title: High resolution analysis of epigenetic changes associated with X inactivation, ChIP-chip

status: Public on Jun 06 2009

city: Nijmegen

last: Stunnenberg

address: Geert Grooteplein 28

department: Department of Molecular Biology (274)

institute: Radboud University

summary: This study describes the epigenetic profiling of the X chromosome during X inactivation. It includes H3K4me3 and H3K27me3 ChIP-Seq profiles of male (E14) and female (LF2 and XT67E1) mouse ES cells, together with their differentiated derivatives (either 4d atRA or 10d EB). It also includes ChIP-chip profiles around the Xic on chromosome X of H3K4me3, H3K27me3, H3K9me2, H3K36me3, Pol II, TBP, H3-Core as well as expression, using male (E14) and female (LF2) mouse ES cells, together with their differentiated derivatives (either 4d atRA or 10d EB).

overall_design: Examination of two different histone modifications in 3 cell lines under 3 conditions using ChIP-Seq. Examination of five different histone modifications two transcription factors and gene expression under three conditions in 2 cell lines using ChIP-chip.


title: Methylketone Biosynthesis in Tomato F2 Segregant Populations

status: Public on Jun 05 2009

city: Rehovot

first: Imri

last: Ben-Israel

address: P.O box 12

department: Institute of Plant Sciences and Genetics in Agriculture

country: Israel

institute: Hebrew University

summary: Genetic analysis of interspecific populations derived from crosses between the wild tomato species Solanum habrochaites f glabratum, which synthesizes and accumulates insecticidal methylketones (MKs) such as 2-undecanone and 2-tridecanone in glandular trichomes, and Solanum lycopersicum (cultivated tomato), which does not, demonstrated that MK metabolism in the wild species can be attributed to several loci. Comparative trascriptome analysis of the glandular trichomes of F2 segregants bulked into low- and high-MK plants identified several genes whose transcripts were either more or less abundant in the high-MK plants.

sample_organism: Solanum lycopersicum x Solanum habrochaites

overall_design: The total RNA was extracted from isolated trichomes of bulked segregants; high and low methylketones accumulators, from the F2 population (5 plants in each bulk). The samples for Gene expression were labeled using Agilent Low RNA Input Linear amplification Kit (Part number:5184-3523).  Five hundred nanograms each of the Control and test samples were incubated with reverse trancription mix at 42°C and  converted to double stranded cDNA primed by oligodT with a T7 polymerase promoter. The cleaned up double stranded cDNA were used as template for aRNA generation. aRNA was generated by in vitro transcription and the dyes Cy3 CTP(Agilent) for control samples and Cy5 CTP(Agilent) for test samples were incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled aRNA was cleaned up and quality assessed for yields and specific activity.  The labeled aRNA samples were hybridized on to a Custom Tomato Gene Expression 4x44k (AMADID:19003). 825ng of cy3 labeled  samples and 825ng of cy5 labeled samples were pooled, fragmented and hybridized and data from these channels were used for comparison to test hybridizations of respective channels.  Fragmentation of labeled aRNA and hybridization were done using the Gene Expression Hybridization kit of Agilent (Part Number 5188-5242). Hybridization was carried out in Agilent’s Surehyb Chambers at 65º C for 16 hours. The hybridized slides were washed using Agilent Gene Expression wash buffers (Part No: 5188-5327) and scanned using the Agilent Microarray Scanner G Model G2565BA at 5 micron resolution.  Four arrays were used and dye swap experiments were included in the final analysis. The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from all four replicates.

first: Eyal

last: Fridman

first: Eran

last: Pichersky


title: Interactions of cytokines in peripheral blood cells from Systemic Lupus Erythematosus

status: Public on Jun 01 2009

city: Ibaraki

first: Norihiro

last: Nishimoto

state: Osaka

address: 7-7-20, Saito-Asagi

country: Japan

institute: Wakayama Medical University

summary: SLE patients are always with various disease manifestation. Various cytokines are pointed interacting and playing pathological roles in SLE although the etiopathology is still obscure. In this study, we aimed to investigate the effects of cytokine interactions in the immune response of SLE patients. Overexpressed interferon-inducible(IFI) genes were confirmed in peripheral blood from SLE patients. Using network-based analysis on the immune response-related genes, several networks including cytokines such as TNF and IFN-γ, or beta-estradiol(E2), were constructed. TNF-regulated genes were dominant in these networks but in vitro TNF stimulation on PBMCs showed no different responses in the expressions of these genes between SLE and healthy individuals. Co-stimulating experiments by TNF, IFN-γ, and E2 with IFN-α, revealed that TNF has repressive while IFN-γ essentially has synergistic effect with IFN-α on IFI gene expressions in vitro. E2 showed different effects on IFI gene expressions among 3 individuals.

overall_design: Peripheral blood was obtained from patients with SLE (n=11) and healthy women (n=6). Gene expression profile was analyzed using DNA microarray covering 30,000 human genes. Differentially expressed immune response-related genes were selected and analyzed by using Expression Analysis Systemic Explorer (EASE) based on Gene Ontology (GO) followed by network pathway analysis with Ingenuity Pathways Analysis (IPA).

first: HooiMing

last: Lee

first: Toru

last: Mima

first: Hidehiko

last: Sugino

first: Chieko

last: Aoki

first: Yasuo

last: Adachi

first: Naoko

last: Yoshio-Hoshino

first: Kenichi

last: Matsubara


title: Clostridium beijerinckii BA101 mutant fermentation time course

status: Public on Jun 02 2009

city: Urbana

first: Zhen

last: Shi

state: IL

address: 1206 West Gregory Drive

department: Institute for Genomic Biology

country: USA

institute: University of Illinois

summary: The fermentation culture of Clostridium beijerinckii mutant BA101 was monitored from exponential growth to stationary phase. During this period the culture underwent a shift from acidogenesis to solventogenesis. Acetone and butanol production was initiated with the onset of the solventogenic phase. Using DNA microarray changes in gene expression were examined during the transitional period.

sample_organism:
Clostridium beijerinckii
Clostridium beijerinckii NCIMB 8052

overall_design: RNA samples were taken from Clostridium beijerinckii mutant BA101 fermentation culture at individual time points during the acidogenic phase and the solventogenic phase. The samples were used for microarray hybridization.

first: Hans

last: Blaschek


title: Overlapping and unique responses of E. coli to adverse conditions

status: Public on Jun 03 2009

city: Ås

first: Birgitte

last: Moen

address: Osloveien 1

country: Norway

institute: Nofima Mat

summary:
In the present study we have determined the global gene expression and biomolecular composition in an Escherichia coli model strain exposed to ten adverse conditions (sodium chloride, ethanol, glycerol, two acids (hydrochloric acid and acetic acid), sodium hydroxide, heat (46°C) and cold (15°C) as well as ethidium bromide and the disinfectant benzalkonium chloride). The large variation in responses and few common genes illustrates the adaptation potential of E. coli and its ability to survive and colonize a wide range of environments.
Keywords: gene expression study, stress response

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design:
The strain used in this study was the genome-sequenced strain E. coli K-12 MG1655 (1) and the following concentrations/conditions were used; CH3COOH (pH 5.9 ± 0.05); HCl (pH 4.75 ± 0.05); NaOH (pH 9.6 ± 0.05); EtOH (5%); NaCl (4.5%); Glycerol (15%); BC (7 and 9 mg/ml); EtBr (150 mg/ml); 46°C; 15°C.
The cultures were prepared by inoculating one colony from tryptone soya agar (TSA) (Oxoid) (overnight growth at 37°C) to 5 ml TSB and incubating overnight at 37°C, with shaking (200 rpm). This culture was initially diluted 1:10 in medium and used to inoculate room-temperature TSB for each of the different stress factors (40 ml total volume) to a final concentration of approx. 1X10^7 CFU/ml (1:100 dilution of overnight culture). For cold stress experiment the medium was chilled to 15°C prior to inoculation. The cultures were incubated at 37°C unless otherwise stated (Table 1), shaking at 200 rpm and samples for global gene expression and biomolecular analyses were collected during exponential growth at a cell density of approx. 1X10^7 CFU/ml. All the stress conditions, including the control, were inoculated with the same overnight culture and started at the same time point. The experiment was performed 3 times at different days and with freshly prepared solutions, resulting in 3 biological replicates.
Total RNA was extracted from 1 ml cells using the RNeasy Protect Bacteria Mini Prep kit (Qiagen) as recommended by the manufacturer including the "on-column" DNase treatment. The concentration and purity of the total RNA was analyzed using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc.) and the RNA 600 Nano LabChip system (Agilent Technologies).
Total RNA (5 ug) and 0.5 ul test/reference Lucidea spike controls RNA (Amersham Biosciences) were reverse transcribed by using random hexamers in the presence of aminoallyl-dUTP (2 aa-dUTP  (Ambion): 3 dTTP (Invitrogen)) as described in standard operating procedure no. M007 (The Institute for Genomic Research [TIGR] protocol). The removal of unincorporated aa-dUTP and free amines was performed according to the TIGR protocol using the Qiagen QIAquick PCR purification kit. After drying in a Speed vac, the samples were stored at -20°C. Coupling aminoallyl-labeled cDNA to Cy3/Cy5 mono reactive dyes (Amersham Biosciences) was done according to the TIGR protocol. The E. coli microarrays consisted of E. coli oligos (Operon) printed on Corning Ultra GAPS slides as described previously (2, 6, 7). Microarray slides were treated with a pre-soak solution (Promega/Corning) before incubation in a pre-hybridization solution (5XSSC [1XSSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1% dodecyl sulfate [SDS], 1 mg/ml bovine serum albumin) at 42°C for 30-60 minutes. After pre-hybridization, the slides were washed briefly in distilled water, followed by a brief wash in isopropanol. The dried Cy3- and Cy5-labeled cDNA was resuspended in 55 ul hybridization solution (30% formamide, 5XSSC, 0.1%SDS and 0.1 mg/ml Salmon Sperm DNA), denatured and applied to the prehybridized microarray underneath a 25- by 60-mm mSeries LifterSlip coverslip (Erie Scientific Company). The slides were hybridized at 42°C overnight (~16 h). After hybridization the slides were washed twice in 2XSSC buffer with 0.1% SDS at 42°C for 5 minutes, followed by two washes in 0.1XSSC buffer with 0.1% SDS for 10 min at room temperature and four washes in 0.1X SSC buffer for 1 minute at room temperature. The slides were dried by centrifugation (90xg for 12 min). The reference used in the microarray experiments was pooled RNA from all the growth conditions including the control.
Slides were scanned with the ScanArray Express 1.0 scanner (Packard BioScience) following the manufacturers guidelines. The fluorescent spot intensities were quantified using ImaGene 5.6.1 (BioDiscovery Inc.) software. Background subtraction and normalization (LOWESS (3, 5)) was performed in GeneSpring 7 (Silicon Genetics). (A lowess curve was fit to the log-intensity versus log-ratio plot. 20% of the data was used to calculate the lowess fit at each point. The curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then the value was set to 10). Only data from spots representing E. coli K-12 MG1655 genes were analyzed in our studies. All analyses were based on 3 biological replicates with the exception of the second biological replicate from conditions HCl and NaOH. These two observations were removed due to bad hybridization giving a total of 34 array hybridizations (observations). Genes not present in any of the 34 observations were filtered out, resulting in the analysis of 4279 out of 4289 MG1655 genes on the array. Missing values (data points) were replaced by using the KNNimpute procedure (4) (k=10) on log2 transformed data.
1.	Blattner, F. R., G. Plunkett, 3rd, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y. Shao. 1997. The complete genome sequence of Escherichia coli K-12. Science 277:1453-74.
2.	Constantinidou, C., J. L. Hobman, L. Griffiths, M. D. Patel, C. W. Penn, J. A. Cole, and T. W. Overton. 2006. A reassessment of the FNR regulon and transcriptomic analysis of the effects of nitrate, nitrite, NarXL, and NarQP as Escherichia coli K12 adapts from aerobic to anaerobic growth. J Biol Chem 281:4802-15.
3.	Quackenbush, J. 2002. Microarray data normalization and transformation. Nat Genet 32 Suppl:496-501.
4.	Troyanskaya, O., M. Cantor, G. Sherlock, P. Brown, T. Hastie, R. Tibshirani, D. Botstein, and R. B. Altman. 2001. Missing value estimation methods for DNA microarrays. Bioinformatics 17:520-5.
5.	Yang, Y. H., S. Dudoit, P. Luu, D. M. Lin, V. Peng, J. Ngai, and T. P. Speed. 2002. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30:e15.
6.	Zhang, L., R. R. Chaudhuri, C. Constantinidou, J. L. Hobman, M. D. Patel, A. C. Jones, D. Sarti, A. J. Roe, I. Vlisidou, R. K. Shaw, F. Falciani, M. P. Stevens, D. L. Gally, S. Knutton, G. Frankel, C. W. Penn, and M. J. Pallen. 2004. Regulators encoded in the Escherichia coli type III secretion system 2 gene cluster influence expression of genes within the locus for enterocyte effacement in enterohemorrhagic E. coli O157:H7. Infect Immun 72:7282-93.
7.	Zheng, D., C. Constantinidou, J. L. Hobman, and S. D. Minchin. 2004. Identification of the CRP regulon using in vitro and in vivo transcriptional profiling. Nucleic Acids Res 32:5874-93.


