title: Gene expression during early sporulation in Phytophthora infestans

status: Public on Jan 27 2009

city: Riverside

first: Howard

last: Judelson

state: CA

address: Boyce Hall

department: Plant Pathology

institute: University of California

summary: Transcriptional changes during asexual sporangia formation by the late blight pathogen Phytophthora infestans were identified using microarrays representing 15,650 genes and RNA from sporulation time-courses, purified spores, and sporulation-defective strains.  Results were confirmed by reverse transcription-polymerase chain reaction analyses of sporulation on artificial media and infected tomato.  During sporulation, about 12% of genes were found to be up-regulated and 5% down-regulated.  The most prevalent induced genes had functions in signal transduction, flagella assembly, cellular organization, metabolism, and molecular or vesicular transport.  Distinct patterns of expression were discerned based on the kinetics of mRNA induction and their persistence in sporangia.  For example, most flagella-associated transcripts were induced very early in sporulation and maintained in sporangia, while many participants in metabolism or small molecule transport were also induced early but had low levels in sporangia.  Data from this study are a resource for understanding sporogenesis, which is critical to the pathogenic success of P. infestans and other oomycetes.

overall_design: Two arrays were analyzed for each type of biological material.  For each of the replicates, RNA was extracted from 2-3 different cultures and pooled.  Therefore, a total of 4-6 biological replicates were analyzed for each type of tissue.

first: Reena

last: Narayan

first: Audrey

last: Ah-Fong

first: KyoungSu

last: Kim


title: aCGH study of invasive duct carcinoma (IDC) of the breast without axillary lymph node LN and with sentinel LN metastasis

status: Public on Feb 01 2009

city: Toronto

first: Susan

last: Done

address: 610 University Ave

country: Canada

institute: Ontario Cancer Institute

summary:
22 patients with IDC: 8 without LN metastases (IDC) and 14 with sentinel LN metastases (IDCM).  Sampling was limited to 29 paraffin blocks which could be used up without interference with diagnostic procedures: 8 IDCs, 11 IDCMs and 10 ipsilateral LN metastases [5 to the sentinel LN (first in lymph drainage chain, MS) and 5 to more distal (MD)].  7 IDCMs had samples of their LN metastases (IDCM9,13, 14, 15, 17, 18, 19). 3 samples of metastases (MS20, 21, 22) did not have paired primary IDCM samples and 4 IDCMs (IDCM10, 11, 12, 16) did not have samples of their LN metastases.
Keywords: Comparative clinical study

overall_design: Histological sections microdissected, DNA extracted, subjected to whole genome amplification protocol (SCOMP) and aCGH.

first: Vladimir

last: Iakovlev


title: Transcription profiling of RaxR-regulon members of Xanthomonas oryzae pv. oryzae PXO99A

status: Public on Jan 31 2009

city: Davis

first: Malinee

last: Sriariyanun

address: 451 East Health Sciences Drive

country: USA

institute: University of California, Davis

summary:
Xanthomonas oryzae pv. oryzae strain PXO99A, so called Xoo, is disable to infect in rice cultivar carrying Xa21 gene. Disrupted mutant of raxR gene, response regulator of two-component regulatory system (TCS), in Xoo was previously shown to partially retrieve back the bacterial capability to establish in Xa21 rice. RaxR was shown to mediate the expression of other rax gene operon members and also its expression is changed dependent on cell population density. In this study, we investigated the regulatory mechanisms mediated by RaxR using whole-genome transcriptional profiling analysis in comparison of (i) PXO99R (PXO99 strain lacking RaxR) vs. PXO99, (ii) PXO99Rox (PXO99 strain overexpressing RaxR) vs. PXO99, and (iii) PXO99Rox vs. PXO99R. As a result of array analysis, we revealed that RaxR is not only required for AvrXa21 activitiy, it also plays roles in regulatory functions, for example, pathogenicity, motility, and stress tolerance. Then, we generated knock out mutants of RaxR regulon members to validate regulatory functions of RaxR and to extend other biological impacts of RaxR beyond the Xoo AvrXa21 activity. The combined interpretation from array analysis and mutant functional validation presents the complexity of regulatory pathways between AvrXa21 activity and other biological activities in Xoo.

Keywords: Comparative transcription profiling between modified genetic mutant and wild type

platform_organism: Xanthomonas oryzae pv. oryzae KACC 10331

overall_design: Three biological and dye-swap replicates, total six samples for each dataset. Three datasets contained; (i) raxR knockout mutant vs. wildtype PXO99A in PSB, nutrient rich media (ii) RaxR constitutively express mutant vs. wildtype PXO99A in PSB, nutrient rich media, and (iii) raxR knockout mutant vs. RaxR constitutively express mutant in PSB, nutrient rich media

first: Pamela

last: Ronald


title: DNA copy number analysis of myxoinflammatory fibroblastic sarcoma, and morphologically similar lesions

status: Public on Jan 26 2009

city: Lund

first: Karolin

middle: H

last: Nord

address: BMC C13

department: Dept of Clinical Genetics

country: Sweden

institute: Lund University

type: Genome variation profiling by genome tiling array

summary: DNA copy number analysis of myxoinflammatory fibroblastic sarcomas, and related lesions, using 32k BAC array CGH. All four cases analyzed with array CGH showed amplification of the region 86.30-87.74 Mb in 3p11.1-12.1. No additional aberration was detected in more than two tumors, except for the deletion distal to the amplification on chromosome 3. Case 1 showed deletion affecting the region from the centromere of chromosome 1 to the 5'-end of TGFBR3. Homozygous deletion was found in one case (case 6) affecting the region 21.08-22.15 Mb on chromosome arm 9p harboring the CDKN2A and CDKN2B genes.

overall_design: DNA copy numbers in cases 1 and 6-8 were analyzed using tiling microarrays containing more than 32 000 partly overlapping BAC clones, generating complete coverage of the human genome (Jönsson et al., 2007, Genes Chromosomes Cancer 46: 543-58.). The arrays were produced at the Swegene DNA Microarray Resource Center, Department of Oncology, Lund University (http://swegene.onk.lu.se), as previously described (Jönsson et al., 2007), using BAC clones mapped to the hg17 genome build. Extraction, labeling and hybridization of genomic DNA from freshly frozen tumor biopsies, as well as pretreatment and washing of slides were performed as described (Heidenblad et al., 2006, Oncogene 25: 7106-16). As a control for normal copy number, a DNA pool derived from multiple healthy male donors was used (Promega, Madison, WI, USA). Primary array CGH data were collected using the GenePix Pro 4.0 (Axon Instruments Inc., Foster City, CA, USA). The quantified data matrix was deposited into the web-based database BioArray Software Environment (BASE) (Saal et al., 2002, Genome Biol 3: software0003.1-0003.6), and data analyses were done as described (Hallor et al., 2008, Br J Cancer 98: 434-42). In brief, following background correction the log(2) ratios were calculated for each spot, and unreliable features and spots not showing signal-to-noise ratios ?5 for both channels were eliminated. Normalization of data was performed using pin-based Lowess normalization and single outlier probes were removed. Thereafter, the log(2) ratios for each sample were segmented, followed by elimination of segments less than 500 kb in size. Copy number alterations were determined by comparing the segmented log(2) ratios to gain/loss thresholds obtained by an adaptive scaling method (Staaf et al., 2007, BMC Genomics 8: 382).

first: KH

last: Hallor

first: R

last: Sciot

first: J

last: Staaf

first: M

last: Heidenblad

first: A

last: Rydholm

first: HCF

last: Bauer

first: K

last: Åström

first: HA

last: Domanski

first: JM

last: Meis

first: LG

last: Kindblom

first: I

last: Panagopoulos

first: N

last: Mandahl

first: F

last: Mertens


title: Late response to boron toxicity in barley leaves

status: Public on Jan 24 2009

city: Ames

first: PLEXdb Curator

middle: Sudhansu

state: IA

address: 1023  Crop Genomes Informatics Lab (Iowa State University)

department: Virtual Reality Application Center

institute: Iowa State University

summary: Barley1 GeneChip was used to investigate transcriptome changes associated with boron (B) toxicity in a sensitive barley cultivar (Hordeum vulgare L. cv. Hamidiye). Eight day old aseptically grown seedlings were subjected to 5 or 10 mM boric acid (B(OH)3) treatments for 5 days and expression profiles were determined with DNA microarrays using total RNA from leaf tissues. Surface sterilized seeds were aseptically germinated and grown on half strength Hoagland’s solution (Hoagland and Arnon, 1950) (pH 5.8) solidified with Phytagel® for 8 days at 23±2ºC with 16 h light (400 µmol m–2 s–1) and 8 h dark photo-cycle with 70% relative humidity. Seedlings were transferred to sterile hydroponic cultures for B treatment, which was applied immediately after transfer as half strength Hoagland’s solution containing either 5 mM B(OH)3 (5B) or 10 mM B(OH)3 (10B) for another 5 days under the same physical conditions. Control (C) groups were transferred to half strength Hoagland’s solution without extra B(OH)3.   ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor,  Mehmet Tufan OZ.  The equivalent experiment is BB63 at PLEXdb.]

overall_design: treated or untreated: Control (C)(3-replications);  treated or untreated: 5 mM B(OH)3 treatment (5B)(3-replications);  treated or untreated: 10 mM B(OH)3 treatment (10B)(3-replications)

first: Mehmet

middle: T

last: Oz


title: Comparative Genomic Hybridization between S. cerevisaie strains JAY270 and S288c.

status: Public on Jan 29 2009

city: Durham

first: Juan Lucas

last: Argueso

state: SP

address: 371 CARL Building, Box 3054

department: Molecular Genetics and Microbiology

institute: Duke University Medical Center

summary: The arrays in this series correspond to a comparative genomic analysis between S. cerevisiae strain JAY270 and the reference laboratory strain S288c. JAY270 is a heterothalic diploid used in bioethanol production from sugar cane feedstock in Brazil. This strain has several chromosomal length polymorphisms between homologous chromosomes. The two Chr6 homologs, Chr6 short and Chr6 long, were examined using microarrays to determine the genomic regions which are rearranged.

overall_design: Three arrays are provided in this series. In all three total genomic DNA from a S288c isogenic strain was labeled with Cy3 and provides a reference for competitive hybridization to the arrays. The first array in the series is a full CGH-array comparing the complete JAY270 diploid genome to S288c. The second and third arrays correspond the PFGE-purified Chr6 short and Chr6 long DNA from JAY270 compared to the S288c genome (Band-arrays). The arrays used in this study were designed using the genome sequence of the reference S288c strain.

first: Juan

middle: L


title: miRNA profiles of diffuse large B cell lymphoma (DLBCL) derived cell lines

status: Public on Jan 28 2009

city: Pamplona

first: Raquel

last: Malumbres

state: Navarra

address: Avenida Pio XII 55

department: Hemato-Oncology

country: Spain

institute: University of Navarra/ Clínica Universidad de Navarra

summary:
miRNA profiling of human Diffuse Large B Cell Lymphoma (DLBCL) cell lines derived from the two main subtypes of this disease: Activated B Cell like (ABC-DLBCL) and Germinal Center B cell like  (GCB-DLBCL), analyzing the 711 human miRNAs present in miRBase V10.0. Five ABC-like DLBCL cell lines (RIVA, Oci-Ly3, Oci-Ly10, HBL1 and U2932) and three GCB-like DLBCL cell lines (Oci-Ly7, Oci-Ly19 and SUDHL-6) were cultured in IMDM (Cellgro) with 20% human plasma, 1% penicillin/streptomycin/L-glutamine (Cellgro,) and 0,2% beta mercaptoethanol (Invitrogen). Total RNA was extracted from cell pellets using the mirVana™ miRNA Isolation Kit (Ambion), and sent to LC Sciences facility for microarray hybridization using microfluidics technology. FirstChoice® Human Skeletal Muscle Total RNA (Ambion) was used as common reference for all hybridizations. Background substracted and normalized data from each channel was used to calculate log ratios sample/reference.

