title: S288c Quiescent and Non-quiescent replicates

summary:
Cells in glucose-limited Saccharomyces cerevisiae cultures differentiate into quiescent (Q) and non-quiescent (NQ) fractions prior to entering stationary phase.  To identify genes involved in this differentiation, Q and NQ cells from 101 deletion-mutant strains were tested for viability and reproductive capacity.  Twenty-one mutants were identified, including 7 that affected reproductive capacity of both cell types.  Thirteen affected only Q or NQ cells, indicating significant differentiation of these cell types.  doa4 strains, lacking ubiquitin hydrolase, affected viability and reproductive capacity in both cell types.  More than 1300 mRNAs differentiating Q and NQ cell fractions were identified by microarray analysis.  Gene-ontology analysis of Q-cell mRNAs showed significant increases in protein-encoding mRNAs involved in membrane maintenance, oxidative stress response, and signal transduction.  NQ-cell mRNAs encode proteins involved in Ty-element transposition and DNA recombination, consistent with apoptosis in these cells.  Consistent with preparation for rapid response to environmental stimuli, approximately 2000 protease-labile mRNAs were identified in Q cells.  The differentiation of these cell types and the ability of genes to selectively affect the survival of Q or NQ cells in yeast are relevant to chronological aging, cell-cycle, genome-evolution, and stem-cell research and provides insight into complex responses that even simple organisms have to starvation.
Keywords: cell type comparison

overall_design: 10 replicates per cell-type. Channel 1 (Cy3) was the control of mixture of exponential and stationary phase cells. Channel 2 (Cy5) was the experimental cell-types of wild-type strain S288c.


title: Proteinase K treatment of Q and NQ cells

status: Public on Dec 27 2007

city: Albuquerque

first: Sushmita

last: Roy

state: NM

country: USA

institute: Biology, University of New Mexico

overall_design: Samples were separeted into Q and NQ fractions using percoll density gradients.  During RNA isolation samples were treated with proteinase K or 10 mM tris.  This was done to test of any differences in portease labile mRNAs in Q and NQ cells.

first: Anthony

middle: D

last: Aragon

first: Maragaret

last: Werner-Washburne


title: Mutant1

summary:
Cells in glucose-limited Saccharomyces cerevisiae cultures differentiate into quiescent (Q) and non-quiescent (NQ) fractions prior to entering stationary phase.  To identify genes involved in this differentiation, Q and NQ cells from 101 deletion-mutant strains were tested for viability and reproductive capacity.  Twenty-one mutants were identified, including 7 that affected reproductive capacity of both cell types.  Thirteen affected only Q or NQ cells, indicating significant differentiation of these cell types.  doa4 strains, lacking ubiquitin hydrolase, affected viability and reproductive capacity in both cell types.  More than 1300 mRNAs differentiating Q and NQ cell fractions were identified by microarray analysis.  Gene-ontology analysis of Q-cell mRNAs showed significant increases in protein-encoding mRNAs involved in membrane maintenance, oxidative stress response, and signal transduction.  NQ-cell mRNAs encode proteins involved in Ty-element transposition and DNA recombination, consistent with apoptosis in these cells.  Consistent with preparation for rapid response to environmental stimuli, approximately 2000 protease-labile mRNAs were identified in Q cells.  The differentiation of these cell types and the ability of genes to selectively affect the survival of Q or NQ cells in yeast are relevant to chronological aging, cell-cycle, genome-evolution, and stem-cell research and provides insight into complex responses that even simple organisms have to starvation.
Keywords: Cell type comparison

overall_design: Yeast deletion mutants (BY4742 background) were grown to stationary-phase (7 days) and then separted into Q and NQ cells using Percoll density gradients.  Microarrays were carried out on these different cell fractions.

first: Margaret


title: Regulation of gene expression by MafA phosphorylation in transformed cells.

status: Public on Dec 25 2007

city: ORSAY Cedex

first: CELIO

last: POUPONNOT

address: Bat 110, Centre Universitaire

department: CNRS UMR 146

country: France

institute: Institut Curie

summary:
Transformation of Chicken Embryo fibroblast by MafA is dependent on its phosphorylation status. Using microarray analysis, we identify a gene expression subprogram regulated by MafA phosphorylation.
Keywords: phosphorylation mediated transcriptional regulation

overall_design:
Total RNA was extracted from Chicken Embryo Fibroblasts expressing control (RCAS), MafA-WT, MafA-4A using RNeasy kit (Qiagen). RNA from two independent cultures for each condition were analyzed and processed by the genomic facility of the Fred Hutchinson Cancer Research Center (Seattle) using a 13K chicken microarray
(http://www.fhcrc.org/science/shared_resources/genomics/dna_array). For one set of sample, dye swap experiments were performed.

first: Nathalie

last: Rocques

first: Alain

last: Eychene

first: Celio

last: Pouponnot


title: BY4742 Quiescent and Non-quiescent replicates

summary:
Cells in glucose-limited Saccharomyces cerevisiae cultures differentiate into quiescent (Q) and non-quiescent (NQ) fractions prior to entering stationary phase.  To identify genes involved in this differentiation, Q and NQ cells from 101 deletion-mutant strains were tested for viability and reproductive capacity.  Twenty-one mutants were identified, including 7 that affected reproductive capacity of both cell types.  Thirteen affected only Q or NQ cells, indicating significant differentiation of these cell types.  doa4 strains, lacking ubiquitin hydrolase, affected viability and reproductive capacity in both cell types.  More than 1300 mRNAs differentiating Q and NQ cell fractions were identified by microarray analysis.  Gene-ontology analysis of Q-cell mRNAs showed significant increases in protein-encoding mRNAs involved in membrane maintenance, oxidative stress response, and signal transduction.  NQ-cell mRNAs encode proteins involved in Ty-element transposition and DNA recombination, consistent with apoptosis in these cells.  Consistent with preparation for rapid response to environmental stimuli, approximately 2000 protease-labile mRNAs were identified in Q cells.  The differentiation of these cell types and the ability of genes to selectively affect the survival of Q or NQ cells in yeast are relevant to chronological aging, cell-cycle, genome-evolution, and stem-cell research and provides insight into complex responses that even simple organisms have to starvation.
Keywords: Cell-type comparison

overall_design: 10 replicates per cell-type. Channel 1 (Cy3) was the control of mixture of exponential and stationary phase cells. Channel 2 (Cy5) was the experimental cell-types of parental strain BY4742.


title: Liver transcript expression patterns in the sparid striped seabream (Lithognathus mormyrus) upon exposure to cadmium

status: Public on Dec 26 2007

city: Haifa

first: Moshe

last: Tom

department: Marine Biology and Biotechnology

country: Israel

institute: Israel Oceanographic and Limnological Research

summary:
Changes in hepatic gene expression profiles upon exposures to cadmium applied by feeding or intra-peritoneal injections were identified in the striped sea bream (Lithognathus mormyrus) using cDNA microarray platform. Two groups of four fish were exposed to fed and injected CdCl2, respectively. Additional eight untreated fish were used as a reference group, pooled into 4 hepatic RNA preparations. The isolated hepatic mRNAs were hybridized, after conversion to labeled cDNAs, onto the microarray followed by slide scanning, imaging and analysis. Four parameters: M, meanA, P-value and B were calculated for each unique spot, for both cadmium-fed vs. reference fish (sample1) and cadmium-injected vs. reference fish (sample2). These parameters enable evaluation of hybridization intensity as well as statistical testing of differential expression.
Keywords: Response to exposure to cadmium

overall_design: Four biological replicate hybridizations were designed, each composed of labeled hepatic cDNAs of three individual fish, each taken from the two experimental conditions and the reference, respectively. The three cDNAs included in each biological replicate, were dual-hybridized in all three possible alternatives, ending with 12 slides. The two dyes were swaped for each pair of biological replicates to reduce putative dye effects. Graphic presentation of the hybridization scheme is provided as supplementary file.

first: Meirav

last: Auslander

first: Yana

last: Yudkovski

first: Vered

last: Chalifa-Caspi

first: Ron

last: Ophir


title: The contribution of different nutrients to spore germination in Saccharomyces cerevisiae

status: Public on Dec 23 2007

city: Rehovot

first: Naama

last: Barkai

address: P.O box 26

department: Molecular Genetics

institute: The Weizmann Institute of Science

summary:
To study the signals and pathways underlying spore germination we examined the global changes in gene expression during this process.
We find that the germination process can be divided into two distinct stages. During the first stage, the induced spores respond only to glucose. The transcription program during this stage recapitulates the general transcription response of yeast cells to glucose. Only during the second phase are the cells able to sense and respond to other nutritional components in the environment. Components of the mitotic machinery are involved in spore germination but in a distinct pattern. In contrast to the mitotic cell cycle, growth related events during germination are not coordinated with nuclear events and are separately regulated.
Genome-wide expression profiling enables us to follow the progression of spore germination, thus dividing this process into two major stages and to identify germination-specific regulation of components of the mitotic cell cycle machinery.
Keywords: Time course

overall_design: Wild type SK1 cells (DS1) were grown to saturation in YPD (2% yeast extract, 4% bactopeptone, 4% glucose) at 30 C for 24 hours, washed in sterile water and plated on sporulation plates (0.25% yeast extract, 1.5% Potassium acetate, 0.05% glucose supplemented with all amino acids).  3 days old asci were harvested and suspended in either 2% glucose or synthetic minimal medium without glucose (0.17% yeast nitrogen base,0.5% ammonium sulfate, supplemented with amino acids). RNA for microarray hybridization was extracted at the indicated times (23 samples). Following two hours of incubation part of the culture was transferred to rich medium (YPD) to allow spore germination and RNA for microarray hybridization was extracted at the indicated times (15 samples).

first: D

last: Joseph-Strauss

first: N

first: G

last: Simchen


title: Differential Gene Expression Between Brown Norway and Sprague Dawley Rats in Response to Renal Ischemia Reperfusion

status: Public on Dec 28 2007

city: Barcelona

first: Jose Luis

last: Viñas

address: rosellon 161 7ª

department: Experimental Pathology

country: Spain

institute: IIBB-CSIC

summary:
Renal ischemia reperfusion injury (I/R) has great clinical relevance and many treatments and hospitalizations are associated with this pathological process. Animal models are widely used in the development of kidney I/R studies and numerous papers have been published in this field in recent years suggesting that the nature of the animal species used in these models can lead to a different response to ischemic insult.
The Brown Norway (BN) rat strain is known to show endogenous resistance to the ischemic process and several studies have sought to identify the cell mechanism present in this resistance. Despite of all the previously reported works, little is know about the genetic response of BN in front of ischemic insult.
By means of a microarray comparative genetic study between BN and the ischemia susceptible rat strain Sprague Dawley (SD), the present work determine the genetic response to the I/R process in both rat strains and the genes that are differentially expressed during the I/R process and link them with some of the main cytoprotective processes responsible for the endogenous resistance to renal ischemic injury in BN rats.
Keywords: comparative genomics hybridization

overall_design:
The following experimental groups for BN and SD rats  (n=3 per group) were studied:
Group 1: Control (C): Animals subjected to identical maneuvers as the I/R group except that the renal pedicles were not clamped.
Group 2: Ischemia/Reperfusion (I/R): Animals were subjected to 45 min of bilateral ischemia and 24 h of reperfusion.
Gene expression profiles were obtained from control and I/R groups in the two rat strains in three biological replicates, resulting in 12 hybridizations from 2 groups × 2 strains × 3biological replicates. To evaluate the technical reliability of microarrays, replicate microarrays were performed on several RNA samples. In total, 12 hybridizations were performed for this study.

