome of Thalassiosira pseudonana under environmentally relevant growth conditions

city: Seattle

first: E.

last: Armbrust

summary:
T. pseudonana gene-specific arrays were used for differential gene expression. Seven hundred nine genes were differentially expressed by more than 2-fold (Bayesian t-test, p< 0.001) under at least one growth-limiting condition relative to nutrient-replete conditions. A striking result of the hierarchical cluster analysis was the identification of a common set of genes that were upregulated by both iron and silicon limitation, but no other treatment. Together, these two treatments accounted for about one fourth of all differentially expressed genes but almost two thirds of the differentially expressed novel genes, further emphasizing the distinctive aspects of silicon manipulation in diatoms.
Keywords: silicon, stress response, cell wall, iron, temperature, carbon dioxide

overall_design: We analyzed 6 different sets of arrays from 6 growth conditions. Four biological replicates (=4 arrays) were used for each growth condition.

first: Thomas

last: Mock

first: Manoj

last: Samanta

first: Vaughn

last: Iverson

first: Chris

last: Berthiaume

first: Matthew

last: Robison

last: Rodesch

first: Karie

last: Holtermann

first: Colleen

last: Durkin

first: Sandra

last: SplinterBonDurant

first: Kathryn

last: Richmond

first: Toivo

last: Kallas

first: Edward

last: Huttlin

first: Francesco

last: Cerrina

middle: R

last: Sussman

first: Elizabeth


title: Complete Gene Expression Profiling of Saccharopolyspora erythraea NRRL 2338

status: Public on Jan 02 2008

city: Segrate - Milan

first: Giorgio

last: Corti

address: via Fratelli Cervi 93

department: Institute of Biomedical Technologies

country: Italy

institute: National Research Council

summary:
A DNA microarray was designed and constructed using the genome sequence of Saccharopolyspora erythraea strain NRRL 2338. Following growth in liquid medium, we analysed the expression of 6494 ORFs along the time course. The results indicated that the 404 genes, whose expression significatively correlated with the time course, identify three distinct growth phases: a rapid growth until 32 h (phase A); a growth slowdown until 52 h (phase B); another rapid growth phase from 56 h to 72 h (phase C) before entering the stationary phase. We experimentally determined regional organization of transcription along the chromosome, highlighting differences between core and non-core regions, but also strand specific patterns of expression, and the behavior of major functional classes. Temporal expression of all the gene clusters for secondary metabolism was analyzed, confirming ery cluster up-regulation during the first growth phase, and finding out six secondary metabolism clusters that are clearly regulated during growth. The use of a DNA microarray, specifically designed on the Sac. erythraea genome sequence, improved specificity and sensitivity of gene expression analysis, giving a global and at the same time detailed picture of how Sac. erythraea genes are modulated.
Keywords: time course

overall_design: Spores of S. erythraea strain NRRL2338 were inoculated into SCM liquid medium. Samples were withdrawn at different time points: after 12h, 16h, 20h, 32h, 36h, 40h, 52h, 56h, 60h and 72h from the initial inoculum. Erythromycin production and wet cell weight were determined. RNA samples were extracted from two independent cultures, processed and hybridized to custom made GeneChips containing DNA oligonucleotide probes corresponding to 6494 predicted Sac. erythaea ORFs.

first: C

last: Peano

first: S

last: Bicciato

first: G

first: F

last: Ferrari

first: E

last: Rizzi

first: RJ

last: Bonnal

first: R

last: Bordoni

first: A

last: Albertini

first: L

last: Rossi Bernardi

last: Donadio

last: De Bellis


title: Allele-Specific Expression 1

first: Aik Choon

last: Tan

summary:
Physiologic allele-specific expression (ASE) in germline tissues occurs during random X-chromosome inactivation and in genomic imprinting, wherein the two alleles of a gene in a heterozygous individual are not expressed equally. Recent studies have confirmed the existence of ASE in apparently non-imprinted autosomal genes; however, the extent of ASE in the human genome is unknown.  We explored ASE in lymphoblastoid cell lines of 145 individuals using an oligonucleotide array based assay.  ASE of autosomal genes was found to be a very common phenomenon in ~20% of heterozygotes at 78% of SNPs at 84% of the genes examined.  Comparison of 100 affected individuals from familial pancreatic cancer kindreds and 45 controls revealed three types of changes in the germline: (a) loss of ASE, (b) gain of ASE, and, (c) rare instances of "extreme" (near monoallelic) ASE. The latter changes identified heterozygous deleterious mutations in a subset of these genes.  Consequently, an ASE assay efficiently identifies candidate disease genes with altered germline expression properties as compared to controls, and provides insights into mechanisms that confer disease risk as for pancreatic cancer.
Keywords: allele-specific expression

first: Aik


title: Gene expression response to the antifungal compound sampangine in C. albicans

