title: Quantification of vascular lineage-specific differentiation, psoriasis (chronic inflammation) study

city: Zurich

first: Jay

middle: Woo

last: Shin

state: Zurich

address: Wolfgang-Paulistrasse 10; HCI H394

department: Institute of Pharmaceutical Sciences

country: Switzerland

institute: ETH Zurich

summary:
Angiogenesis and lymphangiogenesis have important roles in cancer progression and chronic inflammatory diseases, but efficient therapies against these diseases have been hampered by the lack of identified vascular lineage-specific markers and growth factors. Using transcriptional profiling of matched pairs of human dermal blood vascular and lymphatic endothelial cells, we first identified 236 lymphatic and 342 blood vascular signature genes. In silico analyses of the biologic pathways associated with these genes revealed lineage-specific functions for each cell type. Using a selection of 85 identified vascular lineage-specific genes, we developed a TaqMan RT-PCR-based, microfluidic card-formatted low-density microvascular differentiation array (LD-MDA) that was used to reliably identify and quantify the degree of lineage-specific differentiation in different types of endothelial cells, and to detect admixture of lymphatic endothelial cells in commercial preparations of microvascular endothelial cells. Application of Prediction Relevance Ranking and analysis of variance of LD-MDA expression profiles of 43 lesional skin samples obtained from patients with the chronic inflammatory disease psoriasis led to identification of cytokines which are significantly associated with angiogenesis or lymphangiogenesis in vivo. In particular, interleukin-7 and fibroblast growth factor-12 were identified as novel (lymph)angiogenic factors. This technology provides a novel tool to quantify lineage-specific vascular differentiation and to characterize (lymph)angiogenesis in clinical samples obtained from angiogenic diseases.
Keywords: Quantitative real time RT-PCR, psoriasis (chronic inflammation) study

overall_design: Guided by the gene array results, we selected 54 LEC-specific genes and 31 BEC-specific genes, based upon their consistent and strong specific expression in LEC or BEC, as well as on their assignment to important biological pathways. In addition, the five pan-endothelial cell marker genes PECAM-1, vWF, KDR, TEK, CDH5 and the six endogenous control genes ACTB, GAPDH, PGK1, PPIA, RPLP0 and S18 were included in the design of the LD-MDA. After extraction of total RNA, the mRNA expression levels of the 96 genes were analyzed by quantitative RT-PCR using the 7900HT Real-Time PCR System (Applied Biosystems).

first: Rainer

last: Kunstfeld

first: Ulrich

last: Mrowietz

first: Sascha

last: Gerdes

first: Michael

last: Detmar


title: Quantification of vascular lineage-specific differentiation (cell type comparison)

status: Public on Oct 04 2008

summary:
Angiogenesis and lymphangiogenesis have important roles in cancer progression and chronic inflammatory diseases, but efficient therapies against these diseases have been hampered by the lack of identified vascular lineage-specific markers and growth factors. Using transcriptional profiling of matched pairs of human dermal blood vascular and lymphatic endothelial cells, we first identified 236 lymphatic and 342 blood vascular signature genes. In silico analyses of the biologic pathways associated with these genes revealed lineage-specific functions for each cell type. Using a selection of 85 identified vascular lineage-specific genes, we developed a TaqMan RT-PCR-based, microfluidic card-formatted low-density microvascular differentiation array (LD-MDA) that was used to reliably identify and quantify the degree of lineage-specific differentiation in different types of endothelial cells, and to detect admixture of lymphatic endothelial cells in commercial preparations of microvascular endothelial cells. Application of Prediction Relevance Ranking and analysis of variance of LD-MDA expression profiles of 43 lesional skin samples obtained from patients with the chronic inflammatory disease psoriasis led to identification of cytokines which are significantly associated with angiogenesis or lymphangiogenesis in vivo. In particular, interleukin-7 and fibroblast growth factor-12 were identified as novel (lymph)angiogenic factors. This technology provides a novel tool to quantify lineage-specific vascular differentiation and to characterize (lymph)angiogenesis in clinical samples obtained from angiogenic diseases.
Keywords: Quantitative real time RT-PCR, cell type comparison

overall_design: Guided by the gene array results, we selected 54 LEC-specific genes and 31 BEC-specific genes, based upon their consistent and strong specific expression in LEC or BEC, as well as on their assignment to important biological pathways. In addition, the five pan-endothelial cell marker genes PECAM-1, vWF, KDR, TEK, CDH5 and the six endogenous control genes ACTB, GAPDH, PGK1, PPIA, RPLP0 and S18 were included in the design of the LD-MDA. The LD-MDA was then used to evaluate the lineage-specific differentiation of a total of 10 independent lines of primary human dermal LEC, of 8 independent lines of primary human dermal BEC, of two independent lines of HUVEC cells, of the immortalized human microvascular endothelial cell line HMEC-1, of the immortalized human epidermal keratinocyte line HaCaT, and of primary human dermal fibroblasts. After extraction of total RNA, the mRNA expression levels of the 96 genes were analyzed by quantitative RT-PCR using the 7900HT Real-Time PCR System (Applied Biosystems).

first: Kentaro

last: Kajiya

first: Giorgia

last: Jurisic


title: Expression profile of potato plants after inoculation with endophytes

status: Public on Oct 01 2008

city: Seibersdorf

first: Friederike

last: Trognitz

state: NÖ

address: Seibersdorf

department: Health & Environment

country: Austria

institute: AIT Austrian Institute of Technology GmbH

summary: Two potato cultivars, Russet Burbank and Bionta, were inoculated with three different endophytes containing different AHL types. The impact of the endophytes to the different cultivars was measured by gene expression analysis with a customized microarray

overall_design: B. phytofirmans type strain PsJN was originally isolated as a contaminant from surface-sterilized, Glomus vesculiferum-infected onion roots (Nowak et al., 1998), whereas strain P6 RG6-12 was isolated from the rhizosphere of a grassland in the Netherlands (Salles et al., 2006). This strain was selected based on its similarity to strain PsJN based on 16S rRNA gene homology, and similar phenotypic features. Both strains were generally cultivated on King's medium (King et al., 1954).  For the mutant AHL to the strain  B. phytofirmans PsJN a quorum quenching approach as described by Wopperer et al., 2006 was employed. Plasmid pMLBAD-aiiA, which contains aiiA, the Bacillus sp. 240B1 lactonase gene, was transferred to B. phytofirmans PsJN by triparental mating as described by de Lorenzo and Timmis (1994). 2 cultivars, 3 endophytes

first: F

first: K

last: Scherwinski

first: S

last: Schoethaler

first: B

first: A

last: Sessitsch


title: Porphyromonas gingivalis comparative genomic hybridization

status: Public on Oct 09 2008

city: Columbus

first: CHRISTINA

last: IGBOIN

state: OH

address: 305 W 12th Ave

country: USA

institute: Ohio State University

summary:
Genotyping studies suggest that there is genetic variability among P. gingivalis strains, however the extent of variability remains unclear, and the regions of variability have only partially been identified. We previously used heteroduplex analysis of the ribosomal operon intergenic spacer region (ISR) to type P. gingivalis strains in several diverse populations, identifying 6 predominant heteroduplex types and many minor ones. In addition we used ISR sequence analysis to determine the relatedness of P. gingivalis strains to one another, and demonstrated a link between ISR sequence phylogeny and the disease-associated phenotype of P. gingivalis strains.  The availability of whole genome microarrays based on the genomic sequence of strain W83 has allowed a more comprehensive analysis of P. gingivalis strain variability, using the entire genome. The objectives of this study were to define the phylogeny of P. gingivalis strains using the entire genome, to compare the phylogeny based on genome content to the phylogeny based on a single locus (ISR), and to identify genes that are associated with the strongly disease-associated strain W83 that could be important for virulence.

Keywords: Comparative genomic hybridization

overall_design: Comparative genomic analysis of 7 clinically prevalent P. gingivalis strains was performed, using whole genome microarrays based on the sequence of strain W83. Strain W83 was the reference strains and there were 6 test strains. Flip-dye replicates were performed.

first: Christina

middle: O

last: Igboin

first: Eugene

middle: J

last: Leys

first: Ann

middle: L

last: Griffen


title: Gene expression profiling in hepatic tissue of newly weaned pigs fed pharmacological zinc supplemented diets

status: Public on Oct 02 2008

city: East Lansing

first: Catherine

middle: W.

last: Ernst

state: MI

address: 3330 Anthony Hall

department: Animal Science

institute: Michigan State University

summary:
Zinc (Zn) is an essential trace element for all life forms. Zn supplementation has been used to treat diarrheal disease in children, and in the U.S. swine industry at pharmacological levels to promote growth and fecal consistency, but underlying mechanisms explaining these beneficial effects remain unknown. Thus, we hypothesized that the benefits of pharmacological Zn supplementation were a result of changes in gene expression.

For this study, liver RNA from newly weaned pigs fed dietary Zn as Zn oxide for 14 days at either adequate (150 Zn/kg) or pharmacological (2000 mg Zn/kg) levels was evaluated using a 70-mer oligonucleotide microarray.  Interrogation of this microarray revealed 658 annotated transcripts (FDR ≤ 0.05) affected by pharmacological Zn supplementation.  Relative real-time RT-PCR was used to confirm differential expression of two genes.  Results suggest that feeding pharmacological Zn (2000 mg Zn/kg) affects genes involved in reducing oxidative stress and in amino acid metabolism, which are essential for cell detoxification and proper cell function.

overall_design: Oligonucleotide microarrays used for this study consisted of 13,297 70-mer oligos (Pig Array-Ready Oligo Set v. 1.0 and Pig Oligo Extension Set v. 1.0, Qiagen, Inc., Valencia, CA) each spotted once on a single slide.  Slides were printed at the Michigan State University Research Technology Support Facility.  Oligonucleotides spotted in multiple locations for use as potential controls included 76 Arabidopsis thaliana gene spots, 17 beta tubulin spots, 17 glyceraldehyde-3-phosphate dehydrogenase spots, 85 heat shock protein gene spots, 69 ribosomal protein gene spots, 112 randomly generated negative control spots and 470 blanks.  The microarray was screened with the Zn150 and Zn2000 samples, and four microarray slides were screened.  Zn150 samples were randomly paired with Zn2000 samples.  Two samples from each treatment were labeled with Cy3 and the other two were labeled with Cy5.

first: Michelle

middle: M

last: Martínez-Montemayor

first: Gretchen

last: Hill

first: Nancy

middle: E

last: Raney

first: Valencia

middle: D

last: Rilington

first: Robert

last: Tempelman

first: Jane

last: Link

first: Christopher

middle: P

last: Wilkinson

first: Antonio

last: Ramos

middle: W


title: Experimental Malaria Infection Triggers Early Expansion of Natural Killer Cells - NK cell response

city: San Francisco

first: Sajeev

last: Batra

state: CA

institute: UCSF

summary:
In order to gain a better understanding of gene expression during early malaria infection, we conducted microarray analysis of early blood responses in mice infected with erythrocytic stage Plasmodium chabaudi.  Immediately following infection, we observed coordinated and sequential waves of immune responses, with interferon-associated gene transcripts dominating by 16 hours post-infection, followed by strong increases in natural killer (NK) cell-associated and MHC class I-related transcripts by 32 hours post-infection.  We hypothesized that the observed elevation in NK cell-associated transcripts could be the result of a dramatic increase in the proportion of NK cells in the blood during infection, which we confirmed by flow cytometry.  Subsequent microarray analysis of NK cells isolated from the peripheral blood of infected mice revealed a cell proliferation expression signature consistent with the observation that NK cells replicate in response to infection.  Early proliferation of NK cells was directly observed in studies with adoptively transferred cells in infected mice.  These data indicate that the early response to P. chabaudi infection of the blood is marked by a primary wave of interferon with a subsequent response by NK cells.

