title: Direct transcriptional targets of the HHV 8/KSHV Rta Lytic Switch Protein

city: Newark

last: Lukac

address: 225 Warren St.; ICPH E350C; PO Box 1709

department: Microbiology, Biochemistry and Molecular Genetics

institute: Rutgers Univ./NJ Medical School

summary:
Lytic reactivation from latency is critical for the pathogenesis of KSHV.  We previously demonstrated that the 691 amino acid KSHV Rta transcriptional transactivator is necessary and sufficient to reactivate the virus from latency.  Viral lytic cycle genes, including those expressing additional transactivators and putative oncogenes, are induced in a cascade fashion following Rta expression.  In this study, we sought to define Rta’s direct targets during reactivation by generating a conditionally nuclear variant of Rta. WT Rta protein is constitutively localized to cell nuclei, and contains two putative nuclear localization signals (NLSs).  Only one NLS (NLS-2; aa 516-530) was required for nuclear localization of Rta, and relocalized eGFP exclusively to cell nuclei.   Analyses of Rta NLS mutants demonstrated that proper nuclear localization of Rta was required for transactivation and stimulation of viral reactivation.  Fusion of Rta_NLS-1,2 to the hormone binding domain of the murine estrogen receptor generated a variant of Rta whose nuclear localization and ability to transactivate and induce reactivation were tightly controlled post-translationally by the synthetic hormone tamoxifen.  We used this strategy in KSHV-infected cells treated with protein synthesis inhibitors to identify direct transcriptional targets of Rta.   Only eight KSHV genes were activated by Rta in the absence of de novo protein synthesis.  These direct transcriptional targets of Rta were transactivated to different magnitudes, and included the genes nut-1/PAN, ORF57/Mta, ORF56/Primase, K2/vIL-6, ORF37/SOX, K14/vOX, K9/vIRF1, and ORF52.   Our data suggest that induction of most of the KSHV lytic cycle genes requires additional protein expression post-Rta.
Keywords: Comparative transcriptome analysis by oligonucleotide microarray

sample_organism:
Chlorocebus aethiops
Human gammaherpesvirus 8

overall_design: KSHV infected cells were transfected with empty expression vector or with vector expressing RtadeltaNLS1,2-ER (RtadeltaNLS1,2 fused to murine hormone binding domain of estrogen receptor (ER)).  Cells were treated with 4-hydroxytamoxifen (4-OHT) to induce nuclear re-localization of Rta fusion protein.  Cells were pre-treated with hygromycin to eliminate de novo cellular and viral protein expression.  Direct transcriptional targets of Rta were identified by comparing Rta +/- 4-OHT to vector (negative control) +/- 4-OHT.

first: Wei

last: Bu

first: Diana

last: Palmeri

first: Virginie

last: Aris

first: Patricia

last: Soteropoulos


title: Effect of dietary immunostimulation in head kidney and spleen of rainbow trout

status: Public on Aug 27 2008

city: Barcelona

first: Joan

middle: C

last: Balasch

address: UAB Bellaterra faculty of Biosciences

department: Dpt. Cell Biology, Physiology and Immunology

country: Spain

institute: Universitat Autonoma Barcelona

summary:
The effect of dietary immunostimulation in the immune organs, head kidney and spleen, of rainbow trout (Oncorhynchus mykiss), was investigated using a salmonid-specific microarray platform enriched with immune-related genes. Immunostimulant-diet feeding significantly changed transcriptomic expression profiles: larger reduction rather than induction was observed, with significant regulation in genes and functional GO categories related to remodeling processes and immune and hematopoietic activities. The results revealed that Immunostimulant-diets hava effect in the transcriptome of cultured fish.
Keywords: spleen, head kidney, immunostimulats, transcriptomic response, trout

overall_design:
Adult rainbow trout (Oncorhynchus mykiss) were randomly distributed in four fibreglass tanks (25 fish per tank). During the feeding trial, trouts were hand-fed once a day with either the control diet or the immunodiet, in duplicate tanks, following manufacturer’s indication of 1 g food/fish for 4 weeks. After the 4 weeks, 4 fish of each tank (8 for each diet) were sampled for microarray analysis. Tissues removed for RNA extraction were frozen in liquid nitrogen and stored at -80 ºC. Total RNA was extracted from the organs using 1 ml of Tri Reagent (Sigma) per 100 mg of tissue, following the manufacturer’s instructions and quantity and integrity was analyzed by Experion RNA StdSens Analysis Kit (Bio-Rad). The design of the microarray posses 1818 genes printed in six replicates each, including random clones from common and subtracted cDNA libraries which were compared with known vertebrate proteins using blastx. The platform was enriched in immune-related genes.
RNA was pooled within treatments (n=8), and 15 µg of control and test were labeled with Cy3- and Cy5-dCTP (Amersham Pharmacia) using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) primer (Promega), and cDNA was purified with Microcon YM30 (Millipore). Dye swap method was used, therefore each sample was hybridized to two microarrays. For the first slide, test and control cDNA were labeled with Cy5 and Cy3 respectively, and for the second array dye assignments were reversed. Scanning was performed with Axon scanner 4000B and images were processed with GenePix Pro 6.0. After subtraction of mean background, and LOWESS normalization was performed. To assess differential expression of genes, the normalized log intensity ratios were analyzed with Student’s t-test (p<0.01) and genes were ranked by log(p-level). The Bayesian modification to the false discovery rate (FDR) was used to correct for multiple comparison tests, estimating the q-value for the set of differentially expressed genes. The functional categories of Gene Ontology were compared with regulated genes (p<0.01) by the sums of ranks (Student’s t-test, p<0.05) and the statistical significance of over represented functional categories was assessed using the Yates correction to Chi square test (corrected p<0.05).

first: Carmen

last: Doñate

first: Simon

last: MacKenzie


title: Comparative Genomics of D. ethenogenes 195 and an Unsequenced Dehalococcoides-Containing Enrichment Culture

status: Public on Sep 01 2008

city: Berkeley

first: Kimberlee

last: West

state: CA

address: 207 O'Brien Hall

department: Civil and Environmental Engineering

country: USA

institute: University of California, Berkeley

summary:
Tetrachloroethene (PCE) and trichloroethene (TCE) are prevalent groundwater contaminants that can be completely reductively dehalogenated by Dehalococcoides organisms. A Dehalococcoides-containing microbial consortium (ANAS) with the ability to degrade TCE to ethene, an innocuous end-product, was previously enriched from contaminated soil. A whole-genome photolithographic microarray was developed based on the genome of Dehalococcoides ethenogenes 195 (strain 195). This microarray contains probes designed to hybridize to >99% of the predicted protein-coding sequences in the strain 195 genome. DNA from ANAS was hybridized to the microarray to characterize the genomic content of the ANAS enrichment. The microarray revealed that the genes associated with central metabolism including an apparently incomplete carbon fixation pathway, cobalamin salvaging system, nitrogen fixation pathway, and five hydrogenase complexes are present in both strain 195 and ANAS. Although the gene encoding the TCE reductase tceA was detected, 13 of the 19 reductive dehalogenase genes present in strain 195 were not detected in ANAS. Additionally, 88% of the genes in predicted integrated genetic elements in strain 195 were not detected in ANAS, consistent with these elements being genetically mobile. Sections of the tryptophan operon and an operon encoding an ABC transporter in strain 195 were also not detected in ANAS. These insights into the diversity of Dehalococcoides genomes will improve our understanding of the physiology and evolution of these bacteria which is essential in developing effective strategies for bioremediation of PCE and TCE in the environment.
Keywords: comparative genomic hybridization

sample_organism:
Dehalobacter restrictus
Dehalococcoides
Dehalococcoides mccartyi 195

overall_design: Genomic DNA from each culture was divided into replicate samples which were independently fragmented, labeled, and hybridized to arrays. Two microarrays were processed for the positive control (strain 195), two for the negative control (D. restrictus), and five for the ANAS enrichment culture(two analyses from one biological sample followed one year later by three analyses of a second biological sample).

middle: A

first: David

middle: R

last: Johnson

first: Ping

last: Hu

first: Todd

middle: Z

last: DeSantis

first: Eoin

middle: L

last: Brodie

first: Patrick

middle: K

last: Lee

first: Helene

last: Feil

first: Gary

last: Andersen

first: Stephen

middle: H

last: Zinder

first: Lisa

last: Alvarez-Cohen


title: Inducible expression of PrPC in mouse skeletal muscles results in myopathy characterized by changes in gene expression

city: Winnipeg

first: Debra

last: Parchaliuk

state: Manitoba

address: 1015 Arlington Street

department: Prion Diseases Program

country: Canada

institute: Public Health Agency of Canada

summary: Over-expression of wild type PrP in skeletal muscles is sufficient to cause a primary myopathy with no signs of peripheral neuropathy, possibly due to accumulation of a cytotoxic truncated form of PrP and/or PrP aggregation.  In this study we used DNA microarrays to identify 1499 transcripts that are temporally deregulated concomitant with inducible PrPC over-expression in the skeletal muscles of transgenic mice.  Examination using microarrays revealed a transcriptional profile with both similarities and differences to previously investigated models of myopathies.  Down-regulation of genes coding for the myofibrillar proteins MYH2, MYH6, MYH7, MYL2, MYL3 and up-regulation of lysosomal genes CTSS, CTSD, CTSZ, DPEP2, HEXA, HEXB and LAMP1 coincide with the observed myopathy and lysosome accumulation on over-expression of PrPC.  Down-regulation of the MEF2C gene, a key regulatory transcriptional factor muscle development and remodeling of adult muscles in response to physiologic and pathologic signals, may contribute to the centrally placed nuclei in the skeletal muscles.  Significantly, up-regulation of genes involved in p53 signaling and the induction of p53 protein, suggest a central role for this molecule in the myopathy.  Several p53-regulated genes involved in cell cycle arrest (CDNK1A, GADD45a and GADD45b) and apoptosis (BAK1, PMAIP1, BBC3, and BAX) are induced. We suggest that PrPC over-expression in skeletal muscles, possibly in response to accumulation of a cytotoxic truncated form of PrP, causes a primary myopathy involving the induction of p53-dependent pathways.

overall_design: Wild type (WT), PrP-null (KO), and Tg(HQK) mice were fed food pellets either lacking or containing 6g doxycycline (Dox)/kg food to induce PrPC expression. Skeletal muscles from the quadriceps of hind legs were removed at day 0, 4, 7, 14, 30 and 60 days following administration of Dox.  Total RNA was isolated from these tissues for use in subsequent microarray analysis. Mouse gene expression was analysed by two-colour microarray experiments using an inhouse manufactured 16K mouse cDNA microarray. Age matched reference mice (WT) and experimental (KO and HQK) Alexa Flour labeled aRNA were used in each competitive hybridization.  Each sample was labeled individually with both Alexa Fluor 555 and 647 for subsequent dye-swapped hybridizations to account for intensity bias. 3 individual mice from each experimental group at each time point were individually processed for separate microarrays.  We used the program EDGE to identify genes that were differentially expressed in mouse skeletal muscle in either transgenic HQK mice over expressing PrP, or PrP knock out (KO) mice  after administration of Dox.  We used a P value cut-off of 0.05 as the criteria of selection of significantly differentially expressed genes.