title: Culture induced changes in Blood-Brain Barrier transcriptome: implications for amino acid transporters in vivo

city: Zurich

first: Francois

last: Verrey

address: Winterthurerstr. 190

department: Institute of Physiology

country: Switzerland

institute: University of Zurich

summary:
Tight homeostatic control of brain amino acids (AA) depends on transport via solute family carrier proteins expressed by the Blood-Brain Barrier (BBB) microvascular endothelial cells (BMEC). To characterize the mouse BMEC transcriptome and probe culture-induced changes microarray analyses of PECAM-1+ endothelial cells (ppMBMECs) were compared with primary MBMECs (pMBMEC) cultured in the presence or absence of glial cells, and with b.End5 endothelioma cell-line. Selected cell marker and AA transporter mRNA levels were further verified by real-time RT PCR.
Regardless of glial co-culture expression of a large subset of genes was strongly altered by a brief culture step. This is consistent with the known dependence of BMECs on in vivo interactions to maintain physiological functions, e.g. tight barrier formation, and their consequent de-differentiation in culture. Seven (4F2hc, Lat1, Taut, Snat3, Snat5, Xpct, Cat1) of nine highly in vivo expressed AA transporter mRNAs were strongly down-regulated for all cultures and two (Snat2, Eaat3) were variably regulated. In contrast, five AA transporter mRNAs with low in vivo expression, including y+Lat2, xCT, and Snat1, were strongly up-regulated by culture.   We hypothesized that the AA transporters highly expressed in ppMBMECs and strongly down-regulated in culture play a major in vivo role for BBB transendothelial transport.

Keywords: culture vs non-cultured

overall_design: RNA was prepared from 4 sources of endothelial cells: 1) isolated mouse brain microvascular endothelial cells that were cultured in transwells with and 2) without non-contact co-culture with glial cultures or 3) further purified using anti-PECAM1 magnetic bead sorting and 4) the endothelioma b.End5 cell-line.

first: Victoria

last: Makrides

first: Ruth

last: Lyck

first: Nadine

last: Ruderisch

first: Clemens

middle: D

last: Cohen

first: Anton

middle: G

last: Moll

first: Britta

last: Engelhardt


title: Blood gene expression of rats exposed to acetaminophen or methyl parathion

city: Morgantown

first: Christina

middle: M

last: Umbright

state: WV

address: 1095 Willowdale Road

department: HELD

institute: DHHS/PHS/CDC/NIOSH

summary:
The purpose of this study was to investigate whether expression of specific genes in peripheral blood can be used as surrogate marker(s) to detect and distinguish target organ-specific chemical toxicity in rats.  Rats were intraperitoneally administered a single, acute dose of a well-established hepatotoxic (acetaminophen) or neurotoxic (methyl parathion) chemical. Administration of acetaminophen (AP) or methyl parathion (MP) in the rats resulted in hepatotoxicity as evidenced from elevated blood transaminase activities or neurotoxicity as evidenced from the inhibition of acetyl cholinesterase activity in the blood, respectively. Microarray analysis of the global gene expression profile of rat blood identified distinct gene expression markers capable of detecting and distinguishing hepatotoxicity and neurotoxicity induced by AP and MP, respectively. Differential expressions of the marker genes specific for hepatotoxicity and neurotoxicity were detectable in the blood much earlier than the appearance of the commonly used clinical markers, serum transaminases and acetyl cholinesterase. In summary, our results demonstrated that blood gene expression markers can detect and distinguish target organ toxicity non-invasively and with higher sensitivity than traditional surrogate markers.

Keywords: blood, acetaminophen, methyl parathion, rat, hepatotoxicity, neurotoxicity

overall_design:
38 samples were analyzed in this experiment. 15 rats were intraperitoneally administered acetaminophen (AP), 15 rats were intraperitoneally administered methyl parathion (MP), and 8 rats were intraperitoneally administered vegetable oil. At time intervals of 4, 12, and 24 hours following administration of acetaminophen or methyl parathion, 4 rats per chemical were sacrificed and blood was collected. At the 168-hour time interval, 3 rats per chemical were sacrificed and blood was collected. The control animals were sacrificed at the 24-hour time interval. Therefore, there were four rats in each of the 4-, 12-, and 24-hour time points for each chemical and four control rats for each chemical. Similarly, there were three rats in the 168-hour time interval for each chemical. The groups for each chemical and time interval are labeled as follows:
AP—control—4 rats: blood_AP_0_4, blood_AP_0_5, blood_AP_0_33, and blood_AP_0_34
AP—4-hours—4 rats: blood_AP_4_35, blood_AP_4_38, blood_AP_4_39, and blood_AP_4_40
AP—12-hours—4 rats: blood_AP_12_41, blood_AP_12_42, blood_AP_12_43, and blood_AP_12_45
AP—24-hours—4 rats: blood_AP_24_47, blood_AP_24_48, blood_AP_24_49, and blood_AP_24_51
AP—168-hours—3rats: blood_AP_168_53, blood_AP_168_54, and blood_AP_168_56
MP—control—4 rats: blood_MP_0_1, blood_MP_0_2, blood_MP_0_3, and blood_MP_0_ 31
MP—4-hours—4 rats: blood_MP_4_11, blood_MP_4_12, blood_MP_4_13, and blood_MP_4_22
MP—12-hours—4 rats: blood_MP_12_20, blood_MP_12_23, blood_MP_12_24, and blood_MP_12_25
MP—24-hours—4 rats: blood_MP_24_6, blood_MP_24_7, blood_MP_24_9, and blood_MP_24_21
MP—168-hours—3rats: blood_MP_168_26, blood_MP_168_27, and blood_MP_168_29

first: Rajendran

last: Sellamuthu

first: Shengqiao

last: Li

first: Michael

last: Kashon

last: Luster

first: Joseph

last: Pius


title: Global Transcription Profiling and Virulence Potential of Streptococcus pneumoniae after Serial Passage

status: Public on May 29 2009

city: Galveston

first: Utpal

last: Pandya

state: TX

address: 301 University Blvd

department: Microbiology and Immunology

institute: University of Texas Medical Branch

summary:
Alterations in global transcriptional profiles of S. pneumoniae 6304 serotype 4 after 50 passages (50P) and 100 passages (100P) on laboratory media

Keywords: Adaptation to unique growth environment

platform_organism:
Streptococcus pneumoniae TIGR4
Streptococcus pneumoniae R6
Streptococcus pneumoniae G54

overall_design:
Three conditions experiment:
Control, single passage strain (1P), 2 biological replicates
Experimental 50 passage strain (50P), 2 biological replicates
Experimental 100 passage strain (100P), 2 biological replicates
All strains independently grown and harvested at OD600 ~ 0.5.
Four spot replicates per array.

first: David

last: Niesel


title: TBP ChIP-chip on Jurkat cells

status: Public on May 28 2009

city: Lexington

first: hongyan

last: xing

state: KY

address: 741 S. Limestone

department: Biochemistry

institute: University of Kentucky

type: Genome binding/occupancy profiling by genome tiling array

summary: During mitosis, TATA-binding protein(TBP), which remains bound to DNA during mitosis, recruits PP2A and also interacts with a subunit of the condensin complex to allow efficient dephosphorylation and inactivation of condensin near these promoters to inhibit their compaction. These result suggest that TBP function is not only important for expression of genes transcribed bDuring mitosis, TATA-binding protein(TBP), which remains bound to DNA during mitosis, recruits PP2A and also interacts with a subunit of the condensin complex to allow efficient dephosphorylation and inactivation of condensin near these promoters to inhibit their compaction. These result suggest that TBP function is not only important for expression of genes transcribed by all three RNA polymerase during interphase, but also for transmitting the memory of gene activity through mitosis to daughter cellsy all three RNA polymerase during interphase, but also for transmitting the memory of gene activity through mitosis to daughter cells

overall_design: We use ChIP-chip approach to identify TBP-bindind sites in mitotic Jurkat cells at genome-wide level. Input DNA fragments and  DNA fragments immunoprecipitated by TBP antibodies by ChIP assay on mitotic Jurkat cells were used to probe the Affymetrix Genechip human tiling 2.0c to identift TBP-binding sites in mitotic Jurkat cells. Input and ChIP sample were replicated three times as following: Input rep1, TBP ChIP1, Input rep2, TBP ChIP2, Input rep3, TBP ChIP3.

first: Hongyan

last: Xing

first: Kevin

last: Sarge


title: Transcriptome adaptation of group B Streptococcus to growth in human amniotic fluid

city: Warsaw

first: Izabela

last: Sitkiewicz

address: Chełmska 30/34

department: Epidemiology and Clinical Microbiology

country: Poland

institute: National Medicines Institute

summary: We have discovered that GBS significantly remodels its transcriptome in response to exposure to human amniotic fluid.  A large number of the affected genes are of unknown function, which means that much remains to be learned about the full influence of amniotic fluid on GBS. The majority of the observed changes in transcripts affects genes involved in basic bacterial metabolism and is connected to AF composition and nutritional requirements of the bacterium. The observation that many genes encoding adhesions are down-regulated, and genes encoding known virulence factors such as a hemolysin and a potent IL-8 proteinase are up-regulated likely have consequences for the outcome of host-pathogen interactions.

