title: 1.5 and 3 hours macrophage exposure

status: Public on Oct 01 2006

city: Madrid

first: Concha

last: Gil

state: Madrid

address: Pza. Ramón y Cajal s/n

department: Departamento Microbiología II

institute: Universidad Complutense de Madrid

summary:
Purpose of the study

The encounter between Candida albicans and phagocytes (macrophages) is considered the initial step in the development of host immune defenses. The single utility of proteomic and genomic approaches to study host - pathogen interaction has been previously described in the literature (1, 2, 3); but up to now few studies have employed both techniques as complementary approaches. Because of the importance of the macrophages in the innate immune response against fungal infectious, we have investigated the transcriptional profiling and the differential protein expression (using a proteomic approach) of C. albicans after the interaction with these phagocytes.
Description
We have developed an in vitro system by employing the murine macrophage cell line RAW264.7 and the wild-type Candida albicans yeast strain SC5314. The study of the differential C. albicans protein expression in these conditions (3h. of interaction) has been performed by 2D-PAGE. The comparative analyse of the bidimensional  silver stained gels obtained, was performed using the bioinformatic software Melanie 3. A complementary approach for this proteomic work is the study of the yeast transcriptional profiling after two time points of interaction (1.5 and 3h). We have analyzed  differential C. albicans gene expression using Microarrays from  Eurogentec.
Results and conclusions
A total of 132 protein species differentially expressed have been detected; 79 with increased expression and 53 with their expression decreased. The most interesting proteins up and down regulated, have been identified by MALDI-TOF TOF (59 proteins) and classified according to their biological function.The majority of these proteins belong to different metabolic pathways. Up-regulated proteins belong to oxidation of fatty acids and detoxification, meanwhile, other metabolic proteins belonging to glycolysis, fermentation and gluconeogenesis are clearly down-regulated. Proteins belonging to cell rescue, defence and virulence, proteosome and oxidative stress response are also up-regulated. A total of 240 C. albicans differentially expressed genes have been detected after 1,5 and 3h of macrophage interaction (122 over-expressed and 118 under-expressed). The yeast transcriptional response pointed out an increase in genes belonging to oxidative stress, metal homeostasis, DNA damage repair, lipid metabolism and pathogenesis and a down-regulation profile in genes belonging to cytoskeleton, morphogenesis, metabolism (carbohydrate, amino acid and nucleotide) and vesicular-vacuolar transport.
The results obtained after this proteomic/genomic analyses indicate that a great percent of these proteins/genes belong to different cellular pathways showing the attempt of C. albicans to avoid being engulfed by macrophages, and once inside, points out the hostile environment that surrounds the yeast.
References
1-Lorenz MC, Bender JA, Fink GR. Transcriptional response of Candida albicans upon internalization by macrophages. Eukaryot Cell. 2004 Oct;3(5):1076-87.
2-Fradin C, De Groot P, MacCallum D, Schaller M, Klis F, Odds FC, Hube B. Granulocytes govern the transcriptional response, morphology and proliferation of Candida albicans in human blood. Mol Microbiol. 2005 Apr;56(2):397-415.
3- Martínez-Solano L, Nombela C, Molero G, and Gil C. Differential protein expression of murine macrophages upon interaction with Candida albicans. Proteomics, in press.
Keywords: stress response

overall_design:
Hybridization protocol cDNA was synthesized from 20-30 microgrammes of total RNA by reverse transcription using CyScribe Post-Labeling kit, incorporating Cy3-cUTP or Cy5-dUTP (Amersham Biosciences) into the cDNA corresponding to each sample to be compared. The amount of cDNA as well as the incorporation of Cy3 and Cy5 dyes into cDNA targets was quantified on an Ultrospec 3300 Pro UV/ Visible spectrophotometer (Amersham Biosciences). Both labeled cDNA populations were combined, dried in a vacuum trap, and used as a hybridization probe after resuspension in hybridization solution and 100 micrograms Salmon Sperm. Slides were hybridized overnight with labeled probe at42 degrees in a water bath. Before scanning, the chips were washed and dried following the manufactures instructions

Scan protocol Microarrays were scanned with a GenePix 4000B scanner (Axon Instruments. Union City, CA) at a resolution of 5um (PMT values ranging from 550 to 700 and laser power 100%)

Description 18-24 hrs prior to initiation of the co-culture experiment, macrophages were collected and counted with a Neubauer chamber (hemocytometer). A total of 3,5 x 107 cells were plated in complete culture media in 750-ml cell culture flask and grown overnight at 37ºC in a humidified atmosphere with 5% CO2. C. albicans strain SC5314 was grown overnight at 30ºC on solid YED medium (to maintain cells in the yeast form). On day 1, these yeast cells were harvested, washed twice with phosphate buffered saline (PBS), counted and diluted to the desired density in 50 ml of complete culture media. 5 x 107 yeast cells were added per flask to obtain a fungus-macrophage ratio 1:1, (since repeated cell counts of the overnight macrophage cultures indicated a growth rate of 1,4) and incubated for 1.5 h. (for microarray experiments) and 3h. at 37°C and 5% CO2 . Non ingested and unbound Candida cells were removed by washing 3 times with ice-cold PBS. The macrophages and bounded/ingested yeast cells were dislodged by scraping the flask with rubber scrapers in ice-cold water, and pooled by centrifugation for 10 min at 4000 rpm.The resulting cell pellet was resuspended in a 0.25 % Triton X114 solution (50 mM Tris-HCl, 2mM EDTA, pH 7.5), vortexed 3 times and left/chilled on ice in order to eliminate most of the lysated macrophages. After 30 min, the sample was washed 4-6 times with ice-cold MilliQ water, in order to eliminate any trace of the detergent employed.

Data processing GenePix Pro 4.0 analysis software (Axon Instruments) was used to locate spots in the microarray with the appropriate grid and to obtain the two Cy3/Cy5 image TIFF files. All images were further processed using GenePix 4.0 software according to manufacturer instructions. Flagged spots and spots with an average intensity minus background below the media of the intensity minus background for all the non flagged spots in any channel (Cy3 or Cy5) were not retained for further analysis. Within this group those spots which showed in at least one channel a value of intensity minus background higher than 5 times the media of the background for all spots in the microarray for this channel were recovered.

Two biological samples. Dye swap. Four microarrays per condition

first: Elena

last: Fernández-Arenas

first: Virginia

last: Cabezón

first: Clara

last: Bermejo

first: Javier

last: Arroyo

first: César

last: Nombela

first: Rosalía

last: Díez-Orejas


title: Mild exercise training favorably modulates gene expression in human skeletal muscle

status: Public on Sep 09 2006

city: Hershey

first: Yuichiro

last: Nishida

state: PA

address: 500 University Drive

department: Department of Anatomy and Physiology

country: USA

institute: Penn State College of Medicine

summary:
Changes and plasticity in both gene expression and protein signaling in skeletal muscle is considered to be a major cause of metabolic syndrome, while it has been shown that mild exercise training at lactate threshold (LT) intensity is a safe and effective for prevention of metabolic syndrome. To elucidate the molecular mechanisms related to the beneficial effects of LT training for 60 min/day for 5 days/wk for 12 wk, we performed serial analysis of gene expression (SAGE) to examine global mRNA expression in human skeletal muscle. Approximately 57000 SAGE tags were analyzed for before training, as well as 5 days, 6 and 12 wk after the training. The LT training has coordinately induced many genes involved in mitochondrial energy metabolism, fat oxidation, glycolysis and creatine metabolism. Another molecular feature associated with this mild exercise regimen has been an induction of many genes encoding for potent antioxidant enzymes and molecular chaperons. Furthermore, the training modulated the expression levels of 233 novel transcripts. Thus, the current study reveals that LT exercise has favorably altered gene expression in human skeletal muscle to the prevention of metabolic syndrome.
Keywords: transcriptome, serial analysis of gene expression, metabolic syndrome, exercise training, lactate threshold

overall_design: Healthy men performed cycle ergometer training at the lactate threshold level for 60 min/day, five days/wk for 12 wk. After subjects were fasted more than 12 h, muscle biopsies were obtained between 8:00 to 10:30 under local anesthesia with lidocaine from the middle portion of the right vastus lateralis. Muscle biopsies were performed before training, as well as at 5 days, 6 wk and 12 wk during the execution of the program. At 5 day, muscle biopsies were performed 1 h after the training session, since we expected that 4 times training from day 1 to 4 would augment the acute effects of the training at 5 day. To assess the sustained effect of the training program, muscle biopsies were performed 2 days after the last training session, at 6 and 12 wk. The muscle sample was quickly dissected from fat and connective tissues, immediately frozen with liquid nitrogen and stored at –80℃ until analysis.

first: Hiroaki

last: Tanaka

first: Takuro

last: Tobina

first: Kiyohide

last: Murakami

first: Naoko

last: Shono

first: Munehiro

last: Shindo

first: Mayumi

last: Yoshioka

first: Jonny

last: St-Amand


title: Comparison of expression profile between human Müller cells, HMCL-I and HMCL-II

status: Public on Aug 12 2006

city: Ste-Foy

first: Caroline

last: Lupien

state: Québec

address: 2705, boul. Laurier

department: Centre de Recherche du CHUL

country: Canada

institute: Université Laval

summary:
Müller cells are the principal glial cells in the retina. Alterations in Müller cell behaviour are observed in retinal tissue from patients with proliferative diabetic retinopathy. The purpose of this study was to compare gene and protein expression profile of normal human Müller cells (NHMC) with two spontaneously human Müller cell lines generated from type 1 (HMCL-I) and type 2 (HMCL-II) diabetic donors using Serial Analysis Gene Expression (SAGE). Approximatively 50 000 tags were sequenced for each of the three SAGE libraries. Identification of the transcripts was obtained by matching the 15bp (CATG + 11bp) with the UniGene and GenBank databases. Classification of the genes was based upon the updated information of the genome directory found at the NCBI website. SAGE allowed us to characterize the entire transcriptome of human Müller cells and to compare these data with those from Müller cells of type 1 and 2 diabetic donors.
Keywords: Müller cells, SAGE, HMCL-I, HMCL-II, expression profile comparison

overall_design:
Polyadenylated RNA was extracted with the Oligotex mRNA Purification System (Qiagen), annealed with the biotin-5 -T18-3 primer and converted to cDNA with the cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). The resulting cDNA library was digested with NlaIII (anchoring enzyme), and the 3’ restriction fragments were isolated with streptavidin-coated magnetic beads (Dynal Biotech, Oslo, Norway) and separated into two populations. Each population was ligated to one of the two annealed linker pairs and extensively washed to remove unligated linkers. The tag beside the most 3’ NlaIII restriction site (CATG) of each transcript was released by digestion with BsmFI (tagging enzyme). The blunting kit from Takara Co. (Otsu, Japan) was used for the blunting and ligation of the two tag populations. The resulting ligation products containing the ditags were amplified by PCR with an initial denaturation step of 1 min at 950C, followed by 22 cycles of 20 s at 940C, 20 s at 600C and 2 s at 720C with 27 bp primers. The PCR product was digested with NlaIII, and the band containing the ditags was extracted from the acrylamide gel. The purified ditags were self-ligated to form concatemers. The concatemers of 500 to 1800bp were isolated by agarose gel. The resulting DNA fragments were ligated into the SphI site of pUC19 and cloned into UltraMAX DH5-alpha-FT (Invitrogen). White colonies were screened by PCR to select long inserts for automated sequencing.

