title: FJ001:  Retinoic acid-dependent and -independent gene-regulatory pathways of Pitx3 in meso-diencephalic dopaminergic neurons

city: Utrecht

first: Marian

last: Groot Koerkamp

state: Utrecht

address: Heidelberglaan 25

institute: Princess Maxima Center for Pediatric Oncology

summary: Development of meso-diencephalic dopamine (mdDA) neurons requires the combined actions of the orphan nuclear receptor Nurr1 and the paired-like homeobox transcription factor Pitx3. Whereas all mdDA neurons require Nurr1 for expression of Th and survival, dependence on Pitx3 is only displayed by the mdDA subpopulation that will form the substantia nigra (SNc). Previously, we demonstrated that Pitx3-/- embryos lack the expression of the retinoic acid (RA)-generating enzyme Ahd2, which is normally selectively expressed in the Pitx3-dependent DA neurons of the SNc. Restoring RA-signaling in Pitx3-/- embryos revealed a selective dependence of SNc neurons on the presence of RA for differentiation into Th-positive neurons and maintenance throughout embryonic development. Whereas these data are suggestive of an important developmental role for RA in neurons of the SNc, it remained unclear whether other Nurr1 and Pitx3 target genes depend on RA signaling in a manner similar to Th. In search for genes that were affected in Pitx3-deficient mdDA neurons and restored upon embryonic RA treatment, we provide evidence that Delta-like 1, D2R (Drd2) and TH are regulated by Pitx3 and RA signaling, influencing the mdDA terminal differentiated phenotype. Furthermore, we show that regulation of Ahd2-mediated RA-signaling represents only one aspect of the Pitx3 downstream cascade, since Vmat2, Dat,  Ahd2 (Aldh1a1), En1, En2 and Cck were unaffected by RA treatment and are (subset) specifically modulated by Pitx3. In conclusion, our data reveal several RA-dependent and -independent aspects of the Pitx3-regulated gene cascade suggesting that Pitx3 acts on multiple levels in the molecular subset-specification of mdDA neurons.

overall_design: RNA was isolated from dissected ventral midbrains of E14.5 Pitx3-/- and Pitx3+/+  mouse embryos. 3 Experimental samples each consisting of 3 Pitx3-/- ventral midbrains were hybridized to reference RNA derived from 10 Pitx3+/+ ventral midbrains

first: Frank

last: Jacobs

first: Jesse

middle: V

last: Veenvliet

first: Wadia

last: Almirza

first: Elisa

last: Hoekstra

first: Lars

last: von Oerthel

first: Annemarie

last: van der Linden

first: Roel

last: Neijts

last: Koerkamp

first: Dik

last: van Leenen

last: Holstege

first: J

last: Burbach

first: Marten

last: Smidt


title: Isolation and in vitro expansion of human colonic stem cells [CGH]

status: Public on Nov 22 2011

city: Barcelona

first: Eduard

last: Batlle

state: Barcelona

address: Baldiri i Reixac 10

department: Oncology

country: Spain

institute: Institute for Research in Biomedicine Barcelona

type: Genome variation profiling by genome tiling array

summary:
Tissue for normal colonic stem cell isolation was obtained via colectomy from a colorectal cancer patient. It was shown that the resected tissue and the in vitro-cultured organoids grown from tissue-derived adult stem cells do not harbor chromosomal alterations.
For that purpose, genomic DNA from resected mucosa or genomic DNA from adult stem cell-derived organoids grown in a 3-dimensional culture system was compared to white blood cell genomic DNA obtained from the same individual. Furthermore, in vitro organoids which were cultured and serially passaged for several weeks did not acquire chromosomal alterations.

overall_design: Analysis of chromosomal stability of colonic tissue or in vitro-cultured organoids via comparison with blood cells derived from the same individual.

first: Peter

last: Jung


title: Development and Preclinical Efficacy of Novel Transforming Growth Factor-β1 (TGFβ1) siRNAs for Pulmonary Fibrosis

city: Kamakura

first: Satoshi

last: Kondo

state: Kanagawa

address: Tebiro 6-10-1

department: New Projects Development Division

country: Japan

institute: Toray Industries,Inc.

summary: Idiopathic pulmonary fibrosis is a chronic devastating disease of unknown etiology. No therapy is currently available. A growing body of evidence supports the role of TGFβ1 as the major player in the pathogenesis of the disease. This study designed novel human- and mouse-specific siRNAs and siRNA/DNA chimeras targeting both human and mouse common sequences and evaluated their inhibitory activity in pulmonary fibrosis induced by bleomycin and lung-specific transgenic expression of human TGFβ1. Selective novel sequences of siRNA and siRNA/DNA chimeras efficiently inhibited pulmonary fibrosis, indicating their applicability as tools for treating fibrotic disease in humans.

overall_design: Total RNA was extracted from lung tissue from mice with bleomycin (BLM)-induced lung fibrosis treated with mouse TGFβ1 siRNAs or vehicle on different days after BLM infusion.

first: Corina

middle: N

last: D’Alessandro-Gabazza

first: Tetsu

last: Kobayashi

first: Daniel

last: Boveda-Ruiz

first: Takehiro

last: Takagi

first: Masaaki

last: Toda

first: Paloma

last: Gil-Bernabe

first: Yasushi

last: Miyake

first: Atsushi

last: Yasukawa

first: Yoshikazu

last: Matsuda

first: Noboru

last: Suzuki

first: Hiromitsu

last: Saito

first: Yutaka

last: Yano

first: Ayako

last: Fukuda

first: Tetsuya

last: Hasegawa

first: Hidekazu

last: Toyobuku

first: Stephen

middle: I

last: Rennard

middle: D

last: Wagner

first: John

last: Morser

first: Yoshiyuki

last: Takei

first: Osamu

last: Taguchi

first: Esteban

middle: C

last: Gabazza


title: Progressive genomic instability in the FVB/KrasLA2 mouse model of lung cancer

city: San Francisco

first: David

last: Quigley

state: CA

address: 1450 Third St. Room 207

department: Helen Diller Comprehensive Cancer Center

country: USA

institute: UCSF

summary: Alterations in DNA copy number contribute to the development and progression of cancers and are common in epithelial tumors.  We have used array Comparative Genomic Hybridization (aCGH) to visualize DNA copy number alterations across the genomes of lung tumors in the KrasLA2 model of lung cancer.  Copy number gain involving the Kras locus, as focal amplification or whole chromosome gain, is the most common alteration in these tumors, and with a prevalence that increased significantly with increasing tumor size.  Furthermore, Kras amplification was the only major genomic event among the smallest lung tumors, suggesting that this alteration occurs early during the development of mutant Kras driven lung cancers.  Recurring gains and deletions of other chromosomes occur progressively more frequently among larger tumors.  These results are in contrast to a previous aCGH analysis of lung tumors from KrasLA2 mice on a mixed genetic background, in which relatively few DNA copy alterations were observed regardless of tumor size.  Our model features the KrasLA2 allele on the inbred FVB/N mouse strain, and in this genetic background there is a highly statistically significant increase in level of genomic instability with increasing tumor size.  These data suggest that recurring DNA copy alterations are important for tumor progression in the KrasLA2 model of lung cancer, and that the requirement for these alterations may be dependent on the genetic background of the mouse strain.

overall_design: The KrasLA2 allele, originally on a C57BL6/129svJae mixed background, was backcrossed into the FVB/N background for more than 10 generations in order to minimize the effect of genetic heterogeneity on lung tumor development. Mice were sacrificed at 6 months of age.

first: Minh

last: To

middle: A

first: Jian-Hua

last: Mao

first: Reyno

last: Del Rosario

first: Jeff

last: Hsu

first: Graeme

last: Hodgson

first: Tyler

last: Jacks

first: Allan

last: Balmain


title: Unveiling the transcriptional features associated with coccolithovirus infection of natural Emiliania huxleyi blooms.

city: Bergen

first: António

last: Pagarete

address: Thormøhlensgate 53 A & B

department: Department of Biology

country: Norway

institute: University of Bergen

summary: Lytic viruses have been implicated in the massive cellular lysis observed during algal blooms, through which they assume a prominent role in oceanic carbon and nutrient flows. Despite their impact on biogeochemical cycling, the transcriptional dynamics of these important oceanic events is still poorly understood. Here, we employ an oligonucleotide microarray to monitor host (Emiliania huxleyi) and virus (coccolithovirus) transcriptomic features during the course of E. huxleyi blooms induced in seawater-based mesocosm enclosures. Host bloom development and subsequent coccolithovirus infection was associated with a major shift in transcriptional profile. In addition to the expected metabolic requirements typically associated with viral infection (amino acid and nucleotide metabolism, as well as transcription- and replication-associated functions), the results strongly suggest that the manipulation of lipid metabolism plays a fundamental role during host-virus interaction. The results herein reveal the scale, so far massively underestimated, of the transcriptional domination that occurs during coccolithovirus infection in the natural environment.

platform_organism:
Emiliania huxleyi
Emiliania huxleyi virus 86
Emiliania huxleyi virus 163

overall_design: Six mesocosm enclosures were placed in the Raunefjorden (Western Norway coast) and filled with natural community water (in June 2008). Nutrient enrichment was applied in order to trigger the development of E. huxleyi blooms. The major transcriptomic features of those blooms and consequent viral infections were monitered through the use of an oligo microarray containing a total of 3571 gene probes; 2271 (63.6%) matching E. huxleyi ESTs, and 1300 (36.4%) matching EhV-86 and EhV-163 genomic sequences. Each microarray contains 5 technical replicates. Sampling of total RNA present  in 2L of water (from each enclosure) was performed once a day from day 8 to day 16. For enclosures 2 and 3 other sampling points were taken, covering the complete dial-cycle (6h,12h,18h, and 24h).

first: Antonio

first: Michael

middle: J

last: Allen


title: Genotype-dependent gene expression in barley

city: Ames

first: PLEXdb Curator

middle: Sudhansu

state: IA

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

department: Virtual Reality Application Center

institute: Iowa State University

summary: Comparison of mRNA accumulation in the seedling leaves of 8 unstressed barley cultivars   ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor,  .  The equivalent experiment is BB20 at PLEXdb.]

overall_design: genotype: Morex(3-replications);  genotype: Steptoe(3-replications);  genotype: OWB REC(3-replications);  genotype: OWB DOM(3-replications);  genotype: Barke(3-replications);  genotype: Haruna Nijo(3-replications);  genotype: Golden Promise(3-replications);  genotype: Optic(3-replications)


title: Estrogen-dependent gene expression in human breast cancer cells relies upon proteosome-dependent monoubiquitination of histone H2B

city: Goettingen

first: Frank

last: Kramer

address: Humboldtallee 32

department: Department of Medical Statistics

country: Germany

institute: University Medical Center Göttingen

summary: The estrogen receptor-alpha (ERα) determines breast cancer cell phenotype and is a prognostic indicator. A better understanding of the mechanisms controlling ERα function may uncover improved strategies for the treatment of breast cancer. Proteasome inhibition was previously reported to regulate estrogen-induced transcription but the mechanisms by which it influences ERα function remain controversial. In this study we investigated the transcriptome-wide effects of the proteasome inhibitor Velcade on estrogen-regulated transcription in MCF7 human breast cancer cells and demonstrate a specific global decrease in estrogen-induced transcription.

