title: TRANSCRIPTIONAL PROFILING OF FUR– AND IRON- REGULATED GENE EXPRESSION IN LISTERIA MONOCYTOGENES

status: Public on Dec 25 2008

city: Normal

first: Arunachalam Muthaiyan

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

address: Julian Hall

department: Biological Sciences

institute: Illinois State University

summary: Iron is required by almost all bacteria but concentrations of iron above the physiological levels are toxic. In bacteria, intracellular iron is regulated mostly by ferric uptake regulator, Fur or a similar functional protein. Iron limitation results in regulation of number of genes, including those involved in iron uptake process. A subset of the genes under the control of Fur is called Fur regulon. In this study we have identified the Fur- and iron- regulated genes in Listeria monocytogenes by DNA microarray analysis using fur mutant and its isogenic parent. In order to identify genes exclusively regulated in response to iron limitation, fur mutant and its isogenic parent grown in iron-deficient KRM media were compared. Similarly, to identify Fur regulated genes, fur mutant was compared with the parent under iron-deficient and excess-iron conditions. Listeria in low-iron resulted in up-regulation of 180 and down-regulation of 200 genes whereas in the fur mutant, 79 genes were up-regulated and 49 genes down-regulated.  Our studies have identified at least 12 genes that are negatively controlled by Fur. In iron-deficient condition, these genes were up-regulated, while expression of fur was down-regulated. To further investigate the fur regulation, the fur promoter-lacZ transcriptional fusion strains were constructed in fur and perR mutant background. In the  perR mutant, the regulation of fur, svpA, and feoB was inhibited in the iron-limited conditions. Our results indicate that fur is negatively regulated by Fur and PerR. Furthermore, these results demonstrate that regulation of some of the genes in iron-limited conditions require PerR.

overall_design: Experiments were aimed to determine significant transcriptional changes of (a) EGDe strain in the absence of iron (b) fur mutant compared to EGDe in the absence of iron (c) fur mutant compared to EGDe in the presence of iron and (d) fur mutant in the absence of iron. In each transcriptional profiling experiment, labeled cDNA prepared from total RNA was hybridized to L. monocytogenes array slides. The data from the hybridizations for each of the above experiments were  normalized using MIDAS software using LOWESS algorithm to minimize biological and experimental variations. Significantly expressed or repressed genes were identified by estimating the false discovery rate (FDR) using significance analysis of microarrays (SAM) (Δ = 1.01; False discovery rate (FDR) at 5%; q < 0.05).

first: Nagender

last: Ledala

first: Arunachalam

last: Muthaiyan

first: Brian

last: Wilkinson

first: Radheshyam

last: Jayaswal


title: Effects of trenbolone, flutamide and a mix of both in fathead minnow ovaries

status: Public on Dec 23 2008

city: Vicksburg

first: Natalia

last: Vinas

state: MS

address: 3909 Halls Ferry Rd

country: USA

institute: Mississippi State University

summary: Trenbolone and flutamide are prototypical model compounds for respectively androgen and anti-androgen modes of action.  Trenbolone is an anabolic steroid used in cattle industry to increase weight gain and feed efficiency, and flutamide is a pharmaceutical used to treat prostate cancer.  Androgens exert profound effects on the organization, development, and function of the male reproductive system through activation of androgen receptors (ARs).  Flutamide, as an antiandrogen, was expected to temper the effects of trenbolone on androgen receptor (AR)-mediated gene expression.  Female fathead minnows (Pimephales promelas) were exposed via the water to 0.05, 0.5 or 5 µg 17β-trenbolone/L; 50, 150 or 500 µg flutamide/L; or to a mixture of 0.5 μg/L trenbolone and 500 μg/L flutamide.  After a 48 hr constant exposure gene expression in gonads was analyzed using a fathead minnow-specific 22,000-gene microarray.  Trenbolone significantly changed the expression (P < 0.05) of roughly 750 different genes, while flutamide changed the expression of 1420 genes.  Most of the genes that were regulated were distinct for the two chemicals.  However, 70 genes were reciprocally regulated by the two treatments.  Myelocytomatosis viral oncogene homolog (Myc), Yin Yang 1 (YY1) and interferon regulator factor 1 (IRF1) were among the important transcription factors reciprocally regulated by trenbolone and flutamide, suggesting they may be specifically regulated by the AR.  These regulatory molecules, in conjunction with other reciprocally regulated transcription factors, may function as early molecular switches to control phenotypic changes in gonad tissue architecture and function.

overall_design: Fathead minnows exposed for 48h to 0.5 ug trenbolone, 500 ug flutamide, or  a mixture of both.

middle: G

last: Reyero Vinas

first: Daniel

middle: L

last: Villeneuve

first: Kevin

middle: J

last: Kroll

first: Li

last: Liu

first: Edward

last: Orlando

first: Karen

middle: H

last: Watanabe

first: Maria

last: Sepulveda

first: Gerald

middle: T

last: Ankley

first: Nancy

middle: D

last: Denslow


title: A library of yeast transcription factor motifs

status: Public on Dec 26 2008

city: Toronto

first: Lourdes

last: Pena-Castillo

state: Ontario

address: 160 College St. Room 1350

department: Banting and Best Department of Medical Research

country: Canada

institute: University of Toronto

type: Genome binding/occupancy profiling by genome tiling array

summary:
The sequence specificity of DNA-binding proteins is the primary mechanism by which the cell recognizes genomic features. Here, we describe systematic determination of yeast transcription factor DNA-binding specificities. We obtained binding specificities for 112 DNA-binding proteins representing 19 distinct structural classes. One-third of the binding specificities have not been previously reported. Several binding sequences have striking genomic distributions relative to transcription start sites, supporting their biological relevance and suggesting a role in promoter architecture. Among these are Rsc3 binding sequences, containing the core CGCG, which are found preferentially ~100 bp upstream of transcription start sites. Mutation of RSC3 results in a dramatic increase in nucleosome occupancy in hundreds of proximal promoters containing a Rsc3 binding element, but has little impact on promoters lacking Rsc3 binding sequences, indicating that Rsc3 plays a broad role in targeting nucleosome exclusion at yeast promoters.

Keywords: Protein binding microarrays, DNA, proteins

platform_organism:
Saccharomyces cerevisiae
synthetic construct

overall_design:
Protein binding microarray (PBM), ChIP-chip and DIP-chip experiments of yeast transcription factor DNA-binding domains were performed. Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to custom-designed, double-stranded 44K Agilent microarrays in order to determine their sequence preferences. The method is described in Berger et al., Nature Biotechnology 2006. A key feature is that the microarrays are composed of de Bruijn sequences that contain each 10-base sequence once and only once, providing an evenly balanced sequence distribution. Individual de Bruijn sequences have different properties, including representation of gapped patterns.  Here we provide the data transformed into median intensities for all 32,896 8-base sequences, Z-scores for these intensities, and E-scores. E-scores are a modified version of AUC, and describe how well each 8-mer ranks the intensities of the spots. In general the E-scores are slightly more reproducible than Z-scores, but contain less information about relative binding affinity. Additional experimental details are found in Berger et al., Nature Biotechnology 2006, Berger et al., Cell 2008, and the accompanying Supplementary information. Raw 35-mer array data is available on the web link provided.

For the DIP-chip experiments [GSM345371, GSM345403, GSM345414-GSM345421, GSM345429-GSM345432], genomic DNA isolated from S288C yeast was incubated with 40nM of the MBP-tagged DNA binding domain (DBD) of either Cbf1, Pho2, Pho4, Leu3, Rap1, or Swi5 and incubated for 30 minutes prior to purification of protein-DNA complexes.  The bound DNA was then isolated, amplified via Invitrogen's WGA protocol, and hybridized against input DNA on NimbleGen 385k 32bp-tiling whole genome arrays.  ChIPOTle was used to identify peaks of binding from the data and motifs were identified by BioProspector and MDScan and then scored for their ability to predict the identified peaks by GOMER.  Motifs with the best ROC AUC are reported in the paper.

For the ChIP-chip experiments [GSM346493 and GSM346494], isogenic wildtype and rsc3-1 strains carrying Rsc8-TAP were grown in parallel under rsc3-1 restrictive growth conditions (37°C). Following formaldehyde crosslinking, cells were homogenized and extracts were sonicated to shear the chromatin to an average size of ~500 bp. A single pulldown was then performed with IgG sepharose beads and after decrosslinking and LM-PCR amplification of purified IP DNA, samples were labeled and hybridized on Nimblegen 32bp whole genome tiling arrays, comparing the pulled-down DNA to input genomic DNA.

first: Gwenael

last: Badis

first: Esther

last: Chan

first: Harm

last: van Bakel

first: Desiree

last: Tillo

first: Kyle

last: Tsui

first: Clayton

last: Carlson

first: Andrea

last: Gossett

first: Michael

last: Hasinoff

first: Christopher

last: Warren

first: Marinella

last: Gebbia

first: Shaheynoor

last: Talukder

first: Ally

last: Yang

first: Sanie

last: Mnaimneh

first: Dimitri

last: Terterov

first: David

last: Coburn

first: Ai

last: Yeo

first: Zhen

middle: X

first: Neil

last: Clarke

first: Jason

last: Lieb

first: Aseem

middle: Z

last: Ansari

first: Corey

last: Nislow

first: Timothy

middle: R

last: Hughes


title: A Genetic Interaction Map of RNA Processing Factors Reveals Links Between mRNA Processing Complexes

status: Public on Dec 31 2008

city: San Francisco

first: Megan

last: Bergkessel

state: CA

department: Biochemistry

institute: University of California, San Francisco

summary:
All classes of RNA undergo processing. The two largest RNA processing complexes are those involved in rRNA biogenesis and mRNA splicing. Because of their complexity and essential roles, it has been difficult to dissect the functional interactions within these machines as well as to identify possible cross-talk with other steps in gene expression. Here we report the results of a high-density, quantitative genetic interaction map comprising 107,155 individual interactions involving 552 mutants, 166 of which are hypomorphic alleles of essential genes (This data is available at The Krogan Lab Interactome Database). Our data allow unique insights into the functional organization of the RNA processing machines. For example, we identify unexpected connections involving a component of the 19S proteasome, Sem1/Dss1. We show that Sem1 has genetic, physical and functional interactions with the mRNA export complex, Thp1-Sac3; it also interacts physically with a putative component of the COP9 signalosome, Csn12. We show using splicing-specific microarrays that Csn12 has an unanticipated role in mRNA splicing. Finally, we demonstrate the utility of this dataset in determining functions for uncharacterized ORFs.  We show that deletion of the uncharacterized YNR004W causes a very similar splicing defect to that caused by deletion of TGS1, the enzyme that trimethylates the caps of snRNAs and snoRNAs.