first: Jose

middle: L

first: Anna

last: Sola

first: Georgina

last: Hotter


title: Spore Germination in Saccharomyces cerevisiae : transfer of wild type spores to rich (YPD) medium

overall_design: Wild type SK1 cells (DS1) were grown to saturation in YPD (2% yeast extract, 4% bactopeptone, 4% glucose) at 30 C for 24 hours, washed in sterile water and plated on sporulation plates (0.25% yeast extract, 1.5% Potassium acetate, 0.05% glucose supplemented with all amino acids).  3 days old asci were harvested and suspended in YPD at 30 C to initiate spore germination. RNA for microarray hybridization was extracted at high temporal resolution (15-minutes intervals for 7.5 hours, total of 31 samples)


title: Pseudomonas aeruginosa lasR mutants in young cystic fibrosis patients

status: Public on Dec 22 2007

city: Seattle

first: David

last: Dargenio

state: WA

department: Microbiology

institute: University of Washington

summary:
Pseudomonas aeruginosa undergoes genetic change during chronic infection of the airways of cystic fibrosis (CF) patients.  One common change is mutation of lasR.  LasR is a transcriptional regulator that responds to one of the quorum sensing signals in P. aeruginosa, and regulates acute virulence factor expression as well as central metabolic functions.  P. aeruginosa mutants in which lasR was inactivated emerged in the airways of CF patients early during chronic infection, and during growth in the laboratory on Luria-Bertani agar.  Both environments are rich in amino acids.  Inactivation of lasR in these isolates conferred a growth advantage with amino acids, a phenotype that could account for selection of lasR mutants both in vivo and in vitro.  P. aeruginosa lasR mutants were identified by their distinctive colony morphology, including autolysis that correlated with an imbalance in 4-hydroxy-2-alkylquinolines (HAQs), and an iridescent metallic sheen likely caused by the accumulation of one such HAQ.  The alterations in transcriptional profile due to inactivation of lasR were conserved in isolates from multiple young CF patients.  P. aeruginosa lasR mutations may represent surrogate markers to delineate stages in the natural history of CF airway disease, each with different prognostic and therapeutic implications, analogous to the markers used to direct cancer treatment.  Similar to cancer cell mutations that promote unrestricted growth, lasR mutations may promote unrestricted growth of P. aeruginosa in the CF airway by enabling more efficient utilization of available amino acids.
Keywords: disease state analysis

overall_design: Analyse the effects of mutation of the lasR gene in Pseudomonas aeruginosa isolates from cystic fibrosis patients by comparing the transcriptional profile of an isolate from a young patient with that of an isogenic engineered lasR mutant.

middle: A

first: Lucas

middle: R

last: Hoffman

first: Hemantha

last: Kulasekara

first: Robert

middle: K

last: Ernst

first: Samuel

middle: I

last: Miller


title: Gene expression changes induced by paclitaxel in MCF7 breast cancer cells

status: Public on Dec 21 2007

city: Bron

first: Hector

last: Hernandez-Vargas

state: Rhone

address: 36 bis Rue de la Batterie

institute: Genomics Consulting

summary:
Time-dependent changes in gene expression induced by paclitaxel in the breast adenocarcinoma cell line MCF7.
Keywords: genetic modification

overall_design: The Oncochip microarray platform v1.1 contains 9,726 clones corresponding to 6,386 different genes, and it includes 2,489 duplicate clones. Duplicate samples from each condition (no treatment control, paclitaxel 8h, paclitaxel 48h) were labeled with dUTP-Cy5 and hybridized against dUTP-Cy3-labeled Universal Human Reference RNA.

last: Palacios

first: Gema

last: Moreno-Bueno


title: Transcriptional Profiling of Daptomycin Induced Staphylococcus aureus

city: Normal

first: Arunachalam Muthaiyan

email: amuthai@gmail.com, bjwilkin@ilstu.edu

state: IL

address: Julian Hall

department: Biological Sciences

institute: Illinois State University

summary:
Daptomycin is a  lipopeptide antibiotic that has recently been approved for treatment of Gram-positive bacterial infections.  The mode of action of daptomycin is not yet entirely clear.  To further understand the mechanism transcriptomic analysis of changes in gene expression in daptomycin-treated Staphylococcus aureus was carried out.  The expression profile indicated that cell wall stress stimulon member genes (B. J. Wilkinson, A. Muthaiyan, and R. K. Jayaswal.  2005.  Curr. Med. Chem. Anti-Infective Agents 4: 259-276) were significantly induced by daptomycin, and by the cell wall-active antibiotics vancomycin and oxacillin.  Comparison of  the daptomycin response of a two-component cell wall stress stimulon regulator VraSR mutant, S. aureus  KVR, to its parent N315 showed diminished expression of the cell wall stress stimulon  in the mutant.  Daptomycin has been proposed to cause membrane depolarization, and the transcriptional responses to carbonyl cyanide m-chlorophenylhydrazone (CCCP) and nisin were determined.  Transcriptional profiles of the responses to these antimicrobial agents showed significantly different patterns compared to those of  the cell wall-active antibiotics, including little or no induction of the cell wall stress stimulon.  However, there were a significant number of genes induced by both CCCP and daptomycin that were not induced by oxacillin or vancomycin, such that the daptomycin transcriptome was probably reflecting a membrane depolarizing activity of this antimicrobial also.  The results indicate that inhibition of peptidoglycan biosynthesis, either directly or indirectly, and membrane depolarization are parts of the mode of action of daptomycin.
Keywords: mode of action, transcriptional profiling

overall_design:
S. aureus strain ATCC 29213 was provided by Cubist Pharmaceuticals and S. aureus vraSR- mutant KVR derived from S. aureus N315 was provided by K. Hiramatsu. Strain SH1000 was also used in some experiments. Daptomycin (Cubist Pharmaceuticals, Lexington, MA), CCCP and oxacillin (Sigma Chemicals, St. Louis, MO), vancomycin (Lilly Laboratories), nisin (Danisco Beaminster Ltd, UK.) were used for transcriptional profiling.  Overnight grown S. aureus ATCC 29213 was inoculated in MHBc medium (20 ml) in a 50 ml Erlenmeyer flask and incubated at 37°C, with shaking at 200 rpm. Growth was measured at regular intervals at 600 nm until OD= ~0.4.  Based on the GIC study the following concentrations of antimicrobial agents were added separately for 15 min of challenge: daptomycin,  4 µg ml-1; vancomycin , 10 µg ml-1 ; CCCP, 2 µg ml-1 ; nisin, 20 µg ml-1 .  These concentrations were chosen because they gave a similar degree of inhibition of growth. For comparison purposes strain SH1000 was challenged with 1.2 µg oxacillin ml-1  for 15 min.  Hybridization signals were scanned using an Axon4000B scanner (Molecular Devices, Sunnyvale, CA ) with Acuity 4.0 software and scans were saved as TIFF image.  Scans were analyzed using TIGR-Spotfinder (www.tigr.org/software/) software and the local background was subsequently subtracted.   The data set was normalized by applying the LOWESS algorithm using TIGR-MIDAS (www.tigr.org/software/) software. The normalized log2 ratio of test/reference signal for each spot was recorded.  Genes with less than three data points were considered unreliable, and their data points were discarded.  The averaged log2 ratio for each remaining gene on the four replicate slides was ultimately calculated.  Significant changes of gene expression were identified with SAM (significance analysis of microarrays; www.tat.stanford.edu/~tibs/SAM/index.html. Genes analyzed using these programs were further sorted and grouped based on their function using our in-house software Staphylococcus aureus Gene Sorter (SAGS). Several controls were employed to minimize the technical and biological variations and to ensure that the data obtained were of good quality. First, each ORF was present in triplicate on the array.  Second, each RNA preparation was used to make probes for at least two separate arrays for which the incorporated dye was reversed.

Contents of raw data files:

Channel A = Cy3 dye
Channel B = Cy5 dye

File names ending with S1,S3 = Cy3 untreated, Cy5 treated
File names ending with S2,S4 = Cy5 untreated, Cy3 treated

first: Arunachalam

last: Muthaiyan

first: Jared

last: Silverman

first: Radheshyam

last: Jayaswal

first: Brian

middle: J

last: Wilkinson


title: Androgen treatment in female rainbow trout leads to a marked dysregulation of gonadal gene expression profiles

status: Public on Dec 29 2007

city: Nantes

first: Daniel

last: BARON

state: Loire-Atlantique (44)

address: 30 Bd Jean Monnet

department: UMR1064 - Centre de Recherche en Transplantation & Immunologie

institute: Institut national de la santé et de la recherche médicale-INSERM

summary:
The rainbow trout, Oncorhynchus mykiss, has a male heterogametic XY genetic system, and this knowledge can be used to produce experimentally all male or all female genetic populations using males with new genotypes (XX and YY males). These monosex populations have been widely used for sex differentiation studies because they give the opportunity to work on undifferentiated gonads for which the natural fate as testis or ovary is known a priori. Using as a resource the availability of a lot of expressed sequenced tags (ESTs) sequencing projects in trout, we designed and built a micro-array in order to characterize, at the pangenomic scale, rainbow trout natural gonadal differentiation as well as the mechanisms by which androgen masculinize the embryonic ovary. We choose a Nylon membrane array technique used for large-scale gene expression profiling with low cost, easy customization and high sensitivity, which is important when a limiting amount of RNA is available.
Keywords: time course of natural and androgen induced gonadal sex differentiation

overall_design:
Animals and samplings : all-male (male control group ‘Male’) and all-female (female control group ‘Female’) rainbow trout populations were obtained at the INRA experimental fish farm (Sizun, France) as previously described [10471475] Treatment with androgens (female treated Group ‘Female11ß’) was carried out as previously described [11748604]. In each group, 20 to 100 gonads, according to the age of the fish, were sampled and pooled in duplicates at various stages of development [16014816]: onset of the free swimming period after complete yolk resumption (Day 0 = D0), D0+7days (D7), occurrence of oocyte meiosis (D12), beginning of ovarian lamellar structures development (D27), occurrence of previtellogenic oocytes (D60), D90 and D110. They were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction.
Total RNA extraction : total RNA was extracted using TRIzol reagent (Invitrogen, Cergy Pontoise, France) as previously described [11316749]. The total RNA concentration was determined with an Agilent 2100 Bioanalyzer and the RNA 6000 LabChip® kit (Agilent Technologies, Stockport, UK) according to the manufacturers’ instructions.
DNA microarray hybridizations : microarrays were hybridized with two types of 33P-labeled probes. The first one was an oligonucleotide having a common sequence to all spotted PCR-products (called vector hybridization) in order to determine the amount of target DNA accessible to hybridization in each spot. After stripping, a second hybridization was performed with complex probes made from 1 μg of retrotranscribed total RNA [10441335; 10445855; 11971868]. Protocol descriptions for probes preparation, hybridizations and washes are available at http://tagc.univ-mrs.fr/oncogenomics/Nylon_microarrays.php. After stringent washes, arrays were exposed to phosphor-imaging plates that were scanned with a FUJI BAS 5000 at 25 μm resolution. Hybridization signals were quantified using ArrayGauge software (Fuji Ltd, Tokyo, Japan). Signal intensities were corrected for the amount of spotted DNA and the variability of experimental conditions as previously described [10441335; 10445855].

