title: Induction of Dendritic Cell-like Phenotype in Macrophages during Foam Cell Formation

status: Public on Mar 28 2007

city: Charlottesville

first: Christian

middle: Albert

last: Gleissner

state: VA

address: MR5 Building, Rm. 1133

department: Cardiovascular Research Center

institute: University of Virginia

summary:
Foam cell formation from monocyte-derived macrophages is a hallmark of atheroscle-rotic lesions. Aspects of this process can be recapitulated in vitro by exposing MCSF-induced or platelet factor4 (CXCL4)-induced macrophages to oxidized (ox) or minimally modified (mm) low density lipoprotein (LDL). We measured gene expression in periph-eral blood mononuclear cells (PBMCs), monocytes and macrophages treated with CXCL1 (GRO-α) or CCL2 (MCP-1) as well as foam cells induced by native LDL, mmLDL or oxLDL using 22 Affymetrix gene chips. Using an advanced Bayesian error-pooling approach and a heterogeneous error model (HEM) with a false discovery rate (FDR) <0.05, we found 5,303 of 22,215 probe sets to be significantly regulated in at least one of the conditions. Among a subset of 917 candidate genes that were preselected for their known biological functions in macrophage foamcell differentiation, we found that 290 genes met the above statistical criteria for significant differential expression patterns. While many expected genes were found to be upregulated by LDL and oxLDL, very few were induced by mmLDL. We also found induction of unexpected genes, most strikingly MHC-II and other dendritic cell markers such as CD11c. The gene expression patterns in response to oxLDL were similar in MCSF-induced and CXCL4-induced macrophages. Our findings suggest that LDL and oxLDL, but not mmLDL, induce a dendritic cell-like phenotype in macrophages, suggesting that these cells may be able to present antigens and support an immune response.
Keywords: PBMC, monocytes, macrophages, foam cells, chemokines

overall_design:
Human blood was drawn from the antecubital veins of healthy blood donors and provided as buffy coats by the Virginia Blood Services (Richmond, VA). The mononuclear fractions were pooled from four unidentified donors to decrease individual variations in monocytes. Mixed peripheral blood mononuclear cells (PBMCs) were isolated by Histopaque 1.077 (Sigma Diagnostics, Inc., St. Louis, MO). Following centrifugation, the mononuclear layer was removed and washed with PBS containing 0.02% ethylenediaminetetraacetate (EDTA). The pellet was resuspended in 1X H-lyse Buffer (R&D Systems Inc., Minneapolis, MN), and washed with wash buffer. PBMCs contain mainly monocytes and lymphocytes as well as platelets that tend to be associated with blood monocytes. From these PBMCs, monocytes were isolated using a negative selection monocyte isolation kit and LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the isolated fraction was > 97% as estimated by flow cytometry using anti-CD14 (data not shown). Although these cells are often called “untouched” monocytes and thought to show little activation, the gene chip analysis conducted on these cells shows massive changes in gene expression compared to PBMCs (see below).
Monocytes were cultured in Macrophage Serum-Free Medium (MSFM, Invitro-gen, Carlsbad, CA) in the presence of 1% media supplement nutridoma-HU (Roche Molecular Biochemicals, Indianapolis, IN) and 100 nM M-CSF for 6 days, after which the cells showed the expected morphological signs of macrophage differentiation. These macrophages were incubated either with 100 nM CCL2 or CXCL1 for 5 hours. CXCL1 was selected because we have previously found that it is an important arrest chemokine for monocytes in vitro and in atherosclerotic arteries in vivo. CCL2 was chosen because mice lacking CCL2 or its receptor CCR2 are relatively resistant to atherosclerosis, suggesting a role in macrophage recruitment, differentiation and/or survival. Human monocyte-derived macrophages (MDM) were also incubated with native LDL, oxidized LDL (oxLDL) or minimally modified LDL (mmLDL) (each at a concentration of 100 ug/ml) for 2 days to induce foam cell formation. Foam cell formation was verified by oil red O staining (figure 1) and by determining their cholesterol and cholesterol ester content. OxLDL and mmLDL were prepared from the same native LDL for each experiment as described. Control experiments were conducted on macrophages cultured in M-CSF without LDL for an additional 2 days. Two separate sets of monocytes were incubated with CXCL4 (100 nM) for 6 days, another procedure known to induce macrophage differentiation (29), with and without oxLDL to induce foam cell formation. RNA was extracted from cells in all 11 conditions (table 1) and gene expression was measured in duplicates at the University of Virginia Gene Expression Core Facility using Affymetrix equipment.

first: HyungJun

last: Cho

first: Pavel

last: Sashkin

first: Dane

last: Dunson

first: Nitin

last: Jain

first: Jae

middle: K

last: Lee

first: Yuri

last: Miller

first: Klaus

last: Ley


title: Early GA response genes in Arabidopsis thaliana

status: Public on Mar 24 2007

city: Freising

first: Claus

last: Schwechheimer

address: Emil-Ramann-Str. 4

department: Plant Systems Biology

country: Germany

institute: TUM

summary:
The phytohormone GA controls multiple important developmental processes in plants such as germination, elongation growth and flowering time. In this experiment, we look for early GA response genes in 7 day-old light-grown Arabidopsis seedlings. To this end we compare four data sets: (1) a GA biosynthesis mutant ga-1 (SALK_109115) mock treated for 1 hr; (2) a GA biosynthesis mutant ga-1 (SALK_109115) treated for 1 hr with 100 µM GA3; (3) a gid1a-1 gid1b-1 gid1c-2 GA receptor triple mutant mock treated for 1 hr; (4) a gid1a-1 gid1b-1 gid1c-2 GA receptor triple mutant treated for 1 hr with 100 µM GA3. In a comparison of the two ga-1 samples, GA regulated genes can be identified, and the assumption is that bona fide GA regulated genes are not responding in the gid1a-1 gid1b-1 gid1c-2 GA receptor mutant.

Keywords: phytohormone response

overall_design: We compare four samples of 7 day-old light-grown growth-medium grown seedlings (3 independent biological replicates each): (1) a GA biosynthesis mutant ga-1 (SALK_109115) mock treated for 1 hr; (2) a GA biosynthesis mutant ga-1 (SALK_109115) treated for 1 hr with 100 µM GA3; (3) a gid1a-1 gid1b-1 gid1c-2 GA receptor triple mutant mock treated for 1 hr; (4) a gid1a-1 gid1b-1 gid1c-2 GA receptor triple mutant treated for 1 hr with 100 µM GA3. In a comparison of the two ga-1 samples, GA regulated genes can be identified, and the assumption is that bona fide GA regulated genes are not responding in the gid1a-1 gid1b-1 gid1c-2 GA receptor mutant.

first: E

middle: M

last: Dohmann

first: C

last: Nill

first: B

middle: C

last: Willige

first: S

last: Ghosh

first: M

last: Zourelidou

first: A

last: Maier


title: Expression data of germinating ahg1, ahg3 and WT seedling in the presence of ABA

status: Public on May 01 2007

city: Kurashiki

first: Takashi

last: Hirayama

state: Okayama

department: Institute of Plant Science and Resources

country: Japan

institute: Okayama University

summary:
The effect of ahg1 and ahg3 on the gene expression profiles is similar but some genes are differentially affected.
Keywords: Strain Comparison

overall_design: To investigate the effect of ahg1 and ahg3 mutations, the genome wide gene expression profiles of germinating seedlings were examined. These mutations occurred in close but distinct PP2C genes. Stratified seeds were transferred to plates containing 0.5 uM ABA and allowed germinate at 23 °C under 16hr light 8hr dark conditions. After two days samples were collected for RNA extraction.


title: Gene Expression Analysis of Soft Tissue Sarcomas: Characterization & Reclassification of Malignant Fibrous Histiocytoma

status: Public on Apr 01 2007

city: Tokyo

first: Hitoshi

last: Ichikawa

address: 5-1-1 Tsukiji, Chuo-ku

institute: National Cancer Center Research Institute

summary:
In soft tissue sarcomas, diagnosis of malignant fibrous histiocytoma (MFH) has been a very controversial issue, and MFH is now considered to be reclassified into pleomorphic subtypes of other sarcomas. To characterize and reclassify MFH genetically, we analyzed gene expression in 105 samples from ten types of soft tissue tumors.
Keywords: myxofibrosarcoma, leiomyosarcoma, malignant peripheral nerve sheath tumor, fibrosarcoma, malignant fibrous histiocytoma

overall_design: Gene expression of 105 soft tissue tumor samples consisting of synovial sarcoma (n=16), myxoid liposarcoma (n=19), lipoma (n=3), well-differentiated liposarcoma (n=3), dedifferentiated liposarcoma (n=15), myxofibrosarcoma (n=15), leiomyosarcoma (n=6), malignant peripheral nerve sheath tumor (n=3), fibrosarcoma (n=4) and malignant fibrous histiocytoma (n=21) were analyzed using an Affymetrix HG-U133A array.

first: Robert

last: Nakayama

first: Takeshi

last: Nemoto

first: Tsutomu

last: Ohta

first: Teruhiko

last: Yoshida

first: Tadashi

last: Hasegawa


title: Pof D119 mutant expression analysis

status: Public on Mar 22 2007

city: Umea

first: Jan

last: Larsson

address: Umea University

department: Molecular Biology

country: Sweden

institute: Umea University

summary:
Analysis of gene expression in pof deletion mutants. Chromosome 4 genes are down-regulated in pof mutants compared to wildtype control. 200 Drosophila melanogaster first instar larvae were used for each of three biological replicates of y1 w67c23; PofD119/PofD119 and three biological replicates of y1 w67c23 as controls.
Keywords: Mutant vs Wt expression

overall_design: 200 1st instar larvae were used for each of 3 biological replicates of pof mutants and 3 replicates of wt larvae.

first: Per

last: Stenberg

first: Anna-Mia

last: Johansson


title: Two distinct gene signatures identify malignant Neuroblast and Schwannian stromal cells of Neuroblastic Tumors

status: Public on Apr 17 2007

city: genoa

first: Domenico

last: Albino

address: L.go R. Benzi, 10

country: Italy

institute: National Institute for Cancer Research (IST)

summary:
Tumor tissue heterogeneity is a well known feature of several solid tumors. Neuroblastic Tumors (NTs) is a group of paediatric cancers with a great tissue heterogeneity. Most of NTs are composed of undifferentiated, poorly differentiated or differentiating neuroblastic (Nb) cells with very few or absent Schwannian stromal (SS) cells: these tumors are grouped as Neuroblastoma (Schwannian stroma-poor). The remaining NTs are composed of abundant SS cells and classified as Ganglioneuroblastoma (Schwannian stroma-rich) intermixed or nodular and Ganglioneuroma. The importance to understand Nb and SS gene signatures in NTs, is to clarify the complex network mechanism of tumor growth and progression. In order to identify the Nb and SS cells gene signatures, we analyzed the gene expression profiling of 19 cases of neuroblastic tumors: 10 stroma poor (NTs-SP) and 9 stroma rich (NTs-SR), by high density oligonucleotide microarrays. Moreover, the analysis was performed in parallel on both whole and laser microdissected tumor samples: from 4 of 19 cases, was isolated different areas all composed of pure cellular populations.