Sequence files were analyzed by the SAGEana program, a modification of SAGEparser (ftp://ftp.pbrc.edu/public/eesnyder/SAGE/). Tags corresponding to linker sequences were discarded, and duplicate concatemers were counted only once.

We used the comparative count display (CCD) test to identify the transcripts that were differentially expressed significantly (P<0.05) between the groups with more than a twofold change. The CCD test makes a key-by-key comparison of two key-count distributions by generating a probability that the frequency of any key in the distribution differs by more than a given fold factor from the other distribution.

middle: B

first: Carl

last: Bolduc

first: Christian

last: Salesse


title: Expression profiling of normal lung parenchyma

status: Public on Nov 21 2006

city: Vancouver

first: Kim

middle: Marie

last: Lonergan

state: British Columbia

address: 675 West 10th Avenue

department: Cancer Genetics and Developmental Biology

institute: British Columbia Cancer Research Centre

summary:
2 normal lung parenchyma SAGE libraries, generated from 2 pools of 4 individuals each

Discussed in the study:

IDENTIFICATION OF NOVEL LUNG GENES IN BRONCHIAL EPITHELIUM BY SERIAL ANALYSIS OF GENE EXPRESSION Kim M. Lonergan1, Raj Chari1, Ronald J. deLeeuw1, Ashleen Shadeo1, Bryan Chi1, Ming-Sound Tsao2, Steven Jones3, Marco Marra3, Victor Ling1, Raymond Ng1,4, Calum MacAulay5, Stephen Lam5 and Wan L. Lam1
From the 1Department of Cancer Genetics & Developmental Biology,
5Department of Cancer Imaging, 3Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Research Centre, Vancouver, BC, Canada, 2Ontario Cancer Institute / Princess Margaret Hospital, Toronto, ON, Canada, 4the Department of Computer Science, University of British Columbia, Vancouver, BC, Canada

A description of the transcriptome of human bronchial epithelium should provide a basis for studying lung diseases including cancer.  We demonstrate here that minute epithelial specimens obtained by bronchial brushings afford reliable profiling by serial analysis of gene expression (SAGE) leading to lung gene discovery.  We have deduced global gene expression profiles of bronchial epithelium and lung parenchyma, based upon a vast data set of nearly two million sequence tags from 21 SAGE libraries generated from individuals with a history of smoking.  Cluster and linear regression analysis demonstrate the repeatability and reproducibility of bronchial SAGE libraries, and suggest that the transcriptome of the bronchial epithelium is distinct from that of lung parenchyma and other tissue types.  This distinction is highlighted by the abundant expression of signature genes that reflect tissue-specific and region-specific functions.  Through our analysis we have identified novel bronchial-enriched genes and a novel transcript variant for surfactant, pulmonary-associated protein B in lung parenchyma.  Conspicuously, gene expression associated with ciliogenesis is evident in bronchial epithelium.  Additionally, it is noted that a large number of unmapped tags awaits further investigation.
This study represents a comprehensive delineation of the bronchial and parenchyma transcriptomes, identifying more than 20,000 known and hypothetical genes expressed in the human lung, constituting one of the largest human SAGE studies reported to date.
Keywords: lung parenchyma

middle: M

first: Raj

last: Chari

first: Ronald

middle: J

last: deLeeuw

first: Ashleen

last: Shadeo

first: Bryan

last: Chi

first: Ming-Sound

last: Tsao

first: Steven

last: Jones

first: Marco

last: Marra

first: Victor

last: Ling

first: Raymond

last: Ng

first: Calum

last: MacAulay

first: Stephen

last: Lam

first: Wan

middle: L


title: Squamous esophagus

status: Public on Jun 28 2006

city: Utrecht

first: Jantine

middle: Wilhelmina Paula Maria

last: van Baal

address: Heidelberglaan 100

department: Gastroenterology and Hepatology

country: Netherlands

institute: University Medical Center Utrecht

summary:
SAGE libraries made of squamous esophagus tissue, primary cell culture or esophageal squamous cell carcinoma
Keywords: SAGE analysis of different tissues

overall_design:
squamous esophagus biopsy was taken from 1 male metaplastic Barrett's esophagus patient. primary cell culture was from 1 male Barrett's esophagus patient.
Esophageal squamous cell carcinoma was from a patient known to have ESCC

middle: W

first: Francesca

last: Milano

first: Agnieszka

last: Rygiel

first: Jacques

last: Bergman

first: Maikel

middle: P

last: Peppelenbosch

first: Kausilia

middle: K

last: Krishnadath


title: Translation State Array Analysis of Insulin Stimulated Human Endothelial Cells

status: Public on Jul 27 2006

city: Salt Lake City

first: Douglas

middle: I.

last: Schmid

state: UT

department: HMBG

institute: University of Utah

summary:
Confluent human umbilical vein endothelial cells (HUVECs) were exposed to Insulin (100 micM) for 2 hours. Ribosomal profiling via gradient centrifugation and fractionation was used to separate monosome, or under-translated, and polysome, or actively-translated, mRNA species that were then used to probe cDNA arrays, a process known as Translation State Array Analysis (TSAA). Four samples were obtained from these experiments: control monosome, control polysome, Insulin monosome and Insulin polysome. using the normalized signal intensities from the GeneFilters, we calculated a translation index, or measure of movement of an mRNA molecule from the monosome to the polysome fraction upon stimulation. This calculation was made as follows: (Insulin polysome/Insulin monosome)/(control polysome/control monosome). Translational indices greater than 2.5 (upregulated) or lower than 0.4 (downregulated) were chosen for further study.
Keywords: Translation State Array Analysis

overall_design:
HUVECs were stimulated with Insulin (100 micM) for 2 hours.  Cycloheximide (CHX) was added to a final concentration of 100 ng/mL for 5 minutes and cells were then rinsed with cold HBSS containing CHX (100 ng/mL) and scraped from the dish and pelleted by centrifugation (2000 x g, 5 minutes).  The supernatant was removed and cells were carefully resuspended in 375 uL Low Salt Buffer (LSB; 20 mM Tris, 10 mM NaCl, 3 mM MgCl2, pH 7.4) + RNasin (40 U/mL) + DTT (10 nM) for 3 minutes on ice.  125 uL LSB Lysis buffer (LSB containing 200 mM sucrose and 1.2% Triton-X100) was then added and cells disrupted by pipetting.  The mixture was then transferred to 1.7 mL microfuge tubes and centrifuged (20,000 x g, 1 minute, 4°C) to remove nuclei and cellular debris.  The supernatant was transferred to a new tube containing 50 uL LSB + RNasin (40 U/mL) + DTT (10 nM) and 15 uL 5M NaCl.  This mixture was then layered onto prepoured gradients (15-50% sucrose in LSB).  Gradients were spun at 43,700 rpm for 90 minutes (4° C) and then run on a density gradient flow cell fractionator (Isco) to obtain ribosomal profiles.  Ribosomal fractions corresponding to monosomes (M) and polysomes (P) (Figure 1) were collected into Trizol LS (Invitrogen) and total RNA isolated according to the directions of the manufacturer.  Total RNA was reverse transcribed using oligo dT primers in the presence of 33P dCTP to generate labeled cDNA probes from the M and P associated mRNA fractions of control and stimulated cells and these probes used to detect specific array elements on four identical Research Genetics (ResGen) Named Human Gene cDNA GeneFilters (GF 211, Invitrogen).  These filters were washed using high stringency conditions and exposed to phosphorimaging screens for analysis using Pathways 3, proprietary software included with the ResGen filters.
Hybridization signals were normalized to the average intensity of the hybridization signals from the particular array filter to correct for differing global hybridization efficiencies between filters.  To determine if a particular hybridization signal was significantly above background levels, an initial screen of the array elements was conducted by dividing the hybridization signal of an individual cDNA spot by the background signal on particular filter.  Background signal levels were determined using the Pathways 3 analysis software that measures the signal intensity of the area between cDNA spots and averages that signal over the area of the entire array.  Array elements were considered to be distinguishable from background if the hybridization signal/background ratio was greater than 1.1.  Signal intensities from each filter were normalized by dividing the raw signal intensity of the cDNA spot by the average signal intensity for the entire filter.  The translation state (TS) of each qualifying array element was determined by dividing the normalized signal intensity of the element in the polysome fraction by the normalized signal intensity of the element in the monosome fraction:  TS=P/M.  The Translation Index (TLI), a measure of the change of translation states of individual genes, was determined by calculating the ratio of the treated cell TS to the control cell TS; TLI = TSe/TSc where e represents experimental or treated conditions and c represents controls.  In this scenario, a gene that is not regulated by translation would have a TLI value of 1, actively translated genes would have a TLI value greater than 1, whereas a poorly translated or under-translated gene would have a TLI value less than 1.  Arbitrary TLI values of 2.5 and 0.4 were used to categorize individual array elements as being translationally regulated.

middle: I

first: Huimiao

last: Jiang

first: Larry

last: Kraiss


title: A SAGE approach to discovery of apolipoprotein E4 allele-specific genes involved in increased risk in Alzheimer disease

status: Public on Jan 13 2007

city: Durham

first: Puting

last: Xu

state: NC

address: 595 LaSalle St

department: Medicine

institute: Duke

summary:
     APOE4 allele is a major risk factor for late-onset Alzheimer disease (AD).  The mechanism of action of APOE in AD remains unclear. To study the effects of APOE alleles on gene expression in AD, we have analyzed the gene transcription patterns of human hippocampus from APOE3/3, APOE3/4, APOE4/4 AD patients and normal control using Serial Analysis of Gene Expression (SAGE).  Using SAGE, we found gene expression patterns in hippocampus of APOE3/4 and APOE4/4 AD patients differ substantially from those of APOE3/3 AD patients.  APOE3/4 and APOE4/4 allele expression may activate similar genes or gene pools with associated functions.  APOE4 AD alleles activate multiple tumor suppressors, tumor inducers and negative regulator of cell growth or repressors that may lead to increased cell arrest, senescent and apoptosis.  In contrast, there is decreased expression of large clusters of genes associated with synaptic plasticity, synaptic vesicle trafficking (metabolism) and axonal/neuronal outgrowth.  In addition, reduction of neurotransmitter receptors and Ca++ homeostasis, disruption of multiple signal transduction pathways, and loss of cell protection and notably mitochondrial oxidative phosphorylation/energy metabolism are associated with APOE3/4 and APOE4/4 AD alleles.  These findings help define the mechanisms that APOE4 contribute increased risk for AD and identify new candidate genes conferring susceptibility to AD.
Keywords: Serial Analysis of Gene Expression (SAGE); Apolipoprotein E (APOE3/3, APOE3/4, APOE4/4); Alzheimer disease; Hippocampus; apoptosis; signal pathways

overall_design:
Human brain samples and pathological assessment
Human brain tissues were collected in the Kathleen Price Bryan Brain Bank, in the Duke University Alzheimer Disease Research Center, and the Center for Human Genetics’ Brain Bank, Duke University Medical Center, following the rapid autopsy protocol (Hulette et al., 1997). The hippocampus was dissected at the time of autopsy and matching 100-200 mg portions of CA 1-4 was removed and used for RNA isolation and expression studies.  We chose AD patients of apolipoprotein E4/4 (APOE4/4), APOE3/4, and APOE3/3 genotypes and controls with the APOE 3/3 genotypes judged cognitively and pathologically normal.  Clinical information on all patients was collected from clinic and hospital records.  The pathological diagnosis of AD was established according to CERAD criteria (Mirra et al., 1991) and the degree of AD pathological changes was staged according to Braak (Braak and Braak, 1991).   We analyzed selected AD patients with pathological changes at the Braak and Braak stage IV or V (B&B stage IV or V) and compared them with cognitively and pathologically normal controls (B&B stage I) (Table 1). We chose hippocampal samples with relatively short post-mortem delay.