last: Baron

first: Jérome

last: Montfort

first: Rémi

last: Houlgatte

first: Alexis

last: Fostier

first: Yann

last: Guiguen


title: Transcriptional profiling of inductive prostatic mesenchyme

city: Edinburgh

first: Axel

last: Thomson

address: 47 Little France Crescent

country: United Kingdom

institute: MRC Human Reproductive Sciences Unit

summary:
Background: The mesenchymal compartment plays a key role in organogenesis and cells within the mesenchyme/stroma are a source of potent molecules that control epithelia during development and tumourigenesis. We have used Serial Analysis of Gene Expression (SAGE) to profile a key subset of prostatic mesenchyme that regulates prostate development and is enriched for growth-regulatory molecules.
Results: SAGE libraries were made of prostatic inductive mesenchyme (VMP) and the complete prostatic rudiment (including inductive mesenchyme, epithelium and smooth muscle; VSU). By comparing these two SAGE libraries we generated a list of 219 transcripts that were enriched or specific to inductive mesenchyme and which may act as mesenchymal regulators of organogenesis and tumourigenesis.
Conclusions: The use of a precisely defined subset of cells, and a back-comparison approach, allowed us to identify rare mRNAs that might be overlooked using other approaches.
Keywords: SAGE, gene profiling

overall_design: Two SAGE libraries were prepared and compared to each other. The VMP forms a subset of the VSU.

first: Brigid

last: Orr


title: Comparison of linear and exponential protocols using moderate amplifications

status: Public on Dec 20 2007

city: Jouy en Josas

first: Isabelle

last: Hue

address: Domaine de Vilvert

department: PHASE

institute: INRA

summary:
Gene expression profiling has become a tool of choice to study pathological or developmental questions but in most cases the material is scarce and requires sample amplification. Two main procedures have been used: in vitro transcription (IVT) and polymerase chain reaction (PCR), the former known as linear and the latter as exponential. Previous reports identified enzymatic pitfalls in PCR and IVT protocols; however the possible differences between the sequences affected by these amplification defaults were only rarely explored. We thus aimed at characterizing deviating gene expressions due to amplification defaults and further checked that differential expressions identified between embryonic stages were both reliable (minor intersect with biased expressions) and relevant (biologically validated on unamplified material)
Keywords: Amplification, in vitro transcription (IVT),  polymerase chain reaction (PCR), biases, molecular features of the affected sequences, relevance of differential expressions,  biological validation

overall_design: 96 samples - Amplified material from each tissue (brain, ovary, ovoid embryos, tubular embryos and filamentous embryos) was indirectly labelled using "random" hexamers. Three or two independent targets for each tissue and each protocol (target replicates) were thus generated and hybridised to 4 replicates of the same array (array replicates), so that 48 measurements per probe were generated for somatic (3 targets x 2 tissues x 2 protocols x 4 arrays) and embryonic samples (2 targets x 3 tissues x 2 protocols x 4 arrays).

first: Severine

last: Degrelle

first: Christelle

last: Hennequet-Antier

first: Helene

last: Chiapello

first: Karine

last: Piot-Kaminski

first: Francois

last: Piumi

first: Stephane

last: Robin

first: Jean-Paul

last: Renard


title: Analgesia of naloxone and morphine in wild-type and NY1DD sickle cell mice using BMAP cDNA microarrays

city: Minneapolis

first: Ajay

last: Yekkirala

state: MN

address: 308 Harvard St 8-120

institute: University of Minnesota

summary:
Sickle cell disease is the most common genetic disorder in African-Americans.  The opioid analgesic, morphine, has long been the treatment for the severe pain associated with this disease.  Here we reveal that the opioid antagonist, naloxone, possesses potent analgesic activity in two strains of sickle cell mice (NY1DD and hBERK1) and not in their respective controls (ICR-CD1 and C57BL/6J) when administered by three parenteral routes.  In the NY1DD sickle mice, naloxone (i.c.v.) possessed ~300-fold greater potency than morphine (i.c.v.).  Other opioid antagonists (naltrexone, norbinaltorphimine, naltrindole) were substantially less effective in producing analgesia. Naloxone and morphine were synergistic in NY1DD mice, suggesting that analgesia was mediated via different receptor systems. Since microarray analysis suggested naloxone-induced down-regulation of the CCR5 chemokine receptor in NY1DD mice but not in control mice, the role of its endogenous ligand, CCL5 (RANTES), was investigated.
Keywords: Comparison of drug induced gene expression

overall_design:
In total 12 samples were analyzed. We used two strains of mice:
Wild-type: C57BL/6J
Sickle cell: NY1DD

To compare the gene expression patterns of naloxone and morphine, 4 treatments were used:
i) Naloxone on sickle cell mice - 3 replicates
ii) Morphiine on sickle cell mice - 3 replicates
iii) Naloxone on wild-type mice - 3 replicates
iv) Morphine on wild-type mice - 3 replicates

As we used dual channel cDNA microarrays, the experimental (Cy5) channel was used for the drugs (either morphine or naloxone) and the control (Cy3) was always saline treated.

first: Mary

middle: M

last: Lunzer

first: Robert

middle: P

last: Hebbel

first: Philip

middle: S

last: Portoghese


title: Comparative Genomic Analysis of Clinical Strains of Campylobacter jejuni from South Africa

status: Public on Dec 19 2007

city: Albany

first: Craig

middle: T

last: Parker

state: CA

address: 800 Buchanan St

department: PSM

institute: USDA ARS

summary:
Background.  The bacterial foodborne pathogen Campylobacter jejuni is a common cause of acute gastroenteritis and is also associated with the postinfectious neuropathies, Guillain-Barré and Miller Fisher syndromes.  This study described the use of multilocus sequence typing and DNA microarrays to examine the genetic content of a collection of South African C. jejuni strains, recovered from patients with enteritis, Guillain-Barré or Miller Fisher syndromes.
Methodology/Principal Findings.  The comparative genomic analysis by using multilocus sequence typing and DNA microarrays demonstrated that the South African strains with Penner heat-stable (HS) serotype HS:41 were clearly distinct from the other South African strains.  Further analysis of the DNA microarray data demonstrated that the serotype HS:41 strains from South African GBS and enteritis patients are highly similar in gene content.  Interestingly, the South African HS:41 strains were distinct in gene content when compared to serotype HS:41 strains from other geographical locations due to the presence of genomic islands, referred to as Campylobacter jejuni integrated elements.  Only the genomic integrated element CJIE1, a Campylobacter Mu-like prophage, was present in the South African HS:41 strains whereas absent in the closely-related HS:41 strains from Mexico.  A more distantly-related HS:41 strain from Canada possessed both genomic integrated elements CJIE1 and CJIE2.
Conclusion/Significance.  These findings demonstrated that these C. jejuni integrated elements may contribute to the differentiation of closely-related C. jejuni strains.  In addition, the presence of bacteriophage-related genes in CJIE1 may probably contribute to increasing the genomic diversity of these C. jejuni strains.  This comparative genomic analysis of the foodborne pathogen C. jejuni provides fundamental information that potentially could lead to improved methods for analyzing the epidemiology of disease outbreaks and their sources.
Keywords: comparative genomic indexing analysis

overall_design: The genomic diversity of 33 South African, 2 Mexican and 1 Canadian Campylobacter jejuni strains from humans were examined by microarray-based comparative genomic indexing (CGI) analysis. The CGI analysis allowed the assessment of CDS content for each C. jejuni strain relative to the C. jejuni DNA microarray, which comprises ORFs from strains NCTC 11168, RM1221. ORFs were spotted in duplicate. Genomic DNA from strains NCTC 11168/RM1221 were used as a reference DNA and competitively hybridized with genomic DNA from each of the other C. jejuni strains. Two or three replicates for each strain were performed. Data normalization was performed as in Parker et al. J Clin Microbiol 2006, 44(11):4125-4135.

first: Beatriz

last: Quiñones

first: Magalie

last: Guilhabert

first: William

middle: G

last: Miller

middle: E

last: Mandrell

first: Albert

last: Lastovica


title: 42 days old adult worms: adult worms vs. adult worms (self-self)

city: Campos dos Goytacazes

first: Thiago

middle: Motta

last: Venancio

state: RJ

address: Av Alberto Lamego 2000

country: Brazil

institute: UENF

summary:
Recent advances in the study of Schistosoma mansoni genome and transcriptome have led to a better description of the S. mansoni gene complement. In this work, we report the design and use of a new S. mansoni 60-mer oligonucleotide microarray platform with approximately 44,000 probes, based on all publicly available cDNA sequence data for S. mansoni and S. japonicum. The large number of probes combined with the extensive sequence annotation available allowed a comprehensive approach, where most of the S. mansoni transcriptome is represented. Hybridization with adult worm RNA pointed to a set of genes transcriptionally active in this stage of the parasite life cycle. Interestingly, a large proportion (43%) of genes for which transcription was detected in adults is comprised of no match genes, i.e. S. mansoni genes with unknown function and no identifiable orthologs in GenBank. Moreover, detection of bi-directional transcription for several genes leads us to hypothesize a widespread production of antisense RNA in S. mansoni.
Keywords: Expressed genes in adult worms

overall_design: Self-self experiment, adult vs adult. Biological replicates: 2. Independently harvested. The two channel values were separately analyzed, resulting in 4 data points.