We performed genome wide expression analysis by using Affymetrix technology and we used two different approaches for data analysis: SAM (Significance Analysis of Microarrays) and a method based on Game Theory (GT), to identify genes differently expressed in SS and Nb cells. Differently from the SAM method, the analysis based on GT (Moretti et al. (2006)) gives the advantage of selecting relevant genes not only according to the expression profile of each single gene, but considering also gene interaction.
Keywords: Gene signature of Neuroblastic and Schwannian stromal cells.

overall_design: Tissue samples from 10 primary neuroblastoma stroma-poor (NTs-SP) and 9 ganglioneuroblastoma/ganglioneuroma stroma-rich (NTs-SR) tumors, were obtained at diagnosis from patients at different clinical stage. Frozen samples of NTs-SP had a minimum content of 90% of malignant cells, while NTs-SR had more than 80-90% of schwannian stromal cells. Four out of 19 NTs: 2 neuroblastomas (Schwannian stoma-poor) and 2 ganglioneublastomas (Schwannian stroma-rich), were further analyzed with laser microdissection.

first: Stefano

last: Moretti


title: Synergy between PPARgamma ligands and platinum-based drugs in cancer

status: Public on Apr 19 2007

city: Boston

first: Geoffrey

last: Girnun

state: MA

address: One Jimmy Fund Way

department: Cancer Biology

country: USA

institute: Dana-Farber Cancer Institute

summary:
PPARγ is a member of the nuclear receptor family for which agonist ligands have anti-growth effects.  However, clinical studies using PPARγ ligands as a monotherapy failed to show a beneficial effect.  Here we have studied the effects of PPARγ activation with chemotherapeutic agents in current use for specific cancers.  We observed a striking synergy between rosiglitazone and platinum-based drugs in several different cancers both in vitro and using transplantable and chemically induced “spontaneous” tumor models.  The effect appears to be due in part to PPARγ-mediated downregulation of metallothioneins, proteins that have been shown to be involved in resistance to platinum-based therapy.  These data strongly suggest combining PPARγ agonists and platinum-based drugs for the treatment of certain human cancers
Keywords: Gene expression, change, synergy of interaction

overall_design: Cells were treated with either DMSO/control, rosiglitazone, carboplatin or combination or rosiglitazone and carboplatin in duplicate for 24 hr. RNA was isolated and microarray analysis carried out by the Dana-Farber Cancer Institute Microarray Core.

middle: D

first: Elnaz

last: Naseri

first: Scott

middle: B

last: Vafai

first: Lishu

last: Qu

first: Jeffrey

last: Szwaya

first: Roderick

last: Bronson

first: John

middle: A

last: Alberta

first: Bruce

last: Spiegelman


title: The prognostic role of a gene signature from tumorigenic breast-cancer cells.

status: Public on Mar 10 2007

city: Redwood City

first: Xinhao

last: Wang

state: CA

address: 800 Chesapeake

institute: OncoMed Pharmaceuticals Inc

summary:
Breast cancers contain a minority population of cancer cells characterized by CD44 expression but low or undetectable levels of CD24 (CD44+CD24-/low) that have higher tumorigenic capacity than other subtypes of cancer cells. METHODS: We compared the gene-expression profile of CD44+CD24-/low tumorigenic breast-cancer cells with that of normal breast epithelium. Differentially expressed genes were used to generate a 186-gene invasiveness gene signature (IGS), which was evaluated for its association with overall survival and metastasis-free survival in patients with breast cancer or other types of cancer. RESULTS: There was a significant association between the IGS and both overall and metastasis-free survival (P<0.001, for both) in patients with breast cancer, which was independent of established clinical and pathological variables. When combined with the prognostic criteria of the National Institutes of Health, the IGS was used to stratify patients with high-risk early breast cancer into prognostic categories (good or poor); among patients with a good prognosis, the 10-year rate of metastasis-free survival was 81%, and among those with a poor prognosis, it was 57%. The IGS was also associated with the prognosis in medulloblastoma (P=0.004), lung cancer (P=0.03), and prostate cancer (P=0.01). The prognostic power of the IGS was increased when combined with the wound-response (WR) signature. CONCLUSIONS: The IGS is strongly associated with metastasis-free survival and overall survival for four different types of tumors. This genetic signature of tumorigenic breast-cancer cells was even more strongly associated with clinical outcomes when combined with the WR signature in breast cancer.
Keywords: cell type comparison

overall_design: Expression profling were performed on 6 tumorigenic, 3 non tumorigenic samples of breast tumors and 3 normal breast samples on two different platforms GPL96 and GPL97. A gene signature was derived by comparing the gene expressions of 6 tumorigenic samples with 3 normal breast samples

first: R

last: Liu

first: X

first: GY

last: Chen

first: P

last: Dalerba

first: A

last: Gurney

first: T

last: Hoey

first: G

last: Sherlock

first: J

last: Lewicki

first: K

last: Shedden

first: MF

last: Clarke


title: Comparison of gene expression data from human and mouse breast cancers

status: Public on Mar 13 2007

city: Berlin

first: Andreas

last: Klein

address: Charitéplatz 1

department: Institute of Biochemistry

institute: Charité (CCO)

summary: This SuperSeries is composed of the SubSeries listed below.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series

first: Susanne

last: Seitz

first: Rita

last: Schmutzler

first: Dieter

last: Niederacher

first: Norbert

last: Arnold

first: Alfons

last: Meindl

first: Siegfried

last: Scherneck

first: Adolf

last: Graessmann


title: Abscisic acid effect on wild type and the abh1 mutant

status: Public on Mar 01 2007

city: San Diego

first: Julian

last: Schroeder

address: 9500 Gilman Drive

institute: University of California San Diego

summary:
Analysis of the abh1 mutant Arabidopsis plants following treatment with 50 uM abscisic acid (ABA). ABH1 encodes the large (80kDa) subunit of the nuclear mRNA cap binding complex and affects early ABA signal transduction events (Hugouvieux et al., 2001, Cell 106, 477).
Keywords: stress response

overall_design: 4 week old wild-type and abh1 plants were mock-treated or treated with 50uM ABA by spraying. After 4 hours, samples were collected. Total RNA from three biological replicates was pooled for each array hybridization. 2 repliclates were performed. cRNA synthesis and hybridizations were performed  according to standard Affymetrix procedures. Data analysis was run with Affymetrix Micro Array Suite 5.0

first: Veronique

last: Hugouvieux

first: Josef

last: Kuhn

middle: I


title: Comparison of human embryonic stem cell (hESC) derived mesenchymal precursor cell populations

status: Public on May 17 2007

city: New York

first: Agnes

last: Viale

state: NY

address: 1275 York Avenue

institute: Memorial Sloan Kettering Cancer Center

summary:
We have developed efficient protocols for the derivation of mesenchymal precursors from hESCs. While previous protocols were based on mesodermal induction via co-culture of hESCs on OP9 mouse stroma (Barberi et al., PLoS Biology, 2005), our recent work shows the derivation of hESC derived mesenchymal precurors under feeder-free conditions. The data presented here show a large and highly signficant overlap in global gene expression profiles between hESC derived mesenchymal precursors derived under feeder-free conditions with those derived via OP9 co-culure and mesenchymal precurosrs isolated directly from the adult bone marrow.
Keywords: cell type comparison

overall_design: These data compare the gene expression profiles of hESC derived mesenchymal precursors derived via the OP9 protocol, with those obtained under feeder-free conditions and adult bone marrow derived mesenchymal precursors. Three samples of undifferentiated hESCs are used for subtracting genes to define mesenchymal precurosr cell specific signatures (see Barberi et al. Nature Medicine 2007 for details).

first: Tiziano

last: Barberi

first: Lorenz

last: Studer


title: The human reticulocyte transcriptome

status: Public on Mar 26 2007

city: Bethesda

first: Jeffery

middle: Lynn

last: Miller

state: MD

address: 10 Center Drive

department: Molecular Medicine Branch

institute: NIH/NIDDK

summary:
RNA from circulating blood reticulocytes was utilized to provide a robust description of genes transcribed at the final stages of erythroblast maturation. After depletion of leukocytes and platelets, Affymetrix HG-U133 arrays were hybridized with probe from total RNA isolated from blood sampled from 14 umbilical cords and 14 healthy adult humans.
Keywords: Adult vs. fetal reticulocyte transcriptome comparison

overall_design: Peripheral blood from 14 normal adult and 14 human umbilical cord were purified to obtain packed red blood cells and their purity was assessed by CELL-DYN4000. The contaminated WBC count was less than 1 out of 1 million cells. Total RNA from purified reticulocytes included in the purified red blood cells was extracted, labeled, and hybridized onto Affymetrix HG-U133 A and B arrays.

first: Sung-Ho

last: Goh

first: Matthew

last: Josleyn

first: Y.

middle: T

last: Lee

first: Robbert

middle: L

last: Danner

first: Robert

last: Gherman

first: Maggie

last: Cam


title: Effect of insulin infusion on human skeletal muscle

city: Malmö

first: Hemang

last: Parikh

address: Building 91, Plan 12, CRC, Entrance 72, Malmö University Hospital

department: Department of Clinical Sciences-Diabetes and Endocrinology

institute: Lund University

summary:
To identify insulin responsive genes in humans, in the first protocol, skeletal muscle biopsies from six non-diabetic subjects were obtained before and after a two-hour of hyperinsulinaemic (infusion rate 40 mU/m2/min) euglycemic clamp. A variable infusion of glucose (180 g/l) enriched with tritiated glucose (100 μCi/500 ml) maintained euglycemia during insulin infusion, with monitoring of plasma glucose concentration every 5 to 10 min during the basal and clamp periods using an automated glucose oxidation method (Glucose Analyzer 2, Beckman Instruments, Fullerton, CA).

In the second protocol, skeletal muscle biopsies from six non-diabetic subjects were obtained before and after a 3-hour hyperinsulinemic (infusion rate 40 mU/m2/min) euglycemic clamp in order to increase the effects of insulin on gene expression. A variable infusion of glucose (180 g/l) was used to maintain euglycemia during insulin infusion with monitoring of plasma glucose concentration every 5 to 10 min using an automated glucose oxidation method (Glucose Analyzer 2, Beckman Instruments, Fullerton, CA).
Keywords: dose response

overall_design:
The muscle biopsies were obtained from the vastus lateralis
muscle under local anesthesia before and after hyperinsulinaemic (infusion rate 40 mU/m2/min) euglycemic clamp

first: Leif

last: Groop

first: Vamsi

last: Mootha


title: GAPF profiles from HFF2 and Colo320DM cells

city: Seatttle

first: Yi

last: Cao

state: WA

address: 1100 Fairview Ave. N

department: Human Biology

institute: Fred Hutchinson CRC

summary:
Amplification of large chromosomal regions (gene amplification) is a common somatic alteration in human cancer cells and often is associated with advanced disease. A critical event initiating gene amplification is a DNA double strand break (DSB), which is immediately followed by the formation of a large DNA palindrome. Large DNA palindromes are frequent and non-randomly distributed in the genomes of cancer cells and facilitate further increase in copy number. Although the importance of the formation of large DNA palindromes as a very early event in gene amplification is widely recognized, it is not known 1) how a DSB is resolved to form a large DNA palindrome; and 2) whether any local DNA structure determines the location of large DNA palindromes. We show here that intra-strand annealing following a DNA double-strand break leads to the formation of large DNA palindromes and that DNA inverted repeats in the genome determines the efficiency of this event. Furthermore, in human Colo320DM cancer cells, a DNA inverted repeat in the genome marks the border between amplified and non-amplified DNA. Therefore, an early step of gene amplification is a regulated process that is facilitated by DNA inverted repeats in the genome.
Keywords: cancer vs. normal sample comparison