Keywords: E-MAP, RNA Processing, splicing, splicing-specific microarray, CSN12, COP9 signalosome

overall_design: Splicing-specific microarrays were used to assay the changes to splicing caused by deletion of several non-essential genes which had no previously characterized role in mRNA splicing but were suggested to be involved in this process by high-throughput genetic and/or protein-protein interaction data in Saccharomyces cerevisiae. The data includes samples collected from some mutants in log phase growth at 30 degrees C and from some mutants shifted to 16 degrees C, all competitively hybridized against wild type samples collected under the same conditions. .

first: Gwendolyn

middle: M

last: Wilmes

first: Sourav

last: Bandyopadhyay

first: Anna

last: Chan

first: Hannes

last: Braberg

last: Shales

first: Sean

last: Collins

first: Gregg

middle: B

last: Whitworth

first: Tracy

last: Kress

first: Jonathan

middle: S

last: Weissman

first: Trey

last: Ideker

first: Christine

last: Guthrie

first: Nevan

last: Krogan


title: Nutrient-Regulated Antisense and Intragenic RNAs Modulate a Signal Transduction Pathway

city: Bunkyo-ku

first: Katsuhiko

last: Shirahige

state: Tokyo

address: 1-1-1 Yayoi

department: Research Center for Epigenetic Disease

country: Japan

institute: The University of Tokyo

summary: The budding yeast Saccharomyces cerevisiae alters its gene expression profile in response to a change in nutrient availability. The PHO-system is a well-studied case in the transcriptional regulation responding to nutritional changes in which a set of genes (PHO genes) is expressed to activate phosphate (Pi) metabolism for adaptation to Pi-starvation. Pi-starvation triggers an inhibition of Pho85 kinase, leading to migration of unphosphorylated Pho4 transcriptional activator into the nucleus enabling expression of PHO genes. When Pi is sufficient, the Pho85 kinase phosphorylates Pho4 thereby excluding it from the nucleus and resulting in repression (i.e., lack of transcription) of PHO genes. The Pho85 kinase has a role in various cellular functions other than regulation of the PHO system, in that Pho85 monitors whether environmental conditions are adequate for cell growth, and represses inadequate (untimely) responses in these cellular processes. In contrast, Pho4 appears to activate some genes involved in stress-response and is required for G1 arrest caused by DNA damage. These facts suggest the antagonistic function of these two players on a more general scale when yeast cells must cope with stress conditions. To explore general involvement of Pho4 in stress-response, we tried to identify Pho4-dependent genes by a genome-wide mapping of Pho4- and Rpo21-binding (Rpo21 being the largest subunit of RNA polymerase II) using a yeast tiling array. In the course of this study, we found Pi- and Pho4-regulated intragenic and antisense RNAs that could modulate the Pi-signal transduction pathway. Low-Pi signal is transmitted via certain inositol polyphosphate (IP) species (IP7) that are synthesized by Vip1 IP6 kinase. We have shown that Pho4 activates transcription of antisense and intragenic RNAs in the KCS1 locus to downregulate the Kcs1 activity, another IP6 kinase, by producing truncated Kcs1 protein via hybrid formation with the KCS1 mRNA and translation of the intragenic RNA, thereby enabling Vip1 to utilize more IP6 to synthesize IP7 functioning in low-Pi signaling. Since Kcs1 also can phosphorylate these IP7 species to synthesize IP8, reduction in the Kcs1 activity can ensure accumulation of the IP7 species, leading to further stimulation of low Pi-signaling, i.e., forming a positive feedback loop. We also report that genes apparently not involved in the PHO system are regulated by Pho4 either dependent upon or independently of the Pi conditions, and many of the latter genes are involved in stress-response. In S. cerevisiae, a large-scale cDNA analysis and mapping of RNA polymerase II binding using a high-resolution tiling array have identified a large number of antisense RNA species whose functions are yet to be clarified. Here we have shown that nutrient-regulated antisense and intragenic RNAs, as well as direct regulation of structural gene transcription, function in the response to nutrient availability. Our findings also imply that Pho4 is present in the nucleus even under high-Pi condition to activate or repress transcription, which challenges our current understanding of Pho4 regulation.

overall_design: Wild type and pho4 deletion mutant were grown in low or high phosphate medium and distribution of Rpo21 was analyzed.

first: Masafumi

last: Nishizawa

first: Tae

last: Komai

first: Yuki

last: Katou

first: Takehiko

last: Itoh

first: Akio

last: Toh-e


title: Characterization of human embryonic stem cells with features of neoplastic progression

city: Hamilton

first: Mickie

last: Bhatia

address: 1200 Main Street West

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

type:
Expression profiling by array
Genome variation profiling by array

summary:
Cultured human embryonic stem (hES) cells can acquire genetic and epigenetic changes that make them vulnerable to transformation. As hES cells with cancer-cell characteristics share properties with normal hES cells, such as self-renewal,
teratoma formation and the expression of pluripotency markers, they may be misconstrued as superior hES cells with enhanced ‘stemness’. We characterize two variant hES cell lines (v-hESC-1 and v-hESC-2) that express pluripotency
markers at high levels and do not harbor chromosomal abnormalities by standard cytogenetic measures. We show that the two lines possess some features of neoplastic progression, including a high proliferative capacity, growth-factor
independence, a 9- to 20-fold increase in frequency of tumor initiating cells, niche independence and aberrant lineage specification, although they are not malignant. Array comparative genomic hybridization revealed an amplification
at 20q11.1-11.2 in v-hESC-1 and a deletion at 5q34a-5q34b;5q3 and a mosaic gain of chromosome 12 in v-hESC-2. These results emphasize the need for functional
characterization to distinguish partially transformed and normal hES cells.

overall_design:
Custom oligonucleotide array gene expression analysis was performed on total RNA from low passage normal and variant human embryonic stem cell (hESC) lines, all cultured in our laboratory under the same conditions. Furthermore, array-based comparative genomic hybridization was also performed on the normal and variant hESC lines.

v-hESC-1 and v-hESC-2 represent two independent lines that display neoplastic characteristics with distinctive copy number alterations.

v-hESC-1_A2B5 represents neural precursor cultures differentiated from variant hES cells.

GSM349016-GSM349022: Gene expression analysis
GSM351500-GSM351511: aCGH analysis

first: Tamra

middle: E

last: Werbowetski-Olgilvie

first: Marc

last: Bosse

first: Morag

last: Stewart

first: Angelique

last: Schnerch

first: Veronica

last: Ramos-Mejia

first: Anne

last: Rouleau

last: Wynder

first: Mary-Jo

last: Smith

first: Steve

last: Dingwall

first: Tim

last: Carter

first: Christopher

last: Williams

first: Charles

last: Harris

first: Joanna

last: Dolling

first: Doug

last: Boreham


title: Transcriptome analysis of the Aspergillus fumigatus calcineurin

city: Ribeirão Preto

first: Gustavo

middle: Henrique

last: Goldman

state: São Paulo

address: Av do Café, s/n

country: Brazil

institute: Faculty of Pharmaceutical Sciences of Ribeirão Preto

summary: Calcineurin plays an important role in the control of cell morphology and virulence in fungi. Calcineurin is a serine/threonine-specific protein phosphatase heterodimer consisting of a catalytic subunit A and a regulatory Ca2+/Calmodulin binding subunit. A mutant of A. fumigatus lacking the calcineurin A (calA) catalytic subunit exhibited defective hyphal morphology related to apical extension and branching growth, which resulted in drastically decreased filamentation. Here, we investigated which pathways are influenced by A. fumigatus calcineurin during proliferation by comparatively determining the transcriptional profile of A. fumigatus wild type and delta calA mutant strains. Our results showed that although the mitochondrial function is reduced in the delta calA mutant strain, its respiratory chain is functional and the mutant has increased alternative oxidase (aoxA) mRNA accumulation and activity. Furthermore, we identified several genes that encode transcription factors that have increased mRNA expression in the delta calA mutant and that could be involved in the Cal-CrzA pathway. Deletion mutants for these transcription factors had also reduced susceptibility to itraconazole, caspofungin, and sodium dodcyl sulfate.

overall_design:
For the time course microarray experiments, 1.0 x 109 conidia of both A. fumigatus wild type [CEA17 delta akuB(KU80)] and mutant strain (delta calA) were used to inoculate 400 ml of pre-warmed liquid cultures in complete medium (YG) in  1 L erlenmeyer flasks. Cultures were allowed to grow for 6, 8, 12, 18 and 24 hours in a reciprocal shaker (250 rpm) at 37°C. At each time point, germilings were harvested by centrifugation or filtration and total RNA was extracted. Hybridization experiments were competitive using Cy3 or Cy5-labeled probes derived from both wild type and delta calA strain grown for 6, 8, 12, 18 and 24 hours. Hybridizations were performed using the wild type RNA as reference in comparison to the mutant strain (test RNA) for the same time point. Normalized signal intensities were used to generate relative hybridization ratios (query/reference).

Following normalization, the values for each gene in-slide replicates were condensed (median variance <0.01), and corresponding flip-dye replicates were averaged to compensate for dye-specific effects (see Supplementary files linked below).

first: Iran

last: Malavazi

first: Marcia

last: Silva Ferreira

first: Frederico

last: Soriani

first: Marcela

last: Savoldi

first: Sérgio

middle: A

last: Uyemura

first: Maria

last: Souza Goldman


title: A single SR-like protein, Npl3, promotes pre-mRNA splicing in budding yeast

first: Tracy

last: Kress

address: 600 16th Street, Box 2200

department: Biochemisty and Biophysics

summary: Mammalian SR proteins are a family of reversibly phosphorylated RNA binding proteins primarily studied for their roles in alternative splicing. While budding yeast lack alternative splicing, they do have three SR-like proteins: Npl3, Gbp2, and Hrb1. However, these have been primarily studied for their roles in mRNA export, leaving their potential roles in splicing largely unexplored. Here we combined high-density genetic interaction profiling and genome-wide splicing-sensitive microarray analysis to demonstrate that a single SR-like protein, Npl3, is required for efficient splicing of a large set of pre-mRNAs in Saccharomyces cerevisiae. We tested the hypothesis that Npl3 promotes splicing by facilitating co-transcriptional recruitment of splicing factors. Using chromatin immunoprecipitation, we showed that mutation of NPL3 reduces the occupancy of U1 and U2 snRNPs at Npl3-stimulated genes. This provides the first evidence that an SR protein can promote recruitment of splicing factors to chromatin.

overall_design: Splicing-specific microarrays were used to assay changes to splicing in single and double deletion mutants of non-essential SR proteins, in a deletion mutant of a non-essential component of the nonsense-mediated decay pathway, and in a double deletion mutant of in an SR protein plus a non-sense mediated decay factor in Saccharomyces cerevisiae.  The data includes both samples obtained at the permissive temperature and also shifts to the non-permissive temperature for some mutants, as well as dye-flipped technical replicates.

