title: Effect of dietary immunostimulation in gills and intestine of rainbow trout

city: Barcelona

first: Joan

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 portals of entry, intestine and gills, of rainbow trout (Oncorhynchus mykiss), was investigated using a salmonid-specific microarray platform enriched with immune-related genes. IS-diet feeding significantly changed transcriptomic expression profiles: larger reduction rather than induction was observed, with significant changes in genes and functional GO categories related to remodeling processes and antigen presentation.  The results revealed that one of the main effects of IS-diets in trout is the increase of genes involved in antigen recognition in epithelial cells of gills.
Keywords: gills, intestine, immunostimulats, transcriptomic response, ISH, 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: Genomic comparison in Vibrio parahaemolyticus strains by comparative genomic hybridization.

status: Public on Mar 31 2008

city: Suita

first: Kaori

last: Izutsu

state: Osaka

address: 1-1 Yamadaoka

department: Research Institute for Microbial Diseases

country: Japan

institute: Osaka University

summary:
Vibrio parahaemolyticus is a Gram-negative marine bacterium. A limited population of the organisms causes acute gastroenteritis in humans. Almost all of the clinical V. parahaemolyticus isolates exhibit a beta-type hemolysis on Wagatsuma agar, known as the Kanagawa phenomenon (KP). KP is induced by the thermostable direct hemolysin (TDH) produced by the organism, and has been considered a crucial marker to distinguish pathogenic strains from non-pathogenic ones. Since 1996, so-called “pandemic clones”, the majority of which belong to serotype O3:K6, have caused worldwide outbreaks of gastroenteritis. In this study, we used a DNA microarray constructed based on the genome sequence of a pandemic V. parahaemolyticus strain RIMD2210633 to examin the genomic composition of 22 strains of V. parahaemolyticus, including both pathogenic (pandemic as well as non-pandemic) and non-pathogenic strains. Over 85% of the RIMD2210633 genes were conserved in all the strains tested. Many of variably present genes formed gene clusters on the genome of RIMD2210633 and were probably acquired through lateral gene transfer. At least 70 genes over 10 loci were specifically present in the pandemic strains when compared with any of the non-pandemic strains, suggesting that the difference between pandemic and non-pandemic strains is not due to a simple genetic event. Only the genes in the 80-kb pathogenicity island (Vp-PAI) on chromosome II, including two tdh genes and a set of genes for the Type III secretion system, were detected only in the KP-positive pathogenic strains. These results strongly suggest that acquisition of this Vp-PAI was crucial for the emergence of V. parahaemolyticus strains that are pathogenic for humans.
Keywords: comparative genomic hybridization, CGH

overall_design: Total 66 test samples were analyzed. Genomic DNA from each test strain and a reference strain (RIMD2210633) were labeled with Cy3 and Cy5, respectively, and were cohybridized on a single array. Labeling and hybridization were performed three times independently.

first: K

first: T

last: Iida


title: mRNA expression in TTHA1939 deletion mutant of Thermus thermophilus HB8 strain grown on minimum medium

city: Toyonaka

first: Takushi

last: Ooga

address: Machikaneyama 1-1

department: Department of Biology

summary:
We compared the expression profile of TTHA1939 deletion mutant strain of Thermus thermophils HB8 with that of wild-type.  The mutant strain grown in minimum essential medium (CS medium) exhibited growth arrest and cellular aggregation during the logarithm growth phase.  In this phase, 99 genes were suppressed and 63 genes were activated for the mutant strain.  The suppressed genes included genes of ribosomal proteins, TCA cycle, amino acid biosynthesis, fatty acid biosynthesis, cobalamin biosynthesis, transporters, and cell division including dnaB and ftsZ.  The activated genes included genes of nitrogen metabolism, stress proteins, and proteases.  We suggested that these transcriptional alterations in the mutant cell were induced by the ppGpp accumulation which was prevented by ppGpp degradation activity of TTHA1939 in WT cell.
Keywords: gene deletion, minimum essential medium, logarithm growth phase

overall_design: Wild-type and the mutant strains were precultured in rich medium (TR medium) for two times at 70 oC and then subcultured to minimum essential medium (CS medium).  These cells were harvested during the logarithm growth phase (800 minutes from the start of culture).  Total RNA were extracted from each strain and used for the cDNA synthesis.  For the biological replication, above experiments were performed four times independently.  The cDNA fragments were labeled with biotin-dideoxy UTP, using terminal transferase according to the manufacturer’s instructions (ENZO Biochem. Inc., Farmingdale, NY).  The 3’-terminal-labeled cDNA (2 micro g) was hybridized to a TTHB8401 GeneChip (Affymetrix, Santa Clara, CA).  The Probe Array was scanned with a GeneArray Scanner (Affymetrix), and then, the image data was scaled to the target intensity by one-step Tukey’s biweight algorithm using GeneChip Operating software, version 1.0 (Affymetrix, Santa Clara, CA).  The data analysis was performed by using GeneSpring GX (Agilent Tech.).  The genes which had detection call of “presence” more than 4 times from 8 samples were used for following analysis.  The normalized intensities for the mutant strains were calculated by using the intensities for wild-type strains of each data set, and then, the average of these intensities were used.  The genes which produced P value < 0.1 in the Student’s t-test were extracted.  Among them, the genes whose expression level was different more than 2 fold between two strains were considered as significant.

first: Yoshiaki

last: Ohashi

first: Ryoji

last: Masui

first: Tomoyoshi

last: Soga

first: Masaru

last: Tomita

first: Seiki

last: Kuramitsu


title: Co-activator function of RIP140 for NFκB/p65-dependent cytokine gene

city: Heidelberg

first: Inka

last: Zschiedrich

address: Im NeuenheimerFeld 280

department: Molecular Metabolic Control

country: Germany

institute: German Cancer Research Center Heidelberg

summary:
High- and low-grade inflammatory responses represent a hallmark of numerous pathologies such as sepsis and bacterial infection or insulin resistance and obesity, respectively. Here we describe an unexpected co-activator function of receptor interacting protein (RIP) 140 for
nuclear factor (NF) kB, a master transcriptional regulator of inflammation in multiple tissues. Genetic as well as acute deficiency of RIP140, which has been characterized as a co-repressor of various nuclear receptors on metabolic target genes, led to the inhibition of the proinflammatory
program in macrophages, mediated by RIP140’s direct impact on cytokine gene promoter activity. Intriguingly, RIP140’s co-activator function in this setting was found to rely on direct protein-protein interactions with the NFκB subunit p65 and histone acetylase CREB-binding protein (CBP). RIP140-dependent control of proinflammatory gene expression via p65/CBP may, therefore, represent a molecular rational for the cellular integration of metabolic and inflammatory pathways.
Keywords: expression profiling

overall_design:
Gene expression profiling was done
for macrophages from RIP140 knockout or wild-type mice.
RNA isolation, cDNA and cRNA synthesis, and hybridization to
arrays of type Mouse Genome 430 2.0 from Affymetrix
(Santa Clara, CA, USA) were performed according to manufacturer’s
recommendations. 3 arrays per genotype were hybridized.
Microarray data were analysed based on ANOVA using a commercial
software package (Micro Array Solution, version 1.0, SAS Institute,
Cary, NC, USA). Standard settings were used, except the following
specifications: log-linear mixed models were fitted for values of
perfect-matches, with genotype considered to be constant and the
array-id random. Custom CDF with Unigene based gene/transcript
definitions was used to annotate the arrays.

first: Ulrike

last: Hardeland

first: Anja

last: Krones-Herzig

first: Mauricio

last: Diaz

first: Alexandros

last: Vegiopoulus

first: Johannes

last: Müggenburg

first: Dirk

last: Sombroek

first: Thomas

last: Hofmann

first: Rainer

last: Zawatzky

first: Xiaolei

last: Yu

first: Norbert

last: Gretz

first: Mark

last: Christian

first: Roger

last: White

first: Malcom

last: Parker

first: Stephan

last: Herzig


title: The sigma factor E of Mycobacterium tuberculosis modulates the inflammatory response of macrophages

status: Public on Mar 27 2008

city: Newark

first: Virginie

middle: Marie

last: Aris

state: NJ

address: W420M, 225 Warren St

department: Center For Applied Genomics

country: USA

institute: PHRI-UMNDJ

summary:
Mycobacterium tuberculosis (Mtb) is well adapted to survive in macrophages and usually subverts the bactericidal mechanisms of these professional phagocytes.
The adaptation of Mtb to the intracellular life depends on its ability to regulate the expression of its genes.  Among the most important bacterial transcription activators are the sigma factors that bind to the RNA polymerase and give it promotor specificity. Sigma factor E (SigE) controls the expression of genes that are essential for Mtb virulence. Analysis of the macrophage transcriptional response indicated that proteins encoded by the sigE regulon are involved in the modulation of the macrophage inflammatory response.
Keywords: Comparison of responses to infections

overall_design: We compared the global gene expression of mouse bone marrow macrofages infected with H37Rv and SigE to the gene expression profile of the uninfected macrophages.