overall_design: Genomic DNA samples from HFF2 and Colo320DM cells were collected and taken through the GAPF procedure. Their GAPF profiles were analyzed using Affymetrix HGU 133A arrays, and the differentially hybridized genes were determined to be significant according to a FDR<0.05

first: Hisashi

last: Tanaka

first: Donald

last: Bergstrom

first: Charles

last: Kooperberg

first: Stephen

middle: J

last: Tapscott

first: Meng-Chao

last: Yao


title: TGF-beta1 and 2 treatment of human trabecular meshwork cells (Russell-3U24NS043571-01S1-2)

status: Public on Feb 28 2007

city: Phoenix

first: Winnie

last: Liang

state: AZ

address: 445 N. Fifth Street

institute: Translational Genomics

summary:
TGF-beta levels are known to increase in the aqueous humor of eye cells in patients with glaucoma.  Increase TGF-beta is assumed to have a biochemical impact on the trabecular meshwork, and an increase in extracellular matrix formation, which may be responsible for decrease outflow facility of the eye.  This may increase extracellular pressure, causing glaucoma.  TGF-beta 1 may be the cause of abnormal accumulation of extracellular matrices in trabecular meshwork of eyes with primary open angle glaucoma.  Transforming growth factor (TGF)-beta2 regulates the expression of proteoglycans in aqueous humor from human glaucomatous eyes.
To identify gene expression changes as a result of TGF-beta1 and 2 treatment of human trabecular meshwork cells.
We expect to see a change in expression of the proteoglycans in HTM cells as a response to TGF-beta treatment.
Human Trabecular Meswork cells in the eye were bathed by aqueous humor.  TM cells were removed from individuals with the following ages: 16,66,67,73, and 76.  Each individual was treated with EtOH (control), TGF-beta1, or TGF-beta2.  Total RNA from each individual was pooled for each chip.  Technical replicates were created for each treatment type, for a total of 6 chips.
Keywords: dose response

first: Paul

last: Russell


status: Public on May 31 2007

city: Cambridge

first: Todd

last: Sangster

address: Nine Cambridge Center

institute: Whitehead Institute

summary:
The molecular chaperone HSP90 aids the maturation of a diverse but select set of metastable protein clients, many of which are key to a variety of signal transduction pathways.  HSP90 function has been best investigated in animal and fungal systems, where inhibition of the chaperone has exceptionally diverse effects, ranging from reversing oncogenic transformation to facilitating the acquisition of drug resistance.  Inhibition of HSP90 in the model plant Arabidopsis thaliana uncovers novel morphologies dependent on normally cryptic genetic variation and increases stochastic variation inherent to developmental processes.  The biochemical activity of HSP90 is strictly conserved between animals and plants.   However, the substrates and pathways dependent on HSP90 in plants are poorly understood.  Progress has been impeded by reliance on light-sensitive HSP90 inhibitors due to redundancy in the A. thaliana HSP90 gene family.  Here we present phenotypic and genome-wide expression analyses of A. thaliana with constitutively reduced HSP90 levels achieved by RNAi targeting.  HSP90 reduction affects a variety of quantitative life-history traits, including flowering time and total seed set, and decreases developmental stability.  Further, by quantitative analysis of morphological phenotypes, we demonstrate that HSP90-reduction increases phenotypic diversity in both seedlings and adult plants.  Several morphologies are synergistically affected by HSP90 and growth temperature.  Genome-wide expression analyses also suggest a central role for HSP90 in the genesis and maintenance of plastic responses.  The expression results are substantiated by examination of the response of HSP90-reduced plants to attack by caterpillars of the generalist herbivore Trichoplusia ni.  HSP90 reduction potentiates a more robust herbivore defense response.  In sum, we propose that HSP90 exerts global effects on the environmental responsiveness of plants to many different stimuli.  The comprehensive set of HSP90-reduced lines described here is a vital instrument to further examine the role of HSP90 as a central interface between organism, development, and environment.
Keywords: HSP90, mutant comparison

overall_design: Three differerent experiments were performed.  One experiment included lines RNAi-A1, RNAi-A3, and Control-2, with two biological replicates and one technical replicate per line.   The second included lines RNAi-A2, RNAi-B1, RNAi-C1, Control-1, and Control-3, with three biological replicates per line.  The third included the three single-isoform T-DNA insertion lines, along with the Col-0 control, with three biological replicates per line.  See Sangster et al. "Phenotypic Diversity and Altered Environmental Plasticity in Arabidopsis thaliana with Reduced HSP90 Levels" for details of construct construction and further experimental rationale.

first: Adam

last: Bahrami

first: Amity

last: Wilczek

first: Etsuko

last: Watanabe

first: Kurt

last: Schellenberg

first: Catherine

last: McLellan

first: Alicia

last: Kelley

first: Sek-Won

last: Kong

first: Christine

last: Queitsch

first: Susan

last: Lindquist


title: HNF1-alpha inactivation promotes lipogenesis in human hepatocellular adenoma independently of SREBP1 & ChREBP activation

city: Paris

first: Sandrine

last: Imbeaud

address: 15 rue de l'Ecole de Médecine

department: INSERM UMRS1138-EQUIPE 28, Sorbonne Université-Inserm-Université de Paris

country: France

institute: Centre de Recherche des Cordeliers

summary:
Biallelic inactivating mutations of the transcription factor 1 gene (TCF1), encoding hepatocyte nuclear factor 1a (HNF1a), were identified in 50% of hepatocellular adenomas (HCA) phenotypically characterized by a striking steatosis. To understand the molecular basis of this aberrant lipid storage, we performed a microarray transcriptome analysis validated by quantitative RT-PCR, western-blotting and lipid profiling. In mutated HCA, we showed a repression of gluconeogenesis coordinated with an activation of glycolysis, citrate shuttle and fatty acid synthesis predicting elevated rates of lipogenesis. Moreover, the strong dowregulation of L-FABP suggests that impaired fatty acid trafficking may also contribute to the fatty phenotype. In addition, transcriptional profile analysis of the observed deregulated genes in non-HNF1a-mutated HCA as well as in non-tumor livers allowed us to define a specific signature of the HNF1a-mutated HCA. In theses tumors, lipid composition was dramatically modified according to the transcriptional deregulations identified in the fatty acid synthetic pathway. Surprisingly, lipogenesis activation did not operate through SREBP-1 and ChREBP that were repressed. We conclude that steatosis in HNF1a-mutated HCA results mainly from an aberrant promotion of lipogenesis that is linked to HNF1a inactivation and that is independent of both SREBP-1 and ChREBP activation. Finally, our findings have potential clinical implications since lipogenesis can be efficiently inhibited by targeted therapies.
Keywords: Disease state analysis

overall_design:
In a first experiment, microarray analyses were applied to 8 HNF1-alpha mutated HCA and their corresponding non-tumor livers using cDNA in-house manufactured arrays following a randomized and blinded unbalanced design. RNA labelling, hybridization and analysis of fluorescence was carried out, as described in [Graudens et al., Genome Biol 2006, 7: R19; PMID: 16542501], considering that uneven numbers of samples were randomly allocated to each of the engineers who were not aware of sample phenotypes. To assess data reproducibility and minimize dye bias effects, each of the samples was measured four times, twice with Cy3 and twice with Cy5. To ensure robustness and flexibility in data analysis, a reference design was used with a universal reference sample (Stratagene, USA) serving as a baseline for the comparisons of tumor samples. Hybridizations were performed onto an 11K human array (11K_VJF-ARRAY, GPL3282), which provides a genome-wide coverage of functional pathways. Raw data were obtained using the ArrayVision™ 7.0 software (Imaging Research Inc., USA); the resulting 3.2x106 hybridization data points collected from 32 arrays were stored in a MIAME-compliant database and pre-processed for normalization and filtering as described in [Graudens et al., Genome Biol 2006, 7: R19; PMID: 16542501].

In a second experiment, HG-U133A Affymetrix GeneChipTM arrays were used to compare the expression profiles of 5 HNF1-alpha mutated HCA and 4 non related non-tumor livers. RNA labelling, hybridization and analysis were carried out following the manufacturer’s instructions(Affymetrix, Santa Clara, CA). Raw data were obtained by using Microarray Suite 5.0 (MAS5) software, embedded in the Affymetrix GeneChip Operating Software (Santa Clara, USA); the resulting raw numerical data (CEL files) - available as supplementary files - collected from 9 Affymetrix GeneChips were pre-processed for normalization and filtering as described in [Rebouissou et al., J Biol Chem 2007, in press; PMID: 17379603].

A platform-dependant statistical comparison was performed considering HNF1 alpha-mutated HCA versus non tumor liver tissues, using multiple testing procedures to evaluate statistical significance for differentially expressed genes, as described in [Graudens et al., Genome Biol 2006, 7: R19; PMID: 16542501] and in the supplemental experimental procedures in [Rebouissou et al., J Biol Chem 2007, in press; PMID: 17379603]. Thus, a combined cross-platform analysis was done considering the UGCluster Identifiants as a common primary key (Unigene Build184-June05).

Additional information on the procedures is provided in the supplemental experimental procedures in [Rebouissou et al., J Biol Chem 2007, in press; PMID: 17379603].

first: Sandra

last: Rebouissou

last: Balabaud

first: Virginie

last: Boulanger

first: Justine

last: Bertrand-Michel

first: François

last: Tercé

last: Auffray

first: Paulette

last: Bioulac-Sage

first: Jessica

last: Zucman-Rossi


title: Stonewalling Drosophila stem cell differentiation by epigenetic controls

status: Public on Apr 15 2007

city: Dallas

first: Dennis

last: McKearin

state: TX

address: 6000 Harry Hines Blvd., NA5.214

institute: UT Southwestern Med. Ctr.

summary:
Abstract: During Drosophila oogenesis, germline stem cell (GSC) identity is maintained largely by preventing the expression of factors that promote differentiation. This is accomplished via the activity of several genes acting either in the GSC or its niche. The translational repressors, Nanos and Pumilio, act in GSCs to prevent differentiation, likely by inhibiting translation of early differentiation factors, while niche signals prevent differentiation by silencing transcription of the differentiation factor Bam. We have found that the DNA-associated protein Stonewall (Stwl) is also required for GSC maintenance. stwl is required cell-autonomously; clones of stwl- germ cells were lost by differentiation, and ectopic Stwl caused an expansion of GSCs. stwl mutants acted as Suppressors of Variegation, indicating stwl normally acts in chromatin-dependent gene repression. In contrast to several previously described GSC maintenance factors, Stwl likely functions epigenetically to prevent GSC differentiation. Stwl-dependent transcriptional repression does not target bam, but rather Stwl represses the expression of many genes, including those that may be targeted by Nanos/Pumilio translational inhibition.
Keywords: Genetic modification; gene chip hybridization (Affymetrix Drosophila Genome 2.0)

overall_design: Genetic variation comparison between germ cells of Drosophila ovaries from bam mutant and stwl bam double mutant females. Six total samples were analyzed, with each genotype performed in triplicates.