Keywords: miRNA profiling

overall_design: Dual channel experiments using FirstChoice® Human Skeletal Muscle Total RNA (Ambion) as common reference for all hybridizations. Reference was labeled with Cy5 and the cell line extracted RNA with Cy3. Biological replicates: 5 ABC-DLBCL cell lines and 3 GCB-DLBCL cell lines. Microarray hybridization was performed at LC Sciences, using slides containing in situ synthesized oligonucleotides to detect the 711 miRNAs present in miRBase V10.0. Probes hsa-miR-337 and hsa-miR-542-5p were excluded due to systematic dye bias, according to the manufacturer intructions. Background substracted and normalized detectable data was used to calculate log ratios sample/reference. Background is determined using a regression-based background mapping method.  The regression is performed on 5% to 25% of the lowest intensity data points excluding blank spots.  Raw data matrix is then subtracted by the background matrix. Normalization was carried out using a LOWESS (Locally-weighted Regression) method on the background-subtracted data.   A transcript to be listed as detectable must meets at least two conditions: signal intensity higher than 3×(background standard deviation) and spot CV < 0.5.  CV is calculated by (standard deviation)/(signal intensity).  When repeating probes are present on an array, a transcript is listed as detectable only if the signals from at least 50% of the repeating probes are above detection level.

first: Izidore

middle: S

last: Lossos


title: Transcriptomic and metabolomic profiles of the Plasmodium falciparum developmental cycle

city: Princeton

first: Kellen

last: Olszewski

state: NJ

address: Carl Icahn Lab

department: Molecular Biology

institute: Princeton University

summary: In order to further our understanding of the metabolic network of the malaria parasite, Plasmodium falciparum, we carried out a concurrent transcriptomic and metabolomic study of the parasite's intraerythrocytic developmental cycle.  These microarray data were generated to compare the expression levels of metabolic enzymes to the concentrations of their associated metabolites over the 48-hour life cycle.

overall_design: Sorbitol-synchronized Plasmodium falciparum (3D7 strain) was grown in tissue culture flasks in incubators according to standard protocols.  Immediately after reinvasion, and at 8-hour intervals thereafter, parasites were harvested by centrifugation.  For each timepoint, 0.5 mL of packed RBC (10% parasitemia) was pelleted by centrifugation, washed once in PBS and flash-frozen in liquid nitrogen.  Total RNA isolation and amino-allyl cDNA labeling were as previously described (Bozdech et al., 2003).  A pool of 3D7 total RNA from all intraerythrocytic developmental stages was generated and used as the reference sample.  For DNA microarray hybridization, pool cDNA was coupled to Cy3 dye, while cDNA from an individual timepoint was coupled to Cy5 dye.  DNA microarrays were scanned using an Axon 4200A scanner and images analyzed using Axon GenePix software (Axon Instruments, Union City, CA, USA).  Microarray data were stored and analyzed using our in-house database PUMAdb (Princeton University MicroArray database).  All data for individual arrays were normalized by a global normalization using unflagged features with >= 65% of pixels one or more standard deviations over local background.  All unflagged spots were selected and extracted for further analysis.  Data were filtered to remove oligos more than 1 datapoint missing across the timeseries, log2 transformed, mean centered, ordered by the timing of their peak expression level, and visualized with Java Treeview (Saldanha, 2004) (Table S3).

first: Joanne

middle: M

last: Morrisey

first: Daniel

last: Wilinski

first: James

last: Burns

first: Akhil

middle: B

last: Vaidya

first: Joshua

middle: D

last: Rabinowitz

first: Manuel

last: Llinás


title: MicroRNA expression from bronchial epithelial cell samples of current and never smokers.

city: Gaithersburg

first: Sriram

last: Sridhar

state: MD

address: 1 MedImmune Way

department: Translational Medicine

institute: MedImmune

summary:
MicroRNA expression was assayed from bronchial epithelial cells collected via bronchoscopy from healthy current and never smoker volunteers in order to determine the effect of cigarette smoke exposure on airway epithelial microRNA expression

Keywords: Global microRNA expression profiling

platform_organism:
Caenorhabditis elegans
Drosophila melanogaster
Danio rerio
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: Bronchial epithelial cells were collected from current and never smokers via bronchoscopy. Low molecular weight RNA ( < 200 nucleotides) was isolated and hybridized to Invitrogen NCode microRNA microarrays to determine which microRNAs detected in bronchial epithelial cells were differentially expressed in the airways of smokers.

first: Avrum

last: Spira


title: Genomic distribution of CHD7 on chromatin tracks H3K4 methylation patterns

status: Public on Jan 23 2009

city: Cleveland

first: Mike

middle: Paul

last: Schnetz

state: OH

address: 10900 Eulid Ave

department: Genetics

institute: Case Western Reserve University

type: Genome binding/occupancy profiling by genome tiling array

summary: CHD7 is a member of the chromodomain helicase DNA binding domain family of ATP-dependent chromatin remodeling enzymes.  De novo mutation of the CHD7 gene is a major cause of CHARGE syndrome, a genetic disease characterized by a complex constellation of birth defects.  To gain insight to the function of CHD7, we mapped the distribution of the CHD7 protein on chromatin using the approach of chromatin immunoprecipitation on tiled microarrays (ChIP-chip).  These studies were performed in human colorectal carcinoma cells, human neuroblastoma cells, and mouse embryonic stem (ES) cells before and after differentiation into neural precursor cells. The results indicate that CHD7 localizes to discrete locations along chromatin that are specific to each cell type, and that the cell-specific binding of CHD7 correlates with a subset of histone H3 methylated at lysine 4 (H3K4me). The CHD7 sites change concomitantly with H3K4me patterns during ES cell differentiation, suggesting that H3K4me is part of the epigenetic signature that defines lineage-specific association of CHD7 with specific sites on chromatin.  Furthermore, the CHD7 sites are predominantly located distal to transcription start sites, most often contained within DNase hypersensitive sites, frequently conserved, and near genes expressed at relatively high levels.  These features are similar to those of gene enhancer elements, raising the possibility that CHD7 functions in enhancer mediated transcription, and that the congenital anomalies in CHARGE syndrome are due to alterations in transcription of tissue-specific genes normally regulated by CHD7 during development.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: ChIP-chip experiments were performed for CHD7 and H3K4 mono-,di-, and trimethylation modifications in 4 cells types: human DLD1 and SH-SY5Y; mouse ES and differentiated neural precursor cells derived from mouse ES cells.  Microarrays used in these experiments tiled all or subset of ENCODE regions (in mouse, analogous ENCODE regions were assayed).  At least two biological replicates were performed for each CHD7 ChIP experiment; H3K4 ChIP's were performed once in each cell type.

first: Michael

middle: P

first: Cynthia

middle: F

last: Bartels

first: Kuntal

last: Shastri

first: Dheepa

last: Balasubramanian

first: Gabriel

middle: E

last: Zentner

first: Ravishankar

last: Balaji

first: Xiaodong

last: Zhang

first: Lingyun

last: Song

first: Zhenghe

last: Wang

first: Thomas

last: LaFramboise

first: Gregory

last: Crawford

first: Peter

middle: C

last: Scacheri


title: per8::vip micro-array-Identification of the VIP1 targets

city: BEAUCOUZE

first: Sandra

last: Pelletier

address: 42, rue Georges Morel - BP 60057

country: France

institute: INRA

summary:
au07-04_vip1 - per8::vip micro-array - VIP1 early targets - estradiol-induced expression of VIP1-YFP and VIP1_D-YFP; 3h estradiol treatment. First comparison: non-induced versus induced. Second comparison: VIP1 lines versus YFP control line

Keywords: treated vs untreated comparison

overall_design: 8 dye-swap - CATMA arrays

first: Jean-Pierre

last: Renou

first: Heribert

last: Hirt

first: Marie-Laure

last: Martin-Magniette


title: Group B Streptococcus genes controlled by the CiaR response regulator

status: Public on Jan 22 2009

city: Shreveport

first: Daniel

middle: Wilson

last: Shelver

state: LA

address: 1501 Kings Highway

department: Microbiology and Immunology

institute: LSU Health Sciences Center in Shreveport

summary: The purpose of this study was to identify Group B Streptococcus (GBS) genes that are controlled by the CiaR response regulator.  Deletion of the GBS ciaR gene resulted in a significant decrease in intracellular survival within neutrophils, murine macrophages, and human BMEC, which was linked to increased susceptibility to killing by antimicrobial peptides, lysozyme, and reactive oxygen species.  Furthermore, competition experiments in mice showed that wild-type GBS had a significant survival advantage compared to the isogenic ciaR mutant.  Microarray analysis comparing gene expression between the wild-type and ciaR mutant strains revealed several CiaR-regulated genes that may contribute to GBS stress tolerance and subversion of host defenses.

overall_design: Two cultures each of the wild-type GBS strain (COH1) and the isogenic ciaR mutant were grown in Todd-Hewitt broth to an optical density of 0.3.  Cells were disrupted by shaking with glass beads and RNA was isolated by a Trizol method.  A custom Affymetrix chip with a design based on the COH1 genomic sequence was used to analyze gene expression.

first: Darin

last: Quach

first: Nina

last: van Sorge

first: Sascha

middle: A

last: Kristian

first: Joshua

last: Bryan

middle: W

first: Kelly

last: Doran


title: Wild type V. cholerae strain A1552 growth time course in AKI media

status: Public on Jan 21 2009

city: Stanford

last: Stanford Microarray Database

address: 300 Pasteur Drive

department: Stanford University, School of Medicine

institute: Stanford Microarray Database (SMD)

summary:
We identified the mutated gene locus in a pigment overproducing Vibrio cholerae mutant of strain A1552. The deduced gene product is suggested to be a oxidoreductase based on partial homology to putative homogentisate 1,2-dioxygenase in Pseudomonas aeruginosa and Mesorhizobium loti and we propose that the gene VC1345 in the V. cholerae genome be denoted hmgA in accordance with the nomenclature for other species. The hmgA::miniTn5 mutant showed a non-pigmented phenotype after complementation with a plasmid clone carrying the wild type hmgA+ locus. Microarray transcription analysis revealed that expression of hmgA, and the neighboring genes encoding a postulated two component sensor system, was growth phase dependent. Results from qRT-PCR analysis showed that hmgA operon expression was reduced in the rpoS mutant, but pigment production by the wild type V. cholerae or the hmgA mutant was not detectably influenced by the stationary phase regulator RpoS. The pigmented mutant showed increased UV resistance in comparison with the wild-type strain. Interestingly, the pigment producing mutant expressed more toxin co-regulated pili and cholera toxin in comparison with wild type V. cholerae. Moreover, the hmgA mutant showed a 5-fold increase in the ability to colonize the intestines of infant mice. A possible mechanism by which pigment production might cause induction of the ToxR regulon due to generation of hydrogen peroxide was supported by results from tests showing that externally supplied H2O2 led to higher TcpA levels. Taken together, our findings suggest that melanin pigment formation may play a role in V. cholerae virulence factor expression.
Groups of assays that are related as part of a time series.
Elapsed Time: Wild type V. cholerae strain A1552 bacteria were cultured statically for 4 h in AKI medium at 37C and then shifted to aerobic growth for 6 h using shaken culture flasks. Samples were taken at hourly during the this process.

