title: comparison of virulence factors and expression of specific genes of UPEC and APEC in vivo and in vitro

city: yangzhou

first: zhao

last: lixiang

address: 88 Daxue road

department: verterinary school

institute: Yanzhou university

summary: To gain further information of the correlation and pathogenisis of UPEC and APEC, the in vivo expression of 152 specific genes in both murine urinary tract infection (UTI) model and chicken challenge model were compared to that of UPEC U17 and APEC E058 grown statically to exponential phase in rich medium, respectively.

overall_design: DNA microarrays were applied to analyze the transcriptomes of UPEC U17 and APEC E058 grown under both in vivo and in vitro conditions.  The source for in vivo-grown bacterial RNA was obtained from bacteria of the infected chicken and inoculated mice, and the source for in vitro-grown bacterial RNA was cultures of UPEC U17 and APEC E058 grown statically to mid-exponential phase at 37°C in LB.

first: Zhao

last: Lixiang

first: Chen

last: Xiang

first: Xu

last: Xiaojing

first: Huan

last: Haixia

first: Gao

last: Song

first: Liu

last: Xiufan


title: In vivo polarization of Ovalbumin (OVA)-specific CD4 (OTII) T cells

status: Public on Jun 27 2008

city: Birmingham

first: Karine

last: Serre

state: West Midland

address: Wolfson drive, Edgbaston

department: Infection and Immunity

country: United Kingdom

institute: University of Birmingham

summary:
There remains a need for analysis of CD4 helper T cells differentiation in vivo. To this end ovalbumin (OVA)-specific CD4 (OTII) T cells transferred into congenic mice were studied. Live attenuated OVA-expressing Salmonella (SalOVA) induce T-bet and IFN-g in OTII cells, while alum-precipitated OVA (alumOVA) induces GATA-3 and IL-4. Although 70% of alumOVA-responding OTII cells express GATA-3, only 7% produce IL-4. Thus Th2-polarization defined solely by IL-4 production does not recognize the diversity of GATA-3-expressing effectors. Low-density arrays were designed to assess the expression of 384 genes by real-time RT-PCR. Extensive early diversification occurred in both responses. SalOVA selectively induced many chemokines and pro-inflammatory cytokines, while alumOVA induced few Th2-associated cytokines. Several cytokines and molecules associated with Th17 cells and follicular helper cells were also induced by both antigens. The transcription factor Helios was exclusively induced in alumOVA-responding OTII cells, and critically not in standard in vitro Th2-polarization systems. Early synchronous up-regulation of Helios and GATA-3 mRNA is paralleled at protein level with largely coincident localization in specific nuclear foci of OTII cells responding to alumOVA. This appears to be consistent with a key role for both transcription regulators in the direction of Th2 responses in vivo.
Keywords: In vivo T cell polarization

overall_design:
Ovalbumin (OVA)-specific CD4 (OTII) T cells were transferred into C57BL/6 mice that were immunized either with live attenuated OVA-expressing Salmonella (Sal) or with alum-precipitated OVA (alum), or not (Naïve).
Gene expression assay was performed on FACS sorted OTII cells (Naïve, Sal, Alum).
OTII cells were purified from three independent groups of ten naïve, or SalOVA-immunized or alumOVA-immunized mice.

first: Elodie

last: Mohr

first: Kai

middle: M

last: Toellner

first: Adam

middle: F

last: Cunningham

first: Samuel

last: Granjeaud

first: Roger

last: Bird

first: Ian

middle: C

last: MacLennan


title: Norepinephrine transporter-deficient neural crest cells

status: Public on Jun 23 2008

city: Newcastle upon Tyne

first: Maya

last: Sieber-Blum

address: Central Parkway Road

department: Institute of Human Genetics and

institute: Newcastle University

summary:
Background: The goal of this study was to determine the transcriptional consequences of norepinephrine transporter (NET) gene deletion in noradrenergic neuron differentiation. The norepinephrine transporter (NET) is the target of powerful mind-altering substances, such as tricyclic antidepressants and the drug of abuse, cocaine. NET function in adult noradrenergic neurons of the peripheral and central nervous systems is that of a scavenger that internalizes norepinephrine from the synaptic cleft. By contrast, norepinephrine (NE) transport has a different role in embryogenesis. It promotes differentiation of neural crest cells and locus ceruleus progenitors into noradrenergic neurons, whereas NET inhibitors, such as the tricyclic antidepressant desipramine and the drug of abuse, cocaine, inhibit noradrenergic differentiation. While NET structure und regulation of NET function is well described, little is known about downstream targets of NE transport.
Results: We have determined by long serial analysis of gene expression (LongSAGE) the gene expression profiles of in vitro differentiating wild type and norepinephrine transporter-deficient (NETKO) neural crest derivatives. Comparison analyses with the wild type library (GSM 105765) have identified a number of important differentially expressed genes, including genes relevant to noradrenergic neuron differentiation and to the phenotype of NETKO mice. Furthermore we have identified novel differentially expressed genes.
Conclusions: Loss of NET function during embryonic development deregulates signaling pathways that are critically involved in neural crest formation and noradrenergic neuron differentiation.

overall_design: The library was constructed from total RNA of 60 neural crest culture at culture day 7. The neural tubes were dissected from E9.5 embryos that lack the norepinephrine transporter gene.

first: Yao

last: Hu

first: Marc

middle: G

last: Caron


title: Circadian Transcriptome of the Chicken Pineal In Vitro

status: Public on Jul 04 2008

city: College Station

first: Phillip

middle: D.

last: Beremand

state: TX

department: Biology

country: USA

institute: Texas A&M University

summary:
Chick pinealocytes exhibit all the characteristics of a complete circadian system, comprising photoreceptive inputs, molecular clockworks and an easily measured rhythmic output, melatonin biosynthesis.  We used microarray analysis to investigate the expression of approximately 8000 genes within cultured pinealocytes subjected to both LD and DD cycles.  We report that a reduced subset of genes were rhythmically expressed in vitro compared to those previously published in vivo, and that gene expression rhythms were lower in amplitude, although the functional distribution of the rhythmic transcriptome was largely similar.  We also investigated the effects of 6-hour pulses of light or of norepinephrine on gene expression in free-running cultures during both subjective day and night.  As expected, both light and norepinephrine inhibited melatonin production; however, the two treatments differentially up- or down-regulated specific sets of genes in a fashion that was dependent upon time of day.  Our combined approach of utilizing a time of day study and a light/NE pulse microarray experiment allowed us to identify novel genes linking clock input to clock function within the pineal.  We identified approximately 30 rhythmic, light-responsive, NE-insensitive genes with no previously known clock function, which may play a role in circadian regulation of the pineal. These are candidates for future functional genomics experiments to elucidate their potential role in pineal physiology.
Keywords: circadian; avian; pineal; light; norepinephrine

overall_design:
Microarray analysis was performed on pineal samples subjected to one of four experimental groups: cultures placed within a 12-hour light:12-hour dark cycle (LD); cultures placed in continuous darkness (DD); cultures  subjected to a 6-hour light pulse beginning at either CT0 or CT12 (i.e., zero or 12 hours after the beginning of subjective day) or that received no light pulse; and cultures subjected to 6 hours of NE treatment beginning at either CT0 or CT12 or that received similarly administered vehicle.

For LD and DD groups, time-course sampling was performed such that 6 samples were collected over the course of a day at 4-hour intervals.  For the light pulse and NE administration groups, 2 samples were collected over the course of a day, either at CT6 or CT18 (i.e., 6 or 18 hours after the beginning of subjective day).

Four biological replicates and two technical replicates (for each biological repeat) were performed for each sample.  For the LD and DD groups, time-points ZT18 and CT18 served as the control reference for the time-course analysis.  For the light pulse experiment, cultures that did not receive light served as the control.  For the NE administration experiment, cultures that received vehicle served as the control.  Dye swaps for performed for experimental samples from the light pulse and NE administration experiments.

first: Stephen

middle: P

last: Karaganis

first: Vinod

last: Kumar

middle: D

first: Michael

middle: J

last: Bailey

first: Terry

middle: L

last: Thomas

first: Vincent

last: Cassone


title: Transcriptome data - Respiratory S. cerevisiae Strain Phenotype is Glucose Insensitive and vital TFs are Hap4 Cat8 Mig1

status: Public on Jun 20 2008

first: Nicklas

last: Bonander

address: Aston University

department: LHS -Life and Health Science

institute: Aston university

summary:
Background
We previously described the first respiratory Saccharomyces cerevisiae strain, KOY.TM6*P, by integrating the gene encoding a chimeric hexose transporter, Tm6*, into the genome of an hxt null yeast. Subsequently we demonstrated the transferability of this respiratory phenotype in the presence of up to 100 g/L glucose to a yeast strain in which only HXT1-7 had been deleted. In this study, we wanted to examine the basis of the respiratory phenotype of the resultant strain, V5.TM6*P, by comparing its transcriptome with that of its parent, V5, at different glucose concentrations.
Results
cDNA array analyses revealed that alterations in gene expression that occur when transitioning from a respiro-fermentative (V5) to a respiratory (V5.TM6*P) strain, are very similar to those in cells undergoing a diauxic shift. Highly complete collections of known genes of the TCA cycle, glyoxylate cycle and respiratory chain were identified, consistent with a respiratory metabolism. We also undertook an analysis of transcription factor binding sites in our dataset by examining previously-published biological data for Hap4, Cat8 and Mig1, and using this in combination with verified binding consensus sequences, to identify genes likely to be regulated by one or more of these transcription factors. Of the induced genes of our dataset, 77 % had binding sites for Hap2/3/5 (Hap4 is an activator of this complex), with 72 % having at least two (the latter set being more induced than the former). This is relevant since Hap4 is known to be involved in the transcriptional activation of respiratory genes and other mitochondrial functions. In addition, 13 % of genes were found to have a binding site for Cat8, which together with its complexes with Sip4 have previously been identified as mediating de-repression of a number of genes during the diauxic shift. Finally, 21 % of genes had a binding site for Mig1 which is a transcriptional repressor involved in glucose repression. Unexpectedly, both the up- and down-regulation of many of the genes in our dataset had a clear glucose dependence in the parent V5 strain that was not present in V5.TM6*P. This important result indicates that the relief of glucose repression is already operable at much higher glucose concentrations than is widely accepted and suggests that glucose sensing might occur inside the cell.
Conclusions
Our dataset gives a remarkably complete view of the involvement of genes in the TCA cycle, glyoxylate cycle and respiratory chain in the expression of the phenotype of V5.TM6*P. Furthermore, 88 % of the transcriptional response of the induced genes in our dataset can be related to the potential activities of just three transcription factors; Hap2/3/5, Cat8 and Mig1. Overall, our data support genetic remodelling in V5.TM6*P consistent with a respiratory metabolism which is insensitive to external glucose concentrations.