Construction of human SAGE libraries
Total RNA was isolated from frozen hippocampal samples of AD patients and controls using TRIzol reagent (Invitrogen, Carlsbad, CA) as previously described ( Xu 2006).  For SAGE library construction, we used standard protocols as described by Velculescu et al (1995) and Basrai and Hieter (2002) with minor modifications.  Briefly, SAGE was performed with 10 ug total RNA isolated from human brain hippocampus samples as outlined above.  The cDNA was prepared using the SuperscriptII cDNA synthesis kit (Invitrogen) with gel-purified 5’-biotinylated Oligo(dT)18 (Integrated DNA Technologies, Coralville, IA), according to the manufacturer’s protocol.  NlaIII and BsmFI restriction enzymes (New England Biolab, Beverly, MA) were used for tag generation.  BsmFI digestion was per¬formed at 37°C for 2.5 h (instead of 65 °C) using 40 units BsmFI in a 300 ul reaction volume with supplied buffer.  After a 3-hour concatemerization step, concatemers were heated at 65 °C for 10 minutes, following by 2 minutes on ice to enhance cloning efficiency.  Purified concatemers were subsequently cloned in the SphI site of pZero-1 (Invitrogen) and transformed in competent ElectroMax  DH10B cells (Invitrogen) using a 0.1 cm cuvette and the Gene Pulser II (BioRad).   Individual SAGE library clones were selected and PCR amplified using 96-well format Qiagen Real minipreps, and sequenced with ABI 3700 capillary sequencer using BigDye chemistry.

SAGE tag extract and statistical analysis
SAGE tags (10 bp) were extracted from the PHD files with eSAGE software using a threshold value of PHRED 20 for each base (Margulies and Innis, 2000).  The SAGE tags were extracted from individual sequence files from each SAGE library (Table 2).  The SAGE tag databases were compared with each other, such as AD vs. control and APOE4/4 AD vs. APOE3/3 AD SAGE tag databases, etc. using eSAGE software (Table 3).  The compared SAGE databases were mapped to UniGene Homo Sapiens tag-to-gene mapping set (build 182, www.ncbi.nlm.nih.gov/UniGene).

The Chi-square test and Fisher exact test were employed to test the difference in tag counts between two samples as previously described (Man et al., 2000;Hauser et al., 2003).

Functional categorization
We assigned differentially expressed gene transcripts to functional categories by two methods. One method assigned transcripts to broad functional categories defined on the basis of gene function derived from literature searches.  The other method used the software tool, EXPRESSION ANALYSIS SYSTEMATIC EXPLORER (EASE; http://david.niaid.nih.gov/david/ease.htm), to assign identified gene transcripts to “GO: molecular function” categories of the GeneOntology Consortium (www.geneontology.org).   We used the GO system to quantify over- and under-represented (up- and down-regulation) transcripts relative to total genes within a functional category and compare between different SAGE libraries.  Since the GO system does not provide a functional designation for all genes, our functional interpretations relied primarily on the extensive literature search in PubMed and sequence search in NCBI and other HGP databases.

Quantitative real-time PCR
We confirmed selected SAGE data in individual APOE allele-specific ADs and controls by quantitative real-time PCR (qRT-PCR).  Hippocampal total RNA (10 ug) was converted to cDNA using the High Capacity cDNA Archive Kit (Applied Biosystems, ABI, Foster City, CA) according to the manufacturer’s protocol. Assays-on DemandTM Gene Expression primer sets for qRT-PCR were purchased from Applied Biosystems (Table 6).  The primer sequences can be obtained from the authors upon request.  Four housekeeping genes (GAPD, GUSB, HPRT1, APLPO) were used as endogenous controls (or references).  Real-time PCR was performed using TaqMan Universal PCR Mater Mix according to TaqMan Gene Expression Assay protocol (ABI).  Each reaction was performed in quadruplicates, and non-RT (without reverse transcriptase) and non-RNA template controls were included.  The relative amounts of specifically amplified cDNAs in APOE allele-specific ADs compared with APOE3/3 controls were calculated using comparative CT method.

first: Pu-Ting

first: Charles

last: Kroner

first: Yi-Ju

last: Li

first: Jonathan

last: Haines

first: Margaret

middle: A

last: Pericak-Vance

first: John

middle: R

last: Gilbert


title: Barrett's esophagus

summary:
Barrett's esophagus transcriptome was analysed and compared with Barrett's esophagus primary cell culture and esophageal adenocarcinoma.
Keywords: SAGE analysis to compare tissues

overall_design:
Barrett's esophagus biopsy was taken from 1 male metaplastic Barrett's esophagus patient. Barrett's esophagus primary cell culture was cultures from a biopsy taken from a Barrett's esophagus patient and cultured for about 4 to 5 weeks.
Esophageal adenocarcinoma was taken from a patient known to have cancer and previously Barrett's esophagus


title: Gene regulation by IL-1β in IL-1R1-deficient mixed glia

status: Public on Mar 25 2006

city: Manchester

first: Ralph

last: Andre

address: Michael Smith Building, Oxford Road

department: Faculty of Life Sciences

country: United Kingdom

institute: University of Manchester

summary:
Interleukin-1 (IL-1) is a key pro-inflammatory cytokine, which has diverse actions in the brain as a regulator of host defence responses and a mediator of inflammation. Two major agonists, IL-1α and IL-1β bind to a single known functional (type-1) IL-1 receptor (IL-1RI), which associates with an accessory protein (IL-1RAcP) leading to signal transduction. However, recent evidence suggests that some actions of IL-1 in the brain may be independent of IL-1R1 and its classical signalling pathways, pointing to an as yet unidentified functional receptor for IL-1 expressed in the CNS. In this study, cDNA array based gene expression profiling was used to identify possible genes induced by IL-1β independently of IL-1R1. The results show that IL-1β may indeed regulate some genes independently of IL-1R1, suggesting the presence of additional functional IL-1 receptors in the mouse brain.
Keywords: experimental treatment (IL-1β), gene ablation (IL-1R1), glia

overall_design: Wild-type and IL-1R1-/- primary mixed glial cells were treated for 4 hours with 10 ng/ml rrIL-1β or vehicle alone. Paired treatments were undertaken in three independent biological replicates for each cell type.

middle: G

last: Moggs

first: FeiLing

last: Lim

first: Ian

last: Kimber

first: Nancy

last: Rothwell

first: Emmanuel

last: Pinteaux


title: Transformation of stellate cells to myofibroblasts

status: Public on Jul 01 2006

city: Amsterdam

first: Willem

last: Boers

address: Meibergdreef 69

department: AMC Liver Center

institute: Academic Medical Center

summary:
Activated hepatic stellate cells (HSC) that transdifferentiate to myofibroblasts in the injured liver are responsible for scar formation that leads to fibrosis and eventually cirrhosis. To investigate the gene expression profile during different stages of this process, we performed serial analysis of gene expression (SAGE), representing a quantitative and qualitative description of all expressed genes. Stellate cells were isolated from human livers and cultured. SAGE was performed on RNA isolated from quiescent, activated and transdifferentiated HSC. Comparison of the three resulting transcriptomes showed that less than 5% of all genes changed significantly in expression. Established markers of liver fibrosis showed enhanced expression in accordance with the transdifferentiation process. In addition, induction was seen for several genes not yet recognized to be involved in liver fibrosis, such as insulin-like growth factor binding proteins (IGFBP) and antagonists of bone morphogenic proteins: follistatin and gremlin. The induction of these genes was validated in vivo in mice developing liver fibrosis. The expression of IGFBPs and gremlin was measurable in the livers of these mice while it was low or undetectable in control mice without liver fibrosis. Since gremlin modulates the activity of bone morphogenic growth factors, it may represent a novel pathway and a target for therapeutic intervention and together with IGFBPs as a specific marker of liver fibrosis.  In conclusion, the comparison of the three transcriptomes of (activated) stellate cells reveals novel genes involved in fibrogenesis and provides an appreciation of the sequence and timing of the fibrotic process in liver.
Keywords: cell type comparison

overall_design: Human hepatic stellate cells (HSC) were isolated from wedge sections of normal human liver unsuitable for transplantation or from tumor-free human liver after partial hepatectomy as previously reported. Briefly, after a combined digestion with collagenase/pronase, HSC were separated from other liver nonparenchymal cells by ultracentrifugation over gradients of Larcoll (Sigma). The percentage of HSC in these isolates was > 90% as assessed by transmission microscopy, autofluorescence of vitamin A and immunofluorescence of vimentin. Immediately after isolation RNA was extracted for the construction of the SAGE library of quiescent HSC. To obtain activated stellate cells, these cells were cultured for 15 days on plastic culture dishes in modified Dulbecco's medium supplemented with 0.6 U/ml insulin, 2.0 mmol/L glutamine, 0.1 mmol/L nonessential amino acids, 1.0 mmol/L sodium pyruvate, antibiotic antifungal solution (Gibco) and 20% fetal bovine serum. To obtain fully transdifferentiated hepatic stellate cells, or myofibroblasts, the cells were cultured until they had reached passage 6 to 7. Myofibroblast phenotype was confirmed by detection of vimentin and α-smooth muscle actin using immunofluorescence with monoclonal antibodies from Dako and Sigma, respectively. Medium was refreshed twice a week and at the indicated period RNA was extracted using Trizol (Quiagen)

first: Saida

last: Aarrass

first: Chris

last: Linthorst

first: Massimo

last: Pinzani

first: Ronald

last: Oude Elferink

first: Piter

last: Bosma


title: Transcriptome of Nitrosomonas europaea with a disrupted nitrite reductase (nirK) gene

status: Public on Mar 23 2006

city: Riverside

first: Lisa

middle: Yael

last: Stein

state: CA

address: Geology 2207, 900 Univ. Ave.

