title: miR-206 integrates multiple components of differentiation pathways to control the transition from growth to differentiation in rhabdomyosarcoma cells (miRNA)

status: Public on Apr 02 2012

city: Seattle

first: Janet

middle: M.

last: Young

state: WA

address: 1100 Fairview Avenue N., C3-168,  P.O. Box 19024

department: Division of Human Biology

institute: Fred Hutchinson Cancer Research Center

summary: Background: Similar to replicating myoblasts, many rhabdomyosarcoma cells express the myogenic determination gene MyoD. In contrast to myoblasts, rhabdomyosarcoma cells do not make the transition from a regulative growth phase to terminal differentiation. Previously we demonstrated that the forced expression of MyoD with its E-protein dimerization partner was sufficient to induce differentiation and suppress multiple growth-promoting genes, suggesting that the dimer was targeting a switch that regulated the transition from growth to differentiation. Our data also suggested that a balance existed between various inhibitory transcription factors and MyoD activity that kept rhabdomyosarcomas trapped in a proliferative state.   Methods: Potential myogenic co-factors identified by analysis of high-throughput sequencing of chromatin immunoprecipitation experiments in normal myogenic cells were tested for their ability to drive differentiation in rhabdomyosarcoma cell culture models, and their relation to MyoD activity determined through molecular biological experiments.   Results: Modulation of the transcription factors RUNX1 and ZNF238, factors with poorly delineated roles in myogenic development, can induce differentiation in rhabdomyosarcoma cells and their activity is integrated, at least in part, through the activation of miR-206, which acts as a genetic switch to transition the cell from a proliferative growth phase to differentiation. The inhibitory transcription factor MSC also plays a role in controlling miR-206, appearing to function by occluding a binding site for MyoD in the miR-206 promoter.   Conclusions: These findings suggest that nested feed-forward circuits that proceed from MyoD, to RUNX1, to ZNF238, and finally to miR-206 function in both rhabdomyosarcomas as well as normal myogenesis to control the decision point of proliferation versus differentiation.

platform_organism:
Physcomitrium patens
Populus trichocarpa
Arabidopsis thaliana
Glycine max
Medicago truncatula
Oryza sativa
Saccharum officinarum
Sorghum bicolor
Zea mays
Caenorhabditis briggsae
Caenorhabditis elegans
Bombyx mori
Anopheles gambiae
Drosophila melanogaster
Drosophila pseudoobscura
Apis mellifera
Danio rerio
Xenopus laevis
Xenopus tropicalis
Gallus gallus
Lemur catta
Ateles geoffroyi
Lagothrix lagotricha
Macaca mulatta
Macaca nemestrina
Gorilla gorilla
Pan paniscus
Pan troglodytes
Pongo pygmaeus
Homo sapiens
Canis lupus familiaris
Sus scrofa
Bos taurus
Ovis aries
Mus musculus
Rattus norvegicus
Human betaherpesvirus 5
human gammaherpesvirus 4
Gallid alphaherpesvirus 2
Monodelphis domestica
Takifugu rubripes
Murid gammaherpesvirus 4
Human gammaherpesvirus 8
Macacine gammaherpesvirus 4
Saguinus labiatus
Schmidtea mediterranea
Tetraodon nigroviridis
Betapolyomavirus macacae

overall_design: Two biologically independent sets of RD cells were transduced with either MD~E or empty vector pCLBabe retroviruses. After RNA extraction pairs of samples (MD~E versus pCLBabe) were hybridized to arrays, incorporating both dye-swap and technical replicates (i.e. 1 comparison x 2 dye-swaps x 2 biological replicates x 2 technical replicates = 8 arrays)

first: Kyle

last: MacQuarrie

first: Zizhen

last: Yao

middle: M

first: Yi

last: Cao

first: Stephen

middle: J

last: Tapscott


title: Array comparative genomic hybridization analysis reveals a limited number of genomic alterations in familial adenomatous polyposis-associated and sporadic desmoid tumors

status: Public on Apr 01 2012

city: Leiden

first: Els

last: Robanus-Maandag

address: Einthovenweg 20

department: Human Genetics

country: Netherlands

institute: LUMC

type: Genome variation profiling by genome tiling array

summary: Desmoid tumors (also called deep or aggressive fibromatoses) are potentially life-threatening fibromatous lesions. Hereditary desmoid tumors arise in individuals affected by either familial adenomatous polyposis (FAP) or hereditary desmoid disease (HDD) carrying germline mutations in APC. Most non-FAP (sporadic) desmoids carry somatic mutations in the beta-catenin gene. Previous studies identified losses on 5q and 6q, and gains on 8q and 20q as recurrent genetic changes in desmoids. However, virtually all genetic changes were derived from sporadic tumors. To investigate the somatic alterations in FAP-associated desmoids and to compare them with changes occurring in sporadic tumors, we analyzed 17 FAP-associated and 38 sporadic desmoids for copy number abnormalities (CNAs) by means of array comparative genomic hybridization and multiple ligation-dependent probe amplification.   Overall, the desmoids displayed only a limited number of genetic changes, occurring in 44% of cases. Common gains at 8q (7%) and 20q (5%) were almost exclusively found in sporadic tumors. Frequent common losses were observed within a 700 kb region at 5q22.2, comprising the APC gene (11%), in a 2 Mb region at 6p21.2-p21.1 (15%), and in a relatively large region at 6q15-q23.3 (20%). The FAP-associated desmoids displayed a significantly higher frequency of CNAs (59%) than the sporadic tumors (37%). As predicted by the APC germline mutations among these patients, a relatively high percentage (29%) of the FAP-associated desmoids showed loss of the APC region at 5q22.2, which was infrequently (3%) seen among sporadic tumors. Our data suggest that loss of region 6q15-q16.2 is an important event in FAP-associated as well as sporadic desmoids, most likely of relevance for desmoid tumor progression.

overall_design: Fifty-three fresh frozen tumor samples were collected at four institutes: Center for Human Genetics, University of Leuven, Belgium (21 samples); INSERM U674, Fondation Jean Dausset-CEPH, Paris, France (15 samples); Hospital for Sick Children, Toronto, Canada (13 samples); and the Italian Registry of Hereditary Colorectal Cancer (Dr. L. Bertario, 4 samples). In addition, DNA of two fresh frozen tumors, HDD-H of patient III:2 and HDD-I of patient III:6, of our hereditary desmoids disease (HDD) family10 was available (Table 1). In this family, multifocal desmoid tumors were inherited as an autosomal dominant trait, and HDD segregated with a 3' APC mutation at codon 1924. For the latter reason, they were classified as FAP tumors in this study. Three FAP tumors were derived from a family with 2 relatives in the study (D15-1 and D15-2 of a male patient and D16 of his sister). Colonic polyposis had been observed in 12 FAP patients, not in the 2 HDD-FAP patients and not in patient D15. The germline APC mutation was known in 14 of 17 FAP-associated tumors. In 28 of 38 non-FAP-associated desmoid tumors, the beta-catenin gene (CTNNB1) mutation in exon 3 was known and in 1 of 38 tumors an APC mutation was present (data not shown). The remaining tumors were characterized as FAP or non-FAP based on clinical data and positivity of tumor cells upon immunostaining for beta-catenin. DNA was extracted from the tumor samples according to standard methods.

first: Jan

last: Oosting

middle: C

first: Cathy

middle: A

last: Bosch

first: Karoly

last: Szuhai


title: miR-155 regulates the ability of circulating cancer cells to colonize and establish metastasis

status: Public on May 03 2012

city: Lund

first: Rolf

last: Søkilde

address: Scheelevägen 2

department: Department of Oncology

country: Sweden

institute: Lund University, Sweden

summary: Abstract: Metastases are established through a complex multistep process by which tumor cells disseminate from the primary tumor and expand at distant sites. Traditionally, studies on metastasis have focused on proteins and protein-coding genes, but recently microRNAs (miRNAs) have achieved increasing interest. miRNAs involved in the metastatic process are poorly defined, and while the use of clinical tissue samples or in vitro assessment for study of the first phases of the metastatic process is not feasible; such processes may be studies using in vivo models created by inoculation of human cell lines into mice. We have used such a model system consisting of three isogenic human cancer cell lines that are equally tumorigenic in mice, but while two of these cell lines give rise to metastasis at different rates, the last one disseminates single cells that remain dormant in distant organs. Using a global LNA- based miRNA microarray platform, 28 miRNAs were found to be significantly altered between the metastatic and non-metastatic cell lines, with mir-155 being the miRNA exhibiting the largest difference in expression level and central in a network analysis. Examining the potential targets of miR-155 identified calumenin, solute carrier family 26 (sulfate transporter) member 2 (SLC26A2), Integrin αV, and CD73, which showed an inverse expression pattern to miR-155 in the metastatic and non-metastatic cell lines as determined by immunocytochemical staining. The miRNAs identified in this study could be markers of the ability of cancer cells to colonize at distant organs.

overall_design: global microRNA profiling of three breast cancer cell lines with different metastatic potential derived from MDA-435

first: Karina

middle: H

last: Gravgaard

first: Maria

middle: B

last: Lyng

first: Daniel

last: Elias

first: Anne-Vibeke

last: Laenkholm

first: Thomas

last: Litman

first: Henrik

last: Ditzel


title: comparison_cul4_as_dark_light-Functional analysis of the SKP2 and CUL4 proteins

status: Public on Mar 27 2012

city: evry

first: Soubigou-Taconnat

last: Ludivine

address: 2 rue gaston crémieux

department: URGV

country: France

institute: INRA

summary: rs04-04_skp2_cul4 - comparison_cul4_as_dark_light - Antisense and mutants of the SKP2 and CUL4 genes are compared to the Wt in order to identify the genes controlled by those SCF proteins. - The seeds have been sown every two days on MS 255 (Duschefa) without adding sugar. These plates stayed 2 days in the dark at 4°C. Then they got a 8 hours light induction (light induction of germination) and then back to the dark for 5 more days.

overall_design: 4 dye-swap - gene knock out,normal vs antisense mutant comparison

first: Pascal

last: Genschik

first: Jean-Pierre

last: Renou

first: Hajer

last: Hadji

first: Sandrine

last: Balzergue

first: Marie-Laure

last: Martin-Magniette


title: sphingo-1-Identification of Sphingolipid regulated genes in cold stress response.  Sphingolipids regulated genes.

status: Public on Mar 28 2012

summary: rs07-05_sphingolipids-cold - sphingo-1 - The cold choc response seems to be partly triggered by Sphingolipid species. To date no gene response as been associated to sphingolipid signaling pathway in plant. Our aim is to identify among the cold induced genes the ones regulated by sphingolipids and to try to define a sphingolipid pathway specific group of genes. - 7ml of 5 days-old cells suspensions were incubated in presence of different sphingolipid pathway inhibitors, 30 min to 2 hours depending in the coumpound (all were resuspended in DMSO and control were done with DMSO). Then a 30 min cold choc was applied before cells were harvested and frozen in cold nitrogen. RNA were then extracted. FB1 and DMS were from Alexis , Myr from Cayman, TSP from matreya.