city: University

first: Ameeta

last: Agarwal

state: MS

address: NCNPR, Room 2049

department: National Center for Natural Products Research

institute: University of Mississippi

summary:
Sampangine, a plant-derived alkaloid found in the Annonaceae family, exhibits strong inhibitory activity against the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus.  In the present study, transcriptional profiling experiments coupled with the analysis of mutants were performed in an effort to elucidate its mechanism of action.  Using Saccharomyces cerevisiae as a model organism, we show that sampangine produces a transcriptional response indicative of hypoxia, altering the expression of genes known to respond to low oxygen conditions.  Several additional lines of evidence obtained suggest that these responses could involve effects on heme.   First, the hem1 deletion mutant lacking the first enzyme in the heme biosynthetic pathway showed increased sensitivity to sampangine, and exogenously supplied hemin partially rescued the inhibitory activity of sampangine in wild-type cells.  In addition, heterozygous mutants with deletions in genes involved in five out of eight steps in the heme biosynthetic pathway showed increased susceptibility to sampangine.  Furthermore, spectral analysis of pyridine extracts indicated significant accumulation of free porphyrins in sampangine-treated cells.  Transcriptional profiling experiments were also performed in C. albicans to investigate the response of a pathogenic fungal species to sampangine.  Taking into account the known differences in the physiological responses of C. albicans and S. cerevisiae to low oxygen, significant correlations were observed between the two transcription profiles suggestive of heme-related defects.  Our results indicate that the antifungal activity of the plant alkaloid sampangine is due, at least in part, to perturbations in the biosynthesis or metabolism of heme.
Keywords: antifungal compound, transcriptional profiling, C. albicans

overall_design: C. albicans SC5314 cells at OD 0.2 were treated with either sampangine (SMP) at IC50 concentration (6.13 ug/ml), or solvent (0.25% DMSO), allowed to grow to OD 0.5, then harvested and frozen.  Three biological replicate samples were analyzed for each treatment.

first: Tao

last: Xu

first: Melissa

last: Jacob

first: Qin

last: Feng

first: Michael

last: Lorenz

first: Larry

last: Walker

first: Alice

middle: M

last: Clark


title: Gene expression of GidA mutant Streptococcus pyogenes

city: Carbondale

first: KyuHong

last: Cho

address: 1125 Lincoln Dr. Room 131

department: Microbiology

institute: Southern Illinois University Carbondale

summary:
In Streptococcus pyogenes, mutation of GidA results in avirulence despite the same growth rate as the wild type.  To understand the basis of this effect, global transcription profiling was conducted.
Keywords: Wild type vs. GidA mutant Streptococcus pyogenes

overall_design:
A S. pyogenes genespecific microarray, consisting of 60 to 80-mer oligonucleotides representing 1517 genes out of a total of 1752 predicted open reading frames in the SF370 genome, was constructed. S. pyogenes wild type strain (HSC5) or TM-GidA2 strain (GidA mutant derived form the wild type strain HSC5) were grown in Todd-Hewitt broth supplemented with 0.2% yeast extract (THY medium) to mid-logarithmic phase of growth (OD600=0.3). RNA was extracted, amplified, and hybridized to the arrays.
Each experiment consisted of a pair-wise competitive hybridization of amplified RNA samples from the wild type and GidA mutant derived RNA. Since 2 biological replicates were tested in duplicate with a reciprocal dye-flip, a total of eight DNA microarrays were used for comparison for each set of RNA amplifications.

first: S

middle: D

last: Crosby

last: Caparon


title: Impact of CSN1S1 polymorphism on mammary transcriptome of food-deprived lactating goats

city: St Genes-Champanelle

first: Christine

last: Leroux

address: Centre INRA de Theix

department: PHASE

summary:
Genetic (animal species, breed and genotype) has a considerable effect on milk composition. In particular, goats present a remarkable polymorphism at the alpha-S1-casein (CSN1S1) locus which results in large differences in milk protein content and indirectly in milk fat content and its fatty acids composition. In order to decipher the  mammary metabolic pathways involved, we examined the effect of CSN1S1 polymorphism on the expression of 8,379 genes in caprine mammary gland using a bovine oligonucleotide microarray. Six 48-h food-deprived lactating goats were assigned to 2 groups based on their genotype at the CSN1S1 locus: High vs. Low genotype goats carrying, respectively, two reference alleles associated with high CSN1S1 synthesis and two defective alleles associated with low CSN1S1 synthesis.
Keywords: Genotype comparison

overall_design: 6 slides were performed for a total of 3 independent comparisons (CompA, CompB, CompC): each microarray was co-hybridized with one High CSN1S1 genotype goat sample and one Low CSN1S1 genotype goat sample; each hybridization was repeated in a dye-swap manner for a total of 4 spots per oligonucleotide (2 intra- and 2 inter-slides).

first: Séverine

last: Ollier

first: Stéphanie

last: Chauvet

first: Patrice

last: Martin

first: Yves

last: Chilliard