title: Reciprocal communication between endometrial stromal cells and macrophages

status: Public on Jul 21 2010

city: Vermillion

first: Kathleen

middle: M

last: Eyster

state: SD

address: 414 E. Clark St.

department: Basic Biomedical Sciences

institute: University of South Dakota

summary: Endometriosis is an inflammatory disease and bone marrow-derived cells are abundant in endometriotic lesions and in the peritoneal fluid of women with the disease.  This study tested the hypothesis that reciprocal communication occurs between macrophages and cultured human endometrial stromal cells and that this communication contributes to the pathology of endometriosis.  Changes in gene expression elicited by exposure to factors secreted by the opposing cell type were measured by DNA microarray to test this hypothesis.  716 named genes were differentially expressed in cultured endometrial stromal cells in response to factors secreted by macrophages.  Genes that were up-regulated included IL8/CXCL8, MMP3, phospholamban, CYR61/CCN1, CTGF/CCN2, tenascin C, and NNMT, whereas integrin alpha 6 was down-regulated.  In contrast, 15 named genes were differentially expressed in macrophages in response to factors secreted by cultured endometrial stromal cells.   The data document reciprocal communication between macrophages and endometrial stromal cells and suggest that interaction with macrophages stimulates the expression of genes in endometrial stromal cells that contribute to migration, adhesion, invasion, neovascularization and mitosis of endometrial cells that may support the establishment of endometriosis.

overall_design:
Experiment Design:
Goal of the experiment:  To examine reciprocal communication between a telomerase-immortalized human endometrial stromal cell line (THESC, ATCC #CRL-4003) and U-937 macrophages (ATCC #CRL-1593.2 treated with phorbol ester to stimulate differentiation), and also to assess the response of THESC cells to estrogen plus progesterone.

 Keywords:  human, cultured endometrial stromal cell line, macrophage

Experimental factors:  hormone treatment and treatment with macrophage-conditioned medium of endometrial stromal cells, treatment of macrophages with endometrial stromal cell conditioned medium

Experimental design:  T HESC cells were grown in phenol red-free DMEM/F12 medium supplemented with 1% ITS+, 10% charcoal-dextran treated fetal bovine serum albumin, 1% pen/strep, and 500 ng/ml puromycin.  THESC cells were treated for 8 days with vehicle (0.1% ethanol) or estradiol (10-8 M) plus progesterone (10-7 M medroxyprogesterone acetate).  On day 6, serum was removed from the culture medium.  On day 7, half of the cells were treated with U-937 macrophage-conditioned medium for 24 hours.  The cells were harvested and RNA extracted on day 8.

The experiment was repeated with three different passages of cells (passages 9, 11, and 13) to yield an experimental n of 3.  The four groups of cells were vehicle-treated control, estradiol + progesterone, control + macrophage-conditioned medium, and estradiol + progesterone + macrophage-conditioned medium.

The monocytic cell line, U-937, was maintained in suspension in RPMI-1640 with 10% fetal bovine serum and 1% pen/strep.  The U-937 cells were treated with 32 nM phorbol myristate acetate (PMA) for 48 h to stimulate differentiation into macrophages.  The U-937 macrophages were treated with THESC conditioned medium or with non-conditioned control medium.  Total RNA was collected after 24 hours.  The experiment was repeated with three different passages of cells (passages 7, 9, and 12) to yield an experimental n of 3.  The two experimental groups were macrophages treated with non-conditioned control medium and macrophages treated with THESC-conditioned medium.

Quality control steps:  The cRNA that was synthesized from each cultured cell passage and treatment was used for hybridization to a single CodeLink (Applied Microarrays) whole human genome microarray.  Only one sample was hybridized with each slide and only one dye (streptavidin-Alexa 647) was used so no dye swaps were necessary.  Bacterial control spikes were used per manufacturer's instructions.

Samples used, extract preparation and labelling:
The origin of each biological sample:  The samples were telomerase-immortalized human endometrial stromal cells and human U-937 monocytic cell line differentiation into macrophages by treatment with phorbol ester.  The cell lines were obtained from American Type Culture Collection (#CRL-4003 and #CRL-1593.2).

Manipulations of biological samples and protocols used:  T HESC cells were grown in culture in phenol red-free DMEM/F12 medium supplemented with 1% ITS+, 2% charcoal/dextran treated fetal bovine serum, 1% penicillin/streptomycin, and 500 ng/ml puromycin.  The cells were treated for 8 days with vehicle (0.1% ethanol), estradiol (10-8 M) plus progesterone (10-7 M medroxyprogesterone acetate), vehicle + macrophage-conditioned medium, or estradiol (10-8 M) + progesterone (medroxyprogesterone acetate 10-7 M) + macrophage-conditioned medium.   On day 6, serum was removed from the culture medium of all cells.  On day 7, half of the cells were treated with macrophage-conditioned medium for 24 hours.  The cells were harvested and RNA extracted on day 8.

The experiment was repeated with three different passages of cells (passages 9, 11, and 13) to yield an experimental n of 3.  The four groups of cells were vehicle-treated control, estradiol + progesterone, control + macrophage-conditioned medium, and estradiol + progesterone + macrophage-conditioned medium.

To prepare macrophage-conditioned medium, U-937 (ATCC #CRL-1593.2) cells were grown in RPMI-1640 medium with 10% fetal bovine serum until the cells covered 80% of the flask surface.  These U-937 cells were treated with 32 nM PMA for 48 h to stimulate differentiation into macrophages.  Differentiated macrophages were maintained in phenol red-free, fetal bovine serum-free DMEM/F12 medium supplemented with 1% penicillin/streptomycin for 48 hours.  The cell suspension was centrifuged and the conditioned medium was collected, filtered, and stored at -80C.  Before use, macrophage-conditioned medium was supplemented with 1% ITS+ and puromycin.

The monocytic cell line, U-937, was maintained in suspension in RPMI-1640 with 10% fetal bovine serum and 1% pen/strep.  The U-937 cells were treated with 32 nM PMA for 48 h to stimulate differentiation into macrophages.  The differentiated macrophages were treated with THESC conditioned medium or with non-conditioned control medium.  Total RNA was collected after 24 hours.

Experimental factor:  hormone treatment, treatment with macrophage-conditioned medium, and treatment with T-HESC cell conditioned medium

Technical protocols:  The cells were rinsed and TRI reagent (MRC) was added to the culture flasks.  The cells were scraped into centrifuge tubes, bromochloropropane and sodium acetate were added, and the samples were centrifuged to separate the phases.  The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen).  The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen).  The purity and quantity were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip.

	Labelled cRNA was prepared using the Message Amp II-Biotin Enhanced Kit from Ambion, following the manufacturer's Instruction Protocol.  1.0 microgram of T-HESC cell total RNA or 2 micrograms of macrophage total RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C.  (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.)  The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C.  The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C.  The double-stranded cDNA was purified on spin columns included in the Ambion kit.  The double-stranded cDNA was mixed with T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA.  The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on spin columns from the Ambion kit.  The concentration of cRNA was determined by spectrophotometry.

Hybridization procedures and parameters:  10 micrograms of biotinylated cRNA was mixed with fragmentation buffer and heated to 94C for 20 min.  The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides, and hybridized for 18 hours at 37C.
	The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature.  The streptavidin-Alexa 647 fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer)  The slides were then washed 4 times for 5 min each in 1x TNT and twice in 0.05% Tween 20 for 5 sec each.  The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.

first: Keith

last: Hansen

first: Emily

last: Winterton

first: Olga

last: Klinkova

first: Donis

last: Drappeau

first: Connie

last: Mark-Kappeler


title: Differential Role of Sall4 isoforms in ES cell pluripotency: ChIP-chip

status: Public on Sep 13 2010

city: Boston

first: Sridhar

last: Rao

state: MA

address: 1 Blackfan Circle

department: Pediatric Oncology

country: USA

institute: Dana Farber Cancer Institute and Children's Hospital, Boston

type: Genome binding/occupancy profiling by genome tiling array

summary: Murine embryonic stem cells (ESCs) are defined by continuous self-renewal and pluripotency. A diverse repertoire of protein isoforms arising from alternative splicing are expressed in ES cells without defined biological roles. Sall4, a transcription factor essential for pluripotency, exists as two isoforms  (Sall4a and Sall4b). By genome-wide location analysis, we have determined that Sall4b, and not Sall4a, binds preferentially to highly expressed loci in ES cells. Sall4a and Sall4b binding sites are distinguished by both epigenetic marks at target loci and their clustering with binding sites of other pluripotency factors.  When ESCs expressing a single isoform of Sall4 are generated, Sall4b alone could maintain the pluripotent state, although it could not completely suppress all differentiation markers. Sall4a and Sall4b collaborate in maintenance of the pluripotent state, but play distinct roles.  Our work is novel in establishing such isoform-specific differences in ES cells.

overall_design: ES cells expressing the BirA ligase, which attaches biotin to a specific target sequence, were transfected with either Sall4a or Sall4b that had been engineered to have the biotinylation sequence at the N-terminus.  ChIP-on-Chip was then performed to identify loci bound by Sall4a or Sall4b.

first: Stuart

middle: H

last: Orkin


title: Gene expression profiles of cervical cancers for novel molecular classification

status: Public on Sep 28 2010

city: Chiba

first: Shuhei

last: Noda

state: Chiba

address: Anagawa 4-9-1

department: Frontier research center

country: Japan

institute: National institute of radiological sciences

summary: Cervical cancers is the second most malignancy in women. It has been clinically important histological variants such as squamous cell carcinoma (SCC) and adenocarcinoma (AC) and adenosquamous carcinomas (ASC). It has been postulated that AC and ASC has a worse prognosis than pure SCC. However, many of the mixed or other types confuses its diagnosis and aggressive/resistant behavior of some tumors has resulted in debate for prognostic role of empirical pathological classification. In addition, the prognosis of adenosquamous carcinoma is still under debate. To establish a novel molecular classification of cervical cancer, we investigated intrinsic characteristics using expression profile.

overall_design: A total of 80 cevical cancer samples of following histology were included in this study:  54 SCC, 18 ASC, 6 AC, and 2 others. Replicate number: 2. No control/no reference sample was included. One-color.

first: Kaori

last: Imadome

first: Mayumi

last: Iwakawa


title: Granulosa cell responses to Gonal vs Menopur

status: Public on Mar 22 2010

summary:
Goal of the experiment:  To examine differential gene expression in granulosa cells of women undergoing ovarian stimulation with either recombinant human follicle stimulating hormone or purified human menopausal gonadotropin for in vitro fertilization.