first: Patricia

last: Fontan

middle: M

first: Saleena

middle: S

last: Ghanny

last: Soteropoulos

first: Jeff

last: Chang

first: Eugenia

last: Alvarez

first: Analia

last: Trevani

first: Richard

last: Pine

first: Issar

last: Smith


title: Reduced DNA methylation in Arabidopsis allopolyploids induces genome-specific changes in gene expression

status: Public on Mar 30 2008

city: Bethesda

first: Misook

last: Ha

state: MD

address: NIH

institute: National Heart Lung Blood Institute

summary:
Changes in genome structure and gene expression have been documented in both resynthesized and natural allopolyploids that contain two or more divergent genomes.  The underlying mechanisms for rapid and stochastic changes in gene expression are unknown.  Arabidopsis suecica is a natural allotetraploid derived from the extant species A. thaliana and A. arenosa. Here we report that reduced DNA methylation in met1-RNAi A. suecica lines altered the expression of ~200 genes encoding transposons, centromeric and heterochromatic RNAs, and predicted proteins.  Reduced DNA methylation occurred frequently in promoter regions of the upregulated genes but not of the repressed genes and led to increased mobility of En/Spm-like transposons in met1-RNAi A. suecica lines.  Compared to A. arenosa centromeres, A. thaliana centromeres were hypermethylated, which correlates with higher levels of small RNA accumulation in A. thaliana centromeres than that in A. arenosa centromeres.  Derepression of the genes examined was primarily derived from A. thaliana subgenome, and A. arenosa genes are less affected by methylation defects.  Moreover, non-CG (CC) methylation in the promoter region of A. thaliana At2g23810 was maintained in resynthesized allotetraploids, and the methylation spread within the promoter region in natural A. suecica, leading to silencing of At2g23810, which was demethylated and reactivated in met1-RNAi A. suecica lines.  We suggest that a subset of A. thaliana and A. arenosa genes are differentially methylated in natural allopolyploids, and some A. thaliana genes including centromeres are subjected to transcriptional repression and genome-specific RNA-mediated DNA methylation in Arabidopsis allopolyploids.
Keywords: gene expression in reduced DNA methylation lines in Arabidopsis allotetraploids

overall_design:
Spotted oligo-gene microarrays were used for genome-wide gene expression assays.  A total of 8 slides were used for 2 biological and 4 technical replications.  Each dye-swap (technical replication) consists of two slides. One slides was hybridized with an equal amount of Cy3-labled A. suecica control cDNA and Cy5-labeled met1-RNAi cDNA as probes, and another with the same two cDNA samples labeled in a reverse Cy3- and Cy5-combination.  We used 500 ng of mRNA in each labeling reaction using Cy3- or Cy5-dCTP (Amersham Biosciences).  The dye-swap experiment was replicated using an independently isolated RNA sample.  Raw data were collected using Genepix Pro4.1  after the slides were scanned using Genepix 4000B (Axon Instruments/Molecular Devices, Sunnyvale, CA).  The data were processed using a lowess function to remove non-linear components and analyzed using a linear model.  This linear model was employed to partition variation in the observed data relative to technical and biological variation.  We selected the genes that were differently expressed at a statistically significant level (FDR, α=0.05) under both common variance and per-gene variance.  The microarray data were deposited in GEO (http://www.ncbi.nlm.nih.gov/geo/) and an accession number is being assigned.
Functional categories of up- and down-regulated genes were classified using (ftp://ftp.arabidopsis.org/home/tair/Ontologies/Gene_Ontology/OLD/ATH_GO_GOSLIM.20070317.txt) (TAIR release on 17 March 2007) and compared using Venn diagrams.  The differentially expressed genes (identities) in met1-RNAi lines compared to A. suecica control were mapped to oligonucleotide sequences using Perl scripts. The oligos were mapped to genomic coordinates using high (red) and low (blue) gradients corresponding to gene densities. Vertical lines above and below the chromosomes showed up- and down-regulation, respectively, and the length was proportional to the logarithm-fold changes in differential gene expression.

first: Meng

last: Chen

first: Erika

last: Lackey

first: Jianlin

last: Wang

first: Jeffrey


title: Sigma factor E of Mycobacterium tuberculosis controls the expression of bacterial components that modulate macrophages

summary:
Sigma factor E (SigE) controls the expression of genes that are essential for Mtb virulence. In this work, we have identified the SigE regulon during infection of macrophages
Our results indicate that SigE regulates the expression of genes involved in the maintenance of Mtb cell envelope integrity and function (i.e., detoxification and secretion).
Keywords: strains comparison

overall_design: We compared the global gene expression of the H37Rv strain and the sigE mutant strain of Mtb  in different conditions: growing exponentially in liquid media; after 2h of incubation in RPMI; or after 24 hours of infection of human macrophage-like THP-1 cells.

first: María

middle: E

last: Cheng


title: The sigma factor E of Mycobacterium tuberculosis modulates the inflammatory response of human THP1 cells

overall_design: We compared the global gene expression of THP1 macrophages infected with H37Rv and SigE to the gene expression profile of uninfected macrophages.


title: Exon and junction microarrays detect widespread mouse strain- and sex-bias expression differences

city: Seattle

first: Amit

middle: V

last: Kulkarni

state: WA

address: 401 Terry Ave N

department: Genomics

institute: Rosetta Inpharmatics / Merck Pharmaceuticals

summary:
71 liver RNA samples from three mouse strains - DBA/2J, C57BL/6J and C3H/HeJ - were profiled using a custom-designed microarray monitoring exon and exon-junction expression of 1,020 genes representing 9,406 exons.  Gene expression was calculated via two different methods, using the 3'-most exon probe ("3' gene expression profiling") and using all probes associated with the gene ("whole-transcript gene expression profiling"), while exon expression was determined using exon probes and flanking junction probes that spanned across the neighboring exons("exon expression profiling").  Widespread strain and sex influences were detected using a two-way Analysis of Variance (ANOVA) regardless of the profiling method used.  However, over 90% of the genes identified in 3' gene expression profiling or whole transcript profiling were identified in exon profiling, along with 75% and 38% more genes, respectively, showing evidence of differential isoform expression. Overall, 55% and 32% of genes, respectively, exhibited strain- and sex-bias differential gene or exon expression.
Keywords: Grouped

overall_design: Hybridization material was generated through a random-priming amplification of poly[A]+ purified RNA using primers with a random sequence at the 3' end and a fixed motif at the 5' end that was optimized to generate strand-specific cDNA copies of full-length mRNA transcripts [32].  Since the region used for exon and junction probe selection is constrained to a smaller region, more probes contain sequence with suboptimal characteristics (e.g. high GC content or higher homology to other genes).  The hybridization of cDNA, rather than cRNA as commonly done, partially mitigates this issue due to higher specificity and lower background levels [24]. Hybridization conditions were as previously described [33].  All 71 samples were hybridized in a two-channel experiment, where one channel was a common reference, generated by pooling all 71 samples in equal mass.  Array hybridizations were done in duplicate with fluor reversal to systematically correct for Cy3/Cy5 dye bias.  Array images were processed as described to obtain background noise, single channel intensity and associated measurement error estimates [34]. Expression changes between samples and pool were quantified as mean log10(expression ratio), and associated error.

first: Wan-Lin

last: Su

first: Barmak

last: Modrek

first: Debraj

last: GuhaThakurta

first: Stephen

last: Edwards

first: Jyoti

middle: K

last: Shah

first: Archie

last: Russell

first: Eric

last: Schadt

first: Jason

last: Johnson

first: John

middle: C

last: Castle


title: Gene expression profiling of human skeletal muscle in response to stabilized weight loss.

status: Public on Mar 26 2008

city: Paris

first: Karine

last: Clement

address: place du parvis Notre-Dame

department: Service de Medecine et Nutrition

country: France

institute: Hotel-Dieu

summary:
Background: Diet induced weight reduction promotes a decrease in resting energy expenditure that could partly explain the difficulty to maintain reduced body mass. Whether this reduction remains after stabilized weight loss is still controversial. The molecular mechanisms are unknown.
Objective: To investigate the effect of a stabilized 10%-weight loss on resting metabolic rate, body composition and skeletal muscle gene expression profile in obese women.
Design: Obese women were successively submitted to a 4-w very low-calorie diet, a 3-6-wk low-calorie diet, and a 4-wk weight maintenance program to achieve a 10% weight loss. Resting energy expenditure, body composition, plasma parameters and skeletal muscle transcriptome were compared before weight loss and during stabilized weight reduction.
Results: Energy restriction caused an 11% weight loss. Stabilization to the new weight was accompanied by an 11% decrease of the resting metabolic rate normalized to the body cellular mass which was below that of lean subjects. The range of the changes in the skeletal muscle transcriptome was modest. The main regulated genes were that of slow/oxidative fiber markers which were overexpressed and the gene encoding the glucose metabolism inhibitor PDK4 which was down-regulated. The knowledge based approach, gene set enrichment analysis, identified pathways related to insulin and interleukin 6 and long term calorie restriction adaptations during weight loss.
Set of arrays that are part of repeated experiments
Keywords: Biological Replicate