first: Sergio

last: Verjovski-Almeida

first: Katia

middle: C

last: Oliveira

first: Giulliana

last: Almeida

first: Ricardo

last: DeMarco


title: Regulation of pituitary gene expression by adrenalectomy

city: Hershey

first: Yuichiro

last: Nishida

state: PA

address: 500 University Drive

department: Department of Anatomy and Physiology

institute: Penn State College of Medicine

summary:
Overproduction of adrenal hormones causes obesity and hypertension, whereas adrenalectomy (ADX) leads to weight loss and hypotension. The pituitary gland is a major target organ for the adrenal hormones. To identify potential mediators for the effect of ADX on pituitary system regulating weight loss and hypotension, we investigated the effect of ADX on pituitary gene expression using serial analysis of gene expression (SAGE). The numbers of analyzed SAGE tag were 126688 tags for intact mice (n = 51) and 59482 tags for ADX mice (n = 12), which corresponded to 45151 and 21790 distinct tag species, respectively. Thirty-three pituitary genes were differentially expressed between intact and ADX mice. Three genes encoding for pro-opiomelanocortin have been upregulated by ADX. The ADX has induced the gene expression of growth hormone while downregulating an expressed sequence tag similar to (EST) growth hormone. The ADX reduced the expression of genes involved in mitochondrial oxidative phosphorylation, such as NADH dehydrogenase 3 and cytochrome c oxidase 3, whereas neuromedin B gene were dramatically induced. In addition, the ADX increased the expression levels of other three genes involved in hormone release (response to metastatic cancers 1), extracellular matrix (matrix gamma-carboxyglutamate gla protein), and cation transport (solute carrier family member 17). Moreover, one functionally-uncharacterized and 20 novel transcripts were significantly modulated by ADX. Thus the current study has revealed the alterations in the pituitary gene expression that may play key roles in the mechanisms of ADX-induced weight loss and hypotension.
Keywords: adrenalectomy, serial analysis of gene expression

overall_design:
Rodents and pituitary gland sampling. Male C57BL6 mice were obtained from Charles River Laboratories (St. Constant, QC, Canada), at 12-14 weeks of age. Mice were housed in an air-conditioned room (19-25℃) with controlled lighting from 07:15 to 19:15 h and were given free access to food (Lab Rodent Diet No. 5002) and water.
One week prior to sampling of pituitary gland, adrenalectomy was performed in mice of ADX groups (n = 12). The ADX mice received sodium chloride (0.9g/dl) in their drinking water after the surgery. All mice were killed between 08:30 and 12:30 by decapitation under isoflurane anesthesia. Brain was removed from the skull and the pituitary gland was immediately dissected. The pituitary gland was immediately frozen in liquid nitrogen, pooled together for each group, and stored at -80℃ until RNA extraction.

first: Chester

last: Ray

first: Mayumi

last: Yoshioka

first: Jonny

last: St-Amand


title: Host genome surveillance for retrotransposons by transposon-derived proteins

city: Bethesda

first: Shiv

last: Grewal

state: MD

address: NCI bldg 37 Rm 6068 9000 Rockville Pike

department: LBMB

institute: NCI

type: Genome binding/occupancy profiling by genome tiling array

summary:
ChIP-chip analyses of Abp1 and Cbh1 across the S. pombe genome.
Keywords: ChIP-chip

overall_design: Chromatin immunoprecipitated DNA recovered with antibody against FLAG-tagged protein from wild-type asynchronous growing fission yeast cells and whole cell extracts DNA were amplified by random priming and respectively labeled with Cy5 and Cy3. Labeled DNA samples were mixed and hybridized onto a 44k pombe Agilent oligo array. Data were processed using Agilent scanner and Feature Extraction software

first: Hugh

last: Cam

first: Kenichi

last: Noma

first: Hirotaka

last: Ebina

first: Henry

last: Levin


title: Matrix metalloproteinase-3 mediates cardiac differentiation of mouse embryonic stem cells

status: Public on Dec 18 2007

city: Seoul

first: Su

last: Hong

department: Life Science Institute

country: South Korea

institute: Macrogen Inc.

summary:
Recently, it was shown that the Bmp antagonist Noggin could strongly induce cardiomyocyte differentiation by transient treatment of undifferentiated ES cells. In order to determine how Noggin may induce cardiac differentiation, we compared differentially expressed genes during Noggin treatment of ES cells using microarray analysis and found that matrix metalloproteinase (Mmp)-3 is the only gene whose expression is increased by Noggin treatment.
Keywords: embryonic stem cells, cardiac differentiation, matrix metalloproteinase-3, Noggin

overall_design:
To induce differentiation, 150ng/ml of Noggin (Sigma, St. Louis, MO) was added to the culture medium for 3 days. Then the cells were trypsinized and cultured in culture medium without Lif on uncoated Petri dishes to induce EB formation. On the second day after EB formation, Noggin was removed from the culture medium.
For the Noggin treated group, total RNA from undifferentiated ES cells, cells treated with Noggin for 3 days, and EBs either 2 or 4 days after EB formation were isolated using Trizol reagent (Invitrogen, Calsbard, CA). For the non-treated control group, undifferentiated ES cells and EBs either 2 or 4 days after EB formation were isolated.

first: Jaeku

last: Kang

first: Eunsook

last: Ryu

first: Jungjun

last: Park

first: Sangho

last: Lee

first: Jongho

first: Jeongsun

last: Seo


title: Glyoxylate pathway elements involved in the functional transition of soybean cotyledons during seedling development

city: Urbana

first: Lila

middle: O.

last: Vodkin

address: 1201 W. Gregory Dr.

department: Crop Sciences

institute: University of Illinois

summary:
Background
The soybean (Glycine max) cotyledon is a specialized tissue whose main function is to serve as a nutrient reserve that supplies the needs of the young plant throughout seedling development.  During this process the cotyledons experience a functional transition to a mainly photosynthetic tissue.  To identify at the genetic level the specific active elements that participate in the natural transition of the cotyledon from storage to photosynthetic activity, we studied the transcript abundance profile at different time points using a new soybean oligonucleotide chip containing 19,200 probes (70-mer long).
Results
After normalization and statistical analysis we determined that 3,594 genes presented a statistically significant altered expression in relation to the imbibed seed in at least one of the time points defined for the study.    Detailed analysis of this data identified individual, specific elements of the glyoxylate pathway that play a fundamental role during the functional transition of the cotyledon from nutrient storage to photosynthesis. The dynamics between glyoxysomes and peroxisomes is evident during these series of events.   We also identified several other genes whose products could participate co-ordinately throughout the functional transition and the associated mechanisms of control and regulation and we described multiple unknown genetic elements that by association have the potential to make a major contribution to this biological process.
Conclusions
We demonstrate that the global transcript profile of the soybean cotyledon during seedling development is extremely active, highly regulated and dynamic.  We defined the expression profiles of individual gene family members, enzymatic isoforms and protein subunits and classified them accordingly to their involvement in different functional activities relevant to seedling development and the cotyledonary functional transition in soybean, especially the ones associated with the glyoxylate cycle. Our data suggests that in the soybean cotyledon a very complex and synchronized system of control and regulation of several metabolic pathways is essential to carry out the necessary functions during this developmental process.
Keywords: Time Course

overall_design:
Glycine max Williams dry seeds were planted per small pot containing pre-wetted Universal Mix SB300 soil in the green house.  Two days after planting, Cotyledons from 10 imbibed seeds were collected and pooled, washed several times with distilled water, frozen in liquid nitrogen and freeze dried for 48 hours.  Total RNA was extracted from the cotyledonary tissue and four different preparations from the same tissue were pooled and quantified.  The imbibed cotyledon tissue (Stage 1) was used as the reference time point to compare a total of 6 more subsequent cotyledonary developmental stages (Stages 2-7) during the process of germination and emergence.   A second set of seeds was planted at a different time and cotyledons from each stage were collected in a similar manner to obtain data from a biological replicate.
A total of 20 µg of the reference Stage 1 RNA was used to be labeled by reverse transcription with Cy3-dUTP and compared to 20 µg of each of stages 2-7 labeled by reverse transcription with Cy5-dUTP following the protocol by Hedge et al., 2000.  Two 70-mer Oligo chips containing 19,200 features were hybridized to compare differential transcript abundance between each stage and the reference.  The dyes were swapped in two experimental replicates to avoid potential dye bias for a total of 4 Oligo chips per biological replicate.  Since two biological replicates were set up, a total of 8 chips were used to compare transcript abundance per germination stage defined. The corresponding GPR files containing raw data were uploaded into GeneSpring 7.2 (Silicon Genetics, CA), transformed for dye swap and normalized by using the Per spot and Per chip intensity dependent (Lowess) normalization tool.  Time was defined as the only parameter to be taken into account for analysis and 6 different conditions were defined: one for each time point.  A Welch analysis of variance (ANOVA) of these genes in 48 samples with a P-value cutoff 0.05, a false discovery rate (FDR) of 0.05 and between 0.1 and 100 fold expression change in at least 1 out of 6 conditions, defined a gene list of 3,594 genes with statistical significant data.  K-means clustering analysis on this list grouped genes with similar transcript abundance profiles during germination and emergence in 12 clusters using standard correlation as similarity measure

first: Delkin

middle: O

last: Gonzalez


title: Heat stress response in Gillichthys mirabilis

status: Public on Dec 14 2007

city: Portland

first: Brad

last: Buckley

state: OR

address: 1719 SW 10th Ave

department: Biology

institute: Portland State University

summary:
The goal of this study was to measure the effect of heat stress on the transcriptome of a temperate fish species - Gillichthys mirabilis.
Keywords: Stress response

overall_design: Three control fish are compared to three heat-shocked fish.

first: Bradley

first: George

middle: N

last: Somero


title: Differential Gene Expression Profile in Pig Adipose Tissue Treated with/without Clenbuterol

status: Public on Dec 17 2007

city: Beijing

first: Jin

last: Zhang

address: 2#, Yuanmingyuan West Road

country: China

institute: China Agricultural University

summary:
Background
Clenbuterol, a beta-agonist, can dramatically reduce pig adipose accumulation at high dosages. However, it has been banned in pig production because people who eat pig products treated with clenbuterol can be poisoned by the clenbuterol residues. To understand the molecular mechanism for this fat reduction, cDNA microarray, real-time PCR, two-dimensional electrophoresis and mass spectra were used to study the differential gene expression profiles of pig adipose tissues treated with/without clenbuterol. The objective of this research is to identify novel genes and physiological pathways that potentially facilitate clenbuterol induced reduction of adipose accumulation.
Results
Clenbuterol was found to improve the lean meat percentage about 10 percent (P< 0.5). The adipose cells became smaller and the muscle fibers became thicker with the administration of clenbuterol. The mRNA abundance of 82 genes (ESTs) were found to be statistically differentially expressed based on the Student t-test (P<0.5) in the microarray analyses which containing 3358 genes (ESTs).Theses 82 genes (ESTs) were divided into four groups according to their Gene Ontology Biological Process descriptions. 16 genes were cellular metabolism related genes (including five lipids metabolism related such as apolipoprotein D and apolipoprotein R), 10 were signal transduction related genes, 45 were expressed sequence tags (ESTs) and other 11 others were of various categories. Eleven of the 82 genes (ESTs) were chosen for real-time PCR analysis, with eight of them induced to express similar magnitudes as in the microarray analyses, two could not be detected and one expressed magnitudes that disagreed with the microarray results. Apolipoprotein R was also found to be up-regulated by the proteomic analysis.
Conclusions
Profound changes were observed in adipose cells after administration of clenbuterol in pigs by histological section and cDNA microarray techniques. The adipose cells became smaller with stimulation by clenbuterol. Changes in mRNA abundance indicated that DNA transcription and protein translation were enhanced in the adipose cells with clenbuterol stimulation. Five lipids metabolism related genes, such as apoD and apoR, were significantly up-regulated. ApoR was up-regulated in both the mRNA transcription and the protein synthesis. ApoR may be one of the critical molecules through which clenbuterol reduces fat accumulation.
Keywords: dose response