overall_design:
Compare a wild type Saccaromyces cerevisise strain V5 strain (=V), with a mutant strain where glucose transport has been reduced by at least 30% in the strain V5.TM6*P (=T), mutant is devoided of 7 hexose transporters HXT1, HXT2, HXT3, HXT4, HXT5, HXT6, HXT7, the plasma membrane contains a HXT1-HXT7 fusion protein. This construct is a combination of a low and high affinity glucose transporter.
The glucose concentration in the culture media of each array is the parameter that can be how the arrays are sorted.
Accession	Strain	Glucose [g/L]
GSM298426	V5.TM6*P	36.5
GSM298477	V5.TM6*P	36.5
GSM298478	V5.TM6*P	34.8
GSM298480	V5.TM6*P	34.8
GSM298483	V5.TM6*P	26.3
GSM298484	V5.TM6*P	26.3
GSM298485	V5.TM6*P	25.1
GSM298487	V5.TM6*P	25.1
GSM298489	V5.TM6*P	13.5
GSM298491	V5.TM6*P	13.5
GSM298492	V5.TM6*P	 5.6
GSM298494	V5.TM6*P	 5.6
GSM298496	V5-wild type	38.4
GSM298497	V5-wild type	38.4
GSM298500	V5-wild type	  35
GSM298501	V5-wild type	  35
GSM298502	V5-wild type	25.5
GSM298503	V5-wild type	25.5
GSM298504	V5-wild type	24.4
GSM298505	V5-wild type	24.4
GSM298506	V5-wild type	10.5
GSM298508	V5-wild type	10.5
GSM298509	V5-wild type	 7.4
GSM298510	V5-wild type	 7.4

first: Roslyn

last: Bill


title: Global Transcriptional Response of Nitrosomonas europaea to Cyanide

status: Public on Jun 21 2008

city: Corvallis

first: Sunhwa

last: Park

state: OR

address: 116 Gilmore

department: Biological & Ecological Eng.

institute: Oregon State University

summary:
Pure cultures of ammonia oxidizing bacterium, Nitrosomonas europaea, are exposed to cyanide in pseudo-steady state batch reactor in presence of ammonia. Nitrosomonas europaea are generally regarded as the most sensitive organism to various inhibitors commonly encountered in the wastewater treatment plants (WWTP). To find stress genes of Nitrosomonas europaea to cyanide known as inhibitor of respiratory process, whole-genome transcript response to cyanide was determined in this research using microarray and qRT-PCR. When 1 uM NaCN inhibits nitrification about 50 %, transcript levels of 35 genes were increased while transcript levels of 29 genes were decreased, showing more than 2-fold in total 2460 genes. moeZ (NE2353), homologue with rhodanases related to detoxification of CN-, showed 7-fold up regulation and gene cluster including moeZ also showed significant up regulation.
Keywords: cyanide, stress response, moeZ

overall_design: The 1 uM NaCN caused more than 50 % inhibition in physiological response for 1 hour incubation. Transcriptional levels of the cells inhibited by cyanide were compared with the cells under control condition.

last: Ely


title: Variation in homeodomain DNA-binding revealed by high-resolution analysis of sequence preferences

city: Toronto

first: Lourdes

last: Pena-Castillo

state: Ontario

address: 160 College St. Room 1350

department: Banting and Best Department of Medical Research

country: Canada

institute: University of Toronto

summary:
Most homeodomains are unique within a genome, yet many are highly conserved across vast evolutionary distances, implying strong selection on their precise DNA-binding specificities. We determined the binding preferences of the majority (168) of mouse homeodomains to all possible 8-base sequences, revealing rich and complex patterns of sequence specificity, and showing for the first time that there are at least 65 distinct homeodomain DNA-binding activities. We developed a computational system that successfully predicts binding sites for homeodomain proteins as distant from mouse as Drosophila and C. elegans, and we infer full 8-mer binding profiles for the majority of known animal homeodomains. Our results provide an unprecedented level of resolution in the analysis of this simple domain structure and suggest that variation in sequence recognition may be a factor in its functional diversity and evolutionary success.
Keywords: Mouse homeodomain protein binding microarrays

overall_design: 178 Protein binding microarray (PBM) experiments of mouse homeodomains were performed, with 10 proteins done in replicate. Briefly, the PBMs involved binding GST-tagged mouse homeodomains to custom-designed, double-stranded 44K Agilent microarrays in order to determine their sequence preferences. The method is described in Berger et al., Nature Biotechnology 2006.  A key feature is that the microarrays are composed of de Bruijn sequences that contain each 10-base sequence once and only once, providing an evenly balanced sequence distribution. Individual de Bruijn sequences have different properties, including representation of gapped patterns. The array sequences as well as the primary array data are available via a EULA at http://the_brain.bwh.harvard.edu/pbms/webworks2/. Here we provide the data transformed into median intensities (after normalization and detrending of the original array data) for all 32,896 8-base sequences, Z-scores for these intensities, and E-scores. E-scores are a modified version of AUC, and describe how well each 8-mer ranks the intensities of the spots. In general the E-scores are slightly more reproducible than Z-scores, but contain less information about relative binding affinity. Additional experimental details are found in Berger et al., Nature Biotechnology 2006, Berger et al., Cell 2008, and the accompanying Supplementary information.

last: Berger

first: Gwenael

last: Badis

first: Andrew

middle: R

last: Gehrke

first: Shaheynoor

last: Talukder

first: Anthony

middle: A

last: Philippakis

first: Trevis

last: Alleyne

first: Sanie

last: Mnaimneh

first: Savina

last: Jaeger

first: Esther

middle: T

last: Chan

first: Olga

middle: B

last: Botvinnik

first: Faiqua

last: Khalid

first: Wen

last: Zhang

first: Daniel

last: Newburger

first: Quaid

last: Morris

first: Martha

last: Bulyk

first: Timothy

last: Hughes


title: HT29 colon cancer cells: siRNA targeted gene knockdowns vs scrambled siRNA control or GFP siRNA control

city: Washington, DC

first: Norman

middle: H

last: Lee

state: DC

address: 2300  I Street, N.W.

department: Pharmacology & Physiology

institute: The George Washington University Medical Center

summary:
Transcriptional profiling of HT29 colon cancer cells comparing siRNA-targeted knockdowns of glutaminase (GLUT), cytokeratin 20 (KER), DEAD/H box polypeptide 32 (DEAD), ribosomal protein L32 (RPL32) and transcription factor DP1 (TFDP1) versus scrambled siRNA control and GFP siRNA control

Keywords: two-color spotted cDNA microarray, siRNA-targeted gene knockdowns

overall_design: 10-condition, two-color hybridization experiment employing a common reference design (Stratagene Universal Human Reference RNA), Scrambled vs. GLUTi-1356 siRNA vs. GLUTi-774 siRNA vs. KERi-1084 siRNA vs. KERi-660 siRNA vs. DEADi-1261 siRNA vs. RPL32i-378 siRNA vs. TFDPi-1196 siRNA vs. TFDPi-680 siRNA vs. Control GFPi-122 siRNA. Biological replicates: 2 for each condition. One replicate per array.

first: Truong

middle: V

last: Luu

first: Bryan

last: Frank

first: Josh

last: Stuart


title: Molecular Profiling of Primitive Hematopoietic Cells Generated In Vitro from Human Embryonic Stem Cells and In Vivo during Embryogenesis

status: Public on Jun 18 2008

city: Vancouver

first: BCGSC

last: BC Cancer Agency

state: BC

address: 675 West 10th Avenue

department: Gene Expression

institute: Canada's Michael Smith Genome Sciences Centre

summary: Human embryonic stem cells (hESCs) offer an important model for investigating the human hematopoietic celldevelopment.  Here, we used long serial analysis of gene expression and quantitative real-time PCR to characterize two subsets of primitive hematopoietic cells derived in vitro from hESCs.  This revealed differences in their expression of genes associated with lymphoid and myeloid development, cellular biosynthetic processes, and cell cycle regulation.  Further comparisons with analogous data for primitive hematopoietic cells isolated from first trimester human fetal liver and newborn cord blood showed a strong similarity between the transcriptomes of the most primitive hESC- and in vivo-derived populations, with the main differences involving genes that regulate HSC development, self-renewal and homing, chromatin remodeling, AP1 transcription complex genes, and non-coding RNAs.  These data suggest that primitive hematopoietic cells are generated from hESCs in vitro by processes similar to those operative during human embryogenesis in vivo, although some differences were also detected.

overall_design: Human embryonic stem cells (hESCs) are capable of indefinite self-renewal but can also be induced to undergo a stepwise process of differentiation into a spectrum of recognizable mature blood cell types. However, a clear understanding of the molecular mechanism by which the first hematopoietic stem cells (HSCs) acquire their unique defining properties of self-renewal and repopulating potential is lacking.  As a first step towards obtaining the information needed to close this gap, we have undertaken a comparative gene expression analysis of different highly purified primitive human hematopoietic subpopulations (erythroid-megakaryocytic progenitor enriched CD43+CD235a+CD41a+/- cells, mutiplepotent progenitor enriched lin-CD34+CD43+CD45-, and lin-CD34+CD43+CD45+ cells) generated either in vitro from hESCs or in vivo from fetal (human fetal liver lin-CD34+CD38- cells) or neonatal hematopoietic primitive cells (human cord blood lin-CD34+CD38- and lin-CD34+CD38+ cells). This involved preparing a long serial analysis of gene expression (LongSAGE) library from an extracts of each prospectively isolated subpopulation and then sequencing each library to a depth of 200,000 tags.

first: Giorgia

last: Salvagiotto

first: Yun

last: Zhao

first: Maxim

last: Vodyanik

first: Victor

last: Ruotti

first: Ronald

last: Stewart

first: Marco

last: Marra

first: James

last: Thomson

first: Connie

last: Eaves

first: Igor

last: Slukvin


title: C. elegans PRG-1 co-IP small RNAs, 5p-end-independent

status: Public on Jun 16 2008

city: San Francisco

middle: Graham

last: Ruby

state: CA

address: 1700 4th Street QB3 s404

department: Biochemistry

institute: UCSF

summary:
These datasets examine the association of small RNAs with 3p hydroxyls (including endo-siRNAs, miRNAs, and 21U-RNAs) with the PRG-1 protein through co-immunoprecipitation.  Two datasets are provided; one sequences small RNAs that co-IP with PRG-1; the other sequences small RNAs from the IP input sample. These RNA samples were prepared using an approach that captures small RNAs independent of the covalent status of their 5p termini, as described in Ambros et al. (2003) Curr Biol 13:807-18.
These datasets were prepared in the Craig C. Mello laboratory.

Keywords: identification of PRG-1-associated small RNAs

overall_design:
CE-PRG1-coIP-Illumina
1 flowcell each for input, PRG-1 co-IP

first: Pedro

last: Batista

first: J

first: Julie

last: Claycomb

first: Rosaria

last: Chiang

first: Noah

last: Fahlgren

first: Kristin

last: Kasschau

first: Daniel

last: Chaves

first: Weifung

last: Gu

first: Jessica

last: Vasale

first: Shenghua

last: Duan

first: Darryl

last: Conte

first: Shujun

last: Luo

first: Gary

last: Schroth

last: Carrington

first: David

last: Bartel

first: Craig

last: Mello


title: Mutant analysis of C. elegans small RNAs with 5p monophosphates

first: James

type: Non-coding RNA profiling by high throughput sequencing

summary:
These datasets profile the expression of small RNAs with 5p monophosphates and 3p hydroxyls (including miRNAs, 21U-RNAs) in young adult worms from the wt, prg-1 mutant, and fog-2 mutant backgrounds.
These datasets were prepared in the Craig C. Mello laboratory using T4 RNA ligase 1.

