title: Evolution of E. coli transcriptome under osmotic-stress with and without RpoS

city: Dublin

first: Daniel

middle: M

last: Stoebel

address: Moyne Institute, Trinity College

department: Department of Microbiology

country: Ireland

institute: Trinity College, Dublin

summary:
The RpoS sigma factor protein of Escherichia coli is the master transcriptional regulator of physiological responses to a variety of stresses.  This stress response comes at the expense of scavenging for scarce resources, causing a trade-off between stress tolerance and nutrient acquisition.  This trade-off favors non-functional rpoS alleles in nutrient-poor environments. We used experimental evolution to explore how natural selection modifies the regulatory network of strains lacking RpoS when they are evolved in an osmotically stressful environment. We found that strains lacking RpoS adapt less variably, in terms of both fitness increase and changes in patterns of transcription, than strains with functional RpoS.  This phenotypic uniformity was caused by the same adaptive mutation in every independent population: the insertion of IS10 into the promoter of otsBA.  OtsA and OtsB are required to synthesize the osmoprotectant trehalose, and transcription of otsBA requires RpoS in the wild-type genetic background. The evolved IS10 insertion rewires expression of otsBA from RpoS-dependent to RpoS-independent, allowing for partial restoration of wild-type response to osmotic stress.  Our results show that the regulatory networks of bacteria

Keywords: evolution; expression

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

overall_design: This study started with wild-type and rpoS- strains, and evolved five lines from each, for a total of 12 strains.  Expression was measured for each strain with two separate biological replicates of RNA

first: Karsten

last: Hokamp

first: Michael

middle: S

last: Last

first: Charles

last: Dorman


title: DNase hypersensitivity mapping (DNase-chip) using Nimblegen ENCODE arrays in a variety of human cell types

status: Public on Nov 01 2009

city: Chicago

first: Christopher

middle: John

last: Ott

state: IL

address: 2430 N Halsted St. Rm C357

department: Pediatrics

country: USA

institute: Northwestern University

type: Genome binding/occupancy profiling by genome tiling array

summary:
DNase hypersensitivity using Nimblegen ENCODE arrays (DNase-chip) in primary human trachea epithelia (pHTE), normal human bronchial epithelia (NHBE), Caco2, HT29, human skin fibroblasts, primary human male epididymis.
DNase hypersensitivity mapping is used to detect putative regulatory elements of the human genome. We digested chromatin from the above cell types with DNaseI using the DNase-chip protocol devised by Crawford et al. (Nat. Methods, 2006). Briefly, cells are lysed, chromatin is digested with increasing amounts of DNase I, digested ends are blunted in adequetely digested samples, ligated to biotinylated linkers, purified with streptavidin beads, amplified by LM-PCR. As control, randomly sonicated genomic DNA is used. LM-PCR material was labeled and hybridized to hg_17 ENCODE arrays at Nimblegen facility.

overall_design: DNaseI-digested chromatin was hybridized to Nimblegen ENCODE arrays (build hg17). Randomly sonicated genomic DNA was used as control. Three biological replicates on three arrays were performed for Caco2 cells, three replicates were done with skin fibroblasts (two digestions were pooled and hybridized to a single array [Sample: SkinFibro.sample2]), two replicates on two arrays were performed for pHTE, NHBE, and HT29, and a single experiment for primary male epididymis.

middle: J

first: Ann

last: Harris


title: miniChIP-chip of murine hematopoietic stem cells and progeny with H3K4me3, H3K79me2, H3K9/14ac, H3K27me3, H3K9me3, PolII

status: Public on Nov 09 2009

city: Lund

first: Joanne

last: Attema

address: Tornavagen 10

department: Immunology

country: Sweden

institute: Lund University

summary:
This study describes the changes in epigenetic chromatin modifications during murine hematopoietic stem cell differentiation in vivo using a modified miniChIP-chip technology.
We have addressed issues including bivalent (H3K4me3/H3K27me3) modifications, lineage priming hypothesis, and stem cell chromatin properties in our study described in Weishaupt et al., 2009 (Blood)

overall_design: Comparison of 4 murine hematopoietic stem, progenitor and mature cell types directly isolated from primary tissues using FACS. HSCs are phenotypically identified in bone marrow as lineage-, cKit+, Sca1+, CD150+, Flk2/Flt3- (LSKCD150+ cells). MPPs are phenotypically identified in bone marrow as lineage-, cKit+, Sca1+, CD150-, Flk2/Flt3+ (LSKCD150- cells). PreMegEs are phenotypically identified in bone marrow as lineage-, cKit+, Sca1-, CD150+, CD105-, FcgRI/IIlo and CD41- cells as described in Pronk et al., 2007. Splenic-derived CD4+ T cells are phentypically identified as CD4+, CD8-, B220-, Nk1.1- cells as described in Rolf et al., 2008.

first: Holger

last: Weishaupt


title: Global gene-expression analyses of the Eset knock-down ES cells

status: Public on Nov 04 2009

city: Singapore

first: Ping

last: Yuan

address: 60 Biopolis Street #02-01

department: Stem Cell

country: Singapore

institute: Genome Institute of Singapore

summary:
The histone H3 lysine 9 (H3K9) methyltransferase Eset is an epigenetic regulator critical for the development of the inner cell mass (ICM). Although ICM-derived embryonic stem (ES) cells are normally unable to contribute to the trophectoderm (TE) in blastocysts, we find that depletion of Eset by shRNAs leads to differentiation with the formation of trophoblast-like cells and induction of trophoblast-associated gene expression. Using ChIP-seq analyses, we identified Eset target genes with Eset-dependent H3K9 trimethylation. We confirmed that genes that are preferentially expressed in the TE (Tcfap2a and Cdx2) are bound and repressed by Eset. Single cell PCR analysis shows that the expression of Cdx2 and Tcfap2a is also induced in Eset-depleted morula cells. Importantly, Eset-depleted cells can incorporate into the TE of a blastocyst and subsequently placental tissues. Co-immunoprecipitation and ChIP assays further demonstrates that Eset interacts with Oct4, which in turn recruits Eset to silence these trophoblast-associated genes. Our result suggests that Eset restricts the extraembryonic trophoblast lineage potential of pluripotent cells and links an epigenetic regulator to key cell fate decision through a pluripotency factor.
We used microarrays to detail the global programme of gene expression of  control shRNA knock-down ES cells and Eset shRNA knock-down ES cells

overall_design: Three biological replicates for control shRNA knock-down ES cells and Eset knock-down ES cells. The global gene expression profiles of the control shRNA knock-down ES cells were compared to  Eset knock-down ES cells.

first: P

first: J

last: Han

first: G

last: Guoji

first: Y

last: Orlov

first: M

last: Huss

middle: H

last: Loh

first: N

last: Clarke

last: Robson

first: B

last: Lin

first: H

last: Ng


title: Regional brain expression maps for microRNAs in rats

status: Public on Nov 17 2009

city: Roskilde

first: Thomas

last: Werge

address: Boserupvej 2

department: Institute of biological Psychiatry

country: Denmark

institute: Psychiatric Center, Sct. Hans Hospital

summary: MicroRNA transcriptional profiling of male Sprague-Dawley rats rat brains comparing microRNA abundance in five different brain regions.