first: Jingjing

last: Liang

first: Sarah

last: Medina

first: Garrett

last: Sorensen

first: Laura

last: Landry

first: Qingzhong

last: Kong

first: Stephanie

last: Booth


title: Global analysis of metastasis-associated gene expression in the primary cultures from clinical specimens of ccRCC

city: Shanghai

first: Guangwen

last: Cao

state: Shanghai

address: No.800,Xiangyin Road

department: Department of Epidemiology,Faculty of Health Service

country: China

institute: Second Military Medical University

summary:
Metastatic clear cell renal cell carcinoma (ccRCC) has a poor prognosis and unpredictable course and there are no molecular markers that reliably predict ccRCC metastasis. In this study, specimens from 84 patients with ccRCC were directly cultured in vitro. The primary cultures from 38 of 94 specimens contained more than 90% tumor cells at 4th passages. After identified by immunostaining, the primary cultures of metastatic- and nonmetastatic ccRCC specimens from the age-, gender-matched patients were subjected to cDNA microrray assays. A total of 842 differentially expressed genes with FDR (false discovery rate) = 4.79% were identified by using SAM (Significance Analysis of Microarray) software. Pathway enrichment and co-occurrence with “cancer”, “metastasis” and “invasion” in the literature annotations enriched the functions of the 842 genes and indicated the reliability of our microarray assays. Novel genes associated with metastasis were selected based on intra-molecular interaction between 205 differentially expressed genes co-occurred with “metastasis” and those not appeared with “metastasis” on Medline, as well as co-expression analysis between 205 differentially expressed genes co-occurred with “metastasis” and those not appeared on Medline in 12 microarray data. FSTL1, AV722783, SLC15A1, DDX17, ORC2L and PKMYT1 were proved to be potential ccRCC metastasis-associated novel genes, based on its expression patterns in the cultures and tumor tissues. Interestingly, the up-regulated genes (CAV1, PKMYT1 and ORC2L) were up-regulated and the down-regulated genes (FSTL1, GSTM3, CYR61, SLC15A1 and AV722783) were down-regulated, in the primary ccRCC specimens as compared with its adjacent normal kidney in 37 patients (determined by semi-quantitative RT-PCR). This study provides potential biomarkers and novel molecular targets for the early diagnosis and treatment of ccRCC metastasis.
Keywords: Homo sapiens

overall_design:
Because of limited cell number of primary cultures, total RNA of the primary cultures from 2 age and gender matched patients was pooled for each microarray assay. Each assay was technically repeated once. Cy3-labeled 1st cDNA synthesized from total RNA of the metastatic ccRCC pool was mixed with Cy5-labeled 1st cDNA synthesized from total RNA of the non-metastatic ccRCC pool, and hybridized to 16K cDNA chips (GEO Platform ID: GPL6259; SBC-R-HC-100-22, National Engineering Center for Biochip, Shanghai, China) that included 14784 unigenes, 241 negative controls and 527 positive controls.
In our study we used a co-expression analysis for the relationship between genes; three gastric cancer Samples were only introduced to this analysis methods.

first: Xiaojie

last: Tan

first: Yujia

last: Zhai

first: Wenjun

last: Chang


title: Activity of the Bacillus anthracis 20 kDa protective antigen component

status: Public on Aug 26 2008

city: Silver Spring

first: Rasha

last: Hammamieh

state: MD

address: 503 Robert Grant Ave

department: Molecular Pathology

institute: Walter Reed Army Institute of Research

summary:
Edema factor of anthrax binds to the protective antigen (PA83) which cleaves to PA63 and PA20. The role of PA20 was examined using highthroughput gene expression analysis of human peripheral blood mononuclear cells (PBMC) exposed to PA20.

Keywords: Toxicity determination

overall_design: PBMC obtained from  different individuals over 6 monthes were incubated with and without rPA20

first: R

first: W

last: Ribot

first: G

last: Abshire

first: M

last: Jett

first: J

last: Ezzell


title: Remodeling of the Streptococcus agalactiae transcriptome in response to growth temperature

city: Warsaw

first: Izabela

last: Sitkiewicz

address: Chełmska 30/34

department: Epidemiology and Clinical Microbiology

country: Poland

institute: National Medicines Institute

summary: To understand the extent to which GBS modify gene expression in response to temperatures encountered in the various hosts, we conducted a whole genome transcriptome analysis of organisms grown at 30°C and 40°C. We identified extensive transcriptome remodeling at various stages of growth, especially in the stationary phase (significant transcript changes occurred for 25% of the genes). A large proportion of genes involved in metabolism was up-regulated at 30°C in stationary phase, which reflects a slowing of bacterial metabolism in the early stages of its shift to growth at lower temperature followed by an acceleration in the later stages. Conversely, genes up-regulated at 40°C relative to 30°C include those encoding virulence factors such as hemolysins and extracellular secreted proteins with LPXTG motifs. Over-expression of hemolysins was linked to larger zones of hemolysis and enhanced hemolytic activity at 40°C. A key theme identified by our study was that genes involved in purine metabolism and iron acquisition were significantly up-regulated at 40°C.

overall_design: Three cultures of GBS grown in rich Todd-Hewitt Yeast extract medium at 30°C and three cultures grown at 40°C served as a source of RNA samples collected at mid-logarithmic phase, late-logarithmic phase, and stationary growth phase. RNA was isolated using a modified Trizol method, and targets for hybridization with custom Affymetrix chip were generated as we have described previously. Genes with 30°C/40°C ratio greater than 2 and less than 0.5 (P value less than 0.05), were considered differentially expressed.

first: Laurent

last: Mereghetti

first: Nicole

middle: M

last: Green

first: James

last: Musser


title: Benign Copy Number Changes in Clinical Cytogenetic Diagnostics by Array CGH

status: Public on Aug 31 2008

city: Santa Clara

first: Peter

last: Tsang

address: 5301 Stevens Creek Blvd.

department: Agilent Labs

institute: Agilent Technologies

type: Genome variation profiling by genome tiling array

summary: A database of apparently benign copy number variants (bCNVs) detected by a Spectral Genomics Inc./PerkinElmer BAC array platform has been maintained through the University of Utah Comparative Genomic Hybridization laboratory since 2005.  The target population for this database represents 1275 patients with abnormal phenotypes, primarily children referred for developmental delay and mental retardation.  These bCNVs are independent of any identified copy number abnormality detected.  The most common 35 bCNVs observed and their frequencies are reported here, and a subset of ten of the patients studied was evaluated on a new oligonucleotide CNV array set designed by Agilent Technologies.  There was a 76% concordance of calls detected by both array platforms in the same patients; the discordant regions may be due to differences in reference DNAs, or false positive/negative results on either array.  The higher resolution of the Agilent oligonucleotide array compared to the BAC array allowed for further characterization to determine precise breakpoints of the observed CNVs, in addition to documenting additional CNVs of smaller sizes.  As expected, observed CNVs and their frequencies were generally consistent with those of other previously published and available databases, including the Database of Genomic Variants (http://projects.tcag.ca/variation/).  The availability of these data should assist other clinical laboratories in the evaluation of CNVs of unknown clinical significance.

overall_design: The Cytogenetics Laboratory at the University of Utah has been using aCGH to evaluate DNA derived from blood of patients with developmental delay, mental retardation and “syndromic” or dysmorphic phenotypes since 2005.  From this database, we were able to determine which clones represented sequences which were frequently deleted or duplicated in our patient population.  We identified our most common CNV regions and then selected DNAs from ten patients who exhibited copy number changes of those common regions for further study with the Agilent CNV microarray set.  Normal male or female DNA from Promega was used as a sex-mismatched control.  In addition to the ten clinical specimens, two self-self experiments for two patient samples were analyzed to estimate a false positive rate and to fine tune the parameters used for data analysis.  A replicate hybridization was performed on one patient to determine reproducibility.

first: Heidi

last: Whitby

first: Anya

last: Tsalenko

first: Emily

last: Aston

first: Sheri

last: Mitchell

first: Pinar

last: Bayrak-Toydemir

first: Christopher

last: Hopkins

first: Garrick

last: Peters

first: Dione

last: Bailey

first: Laurakay

last: Bruhn

first: Arthur

last: Brothman


title: Expression profiles and ChIP on chip genomewide experiments with deltaCR2 mutant virus

city: Los Angeles

first: Siavash

last: Kurdistani

address: 615 Charles E Young Dr South

department: Biological Chemistry

institute: UCLA

type: Genome binding/occupancy profiling by genome tiling array

summary: Adenovirus e1a induces quiescent human cells to divide. We found that e1a causes global relocalization of the RB-proteins (RB, p130, p107) and p300/CBP histone acetyltransferases on promoters that restricts H3K18ac to a limited set of genes to stimulate cell cycling and inhibit antiviral responses and cellular differentiation. Soon after expression, e1a binds transiently to cell cycle/growth genes, causing enrichment of p300/CBP, PCAF, H3K18ac, depletion of RB-proteins, and transcriptional activation. e1a also associates transiently with antiviral genes, causing enrichment for RB, p130, H4K16ac, increased nucleosome density, and repression. At later times, e1a and p107 bind mainly to development/differentiation genes, repressing transcription. The temporal order of e1a binding required its interactions with p300/CBP and RB-proteins. Our data uncover a defined transcriptional and epigenetic reprogramming leading to cellular transformation.

first: Roberto

last: Ferrari

first: Matteo

last: Pellegrini

first: Gregory

last: Horwitz

first: Wei

last: Xei

first: Arnold

last: Berk


title: Expression profiles and ChIP on chip genomewide experiments with dl1500 virus (expressing WT small e1a).


overall_design: Expression profiles and ChIP on chip genomewide experiments with R2G mutant virus (expressing a mutated version of small e1a).


title: DsbA plays a critical and multi-faceted role in the production of secreted virulence factors by Erwinia carotovora

city: Dundee

first: Pete

last: Hedley

address: Invergowrie

department: Genetics

country: United Kingdom

institute: SCRI

summary:
Erwinia carotovora subsp. atroseptica (Eca) is an enterobacterial phytopathogen causing economically-significant soft rot disease. Pathogenesis is mediated by multiple secreted virulence factors, many of which are secreted by the Type II (Out) secretion system. DsbA catalyses the introduction of disulphide bonds into periplasmic and secreted proteins. In this study, the extracellular proteome (secretome) of wild type Eca SCRI1043 and dsbA and out mutants was analysed by spectral counting mass spectrometry. This revealed that dsbA inactivation had a huge impact on the secretome and identified diverse DsbA- and Out-dependent secreted proteins, representing known, predicted and novel candidate virulence factors. Further characterisation of the dsbA mutant showed that secreted enzyme activities, motility, production of the quorum sensing signal and virulence were absent or greatly reduced. The impact of DsbA on secreted virulence factor production was mediated at multiple levels, including impacting on the Out secretion system and the virulence gene regulatory network. Transcriptome analyses revealed that the abundance of a broad, but defined, set of transcripts, including many virulence factors, was altered in the dsbA mutant, identifying a new virulence regulon responsive to extracytoplasmic conditions. In conclusion, DsbA plays a crucial, multi-faceted role in the pathogenesis of Eca.
Keywords: Mutant comparison

overall_design: RNA samples used for genome-wide transcriptional profiling using microarrays: RNA was hybridised to four Eca genomic arrays, with each array hybridising one wild type and one dsbA mutant sample (from four independent cultures of each strain) and incorporating a dye-swap (i.e. wild type labelled with Cy3 in 2/4 and with Cy5 in 2/4).