overall_design:
Eight cDNA microarray slides were hybridized for the eight pigs.
Eight Chinese miniature pigs were used in the experiments. Four hogs and four sows, all 4 weeks old, were housed in the Nutrition and Metabolism Laboratory at the China Agriculture University. They were raised under exactly the same conditions and were fed the same diets until 8 weeks (average body weight 17 kg). They were randomly divided into 4 groups with each group having two pigs with the same gender and the same parents. For the following 4 weeks, one pig in each group was fed 25 mg/kg clenbuterol twice daily in their diets as the test pig, while the other was fed the same diet without clenbuterol as the control. Then one group of hogs and one group of sows were slaughtered for analysis. These two groups are referred to as the 3 month-old pigs. The other two groups were fed with/without 50 mg/kg clenbuterol twice daily in their diets for another 4 weeks and slaughtered for analysis. These two groups are referred to as the 4 month-old pigs. Approximately 1 g biopsies were taken from the back fat adipose tissues (at the fifth lumbar vertebra level) of each pig. The adipose tissue samples were washed in sterile water, snap frozen in liquid nitrogen and stored at -80℃.
Total RNA of the adipose tissue was extracted with TRIZOL reagent (Invitrogen, Gaithersburg, MD, USA) and further purified with an RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturers instructions.
The porcine cDNA microarray was produced at the China Agricultural University. A total of 11520 spots representing 3358 genes (ESTs) and 1446 quality control spots were included on the microarray slide. 3358 genes (ESTs) were cloned from the procine adipose cDNA library and were printed in triplicate on each slide. (The cDNA microarray gene list and clone numbers are listed in additional file 3. More details on the specific genes and probe sequences are given by Guo et al [39]).
A cDNA microarray hybridization analysis was performed with Pronto!TM Universal Microarray Kits (Corning, MA, USA) according to the manufacturers instructions. Briefly, reverse transcription was done with 10 µg total RNA as the template to synthesize cDNA incorporated with flourence dye Cy3-dCTP or Cy5-dCTP. Probes were purified on a Qiagen spin column (Qiagen, Valencia, CA, USA). Mix dye-labeled cDNA together and dry dye-labeled cDNA were used for hybridization (see Pronto Universal hybridization kit Quick Reference Guide).
To avoid dye bias, the experiments were performed in duplicate by dye swap with Cy5-dCTP first used with the test pig and Cy3-dCTP used with the control pig and then Cy3-dCTP used with the test pig and Cy5-dCTP used with the control pig.

first: Wei

last: Guo

middle: W

first: Xue

middle: X

last: Deng

first: Xiao

last: Hu

first: Ning

last: Li


title: Distinct physiological states of Plasmodium falciparum in malaria infected patients

status: Public on Dec 13 2007

city: Bronx

first: Johanna

middle: Patricia

last: Daily

state: NY

address: 1301 Morris Park Avenue, Price Center

department: Medicine (Infectious Disease)

institute: Albert Einstein College of Medicine

summary:
Patients infected with Plasmodium falciparum sustain diverse disease outcome; we wanted to determine if parasites derived ex vivo have diverse biological states.
To explore parasite in vivo biology we measured parasite steady state whole genome transcription using a previously reported custom made Affy array (Scrmalaria) based on the laboratory strain 3D7.  Extensive bioinformatic analysis was carried out including cross species comparisions to yeast.
For sample cluster analysis, a filtered gene list containing 3937 genes was generated by thresholding gene expression levels to a minimum of 50 EU and removing any genes that varied less than 3 fold or 100 EU across the dataset. To minimize potential effects of different dates of collection and hybridizations, the data was rank ordered by expression level and each gene was given an ordinal value.
Keywords: ex vivo

overall_design: 43 symptomatic patients from Senegal with P. falciparum underwent venipuncture; parasite RNA was derived directly from this sample.


title: Phagocytic receptor type affects phagosome maturation, gene expression and phagosome protein composition in macrophages

status: Public on Dec 12 2007

city: Rostock

first: Eik

last: Hoffmann

address: Albert-Einstein-Str. 3

department: Institute of Biological Sciences

country: Germany

institute: University of Rostock

summary:
The receptors engaged during phagocytic particle uptake determine the  signalling events that occur during phagosome formation and maturation.However, pathogens generally have multiple ligands, making it difficult to dissect the roles of individual receptors in these processes. Here,we used latex beads coupled to single ligands, focusing primarily on IgG, mannan and avidin, and for some experiments on LPS, and monitored 1. phagocytic uptake rates, 2. fusion with lysosomal compartments,  3.macrophage gene expression 4. proteomic composition of isolated phagosomes and 5. the binding of phagosomes to actin filaments. Phagosomes formed in response to mannan fused with lysosomes earlier after uptake than the ones formed by the other ligands. The pattern of gene expression and the protein composition of isolated phagosomes showed that each bead ligand upregulated a distinct pattern of genes and led to a different composition of phagosomes. Furthermore, isolated phagosomes also exhibited differences in binding to F-actin in vitro.These data argue that activation of each receptor initiates a specific signature of signalling events that last many hours and influences several phagocytosis functions.
Keywords: receptor-ligand interactions

overall_design:
J774.A1 cells (mouse macrophage-like cell line) were seeded onto 6-well  plates one day before the experiment. Duplicates were incubated with serum-free DME medium containing 0.01 % latex beads of 1 µm diameter coupled to the following ligands: avidin (Av), Fc fragment of mouse IgG (Fc), lipopolysaccharides from Klebsiella pneumoniae (LPS) and mannan from Saccharomyces cerevisae (Man) for 30 and 60 minutes. After incubation samples as well as untreated controls in duplicates were washed twice in PBS and total RNA was extracted using RNAeasy kit (Qiagen) following manufacturer's instructions. Each sample was  hybridized to CodeLink Mouse whole genome bioarray slides (Amersham). This study was intended to analyze the role of receptor-ligand interactions on phagosome maturation and gene expression after receptor-mediated phagocytosis in macrophages.

Note that CodeLink EXP v4.0-processed data are represented in the Sample tables; GeneSpring-processed data are linked as a supplementary file at the foot of this record.

first: Bibhuti

middle: B

last: Mishra

first: Gareth

last: Griffiths

first: Sergei

last: Kuznetsov


title: Kawasaki disease Cohort2 Acute

city: Stanford

first: Stephen

last: Popper

department: Division of Infectious Diseases, Dept. of Medicine

institute: Stanford University

summary:
Background: Kawasaki disease (KD) is an acute self-limited vasculitis and the leading cause of acquired heart disease in children in developed countries. No etiologic agent(s) has been identified, and the processes that mediate formation of coronary artery aneurysms and abatement of fever following treatment with intravenous immunoglobulin (IVIG) remain poorly understood.
Results: In an initial survey, we used DNA microarrays to examine patterns of gene expression in peripheral whole blood from 20 children with KD; each was sampled during the acute, subacute, and convalescent phases of the illness. Acute KD was characterized by increased relative abundance of gene transcripts associated with innate immune and proinflammatory responses and decreased abundance of transcripts associated with natural killer cells and CD8+ lymphocytes. There was significant temporal variation in transcript levels during the acute disease phase and stabilization thereafter. We confirmed these temporal patterns in a second cohort of 64 patients, and identified additional inter-individual differences in transcript abundance. Notably, higher levels of transcripts of the gene for carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) were associated with an increased percentage of unsegmented neutrophils, fewer days of illness, higher levels of C-reactive protein, and subsequent non-response to IVIG; this last association was confirmed by quantitative reverse transcription PCR in a third cohort of 33 patients, and was independent of day of illness.
Conclusions: Acute KD is characterized by dynamic and variable gene-expression programs that highlight the importance of neutrophil activation state and apoptosis in KD pathogenesis. Our findings also support the feasibility of extracting biomarkers associated with clinical prognosis from gene-expression profiles of individuals with systemic inflammatory illnesses.
Whole blood (PAXgene tubes) was obtained from 64 Kawasaki disease patients prior to treatment.  RNA was amplified using the MessageAmp kit (Ambion), and reverse transcribed. Each sample was labelled with Cy5, and hybridized to a Lymphochip cDNA array along with amplified human reference RNA labelled with Cy3(Universal Human Reference RNA, Stratagene). Analysis was restricted to those array elements (LUIDs) with signal intensity/background of at least 2.5 in either channel for at least 80% of the samples, and a regression correlation of 0.6.  Patterns of gene expression were correlated with clinical parameters, including subsequent response to treatment with intravenous immunoglobulin (IVIG).
A disease state experiment design type is where the state of some disease such as infection, pathology, syndrome, etc is studied.
Phenotype: Patient responded (R) or not (NR) to IVIG therapy. Some patients were not classified because they were treated more than 10 days after onset of fever
Keywords: disease_state_design

overall_design: Complex


title: Cooperative Signaling Through the STAT3 and NF-kB Pathways in Subtypes of DLBCL

first: Louis

middle: M.

last: Staudt

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

department: Lymphoid Malignancies Branch

institute: National Cancer Institute

summary:
The activated B cell-like (ABC) subgroup of diffuse large B cell lymphoma (DLBCL) is characterized by constitutive activation of the NF-êB pathway. Here we show that the NF- êB pathway induces the expression of the cytokines IL-6 and IL-10 in ABC DLBCL cell lines, which also have high levels of total and phosphorylated STAT3 protein, suggesting autocrine signaling. Using RNA interference for STAT3, we defined a gene expression signature of IL-6 and IL-10 signaling through STAT3. Based on this signature, we constructed a molecular predictor of STAT3 signaling that defined a subset of ABC DLBCL tumors with high expression of STAT3, IL-6 and/or IL-10, and their downstream targets. Although the STAT3-high and STAT3-low subsets had equivalent expression of genes that distinguish ABC DLBCL from GCB DLBCL, STAT3-high ABC DLBCLs had higher expression of signatures that reflected NF-kB activity, proliferation, and glycolysis. A smallmolecule inhibitor of JAK signaling, which blocked STAT3 signature expression, was toxic only for ABC DLBCL lines, and synergized with an inhibitor of NF-kB signaling. These findings suggest that the biological interplay between the STAT3 and NF-kB pathways may be exploited for the treatments of a subset of ABC DLBCLs.
Keywords: time series design

overall_design: Activated B cell-like (ABC) subgroup of diffuse large B cell lymphoma (DLBCL) cell lines were used as model systems to study the cytokine pathways in these cells. We expressed inducible IkB super-repressor for 1 to 24 hours to identify NF-kB target genes in OCI-Ly3 and OCI-Ly10 cells for a total of 13 arrays with replicates of each time point. We treated OCI-Ly3 cells with IL-10 for 1 to 96 hours for a total of 10 arrays with replicates of each time point. We treated OCI-Ly10 cells with IL-6 for 30 minutes to 24 hours for a total of 8 arrays with replicates of each time point. OCI-Ly10 cells transfected with no siRNA versus STAT3-siRNA for 8, 24, or 48 hours for a total of 5 arrays with replicates for 24 and 48 hours. We treated OCI-Ly10 cells with JAK inhibitor I for 3 and 6 hours for a total of 4 arrays with replicates of each time point.