overall_design: This set contains 21 microarray samples. 3 controls, 3 estrogen stimulated, 3 Bortezomib + estrogen stimulated, 2* 3 siRNA controls, 3 siRNA PSMB3 knockdowns, 3 siRNA PSMB5 knockdowns

first: Tanja

last: Prenzel

first: Yvonne

last: Begus-Nahrmann

first: Magali

last: Hennion

first: Chieh

first: Theresa

last: Gorsler

first: Corinna

last: Hintermayr

first: Dirk

last: Eick

first: Elisabeth

last: Kremmer

first: Mikael

last: Simons

first: Tim

last: Beissbarth

first: Steven

last: Johnsen


title: Cross-species Detection in Barley1 GeneChip Array

summary: This study was conducted to evaluate the efficiency of cross-species detection in Barley1 GeneChip array.  We hybridized cRNA derived from first leaves of barley green seedlings (as a control), as well as the same stage of seedling leaf from representative genotypes of wheat, oat, rice, maize, and sorghum.  Ten to twenty seedlings for each species were harvested and pooled for RNA preparation, labeling, and hybridization.   ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor,  Rico Caldo.  The equivalent experiment is BB1 at PLEXdb.]

sample_organism:
Avena sativa
Hordeum vulgare
Oryza sativa
Sorghum bicolor
Triticum aestivum
Zea mays

overall_design: species: Barley(2-replications);  species: Wheat(2-replications);  species: Oat(2-replications);  species: Sorghum(2-replications);  species: Corn(2-replications);  species: Rice(2-replications)

first: Rico

last: Caldo

first: Roger

middle: P

last: Wise


title: Mis-expression of Tramtrack in Drosophila embryos

status: Public on Nov 21 2011

city: Cambridge

first: Boris

last: Adryan

address: Tennis Court Road

department: Cambridge Systems Biology Centre & Department of Genetics

country: United Kingdom

institute: University of Cambridge

summary: We profiled the transcriptome of Drosophila melanogaster embryos in ttk2D50 embryos or after over-expression using btl-GAL4; UAS-ttk, respectively. We further isolated cells that express btl-enh-RFPmoe (Cabernard and Affolter 2005) and FACS sorting, and profiled their transcriptomes in the same genetic backgrounds.

overall_design:
A total of 19 samples were analysed with 2-3 replicates each.
For each embryos and sorted cells, the genotypes were:
ttk2D50 btl-enh-RFPmoe (mutant; embryos: 2, cells: 2),
btl-enh-RFPmoe (mutant control; embryos: 2, cells: 3),
btl-GAL4; UAS-ttk, btl-enh-RFPmoe (over-expression; embryos: 3, cells: 2),
btl-GAL4; btl-enh-RFPmoe (over-expression control; embryos: 3, cells: 2).

first: Barbara

last: Rotstein

first: Dávid

last: Molnár

first: Marta

last: Llimargas


title: A high resolution analyis of c-Myc-regulated gene expression dynamics.

city: Singapore

first: Chui Sun

last: Yap

address: 8, College Road

country: Singapore

institute: Duke-NUS

summary:
We profiled the transcriptome of HOMycER12 rat fibroblasts whose c-Myc activity was induced to supra-physiological levels with 4-hydroxytamoxifen (OHT).
In this dataset, we included the expression data hourly for the first 6 h, then every 2 or 4 hours over a total of 24 h for 4-hydoxytamoxifen activated cells, and expression data from control samples (no 4-hydroxytamoxifen activation) at 8, 16 and 24 h.

overall_design: 47 Total samples were analyzed. The Probe Logarithmic Intensity Error (PLIER) algorithm, from the Affymetrix Power Tools (APT) software package, was used to generate gene and exon expression scores. To detect differentially expressed genes a 3-way analysis of variance (ANOVA) was applied with the following factors: time, treatment, and experiment (each biological replicate with controls was considered an independent experiment). For each gene, the time factor p-value was calculated based on differential expression between any time point (t ≥ 1 hr) and the t = 0 time point; the treatment factor p-value was calculated based on differential expression between treated and control samples at time points when both treated and control samples were collected (i.e. 8, 16 and 24 h). The thresholds for these p-values were set to a false discovery rate (FDR) of less than 1%.

first: Nicola

last: Neretti

first: John

middle: M

last: Sedivy


title: Tissue specific differential expression in response to a thermal stress in the snakelocks sea anemone.

status: Public on Nov 17 2011

city: NICE

first: Cecile

last: Sabourault

address: Faculte des Sciences, Parc Valrose, BP 71

department: Biology

country: France

institute: University of Nice

summary: Differential expression between endodermal (zooxanthellate) and ectodermal tissue layers in the endosymbiotic sea anemone Anemonia viridis has been analyzed for 3 specimens subjected to a thermal stress (+10°C) for a 2 days period. A symbiosis-dedicated oligonucleotide microarray (2000 selected features) was generated, representing to date the only available oligonucleotide array used for symbiotic cnidarians (GPL10546). We are describing here the preferential expression in ectoderm vs endoderm (also called epidermis and gastroderm, respectively) during the time course of this thermal stress.

overall_design: RNA was extracted from ectodermal or endodermal dissected tissues. 3 different symbiotic anemones were subjected to a thermal and UV stress (+10°C), sampled at T0, T12h and T36h. Tissue dissections were carried out from tentacles immediately after sampling. For each sample, dye-swap hybridizations compared T0 and later time points. For each time point, statistical analysis combines the 3 biological replicates.

first: Philippe

last: Ganot

first: Aurelie

last: Moya


title: Xenopus M-phase extract treated induced pluripotent stem cells

city: MARSEILLE CEDEX 09

first: Christelle

last: Cayrou

address: 27 Boulevard Lei Roure

institute: CRCM

summary: Transfer of somatic cell nuclei to enucleated eggs or ectopic expression of specific transcription factors are two different reprogramming strategies used to generate pluripotent cells from differentiated cells. However, they are poorly efficient and other unknown factors might be required to increase their success rate. Here, we show that Xenopus egg extracts at the metaphase stage (M phase) have a strong reprogramming activity on mouse embryonic fibroblasts (MEFs). First, they reset replication properties of MEF nuclei toward a replication profile characteristic of early development and they erase several epigenetic marks, such as trimethylation of H3K9, H3K4 and H4K20. Second, when MEFs are reversibly permeabilized in the presence of M phase Xenopus egg extracts, they show a transient increase in cell proliferation, form colonies and start to express specific pluripotency markers. Finally, transient exposure of MEF nuclei to M phase Xenopus egg extracts increases the success of nuclear transfer to enucleated mouse oocytes and strongly synergize with the production of pluripotent stem cells by ectopic expression of transcription factors. The mitotic stage of the egg extract is crucial as neither of these effects is detected when using interphasic Xenopus egg extracts.  Our data demonstrate that mitosis is essential to make mammalian somatic nuclei prone to reprogramming and that, surprisingly, the heterologous Xenopus system has features that are conserved enough to remodel mammalian nuclei.

overall_design: MEF cells were infected by retroviruses encoding for Oct4, Sox2, Klf4 and c-Myc and, then reversibly permeabilized and treated with Xenopus M-phase extract. All samples of each cell type (e.g. untreated MEF, ES cells and M-iPS cells) were made in triplicates. Total RNAs were extracted with RNeasy kit and hybridized on Nimblegen MM9 microarrays.

first: Olivier

last: Ganier

first: Aurore

last: Puy

first: Isabelle

last: Peiffer

first: Marcel

last: Mechali


title: Genes modulated by rhBMP2 and rhBMP7 during osteoblastic differentiation of C2C12 cells

city: Campinas

first: Maria Angélica

middle: Lopes de

last: Souza

state: São Paulo

address: Franscisco Otaviano nº 60 sl 52

country: Brazil

institute: Scylla Bioinformática

summary: This study analyses the gene expression profile of pre-osteoblastic C2C12 mice cells during osteodifferentiation in control and induced by rhBMP2 and rhBMP7 using DNA microarrays. Genes which were already known to be activated by BMP2, to be also activated by rhBMP7, indicating that BMP7 induces these genes in a similar fashion as BMP2. Moreover, we identified some new genes, such as Hoxc8, Glis1, Glis3, Ecsit, LrP6, Dvl1, Grn, BMP1, Ltbp2, Plod1, Htra1 and IGFBP-rP10, to be upregulated by both rhBMPs.

overall_design: Total RNA from mouse pre-myoblastic and pre-osteoblastic C2C12 cell line, after 12h of treatment with rhBMP2 and rhBMP7, was hybridized to a microarray and analyzed.

first: Juan

last: Bustos-Valenzuela

first: Andre

last: Fujita

first: Jose

last: Granjeiro

first: Mari

last: Sogayar


title: Developing seeds of M955 low phytic acid barley 7 days after anthesis

first: PLEXdb Curator

summary: Effect of the low phytic acid mutation on gene expression of developing seeds of M955, a low phytic acid barley genotype with >90% reduction in phytic acid in mature seeds. Expression analysis using the Barley1 GeneChip was performed on total RNA from developing seeds (7DAA) of M955 lpa and wt sib-selections.  Sib-selections were from BC2 populations of M955 with Harrington as the recurrent parent. Material was grown in field trials in two years 2003 and 2004. In 2003 expression analysis was done from 2 lpa and 3 wt sib-selections, and in 2004 analysis was done from 2 lpa and 2 wt sib-selections.   ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor,  David Bowen.  The equivalent experiment is BB22 at PLEXdb.]

overall_design: genotype: M955 LPA sib-selection - year: 2003(2-replications);  genotype: M955 LPA sib-selection - year: 2004(2-replications);  genotype: M955 WT sib-selection - year: 2003(3-replications);  genotype: M955 WT sib-selection - year: 2004(2-replications)

first: David

last: Bowen

first: Edward


title: Transcriptional and functional analysis of trifluoromethionine resistance in Entamoeba histolytica

status: Public on Nov 16 2011

city: Shinjuku-ku

first: Gil

middle: Mallari

last: Penuliar

state: Tokyo

address: Toyama 1-23-1

department: Parasitology

institute: National Institute of Infectious Diseases

summary:
Previously, we have shown that in Entamoeba histolytica, methionine γ-lyase (EhMGL) downregulation results in trifluoromethionine resistance. The transcriptional response, however, of this parasite to the drug is not known.
In this study, we used microarray analysis to determine if additional genes are involved.

overall_design: The expression profile of 9,230 genes in wild type and trifluoromethionine resistant strains was compared. Episomal overexpression of EhBspA1 was performed to verify its role in trifluoromethionine resistance. The transcriptomes of trifluoromethionine resistant strain cultured with or without trifluoromethionine, EhMGL-gene silenced strain, metronidazole resistant strain, and wild type strain under cysteine-deprived conditions were compared to determine the specificity of the changes observed in trifluoromethionine resistant strain.