department: Environ. Sciences

institute: University of California

summary:
Global gene expression was compared between Nitrosomonas europaea wild-type and a nitrite reductase-deficient mutant using a genomic microarray.
Keywords: wild-type to mutant global transcriptome comparison

overall_design:
The nirK mutant was generated from the wild-type by integrating a kanamycin resistance gene cassette into the center of the nirK ORF via homologous recombination from an engineered plasmid introduced into N. europaea by conjugation. Batch cultures of wild-type and nirK mutant Nitrosomonas europaea (ATCC 19718) were grown in mineral medium to late exponential phase.  Total cellular RNA was extracted from cells harvested from multiple cultures for each hybridization experiment (AquaPure RNA Isolation; Biorad), treated with RNase-free DNase I (Ambion), and purified (RNeasy Mini Kit; Qiagen). Fluorescently labeled cDNA libraries from total RNA were generated by incorporation of Cy3- or Cy5-dUTP by reverse transcriptase (SuperScript III; Invitrogen). Labeled cDNA was treated with NaOH (1N, 65 °C, 30 min), neutralized with Tris-Cl (1M, pH 7.6), column purified (Qiagen), and concentrated by vacuum centrifugation.  Two sets of triplicate reactions were performed in which the fluorescent dyes were reversed during cDNA synthesis to minimize dye-specific effects that could influence analysis of hybridization signals to the microarrays. Labeled cDNA libraries (0.6-0.9 ug with 20-30% label incorporation frequency) were hybridized to N. europaea genomic microarrays in buffered solution (5x SSC, 0.1% SDS, 50% formamide, 0.1 mg/ml salmon sperm DNA) in a chamber (Corning) under Hybrislips (Sigma-Alrich) at 50 C for 12 to 16 h. The slides were washed with 2x SSC, 0.1% SDS for 5 min at 42 C; 0.1x SSC, 0.1% SDS for 10 min at 42 C; and 4 times with 0.1x SSC for 1 min at room temperature.
Scanned images (Scan Microarray Express, Perkin Elmer) were analyzed using ImaGene version 5.6 (Biodiscovery).  Spot grids were manually fitted to microarray images and averaged signal intensity and local background was calculated for each microarray element.  The quality of the hybridization signals were assessed for each microarray by the numbers of detected genes plus low and consistent background levels. Spots with poor signal quality, irregular shape, and/or high background were flagged and removed from each data set.  Remaining data were transferred to Excel and Access 2001 (Microsoft Corp. Redmond, WA) and GeneSpring version 6.0 (Silicon Genetics). Normalization was done by scaling total fluorescence measured for both Cy3 and Cy5 to an equivalent averaged intensity across each microarray.  Pearson correlation coefficients were calculated for 12 spots for each gene (duplicate spots across 6 microarrays), and genes showing statistically significant and greater than 2 fold differences in Cy3/Cy5 hybridizations from at least 5 spots were considered differentially regulated.

first: Catherine

last: Cho

first: Tingfen

last: Yan

first: Xueduan

last: Liu

first: Liyou

last: Wu

first: Jizhong

last: Zhou

middle: Y


title: Translation State Array Analysis of Histamine Stimulated Human Endothelial Cells

first: Douglas

last: Schmid

summary:
Confluent human umbilical vein endothelial cells (HUVECs) were exposed to histamine (100 mM) for 2 hours.  Ribosomal profiling via gradient centrifugation and fractionation was used to separate monosome, or under-translated, and polysome, or actively translated, mRNA species that were then used to probe cDNA arrays, a process known as Translation State Array Analysis (TSAA).  Four samples were obtained from these experiments, control monosome, control polysome, histamine monosome and histamine polysome.  Using the normalized signal intensities from the GeneFilters, we calculated a translation index, or measure of movement of an mRNA molecule from the monosome to the polysome fraction upon stimulation.  This calculation was made as follows:  (histamine polysome/histamine monosome)/(control polysome/control monosome).  Translational indices greater than 2.5 (upregulated) or lower than 0.4 (downregulated) were chosen for further study.
Keywords: Translation State Array Analysis

overall_design:
HUVECs were stimulated with histamine (100 mM) for 2 hours.  Cycloheximide (CHX) was added to a final concentration of 100 ng/mL for 5 minutes and cells were then rinsed with cold HBSS containing CHX (100 ng/mL) and scraped from the dish and pelleted by centrifugation (2000 x g, 5 minutes).  The supernatant was removed and cells were carefully resuspended in 375 uL Low Salt Buffer (LSB; 20 mM Tris, 10 mM NaCl, 3 mM MgCl2, pH 7.4) + RNasin (40 U/mL) + DTT (10 nM) for 3 minutes on ice.  125 uL LSB Lysis buffer (LSB containing 200 mM sucrose and 1.2% Triton-X100) was then added and cells disrupted by pipetting.  The mixture was then transferred to 1.7 mL microfuge tubes and centrifuged (20,000 x g, 1 minute, 4° C) to remove nuclei and cellular debris.  The supernatant was transferred to a new tube containing 50 uL LSB + RNasin (40 U/mL) + DTT (10 nM) and 15 uL 5M NaCl.  This mixture was then layered onto prepoured gradients (15-50% sucrose in LSB).  Gradients were spun at 43,700 rpm for 90 minutes (4° C) and then run on a density gradient flow cell fractionator (Isco) to obtain ribosomal profiles.  Ribosomal fractions corresponding to monosomes (M) and polysomes (P) (Figure 1) were collected into Trizol LS (Invitrogen) and total RNA isolated according to the directions of the manufacturer.  Total RNA was reverse transcribed using oligo dT primers in the presence of 33P dCTP to generate labeled cDNA probes from the M and P associated mRNA fractions of control and stimulated cells and these probes used to detect specific array elements on four identical Research Genetics (ResGen) Named Human Gene cDNA GeneFilters (GF 211, Invitrogen).  These filters were washed using high stringency conditions and exposed to phosphorimaging screens for analysis using Pathways 3, proprietary software included with the ResGen filters.
Hybridization signals were normalized to the average intensity of the hybridization signals from the particular array filter to correct for differing global hybridization efficiencies between filters.  To determine if a particular hybridization signal was significantly above background levels, an initial screen of the array elements was conducted by dividing the hybridization signal of an individual cDNA spot by the background signal on particular filter.  Background signal levels were determined using the Pathways 3 analysis software that measures the signal intensity of the area between cDNA spots and averages that signal over the area of the entire array.  Array elements were considered to be distinguishable from background if the hybridization signal/background ratio was greater than 1.1.  Signal intensities from each filter were normalized by dividing the raw signal intensity of the cDNA spot by the average signal intensity for the entire filter.  The translation state (TS) of each qualifying array element was determined by dividing the normalized signal intensity of the element in the polysome fraction by the normalized signal intensity of the element in the monosome fraction:  TS=P/M.  The Translation Index (TLI), a measure of the change of translation states of individual genes, was determined by calculating the ratio of the treated cell TS to the control cell TS; TLI = TSe/TSc where e represents experimental or treated conditions and c represents controls.  In this scenario, a gene that is not regulated by translation would have a TLI value of 1, actively translated genes would have a TLI value greater than 1, whereas a poorly translated or under-translated gene would have a TLI value less than 1.  Arbitrary TLI values of 2.5 and 0.4 were used to categorize individual array elements as being translationally regulated.

first: Huimiao

last: Jiang

first: Larry

last: Kraiss


title: Gene expression of disseminated neuroblastoma

status: Public on Sep 06 2006

city: Cologne

first: Matthias

last: Fischer

address: Kerpener Str. 62

department: Pediatric Oncology

country: Germany

institute: University Children's Hospital

summary:
Comprehensive expression profiling of disseminated neuroblastoma with favorable and unfavorable outcome using SAGE. Results provide insight into the molecular pathogenesis of spontaneous regression and progression of metastatic neuroblastoma and may be used for improving risk estimation of patients with disseminated neuroblastoma.
Keywords: gene expression SAGE-based, neuroblastoma, primary tumor, disseminated disease

overall_design: Samples analyzed: 9 (stage 4S neuroblastoma: n=5, stage 4 neuroblastoma: n=3, neuroblastoma cell line: n=1)


title: Single-cell chromosomal imbalances detection by array CGH

status: Public on Feb 16 2006

city: Leuven

first: Joris

last: Vermeesch

address: Herestraat, 49

department: Center for Human Genetics

country: Belgium

institute: Katholic University Leuven

type: Genome variation profiling by genome tiling array

summary:
In this study, we extend array CGH technology by making the accurate detection of segmental aneusomies possible from a single lymphoblast and fibroblast following Phi29 DNA polymerase amplification
Keywords: array CGH, aCGH

overall_design:
Array CGH experiments were performed on two segmental aneusomic cell lines derived from patients. As a proof of principle, an interstitial 4q-deletion (46, XX, del(4)(q13.1q22.3)) fibroblast cell line and an unbalanced reciprocal translocation involving chromosomes 14 and X (46, XX, der (X) t(X;14)(q21.3;q23.1)) EBV cell line were used. Array CGH experiments were performed using gDNA from the two cell lines to define the exact size of each rearrangement. For the 4q-deletion cell line, the size of the deleted region was 34 Mb corresponding to 39 clones spotted on the array (from RP11-340A13 to RP11-44P19). For the EBV cell line, the size of the 14q-duplication was 47 Mb corresponding to 63 clones (from RP11-62H20 to CTC-820M16) and the Xq-deletion was 58 Mb corresponding to 70 clones (from RP3-380C13 to RP11-218L14). For each cell line, three single cells were amplified. Following DNA amplification, all cells showed the expected DNA yields.
Sex-mismatch array CGH experiments were conducted on amplified DNA samples obtained from the 4q-deletion cell line. Using the chromosome specific threshold obtained from our 18 experiments, sex chromosome and autosome ploidy levels were accurately identified with no false negative and no false positive results. Averaging the 39 clones within the chromosome 4q-deleted region enabled the accurate detection of the deletions. For each of the three amplified single-cell DNA of the unbalanced reciprocal translocation involving chromosomes 14 and X, male and female gDNA were used as references. Sex chromosome and autosome ploidy levels were accurately identified. Averaging intensity ratios of the 63 clones within the chromosome 14 duplicated region enabled the accurate detection of the duplications in the three replicate experiments. When, respectively, male or female gDNA were used as a reference, the average log2 mean ratio of the Xq-deleted region was –0.04 or –0.93, close to the theoretical expected value of 0 or minus 1. Interestingly, the log2 mean ratios of chromosomes 1 to 22 were highly similar when the same single cell amplified DNA was used for the two experiments using respectively female and male DNA as a reference. Hence, array CGH intensity ratio profiles were very reproducible.

first: Cedric

last: Le Caignec

first: Bernard

last: Thienpont

first: Paul

last: Van Hummelen


title: Translation State Array Analysis of LPS Stimulated Human Endothelial Cells

summary:
Confluent human umbilical vein endothelial cells (HUVECs) were exposed to LPS (1 micg/mL) for 2 hours.  Ribosomal profiling via gradient centrifugation and fractionation was used to separate monosome, or under-translated, and polysome, or actively-translated, mRNA species that were then used to probe cDNA arrays, a process known as Translation State Array Analysis (TSAA).  Four samples were obtained from these experiments: control monosome, control polysome, LPS monosome and LPS polysome.  using the normalized signal intensities from the GeneFilters, we calculated a translation index, or measure of movement of an mRNA molecule from the monosome to the polysome fraction upon stimulation.  This calculation was made as follows:  (LPS polysome/LPS monosome)/(control polysome/control monosome).  Translational indices greater than 2.5 (upregulated) or lower than 0.4 (downregulated) were chosen for further study.
Keywords: Translation State Array Analysis