first: Nevan

last: Krogan

first: Christine

last: Guthrie


title: The expression profiles analysis of a virulence plasmid-cured strain of shigella flexneri

status: Public on Dec 22 2008

city: Beijing

first: Li

last: Zhu

address: 20 Dongdajie, Fengtai District

department: State Key Laboratory of Pathogens and Biosecurity

country: China

institute: Beijing Institute of Biotechnology

summary: In this study, we analyzed the expression profiles of a virulence plasmid-cured strain and wild-type strain of shigella flexneri. The results showed that the genes of glp regulon were upregulated in mutant bacteria in stationary phase cultures.

overall_design: The two strains were cultured in LB broth into log-phase and stationary phase respectively. Then, the total RNAs were extracted and analyzed by Nimblegen biochips.

first: Hengliang

last: Wang


title: Transcriptional profiling of Escherichia coli after addition of CO-RMs to anaerobically growing cells

city: Sheffield

first: Robert

last: Poole

state: South Yorkshire

address: Firth Court, Western Bank

department: Molecular Biology & Biotechnology

country: United Kingdom

institute: University of Sheffield

summary: Escherichia coli strain MG1655 was grown to mid-log phase in defined anaerobic media. One sample was treated with 100 µM CORM-3, the other sample was a control. The volume (175 ml), temperature (37oC) and stirring (200 rpm) were constant. After 15 min of exposure to CO-RM, samples were taken from treated and control cells, harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats.

overall_design:
Cells were grown in defined minimal medium (anaerobic). Final concentrations are: glycerol (54 mM), K2HPO4 (23 mM), KH2HPO4 (7.3 mM), NH4Cl (18.7 mM), CaCl2 (69 µM), K2SO4 (15 mM), sodium fumarate (50 mM), Luria Broth (5%) and can amino acids (0.1%). Trace elements were added at 10 ml per l; final concentrations are: 134 µM EDTA, 31 µM FeCl3, 6.15 µM ZnO, CuCl2 0.57 µM, CoNo3 0.34 µM, 1.6 µM H3BO3, 1 µM ammonium molybdenate and 1 µM sodium selenite. MgCl2 was added at 1 ml per l. All chemicals were of AnalaR grade of purity or higher.

E. coli strain MG1655 was grown in mini fermenter vessels in 175 ml of the above defined minimal media (anaerobic). The vessel was continually sparged with N2 to ensure anaerobiosis. CORM-3 was added at a final concentration of 100 µM, chosen because it causes only a slight reduction in the growth rate. Cells were grown to mid-log (OD600 of 0.4) before addition of CO-RM. For RNA extraction, 30 ml was harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats.

Equal quantities of RNA from control and CO-RM-treated cells were labelled by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer).  For each microarray slide, one sample was labelled with Cy3-dCTP, while the other sample was labelled with Cy5-dCTP.  RNA (approximately 9 micrograms) was annealed to 4.5 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC and cooling on ice for 2 min.  This was supplemented with 6 microlitres of 5× First-strand buffer (Invitrogen), 2.5 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 3 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1.5 microlitre Superscript III reverse transcriptase (200 U) (Invitrogen).  This was incubated at 42 oC for 3 hours.  The reaction was terminated by the addition of NaOH (7.5 microlitres of 1M) and HCl (7.5 microlitres of 1M) before the addition of TE buffer (300 microlitres).  Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen).

The Cy3-dCTP-labelled sample was mixed with the Cy5-dCTP-labelled sample, vacuum dried and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides).  This was heated at 95 oC for 3 min then cooled on ice for 3 min.  The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 °C.  Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 °C) SSC buffers for 5 min each at 37oC: 2× SSC/0.1 % SDS, 1× SSC, 0.2× SSC and 0.01× SSC.  Microarray slides were dried by centrifugation at 1500 × g for 2 min before scanning.

Slides were scanned on an Affymetrix 428 scanner.  The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5).

Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight.  The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values.  The Cy3/Cy5 fluorescent ratios were calculated from the normalized values.

Biological experiments (i.e. a comparison of control and plus CO-RM cells) were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats.  Data from the independent experiments were combined. Genes that were differentially expressed ≥ twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.


title: Transcriptional profiling of Escherichia coli after addition of CO-RMs to aerobically growing cells

summary: Escherichia coli strain MG1655 was grown to mid-log phase in defined aerobic media. One sample was treated with 30 µM CORM-3, the other sample was a control. The volume (30 ml), temperature (37oC) and shaking (200 rpm) were constant. After 15 min of exposure to CO-RM, samples were taken from treated and control cells, harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats.

overall_design:
Cells were grown in defined minimal medium (aerobic). Final concentrations are: glycerol (54 mM), K2HPO4 (23 mM), KH2HPO4 (7.3 mM), NH4Cl (18.7 mM), CaCl2 (69 µM), and K2SO4 (15 mM). Trace elements were added at 10 ml per l; final concentrations are: 134 µM EDTA, 31 µM FeCl3, 6.15 µM ZnO, CuCl2 0.57 µM, CoNo3 0.34 µM, 1.6 µM H3BO3, 1 µM ammonium molybdenate and 1 µM sodium selenite. MgCl2 was added at 1 ml per l. All chemicals were of AnalaR grade of purity or higher.

E. coli strain MG1655 was grown in 250 ml side arm flasks in 30 ml of the above defined minimal media (aerobic). CORM-3 was added at a final concentration of 30 µM, chosen because it causes only a slight reduction in the growth rate. Cells were grown to mid-log (Klett values of 30-40) before addition of CO-RM. The total 30 ml volume was harvested into ice cold phenol ethanol (187 µl phenol, 3.56 ml ethanol) to stabilize RNA, and total RNA was purified using Qiagen’s RNeasy Mini kit as recommended by suppliers. RNA was quantified using a BioPhotometer (Eppendorf). Biological experiments were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats.

Equal quantities of RNA from control and CO-RM-treated cells were labelled by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer).  For each microarray slide, one sample was labelled with Cy3-dCTP, while the other sample was labelled with Cy5-dCTP.  RNA (approximately 9 micrograms) was annealed to 4.5 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC and cooling on ice for 2 min.  This was supplemented with 6 microlitres of 5× First-strand buffer (Invitrogen), 2.5 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 3 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1.5 microlitre Superscript III reverse transcriptase (200 U) (Invitrogen).  This was incubated at 42 oC for 3 hours.  The reaction was terminated by the addition of NaOH (7.5 microlitres of 1M) and HCl (7.5 microlitres of 1M) before the addition of TE buffer (300 microlitres).  Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen).

The Cy3-dCTP-labelled sample was mixed with the Cy5-dCTP-labelled sample, vacuum dried and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides).  This was heated at 95 oC for 3 min then cooled on ice for 3 min.  The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 °C.  Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 °C) SSC buffers for 5 min each at 37oC: 2× SSC/0.1 % SDS, 1× SSC, 0.2× SSC and 0.01× SSC.  Microarray slides were dried by centrifugation at 1500 × g for 2 min before scanning.

Slides were scanned on an Affymetrix 428 scanner.  The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5).

Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight.  The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values.  The Cy3/Cy5 fluorescent ratios were calculated from the normalized values.

Biological experiments (i.e. a comparison of control and plus CO-RM cells) were carried out four times, and a dye swap performed for each experiment, providing two technical repeats for each of the four biological repeats.  Data from the independent experiments were combined. Genes that were differentially expressed ≥ twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.


title: Gene expression profiling during storage root formation in sweet potato

city: Seibersdorf

first: Silvia

last: Fluch

address: Campus Seibersdorf

department: Biogenetics/Natural Resources

country: Austria

institute: Austrian Research Centers GmbH

summary:
A Microarray experiment was carried out in order to establish the genetic processes and control mechanism involved during storage root formation in Sweetpotato. A Sweetpotato cDNA chip was created from five varieties covering all the growth stages between them. mRNA from primordial root, fibrous root, pencil root and thick storage root was extracted from four varieties of Sweetpotato. The expression profiles were compared between the root growth stages.

Keywords: Transcription profiling

overall_design: One condition experiment with time as a single parameter. Four different varieties of Sweetpotato (SPK004, Beauregard, Tanzania and Feng Shou Bai) with four growth stages (i.e. 3 weeks, 6 weeks, 10 weeks and 16 weeks) each were used for the comparison. Two pairs of biological replicates and one dye swap for each time point and variety were taken.

first: D

last: Desai

first: M

last: Berenyi

first: S

last: Kopecky

first: W

last: Grueneberg

first: J

last: Sarmiento

middle: P

last: Zhang

last: Bonierbale

first: R

last: Schafleitner

first: K

last: Burg


title: AML1/ETO binding pattern on human promoters

city: Milan

first: Myriam

last: Alcalay

address: Via Adamello 16

department: Experimental Oncology

country: Italy

institute: European Institute of Oncology

summary:
Approximately 20% of Acute Myelogenous Leukemia (AML) cases carry the t(8;21) translocation, which involves the AML1 and ETO genes, and express the resulting AML1/ETO fusion protein that functions as a transcriptional repressor by recruiting NCoR/SMRT/HDAC complexes to DNA.
We used microarrays to identify human promoters bound by AML1/ETO in U937 cells.
Keywords: ChIP-chip

overall_design: A U937 cell line that conditionally expresses HA-tagged AML1/ETO under the control of the mouse metallothionine promoter (U937-A1E) (Alcalay et al., J.Clin.Invest, 2003,112, 1751-1761) was used. Cells were treated for 8h with 100uM ZnSO4 to induce transgene expression, and ChIP was performed using an anti-HA antibody. ChIP products were then PCR amplified, labeled with Cy3/Cy5 fluorescent dyes and hybridized to the NimbleGen HG17 Human Promoter 2 Array set, which explores 4 kb upstream and 1 kb downstream the transcription start site (TSS) of 24,434 annotated genes. Two biological replicates were prepared and hybridized to independent array sets. U937-Mt cells, which carry the empty vector, served as control for non-specific binding of the anti-HA antibody.


title: Human colon expression in healthy controls and UC

city: Cincinnati

first: Lee

last: Denson

state: OH

address: 3333 Burnet Avenue

institute: CCHMC

summary:
Activation of inflammatory pathways in human IBD.  Leukocyte recruitment pathways including those for eosiniphils  are activated in the affected colon in IBD. However, the functional implications of this are not known. We hypothesized that pro-inflammatory eotaxin (CCL11) dependent networks would be up regulated in the colon of pediatric patients with Ulcerative Colitis (UC), and that these would regulate eosinophil recruitment to the gut. These experiments tested differential colon gene expression relative to these pathways in healthy and UC samples. Colon biopsy samples were obtained from UC patients at diagnosis, and healthy controls. The global pattern of gene expression was determined using GeneSpring software, and biological networks were identified using Ingenuity software. Data suggested that a leukocyte recruitment network which includeds CCL11 is up regulated in pediatric UC at diagnosis. The degree of up regulation of these genes compared to healthy controls was remarkably conserved within the UC patient group, suggesting common mechanisms of mucosal inflammation.
These experiments tested differential colon gene expression relative to these pathways in healthy and UC samples.
Keywords: Single time point in UC and healthy controls.

overall_design: Colon RNA was isolated from biopsies obtained from UC at diagnosis and healthy controls. Samples were obtained from the most proximal affected segment of colon. Microarray experiments were performed as described in the CCHMC microarray core, and data was analyzed as described above in the summary. The '107' internal control CEL files (for batches 1,2,4,5) used for ormalization of the Sample VALUEs are also contained within this data set.


title: Gene expression profiling of PmrA (lpg1292) and PmrB (lpg1291) mutants of L. pneumophila

city: New York

first: Sergey

last: Kalachikov

state: NY

address: NWC Bld, 550 West 120th Street Rm 1002

department: Chemical Engineering

institute: Columbia University

summary: The purpose of the study was to examine the role of the elements of PmrA/PmrB two-component system in the global regulation of gene expression in L.pneumophia during axenic growth. Towards this goal pmrA and pmrB isogenic mutants were constracted in L. pneumophila strain AA100 using allelic exchange. Gene expression profiles were generated for both mutants during their growth in axenic media and compared to the corresponding gene expression profiles in the ancestral strain.

platform_organism: Legionella pneumophila subsp. pneumophila str. Philadelphia 1

overall_design:
PmrA mutant: three independently axenically grown samples were analyzed in mid-exponential and postexponential phases of growth.