first: Sarah

middle: J

last: Coulthurst

first: Kathryn

middle: S

last: Lilley

first: Peter

first: Hui

last: Liu

first: Ian

last: Toth

first: George

middle: P

last: Salmond


title: Microarray based analysis of temperature and oxidative stress induced messenger RNA in Schistosoma mansoni

status: Public on Aug 25 2008

city: Albuquerque

first: Anthony

last: Aragon

state: NM

address: Castetter Hall Room 214

institute: University of New Mexico

summary:
The body’s defense against schistosome infection can take many forms. For example, upon developing acute schistosomiasis, patients often have fever coinciding with larval maturation, migration and early oviposition. As the infection becomes established, the parasite comes under oxidative stress generated by the host immune system. The most common treatment for schistosomiasis is the anti-helmenthic drug praziquantel. Its effectiveness, however, is limited due to its inability to kill schistosomes 2 - 4 weeks post-infection. Clearly there is a need for new anti-schistosomal drugs. We hypothesize that gene products expressed as part of a protective response against heat and/or oxidative stress are potential therapeutic targets for future drug development. Using a 12,166 element oligonucleotide microarray to characterize Schistosoma mansoni genes induced by heat and oxidative stress we found that 1,878 Schistosoma mansoni elements were significantly induced by heat stress. These included previously reported heat-shock genes expressing homologs of HSP40, HSP70 and HSP86. One thousand and one elements were induced by oxidative stress including those expressing homologs of superoxide dismutase, glutathione peroxidase and aldehyde dehydrogenase. Seventy-two elements were common to both stressors that could potentially be exploited in the development of novel anti-schistosomal therapeutics.

Keywords: Stress response, time course

overall_design: Eight samples performed in duplicate for each temperature and oxidative stresses at time points 0, 30, 60, and 240 min over a common reference sample for each stress

middle: D

first: Charles

last: Cunningham


title: Effect of TCPOBOP on CAR and PXR regulated genes involved in drug metabolism and cholesterol homeostasis

status: Public on Aug 23 2008

city: Ljubljana

last: Juvan

address: Zaloska 4

department: Institute of Biochemistry

country: Slovenia

institute: Faculty of Medicine, University of Ljubljana

summary: The nuclear receptor CAR (constitutive androstane receptor)  mediates the effects of  1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) on gene transcription. To investigate the relative role of CAR and also PXR in the induction response, cDNA arrays were generated containing 120 (Sterolgene V1) genes which are known to be regulated with these or related nuclear receptors (genes involved in drug metabolism, cholesterol biosynthesis, sterol synthesis/transport, heme synthesis). Samples from livers of wild type and CAR-/-, PXR-/- or CAR/PXR-/- knockout mice were tested after treatment with TCPOBOP for gene expression within the  European Framework V program “Steroltalk” (www.steroltalk.net). Results from these experiments show the complex role of CAR receptor in the expression of genes involved in drug metabolism and cholesterol biosynthesis.

overall_design: Animals were injected i.p. 10mg/kg TCPOBOP or vehicle (5% DMSO in corn oil). After 12h they were sacrificed and total RNA was isolated from the livers. Pools of untreated samples were mixed in each genetic variant group (wild type and CAR-/-, PXR-/- or CAR/PXR-/-) with the TCPOBOP treated ones and hybridized to Sterolgene V1 arrays.

first: Viola

last: Tamasi

first: Markus

last: Beer

first: Damjana

last: Rozman

first: Urs

last: Meyer


title: Telomerase inhibitor TMPyP4 induced widespread gene expression

city: Delhi

first: Anjali

last: Verma

state: Delhi

address: Mall road

department: Structural Biology and Genome Informatics Division

country: India

institute: Institute of Genomics and Integrative Biology

summary:
The effect of TMPyP4, the well established anticancer telomerase inhibitor on genome wide expression of genes, analyzed across different cell-lines, was investigated
Keywords: Comparative gene-expression profiling

overall_design: Cells were treated with TMPyP4 for 48 hours before RNA isolation. Untreated cells were taken as controls and the TMPyP4 treated cells as treatments. Experiments in each cell-line (A549 and HeLa S3) were replicated four times.

first: Shantanu

last: Chowdhury


title: Effect of phenobarbital on CAR and PXR regulated genes involved in drug metabolism and cholesterol homeostasis

summary: The nuclear receptors CAR (constitutive androstane receptor) and PXR (pregnane X receptor) mediate the effects of  phenobarbital (PB) on gene transcription. To investigate the relative role of CAR and PXR in the induction response, cDNA arrays were generated containing 120 genes which are known to be regulated with these or related nuclear receptors (genes involved in drug metabolism, cholesterol biosynthesis, sterol synthesis/transport, heme synthesis). Samples from livers of wild type and CAR-/-, PXR-/- or CAR/PXR-/- knockout mice were tested after treatment with PB for gene expression within the  European Framework V program “Steroltalk” (www.steroltalk.net). Results from these experiments unexpectedly revealed that if CAR and PXR are deleted, PB increases the expression of several other nuclear receptors and genes involved in drug metabolism and cholesterol biosynthesis.

overall_design: Animals were injected i.p. 100mg/kg phenobarbital or vehicle (5% DMSO in corn oil). After 12h they were sacrificed and total RNA was isolated from the livers. Pools of untreated samples were mixed in each genetic variant group (wild type and CAR-/-, PXR-/- or CAR/PXR-/-) with the phenobarbital treated ones and hybridized to Sterolgene V1 arrays.


title: Differentially expressed genes in zebrafish vascular development

status: Public on Aug 22 2008

city: Milwaukee

first: Ramani

last: Ramchandran

address: 8701 Watertown Plank Road

institute: Medical College of Wisconsin

summary:
The goal of this experiment was to identify using tissue specific transgenic zebrafish (Fli:EGFP), genes of interest important for embryonic vascular development.

Keywords: time course, GFP cell isolation, zebrafish

overall_design: The experiment contained 6 samples. A18, A24 = total RNA isolated from 18 and 24 hpf zebrafish embryo; B2, C and D2=RNA isolated from GFP positive cells sorted at 18, 24 and 28 hpf respectively from Tg(Fli1:EGFP) embryos; E2 = RNA isolated from GFP positive cells sorted at 18 hpf from Tg(Flk:GRCFP) embryos.

first: Sukhbir

last: Kaur

first: Lyndsay

last: Field


title: High-throughput pyrosequencing of endogenous small RNAs from Solanum lycopersicum leaf and fruit

city: Norwich

first: Frank

last: Schwach

address: University of East Anglia

department: School of Computing Sciences

institute: University of East Anglia

type: Non-coding RNA profiling by high throughput sequencing

summary: Comparison of the endogenous small RNA content of tomato leaves and fruits.

overall_design:
Size fractionated small RNA from total RNA extracts was ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to 454 high throughput pyrosequencing. Please see www.454.com for details of the sequencing technology.

Note: Raw data files were not available from 454 at the time this experiment was carried out.

first: Simon

last: Moxon

first: Runchun

last: Jing

first: Gyorgy

last: Szittya

first: Rachel

last: Rusholme Pilcher

first: Vincent

last: Moulton

first: Tamas

last: Dalmay


title: Gene expression variation to predict 10-year survival in lymph-node-negative breast cancer

status: Public on Aug 29 2008

city: Göteborg

first: Elin

last: Karlsson

address: Blå stråket 2

department: Oncology

country: Sweden

institute: Göteborg University

summary:
Expression profiling of breast cancer tumours, comparing 10 year survivors to deceased patients

Background
It is of great significance to find better markers to correctly distinguish between high-risk and low-risk breast cancer patients since the majority of breast cancer cases are at present being overtreated.

Methods
46 tumours from node-negative breast cancer patients were studied with gene expression microarrays. A t-test was carried out in order to find a set of genes where the expression might predict clinical outcome. Two classifiers were used to evaluate the gene lists on the different data sets, a correlation-based classifier and a VFI (Voting Features Interval) classifier. We then evaluated the predictive accuracy of this expression signature on tumour sets from two similar studies on lymph-node negative patients which had developed gene expression signatures superior to current methods in classifying node-negative breast tumours. These two signatures were also tested on our material.

Results
A list of 51 genes whose expression profiles could predict clinical outcome with high accuracy in our material (96% or 89% accuracy in cross-validation, depending on type of classifier) was developed. When tested on two independent data sets, the expression signature based on the 51 identified genes had good predictive qualities in one of  the data sets (74% accuracy), whereas their predictive value on the other data set were poor, presumably due to the fact that only 23 of the 51 genes were found in that material. We also found that previously developed expression signatures could predict clinical outcome well to moderately well in our material (72% and 61%, respectively).

Conclusion
The list of 51 genes derived in this study might have potential for clinical utility as a prognostic gene set, and may include candidate genes of potential relevance for clinical outcome in breast cancer. According to the predictions by this expression signature, 30 of the 46 patients should have had different adjuvant treatment than they did.

Keywords: Expression Microarray, Lymph-node-negative Breast Cancer, Clinical Outcome, Classification

overall_design:
23 fresh frozen primary node negative breast cancer tumours from 10 year survivors were compared to 23 tumours from deceased patients in order to find genes where the expression differed between the two groups

Microarrays were produced at the Swegene DNA Microarray Resource Center, Department of Oncology, Lund University, Sweden (http://swegene.onk.lu.se).

Frozen tumours were homogenized together with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). From the cell-suspension total RNA was extracted using RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The quality of the RNA was evaluated with the Agilent 2100 BioAnalyser (Agilent Technologies, Palo Alto, CA, USA). Specimens where the 28S/16S ratio was lower than 1.0 or the RIN-value was lower than 6.7 were excluded from the study. For each sample, Cy3-dCTP (Amersham Biosciences, Buckinghamshire, UK) labelled probes were synthesized from 5 µg of the total tumour RNA and Cy5-dCTP (Amersham Biosciences) labelled reference were synthesized from 5 µg of commercial reference RNA (Universal Human Reference RNA, Stratagene, La Jolla, CA, USA) by reverse transcription. The probes were purified using ChipShot™ labelling cleanup system (Promega, Madison, WI, USA). The hybridizations were carried out using Pronto! Micro Array reagent systems (Corning Inc., Corning, NY, USA). For each sample, labelled tumour cDNA and reference cDNA were co-precipitated and hybridised to the microarray slide. The microarray slides were scanned with Agilent microarray scanner G2565AA (Agilent Technologies) and image analysis was performed using the Genepix 6.0.0.45 software (Axon Instruments, Union City, CA, USA).