Keywords: murine NK cell response to Plasmodium chabaudi infection

overall_design: We analyzed a series of 10 MEEBO arrays on which were hybed RNA amplified from NK cells of C57BL/6 mice either mock-infected or infected with P. chabaudi AS.

first: Charles

middle: C

last: Kim

first: Sunil

last: Parikh

first: Joseph

last: Sun

first: Alissa

last: Myrick

first: Lewis

last: Lanier

first: Philip

last: Rosenthal

last: DeRisi


title: Experimental Malaria Infection Triggers Early Expansion of Natural Killer Cells

summary:
In order to gain a better understanding of gene expression during early malaria infection, we conducted microarray analysis of early blood responses in mice infected with erythrocytic stage Plasmodium chabaudi.  Immediately following infection, we observed coordinated and sequential waves of immune responses, with interferon-associated gene transcripts dominating by 16 hours post-infection, followed by strong increases in natural killer (NK) cell-associated and MHC class I-related transcripts by 32 hours post-infection.  We hypothesized that the observed elevation in NK cell-associated transcripts could be the result of a dramatic increase in the proportion of NK cells in the blood during infection, which we confirmed by flow cytometry.  Subsequent microarray analysis of NK cells isolated from the peripheral blood of infected mice revealed a cell proliferation expression signature consistent with the observation that NK cells replicate in response to infection.  Early proliferation of NK cells was directly observed in studies with adoptively transferred cells in infected mice.  These data indicate that the early response to P. chabaudi infection of the blood is marked by a primary wave of interferon with a subsequent response by NK cells.

Keywords: murine whole blood response to Plasmodium chabaudi infection

overall_design: We analyzed a series of 36 MEEBO arrays on which were hybed RNA amplified from whole blood of C57BL/6 mice either mock-infected or infected with P. chabaudi AS.


title: Genomic lesions in CLL: B-CLL vs. PMNs from respective patients

city: Cold Spring Harbor

first: Vladimir

last: Grubor

state: NY

address: 1 Bungtown Road

institute: Cold Spring Harbor Laboratory

type: Genome variation profiling by genome tiling array

summary:
We examined copy number changes in the genomes of B cells from 58 patients with chronic lymphocytic leukemia (CLL) using representational oligonucleotide microarray analysis (ROMA), a form of comparative genomic hybridization (CGH), at a resolution exceeding previously published studies. We observed at least one genomic lesion in each CLL sample and considerable variation in the number of abnormalities from case to case. Virtually all abnormalities previously reported were also observed here, most of whichwere indeed highly recurrent. We observed the boundaries of known events with greaterclarity and identified previously undescribed lesions, some of which were recurrent. Weprofiled the genomes of CLL cells separated by the surface marker CD38, and foundevidence of distinct subclones of CLL within the same patient. We discuss the potential applications of high resolution CGH analysis in a clinical setting.

Keywords: Genetic modification, polymorphonuclear cell (PMN)

overall_design:
Two-condition experiment, B-CLL vs. PMNs from respective patients. Each done in colour reversal.

85K and the 385K data were generated on the overlapping sets of samples.

first: Alex

last: Krasnitz

first: Jennifer

last: Troge

last: Meth

first: B

last: Lakshmi

first: Jude

middle: T

last: Kendall

first: Boris

last: Yamrom

first: Garrick

last: Alex

first: Deepa

last: Pai

first: Nicholas

last: Navin

first: Lisa

middle: A

last: Hufnagel

first: YoonHa

last: Lee

first: Kerry

last: Cook

first: Steven

last: Allen

first: Kanti

middle: R

last: Rai

first: Rajendra

middle: N

last: Damle

first: Carlo

last: Calissano

last: Chiorazzi

first: Michael

middle: H

last: Wigler

first: Diane

last: Esposito


title: DNA damage response pathways after 30 minutes and 4 hours treatment with 500 μM Na2S in intestinal epithelial cells

status: Public on Oct 10 2008

city: Urbana

first: H

middle: Rex

last: Gaskins

state: IL

address: 1207 W Gregory dr

department: Institute for Genomic Biology

institute: University of Illinois

summary:
Hydrogen sulfide (H2S), a metabolic endproduct of sulfate-reducing bacteria in the large intestine, represents a genotoxic insult to the colonic epithelium, which may also be linked with chronic disorders such as ulcerative colitis and colorectal cancer. The present study defined the early (30 minutes) and late (4 hours) response of non-transformed human intestinal epithelial cells (FHs 74 Int) to sulfide.
We measured the genotoxicity of Na2S using the single cell gel electrophoresis (comet) assay. Changes in gene expression were analyzed after exposure to a genotoxic–but not cytotoxic–concentration of sulfide (500 μM Na2S) using pathway specific quantitative RT-PCR gene arrays.

overall_design: 12 Samples were analysed using the Superarray DNA damage signaling APHS-029B QRT-PCR array. The cells were treated for either 30 minutes or 4 hours with 500 μM Na2S (6 samples for  each time). For each time we had 3 samples as a control (no Na2S) 3 samples treated (500 μM Na2S)

first: Matias

middle: S

last: Attene-Ramos

first: Gerardo

last: Nava

first: Mark

middle: G

last: Muellner

first: Elizabeth

last: Wagner

last: Plewa


title: Differential roles of ROR gamma-t in the development of NKp46+ spleen (LTIL) cells and NKp46+ NK cells in gut and skin

status: Public on Oct 22 2008

city: Dallas

first: Damien

last: Chaussabel

state: TX

address: 3434 Live Oak

institute: Baylor Institute for Immunology Research

summary: Natural killer (NK) cells are NKp46+CD3- lymphocytes that can perform granule-dependent cytotoxicity and produce interferon-gamma, when isolated from blood, lymphoid organs, lung, liver and uterus. Here we identify in dermis, gut lamina propria and cryptopatches, very distinct populations of NKp46+CD3- cells with reduced ability to degranulate and to produce interferon-gamma. In gut, the transcription factor RORgamma-t  and CD127 (IL-7R alpha) defined a novel subset of NKp46+CD3- that is reminiscent of lymphoid tissue inducer (LTi)-like cells. Gut ROR gamma t+NKp46+ cells produced IL-22 in contrast to ROR-gamma t-independent lamina propria and dermis NK cells. These data show that LTi-like cells and NK cells share unanticipated similarities and reveal the heterogeneity of NKp46+CD3- cells in innate immunity, lymphoid organization and local tissue repair.

overall_design: A total of 6 samples were included in this study.  Duplicate samples of natural killer cells from mouse skin, spleen, and intestine were analyzed.


title: Regenerating zebrafish fin fold vs. uncut control

city: Yokohama

first: Atsushi

last: Kawakami

address: 4259 Nagatsuta, Midori-ku

department: Biological Information

country: Japan

institute: Tokyo Institute of Technology

summary:
   Teleost fish have the remarkable ability to regenerate their body parts including heart, spinal cord, and the caudal fin, while many higher vertebrates including us humans have only a limited ability. To facilitate molecular and genetic approaches for regeneration, we previously established an assay using the fin fold of early stage larvae, which regenerate their caudal fin folds as in adult regeneration.
   Here, we performed transcriptional profiling of regenerating larval fin folds and identified genes with differential expression during regeneration. Gene expression profiling of zebrafish larval fin-fold regeneration was performed by comparing amputated fin fold and uncut control.
Keywords: Stress response, injury response.

overall_design:
   Two time points, 18-24 hours post amputation (hpa) and 48 hpa, of regenerating fin fold were analyzed. We performed one replicate per each time point.
   For microarray expression profiling, total RNA was extracted from regenerating and uncut caudal fin folds of AB strain larvae. Tail tissues of 16-24 hpa, 48 hpa, and uncut siblings of the respective stages including 3-5 posterior somite segments were collected on ice. Total RNA was extracted by using TRIzol reagent (Invitrogen, Carlsbad, California, United States) according to the manufacturer’s instruction. The quantity and quality of total RNA were assessed by absorbance at 260 nm and 280 nm and by gel electrophoresis. Approx. 9 μg of total RNA was recovered from ~250 tail tissues at 16-24 hpa or uncut control tissues; and approx. 5 μg, from ~130 tail tissues at 48 hpa or uncut control tissues.
    Probes for microarray analysis were labeled with cy3 (amputated fin fold at 16-24 hpa and uncut control at 48 hpa) or cy5 (uncut control at 16-24 hpa and amputated fin fold at 48 hpa), and used for hybridization.


title: Differential SAGE analysis in Arabidopsis uncovers increased transcriptome complexity in response to low temperature

city: Saskatoon

first: Stephen

middle: James

last: Robinson

state: Saskatchewan

address: 107 Science Place

department: Molecular Genetics

country: Canada

institute: Agriculture and agri-Food Canada

summary:
Five SAGE libraries were generated from A. thaliana leaf tissue collected at time points ranging from 30 minutes to one week of low temperature treatment (4°C).  Over 240,000 high quality SAGE tags, corresponding to 16,629 annotated genes, provided a comprehensive survey of changes in the transcriptome in response to low temperature, from perception of the stress to acquisition of freezing tolerance.
Keywords: SAGE; time course; stress response; cold acclimation; freezing tolerance

overall_design: SAGE libraries were generated from A. thaliana leaf tissue collected after exposure to low temperature (4°C) for 0 minutes, 30 minutes, 2 hours, 2 days and one week, the tags were matched to the Arabidopsis genome and statistical analysis was performed to reveal differential gene expression.

middle: J


title: Specific genomic aberrations in squamous cell lung carcinoma with lymph node or distant metastasis

city: Groningen

first: Mirjam

last: Boelens

address: Hanzeplein 1

country: Netherlands

institute: UMCG

summary: The majority of patients with squamous cell lung cancer (SCC) die because of metastatic disease. The genomic mechanisms underlying this metastatic behaviour are underexposed. We analyzed a cohort of patients with primary squamous cell carcinoma (SCC) using array-based comparative genomic hybridization (aCGH) to identify which genomic aberrations were related metastatic behaviour. The cohort consisted of 34 patients with a follow-up of at least 5 years, including 15 without any metastases, 8 with metastases in regional lymph nodes only, and 11 with metastases exclusively in distant organs within two years after surgery. Common alterations observed in at least 40% of all SCC were gains at 3q13-q29, 5p11-p15, 8q24, 19q13, 20p12-p13, 22q11-q13, and losses at 3p12-p14, 3p24, 4p15, 4q33-q35, 5q14-q23, 5q31-q35, 8p21-p22, 9p21-p24. Amplifications were observed at 2p15-p16, 3q24-q29, 8p11-p12, 8q23-q24, and 12p12, containing candidate oncogenes such as BCL11A, REL, ECT2, PIK3CA, ADAM9, MYC, and KRAS. Gains at 7q36, 8p12, 10q22, 12p12, loss at 4p14 and homozygous deletions at 4q33-q34.1 occurred significantly more frequent in SCC from patients with lymph node metastases. SCC from patients with distant metastases showed a significantly higher frequency of gain at 8q22-q24 and loss of 8p23 and 13q21, and a significantly lower frequency of gain at 2p12 and 2p16 and loss at 11q25 as compared to SCC from patients without metastases. In conclusion, we identified specific genomic aberrations in primary SCC that are related to lymph node or distant metastases. These loci can be further explored for their potential use as predictive or prognostic markers.

overall_design:
PATIENTS AND TISSUE SPECIMENS
We selected a total of 34 patients who presented with centrally located primary squamous cell lung carcinoma (SCC), including 15 patients without lymph node or distant organ metastases within 5 years after surgery; 8 patients with lymph node metastases at the time of surgery, but no distant metastases within 5 years after surgery; 11 patients presenting with distant metastases within 2 years after surgery (median 10 months, range 2-19) but without lymph node metastases. One patient of the ‘no metastases’ group died 3 years after surgery, not of disease. In one patient of the distant metastases group, metastasis presented at 28 months. Patients with both lymph node and distant metastases, and patients who were treated with chemotherapy before or directly after surgery were excluded.