Keywords: time_series_design

overall_design: Using regression correlation

first: Nadia

last: Dolganov


title: Mucosal responses of healthy humans to exponentially growing or stationary Lactobacillus plantarum bacteria

city: Wageningen

last: van Baarlen

address: De Elst 1

department: Animal Sciences

country: Netherlands

institute: Wageningen University

summary:
Some commensal bacteria stimulate the immune system but do not present specific antigenicity. Such adjuvant effects have been reported for the bacterial species Lactobacillus plantarum. To study in vivo human responses to L. plantarum, a randomised double-blind placebo-controlled cross-over study was performed. Healthy adults were provided preparations of living and heat-killed L. plantarum bacteria, biopsies were taken from the intestinal mucosa and altered transcriptional profiles were analysed. Transcriptional profiles of human epithelia displayed striking differences upon exposure to living L. plantarum bacteria harvested at different growth phases. Modulation of NF-κB-dependent pathways was central among the major altered cellular responses. This unique in vivo study shows which cellular pathways are associated with the induction of immune tolerance in mucosal tissues towards common adjuvanticity possessing lactobacilli.
Keywords: mucosal response of healthy adult humans to lactic acid bacteria

overall_design: This study was set up according to a randomised double-blind cross-over placebo-controlled design. It contains transcriptional profiles from biopsies from 8 healthy individuals after oral intake of three different growth stages of Lactobacillus plantarum or placebo control. In total, this study includes data from 8 individuals x 4 treatments=32 arrays.

first: Michiel

last: Kleerebezem

first: Robert-Jan

last: Brummer

first: Cindy

last: van der Meer

first: Guido

last: Hooiveld

first: Fred

middle: J

last: Troost

first: Philip

last: de Groot


title: Transcriptional profiling of cattle infected with Trypanosoma congolense using bovine long oligo (BLO) microarray

city: Dublin

first: David

last: MacHugh

address: University College Dublin, Belfield

department: College of Agriculture, Food Science and Veterinary Medicine

country: Ireland

institute: University College Dublin

summary:
Background: African animal trypanosomiasis (AAT) caused by tsetse fly-transmitted protozoa of the genus Trypanosoma is a major constraint on livestock and agricultural production in Africa and is among the top ten global cattle diseases impacting on the poor. Here we show that a functional genomics approach can be used to identify temporal changes in host peripheral blood mononuclear cell (PBMC) gene expression due to disease progression. We also show that major gene expression differences exist between cattle from trypanotolerant and trypanosusceptible breeds. Using bovine long oligonucleotide microarrays and real time quantitative reverse transcription PCR (qRT-PCR) validation we analysed PBMC gene expression in naïve trypanotolerant and trypanosusceptible cattle experimentally challenged with Trypanosoma congolense across a 34-day infection time course.

Results: Trypanotolerant N’Dama cattle displayed a rapid and distinct transcriptional response to infection, with a ten-fold higher number of genes differentially expressed at day 14 post infection compared to trypanosusceptible Boran cattle. These analyses identified coordinated temporal gene expression changes for both breeds in responses to trypanosome infection. In addition, a panel of genes were identified that showed pronounced differences in gene expression between the two breeds, which may underlie the phenomena of trypanotolerance and trypanosusceptibility. Gene ontology (GO) analysis demonstrate that the products of these genes may contribute to increased mitochondrial mRNA translational efficiency, a more pronounced B cell response, an elevated activation status and a heightened response to stress in trypanotolerant cattle.

Conclusions: This study has revealed an extensive and diverse range of cellular processes that are altered temporally in response to trypanosome infection in African cattle. Results indicate that the trypanotolerant N’Dama cattle respond more rapidly and with a greater magnitude to infection compared to the trypanosusceptible Boran cattle.  Specifically, a subset of the genes analyzed by qRT-PCR, which display significant breed differences, could collectively contribute to the trypanotolerance trait in N’Dama.

sample_organism:
Bos taurus
Bos indicus

overall_design:
Animals and experimental infection

The work presented here is based on an experimental infection that was previously described. Briefly, eight Boran (B. indicus, trypanosusceptible) and eight N’Dama (B. taurus, trypanotolerant) female cattle, aged between 19-28 months, were raised together at the International Livestock Research Institute (ILRI) farm at Kapiti Plains Estate, Kenya in an area free from trypanosomiasis. The cattle were each experimentally infected with the T. congolense clone IL1180, which was delivered via the bites of eight infected tsetse flies [Ref. G. morsitans morsitans]

Blood collection, PBMC isolation and RNA extraction and purification

Peripheral blood (200 ml) was collected before infection and at 14, 21, 25, 29 and 34 dpi in heparanized syringes. These time points were chosen to coincide with the first wave of parasitaemia. Peripheral blood mononuclear cells (PBMC) were isolated using Percoll™ gradients (GE Healthcare, www.gehealthcare.com) and previously described methods, washed with sterile PBS and stored immediately at -80°C in Tri-reagent (Molecular Research Center, Inc, www.mrcgene.com). Total RNA was extracted with chloroform and precipitated with isopropanol. Each sample was further DNase-treated with RQ1 RNase-free DNase (Promega Corp., www.promega.com) and purified using the RNeasy® mini kit (Qiagen, www.qiagen.com). RNA quality and quantity was subsequently assessed using the 18S/28S ratio and RNA integrity number (RIN) on an Agilent Bioanalyzer with the RNA 6000 Nano LabChip® kit (Agilent Technologies, Inc., www.agilent.com).

The bovine long oligo (BLO) microarray

The NCBI GEO platform accession for the BLO microarray is GPL3810.  The BLO microarray platform has 17,328 spot features representing approximately 7,920 predicted bovine mRNA’s spotted in duplicate supplemented with positive (GAPDH, RPL19, RPLP2, 28S RNA, 60S RNA, PPIA, ACTB, PGK1, B2M and GUSB), negative and artificial control sequences spotted multiple times. The oligonucleotides (70-mers) were designed and synthesized commercially by Operon Biotechnologies (Operon Biotechnologies, Inc., www.operon.com). The oligonucleotides were designed to the The Institute for Genomic Research (TIGR) tentative consensus (TC) sequences (release October 5th 2004).

cDNA conversion, labelling and hybridization to bovine long oligonucleotide microarrays

Labelled cDNA was generated using the SuperScript™ Plus Direct cDNA labelling System (Invitrogen Corp., www.invitrogen.com) according to the manufacturers’ instructions. Briefly, 8 µg of sample RNA was labelled with Alexa Fluor® 555 and 10 µg of common reference RNA (CRR) was labelled with Alexa Fluor® 647. A common reference RNA pool was generated by combing equal quantities of all samples in the study, which included RNA from pre- and post-infection time points. Labelled samples were purified using the purification module included with the SuperScript™ Plus Direct cDNA labelling System, combined and supplemented with approximately 60-70 µl SlideHyb Glass Hybridization Buffer #3 (Ambion Inc., www.ambion.com) to a final probe volume of 100 µl.
Probes were hybridized to bovine long oligonucleotide (BLO) microarrays, using an automated HS400 hybridization station (Tecan UK Ltd., www.tecan.com) with the following optimized protocol; WASH: 75°C, Runs 1, Wash 10 s, Soak 20 s; PROBE INJECTION: 85°C; Denaturation: 95°C, 2 mins; HYBRIDISATION: 42°C – 4 hrs; 35°C – 4 hrs; 30°C – 4 hrs; agitation frequency medium, WASH: 37°C, Runs 2, Wash 10 s, Soak 20 s; 25°C, Runs 2, Wash 15 s, Soak 30 s; 25°C, Runs 2, Wash 20 s, Soak 40 s and SLIDE DRYING: 25°C for 2 mins.

Data collection, normalization, quality control and statistical analysis

Microarrays were scanned using a GenePix® 4000B scanner (Molecular Devices Corp., www.moleculardevices.com). The microarray hybridizations represented 5-8 animals of each breed at all of the time points (0, 14, 21, 25, 29 and 34 dpi) where the CRR served as the reference sample in each case (for a total of 88 slides). Spot features where foreground was less than background plus two standard deviations were flagged as lowly expressed. The linear models for microarray data (LIMMA) package in Bioconductor was used to identify differential gene expression. Background correction was performed according to the robust multichip average (RMA) method. An intensity-based normalization method was employed: within microarray print-tip dependent locally weighted scatterplot smoothing (LOWESS) normalization, followed by between microarray quantile normalization.

first: Grace

last: O'Gorman

first: Stephen

last: Park

first: Emmeline

last: Hill

first: Kieran

middle: G

last: Meade

first: Paul

last: Coussens

first: Morris

last: Agaba

first: Jan

last: Naessens

last: Kemp


title: Heart-muscle expression ratios in pig obtained by 454-sequencing, iTRAQ-based proteomics and cDNA microarrays

city: Tjele

first: Jakob

last: Hedegaard

address: PO-box 50

department: Department of Genetics and Biotechnology

country: Denmark

institute: Aarhus University, Faculty of Agricultural Sciences

summary:
The recent development within high-throughput technologies for expression profiling has allowed for parallel analysis of transcriptomes and proteomes in biological systems such as comparative analysis of transcript and protein levels of tissue regulated genes. Until now, such studies of have only included microarray or short length sequence tags for transcript profiling. Furthermore, most comparisons of transcript and protein levels have been based on absolute expression values from within the same tissue and not relative expression values based on tissue ratios. Presented here is a novel study of two porcine tissues based on integrative analysis of data from expression profiling of identical samples using cDNA microarray, 454-sequencing and iTRAQ-based proteomics. Sequence homology identified 2.541 unique transcripts that are detectable by both microarray hybridizations and 454-sequencing of 1.2 million cDNA tags. Both transcript-based technologies showed high reproducibility between sample replicates of the same tissue, but the correlation across these two technologies was modest. Thousands of genes being differentially expressed were identified with microarray. Out of the 306 differentially expressed genes, identified by 454-sequencing, 198 (65 %) were also found by microarray. The relationship between the regulation of transcript and protein levels was analyzed by integrating iTRAQ-based proteomics data. Protein expression ratios were determined for 354 genes, of which 148 could be mapped to both microarray and 454-sequencing data. A comparison of the expression ratios from the three technologies revealed that differences in transcript and protein levels across heart and muscle tissues are positively correlated. We show that the reproducibility within cDNA microarray and 454-sequencing is high, but that the agreement across these two technologies is modest. We demonstrate that the regulation of transcript and protein levels across identical tissue samples is positively correlated when the tissue expression ratios are used for comparison. The results presented are of interest in systems biology research in terms of integration and analysis of high-throughput expression data from mammalian tissues.
Keywords: tissue comparison, platform comparison

overall_design: Tissue samples of heart (HEA) and Longissimus dorsi (LDO) were prepared by pooling from five healthy Hampshire gilts at age four to six months and division into six sub-samples, three for each tissue named HEA1, HEA2, HEA3, LDO1, LDO2 and LDO3. The exact same six tissue samples were used for subsequent expression profiling with cDNA microarray, 454-sequencing and iTRAQ-based proteomics. A reference sample for the cDNA microarray experiment and iTRAQ-based proteomics was constructed by combining the six samples. In the microarray experiment, three cDNA microarray slides were used per sample.