overall_design: Streptococcus agalactiae, serotype III strain NEM316 was grown in human amniotic fluid. Samples of bacteria were collected in mid log, late log and stationary growth phases and were used for RNA isolation for microarray analysis

first: James

last: Musser

first: Nicole

last: Green

first: Nina

last: Guo

first: Ann

last: Bongiovanni

first: Steven

middle: S

last: Witkin


title: Comparison of gene expression in embryos in wich Smicl has been knocked-down and in control embryos

status: Public on May 30 2009

city: Cambridge

first: Clara

last: Collart

address: Tennis court road

department: Gurdon Institute

country: United Kingdom

institute: Cambridge University

summary:
Embryos were injected with either control or Smicl antisense morpholino oligonucleotides and harvested at Stage 10.5 for RNA analysis.
Keywords: Knock-down analysis

overall_design: Embryos from three different spawnings where injected at one cell stage with either control or Smicl antisense morpholino oligonucleotides and cultured to early gastrula stage 10.5 for RNA isolation. Some embryos were cultured to further development to check their phenotypes. Each slide was hybridised with a mixture 1:1 of sample and control cDNAs, each labelled with a different dye. Hybridisations were carried out as dye-swapped technical replicates, therefore six microarray slides were hybridized

middle: H

first: Joana

last: Ramis

first: Jim

middle: C

last: Smith


title: Compensatory IKKa activation of classical NF-kB signaling during IKKb inhibition

city: Bethesda

first: Louis

middle: M.

last: Staudt

state: MD

address: 9000 Rockville Pike, Bldg 10, Rm 4N114

department: Lymphoid Malignancies Branch

institute: National Cancer Institute

summary:
A subtype of diffuse large B-cell lymphoma (DLBCL), termed activated B-cell-like (ABC) DLBCL, depends on constitutive NF-kB pathway signaling for survival. Small molecule inhibitors of IkB kinase b (IKKb), a key regulator of the NF-kB pathway, kill ABC DLBCL cells and hold promise for the treatment of this lymphoma type. We conducted an RNA interference genetic screen to investigate potential mechanisms of resistance of ABC DLBCL cells to IKKb inhibitors. We screened a library of small hairpin RNAs (shRNAs) targeting 500 protein kinases for shRNAs that would kill an ABC DLBCL cell line in the presence of a small molecule IKKb inhibitor more effectively than in its absence. Two independent shRNAs targeting IKKa synergized with the IKKb inhibitor to kill three different ABC DLBCL cell lines but were not toxic by themselves. Surprisingly, IKKa shRNAs blocked the classical rather than the alternative NF-kB pathway in ABC DLBCL cells, as judged by inhibition of IkBa phosphorylation. IKKa shRNA toxicity was reversed by coexpression of wild type but not kinase inactive forms of IKKa, suggesting that IKKa may directly phosphorylate IkBa under conditions of IKKb inhibition. These results suggest that therapy for ABC DLBCL may be improved by targeting both IKKa and IKKb.

Keywords: compound treatment design

overall_design: Gene expression profiling of OCI-Ly3 cells with or without expressing IKKa shRNA in the presence or absence of 12.5 uM IKKb inhibitor for 2 and 3 days. Four samples were analyzed.


title: rdi1∆ vs WT during macrophage co-culture

city: Lynchburg

first: Michael

last: Price

state: VA

address: 1971 University Blvd.

department: Biology & Chemistry

institute: Liberty University

summary:
Rho-GDP dissociation inhibitors (RDI) are repressors of Rho-type monomeric GTPases that allow for precise control of their target processes, e.g. cytoskeletal arrangement, vesicle trafficking, and polarized growth.  In the human pathogenic yeast Cryptococcus neoformans, maintenance of normal cell morphology is vital for pathogenicity.  We identified and deleted the gene encoding an RDI homolog in the human fungal pathogen Cryptococcus neoformans and investigated its impact on pathogenicity in animal models of cryptococcosis.  Rdi1 deletion resulted in altered vacuole size in tissue culture medium, with corresponding alterations in expression of vesicle trafficking related genes.  The rdi1∆ mutant strain showed reduced intracellular survival in macrophages, and severe attenuation of virulence in murine models of cryptococcosis. This reduction in virulence of the rdi1 mutant occurs in the absence of major defects in growth, morphology, or classical virulence-associated phenotypes.
Keywords: mutant response, macrophage co-culture

overall_design: WT and rdi1∆ total RNA were compared in a dye-swap experiment.  One culture of each representing a population of each strain were incubated with macrophages at a ratio of 10:1 (yeast:macrophage).  The co-cultures were incubated for 1hour at 37°C, 5% CO2 in tissue culture medium.

first: Connie

middle: B

last: Nichols

first: Meredith

middle: F

last: Clements

first: Andrew

last: Alspaugh


title: Transcriptome Profiles of Medicago Stem Internodes

city: SAINT PAUL

first: John

middle: W

last: Gronwald

state: MN

address: 1991 UPPER BUFORD CIRCLE

institute: USDA-ARS

summary:
Advances in alfalfa [Medicago sativa (L.) subsp. sativa] breeding, molecular genetics and genomics have been slow because this crop is an allogamous autotetraploid (2n = 4x = 32) with complex polysomic inheritance. Increasing cellulose and decreasing lignin in alfalfa stem cell walls would improve this crop as a cellulosic ethanol feedstock. We selected two alfalfa genotypes (252, 1283) that differ in cellulose and Klason lignin concentration in stem cell walls. Analysis of GeneChip expression data files of alfalfa stem internodes of genotypes 252 and 1283 at two growth stages (elongating, post-elongation) revealed 10,887 SFPs in 8,230 probe sets. Validation analysis by PCR-sequencing of a random sample of SFPs indicated a 12% false discovery rate. Functional classification and over-representation analysis showed that both genotypes were highly enriched in SFP-harboring cell wall genes. We mapped 5,833 of the 8,230 SFP-harboring genes onto putative orthologous loci on Medicago truncatula chromosomes. Clustering and over-representation of SFP-harboring genes within the same functional class (e.g. cell wall genes) was observed on some chromosomes. Prior to analysis of expression data for the two alfalfa genotypes, SFP probes were masked to reduce false positives and false negatives. The combination of SFP and gene expression analysis provide a list of candidate cell wall genes that can be used as molecular markers in a breeding program to improve alfalfa as a cellulosic feedstock. The results of this study will also be useful in advancing understanding of genome organization in alfalfa and for comparative genomics research with other legume species.

Keywords: Stem development and genotype comparison

platform_organism:
Sinorhizobium meliloti
Medicago sativa
Medicago truncatula

overall_design: The alfalfa clonal lines 252 and 1283 were propagated from cuttings and grown in the greenhouse. The greenhouse experiments consisted of three replicates arranged in a randomized complete block design. There were eight plants of each clone, in individual pots, in each replicate. Plant material for analysis was composited within each replicate at harvest. Stem internode tissues were harvested at full bloom. Based on tissue pliability and coloration, the internode in transition from elongation to post-elongation cambial growth was identified. The internodes immediately above (elongating internodes) and below this transition internode (post-elongation internodes) were collected for RNA extraction.

first: Samuel

last: Yang

first: Mesfin

last: Tesfaye

first: Wayne

last: Xu

first: JoAnn

last: Lamb

first: Hans-Joachim

last: Jung

first: Deborah

middle: A

last: Samac

first: Carroll

middle: P

last: Vance


title: Impaired Cohesin Loading in Cornelia de Lange Syndrome Results in a Highly Specific Pattern of Dysregulated Genes

city: Bunkyo-ku

first: Katsuhiko

last: Shirahige

state: Tokyo

address: 1-1-1 Yayoi

department: Research Center for Epigenetic Disease

institute: The University of Tokyo

summary:
Heterozygous mutations in the cohesin regulator, NIPBL, or cohesin structural components SMC1A, and SMC3, result in Cornelia de Lange Syndrome (CdLS).  Genome-wide transcription assessment has identified unique profiles of genes dysregulated in CdLS that correlate with different clinical presentations. Cohesin binding analysis demonstrates a preference for intergenic regions suggesting a cis-regulatory function mimicking that of an insulator.  However, the binding sites are enriched within the promoter regions of the dysregulated genes and are significantly decreased in CdLS probands, indicating an alternative role of cohesin as a classic transcription factor.

Keywords: ChIP-chip

overall_design: This series contains two sets of whole genome ChIP-chip data. The experiments were done for the cohesin subunit hScc1.

first: Masashige

last: Bando

first: Takehiko

last: Itoh


title: In vivo transcriptome of Listeria monocytogenes EGDe

city: Porto

first: Didier

last: Cabanes

state: Porto

address: rua do Campo Alegre, 823

country: Portugal

institute: IBMC

summary: Time course study of the mouse infection by comparing the genomic transcriptional patterns of Listeria monocytogenes EGDe grown under laboratory conditions (exponential growth phase) with that of in vivo-grown bacteria (in mouse spleens) over three days of infection.

first: Ana

last: Camejo

first: Carmen

last: Buchrieser

first: Elisabeth

last: Couvé

first: Olga

last: Reis

first: Filipe

last: Carvalho

first: Pierre

last: Ferreira

first: Sandra

last: Sousa

first: Pascale

last: Cossart


title: Detection of microRNA expression during 3T3-L1 pre-adipocyte differentiation using a custom made microarray chip.

status: Public on May 26 2009

city: Houston

first: Scott

middle: Andrew

last: Ochsner

address: One Baylor Plaza

department: Molecular and Cellular Biology

institute: Baylor College of Medicine

summary: Differentiation of 3T3-L1 cells into adipocytes involves a highly orchestrated series of events including  clonal expansion, growth arrest and terminal differentiation. The mechanisms coordinating these  different steps are not yet fully understood. Here we investigated whether micro (mi)RNAs play a role in  this process. Microarray analysis was performed to detect miRNA expression during 3T3-L1 preadipocyte  differentiation. Several miRNAs, including let-7, were up-regulated during 3T3-L1  adipogenesis. Ectopic introduction of let-7 into 3T3-L1 cells inhibited clonal expansion as well as  terminal differentiation. The mRNA encoding high mobility group AT-hook 2 (HMGA2), a  transcription factor that regulates growth and proliferation in other contexts, was inversely correlated  with let-7 levels during 3T3-L1 cell adipogenesis, and let-7 markedly reduced HMGA2 concentrations.  Knockdown of HMGA2 inhibited 3T3-L1 differentiation. These results suggest that let-7 plays an  important role in adipocyte differentiation and that it does so in part by targeting HMGA2, thereby  regulating the transition from clonal expansion to terminal differentiation.