Brief description of the experiment:  The study was designed to test the hypothesis that human granulosa cell (GC) gene expression response differs between pure recombinant FSH and human menopausal gonadotropin (hMG) stimulation regimens.   Women < 35 years-old undergoing in vitro fertilization for tubal or male factor infertility were prospectively randomized to one of two stimulation protocols, Gonadotropin releasing hormone (GnRH) agonist long protocol plus individualized dosages of (1) recombinant follicle stimulating hormone (rFSH) (Gonal-F®) (n=4) or (2) purified human menopausal gonadotropin (hMG; Menopur®; 75 IU FSH/75 IU LH per vial) (n=3).  Follicle development was monitored by ultrasound and serum estradiol levels.  When two follicles were ≥ 17mm, human chorionic gonadotropin (hCG) (10,000 IU; Novarel®) was administered.  Oocytes were retrieved 35 h post-hCG. Following oocyte recovery, GC were immediately collected in RNALater®.  Pooled GC from individual patients were washed, centrifuged over 40% Percoll®, resuspended in RNALater, and snap-frozen in LN.  Total RNA was extracted and stored at -70C.  Biotinylated cRNA was synthesized from total RNA and each sample was run individually on CodeLink Whole Human Genome Bioarrays (Applied Microarrays).  Unnamed genes and genes with <2-fold difference in expression were excluded from further analysis.   After exclusions, 1736 genes exhibited differential expression between groups. Over 400 were categorized as signal transduction genes, ~180 as transcriptional regulators, and ~175 as enzymes/metabolic genes. Expression of selected genes was confirmed by RT-PCR.  Differentially expressed genes included A kinase anchor protein 11 (AKAP11), bone morphogenic protein receptor II (BMPR2), epidermal growth factor (EGF), insulin-like growth factor binding protein (IGFBP)-4, IGFBP-5, basigin, and hypoxia-inducible factor (HIF)-1 alpha.  The results suggest that major differences exist in the mechanism by which pure FSH alone versus FSH/LH regulate gene expression in preovulatory GC that could impact oocyte maturity and developmental competence.

overall_design:
Experimental design:  Women < 35 years-old undergoing in vitro fertilization for tubal or male factor infertility were prospectively randomized to one of two stimulation protocols, Gonadotropin releasing hormone (GnRH) agonist long protocol plus individualized dosages of (1) recombinant follicle stimulating hormone (rFSH) (Gonal-F®) (n=4) or (2) purified human menopausal gonadotropin (hMG; Menopur®; 75 IU FSH/75 IU LH per vial) (n=3).  Follicle development was monitored by ultrasound and serum estradiol levels.  When two follicles were ≥ 17mm, human chorionic gonadotropin (hCG) (10,000 IU; Novarel®) was administered.  Oocytes were retrieved 35 h post-hCG. Following oocyte recovery, GC were immediately collected in RNALater®.  Pooled GC from individual patients were washed, centrifuged over 40% Percoll®, resuspended in RNALater, and snap-frozen in LN.  Total RNA was extracted and stored at -70C.  Biotinylated cRNA was synthesized from total RNA and each sample was run individually on CodeLink Whole Human Genome Bioarrays (Applied Microarrays

Quality control steps:  The cRNA that was synthesized from the granulosa cells from individual patients was used for hybridization to a single CodeLink (Applied Microarrays) Whole Human Genome microarray.  Only one sample was hybridized with each slide and only one dye (Alexa 647) was used so no dye swaps were necessary.  Bacterial control spikes were used as per manufacturer's instructions.

Samples used, extract preparation and labelling:
The origin of each biological sample:    Granulosa cells from women < 35 years-old undergoing in vitro fertilization.

Manipulations of biological samples and protocols used:  Female patients were treated with gonadotropin releasing hormone (GnRH) agonist long protocol plus individualized dosages of (1) recombinant follicle stimulating hormone (rFSH) (Gonal-F®) (n=4) or (2) purified human menopausal gonadotropin (hMG; Menopur®; 75 IU FSH/75 IU LH per vial) (n=3).  Follicle development was monitored by ultrasound and serum estradiol levels.  When two follicles were ≥ 17mm, human chorionic gonadotropin (hCG) (10,000 IU; Novarel®) was administered.  Oocytes were retrieved 35 h post-hCG. Following oocyte recovery, granulosa cells were immediately collected in RNALater®.  Pooled GC from individual patients were washed, centrifuged over 40% Percoll®, resuspended in RNALater, and snap-frozen in LN.

Experimental factor:  treatment with recombinant FSH versus purified human menopausal gonadotropin

Technical protocols:  Granulosa cells were collected into RNAlater (Ambion) and stored at -80C until processed.  The granulosa cells were pelleted by centrifugation to remove the RNAlater.  Each cell pellet was homogenized in TRI reagent (Molecular Research Centers).  Bromochloropropane and sodium acetate were added, and the samples were centrifuged to separate the phases.  The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen).  The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen).  The purity and quantity were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip.

Labelled cRNA was prepared using the manufacturer's Instruction Protocol (www1.amershambiosciences.com, CodeLink Gene Expression System: Manual Labelled cRNA Target Preparation).  The source of reagents was the CodeLink Expression Assay Reagent Kit, Manual Prep, except where specified otherwise.  0.285 micrograms of total granulosa cell RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C.  (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.)  The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C.  The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C.  The double-stranded cDNA was purified on QIAquick columns (Qiagen) and the eluent was dried down in a SpeedVac concentrator.  The double-stranded cDNA was resuspended in a mixture containing T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA.  The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on RNeasy columns (Qiagen).  The concentration of cRNA was determined by spectrophotometry.

Hybridization procedures and parameters:  10 micrograms of cRNA was mixed with fragmentation buffer and heated to 94C for 20 min.  The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides, and hybridized for 18 hours at 37C.
The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature.  The Alexa fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer)  The slides were then washed 4 times for 5 min each in 1x TNT and once in 0.05% Tween 20 for 5 sec.  The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.

first: John

last: Brannian

middle: A

first: Breanne

last: Mueller

first: Mandi

last: Greenway


title: Genome-Wide Sox17 Binding Sites in Mouse Extraembryonic Endoderm and Embryonic Stem Cells

status: Public on Mar 31 2010

city: Cambridge

first: Kathy

last: Niakan

address: 7 Divinity Ave

department: Stem Cell and Regenerative Biology

institute: Harvard University

summary: We investigated whether Sox17 directly or indirectly regulates extraembryonic endoderm gene expression by identifying Sox17 DNA-binding sites using chromatin-immunoprecipitation coupled with whole-genome promoter tiling array analysis (ChIP-Chip). We used the Sox17 and FLAG antibody to ask whether Sox17 was binding directly to the regulatory regions of genes in homogeneous extraembryonic endoderm (XEN) cell lines and in Sox17-inducible mouse embryonic stem (ES) cells. In XEN cells, Sox17 binding sites were located within the promoters or the introns of 2206 (3%) genes. We performed an ontology analysis for the genes with Sox17 binding sites and found that a significant number had adhesion functions in basement membrane establishment and maintenance. In addition to these ECM genes, Sox17 was also bound to promoter regions of a variety of other genes implicated in extraembryonic endoderm development including key transcription factors. Ontology analysis of all the Sox17 ChIP-chip binding targets identified in Sox17-induced ES cells, demonstrated a significant enrichment near genes involved in the cell cycle as well as genes involved in signaling pathways that function in embryonic stem cell maintenance. Sox17 was also observed to directly bind to the regulatory regions of many genes in pathways known to be functionally important for ES cell pluripotency and self-renewal. These studies suggest that one of Sox17’s functions in the differentiation of ICM and ES cells is to bind the regulatory regions of many genes that encode basement membrane components, thus leading to their activation. In addition to directly activating genes required for primitive endoderm differentiation, Sox17 may also function to activate and reinforce the transcriptional network governing differentiation.

overall_design: Extraembryonic endoderm (XEN) mouse cell lines ChIPed with Sox17 vs. XEN cell Input; Sox17-inducible mouse ES cells (induced with doxycycline for 48hrs) ChIPed with Sox17 or FLAG vs. Input

middle: K

first: Hongkai

last: Ji

first: Stephen

last: Vokes


title: Comparative Genome Hybridization Reveals Few Genetic Differences between Aspergillus flavus and A. oryzae

status: Public on Apr 10 2010

city: Raleigh

first: David

middle: Ryan

last: Georgianna

state: NC

address: 851 Main Campus Dr

department: Plant Pathology

institute: North Carolina State University

summary: Aspergillus flavus and A. oryzae represent two unique species predicted to have spent centuries in vastly different environments. A. flavus is an important opportunistic plant pathogen known for contaminating crops with the carcinogenic mycotoxin, aflatoxin and A. oryzae is a domesticated fungus used in food fermentations. Remarkably, the genomes of these two species are still nearly identical. We have used the recently sequenced genomes of A. oryzae RIB40 and A. flavus NRRL3357 along with array based comparative genome hybridization (CGH) as a tool to compare genomes across several strains of these two species. A comparison of three strains from each species by CGH revealed only 42 and 129 genes unique to A. flavus and A. oryzae, respectively. Further, only 709 genes were identified as being polymorphic between the species. Despite the high degree of similarity between these two species, correlation analysis among all data from the CGH arrays for all strains used in this study reveals a species split. However, this view of species demarcation becomes muddled when focused on only those genes for secondary metabolism.

sample_organism:
Aspergillus flavus
Aspergillus oryzae

platform_organism:
Zea mays
Aspergillus flavus
Aspergillus oryzae

overall_design: CGH comparison between 3 strains of A. flavus and 3 strains of A. oryzae, analyzed at the probe level

middle: R

first: Gary

last: Payne

first: Charles

middle: P

last: Woloshuk

first: Nancy

last: Keller

first: Juijang

last: Yu


title: Chromatin localization of Gli1 in granule neuron precursor cells and medulloblastoma

status: Public on Jun 03 2010

city: Stanford

first: Eunice

last: Lee

state: CA

address: 318 Campus Drive

department: Developmental Biology

institute: Stanford University

summary: Sonic hedgehog (Shh) signals via Gli transcription factors to stimulate proliferation of granule neuron precursor cells (GNPs) in the cerebellum. Deregulation of Shh target genes often results in unrestrained GNP proliferation and eventually medulloblastoma, the most common pediatric brain malignancy. Transcription factor binding location analysis (chIP-chip) revealed 510 and 1,060 genomic loci bound by Gli1 with high confidence in murine GNP and medulloblastoma cells, respectively.  In primary tumors, Gli1 associated with only one-third of the Gli1-bound regions in GNPs. Gene expression profiling, coupled with our binding results, indicated that there were more than one hundred target genes in common between the two cell populations, and importantly, there was an equivalent number of tumor-specific targets.  These results indicate that the transformation of normal GNPs into deadly tumor cells is accompanied by some changes in the battery of genes regulated by Gli1.

overall_design:
Murine RosaGli1-FLAG GNPs and RosaGli1-FLAG;Ptc+/- medulloblastoma cells were isolated and chIPed with anti-FLAG antibody vs. total chromatin input. 3 replicates each.