first: Jean

middle: Z

last: Maines

first: Joseph

last: Park

first: Meredith

last: Williams

last: McKeain


title: Gene profiling of responders and non-responders to antiviral therapies peg interferon and ribavirin against hepatitis C

status: Public on Feb 27 2007

city: Bloomington

first: Milton

middle: W

last: Taylor

state: IN

address: 343 Jordan Hall

department: Biology

institute: Indiana University

summary:
Time series of gene expression arrays before and during treatment of Hepatitis C; days 0, 1, 2, 7, 14 and 28 for 69 participants (IDs 1 through 69 are used).
Treating chronic hepatitis C using peginterferon alpha and ribavirin leads to sustained clearance of virus and clinical improvement in approximately 50% of patients. Response rates are lower among patients with genotype 1 than with genotypes 2 and 3 and among African American ( AA)  compared to Caucasian (CA) patients. Using DNA microarrays, gene expression was assessed in a group of 33 African American and 36 Caucasian American patients with chronic hepatitis C, genotype 1 during the first 28 days of treatment. Results were examined with respect to treatment responses and to race. Patients showed a response to treatment at the gene expression level in RNA isolated from PBMC irrespective of degree of decrease in hepatitis C virus (HCV) RNA levels. However, gene expression responses were relatively blunted in patients with poor viral response (< 1.5 log10 IU/ml decrease at 28 days) compared to those in patients with a marked (> 3.5 log10 decrease) or intermediate (1.5 to 3.5 log10 decrease) response. The number of genes that were up- or down-regulated by peginterferon and ribavirin treatment was fewer in patients with a poor response compared to those with an intermediate or marked viral response. However AA patients had a stronger interferon response than CA patients in general.  The induced levels of known ISGs such as OAS, MX1, IRF-7 and the toll like receptor TLR-7 was lower in  poor response patients than in marked or intermediate response patients.  Thus, the relative lack of viral response to interferon therapy of hepatitis C is associated with blunted interferon cell signaling. No specific regulatory gene could be identified as responsible for this global blunting or racial differences.
Keywords: time-course study

overall_design: Eligible patients were naïve to interferon and ribavirin treatment, had detectable HCV RNA in serum, and nearly all had a liver biopsy performed within the previous 18 months showing chronic hepatitis. Only patients who were born in the United States and designated themselves as “African American/Black” or “Caucasian/White” were eligible. The clinical protocol called for participants to be treated for up to 48 weeks with peginterferon alpha-2a (Pegasys™, Roche Pharmaceuticals Nutley, NJ) in a dose of 180 µg weekly by self-administered subcutaneous injection and ribavirin (Copegus™, Roche) orally in a dose of 1000 or 1200 mg daily based on body weight of less than 75 kg or equal to or greater than 75 kg. Peripheral blood mononuclear cells (PBMC) were collected from patients before treatment (day 0) and on days 1 (after the initial supervised injection of peginterferon), 2, 7, 14 and 28. From the Virahep-C cohort, 72 patients who did not have dose reductions of either peginterferon or ribavirin in the first 28 days of treatment were selected such that 12 patients were included in each race (CA, AA) by virological response category.  The three categories of response were: marked, defined as a decrease in HCV RNA levels of more than 3.5 log10 IU/ml or to undetectable on day 28; intermediate, defined as a decrease of 1.4 to 3.5 log10 IU/ml on day 28; and poor, defined as less than 1.4 log10 IU/ml decline on day 28 relative to baseline. These definitions were made a priori in an attempt to analyze the biological basis for virological responses. Of these, adequate RNA was not obtained from 3 patients to provide gene expression information. Of these 72, 69 were analyzed.

first: Takuma

last: Tsukahara

first: Leonid

last: Brodsky

first: Joel

last: Schaley

first: Corneliu

last: Sanda

first: Matthew

last: Stephens

first: Jeanette

middle: N

last: McClintick

first: Howard

last: Edenberg

first: Lang

last: Li

first: John

middle: E

last: Tavis

first: Charles

last: Howell

first: Steven

middle: H

last: Belle


title: Identification and characterization of small molecule inducers of epidermal keratinocyte differentiation

status: Public on Feb 07 2007

middle: R

last: Walker

address: 10675 John Jay Hopkins

institute: Genomics Institute of the Novartis Research Foundation

summary:
An essential function of the human epidermis is the maintenance of a protective barrier against the environment. As a consequence, keratinocytes, which make up this layer of the skin, undergo an elaborate process of self-renewal, terminal differentiation and cell death. Misregulation of these processes can lead to several human diseases including psoriasis and basal cell and squamous cell carcinomas. To identify novel regulators of keratinocyte differentiation, a cell-based screen of small molecule libraries was carried out for molecules that induce terminal differentiation of normal human epidermal keratinocytes (NHEKs). One class of molecules was identified, the 2-(3,4,5-trimethoxy-phenylamino)-pyrrolo[2,3-d]pyrimidines, which were shown to induce differentiation of epidermal progenitor cells to terminally differentiated keratinocytes. These molecules serve as useful mechanistic probes of the cellular differentiation programs that regulate the formation and homeostasis of the epidermis, and may lead to novel therapeutic approaches for the treatment of skin hyperproliferative disorders.
Keywords: time course of compound treatment

overall_design: samples were taken from duplicate treatments of cells with DMSO or PP-2 (compound 9) at various time points

first: Jiyong

last: Hong

first: Jongkook

last: Lee

first: Kyung

last: Min

first: Nathanael

middle: S

last: Gray

middle: Y

last: Cho

first: Peter

middle: G

last: Schultz

first: Eric

last: Peters


title: Gene expression profiling of B lymphocytes and plasma cells from WaldenstrÖm’s macroglobulinemia.

city: Salamanca

first: Norma

middle: Carmen

last: Gutierrez

department: Hematology

country: Spain

institute: University Hospital

summary:
The tumoral clone of Waldenström’s macroglobulinemia (WM) shows a wide morphological heterogeneity which ranges from B-lymphocytes (BL) to plasma cells (PC). By means of genome-wide expression profiling we have been able to identify genes exclusively deregulated in BL and PC from WM, but with a similar expression pattern in their corresponding cell-counterparts from CLL and MM, as well as normal individuals. The differentially expressed genes have important functions in B-cell differentiation and oncogenesis. Thus, two of the genes down-regulated in WM-BL were IL4R, which plays a relevant role in CLL B cell survival, and BACH2 that participates in the development of class-switched PC. Interestingly, one of the up-regulated genes in WM-BL was IL6. A set of 4 genes was able to discriminate clonal B-lymphocytes from WM and CLL: LEF1 (WNT/ßcatenin pathway), MARCKS, ATXN1 and FMOD. We also found deregulation of genes involved in plasma cell differentiation such as PAX5 which was overexpressed in WM-PC, and IRF4 and BLIMP1 which were underexpressed. In addition, three of the target genes activated by PAX5 -CD79, BLNK and SYK- were up-regulated in WM-PC. In summary, these results indicate that both PC and BL from WM are genetically different from the MM and CLL cell-counterpart.
Keywords: Waldenström’s macroglobulinemia, expression profiling, microarrays, Affymetrix.

overall_design: Bone marrow (BM) samples from 10 patients with Waldenström’s macroglobulinemia (WM), 12 with multiple myeloma (MM) and 11 with chronic lymphocytic leukemia (CLL) were included in the study. All samples corresponded to newly diagnosed untreated patients. In addition, 8 normal B lymphocytes samples (NBL) from peripheral blood and 5 normal plasma cells (NPC) from bone marrow of healthy donors were also selected in order to relate the deregulation of GEP of clonal populations to normal condition. The study was approved by the local research ethics committee and written informed consent was obtained from all patients and healthy donors.

middle: C

last: Gutiérrez

first: Enrique

middle: M

last: Ocio

first: Javier

last: De las Rivas

first: Patricia

last: Maiso

first: Manuel

last: Delgado

first: Encarna

last: Fermiñan

first: María

last: Arcos

last: Sánchez

first: Jesús

last: Hernández

middle: F

last: San Miguel


title: CD34+ and Treatment with UTP

status: Public on Feb 14 2007

city: Modena

first: Rossella

last: Manfredini

address: Via Gottardi 100

department: Life Sciences

institute: Centre for Regenerative Medicine

summary:
Hematopoietic stem cell (HSC) untreated and treated with UTP 10uM at 1, 6 and 24 h
Keywords: Time couse

overall_design: Highly purified CD34+ cells from 6 healthy donors were seeded at 1000000 cells/ml in serum free medium (EX vivo 15) w/o cytokines and treated with 10 uM UTP  for 1, 6 and 24 hours. As a control, CD34+ untreated cells were maintened in the same culture conditions for the same times. RNAs originating from the 6 donors were pooled in order to obtain at least 2 mg per sample (untreated and UTP-treated respectively).


title: Expression data from HELA cells subject to EGF stimulation

city: Rehovot

first: Tal

last: Shay

address: Herzel

department: Physics of Complex Systems

country: Israel

institute: Weizmann Institute of Science

summary:
HELA cells derived from human cervical tumor were subjected to EGF stimulation for 0,20,40,60,120,240 and 480 minutes.
We used microarrays to understand the temporal regulation of the cellular EGFR cascade.
Keywords: time course

overall_design: HELA cells were grown in DMEM supplemented with 10% FCS and antibiotics. Cells were serum deprived for 24 hours. Following stimulation with 10 ng/ml EGF for 0,20,40,60,120,240 and 480 minutes.

first: Amit

last: Ido

first: Citri

last: Ami

first: Shay

last: Tal

first: Domany

last: Eytan

first: Yarden

last: Yosef


title: Chorionic villi cells

status: Public on Feb 17 2007

city: Setagaya-ku

first: Akihiro

last: Umezawa

state: Tokyo

department: Center for Regenerative Medicine

institute: National Center for Child Health and Development

summary:
In the chorionic villi of placenta, trophoblasts and endothelial cells are present, and moreover mesenchymal cells (stromal cells) can be obtained. We generated cells with the mesenchymal phenotype from the chorionic mesoderm, and showed that: a) physiologically functioning cardiomyocytes were transdifferentiated from human placenta-derived chorionic villi cells, but these cells did not induce to osteoblasts and adipocytes ; b) the cardiomyogenic induction rate obtained using our system was relatively high compared to that obtained using the previously described method ; c) co-cultivation with fetal murine cardiomyocytes alone without transdifferentiation factors such as 5-azaC or oxytocin is sufficient for cardiomyogenesis in our system; d) Chorionic villi cells have the electrophysiological properties of 'working' cardiomyocytes.  The chorionic mesoderm contained a large number of cells with a cardiomyogenic potential.
Keywords: Cardiomyogenic induction

overall_design: To isolate chorionic villi cells, we used the explant culture method, in which the cells were outgrown from pieces of chorionic villi attached to dishes. Chorionic villi cells were harvested with 0.25% trypsin and 1 mM EDTA, and overlaid onto the cultured fetal cardiomyocytes at 7 x 103/cm2.  Every 2 days, the culture medium was replaced with fresh culture medium that was supplemented with 10% FBS and 1 ug/ml Amphotericin B (GIBCO).  The morphology of the beating chorionic villi cells was evaluated under a fluorescent microscope.


title: Differential gene expression of IMR32 cells expressing 69-kDa or 82-kDa CHAT proteins

status: Public on Feb 01 2007

city: London

first: Sandeep

middle: K

last: Gill

state: Ontario

address: 100 Perth Drive

department: Molecular Medicine

country: Canada

institute: Robarts Research Institute

summary:
The goal of this study was to look at genes that were affected by 69-kDa and/or 82-kDa ChAT proteins in IMR32 cells
Keywords: comparative

overall_design: The gene expression changes of IMR32 cells stably expressing either 69-kDa or 82-kDa ChAT proteins were anaylzed and compared to control IMR32 wild type cells.  3 biological replicates were anaylzed per condition (69-kDa ChAT expressing cells, 82-kDa ChAT expressing cells, or wild type IMR32 cells) for a total of 9 samples altogether.

first: Jane

last: Rylett

first: David

last: Carter


title: Genome-wide cell cycle studies

status: Public on Feb 16 2007

city: Loughborough

last: Nottingham Arabidopsis Stock Centre (NASC)

address: Sutton Bonington Campus

department: School of Biosciences, University of Nottingham

country: United Kingdom

summary:
This experiment was provided by TAIR (http://arabidopsis.org).
Effective analysis of gene expression during the cell cycle depends on achieving a good level of synchronisation. Until recently, analysis of cell cycle processes in plants has been hampered by the lack of synchronizable cell suspensions for Arabidopsis. We have recently developed a cell synchrony system for Arabidopsis cell suspensions MM1 and MM2d, and have developed two methods of synchronization. The first synchronizes cycling cells by blocking cells at the G1/S boundary using aphidicolin. The second uses sucrose removal and resupply to synchronize cells during re-entry into the cell cycle. Cell cycle synchrony in suspension cultured cells: cells can be reproducibly synchronized by blocking at the G1/S boundary or in early S phase using aphidicolin for 24 hr and then reversing the block by washing (Menges and Murray, 2002). On aphidicolin removal, the synchronous resumption of S phase and progression through the cell cycle occur and sequential RNA samples were taken at 2-3 hourly intervals over a 19 hour period. We have carried out a transcriptional profiling analysis with the aim to study gene expression during cell cycle progression after aphidicolin treatment of suspension-cultured cells using the near full genome ATH1 arrays (Menges et al., 2003).