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: 3T3-L1 cells were induced to differentiation into mature adipocytes using a canonical DMI cocktail. The time point at two days after confluency of 3T3-L1 was defined as day 0. Samples were collected at day 0, day 1, day 4, and day 7. The expression of microRNAs at day 1, day 4, and day 7 was compared to that of day 0.

first: Tingwan

last: Sun

first: Mingui

last: Fu

first: Angie

middle: L

last: Bookout

first: Jinchun

last: Zhou

first: Quan-Zhen

last: Li

first: Steven

last: Kliewer

first: David

middle: J

last: Mangelsdorf


title: Gene Expression during the Egg Development of Drosophila melanogaster.

first: Dean

last: Baker

address: Department of Genetics, Downing Street

institute: University of Cambridge

summary: Despite the importance of egg development to the female life cycle in Drosophila, global patterns of gene expression have not been examined in detail primarily due to the difficulty of synchronizing developmental stages.  Entry into vitellogenesis is however an key stage of oogenesis, and by delaying entry past this control point, we have been able to investigate some of the transcriptional dynamics apparent before and after early egg formation over a 72 hour period.

overall_design: A short-day photoperiod (10:14 light:dark photoperiod) and moderate temperatures (11°C) are required for the induction of ovarian diapause.  For our microarray experiments, 0-4 hours post-eclosion females were immediately introduced to the restrictive environmental conditions.  Females were subsequently collected for RNA extraction after 5-7 days, or were alternatively re-introduced to normal environmental conditions to continue egg development.  Time-points were collected before and after the release of reproductive arrest at 24 hour intervals and hybridized in a balanced loop design, including dye-swaps.  Eight biological replicates were subsequently performed for each treatment, including 3 day old male and female controls maintained under normal environmental conditions.

last: Russell


title: Symbiodinium clade content drives host transcriptome more than thermal stress in the coral Montastraea faveolata part 2

status: Public on May 31 2009

city: San Francisco

first: Michael

last: DeSalvo

state: CA

department: Department of Anesthesia

institute: UC San Francisco

summary: A mutualistic relationship between reef-building corals and endosymbiotic algae (Symbiodinium spp.) forms the basis for the existence of coral reefs. Genotyping tools for Symbiodinium spp. have added a new level of complexity to studies concerning cnidarian growth, nutrient acquisition, and stress. For example, the response of the coral holobiont to thermal stress is connected to the host-Symbiodinium genotypic combination, as different partnerships can have different bleaching susceptibilities. If, and to what extent, differences in algal symbiont clade contents can exert effects on the coral host transcriptome is currently unknown. In this study, we monitored algal physiological parameters and profiled the coral host transcriptional responses in acclimated, thermally stressed, and recovered coral fragments using a custom cDNA gene expression microarray. Combining these analyses with results from algal and host genotyping revealed a striking symbiont effect on both the acclimated coral host transcriptome and the magnitude of the thermal stress response. This is the first study that links coral host transcriptomic patterns to the clade content of their algal symbiont community. Our data provide a critical step to elucidating the molecular basis of the apparent variability seen among different coral-algal partnerships.

overall_design:
Sample Collection and Tank Experiment. On July 31 2007, six replicate fragments (9.5 ± 3.5cm2) were collected using hammer and chisel from the upper sun-exposed surface of five different, healthy-looking Montastraea faveolata colonies near Puerto Morelos, Quintana Roo, México (20o52’28.77”N and 86o51’04.53”W). The fragments were transported to the institute within 1 h and divided evenly between two 50 L aquaria (i.e., 3 fragments from each colony were placed into each tank) that received a constant flow of seawater (0.6 L/min). Each aquarium was fit with a water pump connected to a spray-bar to provide constant water movement and aeration. Both aquaria were placed in a common pond with flowing water to buffer diurnal temperature fluctuations, and both aquaria were acclimated to the same shaded ambient light condition. All coral fragments were mounted on plasticene and kept at a depth of 7 cm. From 10 to 19 August 2007, both aquaria received an average water temperature of 27.9 ± 0.6oC (as recorded by HOBO Light/Temperature Data Loggers, Onset Corp.). Beginning on 11 August, dark-adapted maximum quantum yields for charge separation (Fv/Fm) were measured at dusk for all coral fragments using a DIVING-Pulse Amplitude Modulated (PAM) fluorometer (Walz, Germany). Photosynthetically active radiation (PAR) was measured at noon and averaged 318 ± 129 µmol m-2 s-1. From 20 to 21 August 2007, all coral fragments were brought inside during the passage of Hurricane Dean. On 22 August, the experiment was reconstituted, and acclimation continued on 23 August and lasted until 1 September. During this time, both aquaria received a mean water temperature of 28.5 ± 0.8oC, and mean PAR of 371 ± 169 µmol m-2 s-1. On the night of 1 September, control time point samples from five different colonies were collected from each tank (Figure 1), before one 200-Watt aquarium heater was turned on in the treatment aquarium. A second heater was turned on 3 days later. During the thermal stress experiment, the control aquarium received mean water temperature of 28.8 ± 1.2oC; the heated aquarium, 31.5 ± 1.1oC. During the thermal stress experiment, tanks received mean PAR of 420 ± 152 µmol m-2 s-1. On 11 September, and 19 October, one sample each from five different colonies was collected from each tank for the bleaching and recovery time point, respectively.
Microarray Gene Expression Analysis of Coral Host Gene Expression. For the biologically replicated time series experiment, a pool of RNA from all control tank fragments was used as a reference RNA sample, against which each sample from the treatment tank was competitively hybridized. All control and heat-stressed samples were competitively hybridized against the reference RNA. Dye swaps were not performed, as any dye bias present is equal in all comparisons to the reference.

Note: Samples VALUEs used for statistical analysis of differential expression.  GSE15253_shini_cbr_matrix2_norm.txt used for hierarchical clustering.

first: Shinichi

last: Sunagawa

middle: K

first: Paul

last: Fisher

first: Christian

middle: R

last: Voolstra

first: Roberto

last: Iglesias-Prieto

first: Monica

last: Medina


title: Accumulation of sumoylated Rad52 in checkpoint mutants perturbed in DNA replication

city: Kisarazu

first: Kazuto

last: Kugou

state: Chiba

address: 2-6-7 KazusaKamatatri

department: Department of Frontier Research

institute: Kazusa DNA Research Institute

summary:
Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, ChIP on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double deletion of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD51 single deletion. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed.

Keywords: ChIP-chip

overall_design:
• The goal of the experiment
Genome-wide localization of Rad52 binding sites in Saccharomyces cerevisiae
• Experimental factor
Distribution of Rad52 on chromosome III, IV, and V and the right arm of chromosome VI
Strain: wild type, rad53 mutant, and rad53 siz2 mutant (W303 background, expressing myc tagged protein from its native promoter)
Cell condition 1: G1 arrest with alpha-factor
Cell condition 2: HU treatment
• Experimental design
ChIP analyses: ChIP using anti-c-Myc antibody.
ChIP-chip analyses: In all cases, hybridization data of ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F) were used.
• Quality control steps taken
Confirmation of several loci by quantitative real time PCR.
Wild type cells expressing non-tagged Rad52 were used as a negative control of DNA amplification.

first: Takashi

last: Ohuchi

first: Masayuki

last: Seki

first: Shusuke

last: Tada

first: Kunihiro

last: Ohta

first: Takemi

last: Enomoto


title: Transcriptional profiling of Escherichia coli cultured under severe zinc limitation

city: Sheffield

first: Robert

last: Poole

state: South Yorkshire

address: Firth Court, Western Bank

department: Molecular Biology & Biotechnology

institute: University of Sheffield

summary: Escherichia coli strain MG1655 was grown in parallel chemostat cultures in GGM.  In one chemostat the culture was fed adequate Zn; in the other, measures were taken to eliminate Zn from the chemostat vessel and culture medium.  Culture volume (120 ml), temperature (37 oC) and stirring speed (437 rpm) were maintained.  Steady state values for pH and OD600 were 6.9 and 0.6, respectively. After 50 hours, samples from the adequate Zn and Zn-limited chemostats were harvested into RNAprotect and total RNA was purified using Qiagen’s RNeasy Mini kit (using the supplier’s protocol) prior to use in microarray analysis.  Biological experiments (i.e. a comparison of adequate versus low Zn in chemostat culture) were carried out three times, and a dye swap performed for each experiment, providing two technical repeats for each of the three biological repeats.

overall_design:
Cells were grown in glycerol-glycerophosphate medium (GGM).  Final concentrations in GGM are: MES (40.0 mM), NH4Cl (18.7 mM), KCl (13.4 mM),beta-glycerophosphate (7.64 mM), glycerol (5.00 mM), K2SO4 (4.99 mM), MgCl2 (1.00 mM), EDTA (134 microM), CaCl2.2H2O (68.0 microM), FeCl3.6H2O (18.5 microM), ZnO (6.14 microM), H3BO3 (1.62 microM), CuCl2.2H2O (587 nM), CoNO3.6H2O (344 nM), and (NH4)6Mo7O24.4H2O (80.9 nM) in MilliQ water (Millipore).  Bulk elements (MES, NH4Cl, KCl, K2SO4 and glycerol in MilliQ water at pH 7.4 (batch growth) or 7.6 (continuous culture)) were passed through a column containing Chelex-100 ion exchange resin (Bio-Rad) to remove contaminating cations.  Trace elements (with or without Zn as necessary) and a CaCl2 solution were then added to give the final concentrations shown above prior to autoclaving.  After autoclaving, MgCl2 and beta-glycerophosphate were added at the final concentrations shown above.  All chemicals were of AnalaR grade of purity or higher.  Chelex-100 was packed into a Bio-Rad Glass Econo-column (approximately 120 mm × 25 mm) that had previously been soaked in 3.5% nitric acid for 5 d.

E. coli strain MG1655 was grown in parallel custom-built chemostats made entirely of non-metal parts.  One chemostat was fed medium that contained “adequate” Zn (that normally found in GGM), whilst the other contained no added Zn and had been actively depleted of Zn by use of the special precautions listed here.  Glass growth vessels and flow-back traps were soaked extensively (approximately two months) in 10% nitric acid before being rinsed thoroughly in MilliQ water.  Vent filters (Vent Acro 50 from VWR) were connected to the vessel using PTFE tubing.  Metal-free pipette tips were used (MAXYMum Recovery Filter Tips from Axygen).  Culture volume was maintained at 120 ml using an overflow weir in the chemostat vessel.  The dilution rate (and hence the specific growth rate) was 0.1 h-1.   The vessel was inoculated using one of the side-arms.  Flasks were placed on KMO 2 Basic IKA-Werke stirrers (arbitrary setting: 400).  Stirring speed was calibrated (437 rpm) and matched between vessels using a handheld laser tachometer (Compact Instruments Ltd).  Temperature was kept constant at 37°C.  Twice daily, a 2-ml sample was taken and used to test the pH (which stayed constant at pH 6.9), OD600 (0.6 at steady state), glycerol limitation, and contamination on nutrient agar plates.