first: Henrik

last: Hornshøj

first: Emøke

last: Bendixen

first: Lene

middle: N

last: Conley

first: Pernille

middle: K

last: Andersen

first: Frank

last: Panitz

first: Christian


title: Gene Body Hypermethylation of ATM in Peripheral Blood DNA of Bilateral Breast Cancer Patients

city: London

first: James

last: Flanagan

address: Du Cane Rd

department: Dept of Oncology

country: United Kingdom

institute: Imperial College London

type: Methylation profiling by genome tiling array

summary:
Bilaterality of breast cancer is an indicator of constitutional cancer susceptibility, however, the molecular causes underlying this predisposition in the majority of cases is not known.  We hypothesize that epigenetic misregulation of cancer related genes could partially account for this predisposition.  We have performed methylation microarray analysis of peripheral blood DNA from 14 women with bilateral breast cancer compared to 14 unaffected matched controls throughout 17 candidate breast cancer susceptibility genes including BRCA1, BRCA2, CHEK2, ATM, ESR1, SFN, CDKN2A, TP53, GSTP1, CDH1, CDH13, HIC1, PGR, SFRP1, MLH1, RARB and HSD17B4. We show that the majority of methylation variability is associated with intragenic repetitive elements. Detailed validation of the tiled region around ATM was performed by bisulfite modification and pyrosequencing of the same samples and in a second set of peripheral blood DNA from 190 bilateral breast cancer patients compared to 190 controls. We show significant hypermethylation of one intragenic repetitive element in breast cancer cases compared to controls (p=0.0017) with the highest quartile of methylation associated with a three-fold increased risk of breast cancer (OR = 3.20, 95% C.I.=1.78-5.86, p=0.000083). Increased methylation of this locus is associated with lower steady state ATM mRNA level and correlates with age of cancer patients but not controls, suggesting a combined age-phenotype related association. This research demonstrates the potential for gene-body epigenetic misregulation of ATM and other cancer related genes in peripheral blood DNA that may be useful as a novel marker to estimate breast cancer risk.

Keywords: Differential Methylation Hybridisation

overall_design: 14 Case-Control pairs (matched for age and ethnicity), single array per pair, half samples dye-swapped


title: Transcriptional profiling of FeHm effects on Haemophilus influenzae 10810

city: Oklahoma City

first: Timothy

last: VanWagoner

state: OK

address: 940 SL Young Blvd, BMS340

department: Pediatrics

institute: University of Oklahoma Health Sciences Center

summary:
Background: Haemophilus influenzae has an absolute aerobic growth requirement for heme and has developed multiple complex mechanisms to obtain this essential nutrient.  Although an understanding of the heme acquisition mechanisms of H. influenzae is emerging, significant gaps remain to be elucidated.  In a previous study we utilized H. influenzae strain Rd KW20 to demonstrate the utility of transcriptional profiling in defining the genes exhibiting altered transcription in response to environmental iron and heme levels. The current study expands upon those initial observations by determining the iron/heme regulons of two additional H. influenzae clinical isolates, i.e. the type b isolate 10810 and the nontypeable isolate R2866, to characterize the core iron/heme regulon of the species.
Results: A microarray chip was designed to incorporate probes for all of the genes of H. influenzae isolates 10810 and R2866, and microarray studies were performed to compare gene expression under iron/heme-replete and iron/heme-restricted conditions for each isolate.  Of 1820 ORFs on the array corresponding to R2866 genes, 363 were significantly differentially expressed.  Of these 363 genes, 233 were maximally transcribed under iron/heme-replete conditions and 130 were preferentially transcribed in iron/heme-restricted conditions.  Of the 1883 ORFs representing genes of strain10810, 351 were significantly differentially transcribed, 150 of these were preferentially transcribed in iron/heme-replete conditions and 201 were preferentially transcribed in iron/heme-restricted conditions.  Comparison of the data sets indicated that 163 genes were similarly regulated in both isolates and that 74 of these also exhibited similar patterns of regulation in strain Rd KW20.
Conclusion: This study provides evidence for a core of H. influenzae genes that are regulated by the availability of iron and/or heme in the growth environment.  Elucidation of this core regulon provides targets for investigation of genes with an unrecognized role in iron and heme homeostasis, as well as other potential virulence determinants. In addition, the set of core genes potentially provides targets for therapeutic and vaccine designs since these products of these genes are likely to be preferentially expressed during growth in iron/heme restricted sites of the human body.
Keywords: Transcription analyses

overall_design: Three replicate cultures were grown in iron and heme depleted media for 90 minutes and samples taken for expression analyses. Fe and heme were supplemented to the remaining cultures and the cultures allowed to incubate an additional 20 minutes. Microarray analyses were used to determine changes in gene expression resulting from transfer from FeHm-deplete to FeHm-replete conditions.

last: Whitby

first: Thomas

last: Seale

first: Daniel

last: Morton

first: Terrence

middle: L

last: Stull


title: Global analysis of alternative splicing regulation by insulin and wingless signalling in Drosophila cells

city: Barcelona

first: Stephanie

last: Boue

address: Dr Aiguader 88

institute: CMRB

summary:
Background: Despite the prevalence and biological relevance of both signalling pathways and alternative pre-mRNA splicing, our knowledge of how intracellular signalling impacts on alternative splicing regulation remains fragmentary. We report a genome-wide analysis of changes in alternative splicing using splicing-sensitive microarrays, induced by activation of two distinct signalling pathways, insulin and wingless, in Drosophila cells in culture.

Results: Alternative splicing changes induced by insulin affect more than 150 genes and more than 50 genes are regulated by wingless activation. About 40% of the genes showing changes in alternative splicing also show regulation of mRNA levels, suggesting distinct but also significantly overlapping programs of transcriptional and posttranscriptional regulation. Distinct functional sets of genes are regulated by each pathway and, remarkably, a significant overlap is observed between functional categories of genes regulated transcriptionally and at the level of alternative splicing. Functions related with carbohydrate metabolism and cellular signalling are enriched among genes regulated by insulin and wingless, respectively. Computational searches identify pathway-specific sequence motifs enriched near regulated 5’ splice sites.

Conclusion: Taken together, our data indicate that signalling cascades trigger pathway-specific and biologically coherent regulatory programs of alternative splicing regulation. They also reveal that alternative splicing can provide a novel molecular mechanism for cross-talk between different signalling pathways.

overall_design:
To monitor transcriptional and alternative splicing changes induced by activation of the insulin and wingless pathways, a custom-designed microarray platform was employed featuring probes for all Drosophila genes for which different mRNA isoforms generated by alternative splicing have been described (see Blanchette M, Green RE, Brenner SE, Rio DC: Global analysis of positive and negative pre-mRNA splicing regulators in Drosophila. Genes Dev 2005, 19(11):1306-1314.).

Three biological replicates of total RNA isolated after pathway activation or controls (untreated cells for insulin, control dsRNA for wingless) were purified, reverse transcribed into cDNA and labelled with Cy5 or Cy3 fluorochromes and the cDNA was hybridized to the microarray,

first: Britta

last: Hartmann

first: Robert

last: Castelo

first: Marco

last: Blanchette

first: Stéphanie

last: Boué

first: Donald

last: Rio

first: Juan

last: Valcárcel


title: Gene expression profiling of prostate cancer

city: Pozuelo de Alarcon

first: Jose M.

last: Arencibia

state: Madrid

department: Biochemistry and Biotechnology

institute: Universidad Francisco de Vitoria

summary:
To better understand the molecular mechanism that underlay tumorigenesis and progression of prostate cancer (PCa), we studied the gene expression profiles of sixteen PCa compared to their matched normal tissues. Using Significance Analysis of Microarrays, we identified 53 genes differentially expressed that were more than 2 fold up- or down-regulated in at least 50 % of the tumors.
Keywords: disease state analysis

overall_design: Samples from 16 patients with prostate cancer were analyzed. The matched normal tissue from each patient was used as control. The control RNA (normal tissue) was labeled with Cy3 and the test RNA (prostate cancer tissue) with Cy5.

first: Jose

first: Ana

last: Bonnin

first: Susana

last: Martin

first: Francisco

last: Perez Rodriguez


title: Microarray Analysis of Toxicogenomic Effects of Hydrogen Peroxide on Mycobacterium bovis BCG

status: Public on Jan 20 2009

city: ft. meade

first: Hyeung-Jin

last: Jang

address: 701 Mapes Road

department: OPP/BEAD

institute: USEPA

summary:
In the present study, we employed Affymetrix Mycobacterium bovis BCG GeneChip arrays to investigate the dynamics of global gene expression profiles during the cellular response of Mycobacterium bovis BCG to hydrogen peroxide, which involved initial growth inhibition and metabolism.

Keywords: Transcriptome study

sample_organism: Mycobacterium tuberculosis variant bovis BCG str. Pasteur 1173P2

overall_design: We conducted three independent microarray experiments (biological replicates) in the absence (control) and the presence (experimental) of hydrogen peroxide. We calculated fold change as the ratio between the signal averages of three untreated (control) and three hydrogen peroxide-treated (experimental) cultures for 0, 10 and 60 min exposures.

first: Freshteh

last: Toghrol

first: William

last: Bentley


title: Microarray analysis of the interaction between Rhopalosiphum padi and partially resistant or susceptible barley lines

city: Huddinge

first: Gabriele

last: Delp

address: Alfred Nobels allé 7

department: Life Sciences

institute: Södertörns University College

summary: The bird cherry-oat aphid (Rhopalosiphum padi L.) (Homoptera: Aphididae) is an important pest on cereals causing plant growth reduction but no specific leaf symptoms. Breeding of barley (Hordeum vulgare L.) for R. padi resistance shows that there are several resistance genes involved, reducing aphid growth. In an attempt to identify candidate sequences for resistance-related genes, we performed a microarray analysis of gene expression after two days of aphid infestation in two susceptible barley lines and two genotypes with partial resistance. One of the four lines is a descendant of two of the other genotypes. The analysis revealed large differences in gene induction between the four lines, indicating substantial variation in response even between closely related genotypes. Genes induced in the aphid-infested tissue were mainly related to defence, primary metabolism and signalling. Only twenty-four genes were induced in all lines, none of them related to oxidative stress or secondary metabolism. Few genes were down-regulated and none of those was common to all four lines. There were differences in aphid-induced gene regulation between resistant and susceptible lines, and results from control plants without aphids also revealed differences in constitutive gene expression between the two types of lines. Candidate sequences for both induced and constitutive resistance factors have been identified, among them a proteinase inhibitor, a Ser/Thr kinase and several thionins.