overall_design:
HUVECs were stimulated with LPS (1 micg/mL) for 2 hours.  Cycloheximide (CHX) was added to a final concentration of 100 ng/mL for 5 minutes and cells were then rinsed with cold HBSS containing CHX (100 ng/mL) and scraped from the dish and pelleted by centrifugation (2000 x g, 5 minutes).  The supernatant was removed and cells were carefully resuspended in 375 uL Low Salt Buffer (LSB; 20 mM Tris, 10 mM NaCl, 3 mM MgCl2, pH 7.4) + RNasin (40 U/mL) + DTT (10 nM) for 3 minutes on ice.  125 uL LSB Lysis buffer (LSB containing 200 mM sucrose and 1.2% Triton-X100) was then added and cells disrupted by pipetting.  The mixture was then transferred to 1.7 mL microfuge tubes and centrifuged (20,000 x g, 1 minute, 4°C) to remove nuclei and cellular debris.  The supernatant was transferred to a new tube containing 50 uL LSB + RNasin (40 U/mL) + DTT (10 nM) and 15 uL 5M NaCl.  This mixture was then layered onto prepoured gradients (15-50% sucrose in LSB).  Gradients were spun at 43,700 rpm for 90 minutes (4° C) and then run on a density gradient flow cell fractionator (Isco) to obtain ribosomal profiles.  Ribosomal fractions corresponding to monosomes (M) and polysomes (P) (Figure 1) were collected into Trizol LS (Invitrogen) and total RNA isolated according to the directions of the manufacturer.  Total RNA was reverse transcribed using oligo dT primers in the presence of 33P dCTP to generate labeled cDNA probes from the M and P associated mRNA fractions of control and stimulated cells and these probes used to detect specific array elements on four identical Research Genetics (ResGen) Named Human Gene cDNA GeneFilters (GF 211, Invitrogen).  These filters were washed using high stringency conditions and exposed to phosphorimaging screens for analysis using Pathways 3, proprietary software included with the ResGen filters.
Hybridization signals were normalized to the average intensity of the hybridization signals from the particular array filter to correct for differing global hybridization efficiencies between filters.  To determine if a particular hybridization signal was significantly above background levels, an initial screen of the array elements was conducted by dividing the hybridization signal of an individual cDNA spot by the background signal on particular filter.  Background signal levels were determined using the Pathways 3 analysis software that measures the signal intensity of the area between cDNA spots and averages that signal over the area of the entire array.  Array elements were considered to be distinguishable from background if the hybridization signal/background ratio was greater than 1.1.  Signal intensities from each filter were normalized by dividing the raw signal intensity of the cDNA spot by the average signal intensity for the entire filter.  The translation state (TS) of each qualifying array element was determined by dividing the normalized signal intensity of the element in the polysome fraction by the normalized signal intensity of the element in the monosome fraction:  TS=P/M.  The Translation Index (TLI), a measure of the change of translation states of individual genes, was determined by calculating the ratio of the treated cell TS to the control cell TS; TLI = TSe/TSc where e represents experimental or treated conditions and c represents controls.  In this scenario, a gene that is not regulated by translation would have a TLI value of 1, actively translated genes would have a TLI value greater than 1, whereas a poorly translated or under-translated gene would have a TLI value less than 1.  Arbitrary TLI values of 2.5 and 0.4 were used to categorize individual array elements as being translationally regulated.


title: B27 modulation of LPS response

status: Public on Apr 10 2006

city: Cambridge

first: Giles

last: Yeo

address: CIMR, Addenbrookes Hospital

department: Clinical Biochemistry

institute: University of Cambridge

summary:
Objectives
How HLA-B27 contributes towards arthritis susceptibility is still unclear, but effects on the response to bacteria unrelated to the classical antigen presenting role of B27 have been suggested. This study investigated whether HLA-B27 modulates the innate response to LPS, a component shared between all Gram –ve bacteria that can trigger reactive arthritis.
Methods
Pools of U937 transfectants expressing either HLA-B27, HLA-A2, or the expression  plasmid alone were differentiated with PMA and stimulated with LPS.  Supernatants were analysed for TNF-alpha secretion and the gene expression profiles of unstimulated and LPS stimulated cells were determined by microarray analysis. Changes in gene expression that are indicative of an unfolded protein response were also analysed by quantitative PCR.
Results
TNF-alpha secretion, a biological marker of the inflammatory response to LPS, was not significantly different between U937-B27 and U937-control. No differences in gene expression between unstimulated U937- B27 and U937-control lines were detected.  Both U937-control and U937-B27 exhibited a stereotypic response to LPS. Only 1 gene, OAS2, was differentially expressed by these cell lines, and this was confirmed by quantitative PCR.  Analysis of  XBP-1 splicing suggested that a small increase in the unfolded protein response is induced following LPS stimulation, but this increase was seen in all transfectants.
Conclusions
The expression of B27 does not profoundly alter gene expression following LPS stimulation. Therefore, additional signals, such as those provided by cytokines or intracellular infection, may be required to reveal any influence of B27 expression on the inflammatory response.
Keywords: antigen response

first: Jane

middle: C

last: Goodall

first: Lou

last: Ellis

last: Gaston


title: Single-cell aneuploidy detection by array CGH

status: Public on Feb 01 2006

summary:
In this study, we extend array CGH technology by making the accurate detection of chromosomal imbalances possible from a single fibroblast and blastomere following Phi29 DNA polymerase amplification.
Keywords: CGH

overall_design: Firstly, array CGH experiments were performed on four different fibroblast cell lines, derived from patients affected by, respectively, trisomy 13, 18, 21, and monosomy X. For each cell line, three single cells were amplified. Following DNA amplification, all cells showed the expected DNA yields (n=12; 1.87 µg plus:minus 0.39). Sex-mismatch array CGH experiments were conducted on amplified DNA samples obtained from each cell. Sex chromosome ploidy levels, as well as all expected autosomal abnormalities were clearly identified. Secondly, we applied single-cell aneuploidy screening for the detection of chromosomal imbalances in preimplantation embryos. DNA from blastomeres from three 7-8 cell-stage embryos was amplified by Phi29 DNA polymerase. Following DNA amplification, all cells yielded the expected amount of DNA (n=16; 2.45 µg plus:minus 0.41). Chromosomal aneuploidies were accurately detected using a simple and rapid array CGH protocol.

first: Yves

last: Moreau

first: Claudia

last: Spits

first: Karen

last: Sermon

first: Martine

last: De Rycke

first: Jean-Pierre

last: Fryns

first: Inge

last: Liebaers

first: Andre

last: Van Steirteghem


title: Populus tissue analysis

status: Public on Dec 25 2006

city: Gainesville

first: Matias

last: Kirst

state: FL

address: 1376 Mowry Road

department: SFRC

institute: University of Florida

summary:
We analyzed the transcriptome in roots, young and mature leaves, nodes and internodes in the reference genotype Nisqually-1 and identified a core set of approximately 10,000 genes expressed in common among vegetative organs. Quantitative contrasts of transcript levels among organs identified expected patterns of expression associated with organ-specific physiological processes, although a surprisingly high number of defense-related genes were preferentially expressed in young leaves and nodes. Application of a novel runs test established an objective criterion for the identification of genomic regions in which adjacent genes were expressed more frequently than expected by chance, suggesting the presence of chromatin domains. Comparisons between P. trichocarpa Nisqually-1 and the P. tremula X P. alba hybrid INRA 717-1B4 revealed similar expression patterns, particularly in stems. Although a comparison to Arabidopsis thaliana revealed similar proportions of expressed genes in leaves and stems, there was very little conservation between rank correlations of expression patterns.
Keywords: gene expression

sample_organism:
Populus trichocarpa
Populus tremula x Populus alba

overall_design:
Plant material. Branches from the P. trichocarpa reference genotype Nisqually-1 were placed in soil. After budbreak, shoots were rooted in a misthouse for two weeks. Rooted cuttings (cloned biological replicates) were planted in separate pots in a greenhouse equipped with an ebb-and-flow flood bench system with daily supply of Peters Professional 20-10-20 water-soluble fertilizer diluted to a final concentration of 4 mM nitrogen. After 45 days, whole-roots (R), young leaves (YL) - leaf plastochrone index (LPI) 0-5, mature leaves (ML) - LPI 6-9, nodes (N) and internodes (IN) were collected and immediately frozen in liquid nitrogen. RNA was extracted using standard methods (Chang et al. 1993), DNAse-treated and purified in RNAeasy Qiagen columns (Valencia, CA). The P. tremula × P. alba hybrid genotype INRA 717-1B4, used for a comparison of transcript abundance between poplar species was grown under the same conditions as those described above.
Poplar whole-genome oligonucleotide microarrays. This study was based on hybridizations to whole-genome microarrays containing features representing 42,364 predicted transcriptional units from the P. trichocarpa nuclear genome. All transcriptional units were represented by three 60-mer probes, designed by NimbleGen (Madison, WI) in collaboration with Oak Ridge National Laboratory and were synthesized using maskless lithography. cRNA was synthesized from total RNA extracted from individual plants. Labeling, hybridization and scanning were carried out by NimbleGen (Madison, WI) using standard procedures.

first: Tania

last: Quesada

first: Christopher

last: Dervinis

first: Jerry

last: Tuskan

first: John

last: Davis

first: Stephen

last: DiFazio


title: Transcriptome analysis applied to survival of Shewanella oneidensis MR-1 exposed to ionizing radiation

status: Public on Feb 15 2006

city: Boston

first: XiaoYun

last: Qiu

state: MA

address: 200 longwood Ave

department: Microbiology and Molecular Genetics

institute: Harvard Medical School

summary:
The ionizing radiation (IR) dose that yields 20% survival (D20) of Shewanella oneidensis MR-1 is lower by factors of 20 and 200 than for Escherichia coli and Deinococcus radiodurans, respectively. Transcriptome analysis was used to identify the genes of MR-1 responding to 40 Gy (D20). We observed the induction of 170 genes and repression of 87 genes in MR-1 during a one-hour recovery period after irradiation. The genomic response of MR-1 to IR is very similar to its response to ultraviolet radiation (254 nm), which included induction of systems involved in DNA repair and prophage synthesis, and the absence of differential regulation of tricarboxylic acid cycle activity which occurs in IR-irradiated D. radiodurans. Furthermore, strong induction of genes encoding antioxidant enzymes in MR-1 was observed. DNA damage may not be the principal cause of high sensitivity to IR considering that MR-1 encodes a complex set of DNA repair systems and 40 Gy IR induces less than one double strand break (DSB) in its genome. Instead, a combination of oxidative stress, protein damage and prophage mediated cell lysis during irradiation and recovery might underlie this organism’s great sensitivity to radiation.
Keywords: time course, stress response

overall_design: Gene expression profile of S. oneidensis MR-1 was examined over a period of 1h following IR exposure. Briefly, 80 ml of MR-1 culture grown in Davis medium to OD600 of 0.2 was divided in half. Half of the culture (40 ml) was irradiated on ice to a total dose of 40 Gy. The non-irradiated control culture was incubated on ice for the same length of time (about 20 seconds) as the culture being irradiated. After irradiation, the irradiated cell culture (40 ml) and non-irradiated control culture (40 ml) were each transferred to a separate 100-ml flask (without dilution into fresh medium) and incubated at 30 C on a shaker. An aliquot of cells (12 ml) from each flask was collected after 5 (T5), 20 (T20), and 60 min (T60) of incubation for RNA extraction. Three independent cell cultures and irradiation treatments (repA, repB, and repD) were performed and served as biological replicates for gene expression experiments. For each time point, two technical replicates in hybridization (fluorescent-dye reversal) were carried out for each biological replicate. Thus, six data points were available for each time point (a total of eight data points were available for T20) and enabled the use of statistical tests to determine significant changes in gene expression.