title: Macaca fascicularis GeneChip: Four tissues

city: Ibaraki

first: Naoki

last: Osada

state: Osaka

address: 7-6-8 Saito-Asagi

department: Division of Biomedical Research Resources

country: Japan

summary:
RNA samples from brain, cerebellum, liver, and testis of 3-year-old make Macaca fascicularis was hybridized to the M.fascicularis GeneChip, which was designed by the Laboratory of Genetic Resources, National Institute of Biomedical Innovation.
Keywords: Control study

overall_design: The oligo probes were designed using the cDNA sequences of Macaca fascicularis.

first: Katsuyuki

last: Hashimoto

first: Yosuke

last: Kameoka

first: Keiji

last: Terao

first: Jun

last: Kusuda

first: Ichirou

last: Takahashi


title: Phenotypically concordant and discordant monozygotic twins display different DNA copy number variation profiles (SNP)

status: Public on Dec 11 2007

city: Birmingham

first: Jan

last: Dumanski

state: AL

address: 720 20th Street South

department: Department of Genetics

institute: University of Alabama

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

summary:
The exploration of copy number variation (CNV), notably of somatic cells, is an understudied aspect of genome biology. Any differences in the genetic make-up between twins derived from the same zygote represent an extreme example of somatic variation. We studied 19 pairs of monozygotic twins with either concordant or discordant phenotype using two platforms for genome-wide CNV analyses and show that CNVs exist within pairs in both groups. These findings impact our views of genotypic and phenotypic diversity in monozygotic twins, and suggest that CNV analysis in phenotypically discordant monozygotic twins may provide a powerful tool in identifying disease predisposition loci. Our results also imply that caution should be exercised with the interpretation of disease causality of de novo CNVs found in patients based on analysis of a single tissue in routine disease-related DNA diagnostics
Analysis of copy number variability in concordant healthy monozygotic twin pairs as well as three monozygostic twin pairs discordant a Parkinsons disease (PD) phenotype using the Illumina HumanHap 300 dead chips.
Keywords: SNP data

overall_design: Genotyping using the HumanHap300-duo bead chip from Illumina, GEO accession GPL5711

first: Carl

last: Bruder


title: Effect of INH treatment on Mycobacterium tuberculosis gene expression in various dormancy models.

city: Baltimore

first: Petros

last: Karakousis

address: 1550 Orleans St

department: Medicine

institute: Johns Hopkins University School of Medicine

summary:
These data represent the expression patterns of Mycobacterium tuberculosis in progressive hypoxia, nutrient depletion, and in-vivo hollow fiber models of dormancy.  The assumptions are that the set of genes that respond to INH treatment during Log phase growth would not be differentially regulated during INH treatment in the dormancy models, and that the overall number of differentially regulated genes would be reduced do to the low metabolic state of the cells.
Keywords: Dormancy Model Drug Response Comparison

overall_design: Each condition was repeated as multiple biological replicates to reduce the number of false positives caused by temporal and human inconsistencies.  The in-vitro growth experiments represent a positive control and a core set of genes have previously been shown to respond to INH treatment under these conditions.  The KatG mutant represents a negative control.  In this case the drug is not converted to its active form by the catalase and thus any change in gene regulation would be caused by change in osmotic potential by the introduction of a soluble but non-metabolisable substrate.  The progressive hypoxia model is similar to the KatG mutant because oxygen is required for the activity of the gene, but the overall phenotypic state of the cell is non-replicative.  The nutrient depletion model is also non-replicative dormancy model in which the drug should be in its active state.  The hollow fiber model model is an in-vivo model and is hypothesized to be both low nutrient and low oxygen as well as contain a variety of other stresses.  This model is also marked by a different drug delivery system as it must first pass through the host.  Together these models should demonstrate Mycobacterium tuberculosis gene expression under different metabolic cell states as well as different drug states.

first: Ernest

last: Williams


title: Global transcriptional profiling of Saccharomyces cerevisiae treated with dictamnine

status: Public on Dec 08 2007

city: Changchun

first: Xuming

last: Deng

address: Xi'an Street 5333#

institute: College of Animal Science and Veterinary Medicine, Jilin University

summary:
We employed CapitalBio Corporation to investigate the global transcriptional profiling of Saccharomyces cerevisiae  treated with dictamnine.
Keywords: response to dictamnine

overall_design: S. cerevisiae strain L1190 was incubated in a rotary shaker. Subsequently, dictamnine was added to the culture  and DMSO to the control. Then, the yeast cells were further incubated for a certain time and total RNA was extracted by a hot acidic phenol method. The experimental sample and control sample were incorporated and dissolved in  hybridization solution after labelled. Arrays hybridization was preformed in a CapitalBio BioMixerTM II Hybridization Station overnight and washed with two consecutive solutions. Arrays were scanned with a confocal LuxScanTM scanner and the images obtained were then analyzed using LuxScanTM 3.0 software. A space- and intensity-dependent normalization based on a LOWESS program was employed.


title: Phenotypically concordant and discordant monozygotic twins display different DNA copy number variation profiles (BAC)

type: Genome variation profiling by genome tiling array

summary:
The exploration of copy number variation (CNV), notably of somatic cells, is an understudied aspect of genome biology. Any differences in the genetic make-up between twins derived from the same zygote represent an extreme example of somatic variation. We studied 19 pairs of monozygotic twins with either concordant or discordant phenotype using two platforms for genome-wide CNV analyses and show that CNVs exist within pairs in both groups. These findings impact our views of genotypic and phenotypic diversity in monozygotic twins, and suggest that CNV analysis in phenotypically discordant monozygotic twins may provide a powerful tool in identifying disease predisposition loci. Our results also imply that caution should be exercised with the interpretation of disease causality of de novo CNVs found in patients based on analysis of a single tissue in routine disease-related DNA diagnostics
Keywords: copy number variation, concordant and discordant monozygotic twins

overall_design: Analysis of copy number variability in monozygotic twin pairs discordant for a Parkinsons disease (PD) phenotype. One twin sibling was compared to the other. Dye swap repetitions are made for all PD twin comparisons, except for 1961vs1962, where a normal replicate is made instead.  Three twin pairs have been compared to a healthy female subject (F1), with a regular and a dye swaped experiment. 292cy5_F1_cy3 and 702_cy5_F1_c3 have been repeated twice. In total, 32 hybridizations are made.


title: Microarray validation of an alternative splicing database

city: Berkeley

first: Jonathan

middle: L

last: Bingham

address: 733 Allston Way

institute: Jivan Biologics, Inc

summary:
Alternative splicing generates functional diversity in higher organisms through alternative first and last exons, skipped and included exons, intron retentions and alternative donor and acceptor sites. In large-scale microarray studies in human and mouse, emphasis so far has been placed on exon-skip events, leaving the prevalence and importance of other splice types largely unexplored. Using a new human splice variant database and a genome-wide microarray to probes thousands of splice events of each type, we measured differential expression of splice types across 6 pairs of diverse cell lines and validated the database annotation process. Results suggest that splicing in human is more complex than simple exon skip events, which account for a minority of splicing differences. The relative frequency of differential expression of the splice types correlates with what is found by our annotation efforts.  In conclusion, alternative splicing in human cells is considerably more complex than the canonical example of the exon-skip. The complementary approaches of genome-wide annotation of alternative splicing in human and design of genome-wide splicing microarrays to measure differential splicing in biological samples provide a powerful high-throughput tool to study the role of alternative splicing in human biology.
Keywords: alternative splicing

overall_design:
Identification of alternative splicing changes in pairwise sample comparisons with technical replicates as control.
Array Design: The annotated database of human splice isoforms, SpliceExpress Human Spliceome 1 (SEHS1), was created by combining RefSeq, Build 35 of the NCBI genome assembly and Unigene-derived EST alignments from Alternative Splicing Database (ASD). For each gene, exonic regions and exonic portions from 5′ to 3′ were indexed. Using the in-dexes as well as sequence coordinates, a splice type was assigned to every splice event as exon-skips/includes, intron retentions, alternative first exons, alternative last exons, alter-native donor sites, and alternative acceptor sites. A second database of alternative splicing in human, SpliceExpress Human Spliceome 2 (SEHS2), was generated independently using UCSC genome assembly hg17 with genomic alignments of 3,493,559 cDNA/ESTs and 24,297 RefSeq mRNAs. Data were filtered to eliminate likely spurious introns of less than 15 bases and remove questionable spliceoforms based on the number of sequences providing evidence and based on donor and acceptor motifs. High-confidence splice events contributed to the microarray platform SEHS1.5. All events in SEHS1.5 were common to both SEHS1 and SEHS2 databases. To avoid mismatched probes arising from low quality EST sequences, the respective genomic sequences were used. To detect alternative splicing events, the probe set included 3 exon-exon junction probes per exon-skip event; two exon-exon junction probes per alternative first or last exon; one exon-exon junction probe plus two exon-intron junction probes per intron retention; and two exon-exon junction probes per alternative donor or acceptor site; and at least one probe per single exon gene or gene without splice isoforms. Probes had an average length of 38 nucleotides. Since fixed-length probes repre-senting junctions may vary widely in GC content and melting temperature, probe lengths were varied from 34 to 42 bases so that the total melting temperature was optimized to 68 degrees. Junction probes were isothermally balanced across the splice junction to mini-mize disparities in melting temperature between the two portions.
Sense strand oligonucleotides with an additional 10-base polyT linker at the 3′ end as spacer were synthesized in situ by NimbleGen Systems.
Samples: Four human cell lines were tested: Capan 1 (pancreas), HEK 293 (Kidney), MCF7A (breast) and CaCO2 (colon) using an expression protocol.  Cell lines were obtained from the American Tissue Culture Collection (Manassas, VA) and cultured according to ATCC specifications. Replicate data were generated for each cell line. For HEK293, two additional replicates were generated for use as a control to assign statistical significance when comparing replicate samples.

first: Patricia

last: Carrigan

first: Laurence

middle: J

last: Miller

first: Subha

last: Srinivasan


title: Odontoblast-like cells and dental pulp fibroblasts stimulated with LTA, poly(I:C) and LPS.

status: Public on Dec 07 2007

city: Lyon

first: Jean-Christophe

last: Farges

address: 11, G. Paradin Street

department: Faculty of Odontology

institute: University Lyon 1

summary:
Odontoblasts and fibroblasts are suspected to influence the innate immune response triggered in the dental pulp by micro-organisms that progressively invade the human tooth during the carious process. To determine whether they differ in their responses to oral pathogens, we performed a systematic comparative analysis of odontoblast-like cell and pulp fibroblast responses to TLR2, TLR3 and TLR4 specific agonists (lipoteichoic acid [LTA], double-stranded RNA and lipopolysaccharide [LPS], respectively). Cells responded to these agonists by differential up-regulation of chemokine gene expression. CXCL2 and CXCL10 were thus increased by LTA only in odontoblast-like cells, while LPS increased CCL7, CCL26 and CXCL11 only in fibroblasts. These data suggest that odontoblasts and pulp fibroblasts differ in their innate immune responses to oral micro-organisms that invade the pulp tissue.
Keywords: cell type comparaison

overall_design: Dental pulp fibroblasts and Odontoblast-like cells stimulated with lipopolysaccharide, ipoteichoic acid or poly(I:C), or unstimulated. Triplicates.