The supplementary fc.bar files contain log2(IP/Input) fold changes for each probe, the ma.bar files contain a moving average statistic (i.e., the MA statistic in TileMap) for each probe, and the .bed files contain the 510 and 1,060 high-quality Gli1-bound regions in GNPs and medulloblastoma (MB), respectively, reported in the paper.

middle: Y

first: Matthew

last: Scott

first: Wing

last: Wong


status: Public on Mar 11 2010

summary:
Diverse expression patterns for secondary metabolism gene clusters from Aspergillus flavus under different environmental conditions and in genetic mutants:  Insights into regulation of cyclopiazonic acid along with aflatoxin

Species of Aspergillus produce a diverse array of secondary metabolites, and recent genomic analysis predicts that these species have the capacity to synthesize many more compounds. It has been possible to infer the presence of 55 gene clusters associated with secondary metabolism in A. flavus. Presumably, secondary metabolites play important roles in the ecology of the producing species, but functions for most secondary metabolites remain unknown. Only three metabolic pathways have been associated with the predicted clusters in A. flavus. These include aflatoxin, cyclopiazonic acid (CPA), and aflatrem. To gain insight into the regulation of, and infer ecological significance for the 55 secondary metabolite gene clusters predicted in A. flavus, we examined their expression over 28 diverse conditions. Variables included culture media and temperature, fungal development, colonization of developing maize seeds, and misexpression of laeA, the global regulator of secondary metabolism. Hierarchical clustering analysis of expression profiles allowed us to categorize the gene clusters into four distinct clades.  Gene clusters for the production of aflatoxins, CPA, and seven other unknown compound(s) were identified as belonging to one clade. To further explore the relationships found by gene expression analysis, aflatoxin and CPA production were quantified under five different cell culture environments known to be conducive or non-conducive for aflatoxin biosynthesis and during colonization of developing maize seeds. Results from these studies showed that secondary metabolism gene clusters have distinctive gene expression profiles. Aflatoxin and CPA were found to have unique regulation but similar enough that they would be expected to co-occur in commodities colonized with A. flavus.

overall_design: Analysis of 75 different microarrays examining gene expression of A. flavus and A. oryzae over various conditions


title: A new tick Kunitz type inhibitor, Amblyomin-X, induces tumor cell death by modulating genes related to the cell cycle and targeting the ubiquitin-proteasome system

status: Public on Feb 28 2010

city: São Paulo

first: Eduardo

middle: Moraes

last: Reis

state: SP

address: Av. Prof. Lineu Prestes, 748

department: Biochemistry

country: Brazil

institute: University of São Paulo

summary: Amblyomin-X is a recombinant protein with cytotoxic activity on tumor cells featuring little or no activity on normal cells. The aim of this study was to evaluate the antitumoral activity of amblyomin-X (in vivo and ex vivo) and identify its molecular targets. To that end, global gene expression as well as  cytotoxicity, cell cycle modulation and ubiquitin proteasome system function were evaluated in pancreas adenocarcinoma and melanoma human cell lines after treatment with Amblyomin-X. Several genes related to the cell cycle were altered corroborating the G0/G1 phase alteration observed. PSMB2, that encodes a proteasome subunit, was differentially expressed, in agreement with trypsin-like proteasomal decreased activity and increased pool of poli-ubiquitinylated proteins. Altogether, alterations following Amblyomin-X exposure are in agreement with the observed cell death by apoptosis. Furthermore, In vivo treatment with Amblyomin-X induced regression of tumoral mass in mouse murine melanoma model (B16F10) and decreased the number of metastasis events. In conclusion, our results indicate that Amblyomin-X selectively acts on tumoral cells most likely by targeting the ubiquitin-proteasome system.

overall_design:
Global measurements of gene expression from pancreatic (Mia-PaCa-2) and melanoma (SK-Mel-28) tumor cell lines exposed to Amblyomin-X were obtained using human Whole Genome Code Link Bioarrays (GE Healthcare, Chandler, AZ) containing approximately 55,000 30-mer probes.
Six (Mia-PaCa-2) or five (SK-Mel-28) hybridizations were performed for each condition (Amblyomin-X treated or untreated control cells) using cRNA targets obtained from independent preparations. Cy5-labeled hybridized targets were detected with an arrayWoRx scanner (Applied Precision Inc., WA, USA).

last: Chudzinski-Tavassi


title: Temporal ChIP-on-Chip of RNA-Polymerase-II to detect novel gene activation events during photoreceptor maturation

status: Public on Feb 09 2010

city: Rochester

first: Kenneth

last: Mitton

state: MI

address: 4th Floor - Dodge Hall

department: Eye Research Institute

institute: Oakland University

summary: RNA Polymerase-II active regions were mapped comparing mouse neural retina tissue at age P25 and P2 to find novel gene activation predictions during maturation of photoreceptors. Over 800 predictions of increased activation were novel compared to previous mRNA expression array studies.

overall_design: P2 and P25 mouse neural retina (C57BL6) ChIP'ed with anti-RNA-Pol-II, P25 ChIP'ed with anti-FIZ1, all normalized to input genomic DNA control. Promoter Tiling Array 1.0

first: Padmaja

last: Tummala

first: Raghuveer

middle: S

last: Mali


title: Non-invasive detection of molecular biomarkers in subjects with a history of insulin resistance and colorectal adenomas

status: Public on Feb 11 2010

city: College Station

first: Jennifer

last: Goldsby

state: TX

address: 112 Cater-Mattil

department: Nutrition and Food Sciences

institute: Texas A&M University

summary:
We have developed novel molecular methodology utilizing a stool sample, which contains intact sloughed colon cells, in order to quantify colonic gene expression profiles.  In this study, our goal was to identify diagnostic gene sets (combinations) for the noninvasive classification of different phenotypes.  For this purpose, the effects of a legume enriched, low glycemic index, high fermentable fiber diet was evaluated in subjects with four possible combinations of risk factors, including insulin resistance (IR) and a history of adenomatous polyps.  In a randomized crossover design controlled feeding study each participant (a total of 23, 5-12/group) consumed the experimental diet (1.5 cups of cooked dry beans) and a control diet (isocaloric average American diet) for 4 wk with a 3 wk washout period between diets.  Using prior biological knowledge, the complexity of feature selection was reduced in order to perform an exhaustive search on all allowable feature (gene) sets of size 3, and among these, 27 had (unbiased) error estimates of 0.15 or less.  Linear discriminant analysis (LDA) was successfully used to identify the best single genes and two- to three-gene combinations for distinguishing subjects with IR, a history of polyps or exposure to a chemoprotective legume-rich diet.  These results support our premise that gene products (RNA) isolated from stool have diagnostic value in terms of assessing colon cancer risk.

Keywords: Exfoliated cells, legumes, insulin resistance, polyps

overall_design: 86 slides, 4 treatment groups, 23 patients

first: Chen

last: Zhao

first: Ivan

last: Ivanov

first: Edward

last: Dougherty

first: Terryl

middle: J

last: Hartman

first: Elaine

last: Lanza

first: Gerd

last: Bobe

last: Colburn

first: Joanne

last: Lupton

first: Laurie

last: Davidson

first: Robert

last: Chapkin


title: Characterization of microRNA Expression Profiles in human primary differentiated T lymphocytes

status: Public on Dec 31 2009

city: rome

first: Graziella

last: Curtale

address: viale regina elena 324

country: Italy

institute: "Sapienza" University of Rome

summary:
Activation of the T cell-mediated immune response has been associated with changes in the expression of specific miRNAs. However, the role of miRNAs in the development of an effective immune response is just beginning to be explored. Here we report a microRNA expression profile showing a list of microRNAs, which are highly up-regulated or down-regulated in human memory T cells compared to naive T cells, thus suggesting a possible role of these microRNAs in T lymphocyte differentiation.

Keywords: Primary Tissue miRNA Expression profile

overall_design: We used microarrays to profile miRNA expression in human naive and central memory CD4+ and CD8+ T lymphocytes

first: Franca

last: Citarella

first: Claudia

last: Carissimi

first: Marina

last: Goldoni

first: Nicoletta

last: Carucci

first: Valerio

last: Fulci

first: Francesca

last: Meloni

first: Debora

last: Franceschini

first: Vincenzo

last: Barnaba

first: Giuseppe

last: Macino


title: Two-way communication between endometrial stromal cells and monocytes

status: Public on Nov 16 2009

first: Kathleen

last: Eyster

summary: Immune system cells and cells of the endometrium have long been proposed to interact in both physiological and pathological processes.  The current study was undertaken to examine communication between cultured monocytes and endometrial stromal cells and also to assess responses of endometrial stromal cells to treatment with estradiol (E) in the absence and presence of medroxyprogesterone acetate (P).  A telomerase-immortalized human endometrial stromal cell line (T-HESC) and the U-937 monocyte cell line were used.  T-HESC were treated with E ± P ± monocyte conditioned medium; U-937 were treated ± T-HESC conditioned medium.  Gene expression in response to treatment was examined by DNA microarray.  Bi-directional communication, as demonstrated by changes in gene expression, clearly occurred between U-937 monocytes and T-HESC endometrial stromal cells.

overall_design:
Experiment Design:
Goal of the experiment:  To examine two-way communication between a telomerase-immortalized human endometrial stromal cell line (THESC, ATCC #CRL-4003) and U-937 monocytes (ATCC #CRL-1593.2), and also to assess the response of THESC cells to estrogen in the absence and presence of progesterone.

Brief description of the experiment:  Interactions of immune system cells and of the cells of the uterine endometrium have long been proposed to interact in both physiological and pathological processes (i.e., implantation and development of endometriosis).  The current study was undertaken to examine communication between cultured monocytes and endometrial stromal cells and also to assess responses of endometrial stromal cells to treatment with estradiol (E) in the absence and presence of medroxyprogesterone acetate (P).  A telomerase-immortalized human endometrial stromal cell line (T-HESC) and the U-937 monocyte cell line were used.  T-HESC were treated with E ± P ± monocyte conditioned medium; U-937 were treated ± T-HESC conditioned medium.  Gene expression in response to treatment was examined by DNA microarray.  Bi-directional communication, as demonstrated by changes in gene expression, clearly occurred between U-937 monocytes and T-HESC endometrial stromal cells.

Keywords:  human, cultured endometrial stromal cell line, monocyte

Experimental factors:  hormone treatment and treatment with monocyte-conditioned medium of endometrial stromal cells, treatment of monocytes with endometrial stromal cell conditioned medium

Experimental design:  T HESC cells were grown in phenol red-free DMEM/F12 medium supplemented with 1% ITS+, 10% charcoal-dextran treated fetal bovine serum albumin, 1% pen/strep, and 500 ng/ml puromycin.  THESC cells were treated for 8 days with vehicle (0.1% ethanol), estradiol (10-8 M), or with estradiol (10-8 M) plus progesterone (10-7 M medroxyprogesterone acetate).  On day 6, serum was removed from the culture medium.  On day 7, half of the cells were treated with U-937 monocyte-conditioned medium for 24 hours.  The cells were harvested and RNA extracted on day 8.