Keywords: miRNA and 21U-RNA discovery and mutant expression profiling

overall_design:
CE-mutProfile-5pDependent-Illumina
1 flowcell each for the wt, prg-1 mutant, and fog-2 mutant


title: Exon array analysis of resting and activated primary murine CD4+ T-cells

city: Stockholm

first: Rickard

last: Sandberg

address: Berzelius vag 35

department: Department of Cell and Molecular Biology

country: Sweden

institute: Karolinska Institutet

summary:
The stability, localization and translation of mammalian mRNAs are largely determined by sequences in the 3' untranslated region (UTR).  Here, we describe a conserved program of increased upstream polyadenylation site usage following activation of primary murine CD4+ T lymphocytes.  This program, resulting in shorter 3' UTRs, is a characteristic of immune cell activation and strongly correlates with proliferation across diverse cell types and tissues.  Enforced expression of full-length 3' UTRs confers differences in protein expression that can in some cases be eliminated by deletion of predicted microRNA target sites in the variably included region. Together, our data indicate that polyadenylation site usage is coordinately regulated such that states of increased proliferation are associated with widespread reduction of 3' UTR-based regulation.
Keywords: activation, exon array, time course

overall_design: Two or three biological replicates per time point

first: Joel

last: Neilson

first: Philip

last: Sharp

first: Christopher

last: Burge


title: Mutant analysis of C. elegans small RNAs, 5p-end-independent

last: Ruby

summary:
These datasets profile the expression of small RNAs with 3p hydroxyls (including endo-siRNAs, miRNAs, and 21U-RNAs) in young adult worms from the wt and prg-1 mutant backgrounds.  They were prepared using an approach that captures small RNAs independent of the covalent status of their 5p termini, as described in Ambros et al. (2003) Curr Biol 13:807-18.
These datasets were prepared in the Craig C. Mello laboratory.

Keywords: endo-siRNA mutant expression profiling

overall_design:
CE-prg1-endoSiRNAs-Illumina
1 flowcell each for the wt and prg-1 mutant

first: Pedro

last: Batista

first: J

first: Julie

last: Claycomb

first: Rosaria

last: Chiang


title: Genes Differentially Expressed by Aspergillus flavus Strains After Loss of Aflatoxin Production by Serial Transfers

city: Mississippi State

first: Jeff

last: Wilkinson

state: MS

department: Biochemistry and Molecular Biology

institute: Mississippi State University

summary:
Aflatoxins are carcinogenic fungal secondary metabolites. Levels of aflatoxins in agricultural commodities are stringently regulated by many countries. A cluster of genes is responsible for aflatoxin biosynthesis by Aspergillus flavus and other closely related species. Expression of the clustered aflatoxin genes is governed by a complex network of regulatory mechanisms. To better understand the molecular events that are associated with aflatoxin production, transcription profiling by microarray analyses which compared three independent aflatoxigenic A. flavus strains to individual isogenic progenies that no longer produced aflatoxins after serial transfers was carried out. Twenty-two significantly differentially expressed features were identified. After physical mapping using the A. oryzae genome sequence as the reference, the number of unique genes was reduced to 16. Compared to the parental strains, changes in the aflatoxin gene expression levels in the progenies were not significant, which suggests that the inability to produce aflatoxins is not caused by decreased expression. The only gene showing higher expression levels in the progenies is homologous to glutathione S-transferease genes. Overexpression of this gene, named hcc, at six- to nine-fold in an aflatoxigenic A. flavus did not cause discernible changes in colony morphology or aflatoxin production. Loss of aflatoxin production after serial transfers may not result from a single event but caused by multiple factors.
Keywords: Compartiave hybridization toxigenic and atoxigenic lines of Aspergillus

overall_design: Aspergillus flavus NRRL 29459, NRRL 29474, and NRRL 29490 are aflatoxigenic strains originated from soil collection in a peanut field (Terrell Co., Georgia, USA). Strains 459B-20-2, 474A-20, and 499A-20 were nonaflatoxigenic isolates obtained after 20 serial transfers of the parental strains on potato dextrose agar slants (Horn and Dorner 2002).  Comparsions in each experiment consisted of one aflatoxigenic parental strain and one nonaflatoxigenic progeny, compared after 48- or 72-hr growth. Each comparison was repeated with duplicate dye-flip.

first: Perng-Kuang

last: Chang

first: Jeffery

first: Bruce

middle: W

last: Horn

first: Jiujiang

last: Yu

first: Deepak

last: Bhatnagar

first: Thomas

middle: E

last: Cleveland


title: Identification of markers of responsiveness in rheumatoid arthritis patients

status: Public on Jun 19 2008

city: ROUEN

first: Jean

middle: Philippe

last: SALIER

address: Bvd Gambetta

department: Inserm Unit 519

country: France

institute: Faculty of Medicine and Pharmacy

summary:
Despite the increasing of available drugs in RA, the overall rate of non response is 30 to 40% of treated patients. To optimize the drug prescription and better elaborate the therapeutic strategies, gene profiling in PBMCs was used to predict the responsiveness to anakinra. Thirty two patients treated by anakinra (100mg/day s.c.) and méthotrexate, were categorized as responders whenever a change of Disease Activity Score 28 > 1.2 was obtained at 3 months. Blood from treated patients was collected at baseline. In 7 responders and 7 non responders, 51 mRNAs identified by microarray were expressed as a function of the response to treatment and an unsupervised hierarchical clustering perfectly separated responders from non responders.

Keywords: anakinra effect

overall_design: PBMC then RNA were extracted from blood samples of  7 anakinra responders,  7 anakinra non responders and 1 pool of healthy subjects used as control. Each sample was hybridized twice in different nylon membrane. The control sample was hybridized twice in each nylon membrane.

first: C

last: Bansard

first: T

last: Lequerré

first: M

last: Hiron

last: Derambure

first: O

last: Vittecoq

first: A

last: Daragon

first: S

last: Pouplin

first: F

last: Tron

last: Boyer

last: Daveau

first: X

last: LeLoët

last: Salier


title: Comparison between saprotrophic and biotrophic-like mycelia of cacao WBD causal agent Moniliophthora perniciosa I

status: Public on Jun 13 2008

city: Campinas

first: Jorge

middle: Maurício Costa

last: Mondego

state: SP

address: Cidade Universitária Zeferino Vaz s/n

department: Genetics and Evolution

country: Brazil

institute: UNICAMP

summary:
Moniliophthora perniciosa is the causal agent of cacao Witche´s broom disease. This disease has been causing extensive damages to Brazilian cacao plantation, especially in Southern Bahia. Using glass slides microarrays, we analyzed the expression profile of 3872 whole genome shotgun reads from M. perniciosa genome, comparing two stages of development (Biotrophic-like mycelia and saprotrophic mycelia).
Keywords: Moniliophthora perniciosa, Witches Broom Disease, Pathogenesis, Cacao

overall_design: Pools from RNA of axenic cultures were amplified by RNA technique and labeled with Cyanine-3 CTP and Cyanine-5 CTP. Comparison was performed between biotrophic and necrotrophic mycelia, both grown in glycerol with cacao meristem extracts, using saprotrophic mycelia grown in glycerol as a control.

first: Johana

last: Rincones

first: Leandra

middle: M

last: Scarpari

first: Marcelo

middle: F

last: Carazzolle

first: Joan

last: Barau

first: Gustavo

last: Costa

first: Laurival

last: Vilas-Boas

first: Eduardo

last: Formighieri

first: Maricene

last: Sabha

first: Ricardo

last: Azevedo

first: Robson

last: Dias

first: Júlio

last: Cascardo

first: Lyndel

last: Meinhardt

first: Goncalo

last: Pereira


title: Comparison between saprotrophic and biotrophic-like mycelia of cacao WBD causal agent Moniliophthora perniciosa II

middle: G


title: Heterosis(II, Low scan)

city: Ames

first: Patrick

middle: S.

last: Schnable

state: IA

address: 2035B Roy J Carver Co-Lab

institute: Iowa State University

summary:
Heterosis is the phenomenon whereby the progeny of particular inbred lines have enhanced agronomic performance relative to both parents.  Although several hypotheses have been proposed to explain this fundamental biological phenomenon, the responsible molecular mechanisms have not been determined.  The maize inbred lines B73 and Mo17 produce a heterotic F1 hybrid.  Global patterns of gene expression were compared in seedlings of these three genotypes using a microarray that contains 13,999 cDNAs.  Using an estimated 15% false discovery rate as a cut-off, 1,367 ESTs (9.8%) were identified as being significantly differentially expressed among genotypes.  All possible modes of gene action were observed, including additivity, high- and low-parent dominance, under-dominance, and over-dominance.  The largest proportion of the ESTs (78%, 1,062/1,367) exhibited expression patterns that are not statistically distinguishable from additivity.  Even so, 22% of the differentially regulated genes exhibited non-additive modes of gene expression.  Classified on the basis of significant pair-wise comparisons of genotype means, 181 of these 305 genes exhibited high-parent dominance and 23 exhibited low-parent dominance.  In addition, 44 genes exhibited under- or over-dominant gene action.  These findings are consistent with the hypothesis that multiple molecular mechanisms contribute to heterosis, including over-dominance
Keywords: Genotype Comparison

overall_design: The inbreds B73 (Schnable Lab Accession #660) and Mo17 (Schnable Lab Accession #2618) used in this study were derived by self-pollination from stocks originally obtained from Don Robertson (Iowa State University) and Mike Lee (Iowa State University), respectively.  Mo17 was crossed as a female by B73 to generate the F1.  Kernels from three different seed sources (ears) per genotype were used in the experimental design.  Prior to microarray analyses genotypes were confirmed by genotyping DNA extracted from each seed source using co-dominant IDP genetic markers that distinguish B73 from Mo17 (Fu et al., in preparation).  Ten biological replications of B73, Mo17, and their F1 (Mo17xB73) were grown under highly controlled conditions in a randomized complete block design.  For each replication, the B73, Mo17, and F1 samples were hybridized to three two-color cDNA microarrays using a loop design such that each loop included all pairwise comparisons between genotypes.  RNA pools for each genotype were alternately labeled providing dye balance within each loop.  After hybridization, one biological replicate was removed due to poor quality.  The final analysis incorporated 27 microarray slides (3 slides for each of nine high-quality biological replicates).


title: Transcriptomic Analysis of the Effects of Cyclopamine Exposure on Xenopus Limb Development

status: Public on Jun 14 2008

city: New Haven

first: Geffrey

middle: Foresman

last: Stopper

state: CT

address: 165 Prospect St.