LASER DISSECTION MICROSCOPY
For two SCC samples containing >90% tumour cells in the tissue section (samples T02 and T28), genomic DNA was isolated from the total tissue, in all other cases laser microdissection microscopy (LDM) was performed to obtain pure cell populations. Only vital tumour cells without apparent admixture of inflammatory cells through the tumour fields were selected for LDM. An area of approximately 25x106 μm2 was microdissected from 8μm sections by P.A.L.M. Microlaser Technology system (P.A.L.M., Bernried, Germany). Microdissected cells were immediately collected in SE buffer (75mM NaCl, 0.1mM EDTA) for DNA isolation.

DNA ISOLATION
Genomic DNA was isolated and purified using a standard salt-chloroform extraction protocol. The DNA concentration was measured using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). For each sample, 100 ng genomic DNA was amplified using the BioScore kit (Enzo Life Sciences, Farmingdale, NY). Only SCC cases for which a high yield of amplified DNA was obtained in the amplification reaction were used for aCGH experiments.

ARRAY-BASED CGH (ACGH)
We started with a number of test hybridizations to determine if aCGH profiles of amplified DNA were comparable to those of non-amplified total DNA. We observed consistent profiles for amplified and non-amplified DNA indicating that amplification does not affect the aCGH results (data not shown).
An amount of 600 ng of (amplified) genomic DNA was labeled with Cy3-dUTP (Perkin Elmer, Langen, Germany) using the BioPrima DNA labeling System (Invitrogen Inc., Carlsbad, CA)11. Samples were inversely sex-matched with a normal reference DNA (labeled with Cy5-dUTP) as an internal control for gain or loss at the X chromosome. Hybridizations were included for further analysis only if the calculated ratio for the X-chromosomal BACs were as expected. The design and construction of the BAC-microarray, containing 6465 BACs, has been previously described. The array slides were processed according to the manufacturer’s instructions and as described previously. Arrays were scanned using an Agilent scanner (Agilent, Santa Clara, CA).

first: Klaas

last: Kok

first: Pieter

last: van der Vlies

first: Gerben

last: van der Vries

first: Hannie

last: Sietsma

first: Wim

last: Timens

first: Dirkje

last: Postma

first: Harry

last: Groen

first: Anke

last: van den Berg


title: Characterizing the expression of Pan troglodytes olfactory receptor genes

city: Chicago

first: Omar

last: De la Cruz

address: 5734 S University Ave

department: Department of Statistics

institute: University of Chicago

summary:
We aim, among other things, to characterize olfactory receptor (OR) genes wich are differentially expressed between olfactory epithelium (OE) and other organ tissues, in chimpanzees (Pan troglodytes), and to make comparisons with human data from a previous series using the same platform.
Keywords: Comparative

platform_organism:
Pan troglodytes
Homo sapiens
Canis lupus familiaris
Rattus norvegicus

overall_design: Four OE samples and eight non-OE samples, including two samples of the following tissues: heart, liver, lung, and testis.

first: Xiaohong

last: Zhang

first: Jayant

last: Pinto

first: Dan

middle: L

last: Nicolae

first: Stuart

last: Firestein

first: Yoav

last: Gilad


title: Gene expression profiles of RAW264.7 macrophage cells treated with Paeony root-derived compounds

city: Seoul

first: Hoog-Hee

last: Kim

address: 28 Yeongon-Dong, Chongno-Gu,

department: Cell and Developmental Biology

country: South Korea

institute: Seoul National University

summary:
Paeony root has long been used for its anti-inflammatory effects. In this study, the effects of albiflorin, paeoniflorin, and paeonol, compounds from paeony root, on gene expression profiles were examined in macrophages. The RAW264.7 macrophages were treated with LPS in the presence or absence of albiflorin, paeoniflorin, or paeonol. Global mRNA expression levels were detected by using an oligonucleotide microarray platform covering the mouse whole genome. Our results demonstrate that paeonol has extensive inhibitory effects on the regulation of inflammation-associated gene expression by LPS in macrophages. In addition, the predominant effect of paeonol among the tested compounds suggests that paeonol may be a major ingredient for the anti-inflammatory effect of paeony root.
Keywords: RAW264.7; macrophage; inflammation; LPS; Paeony root components; dose response

overall_design: The RAW264.7 macrophages were treated for 6 h with LPS (1ug/ml) in the presence or absence of albiflorin, paeoniflorin, or paeonol (1, 10, and 100 ug/ml). Cells treated with vehicle (0.1%DMSO) were used as control. Three biological replicates were analyzed for each treatment. Total 33 samples from 11 different treatment regimens were analyzed. The gene expression levels were detected by using an oligonucleotide microarray (Applied Biosystems, cat # MGABI-002) covering the mouse whole genome.

first: Hao

last: Huang

first: Eun

last: Chang

first: Young

middle: K

last: Lee

first: Ju

first: Sam

last: Kang

first: Hong


title: Analysis of growth-phase regulated genes in Streptococcus agalactiae by global transcript profiling

city: Warsaw

first: Izabela

last: Sitkiewicz

address: Chełmska 30/34

department: Epidemiology and Clinical Microbiology

country: Poland

institute: National Medicines Institute

summary: We determined dynamic behavior of S. agalactiae transcriptome during growth in THY medium and detected growth phase regulated genes

overall_design: Three independent cultures (three biological replicates) of S. agalactiae NEM316 strain in rich laboratory medium were sampled at mid log, late log early and late stationary growth phase.

first: James

middle: M

last: Musser


title: Ago2 stable cell line

city: Minneapolis

first: Xiaoxiao

address: 312 Church St. SE

institute: University of Minnesota

summary:
Our genome wide analyses of microRNA expression profiles involve the hybridization of fluorescently labeled RNA samples to custom made, DNA microarrays based on the GAPSII coated slides.  We describe the differences in genome wide miRNA expressions between wild type 293 T  mammalian cells and Ago2 transformed stable cell lines.
Keywords: Expression

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Caenorhabditis elegans
Drosophila melanogaster
Danio rerio
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: Comparing one control and three Ago2-overexpressing stable cell lines (A, B, C). One replicate per array.

first: Yan

last: Zeng


title: Histone H2A.Z and DNA methylation are mutually antagonistic chromatin marks

status: Public on Oct 03 2008

city: Seattle

first: Jorja

last: Henikoff

state: WA

address: 1100 Fairview AV N, A1-162

department: Basic Sciences

institute: Fred Hutchinson Cancer Research Center

type: Methylation profiling by genome tiling array

summary:
Eukaryotic chromatin is separated into functional domains differentiated by posttranslational histone modifications, histone variants, and DNA methylation.  Methylation is associated with repression of transcriptional initiation in plants and animals, and is frequently found in transposable elements. Proper methylation patterns are critical for eukaryotic development, and aberrant methylation-induced silencing of tumor suppressor genes is a common feature of human cancer.  In contrast to methylation, the histone variant H2A.Z is preferentially deposited by the Swr1 ATPase complex near 5' ends of genes where it promotes transcriptional competence.  How DNA methylation and H2A.Z influence transcription remains largely unknown.  Here we show that in the plant Arabidopsis thaliana, regions of DNA methylation are quantitatively deficient in H2A.Z.  Exclusion of H2A.Z is seen at sites of DNA methylation in the bodies of actively transcribed genes and in methylated transposons.  Mutation of the MET1 DNA methyltransferase, which causes both losses and gains of DNA methylation, engenders opposite changes in H2A.Z deposition, while mutation of the PIE1 subunit of the Swr1 complex that deposits H2A.Z17 leads to genome-wide hypermethylation.  Our findings indicate that DNA methylation can influence chromatin structure and effect gene silencing by excluding H2A.Z, and that H2A.Z protects genes from DNA methylation.

Keywords: Affinity-purification on microarray

overall_design: All experiments were done using two channels per chip. DNA methylation experiments compared immunoprecipitated, methylated DNA to control genomic DNA. H2A.Z experiments compared whole micrococcal nuclease-treated affinity-purified chromatin to input chromatin used for affinity purification. Affinity purification was performed using either biotin-tagged H2A.Z, pulled down using streptavidin, or endogenous H2A.Z pulled down using an anti-H2A.Z antibody.

first: Zilberman

last: Daniel

first: Henikoff

last: Steven


title: Characterization of the SigB-PrfA regulatory network in Listeria monocytogenes

city: Ithaca

first: Teresa

last: Bergholz

address: 405 Stocking Hall

department: Food Science

institute: Cornell University

summary:
Listeria monocytogenes SigB and PrfA are pleiotropic regulators of stress response and virulence gene expression, which have been shown to co-regulate genes in L. monocytogenes.  We performed whole genome transcriptional profiling in the presence of PrfA* and active SigB, to identify the overlaps between the PrfA virulence regulon and the SigB stress response regulon. In L. monocytogenes, the PrfA* allele contributes to the activation of virulence genes to a level comparable to that of intracellular growing L. monocytogenes. Our results showed that the core PrfA regulon consists of 12 genes previously described as PrfA regulated.  Furthermore, we found that the role of SigB during virulence gene regulation changes, dependent on the presence or absence of PrfA*. In the absence of PrfA*, SigB activated the transcription of virulence genes such as inlA and inlB. In the presence of PrfA*, SigB negatively influenced the transcription of genes in the PrfA core regulon.  The observed effect of SigB on the transcript level of PrfA regulated genes was shown to reduce the cytotoxic effect of the PrfA* allele in HepG-2 cells.  Our results indicate that the SigB-PrfA regulatory network is important for the adjustment of virulence gene transcription to ensure L. monocytogenes success as an intracellular pathogen.
Keywords: comparison of gene expression of regulatory mutants

platform_organism:
Listeria monocytogenes
Listeria innocua

overall_design: The experimental design included 4 mutant strains of L. monocytogenes 10403S (PrfA*, delta prfA, delta prfA delta sigB, and PrfA* delta sigB), of which cDNA generated from 4 biological replicates were hybridized in all possible pairwise comparisons.  Data were analyzed using a one way ANOVA in R/MAANOVA to determine significant differences in gene expression among the different strains.  A two way ANOVA implemented in R/MAANOVA determined significant differences in gene expression due to the presence or absence of SigB and PrfA.