overall_design: 6 dye-swap - treated vs untreated comparison

first: Isabelle

last: Guillas

first: Ludivine

last: Taconnat


title: Effect of ribosomal protein intron deletion on gene expression

status: Public on Mar 01 2012

city: San Francisco

first: Alex

last: Plocik

state: CA

country: USA

institute: UCSF

summary: Ribosomal proteins are essential to life. While the functions of ribosomal protein-encoding genes (RPGs) are highly conserved, the evolution of their regulatory mechanisms is remarkably dynamic. In Saccharomyces cerevisiae, RPGs are unusual in that they are commonly present as two highly similar gene copies and that they are over-represented among intron-containing genes. To investigate the role of introns in the regulation of RPG expression, we constructed 16 S. cerevisiae strains with precise deletions of RPG introns. We found that several yeast introns function to repress rather than to increase steady-state mRNA levels. Among these, the RPS9A and RPS9B introns were required for cross-regulation of the two paralogous gene copies, which is consistent with the duplication of an autoregulatory circuit.

overall_design: Splicing specific microarrays were used to assess the genome-wide defects in gene expression and pre-mRNA splicing that result from a deletion of a single ribosomal protein gene intron.

first: Christine

last: Guthrie


title: Expression analysis of WS4303 (wild type)  vs WS5041 (mir-58 mutants) total mRNA

city: Cambridge

first: Marko

last: Jovanovic

state: MA

address: 7 Cambridge Center

department: Core Group (Regev group) member

institute: Broad Institute of MIT and Harvard

summary:
To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein (C. elegans strain WS4303). We crossed the TAP::ALG-1 transgene into the mir-58(n4640) mutant background to generate the strain WS5041. For simplicity, we will hereafter term the TAP::ALG-1 transgenic animals as “wild type” and the transgenic WS5041 animals as “mir-58”.
In addition to immunopurifying the TAP::ALG-1 and associated RNAs from these strains, we also compared total mRNA levels between these two strains.

overall_design: Total RNA was isolated from the same WS4303 and WS5041 total extracts that was further used for the TAP::ALG-1 RIP. Three independent biological replicates were analyzed. Long-oligo whole-genome C. elegans arrays, produced by the Genome Sequencing Center at Washington University in St. Louis (http://genome.wustl.edu/genome/celegans/microarray/ma_gen_info.cgi), were used for these experiments. A total of 10 µg of total RNA was used for cDNA synthesis.

first: Jovanovic

last: Marko

first: Hengartner

middle: O

last: Michael

first: Gerber

middle: P

last: Andre

first: Aebersold

last: Ruedi

first: Rehrauer

last: Hubert


title: Real-time quantitative PCR analysis of 88 microRNAs involved in human cell differentiation and development  in normal fibroblasts stimulated with exogenous TGF-β1 and systemic sclerosis (SSc) dermal fibroblasts

city: kumamoto

first: Noritoshi

last: Honda

state: kumamoto

address: 1-1-1 Honjo

department: Department of Dermatology and Plastic Surgery

country: Japan

institute: Kumamoto University

summary: Normal fibroblasts and SSc fibroblasts between the third and six subpassages were used for experiments. Normal and scleroderma fibroblasts were serum-starved for 24 hours and incubated in the presence or absence of TGF-β1 (2ng/ml) for 6 hours. Total RNA was extracted from culture cells with ISOGEN (Nippon Gene, Tokyo, Japan). MicroRNA isolation from total RNA was performed using RT2 qPCR-Grade miRNA Isolation Kit (SA Bioscience). For RT2 Profiler PCR Array (SABioscience), microRNAs were reverse-transcribed into first strand cDNA using RT2 miRNA First Strand Kit (SABiosciences). A mixture of equal amounts of cDNAs from 5 normal fibroblasts or 5 SSc fibroblasts was prepared. The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added into a 96-well RT2 miRNA PCR Array (SABiosciences) that included primer pairs for 88 human microRNAs.

overall_design: Human dermal fibroblasts were obtained by skin biopsy from the affected areas (dorsal forearm) of 5 patients with diffuse cutaneous SSc and <2 years of skin thickening. Control fibroblasts were obtained by skin biopsies from 5 healthy donors. Control donors were each matched with a SSc patient for age, sex, and biopsy site.


title: Purification of TAP::ALG-1 complexes and associated RNA from mixed-stage TAP::ALG-1 transgenic (WS4303) and wild-type (N2) animals

summary:
To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein
We purified TAP::ALG-1 complexes from mixed-stage TAP::ALG-1 transgenic (WS4303) and wild-type (N2, serving as a mock control) animals and hybridized the associated mRNAs to two-color microarrays

overall_design: RNA isolated from WS4303 and N2 (mock control) animals was analyzed for each IP sample and for each total worm extract sample. Three independent biological replicates have been performed. Long-oligo whole-genome C. elegans arrays, produced by the Genome Sequencing Center at Washington University in St. Louis (http://genome.wustl.edu/genome/celegans/microarray/ma_gen_info.cgi), were used for analysis. Log2 ratios (immunopurified RNA over total RNA from extract) were calculated for each gene. Microarrays generated from WS4303 animals were compared to microarrays generated from N2 mock control animals by a Student’s two sample t test.


title: Cardiac transcriptomic shifts during first 24h of thermal acclimation in the porcelain crab, Petrolisthes cinctipes

city: Tiburon

first: Jonathon

last: Stillman

address: 3152 Paradise Drive

department: Romberg Tiburon Center

institute: San Francisco State University

summary:
Intertidal zone organisms can experience transient freezing temperatures during winter low tides, but their extreme cold tolerance mechanisms are not known.  Petrolisthes cinctipes is a temperate mid-high intertidal zone crab species that can experience wintertime habitat temperatures below the freezing point of seawater.  We examined how cold tolerance changed during the initial phase of thermal acclimation to cold and warm temperatures, as well as the persistence of cold tolerance during long-term thermal acclimation.  Thermal acclimation for as little as 6 hours at 8˚C enhanced crab tolerance during a 1h exposure to -2°C relative to crabs acclimated to 18˚C.  Potential mechanisms for this enhanced tolerance were elucidated using cDNA microarrays to probe for differences in gene expression in cardiac tissue of warm and cold acclimated crabs during the first day of thermal acclimation.  No changes in gene expression were detected until 12h of thermal acclimation.  Genes strongly upregulated in warm acclimated crabs represented immune response and extracellular / intercellular processes, suggesting that warm acclimated crabs had a generalized stress response and may have been remodelling tissues or altering intercellular processes.  Genes strongly upregulated in cold acclimated crabs included many that are involved in glucose production suggesting that cold acclimation involves increasing intracellular glucose as a cryoprotectant.  Structural cytoskeletal proteins were also strongly represented among the genes upregulated in only cold acclimated crabs.   There were no consistent changes in composition or the level of unsaturation of membrane phospholipid fatty acids with cold acclimation, which suggests that neither short- nor long-term changes in cold tolerance are mediated by changes in membrane fatty acid composition.  Overall, our study demonstrates that initial changes in cold tolerance are likely not regulated by transcriptomic responses, but that gene expression-related changes in homeostasis begin within 12 hours – the length of a tidal cycle.
all array data and raw images archived at the Porcelain Crab Array Database (http://array.sfsu.edu)

overall_design:
n=264 specimens were divided into warm (18°C, n=96), cold (8°C, n=96), and control (13°C, n=72) acclimation groups.   Crabs were sampled from the 13°C group at 0 h (the start of the experiment) and 24 h, the termination of the experiment.  Crabs were sampled from the warm and cold acclimation groups at 6, 12, 18, and 24 hours following the start of thermal acclimation. At each time point, heart tissue from n=16 crabs from each group was dissected, flash frozen and stored at −80°C.
A pooled total aRNA sample was prepared for each group by mixing equal quantities of total RNA from n=5 individuals in each group in order to have the same amount of biological diversity within each pooled RNA sample.  For microarray hybridizations we used n=25 slides in an incomplete loop design where each sample was hybridized n=5 times, 2-3 times labelled with each Cy dye

first: Daria

last: Ronges

first: Brent

last: Sinclair

first: Jillian

last: Walsh


title: Developmental milestones punctuate gene expression in the Caenorhabditis embryo

city: Haifa

first: Itai

last: Yanai

address: Technion City

department: Biology

country: Israel

institute: Technion - Israel Institute of Technology

summary: The complex process by which a metazoan develops from a single cell to a multi-cellular differentiated organism is typically organized by biologists into stages. For example, in the chordate embryo, the gastrula, the neurula and the tail-bud constitute characteristic processes. A debate runs through the history of embryology questioning whether such stages hint at the modularity of embryonic development1-3, or rather, that “stages exist in the mind of the biologist, and not in the larva”4 or embryo. It thus remains unclear which position accurately reflects the nature of development on a molecular, gene regulatory level. Here we demonstrate that development of five Caenorhabditis species proceeds through multiple distinct stages in which the transcriptome is resistant to differences in species-specific developmental timings. By comparing the complete protein-coding transcriptomes of individually staged embryos across ten morphological markers, we found that time-invariant stages occur throughout development, including a stage we identify as the nematode phylotypic stage. Between such stages, embryos follow transitory states characterized by an acceleration of transcriptional activity. Therefore, on a molecular level, development cannot be viewed as a single process continuously proceeding through time, but rather as a succession of discontinuous stages, or ‘milestones’. Comparing the nematode developmental transcriptome with that of the chordate we report on the macro evolution of a milestone by duplication and diversification. This modular view of development by milestones will allow for a more complete understanding of how the functional organization of the embryo has influenced the evolution of animal morphology and diversity.

sample_organism:
Caenorhabditis briggsae
Caenorhabditis elegans
Caenorhabditis remanei
Caenorhabditis brenneri
Caenorhabditis japonica

overall_design: 150 microarrays, across 10 morphological markers of C. remanei, C. briggsae, C. brenneri, C. elegans and C. japonica wildtype embryos. For each of the two timecourse independent triplicates were generated.

first: Michal

last: Levin

first: Tamar

last: Hashimshony

first: Florian

last: Wagner


title: Transcriptional study of oxLDL-induced foam cell formation in WT and Atf3-/- murine bone marrow macrophages

status: Public on Mar 23 2012

city: Corvallis

first: Stephen

last: Ramsey

state: OR

address: Dryden Hall 208A

department: Biomedical Sciences

institute: Oregon State University

summary:
Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-loaded macrophages in the arterial wall. Intimal macrophages internalize modified lipoproteins such as oxidized LDL (oxLDL) through scavenger receptors, leading to storage of excess cholesteryl esters in lipid bodies and a "foam cell" phenotype. In addition, stimulation of macrophage Toll-like receptors (TLRs) has been shown to promote lipid body proliferation.  We investigated the possibility that there are transcriptional regulators that are common to both pathways for stimulating foam cell formation (modified lipoproteins and TLR stimulation), and identified the transcription factor ATF3 as a candidate regulator.