Experimenter name = Jim Murray
Experimenter phone = 44 1223 334166
Experimenter fax = 44 1223 334162
Experimenter department = J Murray Laboratory
Experimenter institute = University of Cambridge
Experimenter address = Institute of Biotechnology
Experimenter address = Tennis Court Road
Experimenter address = Cambridge
Experimenter zip/postal_code = CB2 1QT
Experimenter country = United Kingdom
Keywords: compound_treatment_design; time_series_design

overall_design: 10 samples were used in this experiment

first: Jim

last: Murray

first: Henrik

last: Townsend

first: Zoe

last: Emmerson

first: Beatrice

last: Schildknecht


title: Expression data from retinoblastoma

status: Public on Jan 29 2007

city: Essen

first: Ludger

last: Klein-Hitpass

address: Virchowstr. 173

department: Universitaetsklinikum

institute: Institut fuer Zellbiologie

summary:
Genomic losses on chromosome 16q are among the most frequent alterations found in retinoblastoma. In this study, Affymetrix GeneChip analyses along with LOH analysis of microsatellie markers was used to identify candidate tumor suppressor loci in a set of retinoblastoma.
We used microarrays to identify genes differentially expressed in retinoblastoma with LOH on 16q (M19484, M22590, M22641, M22860) compared to retinoblastoma without alterations in this region (M20517, M22067, M22233, M23209, M23449, M23818, M23896, M23978)
Keywords: Disease progression

overall_design: To compare the transcriptosomes of retinoblastoma with and without LOH on 16q, total RNA was isolated from a 30 mg block of each tumor and subjected to Affymetrix microarray analysis on HG-U133A arrays.

first: Sandrine

last: Gratias

first: Harald

last: Rieder

first: Reinhard

last: Ullmann

first: Stephanie

last: Schneider

first: Réka

last: Bölöni

first: Martin

last: Kappler

first: Dietmar

last: Lohmann


title: Genes regulated in MPTP-treated macaques

status: Public on Feb 13 2007

city: Kuopio

first: Markus

last: Storvik

address: University of Eastern Finland

department: Pharmacology

country: Finland

institute: University of Eastern Finland

summary:
MPTP treated Macaca fascicularis prefrontal cortices compared to saline treated and untreated controls
Keywords: Disease model

sample_organism:
Macaca fascicularis
Homo sapiens

overall_design:
Macaques injected daily with
MPTP hydrochloride (0.2 mg/kg, or with saline.  Each group consisted of

first: Jean-Luis

last: Nahon

first: Garry

last: Wong

first: Erwan

last: Bezard

first: Marie-Jeanne

last: Fauvergue

first: Audrey

last: Audegond

first: Qin

last: Li

first: Jean-Marie

last: Stutzmann

first: Jean

last: Menager

first: Chuan

last: Qin

first: Bernard

last: Bioulac

first: Christian

last: Gross

first: Anne

last: Vital


title: Cord Blood-Derived Mesenchymal Stem Cells with Distinct Growth Kinetics, Differentiation Potentials, Expression Profiles

city: Tempe

first: Kenro

last: Kusumi

department: School of Life Sciences

institute: Arizona State University

summary:
Phenotypic heterogeneity has been observed among mesenchymal stem/stromal cell (MSC) populations, but specific genes associated with this variability have not been defined. To study this question, we analyzed two distinct MSC populations isolated from the same umbilical cord blood (UCB) sample.  These populations (UCB1 and UCB2) are from a single donor, minimizing differences contributed by genetic background.  We characterized these UCB-MSCs for cell morphology, growth kinetics, immunophenotype and differentiation potential. UCB1 displayed rapid growth kinetics, higher population doublings, and increased adipogenic lineage differentiation compared to UCB2.  To identify the MSC-specific and developmental genes associated with these phenotypic differences, we performed expression analysis using Affymetrix HG-U133 microarrays and compared them to bone marrow (BM) MSCs. First, hepatocyte growth factor (HGF) and stromal derived factor 1 (SDF1/CXCL12) were up -regulated in UCB1 cells, potentially contributing to the higher growth kinetics observed in this circulating cell population.  Second, we observed that peroxisome proliferation activated receptor gamma (PPARG), a marker for adipogenic differentiation, was significantly increased in undifferentiated UCB1 cells.  Moreover, significant expression of gene markers of blastocyst and gatrulation embryonic stages were detected in UCB1 and UCB2 cells, as were selected markers of early hematopoiesis, chondrogenesis, and cardiac differentiation.  Comparison of UCB1, UCB2, and BM by microarray analysis clearly demonstrated clusters of developmental genes that displayed significant differences among these cells. Quantitative PCR analysis of selected genes validated the microarray results.  Comparison of different UCB-derived adherent cells from a single donor has identified gene profiles potentially useful for therapeutic evaluation of MSC populations.
Keywords: cell type comparison

overall_design: In order, to identify developmental genes whose expression differences distinguished UCB1 and UCB2 cell MSCs, we carried out microarray analysis using Affymetrix HG-U133 microarrays.  For comparison, adult BM-derived MSCs (Cambrex, Lot #4F0312) were analyzed using the same cell culture conditions and confluence as UCB-MSCs.  RNA was prepared from three biological replicates for UCB1, UCB2, and BM MSCs, for a total of nine samples analyzed on the HG-U133A and HG-U133B arrays.

first: Vladimir

last: Markov

first: Mahlet

last: Tadesse

first: Dilusha

middle: A

last: William

first: Dorian

last: Hall

first: Vitali

last: Lounev

first: Arlene

last: Carlton

first: Jay

last: Leonard

first: Rick

middle: I

last: Cohen

first: Eric

last: Rappaport

first: Biagio

last: Saitta


title: Comparison of transcriptional profiles of CD4+ and CD8+ T cells from HIV-infected pateints and uninfected control group

status: Public on Feb 23 2007

city: Toronto

first: Martin

last: Hyrcza

address: 1 King's College Circle, Rm 6356

institute: University of Toronto

summary:
We examined the gene expression profiles in ex vivo human CD4+ and CD8+ T cells from untreated HIV-infected individuals at different clinical stages and rates of disease progression. Profiles of pure CD4+ and CD8+ T cells subsets from HIV-infected nonprogressors who controlled viremia were indistinguishable from HIV-uninfected individuals. Similarly, no gene clusters could distinguish T cells from individuals with early from chronic progressive HIV infection, whereas differences were observed between uninfected or nonprogressors versus early or chronic progressors. In early/chronic HIV infection, three characteristic gene expression signatures were observed: (1) CD4+ and CD8+ T cells showed increased expression of interferon stimulated genes (ISGs).  However, some ISGs including CXCL9, CXCL10, and CXCL11, and the IL15Rα in both CD4+ and CD8+ T cells and the anti-HIV ISG APOBEC3G in CD4+ T cells, were not upregulated. (2) CD4+ and CD8+ T cells showed a cluster similar to that observed in thymocytes, and (3) more genes were differentially regulated in CD8+ T cells than in CD4+ T cells, including a cluster of genes downregulated exclusively in CD8+ T cells. In conclusion, HIV infection induces a persistent T cell transcriptional profile, early in infection, characterized by a dramatic but potentially aberrant interferon response, and a profile suggesting an active thymic output.
Keywords: disease state analysis

overall_design: We studied a cohort of HIV infected individuals with various clinical stages of HIV infection and healthy uninfected volunteers as a control group (Table 1). We included 5 individuals with early HIV infection (A), five with chronic progressive HIV infection (C), five individuals with non-progressive HIV infection with low or undetectable viral loads (L) and five HIV uninfected individuals (N). The HIV infected individuals were never on therapy prior to entering the study. Samples were taken once from each donor.

middle: D

first: Mario

last: Ostrowski

first: Sandy

last: Der

first: Olivia

last: Wilkins

first: Stuart

last: Yang

first: Lawrence

last: Heisler

first: Colin

last: Covacs

first: Roberta

last: Halpenny

first: Mona

last: Loufty


title: Identification of molecular predictors of response in a study of tipifarnib treatment in relapsed and refractory AML

status: Public on Jan 31 2007

city: San Diego

first: Jack

last: Yu

address: 3210 Merryfield Row

institute: Veridex, LLC

summary:
Gene signatures were derived to separate responders from nonresponders by tipifarnib treatment.
Keywords: response analysis

overall_design: 58 samples from 58 patients


title: Gene Expression in Human Conjunctiva and Cornea

status: Public on Feb 10 2007

city: New York

first: Jose

middle: Mario

last: Wolosin

address: 1 Gustave L. Levy Place

department: Ophthalmology

institute: Mount Sinai School of Medicine

summary:
PURPOSE. To determine global mRNA expression levels in the corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression.
METHODS. cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing > 22,000 transcripts. The resulting signal intensities and transcript present/absent calls by the primary microarray analyzing software were used in conjunction with a statistical method to identify transcripts that are preferentially or exclusively (transcripts absent in all three replicates in the other tissue) and significantly (expression >1% of the ß-actin level) expressed in one of the two tissues. EASE was used to identify biological systems comparatively over-represented in either epithelium. Immuno-, and cyto-histochemistry was performed to validate or expand on selected the results of interest.
RESULTS. Our analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding conjunctival epithelium values were 592 and 211. The over-represented biological processes in the cornea were related to cell adhesion and oxi-redox equilibria and cytoprotection activities. In the conjunctiva the biological processes that were most prominent were related to innate immunity and to melanogenesis. Immunohistochemistry for antigen presenting cells and melanocytes were consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not previously been identified or not known to be highly expressed in these two epithelia, including, testican-1, ECM1, a forming isoform, CRTAC1 and NQO1 in the cornea and  sPLA2-IIA, multiple MCH class II proteins, IGFBP3, and Na-Pi cotransporter type IIb in the conjunctiva.
CONCLUSION. Comparative gene expression profiling led to the identification of many biological processes and previously unknown genes potentially important for the function of corneal and conjunctival epithelia.
Keywords: Cell type comparison

overall_design: Whole human conjunctivae and human corneas were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA) and processed between 48 - 72 hr of death. No donor details apart from age, sex and cause of death were released. Criteria for inclusion were donor ages between 25 - 65 years old and overt physical integrity of the epithelium over the central cornea as revealed by a quick stain with 0.1 % Trypan blue. For each replicate experiment (Conjunctiva 1 and Cornea 1; Conjunctiva 2 and Cornea 2;Conjunctiva 3 and Cornea 3), RNA from two different corneal or conjunctival specimens was pooled and converted into cDNA. Appropriately fragmented biotin-labeled cRNA was hybridized to the HG-U133A GeneChips® (Affymetrix) oligonucleotide microarray following the manufacture protocol. Three separate experiments each including one conjunctival and one corneal cRNA pool were performed (Conjunctiva 1 and Cornea 1; Conjunctiva 2 and Cornea 2;Conjunctiva 3 and Cornea 3).