first: Ulla

last: Delle

first: Khalil

last: Helou


title: Dietary folate depletion and repletion in C57BL/6J mice, ApoE knockout mice, and choline supplemented C57BL/6J mice

city: Cleveleand

first: Joseph

last: Nadeau

state: OH

address: 10900 Euclid Avenue

institute: Case Western Reserve University

summary:
Defects in homocysteine and folate metabolism are associated with increased risks for neural tube and congenital heart defects, cardiovascular disease and stroke, cancers, and neurodegeneration.  In many but not all cases, dietary supplementation with folate significantly reduces the severity and incidence of these conditions. Common polymorphisms modulate these metabolic pathways and disease risks, but do not fully account for the particular birth defects and adult diseases that occur in at-risk individuals.  To test whether other pathways contribute to disease pathogenesis, we analyzed global and pathway-specific changes in gene expression and levels of selected metabolites after depletion and repletion of dietary folate in two genetically distinct inbred strains of mice.  Compared to the C57BL/6J strain, A/J showed greater homeostatic response to folate perturbation by retaining a higher serum folate level and minimizing global gene expression changes.  Remarkably, folate perturbation led to systematic strain-specific differences only in the expression profile of the cholesterol biosynthesis pathway and translated to changes in levels of serum and liver total cholesterol.  By genetically increasing serum and liver total cholesterol levels in APOE deficient mice, we modestly but significantly improved folate retention during folate depletion, suggesting an interplay between homocysteine and folate metabolism and cholesterol metabolism.  Absence of measurable changes in global methylation patterns or amelioration of effects with supplementation with an alternative methyl donor suggest that dietary folate perturbations do not act through large-scale or general changes in methylation. These results suggest that homeostatic responses in cholesterol metabolism contribute to the beneficial effects of dietary folate supplementation.
Keywords: time course, stress response, diet, genetic, homeostasis

overall_design: Six-week old female C57BL/6J and B6.129P2-Apoetm1Unc/J (Piedrahita et al. 1992) mice were purchased from the Jackson Laboratory. All mice were raised on a control diet containing four ppm folic acid (Basal Diet 5755, TestDiet) for one week before the start of studies. Mice were then placed on folic acid deficient diet (58C3, TestDiet) containing 1% succinylsulfathiazole, a non-absorbable antibiotic commonly used to suppress folate production by bacteria in the intestine, for 14 days. A subset of these mice were placed back on control diet for 7 days after 14 day depletion. There were three groups of mice that underwent folic acid depletion and repletion. ApoE knockout mice on C57BL/6J background, C57BL/6J mice supplemented with 25mM choline and 50mM saccharine in drinking water, and C57BL/6J mice supplemented with 50mM saccharine.  Saccharine was used to reduce the bitter taste of choline in the drinking water.  We also had C57BL/6J mice on the control diet for 14 days and 21 days.  These mice served as controls for treated mice from each time point during hybridization.  The folate level in the control diet for this study was significantly lower than the previous study (GSE9242) due to greater loss of folic acid by irradiation of the diet. There were eight replicate mice per treatment group per folic acid perturbation protocol. An equal amount (by weight) of liver tissue from eight replicate mice was separated into two pools of four replicate tissues each. Pooled RNA from treated mice and pooled RNA from control mice for each time point were aminoallyl labeled with Cy3 and Cy5 in duplicate with reversing of dyes.

first: Toshimori

last: Kitami

first: Renee

last: Rubio

first: William

last: O'Brien

first: John

last: Quackenbush


title: The Fusidic acid Stimulon of Staphylococcus aureus

city: Las Cruces

middle: E

last: Gustafson

email: jgustafs@nmsu.edu, amuthai@gmail.com

address: PO Box 30001 Dept. 3AF

department: Department of Biology and Molecular Biology Program

institute: New Mexico State University

summary:
Genome-wide transcriptional profiling studies of the growth of bacteria
 with antimicrobial agents often reveals aspects of the drug-specific
protective bacterial response. Fusidic acid is a steroid antimicrobial that
inhibits protein synthesis by interfering with the release of elongation
factor G (EF-G) after it has functioned in the translocation step. Two
 hundred and seventy two genes were both up- and down-regulated in a
 fusidic acid-susceptible strain of Staphylococcus aureus following
 challenge with 2 mg/L-1 fusidic acid for 15 min. Many genes altered by
 fusidic acid challenge are associated with protein synthesis such as fusA,
 which encodes EF-G which was up-regulated following exposure to the
 drug. The Staphylococcus microarray meta-database which curates and
 compares S. aureus transcriptome data revealed that the fusidic acid
 stimulon has the greatest overlap with the S. aureus cold shock- and
 stringent-responses. Six out of 9 autolysin genes making up the two
 component YycFG regulon (ssaA1-ssaA4, isaA and sceD) were also
 upregulated by fusidic acid; as were a carboxylesterase (est) and two
 putative Emr-Qac-like multidrug efflux pumps (emr-qac1 and emr-qac2).
 Genes down-regulated by fusidic acid induction encode a putative
 secreted acid phosphatase (sapS) and a number of protease genes
 (yjbG1, yjbG2, htrA1 and htrA2). Transcriptional analysis in conjunction
with mutant fusidic acid susceptibility experiments revealed that th45 e
 virulence gene regulatory agr operon, a YycFG controlled peptidoglycan
 hydrolase gene isaA and the proteases htrA1 and htrA2 are required for
 the expression of wild-type levels of fusidic acid susceptibility.

overall_design: S. aureus pangenome microarrays were utilized to determine transcriptome alterations occurring in S. aureus grown in the presence of  fusidic acid. Total bacterial RNA was isolated as previously described from S. aureus strain SH1000 grown in Muellar-Hinton broth to an OD580nm of 1.0 (37°C, 200 rpm) and then induced with 2 mg/L-1 fusidic acid for 15 min. This RNA and RNA isolated from untreated SH1000 cultures were then converted to fluorescently-labeled cDNA and hybridized to S. aureus microarrays version 4 (NIAID's Pathogen Functional Genomics Resource Center).  The Staphylococcus aureus microarray meta-database (SAMMD) curates data from various microarray  experiments and allows for the comparison of microarray data obtained from different protocols and experiments. SAMMD was used to analyze the transcriptome generated from SH1000 grown in the presence of  fusidic acid. Real-time PCR to confirm microarray data was performed. Following  growth of SH1000 with and without fusidic acid addition at various time points, the cultures were serial diluted with fresh MHB, inoculated onto Muellar Hinton agar and the total surviving CFUs/ml were determined after 24 h.

first: Alejandro

last: Delgado

first: Shahrear

last: Zaman

first: Arunachalam

last: Muthaiyan

first: Vijayaraj

last: Nagarajan

first: Mohamed

middle: O

last: Elasri

first: Brian

last: Wilkinson


title: Effects of diet and age on canine skeletal muscle (biceps femoris) gene expression

status: Public on Aug 21 2008

city: Urbana

first: Kelly

middle: Scott

last: Swanson

state: IL

address: 1207 W. Gregory Dr.

institute: University of Illinois

summary:
Aging animals display a decline in a multitude of physical and physiological functions, including muscle function and strength.  Muscle gene expression in dogs has been evaluated for a few select genes under pathogenic or varying dietary conditions, but global gene expression profiles of aged animals has not been performed.  Because the mechanisms contributing to age-related decline in muscle function are poorly defined, we used canine microarrays to compare gene expression profiles of muscle tissue from geriatric and young adult dogs.  Skeletal muscle (biceps femoris) samples were collected from 6 geriatric (12 yr-old) and 6 young adult (1 yr-old) female beagles after being fed one of two diets (animal protein-based versus plant-protein based) for 12 months.  RNA samples were hybridized to Affymetrix GeneChip Canine Genome Arrays.  Statistical analyses indicated that age had the greatest impact on gene expression, with 262 genes differentially expressed in geriatric dogs.  Although not as robust as age, diet affected mRNA abundance of 22 genes.  The effect of age was most notable in genes related to metabolism, cell cycle and cell development, and transcription function, with all of these functional groups being predominantly down-regulated in older animals.  The effect of diet on gene expression was mostly limited to the geriatric animals, but interactions between age and diet do not allow for a clear-cut pattern of gene expression to be observed.

Keywords: age; diet

overall_design: Six geriatric (11.1 yr old) and 6 weanling (8 wk old) female beagles were used.  Three dogs of each age were assigned to one of two dietary treatments and fed for 12 months.  Diets tested in this experiment were previously shown to manipulate energy metabolism.  One diet was an animal-protein based diet (APB) and was composed primarily of highly digestible ingredients and animal-derived protein and fat sources (brewer’s rice, poultry by-product meal, poultry fat) and was formulated to contain 28% protein, 23% fat, and 5% dietary fiber.  The other diet was a plant-protein based diet (PPB) and was composed primarily of moderately digestible plant-derived ingredients (corn, soybean meal, wheat middlings, and meat and bone meal) and was formulated to contain 26% protein, 11% fat, and 15% dietary fiber.  Although the two diets were very different in terms of ingredient and chemical composition, both were formulated to meet or exceed all nutrient requirements for canine growth according to the Association of American Feed Control Officials.  Young dogs were fed ad libitum to allow for adequate growth, while geriatric dogs were fed to maintain baseline BW throughout the experiment.  To produce the desired metabolic effects, the PPB diet was formulated to contain a lower caloric density (APB = 5.38 kcal/g; PPB = 4.75 kcal/g) and have a lower nutrient digestibility than the APB diet.  Thus, dogs fed the PPB diet needed to consume a greater (P<0.05) quantity of food (237 g/d; 1123 kcal/d) than dogs fed the APB diet (166 g/d; 893 kcal/d) to grow (young) or maintain BW (geriatrics).  Even though metabolic indices were altered, mean BW among dietary treatments was not different at any time over the course of the study for young or geriatric dogs.  After 12 months on experiment, animals were fasted for 12 hr and then given a lethal dose (130 mg/kg BW) of sodium pentobarbital (Euthasol, Virbac Corp., Fort Worth, TX) intravenously into the left forearm.  Death was confirmed by lack of respiration and a corneal reflex, and absence of a heartbeat detected with a stethoscope placed under the left elbow.   Skeletal muscle samples were collected immediately after death was confirmed, flash frozen using liquid nitrogen, and stored at -80oC until further analysis.

first: Lawrence

middle: B

last: Schook


title: Retinal gene expression in chicks during imposed myopic defocus

city: Tuebingen

first: Marita

middle: Pauline

last: Feldkaemper

address: Calwerstrasse 7/1

department: Section of Neurobiology of the Eye

country: Germany

institute: University Eye Hospital Tuebingen, Institute for Ophthalmic Research

summary:
The retina plays an important regulatory role in ocular growth. To screen for new retinal candidate genes that could be involved in the inhibition of ocular growth, we used chick microarrays to analyze the changes in retinal mRNA expression after myopic defocus was imposed by positive lens-wear.
Chicks were raised under a 12h-light/12h-dark cycle (light-onset: 8:00 am and light-offset: 8:00 pm) with unrestricted access to water and food. On the day prior to the experiment, velcro rings were glued to the feathers around the eye under diethylether anaesthesia. Four male white leghorn chicks, aged 9 days, wore +6.9D spectacle lenses over both eyes for 24 hours. Four untreated age-matched male chicks from the same batch served as controls. After decapitation of the chicks, the eyes were enucleated and the retina was prepared. The retinae from both eyes of each chick were pooled for RNA isolation.
Keywords: gene expression comparison

overall_design: Four samples each: Retina of both eyes of +6.9 diopter lens-treated chicks (plus_lens), Retina of both eyes of untreated control chicks (control)

first: Ruth

last: Schippert

first: Frank

last: Schaeffel


title: Dietary folate depletion and repletion in A/J and C57BL/6J mice

last: Nadeau

overall_design: Six-week old female A/J and C57BL/6J mice were purchased from the Jackson Laboratory. All mice were raised on a control diet containing four ppm folic acid (Basal Diet 5755, TestDiet) for one week before the start of studies. Selected mice were then placed on folic acid deficient diet (58C3, TestDiet) containing 1% succinylsulfathiazole, a non-absorbable antibiotic commonly used to suppress folate production by bacteria in the intestine. We had nine different treatment plans per strain with eight replicate mice per treatment.  There were four folic acid depletion treatment in which mice were placed on folic acid deficient diet for 1, 2, 7, or 14 days.  There were two folic acid repletion treatment in which mice were placed on folic acid deficient diet for 14 days followed by 1 day on control diet and another set of mice on 14 days of folic acid deficient diet followed by 7 days of control diet.  There were three control time points in which mice were placed on the control diet for 0, 9, or 22 days.  Eight biological replicate liver tissue from each treatment was pooled and total RNA from each pool and total RNA from Universal Mouse Reference RNA (Stratagene) were aminoallyl labeled with Cy3 and Cy5 in duplicate, with reversing of dyes.