The experiment was repeated with three different passages of cells (passages 5, 7, and 9) to yield an experimental n of 3.  The six groups of cells were vehicle-treated control, estradiol, estradiol + progesterone, control + monocyte-conditioned medium, estradiol + monocyte-conditioned medium, and estradiol + progesterone + monocyte-conditioned medium.

The monocytic cell line, U-937, was maintained in suspension in RPMI-1640 with 10% fetal bovine serum and 1% pen/strep.  The U-937 cells were treated with THESC conditioned medium or with non-conditioned control medium.  Total RNA was collected after 24 hours.  The experiment was repeated with three different passages of cells (passages 7, 9, and 12).  The two experimental groups were non-conditioned control medium and THESC-conditioned medium.

Quality control steps:  The cRNA that was synthesized from each cultured cell passage and treatment was used for hybridization to a single CodeLink (Applied Microarrays) whole human genome microarray.  Only one sample was hybridized with each slide and only one dye (streptavidin-Alexa 647) was used so no dye swaps were necessary.  Bacterial control spikes were used per manufacturer's instructions.

Samples used, extract preparation and labelling:
The origin of each biological sample:  The samples were telomerase-immortalized human endometrial stromal cells and human monocytic cells.  The cell lines were obtained from American Type Culture Collection (#CRL-4003 and #CRL-1593.2).

Manipulations of biological samples and protocols used:  T HESC cells were grown in culture in phenol red-free DMEM/F12 medium supplemented with 1% ITS+, 2% charcoal/dextran treated fetal bovine serum, 1% penicillin/streptomycin, and 500 ng/ml puromycin.  The cells were treated for 8 days with vehicle (0.1% ethanol), estradiol (10-8 M), estradiol (10-8 M) plus progesterone (10-7 M medroxyprogesterone acetate), vehicle + monocyte-conditioned medium, estradiol (10-8 M) + monocyte-conditioned medium, or estradiol (10-8 M) + progesterone (medroxyprogesterone acetate 10-7 M) + monocyte-conditioned medium.   On day 6, serum was removed from the culture medium of all cells.  On day 7, half of the cells were treated with monocyte-conditioned medium for 24 hours.  The cells were harvested and RNA extracted on day 8.

The experiment was repeated with three different passages of cells (passages 5, 7, and 9) to yield an experimental n of 3.  The six groups of cells were vehicle-treated control, estradiol, estradiol + progesterone, control + monocyte-conditioned medium, estradiol + monocyte-conditioned medium, and estradiol + progesterone + monocyte-conditioned medium.

To prepare monocyte-conditioned medium, U-937 (ATCC #CRL-1593.2) cells were grown in RPMI-1640 medium with 10% fetal bovine serum until the cells covered 80% of the flask surface.  Cells were then maintained in phenol red-free, fetal bovine serum-free DMEM/F12 medium supplemented with 1% penicillin/streptomycin for 48 hours.  The cell suspension was centrifuged and the conditioned medium was collected, filtered, and stored at -80C.  Before use, monocyte-conditioned medium was supplemented with 1% ITS+ and puromycin.

The monocytic cell line, U-937, was maintained in suspension in RPMI-1640 with 10% fetal bovine serum and 1% pen/strep.  The U-937 cells were treated with THESC conditioned medium or with non-conditioned control medium.  Total RNA was collected after 24 hours.

Experimental factor:  hormone treatment, treatment with monocyte-conditioned medium, and treatment with endometrial stromal cell conditioned medium

Technical protocols:  The cells were rinsed and TRI reagent (MRC) was added to the culture flasks.  The cells were scraped into centrifuge tubes, bromochloropropane and sodium acetate were added, and the samples were centrifuged to separate the phases.  The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen).  The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen).  The purity and quantity were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip.

	Labelled cRNA was prepared using the manufacturer's Instruction Protocol (www1.amershambiosciences.com, CodeLink Gene Expression System: Manual Labelled cRNA Target Preparation).  The source of reagents was the CodeLink Expression Assay Reagent Kit, Manual Prep, except where specified otherwise.  1.0 microgram of total endometrial cell RNA or monocytic cell RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C.  (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.)  The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C.  The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C.  The double-stranded cDNA was purified on QIAquick columns (Qiagen) and the eluent was dried down in a SpeedVac concentrator.  The double-stranded cDNA was resuspended in a mixture containing T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA.  The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on RNeasy columns (Qiagen).  The concentration of cRNA was determined by spectrophotometry.

Hybridization procedures and parameters:  10 micrograms of cRNA was mixed with fragmentation buffer and heated to 94C for 20 min.  The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides, and hybridized for 18 hours at 37C.
	The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature.  The streptavidin-Alexa 647 fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer)  The slides were then washed 4 times for 5 min each in 1x TNT and twice in 0.05% Tween 20 for 5 sec each.  The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.

first: Keith

last: Hansen

first: Olga

last: Klinkova

first: Connie

last: Mark

first: Emily

last: Winterton


title: Comparison of genetic differences between Streptomyces scabies and turgidiscabies strains

status: Public on Feb 01 2010

city: Helsinki

first: Panu

last: Somervuo

address: Viikinkaari 4

country: Finland

institute: University of Helsinki

summary: Bacteria isolated from potato scab lesions in Finland or northern Sweden were analyzed using microarrays, PCR, and sequencing. Data indicate wide genetic variability in pathogenicity islands among S.turgidiscabies and S.scabies strains.

sample_organism:
Streptomyces scabiei
Streptomyces turgidiscabies
Streptomyces reticuliscabiei

overall_design: Thirteen Streptomyces scabies and turgidiscabies strains from two different growings, Streptomyces reticulisabiei reference strain DSM41804 and Streptomyces scabies reference strain ATCC49173 were hybridized. Data were analyzed in single channel mode.

first: Marja

last: Aittamaa

first: Jari

last: Valkonen


title: MacroH2A1 ChIP-chip

status: Public on Nov 13 2009

city: Dallas

first: W. Lee

last: Kraus

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

summary:
MacroH2A1 ChIP-chip was performed on custom Nimblegen genomic tiling arrays, to understand the genomic binding patterns of this histone variant and its relationship to gene expression
Keywords: ChIP-chip

overall_design: Two macroH2A biological replicates are included. An H3 ChIP-chip sample is included as a control

first: Matthew

last: Gamble

first: Raga

last: Krishnakumar

first: William

middle: L


title: Aging of Human Fibroblast

status: Public on Feb 26 2010

city: Philadelphia

first: Andres

last: Kriete

state: PA

address: 3141 Chestnut St

department: School of Biomedical Engineering

institute: Drexel University

summary: Human fibroblast of different age were investigated for their gene expression profiles in quiescence. Cultures were established from skin samples derived from young and old donors, obtained from the NIA Aging Cell Repository (Coriell Institute for Medical Research, Camden, NJ). All cell lines originated from 2 mm punch biopsies taken from the medial aspect of the upper arm. The donors were members of the Baltimore Longitudinal Study of Aging (BLSA) where they were characterized as "healthy" indviduals. The cell lines investigated had normal karyotypes.

overall_design: RNA was isolated from the cell lysate using Qiagen RNeasy mini kit according to the manufacturer's instructions. Gene expression analysis was performed using the Codelink human bioarray containing single-stranded 30-mer oligonucleotide probes (Applied Microarrays, Tempe, AZ) and chips were run in duplicate. Details of this platform are available on the vendor's homepage website. Slides were scanned at 5 μm resolution with a ScanArray 4000 × l (Perkin Elmer, Waltham, Ma) and analyzed with the CodeLink Analysis Software, providing an integrated optical density (IOD) value for each hybridization spot, which is a measurement of an integrated background intensity value subtracted from the total pixel intensities within the area of the spot.

first: Kelli

last: Mayo

last: Beggs


title: Comparative transcriptional analysis of pre-erythrocytic stages

status: Public on Nov 10 2009

city: Baltimore

first: Calvin

middle: Williams

last: Williams

state: MD

address: HSF I Suite 380, 685 W. Baltimore St.

department: Microbiology and Immunology

institute: University of Maryland

summary: To gain insight into the molecular mechanisms at work during progression through the pre-erythrocytic stages, a comparative microarray based transcriptional study was under taken on radiation attenuated (RAS) and wild type sporozoites (wtSPZ) as well as, and liver stage parasites collected 24 hours (24hrLS) and 48 hours (48hrLS) after wild type sporozoite infection. We were able to identify ~1100 genes significantly differentially expressed during one or more of the pre-erythrocytic stages relative to the mixed blood stages.

overall_design: This study compared the gene expression profiles of radiation attenuated sporozoites (RAS), wild type sporooites (wtSPZ), and infected hepatocytes collected 24hr (24hrLS) and 48hr (48hrLS) after sporozoite infection, using a common reference design. Each sample was hybridized with an equal amount of reference RNA. 48hrLS: 4 biological replicates, with 2 total technical replicates and one dye swap; 24hrLS: 4 biologicla replicates, with 3 total technical replicates; RAS: 2 biological replicates, with 2  technical replicates total, and two tota dye swaps; wtSPZ: 2 biological replicates, with 2 total technical replicates and 2 total dye swaps

first: Abdu

last: Azad


title: Global gene expression of NASH patients

status: Public on Aug 06 2009

city: Buffalo

first: Susan

last: Baker

state: NY

address: 3435 Main street, 422 BRB

department: Pediatrics

institute: SUNY at Buffalo

summary: Background & Aims: Little is known about the molecular pathophysiology of non-alcoholic steatohepatitis (NASH).  Methods: Total RNA from NASH livers and normal healthy livers were analyzed on a whole genome DNA oligo microarray.  The microarray data were validated by real-time quantitative PCR analysis for many of the genes of interest.  Results: Genes related to liver inflammation and fibrosis were found to be elevated in NASH livers compared to normal livers.  The most striking finding is the increased gene transcription of alcohol dehydrogenase (ADH) genes, genes for catalase and cytochrome P450 2E1, and aldehyde dehydrogenase genes.  Transcription of genes TLR4, LBP, CD14 and MD-2 were not significantly elevated in NASH livers.  Conclusions: The augmented activity of all the available genes of the pathways for alcohol catabolism suggest that 1) there is a significant increase of alcohol in the circulation of NASH patients; 2) there is a high priority for the NASH livers to scavenge alcohol from the circulation.  It was reported that gastrointestinal bacteria are the major cause of elevated alcohol in the circulation of obese animals.  Mechanisms for alcohol to induce steatosis and oxidative stress were established decades ago.  Our data is the first human evidence to support Diehl et al’s hypothesis that alcohol is an important factor in the development of non-alcoholic fatty liver diseases.

overall_design: Six NASH patients exhibiting significant insulin resistence were included in this study.  For control groups, total RNA from 4 different subjects were purchased from ADMET.  These subjects are free from liver disease.

first: Robert

middle: D

first: Wensheng

last: Liu

first: Norma

last: Nowak

first: Lixin

last: Zhu


title: Rapamycin-insensitive up-regulation of MMP2 and other genes in TSC2- deficient LAM-like cells

status: Public on Oct 21 2009

first: Po-Shun

last: Lee

address: 1 Blackfan Circle, Karp 6

department: Medicine

institute: Brigham & Women's Hospital

summary:
Rationale:
Increased matrix metalloproteinase (MMP) activity has been implicated in the pathogenesis of lymphangioleiomyomatosis (LAM).