In this specific microarray experiment, we studied the effect of genetic knockout of ATF3 on the transcriptional response of macrophages to oxLDL. Murine bone marrow-derived macrophages from two different mouse strains (Atf3-/- and WT) were incubated in the presence or absence of oxidized low-density lipoprotein (oxLDL), and then transcriptionally profiled using the Affymetrix Mouse Exon Array 1.0 ST. The goal was to study the pattern of differential expression between WT and Atf3-/- strains, in oxLDL-induced foam cells and in non-foamy control cells, to identify cellular pathways that may be dysregulated under loss of the transcription factor ATF3 in macrophage foam cells.

overall_design: Twelve female mice (six Atf3-/-, and six WT control mice) were sacrificed at 8-12 weeks of age, and macrophages were derived from the femoral bone marrow using rhM-CSF. oxLDL was introduced into the medium for six samples (3 WT and 3 Atf3-/-) at 25 ug/mL on day seven, and after 24 h of incubation, RNA was isolated using Trizol. Labeled cRNA derived from the RNA samples was hybridized to Affymetrix Mouse Exon Array 1.0 ST GeneChips. Microarray data were processed using transcript-level probesets, and are in log2 scale.

first: Elizabeth

middle: S

last: Gold

first: Irina

last: Podolsky

first: Kathleen

last: Kennedy

first: Garnet

last: Navarro

first: Vladimir

last: Litvak

first: Aaron

last: Lampano

first: Alan

last: Aderem


title: Resolution of ntla-dependent transcriptome at 16 hpf using caged molecules

city: Stanford

first: James

last: Chen

address: 269 Campus Dr

department: Chemical and Systems Biology

institute: Stanford University

summary:
Transcription factors play diverse roles during embryonic development, combinatorially controlling multiple cellular states in a spatially and temporally defined manner.  Resolving the dynamic transcriptional profiles that underlie these patterning processes is essential for understanding embryogenesis at the molecular level.  Here we show how temporal, tissue-specific changes in embryonic transcription factor function can be discerned by integrating caged morpholinos (cMOs) with photoactivatable fluorophores, fluorescence-activated cell sorting (FACS), and microarray technologies.  As a proof of principle, we have dynamically profiled No tail-a (Ntla)-dependent genes at different stages of axial mesoderm development in zebrafish, discovering and characterizing discrete sets of transcripts that are coincident with either notochord cell fate commitment or differentiation.
Our studies demonstrate how optically controlled chemical tools can be use to probe developmental processes with spatiotemporal precision and reveal the sequential activation of distinct transcriptomes within a cell lineage by a single transcriptional factor.
Zebrafish zygotes were injected with a mixture of ntla caged morpholino (cMO) and caged fluorescein dextran (cFD), and a 100 µm-diameter region of the posterior chordamesoderm 125 μm anterior to the center of Kupffer’s vesicle was UV-irradiated at 12 hours post fertilization (hpf).  The UV irradiation generated an active morpholino targeting the ntla 5'UTR and simultaneously labeled the cells with green fluorescence.  By 36 hpf, the irradiated, green-fluorescent cells contributed to both notochord and floor plate, but the notochord cells were partially vacuolated and disorganized.  Combining these caged reagents with microarray analysis identified transcriptional changes coincident with loss of ntla at 16 hpf and subsequent notochord differentiation.

overall_design:
Zebrafish zygotes were injected with a mixture of ntla cMO/cFD, and a 100 µm-diameter region of the posterior chordamesoderm 125 μm anterior to the center of Kupffer’s vesicle was UV-irradiated at 12 hpf. Control embryos were injected with cFD and similarly irradiated at 12 hpf.  Experimental and control sets of embryos were enzymatically dissociated at 16 hpf, green-fluorescent cells were isolated by FACS, and collected in trizol.  Thirty embryos were used for each experiment or controls, each yielding approximately 8,000 green fluorescent cells.  Five biological replicates were performed along with five paired controls.  Total RNA was isolated from each sample, reverse transcribed, and amplified using WTA2 TransPlex Complete Whole Transcriptome Amplification kit (Sigma) using a miniaturized procedure and manufacturer-recommended incubation steps.  The synthesized cDNA was sent to Nimblegen for dye labeling and hybridization.
Samples were hybridized to the Nimblegen 2007 (Zv 7) Danio rerio Gene Expression Array chip using one dye per chip.  Raw probe data (.pair file) was subjected to RMA, normalization, background correction, and generation of gene expression summary (.calls file) by Nimblegen.  Processed data was analyzed in ArrayStar software (DNAStar).  Significantly affected genes (fold change > 2 and p-value < 0.10) were identified using a moderated t-test.

first: Ilya

last: Shestopalov

first: Cameron

middle: L

last: Pitt

middle: K


title: Superfolder GFP reporters validate diverse new mRNA targets of the classic porin regulator, MicF RNA

status: Public on Feb 17 2012

city: Würzburg

first: Kai

last: Papenfort

state: Würzburg

address: Josef-Schneider Str.2/BAU D15

department: RNA Biology

country: Germany

institute: IMIB Würzburg

summary: MicF is a textbook example of a small regulatory RNA (sRNA) that acts on a trans-encoded target mRNA through imperfect base paring. The discovery of MicF as a post-transcriptional repressor of the major Escherichia coli porin OmpF established the paradigm for a meanwhile common mechanism of translational inhibition, through antisense sequestration of a ribosome binding site. However, whether MicF regulates additional genes has remained unknown for almost three decades. Here, we have harnessed the novel superfolder variant of GFP for reporter-gene fusions to validate newly predicted targets of MicF in Salmonella. We show that the conserved 5’ end of MicF acts by seed pairing to repress the mRNAs of global transcriptional regulator Lrp, periplasmic protein YahO, and lipid A-modifying enzyme LpxR. Whilst MicF binds lrp and yahO in the 5’ UTR, it targets lpxR at both the ribosome binding site and deep within the coding sequence. Repression in the coding sequence of lpxR may be achieved by decreasing mRNA stability through exacerbating the use of a native RNase E site proximal to the short MicF-lpxR duplex. Altogether, this study assigns the classic MicF sRNA to the growing class of Hfqassociated regulators that use diverse mechanisms to impact multiple loci.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

overall_design: To determine the targets of the small regulatory RNA MicF in S. Typhimurium, we looked at the effect of a short pulse of MicF over-expression on the Salmonella transcriptome. To achieve over-expression, the micF gene was cloned in the pBAD plasmid and induced with 0.2% L-arabinose for 10 min. We then extracted the total RNA for transcriptional profiling. A strain carrying the pBAD plasmid w/o insert was used as negative control (also induced by L-arabinose). 3 biological replicates were performed. This sRNA target identification strategy has been described in Papenfort et al; Molecular Microbiology (2006) 62(6), 1674?1688.

first: Colin

last: Corcoran

first: Dimitri

last: Podkaminski

first: Johannes

last: Urban

first: Jay

last: Hinton

first: Jörg

last: Vogel


title: Expression in AtBBX32 Transgenic Soybean

city: Saint Louis

first: Jim

last: Morrell

state: Missouri

address: 800 North Lindbergh Blvd

department: Biotechnology

institute: Monsanto Co.

summary:
Transgenic soybean plants expressing AtBBX32 are taller, and have more nodes, flowers, pods, and seed. The effects of expression of AtBBX32 are most evident in altering expression of a subset of genes in the early morning hours.  In particular, AtBBX32 alters expression of the soybean clock genes TOC1 and LHY1
This experiment compares the response of AtBBX32 transgenic plants with a control plant under field grown conditions at five time points.

overall_design:
Four lines, five timepoints and three biological replicates for a total of 60 samples. Of the four lines, three were AtBBX32 transgenic lines (of which two were high yielding [Event1 and Event2], one was not [Event4]), and a non-transgenic control event [A3244].
Sample times: 3AM, 6AM, 9AM, Noon, and 3PM (ZT21, ZT0, ZT3, ZT6 and ZT9) (Sunrise was at 6:08 AM and Sunset was at 8:08 PM)
Development Stage and Tissue:  Trifoliate leaf at R5 stage (80 days post-emergent)
Each of the three bioreps was a pool of three plants.
The field was located in central Illinois


title: Timecourse of mAbs 18B7 or MOPC-21 (control) binding to Cryptococcus neoformans serotype D strains JEC21 or 24067 (52D) after 1, 2 or 4 hours at 37 C.

status: Public on Feb 23 2012

city: Murfreesboro

first: Erin

last: McClelland

state: TN

address: 1301 East Main Street, P.O. Box 60

institute: Middle Tennessee State University

summary: Changes in gene expression of serotype D strains JEC21 or 24067 (52D) were examined after incubation with mAb 18B7 or the control MOPC-21 for 1, 2 or 4 hours at 37 C.

platform_organism:
Cryptococcus neoformans
Cryptococcus deneoformans

overall_design: Analysis used incubation with a non-binding IgG1 mAb as a control for incubation with a protective IgG1 mAb 18B7 at different timepoints.

middle: E


title: Resolution of ntla-dependent transcriptome at 9 hpf using caged molecules

summary:
Transcription factors play diverse roles during embryonic development, combinatorially controlling multiple cellular states in a spatially and temporally defined manner.  Resolving the dynamic transcriptional profiles that underlie these patterning processes is essential for understanding embryogenesis at the molecular level.  Here we show how temporal, tissue-specific changes in embryonic transcription factor function can be discerned by integrating caged morpholinos (cMOs) with photoactivatable fluorophores, fluorescence-activated cell sorting (FACS), and microarray technologies.  As a proof of principle, we have dynamically profiled No tail-a (Ntla)-dependent genes at different stages of axial mesoderm development in zebrafish, discovering and characterizing discrete sets of transcripts that are coincident with either notochord cell fate commitment or differentiation.
Our studies demonstrate how optically controlled chemical tools can be use to probe developmental processes with spatiotemporal precision and reveal the sequential activation of distinct transcriptomes within a cell lineage by a single transcriptional factor.
Zebrafish zygotes were injected with a mixture of ntla caged morpholino (cMO) and caged fluorescein dextran (cFD), and a 100 µm-diameter region of the shield was UV-irradiated at 6 hours post fertilization (hpf).  The UV irradiation generated an active morpholino targeting the ntla 5'UTR and simultaneously labeled the cells with green fluorescence.  By 36 hpf, the irradiated, green-fluorescent cells contributed to the medial floor plate rather than the notochord, consistent with earlier proposals that Ntla acts as a transcriptional switch between these two cell fates.  Combining these caged reagents with microarray analysis identified transcriptional changes coincident with loss of ntla at 9 hpf and subsequent notochord fate respecification.

overall_design:
Zebrafish zygotes were injected with a mixture of ntla cMO/cFD, and a 100 µm-diameter region of the shield was UV-irradiated at 6 hpf. Control embryos were injected with cFD and similarly irradiated at 6 hpf.  Experimental and control sets of embryos were enzymatically dissociated at 9 hpf, green-fluorescent cells were isolated by FACS, and collected in trizol.  Thirty embryos were used for each experiment or controls, each yielding approximately 8,000 green fluorescent cells.  Five biological replicates were performed along with five paired controls.  Total RNA was isolated from each sample, reverse transcribed, and amplified using WTA2 TransPlex Complete Whole Transcriptome Amplification kit (Sigma) using a miniaturized procedure and manufacturer-recommended incubation steps.  The synthesized cDNA was sent to Nimblegen for dye labeling and hybridization.
Samples were hybridized to the Nimblegen 2007 (Zv 7) Danio rerio Gene Expression Array chip using one dye per chip.  Raw probe data (.pair file) was subjected to RMA, normalization, background correction, and generation of gene expression summary (.calls file) by Nimblegen.  Processed data was analyzed in ArrayStar software (DNAStar).  Significantly affected genes (fold change > 2 and p-value < 0.10) were identified using a moderated t-test.