PmrB mutant: three independently axenically grown samples were analyzed in mid-exponential and postexponential phases of growth.

A100 strain: three independently axenically grown samples were analyzed in mid-exponential and postexponential phases of growth as a reference for the PmrA and PmrB mutants.

first: Souhaila

last: Al-Khodor

first: Irina

last: Morozova

first: Christopher

last: Price

first: Yousef

last: Abu Kwaik


title: Expression Profiling in Selenite-induced Proliferation of HuH7 cells

city: Taipei

first: Ann-Ping

last: Tsou

address: 155, Li-Nong St., Sec.2

department: Inst. Biotechnology in Medicine

country: Taiwan

institute: National Yang-Ming University

summary:
Experimental studies confirmed that seleium induced a global change of gene expression in HuH7 cells. Several novally induced secretory growth factors may contribute to the autocrine mode of growth elicited by selenium treatment.
Keywords: Treatment

overall_design: HuH7 were cultured in 0.1 μM sodium selenite-containing medium for 15 passages (SELT) or in serum-free medium for 30 h (SF30) or in the regular complete medium containing 10 % fetal calf serum (HUH7) for 30 h. The total RNA were reverse transcried and hybridized to Human 8K-1 (Egenomix Technology Corp., Taiwan). The hybridization time and temperature was 16 h and 62℃, respectively. SELT and HUH7 samples were labeled with Cy5 while SF30 sample was labeled with Cy3. Local background correction and interchip global normalization were accomplished by GenePix Pro 5.0 (Molecular Devices, Sunnyvale, CA ).  Genes with differential up- or down-regulation by 1.5 fold with respect to the SF30 cells were obtained using GeneSpring software 7.0 (Silicon Genetics, Agilent Technologies).

first: Yi-Hsien

last: Chen

first: Yu-Chun

last: Lin

first: Cha-Ming

last: Chang

first: Yu-Chi

last: Lee


title: Transcriptional profiling of Zea mays 10 DAP(days after pollination) kernels :  water stress vs well-watered control

city: Milano

first: Rosanna

last: Marino

state: Milano

address: Via celoria 26

department: Department of biomolecular sciences and biothecnology

institute: Universita' degli studi di Milano

summary:
A customized targeted oligoarray was used to monitor the expression levels of 1000 genes, representative of the immature kernel transcriptome.
Using this oligoarray we compared transcripts from 10 DAP kernels of two susceptible and two drought tolerant genotypes.
These four genotypes were extracted from our RIL population (B73xH99) and grown under water stress and well watered field conditions.

Keywords: Stress response

overall_design: Two-condition experiment: water stress field condition vs. well watered field condition. Transcriptional profiling of 10 DAP kernel (two susceptible and two drought tolerant genotypes) in the first condition vs 10 DAP kernel (two susceptible and two drought tolerant genotypes) in the second condition. Hybridization replicates: 4 in dye swap mode. Two probes per gene per array.

first: Maharajah

last: Ponnaiah

first: Pawel

last: Krajewski

first: Carla

last: Frova

first: Luca

last: Gianfranceschi

first: Enrico

last: Pè

first: Mirella

last: Sari-Gorla


title: Analysis of the CcpA regulon in Staphylococcus aureus

status: Public on Dec 20 2008

city: Geneva

first: FRANCOIS

last: Patrice

state: Ge 4

address: Rue Gabrielle-Perret-Gentil, 4

department: Service of Infectious Diseases

country: Switzerland

institute: Genomic Research Laboratory

summary:
Background
The catabolite control protein A (CcpA) is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram positive bacteria. It functions as a catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in S. aureus wild type and ccpA-deleted mutant, focussing on short-time effects of glucose under stable pH conditions.

Results
The addition of glucose to exponentially growing S. aureus increased enzymes of glycolytic pathway, indicating a higher glycolytic activity, while proteins required for the complete oxidation in the TCA cycle were repressed via CcpA.
Phosphotransacetylase and acetate kinase, converting acetylCoA to acetate with a concomitant substrate-level phosphorylation were neither regulated by glucose nor by CcpA. Most CcpA directly repressed genes were involved in utilization of amino acids as secondary carbon sources. More genes were found to be differentially expressed by CcpA in a glucose-independent manner than in the classical, glucose dependent way, suggesting that glucose-independent regulation by CcpA may be of
particular importance in S. aureus. In the presence of glucose, CcpA was
found to regulate expression of genes involved in metabolism, but that
of genes coding for virulence determinants.

Conclusions
This study identified the CcpA regulon of exponentially growing S. aureus, for the first time. As in other bacteria, the CcpA-regulon of S. aureus comprised a large amount of metabolic genes but also some 50 genes associated with virulence. CcpA seemed to work in a glucose- as well as glucose-independent way.

sample_organism: Staphylococcus aureus subsp. aureus str. Newman

overall_design:
The transcriptomes of strain Newman and its isogenic ccpA-deleted mutant were determined in early exponential growth and 30 min after the addition of 10 mM glucose, under controlled pH conditions. In the absence of glucose, the wild type grew slightly faster than the mutant, reaching an OD600 of 1 approximately 20 min earlier than the mutant.
Adding 10 mM glucose at OD600 1 increased the growth rate of the wild type but had only a minor effect on that of the mutant. 60 min after glucose addition, glucose was depleted down to 0.3 mM by the wild type, while still 3 mM glucose was left in the culture of the mutant. Despite increased glucose consumption rates in the wild type, acetate production was only slightly enhanced compared to the  mutant. No lactate was excreted at any time point sampled. Acidification of the  medium upon glucose metabolism was prevented by buffering, maintaining a pH of
7.5 for both strains and under both growth conditions for at least 2 h after glucose addition, allowing to rule out any pH effects.

first: Kati

last: Seidl

first: Susanne

last: Muller

first: Patrice

last: Francois

first: Jacques

last: Schrenzel

last: Engelmann

first: Markus

last: Bischoff

first: Brigitte

last: Berger-Bachi


title: CRCNA_ESgdIL21A2322

city: Nantes

first: Emmanuel

last: Scotet

address: 9 quai moncousu

country: France

institute: INSERM

summary:
Comparative gene expression analysis (PIQOR Immunology)
Human Vg9Vd2 T cells amplified (soluble phosphoantigen) from ex vivo fresh PBMC of healthy human donors
3 weeks of culture in the presence of either IL-2 or IL-2+IL-21
Total RNA extracted from resting purified gamma delta T cells
PIQOR microarrays

overall_design:
PIQOR Human Immunology Service Topic-defined microarrays (Miletnyi Biotec)
Comparison IL-2+IL-21 (Cy5) versus IL-2(Cy3)
T7-amplified RNA

first: Aurélie

last: Thedrez

first: Marc

last: Bonneville


title: Identification of Candidate Genes for Sporadic Amyotrophic Lateral Sclerosis by Array Comparative Genomic Hybridisation

city: Berlin

first: Reinhard

last: Ullmann

department: Human Molecular Genetics

country: Germany

institute: MPIMG

type: Genome variation profiling by genome tiling array

summary:
Background: Amyotrophic lateral sclerosis (ALS) is a devastating disorder of the central nervous system that leads to progressive loss of upper and lower motor neurons. Most cases are sporadic and of unknown aetiology. In this study, we screened 71 patients with sporadic ALS for the presence of DNA copy number variations, in order to identify novel candidate disease genes.
Methods: We have used sub-megabase resolution BAC array comparative genomic hybridisation to detect genomic imbalances in our ALS patient cohort. In order to distinguish phenotypically neutral copy number variations occurring frequently in the normal population from disease-associated aberrations, we screened our results against both the Database of Genomic Variants and a reference data set of approximately 700 normal individuals and probands with other disorders analysed in our laboratory. Aberrations with potential relevance for disease aetiology were verified by oligo array CGH.
Findings: In 71 patients with sporadic ALS, we identified a total of six duplications and seven deletions that scored above our threshold. Twelve of these thirteen variations were smaller than 1Mb. Seven were observed exclusively in ALS patients and thus likely play causal roles in the disorder. Analysis of these regions highlighted, for example, the SLC1A7 (EAAT5) glutamate transporter and the kinesin family member KIF9 as good positional and functional candidate genes for ALS.
Interpretation: Non-polymorphic sub-microscopic duplications and deletions observable by array CGH are frequent in patients with sporadic ALS. Analysis of such aberrations serves as an excellent starting point in deciphering the aetiology of this complex disease.