first: Xiaoyun

first: Michael

last: Daly

first: Alexander

last: Vasilenko

first: Marina

middle: V

last: Omelchenko

first: Elena

last: Gaidamakova

first: Liyou

last: Wu

first: Jizhong

last: Zhou

first: George

last: Sundin

first: James

last: Tiedje


title: Effect of high or low chronic shear stress on microvascular endothelial cells cultured on native- versus AGE-fibronectin

status: Public on Jan 01 2006

city: Vienna

first: Gürkan

last: Sengoelge

address: Waehringer Guertel 18-20

department: Medicine/Division of Nephrology

country: Austria

institute: Medical University Vienna

summary:
Pulmonary microvascular endothelial cells were cultured on the inner surface of fibers (each 700 µm inner diameter) coated with either with native- or advanced glycation endproduct (AGE) modified fibronectin in hollow-fiber cartridges (FiberCell Systems, Inc.). Duration of shear stress application was 10 days. Shear stress was gradually increased to 17 dyne/cm2 in 2 samples (GSM32266: native fibronectin high shear stress or GSM45608: AGE-fibronectin) or was kept at constant low level of 1 dyne/cm2 in 1 sample (GSM41248: native fibronectin low shear stress).
Keywords: parallel sample

first: Sabine

last: Wohlfart

first: Agnes

last: Perschl

first: Sidrah

last: Ul-Haq


title: Gene expression profiles during the development of compensated or decompensated hypertrophy during pressure overload

city: Leiden

first: Henk

last: Buermans

state: ZH

address: Albinusdreef 2

department: Human Genetics

institute: Leiden University Medical Center

summary:
We recently introduced a modification of the established monocrotaline (MCT) model for pulmonary hypertension (PH) and subsequent right ventricular (RV) hypertrophy, which allows for the selective induction of either a compensate or decompensated RV hypertrophic phenotype within four weeks after a single subcutaneous MCT injection. Both doses of 30 or 80 mg/kg body weight lead to an intermediate phase of compensated RV hypertrophy (day 14-19), while the former dose leads to a stable compensated phenotype (HYP) and the latter dose progresses towards decompensated ventricular hypertrophy and RV failure (CHF) around day 25-28 (Buermans et. al, Physiological Genomics 2005). This model provides the unique opportunity to characterize the development of either hypertrophic phenotype during the very early stages after imposition of RV pressure overload remodeling, well before phenotypical differences have become apparent. This experiment series describes gene expression profiles, generated by spotted oligonucleotide microarrays, to characterize and compare the expression of ~4800 genes from the RV of HYP, CHF and time matched control rats at 10, 19 and 25 days after injections. RV samples were hybridized against a common reference pool, prepared from total RNA isolated from the interventricular septum wall from animals at day 19 and 25.
Keywords: dose response x time course

overall_design: Comparison of three groups (Controls, 30mg MCT/kg bw, 80 mg MCT/kg bw) on three time points (day10, 19, 25). Three samples pee groups per time point = 27 arrays. Hybridized against a common reference pool.

first: Warner

last: Simonides


title: Characterization of mRNA transcripts in spermatozoa of fertile men

status: Public on Nov 11 2005

city: Shanghai

first: Yangxing

last: Zhao

address: 800 Dongchuan Rd.

department: School of Medicine

country: China

institute: Shanghai Jiao Tong University

summary:
Accumulated evidence proves that mature spermatozoa contain a complex yet specific mRNA, which could provide information of the past events of spermatogenesis. Our aim was to quantitatively microdissect these mRNA transcripts by a digital approach. SAGE was used to study the mRNA transcripts from ejaculate of an individual and of a pool of 10 fertile men. SAGE library from individual produced 20237 raw tags representing 2459 unique tags (those tags that show at least twice in the library), and that from pooled 10 men generated 21052 raw tags representing 2712 unique tags. The SAGE view of ejaculate of fertile men has revealed a large number of transcripts, which occur in steady frequencies and probably have important implications in the spermatogenesis and fertilization.
Keywords: normal gene expression profile

overall_design: To study the transcripts expression level and their variation in human ejaculate spermatozoa, total RNA extracted from single ejaculate of 10 volunteers were mixed and assigned as “pooled”; for the “individual”, the extracted RNA from 5 separate ejaculate of the same eleventh volunteer were mixed together. And two SAGE libraries were constructed based on these starting RNA materials.

first: Qiaoli

last: Li

first: Chenjiang

last: Yao

first: Zhaoxia

last: Wang

first: Ying

first: Yajing

first: Limin

last: Liu

first: Yifei

first: Lianyun

first: Zhongdong

last: Qiao


title: Inventory of genes expressed in human control hippocampus

status: Public on Dec 30 2005

first: Aronica

last: Eleonora

address: Meibergdreef 9

department: Neuropathology

institute: AMC

summary:
Serial analysis of gene expression (SAGE) was used to get a global view of the gene profile in human hippocampus. A library were generated from control hippocampus, obtained by rapid autopsy.
Keywords: hippocampus human inventory genes

overall_design: Control hippocampus (used to construct the SAGE control library) was obtained from a 48 years old man without history of seizures or other neurological diseases and no brain abnormalities at autopsy and histologically normal hippocampus. Autopsy was performed within 4 hours after death. Tissue was snap-frozen and stored at –80 0C until use. Total RNA was isolated from control hippocampus and hippocampal surgical specimens, using the Trizol method according to the manufacturer’s instructions (Invitrogen - Life Technologies, The Netherlands). Part of the anterior hippocampus of control (including sectors CA1- CA4 and dentate gyrus, DG) was used.  Poly(A)+ RNA isolation, cDNA synthesis and all subsequent steps of the SAGE procedure were essentially performed as described (Velculescu et al., 1995) with minor modifications given previously (Michiels et al., 1999).

first: Sandra

last: Redeker

first: Eleonora

last: Aronica


title: Translation State Array Analysis of Serum Stimulated Human Endothelial Cells

first: Douglas

last: Schmid

overall_design:
HUVECs were stimulated with Serum (20% pooled human serum) for 6 hours.  Cycloheximide (CHX) was added to a final concentration of 100 ng/mL for 5 minutes and cells were then rinsed with cold HBSS containing CHX (100 ng/mL) and scraped from the dish and pelleted by centrifugation (2000 x g, 5 minutes).  The supernatant was removed and cells were carefully resuspended in 375 uL Low Salt Buffer (LSB; 20 mM Tris, 10 mM NaCl, 3 mM MgCl2, pH 7.4) + RNasin (40 U/mL) + DTT (10 nM) for 3 minutes on ice.  125 micL LSB Lysis buffer (LSB containing 200 mM sucrose and 1.2% Triton-X100) was then added and cells disrupted by pipetting.  The mixture was then transferred to 1.7 mL microfuge tubes and centrifuged (20,000 x g, 1 minute, 4 deg C) to remove nuclei and cellular debris.  The supernatant was transferred to a new tube containing 50 uL LSB + RNasin (40 U/mL) + DTT (10 nM) and 15 micL 5M NaCl.  This mixture was then layered onto prepoured gradients (15-50% sucrose in LSB).  Gradients were spun at 43,700 rpm for 90 minutes (4 deg C) and then run on a density gradient flow cell fractionator (Isco) to obtain ribosomal profiles.  Ribosomal fractions corresponding to monosomes (M) and polysomes (P) (Figure 1) were collected into Trizol LS (Invitrogen) and total RNA isolated according to the directions of the manufacturer.  Total RNA was reverse transcribed using oligo dT primers in the presence of 33P dCTP to generate labeled cDNA probes from the M and P associated mRNA fractions of control and stimulated cells and these probes used to detect specific array elements on four identical Research Genetics (ResGen) Named Human Gene cDNA GeneFilters (GF 211, Invitrogen).  These filters were washed using high stringency conditions and exposed to phosphorimaging screens for analysis using Pathways 3, proprietary software included with the ResGen filters.
Hybridization signals were normalized to the average intensity of the hybridization signals from the particular array filter to correct for differing global hybridization efficiencies between filters.  To determine if a particular hybridization signal was significantly above background levels, an initial screen of the array elements was conducted by dividing the hybridization signal of an individual cDNA spot by the background signal on particular filter.  Background signal levels were determined using the Pathways 3 analysis software that measures the signal intensity of the area between cDNA spots and averages that signal over the area of the entire array.  Array elements were considered to be distinguishable from background if the hybridization signal/background ratio was greater than 1.1.  Signal intensities from each filter were normalized by dividing the raw signal intensity of the cDNA spot by the average signal intensity for the entire filter.  The translation state (TS) of each qualifying array element was determined by dividing the normalized signal intensity of the element in the polysome fraction by the normalized signal intensity of the element in the monosome fraction:  TS=P/M.  The Translation Index (TLI), a measure of the change of translation states of individual genes, was determined by calculating the ratio of the treated cell TS to the control cell TS; TLI = TSe/TSc where e represents experimental or treated conditions and c represents controls.  In this scenario, a gene that is not regulated by translation would have a TLI value of 1, actively translated genes would have a TLI value greater than 1, whereas a poorly translated or under-translated gene would have a TLI value less than 1.  Arbitrary TLI values of 2.5 and 0.4 were used to categorize individual array elements as being translationally regulated.

first: Huimiao

last: Jiang

first: Larry

last: Kraiss


title: Genome-scale profiling of histone H3.3 replacement patterns in Drosophila S2 cells

status: Public on Sep 11 2005

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: Genome binding/occupancy profiling by array

type: Genome binding/occupancy profiling by genome tiling array

summary:
Histones of higher eukaryotes are assembled into chromatin primarily during DNA replication, but at other times the histone H3.3 variant replaces canonical H3. We introduce a novel strategy for profiling epigenetic patterns based on H3.3 replacement, using microarrays covering about one third of the Drosophila melanogaster genome at 100-bp resolution. Striking patterns of H3.3 replacement were found over active genes and transposons. H3.3 replacement occurs prominently at sites of abundant RNA polymerase II and methylated H3 lysine-4 throughout the genome, and is enhanced on the dosage-compensated male X chromosome. Active genes are depleted of histones at promoters and are enriched in H3.3 from upstream to downstream of transcription units. Based on these findings, we propose that deposition and inheritance of actively modified H3.3 in regulatory regions maintains transcriptionally active chromatin.
Keywords: Chromatin affinity-purification on microarray

overall_design: Chromatin affinity profiling of histones H3 and H3.3 on Drosophila cDNA and tiling arrays

first: Yoshiko

last: Mito

first: Steven


title: Response and adaptation to growth with low glucose concentration

status: Public on Apr 26 2006

city: Dübendorf

first: Alessandro

middle: Guido

last: Franchini

address: Ueberlandstrasse 133

department: Environmental Microbiology

country: Switzerland

institute: EAWAG

summary:
The faecal indicator bacterium Escherichia coli K12 was used to study the cellular events that take place at the transcription level using the microarray technology during short-term (physiological) and long-term (genetic) adaptation to slow growth under limited nutrient supply. Short-term and long-term adaptation were assessed by comparing the mRNA levels isolated after 40 or 500 hours of glucose-limited continuous culture at a dilution rate of 0.3 h-1 with those from batch culture with glucose excess.
Keywords: glucose-limited continuous culture, adaptation, microarray, high affinity transport systems, transcriptome, Escherichia coli