sample_organism:
Hordeum vulgare
Hordeum vulgare subsp. spontaneum

overall_design: Four barley genotypes, two (Lina, Kara) with susceptibility to the bird cherry-oat aphid (Rhopalosiphum padi) and two (Hsp5, 28:4) with partial resistance, were infested with R. padi. After 48 h of aphid feeding, changes in transcript accumulation were analysed by comparing aphid-infested plants with control (uninfested) plants for each genotype. For each treatment, 3 biological replicates were analysed. Overlaps and differences in regulation of RNA abundance were identified between the four genotypes. 28:4 is a descendant of Hsp5 and Lina.

first: Therese

last: Gradin

first: Inger

last: Åhman

first: Lisbeth

last: Jonsson


title: Compartmental Localization and Clinical Relevance of MICA Antibodies after Renal Transplantation

status: Public on Jan 16 2009

first: Li

last: Li

address: 300 pastuer dr.,

department: Pediatric

institute: Stanford University

summary:
Background: Studies recently support that non-HLA antigens could be additional targets of injury in organ transplant recipients, and MICA was associated with an increased risk of graft loss.
Methods: A ProtoArray platform was used to study 37 serum samples from 22 unique patients (15 renal recipients and 7 healthy controls). Thirty paired pre- and post-transplant serum samples were analyzed for detection of de novo post-transplant antibody responses in the 15 patients (10 acute rejection (AR), 5 Stable). Probes on ProtoArray and cDNA platforms (GSE: 3931) were re-annotated and compartment specific gene lists were analyzed using the integrated genomics method. Normal and transplant kidney IHC were performed for MICA antigen localization.
Results: Mean MICA-Ab (antibody) signal intensity was significantly higher in transplant patients compared with healthy controls and de novo MICA-Ab were detected in 73% transplant patients. The mean post-transplant signal intensity of MICA-Ab was the highest in C4d+AR. Detection of MICA-Ab responses did not correlate with time post-transplantation, but significantly correlated with decline in graft function over the subsequent year. Integrative genomics predicted localization of the MICA antigen to the glomerulus.  IHC confirmed cytoplasmic MICA staining solely in glomerular podocytes in normal kidney. In the transplant kidney, infiltrating mononuclear lymphocytes (T, B and NK) in AR had additional MICA staining.
Conclusions: MICA can be highly detected regardless of graft dysfunction or AR. The intensity signal of the MICA antibody correlates with subsequent decline in graft function. The MICA antigen localizes to the glomerulus and infiltrating mononuclear cells in AR.

overall_design: Pre- and post-transplant serum antibodies were profiled for each patient, using the Invitrogen ProtoArray® Human Protein Microarray v3.0 platform (Invitrogen, Carlsbad, CA). This platform contains 5,056 non-redundant human proteins expressed in a baculovirus system, purified from insect cells and printed in duplicate onto a nitrocellulose-coated glass slide. Each protein is spotted twice on each array, to measure the quality of the signal intensity. Details for experiment processing and analysis follow the previous publication from our group (13). Prospector software was used to retrieve the expression based on immune response profiling of the .gal files.

first: Amery

last: Chen

first: Abanti

last: Chaudhuri

first: Neeraja

last: Kambham

first: Minnie

last: Sarwal


title: Comparative transcriptome analysis of two S. aureus strains with different susceptibilities to mersacidin.

status: Public on Jan 17 2009

city: Bonn

first: Peter

last: Sass

state: NRW

address: Sigmund-Freud-Straße 25

department: Institute for Microbiology (IMMIP)

country: Germany

institute: University of Bonn

summary:
The lantibiotic mersacidin is an antimicrobial peptide of 20 amino acids that is produced by Bacillus sp. strain HIL Y-85,54728. Lantibiotics are antibiotics containing nonproteinogenic amino acids like lanthionine and/or 3-methyllanthionine. Mersacidin interferes with cell wall biosynthesis by targeting the peptidoglycan precursor lipid II and inhibits the growth of MRSA and other gram-positive bacteria. Therefore, mersacidin could be a lead substance for the development of new antibacterial agents. The clinical VISA isolates S. aureus 137/93A and its spontaneous mutant S. aureus 137/93G show reduced sensitivity towards mersacidin. This phenotype could not be traced to factors being responsible for vancomycinresistance (i.e. the thickened cell wall).
Here, we focussed on the comparative transcriptome analysis of S. aureus 137/93A and S. aureus SG511 (sensitive strain) via full genome S. aureus microarrays to identify genes involved in the reduced sensitivity towards mersacidin.

platform_organism: Staphylococcus aureus subsp. aureus N315

overall_design: In this experiment, the transcriptomes of S. aureus 137/93A and S. aureus SG511 (sensitive strain) should be compared via microarray analysis. To this end, S. aureus 137/93A and S. aureus SG511 were grown without mersacidin to determine the genes which are differently expressed in these strains. Each experiment was performed 2 times including a dye swap resulting in two chips per competitive comparison with the incorporated dye reversed. All hybridizations were done with equal amounts of fluorescence-labeled cDNA probes or picomols of dye.


title: Global transcriptional analysis of S. mutans metalloregulation

status: Public on Jan 18 2009

city: Burlington

first: Jeffrey

last: Bond

state: VT

address: 95 Carrigan Dri e

department: Microbiology and Molecular Genetics

institute: University of Vermont

summary:
Multiple S. mutans genes that are subject to SloR control are likely contributors to virulence.  The regulation of these genes in response to metal ion limitation vs. availability in the oral cavity can significantly facilitate the caries-forming process and hence continued analysis of these data can reveal novel candidate genes for the purposes of therapeutic intervention and/or prevention.
Keywords: Genetic modification, dose response, metalloregulation

overall_design: Analyses were based on a comparison between low Mn and high Mn groups (paired comparisons A, B, and C) and the monitoring of gene expression in the presence (WT) or absence (mutant) of SloR under moderate Mn content. Analysis of significance amongst all genes and the formulation of hypotheses that are relevant to the cariogenic state were performed.

first: Grace

last: Spatafora

first: Nathaniel

last: Langer

first: Jeremy

last: Shaw


title: Liver tumor/normal pairs

city: Santa Clara

first: Yuker

last: Wang

address: 3380 Central Expressway

institute: Affymetrix

summary: A major challenge to the study of tumor DNA copy number (CN) in clinical specimens is the lack of appropriate fresh frozen samples and thus a dependence on Formalin-Fixed Paraffin Embedded (FFPE) banked samples, which typically have more extensive clinical follow up information.  However, on most high density CN platforms, DNA from FFPE tissues generally underperforms or suffers high failure rates compared to fresh frozen samples because of DNA degradation and cross-linking.  Molecular Inversion Probe (MIP) technology has been applied successfully to obtain high quality CN and genotype data from DNA isolated from cell lines and frozen tumor samples.  Since the MIP probes require only a small (~40 bp) target binding site, we reasoned they may be well suited to assess FFPE samples.  In this study, we successfully applied MIP technology with a panel of 50,000 markers to CN determination in FFPE samples.  Using an input of 37 ng genomic DNA, we demonstrated high quality CN data with MIP technology from 93 FFPE samples from seven diverse collections.  We found that the performance of FFPE DNA for CN determination was comparable to that of DNA obtained from matched frozen tumor, with only a modest loss in performance of DNA.

overall_design: Tumor FFPE were analyzed using matched normal FFPE as the reference. Tumor and normal pairs: 01-02, 12-13, 16-17, 08-09, 20-21, 24-25, 28-29, and 35-36.  Tumor 32 has no matched normal sample.

first: Victoria

last: Carlton

first: Robert

last: Warren

first: Malek

last: Faham


title: Glucocorticoid-regulated microRNAs and mirtrons in human acute lymphoblastic leukemia

city: Bolzano

first: Johannes

last: Rainer

address: Via A. Volta 21

department: Institute for Biomedicine

country: Italy

institute: Eurac Researc

summary:
Glucocorticoids (GC) have a major impact on the biology of normal and malignant cells of the lymphoid lineage. This includes induction of apoptosis which is exploited in the therapy of acute lymphoblastic leukemia (ALL) and related lymphoid malignancies. MicroRNAs (miRNAs), and the related mirtrons, are ~22 nucleotide RNA molecules implicated in the control of essential biological functions including proliferation, differentiation and apoptosis. They derive from polymerase-II transcripts but whether GCs regulate miRNA/mirtron-encoding transcription units is not known. We investigated miRNA/mirtron expression and GC regulation in 8 ALL in vitro models and 13 ALL children undergoing systemic GC monotherapy using a combination of expression profiling techniques, real time RT-PCR and northern blotting to detect mature miRNAs/mirtrons and/or their precursors. We identified a GC-regulated mirtron  (miR-1233) and a number of GC-regulated miRNAs, including the myeloid-specific miR-223 and the apoptosis and cell cycle arrest-inducing mir15~16 cluster. However, the response was heterogeneous in individual patients and cell lines, the extent of regulation was moderate, and functional analyses of the most promising candidates (miR223 and the miR15~16 cluster) revealed little, if any effect, on cell cycle progression and survival. Thus, although GCs affect miRNA expression in ALL cells, these regulations may not be major contributors to the anti-leukemic GC effects.
Keywords: exon analysis

overall_design: Glucocorticoid-sensitive T-ALL cell line CCRF-CEM-C7H2 was treated in 3 independent experiments either with 100nM dexametasone (a glucocorticoid) or 0.1% ethanol as an empty carrier control. Samples were taken at various timepoints (30 minutes, 2, 6 and 24 hours), RNA extracted and hybridized onto Affymetrix Exon microarrays (HuEx 1.0 st).


title: LAD coronary atherosclerotic plaque gene expression

city: Padova

first: Gerolamo

last: Lanfranchi

address: Via U. Bassi, 58/B

department: CRIBI - Biotechnology Center and Biology Department

institute: University of Padova

summary:
We used microarray analyses in human atherosclerotic plaque to identify network imply in the maintenance and progression of the pathology. To obtain a wide dynamic range we used the scanning method proposed by Skibbe et al. (Bioinformatics. 2006;22(15):1863-1870) performing three subsequent scansions for the same slide. We performed before low than medium and high PMT and laser intensity scansions to permit to detect differences in expression of high expressed genes from the first scansion and from low expressed genes from the last scansion. Data were total and lowess normalized, filtered and used to detect differentially expressed genes separately (low, medium and high). Differentially expressed genes were than integrated. Our data enhance the importance of both the inflammatory stress responses, controlled by central node STAT1 (inducing also the over-expression of the heat shock proteins HSP47 and HO-1), and caveolae system related to steroid hormone receptors.
Since atherosclerosis affects aorta, coronary, carotid, and iliac arteries most frequently than any other body vessel there may be common molecular pathways involved in sustenance of this process. We integrated our DNA microarrays data with plaque carotid gene expression (Array Express databases E-MEXP-268) by meta-analysis method. We identified a series of common potential human atherogenic genes and were able to integrate them in functional networks involved in atherosclerosis.
Activation of the JAK/STAT pathway was confirmed by the up-regulation of IL-6, STAT1, ISGF3G and IL10RA genes in coronary and carotid plaques. These results and constructed functional network could be related to a central role for STAT protein and caveolae system to regulate the hypertension state.
Keywords: disease state analysis, Plaque atherosclerotic, LAD coronary

overall_design:
18 left anterior descendent (LAD) coronaries were dissected from diseased hearts immediately after surgical removal, cleaned from fatty and cardiac muscle tissues and starved in RNAlater Solution (Ambion) for further analysis. Patients were divided in 8 atherosclerotic with plaque (stenosis ≥ 75%) and 10 pooled controls without plaque. At least 2 microarray experiments were performed for each patient (technical replicate) and for patients with sufficient biological material we performed also a biological replicate with its own technical replicate.
Each slide was scanned three times at low, medium and high PMT and laser intensity producing three data batch (low, medium and high) related to the same patients. Each data batch was normalized and analyzed separately. Detected differentially expressed genes were integrated to produce plaque differentially expressed genes (see Supplementary files below).