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

overall_design: Two-condition experiment, regional brain samples vs. universal brain reference. Biological replicates: 4 samples from each region. One sample per array. Each array is divided into 3 replicate subarrays.

first: Line

last: Olsen

first: Mikkel

last: Klausen

first: Lone

last: Helboe

first: Finn

middle: C

last: Nielsen


title: Expression analysis of N. europaea (ATCC 19718) wildtype and pFur::Kan mutant grown in Fe-replete and Fe-limited media

status: Public on Nov 10 2009

city: Corvallis

first: Neeraja

last: Vajrala

state: OR

address: 2082 Cordley Hall

department: Botany & Plant Pathology

institute: Oregon State University

summary:
Investigation of whole genome gene expression level changes in a Nitrosomonas europaea (ATCC 19718) wildtype and pFur::Kan mutant [kanamycin resistance cassette insertion in the promoter region of the fur gene (NE0616)] strains grown in Fe-replete and Fe-limited media.
The Nitrosomonas europaea (ATCC 19718) wiltype cells grown in Fe-limited media were compared to cells grown in Fe-replete media to gain a better understanding of the metabolic changes occurring in response to iron stress. The Nitrosomonas europaea (ATCC 19718) pFur::Kan mutant strain grown in Fe-replete & Fe-limited media were compared to wildtype cells grown in Fe=replete & Fe-limited media to gain a better understanding of the role Fur (NE0616) plays in iron homeostasis control.

overall_design: A 4-plex 3 chip study using total RNA recovered from three separate wild-type cultures each of N. europaea grown in Fe-replete media and Fe-limited media and three seperate cultures each of N. europaea pFur::Kan mutant strain grown in Fe-replete and Fe-limited media. Each chip measures the expression level of 2368 genes from Nitrosomonas europaea (ATCC19718)  with 4 X 72,000  60-mer 14 probe pairs per gene, with two-fold technical redundancy.

first: Daniel

last: Arp


title: Positioning of Necrotic Lobular Intraepithelial Neoplasias within the sequence of breast carcinoma progression

city: Berlin

first: Reinhard

last: Ullmann

department: Human Molecular Genetics

country: Germany

institute: MPIMG

type: Genome variation profiling by genome tiling array

summary: Lobular intraepithelial neoplasia grade 3 (LIN) is a recently recognized variant of intraepithelial lobular neoplasia of the breast composed of either uniform, generally small, cells (classic) with massive lobular distension, pleomorphic cells, signet-ring cells, or any cell type with necrosis.  In contrast to classic forms of LIN, there is no consensus on therapeutic strategies for LIN3. In part this is due to the paucity of molecular data that could assist in defining the relationship of LIN3 to classic LIN and carcinomas. In this study we have employed array CGH to determine the patterns of chromosomal aberrations in 9 LIN3 lesions. By comparison to array CGH data of 13 classic LIN lesions, we demonstrate that LIN and LIN3 share several recurrent changes, in particular gains of 1q and losses of 16q. Both aberrations are known to appear early in tumorigenesis and to be associated with good prognosis. However, apart from this overlap, there were a number of karyotypic features that were observed exclusively in LIN3. Clearly, this lesion was characterized by a significantly higher number of DNA copy number changes (9 vs. 31 on average), a considerable complexity of chromosomal rearrangements with up to 16 breakpoints in one chromosome and overlapping high copy amplifications. The amplicons encompassed a number of known oncogenes such as ERBB2, CyclinD1 and FGF3 that have already been reported to be overexpressed in breast carcinomas. Our data suggest that, at the genetic level, LIN3 represents a highly advanced lesion with considerable resemblance to carcinomas and, therefore, reflects a lesion on the verge of invasion, and might represent the transition state from an intraepithelial neoplasm  to breast carcinoma.

overall_design:
In this study, nine cases of pleomorphic/necrotic LIN lesions as well as one classic LIN and the concurrent invasive lobular carcinoma (ILC) were analyzed by array CGH. Sections of formalin-fixed paraffin-embedded (FFPE) specimens cut at 7-8µm were mounted on special foil-coated slides (Molecular Devices, San Diego, USA). The sections were then deparaffinized with Xylene, processed in decreasing concentrations of ethanol and stained with haematoxylin. Lasercapture microdissection for both lesions was performed at multiple sites using Veritas Arcturus. For reference-DNA, female mammary tissue without histomorphological changes obtained from reduction mammoplasty specimens was procecessed and laser-microdissected as explained above.

Cells were digested in 10µl TE, pH 9, and 0.5µl proteinase K (20mg/ml) for 48h at 55°C. After inactivation of proteinase K at 99°C for 10min, the digest was stored at -20°C.  Without any further purification, the complete digest was used for whole genome amplification by means of the WGA (Whole Genome Amplification) kit from Sigma following the manufacturer’s recommendations. Array CGH was performed as described previously. In brief, two µg of amplified tumor and reference DNA were labelled by random priming (BioPrime® Total Genomic Labeling System, Invitrogen, Carlsbad, CA) with Alexa Fluor® 3 and Alexa Fluor® 5, respectively, and hybridized onto a tiling path BAC array, consisting of the human 32k BAC Re-Array Set (BACPAC Resources Center; http://bacpac.chori.org/pHumanMinSet.htm; DNA kindly provided by Pieter de Jong) and a 1Mb Resolution BAC set (clones kindly provided by Nigel Carter, Wellcome Trust Sanger Centre). All protocols are provided in detail on our website (http://www.molgen.mpg.de/~abt_rop/molecular_cytogenetics/) and more information concerning this platform have been submitted to the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/; GLP: 5114). For the analysis and visualization of array CGH data, our software-package CGH-PRO was employed. No background subtraction was applied. Raw data were normalized by “Subgrid LOWESS”. For the assessment of copy number gains and losses, we used circular binary segmentation in combination with log2 ratio thresholds of 0.15 and -0.15, respectively. High copy amplifications were defined by focal appearance and a log2 ratio exceeding 0.45. In order to statistically investigate the similarity of aberration profiles we applied Pearson's product-moment correlation to cbs ratios of each of the corresponding arrays.


title: G9a selectively represses a class of late-replicating genes at the nuclear periphery (WG_CGH)

status: Public on Nov 03 2009

city: Tallahassee

first: Ichiro

last: Hiratani

state: FL

address: 319 Stadium Dr

department: Biological Science

institute: Florida State University

summary: We have investigated the role of the histone methyltransferase G9a in the establishment of silent nuclear compartments. Following conditional knockout of the G9a methyltransferase in mouse ESCs, 167 genes were significantly up-regulated, and no genes were strongly down-regulated. A partially overlapping set of 119 genes were up-regulated after differentiation of G9a-depleted cells to neural precursors. Promoters of these G9a-repressed genes were AT rich and H3K9me2 enriched but H3K4me3 depleted and were not highly DNA methylated. Representative genes were found to be close to the nuclear periphery, which was significantly enriched for G9a-dependent H3K9me2. Strikingly, although 73% of total genes were early replicating, more than 71% of G9a-repressed genes were late replicating, and a strong correlation was found between H3K9me2 and late replication. However, G9a loss did not significantly affect subnuclear position or replication timing of any non-pericentric regions of the genome, nor did it affect programmed changes in replication timing that accompany differentiation. We conclude that G9a is a gatekeeper for a specific set of genes localized within the late replicating nuclear periphery.

overall_design: 4 cell states each in duplicate (i.e. a total of 8 individual replicates)

first: Tomoki

last: Yokochi


title: Analysis of fetal livers from male mice after protein-restricted diet in mothers

city: Groningen

first: vincent

last: bloks

country: Netherlands

institute: UMCG

type: Methylation profiling by genome tiling array

summary:
Prenatal nutrition as influenced by nutritional status of the mother has been identified as a determinant of adult disease. Feeding low-protein diets during pregnancy in rodents is a well-established model to induce “programming” events in offspring. We hypothesized that protein restriction would influence fetal lipid metabolism by inducing epigenetic adaptations.
Methodology/Principal Findings: Pregnant C57BL/6J OlaHsd mice were exposed to a protein restriction protocol (9% vs. 18% casein). Shortly before birth, dams and fetuses were sacrificed. To identify putative epigenetic changes, CpG island methylation microarrays were performed on DNA isolated from fetal livers.

overall_design:
10 fetal livers: 5 mice from protein-restriction group (mice 1-5, labeled with Alexa 647) and 5 mice from control group (mice 6-10, labeled with Alexa 555).
5 arrays were used for the mice combinations 1/6, 2/7, 3/8, 4/9, 5/10.
All arrays were run at the UHN Microarray Centre, Toronto.


title: Time course of the mRNA expression after phage ϕYS40 infection in crp deletion mutant of Thermus thermophilus HB8.