overall_design:
Overall design: Subjects and study design: Six obese Caucasian premenopausal women were submitted to a weight loss program: including three phases. The first one was a 4-wk very low calorie liquid formula diet (3.3 MJ/day). The second phase was a 3 to 6-wk low calorie diet (4 to 5 MJ/day) until reaching a loss of 10% of the initial body weight. The last phase was a 4-wk isocaloric diet (mean 5.8 MJ/day), planned to stabilize the new body weight. Percutaneous biopsy of the vastus lateralis muscle was obtained in obese women before and after the weight loss program.
Biological Replicate
Complex


title: Elucidating a normal function of huntingtin by analysis of huntingtin-null mouse embryonic fibroblasts

status: Public on Apr 12 2008

city: dallas

first: hua

last: zhang

state: TX

address: 5323 harry hines

department: physiology

institute: utsw

summary:
The polyglutamine expansion in huntingtin (Htt) protein is a cause of Huntington’s disease (HD).  Htt is an essential gene as  deletion of the mouse Htt gene homolog (Hdh) is embryonic lethal in mice. Therefore, in addition to elucidating the mechanisms responsible for polyQ-mediated pathology, it is also important to understand the normal function of Htt protein for both basic biology and for HD. To systematically search for a mouse Htt function, we took advantage of the Hdh +/- and Hdh-floxed mice and generated four mouse embryonic fibroblast (MEF) cells lines which contain a single copy of the Hdh gene (Hdh-HET) and four MEF lines in which the Hdh gene was deleted (Hdh-KO).   The function of Htt in calcium (Ca2+) signaling was analyzed in Ca2+ imaging experiments with generated cell lines.   We found that the cytoplasmic Ca2+ spikes resulting from the activation of inositol 1,4,5-trisphosphate receptor (InsP3R) and the ensuing mitochondrial Ca2+ signals were suppressed in the Hdh-KO cells when compared to Hdh-HET cells.  Furthermore,  in experiments with permeabilized cells we found that the InsP3-sensitivity of Ca2+ mobilization from endoplasmic reticulum was reduced in Hdh-KO cells.  These results indicated that Htt plays an important role in modulating InsP3R-mediated Ca2+ signaling.  To further evaluate function of Htt, we performed genome-wide transcription profiling of generated  Hdh-HET and Hdh-KO cells by microarray.  Our results revealed that 106 unique transcripts were downregulated by more than two-fold with p < 0.05 and 173 unique transcripts were upregulated at least two-fold with p < 0.05 in Hdh-KO cells when compared to Hdh-HET cells.  The microarray results were confirmed by quantitative real-time PCR for a number of affected transcripts.  Several signaling pathways affected by Hdh gene deletion were identified from annotation of the microarray results.   The unbiased approach used in our study provides novel and unique information about the normal function of Htt in cells, which may contribute to our understanding and treatment of HD.
Keywords: cell type comparison

overall_design: we generate four Hdh-HET MEF cell lines and four Hdh-KO MEF cell lines, and performed genome-wide transcription profiling of generated  Hdh-HET and Hdh-KO cells by microarray.

first: Hua

last: Zhang

first: Sudipto

last: Das

first: Quan-Zhen

last: Li

first: Ioannis

last: Dragatsis

first: Joyce

middle: J

last: Repa

first: Scott

last: Zeitlin

first: György

last: Hajnóczky

first: Ilya

last: Bezprozvanny


title: Pentylenetetrazol treatment and its withdrawal induced gene expression changes in female Drosophila head

status: Public on Mar 25 2008

city: Delhi

first: Abhay

last: Sharma

state: Delhi

address: Delhi University campus, Mall Road

department: Functional Genomics Unit

country: India

institute: CSIR-Institute of Genomics and Integrative Biology (IGIB)

summary:
We have recently developed a Drosophila behavioral- and transcriptomic- based (systems) model of chronic pentylenetetrazole (PTZ) induced locomotor plasticity. Pharmacological validation using antiepileptic drugs (AEDs) shows that the model is predictive of antiepileptic, antiepileptogenic, disease-modifying and neuroprotective activities. This model is however developed using male flies. The present submission relates to microarray gene expression profiling of fly heads after treatment of female Drosophila adults with PTZ for seven days subsequent withdrawal of PTZ for next seven days. Expression profiles have been generated at three time points during chronic PTZ treatment, namely 12 hrs, 2nd day and 7th day and at the end of withdrawal period, i.e., 14th day from the beginning of the treatment.
Keywords: Drug response

overall_design: D. melanogaster Oregon-R wild type flies were grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin.  The cultures were grown at 24 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. In chronic PTZ experiment, three to four days old virgin adult females were grown in either normal food (NF) or food containing 8 mg/ml of PTZ for 12 hrs, two days or seven days. In withdrawal experiment, the control and PTZ flies treated with PTZ for seven days in above manner were shifted to vials containing normal food and maintained further for seven days. Each vial contained 30 flies in the beginning of the treatment. Heads were harvested at three time points during PTZ treatement – 12 hrs, 2nd day, and 7th day – and at one time point (14th day from the beginning of the treatment, i.e., seven days after PTZ withdrawal) after withdrawal. Flies frozen in liquid nitrogen were shaken and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four PTZ exposed individuals, during treatment or withdrawal period, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of control and treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and PTZ treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto, Canada). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The preprocessing and quantification of the 16 bit TIFF images were carried out using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 3.0, Excel Add-In, Stanford) under the conditions of one class response and 100 permutations.

first: Mohammad

last: Farhan

first: Priyanka

last: Singh


title: 3-Deazaadenosine treated hCASMC

city: Giessen

first: Alexander

middle: Claus

last: Langheinrich

state: Hessen

address: Klinikstrasse 36

department: Radiology

institute: University of Giessen

summary:
cDNA expression prfiles of purified human smooth muscle cells treated with c³Ado were compared to those of untreated cells.
Keywords: treatment-versus-control

overall_design: Cells were treated with c³Ado or left untreated (controls). Five cultures each were prepared. Total RNA was reverse transcribed, labeled with 32P, and hybridized to nylon filters spotted with 1200 human cDNAs.

first: Daniel

middle: G

last: Sedding

first: Mariam

last: Kampschulte

first: Regina

last: Moritz

first: Jochen

last: Wilhelm

first: Werner

last: Haberbosch

first: Erik

middle: L

last: Ritman

first: Rainer

last: Bohle


title: Identification of protein-coding and intronic noncoding RNAs down-regulated in clear cell renal carcinoma

city: Sao Paulo

first: Glauber

middle: Costa

last: Brito

state: Sao paulo

address: Av. Prof. Lineu Prestes, 748 - sala 1.200

department: Instituto de Quimica - Dep Bioquimica

country: Brazil

institute: Universidade de Sao Paulo

summary:
The clear cell subtype of renal cell carcinoma (RCC) is the most lethal and prevalent cancer of the urinary system. To investigate the molecular changes associated with malignant transformation in clear cell RCC, the gene expression profiles of matched samples of tumor and adjacent non-neoplastic tissue were obtained from 6 patients. A custom-built cDNA microarray platform was used, comprising 2,292 probes that map to exons of genes and 822 probes for noncoding RNAs mapping to intronic regions. Intronic transcription was detected in all normal and neoplastic renal tissues. A subset of 55 transcripts was significantly down-regulated in clear cell RCC relative to the matched non-tumor tissue as determined by a combination of two statistical tests and leave-one-out patient cross-validation. Among the down-regulated transcripts, 49 mapped to untranslated or coding exons and 6 were intronic relative to known exons of protein-coding genes. Lower levels of expression of SIN3B, TRIP3, SYNJ2BP and NDE1 (p < 0.02), and of intronic transcripts derived from SND1 and ACTN4 loci (p < 0.05), were confirmed in clear cell RCC by Real-time RT-PCR. A subset of 25 transcripts was deregulated in additional 6 non-clear cell RCC samples, pointing to common transcriptional alterations in RCC irrespective of the histological subtype or differentiation state of the tumor. Our results indicate a novel set of tumor suppressor gene candidates, including noncoding intronic RNAs, which may play a significant role in malignant transformations of normal renal cells.
Keywords: Disease state analysis

overall_design: Fluorescently labeled cDNA targets derived from tumor and non-tumor tissue from each patient were combined and hybridized to spotted cDNA microarrays. For each patient, a replicate hybridization with dye-swap of labeled targets was performed. For each tissue (tumor and adjacent non-tumor tissue), there are 4 replicate mesurements for each probe.

first: Angela

middle: A

last: Fachel

first: Andre

last: Vettore

first: Giselle

last: Vignal

first: Etel

middle: R

last: Gimba

first: Franz

last: Campos

first: Marcello

last: Barcinski

first: Sergio

last: Verjovski-Almeida

first: Eduardo

last: Reis


title: Genomic Comparison of the O-antigen Biosynthesis Gene Clusters of E. coli O55 Strains Belonging to Three Lineages.