first: Helen

last: Turner

first: Murat

last: Budak

last: Akinci


title: Systemic response to avirulent bacterial infection

status: Public on Jan 22 2007

last: Nottingham Arabidopsis Stock Centre (NASC)

summary:
In the absence of adaptive immunity displayed by animals, plants respond locally to biotic challenge via inducible basal defense networks activated through recognition and response toconserved pathogen associated molecular patterns (PAMPs). In addition, immunity can be induced in tissues remote from infection sites via systemic acquired resistance (SAR), initiated following gene-for-gene recognition between plant resistance proteins and microbial effectors.The nature of the mobile signal and remotely activated networks responsible for establishing SAR remain unclear.
Here we show that despite the absence of PAMP contact, systemically responding leaves rapidly activate a SAR transcriptional signature with strong similarity to local basal defense. Jasmonates have previously been implicated in systemic signalling in response to wounding and plant herbivory but not SAR. We present several lines of evidence that suggest jasmonates may also be central to SAR. Jasmonic acid (JA) rapidly accumulates in phloem exudates of leaves challenged with an avirulent strain of Pseudomonas syringae. In systemically responding leaves transcripts associated with jasmonate biosynthesis are upregulated and JA increases transiently. SAR can be mimicked by foliar JA application and is abrogated in mutants impaired in jasmonate synthesis or response.
We conclude that, jasmonate signalling appears to mediate long-distance information transmission. Moreover, the systemic transcriptional response shares extraordinary overlap with local herbivory and wounding responses, indicating that jasmonates may be central to an evolutionarily conserved signalling network, which decodes multiple abiotic and biotic stress signals.
Experimenter name: William Truman
Experimenter phone: +44 (0)1392 263789
Experimenter fax: +44 (0)1392 263434
Experimenter address: School of Biosciences
Experimenter address: Geoffrey Pope Building
Experimenter address: Stocker Road
Experimenter address: Exeter
Experimenter address: Devon
Experimenter zip/postal_code: EX4 4QD
Experimenter country: UK
Keywords: infection

overall_design: 9 samples were used in this experiment

first: William

last: Truman

first: Murray

last: Grant

first: Henrik

last: Townsend

first: Zoe

last: Emmerson

first: Beatrice

last: Schildknecht


title: Gene expression data from hypothalamic hamartomas (HH) obtained from patients with or without precocious puberty (CPP)

city: Beaverton

first: Valerie

last: Matagne

state: OR

address: 505 nw 185th Ave

department: Neuroscience

institute: OHSU

summary:
Hypothalamic hamartomas (HHs) are congenital lesions of the neuroendocrine brain composed of neurons and astroglia. Frequently, HHs are associated with central precocious puberty (CPP) and/or gelastic seizures. Because HHs might express genes similar to those required for the initiation of normal puberty we used cDNA arrays to compare the gene expression profile of a HH associated with CPP with three HHs not accompanied by sexual precocity. Our aim was to identify genes whose expression may be selectively altered in the HH with CPP and hence, involved in the onset of puberty.
Keywords: comparison between HH with and without CPP

overall_design: Affymetrix arrays were used to detect global changes in gene expression. The results of this analysis were confirmed by semi-quantitative PCR.

first: Anne-Simone

last: Parent

first: Manfred

last: Westphal

first: Heike

last: Jung

first: Sergio

last: Ojeda


title: Gene expression associated with gemcitabine resistance and its reversal by bexarotene

first: Thomas

last: Hermann

address: 10275 Science Center Dr.

department: Molecular Oncology

institute: Ligand Pharmaceuticals

summary:
Resistance of Calu3 NSCLC cells to the cytotoxic nucleoside analog gemcitabine (2',2'-difluorodeoxycytidine) can be prevented as well as reversed by the rexinoid X receptor selective agonist bexarotene.  This study was designed to investigate the changes in gene expression associated with gemcitabine resistance and its reversal by bexarotene.  In addition to the parental Calu3 cells and the 10 cycles of treatment of the gemcitabine resistant Calu3 cells with vehicle or bexarotene, analogous treatment paradigms with gemcitabine alone as well as the combination of both compounds have been included as controls.  (However, it has to be noted that in the combination treatment, cells that were re-sensitized by bexarotene have largely been removed from the culture before harvest due to the cytotoxic activity of gemcitabine.)
Keywords: cell type comparison, compound effects

overall_design: From each of the different Calu3 cultures (parental, gemcitabine-resistant treated with vehicle, bexarotene, gemcitabine, or with the gemcitabine/bexarotene combination for 10 cyclesof ten days each) 4 replicate RNA samples were prepared and individually subjected to microarray analysis.

first: Patricia

last: Tooker

first: Yen

last: Wan-Ching

first: Rene

last: Prudente

first: Jen

last: Sanders

first: Jorge

last: Valencia

first: Wen

last: Luo


title: UPF1-depleted HeLa cells - effect on physiological targets

status: Public on Mar 01 2007

city: Heidelberg

first: Marcelo

last: Viegas

address: IM NEUENHEIMER FELD 156

institute: EMBL-MMPU

summary:
Nonsense-mediated mRNA decay (NMD) is a molecular pathway of mRNA surveillance that ensures rapid degradation of mRNAs containing premature translation termination codons (PTCs) in eukaryotes. Originally, NMD was thought of as a quality control pathway that targets non-functional mRNAs arising from mutations and splicing errors. More recently, NMD has been shown to also regulate normal gene expression and NMD thus emerged as one of the key post-transcriptional mechanisms of gene regulation. We have now systematically analyzed the molecular mechanism of variable NMD efficiency and used different HeLa cell strains as a model system. The results of this analysis show that NMD efficiency can be remarkably variable and represents a stable characteristic of these strains. Low NMD efficiency is shown to be functionally related to the reduced abundance of the exon junction component RNPS1 in one of the HeLa strain analyzed. Furthermore, restoration of functional RNPS1 expression, but not of NMD-inactive mutant proteins, also restores efficient NMD in the RNPS1 deficient cell line. We conclude that cellular concentrations of RNPS1 modify NMD efficiency and propose that the cell type specific co-factor availability represents a novel principle that controls NMD.
Keywords: NMD UPF1 knock down

overall_design: HeLa cells were treated with UPF1 siRNA or Luciferase siRNA as a negative control. After 72 hs, cytoplasmic RNA was isolated and the integrity of the RNA was assessed using a Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA). We performed preparation, processing, and hybridisation of labelled and fragmented cRNA targets to Affymetrix HG_U133A GeneChipsTM according to the manufacturer’s protocols (Affymetrix Inc., Santa Clara, CA). Oligonucleotide arrays were scanned using a confocal laser scanner (GeneArrayTM, Hewlett Packard, Palo Alto, CA). We used the Affymetrix GeneChip Suite 5.0 software (MAS 5.0) to calculate raw expression values for each of the 22,283 probe sets on the U133A oligonucleotide array. Signal intensities were calculated as average intensity difference (AID) between perfect and mismatch probes. Approximately 8,800 probe sets continuously resulting in absent calls were excluded from the analyses. Next, we used GeneSpring 4.2.1 (Silicon Genetics, Redwood City, CA) for scaling, normalisation and background correction of all genes and arrays. We performed Student´s t-test on normalised relative expression ratios to identify significant differentially expressed genes with a minimum factor of difference of >2-fold, within the 95% confidence interval (p<0.05).

first: Niels

last: Gehring

first: Stephen

last: Breit

first: Matthias

middle: W

last: Hentze

first: Andreas

last: Kulozik


title: Cytokinin treatment on aerial parts of seedlings

last: Nottingham Arabidopsis Stock Centre (NASC)

summary:
In Arabidopsis thaliana, the immediate early response of plants to cytokinin is formulated as the multistep AHK-AHP-ARR phosphorelay signaling circuitry, which is initiated by the cytokinin-receptor histidine protein kinases. In the hope of finding components (or genes) that function downstream of the cytokinin-mediated His-Asp phosphorelay signaling circuitry, we carried out genome-wide microarray analyses. To this end, we focused on a pair of highly homologous ARR10 and ARR12 genes by constructing an arr10 arr12 double null mutant. The mutant alleles used in this study were arr10-5 and arr12-1. arr10-5 is the SALK_098604 T-DNA insertion line, whose mutation was determined to be located in the fifth exon of the ARR10 coding sequence. Arr12-1 is the SALK_054752 T-DNA insertion line, whose mutation was determined to be located in the third exon of the ARR12 coding sequence. The resulting mutant exhibits a characteristic phenotype with regard to the cytokinin-mediated His-Asp phosphorelay. Here we, therefore, compared response to cytokinin in wild type with that in arr10 arr12 double mutant. In this study, wild type and the arr10 arr12 double mutant grown vertically on MS agar plates for 2 weeks were treated with 20uM t-zeatin or 0.02% DMSO (solvent for t-zetion solution) for 1h. These treated plant samples were divided into three portions, from which RNA samples were prepared separately from aerial parts of seedlings with use of RNeasy Plant Mini Kit (Qiagen, Valencia, CA, U.S.A.). The Quality of RNAs prepared was analyzed by Bioanalyzer 2100 (Agilent Technologies). These RNA samples were processed as recommended by the Affymetrix instruction (Affymetrix GeneChip Expression Analysis Technical Manual, Affymetrix). These dataset will provide us with bases for understanding the early response to cytokinin on aerial parts of seedlings in Arabidopsis thaliana.
Experimenter name: Hitoshi SAKAKIBARA
Experimenter phone: 81455039576
Experimenter fax: 81455039609
Experimenter address: Biodynamics Research Team
Experimenter address: RIKEN Plant Science Center
Experimenter address: 1-7-22 Suehiro, Tsurumi
Experimenter address: Yokohama
Experimenter zip/postal_code: 230-0045
Experimenter country: JAPAN
Keywords: compound_treatment_design;

overall_design: 12 samples were used in this experiment

first: Hitoshi

last: SAKAKIBARA

first: Henrik

last: Townsend

first: Zoe

last: Emmerson

first: Beatrice

last: Schildknecht


title: Effect of Age on Gene Expression Profiles in Rhesus Monkey Bone Marrow-Derived Mesenchymal Stem Cells

status: Public on Jan 24 2007

city: Los Angeles

first: Joseph

middle: Gerard

last: Hacia

address: 2250 Alcazar Street, IGM 261

department: Biochemistry and Molecular Biology

institute: University of Southern California

summary:
The objective of this study was to elucidate age-related differences in gene expression profiles of rhesus monkey bone marrow-derived mesenchymal stem cells (rhMSC) obtained from fetal, infant, and adult donors relevant to their growth and other properties. Although a high degree of similarity was observed in the rhMSC gene expression profiles when comparing the three age groups, significant differences were found that strongly parallel gene expression profiles of human MSC. The potential functional relevance of differential gene expression was most apparent when comparing fetal and adult rhMSC transcript profiles.  Overall, the observed gene expression profiles are consistent with a loss of rhMSC pluripotency and proliferative capacity with advancing donor age.  In addition, these data highlight the importance of use of non-human primates as a model system for studying the properties of human stem cells.
Keywords: age of donor

overall_design: A total of eleven bone marrow-derived mesenchymal stem cell cultures were established from rhesus monkeys of diverse ages (ranging from gestation day 120 to 8 year old adults).  Total RNA was harvested from these cultures and gene expression analyses were conducted using oligonucleotide microarrays. The expression profiles of a subset of genes was confirmed by qRT-PCR analysis.  A detailed manuscript that details the results of our studies is currently under review.