city: Hyogo

first: Akeo

last: Shinkai

address: 1-1-1, Kouto, Sayo

department: SPring-8 Center

country: Japan

institute: RIKEN Harima Institute

summary:
We observed the expression profile of the total mRNA in crp (TTHA1437) deletion mutant strain of Thermus thermophilus HB8 during infection of bacteriophage ϕYS40.
Keywords: time course, bacteriophage, infection, CRP, cAMP receptor protein, deletion mutant

overall_design: Three crp (TTHA1437) delection mutant strain of T. thermophilus HB8 were pre-cultured at 70 C for 16 h in 3 ml of TT medium containing 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl, 0.4 mM CaCl2, and 0.4 mM MgCl2, which was adjusted to pH 7.2 with NaOH. The cells (2 ml) were inoculated into 1 liter of the same medium and then cultivated at 70°C until A600 value being ~0.8 (1.7   108 cells/ml) that corresponds to logarithmic growth phase. Then the FYS40 phage was infected to the medium at multiplicity of infection (m.o.i.) being ~1, and continued cultivation. Cells were collected after 0, 75 and 100 min infection, and then crude RNA was extracted. The expression of each mRNA at each time point was analyzed on a GeneChip as described under Sample Description Sheet of each sample.

first: Yoshihiro

last: Agari

first: Keiko

last: Sakamoto

first: Aimi

last: Osaki

first: Miwa

last: Ohmori

first: Seiki

last: Kuramitsu


title: Androgen repsonsive microRNAs in LAPC-4 cell lines

city: Baltimore

first: Shawn

middle: E

last: Lupold

state: MD

address: 600 N Wolfe St

department: Urology

institute: Johns Hopkins University School of Medicine

summary: The goals of this study were to evaluate changes in microRNA expression following androgen treatment.

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: Four treatment groups were studied:  untreated and those treated with 0.1, 1.0, and 10.0 nM of synthetic androgen (R1881).  Data is relative to the untreated sample.

first: Judit

last: Ribas

first: Xiaohua

last: Ni

first: Michael

last: Haffner

first: Erik

middle: A

last: Wentzel

first: Amirali

last: Salmasi

first: Wasim

last: Chowdhury

first: Tarana

last: Kudrolli

first: Srinivasan

last: Yegnasubramanian

first: Jun

last: Luo

first: Ron

last: Rodriguez

first: Joshua

middle: T

last: Mendell


title: Gene expression profiles of canine osteosarcoma

city: Bethesda

first: Sean

last: Davis

address: 37 Convent Drive, Room 6138

institute: National Cancer Institute

summary: Pulmonary metastasis continues to be the most common cause of death in osteosarcoma.  Indeed, the 5-year survival for newly diagnosed osteosarcoma patients has not significantly changed in over 20 years. Further understanding of the mechanisms of metastasis and resistance for this aggressive pediatric cancer is necessary. Pet dogs naturally develop osteosarcoma providing a novel opportunity to model metastasis development and progression. Given the accelerated biology of canine osteosarcoma, we hypothesized that a direct comparison of canine and pediatric osteosarcoma expression profiles may help identify novel metastasis-associated tumor targets that have been missed through the study of the human cancer alone.  Collectively, these data support the strong similarities between human and canine osteosarcoma and underline the opportunities provided by a comparative oncology approach as a means to improve our understanding of cancer biology and therapy.

overall_design: Profiles of dog osteosarcoma and several normal tissues, single channel design, tumor versus normal

first: Melissa

last: Paoloni


title: Fetal malformations and early embryonic gene expression response in Cynomolgus monkeys maternally exposed to thalidomide

city: Louisville

first: Thomas

middle: B

last: Knudsen

state: KY

address: 501 S Preston St

department: Birth Defects Center

institute: University of Louisville

summary:
EXPERIMENT: The animal experiments were performed at Shin Nippon Biomedical Laboratories (SNBL), Ltd. (Kagoshima, Japan) in compliance with the Guideline for Animal Experimentation (1987), and in accordance with the Law Concerning the Protection and Control of Animals (1973) and the Standards Relating to the Care and Management of Experimental Animals (1980). This study was approved by the Institutional Animal Care and Use Committee of SNBL and performed in accordance with the ethics criteria contained in the bylaws of the SNBL committee.
Each female monkey was paired with a male of proven fertility for one day between day 11 and day 15 of the menstrual cycle. Pregnant females, aged 5-8 years and weighing 2.84-3.76 kg on day 22 of gestation, were allocated randomly to two groups, each with three monkeys, and housed individually. The monkeys were orally dosed with (±)-thalidomide (Lot no. SDH7273/SDJ3347, Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 0 or 20 mg/kg by oral intubation on day 26 of gestation, which was during the critical period for thalidomide-induced teratogenesis [Delahunt and Lassen, 1964; Hendrickx, 1973]. Dosage was adjusted to the body weight on day 22 of gestation and the drug was packed in a gelatin capsule. Control monkeys received the capsule only.
ANIMAL MODEL: Macaca fascicularis
INTERVAL: NON.
PLATFORM: Proprietary Affymetrix NHP GeneChip® Array for Cynomolgus genome derived from Biogen Idec
Keywords = Developmental
Keywords = Monkeys
Keywords = Thalidomide

overall_design: TFetal malformations and early embryonic gene expression response in cynomolgus monkeys maternally exposed to thalidomidechannel oligonucleotide (Affymetrix) platform.

first: Makoto

last: Ema

first: Ryota

last: Ise

first: Hirohito

last: Kato

first: Satoru

last: Oneda

first: Akihiko

last: Hirose

first: Mutsuko

last: Hirata-Koizumi

first: Toshio

last: Ihara

first: Amar

middle: V

last: Singh


title: Dark and light - Analysis of the effect of NAD concentration on the transcriptome

city: evry

first: Soubigou-Taconnat

last: Ludivine

address: 2 rue gaston crémieux

department: URGV

country: France

institute: INRA

summary:
rs08-02_nad. Dark and light. Establish which transcripts are affected by changes in NAD concentration, and whether this depends on light/dark. Three genotypes: Two lines of Arabidopsis transformed to overexpress QPRT, an enzyme involved in NAD synthesis (NadC4.11 and NadC15.3) and a third control line transformed with an empty vector (162). Leaf discs from the two lines of the QPRT+ plants were incubated with quinolinate, the QPRT substrate, to boost leaf NAD. Discs from the empty vector control were incubated on a buffer withour quinolinate. The experiment was performed in the light and the dark. The aim is to compare the QPRT+ quinolinate samples with the empty vector samples, both for the sample series generated in the light and that generated in the dark.

Keywords: treated vs untreated comparison

overall_design: 8 dye-swap - CATMA arrays

first: Ludivine

last: Taconnat

first: Jean-Pierre

middle: P

last: Renou

first: Graham

last: Noctor

first: Marie-Laure

middle: L

last: Martin-Magniette


title: A microRNA microarray analysis of C.elegans Parkinsons disease models

city: Stockholm

first: Suvi

middle: Tuulia

last: Asikainen

state: Huddinge

address: Karolinska University Hospital

department: CLINTEC

institute: Karolinska Institutet

summary: MicroRNAs (miRNAs) play an important role in human brain development and maintenance. To search for miRNAs potentially involved with molecular mechanisms in the pathogenesis of Parkinsons disease (PD), we utilized miRNA microarrays to identify expression changes of 115 annotated Caenorhabditis elegans (C.elegans) miRNAs in human α-synuclein A53T transgenic, dopamine deficient catecholamine transporter gene cat-1 mutant and parkin gene pdr-1 mutant C.elegans strains. Twelve miRNAs were found regulated differentially in α-synuclein transgenic C. elegans, five in cat-1 mutants and three in pdr-1 mutants. The family of miR64/65 appeared co-under-expressed in α-synuclein and cat-1 strains and members of let-7 family co-under-expressed (except miR-241 over-expressed) in a-synuclein and pdr-1 strains. Class H serpentine receptor (srh) family of G-protein-coupled receptor genes were computationally identified to be highly over-represented target candidates for regulated miRNAs.These results indicate that miRNAs are misregulated in C. elegans PD models and suggest a role for these molecules in the disease pathogenesis.

overall_design: Two color NCode microRNA microrarrays were used (Invitrogen) to measure miRNA expression changes in human alpha-synuclein trangenic, cat-1 mutant, and pdr-1 mutant C.elegans strains. The experiment contains four independent biological replicates (worm populations)/strain. Strain N2 was used as reference channel, also prepared from four independently isolated small RNA samples.