first: Atsushi

last: Furukawa

first: Kumiko

last: Nakada-Tsukui

first: Afzal

last: Husain

first: Dan

last: Sato

first: Tomoyoshi

last: Nozaki


title: Redefining the Relevance of Established Cancer Cell Lines to the Study of Clinical Anti-Cancer Drug Resistance

city: Namur

first: jean-pierre

last: gillet

address: 61 Rue de Bruxelles

department: URPhyM

country: Belgium

institute: University of Namur

summary: Although in vitro models have been a cornerstone of anti-cancer drug development, their direct applicability to clinical cancer research has actually been uncertain. Using a state-of-the-art Taqman-based qRT-PCR assay, we investigated the multidrug resistance (MDR) transcriptome of six cancer types, in both in vitro and in vivo samples.

overall_design: We studied 380 MDR genes, selected a priori based on an extensive curation of the literature published during the last three decades, in established cancer cell lines and clinical samples, either containing greater than 75% tumor cells or microdissected.

first: Jean-Pierre

last: Gillet

first: Anna

last: Calcagno

first: Sudhir

last: Varma

first: Miguel

last: Marino

first: Lisa

last: Green

first: Meena

last: Vora

first: Chirayu

last: Patel

first: Josiah

last: Orina

first: Tatiana

last: Eliseeva

first: Vineet

last: Singal

first: Raji

last: Padmanabhan

first: Ben

last: Davidson

first: Ram

last: Ganapathi

first: Anil

last: Sood

first: Bo

last: Rueda

first: Suresh

last: Ambudkar

first: Michael

last: Gottesman


title: Transcriptional glucocorticoid-response at the exon level and NR3C1 DNA-binding in childhood leukemia models; NR3C1-DNA binding in NALM6 precursor B-ALL cells.

status: Public on Nov 15 2011

city: Bolzano

first: Johannes

last: Rainer

address: Via A. Volta 21

department: Institute for Biomedicine

country: Italy

institute: Eurac Researc

type: Genome binding/occupancy profiling by genome tiling array

summary: Glucorticoids (GCs) are steroid hormones with a wide range of physiological actions in different cell types and tissues. Their ability to induce apoptosis in malignant cells of the lymphoid lineage is exploited since decades in the treatment of childhood acute lymphoblastic leukemia (ALL), the molecular mechanism of this cell death induction being however still unknown.  Using an integrative approach we delineated the transcriptional response of a preB- and a T-ALL model system employing Exon microarrays and identified in vivo interactions of the transcription factor GC receptor (GR) with genes' promoters in the same samples using the ChIP-on-chip technology. We did find differences in GR-DNA interaction in the genes' promoters between the two cell systems and also in extent and kinetics of GC mediated gene regulation. The overall response was stronger in CEM-C7H2 T-ALL cells where it was also accompanied by a stronger autoup-regulation of the GR gene. Considering the differing kinetics we furthermore defined a set of  common response genes. These response genes were enriched in apoptosis related gene ontology processes, and about half of them yielded also GR enrichment in the respective promoter regions.  Globally, the presence of GR at gene's promoters was higher for GC induced compared to repressed genes, indicating that GR mediates gene repression by interaction with distant enhancer regions or by cross-talk with other transcription factors.  In order to identify the genes' GC regulated transcript variants we analyzed the data set also at the level of individual exons and predicted first exons of GC induced isoforms. We could thus define GC regulated variants of response genes, did however also identify first exons not corresponding to any known transcript.  We reconfirmed these results for the genes ATP4B, GPR98, TBCD and ZBTB16 and identified for the latter also a novel exon employing 5' nested amplification of cDNA ends. Expression and regulation of this and all downstream exons of ZBTB16 was further verified by real time RT-PCR in 4 different preB-ALL cell lines. Thus, besides potential other novel gene isoforms, GCs induce a novel short transcript variant of ZBTB16 in preB-ALL cells.  Finally, the implemented bioinformatic methods may be useful also for other high density microarray data sets to identify alternative splice events and transcript variant expression.

overall_design:
This data set was used for the identification of GC receptor (GR/NR3C1) DNA interactions in NALM6 precursor B-ALL cells.
For each probe t-statistics were calculated between signals in test against control samples using the moderated t-test. These t-scores were then smoothed using a moving window approach averaging values of neighboring probes along the chromosome (window half site of 300nt). Windows with less than 4 probes were discarded. To estimate the p-value for enrichment in the test versus the control sample an aproach similar to MAT (Johnson et al. 2006) was used. Resulting p-values were subsequently adjusted for multiple hypothesis testing using the q-value method (Storey and Tibshirani, 2003). Windows with a q-value smaller 0.05 were considered to be significantly enriched, overlapping significant windows were merged.

first: Julien

last: Lelong

first: Daniel

last: Bindreither

first: Christine

last: Mantinger

first: Christian

last: Ploner

first: Stephan

last: Geley

first: Reinhard

last: Kofler


title: Transcriptional glucocorticoid-response at the exon level and NR3C1 DNA-binding in childhood leukemia models; NR3C1-DNA binding in CCRF-CEM-C7H2 T-ALL cells.

overall_design:
This data set was used for the identification of GC receptor (GR/NR3C1) DNA interactions in CCRF-CEM-C7H2 T-ALL cells.
For each probe t-statistics were calculated between signals in test against control samples using the moderated t-test. These t-scores were then smoothed using a moving window approach averaging values of neighboring probes along the chromosome (window half site of 300nt). Windows with less than 4 probes were discarded. To estimate the p-value for enrichment in the test versus the control sample an aproach similar to MAT (Johnson et al. 2006) was used. Resulting p-values were subsequently adjusted for multiple hypothesis testing using the q-value method (Storey and Tibshirani, 2003). Windows with a q-value smaller 0.05 were considered to be significantly enriched, overlapping significant windows were merged.


title: HIF-1 and SKN-1 Coordinate the Transcriptional Response to Hydrogen Sulfide in C. elegans

city: Seattle

first: Dana

middle: L

last: Miller

state: WA

department: Biochemistry

institute: University of Washington

summary: Hydrogen sulfide (H2S) has dramatic physiological effects on animals that are associated with improved survival in changing conditions. C. elegans grown in H2S are long-lived and thermotolerant (1). To begin to identify mechanisms by which adaptation to H2S effects fundamental physiological functions, we have defined transcriptional changes associated with the response to H2S. Using microarray analysis we observe rapid changes in the abundance of specific mRNAs. The number and magnitude of transcriptional changes increased with the duration of H2S exposure. Functional annotation suggests that genes associated with protein homeostasis are upregulated upon prolonged exposure to H2S. Previous work has shown that the hypoxia-inducible transcription factor, HIF-1, is required for survival in H2S (2). We show that hif-1 is required for all early transcriptional changes in H2S. However, our data suggest that other factors may also regulate transcriptional effects of H2S exposure, and we demonstrate that SKN-1, the C. elegans homologue of NRF2, is also required for some H2S-dependent changes in transcription. Moreover, we have found that, like hif-1, skn-1 is essential to survive exposure to H2S. Our results support a model in which HIF-1 and SKN-1 coordinate a broad transcriptional response to H2S, with the end result to affect a global reorganization of protein homeostasis networks. We propose that these transcriptional responses contribute to the effects of adaptation to H2S on lifespan and thermotolerance.

overall_design: A 14 chip study using total RNA isolated independently for each chip. 5 replicates of control untreated samples and 3 biological replicates for each treatement regimen were included

first: Mark

last: Budde

last: Roth


title: Gene Expression Profiling of the Retina after Transcorneal Electrical Stimulation in Wildtype Brown Norway Rats

city: Tübingen

first: Gabriel

last: Willmann

address: Schleichstrasse 12-16

institute: University Eye Hospital and Centre for Ophthalmology Tübingen

summary:
Purpose: To investigate the effect of transcorneal electrical stimulation (TES) on the retina of wildtype Brown Norway (BN) rats by gene expression profiling.
Methods: TES was applied to BN adult wildtype rat retina in vivo for 1h (1ms biphasic pulses at 20Hz; current: 200 µA). RNA was isolated, processed and used for microarray-based expression profiling 4hrs after initial TES. An expression profile was generated for genes differentially expressed at 4hrs after TES vs. sham stimulated animals using a fold change cutoff of 1.2. We validated the profile by real-time quantitative reverse transcription-polymerase chain reaction (qPCR). In addition, the application of TES was verified at the structural and functional level.
Results: Transcriptome changes associated with TES vs. sham stimulated BN wildtype retina were identified. 490 genes were differentially expressed in TES and included well-known genes as well as a large number of novel genes. Electrophysiological recordings showed physiological retinal function after TES and structural in vivo and ex vivo studies revealed intact retinal layers.
Conclusion: Our results demonstrate that TES applied to the retina of wildtype BN rats induce a variety of transcriptome level changes and may help to understand the mechanisms underlying TES. In addition TES has no negative effect on structure and function of wildtype BN retina 24hrs after application.

overall_design:
RNA isolation and Microarray studies
4 animals were euthanized 4h after TES or respective sham treatment. Extraction of total RNA was performed using the RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. QIAshredder mini-spin columns (Qiagen) as well as needle and syringe homogenization were applied for tissue homogenization. To ensure proper integrity all RNA used in our experiments showed single peaks for the 18S and 28S bands as determined by the Agilent 2100 Bioanalyzer using the RNA 6000 Nano LabChip Kit (both Agilent Technologies Inc., CA, USA) following the manufacturer's instructions.
The purity and concentration of total RNA was determined by absorbance measurements at 260 and 280nm using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). All RNA that did not have a 260/280 ratio between 1.9 and 2.1 was discarded to ensure high quality of samples. Respective RNAs from stimulated and sham samples were used to generate double-stranded cDNA with the T7-oligo (dT) primer according to GeneChip® (Affymetrix Inc., Santa Clara, CA, USA) two cycle target labeling protocol as described by the manufacturer. The processed cDNA was hybridized to Affymetrix GeneChip® Rat Gene 1.0 ST arrays for 18–24h (Affymetrix Inc.). Four TES and four sham replicates were used for hybridization to Microarray GeneChips®. Each microarray was washed and stained with streptavidin–phycoerythrin and scanned at a 6μm resolution with Agilent Technologies model G2500A GeneArray scanner. A visual quality control measurement was performed to ensure proper hybridization after each chip was scanned. Additional quality control parameters such as scaling factors used to normalize the chips, average background, and noise were also evaluated.