first: Marie-Jeanne

last: Staquet

first: Stéphanie

middle: H

last: Durand

first: Evelyne

last: Colomb

first: Annick

last: Romeas

first: Claude

last: Vincent

first: Françoise

last: Bleicher

first: Serge

last: Lebecque


title: Kawasaki disease Cohort1 sequential samples

first: Stephen

last: Popper

summary:
Background: Kawasaki disease (KD) is an acute self-limited vasculitis and the leading cause of acquired heart disease in children in developed countries. No etiologic agent(s) has been identified, and the processes that mediate formation of coronary artery aneurysms and abatement of fever following treatment with intravenous immunoglobulin (IVIG) remain poorly understood.
Results: In an initial survey, we used DNA microarrays to examine patterns of gene expression in peripheral whole blood from 20 children with KD; each was sampled during the acute, subacute, and convalescent phases of the illness. Acute KD was characterized by increased relative abundance of gene transcripts associated with innate immune and proinflammatory responses and decreased abundance of transcripts associated with natural killer cells and CD8+ lymphocytes. There was significant temporal variation in transcript levels during the acute disease phase and stabilization thereafter. We confirmed these temporal patterns in a second cohort of 64 patients, and identified additional inter-individual differences in transcript abundance. Notably, higher levels of transcripts of the gene for carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) were associated with an increased percentage of unsegmented neutrophils, fewer days of illness, higher levels of C-reactive protein, and subsequent non-response to IVIG; this last association was confirmed by quantitative reverse transcription PCR in a third cohort of 33 patients, and was independent of day of illness.
Conclusions: Acute KD is characterized by dynamic and variable gene-expression programs that highlight the importance of neutrophil activation state and apoptosis in KD pathogenesis. Our findings also support the feasibility of extracting biomarkers associated with clinical prognosis from gene-expression profiles of individuals with systemic inflammatory illnesses.
Whole blood RNA (PAXgene tubes) was collected from 20 Kawasaki patients during the acute, sub-acute (post-IVIG treatment) and convalescent phases of the disease. RNA was amplified using the MessageAmp kit (Ambion), and reverse transcribed. Each sample was labelled with Cy5, and hybridized to a Lymphochip cDNA array along with amplified human reference RNA labelled with Cy3(Universal Human Reference RNA, Stratagene). Analysis was restricted to those array elements (LUIDs) with signal intensity/background of at least 2.5 in either channel for at least 80% of the samples, and a regression correlation of 0.6.
A disease state experiment design type is where the state of some disease such as infection, pathology, syndrome, etc is studied.
Keywords: disease_state_design

overall_design: Using regression correlation


title: Closing Gaps in the Human Genome with Fosmid Resources Generated from Multiple Individuals

city: Santa Clara

first: Nick

last: Sampas

address: 5301 Stevens Creek Blvd

department: Life Science and Nanotechnology Department

institute: Agilent Technologies

summary:
The published finished human genome contained 340 gaps including 250 gaps in the euchromatic region.  The reasons for these gaps were not fully understood, although subsequent analysis revealed that presence of segmentally duplicated sequences were a good predictor for the presence of gaps.  However, not all segmentally duplicated regions contained gaps.  We made a systemic effort to close euchromatic gaps and understand the nature of gap closing sequences.  Our studies clearly demonstrate that the gap closing sequences analyzed were over 2.3-fold more enriched in segmental duplications and that about 40% of the gap closing sequences were structurally variant.  The structural variant nature of gap closing sequences was verified by aCGH analysis, and by paired-end-sequence and fingerprint analysis of gap spanning clones from recently available human genome fosmid libraries from eight individuals.  Identification and characterization of gap closing sequences provides an effective approach for closing the remaining euchromatic gaps in the human genome.
Keywords: comparative genomic hybridization

overall_design: The DNA samples are a panel of 8 Hapmap samples, described by E. Eichler et al. (2007, Nature 447, 161-165).  This set of 7 female, and one male samples are from from the Coriell Cell Repository, and is comprised of samples from four populations: four Yoruban, two CEPH, one Chinese, and one Japanese.  The reference sample, NA15510, is female and also from the Corriel Cell Repository.  This sample has been extensively characterized, (for example in Tuzan et al. 2005, Nature Genetics 10, p1038) and has been recommended for use in CNV detection programs to allow meaningful comparison of data between studies (discussed in Scherer, et al. 2007, Nature Genetics Supplement 39: S7-S15). 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, NA15510, one on each hybridization date.

first: Donald

last: Bovee

first: Yang

last: Zhou

first: Eric

last: Haugen

first: Zaining

last: Wu

first: Hillary

last: Hayden

first: Will

last: Gillett

first: Eray

last: Tuzun

first: Gregory

middle: M

last: Cooper

first: Karen

last: Phelps

first: Ruth

last: Levy

last: Morrison

first: James

last: Sprague

last: Jewett

first: Danielle

last: Buckley

first: Sandhya

last: Subramaniam

first: Jean

last: Chang

first: Douglas

middle: R

last: Smith

first: Maynard

middle: V

last: Olson

first: Evan

last: Eichler

first: Rajinder

last: Kaul


title: Global response of Silicibacter pomeroyi strain DSS3 to dimethyl sulfoniopropionate (DMSP)

status: Public on Dec 09 2007

city: Athens

first: Shulei

last: Sun

state: GA

address: Department of Marine Science

department: Marine Science

institute: UGA

summary:
Dimethylsufoniopropionate (DMSP) is an important and abundant organic sulfur compound and an important substrate for marine bacterioplankton. The Roseobacter clade of marine alpha-proteobacteria, including Silicibacter pomeroyi strain DSS3, are known to be a key phylogenetic group involved in DMSP degradaton. The fate of DMSP has important implications for the global sulfur cycle, but the genes involved in this process and their regulation are largely unknown. S. pomeroyi is capable of performing two major pathways of DMSP degradation, making it an interesting model organism. Based on the full genome sequence of this strain we designed an oligonucleotide-based microarray for the detection of transcripts of nearly all genes. The array was used to study the transcriptional response of S. pomeroyi cultures to additions of DMSP or Acetate in a time series experiment. We identified a number of DMSP-upregulated genes that could be assigned to potential roles in the metabolization of DMSP. DMSP also affected the transcription of other groups of genes, including genes for transport and metabolization of peptides, amino-acids and polyamines. High DMSP concentrations may be a chemical signal indicating phytoplankton abundance and elicit a regulatory response aimed at making maximum use of the available nutrients under these conditions.
Keywords: Microarray, marine bacterium, messenger RNA, transcription, sulfur metabolism

sample_organism:
Ruegeria pomeroyi
Ruegeria pomeroyi DSS-3

overall_design: The array design is based on the complete genome sequence of S. pomeroyi strain DSS 3 and available from Genbank (Accession numbers CP000031 and CP000032). Probes for all identified potential genes were designed by Combimatrix using proprietary software. A total of 4161 genes out of the 4348 identified potential genes on the S. pomeroyi genome are represented on the array. When possible, two probes per gene were designed.


title: Polyglutamine expansion in TBP alters transcription in cellular model of SCA17

city: New Delhi

first: TANAY

last: GHOSH

email: tghosh@igib.res.in, gtanay@yahoo.com

state: Delhi

address: Mall Road

department: Functional Genomics Unit

country: India

institute: Institute of Genomics and Integrative Biology

summary:
The poorly conserved N-terminal half of TATA binding protein (TBP) harbours a polyglutamine stretch of 29 to 42 in normal individuals.  Expansion of this stretch leading to more than 42 is associated with a neurodegenerative disorder spinocerebellar ataxia 17 (SCA17).  This study compares the espression profile of mouse neuro 2a cells expressing a vector control and TBP varient with 59 polyglutamines known to form intra nuclear aggregates.
Keywords: Neuro 2a, SCA17, TBP, polyglutamine

overall_design: Mouse Neuro 2a cells transfected with expression vector and clones expressing TBP varients having stretch of 59 glutamines were compared by cDNA microarray analysis.  The study includes four biological replicates and two pairs of flip dye replicates.  RNA was extracted and labelled with TSA labeling system (Perkin-Elmer Life Sciences, USA) before hybridization.

first: Tanay

last: Ghosh

first: Beena

last: Pillai


title: PGC1alpha overexpression in human white adipose cells

status: Public on Dec 10 2007

city: Paris

first: Karine

last: Clement

address: place du parvis Notre-Dame

department: Service de Medecine et Nutrition

institute: Hotel-Dieu

summary:
Subcutaneous abdominal adipose tissue was obtained from female subjects undergoing plastic surgery in agreement with French laws on biomedical research. Stromal cells prepared from WAT were cultured for 13 days in a chemically defined medium. At day 13, 60–80% of cells were differentiated into lipid droplet-containing adipocytes. The cells were infected at a m.o.i. of 200 for 6 h. The day after infection, cells were treated with the following drugs at 1 µM unless otherwise indicated: rosiglitazone (BRL49653, Smith Kline and French, Harlow, UK) and 9-cis-RA (Sigma). Cells were harvested after 48 h of treatment. PPAR agonists and 9-cis-RA were prepared in dimethylsulfoxide, and the remaining compounds were diluted in medium. Dimethylsulfoxide or medium was added to control cells) to obtain the same concentration as in compound-treated conditions.