department: Department of Ecology & Evolutionary Biology

institute: Yale University

summary:
Study of the tetrapod limb has contributed a great deal to our understanding of developmental pathways and how changes to these pathways affect morphology.  Most data on tetrapod limb development is known from amniotes, with far less known about genetic mechanisms of limb development in amphibians.  To better understand the mechanisms of limb development in anuran amphibians, we use cyclopamine to inhibit Hedgehog signaling at various stages of limb development in Xenopus.  We use transcriptomic analysis following cyclopamine exposure to understand the downstream effects of Hedgehog inhibition on gene expression.  We find many aspects of Hedgehog function appear to be conserved with respect to amniotes, including the responses of ptc genes, gremlin, bmp2, and the autoregulatory property of shh.  We show that, as was proposed based on experiments in chick, Sonic hedgehog plays two distinct roles in limb development – specification of digit number and specification of digit identity.  In contrast to these points of conservation, we find that Hedgehog signaling is required for the maintenance of early limb bud outgrowth in Xenopus, a requirement not known for any other tetrapod.
Keywords: pharmacological signal inhibition response

overall_design: Eight microarray experiments were performed using RNA extracted from batches of hindlimbs dissected from Xenopus laevis tadpoles. Four of these were experimental groups exposed to cyclopamine at 1 µg/ml for 24 hours before animal fixation, and four were experimental groups exposed to the ethanol, the cyclopamine vehicle, at the same concentration used for cyclopamine vehicle for 24 hours. Each pool of RNA consisted of material from 18-22 hindlimb buds. Two experimental groups and two control groups were from one clutch of tadpoles, and the other four pools were from another clutch. Both samples within each of four paired groups (experimental vs. control) were exposed and processed simultaneously.

first: Kathryn

last: Richards-Hrdlicka

first: Günter

middle: P

last: Wagner


title: Ecdysone Response Timecourse in Kc Cells

status: Public on Jun 12 2008

first: Zareen

last: Gauhar

address: 333 Cedar St, 386 NSB

department: Genetics

institute: Yale

summary:
Kc167 Cells were plated at 0.5x106/ml and grown at 23.50C. They were treated with 20-HE (Sigma) at a final concentration of 5x10-7M. The reference sample was total RNA isolated at 0 hr. after treatment.
The experimental samples were total RNA isolated from cells treated for 1, 3, 6, 12, 24, and 48 hours.

overall_design: Samples were taken at 0hr, 1hr, 3hr, 6hr, 12hr, 24hr and 48hr. Briefly, cells were spun down and Total mRNA was extracted with OligodT (Ambion) nd reverse transcribed in the presence of oligodeothymidine and random hexamers and labeled to the whole genome in-house cDNA microarray and ecdysone responsive genes were identified. Ecdysone responsive genes were identified at each timepoint using a Limma t-test.

first: Ling

last: Sun

first: Sujun

last: Hua

first: Tong-Ruei

last: Li

first: Christopher

last: Mason

first: Kevin

last: White


title: Ribosomal protein S6 Rps6 heterozygous null deletion effect on footpad epidermis

city: Stanford

first: Greg

last: Barsh

address: Beckman Center B271A

institute: Stanford University

summary:
Mutant and non-mutant footpad.
McGowan et al. in press
Keywords: Mutant vs. non-mutant

The tissue (footpad epidermis) is from a conditional heterozygous null deletion of Rps6. One copy of Rps6 was deleted from keratinocytes in the skin using the K5.Cre transgene.

overall_design: K5Cre x Rps6^loxP


title: A comprehensive synthetic genetic interaction network governing yeast histone acetylation and deacetylation

city: Baltimore

first: Daniel

last: Yuan

state: MD

address: 733 N. Broadway, Suite 351

department: Molecular Biology and Genetics

institute: Johns Hopkins Univ School of Medicine

summary:
Histone acetylation and deacetylation are among the principal mechanisms by which chromatin is regulated during transcription, DNA silencing, and DNA repair. We analyzed patterns of genetic interactions uncovered during comprehensive genome-wide analyses in yeast to probe how histone acetyltransferase (HAT) and histone deacetylase (HDAC) protein complexes interact. The genetic interaction data unveil an underappreciated role of HDACs in maintaining cellular viability, and led us to show that deacetylation of the histone variant Htz1p at lysine 14 is mediated by Hda1p. Studies of the essential nucleosome acetyltransferase of H4 (NuA4) revealed acetylation-dependent protein stabilization of Yng2p, a potential nonhistone substrate of NuA4 and Rpd3C, and led to a new functional organization model for this critical complex. We also found that DNA double-stranded breaks (DSBs) result in local recruitment of the NuA4 complex, followed by an elaborate NuA4 remodeling process concomitant with Rpd3p recruitment and histone deacetylation. These new characterizations of the HDA and NuA4 complexes demonstrate how systematic analyses of genetic interactions may help illuminate the mechanisms of intricate cellular processes.
Keywords: genetic modification

overall_design: The 44 datasets in this Series profiled the genome-wide genetic interactions for query genes encoding either HAT and HDAC catalytic subunits or subunits of the associated protein complexes. Of the 32 query genes, 5 were essential and were tested as temperature-sensitive (ts) alleles at three or more temperatures. (ESA1 was also tested as a hypomorphic allele.) The other query genes were tested as null deletion alleles derived from the Yeast Knockout strain collection.

first: Yu-yi

last: Lin

first: Yan

last: Qi

first: Jin-ying

last: Lu

first: Xuewen

last: Pan

middle: S

first: Yingming

last: Zhao

first: Joel

last: Bader

first: Jef

middle: D

last: Boeke


title: Expression profiling of cotton (Gossypium barbadense L. Pima S7) fiber transcriptome during the development

city: Lubbock

first: Thea

middle: A

last: Wilkins

address: 2500 Broadway,Campus Box 42122

department: Plant and Soil Sciences

institute: Texas Tech University

summary: To gain novel insights into the molecular mechanisms underlying Pima cotton fiber quality, expression profiling of the Pima fiber transcriptome was performed as a function of development. The profiling was performed at 6 developmental time points: 8, 11, 14, 17, 21, and 24dpa. The expression profiling using long oligonucleotide microarray showed dynamic changes to the fiber transcriptome that correlate to morphogenesis, and biological cellular processes. Differentially expressed genes were identified by being differentially regulated at various stages of fiber development, and they were used to develop stage-specific regulation patterns that correlate the dynamics of gene expression to fiber morphogenesis.

overall_design: A bi-directional double loop hybridization (Kerr and Churchill, 2001) with dye swap experimental design encompassing a total of 28 hybridizations was employed to maximize discovery of significant changes between developmental stages. Self-hybridization controls were conducted to evaluate both within-array correlation and dye bias.

first: Magdy

last: Alabady

first: Bulak

last: Arpat

first: Osama

last: Elshihy


title: Exploiting orthologue diversity for systematic detection of gain-of-function phenotypes

status: Public on Jun 10 2008

city: Candiolo

first: Enzo

last: Medico

state: TO

address: Strada Prov. 142, km 3,95

department: Oncology

country: Italy

institute: Candiolo Cancer Institute, University of Torino

summary:
Background  Systematic search for genes whose gain-of-function by exogenous expression confers an advantage in cell-based selective screenings is a powerful method for unbiased functional exploration of the genome, and has the potential to disclose new targets for cancer therapy. A major limit of this approach resides in the labor-intensive cloning of resistant cells, identification of the integrated genes and validation of their ability to confer a selective advantage. Moreover, the selection has to be drastic and genes conferring a limited advantage are typically missed.     Results  We developed a new functional screening strategy based on transduction of mammalian cells of a given species with an expression library from another species, followed by one-shot quantitative tracing with DNA microarrays of all library-derived transcripts before and after selection. In this way, exogenous transcripts enriched after selection, and therefore likely to confer resistance, are readily detected. We transduced a retroviral cDNA expression library from mouse testis into human and canine cells, and optimized the use of commercial murine gene expression arrays for species-specific detection of library-derived transcripts. We then conducted a functional screening by growing library-transduced canine MDCK cells in suspension, to enrich for cDNAs conferring anchorage independence. Notably, these cells show partial resistance to loss of anchorage, and the selection can be of limited stringency, compromising approaches based on clonal selection or anyway requiring high stringency. Microarray analysis revealed reproducible enrichment after three weeks of growth on polyhema for seven genes, among which the Hras proto-oncogene and Sox5. When individually transduced into MDCK cells, Sox5 specifically promoted anchorage-independent growth, thereby confirming the validity and specificity of the approach.    Conclusions  The procedure described here brings substantial advantages to the field of expression cloning, being faster, more systematic and more sensitive. Indeed, this strategy allowed identification and validation of genes promoting anchorage-independent growth of epithelial cells under selection conditions not amenable to conventional expression cloning.

Keywords: Xenoarray - a barcode-type microarray-based functional screening.

overall_design: 7 samples: one control sample transduced with GFP, and six samples transduced with a mouse testis retroviral expression library, of which three selected for growth in the absence of anchorage. The cells are canine, the expression arrays are murine to trace the abundance of library-derived murine transcripts.

first: Claudio

last: Isella


title: megamiR, microRNA expression profiles during megakaryocyte-erythrocyte progenitor (MEP) differentiation

first: Jun

address: 10 Amistad St

department: Yale Stem Cell Center and Department of Genetics

summary:
This collection contains microRNA expression profiling data for samples purified from umbilical cord blood, belonging to one of the followiing populations: MEP, MEGA1, MEGA2, ERY1, ERY2, ERY3.
MEP: megakaryocyte-erythrocyte precursors (defined by CD34+ CD38+ IL-3Ra- CD45RA- );
MEGA1: megakaryocyte population 1 (defined by CD34+ CD61+ CD41+ CD45- );
MEGA2: megakaryocyte population 2 (defined by CD34- CD61+ CD41+ CD45- );
ERY1: erythrocyte population 1 (defined by CD34+ CD71+ GlyA- );
ERY2: erythrocyte population 2 (defined by CD34- CD71+ GlyA- );
ERY3: erythrocyte population 3 (defined by CD34- CD71+ GlyA+ ).
Keywords: microRNA, miRNA, MEP, megakaryocyte, erythrocyte, lineage specification

overall_design:
Varied numbers of samples were analyzed per population. Each sample came from one donor.