first: Juliane

last: Ollinger

first: Martin

last: Wiedmann

first: Kathryn

last: Boor


title: MR-1 nitrate sample to fumarate sample

city: Hangzhou

first: haichun

last: gao

address: 388 Yuhangtang Rd.

country: China

institute: Zhejiang University

summary:
One of the most striking observations in the S. oneidensis genome regarding nitrate respiration is that napC is missing and nrfBCD is degenerated. To gain insights into candidate genes encoding the protein(s) in place of NapC and NrfBCD function, i.e., delivering electrons to NapA and NrfA, transcriptional profiling was carried out using the S. oneidensis whole-genome cDNA microarray. Cells of MR-1 grown on nitrate or fumarate under anaerobic conditions were sampled at the exponential phase for the analysis. The quality of the array data was statistically assessed using the method reported previously.
Keywords: Compararive microarray study

overall_design: Compare transcriptional profiles of MR-1 grown on nitrate and Fumarate aiming at finding the electron transfer proteins to NapA and NrfA.

first: Haichun

last: Gao


title: A method of stripping custom microarrays

last: Zhang

summary:
Our genome wide analyses of microRNA expression profiles involve the hybridization of fluorescently labeled RNA samples to custom made, DNA microarrays based on the GAPSII coated slides.  We describe a simple and effective method to regenerate such custom microarrays.  Our protocol entails the use of a very low concentration of sodium hydroxide in a low salt buffer to strip RNA molecules from the arrays.  The solution is also capable of removing DNA molecules hybridized to the slides, while preserving the slide coating and printed DNA probes.  Slides can be stripped and reused at least twice without significantly sacrificing data quality.
Keywords: expression study, new vs. stripped array comparison

overall_design: There are two stripping conditions in this study (1mM NaOH and 2mM NaOH in SSC buffer). For 1mM NaOH treatment, new, once-, twice-, and triple-stripped arrays are studies. For 2mM NaOH treatment, new and once-stripped arrays are measured.


title: Sustained R848 treatment of Mice

status: Public on Sep 30 2008

city: Zuerich

first: Stefan

last: Baenziger

address: Raemisstrasse 100

department: Department of internal Medicine

institute: University Hospital Zurich

summary:
PCR Array Profiling - R848 does not induce TLR-signaling related genes in TLR7-/- mice.
Aim: To corroborate TLR7-dependency of R848 in mice

"Triggering TLR7 in mice induces immune activation and lymphoid system disruption, resembling HIV-mediated pathology", Baenziger et al. Blood 2008

overall_design:
Mice (n=3/sample) were treated with 1mg/kg/d R848 or PBS (Mock) for 7 days. Thereafter, spleen was removed, mRNA extracted from whole splenic tissue and subsequently subjected to microarray RT² Profiler PCR array (Bioscience Corporation) on a Biorad iCycler. The PCR Array system examines the expression of 84 genes related to TLR-mediated signal transduction.

Raw Data (i.e., Ct values) are shown. Ct values >35 or blank was set to 35.
The supplementary file show data generated by real-time quantitative PCR normalized to Hypoxanthine guanine phosphoribosyl transferase 1, Heat shock protein 90kDa alpha, and Glyceraldehyde-3-phosphate dehydrogenase, displayed as fold difference in the expression of each gene between R848- and Mock-treated samples. Two independent experiments are summarized for wild-type mice. TLR7- and IFNAR-/- animals were tested once.


title: Effect of dietary immunostimulation in the portals of entry of rainbow trout after LPS challenge

city: Barcelona

first: Joan

last: Balasch

address: UAB Bellaterra faculty of Biosciences

department: Dpt. Cell Biology, Physiology and Immunology

country: Spain

institute: Universitat Autonoma Barcelona

summary:
Rainbow trout (Oncorhynchus mykiss) were fed during 4 weeks with either a control diet or an immunostimulant diet and then injected with LPS to investigate the effect of dietary immunostimulation in the portals of entry, intestine and gills, using a salmonid-specific microarray platform enriched with immune-related genes. IS-diet feeding significantly changed transcriptomic expression profiles in response to LPS:  significant changes in genes and functional GO categories related to remodeling processes and antigen presentation were different for both diets. The results revealed that one of the main effects of IS-diets in trout is the increase of genes involved in antigen recognition and in adaptive immunity.
Keywords: gills, intestine, immunostimulats, transcriptomic response, trout

overall_design:
Adult rainbow trout (Oncorhynchus mykiss) were randomly distributed in four fibreglass tanks (25 fish per tank). During the feeding trial, trouts were hand-fed once a day with either the control diet or the immunodiet, in duplicate tanks, following manufacturer’s indication of 1 g food/fish for 4 weeks. After the 4 weeks, 5 fish were injected with LPS and 5 fish were injected with PBS. After 24h, samples were taken for microarray analysis. Tissues were removed for RNA extraction and frozen in liquid nitrogen, then stored at -80 ºC. Total RNA was extracted from the organs using 1 ml of Tri Reagent (Sigma) per 100 mg of tissue, following the manufacturer’s instructions and quantity and integrity was analyzed by Experion RNA StdSens Analysis Kit (Bio-Rad). The design of the microarray posses 1818 genes printed in six replicates each, including random clones from common and subtracted cDNA libraries which were compared with known vertebrate proteins using blastx. The platform was enriched in immune-related genes.
RNA was pooled within treatments (n=5), and 15 µg of control and test were labeled with Cy3- and Cy5-dCTP (Amersham Pharmacia) using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) primer (Promega), and cDNA was purified with Microcon YM30 (Millipore). Dye swap method was used, therefore each sample was hybridized to two microarrays. For the first slide, test and control cDNA were labeled with Cy5 and Cy3 respectively, and for the second array dye assignments were reversed. Scanning was performed with Axon scanner 4000B and images were processed with GenePix Pro 6.0. After subtraction of mean background, and LOWESS normalization was performed. To assess differential expression of genes, the normalized log intensity ratios were analyzed with Student’s t-test (p<0.01) and genes were ranked by log(p-level). The Bayesian modification to the false discovery rate (FDR) was used to correct for multiple comparison tests, estimating the q-value for the set of differentially expressed genes. The functional categories of Gene Ontology were compared with regulated genes (p<0.01) by the sums of ranks (Student’s t-test, p<0.05) and the statistical significance of over represented functional categories was assessed using the Yates correction to Chi square test (corrected p<0.05).

first: Carmen

last: Doñate

first: Simon

last: MacKenzie


title: Serial hepatic gene expression profiling in Angus steers during feed restriction and realimentation

city: Beltsville

first: Erin

middle: E

last: Connor

state: MD

address: 10300 Baltimore Avenue

department: Animal and Natural Resources Institute

institute: USDA-ARS, BARC

summary: Growing ruminants maintained under dietary restriction for extended periods will exhibit compensatory growth when reverted to ad libitum feeding. This period of compensatory growth is associated with increased feed efficiency, lower basal energy requirements, and changes in circulating concentrations of metabolic hormones. To identify genetic mechanisms contributing to these physiological changes, 8 month-old steers were fed either ad libitum (control; n = 6) or 60-70% of intake of control animals (feed-restricted; n=6) for a period of 12 weeks. All steers were then fed ad libitum for the remaining 8 weeks of the experiment (realimentation period). Liver was biopsied from each animal at days -14, +1 and +14 relative to realimentation for RNA extraction and gene expression analysis by microarray hybridization.

overall_design: Steers were assigned randomly to one of two treatment groups, control or feed-restricted, and housed indoors in individual pens. Steers were acclimated to their pens for 5 d prior to starting the experimental treatments. Feed was offered once daily between 0630 and 0930 and orts from the previous day's feeding were collected and weighed to estimate actual intake. Control animals were fed ad libitum throughout the 20-wk experimental period. Feed-restricted steers were offered 60-70% of intake of control animals for 12 wks to target a limited rate of gain of approximately 0.5 kg/d. Restricted steers were then fed ad libitum for the remaining 8 wks of the experiment (realimentation period). During the first 3 d of realimentation, feed offered to both treatment groups was divided into two equal rations to gradually adjust restricted animals to full intake. Water was offered ad libitum throughout the experimental period. Approximately 200 mg of liver tissue was collected from each steer by needle biopsy using a Tru-Cut biopsy needle at -14, +1, +14 d relative to realimentation. Liver samples were immediately frozen in liquid nitrogen and stored at -80C until RNA isolation. Total RNA was isolated from 36 liver samples using TRIZOL Reagent (Invitrogen Corp., Carlsbad, CA). Samples were DNase-treated using the TURBO DNA-free kit (Ambion, Inc., Austin, TX) according to manufacturer’s instructions, followed by column purification using the RNeasy Mini Kit (Qiagen, Valencia, CA). Quality and concentration of RNA were assessed using a 2100 Bioanlayzer (Agilent Technologies, Palo Alto, CA) and ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Probe labeling, hybridizations of probes to the oligo microarray, and array scanning were performed by the Roche NimbleGen Systems, Inc. Microarray Core Facility in Reykjavik, Iceland according to standard procedures (Madison, WI; http://www.nimblegen.com).

first: Stanislaw

last: Kahl

first: Theodore

last: Elsasser

first: Robert

last: Li

first: Scott

last: Barao


title: Brain_22A-Scrapie_End-point_PR04-39-to-46_using_GPL7118

city: Winnipeg

first: Reuben

last: Saba

state: Manitoba

address: 1015 Arligton Street

department: Medical Microbiology

institute: University of Manitoba: Public Health agency of Canada

summary: Examining the transcriptional change in whole mouse (VM) brain miRNA expression during prion (22A) induced neurodegeneration in order to identify miRNAs that are differentially expressed. MicroRNAs (miRNAs) are small, non-coding RNA molecules which are emerging as key regulators of numerous cellular processes.  Compelling evidence links miRNAs to the control of neuronal development and differentiation, however, little is known about their role in neurodegeneration. De-regulation of a unique subset of miRNAs may suggest a conserved, disease-specific pattern of differentially expressed miRNAs is associated with prion–induced neurodegeneration.

overall_design: MiRNA extracted from the whole brains of 3 VM mice treated with mouse-adapted 22A scrapie strain were analyzed on GPL7118 (array was divided into top and bottom) for differential expression. Age-matched, PBS treated control mice were used for comparison (competitive hybridization). A technical replicate for each mouse was performed as well as a technical replicate for each of the dye-swaps.


title: Brain_22A-Scrapie_End-point_PR06-11-to-14_using_GPL7121

overall_design: MiRNA extracted from the whole brains of 4 VM mice treated with mouse-adapted 22A scrapie strain were analyzed on GPL7121 (array was divided into top and bottom) for differential expression. Age-matched, PBS treated control mice were used for comparison (competitive hybridization). A technical replicate for each mouse was performed as well as a technical replicate for each of the dye-swaps.