Human subcutaneous abdominal adipose tissue was obtained from female subjects undergoing plastic surgery in agreement with French laws on biomedical research. In brief, on 100 ml of tissue, all visible fibrous material and blood vessels were discarded. Adipose tissue was subjected to collagenase digestion. After centrifugations, stromal cells were suspended in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium supplemented with 10% fetal calf serum (FCS) and antibiotics. Cells were seeded at 30,000 cells/cm2. After 20h incubation, the medium was changed to a chemically defined differentiation medium, consisting of DMEM/Ham's F-12 supplemented with biotin (33 5mol/l), panthotenate (17 5mol/l), insulin (66 nmol/l), triiodothyronine (1 nmol/l), cortisol (100 nmol/l), transferrin (100 ng/ml), (50 5g/ml) gentamicin and (0.2u/ml) penicillin/streptomycin and ciglitazone (1 5g/ml) the first 3 days. Cells then received the same medium without ciglitazone which was changed every 3rd day. At day 13, 60 80% of cells were differentiated into lipid droplet-containing adipocytes.
The full-length human PGC-1alpha; cDNA was cloned into the pAdEasy parent plasmid. Recombination between the pAdEasy and pAdTrack vectors and production of the PGC-1alpha; adenovirus was performed at the Laboratoire de Therapie Genique de Nantes. The virus contains, in tandem, the green fluorescent protein (GFP) gene and the PGC-1alpha; cDNA downstream of separate cytomegalovirus promoters. An adenovirus containing only the GFP gene was used as control. Viral titers were 1011 infectious particles per ml.
For experiments with agonist treatments, cells were infected with PGC1alpha; adenovirus at a m.o.i. of 200 for 6 h. The day after infection, cells were treated as indicated in text and figure legends with the following drugs at 1 5M unless otherwise indicated: rosiglitazone (BRL49653, Smith Kline and French) and 9-cis-retinoic acid (Sigma). Cells were harvested after 48 h of treatment. PPAR agonists and 9-cis-RA were prepared in dimethylsulfoxide then diluted in medium. A similar dimethylsulfoxide was added to control cells as in drug-treated cells.
Total RNA was extracted using the Qiagen RNeasy kit (Qiagen, Courtaboeuf, France, RNA concentration and integrity were assessed using Agilent 2100 Bioanalyzer (Agilent Technologies, Massy France). In order to compare microarray experiments, we used a common reference pool which was generated by mixing equal amounts of total RNA extracted from control samples. One 5g of total RNA from each total RNA sample preparation was amplified by MessageAmp RNA Kit  (Ambion, Huntington, Cambridgeshire, UK) and amplified RNA quality checked using Agilent 2100 Bioanalyser (Agilent Technologies, Massy France). 35g of amplified RNA was labeled with cyanin dyes (Cy) using the CyScribe First-Strand cDNA labeling kit (Amersham Biosciences, Orsay, France). Amplified RNA from the reference pool was labelled with Cy3 and those from the testing samples were labelled with Cy5. The labelled cDNA mixtures were hybridized according to the protocol described at http://cmgm.stanford.edu/pbrown/protocols/index.After scanning the arrays, the images were analyzed using Genepix Pro 5.1 software and entered into SMD.
Keywords: treatment comparison

overall_design:
4 types of tratment were tested:
control (vehicle) cells
cells overexpressiong PGC1 alpha
cells overexpressiong PGC1 alpha and treated with BRL 49653
cells treated with BRL 49653

An amplification and labeling experiment design type is where different amplification and/or labeling methods are compared.

first: Anne

last: Mazzucotelli

first: Nathalie

last: Viguerie

first: Emmanuelle

last: Lepin

first: Dominique

last: Langin

last: Clément


title: Evolution of gene expression during an adaptive diversification event

status: Public on Dec 15 2007

city: Vancouver

first: Mickael

last: Le Gac

address: University Boulevard

country: Canada

institute: University of British Columbia

summary:
During a 1,000 generations evolution experiment, two types of bacteria (S and L) repeatedly diverged from a common ancestor (A) in a fully sympatric seasonal environment containing glucose and acetate. We compare the transcription profile of the two derived types and the common ancestor in order to investigate the metabolic modifications associated with this adaptive diversification event.
Keywords: time point, experimental evolution

overall_design: Total RNA was extracted at 4 different time points of the 24H kinetic growth curve of each of the 3 types of bacteria (S, L and A). At each time point, S and L transcription profiles were compared to A. Each comparison was performed in 5 biological replicates.

first: Michelle

last: Brazas

first: Melanie

last: Bertrand

first: Jabus

last: Tyerman

first: Chrissy

last: Spencer

first: Robert

last: Hancock

first: Michael

last: Doebeli


title: Tannin resistance strategies of Escherichia coli in anaerobic conditions

status: Public on Dec 06 2007

city: Waukesha

first: Alexandra

last: Smith

state: WI

address: W227 N752 Westmound Dr

department: Ruminant Research

institute: Agtech Products

summary:
Gene expression profiles of Escherichia coli, grown anaerobically, with or without Acacia mearnsii (Black wattle) extract were compared to identify tannin-resistance strategies. The cell envelope stress protein, spy, and the multidrug transporter-encoding mdtABCD, both under the control of the BaeSR two-component regulatory system, were significantly up-regulated in the presence of tannins. BaeSR mutants were more tannin-sensitive than their wild-type counterparts.
Keywords: tannin resistance

overall_design: E. coli BW13711 cells were grown anaerobically in continuous culture (d = 0.14h-1) in the presence and absence of Acacia mearnsii (Black Wattle) tannin extract (WTE) at 37°C. Under both steady state conditions, E. coli reached an OD600 of ~0.2. The pH at steady-state was 5.7 and 5.3 without and with WTE, respectively. These conditions indicated that the presence of tannins in the medium did not inhibit the growth of E. coli. E. coli BW13711 cells were harvested from continuous culture after steady-state conditions were reached. Duplicate samples were collected after an additional four volume turnover of the culture medium and total RNA was isolated from these samples using the RNeasy mini kit (Qiagen Inc., Valencia, CA). The presence of residual DNA was checked by PCR using the primers for amplification of cca. If necessary, the remaining DNA was removed by RQ1 RNase-Free DNase (Promega corp, Madison, WI) according to the manufacturer’s instructions. E. coli whole genome transcriptional profiling was performed by hybridization on the GeneChip® E. coli Antisense Genome Array (Affymetrix Inc., Santa Clara, CA) according to the manufacturer’s instructions. Microarray data were analyzed using MicroArray Suite 5.0 (Affymetrix Inc.) and GeneSpring 5.0 (Silicon Genetics, Redwood City, CA) software. The 95% confidence interval of the WTE array duplicates demonstrated that genes >1.9 times up- or down-regulated could be considered as significantly differentially regulated. After removing significantly up- or down-regulated genes which had large variability in expression on the duplicate arrays, it was found that in total 1,498 genes were up- or down-regulated

first: Erwin

middle: G

last: Zoetendal

first: Monica

middle: A

last: Sundset

first: Roderick

middle: I

last: Mackie


title: Endogenous genes regulated by telomere length

city: Dallad

first: zhenjun

last: Lou

state: TX

address: 5323 Harry Hines Blvd.

department: Cell Biology

institute: The University of Texas Southwestern Medical Center at Dallas

summary:
Reporter genes inserted immediately adjacent to telomeres are frequently repressed in cells of model organisms as well as human cells, a phenomena referred to as telomere position effect (TPE). TPE has been proposed to be involved in regulation of cellular senescence, cancer and aging. However, the identification of endogenous genes in human cells regulated by natural telomeres has not been reported. Here we show that the expression of interferon simulated gene 15 (ISG15, located on 1p36.33, G1P2) varies with telomere length. ISG15 expression (RNA and protein) increases in human cells with short telomeres, and decreases with the elongation of telomeres by human telomerase reverse transcriptase (hTERT). The up-regulation of ISG15 is independent of p53 and type I interferon, and is not due to replicative senescence and DNA damage. In human skin specimens obtained from various aged individuals, ISG15 was up-regulated in a subset of cells in older individuals. Our results demonstrate that endogenous human genes can be regulated by the length of telomeres prior to the onset of DNA damage signals, and it suggests that telomeres may be involved in the regulation of human physiology that contributes to the process of aging.
Keywords: cell type comparison

overall_design: To identify endogenous genes regulated by telomere length, we prepared a microarray chip that we call the “telo-array”. The telo-array contains 1,323 subtelomeric genes (within 1,000kb from telomere) representing all 92 telomere ends. In addition, the telo-array has 92 random control genes, 12 housekeeping genes and 198 other genes. Total RNA isolated from human skin fibroblasts (BJ), lung fibroblasts (IMR90) and mammary epithelial cells (HME31) (Table 1) with different telomere lengths were hybridized to the telo-array, and the data were analyzed with GeneSpring software

first: Zhenjun

first: Jun

last: Wei

first: Harold

last: Riethman

first: Jerry

last: Shay

first: Woodring

middle: E

last: Wright


title: Global gene expression in Moraxella catarrhalis ATCC43617 cells grown in the planktonic and in biofilm states

city: Dallas

first: Eric

last: Hansen

address: 5323 Harry Hines Boulevard

department: Microbiology

institute: University of Texas Southwestern Medical Center

summary:
Total RNA samples were isolated from M. catarrhalis grown in the planktonic state (i.e., in broth) and in a biofilm.  Biofilms were generated by growing this strain in the continuous-flow Sorbarod filter system for three days. Total RNA samples and M. catarrhalis genome-directed primers (GDPs) were used for synthesis of Cy3- or Cy5-labeled cDNA samples.  Five independent M. catarrhalis growth experiments and total RNA isolations were used for the synthesis of cDNA samples, and a total of six DNA microarray hybridizations were performed in this study.
Keywords: Effect of Growth Environment on Global Gene Expression by Moraxella catarrhalis

overall_design: The Moraxella catarrhalis DNA microarray was constructed based on the nucleotide sequence of the genome of M. catarrhalis strain ATCC 43617.  The ORFs in the genome were identified by using GLIMMER 2.02.  The 1,761 annotated ORFs were used to design 70-mer oligonucleotides that would specifically anneal to each ORF.  Because genome-directed primers that annealed within the 3' 30% of each ORF were used to synthesize cDNA, each 70-mer probe was designed to anneal to a region within the 5' 70% of each ORF.

first: Wei

last: Wang

first: Rob

last: Blick


title: PrV/PK15 kinetics_QiagenNRSP8 slides

status: Public on Dec 01 2007

city: Jouy en Josas

first: Laurence

last: Flori

address: Domaine de Vilvert

department: Génétique Animale

institute: INRA

summary:
To study in vitro the epithelial cells and PrV interactions during infection, we followed PrV and PK15 cells transcriptome modifications during time-course infection (I) and mock-infection (MI). Four time points were studied: 1h, 2h, 4h and 8h post-I and MI. Four replicates of I and MI were analysed.
Keywords: Pig, PrV, Pk15 cells, kinetics

overall_design: 32 samples - The hybridization scheme which can be define as dye-switch was chosen. A balanced loop design with two independent loops, each loop containing 2 replicates of PrV infection and MI, was used. In total, 32 slides were used in this experiment.

first: Claire

last: Rogel-Gaillard

first: Marielle

last: Cochet

first: Gaetan

last: Lemonnier

first: Karine

last: Hugot

first: Patrick

last: Chardon

first: Stéphane

last: Robin

first: François

last: Lefèvre


title: PrV/PK15 kinetics_SLA/PrV slides

status: Public on Dec 03 2007

summary:
To study in vitro the epithelial cells and PrV interactions during infection, we followed PrV and PK15 cells transcriptome modifications during time-course infection (I) and mock-infection (MI).Six time points were studied: just after I and MI, 1h, 2h, 4h, 8h and 12h post-I and MI. For this study, a pig DNA/cDNA microarray containing genes of the SLA region, additional genes encoding other important immunological molecules and all the PrV genes was constructed.
Keywords: infection time course

overall_design: 48 samples  - The hybridization scheme which can be define as dye-switch was chosen. A balanced loop design with two independent loops, each loop containing 2 replicates of PrV infection and MI, was used. In total, 24 SLA/PrV slides (containing 2 networks)  were used in this experiment.