overall_design:
Wild-type Escherichia coli K12 MG 1655 (ATCC 700926) was grown at 37 °C in mineral medium. Growth was followed by measuring OD546 in regular intervals to confirm that cells were growing at umax (0.63 ± 0.02 h-1). A sample for transcriptome analysis was taken from the batch exponential phase at an OD546 of ~0.4, and immediately after sampling the medium flow was started for continuous cultivation with a dilution rate of 0.3 h-1. Steady-state with respect to biomass and residual glucose concentrations was reached approximately 15 hours after this manipulation. 40 and 500 hours after switching to the glucose-limited continuous culture mode 800 ml of culture liquid was withdrawn from the chemostat, immediately cooled down on crushed ice and cells were collected by centrifugation at 4 °C.
Total RNA from E. coli cells was isolated as described earlier (Sambrook et al., 1989). RNA in samples was quantified spectrophotometrically by measuring extinction at 260 nm and purity (free from DNA) was checked by gel electrophoresis. Synthesis of cDNA from RNA was performed with the CyScribe First-Strand cDNA Labelling Kit (Amersham Bioscience, Little Chalfont, England). The purified cDNA was concentrated to 5 ul and was mixed with 120 ul of hybridization buffer (MWG-Biotech AG), heated to 95 °C for 3 min and cooled down on ice for 3 min. The hybridization mixture was then added to the microarray slide and covered with a coverslip. The hybridisation slide was incubated over night at 42 °C. After the hybridization step the slide was washed three times, the first time for 5 min in 2x (times concentrated) SSC - 0.1 % SDS, the second time for 5 min in 1x SSC, and finally for 5 min in 0.1x SSC. SSC buffer was prepared as 20x solution containing 0.3M Na-citrate and 3M NaCl at pH 7.0. The slides were dried by centrifugation at room temperature for 2 min at 500g.
Microarray slides were scanned using the Affymetrix 428TM Array Scanner (High Wycombe, UK). Spot intensities and corresponding background signals were quantified with the Affymetrix JaguarTM software version 2.0. Further data analysis was performed with the program GeneSpring from Silicon Genetics (Redwood City, USA). Induction factors were calculated from the Cy3 and Cy5 signal intensities of the spot. Spots with signal intensity below a value of 50 were excluded from the analysis and the minimal induction factor was set to 0.01. The normalization was performed with the 50th percentile distribution of remaining spots after background correction. The mean value of the induction factors of a specific gene was calculated from three replicates. Biological experiments were carried out three times, which provided three biological repeats. Data from the independent experiments were combined, genes that were differentially regulated > 2 and < 0.5 (t-test, p <= 0.05) were defined as being statistically significant.

first: Thomas

last: Egli


title: Early dexamethasone-inducible genes in M-1 Cortical Collecting Cells

status: Public on Aug 26 2005

city: Baltimore

first: Djikolngar

last: Maouyo

state: MD

institute: University of Maryland Baltimore

summary:
Precise control of blood pressure and salt balance is ultimately dependent on the regulation of epithelial sodium channels, ENaC, in the distal nephron. Ligand activation of mineralocorticoid and glucocorticoid receptors induces a transcriptional response that leads to an augmentation of ENaC cell surface density and activity, but a complete inventory of the network of genes involved in the response is incomplete. In the present study, we used high-density cDNA microarrays to examine changes in the transcriptional profile of a mouse cortical collecting duct cell line, M-1, in response to a potent glucocorticoid agonist, dexamethasone. Here we identify 64 gene transcripts that are consistently co-regulated with ENaC, increasing within 3 hours of dexamethasone treatment. Within the same early response period, the abundance of 58 diiferent gene transcripts significantly decreased. A subset of up-regulated genes (12) was selected for further validation and the majority (10) of these were confirmed as dexamethone-regulated transcripts by real time RT-PCR. The products of these genes are known to function in intracellular trafficking operations (Numb, tetraspanin CD63, and Vps41), signal transduction, or to operate as modifiers of the cytoskeleton (gelsolin, tektin 2 and transgelin). These studies have identified new group of candidate genes that potentially regulate sodium transport in the collecting duct.
Keywords: Gene identification in a cultured kidney cell model

overall_design: Experimental design. Cells were then treated with 100 nM hydrosoluble dexamethasone or steroid-free medium for 3 hours at 37°C with 5% CO2. RNA samples from dexamethasone-treated and control cells were extracted with Trizol reagent, and DNA-decontaminated using DNA-free kit, commercially available (Ambion, Austin, TX). Yield and concentration was quantified by spectrophotometry. RNA quantity and integrity was confirmed by denaturing formaldehyde agarose gel electrophoresis and ethidium bromide staining. These samples were used for microarray labeling. Microarrays, RNA labeling, and hybridization. High quality spotted cDNA on glass slide microarrays were purchased from the Microarray Centre of the University Health Network (Toronto, Canada). These arrays contained PCR-generated cDNA inserts of all 15,264 sequence-verified mouse ESTs, from the National Institute of Aging collection. Duplicate 100-mm diameter cDNA spots are represented on GAPS II slides (Corning, Inc.). Spot cDNA identity is tracked by composed identifiers including clone identifier, description, and Unigene, and catalogued as “M15Kv1”. Briefly, 5 µg total RNA was reverse-transcribed using oligo-dT flanked at 5’ end with capture sequences specific to fluorescent Cy3- or Cy5-coupled dendrimer complexes. The resulting cDNAs from the control and test samples were mixed and hybridized with the mouse cDNA arrays in humidified hybridization chambers at 60°C for 18 hours. Following hybridization, microarrays were washed three times as described above. Quantitation. Slides were read using a laser confocal scanner, the GSI Lumonics ScanArray 5000 imaging system, at 10-µm resolution set up for excitation/emission at 543/570 and 649/670 nm, and for acquisition of Cy3 and Cy5-labeled spots respectively. Scanned image data were saved in 16-bit tiff files and quantitated with Imagene 4.0 (BioDiscovery, Marina del Rey, CA). The mean intensities of fluorescent spots were measured.

first: Paul

last: Welling


title: A Focused Microarray Approach to Functional Glycomics: Transcriptional Regulation of the Glycome

status: Public on Jun 20 2005

city: La Jolla

first: Jennifer

last: Hammond

address: 10550 N. Torrey Pines Rd.

institute: The Scripps Research Institute

summary:
Glycosylation is the most common post-translational modification of proteins, yet genes relevant to the synthesis of glycan structure and function are incompletely represented and poorly annotated on the commercially available arrays.  To fill the need for expression analysis of such genes we employed the Affymetrix technology to develop a focused and highly annotated glycogene chip representing human and murine glycogenes, including glycosyltranferases, nucleotide sugar transporters, glycosidases, proteoglycans and glycan-binding proteins. In this report the array has been used to generate glycogene expression profiles of nine murine tissues.  Global analysis with a hierarchical clustering algorithm, reveals that expression profiles in immune tissues (thymus, spleen, lymph node and bone marrow) are more closely related, relative to those of non-immune tissues (kidney, liver, brain and testes). Of the biosynthetic enzymes, those responsible for synthesis of the core regions of N-and O-linked oligosaccharides are ubiquitously expressed, while glycosyltransferase that elaborate terminal structures are expressed in a highly tissue-specific manner, accounting for tissue and ultimately cell type-specific glycosylation.  Comparison of gene expression profiles with MALDI-TOF profiling of N-linked oligosaccharides suggested that the alpha-1-3 fucosyltransferase IX, Fut9, is the enzyme responsible for terminal fucosylation in kidney and brain, a finding validated by analysis of Fut9 knockout mice. Two families of glycan-binding proteins, C-type lectins and siglecs, are predominately expressed in the immune tissues, consistent with their emerging functions in both innate and acquired immunity. The glycogene chip reported in this study is available to the scientific community through the Consortium for Functional Glycomics (http://www.functionalglycomics.org).
Keywords: Glycogenes, Glycomics, Glycosyltransferase, Lectin, Glycosylation, Glycome, Microarray, Kidney, Fut9

platform_organism:
Homo sapiens
Mus musculus

overall_design: Total RNA was extracted from 9 different wild-type mouse tissues comprising of liver, brain, kidney, spleen, thymus, lymphnodes, testis, lung and femurs for bone marrow.  3 samples were collected from each tissue type, resulting in a total of 27 samples (9 sets of 3 replicates).  Glycogene expression profiling was performed on each sample using the Affymetrix custom-designed Glycov1 array.

first: Elena

middle: M

last: Comelli

last: Head

first: Tim

last: Gilmartin

last: Whisenant

first: Stuart

last: Haslam

first: Simon

last: North

first: Nyet-Kui

last: Wong

first: Takashi

last: Kudo

first: Hisashi

last: Narimatsu

first: Jeffrey

middle: D

last: Esko

first: Kurt

last: Drickamer

first: Anne

last: Dell

first: James

last: Paulson


title: Transcriptome analysis of Shewanella oneidensis MR-1 in response to cold shock

status: Public on Mar 22 2006

city: Hangzhou

first: haichun

last: gao

address: 388 Yuhangtang Rd.

institute: Zhejiang University

summary:
This study presents a global transcriptional analysis of the cold shock response of Shewanella oneidensis MR-1 after a temperature downshift from 30 to 8 or 15oC based on time-series microarray experiments.  More than 700 genes were found to be significantly affected (p < 0.05) upon cold shock challenge, especially at 8oC. The temporal gene expression patterns of the classical cold-shock genes varied and only some of them, most notably so1648 and so2787, were differentially regulated in response to temperature downshift. The global response of S. oneidensis to cold shock was also characterized by the up-regulation of genes encoding membrane proteins, DNA metabolism and translation apparatus components, metabolic proteins, regulatory proteins, and hypothetical proteins. Most of the metabolic proteins affected are involved in catalytic processes that generate NADH or NADPH.
Keywords: time course, stress response

overall_design: To prepare cells for a microarray analysis, a single colony of MR-1 was used to inoculate 3 ml of LB in 10 ml plastic tubes and grown overnight at 30oC (optimal growth temperature) on a rotary platform (200 rpm). 150 ml of LB medium prewarmed to 30oC in a 500-ml flask was inoculated with an aliquot of a fresh overnight culture to an OD600 of 0.01 and then was shaken on a rotary platform (200 rpm) until an OD600 of a mid-log optical density 0.59 was attained. A 25-ml aliquot of cells was transferred to a 250-ml flask pre-cooled to 8 or 15oC and then incubated in an 8 or 15oC shaking water bath. Samples for microarray analysis were taken at 0 (control), 5, 10, 20, 40, 80 and 160 min, pelleted by centrifugation at maximum speed in a 5415R centrifuge (Eppendorf, Germany) for 10 s, and placed in liquid nitrogen.

first: zamin

last: yang

first: liyou

last: wu

first: jizhong

last: zhou


title: human peripheral nervous system SAGE

status: Public on Apr 16 2005

first: Anneloor

last: ten Asbroek

summary:
expression profiles generated from sciatic nerve as well as from Schwann cells cultured thereof
Keywords: other

first: Rosalein

last: de Jonge

first: Jeroen

last: Vreijling

first: Asker

last: Meijntjes

first: Marcel

middle: S

last: Kwa

first: Antoine

middle: H

last: van Kampen

first: Ivo

middle: N

last: van Schaik

first: Frank

last: Baas


title: Multi-center validation of a gene expression based prognostic signature in lymph node-negative primary breast cancer.