Data were normalized as described (Lu et al., Nature 435, 834-838, 2005) with modifications. Average readings from water-only labeled samples were used for probe-specific background subtraction. Linear normalization among different bead sets for the same sample was performed using readings from 2 post-control probes with equal contribution. Sample normalization was subsequently carried out assuming equal total fluorescence readings.

first: Benjamin

middle: L

last: Ebert

first: Todd

middle: R

last: Golub


title: Gene expression level of six rice lineages

city: NEW HAVEN

first: zhiyu

last: peng

address: 165 Prospect

department: Molecular, Cellular & Developmental Biology

summary: Towards understanding gene expression variation among related rice lineages on a genome-wide scale, we sought to assess global gene expression in the heading-stage panicle using a whole genome oligonucleotide microarray designed to represent 36,926 annotated indica genes.

overall_design:
Using a loop-design, we interrogated gene expression patterns in six related rice lineages, including O. sativa (two Asian cultivars indica and japonica), O. nivara (Asian annual wild rice), O. rufipogon (Asian perennial wild rice) and O. glaberrima (African cultivated rice).
Series_sample_order: Sample 1-12 Slide A; Sample 13-24 Slide B


title: The HAT Inhibitor Anacardic Acid Leads to Changes in Global Gene Expression During in vitro P.falciparum Development

city: Rockville

first: Tetsuya

last: Furuya

institute: National Institutes of Health

summary: To better understand the role of histone lysine acetylation in transcription in Plasmodium falciparum, we sought to attenuate the histone acetyltransferase (HAT) activity of PfGCN5 using anacardic acid (AA). We showed that AA reversibly and noncompetitively inhibited the HAT activity of recombinant PfGCN5. To a lesser extent, AA inhibited the PfGCN5 activity in parasite nuclear extracts, but did not affect the histone deacetylase activity. AA blocked the growth of both chloroquine-sensitive and -resistant strains with a 50% inhibitory concentration of ~30 uM. Treatment of the parasites with 20 uM of AA for 12 h had no obvious effect on parasite growth or gross morphology, but induced hypoacetylation of histone H3 at K9 and K14, but not H4 at K5, K8, K12, and K16, suggesting of specific inhibition of the PfGCN5 HAT. Microarray analysis showed that this AA treatment disturbed the parasite transcription program, resulting in â¥2-fold change in the expression of 271 (~5%) parasite genes in late trophozoites, among which 207 genes were down-regulated. Interestingly, a significant proportion of the down-regulated genes is potentially associated with signal transduction and transcription. Furthermore, cluster analysis of gene expression indicated that AA mostly down-regulated active genes, and this gene pool significantly overlapped with that enriched for H3K9 acetylation. We further demonstrated by chromatin immunoprecipitation and real-time polymerase chain reaction that AA treatment reduced acetylation near the putative promoters of a set of down-regulated genes. The identification of AA as a PfGCN5 inhibitor offers a useful tool for further functional analysis of epigenetics in transcription regulation in malaria parasites.

overall_design: Late trophozoits were selected after AA treatment or untreated for RNA extraction and hybridization on  microarrays. We sought to obtain the expression level changing after AA treatment.

first: Long

last: Cui


title: Developmental timecourse of C. elegans small RNAs with 5p monophosphates

first: James

last: Ruby

summary:
These datasets profile the expression of small RNAs with 5p monophosphates and 3p hydroxyls (including miRNAs, 21U-RNAs) across C. elegans development and in dauer, glp-4 mutant, and mixed-stage wt worms.
These datasets were prepared in the David P. Bartel laboratory using T4 RNA ligase Rnl2(1-249).

Keywords: miRNA and 21U-RNA discovery and developmental expression profiling

overall_design:
CE-devProfile-5pDependent-Illumina
1 flowcell each for Embryo, L1, L2, L3, L4, Adult, Dauer, Glp-4 mutant; 6 flowcells mixed stage

first: Pedro

middle: J

last: Batista

first: J

first: Julie

last: Claycomb

first: Rosaria

last: Chiang

first: Noah

last: Fahlgren

first: Kristin

last: Kasschau

first: Daniel

last: Chaves

first: Weifung

last: Gu

first: Jessica

last: Vasale

first: Shenghua

last: Duan

first: Darryl

last: Conte

first: Shujun

last: Luo

first: Gary

last: Schroth

middle: C

last: Carrington

first: David

last: Bartel

first: Craig

last: Mello


title: Bortezomib resistance in THP-1 cell line

city: Amsterdam

first: Daoud

last: Sie

address: De Boelelaan 1117

department: Pathology

country: Netherlands

institute: Vrije Universiteit Medical Center

summary: Bortezomib is a proteasome inhibitor used in severel different hematological malignancies. Resistance to this drug is still poorly understood. In order get more insight in the resistance mechanism, we developed several bortezomib resistant subclones of the THP-1 monocytic/macrophage cell line. On these subclones expression arrays were performed.

overall_design: We performed expression array three different bortezomib resistant subclones of the THP-1 cell line. The resistant subclones were spotted against the parental THP-1 wildtype cell line.

first: R

last: Oerlemans

first: NE

last: Franke

first: YG

last: Assaraf

last: Cloos

first: I

middle: v

last: Zantwijk

first: CR

last: Berkers

first: GL

last: Scheffer

first: K

last: Debipersad

last: Vojtekova

first: C

last: Lemos

first: JW

last: Heijden

first: B

last: Ylstra

first: GJ

last: Peters

first: GJL

last: Kaspers

first: BAC

last: Dijkmans

first: RJ

last: Scheper

first: G

last: Jansen


title: BK virus infection induces changes of cellular gene expression in Vero cell

status: Public on Jun 11 2008

city: Hsinchu

first: ChengWei

last: Chang

address: 101, Section 2 Kuang Fu Road

department: bmes

country: Taiwan

institute: nthu

summary:
The general aim of this paper is to explore a little further into molecular biology of the interaction between virus and host cell. We seek to use cDNA microarray technology, one revolutionary methodological advance, to perform a high throughput analysis of cDNA clones with the intent of identifying genes expressed in association with the infection with BKV in Vero cells (green monkey kidney cells). This technology, which can supply quantitative expression information for many thousands of genes simultaneously, has been used to classify the cellular genes at transcript levels in different physiological states of a tissue or cell. Accordingly, in this study, we have constructed a cDNA microarray containing 32448 spots (quadruplication of 7,334 human cDNAs and doping controls) to study that BKV infection of Vero cells. Our purposes were (i) to appraise whether cDNA microarrays could be employed to investigate the expression of cellular genes during the 5 time courses (5, 10, 15, 19 and 25 days post-infection (dpi)) of infection with BKV, (ii) to identify any genes that resulted in up-regulation or down-regulation of cell gene expression transcription, (iii) to determine the etiological role of BKV in nephropathy and/or neoplasia, (iv) to form a clearer picture of virus-associated pathophysiology in kidney.
Keywords: BK virus, BKV, cDNA microarray, Vero cell

sample_organism:
Homo sapiens
Betapolyomavirus hominis

overall_design:
We used a loop design in this study. cDNA microarray experiment consisted of ten RNA samples, including BKV-infected samples and their corresponding uninfected controls in 5 different time points.
See supplementary PDF file for additional information

first: HuaKuo

last: Lin

first: DeChing

first: TsungHsiung

last: Hseu

first: Ian

last: Hsu


title: Concentration dependent gene expression changes in Daphnia magna exposed to metals

status: Public on Jun 09 2008

city: Boston

first: Helen

last: Poynton

state: MA

address: 100 Morrissey Blvd.

department: School for the Environment

institute: UMass Boston

summary:
To determine dose-dependent effects of metal exposure we performed microarray hybridizations with RNA isolated from Daphnia magna following Cu, Cd, and Zn exposures over a range of concentrations that included a tolerated concentration, a sublethal concentration, and a highly toxic concentration.  The gene expression profiles revealed effects to digestion related genes, immune related genes, metallothioneins, and oxidative stress genes at the higher concentrations.  We also observed that the highest concentrations produced less specific gene expression profiles than the lower concentrations suggesting a more general stress response at the higher concentrations.  The lowest concentration tested, representing tolerated concentrations of the metals, caused differential expression of only a few genes and were distinct from the expression profiles of the higher concentrations.  This result provides support for the presence of a No Observed Transcriptional Effect Level (NOTEL) for metal exposure in D. magna and suggests that gene expression analysis may offer a strategy for distinguishing between toxic and nontoxic concentrations of metals in the environment.
Keywords: ecotoxicogenomic exposure study

overall_design: We exposed Daphnia magna to 2 different concentrations of Cd and Zn and 3 different concentrations of Cu for 24 hours. For each exposure condition, we performed 3 exposures and 2 technical replicates (as dye swap) for each exposure (6 microarrays total).  All exposures were compared to a unexposed laboratory control (COMBO media).

first: Julia

last: Varshavsky

first: Bonnie

first: Alex

middle: V

last: Loguinov

first: Chris

last: Vulpe


title: Gene Expression changes induced by exposure to field water samples collected at Greenhorn Mine, CA

summary:
Our research goal is to illustrate the potential of gene expression profiling to discriminate between polluted and non-polluted field sites and predict the presence of a specific contaminant.  Using a gene expression analysis, we challenged our custom Daphnia magna cDNA microarray to determine the presence of a specific metal toxicant in blinded field samples collected from two copper mines in California.  We compared the gene expression profiles from our field samples to previously established expression profiles for Cu, Cd, and Zn.  The expression profiles from the Cu containing field samples clustered with the Cu specific gene expression profiles.  Many of the previously discovered copper biomarkers were also differentially expressed in the field samples, suggesting that gene expression analysis is capable of producing robust biomarkers of exposure, which can be validated in field studies.  In addition, our study revealed that upstream field samples containing undetectable levels of Cu caused the differential expression of only a few genes, lending support for the concept of a No Observed Transcriptional Effect Level (NOTEL).  If confirmed by further studies, the NOTEL may play an important role in discriminating polluted and non-polluted sites in future monitoring efforts.
Keywords: ecotoxicogenomic exposure study

overall_design: We collected water samples from 4 sites at Greenhorn Mine.  Each site was contaminated with a different amount of Cu. For each site, we performed 3 exposures and 2 technical replicates (as dye swap) for each exposure (6 microarrays total).  All field sample exposures were compared to a unexposed laboratory control (COMBO media).

first: Rick

last: Zuzow


title: Focused gene expression microarray of tumors from 596 men with prostate cancer using RNA from archival FFPE tissue

city: Rochester

first: Robert

middle: B.

last: Jenkins

state: MN

address: 200 First Street SW

department: Laboratory Medicine and Pathology

institute: Mayo Clinic

summary:
After therapy for prostate cancer many men develop a rising PSA.  Such men may develop a local or metastatic recurrence that warrants further therapy.   However many men will have no evidence of disease progression other than the rising PSA and will have a good outcome.  A case-control design, incorporating test and validation cohorts, was used to test the association of gene expression results with outcome after PSA recurrence.
Since clinical training and validation analysis requires tissue from patients with sufficient follow-up time, for this study we sampled individuals from the Mayo Radical Retropubic Prostatectomy (RRP) Registry.  The registry consists of a population of men who received prostatectomy as their first treatment for prostate cancer at the Mayo Clinic (For a current description and use of the registry; see PMID: 17418069).  As systemic progression is relatively infrequent, we designed a case-control study nested within a cohort of men with a rising PSA.  Between 1987-2001, inclusive, 9,989 previously-untreated men had RRP at Mayo. On follow-up, 2,131 developed a rising PSA (>30 days after RRP) in the absence of concurrent clinical recurrence.  PSA rise was defined as a follow-up PSA >= 0.20 ng/ml, with the next PSA at least 0.05 ng/ml higher or the initiation of treatment for PSA recurrence (for patients whose follow-up PSA was high enough to warrant treatment).  This group of 2,131 men comprises the underlying cohort from which SYS cases and PSA controls were selected.
Within 5 years of PSA rise, 213 men developed systemic progression (SYS cases), defined as a positive bone scan or CT scan.    Of these, 100 men succumbed to a prostate cancer-specific death, 37 died from other causes and 76 remain at risk.
PSA recurrence controls (213) were selected from those men without systemic progression within 5 years after the PSA rise and were matched (1:1) on birth year, calendar year of PSA rise and initial diagnostic pathologic Gleason score (<=6, 7+).   Twenty of these men developed systemic progression greater than 5 years after initial PSA rise and 9 succumbed to a prostate cancer-specific death.
A set of 213 No Evidence of Disease (NED) Progression controls were also selected from the Mayo RRP Registry of 9,989 men and used for some comparisons. These controls had RRP from 1987-1998 with no evidence of PSA rise within 7 years of RRP.  The median (25th, 75th percentile) follow-up from RRP was 11.3 (9.3, 13.8) years.   The NED controls were matched to the systemic progression cases on birth-year, calendar year of RRP and initial diagnostic Gleason Score.  Computerized optimal matching was performed to minimize the total distance between cases and controls in terms of the sum of the absolute difference in the matching factors (PMID: 8728456).
All paraffin-embedded blocks from eligible men were identified and each block was surveyed for the tissue present (primary and secondary Gleason cancer regions, normal and metastatic lymph nodes, etc.).  Of the 639 eligible patients, paraffin blocks were available on 623 (97.5%).  Similarly, RNA was successfully isolated and the DASL assays successfully performed on a very high proportion of patients/specimens:  Usable RNA was prepared from all 623 blocks, and the Cancer Panel and custom prostate cancer panel DASL arrays were both successful on 596 RNA specimens (95.7% of RNAs; 93.3% of design patients).
Keywords: outcome prediction

overall_design: 596 total cases (n=200, 201, 195 for systemic disease progression, PSA recurrence and no evidence of disease groups).