status: Public on Apr 01 2008

city: Kiyotake

first: Atsushi

last: Iguchi

address: Oaza-kihara 5220

institute: Miyazaki university

summary:
Typical enteropathogenic Escherichia coli (EPEC) O55:H7 is regarded as the closest relative of enterohemorrhagic E. coli (EHEC) O157:H7. Both serotypes usually express the γ1 intimin subclass and trigger actin polymerazation by the Tir-TccP pathway. However, atypical O55:H7 strains capable of triggering actin polymerization via the Tir-Nck pathway have recently been identified. In this study, we investigated the genotypic differences and phylogenetic relationships between typical and atypical O55:H7 strains. We show that the atypical O55:H7 strains, which express the θ intimin subclass and lack both tccP and tccP2, belong to an E. coli lineage distinct from the typical O55:H7 and from the EPEC O55:H6, which also uses the Tir-Nck actin polymerization pathway. We conducted genomic comparisons of the chromosomal regions covering the O-antigen gene cluster and its flanking regions between the three O55 lineages by restriction fragment length polymorphism analysis of PCR products and DNA sequencing analysis of about 65-kb chromosomal regions. This unexpectedly revealed that horizontal transfer of large fragments (≥ 40 kb) encoding the O55-antigen gene cluster and part of neighboring colanic acid gene cluster is involved in the emergence of the three O55 E. coli lineages. The data provide new insights into the mechanisms involved in the generation of a wide variety of O-serotypes in Gram-negative bacteria.
Keywords: comparative genomic hybridization

overall_design: Total 8 test samples were analyzed. Genomic DNA from each test strain and a reference strain (O157 Sakai) were labeled with Cy3 and Cy5, respectively, and were cohybridized on a single array. Labeling and hybridization were performed twice independently.

first: Tetsuya

last: Hayashi


title: Assessment of CcpA-mediated catabolite control of metabolism and enterotoxin production in Bacillus cereus ATCC 14579

status: Public on Mar 28 2008

city: Wageningen

first: Menno

middle: van der

last: Voort

address: Bomenweg 2

department: Agrotechnology and Food Sciences Group

country: Netherlands

institute: Wageningen UR

summary:
In Bacillus cereus the catabolite control protein CcpA was shown to be involved in optimizing the efficiency of glucose catabolism by activating genes encoding glycolytic enzymes including a non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase that mediates conversion of D-glyceraldehyde 3-phosphate to 3-phospho-D-glycerate in one single step, and by repressing genes encoding the citric acid cycle and gluconeogenic enzymes. Two B. cereus-specific CcpA-regulated operons were identified, encoding enzymes involved in the catabolism of fuculose/arabinose and aspartate. In addition, a genome search using the CRE-site consensus predicted the B. cereus CcpA regulon to include 10 PTS-system gene clusters as well as genes coding for overflow metabolic enzymes leading to acetoin and acetate. Notably, catabolite repression of the genes encoding non-hemolytic enterotoxin (Nhe) and hemolytic (Hbl) enterotoxin appeared CcpA-dependent, and for the corresponding enterotoxin operons, putative CRE-sites were identified. This points to metabolic control of enterotoxin gene expression and suggests that CcpA-mediated glucose sensing provides an additional mode of control to PlcR activated expression of nhe and hbl genes in B. cereus.
Keywords: Time course analysis by comparing transriptomes of the wildtype and the ccpA deleton strain.

overall_design: The wildtype (B. cereus ATCC 14579) and ccpA deletion strain were sampled during aerobic growth in Brain heart infusion broth. Samples of wildtype and ccpA deletion strain were compared at 4 time points, i.e. early exponential (OD600 0.2), mid-exponential (OD600 0.8), transition phase (OD600 4), and stationary phase (OD600 8). For each time point biological duplo's were obtained, which were subsequently differently labelled to perform a dye swap.

last: van der Voort

first: Oscar

middle: P

last: Kuipers

first: Girbe

last: Buist

first: Willem

last: de Vos

first: Tjakko

last: Abee


title: Gene expression profiling of peripheral blood mononuclear cells from patients with minimal change nephrotic syndrome

status: Public on Mar 22 2008

city: Akita

last: Komatsuda

address: Hondo 1-1-1

institute: Akita University School of Medicine

summary:
Background: Minimal change nephrotic syndrome (MCNS) is considered to be associated with T cell dysfunction, via unknown mechanisms. Experimental observations suggest that some humoral factors alter the permeability of glomerular filtration barrier. However, the nature of such factors remains still uncertain. Methods: Using cDNA microarrays, we performed gene expression profiling of peripheral blood mononuclear cells (PBMC) from three patients with MCNS during nephrosis and remission phases. To confirm the cDNA microarray results, we performed quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses in nephrosis and remission samples from 20 MCNS patients and six patients with nephrotic syndrome due to membranous nephropathy. Results: Out of 24,446 genes screened, 33 genes were up-regulated (at least 1.5-fold) in PBMC from these MCNS patients during the nephrosis phase. Up-regulated genes mainly encoded proteins involved in signal transduction and cytokine response. For further examination, we selected two genes encoding provable secretary proteins, chemokine (C-C) ligand 13 (also known as monocyte chemotactic protein-4) (CCL13) and a novel galectin-related protein (HSPC159). The results of RT-PCR showed that expressions of CCL13 and HSPC159 mRNA in nephrosis PBMC samples are higher than those in remission PBMC samples from all 20 MCNS patients examined. On the other hand, these mRNA expression patterns were variable among six patients with membranous nephropathy. Conclusions: We conclude that CCL13 and HSPC159 mRNA expressions in PBMC is up-regulated in MCNS patients during the nephrosis phase. These expression changes in PBMC might be involved in the pathophysiologic processes of MCNS.
Keywords: CCL13 - cDNA microarray - gene expression profile - HSPC159 - minimal change nephrotic syndrome


title: Expression Profiling of A. thaliana Plants after Infection with the Xylem-Colonizing Fungus V. longisporum

city: Goettingen

first: Gabriela

last: Salinas

state: Lower-Saxony

address: Kreuzbergring 57

department: Department of Pathology

institute: Universitaetsmedizin Goettingen

summary:
Verticillium longisporum is a soil-borne fungal pathogen causing vascular disease predominantly in oilseed rape. The pathogen enters its host through the roots and entertains a parasitic life stage in the xylem before invading other tissues late in the infection cycle. We have started to approach the question how and when the host plant senses the colonization of the xylem using Arabidopsis thaliana as a model plant. Although the stress-related phytohormones salicylic acid, jasmonic acid and abscisic acid increase only at 28 to 35 days, expression of V. longisporum-induced genes (VliGs) starts in the leaf veins as early as 5 dpi when disease symptoms and fungal DNA cannot yet be detected. It is concluded that an elicitor is transported from the root to the aerial parts. More than one third of the VliGs identified by whole genome expression profiling at 18 dpi encode apoplastically localized proteins involved in cell wall modifications and potential defense responses. The identified VliGs provide a useful tool to elucidate the contribution of the induced genes to the disease phenotype and the defense response. Moreover, they will help to identify the elicitor(s) and the components of the signal transduction chain that shape the V. longisporum – plant interaction.
Keywords: Arabidopsis, cell wall, microarray, phytohormones, Verticillium longisporum, xylem

overall_design: 3 VL treated samples vs. 3 MOCK treated samples (control), DyeSwap Design DRLL (Jobst Landgrebe, Frank Bretz, Edgar Brunner (2004): Efficient two-sample designs for microarray experiments with biological replications. In Silico Biology 4: (2004))

first: Hella

last: Tappe

first: Saskia

last: Fleurl

first: Jan-Gerrit

last: Carsjens

first: Astrid

last: Ratzinger

first: Meik

last: Dilcher

first: Petr

last: Karlovsky

first: Ivo

last: Feussner

first: Andrea

last: Polle

first: Christiane

last: Gatz


title: Bovine adult versus prepubertal cumulus cells

status: Public on Mar 18 2008

city: East Lansing

first: George

middle: W

last: Smith

state: MI

address: 1230 Anthony Hall

institute: Michigan State University

summary:
The present study was undertaken to discover molecular markers in bovine cumulus cells predictive of oocyte competence and elucidate their functional significance.  Differences in RNA transcript abundance in cumulus cells harvested from oocytes of adult versus prepubertal animals (model of poor oocyte quality) were identified by microarray analysis.  Four genes of interest encoding for the lysosomal cysteine proteinases cathepsin B, S, K and Z and displaying greater transcript abundance in cumulus cells surrounding oocytes harvested from prepubertal animals were chosen for further investigation. Greater mRNA abundance for such genes in cumulus cells of prepubertal oocytes was confirmed by real time RT-PCR. Elevated transcript abundance for cathepsins B, S and Z was also observed in cumulus cells surrounding adult metaphase II oocytes that developed to the blastocyst stage at a low percentage following parthenogenetic activation, versus those that developed at a high percentage. Functional significance of cumulus cell cathepsin expression to oocyte competence was confirmed by treatment of cumulus oocyte complexes during in vitro oocyte maturation with a cell permeable cysteine proteinase (cathepsin) inhibitor.  Inhibitor treatment decreased apoptotic nuclei in the cumulus layer and enhanced development of parthenogenetically activated and in vitro fertilized adult oocytes to the blastocyst stage.  Stimulatory effects of inhibitor treatment during meiotic maturation on subsequent embryonic development were not observed when oocytes were matured in the absence of cumulus cells.  Results support a functional role for cumulus cell cathepsins in compromised oocyte competence and suggest that cumulus cell cathepsin mRNA abundance may be predictive of oocyte quality.
Keywords: Bovine, microarray, cDNA, cumulus cells

overall_design:
Differences in RNA transcript abundance in cumulus cells harvested from oocytes of adult versus prepubertal animals (model of poor oocyte quality) were identified by microarray analysis.
Total RNA from pools of cumulus cells (n = 4) collected from adult and prepubertal animals for microarray experiments was amplified.    Two color microarray experiments were conducted using a bovine cDNA array containing expressed sequence tags (ESTs) representing approximately 15200 unique genes.  Hybridizations were performed on duplicate slides (prepubertal versus adult) and incorporated a dye swap. The total number of slides used is eight.