title: microRNA profiles of hematopoietic derivatives

status: Public on Feb 18 2012

city: Hamilton

first: Mickie

last: Bhatia

state: Ontario

address: 1200 Main Street West

department: Stem Cell and Cancer Research Institute (SCC-RI)

country: Canada

institute: McMaster University

summary: Hematopoietic stem cells (HSCs) can regenerate the entire hematopoietic system in vivo, providing the most relevant criteria to measure candidate HSCs derived from human embryonic stem cell (hESC) or induced pluripotent stem cell (hiPSC) sources. Here, we show that unlike primitive hematopoietic cells derived from hESCs, phenotypically identical cells derived from hiPSC are more permissive to graft the bone marrow of xenotransplantation recipients. Despite establishment of bone marrow graft, hiPSC-derived cells fail to demonstrate hematopoietic differentiation in vivo. However, once removed from recipient bone marrow, hiPSC-derived grafts were capable of in vitro multilineage hematopoietic differentiation, indicating that xenograft imparts a restriction to in vivo hematopoietic progression. This failure to regenerate multilineage hematopoiesis in vivo was attributed to the inability to downregulate key microRNAs involved in hematopoiesis. Based on these analyses, our study indicates that hiPSCs provide a beneficial source of pluripotent stem cell-derived hematopoietic cells for transplantation compared with hESCs. Since use of the human-mouse xenograft models prevents detection of putative hiPSC-derived HSCs, we suggest that new preclinical models should be explored to fully evaluate cells generated from hiPSC sources.

overall_design: Human pluripotent stem cell-derived hematopoietic cells were isolated and qPCR-based microRNA profiling was performed.

first: Ruth

last: Risueno

first: Eleftherios

last: Sachlos

first: Jong

last: Lee

first: Jung

first: Seok

last: Hong

first: Eva

last: Szabo


title: Expression analysis of Magnetospirillum magneticum AMB-1 WT, ΔMAI, ΔctrA, ΔdivK, and ΔctrA complemented with CtrA D51E, CtrA D51A, and an empty vector control

city: Berkeley

first: Arash

last: Komeili

address: 111 Koshland Hall #3102

department: Plant and Microbial Biology

institute: UC Berkeley

summary:
Investigation of whole genome expression changes in Magnetospririllum magneticum mutants, probing the role of the CtrA regulatory pathway.
The mutants are further described in a manuscript submitted for publication at J. Bacteriology.
Developmental events across the prokaryotic life cycle are highly regulated at the transcriptional and post-translational levels.  Key elements of a few regulatory networks are conserved among phylogenetic groups of bacteria, although the features controlled by these conserved systems are as diverse as the organisms encoding them.  In this work, we probe the role of the CtrA regulatory network, conserved throughout the Alphaproteobacteria, in the magnetotactic bacterium, Magnetospirillum magneticum strain AMB-1, which possesses unique intracellular organization and compartmentalization.  While we show that CtrA in AMB-1 is not essential for viability, it is required for motility, and its putative phosphorylation state dictates the ability of CtrA to activate the flagella biosynthesis gene cascade.  Gene expression analysis of strains expressing active and inactive CtrA alleles point to the composition of the extended CtrA regulon, including both direct and indirect targets.  These results, combined with a bioinformatic study of the AMB-1 genome, enabled the prediction of an AMB-1 specific CtrA binding site.  Further, phylogenetic studies comparing CtrA sequences from Alphaproteobacteria in which the role of CtrA has been experimentally examined reveals an ancestral role of CtrA in the regulation of motility and suggests that its essential functions in other Alphaproteobacteria were acquired subsequently.

overall_design: Total RNA was recovered from each of the wild-type and mutant strains, reverse transcribed to cDNA, fluorescently labeled, and hybridized to whole genome microarrays. The arrays contain 7 probes/gene, with the entire genome duplicated twice. In addition, the arrays contain 7 probes for 22 unannotated ORFs and tiling of a genomic region from coordinates 977403-1097027.

first: Shannon

last: Greene


title: ChIP-chip of RNA Polymerase (RNAP) and RpoD in Salmonella enterica serovar Typhimurium strain SL1344

status: Public on Feb 16 2012

city: Dublin

first: Shane

last: Dillon

address: Trinity College

department: Department of Microbiology

country: Ireland

institute: Moyne Institute of Preventive Medicine

type: Genome binding/occupancy profiling by genome tiling array

summary: ChIP-on-chip analysis of RNAP and RpoD binding to the Salmonella enterica serovar Typhimurium chromosome demonstrated a high degree of overlap between RNAP and RpoD binding and provided us with important insights into the global distribution of these factors. Furthermore this data was correlated with information on the location of 1873 transcription start sites identified by RNA-Seq technology, thereby providing a detailed transcriptional map of Salmonella Typhimurium.

platform_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

overall_design: Analysis of RNAP, RNAP-Rifampicin and and RpoD binding in Luria Broth (LB)

first: Andrew

middle: D

last: Cameron

first: Carsten

last: Kroger

first: Karsten

last: Hokamp

first: Charles

last: Dorman


title: Effect of lin-28 and lin-46 on miRNA profile in late L1 stage Caenorhabditis elegans

status: Public on Feb 09 2012

city: Stratford

first: Eric

last: Moss

state: NJ

address: 2 Medical Center Dr.

department: Molecular Biology

institute: UMDNJ

summary: Comparison of miRNA profiles of wildtype and lin-28(n719); lin-46(ma164) Caenorhabditis elegans nematodes at the L1 stage

overall_design: Two genotypes, wildtype vs. mutant. Biological replicates: 3 wild type, 3 mutant, independently grown and harvested. One replicate per slide.

first: Bhaskar

last: Vadla

middle: G


title: Gene expression variation in horse placental and fetal tissue, and resting and stimulated horse lymphocytes [Agilent-018932]

status: Public on Feb 14 2012

city: Ithaca

first: Donald

last: Miller

state: NY

address: Hungerford Hill Rd.

department: Baker Institute for Animal Health

institute: Cornell University

summary: We undertook gene expression microarray experiments to identify genes that are differentially expressed in different placental and fetal tissue, and resting and Pokeweed Mitogen (PWM) stimulated horse lymphocytes.  Conceptus tissues were dissected to obtain chorionic girdle, chorion, and fetal tissue.  Freshly isolated horse peripheral blood lymphocytes were split and harvested immediately, or stimulated with PWM and harvested over a five day period. These experiments utilized a custom Agilent horse array designed in house that featured >14,000 probes on an 8x15k array format.  Several genes were selected from the results for validation by quantitative real-time PCR.  QPCR results matched the microarray results very closely.

overall_design: Four Day 33-35 chorionic girdle RNAs were compared to matching chorion RNAs, and fetal tissue from two of the conceptuses.  Gene expression in resting lymphocytes was compared to gene expression in PWM treated lymphocytes.

first: Douglas

middle: F

last: Antczak


title: Gene expression in response to chloroplastic H2O2: IS-GUS-2-17 vs. IS-tAPX-19-23 under estrogen treatment

status: Public on Feb 04 2012

city: Matsue

first: Takanori

last: Maruta

address: 1060 Nishikawatsu

institute: Shimane University

summary: To identify chloroplastic H2O2-responseive genes, an estrogen-inducible RNAi method was used for silencing of tAPX, a H2O2-scavenging enzyme in chloroplasts. At 48 h after treatment with estrogen, the expression of  tAPX was silenced in the IS-tAPX-19-23 plants, but not in the IS-GUS-2-17 plants (negative control), resulting a large change in gene expression. These genes are candidates for chloroplastic H2O2-responseive genes.

overall_design: IS-GUS-2-17 vs. IS-tAPX-19-23 leaves. Biological replicates 2.

first: Masahiro

last: Noshi

first: Aoi

last: Tanouchi


title: Analysis of microRNAs in bovine early embryonic development

status: Public on Feb 08 2012

city: Québec City

first: Marc-André

last: Sirard

state: Quebec

address: Offfice 2732, 2440 Hochelaga Blvd.

department: Sciences Animales

institute: Université Laval

summary: In animals, the maternal-to-embryonic transition (MET) is an important step occurring in the first days of early development. This important transition involves the degradation of maternal transcripts that have been stocked during oogenesis and used until this transition. Moreover, some precise and specific control mechanisms must govern the adequate synchronization of the MET events to promote embryonic genome activation. These mechanisms are not well understood, but microRNAs could be one of the mechanisms involved. MicroRNAs are short non-coding RNAs involved in a growing number of pathways by their post-transcriptional repression of mRNA translation. Thus, we analyzed microRNA expression during oocyte maturation by comparing GV-oocytes with MII-oocytes in cow.

platform_organism:
Lemur catta
Saguinus imperator
Ateles sp.
Lagothrix
Macaca mulatta
Macaca nemestrina
Symphalangus syndactylus
Gorilla gorilla
Pan paniscus
Pan troglodytes
Pongo abelii
Homo sapiens
Canis lupus familiaris
Sus scrofa
Bos taurus
Ovis aries
Cricetulus griseus
Mus musculus
Rattus norvegicus
Monodelphis domestica
Rhinopithecus bieti

overall_design: Groups of 300 Oocytes_GV and Oocytes_MII were used for miRNA expression analysis. The miRNA expressions were determined using the MiRMammalia_11.0 array from LC Sciences.

first: Elise

last: Mondou

first: Isabelle

last: Dufort

first: Marc-Andre


title: Developmental characterization of the microRNAs-specific C. elegans Argonautes alg-1 and alg-2

status: Public on Feb 03 2012

middle: A.

last: Miska

address: Tennis Court Rd

department: Wellcome Trust/Cancer Research UK Gurdon Institute, The Henry We

country: United Kingdom

institute: University of Cambridge

summary: This study analyzes miRNA association with ALG-1 and ALG-2 in different stages during larval development of C. elegans

overall_design: Staged animals (L1, L2, L3, L4) - the alg-2 mutant expressing tagged-ALG-2, alg-1 mutant, and wild type controls - were lysed.  ALG-1 IP or ALG-2 (GFP) IP was performed using tags from all four samples at all four timepoints. Small RNAs were released from antibodies and 5' end-labelled with AlexaFluor532. Labelled RNA was hybridized to a custom microRNA microarray platform to quantify miRNA content.

first: Martin

last: Simard

first: Javier

last: Armisen


title: Expression analysis of Drosophila melanogaster's primordial germ cells and somatic cells in wild type and smaug mutants

city: Toronto

first: Howard

last: Lipshitz

state: ON

address: 1 King's College Circle

department: Molecular Genetics

institute: University of Toronto

summary: Background: During the maternal-to-zygotic transition (MZT) vast changes in the embryonic transcriptome are produced by a combination of two processes: elimination of maternally provided mRNAs and synthesis of new transcripts from the zygotic genome. Previous genome-wide analyses of the MZT have been restricted to whole embryos. Here we report the first such analysis for primordial germ cells (PGCs), the progenitors of the germ-line stem cells.  Results: We purified PGCs from Drosophila embryos, defined their proteome and transcriptome, and assessed the content, scale and dynamics of their MZT. Transcripts encoding proteins that implement particular types of biological functions group into nine distinct expression profiles, reflecting coordinate control at the transcriptional and posttranscriptional levels. mRNAs encoding germ-plasm components and cell-cell signaling molecules are rapidly degraded while new transcription produces mRNAs encoding the core transcriptional and protein synthetic machineries. The RNA-binding protein, Smaug, is essential for the PGC MZT, clearing transcripts encoding proteins that regulate stem cell behavior, transcriptional and posttranscriptional processes. Computational analyses suggest that Smaug and AU-rich element binding-proteins function independently to control transcript elimination.  Conclusion: The scale of the MZT is similar in the soma and PGCs. However, the timing and content of their MZTs differ, reflecting the distinct developmental imperatives of these cell types. The PGC MZT is delayed relative to that in the soma, likely because relief of PGC-specific transcriptional silencing is required for zygotic genome activation as well as for efficient maternal transcript clearance.