Microarray analysis
For statistical analysis of microarray data all cell files were imported to GeneSpring GX software (Agilent Technologies Inc.) where GC-RMA algorithm was applied to yield log signal values on each probe set. All transcripts with a minimum change of 1.2 fold together with a p value less than 0.05 were selected using Significance Analysis of Microarray (SAM) (Tusher VG, 2001).
The visualization of the relationships between the samples by principal component analysis (PCA) based on the GC-RMA signal and hierarchical clustering using the Pearson correlation was obtained using PARTEK Genomics Suite version 6.4.
All differentially regulated genes were imported into Ingenuity Pathway Analysis (Ingenuity Systems, CA, USA) for generation of networks and canonical pathway analysis. The data set containing all significant gene identifiers along with corresponding expression and significance values was uploaded into the application. Each identifier was mapped to its corresponding object in Ingenuity's Knowledge Base. The set fold change cutoff identified all molecules whose expression was significantly differentially regulated. These network eligible molecules were overlaid onto a global molecular network developed from information contained in Ingenuity’s Knowledge Base. Networks were then algorithmically generated based on their connectivity. The functional analysis of a network identified the biological functions using a right-tailed Fisher’s exact test that were most significant to the molecules in the network.
Canonical pathways analysis identified the pathways from the Ingenuity Pathways Analysis library of canonical pathways that were most significant to the data set. The significance of the association between the data set and the canonical pathway was measured in 2 ways: 1) A ratio of the number of molecules from the data set that map to the pathway divided by the total number of molecules that map to the canonical pathway is displayed. 2) Fisher’s exact test was used to calculate a p-value determining the probability that the association between the genes in the dataset and the canonical pathway is explained by chance alone.

first: Karin

last: Schäferhoff

first: Andreas

last: Schatz

first: Dominik

last: Fischer

first: Blanca

last: Arango-Gonzales

first: Tobias

last: Röck

first: Lubka

last: Naycheva

last: Bonin

first: Karl-Ulrich

last: Bartz-Schmidt

first: Eberhart

last: Zrenner

first: Florian

last: Gekeler


title: Transcriptional glucocorticoid-response at the exon level and NR3C1 DNA-binding in childhood leukemia models; transcriptional response in CCRF-CEM-C7H2 T-ALL cells.

first: Johannes

last: Rainer

summary:
Glucorticoids (GCs) are steroid hormones with a wide range of physiological actions in different cell types and tissues. Their ability to induce apoptosis in malignant cells of the lymphoid lineage is exploited since decades in the treatment of childhood acute lymphoblastic leukemia (ALL), the molecular mechanism of this cell death induction being however still unknown.
Using an integrative approach we delineated the transcriptional response of a preB- and a T-ALL model system employing Exon microarrays and identified in vivo interactions of the transcription factor GC receptor (GR) with genes' promoters in the same samples using the ChIP-on-chip technology. We did find differences in GR-DNA interaction in the genes' promoters between the two cell systems and also in extent and kinetics of GC mediated gene regulation. The overall response was stronger in CEM-C7H2 T-ALL cells where it was also accompanied by a stronger autoup-regulation of the GR gene. Considering the differing kinetics we furthermore defined a set of  common response genes. These response genes were enriched in apoptosis related gene ontology processes, and about half of them yielded also GR enrichment in the respective promoter regions.
Globally, the presence of GR at gene's promoters was higher for GC induced compared to repressed genes, indicating that GR mediates gene repression by interaction with distant enhancer regions or by cross-talk with other transcription factors.
In order to identify the genes' GC regulated transcript variants we analyzed the data set also at the level of individual exons and predicted first exons of GC induced isoforms. We could thus define GC regulated variants of response genes, did however also identify first exons not corresponding to any known transcript.
We reconfirmed these results for the genes ATP4B, GPR98, TBCD and ZBTB16 and identified for the latter also a novel exon employing 5' nested amplification of cDNA ends. Expression and regulation of this and all downstream exons of ZBTB16 was further verified by real time RT-PCR in 4 different preB-ALL cell lines. Thus, besides potential other novel gene isoforms, GCs induce a novel short transcript variant of ZBTB16 in preB-ALL cells.
Finally, the implemented bioinformatic methods may be useful also for other high density microarray data sets to identify alternative splice events and transcript variant expression.

overall_design:
This data set was used for the identification of glucocorticoid regulated genes/transcripts in CCRF-CEM-C7H2 T-ALL cells.
For each gene a representative transcript probe set was selected based on a score that combines average expression and variance expression across all samples. Differentially expressed genes/transcripts were identified for each time point by comparing expression values of GC treated to the respective control samples. Raw p-values were calculated employing the moderated t-test, and adjusted for multiple hypothesis testing with the method from Benjamini and Hochberg. Genes with an adjusted p-value smaller 0.05 and an absolute average M (log 2 fold change) value larger 0.7 were considered significantly differentially expressed.

first: Julien

last: Lelong

first: Daniel

last: Bindreither

last: Mantinger

last: Ploner


title: Effect of the Conceptus on Global Gene Expression in the Mouse Uterus During Decidualization

status: Public on Nov 20 2011

city: CARBONDALE

first: BRENT

last: BANY

state: ILLINOIS

address: 1135 LINCOLN DRIVE

department: PHYSIOLOGY

institute: SOUTHERN ILLINOIS UNIVERSITY SCHOOL OF MEDICINE

summary: Analysis of differences in gene expression in the mouse uterus during decidualization in the presence and absence of a conceptus. A deciduomal model previously shown to better mimick natural decidualization was used.  The results reveal some, but very few, genes that are differentially expressed, which is unlike previous reports using other deciduomal models shown previously to not mimick natural decidualization.

overall_design: Total RNA obtained from isolated uterine segment tissue undergoing decidualization from pregnant or pseudopregnant mice. Three replicates per condition.

first: Brent

last: Bany


title: Effect of Rho-associated kinase inhibitor on cryopreservation of hES cells

city: Matsumoto

first: Katsunori

last: Sasaki

state: Nagano

address: 3-1-1 Asahi

department: School of Medicine

institute: Shinsyu University

summary: After thawing, the mechanism of apoptosis advancing in human ES cells was invesitigated mainly by Gene conpath analysis. As time passed after thawing, nuclear fragmented cells increased more and more in cR-/mR- and cR+/mR-, but in cR+/mR+ and cR-/mR+, thawed cells formed cluster markedly, which was seen among three cell lines. Flow cytometry using TUNEL assay supported final colony formation ratio, which defined that such cell death was due to apoptosis.  Gene map analysis 12 hours later after thawing of cR-/mR- showed activation of IL-1β & its receptor IL-1R and TGF-β & its receptor ACVR1C required for activation of caspase-8, initiation of caspase-8 and 10 and marked activation of ARHGDIB promoting actin reorganization comparing to cR+/mR+.  Recovered cell lines in any condition did not lose proliferation and pluripotency regardless of ROCK inhibitor treatment.　These results showed apoptotic events after thawing were more marked in cR- and advanced through autoenhanced cascaded mechanism followed by initiation of self-cytokain activity, and were suppressed by ROCK inhibitor Y-27632.

overall_design: To investigate the protective mechanism caused by a novel cryo-technique of human embryonic stem (hES) cells using ROCK inhibitor Y-27632 against the cell death due to cryopreservation and thawing with Gene map analysis in addition to estimations of survival rate, apoptosis, undifferentiated states and pluripotency, the dissociated hES cells were cryopreserved in a freezing container in the following four conditions: addition of 10µM of Y-27632 to 1: cryoprotection solution and post-thawing medium (cR+/mR+), 2: only addition to cryoprotection (cR+/mR-), 3: only addition to post-thawing medium (cR-/mR+), 4: no addition to any medium (cR-/mR-).

first: Hinako

last: Ichikawa


title: Escherichia coli Gene Expression Profiling in Response to the Interaction with Lettuce Rhizosphere

city: st. paul

first: zhe

last: hou

state: MN

address: 1334 Eckles Ave

department: Food Science and nutrition

institute: University of Minnesota

summary: Leafy green vegetables, such as lettuce, have been increasingly implicated in outbreaks of foodborne illnesses due to contamination by Escherichia coli O157:H7. While E. coli can survive in soils, colonize plants, and survive on produce, very little is known about the interaction of E. coli with the roots of growing lettuce plants. In these studies a combination of microarray analyses and microbial genetics were used to gain a comprehensive understanding of bacterial genes involved in the colonization and growth of E. coli K12 on lettuce roots using a hydroponic assay system. Here we report that after three days of interaction with lettuce roots, 193 and 131 genes were significantly up-regulated and down-regulated at least 1.5 fold, respectively.  Forty-five out of the 193 up-regulated genes (23%) were involved in protein synthesis and were highly induced. Genes involved in stress response, attachment and biofilm formation were up-regulated in E. coli when they interacted with lettuce roots under conditions of hydroponic growth. In particular crl, a gene regulating the cryptic csgA gene for curli production, was significantly up regulated.  The crl, csgA and fliN mutants had a reduced capacity to attach to roots as determined by bacterial counts and by confocal laser scanning microscopy.  Our microarray data showed that E. coli K12 increased the synthesis of proteins indicated that a dramatic change was induced in the physiology of the microorganism. This study indicates that E. coli K12 can efficiently colonize lettuce roots by using attachment and biofilm modulation genes and can readily adapt to the rhizosphere of lettuce plants. Further studies are needed to better characterize this interaction in pathogenic strains of this species.

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

overall_design: Escherichia coli MG1655 strains were grown in the lettuce rhizosphere for three days. Transcriptional profiling of E. coli was compared between cells grown with and without rhizosphere . Three biological replicates of each treatment were prepared, and six microarray slides were used.

first: Zhe

last: Hou

first: Francisco

last: Diez-Gonzalez

first: Michael

last: Sadowsky


title: Effects of UV and temperature on gene expression in the snakelocks sea anemone

first: Cecile

last: Sabourault

summary: We monitored gene expression response of the symbiotic sea anemone Anemonia viridis subjected to a thermal and/or UV stress. A symbiosis-dedicated oligonucleotide microarray (2000 selected features) was generated, representing to date the only available oligonucleotide array used for symbiotic cnidarians (GPL10546). We are describing here the expression evolution during the first phase (5 days) of the stress.

overall_design: Sea anemones were subjected to thermal stress (+10°C) and/or UV stress (+1.25 mW.cm-2), and compared to control. For each stress condition, kinetic sampling of whole tentacles from 3 specimens (biological replicates) was performed over a 5 days period: T0, T24h, T48h and T120h. For each sample, dye-swap hybridizations compared T0 and later time points. Statistical analysis combines the 3 biological replicates of each condition.

first: Philippe

last: Ganot

first: Aurelie

last: Moya


title: Hydroxymethyl DNA-IP of genomic DNA for many different normal human tissues

status: Public on Nov 18 2011

city: Edinburgh

first: Colm

middle: E

last: Nestor

address: Western General Hospital, Crewe Road

department: MRC Human Genetics Unit

institute: Univeristy of Edinburgh

type: Methylation profiling by genome tiling array

summary:
5-hmC is a novel epigenetic mark derived from oxidation of methylcytosine. We profile this mark in several normal human tissues 5-hmC patterns are highly tissue specific and enriched in the body of transcribed genes
5-hmC patterns are highly tissue specific and enriched in the body of transcribed genes

overall_design: comparison of six normal human tissue types

first: Richard

middle: R

last: Meehan


title: PCR array screening for gene expression changes in HCC model comparing sorafenib treated or control samples

status: Public on Nov 11 2011

city: Tianjin

first: Wei

last: Zhang

address: Huan Hu Xi Road

country: China

institute: ianjin Medical University Cancer Institute and Hospital

summary: Candidate genes were successfully screened by Cancer PathwayFinder PCR Array

overall_design: Cancer PathwayFinder PCR Array was applied to compare gene changes in xenograft tumor samples of HCC model. A total of 12 samples were examined with 6 from Sorafenib treated group and 6 from control.