first: Thomas

last: Kollmeyer

first: Tohru

last: Nakagawa

first: Bruce

middle: W

last: Morlan

first: S

middle: K

last: Anderson

first: Eric

last: Bergstrahl

first: Brian

last: Davis

first: Yan

last: Asmann

first: Julie

last: Cunningham

first: George

last: Klee

first: Karla

last: Ballman

middle: B


title: Gene Expression changes induced by exposure to field water samples collected at Walker Mine, CA

first: Helen

overall_design: We collected water samples from 4 sites at Walker Mine.  Each site was contaminated with a different amount of Cu. For each site, we performed 3 exposures and 2 technical replicates (as dye swap) for each exposure (6 microarrays total).  All field sample exposures were compared to a unexposed laboratory control (COMBO media).

middle: G


title: Activation of peroxisome proliferator-activated receptor alpha in human peripheral blood mononuclear cells

status: Public on Jun 06 2008

city: Wageningen

first: Guido

last: Hooiveld

address: HELIX, Stippeneng 4

department: Div. Human Nutrition & Health

institute: Wageningen University

summary:
Background: Peripheral blood mononuclear cells (PBMCs) are relatively easily obtainable cells in humans. Gene expression profiles of PBMCs have been shown to reflect the pathological and physiological state of a person. Recently, we showed that the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα) has a functional role in human PBMCs during fasting. However, the extent of the role of PPARα in human PBMCs remains unclear. In this study, we therefore performed gene expression profiling of PBMCs incubated with the specific PPARα ligand WY14,643. Results: Incubation of PBMCs with WY14,643 for 12 hours resulted in a differential expression of 1,373 of the 13,080 genes expressed in the PBMCs. Gene expression profiles showed a clear individual response to PPARα activation between six healthy human blood donors, which was not the result of the nutritional status of the donors. Pathway analysis showed that genes in fatty acid metabolism, primarily in β-oxidation were up-regulated upon activation of PPARα with WY14,643, and genes in several amino acid metabolism pathways were down-regulated. Conclusions: This study shows that PPARα in human PBMCs regulates fatty acid and amino acid metabolism. In addition, PBMC gene expression profiles show individual responses to WY14,643 activation. We show that PBMCs are a suitable model to study changes in PPARα activation in healthy humans.
Keywords: metabolic state analysis

overall_design: PBMCs from six healthy Caucasian male blood donors, aged between 30 and 48 yr, were isolated directly after arrival of the buffy coat (max. 8 hours after donation) by Ficol-paque Plus density gradient centrifugation (Amersham Biosciences, Roosendaal, the Netherlands). PBMCs were incubated in RPMI1640 medium with 2 mmol/L L-glutamine, 10% fetal bovine serum and antibiotics (penicillin and streptomycin) in the presence of 5% CO2 at 37°C. at 1.0 x 106 cells per ml with either WY14,643 (50 μM) or vehicle (DMSO, 0.1%) for 12 hours. Total RNA from PBMCs was labeled using a one-cycle cDNA labeling kit (Affymetrix Inc, Santa Clara, CA) and hybridized to Affymetrix Human whole genome U133 plus 2.0 arrays (Affymetrix). Sample labeling, hybridization to chips and image scanning was performed according to the manufacturer’s GeneChip Expression Analysis Technical Manual (Affymetrix).

first: Mark

last: Bouwens

first: Lydia

last: Afman

first: Michael

last: Müller


title: Expression profiling of cotton (Gossypium hirsutum L. TM1) fiber transcriptome during the development

first: Thea

last: Wilkins

summary: In order to study gene expression at the genomic level during elongation and secondary cell wall synthesis of upland cotton fiber, oligonucleotide microarrays were employed. RNA was isolated from fibers in 7 different time points beginning prior to peak fiber expansion, continuing through termination of fiber expansion and ending at peak cellulose synthesis (5, 8, 10, 14, 17, 21, and 24dpa). The arrays contained ~25,000 oligonucleotides representing ~12,200 genes designed from a fiber EST database during peak cell expansion. Dynamic changes in gene expression were analyzed in a developmental context to identify stage-specific biological processes and pathways likely to be crucial to cell polar elongation or cellulose biosynthesis and secondary cell wall biogenesis. Genes with significant changes in expression relative to any preceding time point were identified (moderated t-statistics, adjusted p-value <0.05) for each developmental time point  with an expected false discovery rate for multiple testing <5%

overall_design: A bi-directional double-loop experimental design  was adopted for the microarray analysis (Kerr and Churchill, 2001; Glonek, 2004) to analyze all possible significant changes in gene expression between any two developmental time points,. The double loop design guarantees that the two time points before and after each individual time point will have direct comparisons with multiple paths available to compare any two points. Self-hybridization control experiments between independent RNA isolations for each developmental stage demonstrated a high degree of reproducibility

first: Brad

last: Sickler

first: Bulak

last: Arpat


title: Microarray analaysis of adult and childhood renal tumors.

status: Public on Jun 05 2008

city: Grand Rapids

last: Kort

state: MI

address: 333 Bostwick NE

institute: Van Andel Research Institute

summary:
Analysis of adult and childhood tumors reveals activation of an E2F3 signature unique to Wilms tumors.
Keywords: Disease state analysis

overall_design: Clear cell, chromophobe, papillary (types 1 and 2), oncocytoma, and Wilms tumor were compared to normal kidney tissue.  No technical replicates.


title: miRNA profiling of adult and pediatric renal tumors

summary:
Profiling of adult and pediatric renal tumors reveals that genome wide miRNA expression patterns are unique to each tumor subtypes.
Keywords: Disease state analysis

overall_design: Samples from Clear Cell RCC, Papillary RCC, Chromophobe RCC, Oncocytoma, and Wilms Tumor compared to pooled normal kidney samples from these same patients.  No technical replicates.


title: Transcriptional analysis of C. jejuni response to FQ treatment

first: Qijing

last: Zhang

address: 1600 S 16th Street

department: VMPM

summary:
Fluoroquinolones (FQs) are one class of commonly used antimicrobials in clinical therapy of diseases caused by Campylobacter and other foodborne pathogens. The molecular mechanisms underlying the response of Campylobacter to FQ treatment is of interest because unlike other enteric bacteria, Campylobacter lacks many genes encoding components of DNA repair systems and SOS response. To understand how Campylobacter survive antibiotic treatment, DNA microarray was used to compare the gene expression profiles of C. jejuni 11168 in the presence and absence of ciprofloxacin. Our analysis identified 45 genes that showed ≥1.5-fold (p<0.05) changes in expression in the presence of ciprofloxacin. The up-regulated genes (13) are involved in cell membrane biosynthesis and DNA repair or have unknown functions, while the majority of the down-regulated genes (32) are involved in cellular process and energy metabolism. These findings suggest that C. jejuni modulates membrane biosynthesis, increases spontaneous mutation rates, and decreases metabolism in response to fluoroquinolone treatment.
Keywords: 0.06 µg/ml (0.5X MIC) or 1.2 µg/ml (10X MIC) ciprofloxacin treated campylobacter VS. untreated campylobacter, the bacterial cells were treated for 0.5 hour

overall_design: The design utilized a commercially available two color microarray slide for the entire transcriptome of Campylobacter jejuni. Six hybridizations were performed each with independently extracted samples of either untreated C. jejuni cDNA samples or ciprofloxacin-treated (0.06 µg/ml or 1.2 µg/ml) C.jejuni cDNA samples. A dye swap was utilized to help minimize dye dependent bias. Thus there were six biological replicates of each sample.

first: Jing

last: Han


title: Regulation of gene expression in Streptococcus gordonii by coaggregation with Actinomyces naeslundii

status: Public on Jun 02 2008

city: Newcastle upon Tyne

first: Nicholas

last: Jakubovics

address: Framlington Place

department: School of Dental Sciences

institute: Newcastle University

summary:
Oral streptococci, including Streptococcus gordonii, and Actinomyces naeslundii, are consistently found to be the most abundant bacteria in the early stages of dental plaque accumulation. These organisms interact physically (coaggregate) in vitro and in vivo. We hypothesized that coaggregation between S. gordonii and A. naeslundii leads to changes in gene expression in the partner organisms. Furthermore, we predicted that coaggregation-induced changes in phenotype contribute to the success of streptococci and actinomyces in dental plaque. To assess the responses of S. gordonii to coaggregation with A. naeslundii, RNA was extracted from S. gordonii cells 3 h after inducing coaggregation with A. naeslundii or from equivalent S. gordonii monocultures. The two RNA populations were reverse transcribed and compared by competitive hybridization with an S. gordonii genomic microarray. The most striking feature of the response to coaggregation was a profound change in expression of S. gordonii genes involved in arginine biosynthesis and transport. Subsequent experiments demonstrated that coaggregation with A. naeslundii stabilizes arginine biosynthesis in S. gordonii and enables growth under low-arginine conditions, such as those present in human saliva.
Keywords: Cell-cell interaction

overall_design: The S. gordonii microarrays consist of 2195 70-mer oligonucleotides representing 2151 open reading frames, each repeated six times on the array. Chemically defined medium (CDM), was based in Tereleckyj’s FMC with minor modifications (Jakubovics et al., 2008). For coaggregate cultures, concentrated suspensions of S. gordonii DL1 (Challis) and A. naeslundii MG1 in CDM were mixed, vortexed and diluted to 1 x 108 cfu/ml. Monocultures were set up identically, except that A. naeslundii cells were omitted. Cultures were incubated at 37oC for 3 h prior to harvesting and extraction of total RNA. Purified RNA was reverse transcribed and cDNAs were labelled with Cy3 or Cy5 dye. cDNAs from coaggregate cultures and from S. gordonii monocultures were competitively hybridized with the S. gordonii microarray. Three independent sets of cultures were used, and flip dye pairs were included for two of the biological replicates (ie 5 hybridizations in total). In control experiments, cDNA derived from A.naeslundii monocultures did not hybridize with the S. gordonii microarrays. Data represent the ratios of gene expression in coaggregated S. gordonii compared with S. gordonii monocultured cells.