first: Kristiina

last: Louhiranta

first: Martina

last: Rudgalvyte

first: Merja

last: Lakso

first: Richard

last: Nass

first: Garry

last: Wong


title: Metastatic potential of melanoma cells is not affected by electrochemotherapy

city: Ljubljana

first: Vid

last: Mlakar

address: Korytkova 2

department: Department for molecular genetics

country: Slovenia

institute: Faculty of Medicine

summary: Electrochemotherapy is a local treatment combining chemotherapy and application of electric pulses to the tumour. Electrochemotherapy with bleomycin and cisplatin has already been found to be effective in controlling local tumour growth in the treatment of malignant melanoma. However, not much is known about the effect of electrochemotherapy on the metastatic potential of tumour cells. Prevention of metastasis is an important aspect of successful treatment. It is known that metastasis can be induced by different treatment modalities. Therefore, the aim of this study was to evaluate the effect of electrochemotherapy with cisplatin on the metastatic potential of human malignant melanoma cells. The cells treated by electrochemotherapy with cisplatin, were tested for their ability to migrate and invade through Matrigel coated porous membrane. There were no significant changes observed in cell migration and invasion after electrochemotherapy. Also, there were no changes observed in cell adhesion on Matrigel. Gene expression analysis demonstrated that very low number of genes was differentially expressed after electrochemotherapy with cisplatin. Two genes, LAMB3 and CD63 involved in cell migration, were both down-regulated after electrochemotherapy with cisplatin and the expression of metastasis promoting genes was not increased after electrochemotherapy. Our data suggest that electrochemotherapy does not increase metastatic behaviour of human melanoma cells.

overall_design: We analysed expression of genes involved in apoptosis and development of cancer in malignant melanoma cell line treated with cisplatin and cisplatin combined with electroporation in order to identify genes involved in cell line response. We used dual color approach. Each microarray contained at least 4 replicates of each gene and each condition was performed in at least two replicates, obtaining at least 8 measurements for each gene.  Treated cell line was labeled with Cy5 and nontreated cell line was labeled with Cy3.

first: Vesna

last: Todorovic

first: Gregor

last: Sersa

first: Maja

last: Cemazar

first: Damjan

last: Glavac


title: AI versus IVF-ET & AI versus SCNT

city: Jouy-en-Josas

first: Olivier

last: Sandra

address: Domaine de Vilvert

department: UMR BDR

summary:
Implantation is crucial for placental development which directly impacts fetal growth and pregnancy success with possible consequences on post-natal health. We postulated that early perturbations of the conceptus-maternal environment communication may alter the endometrium physiology that could account for the final reproductive outcome. Using cattle as an animal model, we compared gene expression profiles of the endometrial caruncular and intercaruncular areas during the critical period of implantation in three types of pregnancies: artificial insemination (AI), in vitro fertilization with embryo transfer (IVF-ET) or somatic cell nuclear transfer (SCNT). Less than 35% of the differentially expressed genes were found to be common between AI, IVF-ET, and SCNT conditions. Compared to AI, numerous biological functions and several canonical pathways and genes were found to be significantly affected in IVF-ET or SCNT, with a major impact on metabolism and immune function in SCNT. Our data show that the endometrium can fine-tune its physiology and could be considered as a biological sensor in response to pregnancy manipulations. Determining the limits of the endometrial plasticity should bring new insights on the contribution of the maternal compartment to the pregnancy outlet.

Keywords: Fluorescence Microarray - Dye switch loop design

overall_design: 44 samples

first: Nadéra

last: Mansouri-Attia

first: Julie

last: Aubert

first: Séverine

last: Degrelle

first: Robin

last: Evert

first: Corinne

last: Giraud-Delville

first: Yvan

last: Heyman

first: Laurent

last: Galio

first: Isabelle

last: Hue

first: Xiangzhong

last: Yang

first: XCindy

last: Tian

first: Harris

last: Lewin

first: Jean-Paul

last: Renard


title: Time course of the mRNA expression after bacteriophage ϕYS40 infection in wild-type Thermus thermophilus HB8 strain.

first: Akeo

last: Shinkai

summary:
We observed the expression profile of the total mRNA of wild-type Thermus thermophilus HB8 strain during infection of bacteriophage ϕYS40.
Keywords: time course, bacteriophage, infection, wild type

overall_design: Three wild-type T. thermophilus HB8 strains were pre-cultured at 70°C for 16 h in 3 ml of TT medium containing 0.8% polypeptone, 0.4% yeast extract, 0.2% NaCl, 0.4 mM CaCl2, and 0.4 mM MgCl2, which was adjusted to pH 7.2 with NaOH. The cells (2 ml) were inoculated into 1 liter of the same medium and then cultivated at 70°C until A600 value being ~0.8 (1.7 × 108 cells/ml) that corresponds to logarithmic growth phase. Then the ΦYS40 phage was infected to the medium at multiplicity of infection (m.o.i.) being ~1, and continued cultivation. Cells were collected after 0, 25, 50, 75 and 100 min infection, and then crude RNA was extracted. The expression of each mRNA at each time point was analyzed on a GeneChip as described under Sample Description Sheet of each sample.

first: Yoshihiro

last: Agari

first: Keiko

last: Sakamoto

first: Aimi

last: Osaki

first: Miwa

last: Ohmori

first: Seiki

last: Kuramitsu


title: Electric pulses do not change expression profile of genes involved in development of cancer in malignant melanoma cells

first: Vid

last: Mlakar

summary:
Electroporation is a versatile method for in vitro or in vivo delivery of different molecules into cells. However, no study so far has analysed the effects of electric pulses used in electrochemotherapy (ECT pulses) or electric pulses used in electrogene therapy (EGT pulses) on malignant cells. We studied the effect of ECT and EGT pulses in human malignant melanoma cells in vitro in order to understand and predict possible effect of electric pulses on gene expression and their possible effect on cell behaviour.
We used microarrays with 2698 different oligonucleotides to obtain expression profile of genes involved in apoptosis and development of cancer in a malignant melanoma cell line (SK-MEL28) exposed to ECT pulses and EGT pulses. Cells exposed to ECT pulses showed 68.8% average survival rate and 31.4% average survival rate in cells exposed to EGT pulses. Only seven common genes were found differentially expressed in cells 16 h after exposure to ECT and EGT pulses. We found that ECT and EGT pulses induce HSP70 stress response mechanism, repress histone protein H4, a major protein involved in chromatin assembly, and down-regulate components involved in protein synthesis.
Our results show that electroporation does not significantly change expression profile of major tumour suppressor genes or oncogenes of cell cycle. Moreover, electroporation also does not changes the expression of genes involved in stability of DNA, supporting the current evidence that electroporation is a safe method that does not promote tumorigenesis. However, in spite of being considered an isothermal method it does to some extent induce stress that resulted in expression of environmental stress response mechanism, HSP70.

overall_design: The difference in expression of genes involved in development of cancer was obtained by comparison of malignant melanoma cells exposed to EGT or ECT pulses against the same untreated malignant melanoma cells. In our experimental design, microarrays with 2698 different genes were used as a dual colour system in which exposed and non-exposed cells’ mRNA were separately labelled, mixed and hybridised together on each array. Only microarrays expressing at least 50% of genes were used for further analysis. All oligonucleotides on the same array were spotted in quadruplicates and each microarray analysis was performed in duplicates, therefore obtaining eight measurements of the same oligonucleotide. The acquired data were analysed with Acuity 4.0 to select reliable signals. Only genes, present in both duplicated microarrays were considered for further processing.

first: Vesna

last: Todorovic

first: Maja

last: Cemazar

first: Damjan

last: Glavac

first: Gregor

last: Sersa


title: Downregulation of three tumour suppressor genes SLC26A3, DCN and TPM1 in colorectal cancer

summary: Despite identification of major genes and pathways involved in development of colorectal cancer (CRC) it became obvious that several steps in these pathways might be bypassed by other yet unknown genetic events that lead towards CRC. To improve our understanding of the genetic mechanisms of CRC development we used microarrays to identify novel genes involved in development of CRC.

overall_design: In order to identify possible novel genes involved in the development of colorectal cancer we analysed the expression profile of 16 colorectal cancers. We used dual color approach. Tumour tissue was labeled with Cy5 and corresponding normal tissue was labeled with Cy3. Each array contained at least 4 replicate spots for each gene analysed. Expression was obtained by calculating median from replicate spots. Genes not present in at least 100% of all samples were filtered out. Median value for each gene from 16 arrays was calculated.