Keywords: array CGH

overall_design: A cohort of 71 ALS patients was analysed by means of a submegabase resolution BAC array. No replicates were included. Experiments were done without dye swap.


title: Effect of Leptin on Mouse Trophoblast Giant Cells

city: Columbia

first: Laura

middle: Clamon

last: Schulz

state: MO

address: 1 Hospital Dr.

department: Obstetrics, Gynecology and Women's Health

institute: University of Missouri

summary:
Leptin, a hormone produced primarily by adipose tissue, plays a role in both energy homeostasis and reproduction, and is required in early pregnancy.  Leptin stimulates metalloproteinase activity in cultured human trophoblast and stimulates invasiveness of cultured mouse trophoblast.  The goal of the present study was to examine molecular mechanisms of this function in primary cultures of mouse trophoblast.  Leptin was found to stimulate the phosphorylation of MEK, but not STAT3.. It also increased levels of the protein SOCS3.  The ability of leptin to stimulate metalloproteinase activity was blocked by the MEK inhibitor PD98059, but also by the vehicle inhibitor DMSO.  Microarray analysis revealed that leptin stimulated some genes associated with cell motility, such as Stmn1.  In addition, leptin appeared to inhibit changes in gene expression associated with terminal differentiation of trophoblast giant cells, including inhibition of members of the TGFß signaling pathway and of genes associated with endoreduplication.  However, feulgen staining revealed a loss of cells with low ploidy.  We conclude that leptin may be promoting trophoblast invasion by maintaining cells in an intermediate stage of differentiation.
Keywords: time course, response to hormone treatment, primary cell culture

overall_design: The experiments were performed on primary cultures of mouse trophoblast cells which were isolated from placentas on day 10 of pregnancy.   There were two treatments: control (serum-free medium) and recombinant mouse leptin (50 ng/mL).  RNA was collected at two time points, 1 hour and 24 hours, for the controls.  RNA was collected at three time points after leptin treatment: 1 hour, 6 hours, and 24 hours.  Thus, there were 5 samples for each experiment.  The experiment was repeated four times, for a total of 20 arrays.

middle: C

first: Eric

last: Widmaier

first: Jing

last: Qiu

first: R

last: Roberts


title: Caulobacter vibrioides CB15N mRNA expression in M2-Glucose Vs. M2-Inositol

city: East Lansing

first: Sean

last: Crosson

state: Michigan

address: 567 Wilson Rd

department: Dept. Microbiology and Molecular Genetics

institute: Michigan State University

summary: The purpose of this experiment was to examine the differential transcriptional profiles of Caulobacter CB15N grown in M2-Glucose versus M2-Inositol.

overall_design: mRNA expression was compared between Caulobacter CB15N cells grown in M2-Glucose vs. M2-Inositol. Four arrays were run, two of each dye combination.

first: Cara

last: Boutte

first: Balaji

last: Srinivasan

first: Jason

last: Flannick

first: Antal

middle: F

last: Novak

first: Andrew

last: Martens

first: Serafim

last: Batzoglou

first: Patrick

last: Viollier


title: RelA and RelB-dependent transcriptome analysis in lymphotoxin-ß receptor stimulated mouse embryonic fibroblasts

status: Public on Dec 18 2008

city: Jena

first: Daniela

last: Albrecht

address: Wildenbruchstr. 15

institute: BioControl Jena GmbH

summary:
Background: Lymphotoxin signaling via the lymphotoxin-β receptor (LTβR) has been implicated in several biological processes, ranging from development of secondary lymphoid organs, maintenance of splenic tissue, host defense against pathogens, autoimmunity, and lipid homeostasis. The major transcription factor that is activated by LTβR crosslinking is NF-κB. Two signaling pathways have been described that result in the activation of classical p50-RelA and alternative p52-RelB NF-κB heterodimers.
Results: Using microarray analysis, we investigated the transcriptional response downstream of the LTβR in mouse embryoni fibroblasts (MEF) and its regulation by the RelA and RelB subunits of NF-κB. We describe novel LTβR-responsive genes that are regulated by RelA and/or RelB. Interestingly, we found that the majority of LTβR-regulated genes require the presence of both RelA and RelB, suggesting significant crosstalk between the two NF-κB activation pathways. Gene Ontology (GO) analysis confirmed that LTβR-NF-κB target genes are predominantly involved in the regulation of immune responses. However, other biological processes, such as apoptosis/cell death, cell cycle, angiogenesis, and taxis were also regulated by LTβR signaling. Moreover, we show that activation of the LTβR inhibits the expression of a key adipogenic transcription factor, peroxisome proliferator activated receptor-γ (pparg), suggesting that LTβR signaling may interfere with adipogenic differentiation.
Conclusions: Thus, microarray analysis of LTβR-stimulated fibroblasts revealed further insight into the transcriptional response of LTβR signaling and its regulation by the NF-κB family members RelA and RelB.

Keywords: cell type comparison (wt vs relA-/- vs relB-/-) after genetic modification using a time course

overall_design: for each cell type (wt, relA-/-, relB-/-) two time points were analysed (0h as control and 10h) using 3 technical replicates resulting in 18 samples in total

first: Agnes

last: Lovas

first: Dörte

last: Radke

first: Buket

last: Yilmaz

first: Ulrich

last: Möller

first: Andreas

last: Habenich

first: Falk

last: Weih


title: The modulation of GBE50 and Lovastatin to cultured HepG2 cells

city: shanghai

first: Zhang

last: Qinghua

address: 151# libing road

institute: Shanghai shbiochip co

summary:
This study was designed to investigate the modulation of GBE on cellular cholesterol metabolism which has been suggested in recent studies. To explore the molecular mechanisms gain insights into the molecular network, we used microarray to high-throughput measurement of genes concerning both spatial and temporal levels to understand the complexity and dynamics of cells in response to GBE and Lovastatin, the genes expression pattern were similar between GBE and Lovastatin treatment in HepG2 cells, the major changed genes were involved in biosynthesis of steroids and cell cycle. Cholesterol metabolism related genes were further examined by RT-PCR and western blot, showing that the cholesterogenic genes were upregulated in response to the reduction of cholesterol and were consistent with the microarray findings.

Keywords: time course

overall_design:
HepG2 cells were maintained in Minimum Essential Medium supplemented with 10% FBS in humidified atmosphere with 5% CO2 at 37°C. For experiments, cells were seeded in 6-well plates at a density of 4×105 cells per well, when the cells were grown into a 70 % confluence, the medium was replaced with 2%FBS medium for later drug treatment, cells were incubated with 200μg/mL of GBE or 0.5μM Lovastatin for various time periods (2, 6, 12, 24hours). The control groups were treated with 0.1% DMSO that contained in drug as vehicle at most time point. The cells were collected and RNA were used for transcript profiling analysis.

Label protocol:Total RNA were primed with T7 Promoter Primer (from the Agilent Low RNA Input Linear Amplification Kit PLUS, Two-Color) at 65 ℃ for 10 min, then reversed transcribed at 40℃ for 2 h in the presence of 1ul MMLV-RT (Agilent), and 10 mM dNTP, and RNase OUT (Agilent) and labeled with either Cy3-dCTP or Cy5-dCTP. Equal amounts of the RNA from control sample were mixed, and the mixture was labeled with Cy5, while the RNA from each GBE treated HepG2 cells was labeled with Cy3.

Hybridization protocol: After mixed with hybridization buffer(GE healthcare), samples were applied to microarray slide, cover the slip with care. Place the slide in a wet box.Incubate in a hybridization chamber at 42℃  for 16-18 hours, avoid light exposure. After hybridization, slides were washed sequentially with 1 SSC/0.2 SDS(50°C), 0.1 SSC/0.2%SDS(50°C) and 0.1 SSC(room temperature) before scanning.

Scan protocol: Axon GenePix 4000B

Description: Total RNA was prepared by using TRIzol reagent (Life Technologies, USA), further purified with an RNEasy column (Qiagen, Germany), and quantified by using gel electrophoresis and spectrophotometer followed by reverse transcription labeling. Each RNA sample was reverse-transcribed into cDNA primed with oligo(dT) and labeled with either Cy3-dCTP or Cy5-dCTP by using Superscript II reverse transcriptase (Life Technologies, USA). After hybridization under a standard protocol, data acquisition was performed by using a laser scanner (Agilent, USA), and the image data were analyzed using ImaGene 4.2 (Biodiscovery, Santa Monica, CA, USA).

Data processing: LOWESS normalized, background subtracted VALUE data obtained from log of processed Red signal/processed Green signal.

first: Qinghua

last: Zhang

first: Zuoquan

last: Xie

first: Lu

first: Zhidong

last: Zhu

first: Qi

last: Wang


title: Dengue VN Microarray MD and DF study

city: Ho Chi Minh

first: Long

last: Hoang

address: 190 Ben Ham Tu

country: Viet Nam

institute: OUCRU

summary: DNA microarrays and specific RT-PCR assays were used to reveal transcriptional patterns in the blood of children presenting with dengue shock syndrome (DSS) and well-matched patients with uncomplicated dengue. The transcriptome of patients with acute uncomplicated dengue was characterized by a metabolically demanding "host defense" profile; transcripts related to oxidative metabolism, interferon signaling, protein ubiquination, apoptosis and cytokines were prominent. In contrast, the transcriptome of DSS patients was surprisingly benign, particularly with regard to transcripts derived from apoptotic and type I interferon pathways. These data highlight significant heterogeneity in the type or timing of host transcriptional immune responses precipitated by DENV infection independent of the duration of illness. In particular, they suggest that if transcriptional events in the blood compartment contribute to capillary leakage leading to hypovolemic shock, they occur before cardiovascular decompensation occurs, a finding that has implications for rational adjuvant therapy in this syndrome.

overall_design: Whole blood transcriptional profiles of children infected with dengue virus with different clinical outcomes were compared. The subjects including 9 acute dengue shock samples, 9 acute uncomplicated dengue samples, 6 autologous follow up dengue samples and 6 autologous follow up uncomplicated dengue patients. Microarray data was normalised using Genespring GX7 software, statistical analysis was performed in Multiexperiment viewer software. Pathway  analysis was performed using Ingenuity Pathway analysis online software.

first: Martin

middle: L

last: Hibberd

first: Cameron

last: Simmons

first: Jeremy

last: Farra

first: Ling

last: Ling

first: Dung

last: Nguyen

first: Bridget

last: Wills

first: Ngoc

middle: V

last: Tran

first: Hien


title: 14028 Promoter libraries through human PC3 tumor in nude mice

status: Public on Dec 19 2008

city: Irvine

first: Michael

last: McClelland

address: 132 Med Surge I

department: Microbiology & Molecular Genetics

institute: University of California, Irvine

summary:
Salmonella typhimurium 14028s promoter library (180,000 clones, both stable and destabilized GFP versions) recovered from either spleens / PC3 tumors 2 days after IV / IT delivery into nude mice, sorted for green bacteria using FACS, compared to the initial promoter library grown in LB Ampicillin broth, O/N at 37°C with aeration
Keywords: promoter screen

platform_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2

overall_design: used 5 mice each for IT delivery of the 14028 promoter libraries into tumor or IV delivery and subsequent harvest from spleen. Sorted for green bacteria using FACS. Compared various sortings after these environments to the original promoter library after plasmid recovery and Klenow labeling of PCR amplified plasmid inserts by hybridization onto 380k custom-made Nimblegen Salmonella tiling oligo array

first: Nabil

last: Arrach

first: Steffen

last: Porwollik


title: Sm29 is a protective surface protein from the tegument of lung-stage Schistosoma mansoni

city: Campos dos Goytacazes

first: Thiago

middle: Motta

last: Venancio

state: RJ

address: Av Alberto Lamego 2000

institute: UENF

summary:
Schistosomiasis continues to be a significant public health problem1. Although vaccine development against this disease has experienced more failures than successes, encouraging results have recently been obtained using membrane-spanning protein antigens from the tegument of S. mansoni. Our group recently identified Sm29, an antigen that is predominantly recognized by IgG1 and IgG3 antibodies of resistant patients2.  In the present study, we show that Sm29 is located on the surface of adult worms and lung-stage schistosomula. Immunization of mice with recombinant (r) Sm29 engendered 51% reduction in adult worm burdens, 60% reduction in intestinal eggs and 55% reduction in liver granulomas. Protective immunity in mice was associated with high titers of specific IgG1 and IgG2a and elevated production of IFN-γ, TNF-α and IL-12. Further, cellular responses of infected schistosomiasis patients to rSm29 consisted of elevated IFN-γ and an absence of IL-5.  Gene expression analysis of worms recovered from rSm29 vaccinated mice relative to control mice revealed a significant (q< 0.01) down-regulation of 498 genes, while no up-regulation was detected. Among down-regulated genes, many of them encode surface antigens and proteins associated with immune signals suggesting that under immune attack schistosomes reduce the expression of critical surface proteins. This study demonstrates that the membrane-bound Sm29 protein is a new molecule that has great potential as a vaccine candidate against schistosomiasis.