The “Zn-free” chemostat was inoculated with cells that had been sub-cultured in Zn-free medium to deplete internal stores of Zn.  A 0.25 ml aliquot of a saturated culture of strain MG1655 grown in LB was centrifuged and the pellet used to inoculate 5 ml of GGM that was incubated overnight at 37°C with shaking.  A 2.4 ml (i.e. 2% of chemostat volume) aliquot of this was then used to inoculate the chemostat.  The “adequate Zn” chemostat was inoculated with cells treated in essentially the same way but grown in GGM containing an “adequate” level of Zn.  The two cultures (+/- Zn) used to inoculate the chemostats had OD600 readings within 2.5% of each other.  Chemostats were grown for 50 h to allow five culture volumes to pass through the vessel and allow an apparent (pseudo-)steady state to be reached.   At this point, 10 ml samples were removed from the chemostats and total RNA was stabilised using RNAprotect (Qiagen).  Total RNA was purified using Qiagen’s RNeasy mini kit.

Equal quantities of RNA from adequate Zn and Zn-limited chemostats were labeld by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer).  For each microarray slide, one sample was labeld with Cy3-dCTP, while the other sample was labeld with Cy5-dCTP.  RNA (15-20 micrograms) was annealed to 9 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65°C for 10 min, followed by 10 min at 22°C.  This was supplemented with 4 microlitres of 5× First-strand buffer (Invitrogen), 2 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 2 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1 microlitre Superscript II reverse transcriptase (200 U) (Invitrogen).  This was incubated at 42°C for 2 hours.  The reaction was terminated by the addition of NaOH (5 microlitres of 1M) and HCl (5 microlitres of 1M) before the addition of TE buffer (200 microlitres).  Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen).

The Cy3-dCTP-labeld sample was mixed with the Cy5-dCTP-labeld sample and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides).  This was heated at 95°C 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 37°C: 1× SSC/0.1 % SDS, 1× SSC, 0.2× SSC and 0.01× SSC.  Microarray slides were dried by centrifugation at 500 × 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 or Cy5/Cy3 (test/reference) fluorescent ratios were calculated from the normalized values.

Biological experiments (i.e. a comparison of adequate versus low Zn in chemostat culture) were carried out three times, and a dye swap performed for each experiment, providing two technical repeats for each of the three biological repeats.  Data from the independent experiments were combined.  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.

first: Alison

middle: I

last: Graham


title: Gene expression data from human erythroleukemic cells in a time dependent manner after PMA treatment

city: Seoul

first: Jin-Kyung

last: Limb

address: 11-1 Daehyun-dong, Seodaemun-gu,

department: Molecular life science

country: South Korea

institute: Ewha Womans University

summary: The molecular regulation of megakaryocytic differentiation is poorly understood.  Using a myelogenous leukemia cell line, K562, which can partly recapitulate the process in response to phorbol 12-myristate 13-acetate (PMA), we performed a microarray-based gene expression profiling using Illumina HumanRef-8 Expression Beadchip to identify genes that play significant roles in megakaryopoiesis.  Time course microarray data were obtained at 8 time points.  Illumina HumanRef-8 Expression Beadchip has ~over 24,000 transcripts a piece. Duplicate experiments were performed for each time point.  Here, we reported raw data of two independent experiments.  After microarray data analysis, selecting genes with significant detection p-value produced ~14,000 probes out of total 23,920 probes. Quantile normalization was carried out for each dataset at 8 time points using the average expression value.  The K-means clustering method (k=12) was used to classify those probes according to characteristic temporal pattern.  We selected transcription factors shown fold changes in a time dependent manner to examine genes related for megakaryopoiesis.  Finally, we can suggest that FosB which belongs to the Fos family of AP1 transcription factors is related in megakaryopoiesis.

overall_design: The K562, a human chronic myeloid leukemia cell line, was obtained from the American Type Culture Collection and cultured in RPMI containing 10% heat-inactivated fetal bovine serum (Hyclone) 100 unit/ml penicillin and 100ug /ml streptomycin in a humidified 5% CO2 atmosphere at 37 degrees C.  Differentiation toward megakaryocytic lineage was induced by growing the 3 × 105 K562 cells in a six-well plate in media containing 20 nM, PMA dissolved in dimethyl sulfoxide (DMSO) for 0, 0.5, 1, 3, 6, 12, 24, and 48 hour.  Control cultures were treated similarly with the solvent DMSO. Total RNA was extracted from two independent experiments using a RNeasy Mini Extraction Kit (Qiagen, Hilden, Germany).  Biotinylated cRNA was prepared from 550 ng total RNA per sample using the Illumina TotalPrepTM RNA Amplification Kit (Ambion, Austin, TX) and hybridized to the Illumina HumanRef-8 Expression Beadchip (Illumina, Inc., San Diego, CA) according to the manufacturer's instructions.

first: Jaesang

last: Kim

first: Bohee

first: Suhyeon

last: Yoon

first: Sanghyuk

last: Lee

first: YunSoo

last: Bae

first: Gil-Ja

last: Jhon


title: The expression profiles analysis of shigella flexneri incubated in rabbit ileum

status: Public on May 22 2009

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: To find the alterations of expression profiles of shigella flexneri, we performed DNA chip analysis and proteomic analysis at the same time.

overall_design: an overnight culture of the wild-type strain in LB broth was harvested and resuspended in 20 ml PBS buffer and then incubated in PBS at 37 degree and in rabbit ileum for 4 hours respectively.

first: Hengliang

last: Wang


title: MicroRNA signatures predict Estrogen receptor, Progesterone receptor and HER2/neu receptor status in Breast Cancer

status: Public on May 21 2009

city: Nottingham

first: Christophe

last: Lemetre

address: Clifton Lane

department: Bioinformatics

institute: The Nottingham Trent University

summary: Breast cancer is a heterogeneous disease encompassing a number of phenotypically diverse tumours. Expression levels of the estrogen, progesterone and HER2/neu receptors which characterise clinically distinct breast tumors have been shown to change during disease progression and in response to systemic therapies. Mi(cro)RNAs play critical roles in diverse biological processes and are aberrantly expressed in several human neoplasms including breast cancer, where they function as regulators of tumour behaviour and progression. The aims of this study were to identify miRNA signatures that accurately predict the oestrogen receptor (ER), progesterone receptor (PR) and HER2/neu receptor status of breast cancer patients to provide insight into the regulation of breast cancer phenotypes and progression.

overall_design:
Expression profiling of 353 microRNAs was performed in 29 early stage breast cancer specimens. MiRNA signatures associated with ER, PR and HER2/neu status were generated using artificial neural networks (ANN) and expression of specific microRNAs was validated using RQ-PCR.
Results: Stepwise artificial neural network (ANN) analysis identified predictive miRNA signatures corresponding with estrogen (miR-342, miR-299, miR-217, miR -190, miR-135b, miR-218), progesterone (miR-520g, miR-377, miR-527-518a, miR-520f-520c) and HER2/neu (miR-520d, miR-181c, miR-302c, miR-376b, miR-30e) receptor status. MiR-342 and miR-520g expression was further analysed in 95 breast tumours. MiR-342 expression was highest in ER and HER2/neu positive luminal B tumours and lowest in triple-negative tumours. MiR-520g expression was elevated in ER and PR negative tumours.

first: Aoife

last: Lowery

first: Nicola

last: Miller

first: A

last: Devaney

first: Roisin

middle: E

last: McNeill

first: Pamela

last: Davoren

first: Vladimir

last: Benes

first: Sabine

last: Schmidt

first: Jonathon

last: Blake

first: Graham

last: Ball

first: Michael

last: Kerin


title: IFN-alpha Stimulated Gene Expression in Sensitive and Resistant Renal Cell Carcinoma Cell Lines

first: Michelle

last: Holko

address: 45 Center Dr

department: NCBI

institute: GEO

summary: The antiproliferative, antiviral, and immunomodulatory properties of interferons (IFNs) have led to its therapeutic implementation. IFNs effects are mediated by a complex network of signal transducers, culminating in IFN-stimulated gene (ISG) induction. This complexity leads to diverse clinical responses to IFN, from no response to complete regression of disease. Elucidation of ISG induction patterns is, therefore, essential to understand and maximize its therapeutic potential. To correlate ISG expression profiles with IFN responsiveness, two renal cell carcinoma (RCC) cell lines differing in antiviral and apoptotic response to IFN were treated with IFN-alpha  for different times, and expression profiles were analyzed using a customized microarray containing 850 unique putative ISGs. Genes with similar kinetics of induction in both cell lines were clustered and analyzed for gene function. Seven sets of coordinately regulated genes were identified by k-means cluster analysis, and significant functional similarities were identified for five of the seven sets. Strikingly, expression of genes associated with transcription temporally preceded expression of those involved in signal transduction. Enhanced antiviral sensitivity to IFN was coincident with sustained expression of ISGs involved in transcriptional regulation. However, no difference in Stat1 activation was observed between the cell lines. Analysis of ISG expression patterns suggests that subtle differences in transcription profiles contribute to differences in IFN responsiveness.

overall_design: Two human renal cell carcinoma cell lines (ACHN and RCC1) were treated with IFN-alpha for 3, 6, 9, 12, 16, and 24 hours.  Total RNA from treated cells (Cy5) is hybridized with total RNA from untreated control cells (Cy3) to the custom ISG 1.3.1 cDNA array.

first: Bryan

last: Williams


title: Dorsal Ventral Pancreatic Bud Comparison

city: Woods Hole

first: Marko

last: Horb

state: MA

address: 7 MBL Street

institute: MBL

summary:
During embryogenesis, the pancreas develops from separate dorsal and ventral buds, which fuse to form the mature pancreas. Little is known about the functional differences between these two buds or the relative contribution of cells derived from each portion to the pancreas after fusion. To follow the fate of dorsal or ventral bud derived cells in the pancreas after fusion, we produced chimeric Elas-GFP transgenic/wild type embryos in which either dorsal or ventral pancreatic bud cells expressed GFP. We found that ventral pancreatic cells migrate extensively into the dorsal pancreas after fusion, whereas the converse does not occur. Moreover, we found that annular pancreatic tissue is composed exclusively of ventral pancreas derived cells. To identify ventral pancreas specific genes that may play a role in pancreatic bud fusion, we isolated individual dorsal and ventral pancreatic buds, prior to fusion, from stage 38/39 Xenopus laevis tadpoles and compared their gene expression profiles. Morpholino-mediated knockdown of one of these ventral specific genes, transmembrane 4 superfamily member 3 (tm4sf3), inhibited dorsal-ventral pancreatic bud fusion as well as acinar cell differentiation. Conversely, overexpression of tm4sf3 promoted the development of annular pancreas. Our results are the first to define molecular and behavioral differences between the dorsal and ventral pancreas, and suggest an unexpected role for the ventral pancreas in pancreatic bud fusion.