overall_design: There are 26 samples in total, including 1-to-3 hour,  3-to-5 hour, 5-to-7 hour PGCs in wild type and smaug mutant flies and 1-to-3 hour somatic cells in wild type flies. Except for the 1-to-3 hour and 3-to-5 hour smaug mutant PGCs which have three replicates, all the other samples have four replicates.


title: Transcriptional analyses of the ectomycorrhizal (ECM) fungus Paxillus involutus during growth on various lignocellulose soil substrates

status: Public on Feb 01 2012

city: Lund

first: Tomas

last: Johansson

address: Sölvegatan 37

department: MEMEG

institute: Lund University

summary:
In this study, examinations were performed on how the ECM fungus Paxillus involutus degrade complex, plant and litter material by using elemental analyses, FTIR spectroscopy, pyrolysis-GC/MS, and synchronous fluorescence spectroscopy together with microarray analyses screening 12,214 gene models, derived from 454 sequenced cDNA libraries.
Rineau, F., Roth, D., Shah, F., Smits, M., Johansson, T., Canbäck, B., Bjarke Olsen, P., Persson, P., Nedergaard Grell, M., Lange, L., & Tunlid, A. (201X) Expression levels tune enzymatic exploitation of plant litter material by ectomycorrhizal fungi (manuscript in preparation).

overall_design: A 18-subarray  study (data from 18 subarrays collected from two 12-plex microarrays (IDs 468335 and 468400) using total RNA recovered from three separate wild-type glass-bead cultures after amendments of various soil-derived substrates.

first: Johansson

last: Tomas

first: Tunlid

last: Anders


title: Innate immunity and the evolution of resistance to an emerging infectious disease in a wild bird

status: Public on Feb 24 2012

city: Moulis

first: Camille

last: Bonneaud

address: Station d'Ecologie Experimentale

institute: Centre National de la Recherche Scientifique (CNRS)

summary: Innate immunity is expected to play a primary role in conferring resistance to novel infectious diseases, but few studies have attempted to examine its role in the evolution of resistance to emerging pathogens in wild vertebrate populations.  Here we used experimental infections and cDNA microarrays to examine whether changes in the innate and/or acquired immune responses likely accompanied the emergence of resistance in house finches (Carpodacus mexicanus) in the eastern United States subject to a recent outbreak of conjunctivitis-causing bacterium (Mycoplasma gallisepticum- MG).  Three days following experimental infection with MG, we observed differences in the splenic transcriptional responses between House Finches from eastern U.S. populations, with a 12-year history of MG exposure, versus western U.S. populations, with no history of exposure to MG.  In particular, western birds down-regulated gene expression, while eastern finches showed no expression change relative to controls.  Studies involving poultry have shown that MG can manipulate host immunity, and our observations suggest that pathogen manipulation occurred only in finches from the western populations, outside the range of MG.  Fourteen days after infection, eastern finches, but not western finches, up-regulated genes associated with acquired immunity (cell-mediated immunity) relative to controls.  These observations suggest population differences in the temporal course of the response to infection with MG, and imply that innate immune processes were targets of selection in response to MG in the eastern U.S. population.

overall_design: Birds were randomly selected to be kept either as controls or infected via ocular inoculation with 20 μl of culture containing 1 x 104 to 1 x 106 color changing units/ml of an early 2007 Auburn MG isolate.  All infected birds were inoculated with precisely the same volume of the same culture.  Control birds were sham infected using sterile SP4 medium (Whitcomb 1983). Infected birds were euthanized three days (N=6 from Arizona and N=11 from Alabama) and 14 days (N=11 from Arizona and N=12 from Alabama) after treatment.  Control birds were euthanized 14 days after sham-inoculation;   Control (N=11 birds from Arizona and 9 from Alabama) and infected birds were maintained under identical conditions, but in separate rooms of an aviary. Infected birds were euthanized three days (N=6 from Arizona and N=11 from Alabama) and 14 days (N=11 from Arizona and N=12 from Alabama) after treatment.  Control birds were euthanized 14 days after sham-inoculation. We used a common reference design (Yang & Speed 2002), in which we pooled 2 to 6 spleens from birds from the same population in the same treatment to generate enough mRNA for microarray hybridizations and hybridized two pools for each treatment from each population.

first: Susan

last: Balenger

first: Jiangwen

last: Zhang

first: Scott

middle: V

last: Edwards

first: Geoffrey

last: Hill


title: Antibody detection of translocations (ADOT) in Ewing Sarcoma

status: Public on Jan 31 2012

city: Salt Lake City

first: Wen

last: Luo

state: UT

address: 2000 Circle of Hope

institute: Huntsman Cancer Institute

summary: We developed a new approach called antibody detection of translocations (ADOT) which combines a transcriptional microarray-based approach with a novel antibody-based detection method to detect translocations in cancer. ADOT allows for the accurate and sensitive identification of translocations and provides exon-level information about the fusion transcript. ADOT can detect translocations in poor quality unprocessed total RNA. We demonstrate the feasibility of ADOT by examples in which both known and unknown Ewing sarcoma translocations are identified from cell lines, tumor xenografts, and FFPE primary tumors. These results demonstrate that ADOT may be an effective approach for translocation analysis in clinical specimens with significant RNA degradation and may offer a novel diagnostic tool for translocation-based cancers.

overall_design: We designed oligonucleotide probes for each possible exon-exon combination between potential fusion partners and printed the DNA oligonucleotides on custom-designed microarrays. Total RNA from tumor cells or tissues was hybridized on the array. Bound RNA was detected with the S9.6 monoclonal antibody that recognizes RNA-DNA duplexes in a sequence-independent fashion,and detected with Cy3-labeled anti-mouse IgG.

first: Stephen

last: Lessnick

first: Brett

last: Milash

first: Brian

last: Dalley

first: Richard

last: Smith

first: Holly

last: Zhou

first: Natalie

last: Dutrow

first: Bradley

last: Cairns


title: Differential miroRNA expression in round spermatids of GRTH null mice compared to wild type.

city: Bethesda

first: Chon-Hwa

last: Tsai-Morris

state: Md

address: Bldg. 49, Rm 6B03, 49 Convent Drive

department: PDEGEN, NICHD, NIH

summary: miRNA expression profiles for round spermatids of wild type and GRTH knock-out mice were determined by Rodent TaqMan® Low Density miRNA Arrays A v2.0 (TLDA, Applied Biosystem).

overall_design: Purified round spermatids were prepared from the testis of wild type and GRTH null mice (C57BL/6 strain).  Equal amount of total RNA from 20 mice (wild type or GRTH KO)  was pooled prior to gene expression analysis.

first: C

first: M

last: Dufau


title: Changes in gene expression profile during the growth on ethanol in Acetobacter aceti

city: Bunkyo-ku

first: Hiroyuki

last: Arai

state: Tokyo

address: Yayoi 1-1-1

department: Department of Biotechnology

institute: The University of Tokyo

summary: Time-dependent transcriptome change in the A. aceti cells during growth on ethanol was determined by NimbleGen Eukaryotic Expression array (4x72K).

overall_design: Acetobacter aceti NBRC14818 was cultivated in the medium containing ethanol in Erlenmeyer flask with rotary shaking. Total RNA was extracted when optical density at 600 nm was 0.1, 0.2, 0.6, or 1.2. The experiment was performed in duplicate independent cultures.

first: Kenta

last: Sakurai


title: DNA methylation analysis by MeDIP-on-chip in mouse E13.5 PGCs

city: Illkirch

first: Michael

last: Weber

address: 300 Bd Sebastien Brant

department: UMR7242 Biotechnology and Cell Signalling

institute: CNRS

type: Methylation profiling by genome tiling array

summary:
Epigenetic reprogramming, characterized by loss of cytosine methylation and histone modifications, occurs during mammalian development in primordial germ cells (PGCs).
Here we provide a detailed map of cytosine methylation on a large portion of the genome in developing male and female PGCs isolated from mouse embryos.

overall_design: We mapped DNA methylation in E13.5 PGCs isolated by FACS sorting from Oct4-GFP male and female embryos. As a control, we also mapped DNA methylation in E7.5 epiblasts from wich PGCs derive from. MeDIP and Input samples were hybridized to Nimblegen HD2 MM8 promoter deluxe arrays covering 12 kb of all gene promoters. Experiments were performed in duplicates for E7.5 epiblasts and triplicates for E13.5 PGCs.

first: Sylvain

last: Guibert


title: WT vs KO mecA  comparison

city: Louvain-La-Neuve

first: Pascal

last: Hols

address: Place Croix du Sud 5

department: Institut des Sciences de la Vie

country: Belgium

institute: Université catholique de louvain

summary:
Transcriptional profiling of S. thermophilus LMD-9 wild-type compared to the isogenic mutant strain CB052 (deletion of mecA, STER_0216) for the identification of the MecA regulon.
Cells were grown in THB medium supplemented with glucose 1% (THBG) and sampled at OD600 = 0.4 for mRNA extraction

overall_design: wild-type vs  knockout, 2 WT biological replicates (one used as technical replicate for a dye swap), 2 KO biological replicates (one used as technical replicate for a dye swap)

first: Céline

last: Boutry

first: Laetitia

last: Fontaine

first: André

last: Clippe


title: WT vs KO comX comparison

summary:
Transcriptional profiling of S. thermophilus LMD-9 wild-type compared to the isogenic mutant strain CB003 (deletion of comX, STER_0189) for the identification of the ComX regulon.
Cells were grown in CDM medium supplemented with lactose 1% (CDML) and sampled at OD600 = 0.4 for mRNA extraction


title: Differences in the transcriptomes of organically and conventionally grown potatoes.

city: Wageningen

first: Jeroen

last: van Dijk

address: Bornsesteeg 45

institute: RIKILT, institute of food safety

summary: Microarray analysis was performed on a well-defined set of potato tuber samples grown under different, well-recorded environmental conditions. The data were analysed to assess the potential of transcriptomics to detect differences in gene expression as a result of these environmental conditions. Differences were found for both factors, both in PCA and in ANOVA analysis.