title: Diverse routes to oral cancer differing in genome instability and risk for cervical node metastasis

city: San Francisco

first: Taku

last: Tokuyasu

address: 1450 3rd Street, HD555

department: Helen Diller Family Comprehensive Cancer Center

institute: UCSF

summary: Clinically evident oral lesions, oral epithelial dysplasia, precede development of oral squamous cell carcinomas (SCC) and are considered to transform to cancer by acquisition of genetic or epigenetic alterations.  Here, we show that, +3q24-qter, -8pter-p23.1, +8q12-q24.2 and +20 are early events identifying two pathways to oral cancers that differ in clinical behavior.  One or more of these copy number aberrations is present in the major subgroup (3q8pq20 subtype, 75-80% of lesions) that develops with chromosomal instability and risk for metastasis, while they are absent from the smaller and chromosomally stable non-3q8pq20 subgroup (20-25% of lesions) associated with low risk for metastasis.  Thus, +3q, -8p, +8q and +20 is a biomarker for oral SCC metastasis.  On the other hand, while increased numbers of genomic alterations can be harbingers of progression to cancer, dysplastic lesions lacking copy number changes cannot be considered benign as they are potential precursors to non-3q8pq20 oral SCC.

overall_design: 63 oral SCCs and adjacent regions of normal tissue, 44 oral dysplasias


title: NKX3-1, a Novel Transcriptional Factor of AR, Promotes Prostate Cancer Cell Survival via RAB3B GTPase-mediated protein trafficking (mRNA)

city: singapore

first: Peck Yean

last: TAN

address: 60 Biopolis Street #02-01 Genome

institute: Genome Institute of Singapore (GIS)

summary: Androgen receptor (AR) orchestrates an intricate transcriptional regulatory network that governs prostate cancer initiation, development and progression. To understand this network in detail, we generated genome-wide maps of AR occupancy by ChIP-seq in LNCaP cells. We found NKX3-1, an androgen-dependent homeobox protein well-characterized for its role in prostate development and differentiation, being recruited to AR binding sites (ARBS) in response to androgen signaling. We identified 6,359 NKX3-1 binding sites, most of which overlapped with AR. In addition to its novel collaborative transcriptional role at well-known prostate cancer model genes, our binding and knockdown studies further suggested that NKX3-1 potentially regulates AR in a feed-forward manner. Integrative analysis of Oncomine molecular concepts showed that these androgen-regulated AR and NKX3-1 associated genes are significantly overexpressed in prostate carcinoma as well as advanced and recurrent prostate tumors. From our transcriptomic profiling and Gene Ontology analysis, we observed that AR and NKX3-1 co-regulate genes involved in ‘protein trafficking’ processes, which are mandatory events in the integration of oncogenic signaling pathways leading to prostate cancer development and progression. Interestingly, we found that AR and NKX3-1 co-regulate several members of the RAB GTPase family of secretory/trafficking proteins via the involvement of FoxA1 in a ternary complex and we believe that these AR/NKX3-1/FoxA1 co-regulated RAB genes could serve as expression signatures in prostate carcinogenesis.  More specifically, through functional analyses, we showed that NKX3-1, together with AR and FoxA1, could promote prostate cancer cell survival through activation of RAB3B expression. Collectively, our study has provided important insights into the hierarchical transcriptional regulatory network established between AR and NKX3-1 and sought to elucidate the important genetic-molecular-phenotypic paradigm in androgen-dependent prostate cancer.

overall_design: Gene expression profiling of LNCaP in response to ETOH (vehicle) or DHT (5α-dihydrotestosterone) stimulation across different treatment time-points using microarray.

first: Peck

middle: Y

last: Tan

first: Cheng

middle: W

last: Chang

first: Kern

last: Chng

first: Edwin

last: Chueng


title: Transcriptional profiling of 8Br-cAMP treated human H295R adrenal cortex

city: Antwerp

first: Caroline

last: Vanparys

address: Groenenborgerlaan 171

institute: University of Antwerp

summary:
Transcriptional profiling of 8Br-cAMP exposed H295R cells at different time points (10h-24h-48h) and different concentrations (50uM-100uM-300uM) with untreated solvent control cells (blanks)
Analysed on a H295R custom microarray, referred to as platform GPL9718

overall_design: common reference design (pool of control H295R cells of different experiments as reference RNA sample), 3 biological replicates


title: Transcriptional glucocorticoid-response at the exon level and NR3C1 DNA-binding in childhood leukemia models; transcriptional response in NALM6 precursor B-ALL cells.

first: Johannes

last: Rainer

overall_design:
This data set was used for the identification of glucocorticoid regulated genes/transcripts in NALM6 preB-ALL cells.
For each gene a representative transcript probe set was selected based on a score that combines average expression and variance expression across all samples. Differentially expressed genes/transcripts were identified for each time point by comparing expression values of GC treated to the respective control samples. Raw p-values were calculated employing the moderated t-test, and adjusted for multiple hypothesis testing with the method from Benjamini and Hochberg. Genes with an adjusted p-value smaller 0.05 and an absolute average M (log 2 fold change) value larger 0.7 were considered significantly differentially expressed.

first: Julien

last: Lelong

first: Daniel

last: Bindreither

first: Christine

last: Mantinger

first: Christian

last: Ploner

first: Stephan

last: Geley

first: Reinhard

last: Kofler


title: Cervical carcinoma-associated fibroblasts are DNA diploid and do not show evidence for somatic genetic alterations

status: Public on Nov 08 2011

city: Leiden

first: Jan

last: Oosting

address: Albinusdreef 2

department: Pathology

country: Netherlands

institute: Leiden University Medical Center

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

summary:
Cancer-associated fibroblasts (CAFs) have been recognized as important contributors to cancer development and progression. However, opposing evidence has been published whether CAFs, in addition to epigenetic, also undergo somatic genetic alterations and whether these changes contribute to carcinogenesis and tumour progression.
We combined multiparameter DNA flow cytometry, flow-sorting and 6K SNP-arrays to study DNA aneuploidy, % S-phase, loss of heterozygosity (LOH) and copy number alterations (CNAs) to study somatic genetic alterations in cervical cancer-associated stromal cell fractions (n = 58) from formalin-fixed, paraffin-embedded (FFPE) samples. Tissue sections were examined for the presence of CAFs. Microsatellite analysis was used to study LOH.
By flow cytometry we found no proof for DNA aneuploidy in the vimentin-positive stromal cell fractions of any samples (CV G0G1 population 3.7% +/- 1.2; S-phase 1.4% +/- 1.8). The genotype concordance between the stromal cells and the paired normal endometrium samples was > 99.9%. No evidence for CNAs or LOH was found in the stromal cell fractions. In contrast, high frequencies of DNA content abnormalities (43/57), a significant higher S-phase (14.6% +/- 8.5)(p = 0.0001) and substantial numbers of CNAs and LOH were identified in the keratin-positive epithelial cell fractions (CV G0G1 population 4.1% +/- 1.0). Smooth muscle actin and vimentin immunohistochemistry verified the presence of CAFs in all cases tested. LOH hot-spots on chromosomes 3p, 4p and 6p were confirmed by microsatellite analysis but the stromal cell fractions showed retention of heterozygosity only.
From our study we conclude that stromal cell fractions from cervical carcinomas are DNA diploid, have a genotype undistinguishable from patient-matched normal tissue and are genetically stable. Stromal genetic changes do not seem to play a role during cervical carcinogenesis and progression. In addition, the stromal cell fraction of cervical carcinomas can be used as reference allowing large retrospective studies of archival FFPE tissues for which no normal reference tissue is available.

overall_design: Paired experiment, Endometrial (non-tumor) cells vs stromal cells from cervical tumors. Biological replicates: 58 patients. From 5 tumors also the tumor fraction was profiled.

first: Oosting

last: Jan

first: Corver

last: Willem

first: terHaar

middle: T

last: Natalja

first: Fleuren

last: Gert Jan


title: Chicken ovary cancer versus normal

city: Ithaca

first: Wei

last: Wang

state: NY

address: T2-003 Vet Res Tower

institute: Cornell University

summary: Ovarian cancer has a high mortality rate due, in part, to the lack of early detection and incomplete understanding of the origin of the disease.  The hen is the only spontaneous model of ovarian cancer, and can therefore aid in the identification and testing of early detection strategies and therapeutics.  To our knowledge, no studies to date have examined global gene expression in ovarian cancer of the hen.  Our aim was to combine the use of the hen animal model and microarray technology to identify differentially expressed genes in ovarian tissue from normal hens compared to hens with ovarian cancer.

overall_design: Ovarian tissue samples from whole ovaries were collected from hens for RNA extraction and hybridization on Affymetrix microarrays.  Hens were matched for age and laying status.  Normal hens (n=3) showed no gross or histopathological evidence of ovarian cancer, while cancer specimens (n=3) had tumors that were stage 2 (restricted to the ovary and observable at necropsy) or 3 (ovarian tumor with abdominal seeding).  Total RNA was extracted using TRIZOL according to the manufacturer's instructions.

first: Lindsey

middle: S

last: Trevino

first: James

last: Giles

first: MaryEllen

last: Urick

first: Patricia

last: Johnson


title: B-cell KO mice data

city: Bethesda

first: Natalia

last: Shulzhenko

state: MD

address: 9000 Rockville Pike

department: Laboratory of Cellular and Molecular Immunology

institute: NIAID, USA

summary:
Using a systems biology approach, we discovered and dissected a three-way interaction between the immune system, the intestinal epithelium, and the microbiota. We found that mice lacking B lymphocytes, or lacking IgA, have low intestinal expression of lipid metabolism genes regulated by the transcription factor GATA4, and a consequent decrease in fat absorption in the intestine. The defect disappeared in germ free mice, suggesting that it is dependent on the microbiota; and sequencing analysis of the bacteria showed subtle differences between normal and B-cell deficient mice. Analysis of gene expression of gut biopsies from patients with common variable immunodeficiency and intestinal dysfunction revealed a high similarity to mouse B-cell knockout profiles. These data provide an explanation for a longstanding enigmatic association between immunodeficiency and defective lipid absorption in humans.
This series represents first part of the study including: 1) B-cell KO mice of different strains and their controls 2) germ free B-cell Ko mice and their controls 3) B lymphocytes

overall_design: Intensity of each channel was extracted from two-color arrays. We used direct dye swap design and performed paired analysis between small intestine of KO and control mice. Corresponding pairs of KO and control were hybridized on the same array and can be matched by the array name. For comparison between B lymphocytes and intestinal tissue we used B-cell KO intestinal samples and B lymphocytes samples balancing the number of Cy5/3 labeled samples in the groups.