first: Anilkumar

last: Bettegowda

first: Osman

last: Patel

first: Kyung-Bon

last: Lee

first: Ki-Eun

last: Park

first: James

last: Ireland


title: Genome-Wide Transcriptional Responses of Nitrosomonas europaea to cadmium

city: Corvallis

first: Sunhwa

state: OR

address: 116 Gilmore

department: Biological & Ecological Eng.

institute: Oregon State University

summary:
Heavy metals have been postulated as significant nitrification inhibitor in wastewater treatment plant. The effect of heavy metals such as Cd2+, Cu2+ and Hg2+ to nitrifying bacterium, Nitrosomonas europaea, was studied in pseudo-steady state batch reactor. Under incubation of Nitrosomonas europaea with 1 ?M CdCl2 for 1 hour, transcripts for 66 of 2460 genes were found at high level, yet transcripts of 50 genes were found at low level. Mercury resistance genes (merACDPT) showed 277-fold up regulation.
Keywords: cadmium, stress response, global transcription, mercury resistance genes, merA,

overall_design: The 1 uM CdCl2 caused more than 50 % inhibition in physiological response for 1 hour incubation. Transcriptional levels of the cells inhibited by cadmium were compared with the cells under control condition.

first: Roger

last: Ely


title: Latitudinal expression analysis of Mytilus californianus

city: Columbia

first: Sean

last: Place

state: SC

address: 715 Sumter St.

department: Biological Sciences

institute: University of South Carolina

summary:
Transcriptional analysis of the effects of natural environmental variation across the latitudinal range of Mytilus californianus
Keywords: Environmental response

overall_design: 5 Biological replicates from 4 separate populations of mussels were compared using a common reference sample. Dye swap analysis was performed for each replicate.

first: Gretchen

last: Hofmann

first: Michael

last: O'Donnell


title: Gene expression changes in Drosophila head induced by pentylenetetrazole and levetiracetam combination treatment

status: Public on Mar 19 2008

first: Abhay

last: Sharma

summary:
We have recently developed a Drosophila behavioral- and transcriptomic- based (systems) model of chronic pentylenetetrazole (PTZ) induced locomotor plasticity. Pharmacological validation using antiepileptic drugs (AEDs) sodium valproate and levetiracetam (LEV) at behavioral level showed a weaker prophylactic and stronger symptomatic effect of sodium valproate and the opposite, i.e., stronger prophylactic and weaker symptomatic, effect in the model. Microarray gene expression profiling of fly head after sodium valproate and LEV treatment of flies further supported the above therapeutic effect of the two AEDs in the PTZ fly model. Given the strong prophylactic effect of LEV in our model, we hypothesized that time series of transcriptomic alterations caused by PTZ at 12 hrs, 2nd day and 7th day (an earlier GEO submission) may be neutralized by LEV. The present submission relates to microarray expression profiles at 12 hrs, 2nd day and 7th day of a drug regime in which flies were treated with both PTZ and LEV for four days followed by three days of treatment with PTZ alone. Results are consistent with our above hypothesis.
Keywords: Drug combination response

overall_design: D. melanogaster Oregon-R wild type flies were grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin.  The cultures were grown at 24 + 1 C, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 8 mg/ml PTZ and/or 5 mg/ml LEV. Combination treatment was carried out for four days after which flies were shifted to new vials containing LEV mixed food. Each treatment vial contained 30 flies. Heads were harvested at 12 hrs, 2nd day and 7th day. Flies frozen in liquid nitrogen were shaken and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four PTZ and LEV combined or subsequent alone PTZ treated individuals, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of control and treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and drug treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto, Canada). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The preprocessing and quantification of the 16 bit TIFF images were carried out using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 3.0, Excel Add-In, Stanford) under the conditions of one class response, imputation and 100 permutations.

first: Mohammad

last: Farhan

first: Priyanka

last: Singh


title: Profiling BRCA1, BRCA2 and non-BRCA1/2 LCLs post-Irradiation

status: Public on Mar 21 2008

city: Brisbane

first: Nic

last: Waddell

address: Herston Road

country: Australia

institute: QIMR

summary:
The functional consequences of missense variants in disease genes are difficult to predict. We assessed if gene expression profiles could distinguish between BRCA1 or BRCA2 pathogenic truncating and missense mutation carriers and familial breast cancer cases whose disease was not attributable to BRCA1 or BRCA2 mutations (BRCAX cases). 72 cell lines from affected women in high-risk breast-ovarian families were assayed after exposure to ionising irradiation, including 23 BRCA1 carriers, 22 BRCA2 carriers, and 27 BRCAX individuals. A subset of 10 BRCAX individuals carried rare BRCA1/2 sequence variants considered to be of low clinical significance (LCS). BRCA1 and BRCA2 mutation carriers had similar expression profiles, with some subclustering of missense mutation carriers. The majority of BRCAX individuals formed a distinct cluster, but BRCAX individuals with LCS variants had expression profiles similar to BRCA1/2 mutation carriers. Gaussian Process Classifier predicted BRCA1, BRCA2 and BRCAX status with a maximum of 62% accuracy, and prediction accuracy decreased with inclusion of BRCAX samples carrying an LCS variant, and inclusion of pathogenic missense carriers. Similarly, prediction of mutation status with gene lists derived using Support Vector Machines was good for BRCAX samples without an LCS variant (82-94%), poor for BRCAX with an LCS (40-50%), and improved for pathogenic BRCA1/2 mutation carriers when the gene list used for prediction was appropriate to mutation effect being tested (71-100%). This study indicates that mutation effect, and presence of rare variants possibly associated with a low risk of cancer, must be considered in the development of array-based assays of variant pathogenicity.
Keywords: cell type comparison, stress response

overall_design: A cohort of 72 lymphoblastoid cell lines derived from patients who are carriers of different mutations: 23 BRCA1, 22 BRCA2, 27 non-BRCA1/2. 7 of the LCLs were regrown, treated and arrayed as replicate samples A-G

first: Amanda

last: Spurdle


title: c3h10t1/2 with dexamethasone

city: san francisco

first: Alex

middle: Steven (Yick-Lun)

last: So

state: CA

department: cellular and molecular pharmacology

institute: university of california san francisco

summary:
determine genes regulated by dexamethasone in c3h10t1/2 cells after 90 minutes of treatment compared to vehicle
Keywords: treatment comparison microarray

overall_design:
c3h10t1/2 cells were treated with vehicle or 1uM dexamethasone for 90 minutes (3 biological replicates of each)
RNA samples were isolated from RNeasy kit (Qiagen)
hybridized all samples to a pool of RNA from vehicle and treated c3h10t1/2 cells

middle: Y

first: Samantha

middle: B

last: Cooper

first: Brian

last: Feldman

first: Mitra

last: Manuchehri

first: Keith

last: Yamamoto


title: E. coli K-12 wild type with R1drd19 biofilm vs wild type without R1drd19 biofilm

city: College Station

first: Xiaole

last: Yang

address: Chemical Engineering Department

institute: Texas A & M University

summary:
E. coli K-12 wild type strains gene expression in biofilm cells with R1drd19 relative to that in biofilm cells of E. coli K-12 wild type strains without R1drd19. All the samples were cultured in LB or M9C glu on glass wool at different time.
Keywords: biofilm

overall_design:
Strains: E. coli K-12 strains with and without R1drd19 respectively;
Medium: LB was used for BW25113 strains, and M9C glu was used for MG1655 strains;
Biofilm grown on glass wool;
Time: 7h, 15h, 24h respectively;
Cell type: biofilm.

first: Thomas

last: Wood


title: Chronic tetraethylammonium chloride treatment induced gene expression changes in Drosophila head

summary:
Tetraethylammonium chloride (TEA) is an epileptogenic agent. In an effort to develop a Drosophila model of kindling epileptogenesis, we have examined chronic TEA induced alterations in gene expression profile of fly heads. Expression profiling of fly heads after 7 day long TEA treatment revealed perturbations in various functional category of genes.
Keywords: Drug Response

overall_design: D. melanogaster Oregon-R wild type flies were grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin.  The cultures were grown at 24 +/- 1 ºC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 16 mg/ml of TEA for seven days. Following this, the control and TEA treated flies were shifted to vials containing normal food and maintained further for seven days. Each vial contained 30 flies in the beginning of the treatment. Heads were harvested at the end of seven day treatment. Flies frozen in liquid nitrogen were shaken and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four TEA exposed individuals, during treatment or withdrawal period, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of control and treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and TEA treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The preprocessing and quantification of the 16 bit TIFF images were carried out using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 3.0, Excel Add-In, Stanford) under the conditions of one class response and 100 permutations.