Keywords: Developmental genomics

overall_design: We analyzed two samples of dorsal and two samples of ventral pancreatic buds.

first: Lori

middle: D

first: Zeina

last: Jarikji

first: Farhana

last: Shariff

first: Craig

last: Mandato

first: Ken

last: Cho


title: AIN-2::GFP IP of mRNAs associated with miRISC in stage sychronized C. elegans populations

city: Boulder

first: liang

last: zhang

state: CO

address: University of Colorado at Boulder, Han lab, Department of MCD Biology, Campus box 0347

institute: university of colorado at boulder

type:
Other
Expression profiling by array

summary: AIN-2::GFP IP were used to purify miRISC from stage sychronized C.elegans populations (Egg, L1, L2, L3 and L4 stages). The mRNA composition of the IP results and the corresponding total RNA samples were analyzed by WUSTL Caenorhabditis elegans Whole Genome 23k Oligo Array. The miRISC associated mRNAs (miRNA targets) in each stage were identified by measuring the relative enrichment of each mRNA in the IP sample versus the corresponding total RNA sample

overall_design: The mRNAs in AIN-2::GFP IP results (IP) and the corresponding input total lysate (tot) were analyzed for each stage (Egg, L1, L2, L3, and L4 stages). At least three independent biological replicates were analyzed for each stage.

first: Liang

last: Zhang

first: Molly

last: Hammell

first: Brian

last: Kudlow

first: Victor

last: Ambros

first: Min

last: Han


title: The Histone Deacetylase Rpd3p Coordinates Transient Changes in Genomic Expression in Response to Acute Stress

city: Madison

first: Audrey

middle: P.

last: Gasch

state: WI

address: 425-g Henry Mall

department: Laboratory of Genetics

institute: University of Wisconsin Madison

summary:
To characterize the role of chromatin remodeling proteins in mediating stress-activated expression changes, whole-genome expression was
followed in wild-type and mutant cells (including strains lacking the histone deacetylase Rpd3p, associated Rpd3p proteins, and the transcription factors Msn2p and Msn4p) responding to multiple stresses including heat shock, hydrogen peroxide treatment, and salt stress, as well as stress relief.
Keywords: Timecourse and single timepoint expression studies of stress response in yeast

overall_design: Gene expression was done in duplicate for timecourses of stress response and in at least triplicate for single timepoint measurements.  A total of 109 microarrays were done.

first: Adriana

last: Alejandro-Osorio

first: Dana

last: Huebert

first: Dominic

middle: T

last: Porcaro

first: Megan

middle: S

last: Sonntag

first: Jessica

last: Will


title: Rec8 guides canonical Spo11 distribution along yeast meiotic chromosomes

first: Kazuto

last: Kugou

summary:
Spo11-mediated DNA double strand breaks (DSBs) that initiate meiotic recombination are temporally and spatially controlled. The meiotic cohesin Rec8 has been implicated in regulating DSB formation, but little is known about the features of their interplay. To shed light on this point, we investigated the genome-wide localization of Spo11 in budding yeast during early meiosis by chromatin immunoprecipitation using high-density tiling arrays. We found that Spo11 is dynamically localized to meiotic chromosomes. Spo11 initially accumulated around centromeres and thereafter localized to arm regions as premeiotic S-phase proceeded. During this stage, a substantial proportion of Spo11 bound to Rec8 binding sites. Eventually, some of Spo11 further bound to both DSB and Rec8 sites. We also showed that such a change in a distribution of Spo11 is affected by hydroxyurea (HU) treatment. Interestingly, deletion of REC8 influences the localization of Spo11 to centromeres and in some of the intervals of the chromosomal arms. Thereby we observed a lack of DSB formation in a region-specific manner. These observations suggest that Rec8 would prearrange the distribution of Spo11 along chromosomes and will provide clues to understanding temporal and spatial regulation of DSB formation.
Keywords: ChIP-chip

overall_design:
• The goal of the experiment
Genome-wide localization of Spo11, Mre11, Rec8, and DSB sites on meiotic chromosomes in Saccharomyces cerevisiae
• Keywords
Meiosis, Meiotic homologous recombination, Premeiotic DNA replication, cohesin, Saccharomyces cerevisiae, Genome tilling array (chromosome III, IV, V, VI), Spo11, Mre11, Rec8, DSB (Double strand break)
• Experimental factor
Distribution of Spo11, Mre11, and Rec8 in wild type in early meiosis (1.5 hrs, 2 hrs, 3 hrs, 4 hrs, and 5 hrs in sporulation medium)
Distribution of Spo11 in rec8delta cells in early meiosis (1.5 hrs, 2 hrs, 3 hrs, 4 hrs, and 5 hrs in sporulation medium)
Distribution of Spo11 in wild type in the presence of HU (2hrs and 4 hrs in sporulation medium containing HU)
Distribution of DSB sites in rad50S mutant cells at 7 hrs in sporulation medium
Distribution of DSB sites in rec8delta rad50S mutant cells at 7 hrs in sporulation medium
• Experimental design
ChIP analyses: SK1 background cells expressing FLAG tagged protein were used for the ChIP using anti-FLAG M2 antibody.
ChIP-chip analyses: In all cases, hybridization data for ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F for chromosome III, IV, V, VI and rikDACF for chromosome VI) were used.
Mapping of DSB sites: DSB rich fraction was concentrated by ChIP of Spo11-FLAG in rad50S mutant without crosslinking. In the mutant, DSBs ramain unrepaired with covalently attached Spo11.Meiotic cells (at 7 hours in sporulation medium) were used for the analyses.
• Quality control steps taken
Confirmation of several loci by quantitative real time PCR. Southern blotting of several DSB sites.

first: Tomoyuki

last: Fukuda

first: Shintaro

last: Yamada

first: Masaru

last: Ito

first: Hiroyuki

last: Sasanuma

first: Saori

last: Mori

first: Yuki

last: Katou

first: Takehiko

first: Kouji

last: Matsumoto

last: Shibata

first: Katsuhiko

last: Shirahige

first: Kunihiro

last: Ohta


title: Genome-Wide Promoter Analysis of Epigenetic Regulation by Cocaine (MM5 data)

status: Public on May 20 2009

city: Dallas

first: guanghua

last: xiao

address: 5323 Harry Hines Blvd

department: Psychiatry

institute: University of Texas Southwestern Medical Center

summary: Changes in gene expression contribute to the long-lasting regulation of the brain's reward circuitry seen in drug addiction, however, the specific genes regulated and the transcriptional mechanisms underlying such regulation remain poorly understood. Here, we used chromatin immunoprecipitation coupled with promoter microarray analysis to characterize genome-wide epigenetic changes in the mouse nucleus accumbens, a crucial brain reward region, after repeated cocaine administration. Our findings reveal several interesting principles of gene regulation by cocaine and of the role of ΔFosB and CREB, two prominent cocaine-induced transcription factors, in this brain region.

overall_design: Mice were treated with cocaine or saline. Chromatin immunoprecipitation was performed for acetylated histone H3 and H4 as described previously (Kumar et al., 2005) with minor modifications. Immunoprecipitated DNA was amplified via ligation-mediated PCR and hybridized to Nimblgen mouse MM5 promoter arrays.  2 biological replicates per condition.

first: Kumar

last: Arvind

first: Renthal

last: William


title: Integration of tomato reproductive developmental landmarks and expression profiles, and the effect of SUN on fruit shape

city: Wooster

first: Esther

last: van der Knaap

state: OH

address: 1680 Madison Ave.

department: Horticulture and crop science

institute: The Ohio State University

summary: Universally accepted landmark stages are necessary to highlight key events in tomato reproductive development.  In this study, we provide a description of floral and fruit development in a red-fruited closely related wild relative of tomato, Solanum pimpinellifolium accession LA1589. We use established and propose new landmarks as the framework for the characterization of the tomato fruit shape gene SUN in fruit development. SUN controls fruit shape predominantly after fertilization and its effect reaches a maximum at 8 days post anthesis coinciding with fruit landmark 4 representing the globular embryo stage of seed development. We also analyzed gene expression profiles of floral buds 10 days before anthesis (floral landmark 7), anthesis-stage flowers (floral landmark 10 and fruit landmark 1), and 5 days post anthesis fruit (fruit landmark 3).  The expression profiles of the NILs that differ at sun showed that 34 genes were differentially expressed and most of them at a less than 2-fold difference. However, many genes were differentially expressed between the developmental times points, including many genes involved in phytohormone biosynthesis or signaling as well as organ identity and patterning of tomato fruit.

overall_design: Three biological replicates were conducted with three sets of LA1589 sun NILs that differ at sun growing during different time periods resulting in 3 time points x 2 genotypes x 3 replicates = 18 array hybridizations.

first: Han

last: Xiao


title: Symbiodinium clade content drives host transcriptome more than thermal stress in the coral Montastraea faveolata (part 1)

first: Michael

last: DeSalvo

summary: Given the overwhelming evidence that symbiont genotypes differentially affect host processes such as growth, bleaching susceptibility, and nutrient acquisition, we set out to measure gene expression differences in fragments of Montastraea faveolata harboring two different clades of Symbiodinium.  On the reefs near Puerto Morelos, México, colonies of M. faveolata are known to shift algal symbiont clade with depth, often associating with clade A at the top, clade B in the middle, and clade C near the bottom of the colony.  By measuring photosynthetic efficiency and gene expression in control and heat-stressed fragments containing either clade B, clade C, or a mix of both, we found that: 1) the algal response to thermal stress is due to both host and algal factors; 2) fragments of M. faveolata express different genes in response to sub-bleaching thermal stress depending on algal genotype; 3) the overall effect of heat stress on coral gene expression is less significant than the effect of housing different zooxanthellae types.  Overall, we present convincing evidence that different Symbiodinium clades may be functionally distinct, which in turn, greatly influences host gene expression.

overall_design: Six fragments (9.5 ± 3.5cm2) from the top (2.7m), middle (3.7m), and bottom (5.2m) of one massive colony of Montastraea faveolata were collected with a hammer and chisel at “La Bocana” reef near Puerto Morelos, Quintana Roo, Mexico on 31 July 2007.  The fragments were divided evenly between two aquaria (50L) that received a constant flow of seawater (~0.64L/min).  Each aquarium was fit with a water pump connected to a spray-bar to provide constant water movement and aeration.  Both aquaria were placed in a common pond with flowing water to buffer diurnal temperature fluctuations, and both aquaria were exposed to shaded ambient light.  All coral fragments were mounted on plasticene and kept at a depth of ~7cm. From 10 to 19 August 2007 (acclimation #1), both aquaria received an average water temperature of 27.9 ± 0.6oC (as recorded by HOBO Light/Temperature Data Loggers by Onset Corp.).  Effective quantum yield (ΔF/Fm’) measurements were taken at noon, and maximum quantum yield (Fv/Fm) measurements were taken at dusk for all 18 coral fragments using a DIVING-PAM (Walz) beginning on 11 August.  Photosynthetically active radiation (PAR) was measured at noon and averaged 318 ± 129 umol/m2/sec.  From 20 to 21 August 2007, all coral fragments were brought inside during the passage of Hurricane Dean.  On 22 August, the experiment was reconstituted, and a second acclimation period began on 23 August and lasted until 1 September.  During this time, both aquaria received water at an average temperature of 28.5 ± 0.8oC, and PAR at an average of 371 ± 169 umol/m2/sec. On the night of 1 September, one 200-Watt aquarium heater was turned on in the treatment aquarium, and a second heater was turned on 3 days later.  During the thermal stress experiment, the control aquarium received an average water temperature of 28.8 ± 1.2oC; the heated aquarium, 31.5 ± 1.1oC. PAR present during the thermal stress experiment averaged 420 ± 152 uE.  On the night of 7 September, all fragments were frozen in liquid nitrogen.  Eighteen total microarray hybridizations were performed.  A reference RNA sample was created by pooling RNA from all 18 prep’s.  All control and heat-stressed samples were competitively hybridized against the reference RNA to the M. faveolata microarray (1,310 features).