status: Public on Oct 15 2005

city: San Diego

first: Tim

last: Jatkoe

institute: Veridex

summary:
A gene signature predicting survival of breast cancer patients was validated on a custom chip.
Keywords: breast cancer patient survival analysis

overall_design: 180 breast cancer samples

last: Smid

first: John

last: Foekens


title: gene expression profiles of Barrett's esophagus, squamous esophagus and gastric cardia

city: Utrecht

first: Jantine

middle: Wilhelmina Paula Maria

last: van Baal

address: Heidelberglaan 100

department: Gastroenterology and Hepatology

institute: University Medical Center Utrecht

summary:
SAGE performed on biopsies of Barrett's esophagus, squamous esophagus and gastric cardia taken from a metaplastic Barrett's esophagus patient.
Keywords: SAGE

overall_design: comparative analysis of gene expression profiles of Barrett's esophagus, normal squamous esophagus and gastric cardia tissue

middle: W

first: Francesca

last: Milano

first: Agnieszka

last: Rygiel

first: Jacques

middle: J

last: Bergman

first: Wilda

last: Rosmolen

first: Sander

last: van Deventer

first: Kenneth

last: Wang

first: Maikel

middle: P

last: Peppelenbosch

first: Kausilia

last: Krishnadath


title: 2.45 GHz Radiofrequency fields alter gene expression in cultured human cells

status: Public on Jul 30 2005

city: Evanston

first: San Ming

state: IL

address: 1001 University Place

department: Center for Functional Genomics

institute: ENH Research Institute, Northwestern University

summary:
The biological effect of radiofrequency (RF) fields remains controversial. We address this issue by examining whether RF fields can cause changes in gene expression. We observed that 221 genes altered their expression after a 2-hour exposure. The number of affected genes increased to 759 after a 6-hour exposure. Functional classification of the affected genes reveals that apoptosis-related genes were among the up-regulated ones and the cell cycle genes among the down-regulated ones. We observed no significant increase in the expression of heat shock genes. These results indicate that the RF fields at 2.45 GHz can alter gene expression in cultured human cells through non-thermal mechanism.
Keywords: gene expression SAGE

overall_design: We used the pulsed RF fields at a frequency of 2.45 GHz that is commonly used in telecommunication to expose cultured human HL-60 cells. We used the SAGE (serial analysis of gene expression) method to measure the RF effect on gene expression at the genome level.

first: Sanggyu

last: Lee

first: Debra

last: Johnson

first: K

last: Dunbar

first: Hui

last: Dong

first: Xijin

last: Ge

first: Yeong

last: Kim

first: Claudia

last: Wing

first: Nimanthi

last: Jayathilaka

first: Nimmi

last: Emmanuel

first: Chenn

middle: Q

last: Zhou

first: Howard

middle: L

last: Gerber

first: Charles

last: Tseng

first: San


title: The pattern of gene expression in human CD34(+) stem/progenitor cells

status: Public on Mar 01 2005

summary:
We have analyzed the pattern of gene expression in human primary CD34(+) stem/progenitor cells. We identified 42,399 unique serial analysis of gene expression (SAGE) tags among 106,021 SAGE tags collected from 2.5 x 10(6) CD34(+) cells purified from bone marrow. Of these unique SAGE tags, 21,546 matched known expressed sequences, including 3,687 known genes, and 20,854 were novel without a match. The SAGE tags that matched known sequences tended to be at higher levels, whereas the novel SAGE tags tended to be at lower levels. By using the generation of longer sequences from SAGE tags for gene identification (GLGI) method, we identified the correct gene for 385 of 440 high-copy SAGE tags that matched multiple genes and we generated 198 novel 3' expressed sequence tags from 138 high-copy novel SAGE tags. We observed that many different SAGE tags were derived from the same genes, reflecting the high heterogeneity of the 3' untranslated region in the expressed genes. We compared the quantitative relationship for genes known to be important in hematopoiesis. The qualitative identification and quantitative measure for each known gene, expressed sequence tag, and novel SAGE tag provide a base for studying normal gene expression in hematopoietic stem/progenitor cells and for studying abnormal gene expression in hematopoietic diseases.
Keywords: other

first: Guolin

first: Jianjun

last: Chen

first: Terry

last: Clark

first: Janet

last: Rowley


title: Compensated and decompensated hypertrophy after monocrotaline injection

status: Public on Aug 18 2004

first: Henk

last: Buermans

summary:
14 days after a single subcutaneous monocrotaline (MCT) injection we isolated the left and right ventricles from wistar rats.

Factor1: Comparison between control (CON), compensated hypertrophy (HYP), and decompensated hypertrophy (CHF).
Factor2: Comparison between left ventricle (LV) and right ventricle (RV).
Keywords: dose response


title: Targets of c-Jun NH(2)-terminal kinase 2-mediated tumor growth regulation

status: Public on Jul 16 2003

city: New Heaven

first: Nikki

middle: J.

last: Holbrook

state: CT

address: 333 Cedar Street

department: Department of Internal Medicine

institute: Yale University School of Medicine

summary:
Although the c-Jun NH(2)-terminal kinase (JNK) pathway has been implicated in mediating cell growth and transformation, its downstream effectors remain to be identified. Using JNK2 antisense oligonucleotides (JNK2AS), we uncovered previously a role for JNK2 in regulating cell cycle progression and survival of human PC3 prostate carcinoma cells. Here, to identify genes involved in implementing JNK2-mediated effects, we have analyzed global gene expression changes in JNK2-deprived PC3 cells using Serial Analysis of Gene Expression. More than 40,000 tags each were generated from control and PC3-JNK2AS libraries, corresponding to 15,999 and 20,698 unique transcripts, respectively. Transcripts corresponding to transcription factors, stress-induced genes, and apoptosis-related genes were up-regulated in the PC3-JNK2AS library, revealing a significant stress response after the inhibition of JNK2 expression. Genes involved in DNA repair, mRNA turnover, and drug resistance were found to be down-regulated by inhibition of JNK2 expression, further highlighting the importance of JNK2 signaling in regulating cell homeostasis and tumor cell growth.
Keywords: other

first: Olga

middle: U

last: Potapova

first: Sergey

last: Anisimov

first: Myriam

last: Gorospe

first: Ryan

last: Dougherty

first: William

middle: A

last: Gaarde

first: Kenneth

middle: R

last: Boheler


title: Novel estrogen and tamoxifen induced genes

first: Kornelia

last: Polyak

address: 450 Brookline Ave

department: Medical Oncology

institute: Dana-Farber Cancer Institute

summary:
The breast cancer promoting effects of estrogen and the chemopreventive effects of tamoxifen are thought to be mediated by the estrogen receptor, a ligand-dependent transcription factor. Therefore, comprehensive analysis of gene expression profiles following estrogen or tamoxifen treatment may help us better understand the role estrogen plays in tumorigenesis. We utilized SAGE (Serial Analysis of Gene Expression) technology to identify genes regulated by estrogen and tamoxifen in the ZR75-1 estrogen dependent breast cancer cell line. In this manner we have identified several genes that were regulated by estrogen or tamoxifen. Here we report the identification and initial characterization of EIT-6 (Estrogen Induced Tag-6), a novel nuclear protein and a new member of the evolutionarily conserved SM-20 family of growth regulatory immediate-early genes. EIT-6 appears to be a direct transcriptional target of the estrogen receptor and constitutive expression of EIT-6 promotes colony growth in human breast cancer cells. These data indicate that EIT-6 may play a role in estrogen induced cell growth.
Keywords: other

first: Pankaj

last: Seth

first: Ian

last: Krop

first: Dale

last: Porter


title: Pancreas and islet gene expression

status: Public on Feb 05 2003

city: Saint Louis

first: Corentin

last: Cras-Méneur

state: MO

address: 660 S. Euclid

department: Metabolism Division

institute: Washington University School of Medicine

summary:
Gene expression of pancreatic exocrine cells, pancreatic islets and hand-selected pancreatic islets examined.
Keywords: other

first: H

last: Inoue

first: C

first: Y

last: Zhou

first: M

last: Ohsugi

first: E

last: Bernal-Mizrachi

first: D

last: Pape

first: S

last: Clifton


title: Effects of estrogen

status: Public on May 21 2002

city: Smithville

middle: Marcelo

last: Aldaz

state: TX

department: Science Park Research Division, Carcinogenesis

institute: M.D. Anderson Cancer Center

summary:
Effects of estrogen on global gene expression: identification of novel targets of estrogen action.
Keywords: other

first: A

last: Charpentier

middle: K

last: Bednarek

first: R

last: Daniel

first: K

last: Hawkins

last: Laflin

last: Gaddis

middle: C

last: MacLeod


title: Differentiation of cultured THP-1 cells

status: Public on Nov 19 2004

city: London

first: J Max

last: Wilkinson

state: UK

address: Rowland Hill Street

department: Medicine

institute: Royal Free & University College London School of Medicine

summary:
Libraries constructed from cultured THP-1 cells receiving PMA treatment or not.
Keywords: other

first: James

middle: M


title: Young and old muscle comparison

status: Public on Feb 01 2002

city: Rochester

first: Stephen

last: Welle

state: NY

address: 601 Elmwood Avenue

institute: University of Rochester

summary:
RNA was pooled from vastus lateralis biopsies obtained from 8 younger (21-24 yr) and 8 older (66-77 yr)men. Healthy subjects with no neuromuscular disease.
Keywords: other

last: Thornton


title: eye-SAGE

status: Public on Oct 03 2001

city: Boston

first: Dror

last: Sharon

address: 243 Charles St.

institute: Massachusetts Eye and Ear

summary:
Human retinal and RPE SAGE libraries.
Profile of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium determined through serial analysis of gene expression (SAGE).
Keywords: other

first: Seth

last: Blackshaw

first: Constance

last: Cepko

first: Thaddeus

last: Dryja


title: Human Adrenal Cortex affected by Primary Pigmented Nodular Adrenocortical Disease

status: Public on Mar 11 2005

city: Washington

first: Anelia

last: Horvath

state: DC

address: 2300 Eye Street NW

department: Biochemistry and Molecular Medicine

institute: GWU

summary:
Comparison between human adrenal cortex derived from adolescent female and human adrenal cortex derived from adolescent female affected by Primary Pigmented Nodular Adrenocortical Disease caused by germline mutation in PRKAR1a
Keywords: other

middle: D

first: Constantine

last: Stratakis

first: Wai-yee

last: Chan


title: Comparative SAGE Analysis of the Response to Hypoxia in Human Pulmonary and Aortic Endothelial Cells

status: Public on Jun 28 2005

city: Pittsburgh

first: David

middle: Gerard

last: Peters

state: PA

institute: University of Pittsburgh

summary:
Cultures of human aortic (HAEC) and pulmonary artery endothelial cells (HPAEC) were exposured to short-term chronic hypoxia (1% O2) for either 0h, 8h or 24h
Keywords: Time course, cell-type comparison

overall_design: The response of each cell type (HAEC and HPAEC) to short-term chronic hypoxia was determined by a single SAGE library for each of three time points (0h, 8h and 24h)

middle: G

first: Wendy

last: Ning

first: Tian

last: Chu

first: C

last: Li

first: Augustine

last: Choi


title: PBECs stimulated with pro-inflammatory cytokines and P. aeruginosa

status: Public on Feb 03 2005

city: Amsterdam

first: Joost

last: Vos

address: Van der Boechorststraat 7

department: Molecular Cell Biology and Immunology

institute: Vrije Universiteit Medisch Centrum

summary:
Comparison of libraries derived from primary human bronchial epithelial cells which are [i] unstimulated, [ii] stimulated with the pro-inflammatory cytokines IL1beta and TNFalpha and [iii] stimulated with heat-inactivated Pseudomonas aeruginosa (strain PAO1).
Keywords: other

middle: B

first: Marianne

last: van Sterkenburg

first: Klaus

middle: F

last: Rabe

last: Schalkwijk

first: Pieter

middle: S

last: Hiemstra

first: Nicole

last: Datson