title: Chronic pilocarpine hydrochloride treatment induced gene expression changes in Drosophila head

summary:
Pilocarpine hydrochloride (PILO) is an epileptogenic agent. In an effort to develop a Drosophila model of kindling epileptogenesis, we have examined chronic PILO induced alterations in gene expression profile of fly heads. Expression profiling of fly heads after 7 day long PILO treatment revealed perturbations in various functional category of genes.
Keywords: Drug Response

overall_design: D. melanogaster Oregon-R wild type flies were grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin.  The cultures were grown at 24 +/- 1ºC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 1.5 mg/ml of PILO for seven days. Following this, the control and PILO treated flies were shifted to vials containing normal food and maintained further for seven days. Each vial contained 30 flies in the beginning of the treatment. Heads were harvested at the end of seven day treatment. Flies frozen in liquid nitrogen were shaken and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four PILO exposed individuals, during treatment or withdrawal period, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of control and treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and PILO treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The preprocessing and quantification of the 16 bit TIFF images were carried out using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 3.0, Excel Add-In, Stanford) under the conditions of one class response and 100 permutations.


title: Comparing wild type Salmonella Typhimurium to a corA mutant

city: Cleveland

first: Krisztina

middle: Margaret

last: Papp-Wallace

email: kmp12@case.edu, mem6@case.edu

state: OH

address: 2109 Adelbert Road W317

department: Pharmacology

institute: Case Western Reserve Univeristy

summary:
CorA is the primary Mg2+ channel in Salmonella enterica serovar Typhimurium.  A strain lacking corA is attenuated in mice after infection either by oral gavage or intraperitoneal injection.  Microarray studies show that several virulence effectors in Salmonella pathogenicity island 1 and Salmonella pathogenicity island 2 are repressed in the corA strain compared to wild type.  While these results could be sufficient to explain the virulence deficit, the microarray data suggest additional defects that could also contribute.  Motility is significantly reduced in a corA strain whereas enterochelin-dependent iron uptake and curli are upregulated.  A corA strain is defective for invasion of and replication within Caco-2 epithelial cells.  However, a corA strain does not have a significant survival defect in J774A.1 macrophages.  Thus, despite the presence of two other Mg2+ transporters, loss of CorA affects multiple systems which manifests ultimately as a decrease in virulence.
Keywords: strain comparison

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium

overall_design: Cells were collected for RNA isolation on three separate days from wild type or corA strains giving six total samples from three independent cultures for each strain.  A single microarray was run for each sample, three for wild type and three for the corA strain.  Genomic DNA from wild type was used as a reference on each slide.  Each microarray consists 3 subarrays.

middle: M

first: Margaret

last: Nartea

first: David

last: Kehres

first: Steffen

last: Porwollik

last: McClelland

first: Steven

last: Libby

first: Ferric

last: Fang

middle: E

last: Maguire


title: Chronic strychnine treatment induced gene expression changes in Drosophila head

first: Abhay

last: Sharma

summary:
Strychnine (STR) is a convulsant agent. In an effort to develop a Drosophila model of kindling epileptogenesis, we have examined chronic STR induced alterations in gene expression profile of fly heads. Expression profiling of fly heads after 7 day long STR treatment revealed perturbations in various functional category of genes.
Keywords: Drug Response

overall_design: D. melanogaster Oregon-R wild type flies were grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin.  The cultures were grown at 24 +/- 1 ºC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 3.33 mg/ml of STR for seven days. Following this, the control and STR treated flies were shifted to vials containing normal food and maintained further for seven days. Each vial contained 30 flies in the beginning of the treatment. Heads were harvested at the end of seven day treatment. Flies frozen in liquid nitrogen were shaken and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four STR exposed individuals, during treatment or withdrawal period, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of control and treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and STR treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The preprocessing and quantification of the 16 bit TIFF images were carried out using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 3.0, Excel Add-In, Stanford) under the conditions of one class response and 100 permutations.

first: Mohammad

last: Farhan


title: Stress-dependent dynamics of chromatin remodeling in yeast: Dual roles for SWI/SNF in heat shock stress response

status: Public on Mar 17 2008

city: Austin

first: Vishy

last: Iyer

address: 2500 Speedway Dr. MBB 3.212

department: Molecular Biosciences

institute: University of Texas at Austin

type: Genome binding/occupancy profiling by array

summary:
Total H4 and acetyl H4 IP, time course Total H4 and acetyl H4 occupancy, Snf2 occupancy, gene expression analysis
Keywords: stress response, time course

overall_design: S cerevisiae were subjected to heat shock and stationary phase stress. The histone occupancy and H4 acetylation levels were monitored in parallel with gene expression studies, and Snf2 occupancy before and after stress.

first: Sushma

last: Shivaswamy

first: Vishwanath


title: A Chromatin Immunoprecipitation Approach Based on BAC Transgenesis

status: Public on Mar 15 2008

city: Houston

first: Sujun

last: Hua

state: Tx

address: 1901 East Rd

institute: MD Anderson Cancer Center

type: Genome binding/occupancy profiling by genome tiling array

summary:
We have devised a technology that enables chromatin immunoprecipitation (ChIP) experiments in cultured human cells by circumventing both the laborious production of protein-specific antibodies and the optimization of crosslinking/immunoprecipitation conditions for each protein. Our approach utilizes bacterial artificial chromosomes (BACs) to physiologically express chromatinbinding proteins fused to epitope tags that are recognized by widely available and highly specific antibodies. We demonstrate the robustness of our methodology for mapping chromatin binding sites of transcription factors.
Keywords: ChIP-Chip Analysis

overall_design: This series contains ChIP-on-Chip data sets for three transcription factors (FOXA1 (Forkhead box A1), XBP1-S (spliced X-box binding protein 1) and VDR (Vitamin D receptor)) and control samples (INPUT) for partial of the human genome including chromosomes 4, 15, 18, and 20 in MCF7 cells. We mapped the binding sites of these three transcription factors in the BAC transgenic MCF7 cells in which we tagged the transcription factors with a modified LAP (localization and affinity purification) tag. Goat anti-GFP (raised against His-tagged full-length eGFP and affinity-purified with GST-tagged full-length eGFP) was used to perform ChIP experiments in those transgenic lines. For comparison, we also mapped FOXA1 and XBP1-S binding sites with the available antibodies against the endogenous proteins. Goat anti-FoxA1 (Abcam: ab5089) and goat anti-XBP1 (Santa Cruz Biotech: sc-7160X) were used in these studies. Independent biological triplicates were performed for each transcription factor, as well as the control (Input).

first: Ralf

last: Kittler

first: Kevin

last: White


title: Transcriptional control of amino acid biosynthesis

city: Cambridge

first: Joel

last: Moxley

state: MA

address: 77 Massachusettes Ave

institute: MIT

summary:
We transcriptional profiled four transcription factor knockout strains in S288C background growing in YNB media + 2% glucose to understand the link between mRNA levels and our measured C13 fluxes of amino acid biosynthesis.  We conducted this analysis as a follow up to our work on the Gcn4p transcription factor.
Keywords: genetic modification

overall_design: Multiple biological replicates were conducted for four transcription factor knockout strains


title: Gene Expression Analysis of Human Endometrial Endothelial Cells Exposed to Bisphenol A

status: Public on Mar 13 2008

city: Uppsala

first: Carolina

last: Bredhult

address: Uppsala University Hospital

department: Women's and Children's Health

country: Sweden

institute: Uppsala University

summary:
The endocrine disrupting chemical bisphenol A (BPA) can affect reproductive organs, tissues and cells in several species. Treatment of human endometrial endothelial cells (HEECs) with 50 µM BPA decreased their proliferation compared with the control. Microarray analyses revealed that BPA affected biological processes such as the cell cycle, cell division, and cytoskeleton organization, confirming the results of the proliferation assay. Expression of three of the most differentially expressed genes identified in the microarray analysis was verified by real-time quantitative reverse transcription polymerase chain reaction in five HEEC cultures obtained from women in the proliferative phase and in five cultures obtained from women in the secretory phase of the menstrual cycle after treatment with BPA. The present study supports our previous findings of decreased proliferation and increased cell death in response to BPA, and may offer important clues to the mechanisms of action of BPA.
Keywords: Exposure analysis

overall_design: Two-condition experiments, BPA-exposed vs. control HEEC. Biological replicates: 5 HEEC cultures, independently grown, exposed and harvested. One replicate per array. Dye-swaps for exposed and reference samples between replicates.