Keywords: Schistosoma mansoni gene expression in vaccinated mice

overall_design:
Two biological samples were used in the study, each used in two arrays (technical replicates with dye swap) and each array containing two replicates of each spot. Overall, there are eight data points data points for each spot.

Only cases with more than 4 of 8 points are valid ratios were considered. Value and its respective dye swap value [after changing the log(ratio) signal] were averaged, reducing the data point to four. The average values were than used in the significance testing using SAM (Tusher et al., 2001).

The samples were used to get average values using the following combination:

3000139790L with 3000139792L;
3000139790R with 3000139792R;
3000139791L with 3000139795L;
3000139791R with 3000139795R

The normalized, averaged dyeswap values are provided in a supplementary file at the foot of the record.

first: Fernanda

last: Cardoso

first: Gilson

last: Macedo

first: Elisandra

last: Gava

first: Gregory

middle: T

last: Kitten

first: Vitor

last: Mati

first: Alan

last: Melo

first: Marcelo

last: Caliari

first: Luciana

first: Giulliana

last: Almeida

first: Sergio

last: Verjovski-Almeida

last: Oliveira


title: RPL12A/B double deletion strains: Control vs. Wt RPL12B or Mutant RPL12B-R66K Transformed

first: Guang-Huar

last: Young

state: Taiwan (TWN)

address: 6F.-3, No.21, Ln. 583, Ruiguang Rd., Neihu Dist., Taipei City 114, Taiwan (R.O.C.)

department: Scientific Research

summary:
Transcriptional and translational profiling of RPL12A/B double deletion comparing control untransformed RPL12B strains with transformed with a pESC-RPL12B wild type or muated RPL12B-R66K gene.

Keywords: Genetic modification

overall_design: Two-condition experiment, Transcriptional experiments from total mRNA and Translational experiments from polysomal mRNA from three strains including deleted RPL12A/B, retransformed WT RPL12B and mutated RPL12B-R66K strain. Biological replicates: independently grown and harvested. double, triple or more replication per array.


title: The contributions of sex, genotype and age to transcriptional variance in Drosophila melanogaster

status: Public on Dec 17 2008

city: Raleigh

first: Stan

last: Martin

state: NC

address: 1404 Crete Dr.

institute: Science Apps, L3C

summary: Here we present a statistically rigorous approach to quantifying microarray expression data that allows the relative effects of multiple classes of treatment to be compared and incorporates analytical methods that are common to quantitative genetics. From the magnitude of gene effects and contributions of variance components, we find that gene expression in adult flies is affected most strongly by sex, less so by genotype and only weakly by age (for 1-  and 6-wk flies); in addition, sex-genotype interactions may be present for as much as 10% of the Drosophila transcriptome. This interpretation is compromised to some extent by statistical issues relating to power and experimental design. Nevertheless, we show that changes in expression as small as 1.2 fold can be highly significant. Genotypic contributions to transcriptional variance may be of a similar magnitude to those relating to some quantitative phenotypes and should be considered when assessing the significance of experimental treatments.

overall_design: The experimental design consisted of 24 cDNA microarrays, 6 for each combination of 2  genotypes (Oregon R and Samarkand) and the 2 sexes, involving 48 separate labeling reactions. We directly contrasted two time points, 1-wk and 6-wk adult flies, on each microarray. The dyes Cy3 and Cy5 were flipped for two of the six replicates of each genotype and sex combination. A common reference sample was not used. In total, we spotted 4,256 clones, representing a third of the genome, two-thirds of which were verified by resequencing before printing. We excluded 325 clones from the analysis because no consistent expression above background was detected.


title: Gene Expression Profiles of PBMC Subsets in Acute GVHD Following Cord Blood Transplantation (IMS v.2)

city: Minato-ku

first: Naoyuki

last: Takahashi

address: 4-6-1, Shirokanedai

department: Research Hospital

institute: The Institute of Medical Science, The University of Tokyo

summary:
In comparison with allogeneic hematopoietic stem cell transplantation using other sources, umbilical cord blood transplantation (UCBT) offers substantial clinical advantages including a lower incidence and severity of acute graft-versus-host disease (GVHD) despite the use of allogeneic UCB stem cells with more disparate HLA.  However, detailed pathophysiology of acute GVHD developed after UCBT has not yet been clarified.
Here, we examined the roles of inflammatory cells in the pathogenesis of acute GVHD following UCBT, by expression profiling of a total of 615 genes in each of 4 subsets (CD4+, CD8+, CD14+, and CD56+) of peripheral blood mononuclear cells (PBMCs), which were taken from 5 patients with hematologic malignancy, in whom acute GVHD had developed after UCBT.  By comparing expression profiles measured during acute GVHD with those measured upon discharge (recovery phase), for each subset of PBMCs, we identified immuno-regulatory genes which were assumed to be linked to the pathogenesis of acute GVHD.
Keywords: disease state analysis

overall_design: CD4+, CD8+, CD14+, and CD56+ cell subsets were isolated from the peripheral blood mononuclear cells (PBMCs) of 5 patients with hematologic malignancy, in whom acute GVHD had developed after cord blood transplantation.  PBMCs were taken twice – during acute GVHD phase and upon discharge (recovery phase), from each patient.  By using a custom-made fibrous oligonucleotide DNA microarray Genopal (Mitsubishi Rayon), we compared expression profiles of 615 unique genes measured during acute GVHD with those measured upon discharge (recovery phase), for each subset of PBMCs.

first: Noriharu

last: Sato

first: Tatsunobu

last: Fukushima

first: Satoshi

first: Arinobu

last: Tojo


title: Effect of rimonabant on gene expression of visceral and subcutaneous adipose tissues: high fat diet

status: Public on Dec 15 2008

city: Lausanne

first: Carine

last: Poussin

address: Quartier UNIL-Sorge

institute: University of Lausanne

summary:
Mice were fed for 6 months with a normal chow (NC) or a high fat diet (HFD). After 6 months of diet, high fat fed mice with Low, Medium and High body weight, but with similar glucose intolerance, were selected.
These selected mice as well as NC mice were then treated with Rimonabant or Vehicle for 1 month. After treatment, mice were sacrificed and visceral and subcutaneous adipose tissues were collected and immediately frozen in liquid nitrogen.
Total RNA from each sample was further extracted, purified, quality-controled before and after amplification, Cy5-labeled and co-hybridized with a Cy3-labeled mouse Universal Reference total RNA on the mouse 17K microarray.

Keywords: diet response, pharmacological response

overall_design: Each individual RNA sample was hybridized with a mouse Universal Reference Total RNA

first: Diana

last: Hall

first: Kaori

last: Minehira

first: David

last: Tarussio

first: Joel

last: Gyger

first: Bernard

last: Thorens


title: Chromosomal High Affinity Binding Sites for the Drosophila Dosage Compensation Complex.

status: Public on Dec 14 2008

city: Martinsried

first: Tobias

last: Straub

address: Großhadener Str. 9

department: Biomedical Center, Bioinformatics

institute: LMU Munich

summary:
We identified 131 high affinity sites of the Drosophila DCC combining residual ChIP-chip profiles after differential crosslinking and RNAi-mediated knockdown of spreading factors

Keywords: ChIP-chip

overall_design: MSL1 and MSL2 ChIP under various conditions including differential crosslinking and RNAi against MOF, MLE and MSL3. 2-4 replicates per experiment. dye-swaps as indicated in sample description.

first: Charlotte

last: Grimaud

first: Gregor

middle: D

last: Gilfillan

first: Angelika

last: Mitterweger

first: Peter

last: Becker


title: Array CGH analysis for 29 pancreatic ductal adenocarcinoma (PDAC) samples

city: London

first: Tomohiko

last: Harada

address: Charterhouse Square

department: Centre for Molecular Oncology

institute: Cancer Research UK

summary:
Background/Aims: Microarray-based comparative genomic hybridisation (CGH) has allowed high-resolution analysis of DNA copy number alterations across the entire cancer genome. Recent advances in bioinformatics tools enable us to perform a robust and highly sensitive analysis of array CGH data and facilitate the discovery of novel cancer-related genes. Methods: We analysed a total of 29 pancreatic ductal adenocarcinoma (PDAC) samples (six cell lines and 23 microdissected tissue specimens) using 1 Mb-spaced CGH arrays. The transcript levels of all genes within the identified regions of genetic alterations were then screened using our Pancreatic Expression Database. Results: In addition to 238 high-level amplifications and 35 homozygous deletions, we identified 315 minimal common regions of “non-random” genetic alterations (115 gains and 200 losses) which were consistently observed across our tumour samples. The small size of these aberrations (median size of 880 kb) contributed to the reduced number of candidate genes included (on average 12 Ensembl-annotated genes). The database has further specified the genes whose expression levels are consistent with their copy number status. Such genes were UQCRB, SQLE, DDEF1, SLA, ERICH1 and DLC1, indicating that these may be potential target candidates within regions of aberrations. Conclusion: This study has revealed multiple novel regions that may indicate the locations of oncogenes or tumour suppressor genes in PDAC. Using the database, we provide a list of novel target genes whose altered DNA copy numbers could lead to significant changes in transcript levels in PDAC. (Harada et al. Pancreatology)
Keywords: pancreatic ductal adenocarcinima, tissue microdissection, array CGH, genetic alterations

overall_design: A panel of 23 microdissected PDAC tissues and 6 PDAC-derived cell lines were analysed using Sanger's CGH arrays with 1 Mb resolution. Clinical info of the samples used is provided as a supplementary file.