first: Andrey

last: Morgun


title: Genome-wide binding map of the HIV Tat protein to the human genome (ChIP-chip)

status: Public on Nov 06 2011

city: Los Angeles

first: Siavash

middle: K

last: Kurdistani

address: 615 Charles E. Young Dr. South, 337

department: Biological Chemistry

institute: University of California Los Angeles

summary: The HIV-1 Trans-Activator of Transcription (Tat) protein binds to multiple host cellular factors and greatly enhances the level of transcription of the HIV genome.  Here, we report the genome-wide binding map of Tat to the human genome in Jurkat T cells (Jurkat-Tat cells) using chromatin immunoprecipitation combined with next-generation sequencing.  cDNA microarray was used to monitor gene expression changes between Jurkat and Jurkat-Tat cells.  Additionally, we compared distribution of H3K9ac near gene promoters between Jurkat and Jurkat-Tat cells using ChIP-chip method and hybridized onto Agilent promoter array.  Our data reveal that Tat’s interaction with the host genome is more extensive than previously thought, with potentially important implications for the viral life cycle.

overall_design: Expression profiles on Jurkat-Tat cells versus Jurkat cells.   ChIP on chip for H3K9ac in Jurkat-Tat versus Jurkat cells.  ChIP-seq for HIV-1 Tat protein in Jurkat-Tat cells.

first: Celine

last: Marrban

first: Trent

last: Su


title: Patterns of dioxin-altered mRNA expression in livers of dioxin-sensitive versus dioxin-resistant rats

status: Public on Nov 14 2011

city: Toronto

first: Paul

last: Boutros

state: Ontario

address: 101 College Street, Suite 800

country: Canada

institute: Ontario Institute for Cancer Research

summary: The dioxin congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a wide range of toxic effects in rodent species, all of which are mediated by a ligand-dependent transcription-factor, the aryl hydrocarbon receptor (AHR). The Han/Wistar (Kuopio) (H/W) strain shows exceptional resistance to many TCDD-induced toxicities; the LD50 of >9600 µg/kg for H/W rats is higher than for any other wild-type mammal known. We have previously shown that this resistance primarily results from H/W rats expressing a variant AHR isoform that has a substantial portion of the AHR transactivation domain deleted. Despite this large deletion, H/W rats are not entirely refractory to the effects of TCDD; the variant AHR in these animals remains fully competent to up-regulate well-known dioxin-inducible genes. TCDD-sensitive (Long-Evans, L-E) and resistant (H/W) rats were treated with either corn-oil (with or without feed-restriction) or 100 µg/kg TCDD for either four or ten days. Hepatic transcriptional profiling was done using microarrays, and was validated by RT-PCR analysis of 41 genes. . A core set of genes was altered in both strains at all time points tested, including CYP1A1, CYP1A2, CYP1B1, Nqo1, Aldh3a1, Tiparp, Exoc3, and Inmt. Outside this core, the strains differed significantly in the breadth of response: three-fold more genes were altered in L-E than H/W rats. At ten days almost all expressed genes were dysregulated in L-E rats, likely reflecting emerging toxic responses. Far fewer genes were affected by feed-restriction, suggesting that only a minority of the TCDD-induced changes are secondary to the wasting syndrome.

overall_design: Rats from sensitive (Long-Evans, LE) and resistant (Han/Wistar, HW) strains were treated with 100 ug/kg TCDD or corn oil vehicle and sacrificed either 4 or 10 days after treatment. LE control rats were either fed normally or feed-restricted to control for the wasting effects of TCDD treatment. Each treatment group contains four or five animals (biological replicates), each of which was assayed on an individual microarray.

first: Cindy

middle: Q

last: Yao

first: John

middle: D

last: Watson

first: Alexander

middle: H

last: Wu

first: Ivy

last: Moffat

first: Stephenie

last: Prokopec

first: Ashley

middle: B

last: Smith

first: Allan

last: Okey

first: Raimo

last: Pohjanvirta


title: Molecular bases of virulence variability in Staphylococcus aureus mastitis isolates

city: Geneva

first: FRANCOIS

last: Patrice

state: Ge 4

address: Rue Gabrielle-Perret-Gentil, 4

department: Service of Infectious Diseases

country: Switzerland

institute: Genomic Research Laboratory

summary: Background: S. aureus is one of the main pathogen involved in ruminant mastitis worldwide. The severity of staphylococcal infection is highly variable and ranges from subclinical to gangrenous mastitis. Such variability implies host as well as staphylococcal factors. This work is an in-depth characterization of S. aureus mastitis isolates to identify factors involved in mastitis severity.   Methods and findings: We combined three “omic” approaches to comprehensively compare two clonally related S. aureus strains that were isolated from and shown to reproducibly induce severe (strain O11) and milder (strain O46) mastitis in ewes. The genomes of O11 and O46 were sequenced (Illumina technology) to determine their respective gene content and comparative transcriptomic and proteomic analyses were carried out on both strains grown in conditions mimicking mastitis context. High differences were highlighted in mobile genetic elements, iron acquisition and metabolism, transcriptional regulation and exoprotein production. In particular, O11 overproduced exoproteins, including toxins and proteases when compared to O46. This was confirmed in 4 other S. aureus strains isolated from subclinical or clinical mastitis cases. Dose-dependant production of some staphylococcal factors seem to play a role in hypervirulence of strains isolated from severe mastitis. Mobile genetic elements, transcriptional regulators, exoproteins or strain ability to deal with iron starvation constitute good targets for further research to better define the underlying mechanisms of mastitis severity.   Conclusions: Differences observed in mastitis severity likely result from the ability of the strains to adapt and to express virulence factors in the mastitis context rather than from deep variations in gene content.

overall_design: Expression of S. aureus O46 from subclinical mastitis and O11 from a lethal gangrenous mastitis were compared at two different times

first: Caroline

last: Le Marechal

first: Nubia

last: Seyffert

first: Julien

last: Jardin

first: David

last: Hernandez

first: Gwenaël

last: Jan

first: Vasco

last: Azevedo

first: Patrice

last: François

first: Jacques

last: Schrenzel

first: Maarten

last: van de Guchte

first: Sergine

last: Even

first: Nadia

last: Berkova

first: Richard

last: Thiéry

last: Fitzgerald

first: Eric

last: Vautor

first: Yves

last: Le Loir


title: Transcriptome analysis of Salmonella enterica serovar Typhimurium growth in physiological murine bile

status: Public on Nov 10 2011

city: Vancouver

first: L. Caetano

middle: M.

last: Antunes

state: BC

department: Michael Smith Laboratories

institute: The University of British Columbia

summary: Infection with Salmonella enterica serovar Typhi in humans causes the systemic, life-threatening disease typhoid fever. In the laboratory, typhoid fever can be modeled through the inoculation of susceptible mice with Salmonella enterica serovar Typhimurium. The ensuing disease is characterized by systemic dissemination and colonization of many organs, including the liver, spleen and gallbladder. Using this murine model, we previously characterized the interactions between Salmonella Typhimurium and host cells in the gallbladder and showed that this pathogen can successfully invade gallbladder epithelial cells and proliferate. Additionally, we showed that Salmonella Typhimurium can use bile phospholipids to grow at high rates. These abilities are likely important for quick colonization of the gallbladder during typhoid fever and further pathogen dissemination through fecal shedding. To further characterize the interactions between Salmonella and the gallbladder environment we compared the transcriptome of Salmonella cultures grown in LB or physiological murine bile. Our data showed that many genes involved in bacterial central metabolism are affected by bile, with the citric acid cycle being repressed and alternative respiratory systems being activated. Additionally, our study revealed a new aspect of Salmonella interactions with bile through the identification of phoP as a bile-responsive gene. Repression of phoP expression does not involve PhoPQ sensing of a bile component. Due to its critical role in Salmonella virulence, further studies in this area will likely reveal aspects of the interaction between Salmonella and bile that are relevant to disease.

platform_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2

overall_design: Salmonella enterica serovar Typhimurium LT2 glass slide microarrays were obtained through the NIAID’s Pathogen Functional Genomics Resource Center, managed and funded by Division of Microbiology and Infectious Diseases, NIAID, NIH, DHHS and operated by the J. Craig Venter Institute. Two independent cultures of Salmonella enterica serovar Typhimurium SL1344 were grown overnight in Luria-Bertani (LB) broth with streptomycin (100 μg/mL) at 37oC with shaking (225 RPM). Cells were pelleted by centrifugation and resuspended in sterile Dulbecco’s phosphate-buffered saline (PBS). These suspensions were further diluted 1:50 in PBS and used to inoculate 70 μL of either LB or physiological murine bile (in 0.65-mL tubes) at a 1:100 dilution. Cultures were incubated for 24 hours at 37oC with agitation (225 RPM). Then, samples were diluted in PBS and serial dilutions were plated on LB plates containing 100 μg/mL of streptomycin and incubated overnight at 37oC for bacterial growth and enumeration by colony counting. The remaining cells were used in the subsequent steps, as described below. RNA isolation began with the addition of 2 volumes of RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany) to the bacterial cultures and incubation at room temperature for approximately 5 minutes. Cells were pelleted by centrifugation and RNA was isolated using the RNeasy Mini Kit (Qiagen) with the on-column DNase digestion, according to the manufacturer’s recommendations. Synthesis of cDNA was performed as described herein. The entire RNA sample (≥3 μg) was mixed with 6 μg of Random Primers (Invitrogen, Burlington, Canada) and 40 units of RNaseOUT recombinant ribonuclease inhibitor (Invitrogen, Burlington, Canada) in a total volume of 18.5 μL and incubated at 70oC for 10 minutes. Samples were incubated on ice for 30 seconds, centrifuged to bring down condensation and incubated with the following components (final concentrations; Invitrogen): First Strand Buffer (1X), dithiothreitol (DTT; 10 mM), dNTPs (dATP, dCTP, dGTP, dTTP; 0.5 mM each) and 100 units of SuperScript II reverse transcriptase in a total volume of 30 μL. The mixture was incubated at 42oC overnight and the reaction was stopped and the RNA degraded through the addition of 0.5 M EDTA and 1 M sodium hydroxide (10 μL each) and incubation at 65oC for 15 minutes. Then, 25 μL of 1 M Tris (pH 7) was added to neutralize the pH of the cDNA solution. cDNA was purified using the MinElute PCR Purification Kit (Qiagen) according to the manufacturer’s recommendations, except that the Qiagen wash and elution buffers were substituted by phosphate buffers, according to the PFGRC microarray protocol. cDNA was then precipitated using ammonium acetate and ethanol and labeled with Cy3 (LB cultures) and Cy5 (bile cultures) using the ULS aRNA Fluorescent Labeling Kit (KREATECH Biotechnology, Amsterdam, The Netherlands) according to the manufacturer’s instructions. Samples were mixed, dried and saved at -80oC until used. Pre-hybridization, hybridization and washing steps were performed essentially as described in the PFGRC protocols, except that the hybridization buffer contained KREAblock blocking buffer (25%; KREATECH Biotechnology). After hybridization, slides were scanned using an Affymetrix 428 Array Scanner from Eurofins MWG Operon (Huntsville, USA).