title: Expression signatures of human HeLa cells with RNAi targetting A1 hnRNP, FUS, H hnRNP and p68-DDX5

status: Public on Aug 20 2008

city: Badalona

first: Lauro

last: Sumoy

state: Barcelona

address: Ctra. Can Ruti, Camí de les escoles s/n

department: High Content Genomics and Bioinformatics

institute: IGTP

summary:
Transcription profiling of human HeLa cells (cervical cancer cell line) transfected with a plasmid expressing shRNAs cloned into the pSuper expression vector compared to emprty vector negative controls for transfection. Four different RNA interference treatments targetted: A1 hnRNP (HNRNPA1, Heterogeneous nuclear ribonucleoprotein A1); FUS (fusion gene, involved in t(12;16) in malignant liposarcoma); H hnRNP (HNRNPH1); and p68 helicase (DDX5, DEAD (Asp-Glu-Ala-Asp) box polypeptide 5).

Keywords: genetic modification

overall_design: Five-condition experiment, where HNRNPA1, FUS, HNRNPH1 and DDX5 gene product levels were inhibited by siRNA transfection and compared to transfection with the negative control (scrambled siRNA). Biological replicates: 2 of each of the first three treatments and 3 of the treatment against DDX5, all independently grown and harvested. No technical replicates were performed.

first: Maria

last: Camats

first: Sònia

last: Guil

first: Mariette

last: Kokolo

first: Montse

last: Bach-Elias


title: C1 evolved adaptive clones M1-M5

city: Stanford

first: Gavin

last: Sherlock

address: 300 Pasteur Drive

institute: Stanford University

summary:
Chemostat evolution experiment C1 under glucose-limited condition at 30C.  Gene Expression profiles of adaptive clones M1-M5 in evolved conditions compared with original parents.
An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.
Genotype: Evolved clones.  The genotype is not necessarily complete.
Keywords: all_pairs

first: Katy

last: Kao


title: Chromosomes 6 and 18 Induce Neoplastic Suppression in Epithelial Ovarian Cancer Cells

status: Public on Aug 19 2008

city: London

first: Ken

last: Choi

address: 46 Cleveland Street

department: Gynaecological Oncology

institute: University College London

summary: Metaphase comparative genomic hybridisation (CGH) studies indicate that chromosomes 4, 5, 6, 13, 14, 15 and 18 are frequently deleted in primary ovarian cancers (OC). Therefore, we used microcell-mediated chromosome transfer (MMCT) to establish the functional effects of transferring normal copies of these chromosomes into two epithelial OC cell lines. The in vitro neoplastic phenotype (measured as anchorage dependent and independent growth and invasion) was compared between recipient OC cell lines and multiple MMCT hybrids. Chromosomes 6 and 18 showed strong evidence of functional, neoplastic suppression for multiple hybrids in both cell lines. We also found evidence in one cancer cell line suggesting that chromosomes 4, 13 and 14 may also cause functional suppression. Array CGH and microsatellite analyses were used to characterise the extent of genomic transfer in chromosome 6 and 18 hybrids. A 35Mb deletion on chromosome 6 in two hybrids from one cell line mapped the candidate region proximal to 6q15 and distal to 6q22.2; and an approximate 10Mb candidate region spanning the centromere on chromosome 18 was identified in another two hybrids from the other cell line. These data confirm reported functional effects of chromosome 6 in OC cell lines; but to our knowledge, this is the first time that functional suppression for chromosome18 has been reported. This suggests that these chromosomes may harbour genes that behave as tumour suppressors. The future identification of these genes may have a significant impact on the understanding and treatment of the disease and the identification of novel therapeutic targets.

overall_design: Four clones from chr 6 and chr 18 hybrids that were transferred by MMCT successfully were mapped by CGH microarray to identified transferred regions that induced neoplastic suppression in epithelial ovarian cancer cell lines (TOV21G and TOV112D).

first: Dimitra

last: Dafou

first: Susan

last: Ramus

first: Barbara

last: Grun

first: Christopher

last: Jones


title: Altered gene expression in the brain and liver of female fathead minnows exposed to fadrozole

city: Duluth

first: Daniel

middle: L.

last: Villeneuve

state: MN

address: 6201 Congdon Blvd

department: Mid-Continent Ecology Division

institute: US EPA

summary:
The fathead minnow (Pimephales promelas) is a small fish species widely used for ecotoxicology research and regulatory testing in North America.  This study used a 2000 gene oligonucleotide microarray to evaluate the effects of the aromatase inhibitor, fadrozole, on gene expression in the liver and brain tissue of exposed females.  Reproductive measures, plasma vitellogenin, and gene expression data for the brain isoform of aromatase (CYP19B), vitellogenin precursors, and transferrin all provided evidence supporting the efficacy of the fadrozole exposure.  Unsupervised analysis of the microarray results identified 20 genes in brain and 41 in liver as significantly up-regulated and 7 genes in brain and around 45 in liver as significantly down-regulated.  Differentially expressed genes were associated with a broad spectrum of biological functions, many with no obvious relationship to aromatase inhibition.  However, in brain, fadrozole exposure elicited significant up-regulation of several genes involved in the cholesterol synthesis, suggesting it as one potentially impacted pathway.  Gene ontology-based analysis of expression changes in liver suggested overall down-regulation of protein biosynthesis.  While real-time PCR analyses supported some of the microarray responses, others could not be verified.  Overall, results of this study provide a foundation for developing novel hypotheses regarding the system-wide effects of fadrozole, and other chemical stressors with similar modes of action, on fish biology.
Keywords: chemical stress response

overall_design:
--Fadrozole exposure--
Treatments:  0, 60 ug fadrozole/L
Replication:  4 replicate tanks per treatment, 2 replicate pairs (1 male, one female per pair) per tank for a total of 8 male, 8 female per treatment.
Route of exposure:  Waterborne, continuous flow through, without the use of carrier solvents, flow rate approx. 45 ml/min.
Tanks:  20 L tanks containing 10 L of exposure water
Fadrozole (purity ≥ 99%; Dr. H. Cooper Eckhardt, Summit, NJ, USA).
Fish:  Adult (ca. 6 month old) male (4.65±0.93 g) and female (1.90±0.44 g)
Conditions:  (25°C, 16:8 light:dark photoperiod, fed adult brine shrimp twice daily)
Duration: 7 d (± 4 h)
Sampling:  Fish humanely euthanized in tricaine methanesulfonate (Finquel; Argent, Redmond, WA, USA).  Liver, and brain samples transferred directly to pre-weighed vials of RNAlater® (Sigma, St. Louis, MO).  Plasma samples were stored frozen at -80°C.
Average water quality characteristics (±SD):  temperature 25.5±0.1 °C; pH 7.21±0.04; dissolved oxygen 5.74±0.52 mg/L.

--Microarray analysis--
n=3 microarrays per treatment and tissue
Experimental samples:  total RNA pooled from n=2 fish from the same treatment and replicate (except for pooled sample BC3 which included fish from two different replicate tanks).
Reference sample:  pooled liver, brain, and gonad samples from adult male and female fathead minnows
Microarray design:  Two color, reference design
Hybridization:  Experimental samples Cy5, reference sample Cy3

first: Iris

last: Knoebl

first: Patrick

last: Larkin

first: Ann

last: Miracle

last: Carter

first: Nancy

last: Denslow

first: Gerald

middle: T

last: Ankley


title: Comparative analysis of gene expression profiles between the ovary treated with or without Ci-TK

city: Osaka

first: Honoo

last: Satake

address: 1-1-1 Wakayamadai, Shimamoto, Mishima

country: Japan

institute: Suntory Inst. for Bioorg. Res.

summary:
Tachykinins (TKs) and their receptors have been shown to be expressed in the mammalian ovary. However, the biological roles for ovarian TKs have yet to be verified. Ci-TK and Ci-TK-R, characterized from the protochordate (ascidian), Ciona intestinalis, are prototypes of vertebrate TKs and their receptors. In the present study, we show a novel biological function of TKs as an inducible factor for oocyte growth using C. intestinalis as a model animal. Immunostaining demonstrated the specific expression of Ci-TK-R in test cells residing in oocytes at the vitellogenic stage. DNA microarray and real-time PCR substantiated that Ci-TK induced gene expression of several proteases including cathepsin D, chymotrypsin, and carboxypeptidase B2 and functionally unidentified lectins or glycosidases in the ovary. The enzymatic activities of the above proteases in the ovary were also shown to be enhanced by Ci-TK. Of particular significance is that treatment of Ciona oocytes with Ci-TK resulted in progression of growth from the vitellogenic stage to the post-vitellogenic stage, which was completely blocked by a TK antagonist or protease inhibitors. These results led to the conclusion that Ci-TK enhances growth of the vitellogenic oocytes via up-regulation of gene expression and enzymatic activities of the proteases.
Keywords: tachykinins, ovary

overall_design:
two samples
Dye Swap design with 2 arrays

first: Manabu

last: Fujie

first: Nori

last: Satoh


title: Gene expression profiles in liver of pigs with extreme high and low levels of androstenone.

status: Public on Aug 18 2008

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: ABSTRACT: BACKGROUND: Boar taint is the unpleasant odour and flavour of the meat of uncastrated male pigs that is primarily caused by high levels of androstenone and skatole in adipose tissue. Androstenone is a steroid and its levels are mainly genetically determined. Studies on androstenone metabolism have, however, focused on a limited number of genes. Identification of additional genes influencing levels of androstenone may facilitate implementation of marker assisted breeding practices. In this study, microarrays were used to identify differentially expressed genes and pathways related to androstenone metabolism in the liver from boars with extreme levels of androstenone in adipose tissue. RESULTS: Liver tissue samples from 58 boars of the two breeds Duroc and Norwegian Landrace, 29 with extreme high and 29 with extreme low levels of androstenone, were selected from more than 2500 individuals. The samples were hybridised to porcine cDNA microarrays and the 1 % most significant differentially expressed genes were considered significant. Among the differentially expressed genes were metabolic phase I related genes belonging to the cytochrome P450 family and the flavin-containing monooxygenase FMO1. Additionally, phase II conjugation genes including UDP-glucuronosyltransferases UGT1A5, UGT2A1 and UGT2B15, sulfotransferase STE, N-acetyltransferase NAT12 and glutathione S-transferase were identified. Phase I and phase II metabolic reactions increase the water solubility of steroids and play a key role in their elimination. Differential expression was also found for genes encoding 17beta-hydroxysteroid dehydrogenases (HSD17B2, HSD17B4, HSD17B11 and HSD17B13) and plasma proteins alpha-1-acid glycoprotein (AGP) and orosomucoid (ORM1). 17beta-hydroxysteroid dehydrogenases and plasma proteins regulate the availability of steroids by controlling the amount of active steroids accessible to receptors and available for metabolism. Differences in the expression of FMO1, NAT12, HSD17B2 and HSD17B13 were verified by quantitative real competitive PCR. CONCLUSIONS: A number of genes and pathways related to metabolism of androstenone in liver were identified, including new candidate genes involved in phase I oxidation metabolism, phase II conjugation metabolism, and regulation of steroid availability. The study is a first step towards a deeper understanding of enzymes and regulators involved in pathways of androstenone metabolism and may ultimately lead to the discovery of markers to reduce boar taint.