Objectives:
To investigate how TSC1 or TSC2 deficiency alters MMP expression and regulation.

Methods:
We studied immortalized cells that lack TSC2 derived from an angiomyolipoma (AML) of a LAM patient, and a TSC2 add back derivative; and murine embryonic fibroblast cells that lack Tsc1 or Tsc2 and respective controls. Global gene expression analysis was carried out in the AML and derivative cell lines. MMP levels in the conditioned media from these cells were analyzed by zymography and ELISA.

Measurements and Main Results:
We found increased MMP-2 expression in cells lacking TSC1/TSC2 compared to their respective controls by zymography. MMP-2 overproduction by these cells was not affected by rapamycin treatment. Gene expression analysis confirmed increased MMP-2 gene expression that was not affected by rapamycin. Furthermore, multiple other genes were found to be over-expressed in rapamycin-treated TSC2-deficient cells compared to TSC2+ cells.

Conclusions:
We conclude that TSC1/TSC2 deficiency leads to MMP-2 overproduction that is rapamycin insensitive, and that several genes exhibit similar patterns suggesting TSC1/TSC2 dependent but mTOR independent pathways may be involved in the pathogenesis of LAM.

overall_design: We studied immortalized cells that lack TSC2 derived from an angiomyolipoma (AML) of a LAM patient, and a TSC2 add back derivative. Global gene expression analysis was carried out in the AML and derivative cell lines. MMP levels in the conditioned media from these cells were analyzed by zymography and ELISA.

first: Szeman

middle: W

last: Tsang

first: Marsha

last: Moses

first: Zachary

last: Trayes-Gibson

first: Li-Li

last: Hsiao

first: Roderick

last: Jensen

first: Rachel

last: Squillace

first: David

last: Kwiatkowski


title: Ctr9 Promoter Region Binding

status: Public on Jan 31 2009

city: Dresden

first: Li

last: Ding

address: Pfotenhauerstr. 108

country: Germany

institute: MPI-CBG

type: Genome variation profiling by genome tiling array

summary:
To study the function of Paf1C in mouse ESCs, we generated an ES cell line stably expressing a location and affinity purification (LAP)-tagged Ctr9 fusiuon protein using the bacterial astificial chromosome (BAC)-based TransgeneOmics approach. To investigate whether pluripotency and lineage control genes differentially regulated upon Paf1C depletion are direct targets of the Paf1C, we analyzed the binding of the Ctr9-LAP fusion protein by ChIP-chip and identified 2175 promoter regions that were bound by Ctr9.
The supplementary bed file contains promoter regions bound by Ctr9.

Keywords: ChIP-chip

overall_design: GFP-tagged Ctr9 ChIPed with GFP vs. mock in  BAC-transgenic mouse ESCs.

first: Herbert

last: Schulz

first: Maciej

last: Paszkowski-Rogacz


title: Whole genome expression in meningioma

status: Public on Nov 21 2008

city: Homburg

first: Nicole

last: Ludwig

address: Kirrberger Strasse

department: Human Genetics

institute: Saarland University

summary: We performed expression profiling of 24 meningioma and two dura controls analyzing 55000 transcripts including 18300 known genes. We compared expression in meningioma vs. dura, expression of low grade (WHO I) vs. higher-grade (WHO II and WHO IIII) tumors and expression of meningothelial and syncytial meningioma vs. fibroblastic meningioma.

overall_design: Gene expression was analysed in 24 meningioma including eight of each WHO grade and two dura controls analyzing 55000 transcripts including 18300 known genes.


title: Microarray-based species identification of Streptomyces spp. isolated from potato scab lesions in Norway

status: Public on May 01 2010

first: Panu

last: Somervuo

summary: 28 Streptomyces strains isolated from common scab lesions of potato tubers from a wide geographic range in Norway, were selected for microarray analysis. The selected strains were subjected to species identification by microarray, 16S phylogenetic analysis and PCR; and microarray-based comparative genome analysis. To our knowledge, this is the first report of S. turgidiscabies and S. europaeiscabiei in Norway.

sample_organism:
Streptomyces scabiei
Streptomyces turgidiscabies
Streptomyces europaeiscabiei

overall_design: 28 Norwegian Streptomyces strains were hybridized in duplicates, one S.turgidiscabies strain (St32) and one S.scabies strain (ATCC49173) were hybridized in 4 replicates. Two out of 64 hybridizations failed (replicate hybridizations of Norwegian strains 33 and 44), for a total of 62 samples. Normalization was based on log-ratios against reference strain.

first: Merete

last: Dees

first: Jari

last: Valkonen


title: Chromatin immunoprecipitation of C/EBP delta in mouse bone-marrow macrophages stimulated with LPS for 6hr

status: Public on Mar 27 2009

city: Seattle

first: Bruz

last: Marzolf

state: WA

institute: Institute for Systems Biology

summary:
The innate immune system is a two-edged sword; it is absolutely required for host defense against infection, but if left uncontrolled can trigger a plethora of inflammatory diseases.  Here we used systems biology approaches to predict and validate a gene regulatory network involving a dynamic interplay between the transcription factors NF-kB, C/EBPd, and ATF3 that controls inflammatory responses.  We mathematically modeled transcriptional regulation of Il6 and Cebpd genes and experimentally validated the prediction that the combination of an initiator (NF-kB), an amplifier (C/EBPd) and an attenuator (ATF3) forms a regulatory circuit that discriminates between transient and persistent Toll-like receptor 4-induced signals. Our results suggest a mechanism that enables the innate immune system to detect the duration of infection and to respond appropriately.

Keywords: ChIP-chip

overall_design: Two sample types with 3 biological replicates each.

first: Vladimir

last: Litvak

first: Stephen

last: Ramsey

first: Alistair

middle: G

last: Rust

first: Alan

last: Diercks

first: Daniel

middle: E

last: Zak

first: Kathleen

last: Kennedy

first: Aaron

last: Lampano

first: Matti

last: Nykter

first: Ilya

last: Shmulevich

last: Aderem


title: PSF regulates specific subsets of human mRNAs

status: Public on Dec 09 2008

city: Frederick

first: Andrei

last: Zolotukhin

address: P.O. Box B

institute: NCI-Frederick

summary: We sought to identify the transcripts that responded to changes in cellular SFPQ/PSF (polypyrimidine tract-binding protein (PTB)-associated splicing factor) protein levels by expression profile analysis of PSF knockdown cells. We constructed clonal HeLa cell lines stably expressing small hairpin RNAs (shRNA) targeted to various regions within the PSF CDS. For microarray studies, we chose two clones (#s 1_1 and 3_1) harboring independently targeted shRNAs that showed ~50% reduction of the endogenous PSF protein. As controls, we used two independent clones harboring a non-targeting shRNA (#s 6_2 and 6_4). Microarray analysis of total cellular RNA identified the transcripts that were reproducibly upregulated (>1.5 fold, n=284) and downregulated (< 0.66 fold, n=121) in both independent knockdown cell lines, and hence were likely enriched in the nonrandomly responding species. We next studied the effects of PSF depletion on particular gene categories. Analysis of the transcripts with unique RefSeq identifiers (n=15805) using PANTHER tools at http://www.pantherdb.org (Molecular Function) revealed significant nonrandom responses of transcripts encoding ribosomal proteins (RP) (downregulation, n=150, P=2.9E-04), while other subgroups exhibited higher P-values. We wondered whether the nonrandomly affected transcripts shared nucleotide sequence features that might account for their response to PSF depletion. To extract such features, we analyzed the mRNAs representing the top up- and downregulated species (n=100 each) as well as the “ribosomal protein” category retrieved from PANTHER database (n=150)  for the presence of shared sequence motifs, by using Gibbs motif sampling and dscan software at http://bayesweb.wadsworth.org/gibbs/gibbs.html. The Gibbs sampling returned very similar shared signatures for the up- and downregulated species; and a very similar signature (motif #3) was elicited from the combined up and downregulated datasets (n=200). Notably, a related signature was also elicited from RP sequences. In order to assign unbiased probabilities to the nonrandomness of motif occurrence, we used nucleotide frequency matrices representing motif #3 and dscan utility to calculate the hit scores in the up and downregulated mRNAs. As a control, the same procedure was performed on samplings (n=100) randomly extracted from human EST sequence databases, and the hit scores were evaluated using the Mann-Whitney test. We found that the random samplings featured only low scores, while the PSF-repressed and, to even greater extent, the PSF-activated samplings exhibited significantly higher scores (P=0.01 and P=0.0001,  respectively; α=0.05), and similar P-values were obtained when using different control samplings. These data revealed a correlation between PSF-responsiveness and high occurrence of a particular sequence signature, and suggested that the presence of such sequences could contribute to the response. Because PSF can directly bind mRNA in vivo, it was plausible that it recognized the motifs directly, as part of mRNA. Indeed, comparison of motif #3 to the direct PSF-binding sites in pre-mRNA revealed a similarity that included a common UGNAGC signature, whereas the SELEX-derived PSF aptamers shared the core GYYG signature. In addition, the motif #3 also exhibited, albeit to a different extent, direct repeats of a trinucleotide CUG that were not found in the CLIP and SELEX signatures. Since such sequences have not been previously noted in PSF-binding RNA, their significance was not clear. However, the pre-mRNA CLIPs were nonrandomly enriched in CUG‘s constituent dinucleotides CU and UG, possibly reflecting a shared recognition determinant. In summary, our results showed that PSF regulates specific subsets of human genes, and suggested that PSF-mRNA recognition could contribute to the regulation.

overall_design: Two biological replicates were analyzed for the control and knockdown cell lines.

last: Michalowski

first: Irina

middle: V

last: Tretyakova

first: Barbara

last: Felber


title: ChIP-on-chip examination of inducible genes in T cells

status: Public on Jun 01 2009

city: Bruce

first: Kristine

last: Hardy

state: ACT

address: University of Canberra

country: Australia

institute: University of Canberra

summary:
Specific chromatin characteristics, especially the modification status of the core histone proteins, are associated with different gene expression states. For example, active genes are marked by histone acetylation and methylation on specific lysine residues while inactive genes are marked by histones that are methylated on a different set of lysine residues and are not acetylated. There is growing interest in the chromatin characteristics of genes that respond to environmental or developmental signals with evidence that developmentally regulated genes may possess distinct chromatin marks. Using a T cell model, we examined the chromatin characteristics of inducible genes that respond to activation using ChIP-on-chip. We find that inducible genes, especially rapidly induced primary response genes, are more likely to display the chromatin characteristics of active genes, ie high levels of H3K9 acetylation. Consequently, only a small percentage of inducible genes display an increase in chromatin acetylation following activation. Our results provide evidence that inducible genes, especially rapidly induced genes, can be defined by their chromatin signature.