first: Caetano

first: B

last: Finlay


title: Massage therapy attenuates NFκB nuclear accumulation and inflammatory cytokine production following damage in human skeletal muscle

city: Novato

first: Simon

last: Melov

address: 8001 Redwood Blvd

institute: Buck Institute for Research on Aging

summary: Massage therapy is commonly used for the physical rehabilitation of skeletal muscle to ameliorate pain and promote recovery from injury.  While there is some evidence that massage may relieve pain in injured muscle, the cellular effects remain unknown. To assess the effects of massage, we administered either massage therapy or no treatment to separate quadriceps of eleven young, male participants after exercised-induced muscle damage. Muscle biopsies were acquired from the quadriceps (vastus lateralis) at baseline (rest), immediately after 10 minutes of massage treatment (0h), and after a 2.5 hour period of recovery. We found that massage activated the mechanotransduction signaling pathways FAK and Erk1/2, potentiated mitochondrial biogenesis signaling (nuclear PGC-1α), and mitigated the rise in NFkB (p65) nuclear accumulation caused by muscle trauma. Moreover, in spite of having no effect on muscle metabolites (glycogen, lactate), massage attenuated the production of the inflammatory cytokines TNF-α and IL-6 and reduced HSP27 phosphorylation, effectively mitigating cellular stress resulting from myofiber injury. In summary, massage therapy appears to be clinically beneficial when administered to acutely damaged skeletal muscle by reducing inflammation and promoting mitochondrial biogenesis.

overall_design: 55 muscle biopsies were used in this study as material to evaluate the effects of massage on gene expression. These biopsies were taken from the vastus lateralis of eleven healthy recreationally active males with full ethical approval at McMaster's university. Each individual had an initial biopsy at rest (baseline), followed by exercist testing for peak aerobic capacity. On a second visit, the individual performed a bout of exhaustive aerobic exercise, prior to recieving a randomized massage treatment to one leg. Immediatley following the exercise, subjects were allowed to recover for 10 minutes, while massage oil was lightly applied to both quadriceps. Then a single leg was randomized to recieve massage treatment for 10 minutes. Subjects then rested for 10 minutes, and one additional biopsies were carried out (on each leg). Two and half hours later (3 hours after the cessation of the exercise bout), 2 more biopsies were taken (one from each leg). This amounted to 5 biopsies from each individual.

first: Justin

last: Crane

first: Daniel

middle: I

last: Ogborn

first: Colleen

last: Cupido

first: Alan

last: Hubbard

first: Jacqueline

last: Bourgeois

first: Mark

middle: A

last: Tarnopolsky


title: Expression analysis of Arabidopsis peptidoglycan (PGN) recognition mutants treated with PGN

city: Wuhan

first: Kenichi

last: Tsuda

address: No.1 Shizishan Road

institute: Huazhong Agricultural University

summary: Recognition of microbial patterns by host pattern recognition receptors is a key step in immune activation in multicellular eukaryotes. Peptidoglycans (PGN) are major components of bacterial cell walls and possess immunity-stimulating activities in metazoans and plants. Here, we show that PGN sensing in Arabidopsis thaliana requires three LysM domain proteins, LYM1, LYM3 and CERK1.

overall_design: A 24 DNA microarray study using toral RNA from three Arabidopsis mutants (lym1-1, lym3-1, cerk1-2) as well as wild type treated with water or peptidoglycan.

first: Roland

last: Willmann

first: Fumiaki

last: Katagiri

first: Andrea

last: Gust

first: Thorsten

last: Nürnberger


title: The impact of quantitative microarray optimization on gene expression analysis

city: Vienna

first: D

middle: P

last: Kreil

address: Muthgasse 18

department: Dept of Biotechnology

country: Austria

institute: Boku University Vienna

summary: Background: With the growing availability of entire genome sequences, an increasing number of scientists can exploit oligonucleotide microarrays for genome-scale expression studies. While probe-design is a major research area, relatively little work has been reported on the optimization of microarray protocols. Results: As shown in this study, suboptimal conditions can have considerable impact on biologically relevant observations. For example, deviation from the optimal temperature by one degree Celsius lead to a loss of 44% of differentially expressed genes identified. While genes from thousands of Gene Ontology categories were affected, transcription factors and other low-copy-number regulators were disproportionately lost. Calibrated protocols are thus required in order to take full advantage of the large dynamic range of microarrays. For an objective optimization of protocols we introduce an approach that maximizes the amount of information obtained per experiment. A comparison of two typical samples is sufficient for this calibration. We ensure, however, that optimization results are independent of the samples and the specific measures used for calibration. Both simulations and spike-in experiments confirm an unbiased determination of generally optimal experimental conditions. Conclusions: Well calibrated hybridization conditions are thus easily achieved and necessary for the efficient detection of differential expression. They are essential for the sensitive profiling of low-copy-number molecules. This is particularly critical for studies of transcription factor expression, or the inference and study of regulatory networks. Supporting material, including source code and data, is available at http://bioinf.boku.ac.at/pub/optMA2010/.

overall_design: Optimization of hybridization temperature via an assessment of differential expression between two samples (male vs female Drosophila melanogaster) in 6 technical replicates (3 regular + 3 dye-swaps) for hybridizations at different temperatures (in two batches of 50, 52, 54, and 56; and 47, 49, 50, and 51 degree Celsius, with the repeated hybridization at 50 degree Celsius serving to demonstrate batch-to-batch stability).

first: Peter

last: Sykacek

first: David

first: Lisa

last: Meadows

first: Richard

last: Auburn

first: Bettina

last: Fischer

first: Steven

last: Russell

first: Gos

last: Micklem


title: miRNA array of MCF-7 cells

status: Public on Nov 04 2011

city: new orleans

first: Melyssa

last: Bratton

address: 1430 Tulane Ave

institute: Tulane University School of Medicine

summary:
Background: Several environmental agents termed “endocrine disrupting compounds” or EDCs have been reported to bind and activate the estrogen receptor-a (ER).  The EDCs DDT and BPA are ubiquitously present in the environment, and DDT and BPA levels in human blood and adipose tissue are detectable in most if not all women and men.  ER-mediated biological responses can be regulated at numerous levels, including expression of coding RNAs (mRNAs) and more recently non-coding RNAs (ncRNAs). Of the ncRNAs, microRNAs have emerged as a target of estrogen signaling.  Given the important implications of EDC-regulated ER function, we sought to define the effects of BPA and DDT on microRNA regulation and expression levels in estrogen-responsive human breast cancer cells.
Methodology/Principal Findings:  To investigate the cellular effects of DDT and BPA, we used the human MCF-7 breast cancer cell line, which is ER(+) and hormone sensitive.  Our results show that DDT and BPA potentiate ER transcriptional activity, resulting in an increased expression of receptor target genes, including progesterone receptor, bcl-2, and trefoil factor 1.  Interestingly, a differential increase in expression of Jun and Fas by BPA but not DDT or estrogen was observed.  In addition to ER responsive mRNAs, we investigated the ability of DDT and BPA to alter the miRNA profiles in MCF-7 cells.  While the EDCs and estrogen similarly altered the expression of multiple microRNAs in MCF-7 cells, including miR-21, differential patterns of microRNA expression were induced by DDT and BPA compared to estrogen.

overall_design: This experiment includes a total of 4 individual samples with no biological or technical repeats


title: Transcriptional profile of Candida parapsilosis bcr1 knockout.

city: Dublin

first: Geraldine

last: Butler

address: Belfield

department: School of Biomolecular and Biomedical Science

country: Ireland

institute: University Colege Dublin Conway Institute

summary: Whole genome microarrays were used to compare the transcriptional profile of Candida parapsilosis bcr1 knockout to wild type cells.

overall_design: RNA was isolated from CLIB214 (wild type) or bcr1 delete (CDb71 or CDUHB6) grown in SD medium supplemented with 50 mM glucose and 10% fetal calf serum and labeled with Cy3 or Cy5. 7 independent biological replicates were compared. Two dye swaps were perfomed so that five of seven CLIB214 cultures were labeled with Cy3, and two were labeled with Cy5.

first: Chen

last: Ding

first: Alessandro

last: Guida

first: Sarah

last: Maguire


title: Total expression profiling of embryonic regions of EHF mouse embryos

city: new york

first: hui

last: zhao

address: 430 east 67th street

institute: MSKCC

summary: Establishment of left-right (LR) asymmetry occurs shortly after gastrulation and utilizes a cascade of events. In the mouse, LR symmetry is broken at the node, involves signal relay to the lateral plate, and results in asymmetric organ morphogenesis. How information transmits from the node to the lateral plate remains unclear. Noting that embryos lacking Sox17 exhibit defects in both gut endoderm formation and LR patterning, we investigated a connection between these two processes. We noted an endoderm-specific absence of the critical gap junction component, Connexin43, in Sox17 mutants. Dye-coupling experiments revealed planar gap junction coupling across the gut endoderm in wild-type but not mutant embryos. The role for gap junction communication in LR patterning was confirmed by pharmacological inhibition. Collectively, our data demonstrate communication across gap junctions in gut endoderm as a mechanism for information relay between node and lateral plate critical for the establishment of LR asymmetry in mice.

overall_design: Total RNA isolated from embryonic regions of wild-type embryos at EHF stages, three samples per well, in triplicates

first: Manuel

last: Viotti

first: Anna-Katerina

last: Hadjantonakis


title: Differential transcript accumulation in seedling and mature shoots identifies components contributing to corn earworm resistance

status: Public on Nov 03 2011

city: Macomb

middle: Oliver

last: Musser

state: IL

department: Biological Sciences

institute: Western Illinois University

summary: As maize seedlings germinate into the soil, they encounter an environment teeming with insects  seeking rich sources of nutrition. Maize presumably has developed a number of molecular  mechanisms to ensure survival at the beginning of its life cycle. Bioassays indicated maize  seedlings were more toxic to caterpillars than shoots from 3 or 4 leafed plants. Microarray  technology was utilized to document the expression of a number of genes with potential  defensive functions in seedling tissue. In addition to elevated levels of the genes involved in the  biosynthesis of DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one), an anti-insect  resistance molecule, other highly expressed genes in the seedling encode the following putative  proteins: defensin, hydroxyproline and proline-rich protein, thaumatin-like protein, lipase,  cystatin, protease inhibitor, and a variety of proteases. Changes in the levels of gene expression  by the microarray technology were well correlated to results using quantitative RT-PCR. The  potential resistance genes identified occurred mainly on chromosomes 1 and 5 in the B73  genome. Analysis of promoters of four DIMBOA biosynthetic genes determined that a Dof  transcription factor, two of which were differentially expressed in three vegetative stages, is  possibly involved in regulation of the DIMBOA biosynthetic pathway. The results indicate that  maize employs a wide variety of potential resistance mechanisms in seedling tissue to resist a  possible insect attack.

overall_design: Three different Zea mays plant (B73) life stages were sampled: a) seedlings, which had grown just above the soil line, which also served as reference tissue (reference tissue was always labeled Cy5 and consisted of leaf shoots pooled from 12 plants); b) leaf shoots from plants with four leaves; and c) leaf shoots from plants with six leaves. There were 3 samples (biological replications) of each treatment (6 plants per sample) and the samples were labeled Cy3 and which were combined with a reference chip for a total of 9 chips: (3 biological replicates of seedling, 4 leaf stage and 6 leaf stage).