first: Lena

last: Sahlin

first: Matts

last: Olovsson


title: A Gene Expression Network Model of Type 2 Diabetes Links Cell Cycle

city: Madison

first: Mark

middle: Patrick

last: Keller

state: WI

address: 433 Babcock Drive

department: Biochemistry

institute: University of Wisconsin

summary:
Insulin resistance is necessary but not sufficient for the development of type 2 diabetes. Diabetes results when pancreatic beta-cells fail to compensate for insulin resistance by increasing insulin production through an expansion of beta-cell mass or increased insulin secretion. Communication between insulin target tissues and beta-cells may initiate this compensatory response. Correlated changes in gene expression between tissues can provide evidence for such intercellular communication. We profiled gene expression in six tissues of mice from an obesity-induced diabetes-resistant and a diabetes-susceptible strain before and after the onset of diabetes. We studied the correlation structure of mRNA abundance and identified 105 co-expression gene modules. We provide an interactive gene network model showing the correlation structure between the expression modules within and among the six tissues. This resource also provides a searchable database of gene expression profiles for all genes in six tissues in lean and obese diabetes-resistant and diabetes-susceptible mice, at 4 and 10 weeks of age. A cell cycle regulatory module in islets predicts diabetes susceptibility. The module predicts islet replication; we found a strong correlation between ^2 H_2 O incorporation into islet DNA /in vivo/ and the expression pattern of the cell cycle module. This pattern is highly correlated with that of several individual genes in insulin target tissues, including IGF2, which has been shown to promote beta-cell proliferation, suggesting that these genes may provide a link between insulin resistance and beta-cell proliferation.
Keywords: time course, mouse strain comparison, effect of obesity,

overall_design: Type 2 diabetes is a disorder that involves an increased demand for insulin brought about by insulin resistance, together with a failure to compensate with sufficient insulin production. Although Insulin resistance occurs in most obese individuals, diabetes is generally forestalled through compensation with increased insulin. This increase in insulin occurs through an expansion of beta-cell mass and/or increased insulin secretion by individual beta-cells. Failure to compensate for insulin resistance leads to type 2 diabetes. One way to understand the pathophysiology of diabetes is to examine the coordinate changes in gene expression that occur in insulin-responsive tissues and pancreatic islets in obese animals that either compensate for insulin resistance or progress to type 2 diabetes. In each case, there are groups of genes that undergo changes in expression in a highly correlated fashion. By identifying groups of correlated transcripts (gene expression modules) during the compensation and development of diabetes, we can gain insight into potential pathways and regulatory networks in obesity-induced diabetes. We study two strains of mice that differ in obesity-induced diabetes susceptibility. In this study, we surveyed gene expression in six tissues of lean and obese C57BL/6 (B6) and BTBR mice aged 4 wks and 10 wks. B6 mice remain essentially non-diabetic at all ages, irrespective of obesity. When obese, BTBR mice become severely diabetic by 10 weeks of age. By analyzing the correlation structure of the genes under three contrast conditions, obesity, strain, and age, we identified gene expression modules associated with the onset of diabetes and provide an interactive co-expression network model of type 2 diabetes. We found a key module that is comprised of cell cycle regulatory genes. In the islet, the expression profile of these transcripts accurately predicts diabetes and is highly correlated with islet cell proliferation.

first: Eric

last: Schadt

first: Alan

middle: D

last: Attie


title: RNA binding activity of the recessive parkinsonism protein DJ-1

status: Public on Mar 14 2008

city: Bethesda

first: J Raphael

last: Gibbs

address: 35 Convent Drive, Bldg 35/1A1015, MSC3707

department: Computational Biology Core

institute: National Institutue on Aging, NIH

summary:
Parkinson disease (PD) is a major neurodegenerative condition with several rare Mendelian forms. Oxidative stress and mitochondrial function have been implicated in the pathogenesis of PD but the molecular mechanism(s) involved in the degeneration of specific neuronal groups remains unclear. DJ-1 mutations are one cause of recessive parkinsonism, but this gene is also involved in cancer by promoting Ras signaling and suppressing PTEN-induced apoptosis. The specific function of DJ-1 is unclear, although it is responsive to oxidative stress and may play a role in the maintenance of mitochondria. Here we show that DJ-1 associates with specific RNA targets in cells and in the brain including mitochondrial genes, genes involved in glutathione metabolism and members of the PTEN/PI3K cascade. Pathogenic recessive mutants are deficient in this activity. We show that DJ-1 is sufficient for RNA binding at nanomolar concentrations in vitro and that there is some RNA sequence specificity to the association. Oxidative stress causes DJ-1 to dissociate from RNA. Using in vitro and in vivo models of mild oxidative stress, we show that DJ-1 normally suppresses translation in normal circumstances but allows translation after oxidative stress. We tested the hypothesis that these specific RNA targets are responsible for sensitivity to stress by exposing knockout flies to glutathione synthesis inhibitors and saw the predicted increased sensitivity in vivo. These data implicate a single mechanism for the pleiotropic effects of DJ-1 in different model systems, namely that the protein binds and regulates specific groups of RNA targets in an oxidationdependent manner. Furthermore, these results suggest how a small protein might both be an upstream regulator of processes important in parkinsonism and be a modifier of cancer-related processes.
Keywords: Immunoprecipitated RNA comparisons

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: 6-8 biological replicates of RNA immunoprecipitated with DJ-1 protein was compared to RNA isolated with a nonspecific IgG control antibody in human cells, or to nonspecific RNA isolated with the DJ-1 antibody from KO mouse brains.

first: Marcel

last: van der Brug

first: Jeff

last: Blackinton

first: Jayanth

last: Chandran

first: Ling-yang

last: Hao

first: Ashish

last: Lal

first: Myriam

last: Gorospe

last: Cookson


title: Transcriptional response of soybean plants to soybean mosaic virus infection

city: London

first: Mohan

last: babu

state: Ontario

address: 1391 Sandford

department: Virology

country: Canada

institute: AAFC

summary:
Transcriptional changes in soybean plants infected with Soybean mosaic virus (SMV) was assessed at 7, 14 and 21 days post inoculation (dpi).
Keywords: Time course

overall_design: Two separate cDNA labeling reactions (Cy3 and Cy5), one for the RNA from SMV-infected leaf tissues and the other for the RNA from the mock-inoculated leaves, were carried out. A total of 18 microarray slides were used for this study: 6 array hybridizations including three reciprocal labeling experiments from three independent biological replications of either SMV-infected or mock-inoculated leaf at each of the three time points. Dye swaps were performed to ensure that the results were not biased by dye effects.

last: Babu

first: Aiming

last: Wang


title: Profiling the asexual development transcriptome of the late blight pathogen Phytophthora infestans

city: Riverside

first: Howard

middle: S

last: Judelson

address: Boyce Hall

department: Plant Pathology

institute: University of California

summary:
Much of the pathogenic success of Phytophthora infestans, the potato and tomato late blight agent, relies on its ability to generate from mycelia large amounts of sporangia, which release zoospores that encyst and form infection structures. To better understand these critical stages, Affymetrix GeneChips based on 15,650 unigenes were designed and used to profile the life cycle, through an analysis of RNA from hyphae, sporangia, cleaving sporangia, motile zoospores, and germinated zoospore cysts.
Keywords: Developmental study

overall_design: Gene expression in non-sporulating hyphae, asexual sporangia, sporangia undergoing cleavage, swimming zoospores, and germinated cysts containing appressoria were characterized using isolate 88069. Several strategies controlled for experimental error. Firstly, two replicate hybridizations were used for each tissue type. Secondly, RNAs for the two replicates were prepared by research groups in North America and/or Europe. Thirdly, RNA for each hybridization was pooled from two or three independent cultures made by that group. Finally, before using an RNA sample in an experiment the expression of selected well-characterized genes was measured to ensure that the tissue was properly prepared.

first: Audrey

last: Ah Fong


title: Gene expression comparison of human ES cells grown in different media

status: Public on Mar 12 2008

city: Münster

first: Boris

last: Greber

address: Röntgenstraße 20

department: Cell and Developmental Biology

institute: Max Planck Institute for Molecular Biomedicine

summary:
Mouse embryonic fibroblasts can be used to condition basis human ES cell medium to allow feeder-free growth. To investigate the impact of factors released by the MEFs on gene expression in hES cells, global gene expression was analysed in cells grown in MEF-conditioned medium as compared to cells grown in a chemically defined one.
Keywords: Media comparison

overall_design: H1 hES cells were grown in parallel in two types of media, MEF-conditioned medium and N2B27-based defined medium. Both conditions are represented by two biological replicates.

first: Hans

last: Lehrach

first: James

last: Adjaye


title: wuHMM: a robust algorithm to detect DNA copy number variation using long oligonucleotide microarray data

city: Baltimore

first: Patrick

last: Cahan

address: 733 North Broadway, MRB 653

department: ICE and Biomedical Engineering

institute: Johns Hopkins University School of Medicine

type: Genome variation profiling by genome tiling array

summary:
Copy number variants (CNVs) are currently defined as genomic sequences that are polymorphic in copy number and range in length from 1,000 to several million base pairs.  Among current array-based CNV detection platforms, long-oligonucleotide arrays promise the highest resolution.  However, the performance of currently available analytical tools suffers when applied to these data because of the lower signal:noise ratio inherent in oligonucleotide-based hybridization assays.  We have developed wuHMM, an algorithm for mapping CNVs from array comparative genomic hybridization (aCGH) platforms comprised of 385,000 to more than 3 million probes.  wuHMM is unique in that it can utilize sequence divergence information to reduce the false positive rate (FPR).  We apply wuHMM to 385K-aCGH, 2.1M-aCGH, and 3.1M-aCGH experiments comparing the 129X1/SvJ and C57BL/6J inbred mouse genomes.  We assess wuHMM’s performance on the 385K platform by comparison to the higher resolution platforms and we independently validate 10 CNVs.  The method requires no training data and is robust with respect to changes in algorithm parameters.  At a FPR of less than 10%, the algorithm can detect CNVs with five probes on the 385K platform and three on the 2.1M and 3.1M platforms, resulting in effective resolutions of 24 kb, 2-5 kb, and 1 kb, respectively.
Keywords: CNV detection algorithm development and assessment

overall_design: All four samples in this series are hybridizations of genomic DNA from inbred mouse strains 129X1/SvJ versus C57BL6/J.  The experiments were performed at increasing resolutions (one 385K, two 2.1M, and one 3.1M).