first: Alice

last: Tarantal


title: Group II-A WRKY transcription factors and early leaf senescence (2)

institute: Nottingham Arabidopsis Stock Centre (NASC)

summary:
In our laboratory we are interested in studying the functions of WRKY zink finger type transcription factors. There are 74 members of this gene family in Arabidopsis.  WRKY factors are key regulators of distinct plant defense responses and are involved in certain developmental programs e.g. plant senescence.  We would like to determine the functions of a small sub-group (group II-a) of WRKY factors.  Our aim to compare and contrast the gene expression profiles of 35 days-old untreated wild type and WRKY T-DNA knockout plants grown in a growth chamber under long day growth conditions.  All plants chosen at this stage showed slight yellowing of the first two to four leaves.
Experimenter name: Bekir Uelker
Experimenter phone: 49-221-5062-310
Experimenter fax: 49-221-5062-353
Experimenter department: Somsissich Lab
Experimenter institute: Max-Planck-Institute for Plant Breeding Research
Experimenter address: Department of Plant Microbe Interactions
Experimenter address: Carl-von-Linne-Weg 10
Experimenter address: Cologne
Experimenter zip/postal_code: 50829
Experimenter country: Germany
Keywords: genetic_modification_design

overall_design: 8 samples were used in this experiment

first: Bekir

last: Uelker

first: Imre

last: Somssich


title: Group II-A WRKY transcription factors and early leaf senescence

summary:
In our laboratory we are interested in studying the functions of WRKY zinc finger type transcription factors. There are 74 members of this gene family in Arabidopsis. WRKY factors are key regulators of distinct plant defense responses and are involved in certain developmental programs e.g. trichome development and plant leaf senescence. We would like to determine the functions of a small sub-group (group II-a) of WRKY factors in response to bacterial infection. Our aim is to compare and contrast the gene expression profiles of 3-week-old wild type and WRKY T-DNA knockout mutants grown in a growth chamber under long day growth conditions and subsequently challenged for 6 hours with the virulent bacterial pathogen /Pseudomonas syringae/ DC3000.
Experimenter name: Bekir Uelker
Experimenter phone: 49-221-5062-310
Experimenter fax: 49-221-5062-353
Experimenter department: Somsissich Lab
Experimenter institute: Max-Planck-Institute for Plant Breeding Research
Experimenter address: Department of Plant Microbe Interactions
Experimenter address: Carl-von-Linne-Weg 10
Experimenter address: Cologne
Experimenter zip/postal_code: 50829
Experimenter country: Germany
Keywords: genetic_modification_design

overall_design: 7 samples were used in this experiment


title: Transcriptome response to change in ploidy level in Arabidopsis

summary:
By reciprocally crossing 2x and 4x C24 ecotype plants, we have generated 4 types of offspring with various ploidy (2x; 3x; 4x) or parent-of-origin genome dosage (3x from 4xper2x; 3x from 2xper4x). For each offspring generated, total RNA was extracted using Trizol from 8 seedlings 9 days after germination (developmental stage1.02, 2 leaves).
Sample names:
DIP diploid
TET tetraploid
TFE triploid female excess
TME triplod male excess
Experimenter name: Olivier Garnier
Experimenter phone: 00-353-21-490-4028
Experimenter address: Plant molecular genetics lab, Dpt of Biochemistry, UCC
Experimenter address: Lee Maltings Prospect row
Experimenter zip/postal_code: Cork
Experimenter country: Ireland
Keywords: strain_or_line_design

overall_design: 11 samples were used in this experiment

first: Olivier

last: Garnier

first: Charlie

last: Spillane


title: Identification of candidate Arabidillo target genes in Arabidopsis

summary:
Arabidopsis has two genes, Arabidillo-1 and -2, related to animal Armadillo/ beta-catenin (Coates, 2003).  Armadillo/beta-catenin directly activates the expression of developmental and cell proliferation genes, and also independently regulates cell-cell adhesion. Arabidillo proteins are nuclear and promote lateral root development.
We aim to identify candidate Arabidillo target genes by comparing the transcriptomes of wild type, arabidillo-1/2 mutant and Arabidillo-1 overexpressing lines.
The experiment will compare Col-0 (3 slides) with a 35S::Arabidillo-1-YFP overexpressing line (3 slides) and Col-3 with the arabidillo-1/2 mutant (3 slides).  Each RNA prep will be from plate-grown 7-day old  seedlings.
Reference: Coates, JC (2003) Armadillo repeat proteins: beyond the animal kingdom?  Trends in Cell Biology 13 p.463-471
Experimenter name: Juliet Coates
Experimenter phone: 0121 414 5481
Experimenter institute: University of Birmingham
Experimenter address: School of Biosciences
Experimenter address: Birmingham
Experimenter zip/postal_code: B15 2TT
Experimenter country: UK
Keywords: genetic_modification_design

first: Juliet

last: Coates


title: Ewing samples and EWS-FLI-1 inhibited Ewing cell lines

city: Paris

last: Delattre

address: 26 rue d'Ulm

department: Genetics

institute: Institut Curie

summary:
The cellular origin of Ewing tumor (ET), a tumor of bone or soft tissues characterized by specific fusions between EWS and ETS genes, is highly debated. Through gene expression analysis comparing ETs with a variety of normal tissues, we show that the profiles of different EWS-FLI1-silenced Ewing cell lines converge toward that of mesenchymal stem cells (MSC). Moreover, upon EWS-FLI1 silencing, two different Ewing cell lines can differentiate along the adipogenic lineage when incubated in appropriate differentiation cocktails. In addition, Ewing cells can also differentiate along the osteogenic lineage upon long-term inhibition of EWS-FLI1. These in silico and experimental data strongly suggest that the inhibition of EWS-FLI1 may allow Ewing cells to recover the phenotype of their MSC progenitor.
Keywords: expression analysis

overall_design: Ewing tumors and EWS-FLI-1 inhibited cell lines were profiled on Affymetrix U133A (GPL96) arrays.

first: Franck

last: Tirode

first: karine

last: Laud-Duval

first: Alexandre

last: Prieur

first: Bruno

last: Delorme

first: Pierre

last: Charbord


title: Identification of genes involved in nutritional regulation of root architecture

last: Nottingham Arabidopsis Stock Centre (NASC)

summary:
AIM:
1. To identify genes that respond to N-deficiency and N-resupply and
2. To identify the subset of these genes that are under the (direct or indirect) regulatory influence of the ANR1 MADS-box gene and which may therefore participate in the nutritional regulation of root architecture.
BACKGROUND:
The Arabidopsis ANR1 gene is a key regulator of root architecture (Zhang and Forde, 1998): When ANR1 expression is suppressed (by antisense or co-suppression) the resulting lines are no longer able to proliferate their lateral roots in response to localised supplies of NO3- (Zhang and Forde, 1998).
ANR1 encodes a root-specific member of the MADS box family of transcription factors and is thought to be a component of a signalling pathway that links an external NO3- signal to increase meristematic activity in the lateral root meristem (Zhang et al., 1999). ANR1 expression is modulated by changes in the NO3- supply (Zhang and Forde, 1998; Y.Gan and B.G. Forde, unpublished observations).
In this experiment we will study the global changes in gene expression that occur when Arabidopsis plants are exposed to three different N regimes that are known to modulate ANR1 expression.  The treatments will be done on wild-type plants and on an ANR1 knockout mutant as a means of identifying the (direct and indirect) downstream targets of ANR1. This experiment is complementary to another microarray experiment (BBSRC grant no. 89/P13011) which is using a DEX-inducible ANR1 transgene to identify genes that are immediately downstream of ANR1 in the signal transduction cascade
Experimental:
Plants of the two genotypes will be grown in hydroponic culture in a growth cabinet for 4-5 weeks before subjecting them to the different N treatments:
1) Continuous nitrate supply;
2) N deprivation for 2.5 days; and
3) N deprivation followed by 3 h nitrate induction.
All harvesting will be done simultaneously to avoid diurnal effects. Roots from 12 seedlings will be harvested and pooled with roots from 12 seedlings whose treatment has been staggered by 24 h. We propose to replicate the experiment three times.
References: Zhang, H. and Forde, B.G. (1998) Science 279, 407-409. Zhang, H. et al. (1999) Proc. Natl Acad. Sci. USA 96, 6529-6534.
Experimenter name: Yinbo Gan
Experimenter phone: 01524 592935
Experimenter fax: 01524 843854
Experimenter department: Lancaster University
Experimenter address: Department of Biological Sciences
Experimenter address: Lancaster University
Experimenter address: Bailrigg
Experimenter address: Lancaster
Experimenter zip/postal_code: LA1 4YQ
Experimenter country: UK
Keywords: genetic_modification_design;  growth_condition_design

overall_design: 6 samples were used in this experiment

first: Yinbo

last: Gan

first: Brian

last: Forde


title: Comparison of transcriptional profiles between sni1 and wild type

summary:
Microarray experiment was performed using 4-week old plants to compare transcriptional profiles between sni1 (suppressor of npr1) and wild type (Col-0).  Three biological replicates were included.
Experimenter name: Wendy Durrant
Experimenter phone: 1(919)613-8175
Experimenter fax: 1(919)613-8177
Experimenter department: Durrant Lab
Experimenter institute: Duke University
Experimenter address: Rm. B354, LSRC Bldg.
Experimenter address: Research Dr.
Experimenter address: Duke University
Experimenter address: Durham, NC
Experimenter zip/postal_code: 27708
Experimenter country: USA
Keywords: genetic_modification_design;

first: Wendy

last: Durrant

first: Xinnian

last: Dong

first: Henrik

last: Townsend

last: Emmerson

last: Schildknecht


title: Differential gene expression patterns in potassium-starved and Caesium-treated plants

summary:
At high concentrations ceasium (Cs) is toxic to plant growth. This toxic effect may occur when Cs blocks potassium (K) uptake mechanisms in plants. Consequently, plants starved of K and plants exposed to toxic concentrations of Cs should have similar gene expression patterns. To test this hypothesis, Arabidopsis will initially be grown on agar containing 1/10 MS salts before being transferred to either 1/10 MS nutrient solution (control plants), 1/10 MS nutrient solution containing 2 mM Cs, or 1/10 MS nutrient solution with no K. Roots and shoot will then be harvested seven days after transfer and used to challenge ATH1 GeneChips.
Experimenter name: John Hammond
Experimenter phone: 01789 470382
Experimenter fax: 01789 470552
Experimenter institute: Warwick University
Experimenter address: Horticulture Research International
Experimenter address: Wellesbourne
Experimenter address: Warwick
Experimenter zip/postal_code: CV35 9EF
Experimenter country: UK
Keywords: compound_treatment_design