first: Shinichi

last: Sunagawa

first: Paul

last: Fisher

first: Christian

last: Voolstra

first: Roberto

last: Iglesias-Prieto

first: Monica

last: Medina


title: Microarray analysis of gene expression in the kidneys and testes of mice after low-dose-rate irradiation

status: Public on May 27 2009

city: Chiba

first: Mitsuru

last: Nenoi

address: 4-9-1, Anagawa, Inage-ku

department: Research Center for Radiation Protection

institute: National institute of Radiological Sciences

summary:
Measuring global gene expression using cDNA or oligonucleotide microarrays is an effective approach to understanding the complex mechanisms of the effects of radiation. However, few studies have been carried out that investigate gene expression in vivo after prolonged exposure to low-dose-rate radiation. In this study, C57BL/6J mice were continuously irradiated with γ-rays for 485 days at dose-rates of 0.032 – 13 μGy/min. Gene expression profiles in the kidney and testis from irradiated and unirradiated mice were analyzed, and differentially expressed genes were identified. A combination of pathway analysis and hierarchical clustering of differentially expressed genes revealed that expression of genes involved in mitochondrial oxidative phosphorylation was elevated in the kidney after irradiation at the dose-rates of 0.65 μGy/min and 13 μGy/min. Expression of cell cycle-associated genes was not profoundly modulated in the kidney, in contrast to the response to acute irradiation, suggesting a threshold in the dose-rate for modulation of the expression of cell cycle-related genes in vivo following exposure to radiation. We demonstrated that changes to the gene expression profile in the testis were largely different from those in the kidney. The Gene Ontology categories “DNA metabolism”, “response to DNA damage” and “DNA replication” overlapped significantly with the clusters of genes whose expression decreased with an increase in the dose-rate to the testis. These observations provide a fundamental insight into the organ-specific responses to low-dose-rate radiation.

Key Words: kidney, low-dose-rate, radiation, microarray, mitochondrial oxidative phosphorylation, testis

overall_design:
3 biological replicates for each experimental group were analysed.

GSE14290_Kidney_raw_data.csv contains the raw data for GSM357146, GSM357425, GSM357426, GSM357452 to GSM357460.
GSE14290_Testis_raw_data.csv contains the raw data for GSM357471 to GSM357482.
In these files, the sample identifiers are included in the column headers.

first: Keiko

last: Taki

first: Bing

last: Wang

first: Tetsuo

last: Nakajima

first: Jianyu

last: Wu

first: Tetsuya

last: Ono

first: Yoshihiko

last: Uehara

first: Tsuneya

first: Yoichi

last: Oghiso

first: Kimio

last: Tanaka

first: Kazuaki

last: Ichinohe

first: Shingo

last: Nakamura

first: Satoshi

first: Junji

last: Magae

first: Ayana

last: Kakimoto


title: Folsomia candida exposed to soil containing different phenanthrene concentrations

city: Amsterdam

first: Benjamin

last: Nota

state: Noord Holland

address: Plesmanlaan 125

department: Research Facilities

country: Netherlands

institute: Sanquin

summary:
Polycyclic aromatic hydrocarbons are common pollutants in soil, have negative effects on soil ecosystems, and are potentially carcinogenic. The Springtail (Collembola) Folsomia candida is often used as an indicator species for soil toxicity. Here we report a toxicogenomic study that translates the ecological effects of the polycyclic aromatic hydrocarbon phenanthrene in soil to the early transcriptomic responses in Folsomia candida.
Microarrays were used to examine two different exposure concentrations of phenanthrene, namely the EC10 (24.95 mg kg-1 soil) and EC50 (45.80 mg kg-1 soil) on reproduction of this springtail, which evoked 405 and 251 differentially expressed transcripts, respectively. Fifty transcripts were differential in response to either concentration. Many transcripts encoding xenobiotic detoxification and biotransformation enzymes (phases I, II, and III) were upregulated in response to either concentration. Furthermore, indications of general and oxidative stress were found in response to phenanthrene. Chitin metabolism appeared to be disrupted particularly at the low concentration, and protein translation appeared suppressed at the high concentration of phenanthrene; most likely in order to reallocate energy budgets for the detoxification process. Finally, an immune response was evoked especially in response to the high effect concentration, which was also described in a previous transcriptomic study using the same effect concentration (EC50) of cadmium.
Our study provides new insights in the molecular mode of action of the important polluting class of polycyclic aromatic hydrocarbons in soil animals. Furthermore, we present a fast, sensitive, and specific soil toxicity test which enhances traditional tests and may help to improve current environmental risk assessments and monitoring of potentially polluted sites.

overall_design: Folsomia candida was exposed to phenanthrene spiked soil or untreated (reference/control) soil for 2 days. Two different concentrations of phenanthrene were used, 24.95 and 45.80 mg/kg soil which represent the EC10 and EC50 on reproduction, respectively. For each concentration treatment 4 biological replicates were used, replicate samples consisted of total RNA extracted from ~30 animals exposed in the same jar to either reference or phenanthrene spiked soil. Phenanthrene treated samples were always hybridized to reference samples in an evenly distributed dye-swap manner, which resulted in total in 8 hybridizations of 16 samples.

first: Mirte

last: Bosse

first: Bauke

last: Ylstra

first: Nico

middle: M

last: van Straalen

first: Dick

last: Roelofs


title: Structural evidence for consecutive Hel308-like modules in the spliceosomal ATPase Brr2

city: Ithaca

first: Jeffrey

last: Pleiss

state: NY

address: 451 Biotech

department: Molecular Biology and Genetics

institute: Cornell University

summary: Brr2 is a DExD/H-box helicase responsible for U4/U6 unwinding, a critical step in spliceosomal activation.  Brr2 contains an N-terminal domain and two tandem sets of a helicase-like domain followed by a Sec63 domain with unknown function.  We determined the crystal structure of the second Sec63 domain, which unexpectedly resembles domains 4 and 5 of DNA helicase Hel308.  The helicase-like domain upstream of Sec63 has clear sequence similarity with domains 1-3 of Hel308.  In addition modeling indicates that Brr2 is composed of an N-terminal domain and two consecutive Hel308-like modules (Hel308-I and II). Together this provides our first glimpse of the overall structure of this large and unique spliceosomal ATPase and helicase.  Our structural model and mutagenesis data suggest that Brr2 shares a similar helicase mechanism to Hel308, that differs from many DEAD-box proteins.  We demonstrate that Hel308-II interacts with Prp8 and Snu114 in vitro and in vivo, potentially serving as a mediator for the regulation of Brr2’s activity by Prp8.  We further find that the C-terminal region of Prp8 (Prp8-CTR) facilitates the binding of the Brr2/Prp8-CTR complex to U4/U6, suggesting a potential role of Prp8-CTR as an auxiliary substrate binding and specificity domain for Brr2.

overall_design: Splicing specific microarrays were used to assess the genome-wide defects in pre-mRNA splicing that result from a deletion of the second Sec63 domain of yeast Brr2.

first: Lingdi

last: Zhang

first: Tao

last: Xu

first: Corina

last: Maeder

first: Laura-Oana

last: Bud

first: James

last: Shanks

first: Jay

last: Nix

first: Christine

last: Guthrie

first: Rui

last: Zhao


title: Genome-Wide Screen of Promoter Methylation Identifies Novel Markers for Tumor Development in Melanoma

city: New Haven

first: Ruth

last: Halaban

state: CT

address: 15 York St.

department: Dermatology

institute: Yale University

type: Methylation profiling by genome tiling array

summary:
Background:
DNA methylation is an important component of epigenetic modifications that influences the transcriptional machinery and is aberrant in many human diseases. In particular, dysregulation of promoter methylation in cancer has already been shown to be associated with repression of tumor suppressors and activation of oncogenes. In addition, detection of altered promoter methylation status is suitable for the design of diagnostic or prognostic tests. In this study we present a new methodological approach for the robust identification of promoter methylation markers and the first genome-wide study for the detection of methylation markers in melanoma.
Methods and Findings:
Genome-wide promoter methylation and gene expression of ten early passage melanoma cell strains are compared to newborn and adult normal melanocytes. For the identification of markers we applied linear mixed effect models (LME) in combination with a series of filters based on the localization of promoter methylation relative to the transcription start site, overall promoter CpG content, and differential gene expression. The aim of this methodology is to identify markers whose promoter differential methylation is likely to be functionally related to differential expression. We identified 76 markers, 68 hyper- and 8-hypo-methylated in melanomas (LME P<0.05). Promoter methylation profiles and differential expression of five of these markers were successfully validated. In addition, promoter demethylation following Aza treatment consistently restored expression of markers hyper-methylated in melanoma.
Conclusions:
The proposed methodology allows the identification of robust markers and can be applied to other experimental scenarios where promoter methylation is evaluated. More importantly, the list of markers represents the first systematic effort in the identification of methylation markers in melanoma. Many of the identified markers were not previously known to be regulated by promoter methylation and/or associated with this or other types of cancer.

first: Yasuo

last: Koga

first: Mattia

last: Pelizzola

first: Elaine

last: Cheng

first: Michael

last: Krauthammer

first: Mario

last: Sznol

first: Annette

last: Molinaro

first: Sherman

last: Weissman


title: Array-CGH of malignant peripheral nerve sheath tumors (MPNST)

city: Cardiff

first: Kiran

middle: Kumar

last: Mantripragada

address: Heath Park Campus

department: Medical Genetics

institute: Cardiff University

summary: Comparison of copy number changes in MPNST samples against benign neurofibromas in NF1 patients

overall_design: 24 MPNSTs and 3 NF samples were hybridised to the human 32K BAC tiling path array

first: Meena

last: Upadhyaya


title: Copy Number Alterations in HNSCC with or without Oncogene Expressing Human Papillomavirus

first: Daoud

last: Sie

address: De Boelelaan 1117

department: Pathology

institute: Vrije Universiteit Medical Center

type: Genome variation profiling by genome tiling array

summary: Oncogene expressing human papillomavirus type 16 (HPV16) is found in a subset of head and neck squamous cell carcinomas (HNSCC). HPV16 drives carcinogenesis by inactivating p53 and pRb with the viral oncoproteins E6 and E7, reflected by a low level of mutations in TP53 and allelic loss at 3p, 9p and 17p, genetic changes frequently found in HNSCCs of non-viral etiology. We hypothesize that two pathways to HNSCC exist: one determined by HPV16 and one by environmental carcinogens. To define the critical genetic events in these two pathways, we now present a detailed genome analysis of HNSCC with and without HPV16 involvement by employing high resolution micro-array comparative genomic hybridization. Four regions showed alterations in HPV-negative tumors that were absent in HPV-positive tumors: losses at 3p11.2-26.3, 5q11.2-35.2, 9p21.1-24, and gains/amplifications at 11q12.1-13.4. Also, HPV16-negative tumors demonstrated loss at 18q12.1-23, in contrast to gain in HPV16-positive tumors. Seven regions were altered at high frequency (>33%) in both groups: gains at 3q22.2-qter, 5p15.2-pter, 8p11.2-qter, 9q22-34.1, 20p-20q and losses at 11q14.1-qter and 13q11-33. These data show that HNSCC arising by environmental carcinogens are characterized by genetic alterations that differ from those observed in HPV16-induced HNSCC, and most likely occur early in carcinogenesis. A number of genetic changes are shared in both tumor groups and can be considered crucial in the later stages of HNSCC progression.

overall_design: Tumor DNA is isolated from fresh frozen tissue. Tumor DNA is enriched by microdissection. Reference DNA is isolated from blood of normal individuals of the opposit gender.

first: Serge

last: Smeets

first: Boudewijn

last: Braakhuis

first: Saman

last: Abbas

first: Peter

last: Snijders

first: Bauke

last: Ylstra

first: Mark

last: van de Wiel

first: Gerrit

last: Meijer

first: Rene

middle: C

last: Leemans

first: Ruud

middle: H

last: Brakenhoff


title: Histone H3 Lysine 4 mono-, di- and trimethyl and CTCF in CD4+CD25+CD45RA+ regulatory and conventional CD4+CD25- T-cells

city: Regensburg

first: Christian

last: Schmidl

address: Franz-Josef-Strauss-Allee 11

country: Germany

institute: Rengensburg Center for Interventional Immunology

summary:
Analysis of Histone H3 Lysine 4 mono-, di- and trimethyl and the boundary protein CTCF in CD4+CD25+CD45RA+ regulatory T-cells and conventional CD4+CD25- T-cells.