title: Transcript profiling of the pea shoot apical meristem highlights processes underlying its function and maintenance

city: Parkville

first: Chui

last: WONG

state: Victoria

address: University of Melbourne

department: Land and Food

country: Australia

institute: University of Melbourne

summary:
Plant growth is the result of cell proliferation in the meristems, which requires a dynamic balance between the formation of new tissue and the maintenance of a set of undifferentiated stem cells. There is much that remains unknown about this vital developmental process. In this study, we have reported the generation of 2735 ESTs from three cDNA libraries derived from dissected garden pea (Pisum sativum cv Torsdag) shoot apical meristems. Clustering analysis of the resulting sequences gave rise to 1686 non-redundant ESTs. The unique sequences generated together with 500 other pea transcripts randomly selected from the GenBank pea protein database have been utilized to construct a 2K oligonucleotide array. Using this pea array, we have obtained the transcript profiles of pea shoot apical meristems in comparison to non-apical-meristem tissues. A total of 181 or 174 transcripts were identified to be significantly up- or down-regulated in the pea shoot apical meristem, respectively. As expected, close to 61% of the transcripts down-regulated in the shoot apical mersitem are those retrieved from the public database, whereas sequences from the same source only made up 12% of the genes that were expressed at higher levels in SAMs. This revelation highlights the under-representation of transcripts from the meristmatic tissues in the current public protein database. Manual inspection of the list of up-regulated transcripts reveals the presence of sequences predicted to encode products associated with cell division and proliferation, epigenetic regulation, auxin-mediated responses and miRNA regulation. Similar genes have been implicated in the regulation of plant stem cell activity. Taken together, our EST collection as well as the microarray data provides useful starting points for more in depth analysis of the meristem function and maintenance.
Keywords: shoot apical meristem transcript profiling

overall_design:
Total RNA was extracted from dissected SAM (approximately 80 SAMs per extraction) or other plant parts (primary stem, primary roots and mature leaves) using Qiagen RNeasy Mini Kit. Four independent tissue collections and RNA extractions (designated A, B, C and D) were performed for each of the microarray hybridization experiment.The Cy5- or Cy3-labelled cDNA was then hybridized to different sector of the chip according to a balanced block design dual label experiment scheme (Cochran & Cox, 1992):
Sector 1: Cy3-SAM A vs Cy5-NM A
Sector 2: Cy5-SAM B vs Cy3-NM B
Sector 3: Cy3-SAM C vs Cy5-NM C
Sector 4: Cy5-SAM D vs Cy3-NM D

last: Wong

first: Harald

last: Ottenhof

first: Prem

last: Bhalla

first: Mohan

middle: B

last: Singh


title: Progression of heart failure induced by tachycardia

city: Baltimore

first: Zhong

last: Gao

address: Ross 844 720 Rutland Avenue

institute: Johns Hopkins University

summary:
Analysis of changes of gene expression profiles in the left ventricle (LV) during the progression of heart failure (HF) in the canine tachypacing induced HF model. Gene expression profiling was performed on samples collected at different time points representing various stages of LV dysfunction, i.e. tachypaced for 3 days (Day-3), 1 week (Week-1), 2 weeks (Week-2), 3-4 weeks (End stage), and unpaced controls (Day-0). Results provide insights into molecular mechanisms of heart failure progression.
Keywords: time course

overall_design: This work aims to study changes of gene expression profiles in the left ventricle (LV) during the progression of heart failure (HF) in the canine tachypacing induced HF model. LV transmural tissue sections were collected at different time points after initiating rapid pacing representing various stages of LV dysfunction, i.e. tachypaced for 3 days (Day-3), 1 week (Week-1), 2 weeks (Week-2), 3-4 weeks (End stage), and unpaced controls (Day-0). As biological replicates, tissues were collected separately from 3 individual dogs at each of the time points. In total we collected 15 tissue samples for all 5 time points. Three RNA samples were independently isolated from each of the 15 tissue samples as technical replicates. The resulting 45 RNA samples were used for microarray experiment. Affymetrix gene expression profiles of the 45 RNA samples were used for all data analysis in this study.

first: Deborah

last: DiSilvestre

first: Fadi

last: Akar

first: Yanli

last: Tian

first: Antti

last: Tanskanen

first: David

last: Kass

first: Raimond

last: Winslow

first: Gordon

middle: F

last: Tomaselli


title: Regulation of the early embryonic RPE (retinal pigment epithelium) transcriptome by the neural retina

city: New Haven

first: Lawrence

last: Rizzolo

state: CT

address: 333 Cedar St

department: Dept of Surgery

institute: Yale University

summary:
Purpose: The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier. Primary cultures of RPE can model the barrier, but are very sensitive to culture conditions.  We examined how the neural retina regulates the RPE transcriptome in a culture model of embryonic development. Attention focused on the tight junctional genes essential for barrier function.

Methods: Chick RPE from embryonic day 7 (E7) or embryonic day 14 (E14) was cultured on filters in a serum free medium. Media conditioned by organ culture of neural retinas was applied to the apical surface of the cultured RPE. Fetal bovine serum (2%) was included in some experiments.  When the transepithelial electrical resistance (TER) reached a plateau, total RNA was isolated to probe the chick genome on Affymetrix microarrays. The expression tight junctional mRNAs was confirmed by real-time PCR and immunoblotting the protein.

Results: The TER was slightly increased by serum, but increased 2-3X by retinal conditioned medium.  Serum diminished the effect of retinal conditioned medium.  In basal conditions, 86% of the transcriptome expressed during development in vivo was also expressed in culture. Approximately 5% of the transcriptome expressed in culture was absent in vivo.  E14 retinal conditioned medium affected 15% of the transcriptome in E7 cultures (24% if serum was included), but only 1.9% in E14 cultures (12% with serum). Examination of 610 genes important for RPE function revealed that mRNAs for 17% were regulated by retinal conditioned medium alone in E7 cultures, compared to 6.2% for E14. For tight junctions, retinal conditioned medium had the most affect on members of the claudin family. These results were confirmed by quantitative real-time PCR. Besides regulating mRNA levels, immunoblotting and immunocytochemistry suggested additional mechanisms whereby retinal secretions regulated protein expression and localization.

Conclusions: Gene expression in primary cultures of embryonic RPE resembled the native tissue, but expression, and differentiation, is improved when elements of the normal extracellular environment are replicated in culture.  For a small group of proteins, retinal secretions were required to maintain in vivo-like expression in culture.  Albeit insufficient, retinal secretions promoted differentiation of immature RPE and helped maintain the properties of more mature RPE.

Keywords: Tissue interactions, retina, retinal pigment epithelium, blood-retinal barrier, tight junctions

overall_design: RPE were isolated from chicken embryos on embryonic day 7 or 14 and cultured, as described <Rahner C, Fukuhara M, Peng S, Kojima S, Rizzolo LJ. The apical and basal environments of the retinal pigment epithelium regulate the maturation of tight junctions during development. J Cell Sci. 2004;117:3307-18>. E7 is when tight junctions of the RPE begin to form; E14 is when tight junctions achieve their mature morphological appearance in vivo. The cells were cultured in medium that contains or lacks E14 retinal conditioned medium. Because serum inhibits the effect of retinal conditioned medium, serum was added to some cultures, resulting in 4 culture conditions for each age. To isolate total RNA, the RNeasy Protect kit (Qiagen) was used according to the manufacturer's protocols. For each culture, 3 independent preparations were used for analysis on Affymetrix microarrays of the chicken genome (Santa Clara, CA). The quality of the total RNA was assessed by the Keck Center, Yale University using formamide gels and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA)

first: Ru

last: Sun

first: Matthew

last: Weitzman

first: Xiang

last: Chen

first: Heping

last: Zhang


title: Chromosomal clustering of genes in absent, small, or homeotic discs 2 (ash2) mutant larvae

status: Public on Sep 26 2008

first: Sergi

last: Beltran

address: Baldiri Reixac 10

department: Serveis Cientificotècnics

institute: Universitat de Barcelona

summary:
We have performed a systematic examination of genome-wide expression profiles using microarrays to reconstruct a global picture of the trx regulatory gene network in D. melanogaster. Using computational analysis of the microarray data, we have identified 25 clusters of genes potentially regulated by trx, most of them being located in the arm L of chromosome 3. Functional analysis revealed that most clusters are enriched in structural proteins involved in cuticle formation, which are preferently expressed in salivary glands. The same organization in clusters was observed in the transcriptomes of four independent experiments, being a distinctive feature of the regulatory networks of trx and other chromatin regulators (ASH2, NURF, Pc, ASH1). We have also identified many of these clusters in D. simulans, D. yakuba, D. pseudoobscura and partially in A. gambiae.
Keywords: loss of function analysis

overall_design: Total RNA from w1118;+;+ larvae was pooled and used as a common reference in four microarrays against w1118;+;ash2I1 total RNA coming from two different extractions to take biological differences into account. Amplified RNA (aRNA) was obtained with the Amino-Allyl Messageamp II aRNA Amplification Kit (Ambion, Inc) from w1118;+;+ and w1118;+;ash2I1 prior to hybridization.

first: Miguel

last: Pignatelli

first: Enrique

last: Blanco

first: Adrià

last: Punset

first: Florenci

last: Serras

first: Roderic

last: Guigó

first: Montserrat

last: Corominas


title: Chromosomal clustering of genes in trithorax (trx) mutant larvae

overall_design: Total RNA from w1118;+;+ larvae was pooled and used as a common reference in four microarrays against w1118;+;trxE3/trxB11 total RNA coming from two different extractions to take biological differences into account. Two amplifications from w1118;+;+ and one from each w1118;+;trxE3/trxB11 replicate were performed with the Amino-Allyl Messageamp II aRNA Amplification Kit (Ambion, Inc) to obtain amplified RNA (aRNA). The two arrays from each replicate pair were hybridized with the same amplified RNA from sample and common reference but with dyes (Cy3 and Cy5 from Amersham, Inc) swapped to take dye-bias into account.


title: SuperSAGE: The drought stress-responsive transcriptome of chickpea roots

city: Dijon

first: Carlos

middle: Mauricio

last: Molina

state: Bourgogne

address: 17 Rue Sully

department: UMRLEG

country: France

institute: INRA

summary:
Background
Drought is the major constraint to increase yield in chickpea (Cicer arietinum). Improving drought tolerance is therefore of outmost importance for breeding. However, the complexity of the trait allowed only marginal progress. A solution to the current stagnation is expected from innovative molecular tools such as transcriptome analyses providing insight into stress-related gene activity and, combined with molecular markers and expression (e)QTL mapping, may accelerate knowledge-based breeding. SuperSAGE, an improved version of the serial analysis of gene expression (SAGE) technique, currently is the most advanced tool for transcriptome analysis. SuperSAGE generates genome-wide, high-quality transcription profiles from any eukaryote. The method produces 26bp long fragments (SuperTags26bp tags) from defined positions in cDNAs, providing sufficient sequence information to unambiguously characterize the mRNAs. Further, SuperSAGE Tags may be immediately used to produce so called SuperTag microarrays and probes for real-time-PCR thereby overcoming the lack of genomic tools in non-model organisms.
Results
We applied SuperSAGE to the analysis of gene expression in chickpea roots in response to drought. To this end, we sequenced 80,238 26bp SuperTtags representing 17,493 unique transcripts (UniTags) from drought-stressed and non-stressed control roots. A total of 7,532 (43%) UniTags were more than 2.7-fold differentially expressed , and 880 (5.0%) were regulated more than 8-fold upon stress. Their large size enabled the unambiguous annotation of 2,798 (16.3%) UniTags to genes or proteins in public data bases and thus to stress-response processes. We designed a microSuperTag array carrying 3,000 of these SuperTags26bp tags. The chip data confirmed the SuperSAGE results in 79 % of cases whereas RT-PCR confirmed the SuperSAGE data in all cases.
Conclusions
This study represents the most comprehensive analysis of the drought-response transcriptome of chickpea available to date. It demonstrates that - inter alias - signal transduction, transcription regulation, osmolyte accumulation, chromosome organization, and ROS scavenging undergo strong transcriptional remodelling in chickpea roots already 6h after drought stress. Certain transcript isoforms characterizing these processes are potential targets for breeding for drought tolerance. We demonstrate that these can be easily accessed by micro-arrays and RT-PCR assays readily produced downstream of SuperSAGE. Our study proves that SuperSAGE has a good potential is best suited for molecular breeding also in non-model crops.