Keywords: ChIP-chip

overall_design: Cells were stimulated for 0h, 0.5h or 4h. Extracts were immunoprecipitated with either, H3K9ac, H3 or no antibody and total genomic input DNA was also collected. H3K9acetylated ChIP samples were compared to either total input genomic DNA or pan histone 3 (H3)  ChIP samples. Mock ChIP samples were compared to total input DNA. 2 biological replicates were used for all treatments except for non-stimulated EL4 cells where 3 replicates were used.

first: Chloe

last: Lim

first: Frances

last: Shannon

first: Lina

last: Ma

first: Kaimen

last: Peng


title: Genome-wide analysis of H3K79 dimethylation in normal and MLL-AF4 leukemic pre-B cells

status: Public on Nov 04 2008

city: Plantagenet

first: Madeleine

middle: E.

last: Lemieux

state: ON

address: 275 Main St., Suite 252

country: Canada

institute: Bioinfo

summary:
We created a mouse model where conditional expression of physiologic levels of an Mll-AF4 fusion oncogene induces development of acute lymphoblastic (ALL) or acute myeloid leukemias (AML). ChIP-chip analysis demonstrated increased histone H3 Lysine 79 (H3K79) dimethylation that correlated with Mll-AF4 associated gene expression profiles in murine ALLs, and in human MLL-rearranged leukemias. In addition, human MLL-rearranged ALLs can be distinguished from other ALLs by their genome-wide H3K79 methylation profiles.

Keywords: Cell type comparison

overall_design: DNA was immunoprecipitated with anti-dimethyl-H3K79 antibodies from purified marrow preB cells of normal mice or animals made leukemic by activation of an MLL-AF4 fusion. The ChIP DNA was then hybridized to Affymetrix promoter tiling microarrays.


title: Genome-wide analysis of H3K79 dimethylation in MLL-AF4 leukemic bone marrow

overall_design: DNA was immunoprecipitated with anti-dimethyl-H3K79 antibodies from bone marrow of mice made leukemic by activation of an MLL-AF4 fusion. The ChIP DNA was then hybridized to Affymetrix promoter tiling microarrays.


title: Chip-chip from mouse embryonic stem (mES) cells with bioTrim28, and bioCnot3

status: Public on Jun 30 2009

city: Boston

first: guang

last: hu

address: NRB 160, 77 Ave. Louis Pasteur

department: Genetics

institute: Brigham and Women's Hospital, Harvard Medical School

summary:
Targets of putative self-renewal factors Trim28 and Cnot3 in mouse embryonic stem cells were identified by bioChIP-chip (biotin-mediated ChIP-chip).

Keywords: Biotin-mediated Chip-chip, Mouse embryonic stem cells

overall_design: Comparison of targets of Trim28 and Cnot3 transcription factors in mES cells

first: Guang

last: Hu

first: Jonghwan

last: Kim

first: Qikai

last: Xu

first: Yumei

last: Leng

first: Stuart

middle: H

last: Orkin

first: Stephen

last: Elledge


title: Intraspecific variation of recombination rate in maize

status: Public on Sep 05 2013

city: Freising

first: Eva

last: Bauer

address: Liesel-Beckmann-Str. 2

department: Plant Breeding

institute: Technical University of Munich

summary:
The data set submitted here contains the raw SNP genotyping data obtained from the analysis of 24 biparental segregating maize (Zea mays L.) populations and their respective parents. The processed and filtered data were used to construct genetic linkage maps which we used in our study of variation of recombination rate in maize.
In sexually reproducing organisms, meiotic crossovers ensure the proper segregation of chromosomes and contribute to genetic diversity by shuffling allelic combinations. Such genetic reassortment is exploited in breeding to combine favorable alleles, and in genetic research to identify genetic factors underlying traits of interest via linkage or association-based approaches. Crossover numbers and distributions along chromosomes vary between species, but little is known about their intraspecies variation.
In our study, we report on the variation of recombination rates between 22 European maize inbred lines that belong to the Dent and Flint gene pools. We genotyped 23 doubled-haploid populations derived from crosses between these lines with a 50k-SNP array and constructed high-density genetic maps, showing good correspondence with the maize B73 genome sequence assembly. By aligning each genetic map to the B73 sequence, we obtained the recombination rates along chromosomes specific to each population. We identified significant differences in recombination rates at the genome-wide, chromosome, and intrachromosomal levels between populations, as well as significant variation for genome-wide recombination rates among maize lines. Crossover interference analysis using a two-pathway modeling framework revealed a negative association between recombination rate and interference strength.
To our knowledge, the present work provides the most comprehensive study on intraspecific variation of recombination rates and crossover interference strength in eukaryotes. Differences found in recombination rates will allow for selection of high or low recombining lines in crossing programs. Our methodology should pave the way for precise identification of genes controlling recombination rates in maize and other organisms.

overall_design:
We genotyped 2233 maize DH lines from 24 biparental populations, and the 23 parents of these populations using the Illumina MaizeSNP50 BeadChip. We created two large half-sib panels, one each for the Dent and the Flint germplasm. The Dent populations have the prefix CFD, the Flint populations have the prefix CFF. In each panel, a common central parent was crossed to diverse founder lines, and doubled haploids were generated from the respective F1 plants. For a detailed description of the material, see Bauer et al. (2013) Genome Biology (submitted).
We submit here three datasets: 1) Dataset Parents comprises all 23 parental lines. 2) Dataset CFD comprises all 1005 DH lines from Dent crosses, 3) Dataset CFF comprises all 1262 DH lines from Flint crosses.


title: Azacytidine time course on MCF7 cells

status: Public on Jan 12 2008

city: Heidelberg

first: George

last: Reid

address: Meyerhofstrasse 1

institute: European Molecular Biology Organisation

summary:
MCF7 cells at 80% confluency were treated with 5 micromolar azacytidine with gene expression profiling determined at 0, 1, 2, 3, 4, 6 and 12 hours of exposure to compound.
Keywords: time course

overall_design: Approximately 5,000,000 million cells were plated into 10 cm plates from a common pool. Cells were grown in DMEM supplemented with 10% foetal calf serum until 80% confluency was attained. Cells were then exposed to 5 micromolar azacytidine prepared as a 10 millimolar stock in DMSO. Each time point was represented by three biological replicates. Total RNA was extracted from the cells using the trizol method and RNA samples were validated for integrity throughout the isolation and extraction procedure. All gene expression profiles showed similar distributions with similar raw median intensities.

first: Maria

last: Polycarpou-Schwarz

first: Vladimir

last: Benes

first: Tomi

last: Bähr-Ivacevic


title: The effect of homocysteine on human aortic smooth muscle cells

status: Public on Oct 21 2008

city: Townsville

first: Jonathan

last: Golledge

state: Queensland

address: School of Medicine

department: School of Medicine

institute: James Cook University

summary:
Vascular calcification is a common manifestation of atherosclerosis and involves cell-mediated processes similar to the formation of bone (osteogenesis). Elevated plasma levels of homocysteine are an independent risk factor for atherosclerotic vascular disease but the underlying mechanisms remain unclear. We postulated that hcy can modulate the cells involved in atherosclerosis to promote calcification. Cell experiments were performed to assess the effect of homocysteine on the osteogenic differentiation of aortic smooth muscle cells (AoSMC).
Keywords: dose repsonse comparison

overall_design: To study the ability of homocysteine to induce osteogenic differentiation, AoSMC were cultured in T75 flasks (7.5x10E5 /mL) in growth medium with 0, 10 and 100 μmol/L DL-homocysteine. Four biological replicates were prepared for each condition. After 14 days, RNA was extracted from cells and the expression levels of osteogenic genes were compared between the different conditions of homocysteine concentration.

first: Corey

last: Moran

first: Catherine

last: Rush

first: Ann

last: Van Campenhout


title: STAT4 ChIP-on-chip in IL12-Mediated T Helper 1 Cell Differentiation

status: Public on Jun 26 2009

city: indianapolis

first: Narayanan

middle: B

last: Perumal

state: IN

address: 719 Indiana Avenue Suite, WK311

department: School of Informatics

institute: Indiana University-Purdue University Indianapolis

summary:
The Signal Transducer and Activator of Transcription factor STAT4 is a critical component in the development of inflammatory adaptive immune responses. It has been extensively characterized as a lineage-determining factor in Th1 development. However, the genetic program activated by STAT4 that results in an inflammatory cell type is not well defined. In this report we use DNA isolated from STAT4-chromatin immunoprecipitation to perform ChIP-on-chip analysis of over 28,000 mouse gene promoters to identify STAT4 targets. We demonstrate that STAT4 binds multiple gene-sets that program distinct components of the Th1 lineage. While many STAT4 target genes display STAT4-dependent IL-12-inducible expression, other genes displayed IL-12-induced histone modifications but lack induction, possibly due to high relative basal expression. In the subset of genes that STAT4 programs for expression in Th1 cells, IL-12-induced mRNA levels remain increased for a longer time than mRNA from genes that are not programmed. This suggests that STAT4 binding to target genes, while critical, is not the only determinant for STAT4-dependent gene programming during Th1 differentiation.
The supplementary bed file contains all 7504 Stat4 binding sites reported in the supplementary Analysis_results.xls Excel file.

overall_design: IL12-stimulated CD4+ T cell DNA ChIPed with Stat4 vs. Input DNA (2 replicates)

first: Mark

last: Kaplan


title: Gene expression in immortalized B-lymphocytes derived from patients with multiple sclerosis

status: Public on May 08 2008

city: Rensselaer

first: Sridar

last: Chittur

address: One Discovery Drive, CRC 342G

department: Center for Functional Genomics

institute: SUNY-University at Albany

summary:
This study aims to determine if global gene expression and transcription factor networks in B-lympocytes of siblings with MS were different from healthy siblings.
Keywords: Multiple sclerosis, sibling comparisons

overall_design: 10 samples from 3 families were used in this study. Of these 4 were unaffected siblings while 6 were siblings with MS. The total RNA from these samples were processed for hybridization on to Codelink Whole Human Genome arrays.