Meta-analysis supplementary files also linked below.
GSE3526-GSE7307-asym-symp-Meta-analysis.txt is result file of the analysis of Carotid data. From this file, we extracted specific and common carotid and coronary plaque differentially espressed genes comparing lists of differentially expressed genes using EntrezGene symbol. All files with starting name with Specific word fill in the specific category and the other in the common one.

first: Stefano

last: Cagnin

first: Michele

last: Biscuola

first: Cristina

last: Patuzzo

first: Elisabetta

last: Trabetti

first: Alessandra

last: Pasquali

first: Paolo

last: Laveder

first: Giuseppe

last: Faggian

first: Mauro

last: Iafrancesco

first: Alessandro

last: Mazzucco

first: Pier-Franco

last: Pignatti


title: Genome-wide DNA methylation in CEBPA mutant and CEBPA silenced AML

status: Public on Jan 15 2009

city: Miami

first: Maria

middle: Eugenia

last: Figueroa

state: FL

address: 1501 NW 10th Ave, BRB 709A, Locator code C227

department: Human Genetics

institute: University of Miiami

summary:
Acute Myeloid Leukemia (AML) is a heterogeneous disease from the molecular and biological standpoints, and even patients with a specific gene expression profile may present clinical and molecular heterogeneity. We studied the epigenetic profiles of a cohort of patients that shared a common gene expression profile but differed in that only half of them harbored mutations of the CEBPA locus, while the rest presented with silencing of this gene and co-expression of certain T cell markers. DNA methylation studies revealed that these two groups of patients could be readily segregated in an unsupervised fashion based on their DNA methylation profiles alone. Furthermore, CEBPA silencing was associated with the presence of an aberrant DNA hypermethylation signature, which was not present in the CEBPA mutant group. This aberrant hypermethylation occurred more frequently at sites within CpG islands. CEBPA silenced leukemias also displayed marked hypermethylation when compared with normal CD34+ hematopoietic cells, while CEBPA mutant cases showed only mild changes in DNA methylation when compared to these normal progenitors.  Biologically, CEBPA silenced leukemias presented with a decreased response to myeloid growth factors in vitro.

Keywords: DNA methylation profiling

overall_design: Direct comparison of DNA methylation in leukemic blasts from 8 patients with Acute Myeloid Leukemia (AML) carrying a CEBPA mutation and 8 patients with AML without CEBPA mutation but with silencing of CEBPA expression. Two control groups are included: 8 CD34+ bone marrow samples from healthy donors and 9 samples of T Acute Lymphoblastic Leukemia (T-ALL) patients.

first: Bas

last: Wouters

first: Ari

last: Melnick

first: Ruud

last: Delwel


title: Distinct transcriptional outputs associated with mono- and di-methylated histone H3 arginine 2

city: Cambridge

first: Antonis

last: Kirmizis

state: England

address: Gurdon institute, Tennis Court Rd

institute: Antonis Kirmizis

summary: Dimethylation of histone H3 arginine 2 (H3R2me2) maintains transcriptional silencing by inhibiting Set-1 mediated trimethylation of H3K4. Here we demonstrate that arginine 2 is also monomethylated (H3R2me1) in yeast but that its functional characteristics are distinct from H3R2me2: (a) H3R2me1 does not inhibit H3K4 methylation, (b) it is present throughout the coding region of genes and (c) it correlates with active transcription. Collectively, these results indicate that different H3R2 methylated states have defined roles in gene expression.

overall_design: hIP-chip analysis was performed to determine the genomic distribution of histone modifications H3R2me1, H3R2me2a, H3K4me3, H3K36me3, and H3K79me3 in the BY4741 yeast strain.  ChIP samples were amplified, labelled, and hybridized to 50-mer tiling arrays covering the entire yeast genome at a 64 bp resolution.

first: Helena

last: Santos-Rosa

first: Christopher

last: Penkett

first: Michael

last: Singer

first: Roland

last: Green

first: Tony

last: Kouzarides


title: Arabidopsis CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance

city: St Paul

first: lin

last: wang

state: MN

address: 350 Cargill building, 1500 Gortner Ave

department: Plant Biology

institute: University of Minnesota

summary: Salicylic acid (SA)-induced defense responses are important factors during effector triggered immunity and microbe-associated molecular pattern (MAMP)-induced immunity in plants. This article presents evidence that a member of the Arabidopsis CBP60 gene family, CBP60g, contributes to MAMP-triggered SA accumulation. CBP60g is inducible by both pathogen and MAMP treatments. Pseudomonas syringae growth is enhanced in cbp60g mutants. Expression profiles of a cbp60g mutant after MAMP treatment are similar to those of sid2 and pad4, suggesting a defect in SA signaling. Accordingly, cbp60g mutants accumulate less SA when treated with the MAMP flg22 or a P. syringae hrcC strain that activates MAMP signaling. MAMP-induced production of reactive oxygen species and callose deposition are unaffected in cbp60g mutants. CBP60g is a calmodulin-binding protein with a calmodulin-binding domain located near the N-terminus. Calmodulin binding is dependent on Ca2+. Mutations in CBP60g that abolish calmodulin binding prevent complementation of the SA production and bacterial growth defects of cbp60g mutants, indicating that calmodulin binding is essential for the function of CBP60g in defense signaling. These studies show that CBP60g constitutes a calmodulin-dependent link between MAMP recognition and SA accumulation that is important for resistance to P. syringae.

overall_design: This experiment consists of three biological replicates. For each genotype, two leaves per plant were pooled from three pots to prepare total RNA.

first: Lin

last: Wang

first: Kenichi

last: Tsuda

first: Jane

last: Glazebrook


title: Transcriptional response of Candida parapsilosis in low oxygen (hypoxic) conditions in SD media

first: Geraldine

last: Butler

address: Belfield

department: School of Biomolecular and Biomedical Science

institute: University Colege Dublin Conway Institute

summary: Whole genome microarray was used to compare the transcriptional profile of C. parapsilosis growing in normoxic (21% oxygen) and hypoxic (1% oxygen) conditions.

overall_design: RNA was isolated from cells grown in SD media at 37°C for 3 h in atmosphere oxygen of 1% oxygen, and labeled with Cy5 or Cy3. Six independent biological replicates were compared. 4 out of 6 hypoxic samples were labeled with Cy5, and 2 were labeled with Cy3.

first: Chen

last: Ding

last: Guida


title: Plasmodium berghei WT and ap2-o disruptant ookinete culture 12h

status: Public on Jan 13 2009

city: tsu

first: masao

last: yuda

state: mie

address: edobashi 2-174

country: Japan

institute: mie university


title: MEF2D binding sites in rat hippocampal neurons

city: Boston

first: Tae-Kyung

last: Kim

state: MA

address: 300 Longwood avenue, CLS building 12260

department: Neurology

institute: Children's Hospital

summary:
We analyzed the binding profiles of MEF2D, a member of MEF2 family transcription factors, in rat hippocampal neurons.

Keywords: ChIP-chip

overall_design: We used custom-designed rat genome tiling array manufactured by Nimblegen Systems, Inc . This array contains probes that represent the following rat genomic regions: 182 genes identified by mRNA profiling experiments, 86 genes whose expression was decreased by both KCl-mediated depolarization and MEF2 RNAi, and 40 control genes whose expression was not altered by MEF2 RNAi or MEF2-VP16-ER. The array not only covers the entire gene regions but also contains probes that correspond to the 40 kb 5′and 3′ to each gene.  Repeatmasking was conducted by Nimblegen to ensure that repetitive elements were not tiled on the microarray.  Probe length and spacing between the probes were 50-75mer and 50 bp, respectively.


title: Transcription analysis of biofilm grown in different depth

city: Jerusalem

first: Moshe

last: Shemesh

address: Hebrew University-Hadassah POB 12272

department: Institute of Dental Sciences

country: Israel

institute: Hebrew University-Hadassah

summary:
Using DNA-microarrays analysis 39 differentially expressed genes were identified in 200 vs. 100 microns biofilms, which might be associated with thickness of S. mutans biofilm.
Using DNA-microarrays analysis 29 differentially expressed genes were identified in 400 vs. 100 microns biofilms of S. mutans.

overall_design:
200 vs. 100 microns in depth:
The arrays consisted of 1948 70-mer oligonucleotides representing 1960 ORFs from S. mutans UA159 and additional control sequences. The results represent the findings of two independent biological replicate arrays performed with two different RNA samples. In one of the arrays the 100-microns biofilm RNA sample was labeled with Cy5 and the 200-microns biofilm RNA with Cy3, while the second array was reversibly labeled.

400 vs. 100 microns in depth
The arrays consisted of 1948 70-mer oligonucleotides representing 1960 ORFs from S. mutans UA159 and additional control sequences. The results represent the findings of three independent biological replicate arrays. In two of the arrays the RNA sample from 100-microns depths biofilm was labeled with Cy5 and the 400-micron - with Cy3, while the third array was reversibly labeled.


title: Postrecruitment Regulation of RNA Pol II Directs Rapid Signaling Responses at the Promoters of Estrogen Target Genes

status: Public on Jan 12 2009

city: Dallas

first: W. Lee

last: Kraus

state: TX

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

summary: Under current models for signal-dependent transcription in eukaryotes, DNA-binding activator proteins regulate the recruitment of RNA polymerase II (Pol II) to a set of target promoters.  Yet, recent studies, as well as our results herein, show that Pol II is widely distributed (i.e., "preloaded") at the promoters of many genes prior to specific signaling events.  How Pol II recruitment and Pol II preloading fit within a unified model of gene regulation is unclear.  In addition, the mechanisms through which cellular signals activate preloaded Pol II across mammalian genomes remain largely unknown.  Here we show that the predominant genomic outcome of estrogen signaling is the post-recruitment regulation of Pol II activity through phosphorylation, rather than recruitment of Pol II.  Furthermore, we show that negative elongation factor (NELF) binds to estrogen target promoters in conjunction with preloaded Pol II and represses gene expression until the appropriate signal is received.  Finally, our studies reveal that the estrogen-dependent activation of preloaded Pol II facilitates rapid transcriptional and post-transcriptional responses which play important physiological roles in regulating estrogen signaling itself.  Our results reveal a broad use of post-recruitment Pol II regulation by the estrogen signaling pathway, a mode of regulation that is likely to apply to a wide variety of signal-regulated pathways.

overall_design: ChIP-chip analysis for RNA Pol II, Ser5 phosphorylated RNA Pol II and NELF-A in MCF7 breast cancer cells.