overall_design: Liver samples from 116 animals with extreme androstenone values, 29 high and 29 low from each of the two breeds Norwegian Landrace and Duroc, were used for this experiment. Each sample was hybridised together with a common refrence resulting in a total of 116 microarrays.

first: Maren

last: Moe

first: Sigbjørn

last: Lien

first: Christian

last: Bendixen

first: Henrik

last: Hornshøj

first: Ingunn

last: Berget

first: Theo

last: Meuwissen

first: Eli

last: Grindflek


title: RNA exosome depletion reveals ubiquitous transcriptional activity upstream of active human promoters

status: Public on Aug 15 2008

city: Århus

first: Pascal

last: Preker

address: C. F. Møllers Alle, Bldg 1130

institute: Århus University

type: Expression profiling by genome tiling array

summary: Recent high-throughput transcriptome analyses have revealed that >90% of all human DNA is transcribed. The vast majority of these transcripts are non-coding, thus challenging the classical definition of what constitutes a gene and, by association, a promoter. Adding to the complexity, short-lived transcripts have likely gone unnoticed. Here, we couple RNAi-depletion of one of the major RNA degradation activities, the human 3’-5’ exoribonucleolytic exosome with ENCODE microarrays tiling 1% of the human genome, to reveal a new class of short, polyadenylated and highly unstable transcripts. We name these RNAs PROMoter uPstream Transcripts (PROMPTs) due to their production at a restricted distance (0.5-2.5 kb) upstream active transcription start sites. Transcription of individual PROMPTs is bi-directional and detectable at the majority, if not all, active genes. PROMPT DNA bears hallmarks of transcription, including coverage by RNA polymerase II (RNAPII), H3K4me3, and H3K9ac. PROMPT expression requires the presence of the gene promoter and is positively correlated with gene activity. Remarkably, the transcription activity in a given PROMPT region is comparable to that of a similar region immediately downstream the promoter. Interestingly, stabilization of these new transcripts is particularly high in CpG-rich promoters. We propose that PROMPTs are common characteristics of RNAPII driven genes with an intrinsic regulatory potential.

overall_design:
Cell culture and RNAi-mediated knock-downs. HeLa cells were grown in DMEM medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. Transfections were done with 20 nM siRNA for 3 days and repeated for another 3 days both times using Lipofectamin20000 as transfecting agent according to the manufacturers instructions (Invitrogen).

Double-stranded cDNA was generated from 20 μg of total RNA from eGFP and hRrp40 siRNA-treated cells using Invitrogens double-stranded cDNA synthesis kit and an anchored primer (T20VN). Labeling with Cy3 and Cy5, respectively, hybridization to ENCODE NimbleGen HG17 50mer 39K tiling arrays (catalog number C1980001), analysis of primary data and identification of peaks were all paid services by Roche NimbleGen.

first: Jesper

last: Nielsen

first: Susanne

last: Kammler

first: Søren

last: Lykke-Andersen

first: Marianne

last: Christensen

first: Mikkel

last: Schierup

first: Torben

last: Jensen


title: Genomic alterations indicate tumor origin and varied metastatic potential of disseminated cells from prostate cancer

status: Public on Aug 17 2008

city: Seattle

first: Ilona

middle: N

last: Holcomb

state: WA

address: 1100 Fairview Ave N

department: Human Biology

institute: Fred Hutchinson Cancer Research Center

summary:
Disseminated epithelial cells can be isolated from the bone marrow of a far greater frac-tion of prostate-cancer patients than the fraction of patients who progress to metastatic disease. To provide a better understanding of these cells, we have characterized their genomic altera-tions. We first present an array comparative genomic hybridization method capable of detecting genomic changes in the small number of disseminated cells (10-20) that can typically be ob-tained from bone-marrow aspirates of prostate-cancer patients. We show multiple regions of copy-number change, including alterations common in prostate cancer, such as 8p loss, 8q gain, and gain encompassing the androgen-receptor gene on Xq, in the disseminated cell pools from 11 metastatic patients. We found fewer and less striking genomic alterations in the 48 pools of disseminated cells from patients with organ-confined disease. However, we identify changes shared by these samples with their corresponding primary tumors and prostate-cancer altera-tions reported in the literature, evidence that these cells, like those in advanced disease, are disseminated tumor cells (DTCs). We also demonstrate that DTCs from patients with advanced and localized disease share several abnormalities, including losses containing cell-adhesion genes and alterations reported to associate with progressive disease. These shared alterations might confer the capability to disseminate or establish secondary disease. Overall, the spectrum of genomic deviations is evidence for metastatic capacity in advanced-disease DTCs and varia-tion in that capacity in DTCs from localized disease. Our analysis lays the foundation for eluci-dation of the relationship between DTC genomic alterations and progressive prostate cancer.
Keywords: array comparative genomic hybridization, prostate cancer, disseminated cells

overall_design: The aim of this study was to characterize the genomic changes identified in disseminated cells isolated from the bone marrow of prostate cancer patients. As these samples were composed of pools of 10-20 cells, we verified that amplification of this material by ligation mediated PCR was compatible with array CGH. Tests of the method were conducted using nine samples of a small number of normal cells and five LNCaP samples (one bulk sample and four samples of 10-20 cells). Genomic profiles of disseminated cells were produced from 48 samples collected from the bone marrow of 48 patients (i.e. one sample per patient) with localized prostate cancer and from 13 samples collected from the bone marrow of 11 patients with advanced disease (3 biological replicates were collected from one individual with advanced disease).

first: Douglas

middle: I

last: Grove

first: Martin

last: Kinnunen

first: Cynthia

last: Friedman

first: Ian

last: Gallaher

first: Todd

last: Morgan

first: Cassandra

last: Sather

first: Jeffrey

last: Delrow

first: Peter

last: Nelson

first: Paul

last: Lange

first: William

last: Ellis

first: Lawrence

last: True

first: Janet

last: Young

first: Li

last: Hsu

first: Barbara

last: Trask

first: Robert

last: Vessella


title: miRNA Microarray Profiling of Nine Different Normal Human Tissues

city: Santa Clara

last: Ach

address: 5301 Stevens Creek Boulevard

department: Agilent Laboratories

institute: Agilent Technologies

summary: We measured the expression of 470 human miRNAs in nine human tissues using Agilent DNA microarrays.

overall_design: miRNA microarrays were manufactured by Agilent Technologies (Santa Clara, CA), and contain 20-40 features targeting each of 470 human miRNAs (Agilent design ID 016436).  Sequences of the 470 miRNAs were obtained from the Sanger miRBase, release 9.1.  Labeling and hybridization of total RNA samples were performed according to the manufacturer’s protocol.  100ng total RNA was used as input into the labeling reaction, and the entire reaction was hybridized to the array for 20 hours at 55°C.  The labeling and hybridizations of the nine human tissues were done 4-5 times using the same starting RNA sample for each tissue.

middle: A

first: Hui

last: Wang

first: Bo

last: Curry


title: miRNA Microarray Profiling with Three Different Total RNA Prep Methods

summary: We performed miRNA microarray profiling on samples prepared from two different cell lines by three widely-used total RNA sample prep methods.

overall_design:
miRNA microarrays were manufactured by Agilent Technologies (Santa Clara, CA), and contain 20-40 features targeting each of 470 human miRNAs (Agilent design ID 016436). Sequences of the 470 miRNAs were obtained from the Sanger miRBase, release 9.1. Labeling and hybridization of total RNA samples were performed according to the manufacturer’s protocol. 100ng total RNA was used as input into the labeling reaction, and the entire reaction was hybridized to the array for 20 hours at 55°C.

For total RNA preps, frozen cell pellets were resuspended in phosphate buffered saline and divided into equal aliquots of 5x106 (HeLa) or 1x107 (ZR-75-1) cells and refrozen.  Individual aliquots were subsequently thawed just before use.


title: Differential expression of 24,426 human alternative splicing events and predicted cis-regulation in 48 tissues

first: Amit

middle: V

last: Kulkarni

address: 401 Terry Ave N

department: Genomics

institute: Rosetta Inpharmatics / Merck Pharmaceuticals

summary:
Alternative pre-messenger RNA splicing impacts development, physiology, and disease, but its regulation in humans is not well understood, partially due to the limited scale to which the expression of specific splicing events has been measured.  We generated the first genome-scale expression compendium of human alternative splicing events using custom whole-transcript microarrays monitoring expression of 24,426 mutually exclusive alternative splicing event pairs in 48 diverse human samples. Over 11,700 genes and 9,500 splicing events were differentially expressed, providing a rich resource for studying splicing regulation. An unbiased, systematic screen of 21,760 4-mer to 7-mer words for cis-regulatory motifs identified 143 RNA 'words' enriched near regulated cassette exons, including six clusters of motifs represented by UCUCU, UGCAUG, UGCU, UGUGU, UUUU, and AGGG, which map to trans-acting regulators PTB, Fox, Muscleblind, CELF/CUG-BP, TIA-1, and hnRNP F/H, respectively. Each cluster showed a distinct pattern of genomic location and tissue specificity.  For example, UCUCU occurs 110 to 35 nucleotides preceding cassette exons upregulated in brain and striated muscle but depleted in other tissues.  UCUCU and UGCAUG appear to have similar function but independent action, occurring 5' and 3', respectively, of 33% of the cassette exons upregulated in skeletal muscle but co-occurring for only 2%.