overall_design: Factorial design; 1 potato cultivar (Sante); 2 fertilizers (organic, conventional); 2 crop protection treatments (organic, conventional), 4 biological replicates, 16 samples.  Raw data files: columns 1 - 11 is Cy3, 12 - 21 is Cy5

first: Kok

last: Esther

first: Katarina

last: Cankar

first: Beenen

last: Henriek


title: An antibiotic that inhibits a late step in wall teichoic acid biosynthesis induces the cell wall stress stimulon in Staphylococcus aureus

status: Public on Jan 12 2012

city: Normal

first: Arunachalam Muthaiyan

email: amuthai@gmail.com, bjwilkin@ilstu.edu

state: IL

address: Julian Hall

department: Biological Sciences

institute: Illinois State University

summary: Wall teichoic acids (WTAs) are phosphate-rich, sugar-based polymers attached to the cell walls of most Gram-positive bacteria. In Staphylococcus aureus, these anionic polymers regulate cell division, protect cells from osmotic stress, mediate host colonization, and mask enzymatically susceptible peptidoglycan bonds.  Although WTAs are not required for survival in vitro, blocking the pathway at a late stage of synthesis is lethal.  We recently discovered a novel antibiotic, targocil, that inhibits a late acting step in the WTA pathway. Its target is TarG, the transmembrane component of the ABC transporter (TarGH) that exports WTAs to the cell surface. Here we examine the effects of targocil on S. aureus using transmission electron microscopy (TEM) and gene expression profiling. We report that targocil treatment leads to multicellular clusters containing swollen cells displaying evidence of osmotic stress, strongly induces the cell wall stress stimulon, and reduces the expression of key virulence genes, including dltABCD and capsule genes. We conclude that WTA inhibitors that act at a late stage of the biosynthetic pathway may be useful as antibiotics, and we present evidence that they could be particularly useful in combination with beta-lactams.

overall_design: Growth conditions for microarray analysis-For transcriptional profiling, an overnight S. aureus SH1000 culture was diluted (2% (v/v) into 20 mL TSB medium in a 50 mL Erlenmeyer flask and grown at 37 °C with shaking at 200 rpm. For the targocil treatment experiments, S. aureus was grown to an OD600 ~0.4 and challenged with 8x MIC of targocil dissolved in DMSO) for 30 min. Simultaneously, an equal amount of DMSO (final concentration 2% (v/v)) was added to the control culture, which was incubated along with the treated cultures. After 30 min, 5 mL aliquots were collected from control and treated cultures and used for toatl RNA preparation.

first: Jennifer

last: Campbell

first: Atul

last: Singh

first: Jonathan

last: Swoboda

last: Gilmore

first: Brian

last: Wilkinson

first: Suzanne

last: Walker


title: Role of miRNAs in the differentiation of human embryonic stem cells to an endothelial cell lineage

status: Public on Jan 01 2012

city: London

first: Nicole

middle: Margaret

last: Kane

address: 30 Guilford Street

institute: University College London

summary: To assess changes in the miRNA-ome during the early stages of endothelial differentiation, the expression of mature human miRNAs at days 2, 4 and 10 of endothelial differentiation (vs pluripotent time-matched samples) in SA461 hESC cell line was analysed in a two-channel microarray. Expression of the pluripotency-associated miRNAs miR-302a – d and miR-372 and miR-373 was significantly suppressed with progression of differentiation. The expression of miR-99b, miR-181a and miR-181b was, in contrast, increased in a time- and differentiation-dependent manner compared to time-matched pluripotent samples. The expression profiles were validated and good concordance was observed between microarray, Q-PCR and Northern blot analysis.

overall_design: This experiment includes a total of 28 samples which are divided in to 3 groups, each with 4 biological repeats: group 1, Parental pluripotent hESC control sample. group 2, time-matched pluripotent hESC control samples. Group 3, hESC cells directed toward endothelial differentiation at time points 2 days, 4 days and 10 days.

middle: M

first: John

last: McClure

first: Andrew

last: Baker


title: Copy number variation of individual cattle genomes using next-generation sequencing.

status: Public on Jan 25 2012

city: Beltsville

first: George

last: Liu

state: MD

address: 10300 Baltimore Ave

institute: United States Department of Agriculture

summary: Copy number variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often intractable. Using a read depth approach based on next-generation sequencing, we examined genome-wide copy number differences among five taurine (three Angus, one Holstein and one Hereford) and one indicine (Nelore) cattle. Within mapped chromosomal sequence, we identified 1,265 CNV regions comprising ~55.6 Mbp sequence-476 of which (~38%) have not previously been reported. We validated this sequence-based CNV call set with aCGH, qPCR and FISH, achieving a validation rate of 82% and a false positive rate of 8%. We further estimated absolute copy numbers for genomic segments and annotated genes in each individual. Surveys of the top 25 most variable genes revealed that the Nelore individual had the lowest copy numbers in 13 cases (~52%, chi squared test; p value <0.05). In contrast, genes related to pathogen- and parasite-resistance, such as CATHL4 and ULBP17, were highly duplicated in the Nelore individual relative to the taurine cattle, while genes involved in lipid transport and metabolism, including APOL3 and FABP2, were highly duplicated in the beef breeds. These CNV regions also harbor genes like BPIFA2A (BSP30A) and WC1, suggesting that some CNVs may be associated with breed-specific differences in adaptation, health, and production traits. By providing the first individualized cattle CNV and segmental duplication maps and genome-wide gene copy number estimates, we enable future CNV studies into highly duplicated regions in the cattle genome.

overall_design: 5 NimbleGen Bos Taurus UMD3 custom 2.1M whole genome high density aCGH

first: Derek

last: Bickhart

first: Yali

last: Hou

first: Steven

last: Schroeder

first: Can

last: Alkan

first: Maria

last: Cardone

first: Lakshmi

last: Matukumalli

first: Jiuzhou

last: Song

first: Robert

last: Schnabel

first: Mario

last: Ventura

first: Jeremy

last: Taylor

first: Jose

last: Garcia

first: Curt

last: VanTassell

first: Tad

last: Sonstegard

first: Evan

last: Eichler


title: Mesenchymal progenitors from hESCs & profiling vs. BMMSCs

city: Taipei

first: Men-Luh

last: Yen

address: No.1, Sect. 1, Jen-Ai Road

country: Taiwan

institute: National Taiwan University

summary: New potential sources of stem cells for clinical application include bone marrow mesenchymal stem cells (BMMSCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (iPS).  However, each source is not without its own concerns.  While research continues in an effort overcome these problems, the generation of mesenchymal progenitors from existing hESC lines may circumvent many of these issues.  We report here a comparison of the transcriptome of hESC-derived mesenchymal progenitors (EMPs), its parental hESC line, and 2 donors of adult BMMSCs.

overall_design: A custom-designed oligo-microarray of just over 11,000 highly-expressed genes in stem cells was used to profile the transcriptome of BMMSCs, EMPs, and hESCs.

first: Min-Liang

last: Kuo


title: Intracellular transcription by multiple Listeria monocytogenes strains

status: Public on Dec 02 2011

city: Chicago

first: Jess

last: Leber

address: 920 E. 58th Street CLSC 615

department: Microbiology

institute: The University of Chicago

summary: These studies were designed to examine the transcription of Listeria monocytogenes strains 10403S and LO28 during intracellular replication in mammalian macrophages.

platform_organism: Listeria monocytogenes serotype 4b str. F2365

platform_organism: Listeria monocytogenes serotype 1/2a str. F6854

platform_organism: Listeria monocytogenes serotype 4b str. H7858

overall_design: Duplicate WT Listeria monocytogenes (strains 10403S and LO28) were used to infect mouse bone marrow-derived macrophages (BMMs).  Bacterial RNA was harvested at 4 hours post-infection.


title: Microarray-based transcriptomic analysis of differences between long-term gregarious and solitarious desert locusts

status: Public on Nov 25 2011

city: Leuven

first: Jozef

last: Vanden Broeck

address: Naamsestraat 59

department: Animal Physiology and Neurobiology

institute: KU Leuven

summary: Desert locusts (Schistocerca gregaria) show an extreme form of phenotypic plasticity and can transform between a cryptic solitarious phase and a swarming gregarious phase. The two phases differ extensively in behavior, morphology and physiology but very little is known about the molecular basis of these differences. We used our recently generated Expressed Sequence Tag (EST) database derived from S. gregaria central nervous system (CNS) to design oligonucleotide microarrays and compare the expression of thousands of genes in the CNS of long-term gregarious and solitarious adult desert locusts. This identified 214 differentially expressed genes, of which 40% have been annotated to date. These include genes encoding proteins that are associated with CNS development and modeling, sensory perception, stress response and resistance, and fundamental cellular processes. Our microarray analysis has identified genes whose altered expression may enable locusts of either phase to deal with the different challenges they face. Genes for heat shock proteins and proteins which confer protection from infection were upregulated in gregarious locusts, which may allow them to respond to acute physiological challenges. By contrast the longer-lived solitarious locusts appear to be more strongly protected from the slowly accumulating effects of ageing by an upregulation of genes related to anti-oxidant systems, detoxification and anabolic renewal. Gregarious locusts also had a greater abundance of transcripts for proteins involved in sensory processing and in nervous system development and plasticity. Gregarious locusts live in a more complex sensory environment than solitarious locusts and may require a greater turnover of proteins involved in sensory transduction, and possibly greater neuronal plasticity.

platform_organism:
Locusta migratoria
Schistocerca gregaria

overall_design: Two condition experiment: Solitarious central nervous system (CNS) vs gregarious CNS. 6 biological repeats per condition, balanced over 3 male and 3 females samples. The overall experimental design consisted of two identical repeats of an n+2 A-optimal design, organized per sex (n=6 per sex, 3 solitarious - 3 gregarious samples)

first: Liesbeth

last: Badisco

first: Swidbert

middle: R

last: Ott

first: Stephen

last: Rogers

first: Thomas

last: Matheson

first: Dries

last: Knapen

first: Lucia

last: Vergauwen

first: Heleen

last: Verlinden

first: Elisabeth

last: Marchal

first: Matt

last: Sheehy

first: Malcolm

last: Burrows


title: adt08-01_upp-Trancriptome analysis of the Arabidopsis upp-1/2 mutants

status: Public on Dec 31 2011

city: evry

first: Soubigou-Taconnat

last: Ludivine

address: 2 rue gaston crémieux

department: URGV

institute: INRA

summary:
adt08-01_upp - adt08-01_upp - Impact on gene expression of the UPP loss-of-function at early development stage - WT/upp comaparison at the beginning of the development alteration (4d for Col0/upp-1, 5d for WS/upp-2), and 3 days later (7d for Col0/upp-1, 8d for WS/upp-2)

Keywords: wt vs mutant comparison

overall_design: 8 dye-swap - CATMA arrays

first: Samuel

last: Mainguet

first: Jean-Pierre

last: Renou

first: Marie-Laure

last: Martin-Magniette


title: Deciphering the genetic architecture of variation in the immune response to Mycobacterium tuberculosis infection (genotype)

status: Public on Dec 30 2011

city: Montreal

first: luis

last: Barreiro

address: 3175 Chemin de la Côte Sainte-Catherine

institute: luis

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

summary: Identification of genetic polymorphisms associated with inter-individual variation in immune response to Mycobacterium tuberculosis infection.

overall_design: We performed a genome-wide mapping study of loci that are associated with functional variation in immune response to MTB (This dataset). We characterized transcript and protein expression levels and mapped expression quantitative trait loci (eQTLs) in primary dendritic cells from 65 individuals, before and after infection with MTB (see GSE34151).

first: Luis

first: Ludovic

last: Tailleux

first: Athma

middle: A

last: Pai

first: Brigitte

last: Gicquel

first: John

middle: C

last: Marioni

first: Yoav

last: Gilad


title: The effect of TNFa on endothelial cells

status: Public on Nov 29 2011

city: Fukuoka

first: Hiromitsu

last: Araki

address: 744 Motooka Nishi-Ku

institute: Kyushu University

summary: To understand the transcripts induced by TNFa and how these may interact in inflammatory networks, the transcriptome of HUCECs stimulated with TNFa was examined in 3 different pools each cosisting of 10 different HUVEC isolates.