first: Claude

last: Chelala

first: Arshad

last: Khan

first: Gavin

last: Kelly

first: Nicholas

last: Lemoine


title: Comparison between Backfat Tissue of Fat Type Pig and Lean Type Pig, among developmental phases

status: Public on Dec 16 2008

city: Ya'an

first: Mingzhou

last: Li

state: Sichuan

address: No.46  Xinkang Road

department: College of Animal Science and Technology

institute: Sichuan Agricultural University

summary:
The objective of this study was to identify key genes associated with porcine muscle growth and adipose metabolism which different expression in porcine backfat tissue between Fat Type Pig(Taihu pig) and Lean Type Pig (Landrace), among developmental phases(Month 1,2,3,4,5). The gene expression analyses will increase understanding of impact factors of pork quality by identifying key genes and pathways controlling backfat development. Relative real-time RT-PCR was used to confirm differential expression of 3 different expression genes(ME,SCD and UCP3) which normalized by 3 housekeep genes(ACTB, TBP and TOP2B).
Keywords: time course and breed comparison

overall_design:
The different expression experiments between Taihu pig and Landrace used partnership design ( Landrace Month 1 vs Taihu Month 1, 2 vs 2,3 vs 3, 4 vs 4,5 vs 5).
The different expression experiments among developmental phases(Month 1,2,3,4,5)  used  indirect loop design(Landrace Month 2 vs 1,3 vs 2,4 vs 3,5 vs 4,1 vs 5; Taihu Pig  Month 2 vs 1,3 vs 2,4 vs 3,5 vs 4,1 vs 5).

first: Xuewei

first: Li

last: Zhu

first: Yujiao

last: Guo

first: Lei

last: Chen

first: Qiang

first: Surong

last: Shuai

first: Anan

last: Jiang

first: Guangxi

last: Yang

first: Guoqing

last: Tang

first: Yanzhi


title: Comparison between longissimus dorsi muscle tissue of Fat and Lean Type Pig, among developmental phases

summary:
The objective of this study was to identify key genes associated with porcine muscle growth and adipose metabolism which different expression in porcine longissimus dorsi muscle tissue between Fat Type Pig(Taihu pig) and Lean Type Pig (Landrace), among developmental phases(Month 0,1,2,3,4,5). The gene expression analyses will increase understanding of impact factors of pork quality by identifying key genes and pathways controlling longissimus dorsi muscle development. Relative real-time RT-PCR was used to confirm differential expression of 5 different expression genes(PPARGC1A, RYR1, IGF2, IGF1R and IGFBP5) which normalized by 3 housekeep genes(ACTB, TBP and TOP2B).
Keywords: time course and breed comparison

overall_design:
The different expression experiments between Taihu pig and Landrace used partnership design ( Landrace Month 0 vs Taihu Month 0, 1 vs 1,2 vs 2,3 vs 3, 4 vs 4,5 vs 5).
The different expression experiments among developmental phases(Month 0,1,2,3,4,5)  used  indirect loop design(Landrace Month 1 vs 0, 2 vs 1,3 vs 2,4 vs 3,5 vs 4,0 vs 5; Taihu Pig  Month 1 vs 0, 2 vs 1,3 vs 2,4 vs 3,5 vs 4,0 vs 5).


title: Cardiac Gene Expression Profiling in CryAB R120G Transgenic Mice

status: Public on Dec 12 2008

city: Salt Lake City

first: Brett

last: Milash

state: UT

department: Center for High Performance Computing

institute: University of Utah

summary:
Protein aggregation cardiomyopathy is a life-threatening manifestation of a multisystem disorder caused by the exchange mutation in the gene encoding the human small HSP ?B-crystallin (hR120GCryAB).  Genetic studies in mice have established cardiac hR120GCryAB expression causes increased activity of glucose 6-phosphate dehydrogenase (G6PD) and 'reductive stress' (Rajasekeran et al., Cell. 2007;130(3):401-2).  However, the initiating molecular events in the pathogenesis of this novel toxic gain-of-function mechanism remain poorly defined.  In an integrated systems approach using gene expression profiling, we identified a 'biosignature,' whose features can be validated to predict the onset, rate of progression, and clinical outcome of R120GCryAB cardiomyopathy.
Keywords: time-course, genetic modification, disease state analysis

overall_design: 34 total samples were analyzed in in-house spotted microarrays (NIA mouse clone set), 2 time points (3 and 6 month old mice), 3 mouse strains (nontransgenic,  human CryAB wild type transgenic, human R120G CryAB transgenic), 4-7 replicates per group, reference standard design (Stratagene Universal Mouse Reference RNA)

first: Rajasekaran

middle: N

last: Soorappan

first: Matthew

last: Firpo

first: Robert

last: Weiss

first: Ivor

middle: J

last: Benjamin


title: Priming for Th2 differentiation requires IL-2-mediated induction of IL-4 receptor alpha chain expression

status: Public on Dec 11 2008

city: Bethesda

first: Dustin

last: Schones

state: MD

address: Building 10, Room 7B20A, 9000 Rockville Pike

institute: NHLBI/NIH

type: Genome binding/occupancy profiling by high throughput sequencing

summary:
Th2 cells enable humoral immunity and host-defense to parasites. Whereas IL-4 drives Th2 differentiation and IL-2 is important later in this process by augmenting IL4 chromatin accessibility, we demonstrate that IL-2 serves an essential early role in regulating IL-4Rα expression by inducing binding of Stat5a and Stat5b to the IL4ra locus, with sustained binding during Th2 differentiation.  Although IL-4 induces IL-4Rα expression, TCR-induced IL-4Rα expression was unexpectedly normal in ILr-/- but profoundly diminished in IL2-/- T cells. Remarkably, enforced IL-4Rα expression rescued Th2 differentiation in IL2-/- cells. These results reveal a novel function for IL-2, with IL-2 via Stat5 providing an early signal for IL4ra induction, thereby priming cells for Th2 differentiation and promoting/maintaining IL-4Rα expression in Th2-committed cells.
ChIP-seq experiments for Stat5a and Stat5b with an IgG control experiment for 8hr, 13hr and 2 rounds of Th2 differentiation.
Keywords: ChIP-seq, Illumina, Th2 differentiation, Stat

overall_design: ChIP-seq experiments for Stat5a and Stat5b with an IgG control experiment for 8hr, 13hr and 2 rounds of Th2 differentiation. There are a total of 22 lanes of data for the three samples across the three time points.

first: Wei

last: Liao

first: Jangsuk

last: Oh

first: Yongzhi

last: Cui

first: Kairong

first: Tae-Young

last: Roh

first: Keji

last: Zhao

first: Warren

last: Leonard


title: Functional genomics investigation of PfAdoMetDC/ODC co-inhibition of Plasmodium falciparum

city: Pretoria

first: Anna

middle: Catharina (Tharina)

last: van Brummelen

state: Gauteng

address: Lynwood Road

department: Biochemistry

country: South Africa

institute: University of Pretoria

summary:
Polyamines are ubiquitous components of all living cells and their depletion usually causes cytostasis, a strategy employed for treatment of West-African trypanosomiasis. To evaluate polyamine-depletion as an antimalarial strategy, cytostasis caused by the co-inhibition of S-adenosylmethionine decarboxylase/ornithine decarboxylase (PfAdoMetDC/ODC) in Plasmodium falciparum was studied with a comprehensive transcriptome, proteome and metabolome investigation. Highly synchronized cultures were sampled just before and during cytostasis and a novel zero time point definition was used to enable interpretation of results in lieu of the developmentally regulated control of gene expression in P. falciparum. Transcriptome analysis revealed the occurrence of a generalized transcriptional arrest just prior to the growth arrest due to polyamine-depletion. However, the abundance of 538 transcripts was differentially affected and included three perturbation-specific compensatory transcriptional responses: the increased abundance of the transcripts for lysine decarboxylase (LDC) and ornithine aminotransferase (OAT) as well as the decreased abundance of that for S-adenosylmethionine synthetase (AdoMet synthetase). Moreover, the latter two compensatory mechanisms were confirmed on both protein and metabolite levels confirming their biological relevance. In contrast with previous reports, the results provide evidence that P. falciparumrespond to alleviate the detrimental effects of polyamine-depletion via regulation of its transcriptome and subsequently the proteome and metabolome.

Keywords: functional genomics, time course, reference design, drug exposure, stress response, polyamine depletion, cytostasis

overall_design: Transcriptional profiling of PfAdoMetDC/ODC co-inhibited P. falciparum was performed with in-house spotted, oligonucleotide microarray analysis as previously reported [Bozdech, Z., Zhu, J., Joachimiak, M., Cohen, F., Pulliam, B., and DeRisi, J. (2003) Genome Biol. 4(2), R9]. Parasites were simultaneously treated in the schizont stage with the inhibitors, DFMO and MDL73811, alongside untreated controls in duplicate (i.e. 2 biological replicates A, B). A time course study was performed and samples were harvested at three time points in the trophozoite stage [19, 27 and 34 hours post-invasion (hpi)], just before and after cytostasis. RNA was isolated and cDNA synthesis was performed. A reference design microarray experiment was conducted and a composite reference pool was compiled from all the samples included in the experiment. Samples were fluorescently labelled with Cy5 dye and the reference pool with Cy3 and co-hybridized overnight. Where possible, technical replication was performed.

first: Kellen

last: Olszewski

first: Daniel

last: Wilinski

first: Manuel

last: Llinás

first: Abraham

middle: I

last: Louw

first: Lyn-Marie

last: Birkholtz


title: Lungs of ferrets infected with either an H3N2 or H5N1 subtype of influenza A virus.

status: Public on Dec 09 2008

city: Toronto

first: Longsi

last: Ran

state: ON

address: 101 College Street

department: Division of Experimental Therapeutics

institute: Toronto General Research Institute

summary:
A global genomics approach was used to identify patterns of immune dysregulation during H5N1 influenza virus infection as the host response, in particular hyperchemokinemia, is thought to contribute to the extreme pathology associated with this disease.
Keywords: time course

overall_design: Ferrets were inoculated intranasally with 10(6) EID50 of either A/Vietnam/1203/04 (H5N1) or A/Panama/2007/99 (H3N2).  At 2, 4 and 6 days post-infection (DPI), ferrets were euthanized and lung tissue was excised for RNA purification and subsequent gene expression analysis.

first: David

last: Kelvin


title: Transcriptional activity of Bacillus subtilis exposed to lethal heat stress

status: Public on Dec 08 2008

city: Zeist

first: Remco

last: Kort

address: Utrechtseweg 48

department: Microbiology

country: Netherlands

institute: TNO

summary:
Transcriptional profiling of Bacillus subtilis to lethal heat stress by systematic sampling of bacterial cell suspensions exposed to gradually increasing temperatures.