Keywords: multiple tissue comparison

overall_design: PolyA+ purified RNA pooled from multiple donors of a single human tissue type (e.g. cerebellum) were amplified with random primers and hybridized on a two-color ink-jet oligonucletodie microarray (17 array set) against a common reference pool, comprising ~20 normal adult tissue pools, on custom microarray patterns containing probes to monitor every exon and exon-exon junction in transcript databases, patent databases, and predicted from mouse transcripts.  Data were analyzed for gene expression (the average of multiple probes), exon and junction expression, and splice form proportionality (see paper).


title: Mouse colon expression in wild type and STAT5b deficient mice

city: Cincinnati

first: Xiaonan

last: Han

address: 3333 Burnet Ave.

department: Gastroenterology

institute: Cincinnati Children's Hospital Medical Center

summary:
Regulation of epithelial barrier function is dependent upon precise control of cell survival and activation of inflammatory pathways in response to the enteric flora.
These experiments tested differential colon gene expression relative to these pathways in wild type and STAT5b deficient mice.
Keywords: Single time point in wild type and STAT5b deficient mice.

overall_design: Colon RNA was isolated from adult wild type and STAT5b deficient mice.


title: Comparsion of Smad1 and Smad5 Dependent Transcript Profiles in Zebrafish

city: Bronx

first: Lisa

middle: J.

last: McReynolds

state: NY

address: 1300 Morris Park Ave. Chanin 501

department: Developmental and Molecular Biology

institute: Albert Einstein College of Medicine

summary:
The BMP signaling pathway regulates multiple steps of hematopoiesis, mediated through receptor-regulated Smads, including Smad1 and Smad5. Here we use loss-of-function approaches in zebrafish to compare the roles of Smad1 and Smad5 during embryonic hematopoiesis. Microarray experiments revealed that the two proteins regulate redundantly the key initiators of the hemato-vascular program, including scl, lmo2, and gfi1. However, each also regulates a remarkably distinct genetic program, with Smad5 uniquely regulating the BMP signaling pathway itself. Our results suggest that specificity of BMP signaling output, with respect to hematopoiesis, can be explained by differential functions of Smad1 and Smad5.
Keywords: Gene expression transcript profiles

overall_design: The experiment was designed to identify the unique Smad1 and Smad5 dependent transcripts during the somitogenesis stage of development, during which mesoderm is specified to the hematopoietic lineage. Embryos were injected with translational blocking morpholinos for Smad1, Smad5 or both, and then collected at the 1-somite stage for RNA extraction. For every experiment control uninjected wildtype sibling embryo were also collected for comparison. Three biological replicates were done for each knockdown set. Total RNA was sent to Nimblegen for cDNA synthesis, dye labeling and hybridization. Single knockdown samples were hybridized to the Nimblegen 2006 Danio rerio Gene Expression Array chip and the double knockdown samples to the 2007 verison of the chip, which contains the same test genes, but with additional control oligos. Dye swaps were done for each set; for 2 of the 3 hybridization in each set Cy3 was the dye used for the experimental sample and in the 3rd Cy5 was used. The raw hybridization data was obtained from Nimblegen, normalized using NimbleScan and anaylzed using R software.

first: Sunny

last: Gupta

first: Maria

last: Figueroa

first: Mary

middle: C

last: Mullins

last: Evans


title: Stage III serous ovarian adenocarcinomas

status: Public on Aug 12 2008

city: Gothenburg

first: Karolina

last: Partheen

address: Blå Stråket 2

department: department of Oncology

institute: University of Gothenburg

summary:
The clinical outcome for patients with ovarian cancer is difficult to predict. However, the use of biomarkers as additional prognostic factors may improve the outcome for these patients. In order to find novel candidate biomarkers, differences in gene expressions were analyzed in 54 stage III serous ovarian adenocarcinomas with oligonucleotide microarrays containing 27,000 unique probes. The microarray data was verified with quantitative real-time polymerase chain reaction for the genes TACC1, MUC5B and PRAME. With a hierarchical cluster analysis we detected a sub-group including 60% of the survivors. The gene expressions in tumours from patients in this sub-group of survivors were compared to the remaining tumours, and 204 genes were found to be differently expressed. We conclude that the sub-group of survivors might represent patients with favourable tumour biology and sensitivity to treatment. A selection of the 204 genes might be used as a predictive model to distinguish patients within and outside of this group. Alternative chemotherapy strategies could then be offered as first-line treatment, which could improve the clinical outcome for these patients.

Keywords: Comparison between groups of samples

overall_design: 20 fresh frozen tumors from 5 year survivors were compared to 34 tumors from deceased patients in order to find genes where the expression differed between the two groups


title: Genome-wide profiling of follicular lymphoma by array comparative genomic hybridization

status: Public on Aug 09 2008

city: Vancouver

first: K-John

last: Cheung

state: BC

address: 600 West 10th Ave

institute: British Columbia Cancer Agency

summary: The secondary genetic events associated with follicular lymphoma (FL) progression are not well defined.  We applied genome-wide BAC array comparative genomic hybridization to 106 diagnostic biopsies of FL to characterize regional genomic imbalances.   Using an analytical approach that defined regions of copy number change as intersections between visual annotations and a Hidden Markov model-based algorithm, we identified 71 regional alterations that were recurrent in ≥10% of cases.  These ranged in size from ~200 kb to 44 Mb, affecting chromosomes 1, 5, 6, 7, 8, 10, 12, 17, 18, 19, and 22.  We also demonstrated by cluster analysis that 46.2% of the 106 cases could be sub-grouped based on the presence of +1q, +6p/6q-, +7 or +18.  Survival analysis showed that 21 of the 71 regions correlated significantly with inferior overall survival (OS).  Of these 21 regions, 16 were independent predictors of OS using a multivariate Cox model that included the International Prognostic Index (IPI) Score.  Two of these 16 regions (1p36.22-p36.33 and 6q21-q24.3) were also predictors of transformation risk and independent of IPI.  These prognostic features may be useful to identify high-risk patients as candidates for risk-adapted therapies.

overall_design: Array comparative genomic hybridization analysis of 106 follicular lymphoma diagnostic biopsies

middle: J

first: Sohrab

last: Shah

first: Christian

last: Steidl

first: Nathalie

last: Johnson

first: Thomas

last: Relander

first: Adele

last: Telenius

first: Betty

last: Lai

first: Kevin

last: Murphy

first: Wan

last: Lam

first: Abdulwahab

last: Al-Tourah

first: Joseph

middle: M

last: Connors

first: Raymond

last: Ng

first: Randy

last: Gascoyne

first: Douglas

last: Horsman


title: murine MYC-dependent lymphoma cells: Dox vs. NoDox treatment

city: Berlin

first: Lars

last: Bullinger

address: Augustenburger Platz 1

department: Hematology, Oncology and Tumorimmunology

institute: Charité

summary:
miRNA expression profiling of murine MYC-dependent lymphoma cell lines harboring the MYC-transgene in a Tet-off system comparing control untreated lymphoma cells (high MYC expression state) with 18hours Dox treated lymphoma cells (low MYC expression state).

Keywords: inducible expression system

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: Two-condition experiment, Dox vs. NoDox treatment. Biological replicates: 26 distinct cell lines, 1 technical replicate. Four replicates per array.

first: Sandrine

last: Sander

last: Wirth


title: Comparative analysis of the gene expression profiles of M.tuberculosis espR (Rv3849) transposon and knockout mutants

status: Public on Aug 08 2008

city: San Francisco

first: Paolo

last: Manzanillo

department: Microbiology and Immunology

institute: University of California at San Francisco

summary:
Mycobacterium tuberculosis, a pathogen of global importance, utilizes the ESX-1 protein secretion system to export virulence factors that disarm host macrophages. Although this secretory pathway is critical for virulence, how ESX-1 is regulated is completely unknown. Here we show that EspR (Rv3849) is a key regulator of ESX-1. EspR activates transcription of an operon that includes three ESX-1 components, Rv3616c-Rv3614c, whose expression in turn promotes secretion of ESX-1 substrates.

Keywords: Strain comparison

overall_design: Comparison of the global gene expression of three M.tb strains grown in log phase: Erdman strain of M.tb, espR transposon mutant (R-), and an espR knockout (deltaR).

first: Sridharan

last: Raghavan

first: Jeffery

last: Cox


title: Gene expression profile of M.tuberculosis espR (Rv3849) mutants containing N- or C-terminal mutations in the espR gene

overall_design: Comparison of the global gene expression of the following M.tb strains grown in log phase: Erdman strain of M.tb (WT), espR transposon mutant (R-), the espR transposon mutant complemented with the espR gene (R+), the espR mutant (R-) complemented with an espR gene containing an R8A amino acid subsitution (R- R8A), the espR mutant (R-) complemented with an espR gene containing an R21A amino acid subsitution (R- R21A), espR mutant (R-) complemented with an espR gene containing a C-terminal 10-amino acid truncation (R- delta10), and the espR mutant (R-) complemented with an espR gene containing a C-terminal 5-amino acid truncation (R- delta5).


title: Gene expression profile of M.tuberculosis espR (Rv3849) mutant complemented with an N-terminal Flag espR gene

overall_design: Comparison of the global gene expression of four M.tb strains grown in log phase: Erdman strain of M.tb, espR transposon mutant, the espR transposon mutant complemented with the wild type copy of the espR gene, and the espR transposon mutant complemented with an N-terminal FLAG espR gene.


title: Analysis of pvalb-egfp cells from mouse visual cortex.

city: Wako

first: Charles

last: Plessy

state: Saitama

address: 2-1 Hirosawa

department: DRI

institute: RIKEN

summary:
We provide an annotated cDNA clone collection which is particularly suitable for transcriptomic analysis in the mouse brain. Using it on microarrays, we compared the transcriptome of EGFP positive and negative cells in a parvalbumin-egfp transgenic background and showed that more than 30 % of clones are differentially expressed. Our clone collection will be a useful resource for the study of the transcriptome of single cell types.
Keywords: Cell type comparison

overall_design: Comparison between fluorescent and non-fluorescent cells isolated from the visual cortex of parvalbumin-egfp transgenic mice. Using five biological replicates with a dye-swap strategy (10 hybridisations).

first: Michela

last: Fagiolini

first: Akiko

last: Wagatsuma

first: Norihiro

last: Harasawa

first: Takenobu

last: Kuji

first: Atsuko

last: Asaka-Oba

first: Hiroyuki

last: Nakahara

first: Takao

last: Hensch

first: Piero

last: Carninci


title: microRNA  expression profile of activated versus non activated primary B cells

city: Madrid

first: Almudena

middle: R.

last: Ramiro

state: Madrid

address: Calle Melchor Fernandez Almagro 3

department: Molecular Oncology

institute: CNIO

summary:
microRNA  expression profile in 5 indepent sets of paired samples of LPS+IL-4 activated versus non activated primary mouse spleen B cells

Keywords: cell type comparison

platform_organism:
Homo sapiens
Mus musculus

overall_design: Activated B cells for miRNA array analysis were obtained after CFSE labelling (Molecular Probes), LPS+IL4 stimulation for 3 days and sorting of cells that had undergone at least 1 cell division. Sortings were performed with a FACSAria cell sorter (BD Biosciences).

first: R

first: V

middle: G

last: de Yebenes


title: Global gene expression analysis of Arabidopsis ppi2-1 mutant

city: Miyazaki

first: Takehito

last: Inaba

state: Miyazaki

address: 1-1 Gakuenkibanadai-nishi

department: Interdisciplinary Research Organization

institute: University of Miyazaki

summary: This study aims at investigating the gene expression profile of Arabidopsis ppi2-1 mutant.  Using Super-SAGE, we compared the gene expression profile of ppi2-1 mutant with that of wild-type Arabidopsis.

overall_design: To obtain the SuperSAGE libraries, we followed the original SuperSAGE protocol described by Matsumura and colleagues (Matsumura et al., 2003) with some modification.  Total RNA was isolated from the 3rd to 6th leaves of Ws and the ppi2-1 mutant using RNAiso reagent.  The SuperSAGE library was directly sequenced with a large-scale pyrosequencing method.