overall_design: In the study present here, HUVEC isolates from 3 pools of 10 different individuals were cultured until passage 4 in fully supplemented growth conditions. Passage 4 HUVEC isolates were then treated with TNFa (10ng/ml), with RNA extracted at time points 0, 1, 1.5, 2, 4, 5, and 6hrs after TNFa treatment . RNA was extracted and hybridised onto CodeLink microarrays.

first: Daniel

last: Hurley

first: Yoshinori

last: Tamada

first: Ben

last: Dunmore

first: Deborah

last: Sanders

first: Sally

last: Humphreys

first: Muna

last: Affara

first: Seiya

last: Imoto

first: Kaori

last: Yasuda

first: Yuki

last: Tomiyasu

first: Christopher

last: Savoie

first: Vicky

last: Cho

first: Steve

last: Smith

first: Satoru

last: Kuhara

last: Miyano

first: Stephen

last: Charnock-Jones

first: Edmund

last: Crampin

first: Cristin

last: Print


title: Activation of cyclic AMP signaling leads to different pathway alterations in lesions of the adrenal cortex caused by germline PRKAR1A defects versus those due to somatic GNAS mutations

status: Public on Nov 15 2011

first: Madson

middle: Q

last: Almeida

address: 10 Center Drive, MSC 1103

department: Section on Genetics and Endocrinology / NICHD

institute: NIH

summary: PRKAR1A inactivating mutations are responsible for primary pigmented nodular adrenocortical disease (PPNAD) whereas somatic GNAS activating mutations cause macronodular disease in the context of McCune-Albright syndrome (MAS), ACTH-independent hyperplasia (AIMAH) and, rarely, cortisol-producing adenomas (CPA). The whole-genome expression profile (WGEP) of normal (pooled) adrenals, PRKAR1A- (3) and GNAS-mutant (3) was studied.

overall_design: Total RNA obtained from adrenal tumors were compared to those samples obtained from normal adrenal pools

first: M

last: Nesterova

first: C

last: Stratakis

last: Cheadle

first: T

last: Watkins


title: Gene expression profiles of 400 siRNA knocked down on HUVEC

first: Hiromitsu

last: Araki

summary: Gene regulatory networks inferred from RNA abundance data have generated significant interest. Several high-profile studies have suggested that gene networks may be useful for identifying molecular pathways related to development, human disease and drug action. However despite, this potential, gene network approaches are used relatively infrequently and their use generally requires input from specialist bioinformaticians. To make their use and evaluation more accessible to non-specialist bioinformaticians and to laboratory scientists, we have assembled a comprehensive but easily used suite of tools for the generation and evaluation of gene regulatory networks, applicable to both microarray and RNA-seq data. We illustrate the use of these tools with two previously unpublished microarray datasets generated in primary human endothelial cells by 400 siRNA-mediated knockdowns. Using these tools and data sets, we reverse-engineered a range of different types of transcriptional regulatory network model, and compared these networks with known molecular pathways reported in the literature.

overall_design: In this study, we generated microarray data from human umbilical vein endothelial cells (HUVECs) transfected with 400 siRNAs and infered gene networks from them by using five different methods.

first: Daniel

last: Hurley

first: Yoshinori

last: Tamada

first: Ben

last: Dunmore

first: Deborah

last: Sanders

last: Humphreys

last: Affara


title: DNA hypermethylation in colorectal cancer: Colorectal cancer tissues vs. Adjacent non-tumor tissues

status: Public on Nov 14 2011

city: Beijing

first: wang

last: liang

address: 7A North Yongchang Road BDA

country: China

institute: BioChainBJ

summary: To identify novel hypermethylated genes in colorectal cancer (CRC) and to test their potential application in CRC early diagnosis, we performed a genome-wide screening of 57,723 CpG dinucleotides covering 4,010 genes in paired DNA samples extracted from 3 fresh frozen CRC tissues and their matching non-tumor adjacent tissues from a cohort of 3 CRC patients undergoing curative surgery using MIRA-based microarray. We also validated candidate hypermethylated genes screened by MIRA-based microarray in independent CRC samples using combined bisulfite restriction analysis. A total of 297 CpG dinucleotides in CRC covering 211 genes were found to be hypermethylated in CRC tissues. From these 211 candidate methylated genes, seven novel methylated genes were picked up for validation and three genes were confirmed to be methylated in cancer samples but not in non-cancer samples.We also compared the methylation levels of these three novel hypermethylated genes with those of Vimentin and SEPT9, well-known hypermethylated genes in CRC, and found that methylated PHOX2B, FGF12 and GAD2 were better than methylated Vimentin and SEPT9 in differentiating CRC cancer tissue from normal tissue. Significant enrichment analysis of GO terms of the hypermethylated genes showed that a high proportion of hypermethylated genes in tumor tissues are involved in regulation of transcription.

overall_design: Paired experiments, colorectal cancer tissue vs. adjacent non-cancer tissue. Biological replicates: 3 cancer replicates, 3 paired non-cancer replicates.

first: Hai

last: Li

first: Yong

last: Du

first: Dong

last: Zhang

first: Li-Na

last: Wang

first: Chun

last: Yang

first: Bin

last: Liu

first: Wei-Jie

first: Lei

last: Shi

first: Wei-Guo

last: Hong

first: Liang

first: Yin-Xue


title: Expression data in wild type and NsrR-null mutant of B. subtilis

status: Public on Oct 28 2011

city: Beaverton

first: Michiko

last: Nakano

address: 20000 NW Walker Rd

institute: OHSU

summary:
NsrR strongly represses transcription of small numbers of genes in the absence of NO
NsrR moderately represses transcription of large numbers of genes in a less NO-sensitive manner

sample_organism: Bacillus subtilis subsp. subtilis str. 168

overall_design: The wild-type and mutant cells were cultured anaerobically in the presence and absence of NO.  Biological triplicate samples were used to isolate RNA.

first: Alan

last: Grossman


title: Cultured chicken embryonic hepatocytes (CEH) exposed to linear PFOS (L-PFOS),  technical-grade PFOS (T-PFOS), or linear PFUdA

status: Public on Oct 29 2011

city: Ottawa

first: Jason

middle: M.

last: O'Brien

address: 1125 Colonel By Drive, Raven Road

institute: Environment Canada

summary:
This series was used for two studies:
Study 1:
Recently it was discovered that the perfluorooctane sulfonate (PFOS) detected in wildlife, such as fish-eating birds, had a greater proportion of linear PFOS (L-PFOS) than the manufactured technical product (T-PFOS), which contains linear and branched isomers. This suggests toxicological studies based on T-PFOS data may inaccurately assess exposure risk to wildlife. To determine if PFOS effects were influenced by isomer content we compared the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either L-PFOS or T-PFOS using Agilent microarrays. At equal concentrations (10 ?M), T-PFOS altered the expression of more transcripts (340, >1.5 fold change, p<0.05) compared to L-PFOS (130 transcripts). Higher concentrations of L-PFOS (40 ?M) were also less transcriptionally disruptive (217 transcripts) than T-PFOS at 10 ?M. Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, hepatic system development and cellular growth and proliferation. Pathway and interactome analysis suggested that genes may be affected through the RXR receptor, oxidative stress response, TP53 signaling, MYC signaling, Wnt/?-catenin signaling and PPAR? and SREBP receptors. In all functional categories and pathways examined, the response elicited by T-PFOS was greater than L-PFOS. These data show that T-PFOS elicits a greater transcriptional response in CEH than L-PFOS alone and demonstrates the importance of considering the isomer-specific toxicological properties of PFOS when assessing exposure risk.

Study 2:
In many bird populations, concentrations of perfluoroundanoic acid (PFUdA) are second only to perfluorooctane sulfonate (PFOS) among perfluoroalkyl compounds. Here, we used microarrays to characterize the transcriptional response of cultured chicken embryonic hepatocytes (CEH) to PFUdA and compared it to the response induced by PFOS. At non-cytotoxic doses, PFUdA (1 or 10 ?M) disrupted the expression of more genes (854) than PFOS (447, at 10 or 40 ?M) in CEH. Using functional, pathway and interactome analysis we identified several potentially important modes-of-action (MoAs) for PFUdA and some associated key events, including the suppression of the acute-phase response (APR) through peroxisome proliferator activated receptor activation. We then measured the expression of five APR genes, fibrinogen alpha (fga), fibrinogen gamma (fgg), thrombin (f2), plasminogen (plg), and protein C (proC), in the liver of chicken embryos exposed in ovo to PFUdA. The expression of fga, f2, and proC were down-regulated in embryo livers (100 or 1000 ng/g, p<0.1) as predicted from microarray analysis, whereas fibrinogen gamma (fgg) was up-regulated and plg was not significantly affected. Our results demonstrate PFUdA is more transcriptionally disruptive than PFOS in CEH. Additionally, we identified APR suppression as a potentially important and environmentally relevant MoA. These findings suggest in ovo exposure of birds to PFUdA could lead to post-hatch developmental deficiencies, such as impaired immune response.

overall_design: Reference Design. Reference = pool of equal parts of all control and treated samples. Control groups and 7 treatment groups. Control samples were CEH exposed DMSO only (vehicle solvent). Treatments were: CEH exposed to 10 uM and 40 uM L-PFOS, 10 uM T-PFOS, 0.03 and 1 nM TCDD, and 1 uM and 10 uM PFUdA.

first: Aislynn

middle: J

last: Austin

first: Andrew

last: Williams

first: Carole

last: Yauk

first: Doug

last: Crump

first: Sean

middle: W

last: Kennedy


title: Immune related gene expression comparison between human CD14+ HLA-DR-neg/low MDSC vs CD14+ HLA-DR-high control monocytes

status: Public on Sep 09 2011

city: Bethesda

first: Tim

last: Greten

address: 9000 Rockville Pike

institute: NCI

summary: Myeloid-derived suppressor cells (MDSC) is a heterogeneous population of cells that can negatively regulate T-cell function. As opposed to murine MDSC, which are characterized as Gr-1+CD11b+ cells, human MDSC are not so clearly defined due to lack of specific markers. Our lab has previously identified a new subset of MDSC as CD14+HLA-DR-neg/low cells from PBMC. CD14+HLA-DR-neg/low MDSC not only suppress proliferation and IFN-gamma secretion of autologous T cells, but also induce CD25+Foxp3+ regulatory T cells that are suppressive in vitro, whereas the counterpart CD14+HLA-DR-high monocytes don’t have the effect. In this study, we compare the immune-related gene expression between CD14+HLA-DR-neg/low MDSC and CD14+HLA-DR-high monocytes to better characterize the difference between these two populations and to find new potential specific marker for human MDSC.

overall_design: PBMC were isolated from fresh blood healthy donor by density centrifugation. CD14+ cells were isolated by AutoMACS CD14 microbeads using a AutoMACS (Miltenyi), and then stained with CD14 and HLA-DR antibodies. MDSC and monocytes control cells were sorted as CD14+ HLA-DR-neg/low and CD14+HLA-DR-high cells respectively.  The sorted two populations were immediately frozen in liquid nitrogen and shipped to the company on dry ice for RNA isolation and further microarray.