first: Jagannadha

last: Avasarala

first: Ajish

last: George

first: John

middle: A

last: Tine


title: Progerin-induced changes in gene expression

status: Public on Feb 09 2008

city: Bethesda

first: paola

last: scaffidi

address: 41 Library Drive, B508

institute: NCI, NIH

summary:
The premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by constitutive production of progerin, a mutant form of the nuclear architectural protein lamin A1.  Progerin is also sporadically expressed in wild type cells and has been linked to physiological aging.  HGPS cells exhibit extensive nuclear defects including abnormal chromatin structure and increased DNA damage.  At the organismal level, HGPS affects several tissues particularly of mesenchymal origin.  How the cellular defects of HGPS cells lead to the organismal defects has been unclear.  To begin to unravel how progerin leads to disease phenotypes, we analyzed time-dependent changes in transcriptional profiles in response to progerin expression in a hTERT-immortalized skin fibroblast cell line expressing either GFP-progerin or the  GFP-wt lamin A control
Keywords: time course, cell line comparison

overall_design: Three time points (0d, 5d, 10d after induction of the exogenous protein) were analyzed for each cell line (expressing either GFP-progerin or GFP-wt lamin A). Two biological replicates were analyzed for each time point.

first: Paola

last: Scaffid

first: Tom

last: Misteli


title: MELK Leucine Zipper Kinase is a Key Differentiator between Glioblastoma Multiforme and Pilocytic Astrocytomas

status: Public on Mar 10 2008

city: Sao Paulo

first: Oswaldo

middle: Keith

last: Okamoto

address: R. Botucatu, 862.

department: Neurology

institute: UNIFESP

summary:
Purpose: Gene expression analyses comparing tumors of different behaviors have been used for the identification of genes that have a role in the growth and maintenance of tumors, with the aim of both understanding the molecular basis of tumorigenesis and identifying novel potential therapeutic targets. We have here used this approach to identify the underlying gene expression differences between highly invasive glioblastoma multiforme and typically benign pilocytic astrocytomas. Experimental Design: We performed cDNA microarray analyses comparing astrocytomas of polar grade: grade I, pilocytic astrocytoma (PA), the non-invasive and most frequent pediatric tumor, with grade IV, the highly infiltrative glioblastoma multiforme (GBM) Results: Despite the significant clinical and pathological differences between the two tumor types, only 63 genes were found to exhibit two-fold or greater over-expression in GBM as compared with PA. A functional classification of the GBM over-expressed genes indicated that more than 50% are related to the regulation of the cell cycle and mitosis. Microarray data was validated by RQ-PCR testing six over-expressed genes in GBM related to PA: MELK, AUKB, ASPM, PRC1, IL13RA2 and KIAA0101. The differential expression was confirmed for all six genes, showing at least a 5 fold increase in the average expression levels in GBM compared to PA. Of the over expressed genes, that which exhibited the most statistically significant difference is Maternal Embryonic Leucine zipper Kinase (MELK). We undertook a more detailed investigation of the expression of this serine/threonine kinase gene in astrocytomas in the light of its role in the regulation of  multipotent neural progenitor proliferation and previous suggestions that this may be a oncogenic target of general importance. In an examination of more than 100 tumors of the central nervous system we found progressively higher expression of MELK in GBM with astrocytoma grade and a noteworthy uniformity of high level expression in GBM. This latter feature probably explains the lack of observed association of MELK expression with survival of GBM patients. Over-expression at a similar level to GBM was also observed in medulloblastoma. We found neither gene methylation nor amplification to be a factor in MELK expression but were able to demonstrate that MELK knockdown in malignant astrocytoma cell lines caused a reduction in proliferation and anchorage-independent growth in in vitro assays. Conclusions: Our results indicate that GBM and PA differ by the expression of surprisingly few genes. Among them, MELK may play a key role in differentiating these two tumor types and represent an important therapeutic target for the management of the most frequent brain tumors in adult and children.
Keywords: Comparizon of CNS tumors of different grades.

overall_design:
Nine primary astrocytomas (three samples of PA, mean age of 19 yo; and six samples of GBM, mean age of 56 yo, and mean survival time of 7.6 months), and a pool of three non-neoplastic brain tissues (mean age of 47 yo) were used for microarray analyses.
Analyses were carried out with commercial oligonucleotide microarrays (CodeLink Bioarrays-Human , GE Healthcare, Piscataway, NJ), following the manufacturer’s protocol. Independent hybridizations for each sample were carried out in duplicate. Individual gene PA and GBM expression profiles were compared with each other, and with non-neoplastic brain tissue. Differentially expressed genes were identified by calculating the ratio of the mean normalized fluorescence values obtained from each duplicate GBM or PA sample and non-neoplastic brain tissue, and subsequently between duplicate GBM and PA sample.  Results were expressed as fold variation, and genes displaying greater than 2-fold changes in transcript abundance and p<0.01 were selected.

first: Suely

middle: K

last: Marie

first: Miyuki

last: Uno

first: AnaPaula

last: Hasegawa

first: Sueli

middle: M

last: Oba-Shinjo

first: Tzeela

last: Cohen

first: Anamaria

last: Camargo

first: Ana

last: Kosoy

first: Carlos

last: Carlotti Jr

first: Silvia

middle: C

last: Toledo

last: Moreira Filho

first: Marco

last: Zago

first: Andrew

last: Simpson

first: Otavia

last: Caballero


title: Whole Genome Microarray comparison of two Interferon Beta 1a Pharmaceutical Preparations

status: Public on Jun 30 2008

city: Buenos Aires

first: Patricio

last: Yankilevich

address: Constitución 4234

country: Argentina

institute: BioSidus

summary:
We have investigated the in vitro pharmacodynamic genomic effects of interferon beta 1a treatment on a whole genome microarray assay on lymphocytes from Multiple Sclerosis patients comparing the similarity between two biological products manufactured by two pharmaceutical companies.
Keywords: Drug pharmacogenomics

overall_design: Ex vivo whole genome expression profiling of the effect of two preparations of IFNß1a was performed on non-adherent mononuclear cells from five Relapsing-Remitting Multiple Sclerosis patients (RRMS, 2 males and 3 females, aged from 15 to 54 years old) using microarrays analysis (CodeLinkTM Human Whole Genome).

first: Aida

last: Sterin Prync

first: Roberto

last: Diez

first: Marcelo

last: Kauffman

last: Barrero

first: Laura

last: Marangunich

first: Ricardo

last: Bello

last: Criscuolo

first: Alejandro

last: Vidal


title: Genome-wide in vivo binding analysis for PU.1 in RAW264.7 macrophages

status: Public on Oct 11 2007

city: Regensburg

first: Thomas

last: Langmann

address: Franz-Josef-Strauss-Allee 11

department: University of Regensburg

institute: Institute of Human Genetics

summary:
We used Affymetrix Mouse Promoter 1.0R arrays to analyse PU.1-dependent promoters in mouse macrophages
Keywords: PU.1 ChIP versus IgG ChIP, ChIP-Chip

overall_design: 3 biological replicates of PU.1 and IgG Immunoprecipitation were performed each

first: Langmann

last: Thomas


title: Activated Cell Death Pathway

status: Public on Apr 30 2007

city: Chiba

first: Shuhei

last: Noda

address: Anagawa 4-9-1

department: Frontier research center

institute: National institute of radiological sciences

summary:
Identifying key genes to evaluate the therapeutic responses of chemoradiotherapy.
Keywords: Cervical cancer, chemoradiotherapy, biopsy, CDKN1A, Bax

overall_design: Twenty-eight squamous cell carcinoma of cervix from 24 patients were taken as biopsy sample before treatment and during treatment; twice for each patient. Duplicate of each sample were analyzed. Fourteen patients received radiotherapy alone and 10 patients received chemoradiotherapy.

first: Mayumi

last: Iwakawa


title: Epigenetic therapy with hydralazine and magnesium valproate added to chemoradiation in cervical carcinoma.

status: Public on Jul 31 2007

city: México

last: Pérez-Plasencia

address: Av. San Fernando 22

department: BASIC RESEARCH

country: Mexico

institute: INSTITUTO NACIONAL DE CANCEROLOGIA

summary:
As a number of epigenetic alterations have been described in cervical cancer which may participate in the development and progression of this tumor, and the primary treatment for locally advanced disease is concurrent chemoradiation, we wanted to evaluate the efficacy and safety of hydralazine and valproate added to radiation and cisplatin. In addition, the transcriptome changes induced by the epigenetic drugs were also evaluated.
Keywords: Cervical Cancer, epigenetic therapy, valproate, hydralazine, transcriptional response, epigenetic drugs

overall_design:
Previously untreated patients with histological diagnosis of carcinoma of the cervix and FIGO staged IIIB were entered into this phase II study. Patients received a single oral dose of 500 mg of sulphamethazine early in the morning, and urine was collected within the ensuing 6 h for phenotyping of acetylator status as reported.  Afterward, patients began treatment (day –7) with a daily dose of a slow-release formulation of hydralazine tablets containing either 182 mg for rapid, or 83 mg for slow acetylators. Magnesium valproate tablets of 700 mg were also administered as a slow-release formulation at a dose of 30 mg/kg t.i.d. Both were administered until intracavitary therapy was completed. Punch biopsies were taken from primary cervical tumors at diagnosis and at day 8 of treatment with hydralazine and magnesium valproate prior to the first dose of cisplatin and external radiation. Part of the biopsy was sent to the Institution’s Pathology Department for routine hematoxilin and eosin evaluation. The remaining biopsy specimen was immediately frozen at –70°C for biological analyses.
RNA isolation was done in TRIzol according to manufacturer’s instructions. One μg of total RNA was used as input material. All reagents were provided in the CodeLink expression assay kit (Amersham, Piscataway, NJ USA). First-strand cDNA was generated using Superscript II reverse transcriptase and a T7 primer. Subsequently, second-strand cDNA was produced using E. coli DNA polymerase I and RNase H. The resultant double-stranded cDNA was purified on a QIAquick column (Qiagen, Valencia, CA) and cRNA was generated via an in vitro transcription reaction using T7 RNA polymerase and biotin-11-UTP (Perkin-Elmer, Boston, MA). cRNA was purified on an RNeasy column (Qiagen), quantified by UV spectrophotometry, and 10ug were then fragmented by heating at 94°C for 20 minutes in the presence of magnesium. The fragmented cRNA was hybridized overnight at 37°C in hybridization buffer to a CodeLink Whole Human Genome Bioarray in an Innova 4080 shaking incubator (New Brunswick, Edison, NJ) at 300 rpm.

last: Perez-Plasencia

first: Alfonso

last: Duenas-Gonzalez


title: Study of Leukotriene B4 (LTB4) effect on primary human monocytes transcription profile

status: Public on Oct 18 2007

city: San Diego

first: Heng

last: Dai

address: 3210 Merryfield Row

department: Bioinformatics

institute: Johnson & Johnson PRD

summary:
Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation, implicated in numerous diseases including atherosclerosis. We have used primary human monocytes, which express both receptors, to analyze transcriptional responses to LTB4. Comparisons were made between LTB4- and vehicle-treated samples at the same time point.  The analysis showed that expression of 72 genes was upregulated at least two-fold at two time points. These genes include multiple chemokines as well as some genes with unknown function.
Keywords: time course

overall_design: Cells were stimulated with LTB4 for various times. For each time point, there is one treated sample and one vehicle control. After isolation, each RNA sample is hybridized to duplicate CodeLink chips.