Keywords: Stress response

overall_design: Bacterial cell suspensions exposed to increasing temperatures ranging from 30-62ºC (Cy5 channel) versus 30 degrees C reference (Cy 3 channel), by transfer of the bacterial cell suspension from a 30ºC to a 65ºC water bath; sampling and quenching at 30, 40, 50, 57, 58, 59, 60 and 62ºC; total time to reach 62ºC is ~12 minutes . Biological replicates: 2, independently grown cultures. One replicate per array. Strains B subtilis 168 1A1; B subtilis A163 (food product isolate); B. subtilis MC85 (food product isolate).


title: Exon array analysis of two human cell lines (HEK293T Ramos B cell)

city: Berlin

first: Marc

last: Sultan

address: Ihnestr. 73

institute: Max Planck Institute for molecular genetics

summary: In order to compare previously generated transcriptome data by second generation sequencing (Sultan et al. Science 2008), we interrogated the human Affymetrix exon chips 1.0ST using the same source of material.

overall_design: Two biological replicates from HEK293T and B cell lines were hybridized on the    human Affymetrix exon chips 1.0ST

first: Hugues

last: Richard

first: Marcel

middle: H

last: Schulz

first: Stefan

last: Haas

first: Asja

last: Nürnberger

first: Sabine

last: Schrinner

first: Daniela

last: Balzereit

first: Emilie

last: Dagand

first: Axel

last: Rasche

first: Hans

last: Lehrach

first: Martin

last: Vingron

first: Marie-Laure

last: Yaspo


title: Microarray Transcriptome Comparison Subzero Temperature and Optimal Temperature Growth in Psychrobacter arcticus 273-4

status: Public on Dec 10 2008

city: East Lansing

first: Peter

middle: W

last: Bergholz

state: MI

address: 540 Plant and Soil Sciences

department: Center for Microbial Ecology

institute: Michigan State University

summary: Permafrost soils are extreme environments that exert low-temperature, desiccation and starvation stress on bacteria over thousands to millions of years.  To understand how Psychrobacter arcticus 273-4 survived for > 20,000 years in permafrost, transcriptome analysis was performed during growth at 22°C, 17°C, 0°C, and -6°C using a mixed effects ANOVA model.  Genes for transcription, translation, energy production and most biosynthetic pathways were down-regulated at low temperatures.  Evidence of isozyme exchange was detected over temperature for D-alanyl-D-alanine carboxypeptidases (dac1 and dac2), DEAD-box RNA helicases (csdA and Psyc_0943) and energy efficient substrate incorporation pathways for ammonium and acetate.  Specific functions were compensated by up-regulation at low temperature including genes for the biosynthesis of proline, tryptophan, methionine, and histidine.  RNases and peptidases were generally up-regulated at low temperatures.  Changes in energy metabolism, amino acid metabolism, and RNase gene expression were consistent with induction of the stringent response by relA activity.  In contrast to results observed in other psychrophiles and mesophiles, only clpB and hsp33 were up-regulated at low temperature with no up-regulation of other chaperones and peptidyl-prolyl isomerases.  Knockout mutants of relA, csdA, and dac2 were all deficient in low temperature growth, but a mutant in dac1 was deficient in growth at 17°C.  The combined data suggest that the basal biological machinery including translation, transcription and energy metabolism are well adapted to function across the -6°C to 22°C growth range of P. arcticus and temperature compensation by gene expression was employed to address specific challenges to low-temperature growth.

overall_design: Cy-dye labelled cDNA populations were generated from total RNA extracted from mid-exponential growth phase cultures of Psychrobacter arcticus 273-4 grown at 22°C, 17°C (optimal growth rate), 0°C, and -6°C.  All pairwise temperature comparisons were performed in this 4-level single factor experiment with dye swapping nested within biological replicate.  Five biological replicates (20 samples totalled over all temperatures) were included in each comparison for a total of 30 hybridizations.  Data were analyzed using R/MAANOVA with an ANOVA model which included gene, dye, biological replicate, slide, and growth temperature terms (with interactions).  Spike-in mRNAs were included immediate following the cell lysis stage in order to serve as monitors for RNA degradation from lysis through hybridization.

first: Corien

last: Bakermans

first: James

middle: M

last: Tiedje


title: Salmon Spawning Migration: Metabolic Shifts and Environmental Triggers

city: Nanaimo

first: Shaorong

last: Li

state: British Columbia

address: 3190 Hammond Bay Road

department: Salmon and Freshwater Ecosystems Division

institute: Fisheries and Oceans Canada

summary:
A large-scale functional genomics study revealed shifting energy generating processes in white muscle during the final 1,300 km migration of wild sockeye salmon to their spawning grounds in the Fraser River, British Columbia.  In 2006, Lower Adams stock sockeye salmon ceased feeding after passing the Queen Charlotte Islands, 850 km from the Fraser River.  Enhanced protein turnover and reduced transcription of actin, muscle contractile and heme-related proteins were early starvation responses in saltwater.   Arrival to the estuarine environment triggered massive protein turnover through induction of proteasoma and lysosomal proteolysis and protein biosynthesis, and a shift from anaerobic glycolysis to oxidative phosphorylation.  Response to entry into freshwater was modest, with up-regulation of heat shock proteins and nitric oxide biosynthesis.  High river temperatures resulted in a strong defense/immune response and high mortalities in 50% of fish.  Arrival to the spawning grounds triggered further up-regulation of oxidative phosphorylation and proteolysis, down-regulation of protein biosynthesis and helicase activity, and continued down-regulation of muscle proteins and most glycolytic enzymes.  However, sharp up-regulation of PFK-I indicated induction of glycolytic potential at the spawning grounds.   The identification of potential environmental cues triggering genome-wide transcriptional shifts in white muscle associated with migration and the strong activation of proteasomal proteolysis were both novel findings.

Keywords: Functional genomics study on wild-caught fish

platform_organism:
Oncorhynchus mykiss
Salmo salar

overall_design: The experiment was based on expression profiles of white muscle tissue collected from wild migrating adult sockeye salmon during their return spawning migration back to the Fraser River.  Fish were collected from seven sites along the final 1,300 km migration path, and white muscle samples were quickly frozen in liquid nitrogen upon capture.  Marine sampling sites included (from north to south) the Queen Charlotte Islands (QCI), Johnstone Strait (JS), Juan de Fuca Strait (JDFS), and the Strait of Georgia (SOG).  Freshwater sampling sites included Whonnock (W), Savona (SV) and the Lower Adams Spawning Grounds.  Genetically-based stock ID was used to identify the natal sites of fish collected from the wild.  The experiment was designed to profile the transcriptional shifts associated with migration of the Adams River stock complex.  The total experiment included 80 microarray slides, with a minimum biological replicate size per site of 6 (SV), and maximum of 18 (JS) (see supplemental table for details). Additional intra-site variables, which could only be addressed in some sites, included sex (female biased) and river entry timing (for JS, JDFS and W sites; identified through radio-tracking of marine collected fish).  Total RNA was amplified (1 round) with MessageAmpTMII-96 kit (Ambion, TX, USA), and reverse transcribed to cDNA before labelling with ALEXA dyes using the Invitrogen Indirect Labelling Kit.  The experiment was based on a reference design, with the reference containing the combined aRNA of all individuals used in the experiment.  Individual samples were labelled with Alexa 555 and the reference control with Alexa 647, with no dye flips included.  A single technical replicate of one SV fish (replicate 5) was included in the experimental design.  This experiment is part of a larger white muscle experiment containing additional sockeye salmon stocks.

first: Kristina

last: Miller

first: Angela

last: Schulze

first: Norma

last: Ginther

first: David

middle: A

last: Patterson

first: Anthony

last: Farrell

first: Scott

middle: G

last: Hinch


title: Identification of novel neuroendocrine markers by gene expression profiling

city: Trondheim

first: Tom

middle: Even

last: Wheeler

address: Olav Kyrresgt. 3

department: Department of cancer research and molecular medicine

country: Norway

institute: Norwegian University of Science and Technology, NTNU

summary:
(This section will be revisited in good time prior to publication, for final approval)
Research within the field of functional genomics provides a more profound understanding of living organisms, by elucidating the interaction between genes and gene products. This is of increasing importance in many fields, and especially within cancer research.
With the use of microarray technology, the gene expression of a vast number of genes can be measured simultaneously. This allows us insight into tumor biology, and an opportunity to obtain knowledge of diagnostic and prognostic value, as well as hints towards the causes of cancer. In this study the gene expression in a selection of human neuroendocrine (NE) and non-neuroendocrine tumor cell lines were compared. Both qualitative and quantitative methods were applied to get an insight into the NE tumor biology.
NE cells are found in practically every organ and tissue in the body and are assigned to synthesize peptide hormones and take up amino acids and transform these into biogenic amines by carboxylation. NE tumors arise after malignant transformation of neuroendocrine cells and are usually highly differentiated and have a low growth rate, which makes this a high prevalence tumor type. These rather rare tumors characteristically express NE markers, such as chromogranin A, and contains characteristic dense-secretory core granules.
By using cDNA microarray analysis, the gene expression profiles of various NE tumor cell lines (e. g. carcinoid, neuroblastoma, medullary thyroid carcinoma and endocrine pancreatic tumor) were compared with those of non-NE tumor cells. As a reference the commercially available Universal human RNA reference (Stratagene) was used. Verification of the results were done by real time RT-PCR analysis after the NE nature of the cell lines was stated by immunohistochemical, electron microscopic and light microscopic examinations.
A large number of genes were differentially expressed in NE cell lines vs. the non-NE cell lines, and were found to be of interest in NE tumor biology. Some of these genes were previously known in NE tumor biology, whereas the vast majority of the specified genes are novel in the context of NE tumor biology. Genes with little previous information were also identified and thought to be an asset in the search of new NE markers.
A feasible prospective of the study would be expansion with additional samples cell lines and a more thorough verification with real time RT-PCR analysis. This would enhance the credibility and thus the utility of the findings. Future studies on the biological functions of the candidate genes and the mechanisms of their regulation, will hopefully enable suggestions of new NE markers of prognostic or diagnostic value, and eventually new drug targets.
Keywords: neuroendocrine, neuroendocrine markers, gene expression profiling, cDNA microarray

overall_design:
The hybridisation was performed by an experienced laboratory
technician Eli Helge, (Dept. of Laboratory Medicine, Children`s and Women`s health, NTNU) at
the microarray core facility. Three separate runs with 10, 11 and 3 slides was needed to obtain the 20 hybridisations (dyeswap x 10 cell lines) planned for this study. Of the 10 cell lines 6 were neuroendocrine and 4 were non-neuroendocrine. For comparison between these two groups the universal human reference RNA (Stratagene) was used as the "control" on each slide.

An adapted version of the 3DNA Array 350 protocol (Genisphere) was used. The slides were scanned with a
ScanArrayTMExpressHT (Packard BioScience) twice immediately after the
hybridisation was completed to avoid photo bleaching of the fluorescent dyes.

first: Eva

last: Hofsli

middle: E

first: Mette

last: Langaas

first: Turid

last: Follestad

first: Ursula

last: Falkmer

first: Liv

last: Thommesen

first: Sture

first: Astrid

last: Laegreid