overall_design:
Chickpea drought-stressed root material was used to develop 2 SuperSAGE librareis
Control: Normal greenhouse conditions (3-5 weeks old plants)
Stress: Normal greenhouse conditions + 6h dehydration (3-5 weeks old plants)
Biological replicas and confirmation: microarray spotting and qRT-PCR

first: Björn

last: Rotter

first: Ralph

last: Horres

first: Sripada

last: Udupa

first: Bert

last: Besser

first: Luis

last: Bellarmino

first: Michael

last: Baum

first: Hideo

last: Matsumura

first: Ryohei

last: Terauchi

first: Günter

last: Kahl

first: Peter

last: Winter


title: Effect of treatment with H2O2 or DPTA-NONOate on E. histolytica

city: Stanford

first: Gretchen

middle: Marie

last: Ehrenkaufer

address: Grant S-143, 300 Pasteur Dr.

department: Microbiology and Immunology

institute: Stanford Uninversity

summary: E. histolytica of strains HM1:IMSS (virulent) and Rahman (avirulent) were examined for response to oxidative and nitrosative stress.

overall_design: Response to oxidative stress in E. histolytica was assayed by treating Eh (HM1:IMSS) and Eh (Rahman) with 1mM H2O2 for 60 minutes or 90 minutes and comparing transcription to parasites grown in TYI. In addition, response to nitosative stress was assayed in Eh (HM1:IMSS) by treatment for 60 minutes with 200 µM DPTA-NONOate.

first: João

last: Vicente

first: Ligia

last: Saraiva

first: Miguel

last: Teixeira

first: Upinder

last: Singh


title: p53- repressed miRNAs are involved with E2F in a feed forward loop promoting proliferation

status: Public on Sep 25 2008

city: Bethesda

first: Giang Huong

last: Nguyen

address: 37 Convent Drive

department: CCR

institute: NCI

summary:
There is a distinctive miRNA expression profile between early (young) and replicative senescence (old).
We observed a cluster of miRNAs that were repressed upon senescence in both MRC-5 and WI-38 fibroblast cells.

platform_organism:
Homo sapiens
Mus musculus

overall_design: miRNAs profiling in young and senescence human emryonic fibroblasts (MRC-5 and WI-38)

first: Giang


title: Gene expression profiling of human anaplastic thyroid carcinoma cells by microarray

city: Busan

first: Young

middle: Hyun

last: Yoo

address: 3-1 Dongdaesin-Dong

department: Dept Anatomy and Cell Biology

institute: Dong-A Univ Coll Med

summary: In this study, we profiled the change of gene expression by p53 knock-down on anaplastic thyroid carcinoma (ATC) cell line, KAT-18 cells using Applied Biosystem's Microarrays. When compared with the untreated control KAT-18 cells, 2,926 genes were found to have altered expression levels of 2-fold or more in cells treated with antisense p53 oligonucleotide. Microarray analysis revealed that p53 knock-down modified the expression of numerous apoptosis-related genes. Among those genes, 33 apoptosis pathway activators and 20 apoptosis pathway repressors were identified.

overall_design: We analyzed human thyroid carcinoma cell (KAT18) on Applied Biosystem Human 1700 Chip Arrays. There were control and test sample, respectively, KAT18 untreated and treated antisense p53.

first: TaeHyun

last: Kim

first: SangRyull

last: Lee

first: JeeHyun

last: Rho

first: WolSoon

last: Jo

first: YoungHwa

last: Soung

first: Sung-Heun


title: The TSC/mTOR pathway regulates the innate inflammatory response

city: Vienna

first: Thomas

last: Weichhart

address: Waehringerstraße 10

department: Center for Pathobiochemistry and Genetics

institute: Medical University of Vienna

summary:
The TSC/mTOR (tuberous sclerosis complex/mammalian target of rapamycin) pathway has a central role in cell growth and is involved in human tumorigenesis. Here, we demonstrate an unexpected role of TSC2 and mTOR in regulating key inflammatory cytokines in monocytes, macrophages, and dendritic cells after bacterial stimulation. mTOR deficiency promoted IL-12/IL-23 and blocked IL-10 production via the transcription factor NF-kB. Conversely, loss of TSC2, a key negative regulator of mTOR, led to reduced NF-kB activity, limited IL-12 but enhanced IL-10 production. Transcriptional profiling demonstrated that mTOR additionally regulated many mediators important for inflammation and immunoregulation including PD-L1, CCR5, CCL22, and MCP-1. mTOR inhibition in vivo rescued susceptible mice from a lethal Listeria monocytogenes infection by modulating IL-12/IL-10 production. These data identify the TSC2/mTOR pathway as a novel pathway in innate immune responses by controlling NF-kB with profound clinical implications for infectious diseases, cancer, or transplantation.
Keywords: inflammatory response of monocytes to LPS and rapamycin

overall_design: 107 CD14+ monocytes from four different donors were stimulated with 100 ng/ml LPS with or without 100 nM rapamycin for 4 hours. Cells were harvested, washed and pelleted and frozen at –80° C. Sample preparation and hybridization to a PIQORTM Immunology Microarray Human Antisense (quadrupled cDNA fragments for 1070 genes) was performed at the Miltenyi Microarray Service Unit. In brief, RNA was extracted with the TRIzol method (Sigma-Aldrich) and 1mg of total RNA was linear amplified by T7 polymerase. Total RNA from the LPS treated cells were Cy3 labeled, LPS and rapamycin treated cells were Cy5 labeled; both samples of a donor were hybridized on a single microarray. Only genes with signal intensities >2-fold above average background in one of the two channels were regarded as expressed.

first: Giuseppina

last: Costantino

first: Marko

last: Poglitsch

first: Maximillian

last: Zeyda

first: Margit

last: Rosner

first: Karl

last: Stuhlmeier

last: Stulnig

first: Walter

last: Hörl

first: Markus

last: Hengstschläger

first: Mathias

last: Müller

first: Marcus

middle: D

last: Säemann


title: Maize gene expression during infection with Ustilago maydis strain SG200Dpep1

city: Marburg

first: Gunther

last: Doehlemann

state: Hessen

address: Karl von Frisch Str.

department: Organismic Interactions

country: Germany

institute: Max Planck Institue for Terrestiral Microbiology

summary: The basidiomycete Ustilago maydis causes smut disease in maize. Colonization of the host plant is initiated by direct penetration of cuticle and cell wall of maize epidermis cells. The invading hyphae are surrounded by the plant plasma membrane and proliferate within the plant tissue. We identified a novel secreted protein, termed Pep1. Disruption mutants of pep1 are not affected in saprophytic growth and develop normal infection structures. However, Δpep1 mutants fail to penetrate the epidermal cell wall and elicit a strong plant defense response. Using Affymetrix maize arrays we identified about 110 plant genes which are differentially regulated in Δpep1 and wild type infections during the penetration stage.

overall_design: In three independent experiments plants were infected with the strain SG200Dpep1 which is derived from the solopathogenic U. maydis strain SG200. Samples from infected leaves were taken at  24 hours post infection. Samples were treated under the same conditions as described previously.

first: Regine

last: Kahmann


title: Global transcription profiles of Trichophyton rubrum in response to Flucytosine

status: Public on Sep 23 2008

city: Beijing

first: Qi

last: Jin

state: Beijing

address: BDA Rong Jing east street No.6, 3rd floor

department: National Institute for Viral Disease Control and Prevention

institute: Chinese Center for Disease Control and Prevention

summary:
A cDNA microarray was constructed from the expressed sequence tags (ESTs) of different developmental stages, and transcriptional profiles of the responses to 5-Flucytosine were determined.

Keywords: Expression profiling by array

overall_design: The expression profiles of Trichophyton rubrum treated with 5-Flucytosine were compared to those of Trichophyton rubrum without drug. Each treatment has three replicates.

first: Rong

last: Zhao

first: Youjiang

last: Diao

first: Jian

last: Yang


title: PreA overexpression in Salmonella typhimurium

city: Irvine

first: Michael

last: McClelland

address: 132 Med Surge I

department: Microbiology & Molecular Genetics

institute: University of California, Irvine

summary: Comparisons of overexpression of PreA (in wt or preAB mutant Salmonella) to Salmonella lacking preA or preAB in late log phase growth in LB

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium

overall_design: preA was externally supplied to wt or preAB mutant Salmonella typhimurium on a pBAD18 plasmid, which was then induced by 10mM arabinose and grown to late log phase (OD0.74). Transcription profiles were compared with those obtained from preA or preAB mutant Salmonella harboring pBAD18 only as vector control.

first: Max

last: Merighi

first: Alicia

last: Septer

first: Steffen

last: Porwollik

first: Pui

last: Cheng


title: microRNA expression profiling in human medulloblastoma

city: Rome

first: Enrico

last: De Smaele

state: Rome

address: viale Regina Elena 324

department: Experimental Medicine

country: Italy

institute: Sapienza University of Rome

summary:
A high throughput microRNA expression profiles was performed in human primary medulloblastoma specimens and relative controls to investigate microRNA involvement in medulloblastoma carcinogenesis.
Medulloblastoma is an aggressive brain malignancy with high incidence in childhood. MicroRNA expression analysis has emerged as a powerful tool to identify candidate molecules playing an important role in a large number of malignancies. A high throughput microRNA expression profiles was performed in human primary medulloblastoma specimens and relative controls to investigate microRNA involvement in medulloblastoma carcinogenesis.