first: Bastian

last: Hoechst

first: Fei

last: Zhao


title: Molecular mechanisms of hepatocarcinogenesis in c-Myc/Tgfa transgenic mice

status: Public on Sep 28 2011

first: Daekwan

last: Seo

address: 37 Convent Dr. Rm 4140

department: NCI

summary: A complete understanding of molecular mechanisms that underlie cancer onset and progression could provide a basis to improve early diagnosis and more effective treatment. However, this is still a challenge in human, partly due to the difficulty of analyzing the early stages of the disease. In this context, genetically engineered mice represent a valuable alternative to model human carcinogenesis. Here, we studied cancer development in c-Myc/Tgfa transgenic mice that developed liver tumors which closely reproduce a subset of human hepatocellular carcinoma (HCC) with a poor prognosis. By using a functional genomics approach from early to late stages of HCC development, we demonstrated that hepatocarcinogenesis in c-Myc/Tgfa mice resulted from a progressive accumulation of transcriptional alterations due to an active hepatocyte proliferation in a chronic oxidative stress environment generated by a general metabolic disorder. One striking observation was the deregulation of numerous immune-related genes starting from an early stage of the disease. Particularly, we showed that activating ligands for natural killer (NK) cells were specifically induced in dysplastic hepatocytes which simultaneously lost the expression of MHC-I molecules. Besides this early mechanism of NK-mediated immune surveillance, we further reported a drastic decrease in hepatic NK cell population which may indeed contribute to the emergence and clonal expansion of progenitors for liver tumors. In conclusion, our study provided a detailed and comprehensive characterization of hepatocarcinogenesis in c-Myc/Tgfa transgenic mice and emphasized the critical role of the innate immune surveillance disruption at the early stages of liver cancer.

overall_design: In the present study, we reported a detailed and comprehensive dynamic characterization of the cellular and molecular alterations involved in tumor onset and progression in the liver of c-Myc/Tgfa double-transgenic mice (B6CBAxCD1 background). Liver samples from male animals were collected at  various time-points ranging from 3 weeks to 9 months for c-Myc/Tgfa double-transgenic mice, or 18 months for c-Myc and Tgfa single-transgenic mice. Tissue samples  were divided into two parts; one was fixed in 10% formalin for histological evaluation and the other was used for RNA analysis. Total RNAs were isolated from livers with moderate or severe hepatocyte dysplasia (at 3 weeks and 3 months, respectively), as well as from HCC and surrounding non-tumor livers (5-15 mice per group). Total RNAs were also isolated from the livers of B6CBAxCD1 wild-type mice at 3 weeks and 3 months. RNAs isolated from the normal livers of B6CBA WT mice (3 months old, n=10) were pooled and used as a common technical reference for all microarray experiments.

first: Cédric

last: Coulouarn

first: Valentina

last: Factor

first: Elizabeth

last: Conner

first: Snorri

middle: S

last: Thorgeirsson


title: Transcriptomics and metabolomics profiling in Flax stem tissues containing contrasted secondary cell wall architectures.

status: Public on Oct 01 2011

city: Villeneuve d'Ascq

first: Stephane

last: Fenart

address: Cité Scientifique

department: Biology

institute: University Lille 1

summary: We used our previously described gene expression platform (Fenart et al., 2010) to assess gene expression along the stem of flax, both in inner and outer tissues.

overall_design: 32 chips study using total RNA from Internal and external part of Flax stem, sampled on whole stem or in different parts of the stem.

first: R

last: Huis

first: S

last: Fénart


title: Expression data from Burkholderia multivorans cystic fibrosis clinical isolates

status: Public on Aug 22 2011

city: Lisboa

first: Leonilde

middle: Morais

last: Moreira

address: A. Rovisco Pais

department: Bioengineering

country: Portugal

institute: Instituto Superior Tecnico

summary: Burkholderia cepacia complex (Bcc) comprises opportunistic bacteria infecting hosts such as cystic fibrosis (CF) patients. Bcc long-term infection of CF patient airways has been associated with emergence of phenotypic variation. Here we studied two Burkholderia multivorans clonal isolates (D2095 and D2214) displaying different morphotypes from a chronically infected CF patient in order to evaluate traits development during lung infection.

platform_organism:
Burkholderia cenocepacia J2315
Burkholderia multivorans ATCC 17616

overall_design: For expression profiling, overnight cultures of the mucoid D2095 and the nonmucoid D2214 isolates grown in SM medium were diluted to an initial OD640 nm of 0.1 into the same medium. Triplicate samples were cultured at 37ºC with 250 r.p.m. agitation for 17 h and RNA extracted from the two bacterial isolates.

first: Inês

middle: N

last: Silva

first: Ana

last: Ferreira

first: Jorg

last: Becker


title: Hybridization of Burkholderia multivorans D2095 and D2214 genomic DNA

summary: Burkholderia cepacia complex (Bcc) comprises opportunistic bacteria infecting hosts such as cystic fibrosis (CF) patients. Bcc long-term infection of CF patient airways has been associated with emergence of phenotypic variation. Here we studied two Burkholderia multivorans clonal isolates (D2095 and D2214) displaying different morphotypes from a chronically infected CF patient in order to evaluate traits development during lung infection. Since the custom array described in platform GPL13356 was based on Burkholderia multivorans ATCC 17616 genome, here we performed a DNA-DNA hybridization to determine which probes of the array hybridize with our test genomes

overall_design: Total DNA was extracted from overnight cultures of the mucoid D2095 and the nonmucoid D2214 isolates grown in LB medium.

middle: D


title: miRNA profiling of Macaca nemestrina plasma samples: before SIV infection and during acute infection

status: Public on Sep 15 2011

city: Baltimore

first: Kenneth

last: Witwer

address: 733 N Broadway, BRB Rm 820

department: Molecular and Comparative Pathobiology

institute: Johns Hopkins University School of Medicine

summary: This study describes a circulating miRNA signature of acute retroviral infection, which may include candidate diagnostic or prognostic miRNAs for lentivirus-associated neurologic conditions.

overall_design: Plasma samples were taken from six macaques prior to infection and at ten days post-infection. Three macaques each went on to develop no or severe encephalitis. miRNA profiles were generated for each animal before and 10 days after SIV infection.

first: Stephanie

middle: L

last: Sarbanes

first: Janice

middle: E

last: Clements


title: Novel interaction of PHY-906 and Irinotecan on the inflammatory processes in mouse tumors

status: Public on Oct 22 2011

city: Doha

first: Francesco

middle: Maria

last: Marincola

address: Al Nasr Tower, AL Corniche Street, PO Box 26999

country: Qatar

institute: Sidra Medical and Research Center

summary: Traditional Chinese Medicine (TCM) has been used for thousands of years to treat or prevent diseases, including cancer. Good manufacturing practices (GMP) and sophisticated product analysis (PhytomicsQC) to ensure consistency are now available allowing the assessment of its utility. Polychemical Medicines, like TCM, include chemicals with distinct tissue-dependent pharmacodynamic properties that result in tissue-specific bioactivity. Determining the mode of action of these mixtures was previously unsatisfactory; however, information rich RNA microarray technologies now allow for thorough mechanistic studies about complex mixtures effects. PHY906 is a long used four herb TCM formula employed as adjuvant to relieve side effects associated with chemotherapy. Animal studies documented a decrease in global toxicity and an increase in therapeutic effectiveness of chemotherapy when PHY906 was combined. Using a systems biology approach, we studied tumor tissue to identify reasons for the enhancement of the antitumor effect of Irinotecan by PHY-906 in a well-characterized pre-clinical model; PHY-906 and Irinotecan were administered orally to female BDF-1 mice bearing subcutaneous Colon 38 tumors. We observed that 1) individually PHY-906 and Irinotecan induce distinct alterations in tumor, liver and spleen; 2) PHY-906 alone predominantly induces repression of transcription and immune-suppression in tumors; 3) these effects are reverted in the presence of Irinotecan, with prevalent induction of pro-apoptotic and pro-inflammatory pathways that may favor tumor rejection. Most importantly, PHY-906 together with Irinotecan triggers unique changes not activated by each one alone suggesting that the combination creates a unique tissue-specific response.

overall_design: Four groups of BDF-1 mice bearing colon 38 tumors were treated with Phosphate Buffered Saline (PBS) (n=10), PHY-906 (n=10), Irinotecan (CPT-11, Camptosar(TM)) (n=10) or the combination PHY-906 and Irinotecan (n=10). Tumor (38 samples), spleen (38 samples), and liver (35 samples) tissues were removed and frozen for total RNA isolation and subsequent microarray hybridization. There were a total of 111 samples representing 12 treated tissue groups with 8 to 10 biological replicates each. A reference sample was generated from a pool of mixed normal mouse tissue.


title: SNP data from genomic DNA from a subject, fibroblasts, iPSCs and neurons with four copies of SNCA, and genomic DNA from an unaffected first degree relative and equivalent cell lines

city: Edinburgh

first: Tilo

last: Kunath

address: 5 LIttle France Drive

department: Centre for Regenerative Medicine

institute: University of Edinburgh

summary:
A major barrier to research on Parkinson’s disease (PD) is inaccessibility of diseased tissue for study. One solution is to derive induced pluripotent stem cells (iPSCs) from patients with PD and differentiate them into neurons affected by disease. We created an iPSC model of PD caused by triplication of SNCA encoding α-synuclein. α-Synuclein dysfunction is common to all forms of PD, and SNCA triplication leads to fully penetrant familial PD with accelerated pathogenesis. After differentiation of iPSCs into neurons enriched for midbrain dopaminergic subtypes, those from the patient contain double α-synuclein protein compared to those from an unaffected relative, precisely recapitulating the cause of PD in these individuals. A measurable biomarker makes this model ideal for drug screening for compounds that reduce levels of α-synuclein, and for mechanistic experiments to study PD pathogenesis.
This SNP microarray study was carried out to confirm presence of SNCA triplication in the affected subject and the derived cell lines.

overall_design: 11 samples were analysed: genomic DNA from the two subjects in the study, the two parent fibroblast lines (AST denoting alpha-synuclein triplication and NAS denoting normal alpha-synuclein), two iPSC lines from each parent fibroblast line (four in total), a human embryonic stem cell line (SHEF4) and two neuronal samples one each from AST and NAS iPSCs).

first: Michael

last: Devine

first: Mina

last: Ryten

first: Petr

last: Vodicka

first: Alison

last: Thomson

first: Henry

last: Houlden

first: Tom

last: Burdon

first: Fatima

last: Cavaleri

first: Nicola

last: Drummond

first: Masumi

last: Nagano

first: Jan-Willem

last: Taanman

first: Anthony

middle: H

last: Schapira

first: Katrina

last: Gwinn

first: Johm

last: Hardy

first: Patrick

last: Lewis


title: Wine yeast EC1118 and CSM comparative transcriptomics during synthetic grape juice fermentation MS300 at day 6

status: Public on Oct 26 2011

city: Burjassot

first: Agustin

last: Aranda

state: Valencia

address: Moliner 50

department: Biochemistry and Molecular Biology

country: Spain

institute: University of Valencia

summary: CSM is a commercial wine yeast with a long chronological life span, while commercial strain EC1118 displays a short life span in laboratory synthetic complete medium SC. Our aim is compare their expression profile on a high sugar fermentation

overall_design: From a preculture of two days in YPD, MS300 was innoculated at 10exp6 cells/mL in filled-in 50 mL conical centrifuge tubes and they were cultivated at 24ºC with mild shaking for 6 days.

first: Helena

last: Orozco

first: Emilia

last: Matallana