To investigate regulatory functions or potential new transcription start sites in Treg and Tconv cells, we investigated the associated histone modifications. Mono- and dimethylation of histone 3 lysin 4 (H3K4) were previously shown to mark enhancer regions, whereas H3K4 trimethylation generally associates with transcription start sites. At imprinted loci, binding of the insulator protein CTCF, which restricts or directs enhancer-promoter interactions, is often regulated by DNA-methylation. Therefore we performed ChIP-on-chip experiments (chromatin immunoprecipitation followed by microarray hybridization; samples were amplified with ligation mediated PCR [see label protocol for the procedure] prior to labeling) for mono- di- and trimethylation of histone 3 lysin 4 and of CTCF in expanded Treg and Tconv cells.

Keywords: ChIP-on-chip

overall_design: ChIP-on-chip experiments for H3K4 mono-, di- and trimethyl and CTCF in CD4+CD25+CD45RA+ regulatory T-cells and conventional CD4+CD25- T-cells were co-hybridizied  with the input. Three biologiacal replicates (rep1-3) were performed for every histone mark, two CTCF (rep1 and rep2).

last: Rehli


title: Effect of Fasting on Hepatic Gene Expression in Postpubertal Holstein Dairy Heifers

city: Guelph

first: John

middle: Henry

last: Doelman

state: Ontario

address: 50 Stone Road East

department: Animal and Poultry Science

country: Canada

institute: University of Guelph

summary: 102 postpubertal Holstein dairy heifers were randomly assigned to a fed or fasted treatment. Liver biopsies were performed to obtain tissue from which mRNA was extracted and used for microarray analysis.  Transcriptional profiling revealed adaptive mechanisms in response to negative energy balance.

overall_design:
Two condition experiment; fed and fasted.  Common reference design employed; reference sample consisting of RNA derived from bovine liver, spleen and placental tissue.
Biological replicates of 51 Fed (as control) and 51 Fasted (as treatment). One replicate per array.  Dye swaps performed on 7 of the 51 fasted treatment group samples, and on 5 of the 51 fed treatment group samples.


title: Mus musculus hematopoietic stem cell

city: Daegu

first: Sanggyu

last: Lee

address: 1370 Sankyuk-dong, Bukgu

institute: Kyungpook National University

summary: A transcriptome study in mouse hematopoietic stem cells was performed using a sensitive SAGE method, in an attempt to detect medium and low abundant transcripts expressed in these cells. Among a total of 31,380 unique transcript, 17,326 (55%) known genes were detected, 14,054 (45%) low-copy transcripts that have no matches to currently known genes. 3,899 (23%) were alternatively spliced transcripts of the known genes and 3,754 (22%) represent anti-sense transcripts from known genes.

overall_design: Mouse hematopoietic stem cells were purified from bone marrow cells using negative and positive selection with a Magnetic-Activated Cell Sorter (MACS). total RNA and mRNA were purified from the purified cells using Trizol reagent and magnetic oligo dT beads. Double strand cDNAs were synthesized using a cDNA synthesis kit and anchored oligo dT primers. After NlaIII digestion, 3’ cDNAs were isolated and amplified through 16-cycle PCR. SAGE tags were released from the 3’ cDNA after linker ligation. Ditags were formed, concatemerized and cloned into a pZERO vector. Sequencing reactions were performed with the ET sequencing terminator kit. Sequences were collected using a Megabase 1000 sequencer. SAGE tag sequences were extracted using SAGE 2000 software.

first: Hyeng-Soo

last: Kim

first: Junmo

last: Hwang

first: Young-Hun

first: Seonggon

first: Jae-Woong

first: Kil-Soo

first: Ji-Hong

last: Ha

first: Jin-Woong

last: Chung

first: Kyu-Tae

last: Chang

first: Zae-Young

last: Ryoo


title: Study of the response of Aspergillus niger to three levels of ambient pH

status: Public on May 18 2009

city: Kgs. Lyngby

first: Mikael

last: Andersen

address: Søltofts plads, bygn 223

department: BioCentrum-DTU

country: Denmark

institute: Technical University of Denmark

summary: The multiple modes of response to ambient pH were explored and new regulatory structures determined.

platform_organism:
Aspergillus niger
Aspergillus oryzae
Aspergillus nidulans

overall_design: Biological triplicates from the mid-exponential growth phase of controlled bioreactor batch-cultivations of A. niger.

first: Linda

last: Lehmann

first: Jens

last: Nielsen


title: Detection of differentially methylated regions in CD4+CD25+CD45RA+ regulatory T-cells and conventional CD4+CD25- T-cells

summary:
We have previously developed an approach that fractionates genomic DNA fragments depending on their CpG density (methyl-CpG-immunoprecipitation, MCIp), and adapted this approach to identify regions that are differentially methylated in the two closely related regulatory T-cells (Treg cells) and conventional T-cells (Tconv cells). Because Treg cells naturally occur at a relatively low frequency, we used a previously established protocol to expand Treg cells from a stable naïve Treg population that is characterized by the co-expression of CD4, CD25 and CD45RA. We separated gDNA of both expanded T cell lineages (Tregexp and Tconvexp) into unmethylated (CpG) and methylated pools (mCpG) using MCIp and compared cell type-specific differences in DNA methylation by co-hybridization of the two umethylated or the two methylated DNA subpopulations of Treg and Tconv, respectively, to these locus-wide custom tiling arrays. As enriched DNA-fragments from a cell type in the methylated fraction should be depleted in the unmethylated fraction, the signal intensities in CpG pool and mCpG pool hybridizations should complement themselves (“Mirror-Image” approach) and thereby allow the identification of differentially methylated regions (DMR). Because we expected to find lineage-specific methylation differences with greater probability in regions associated with differential transcriptional activity, we limited our analysis to gene loci that showed cell type-specific gene expression in Treg versus Tconv cells plus a handful of control regions that were equally expressed in both cell types. The microarray used in this study covered 12 megabases of the human genome and contained 69 regions (with a median size of 100.000 kb) and 128 proximal promoter regions and 181 genes, which included a number of well known and functionally relevant genes like CD40LG, IFNG, FOXP3, IL2RA and CTLA4.

Keywords: MCIp-on-chip; comparative genomic hybridization

overall_design: With MCIp gDNA from Treg or Tconv cells was separated into hypo- and hypermethylated pools. On each array, wheather the two hypomethylated fractions- one from Treg, the other from Tconv cells- or the two hypermethylated fractions were cohybridized. Two biological replicates.

first: Michael


city: Kiel

first: Paul-Martin

last: Holterhus

address: Campus Kiel, Schwanenweg 20

department: Pädiatrische Endokrinologie und Diabetologie

institute: Universitätsklinikum Schleswig-Holstein

summary:
Human scrotum and labia majora samples treated with DHT and/or AZA
A compound treatment design type is where the response to administration of a compound or chemical (including biological compounds such as hormones) is assayed.
Compound Based Treatment: dihydrotestosterone (DHT), 5-aza-deoxy-cytidine (AZA), none or both
Cell Line: normal scrotum or labia majora derived from complete androgen insensitivity syndrome due to inactivating mutation of the androgen receptor gene

overall_design: Computed


title: Bacillus subtilis: Smc-TAP ChIP-on-chip

status: Public on May 14 2009

city: Newcastle upon Tyne

first: Stephan

last: Gruber

address: Framlignton Place

department: ICaMB

institute: Newcastle University

summary:
Genome-wide analysis of Smc distribution using ChIP-on-chip
ChIP DNA versus Input DNA

Keywords: ChIP-chip

overall_design: Combined ChIP and Input DNA from three independent experiments on single array

first: Jeff

last: Errington


title: Effect of hypoxia on gene expression by human macrophages

city: Singapore

first: Subhra

last: Biswas

address: 8A Biomedical Grove

country: Singapore

institute: Singapore Immunology Network

summary: Ischemia exists in many diseased tissues including arthritic joints, atherosclerotic plaques and malignant tumors. Macrophages accumulate in these sites and upregulate genes  in response to the hypoxia present. We used Illumina beadarrays  to investigate the effect of hypoxia (18h at 0.1% oxygen) on gene expression by human monocyte-derived macrophages in vitro.

overall_design:
Primary macrophages were differentiated for 7 days in vitro from human peripheral blood (monocyte-derived macrophages, MDMs) and were subjected to severe hypoxia (< 0.1% O2) or normoxia (20.9% O2) in 5% CO2 humidified multi-gas incubators. Two replicates per condition.

The supplementary file 'GSE16099_non-normalized_data.txt' contains non-normalized (raw) data for Samples GSM402885-GSM402888.

first: Claire

last: Lewis


title: Genome-Wide Promoter Analysis of Epigenetic Regulation by Cocaine (MM8 data)

first: guanghua

last: xiao

state: TX

summary: Changes in gene expression contribute to the long-lasting regulation of the brain's reward circuitry seen in drug addiction, however, the specific genes regulated and the transcriptional mechanisms underlying such regulation remain poorly understood. Here, we used chromatin immunoprecipitation coupled with promoter microarray analysis to characterize genome-wide epigenetic changes in the mouse nucleus accumbens, a crucial brain reward region, after repeated cocaine administration. Our findings reveal several interesting principles of gene regulation by cocaine and of the role of ΔFosB and CREB, two prominent cocaine-induced transcription factors, in this brain region.

overall_design: Mice were treated with cocaine or saline. Chromatin immunoprecipitation was performed for methylated histone H3 lysine 9 and 27, ∆FosB, and phospho-CREB as described previously (Kumar et al., 2005) with minor modifications. Immunoprecipitated DNA was amplified via ligation-mediated PCR and hybridized to Nimblgen mouse MM8 promoter arrays.  2-3 biological replicates per condition.

first: Kumar

last: Arvind

first: Renthal

last: William