first: Gasper

last: Berginc

first: Zdravko

last: Stor

first: Miran

last: Rems


title: Queen conch (Strombus gigas) testis gene expression profiling:  offshore versus near-shore

city: Providence

first: Daniel

middle: James

last: Spade

state: RI

address: Box G-E5

department: Pathology and Laboratory Medicine

institute: Brown University

summary: This study compares gene expression in the testis of three offshore (Pelican Shoal) and three near-shore (Tingler Island) adult male queen conchs (Strombus gigas) collected from the wild on February 15, 2007.

overall_design: Six samples are analyzed, including three biological replicates from each of two locations (offshore = Pelican Shoal; near-shore = Tingler Island).  The offshore individuals are considered to be reference individuals, as they exhibit successful sexual reproduction, while near-shore conchs do not.  There are no technical replicates.  This is a single-channel (Cy3) microarray experiment.

first: Robert

last: Griffitt

first: Kevin

last: Kroll

last: Glazer

first: David

last: Barber

first: Nancy

middle: D

last: Denslow


title: Comparison between caruncles and intercaruncles in AI, IVF-ET and SCNT pregnancies

first: Olivier

last: Sandra

summary:
Implantation is crucial for placental development whose quality will directly impact fetal growth and pregnancy success with possible consequences on post-natal health. We postulated that early perturbations of the conceptus-maternal environment communication may alter the endometrium physiology that could account for the final reproductive outcome. Using cattle as an animal model, we compared gene expression profiles of the endometrial caruncular and intercaruncular areas at implantation in three types of pregnancies, namely artificial insemination (AI), in vitro fertilization with embryo transfer (IVF-ET) or somatic cell nuclear transfer (SCNT). Less than 35% of the differentially regulated genes were found to be common between AI, IVF-ET, and SCNT conditions. Compared to AI, numerous biological functions and several canonical pathways and genes were found to be significantly affected in IVF-ET or SCNT, with a major impact on metabolism and immune function in SCNT. Our data show that endometrium can fine-tune its physiology and could be considered as a biological sensor in response to pregnancy manipulations. Determining the limits of the endometrial plasticity should bring new insights on the contribution of the maternal compartment to the issue of pregnancy.

Keywords: Fluorescence Microarray

overall_design: 30 samples

first: Nadéra

last: Mansouri-Attia

first: Julie

last: Aubert

first: Séverine

last: Degrelle

first: Robin

last: Evert

first: Corinne

last: Giraud-Delville

first: Yvan

last: Heyman

first: Laurent

last: Galio

first: Isabelle

last: Hue

first: Xiangzhong

last: Yang

first: XCindy

last: Tian

first: Harris

last: Lewin

first: Jean-Paul

last: Renard


title: Identification of isobutanol response network of E. coli

city: Princeton

first: Mark

last: Brynildsen

state: NJ

address: 25 William Street

department: Chemical and Biological Engineering

institute: Princeton University

summary: Isobutanol has emerged as a potential biofuel due to recent metabolic engineering efforts. Here we used gene expression and transcription factor(TF)-gene interaction data, genetic knockouts, and Network Component Analysis (NCA) to map the isobutanol response network of Escherichia coli under aerobic conditions. A transcriptional response network consisting of 2004 genes/TFs and 2600 interactions was identified. Through further investigation ArcA, Fur, and PhoB were demonstrated to be important mediators of this response. In addition, the ethanol, n-butanol, and isobutanol response networks were compared in order to identify common and distinct toxicity features associated with these three alcohol based biofuels.

overall_design: E. coli was grown aerobically at 37C in minimal MOPS media with 0.2% glucose as the sole carbon source. At mid-log, the cultures were split in half with one half receiving a 1% isobutanol, 1% n-butanol, or 3% ethanol (vol/vol) treatment, while the other half remained unperturbed. After 10 minutes of continued growth cultures were harvested. Total RNA was purified using a Qiagen RNeasy midikit, and labeled indirectly with amino-allyl dUTP. Every experiment had a minimum of 4 biological replicates, each with 2 technical replicates (8 arrays/experiment). Normalized log10 expression ratios were obtained from lcDNA implemented with quality filtering (receptor.seas.ucla.edu/lcDNA; Hyduke DR, Rohlin L, Kao KC, Liao JC. 2003 A software package for cDNA microarray data normalization and assessing confidence intervals. OMICS 7(3):227-234.)

first: James

last: Liao


title: M. sedula transcriptional response under heterotrophic, autotrophic, and mixotrophic growth conditions

city: Raleigh

first: Kathryne

middle: Sherlock

last: Auernik

state: NC

address: Partners II, rm 3309, 849 Main Campus Drive

department: Chemical and Biomolecular Engineering

institute: North Carolina State University

summary: To study mixotrophy, it is desirable to have an organism capable of growth in the presence and absence of both organic and inorganic carbon sources, as well as organic and inorganic energy sources. Metallosphaera sedula is an extremely thermoacidophilic archaeon which has been shown to grow in the presence of inorganic carbon and energy source supplements (autotrophy), organic carbon and energy source supplements (heterotrophy), and in the presence of organic carbon and inorganic energy source supplements. The recent elucidation of M. sedula’s inorganic carbon fixation cycle and its genome sequence further facilitate its use in mixotrophic studies. In this study, we grow M. sedula heterotrophically in the presence of organic carbon and energy sources (0.1% tryptone), autotrophically in the presence of inorganic carbon and energy sources (H2 + CO2), and “mixotrophically” in the presence of both organic and inorganic carbon and energy sources (0.1% tryptone + H2 + CO2 ) to characterize the nature of mixotrophy exhibited.

overall_design: Two 3 slide loops joined at equivalent conditions (8 slides total) for Mse cells includes 3 conditions tested in duplicate (biological repeats): heterotrophy (H1 and H2), autotrophy (A1 and A2),  and mixotrophy (M1 and M2). Half of an RNA sample for one condition was labeled with Cy3 while the other half was labeled with Cy5. The two differently labeled samples were run on different slides. Each probe is spotted on each slide 5 times (5 replicates; spot intensities for all replicates on slide provided in associated raw data file).

first: Robert

last: Kelly


title: Oligonucleotide DNA microarray profiling identifies novel genes involved in lung adenocarcinoma

first: Vid

last: Mlakar

summary: The purpose of this study was to find novel gene(s) involved in the development of lung adenocarcinomas (AD) using an oligonucleotide based DNA microarray platform with a selected set of genes from Human Apoptosis Subset v2.0 and Human Cancer Subset v3.0 (Operon).

overall_design: In order to identify possible novel genes involved in the development of lung ADs we analysed the expression profile of 12 lung adenocarcinomas. We used dual color approach. Tumour tissue was labeled with Cy5 and corresponding normal tissue was labeled with Cy3. Each array contained at least 4 replicate spots for each gene analysed. Gene expression was obtained by calculating median from replicate spots.

first: Mojca

last: Strazisar

first: Mihael

last: Sok

first: Damjan

last: Glavac


title: Divergent mechanisms controlling hypoxic sensitivity and lifespan by the DAF-2/Insulin/IGF-receptor pathway

status: Public on Oct 31 2009

city: St. Louis

first: C

middle: Michael

last: Crowder

state: MO

address: 660 S. Euclid Ave Campus Box 8054

department: Anesthesiology

institute: Washington University in St. Louis

summary: To identify genes that selectively regulate hypoxic sensitivity, we compared the whole-organismal transcriptomes of three daf-2 reduction-of-function alleles, all of which are hypoxia resistant, thermotolerant, and long lived but differ in their rank of severities for these phenotypes. The transcript levels of 172 genes were increased in the most hypoxia resistant daf-2 allele, e1370, relative to the other alleles whereas transcripts from only 10 genes were decreased in abundance.

overall_design: Synchronously staged L4 animals were grown at 20C, washed off plates with M9 buffer and total RNA was immediately isolated. This total RNA was the template for the cDNA microarrays.

first: Meghann

last: Mabon

first: Barbara

last: Scott

middle: M


title: Plasmodium falciparum: hyperoxic vs normoxic conditions

status: Public on Oct 30 2009

city: MARSEILLE

first: Charles

last: Chapus

address: Allée du Médecin colonel Eugène Jamot

institute: IMTSSA

summary: Transcriptional profiling of P. falciparum comparing parasites exposed to 5% and 21% oxygen environmental conditions  in the late ring stage.