first: Binggang

last: Sun

first: Jing-Feng

last: Huang

first: Xiaohua

last: Xue

first: Anton

last: Bittner

first: Lars

last: Karlsson

first: Anne

last: Fourie


title: Transcriptome analysis of Stem Cells from Human Umbilical Cord Blood

status: Public on Apr 05 2007

city: Ribeirao Preto

last: Vêncio

address: Av. Bandeirantes, 3900

department: Computing and Mathematics Department

institute: Universidade de São Paulo

summary:
Uncovering the molecular mechanism of stem cell self-renewal is critical to broaden current therapeutic applications and to understand how its de-regulation may lead to pathological conditions. In this work, we report changes in gene expression and related cell processes during conditions of high in vitro expansion of CD133+/CD34+ primitive stem cells from human umbilical cord blood (UCB). Based on DNA microarray assays, we demonstrate that primary metabolism and cell cycle genes are specifically up-regulated, as well as members of the Wnt and Notch signaling pathways. Estradiol (E2) treatment resulted in enhanced cell expansion, which was correlated with activation of MEK/ERK signaling genes and inhibition of GLI2, a member of the Hedgehog pathway. Most notably, E2-induced CD133+/CD34+ cell expansion was highly associated to regulation of genes disrupted in cancer, such as the recently described DOCK4 and SPARCL1 tumor suppressor genes. Quantitative real-time PCR analysis confirmed down-regulation of DOCK4 and SPARCL1 in E2-treated CD133+/CD34+ cells and parallel results were verified when comparing their expression in mononuclear blood cells of chronic myeloid leukemia patients and healthy individuals. The striking differential expression of cancer-associated genes found reveals potential molecular targets for oncogenic transformation of CD133+/CD34+ cells and strengthens the importance of pre-clinical studies assessing safety of stem cell expansion protocols for therapeutic application.
Keywords: dose response

overall_design: Gene expression intensities were measured using CodeLink Human Whole Genome Bioarrays.

first: Oswaldo

last: Okamoto

middle: Z


title: Whole human genome DNA microarray analysis of gene expression in ectopic vs. eutopic endometrium

status: Public on Feb 15 2007

city: Vermillion

first: Kathleen

last: Eyster

address: 414 E. Clark St.

department: Basic Biomedical Sciences

institute: University of South Dakota

summary:
Brief description of the experiment:   The objective of this experiment was to use DNA microarrays to identify differentially expressed genes in eutopic uterine endometrium compared to ectopic endometrium.  174 of the 53,000 genes on the whole human DNA microarrays were changed by 5-fold or greater in ectopic vs. eutopic endometrium.  Families of genes that were differentially expressed include immune system and inflammatory pathway genes,  genes whose cognate proteins code for cell adhesion, junctional proteins, the extracellular matrix and its remodeling, and cytoskeletal proteins, and ligands, receptors, and components of specific signal transduction pathways.  The altered immune environment may allow survival of endometriotic cells that enter the peritoneal cavity.  Alterations of cell adhesion-associated genes may contribute to the adhesive and invasive properties of ectopic endometrium, and changes in signal transduction pathways support a change in the communication among cells of the endometrial explant compared to eutopic endometrium.  These families of differentially expressed genes provide multiple opportunities for the development and testing of new hypotheses regarding endometriosis.
Keywords: disease state analysis, endometriosis

overall_design:
Experimental factors:  Eutopic endometrium from within the uterus (endometrial biopsy) was compared to ectopic endometrium (endometriosis explants, biopsies of endometrial tissue implanted in the peritoneal cavity).

Experimental design:  Eutopic and ectopic endometrium were obtained from the same patients; for each patient, eutopic endometrium from the uterus served as the control for her ectopic endometrial samples.

Quality control steps taken:  The cRNA that was synthesized from each endometrial sample was used for hybridization to a single CodeLink (Amersham/GE) whole human genome microarray.  Only one sample was hybridized with each slide and only one dye (Alexa 647) was used so no dye swaps were necessary.  Bacterial control spikes were used as per manufactorer’s instructions.

Samples used, extract preparation and labelling:

The origin of each biological sample:  All samples were obtained at laparotomy or laparoscopy from women of reproductive age.  All of the patients were diagnosed with endometriosis.  No drug or hormonal treatments were given to the patients.
Manipulation of biological samples and protocols used:  The biopsies of endometriosis and of eutopic endometrium were placed into RNAlater (Ambion) upon surgical removal from patients and frozen at -70C until RNA was extracted.

Experimental factor:  Eutopic endometrium vs. ectopic endometrium.

first: Olga

last: Klinkova

first: Vanessa

last: Kennedy

first: Keith

last: Hansen


title: MSX2 enhances the malignant phenotype and associates with Twist 1 expression in human pancreatic cancer

status: Public on Dec 23 2006

city: Tokyo

first: Wataru

last: Fujibuchi

state: Tokyo

address: 2-42 Aomi, Koto-ku

department: Computational Biology Research Center

institute: Advanced Industrial Science and Technology

summary:
Twist 1 expression was significantly associated with MSX2 expression in human pancreatic carcinomas and was down-regulated in MSX2 pancreatic cancer cells.
Keywords: cell type comparison

overall_design: Cell type comparison. Overexpressoin of MSX2 in human pancreatic cancer cell line BxPC3. B3-EV,	human pancreatic cancer cell line BxPC3 with empty vector. B7, MSX2-overexpressing BxPC3 with expression vectors.

first: Kennichi

last: Satoh

first: Shin

last: Hamada

first: Kenji

last: Kimura

first: Atsushi

last: Kanno

first: Morihisa

last: Hirota

first: Naoki

last: Tanaka

first: Koh

last: Miura

last: Masamune

first: Barbara

last: Vonderhaar

first: Tooru

last: Shimosegawa


title: Expression data from Daudi cells stimulated with anti-CD40 and/or IL-4 for 6 hr

status: Public on Jul 31 2006

city: Bunkyo-ku, Tokyo

first: Hiroshi

last: Fukasawa

address: Kadokawa Hongo Bldg. 4F, 5-24-5 Hongo

institute: Institute of Medicinal Molecular Design, Inc.

summary:
We used microarrays to detail the transcriptome response to CD40-ligation and IL-4 stimulation in Daudi cells.
Keywords: gene expression array-based, response to anti-CD40 and/or IL-4

overall_design: 3 replicates per treatment

first: Toshifumi

last: Wakamatsu


title: Expression data from Daudi cells stimulated with anti-CD40 and/or IL-4 for 6 hr with or without AICAR-pretreatment

summary:
We used microarrays to detail the action of AICAR on the transcriptome response to CD40-ligation and IL-4 stimulation in Daudi cells.
Keywords: gene expression array-based, response to anti-CD40 and/or IL-4 with or without AICAR

overall_design: 3 replicates for each treatment


title: Gene Expression Profiles of Epigenetic treatment in primary breast cancer.

status: Public on Nov 18 2006

first: Carlos

last: Pérez-Plasencia

summary:
Aberrant DNA methylation and histone deacetylation participate in cancer development and progression; hence, their reversal by inhibitors of DNA methylation and histone deacetylases (HDACs) is at present undergoing clinical testing in cancer therapy. As epigenetic alterations are common to breast cancer, in this proof-of-concept study demethylating hydralazine plus the HDAC inhibitor magnesium valproate were added to neoadjuvant doxorubicin and cyclophosphamide in locally advanced breast cancer to assess their safety and biological efficacy.
Keywords: Epigenetic treatment, Hydralazine, valproate, breast cancer

overall_design: Core-needle biopsies were taken before and after patients received for 7 days hydralazine and valproate (before receiving the first course of chemotherapy). Gene expression could only be done in three patients: Patient 1 had analyzed the pre and post-treatment samples. Patients 2 and 3 had only analyzed their post-treatment biopsy as the yield of RNA was insuficient from the pre-treatment biopsies.

last: Perez-Plasencia

first: Claudia

last: Arce

first: Alfonso

last: Duenas-Gonzalez


title: Gene expression induced by Nanosecond pulsed electric fields

status: Public on Feb 01 2006

city: Evanston

first: Xijin

last: Ge

state: IL

address: 1001 University Place

institute: ENH Research Institute

summary:
We use a human whole genome microarray to analyze the effects of nanosecond pulsed electric fields on Jurkat cells with the focus on early response genes to DNA damage.
Keywords: nanosecond pulsed electric fields, jurkat cells, DNA damage

overall_design: The gene expression profiles before, 30min and 60min after electric field radiation are surveyed. Three replicates for each time point.

first: Zheng

last: Wang

first: Ashih

last: Bassi

first: Muhammad

last: Khalid

first: Karl

last: Schoenbach

first: Chenn

middle: Q

last: Zhou

first: Howard

last: Gerber

first: Charles

last: Tseng


title: GAS41 over-expression in Saos2 Tet-Off cells

status: Public on Jul 07 2006

city: Homburg/Saar

first: Sabrina

middle: M.

last: Heisel

address: Kirrbergerstrasse

department: Department of Human Genetics

institute: Saarland University

summary:
Transcription initiation in eukaryotes by RNA polymerase II requires numerous general and regulatory factors including the general transcription factors. Here, we report a new cofactor of the general transcription factor IIF, GAS41, which was previously implicated in tumor development. Microarray analysis of cells with induced overexpression of cofactor GAS41 identifies a significant number of up-regulated transcripts (p-value of 0.01).
Keywords: altered gene expression

overall_design: Saos2 Tet-Off clone B3-Dox, with induced GAS41 overexpression, was compared to Saos2 Tet-Off clone B3+Dox, without overexpression. Additionally, B3-Dox was compared to Saos2 Tet-Off clones F7 +Dox/ -Dox, both transfected with pTRE2pur response plasmid only, to exclude any doxycycline effect. All experiments were done in triplicates, revealing twelve data sets.

first: Nicole

last: Schuetz


title: Osteoblast and Osteocyte Profiles at Different Cell-cummunication Status

status: Public on Jun 05 2005

city: San Antonio

first: Wuchen

last: Yang

address: 7703 Floyd Curl Drive

department: Department of Periodontics

institute: The University of Texas Health Science Center at San Antonio

summary:
Type of Experiment:
1) Profiles of Osteoblast vs. osteocyte in vitro;
2) Profiles of osteoblast low density vs. confluency in vitro;
3) Profiles of osteocyte with gap junction vs. without gap junction.

Experimental factors:
1) 2T3 osteoblast cells at low density expressed extensive filopodia, reminiscent of early osteoblast precursors and similar to MLO-Y4 dendritic processes.
2) MLO-Y4 osteocytes at low vs. high density represent the genes that are changed in a highly connected network vs. low connected network.

The number of hybridizations performed:  Triplicate hybridizations for each status.
Keywords = Osteoblast
Keywords = Osteocyte
Keywords: parallel sample

first: Marie

last: Harris

first: Dayong

last: Guo

first: Lynda

middle: F

last: Bonewald

first: Stephen