first: Miltiadis

last: Kininis

first: William


title: Transcriptional analysis of respiratory pathways in the hyperthermophilic crenarchaeon Pyrobaculum aerophilum

city: Santa Cruz

first: Aaron

last: Cozen

state: CA

address: 1165 High St.

department: Ocean Sciences

institute: University of California, Santa Cruz

summary:
We compared global transcriptional patterns in Pyrobaculum aerophilum cultures with oxygen, nitrate, arsenate and ferric iron as respiratory electron acceptors to identify genes and regulatory patterns that differentiate these pathways.
Keywords: Time course study with different respiratory electron acceptors

overall_design: To focus the microarrays on genes that are specifically affected by changes in terminal electron acceptors we analyzed gene expression in time courses with cultures shifted from aerobic growth to either NO3-, As(V), Fe(III)-citrate, or additional O2. Gene expression and substrate usage were measured at three time points (2.5, 4.5, and 7.5 hours), allowing sufficient time for changes in gene expression, while restricting cultures to a maximum of one or two cell divisions.

first: Matthew

middle: T

last: Weirauch

first: Katherine

middle: S

last: Pollard

first: David

last: Bernick

first: Joshua

middle: M

last: Stuart

first: Todd

last: Lowe


title: Jade1 ChIP-chip on ENCODE Tiling array

status: Public on Jan 09 2009

city: Stanford

first: Tiffany

last: Hung

address: 371 SERRA MALL

summary: Several MYST-family histone acetyltransferase (HAT) enzymes associate with specific ING tumor suppressor proteins. ING complexes containing the HBO1 HAT protein are the major source of histone H4 acetylation in vivo and have been shown to play critical roles in gene regulation and DNA replication. Here, we present a molecular dissection of HBO1/ING HAT complexes that unravels the protein domains required for complex assembly and function. A distinctive characteristic of ING HAT complexes is the presence of multiple PHD finger domains in different subunits. Biochemical and functional analysis of these domains indicate that they interact with histone H3 N-terminal tail region but with different specificity towards the methylation status of lysine 4. They play essential and intricate role in regulating binding to chromatin and substrate specificity. This is achieved in part through expression of subunit isoforms controlling which PHD fingers are present in the complex. Importantly, localization analysis on the human genome indicate that HBO1 complexes are enriched throughout the coding region of genes, supporting a role in transcription elongation. These results also underline the importance and versatility of PHD finger domains in regulating chromatin association and trans-histone acetylation specificity within a single protein complex.

overall_design: ChIP-chip of FLAG-JADE1 +/- doxorubicin treatment in Hela cells

first: Nehme

last: Saksouk

first: Nikita

last: Avvakumov

first: Or

last: Gozani

first: Jacques

last: Cote


title: Transcriptional profiling of FeHm effects on Haemophilus influenzae R2866

city: Oklahoma City

first: Timothy

last: VanWagoner

address: 940 SL Young Blvd, BMS340

institute: University of Oklahoma Health Sciences Center

summary:
Background: Haemophilus influenzae has an absolute aerobic growth requirement for heme and has developed multiple complex mechanisms to obtain this essential nutrient.  Although an understanding of the heme acquisition mechanisms of H. influenzae is emerging, significant gaps remain to be elucidated.  In a previous study we utilized H. influenzae strain Rd KW20 to demonstrate the utility of transcriptional profiling in defining the genes exhibiting altered transcription in response to environmental iron and heme levels. The current study expands upon those initial observations by determining the iron/heme regulons of two additional H. influenzae clinical isolates, i.e. the type b isolate 10810 and the nontypeable isolate R2866, to characterize the core iron/heme regulon of the species.
Results: A microarray chip was designed to incorporate probes for all of the genes of H. influenzae isolates 10810 and R2866, and microarray studies were performed to compare gene expression under iron/heme-replete and iron/heme-restricted conditions for each isolate.  Of 1820 ORFs on the array corresponding to R2866 genes, 363 were significantly differentially expressed.  Of these 363 genes, 233 were maximally transcribed under iron/heme-replete conditions and 130 were preferentially transcribed in iron/heme-restricted conditions.  Of the 1883 ORFs representing genes of strain10810, 351 were significantly differentially transcribed, 150 of these were preferentially transcribed in iron/heme-replete conditions and 201 were preferentially transcribed in iron/heme-restricted conditions.  Comparison of the data sets indicated that 163 genes were similarly regulated in both isolates and that 74 of these also exhibited similar patterns of regulation in strain Rd KW20.
Conclusion: This study provides evidence for a core of H. influenzae genes that are regulated by the availability of iron and/or heme in the growth environment.  Elucidation of this core regulon provides targets for investigation of genes with an unrecognized role in iron and heme homeostasis, as well as other potential virulence determinants. In addition, the set of core genes potentially provides targets for therapeutic and vaccine designs since these products of these genes are likely to be preferentially expressed during growth in iron/heme restricted sites of the human body.
Keywords: Transcription analyses

overall_design: Three replicate cultures were grown in iron and heme depleted media for 90 minutes and samples taken for expression analyses. Fe and heme were supplemented to the remaining cultures and the cultures allowed to incubate an additional 20 minutes. Microarray analyses were used to determine changes in gene expression resulting from transfer from FeHm-deplete to FeHm-replete conditions.

first: Paul

last: Whitby

first: Thomas

last: Seale

first: Daniel

last: Morton

first: Terrence

last: Stull


title: Evaluation of three different fluorescent dyes for cDNA labeling in microarrays

city: Copenhagen

first: Shila

last: Mortensen

address: Bülowsvej 27

department: Dep. for Veterinary Diagnostics and Research

institute: National Veterinary Institute, Technical University of Denmark

summary:
The simultaneous measurement of gene expression for thousands of genes in a single analysis by the microarray technology allows researchers to describe transcriptomes in various samples of interest. Problems with variation in data quality derived from microarray experiments are well known and might result from poor RNA quality, background problems, or sub optimal signal strength. To assess variation due to the fluorescent dye chosen, three different dye pairs were tested for labelling of cDNA in gene expression analysis experiments on a porcine immune focused oligonucleotide microarray (POM3). This in-house oligonucleotide microarray allowed a direct comparison of background fluorescence, Median signal intensities, numbers of spots detected, and resistance to photobleaching between different dye pairs. We tested Alexa Flour 546/647, Cy3/Cy5 as well as Oyster 550/650 all from Genisphere Inc., Hatfield, PA, USA.
Keywords: Comparison of fluorescent dyes

overall_design:
Each dye pairs were hybridized on two slides. The same two samples were compared on all the slides used in the present study. The liver sample from a healthy animal was labeled with the high wavelength dyes (Oyster 650/Alexa 647/Cy5) and the liver sample from the sick animal was labeled with the low wavelength dyes (Oyster 550/Alexa 546/Cy3). Each slide was scanned 3 times, immediately after hybridization (first round), after 1 month of storage in darkness and after 24 hours on the bench (12 hours of daylight and 12 hours of artificial light).

Total RNA from the infected and control liver samples were extracted using RNeasy midi kit (Qiagen, Denmark) and DNase treated using RNase-Free DNase Set (Qiagen).
3DNATM Array 900 expression array detection kits (Genisphere Inc.) were used for the labeling and cDNA synthesis reaction of the RNA in the present study. Three different pairs of fluorescent dyes Oyster 550/650 (Genisphere Inc.), Alexa 546/647 (Genisphere Inc.), and Cy3/Cy5 (Genisphere Inc.) were used in separate labeling reactions. Labeling was done according to the manufacturers protocol for large-scale cDNA synthesis. For the cDNA synthesis 9.2µg total RNA from each liver sample was used and mixed with 20U AmpliQ RTenzyme and 10x first strand buffer (Bie og Berntsen, Rødovre, Denmark).

Hybridization and washing were performed according to the manufacturers instructions (Genisphere) using Corning hybridization chambers. For one cDNA hybridization reaction; 6.5µl cDNA from each synthesis, 0.5µl Salmon sperm (10µg/µl) and 13.5µl 2 x Formamide-Based Hybridization Buffer (3DNA Array 900, Genisphere) was used. A total hybridization mix of 29µl was applied under a 22I x 25 mm LifterSlip (Erie Scientific, Portsmouth, NH, USA) carefully avoiding air bubbles. Slides were incubated at 44oC in a water bath over night. Wash buffer 1 (3DNA Array 900, Genisphere) was preheated to 44oC for the post cDNA hybridization wash. For two 3DNA hybridization reaction mixes; 2.5µl of each Capture reagent and 26µl SDS-Based hybridization buffer was mixed to a final volume of 52µl. 26µl 3DNA hybridization mix was applied to each slide, and incubated in darkness in a water bath at 50oC for 4 hours. Wash buffer 1 was preheated to 60oC for the post 3DNA hybridization wash. Slides were scanned on a CCD ArrayWoRxe auto (Applied Precision, Issaquah, WA, USA) using different exposure times (0.3 for 595 nm and 1.2 for 685 nm).

first: Kerstin

last: Skovgaard

first: Peter

last: Heegaard

first: Mette

last: Boye


title: Transcriptome profiling of Pacific oyster Crassostrea gigas families that differ in tolerance of heat shock

city: Charleston

first: Marine

last: Genomics

state: SC

address: 331 Ft. Johnson Road

institute: Medical University South Carolina

summary:
ESTs in which transcription levels in oyster gill differed among sampling times after heat shock and between families with high or low survival of heat shock were identified through expression profiling of 1,675 spot pairs

Keywords: comparison of families characterized by low or high survival after heat shock

platform_organism:
Crassostrea virginica
Magallana gigas

overall_design: Oyster families were characterized by survival after heat shock, exposed to a sub-lethal heat shock, and gill was collected before and at 1, 3, 6, and 24 h afterwards.  Differences among sampling times and between family types were assessed using a 2-way analysis of variance.  In total, 1,675 ESTs were included in the analysis.

first: R

last: Lang

first: C

last: Bayne

first: M

middle: D

last: Camara

last: Jenny

last: Cunningham

last: Langdon


title: Base and acid resistance of B. subtilis associated with transcriptomic responses

city: Gambier

first: Joan

middle: Lyn

last: Slonczewski

address: 202 N. College Rd

department: Biology Dept

institute: Kenyon College

summary: Gene expression profiles of Bacillus subtilis strain AG174 were compared as a function of steady-state external pH during growth with aeration. Aerated overnight cultures were diluted 1:500 and incubated in 250 mL baffled flasks containing potassium-modified Luria-Bertani medium (LBK) buffered with 50 mM HOMOPIPES at pH 6.0, pH 7.0, and pH 9.0. Tubes were incubated at 37°C with aeration (260 rpm) to an optical density at 600 nm of 0.2. For each of the three pH conditions, RNA was isolated from five independent cultures.

overall_design: Overnight cultures were diluted 1:500 and incubated in 250 mL baffled flasks containing potassium-modified Luria-Bertani medium (LBK) buffered with 50 mM HOMOPIPES at pH 6.0, pH 7.0, and pH 9.0. Cultures were incubated at 37°C with aeration (260 rpm) to an optical density at 600 nm of 0.2. Five replicate cultures were grown at each pH.

first: Sarah

middle: H

last: Cleeton

first: Grace

last: Lee

first: Cheena

last: Ugwu

first: Jessica

last: Wilks

first: Ryan

last: Kitko

first: Brian

last: Jones

first: Sandra

last: BonDurant


title: Whole-genome comparative RNA expression profiling of axenic and intracellular amastigote forms of Leishmania infantum

status: Public on Jan 08 2009

city: Quebec

first: Barbara

last: Papadopoulou

state: Quebec

address: 2705 boulevard Laurier

institute: Centre de Recherche du CHUL

summary:
Transcriptional analysis of L. infantum promastigote compared to L. infantum intracellular amastigote and transcriptional analysis of L. infantum promastigote compared to L. infantum axenic amastigote.
The full-genome DNA microarrays includes one 70-oligonucleotides probe for each gene of L.infantum.