first: Tomohiro

last: Kakizaki

first: Hideo

last: Matsumura

first: Ryohei

last: Terauchi


title: Effect of pH on Cadmium Toxicity in Escherichia coli

city: Oshkosh

first: Craig

last: Worden

state: WI

address: 800 Algoma Boulevard

department: Biology and Microbiology

institute: UW Oshkosh

summary: Metals at high concentrations can exert toxic effects on microorganisms.  It has been widely reported that lowering environmental pH reduces effects of cadmium toxicity in bacteria.  Understanding the effects of pH-mediated cadmium toxicity on bacteria would be useful for minimizing cadmium toxicity in the environment and gaining insight into the interactions between organic and inorganic components of life.  Growth curve analysis confirmed that cadmium was less toxic to Escherichia coli at pH 5 than at pH 7 in M9 minimal salts medium.  To better understand the cellular mechanisms by which lowering pH decreases cadmium toxicity, we used DNA microarrays to characterize global gene expression patterns in E. coli in response to cadmium exposure at moderately acidic (5) and neutral (7) values of pH.  Higher expression of several stress response genes including hdeA, otsA, and yjbJ at pH 5 after only 5 minutes was observed and may suggest that acidic pH more rapidly induces genes that confer cadmium resistance.  Genes involved in transport were more highly expressed at pH 7 than at pH 5 in the presence of cadmium.  Of the genes that showed an interaction between pH and cadmium effects, 46% encoded hypothetical proteins, which may have novel functions involved in mitigating cadmium toxicity.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design:
GROWTH CONDITIONS FOR MICROARRAY EXPERIMENTS:
Two overnight cultures of E. coli K-12 were started in M9 medium.  A 250 mL Erlenmeyer flask containing 100 mL of M9 medium was inoculated with 0.5 mL for each of the two overnight cultures, each of which was considered a biological replicate.  The cultures were grown on a rotary shaker (200 rpm) at 37 °C  until the contents of the flask reached an OD600 of 0.3 (mid-log phase of growth).  Each culture was divided into four 25 mL aliquots, transferred to 50 mL conical tubes (Corning), and centrifuged at 2540 x g for 12 minutes.  The supernatant was decanted, and the cells were resuspended in 25 mL of M9 medium at pH 7 or pH 5 in the presence or absence (two cultures of each) of 5.4 µM (1 µg/mL) total cadmium, added as CdCl2.  The cultures were incubated at 25 °C for either 5 or 15 minutes with manual rotation of the flasks once per minute.  After the appropriate amount of time, 15 mL of RNAProtect Bacteria Reagent (Qiagen) was added to each culture to immediately halt all metabolic processes.  The solutions were vortexed, incubated at 25 °C for 5 minutes, and centrifuged for 12 minutes at 3750 x g.  RNA was extracted from the cell pellets immediately following centrifugation.

RNA EXTRACTION AND HYBRIDIZATION PROCEDURES:
RNA was extracted and purified using a Masterpure RNA purification kit (Epicentre Technologies).  The quantity and quality of the RNA samples were determined spectrophotometrically.  Preparation of the cDNA, labeling with Cy3 and Cy5, and successive hybridizations were accomplished using a 3DNA Array 900MPX kit following the manufacturer’s protocols (Genisphere) with the following modifications.  3DNA reverse transcriptase enzyme (Genisphere # RT300320) rather than SuperScript II was added to 1 µg of RNA and 2 µL of a random primer (1 µg/µL).  The final cDNA hybridization mix contained 29 µL 2X enhanced cDNA hybridization buffer rather than 2X SDS-based hybridization buffer or 2X formamide-based hybridization buffer.  The cDNA mix was hybridized to a cDNA microarray printed by the Microarray and Proteomics Facility at the University of Alberta (Operon version 1.0 oligonucleotides).  The arrays were scanned with a Versarray ChipReader (BioRad) with laser power at 75%, photomultiplier tube (PMT) sensitivity at 800 V, and detector gain at 1.

DATA ANALYSIS:
Each array directly compared transcription at pH 5 and pH 7 for a given cadmium treatment (0 or 5.4 µM cadmium) and exposure time.  Dye swaps were performed for each biological replicate for each of the following treatments (8 total arrays): 5 minutes with cadmium exposure, 5 minutes without cadmium exposure, 15 minutes with cadmium exposure, and 15 minutes without cadmium exposure.  The 0-minute exposure to cadmium treatment was obtained from the 5-minute microarray without cadmium exposure.  Spot intensities and locations were determined using TIGR Spotfinder, Version 3.1.1.  All subsequent analyses were performed using the ma-anova package in the open-source statistical software package, R (www.r-project.org), Version 2.4.1.  The data were normalized using the regional lowess method.  Following normalization the median expression values of genes represented in triplicate on each array were determined for each gene.  A mixed model two-way ANOVA for the main fixed effects of pH, cadmium, and their interaction (array and spot were the random effects) were performed (using Type III F-tests) separately for each time point to identify genes for which pH and cadmium interacted to significantly affect expression (FDR-adjusted p ≤ 0.05).

middle: R

first: William

middle: K

last: Kovac

first: Lisa

last: Dorn

first: Todd

last: Sandrin


title: Effect of heat shock on Rickettsia prowazekii Madrid E from four independent biological replicates

city: Mobile

first: Jonathon

last: Audia

state: AL

address: 307 University Blvd N

institute: University of South Alabama

summary: Here we present the first microarray analysis of global gene expression changes in the obligate intracytoplasmic pathogen Rickettsia prowazekii using temperature upshift as a model stress condition.  We describe a methodology for isolating highly-purified rickettsial RNA where a combination of differential centrifugation and use of Ambion’s MICROBEnrich reagent removed the majority (~90 %) of host cell RNA.  A pool of rickettsiae-infected L-cells grown at 34 oC was split and half of the sample was challenged at 42 oC for 30 minutes, total RNA was isolated and converted to cDNA followed by hybridization to an oligonucleotide DNA array.  We report data (duplicate hybridizations) from four, independent biological replicates.  Twenty-three rickettsial transcripts were significantly increased by temperature upshift greater than two-fold and no transcripts demonstrated reproducible decreases.  Notably, the GroESL, DnaK, and DnaJ members of the family of chaperones as well as the two R. prowazekii proteins annotated as putative heat shock proteins were induced.  In addition, proteins annotated as proteases were also induced indicating that R. prowazekii mounts a response to heat-shock induced protein unfolding.  These data are the first global analysis of the R. prowazekii transcriptome and provide insight into the mechanisms employed by rickettsiae to adapt to temperature upshifts.

overall_design:
Four, independent biological replicates (Control at 34 C versus Heat Shock at 42 C, 30 minutes)
All arrays were performed in duplicate (dye swaps)

middle: P

first: Herbert

middle: H

last: Winkler

first: Mary

last: Patton

first: Seth

middle: D

last: Crosby


title: Global transcriptional response to vancomycin in Mycobacterium tuberculosis

city: Padua

first: Roberta

last: Provvedi

address: Via Gabelli 63

department: Biologia

country: Italy

institute: University of Padua

summary: We characterized the variation of M. tuberculosis transcriptional profile in response to inhibitory and subhinibitory concentrations of vancomycin.

overall_design: Whole genome microarrays were employed to profile gene expression in M. tuberculosis H37Rv in response to inhibitory or subinibitory concentration of vancomycin. Inhibitory conditions were achieved by exposing bacteria to 400 µg/ml of vancomycin, a concentration exceeding 10 times the minimum inhibitory concentration (10X-MIC); in these conditions bacterial replication arrested rapidly without killing. Subhinibitory conditions were obtained growing the cells in the presence of 5 µg/ml of vancomycin (1/8-MIC) which allowed bacteria to grow with the same profile as untreated cells, although at a reduced rate. Following the addition of vancomycin at a concentartion 10X-MIC, the culture was incubated 1 h, or 4 hrs, and then analyzed to identify which genes were expressed differentially.

first: Francesca

last: Boldrin

first: Francesco

last: Falciani

first: Riccardo

last: Manganelli


title: TL2_14028s_M9+arabinose_LibAra_vs_Lib0

city: Irvine

first: Michael

last: McClelland

state: CA

address: 132 Med Surge I

department: Microbiology & Molecular Genetics

institute: University of California, Irvine

summary:
Salmonella typhimurium 14028s Transposon library grown in M9 minimal medium (arabinose 0.4%), O/N at 37°C with aeration, compared to the initial library selected on Luria agar plates + kanamycin (50ug/ml), O/N at 37°C
Keywords: Transposon tag analysis

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium

overall_design: 3 biological replicates analysed after arbitrarily primed sampling (DOPR) + specific PCR amplification + T7 in vitro transcription + RT labeling (Cy3) + hybridization , compared to identically processed standard library (Cy5)


title: Folsomia candida exposed to cadmium spiked soil

city: Amsterdam

first: Benjamin

last: Nota

state: Noord Holland

address: Plesmanlaan 125

department: Research Facilities

country: Netherlands

institute: Sanquin

summary:
Increasing concern about pollution of our environment calls for advanced and rapid methods to estimate ecological toxicity. The use of gene expression microarrays in environmental studies can potentially meet this challenge. We present a novel method to examine soil toxicity. We exposed the collembolan Folsomia candida to soil containing an ecologically relevant cadmium concentration, and found a cumulative total of 1586 differentially expressed transcripts across three exposure durations, including transcripts involved in stress response, detoxification, and hypoxia. Additional enrichment analysis of gene ontology (GO) terms revealed that antibiotic biosynthesis is important at all time points examined. Interestingly, genes involved in the "penicillin and cephalosporin biosynthesis pathway" have never been identified in animals before, but are expressed in F. candida’s tissue. The synthesis of antibiotics can possibly be a response to increased cadmium-induced susceptibility to invading pathogens, which might be caused by repression of genes involved in the immune-system (C-type lectins and Toll receptor). This study presents a first global view on the environmental stress response of an arthropod species exposed to contaminated soil,and provides a mechanistic basis for the development of a gene expression soil quality test.
Keywords: cadmium, soil, Collembola, environmental genomics

overall_design: Folsomia candida was exposed to cadmium spiked soil or untreated (reference/control) soil for 2, 4, and 7 days. For each time point 4 biological replicates were used, replicate samples consisted of total RNA extracted from 30 animals exposed in the same jar to either reference or cadmium spiked soil. Cadmium treated samples were always hybridized to reference samples from the same time point, in an evenly distributed dye-swap manner, which resulted in total in 12 hybridizations of 24 samples.

first: Martijn

last: Timmermans

first: Oscar

last: Franken

first: Kora

last: Montagne-Wajer

first: Janine

last: Marien

first: Muriel

middle: E

last: de Boer

first: Tjalf

first: Bauke

last: Ylstra

first: Nico

last: van Straalen

first: Dick

last: Roelofs


title: Global gene expression profile of small intestine from wild-type and NHE3/CFTR double knockout mice.

first: Gary

last: Shull

address: 231 Albert Sabin Way ML0524

institute: University of Cincinnati

summary:
The objective of this study was to identify changes in gene expression levels between wild-type and NHE3/CFTR double knockout small intestine.
Keywords: gene expression comparison

overall_design: Six wild-type and six NHE3/CFTR double knockout small intestinal RNA samples were compared.  To facilitate statistical analysis and reduce affects of Cy3 and Cy5 labeling, reciprocal dye flips were used for half of the WT and double knockouts.

first: Emily

last: Bradford