overall_design: Two experimental conditions: 5% vs 21%. Biological replicates: 3 normoxia and 3 hyperoxia replicates

first: Marylin

last: Madamet

first: Yannick

last: Lepriol

first: Jérôme

last: Desplans

first: Daniel

last: Parzy


title: Transcriptome reorganization during seasonal wood development in Pinus radiata

city: Canberra

first: Xinguo

last: Li

state: ACT

address: Building 602, Black Mountain Laboratories

department: Plant Industry

country: Australia

institute: CSIRO

summary: Seasonal wood development results in two distinct wood types: earlywood (EW) and latewood (LW), which is the major cause of wood qaulity variation. We investigate transcriptome reorganization during seasonal wood development in radiata pine using a newly developed 18k cDNA microarrays.

overall_design: Three sampling trees each at juvenile (5 yrs), transition (9 yrs) and mature (14 yrs) ages (based on the wood rings at breast height) were selected from a plantation forest of radiata pine at Bondo, NSW , Australia (35º 16' 44.04 S, 148º 26' 54.66 E). The sampling trees at juvenile and mature ages were grown within 50 m distance and under similar environment. Two sampling trees at rotation age (30 yrs) were chosen at Yarralumla, ACT, Australia (35° 18' 27'' S, 149° 7' 27.9'' E).

first: Harry

middle: X

last: Wu

first: Simon

middle: G

last: Southerton


title: Androgen repsonsive microRNAs in LNCaP cell Lines

first: Shawn

last: Lupold

summary: The goals of this study were to evaluate changes in microRNA expression following androgen treatment.

overall_design: Four treatment groups were studied:  untreated and those treated with 0.1, 1.0, and 10.0 nM of synthetic androgen (R1881).  Data is relative to the untreated sample.

first: Judit

last: Ribas

first: Xiaohua

last: Ni

first: Michael

last: Haffner

first: Erik

last: Wentzel

first: Amirali

middle: H

last: Salmasi

first: Wasim

last: Chowdhury

first: Tarana

last: Kudrolli

first: Srinivasan

last: Yegnasubramanian

first: Jun

last: Luo

first: Ron

last: Rodriguez

first: Joshua

last: Mendell


title: Genome Alteration Print (GAP): a tool to visualize and mine complex cancer genomic profiles obtained by SNP-arrays

city: Paris

first: Marc-Henri

last: Stern

address: 26 rue d'Ulm

department: Research Dept

institute: INSERM/Curie Institute

type:
Genome variation profiling by SNP array
SNP genotyping by SNP array

summary: We describe a method for automatic detection of absolute segmental copy numbers and genotype status in complex cancer genome profiles measured by SNP arrays. The method is based on pattern recognition of segmented and smoothed copy number and allelic imbalance profiles. Overall copy number assignments were verified by DNA indexes of breast  carcinomas and karyotypes of cell lines. The method performs well even for poor quality data, low tumor content, and highly rearranged tumor genomes.

overall_design: To investigate the efficiency of the method 19 primary breast carcinoma samples and 2 breast cancer cell lines were analyzed using 300K Illumina SNP arrays (Illumina HumanHap300-Duov2 Genotyping BeadChip)

first: Tatiana

last: Popova

first: Elodie

last: Manie

first: Dominique

last: Stoppa-Lyonnet

first: Guillem

last: Rigaill

first: Emmanuel

last: Barillot


title: Involvement of a chromatin remodeling complex in damage tolerance during DNA replication

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: ATP-dependent chromatin remodeling complexes have been shown to participate in DNA replication in addition to transcription and DNA repair. However, the mechanisms of their involvement in DNA replication remain unclear. Here, we reveal a specific function of the yeast INO80 chromatin remodeling complex in the DNA damage tolerance pathways. Whereas INO80 is necessary for the resumption of replication at forks stalled by methyl methane sulfonate (MMS), it is not required for replication fork collapse after treatment with hydroxyurea (HU). Mechanistically, INO80 regulates DNA damage tolerance during replication through modulation of PCNA (proliferating cell nuclear antigen) ubiquitination and Rad51-mediated processing of recombination intermediates at impeded replication forks. Our findings establish a mechanistic link between INO80 and DNA damage tolerance pathways, indicating that chromatin remodeling is important for accurate DNA replication.

overall_design: INO80 distribution in WT cells was measured.

first: Karina

last: Falbo

first: Yuki

last: Katou

first: Xuetong

last: Shen


title: miRNA profiling from freshly harvested livers

city: Heidelberg

first: Mirco

last: Castoldi

address: Im Neuenheimer Feld 156

department: Department of Pediatric Oncology, Hematology and Immunology

institute: University of Heidelberg

summary: Correlation of miRNA expression patterns with clinically relevant information is greatly facilitated by the retrospective analysis of samples archived in tissue banks. Unfortunately, the quality of samples stored in tissue banks is variable due to heterogeneity in pre-analytical preparation of clinical specimens. Collectively, these variables will impact the reliability of the results of the analysis. To date no systematic studies have been performed to investigate the relationship between total RNA degradation and miRNA profiles determined from snap-frozen collections or freshly harvested tissues. To investigate this question, we compared miRNA expression profiles generated through delaying the extraction of RNA from Liver and Duodenum, to generate different RNA integrities as defined by RIN values.

overall_design:
Liver samples were collected from mice, sliced into five identical pieces, transfered into eppendorf tubes and either processed immediately (T0) or maintained on ice and at latter time points [30 min (T30), 60 min (T60), 120 min (T120) and 240 min (T240)]. RNA was extracted by using Trizol and RNA integrity was assessed by Bioanalyzer electropherograms. We found that liver samples did not show any significant RNA degradation even when samples remained on ice for up to 4 hrs
However, miRNA expression profiling by microarray shows that miRNAs from freshly harvested Liver are partially degraded. Based on these data, we conclude that samples with low RIN values (less than 7) do not merit analysis on miRNA arrays.

first: David

last: Ibberson

first: Vladimir

last: Benes

first: Martina

middle: U

last: Muckenthaler


title: miRNA profiling from freshly harvested duodenums

summary: Correlation of miRNA expression patterns with clinically relevant information is greatly facilitated by the retrospective analysis of samples archived in tissue banks. Unfortunately, the quality of samples stored in tissue banks is variable due to heterogeneity in pre-analytical preparation of clinical specimens. Collectively, these variables will impact the reliability of the results of the analysis. To date no systematic studies have been performed to investigate the relationship between total RNA degradation and miRNA profiles determined from snap-frozen collections or freshly harvested tissues. To investigate this question, we compared miRNA expression profiles generated through delaying the extraction of RNA from liver and duodenum, to generate different RNA integrities as defined by RIN values.

overall_design:
Duodenum samples were collected from mice, sliced into five identical pieces, transfered into eppendorf tubes and either processed immediately (T0) or maintained on ice and at latter time points [30 min (T30), 60 min (T60), 120 min (T120) and 240 min (T240)]. RNA was extracted by using Trizol and RNA integrity was assessed by Bioanalyzer electropherograms. We found that duodenal samples (which are rich in RNases) do show high susceptibility to degradation. These findings suggest that tissues such as duodenum or pancreas should either be processed immediately or snap-frozen and processed individually.
miRNA expression profiling by microarray shows that miRNAs from freshly harvested duodenum are extensively degraded. Based on these data, we conclude that samples with low RIN values (less than 7) do not merit analysis on miRNA arrays.