Keywords: RT-PCR

overall_design:
In the study presented here we analized  expression of a panel of miRNAs in 14 cerebellum controls (9 adult and 5 fetal samples) and in 34 medulloblastoma samples
Q-PCR measurement of miRNA expression levels

first: Elisabetta

last: Ferretti

first: Agnese

last: Po

first: Alberto

last: Gulino


title: Cell cycle- and chaperone-mediated regulation of H3K56ac incorporation in yeast

city: Worcester

first: Oliver

last: Rando

state: MA

address: 364 Plantation St.

institute: UMass Medical School

type: Genome binding/occupancy profiling by genome tiling array

summary: Acetylation of histone H3 lysine 56 is a covalent modification best-known as a mark of newly-replicated chromatin, but has also been linked to replication-independent histone replacement.  Here, we measured H3K56ac levels at single-nucleosome resolution in asynchronously growing yeast cultures, as well as in yeast proceeding synchronously through the cell cycle. We developed a quantitative model of H3K56ac kinetics, which shows that H3K56ac is largely explained by the genomic replication timing and the turnover rate of each nucleosome, suggesting that cell cycle profiles of H3K56ac should reveal most first-time nucleosome incorporation events. However, since the deacetylases Hst3/4 prevent use of H3K56ac as a marker for histone deposition during M phase, we also directly measured M phase histone replacement rates. We report a global decrease in turnover rates during M phase, and a further specific decrease in turnover among early origins of replication, which switch from rapidly-replaced in G1 phase to stable-bound during M phase. Finally, by measuring H3 replacement in yeast deleted for the H3K56 acetyltransferase Rtt109 and its two co-chaperones Asf1 and Vps75, we find evidence that Rtt109 and Asf1 preferentially enhance histone replacement at rapidly-replaced nucleosomes, whereas Vps75 appears to inhibit histone turnover at those loci. These results provide a broad perspective on histone replacement/incorporation throughout the cell cycle, and suggest that H3K56 acetylation provides a positive feedback loop by which replacement of a nucleosome enhances subsequent replacement at the same location.

overall_design:
To characterize incorporation of H3K56ac in yeast, several sets of ChIP-chip experiments were performed, along with a set of gene expression microarrays.  The following describes each individual set, the number of biological and array replicates performed, dye-flip replicates if performed, the total number of arrays, and the applicable figures in the manuscript.  Except for Item D (gene expression), all arrays were ChIP-chip experiments.
A.  Mid-log H3K56ac measurement.  3 biological replicates, 1 array replicate each of K56Ac ChIP with Grunstein Lab antibody.  2 biological replicates, 2 array replicates each of K56Ac ChIP with Upstate antibody.  7 total arrays.  Figure 1A-C, 3, S1, S2A, S6, S7.
B.  Cell cycle - H3K56ac measurements.  1 biological replicate, 3 array replicates each of K56Ac ChIP with Upstate antibody.  17 time points (10 min. unavailable in third array replicate).  50 total arrays.  Figures 2, 3, 4, S4, S5, S7, S8.
C.  G1 phase turnover.  1 biological replicate (3 time points) with 1 dye-flip replicate each time point (45 minutes replaced with 60 minutes for dye-flip replicate).  6 total arrays.  Tables S2, S3.
D.  Cell cycle - mRNA measurements.  2 biological replicates, 1 array replicate each.  34 total arrays.  Figure S3.
E.  M phase turnover rates.  2 biological replicates, second biological replicate has one dye-flip replicate.  17 total arrays.  Figure 5.
F.  Deletion mutants in H3K56ac pathway for G1-arrested cells.  Strains:  wild-type parental strain (PKY4212; 2 biological replicates), asf1D (strain 2 biological replicates), vps75D (3 biological replicates, third biological replicate having two array replicates), rtt109D (2 biological replicates, each with one dye-flip replicate).  12 total arrays.  Figure 6.

first: Tommy

last: Kaplan

last: Liu

first: Judith

last: Erkmann

first: John

last: Holik

last: Grunstein

first: Paul

last: Kaufman

first: Nir

last: Friedman


title: Evaluation of the amplicons for detection of known CNVs using Cs22 HR-CGH

status: Public on Sep 24 2008

first: Xinghua

last: Pan

address: 300 Cedar Street, TAC-S320

department: Department of Genetics

institute: Yale University School of Medicine

summary:
Highly specific amplification of complex DNA pools without bias or template-independent products (TIPs) remains a challenge. We have developed a procedure using phi29 DNA polymerase and trehalose and optimized control of amplification to create micrograms of specific amplicons without TIPs from down to sub-femtograms of DNA. The amplicons from 5 ng and 0.5 ng DNA, which were from originally good quality of gDNA (05-050), or partially degraded gDNA (04-018), faithfully demonstrated all previously known heterozygous segmental duplications and deletions (3 Mb to 18 kb) located on chromosome 22 and even a homozygous deletion smaller than 1 kb with high resolution chromosome-wide CGH. Specifically, HR-CGH with 5 ng-input gDNA-derived amplicon detected all previously known chromosomal segmental aberrations in chromosome 22 in samples from two different probands, and was indistinguishable from the HR-CGH result with native gDNA from the same probands (Fig. 4, Fig. S3 and S4). The break points were also precisely demonstrated. These include a heterozygous genomic segmental duplication (3 copies, 3 Mb in size, sample 05-050, Fig. 4) and 2 different heterozygous deletions (1 copy, 1.4 Mb and 18 kb respectively, sample 04-018, Fig. S4), all of which are located in or bounded by regions of low copy repeats (LCRs). In addition, a previous known homozygous deletion of 975 bp (in 04-018 and 05-050) was again accurately demonstrated (05-050 data showed in Fig. 4c), although sometimes (04-018) the data was a little noisier than with unamplified DNA (Fig. S4d). In contrast, the Wpa-40oC resulted in abundant signal noise and failed in detection of these copy number aberrations (Fig. 4, Fig. S3, S4). Impressively, HR-CGH with 0.5 ng gDNA-derived amplicons via Wpa also clearly detected the known CNVs, although noisier (Fig. S4). The 0.1ng gDNA derived amplicons via Wpa could not unambiguously show CNVs because of higher variability of signals, but the CNVs’ patterns were mostly well maintained (Fig. S4 for 04-018). We did also notice some locus-imbalance in the amplicon, however this was minimized, and was reproducible when the input was above a certain threshold amount, and it could be well compensated if the same amplified reference sample was applied in parallel as showed above.

Keywords: Whole-pool amplification, high resolution comparative genome hybridization (HR-CGH)

overall_design: The overall goal of this part of study was a validation of the quality of the amplicons from different amounts of original starting gDNA, good quality or partially degraded gDNA, with our new procedure Wpa, or our intermediate testing procedure Wpa-40C, with native gDNA as control, in terms of the detectability of the known large-scale heretozygous deletions and duplications (CNVs), or small-scale (sub-kb) CNVs. Amplified or native genomic DNA isolated from patients with known CNVs (Cy3) was co-hybridized with in-parallel amplified or native reference gDNA pool (Promega; Cy5).

first: Alexander

last: Urban

first: Dean

last: Palejev

first: Vincent

last: Schulz

first: Fabian

last: Grubert

first: Yiping

last: Hu

last: Snyder

first: Sherman

last: Weissman


title: Identification of BABY BOOM downstream targets

status: Public on Sep 22 2008

city: Wageningen

last: Passarinho

address: Bornsesteeg 65

department: Business Unit Bioscience

institute: Plant Research International BV

summary:
A glucocorticoid-regulated BBM protein (35S:BBM-GR) was used in combination with microarray analysis to identify genes directly activated by BBM. We employed the system described by (Lloyd et al., 1994) in which dexamethasone (DEX) and cycloheximide (CHX) are applied together to respectively, induce nuclear localization of the BBM-GR protein and prevent translation of the primary targets mRNAs. In this way it is possible to identify direct targets of a transcriptional activator by comparing gene expression profiles between DEX+CHX-treated transgenic and wild-type tissues.
The ability of the 35S:BBM GR construct to induce somatic embryogenesis in Arabidopsis seedlings was determined by phenotypic observation of 35S:BBM GR seeds germinated and grown in the presence of 10 µM dexamethasone (DEX). As in 35S:BBM seedlings, we observed somatic embryo formation on the cotyledons, first leaves and shoot meristem of DEX-treated 35S:BBM GR seedlings. We identified a set of 20 genes (including BBM itself) and our analysis indicates that BBM directly activates a signaling pathway comprising transcription factors and other signaling molecules, but which does not initially include genes known to induce somatic embryogenesis, such as LEC1, LEC2 or WUS. The functions of the BBM target genes are unknown, however a number of them have recently been identified in microarray screens for meristem-expressed genes. The identification of BBM-interacting partners and downstream targets provides new tools for unraveling pathways related to plant cell growth and organogenesis.
Keywords: transcriptional activation

overall_design: To identify candidate BBM targets, we treated 4 day old 35S:BBM-GR seedlings for 8 hours with 10 µM DEX in the presence of 10 µM CHX and compared the mRNA population from these seedlings using Arabidopsis Operon microarrays (http://www.ag.arizona.edu/microarray/) to that of 4 day old wild-type seedlings treated in the same way. The seed batches used in this analysis showed 100% EFS when grown continuously on media with 10 µM DEX. Four day old seedlings were chosen as the experimental material because they are highly responsive to BBM GR activation and provide sufficient RNA for the microarray experiments. The biological reproducibility of the data was monitored using two independent single locus 35S:BBM GR lines. The technical reproducibility of the data was monitored using two independently DEX + CHX-treated samples from each transgenic line. Dye effects were monitored in a dye-swap experiment using one of the four RNA samples from the two biological replicates

first: Hiroyuki

last: Fukuoka

first: Meiqing

last: Xing

first: Jeroen

last: van Arkel

first: Richard

last: Immink

first: Ronny

last: Joosen

first: Chris

last: Maliepaard

first: Michiel

last: Lammers

first: Lydia

last: Herdies

first: Bart

last: den Boer

first: Lonneke

last: van der Geest

first: Kim

last: Boutilier


title: gene profiles in prostate cancer and normal prostate tissues

city: Shanghai

first: Mei

last: Jiang

address: Handan Road

department: Institute of Genetics

institute: Fudan University

summary:
Prostate cancer is one of the most commonly diagnosed cancers among men in the world and the pathogenesis of prostate cancer remains poorly understood. In this study, we intended to disclose genes that might be involved in tumorigenesis using cDNA microarray technology.
Keywords: disease state analysis

overall_design: A total of 28 experiments were performed without replicates, using a pool of unrelated fetal tissues as common references. 25 experiments were done for prostate cancer versus common reference and 3 for normal prostate versus common reference. Prostate cancer and normal prostate samples were labeled with Cy5-dUTP. Common references were labeled with Cy3-dUTP.


title: Gene expression profiling of chemokines and chemokine receptors on B-cells in chronically HIV-1 infected patients

status: Public on Sep 19 2008

city: Stockholm

first: Alberto

last: Cagigi

address: Nobelsvag 16

country: Sweden

institute: Karolinska Institutet

summary:
From the analysis of all genes included in the array a number of individual genes were identified as dys-regulated in HIV-1 infected patients compared to healthy controls. In particular, increased CXCL13 expression in all individual patient samples.