first: Steven

last: Gill

first: Stacey

middle: E

last: Iobst

first: M

middle: M

last: Vickerman

first: Paul

last: Kolenbrander


title: Understanding Vitamin D resistance using Nonsense Mediated Decay arrays

status: Public on Jun 01 2008

first: Daoud

last: Sie

summary:
Vitamin D is the strongest known natural anti-proliferative. A large number of studies in a wide spectrum of cancers, including epidemiological, in vitro and animal models, demonstrate that the active form of Vitamin D has anti-cancer benefits, affecting both progression and metastasis. Alike the role in calcium Vitamin D regulation, its anti-proliferative effect is thought to function through the Vitamin D receptor (VDR), although convincing evidence is lacking. Notwithstanding, separation of the calcemic and the anti-proliferative activity of Vitamin D analogues has been a major obstacle in developing new drugs for the treatment of cancer. The work presented attempts to unveil the molecular mechanism behind the anti-proliferative action of Vitamin D using genomic tools. For that purpose four independently developed Vitamin D sensitive/resistant MCF7 cell line pairs were collected. These unique biological replicates enabled us, both to increase the power of our study and to omit the use of Vitamin D. We deem this omission crucial since in the presence of Vitamin D only downstream genes involved in proliferation and cell cycle would be identified rather than causal resistance genes. The use of a variety of genomic techniques including expression, NMD and oligo CGH arrays reveal in the resistant cell lines the 11q13-14 as a region of DNA copy number loss and an altered expression of EGFR signaling pathway genes. Surprisingly, no genes known from calcium Vitamin D regulation were identified, nor did the VDR silencing by RNAi induce resistance to the sensitive cell lines.
Keywords: Expression microarray, NMD array, cell type comparision

overall_design: Several MCF7 breast tumor cell lines independently derived from the human breast cancer cell line, both resistant and sensitive to Vitamin D, were used. The pairs of cell lines (parental-resistant) were developed in different laboratories and using different methodologies; while some cell lines acquired resistance by long exposure to the physiological concentration of Vitamin D (100nM), others were induced to resistant by being exposed to increasing amounts of Vitamin D. A total of 4 microarray expression experiments were carried out. In all microarray experiments RNA from the Vitamin D resistant cell line treated with Emetine was hybrizided agains RNA from the respsective sensitive/parental cell line treated with emetine.

first: Jose

last: Costa

last: Eijk

last: van de Wiel

first: Derk

last: ten Berge

first: Fernando

last: Schmitt

first: Carmen

last: Narvaez

first: JoEllen

last: Welsh

first: Bauke

last: Ylstra


title: Identification of RNAs that are physically and functionally associated with RNase P

city: Lexington

first: Gregg

middle: Brooks

last: Whitworth

state: VA

address: 204 West Washington St

department: Biology

institute: Washington and Lee Unversity

summary:
Numerous RNAs copurify with RNase P and are affected by temperture sensitive mutations in conserved residues with the essential RNA and protein subunits.  Specifically, RNase P physically interacts with ribosomal protein mRNAS and the intron-encoded box C/D snoRNAs.
Keywords: RNA copurification, temperature sensitive mutants

overall_design: ORF and intergenic regions were analyzed in order to determine which RNAs were affected in RNase P strains.

first: Daniel

last: Coughlin

first: Jeffrey

last: Pleiss

first: Scott

last: Walker

first: David

last: Engelke


title: Gene alterations by PPAR-gamma agonists in human colorectal adenocarcinoma HCT-116 cells

city: Knoxville

first: Seung

middle: Joon

last: Baek

state: TN

address: 2407 River Dr

department: Pathobiology

institute: University of Tennessee

summary:
We examined the alternation of gene expression by MCC-555, rosiglitazone (RGZ) and 15-deoxy-Δ12, 14-prostaglandin J2 (PGJ2) in human colorectal adenocarcinoma HCT-116 cells.
Keywords: effects of PPAR-gamma agonists

overall_design: Common control: DMSO as a vehicle treatement in human colorectal adenocarcinoma HCT-116 cells; Samples: Three PPAR-gamma ligand treatments in HCT-116 cells.

first: Maria

last: Cekanova

first: Joshua

middle: S

last: Yuan

first: Xiuoon

last: Li


title: A compendium of antibiotic-induced transcription profiles reveals broad regulation of P. multocida virulence genes

city: Schwabenheim

first: Elena

last: Melnikow

address: Zur Propstei

department: Drug Discovery

country: Germany

institute: Intervet Innovation GmbH

summary:
Treatment of bacteria with antibiotics at or close to the inhibitory concentration leads to specific transcriptional responses often affecting target genes and targets pathways. A dataset of transcriptional profiles (compendium) induced by antibiotics with known mode-of-action (MoA) can be used to gain information on the putative MoA of novel substances with unknown MoAs.
We used a Pasteurella multocida microarray to generate a compendium of transcriptional profiles and to obtain information on the putative MoA of a novel antibiotic compound. We also show a strong impact of the bacteriostatic antibiotics on P. multocida virulence gene transcription.
Keywords: antibiotica treatment, time course

platform_organism: Pasteurella multocida subsp. multocida str. Pm70

overall_design: Midlog-grown cultures of P. multocida were treated for 10 or 30 min with 8 different antibiotics and one novel compound (thiazin) at minimal inhibitory concentrations (MICs) and were harvested. Control bacteria were not-treated and harvested at approximately the same optical density an OD578 of ~ 0.5. Total RNA was extracted from these samples and labelled with biotin. P. multocida whole genome transcriptional profiling was performed by hybridization on the custom-made Affymetrix microarray according to the manufacturer’s instructions. The experiments were done in triplicates.

first: Caroline

last: Schoenfeld

first: Volker

last: Spehr

first: Ralf

last: Warrass

first: Nikolas

last: Gunkel

first: Michael

last: Duszenko

first: Paul

last: Selzer

first: Heinz

last: Ullrich


title: Norway Spruce Cultured Cells Chitosan & C.polonica Stress

city: Vancouver

first: Joerg

last: Bohlmann

department: Michael Smith Laboratories

institute: University of British Columbia

summary:
Conifer-specific responses to elicitation with the chemical elicitor chitosan have been investigated using a transcriptome analysis in Norway spruce using a cell suspension culture system that has been previously described (Phillips, Walter et al. 2007). This study has demonstrated that the early events following chitosan elicitation include calcium mediated signaling and an oxidative response that have not previously been described in intact trees.

Keywords: stress response

overall_design: A factorial hybridization design with dye balance was chosen to assess gene expression among the untreated control cells, the four time points of cells treated with chitsan (i.e., 15 min, 30 min, 45 min and 2h), and cells treated with the heat-killed Ceratocystis polonica fungus (30 min).  Each of the eight biological replicates for each treatment and time point were hybridized only once for a total of 24 hybridizations.

first: Steven

last: Ralph

first: Dustin

last: Lippert

middle: A

last: Phillips

first: Jonathan

last: Gershenzon

first: Rick

last: White

first: Cristoph

last: Borchers


title: Conserved tissue expression signatures of intronic noncoding RNAs transcribed from human and mouse loci

city: São Paulo

first: Eduardo

last: Reis

address: Av. professor Lineu Prestes, 748

department: Departamento de Bioquímica

institute: Instituto de Química da Universidade de São Paulo

summary:
It has been postulated that noncoding RNAs (ncRNAs) are involved in the post-transcriptional control of gene expression, and may have contributed to the emergence of the complex attributes observed in mammalians. The complement of ncRNAs expressed from intronic regions of the human and mouse genomes comprises at least 78,147 and 39,660 transcriptional units, respectively. To identify conserved intronic sequences expressed in both humans and mice, we used custom-designed human cDNA microarrays to separately interrogate RNA from mouse and human liver, kidney and prostate tissues. An overlapping tissue expression signature was detected for both species, comprising 198 transcripts; among these, twenty two RNAs map to intronic regions with evidence of evolutionary conservation in humans and mice. Transcription of selected human-mouse intronic ncRNAs was confirmed using strand specific RT-PCR. Altogether, these results support an evolutionarily conserved role of intronic ncRNAs in human and mouse, which are likely to be involved in the fine tuning of gene expression regulation in different mammalian tissues.
Keywords: tissue expression pattern

sample_organism:
Homo sapiens
Mus musculus

overall_design:
1.	Experiment Design:

1.1.	Type of experiment:
The experiments described here aim to generate expression profiles from human and mouse  prostate, kidney and liver tissue samples using a custom-designed cDNA microarray platform with 4,608 unique elements in replicate (9,216) enriched in gene fragments that map to intronic regions of known human genes. The platform consists of 3,355 probes of human cDNA fragments that map in the human genome sequence either to intronic regions of known genes (822), to unannotated regions of the human genome (241) or to known exons of RefSeq genes (2,292). We separately compared the expression profiles obtained from human and mouse samples  to identify sequence conserved tissue-specific intronic transcripts.

1.2.	Experimental factors:
Tissues from liver, prostate and kidney from five adult male mice of the Balb/c strain were obtained and frozen in liquid nitrogen. Likewise, five non-tumor samples of human prostate, kidney or liver from our tumor bank were selected. All samples were obtained from patients who signed informed consent, and approval was received from the ethics committees of the hospitals. Total RNA was purified from tissues and treated with DNAse to reduce the levels of genomic DNA contaminant. Either human and or mouse samples from for each tissue from the five individuals were separately pooled using equal quantities of RNA.

1.3.	The number of hybridizations performed in the experiment.
RNA pools from each tissue sample was hybridized twice, performing a total of 18 hybridizations.

1.4.	The type of reference used for the hybridizations, if any.
All hybridizations included external mRNA spikes for quality control of the experiments (detailed below in section 2.5). Hybridizations were performed as single color experiments.

1.5.	Hybridization design:
Three aliquots of 15ug of the pool of total RNA from each tissue was labeled with Cy5-dUTP after reverse transcription (CyScribe first strand cDNA labeling kit, Amershan Biosciences) and hybridizations were carried out using all labeled-target in an Automate Slide Processor (Amershan Biosciences). Images were obtained using a Generation III Scanner (Amersham Biosciences). The human and mouse obtained expression data were independently analyzed using a highly stringent methodology, including different statistical approaches (see below for a detailed description).