title: Physiology and genetic activity of Listeria monocytogenes during multi-hurdle stress induced cell death

city: Hobart

first: John

middle: Phillip

last: Bowman

state: Tasmania

address: College Road, Sandy Bay

department: School of Agricultural Science

institute: University of Tasmania

summary:
The physiological basis of the hypothesis that temperature is the primary governing factor of bacterial cell inactivation under otherwise non-growth permissive conditions was investigated. Application of simultaneous low pH (pH 3.5) and low water activity (aw = 0.9; 2.5 M NaCl) conditions, applied to L. monocytogenes strains Scott A and FW03/0035, and increasing incubation temperature from 25°C up to 45°C resulted in increased permeability to ethidium homodimer-1 and corresponded to accelerated declines in esterase activity and ATP basal levels but did not result in autolysis. Triphasic survival curves were readily observable when sufficiently large cell populations were inactivated at 25°C and 35°C; indicative of a mixture of sensitive and resistant sub-populations. Enrichment-based recovery experiments however indicate that the stress conditions eventually lead to complete loss of reproductive capacity, potentially corresponding to an irreversible collapse of pH homeostasis. Transcriptomic analyses were used to further obtain insights into the physiology of the inactivation process occurring at 25°C. RT-PCR, rifampin-enforced decay and microarray experiments revealed transcripts of tufA and other genes become substantially more stable during inactivation during exposure to combined low pH/aw and during non-growth permissive temperature exposure. Gene transcripts were delineated through K-means clustering that appear to be important for initial survival of combined low pH/aw and include an overrepresentation of SigB-activated genes, the response of which fades with increasing time of inactivation exposure.
The microarray component of the experiments had the aim of determining:  i) Gene expression responses of L. monocytogenes strain Scott A when exposed to  a non-growth permissive environment consisting of a broth system acting as a food simulated environment adjusted to low pH (pH 3.5) and low water activity (2.5 M NaCl).  ii) To examine the trend in gene expression over time under inactivating (killing) conditions by applying gene set (functional and regulatory) expression trend analysis and K-means cluster analysis.  iii) To correlate this data to other physiological and mRNA quantification (real-time-PCR) data.

overall_design: Experimental design:  Two biologically replicated control cultures (each with two technical replicate sper chip) were labelled with Cy3 for each treatment sample for a total of 4 pairs of biological replicates. The treatments consisted of treated (inactivated in brain-heart infusion broth at pH 3.5 and water activity 0.9) L. monocytogenes cells with two biological replicates (each with two technical replicates per chip) labelled with Cy5. The time course (incubation time under inactivating conditions) of treatments was “initial” (20 minutes), 24 h, 48 h, and 72 h.

first: Donglai

last: Zhang

first: Tom

last: Ross


title: Analysis of sex chromosomes effect on hybrid male sterility

city: Hamilton

first: Rama

middle: S.

last: Singh

state: Ontario

address: 1280 Main Street West

department: Biology Department

country: Canada

institute: McMaster University

summary:
Sex chromosomes and more particularely the X chromosomes are known to have a major effect on hybrid male sterility. In this experiment by making use of the reciprocal hybrids between D. simulans and D. sechellia, we are showing the effect of these chromosomes on gene expression in male hybrids
Keywords: X chromosome, hybrid

sample_organism: Drosophila sechellia x Drosophila simulans

overall_design: Testes from for days old individuals (D. simulans, D. sechellia, hybrid D. simulans female x D. sechellia male, hybrid D. sechellia female x D. simulans male) were dissected and RNA was extracted and hybridized along with a reference RNA from the whole body of 4 days old D. melanogaster male. Gene expression in hybrids were compared to parental gene expression in order to isolate misexpressed genes in each hybrids. In order to reveal the cross effect misexpressed genes in hybrids were compared to identify genes commonly misexpressed and genes genes misexpressed in only one hybrid.

first: Wilfried

last: Haerty

middle: S


title: High succinic acid tolerance E.coli (DST160)

city: Bucheon

first: Pil

last: Kim

state: Gyunggi

address: 43-1 Yokkok2-dong

country: South Korea

institute: Catholic Univ. of Korea

summary: To understand cell responses against high succinate stress, a continuous culture was performed for 9 months using a wild Escherichia coli under gradually increasing succinate stress. The final adapted strain against high succinate stress, named DST160, was able to grow with no lag phase in medium containing 1.35 M of succinate stress while wild W3110 showed more than 38 h lag phase. The maximum growth rate and growth yield of DST160 under the stress condition were 0.20 h-1 and 0.192 g-cell/g-glucose, which were 53.8 % and 12.3 % higher than those of W3110 (0.13 h-1 and 0.171 g-cell/g-glucose, respectively).

sample_organism: Escherichia coli str. K-12 substr. W3110

overall_design:
A single colony of E. coli W3110 was inoculated into a 15-mL tube containing 4 mL of LB medium. After 12 h cultivation in a shaking incubator at 37℃ and 220 rpm, pre-culture (1 mL) was transferred to a 250 mL-fermentor (Mini-chemostat fermentor; Biotron Inc., Bucheon, Korea) containing 100 mL medium consisted of M9-glucose minimal medium (3 g glucose , 0.8 g NH4Cl, 0.5 g NaCl, 7.5 g Na2HPO4•2H2O, 3 g KH2PO4 with separately added trace elements of 0.2 g MgSO4•7H2O, 0.1 g CaCl2, 1 mg thiamine) per liter supplemented with 254 mmole of succinate (30 g succinic acid) per liter. The initial pH of the medium was set to be pH 7.5 by adding 6N NaOH. The fermentor was operated at 37℃, 350 rpm with air flushing (100 mL/min.). After 12 h batch mode growth, feeding and outlet pumps were turned on at flow rate of 0.167 mL/min (Dilution rate=0.1 h-1). The fresh medium reservoir (10 L) contained the same contents and pH with the initial medium. Every 10 turnover time (100 h), the reservoir was changed with the same medium composition except succinate concentration. The succinate concentration of the reservoir was gradually increased by 25.4 ~ 84.7 mM (3 ~ 10 g/L of succinic acid) at a time than previous states depending on the cell concentrations at the outlet. When the cell concentration was reduced below O.D.=0.2, succinate feeding pump was temporarily stopped to go back to the slightly lower succinate concentration. The continuous culture was performed until the steady state when succinate in the reservoir reached 1.35 M (160 g/L in succinic acid form). The final adapted cells in 100 mL culture were harvested by centrifuge and frozen rapidly in a liquid nitrogen tank for further analyses including DNA microarray.

To understand the change of up/down regulations in the high-succinate adapted E. coli, total mRNAs of control cells and the adapted E. coli cells were extracted using a RNA extraction kit (RNeasy mini kit; Quiagen, Hilden, Germany), and the transcriptomes were compared using DNA microarray. Hybridization and washing were accomplished by a facility of Digital Genomics Co (Seoul, Korea). DNA microarray were scanned using an Axon GenePix 4000B Scanner (Axon Instruments, Union City, CA, USA), and the scanned images were analyzed with GenePix Pro 3.0 software to obtain gene expression ratios.

first: Yeong-Deok

last: Kwon


title: Transcriptomic analysis of MAPK KOs mutants under normal growing conditions (bb)

city: BEAUCOUZE

first: Sandra

last: Pelletier

address: 42, rue Georges Morel - BP 60057

summary:
au07-10_mpk - bb - Gain insight into the function of mpk genes by characterizing the transcriptional profile of the mpk5, mpk10 and mpk11 KO mutants in comparison with that of the Arabidopsis wt plants. - Transcriptomic analysis of the double mutant bak1-4 bkk1-1 to compare its gene expression profile with that of the single mutants atmekk1 and atmpk4 and elucidate their involvement in the brassinolide signalling.