Abstract from publication: HIV-1 infection is associated with B-cell abnormalities such as hypergammaglobulinemia, poor immunisation responses and loss of serological memory. To determine whether altered expression of chemokine receptors and their ligands may play a role in B-cell dysfunctions during HIV-1 infection, the expression of CXCR4, CXCR5 and CCR7 receptors and their respective ligands on CD19+ B-cells were examined in HIV-1 infected patients and controls. We report a decreased CXCR5 expression on B-cells from patients (p<0.05), a phenomenon associated with a low CD4 T-cell count (<350 cells/µl). Interestingly, an increased expression of CXCL13, the ligand for CXCR5, was found in peripheral B-cells from HIV-1 infected patients. Moreover upon B-cell activation in vitro, CXCL13 was secreted in culture. In addition, CXCL13 positive B-cells were also found in the lymph nodes of HIV-1 infected patients, but not in control tissue. B-cell migration towards CXCL13, CXCL12 and CCL21, ligands for CXCR5, CXCR4 and CCR7, was also evaluated. In patients with a low CD4 Tcell count, migration towards all ligands was increased. Our findings indicate that altered expression of the chemokine receptor-ligand pair, CXCR5/CXCL13 may participate in the establishment of B-cell dysfunctions during HIV-1 infection.

overall_design: Total RNA was extracted from purified peripheral B-cells from 4 controls and 4 patients.

first: Anna

last: Nilsson


title: Comparison of antigen stimulation response in Mycobacterium bovis-infected vs. control cattle using BOTL-5 microarray

status: Public on Sep 20 2008

city: Dublin

first: David

last: MacHugh

address: University College Dublin, Belfield

department: College of Agriculture, Food Science and Veterinary Medicine

country: Ireland

institute: University College Dublin

summary: Bovine tuberculosis (BTB) caused by Mycobacterium bovis continues to cause substantial losses to global agriculture and has significant repercussions for human health. The advent of high throughput genomics has facilitated large scale gene expression analyses that present a novel opportunity for revealing the molecular mechanisms underlying chronic mycobacterial infection. Using this approach, we have previously shown that innate immune genes in peripheral blood mononuclear cells (PBMC) from BTB-infected animals are repressed in vivo in the absence of exogenous stimulation. In the present study, functional genomics methods were used to examine the immune response of PBMC from BTB-infected (n = 6) and healthy control (n = 6) cattle to stimulation with bovine purified protein derivative (PPDb) in vitro. PBMC were harvested before, and at 3h and 12h post in vitro stimulation with PPDb. Gene expression changes were catalogued within each group using a reference hybridization design and a targeted immunospecific cDNA microarray platform (BOTL-5) with 4,800 spot features representing 1,391 genes

overall_design:
Experimental animals and infection status
Sixteen cattle were used for this study. The eight infected animals were chosen from herds with a recent history of chronic infection with M. bovis. The animals were selected on the basis of the skin-fold thickness response to bovine and avian tuberculin in the single intradermal comparative tuberculin test (SICTT). The SICTT reactor animals were selected where the skin thickness response to PPD-bovine exceeded that of PPD-avian by at least 12 mm. All of these animals were also measured positive in a whole blood IFN-assay [ref.52]. The cattle were confirmed positive for tuberculosis following detailed post-mortem pathological examination and/or culture. Bronchial, mediastinal, submandibular, retropharyngeal, mesenteric and hepatic lymph nodes and lungs were examined macroscopically for tuberculosis lesions. Suspected lesions were cultured on Stonebrinks and Lowenstein-Jensen media at 37°C for eight weeks to detect M. bovis [ref.53]. The eight non-infected control animals were selected from a herd without a recent history of tuberculosis and were SICTT and IFN-gamma test negative.
Blood sampling and analysis
400 ml of blood was collected from each animal in sterile heparinised bottles. Five ml of blood was used for haematological analysis and leukocyte cell population subsets were compared between infected and control groups as previously described [ref.24].
PBMC separation, culture, RNA extraction and quality control
PBMC were isolated using the Percoll gradient method as previously described [ref.54].PBMC were seeded at  10^7 per flask and cultured in RPMI 1640 culture medium supplemented with 5% FBS, 0.1% mercaptoethanol and 0.1% gentamicin. A total of 48 tissue culture flasks represented 16 individual PBMC samples (BTB-infected [n = 8] and healthy control [n = 8]) per time point at 0h (pre-stimulation), 3h and 12h post-stimulation with PPDb. All PBMC samples were cultured overnight at 37°C in 5% CO2 to minimize noise in gene expression measurements potentially introduced by the mechanical disruption of cells associated with PBMC isolation. After cells were harvested for time point 0, remaining flasks were stimulated using 50 ug/ml PPD-bovine and incubated for 3h and 12h at 37°C in 5% CO2. Residual cells not seeded for culture in either treatment were immediately dissolved in 3 ml TriReagent (Molecular Research Centre Inc., Cincinnati, OH) and frozen in 1.5 ml cryotubes at -80°C for use as a common reference RNA (CRR) pool. Total RNA was extracted from PBMC harvested after 3h and 12h post-stimulation using a combined TriReagent, DNase treatment and Qiagen RNeasy method (Qiagen Ltd., Crawley, UK) according to the manufacturer instructions. The integrity and stability of RNA samples is crucial for gene expression analyses using microarray technology; therefore, RNA yield and quality were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies Ireland Ltd., Dublin, Ireland). The two-step method for RNA extraction described above was found to produce RNA of high yield and quality (ratios of 18S to 28S ribosomal RNA averaged > 1.6).
Microarray experimental design
The 3,888 feature BOTL-5 immunogenetic microarray system used has been described previously [ref.55]. The NCBI GEO platform accession for the BOTL-5 microarray is GPL5751. The immunobiology-targeted BOTL-5 array contains 1,391 genes or ESTs spotted in duplicate with multiple additional control features (blank spots, negative spots, housekeeper genes) and is an expanded version of the BOTL-4 array described previously by our group [refs.22, 56]. A reference design was used for microarray hybridizations, such that all RNA samples were labelled using Cy3 and co-hybridized with Cy5 labelled common reference RNA (CRR) pool as described previously [ref.24]. Thirty-six arrays were hybridized in total, representing six individual animal PBMC samples from each treatment group pre-stimulation, and at 3h and 12h post stimulation with PPDb, as shown in Fig. 1. It was hypothesized that the CRR pool would display similar mRNA expression levels and gene coverage as the target samples, therefore allowing flexible, accurate and consistent comparison of gene expression data across a time course without arbitrarily pairing animals from different groups [ref.57]. The CRR pool contained equal amounts of total RNA from the treated and control animal groups.

first: Kieran

last: Meade

first: Eamonn

last: Gormley

first: Cliona

last: O’Farrelly

first: Stephen

last: Park

first: Eamon

last: Costello

first: Joseph

last: Keane

first: Yingdong

last: Zhao


title: chromosomal aberrations in prostate cancer

summary:
Prostate cancer is one of the most commonly diagnosed cancers among men in the world and the pathogenesis of prostate cancer remains poorly understood. In this study, we intended to disclose chromosomal aberrations that might be involved in tumorigenesis, using cDNA microarray-based CGH technology.
Keywords: disease state analysis

overall_design: A total of 18 experiments were performed without replicates, using a kidney tissue as normal references. Prostate tumors and normal references were labeled with Cy5-dCTP and Cy3-dCTP, respectively.


title: SAGE tags generated from Triticum aestivum cultivars Scorpion 25 and Xi19 grown under normal and hot/dry conditions

city: Bristol

first: Gary

last: Barker

address: Woodland Road

department: Biological Sciences

country: United Kingdom

institute: University of Bristol

summary: Triticum aestivum cultivars Scorpion 25 and Xi 19 were grown under both normal and hot/dry conditions.  We compared the effect of these two growth conditions on these two closely related varieties.

overall_design: We sequenced two biological replicates each for both Scorpion 25 and Xi19 grown under normal conditions. One library for hot/dry growth conditions was sequenced for each of these two cultivars.

first: Rebecca

last: Poole

first: Kay

last: Werner

first: Gaia

last: Biggi

first: Jane

last: Coghill

first: George

last: Gibbins

first: Simon

last: Berry

first: Jim

last: Dunwell

first: Keith

last: Edwards


title: Probing nucleosome function: A highly versatile library of synthetic histone H3 and H4 mutants

city: Baltimore

first: Daniel

last: Yuan

address: 733 N. Broadway, Suite 351

department: Molecular Biology and Genetics

institute: Johns Hopkins Univ School of Medicine

summary:
The structural integrity of the nucleosome is central to regulation of DNA metabolism and transcription. We describe a library of 486 systematic histone H3 and H4 substitution and deletion mutants in Saccharomyces cerevisiae that probe the contribution of each residue to nucleosome function and can be episomal or genomically integrated. We tagged each mutant histone gene with unique molecular barcodes, facilitating identification of mutant pools through barcode amplification, labeling, and microarray hybridization. We probed fitness contributions of each residue to chemical perturbagens of chromosome integrity and transcription, mapping global patterns of chemical sensitivities and requirements for three forms of transcriptional silencing onto the nucleosome surface. Lethal mutants were surprisingly rare and of distinct types; one set of mutations mapped precisely to the DNA interaction surface. The barcode microarrays were useful for scoring complex phenotypes such as competitive fitness in a chemostat, proficiency of DNA repair, and synthetic genetic interactions.
Keywords: genetic modification

overall_design: These nine datasets characterize a microarray platform repurposed for profiling a barcoded comprehensive library of synthetic histone mutants. The first two datasets establish the wide dynamic range and high sensitivity and specificity of data from this platform. The other seven datasets demonstrate the usefulness of this technology for scoring subtle and complex phenotypes of the histone H3 and H4 alleles in this library.

first: Junbiao

last: Dai

first: Edel

last: Hyland

first: Hailiang

last: Huang

first: Joel

last: Bader

first: Jef

last: Boeke


title: Identification of direct T-box target genes in the developing zebrafish mesoderm

city: Berkeley

first: Aaron

last: Garnett

address: 555 LSA

institute: UC Berkeley

summary:
The zebrafish genes spadetail (spt) and no tail (ntl) encode T-box transcription factors that are important for early mesoderm development. Although much has been done to characterize these genes, the identity and location of target regulatory elements remain largely unknown. Here, we survey the genome for downstream target genes of the Spt and Ntl T-box transcription factors. We find evidence for extensive additive interactions towards gene activation and limited evidence for combinatorial and antagonistic interactions between the two factors. Using in vitro binding selection assays to define Spt- and Ntl-binding motifs, we searched for target regulatory sequence via a combination of binding motif searches and comparative genomics. We identified regulatory elements for tbx6 and deltaD, and, using chromatin immunoprecipitation, in vitro DNA binding assays and transgenic methods, we provide evidence that both are directly regulated by T-box transcription factors. We also find that deltaD is directly activated by T-box factors in the tail bud, where it has been implicated in starting the segmentation clock, suggesting that spt and ntl act upstream of this process.

Keywords: Morpholino Knockdown

overall_design: We measured transcript levels in zebrafish embryos at 75% epiboly.  We compared levels in spadetail (spt) and/or no tail (ntl) morpholino injected embryos to those in control embryos mock injected with Danieau solution plus 0.25% phenol red.  Three biological replicates are included for each treatment.

middle: T

first: Tina

last: Han

first: Michael

last: Gilchrist

first: James

middle: C

last: Smith

last: Eisen

first: Fiona

last: Wardle

first: Sharon

last: Amacher


title: Sexually dimorphic gene expression in the chick brain at stage 29

status: Public on Sep 18 2008

city: Seoul

first: JAE

middle: YONG

last: HAN

address: 1 Gwanak-ro, Gwanak-gu

department: Department of Agricultural Biotechnology

institute: Seoul National University

summary: Biological bases for sexual differences in the brain exist in a wide range of vertebrate species, including the chicken. We examined whether sexually dimorphic gene expression in the brain precedes gonadal differentiation. Using the Affymetrix GeneChip® Chicken Genome Array, we identified  many female- and male-enhanced genes that are differentially expressed in sex-specific brains from stage 29 chicken embryos.  We postulate that these genes have potential roles in the sexual differentiation of neural function and development in chickens.

overall_design: All analyses were performed in triplicate. The Affymetrix data from male and female brain samples at stage 29 were generated. The male and female brain samples by genetic sexing were pooled and homogenized. Pooled samples ranged from 5 to 8 embryos. 4g of total RNA from the pooled samples was used for labeling. Probe synthesis from total RNA samples, hybridization, detection, and scanning were performed according to standard protocols from Affymetrix.