overall_design: 18 samples were used in this experiment

first: John

last: Hammond

first: Philip

last: White

first: Zoe

first: Beatrice


title: The molecular basis of plant insect interactions

summary:
The aim of this study is to identify Arabidopsis genes whose expression is altered by aphid feeding. An understanding of the plant aphid interaction at the level of the plant transcriptome will 1) consolidate current areas of investigation focused on the phloem composition (the aphid diet), 2) open up areas of plant aphid interactions for ourselves and other workers, 3) Contribute to understanding the use of new molecular technologies in an environmental context and  4) contribute to existing and development of novel control strategies.Our Arabidopsis/Myzus persicae system provides a valuable model for the study because of: a) the advantages of using Arabidopsis, b) The ability to use clonal insects, c) phloem feeding aphids facilitate focus on a specific cell type, d) aphid stylectomy allows collection of pure phloem sap to monitor phloem phenotype of the plant and the insect diet, e) we have techniques to monitor the reproductive performance and feeding behaviour aphids.Our strategy has been to test the function of selected genes, particularly those regulating phloem composition (the feeding site of the aphid) based on current phloem models of phloem function. Gene choice is limited the simplicity of current models of phloem aphid interaction.We propose a simple two treatment (aphid infested vs control plants) experiment that will identify novel target genes for future analysis. Arabidopsis plants (variety Columbia) will be grown in 16/8 light/dark in temperature controlled growth rooms. At growth stage 3.90, when rosette growth is complete, 10 clonal adult Myzus persicae will be caged in clip cages on the two largest leaves on each plant. Control plants will be treated identically except that the cages will be empty. Leaves will be harvested 8 h after infestation. This time point is selected as we know that 90% of aphids are plugged into the sieve element within 2h and that a 6h lag phase has period has previously been used when examining gene expression affected by wounding. In subsequent experiments we will examine time courses of expression of relevant genes using other approaches. Pooling two leaves from each of ten plants will generate the RNA sample, ensuring that expression signals are representative of the population of plants.
Experimenter name: Jeremy Pritchard
Experimenter phone: 0121 414 5570
Experimenter fax: 0121 414 5925
Experimenter institute: University of Birmingham
Experimenter address: School of Biosciences
Experimenter address: University of Birmingham, Edgbaston, Birmingham, West Midlands
Experimenter zip/postal_code: B30 2EN
Experimenter country: UK
Keywords: pathogenicity_design

first: Jeremy

last: Pritchard


title: Impact of Type III effectors on plant defense responses

summary:
Our interest lies in how plants respond to bacterial pathogens. Over the past three years we have identified and documented reproducible, landmark biochemical and molecular events following the challenge of Arabidopsis with the phytopathogenic enterobacteria P. syringae. Significantly, our studies revealed 60% of cDNA-AFLP differentials not present on the 8,200 feature GeneChips and 20% absent from public EST databases (de Torres in press). We now seek to exploit this background using carefully defined time-points to analyse global changes in the Arabidopsis transcriptome using challenges selected to define gene targets implicated in (i) expression of basal immunity (ii) the establishment of successful parasitism (resistance) by a virulent pathogen (host). The results will provide a rationale for future functional assays of the identified pathways using transgenic knockouts and mutant analyses. Additionally, data will provide underpinning support for comparative proteomics of the defense response currently in progress with GARNet support using the same experimental parameters (BBSRC 32/P14635).  We propose the following treatments: (i) Mock vs. DC3000hrpA @ 60 min: 60 minutes is subsequent to host immediate-early stress responses and will  catalogue the innate responses induced by pathogen associated molecular patterns. The hrpA lesion will ensure no type III effectors influence transcriptional responses. Gene products induced at this time are predicted to potentiate latter host responses (2 treatments X 3 biological replicates = 6 chips).  (ii) Mock vs. DC3000 vs. DC3000hrpA vs. DC3000::avrRpm1 @ 4 hours: A key time point previously defined where no macroscopic symptoms are visible but significant differences exist between compatible and incompatible interactions at the molecular and physiological levels. These treatments will serve to define the earliest genes induced by the complement of DC3000 type III effector and will specifically define genes/pathways suppressed by virulence factors in addition to those implicated in orchestration of the hypersensitive cell death. We will also include an incompatible interaction on a transgenic line expressing an RPM1 interacting protein, which fails to mount an HR but exhibits hyper-resistance. We predict this challenge will separate the resistance response (pathogen restriction) from that associated with hypersensitive cell death (5 treatments X 3 biological replicates = 15 chips). (iii) Mock vs. DC3000 vs. DC3000hrpA @ 14 hours: At 10 h before phenotypes are apparent in the DC3000 background, Type III effector delivery is well advanced and impact on host transcription maximal (3 treatments X 3 biological replicates = 9 chips).
Keywords: time_series_design;  pathogenicity_design;  compound_treatment_design

overall_design: Number of plants pooled:4 leaves/plant; 18 plants/time point

first: Marta

last: de Torres Zabala


title: Comparative transcriptome analysis between wild-type and gpa1 mutant in response to ABA

summary:
Mutations in the heterotrimeric G-protein a-subunit of Arabidopsis, GPA1, leads to deficiency in ABA-induced stomatal closure (Wang et al., 2001). To further investigate whether GPA1 is involved in the regulation of gene expression in response to ABA, we examined the induction of known ABA-inducible genes in the gpa1 mutant and compared it to wild-type. We found significant differences in levels of ABA-induced expression between wild-type and gpa1 mutant. In order to systematically investigate GPA1 involvement in ABA signalling leading to gene expression, we are requesting the transcriptome analysis of the gpa1 mutant in response to ABA.In detail, 2 week old wild-type and gpa1 plants grown in the 16/8 hrs light and dark cycle will be treated with either ABA or with a control solution for 3 hours.  10 plants will be used per sample to produce RNA, to give 4 samples, one for each chip: gpa1-1 mutant in the presence (chip1) or absence of ABA (chip2) and wild-type (WS2) in the presence (chip3) or absence of ABA (chip4).
Keywords: strain_or_line_design

overall_design: Number of plants pooled:10 plants

first: Haruko

last: Okamoto


title: Clarification of the genetic basis of the iae1 and iae2 phenotypes

summary:
The mutants iae1 and iae2 have been mapped to two distinct loci on chromosome 2.  With the aid of the GetCID service, we believe iae1 has been localised to a relatively small region (~100kb); while mapping has restricted iae2 to a small interval (~50kb) with a very limited number of candidate genes. Although it has proved difficult to determine which gene is mutated in iae1, we belive that use of the transcriptomics service will help identify which genes are affected (directly and indirectly) in this background.  The iae2 locus is sufficiently restricted by genetics and by molecular data that identifying a candidate (and any downstream loci affected) should be straightforward. We require the use of the full genome chips. The data from this experiment should help tie together the data we have for a paper that is currently in preparation.
Keywords: mutant;  transgenic;

overall_design: Number of plants pooled:30 (done)

first: David

last: Stevenson


title: The effect of mutations in AtrbohC on the pattern of gene expression in primary root tissue.

summary:
Aim:  To determine the effect of an AtrbohC mutation on the gene expression pattern in primary root tissue, to identify candidate genes acting downstream of AtrbohC, particularly any encoding antioxidant-related proteins, signal transduction components or proteins known to be required for normal root-hair development. Background:  Root-hairs are a model system for investigating plant cell polarity. The root-hair mutant rhd2 (Schiefelbein and Somerville, 1990. Plant Cell, 2:235) has short hairs that burst at their tips, (Jones and Smirnoff, unpublished). RHD2 has been cloned and is identical to AtrbohC (L. Dolan, pers. comm.), which encodes a homologue of the superoxide-generating neutrophil respiratory burst oxidase catalytic subunit gp91phox (Torres et al., 1998. Plant J., 14:365). Superoxide rapidly dismutates to hydrogen peroxide (H2O2), suggesting that the rhd2 phenotype may result from reduced H2O2 levels in root-hair cells. Low doses of exogenous antioxidants phenocopy the rhd2 root-hair phenotype in wild-type plants (Jones and Smirnoff, unpublished) further supporting a role for H2O2 in root-hair growth. Fluorescent dyes that detect H2O2 show distinct localisation patterns in growing root-hair cells, (Jones and Smirnoff, unpublished). H2O2 may be an important second messenger in plant cell signalling with proposed roles in the development of cotton fibres (Potikha et al., 1999. Plant Physiol., 119: 849) and in ABA-induced stomatal closure (Zhang et al., 2001. Plant Physiol., 126: 1438). In cultured Arabidopsis cells H2O2 induces gene expression, including that of a gp91phox homologue, (Desikan et al., 1998. J. Exp. Bot., 49: 1767; Desikan, et al., 2000. Free Rad. Biol. Med., 28: 773; Baxter-Burrell et al., 2002. Science, 296: 2026) and activates a MAP kinase cascade (Desikan et al., 1999. J. Exp Bot., 50: 1863). cDNA microarray technology has been used previously to examine the effects of H2O2 on gene expression during oxidative stress (Desikan et al., 2001. Plant Physiol., 127: 159). We wish to investigate the effects of H2O2 on gene expression during root development using the rhd2 mutant. We are currently determining the expression pattern of RHD2. By extracting RNA from the small region of the primary root (for wild-type and rhd2 plants grown in sterile conditions) where root hairs are growing we hope to enrich for root-hair RNAs. This may reveal candidate genes that could be examined more closely at the single-cell level. This approach will provide new insights into the role of H2O2 in root-hair development.
Keywords: strain_or_line_design

overall_design: Number of plants pooled:100

first: Mark

last: Jones


title: The molecular basis of chilling and freezing stress

summary:
Our analysis of the sfr6 freezing-sensitive mutant (Knight, H., Veale, E., Warren, G. J. and Knight, M. R. (1999).  Plant Cell 11, 875-886.) and cls8 (unpublished) chilling-sensitive mutant of Arabidopsis, has revealed that the expression of certain cold-regulated genes is aberrant in both these mutants.  In order to understand the molecular basis of chilling and freezing stress in Arabidopsis and also to determine commonalities and differences between these 2 different physiological stress-tolerance processes, we request transcriptome analysis for both of these mutants compared to wild type in one experiment, upon cold treatment and at ambient conditions.  The sfr6 mutant shows the most severe phenotype with respect to cold gene expression, but is tolerant to chilling (Knight, H., Veale, E., Warren, G. J. and Knight, M. R. (1999).  Plant Cell 11, 875-886.).  However, it is unable to cold acclimate and hence is sensitive to freezing.  The cls8 mutant, on the other hand, has a relatively mild phenotype relative to the cold-regulated genes we have examined, but is very sensitive to chilling temperatures (15 to 10 degree centigrade).  It is thus likely that in cls8 we have not yet identified the genes which are most affected, and which account for the physiological phenotype.  Both sfr6 and cls8 have been fine-mapped and are close to being cloned.  The cls8 mutant has an altered calcium signature in response to cold which means it is likely to be affected in early signalling, e.g. cold perception itself.We will compare the expression profiles of genes in sfr6, cls8 and Columbia (parental line for both mutants), both at ambient, and after treatment with cold (5 degrees) for 3 hours.  This timepoint is designed to capture both rapidly responding genes e.g. CBF/DREB1 transcription factors, and also more slow genes e.g. COR genes (KIN1/2 and LTI78).  Pilot northerns confirm that this time point is suitable.This analysis will provide new insight into 2 novel genes required for tolerance to low temperature in Arabidopsis, and additionally will determine the nature of overlap between the separate processes of chilling and freezing tolerance.
Keywords: strain_or_line_design

overall_design: Number of plants pooled:40-60

first: Marc

last: Knight