1.6.	Quality control steps taken:
Integrity of total RNA was checked by electrophoresis using the Bio Sizing  total RNA Nano assay in the 2100 Bioanalyzer (Agilent Technologies).
Three replicates of each experiment were performed, as describe in the previous section.

first: Rodrigo

last: Louro

first: Tarik

last: El-Jundi

first: Helder

middle: I

last: Nakaya

first: Sergio

last: Verjovski-Almeida


title: Understanding Vitamin D resistance using expression microarrays

first: Daoud

last: Sie

summary:
Vitamin D is the strongest known natural anti-proliferative. A large number of studies in a wide spectrum of cancers, including epidemiological, in vitro and animal models, demonstrate that the active form of Vitamin D has anti-cancer benefits, affecting both progression and metastasis. Alike the role in calcium Vitamin D regulation, its anti-proliferative effect is thought to function through the Vitamin D receptor (VDR), although convincing evidence is lacking. Notwithstanding, separation of the calcemic and the anti-proliferative activity of Vitamin D analogues has been a major obstacle in developing new drugs for the treatment of cancer.
	The work presented attempts to unveil the molecular mechanism behind the anti-proliferative action of Vitamin D using genomic tools. For that purpose four independently developed Vitamin D sensitive/resistant MCF7 cell line pairs were collected. These unique biological replicates enabled us, both to increase the power of our study and to omit the use of Vitamin D. We deem this omission crucial since in the presence of Vitamin D only downstream genes involved in proliferation and cell cycle would be identified rather than causal resistance genes. The use of a variety of genomic techniques including expression, NMD and oligo CGH arrays reveal in the resistant cell lines the 11q13-14 as a region of DNA copy number loss and an altered expression of EGFR signaling pathway genes. Surprisingly, no genes known from calcium Vitamin D regulation were identified, nor did the VDR silencing by RNAi induce resistance to the sensitive cell lines.
Keywords: Expression microarray, cell type comparision

overall_design: Several MCF7 breast tumor cell lines independently derived from the human breast cancer cell line, both resistant and sensitive to Vitamin D, were used. The pairs of cell lines (parental-resistant) were developed in different laboratories and using different methodologies; while some cell lines acquired resistance by long exposure to the physiological concentration of Vitamin D (100nM), others were induced to resistant by being exposed to increasing amounts of Vitamin D. A total of 14 microarray expression experiments were carried out, including four biological replicates and 10 technical replicates including dye swaps. In all microarray experiments RNA from the Vitamin D resistant cell line was hybrizided agains RNA from the respsective sensitive/parental cell line.

first: José

middle: L

last: Costa

last: Eijk

first: Mark

last: van de Wiel

first: Derk

last: ten Berge

first: Fernando

last: Schmitt

first: Carmen

last: Narvaez

first: JoEllen

last: Welsh

first: Bauke

last: Ylstra


title: Development of a 37K High-density oligo-nucleotide microarray for rainbow trout

city: Morgantown

first: Mohamed

last: Salem

state: WV

address: Division of Animal and Nutritional Sciences, West Virginia University

institute: West Virginia University

summary:
We have constructed a rainbow trout high-density oligonucleotide microarray by using all the available tentative consensus (TC) sequences from the Rainbow Trout Gene Index database (The Computational Biology and Functional Genomics Lab., Dana Farber Cancer Institute and Harvard School of Public Health).  The Rainbow Trout Gene Index integrates research data from all available international rainbow trout genomic research projects. The newly designed microarray incorporates 37,394 unique transcript-specific oligonucleotide probes, 60-mer long each. The microarray was printed according to our design by Agilent Technologies using the 4 X 44-design format and contains 1417 Agilent control spots.  The performance of the new microarray platform was evaluated by analyzing gene expression associated with the rainbow trout vitellogenesis-induced muscle atrophy.  These chips can be ordered from Agilent using design number 016320. This microarray is anticipated to open new avenues of research that will aid in the development of novel strategies to enhance growth efficiency and quality in salmonid species.
Keywords: Development of an oligo-array for rainbow trout

overall_design: The performance of the new microarray platform was evaluated by analyzing transcriptome response associated with the rainbow trout vitellogenesis-induced muscle atrophy.  Severe muscle deterioration accompanies the physiological responses of the energetic demands of the rainbow trout spawning/vitellogenesis. Atrophying muscle of fertile fish had 11% less extractable muscle (g/bw) and 11% less protein content compared to non-atrophying muscle of sterile fish (p<0.01).  The rainbow trout was used to profile changes in gene expression of atrophying muscles. Gene expression levels were determined by comparing the amount of mRNA transcript present in the experimental sample (fertile fish) to the control (sterile fish). RNAs isolated from each experimental fish were run on separate microarrays in independent experiments, with no pooling.  A total of 8 fish were used in the microarray experiments (4 replicates x 2 groups). Fluorophors (Cy3 and Cy5) were randomly assigned to RNA from each of the atrophying and nonatrophying muscles to limit the dye effect.

first: Caird

last: Rexroad

first: Brett

last: Kenney

first: Jianbo

last: Yao


title: Understanding Vitamin D resistance using array comparative genomic hybridization

summary:
Vitamin D is the strongest known natural anti-proliferative. A large number of studies in a wide spectrum of cancers, including epidemiological, in vitro and animal models, demonstrate that the active form of Vitamin D has anti-cancer benefits, affecting both progression and metastasis. Alike the role in calcium Vitamin D regulation, its anti-proliferative effect is thought to function through the Vitamin D receptor (VDR), although convincing evidence is lacking. Notwithstanding, separation of the calcemic and the anti-proliferative activity of Vitamin D analogues has been a major obstacle in developing new drugs for the treatment of cancer. The work presented attempts to unveil the molecular mechanism behind the anti-proliferative action of Vitamin D using genomic tools. For that purpose four independently developed Vitamin D sensitive/resistant MCF7 cell line pairs were collected. These unique biological replicates enabled us, both to increase the power of our study and to omit the use of Vitamin D. We deem this omission crucial since in the presence of Vitamin D only downstream genes involved in proliferation and cell cycle would be identified rather than causal resistance genes. The use of a variety of genomic techniques including expression, NMD and oligo CGH arrays reveal in the resistant cell lines the 11q13-14 as a region of DNA copy number loss and an altered expression of EGFR signaling pathway genes. Surprisingly, no genes known from calcium Vitamin D regulation were identified, nor did the VDR silencing by RNAi induce resistance to the sensitive cell lines.
Keywords: comparative genomic hybridization, cell type comparision

overall_design: Several MCF7 breast tumor cell lines independently derived from the human breast cancer cell line, both resistant and sensitive to Vitamin D, were used. The pairs of cell lines (parental-resistant) were developed in different laboratories and using different methodologies; while some cell lines acquired resistance by long exposure to the physiological concentration of Vitamin D (100nM), others were induced to resistant by being exposed to increasing amounts of Vitamin D. A total of 4 microarray expression experiments were carried out. In all microarray experiments DNA from the Vitamin D resistant cell line was hybrizided agains DNA from the respsective sensitive/parental cell line.

first: Paul

middle: J


title: Roles for microRNAs, miR-93 and miR-130b, and TP53INP1 tumor suppressor in cell growth dysregulation by HTLV-1

city: Bethesda

first: Man Lung

last: Yeung

address: 9000 Rockville Pike

institute: NIH

summary: This is to study the role of miRNAs in human T-cell leukemia virus-1, HTLV-1.

overall_design: In this study, we profiled the miRNA expression in virus transformed human cell lines and primary peripheral blood mononuclear cells (PBMCs) from Adult-T cell leukemia (ATL) patients using the RNA-primed, array-based Klenow enzyme (RAKE) assay.

first: Man-Lung

first: Jun-ichirou

last: Yasunaga

first: Yamina

last: Bennasser

first: Nelson

last: Dusetti

first: Masao

last: Matsuoka

first: Kuan-Teh

last: Jeang


title: MCMV Infection of Developing Central Nervous System, Developmental and Immunological Response

city: Birmingham

first: William

last: Britt

email: thad007@uab.edu, wbritt@peds.uab.edu

state: AL

address: 1600 6th Ave S

department: Pediatrics, Division Infectious Disease

institute: University of Alabama at Birmingham

summary:
Prenatal Human Cytomegalovirus (HCMV) infection often causes CNS maldevelopment.  In a murine model, we detect Murine Cytomegalovirus (MCMV) in brain following intra-peritoneal inoculation at birth. Infected mice show impaired cerebellar development and impaired neurologic function on a beam balance test as adults. Among developmental genes differentially regulated, hindbrain expression of the homeodomain transcription factor HOXa5 was reduced with infection, and fewer HOXa5 expressing neurons were found in vestibular nuclei.  Based on the hypothesis that immune activation connects focal viral infection and global CNS maldevelopment, we defined the components of CNS immune response.  Flow cytometry showed a large increase in both number and activation of CNS monocytes.  Monocytes were found in close association with infected cells by immunohistochemistry (IHC).  Oligonucleotide microarrays contained herein identified many differentially expressed genes related to innate immune response.  Chemokines, cytokines, cell surface receptors, and proteases are some of the many immunological genes shown to be differentially regulated by MCMV infection. These results together show that MCMV infection induces a complex immune response associated with changes in developmental gene expression and lasting neurologic defecit.
Keywords: disease state comparison (virus infection) and competetive hybridization for expression analysis

overall_design: Previous work empirically determined the kinetics of viral spread to and replication in the Balb-C murine CNS.  Balb-C mice were inoculated intra-peritoneally at birth, and harvested at 5, 8, 12, and 21 days post inoculation for cerebellar experiments. Controls were mock infected with vehicle (DMEM cell growth media) and harvested at the same timepoints. Each microarray was a two channel MEEBO array, upon which labeled and reverse transcribed cDNA from 3 pooled infected cerebella and three pooled uninfected cerebella competetively hybridized.  Three unique biological replicates were performed with day 5 cDNA, 5 unique biological replicates at day 8, 5 unique biological replicates at day 12, and 4 unique biological replicates at day 21.  For the analysis, colors channels were seperated and treated as independent hybridizations. However, since the experiments were dual channel, the primary data may be employed to generate ratio based measurements, and  each sample is provided a corresponding number that can be used to pair the processed data and derive ratios (mock 3, infected 3).  Mice were similarly inoculated and harvested at 5, 15, and 21 days post natal for liver tissue analysis.  Inoculation was performed by injection with a microsyringe of 50ul titered virus delivering 500-1000 PFU per animal.

first: Thadeus

last: Koontz

first: Marina

last: Bralic

first: Jelena

last: Tomac

first: Ester

last: Pernjac-Pugel

first: Glen

middle: R

last: Bantug

first: Stipan

last: Jonjic


title: Kidney of MFP2-/-

city: Neuherberg

first: Martin

last: Irmler

state: Bayern

address: Ingolstaedter Landstrasse 1

department: Institute of Experimental Genetics

institute: Helmholtz Zentrum München GmbH

summary:
The MFP2-/- (multifunctional protein 2) is a multifunctional enzyme with discovery in connection with different metabolic pathways. It is involved in beta-oxidation of branched fatty acids and long chain fatty acids in peroxisomes and in sythesis of bile fatty acids. MFP2 knock out mouse was made with a phenotype only visible in homozygotes (severe growth retardation, 40% mortality during the first week after birht, reduction of fertility in males, less activity). The studies have been performed to identify changes in  gene expression levels in kidney due to clinical chemical changes in the phenotype of this mutant mouse line during a screening in the German Mouse Clinic.
Keywords: dual colour hybridisation on cDNA microarrays, MFP2, Hsd17b4, kidney

overall_design: DNA chip technology has been exploited for RNA expression profiling analyses. Close to genomewide microarrays were used for identification of changes in gene expression levels in kidney of 4 MFP2-/- mice. Individual mutant mice were hybridised in repetitions against a pool of 5 reference animals. In total 16 chip hybridisations were performed including 50% dye swap experiments.

first: Marion

last: Horsch

first: Johannes

last: Beckers

first: Jurek

last: Adamski

first: Gabriele

last: Möller