first: Eric

last: Johnson

first: Pat

last: Dowd

first: Lewis

middle: Z

last: Liu

middle: O


title: Epigenetic profiling of histone H3K4me1, H3K4me3, H3K27me3, H3ac, H4ac, CBP and P300 using ChIP-chip

status: Public on Nov 09 2011

city: Ann Arbor

first: Yongsheng

last: Huang

state: MI

address: 100 Washtenaw Ave

department: Bioinformatics

institute: University of Michigan

summary: The clustered homeobox proteins play crucial roles in development, hematopoiesis and leukemia yet the targets they regulate and their mechanisms of action are poorly understood.  Here, we identified the binding sites for Hoxa9 and the Hox cofactor Meis1 on a genome-wide level and profiled their associated epigenetic modifications and transcriptional targets. Hoxa9 and the Hox cofactor Meis1 co-bind at hundreds of highly evolutionarily-conserved sites, most of which are distant from transcription start sites.  These sites show high levels of histone H3K4 monomethylation and CBP/P300 binding characteristic of enhancers. Furthermore, a subset of these sites shows enhancer activity in transient transfection assays. Many Hoxa9 and Meis1 binding sites are also bound by PU.1 and other lineage-restricted transcription factors previously implicated in establishment of myeloid enhancers.  Conditional Hoxa9 activation is associated with CBP/P300 recruitment, histone acetylation and transcriptional activation of a network of proto-oncogenes including Erg, Flt3, Lmo2, Myb and Sox4. Collectively this work suggests that Hoxa9 regulates transcription by interacting with enhancers of genes important for hematopoiesis and leukemia.

overall_design: Enriched Hoxa9 and Meis1 regions and a selected set of the nearest 360 TSSs were tiled onto Nimblegen custom arrays (Mus musculus 8, Feb 2006) with 50-mer probes (average spacing of 35bp), along with 60 negative control sequences that were selected randomly from the mouse genome

first: Jay

last: Hess


title: COMPARISON OF THE GIARDIA LAMBLIA TROPHOZOITE AND CYST TRANSCRIPTOME

city: North Grafton

first: Giovanni

last: Widmer

address: 200 Westboro Rd

institute: Tufts University

summary: To investigate the magnitude of the transcriptional changes occurring  during the life cycle of Giardia  lamblia we compared the transcriptome of trophozoites and cysts.  Cysts were found to possess a much smaller transcriptome, both in terms of mRNA diversity and abundance. Genes encoding proteins related to ribosomal functions are highly over-represented.  In cysts which have lost infectivity the transcriptome is further depleted. In contrast, exposure of cysts to conditions which promote excystation induced transcription.

overall_design: Six cysts and three trophozoites life cycle stages were analyzed.

first: Zahra

last: Faghiri

first: Zijin

last: Wu


title: Gene expression in broiler livers upon supplementation with selenized yeast

city: Zurich

first: Bruno

last: Dietrich

address: Tannenstrasse 1

department: Department of Agricultural and Food Sciences

institute: ETH Zurich

summary: Selenium (Se) is an essential cofactor of the antioxidant enzyme glutathione peroxidase beside other functions. The evaluation of optimal selenium supplementation in chicken feed and the subsequent effects on animal health and performance requires comprehensive knowledge of the overall metabolic effects of selenium. Therefore the gene expression was measured in the control group with a standard diet and in the group with a Se supplemented diet (0.5mg Se/kg diet) to determine significantly altered gene expression. The selenium was supplemented in the form of selenized yeast (Se-yeast), which mainly consists of organic Se in the form of L-selenomethionine and L-selenocysteine. The control group received a diet, which contained 70μg of Se / kg diet and the Se-yeast group 620μg of Se / kg diet (analyzed).

overall_design: The one-day old broiler chicks were separated into two groups and received the control or the Se-supplemented diet ad libitum for 35 days. After slaughter the gene expression was determined in the liver of four control and five samples from the Se-yeast group. One sample from the control group did not correspond to the quality requirements and was excluded from the analysis.

first: Caspar

last: Wenk


title: Analysis of miRNA profiles in CD4+ T-lymphocytes following exposure to HIV-1

city: Parma

first: Claudio

last: Casoli

address: Vicolo delle Asse 1

institute: GEMIB Laboratory

summary:
Assessment of the extent to which the altered profiles of miRNA expression influence viral replication and latency, as well as the efficiency of host defenses, may be useful for understanding the basis of the HIV-1-related alterations in cellular physiology and immunologic control. To this end, three patient groups were enrolled. One group consisted of subjects who were classified as élite control long-term non-progressors (éLTNP).  A second study group was HIV-1-positive subjects, who were antiretroviral therapy naive.  A third group was multiply exposed to HIV-1, but uninfected (MEU). This study allowed us to investigate the existence of a CD4+T- lymphocytes miRNAs signature able to discriminate among different stages of HIV-1 infection, and to evaluate whether the exposure to HIV-1 antigen is sufficient to change the miRNA profile.

MicroRNAs inhibit HIV-1 expression by either modulating host innate immunity or by directly interfering with viral mRNAs. We evaluated the expression of 377 miRNAs in CD4+ T cells from HIV-1 élite LTNP (éLTNP), naive and multiply exposed uninfected individuals (MEU) and we observed that the éLTNP patients clustered with naive, whereas all MEU subjects grouped together. The discriminatory power of miRNAs showed that 21 miRNAs significantly differentiated éLTNP from MEU and 23 miRNAs distinguished naive from MEU, while only 1 (miR-155) discriminated éLTNP from naive. We proposed that miRNA expression may discriminate between HIV-1 infected and exposed but negative individuals. Analysis of miRNAs expression after exposure of healthy CD4+T cells to gp120 in vitro confirmed our hypothesis that a miRNA profile could be the result not only of a productive infection, but also of the exposure to HIV-1 products that leave a signature in immune cells. The comparison of normalized Dicer and Drosha expression in ex vivo and in vitro conditions revealed that these enzymes did not affect the change of miRNA profiles, supporting the existence of a Dicer-independent biogenesis pathway.

overall_design: We have compared miRNA profiles of CD4+ T-lymphocytes from 18 HIV-1-exposed subjects with healthy CD4+ T-lymphocytes following exposure to gp120 using a RT-qPCR assay.

first: Fabio

last: Bignami

first: Elisabetta

last: Pilotti

first: Linda

last: Bertoncelli

first: Paola

last: Ronzi

first: Mariolina

last: Gulli

first: Nelson

last: Marmiroli

first: Giacomo

last: Magnani

first: Marcello

last: Pinti

first: Lucia

last: Lopalco

first: Cristina

last: Mussini

first: Roberta

last: Ruotolo

first: Massimo

last: Galli

first: Andrea

last: Cossarizza


title: Euchromatic histone modifications associated with rice centromeres

city: Madison

first: Yufeng

last: Wu

state: WI

address: 1575 Linden Drive

institute: UW-Madison

summary: The centromere is defined by the presence of a centromere-specific histone H3 variant, CENH3.  Establishment and maintenance of the centromeric chromatin (CEN chromatin) is determined by poorly understood epigenetic mechanisms.  Interestingly, CEN chromatin in several eukaryotes showed euchromatic characteristics although being embedded within pericentromeric heterochromatin.  Specifically, H3K4me2 appeared to be a unique histone modification mark associated with animal centromeres.  We developed a genomic tiling array for four fully sequenced rice centromeres.  A ChIP-chip approach was used to study the patterns of several euchromatic histone modification marks, including H3K4me2, H3K4me3, H3K36me3, and H3K4K9a, associated with rice centromeres.  We demonstrate that the CENH3 subdomains within the four centromeres are depleted with the four histone H3 marks.  The vast majority of the four histone marks were associated with the genes located in the H3 subdomains within the centromeric cores.  Genes in the centromeres showed similar histone modification patterns as those located outside of the centromeres.  Thus, the euchromatic characteristics of rice CEN chromatin are trademarks of the transcribed sequences embedded in the H3 subdomains of the centromeres.  We propose that the constitutively expressed genes located in rice centromeres may provide a barrier for loading of CENH3 into the H3 subdomains.  The separation of CENH3 into the H3 subdomains is favorable for the three dimensional structure and its associated function of rice centromeres.

overall_design: We developed a genomic tiling array that covers four rice centromeres (Cen4, Cen5, Cen7, and Cen8) using the NimbleGen 3x720K array based on the NimbleGen HD2 platform. We used four antibodies (H3K4me3, H3K4me2, H3K36me3, H3K4K9ac) to perform ChIP-chip experiments. ChIP was conducted using leaf tissue from two-week old rice seedlings. We have 3 biological replicates for each antibody and 6 technological replicates of hybridization with position exchange on the array. Thus, each histone modification included 18 hybridization experiments.

first: Shinji

last: Kikuchi

first: Huihuang

last: Yan

first: Wenli

last: Zhang

first: Heidi

last: Rosenbaum

first: A

middle: L

last: Iniguez

first: Jiming

last: Jiang


title: Mouse Inflammatory Cytokines and Receptors Microarray-RNA profiling in F4/80+ and CD11c+ cells from lungs of WT mice before and after Mycoplasma pulmonis infection

status: Public on Nov 02 2011

city: Fort Worth

first: Jerry

last: Simecka

state: TX

address: 3500 Camp Bowie Blvd

department: Molecular Biology & Immunology

institute: UNT Health Science Center

summary:
RNA profiling of inflammatory chemokines, cytokines and receptors in AUTOMACS sorted F4/80+ and CD11c+ cells from lungs  of 6-12 week old wild-type C3H/HeN mice
before and after 14-day Mycoplasma pulmonis infection

overall_design: RNA pooled from pulmonary F4/80+ & CD11c+ cells of each group- Uninfected and Infected; Experiment performed twice. Comparison-RNA expression within each population of cells in Mycoplasma infected  vs. uninfected mice

first: Sheetal

last: Bodhankar


title: Capicua-dependent transcriptional changes in adult mouse cerebellum

status: Public on Nov 07 2011

city: Houston

first: Peng

last: Yu

address: 1 Baylor Plaza

institute: BCM

summary: Analysis of cerebella from Capicua (Cic) mutant mice and wild-type controls at 28 days of age (P28). Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by expansion of a translated CAG repeat in Ataxin-1 (ATXN1). The transcriptional repressor Cic binds directly to Atxn1 and plays a key role in SCA1 pathogenesis. Two isoforms of Cic, long (Cic-L) and short (Cic-S), are transcribed from alternative promoters. Using embryonic stem cells in which the Cic locus was targeted by an insertion of a genetrap cassette between exon 1 of the Cic-L isoform and exon 1 of the Cic-S isoform, we generated mice that carried this allele and backcrossed these onto a Swiss Webster (CD-1) strain for >6 generations. The resulting Cic-L-/- mice completely lack the Cic-L isoform with ~10% of Cic-S remaining. These data were used to compare with previous microarray data to determine the Cic-depedent pathogenic mechanisms in SCA1.

overall_design: Total RNA from cerebella of wild-type (n=4) and Capicua mutant mice (n=4) at 28 days of age was prepared and labeled according the manufacturer's protocols for the Affymetrix Mouse Gene 1.0 ST Array.

first: John

last: Fryer

first: Huda

middle: Y

last: Zoghbi

first: Chad

last: Shaw