Keywords: stage and culture condition

platform_organism:
Leishmania major
Leishmania infantum

overall_design:
Intracellular amastigote analysis: Two-condition experiment, promastigote stage vs intracellular amastigote stage. Six biological replicates for each stage, independently grown and harvested. One replicate per array
Axenic amastigote analysis: Two-condition experiment, promastigote stage vs axenic amastigote stage. Four biological replicates for each stage, independently grown and harvested. One replicate per array.

first: A

last: Rochette

first: F

last: Raymond

first: J

last: Corbeil

last: Ouellette

first: B


title: Characterization of the Bacterial Diversity of Drinking Water From 3 Parisian Treatment Plants Using Genomic Approaches

city: paris

first: Jean Baptiste

last: Poitelon

address: 9 rue Schoelcher

institute: EaudeParis

summary: We characterized the bacterial diversity of chlorinated drinking water from three surface water treatment plants supplying the city of Paris, France. For this purpose, we used serial analysis of V6 ribosomal sequence tag (SARST-V6) to produce concatemers of PCR-amplified ribosomal sequence tags (RSTs) from the V6 hypervariable region of the 16S rRNA gene for sequence analysis. Using SARST-V6, we obtained bacterial profiles for each drinking water sample, demonstrating a strikingly high degree of biodiversity dominated by a large collection of low-abundance phylotypes. In all water samples, between 57.2-77.4% of the sequences obtained indicated bacteria belonging to the Proteobacteria phylum. Full-length 16S rDNA sequences were also generated for each sample, and comparison of the RSTs with these sequences confirmed the accurate assignment for several abundant bacterial phyla identified by SARST-V6 analysis, including members of unclassified bacteria, which account for 6.3-36.5% of all V6 sequences. These results suggest that these bacteria may correspond to a common group adapted to drinking water systems. The V6 primers used were subsequently evaluated with a computer algorithm to assess their hybridization efficiency. Potential errors associated with primer-template mismatches and their impacts on taxonomic group detection were investigated. The biodiversity present in all three drinking water samples suggests that the bacterial load of the drinking water leaving treatment plants may play an important role in determining the downstream community dynamics of water distribution networks.

overall_design: 3 different drinking water samples (Orly, Ivry, Joinville drinking water sample)

first: Jean-Baptiste

first: Michel

last: Joyeux

first: Bénédicte

last: Welté

first: Jean-Pierre

last: Duguet

first: Eric

last: Prestel

first: Olivier

last: Lespinet

first: Michael

last: DuBow


title: Intramuscular fat variability in pigs

city: saint-gilles

first: marie

last: damon

address: domaine de la prise

institute: INRA

summary:
Intramuscular (i.m.) fat content influencing consumer’s acceptability of pork is considered as a limiting factor for meat quality. To gain insight into the biological basis of individual variability in i.m. fat content, both gene expression profiling and proteomic investigation were associated in pig longissimus muscle (LM).
Keywords: intramuscular fat, gene expression, pigs, proteomics, microarray, pork meat

overall_design: Animals were sampled from a population of 1,000 pigs generated as an F2 intercross between two production sire lines: FH016 (Pietrain type, France Hybrides SA, St Jean de Braye, France) and FH019 (Synthetic line, from Duroc, Hampshire and Large White founders, France Hybrides SA, St Jean de Braye, France).

first: Marie

last: Damon

first: Jingshun

last: Liu

first: Florence

last: Gondret

first: Pierre

last: Cherel


title: Gene analysis of the neonatal rat lung exposed to STZ-induced maternal diabetes

status: Public on Jan 07 2009

city: Turku

first: Anna

last: Koskinen

address: Kiinamllynkatu 10

department: University of Turku

country: Finland

institute: Research centre of Applied and Preventive Cardiovascular Medicine (CAPC)

summary:
Maternal diabetes is associated with a wide range of fetal and neonatal adverse effects including pulmonary disturbances. To investigate the effects of maternal diabetes on neonatal lung gene expression profile, we performed microarray analysis on the lungs of 14-day-old rats born to diabetic dam.

Keywords: disease state analysis

overall_design: Four neonatal lungs exposed to maternal diabetes and four control lungs were analyzed.

first: Hanna

last: Soukka

first: Asta

last: Laiho


title: FOXA1 ChIP-chip in DLD1 cells

city: Lille

first: Jérôme

last: Eeckhoute

address: Bâtiment J&K, Faculté de Médecine de Lille-Pôle Recherche, Boulevard du Professeur Leclerc

institute: CNRS

summary: FOXA1 recruitment sites within chromosomes 8, 11 and 12 were analyzed by ChIP-chip in DLD1 cells

overall_design:
DLD1 colon cancer cells were grown in full media, cross-linked and processed as in (Carroll et al. 2005). DNA was hybridized to Human Affymetrix tiled array F.
Duplicates of FOXA1 and input DNA.


title: Gene expression variations caused by dietary changes in methy-donor content

status: Public on Jan 14 2009

city: Research Triangle Park

first: Kathy

last: Boon

state: NC

address: 111 TW Alexander Dr

department: Division of Intramural Research

institute: NHLBI/NIEHS

summary: In this study we adapted and tested the applicability of Methylation Specific Digital Karyotyping (MSDK), a non-array based method, for the prospective analysis of epigenetic changes after perinatal nutritional modifications in a mouse model of allergic airway disease. As part of the same study we also analyzed the gene expression profiles by SAGE.

overall_design: Pairwise comparison of two pools of two individual mouse lung tissue samples.

first: John

last: Tomfohr

first: Nathaniel

last: Bailey

first: Shuichiro

last: Maruoka

last: Hollingsworth

first: Schwartz

last: David


title: Actinobacillus pleuropneumoniae BHI vs Koromycin

status: Public on Jan 06 2009

city: St-Hyacinthe

first: Vincent

last: Deslandes

address: 3200 rue Sicotte

department: Groupe de Recherche sur les Maladies Infectieuses du Porc

institute: Université de Montréal - Faculté de Médecine Vétérinaire

summary: Actinobacillus pleuropneumoniae is the etiologic agent of contagious pleuropneumonia, an economically important disease of commercially reared swine throughout the world. To cause this disease, A. pleuropneumoniae must rapidly overcome porcine pulmonary innate immune defenses.  Effects of koromycin, an antimicrobial agent that acts as an noncompetitive inhibitor of the interaction of NQR with its quinone substrate, on the transcriptome of A. pleuropneumoniae was investigated.

platform_organism: Actinobacillus pleuropneumoniae serovar 5b str. L20

overall_design:
Labelled cDNA synthetised from tester condition (namely wt strain growing in BHI-koromycin) was co-hybridized on the microarray with labelled cDNA from the control condition (wt BHI). Control was labelled with Cy3.http://www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSE13006

At least four hybridizations were conducted for each sample combination type. Microarray analysis was carried out using the TM4 Suite of softwares (TIGR) and the SAM algorithm, using a false discovery rate (FDR) value of 0%. For the planktonic and adhesion experiments, Cy5 signal (test) was compared to Cy3 signal (control), except for one dye swap, in order to obtain a list of significant differentially expressed genes.

first: Abdul

last: Lone

last: Nash

first: Mario

last: Jacques

first: Janet

middle: I

last: MacInnes


title: Regulation of leukemic cell differentiation and retinoid-induced gene expression by statins

city: Torino

first: Raffaele

last: Calogero

state: To

address: Via Nizza 52

department: Molecular Biotechnology Center

institute: University of Torino

summary: There is emerging evidence that, beyond their cholesterol lowering properties, statins exhibit important antileukemic effects in vitro and in vivo, but the precise mechanisms by which they generate such responses remain to be determined. We have previously shown that statins promote differentiation of acute promyelocytic leukemia (APL) cells and enhance generation of all-trans-retinoic acid (ATRA)-dependent antileukemic responses. We now provide evidence that statin-dependent leukemic cell differentiation requires engagement and activation of the JNK kinase pathway. In addition, in experiments to define the molecular targets and mediators of statin-induced differentiation we found a remarkable effect of statins on ATRA-dependent gene transcription, evidenced by the selective induction of over 400 genes by the combination of atorvastatin and ATRA. Altogether, our studies identify novel statin molecular targets linked to differentiation, establish that statins modulate ATRA-dependent transcription, and suggest that combined use of statins with retinoids may provide a novel approach to enhance antileukemic responses in APL and possibly other leukemias.

overall_design: To determine whether the effects of statin-treatment on ATRA-induced leukemic cell differentiation reflect induction of specific genes, the patterns of gene expression induced by ATRA, atorvastatin, or by the combination of atorvastatin + ATRA were subsequently examined using DNA microarrays. We analyzed three prototypic situations (ATRA, atorvastatin, ATRA + atorvastatin) using Illumina Sentrix® Human-6 Expression BeadChips over three points time course (8, 24, 48 hours), and time course points were replicated in three independent experiments performed at different days. A total of 36 arrays were hybridized. After average probe intensity 9 calculation, log2 transformation and normalization, probes characterized by low quality signal or invariant expression within the experimental conditions were discarded. A total of 11287 out of the 47293 RefSeq BeadChip probes were used to identify significantly differentially expressed genes. Statistical analysis was performed using a two step regression strategy. 1901 RefSeqs were found significantly differentially expressed in at least one condition over the time course treatments. After calculating the log2 (fold change) variation for all treatments with respect to the corresponding control time point, 782 out of 1901 RefSeq probes were characterized by a |log2(fold change)| ≥ 1 for at least one of the time points.

first: Antonella

last: Sassano

first: Marco

last: Lo Iacono

first: Giovanni

last: Antico

first: Alison

last: Jordan

first: Shahab

last: Uddin

first: Leonidas

middle: C

last: Platanias


title: FAIRE-chip in MCF7 and LNCaP

first: Jérôme

last: Eeckhoute

summary:
The compaction degree of chromatin is intimately related to its functionality and active cis-regulatory elements typically exist within open chromatin regions depleted in nucleosomes (Heintzman et al. 2007; Boyle et al. 2008). These domains can be identified using Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) that allows for enrichment of nucleosome-depleted genomic regions when cross-linked chromatin is subjected to phenol-chloroform extraction (Nagy et al. 2003; Hogan et al. 2006)
Here, chromatin structure was analyzed in MCF7 and LNCaP cells using Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) (Nagy et al. 2003; Hogan et al. 2006)

Keywords: FAIRE-chip

overall_design: MCF7 and LNCaP cells were grown in full media, cross-linked and processed as in (Nagy et al. 2003; Hogan et al. 2006). DNA obtained by FAIRE was hybridized to Human Affymetrix tiled array F. Triplicates of FAIRE and input DNA are provided (4 inputs for MCF7 cells).