first: Laura

last: Godfrey

first: Peggy

last: Eis

first: Todd

last: Richmond

first: Rebecca

last: Selzer

first: Michael

last: Brent

last: McLeod

first: Timothy

middle: J

last: Ley

last: Graubert


title: Activin A-driven differentiation timecourse with human embryonic stem cells

summary:
Activin A is known to induce endodermal differentiation in human embryonic stem cells under low-serum or certain defined media conditions. To gain insights into global gene expression changes during Activin A-induced differentiation, gene expression was monitored by means of microarrays in short intervals until 4 days of treatment.
Keywords: Differentiation timecourse experiment

overall_design: hES cells were adapted to N2B27-based chemically defined medium, replated and continuously treated with Activin A in the absense of FGF2. Cells from replicate cultures were harvested 0, 12, 24, 36, 48, 72, and 96 hours after the inititation of Activin A stimulation. Each timepoint was represented by two biological replicates.


title: Comparison of L. interrogans strains 56601 and IPAV at 37°C

city: shanghai

first: xiaokui

last: guo

state: shanghai

address: ChongQing nan Road 280

department: Department of Microbiology and Parasitology

country: China

institute: Shanghai Jiao Tong University

summary:
DNA microarray analysis was used to compare the differential gene-expression profiles between Leptospira interrogans serovar Lai type strain 56601 and its corresponding attenuated strain IPAV. A 22 kb genomic island covering a cluster of 34 genes (LA0186 to LA0219) was actively expressed in both strains but concomitantly up-expressed in strain 56601 in contrast to that of IPAV. RT-PCR assays proved that the gene cluster comprised five transcripts. Gene annotation of this cluster revealed characteristics of a putative prophage-like remnant with at least 8 out of 34 CDSs encoding prophage-like proteins, of which, LA0195 is probably a putative prophage CI-like regulator. The transcription initiation activities of putative promoter-regulatory sequences of transcripts I, II and III, all proximal to LA0195, were further analyzed in the Escherichia coli promoter probe vector pKK232-8 by assaying the reporter CAT enzyme activities. The strong promoter activities of both transcripts I and II indicated by the E. coli CAT assay were well correlated with the in vitro sequence-specific binding of the recombinant LA0195 protein to the corresponding promoter probes detected by the electrophoresis mobility shift assay. On the other hand, the promoter activity of transcript III was very low in E. coli and failed to show active binding to LA0195 in vitro. These results suggested that LA0195 is likely involved in the transcription of transcripts I and II. However, the identical complete DNA sequences of this prophage remnant from these two strains strongly suggests that possible regulatory factors or signal transduction systems resided outside of this region within the genome maybe responsible for the differential expression profiling in these two strains.
Keywords: Differential expression of  L.interrogans 56601 and IPAV in 37°C

sample_organism: Leptospira interrogans serovar Lai str. 56601

overall_design: The microarray chip was prepared as previously described (42). In brief, the 3528 protein coding sequences (CDSs) successfully amplified by PCR were printed in triplicate on the slides as probes. Three expression profiling experiments were performed via reverse transcription using Superscript (Invitrogen) and with one experiment having the cDNA of IPAV labeled by Cy3 and of 56601 labeled by Cy5, while the other two having the cDNA of 56601 labeled by Cy3 and of IPAV labeled by Cy5. The unincorporated dye was removed using a QIAquick Nucleotide Removal Kit (Qiagen) as specified by the manufacturer's protocol. Samples were hybridized at 42°C for 16h, and then washed as described in the manual. Hybridization experiments were performed in triplicate using cDNA derived from three different cultures of virulent strain 56601 and avirulent strain IPAV grown at 37°C. The hybridized slides were scanned and analyzed by Tiffsplit (Agilent) to calculate the signal intensities and to determine the presence or absence of each CDS. The data were then normalized, and their backgrounds were defined using GeneSpring 4.0 (Silicon Genetics). The GeneSpring software was used to further analyze the transcription patterns. To identify genes with significantly altered expression levels, cutoff values for expression level ratios 1.5 were used to filter genes with changes (n-fold) greater than ±1.5 in three independent biological samples (27, 48, 50). Student’s t test analysis of variance was used to compare the mean expression levels of the test and reference samples. Genes with significant differential expression levels (P < 0.05) were selected.

first: Jin-Hong

last: Qin

first: Yue-Ying

last: Sheng

first: Zhi-Ming

last: Zhang


title: Response of D. hansenii to osmotic stress

city: San Juan

first: Sandra

last: Pena de Ortiz

state: PR

address: Gandara Ave., JGD Bldg. Rooms 108, 204

department: Department of Biology

country: Puerto Rico

institute: University of Puerto Rico

summary:
The euryhaline marine yeast Debaromyces hansenii is a model system for the study of genes related to osmotic stress. To study the transcriptional response of this organism to osmotic stress we have used the two color microarray based gene expression analysis of 6211 genes which constitutes the whole genome. Analysis was done at three time points after induction with salt (0.5h, 3h and 6h.) The mRNA level of 64 genes significantly increased at least 3-fold after induction with 2M NaCl whereas that of  45 genes was 3-fold diminished. The induced as well as the repressed genes were grouped into functional categories to identify biochemical processes possibly affected by osmotic shock. 53% of the induced genes encode for ribosomal proteins, 12.5% for mitochondrial and redox, 6% for aminoacid, cell wall and carbohydrate, and 1.5%for heat shock, protein transport and glycerol proteins. The function of 31% of repressed genes is currently unknown. 15% of the repressed genes are involved in carbohydrate metabolism and protective function, 6% are associated with cell wall, redox and signal transduction, and 4% in vacuolar and  lipid functions. Surprisingly, the activity of NAD+ glycerol 3-phosphate dehydrogenase gene (GPD1) involved in the glycerol biosynthesis in response to osmotic stress did not show induction.
Keywords: time course, stress response, mRNA expression

overall_design: The experimental design consisted of six microarray experiments.   Cy-3 labeled cRNA control targets were combined with Cy-5 labeled cRNA experimental targets obtained from 0.5 hrs, 3 hrs and 6 hrs samples.  The combined Cy-3 and Cy-5 cRNAs targets were used for probe hybridization onto the first, third, and fifth microarray, respectively.  Dye swapping was done using different biological replicates for both Cy-5 labeled control targets and the Cy-3 experimental targets (as above) and used for hybridization on the second, fourth and the sixth microarray respectively, respectively.  Dye swaps using different biological replicates were done to correct for any dye-bias.

first: Nancy

last: Arroyo

first: Govind

last: Nadathur

last: Peña de Ortiz

first: Humberto

middle: G

last: Ortiz-Zuazaga

first: Adrinel

last: Vázquez


title: Short-term Activin A and BMP4 stimulation experiments with human ES cells

first: Boris

last: Greber

summary:
This experiment was carried out to identify the short-term effects of Activin A and BMP4 stimulation on gene expression in human embryonic stem cells grown in a chemically defined medium.
Keywords: Growth factor stimulation experiment

overall_design: hES cells were adapted to chemically defined medium, replated, and, three days after passaging, stimulated with either recombinant Activin A or BMP4. Data are from independent experiments (distinct non-stimulated controls for Activin A and BMP4 experiments - two biological replicates each).

first: Hans

last: Lehrach

first: James

last: Adjaye


title: MEF-CM and UM_GTA re-stimulation experiment with human ES cells

summary:
Human embryonic stem cells can be maintained in a basic Serum Replacement (Invitrogen) based medium that has been conditioned on mouse embryonic fibroblasts (MEFs), yielding MEF-CM. Ligands secreted into the medium by the MEFs include Activin A, TGFß1, and Gremlin. This experiment served the purpose of identifying the short-term effects of MEF-CM and its substitute UM_GTA (unconditioned medium plus Activin A, TGFb1, and Gremlin) on gene expression in human embryonic stem cells.
Keywords: Media / growth factor stimulation experiment

overall_design: hES cells were grown in MEF-CM, then in unconditioned medium for 1 day. Cells were then re-stimulated with MEF-CM or UM_GTA for 4h to be compared to non-stimulated samples. The experiment was carried out in the presence of  5+5 (MEF-CM) or 10 ng/ml (UM, UM_GTA) of FGF2. Activin A was used at 20 ng/ml, TGFß1 at 2 ng/ml, and Gremlin at 200 ng/ml. Two biological replicates were hybridised per sample type.