Keywords: gene knock out

overall_design: 1 dye-swap - CATMA arrays

first: Concetta

last: Giuliani

first: Heribert

last: Hirt

first: Jean-Pierre

last: Renou

first: Marie-Laure

last: Martin-Magniette


title: The transcriptomic response to heat stress and bleaching in the Elkhorn coral Acropora palmata

city: San Francisco

first: Michael

last: DeSalvo

state: CA

department: Department of Anesthesia

institute: UC San Francisco

summary: The emergence of genomic tools for reef-building corals and symbiotic anemones comes at a time when alarming losses in coral cover are being observed worldwide. These tools hold great promise in elucidating novel and unforeseen cellular processes underlying the successful mutualism between corals and their algal endosymbionts (Symbiodinium spp.). Since thermal stress triggers a breakdown in the symbiosis (coral bleaching), measuring the transcriptomic response to thermal stress-induced bleaching offers an extraordinary view of the cellular processes specific to coral-algal symbioses. In the present study, we utilized a cDNA microarray containing 2,059 genes of the Caribbean Elkhorn coral Acropora palmata to identify genes differentially expressed upon thermal stress. Fragments from four separate colonies were exposed to elevated temperature (3˚C increase) for two days, and samples were frozen for microarray analysis after 24 and 48 hours. Fragments experienced a 60% reduction in algal cell density after two days. 204 genes were differentially expressed in samples collected one day after thermal stress; in samples collected after two days, 104 genes. Annotations of the differentially expressed genes indicate a conserved cellular stress response in A. palmata involving: 1) growth arrest; 2) chaperone activity; 3) nucleic acid stabilization and repair; and 4) the removal of damaged macromolecules. Other differentially expressed processes include sensory perception, metabolite transfer between host and symbiont, nitric oxide signaling, and modifications to the actin cytoskeleton and extracellular matrix. The results are also compared to those from a previous coral microarray study of thermal stress in Montastraea faveolata.

overall_design:
The field experiment was performed at the Smithsonian Tropical Research Institute’s Bocas del Toro field station in Panamá during September and October 2006. Four colonies of A. palmata were sampled from two separate reefs 21 km apart (two colonies from Isla Solarte – 9o19’56.78” N and 82o12’54.65” W, and two colonies from Cayos Zapatillas – 9o15’08.79” N and 82o02’24.63” W). Each colony was broken into six fragments using a hammer and chisel. For each colony, three fragments were placed in a control aquarium, and three fragments were placed in an experimental aquarium fitted with two 200-Watt aquarium heaters, such that each colony was represented by a pair of aquaria (total of three control and three heated aquaria, all 75-liter). The three control aquaria were placed in one large fiberglass pond with continuous water flow, and the three experimental aquaria were placed in another large pond. All aquaria were exposed to shaded ambient light, and each aquarium was a closed system (but contained a pump to generate continuous water flow). Fragments were kept at a depth of ~25cm. HOBO Pendant Temperature/Light Data Loggers (Onset Corp UA-002-64) recorded temperature and light data every three minutes. These data loggers are not designed to measure photosynthetically active radiation (PAR – 400-700nm), as only ~30% of the measured light is in the range of PAR. For this reason, relative light levels in the aquaria are reported (expressed as the percentage of the average 10am to 2pm light intensity measured on a reef ~4m deep in Bocas del Toro (9o22’68.4” N and 82o18’24.6” W) during September and October 2007). Light intensity differed slightly between the four aquaria fitted with HOBOs (control aquaria 1 – 43%; control aquaria 2 – 46%; heated aquaria 1 – 35%; and heated aquaria 2 – 34% of reef light). After an acclimation period of four days at the natural temperature of the seawater system (mean temperature = 30.29±0.07oC), a fragment from each control and experimental aquaria was sampled (t0C and t0H). After time zero sampling, the heaters in each of the experimental aquaria were turned on. The temperatures of the experimental aquaria increased to ca. 32oC over three hours. The mean temperature of the control aquaria during the entire experiment was 29.74±0.03oC, and the mean temperature of the heated aquaria was 32.72±0.32oC. Control and experimental fragments were sampled again one day (1dC and 1dH) and two days (2dC and 2dH) after turning on the heaters. Heated fragments from one of the colonies (col3) showed extreme bleaching after one day of thermal stress. The remaining fragment of col3 was removed at this time to avoid fouling of the water due to death. Thus, there are four replicates for t0C, t0H, 1dC, and 1dH, and three replicates for 2dC and 2dH. All samples were taken at night. Fragments were frozen in liquid nitrogen.

We employed a reference design where all control and heat-stressed samples were compared to a pooled reference aRNA sample composed of aRNA from the four t0C fragments. Since all RNA samples were compared to the reference sample, direct comparisons of gene expression across all time points and conditions can be performed.

middle: K

first: Shinichi

last: Sunagawa

first: Christian

middle: R

last: Voolstra

first: Monica

last: Medina


title: Transcriptomic analysis of MAPK KOs mutants under normal growing conditions (mpk)

summary:
au07-10_mpk - mpk - Gain insight into the function of mpk genes by characterizing the transcriptional profile of the mpk5, mpk10 and mpk11 KO mutants in comparison with that of the Arabidopsis wt plants. - 1) mpk5 (WS genetic background) versus wt-WS 2) mpk11 (Col0 genetic background) versus wt-Col0 3) mpk10 (Col0 genetic background) versus wt-Col0 Seeds were surface sterilized and grown in MS agar medium (3% sucrose) for 11 days in long day conditions. Entire seedling were harvested and deep frozen in liquid nitrogen.

Keywords: gene knock out

overall_design: 8 dye-swap - CATMA arrays

middle: P


title: Cardiac and pulmonary responses to diesel engine particulate in Apo E(-/-) mice II

city: Honolulu

last: Maresh

state: HI

address: 651 Ilalo

department: Medicine

institute: University of Hawaii

summary:
Mice deficient in Apolipoprotein E (Apoe E(-/-)) vs wild type mice were exposed to diesel engine particulate by single dose intratracheal instillation. The transcript-level response of the hearts after 24h and of the lungs after 24 h were subsequently analyzed by the 2-color microarray method.
Keywords: stress response, disease state analysis

overall_design: Three mice of the Apo E (-/-) strain were exposed to diesel engine particulate from the National Institute of Standards and Technology via intratracheal instillation. Controls consisted of 3 littermates exposed to saline vehicle. Similarly, three wild type mice were similarly exposed to diesel particulate and three to vehicle control. 24 hours after exposure, mice were humanely sacrificed by carbon dioxide asphyxiation and the hearts and lungs were collected and frozen for analysis of total RNA. In each biological replicate, 2 color microarrays were utilized to compare pooled RNA representing all three animals of each strain, and in each condition and in each tissue; each biological replicate was performed with a dye reversal.  Biological replicates of the wild type mice responses to diesel engine particulate were performed three times for heart and lung.

first: Darrow

last: April


title: Murine C2C12 cells: GTP-stimulated cells vs. Control in DM

status: Public on Oct 29 2009

city: Chieti

first: Rosa

last: Mancinelli

state: Chieti

address: via Dei Vestini 29

department: Dept. Basic and Applied Medical Sciences

country: Italy

institute: University "G. d'Annunzio" Chieti-Pescara, Chieti

summary: To define the signalling of extracellular GTP as enhancer of myogenesis, we investigated if the gene expression profile of differentiated C2C12 cells (4 hours in DM) was affected by extracellular GTP.

overall_design: Two-condition experiment, GTP-treated C2C12 cells vs Control C2C12 cells at 4 h of differentiation. Biological replicates: 1 control, 1 GTP-treated cells, independently grown and harvested. One replicate per array.


title: Studies of two mutants for clavata genes (clavata-ralstonia)

institute: INRA

summary:
ra08-02_clavata-ralstonia - clavata-ralstonia - Clavata1 (Clv1.12) and clavata2 (RPL10) mutants display an increased resistance to the bacteria Ralstonia solanacearum. Transcriptome analysis aims to understand resistance mechanisms. - 3 biological repets

Keywords: wt vs mutant comparison

overall_design: 6 dye-swap - CATMA arrays

first: Yves

last: Marco

first: Xavier

last: Barlet

middle: L


title: Early drought-responsive changes in pepper leaves and roots

status: Public on Oct 28 2009

city: Seibersdorf

first: Silvia

last: Fluch

address: Campus Seibersdorf

department: Biogenetics/Natural Resources

country: Austria

institute: Austrian Research Centers GmbH

summary:
Drought is one of the major factor that limits crop production and reduces yield. To understand the early response of plants under nearly natural conditions, pepper plants were grown in a greenhouse and drought stressed by withholding water for one week. Plants adapted to the decreasing water content of the substrate by adjustment of their osmotic potential in roots by accumulation of raffinose, glucose, galactinol and proline. In contrast in leaves levels of fructose, sucrose and also galactinol increased. Due to the water deficit cadaverine, putrescine, spermidine and spermine accumulated in leaves whereas the concentration of polyamines was reduced in roots. These polyamines are suggested to rather act as stress protectants than for osmotic adjustment. To understand the molecular basis of the response to this early drought stress better, four suppression subtractive hybridisation libraries from leaves and roots were constructed. Microarray technique was used to identify differentially expressed genes. A total of 109 unique ESTs were detected. The diversity of the putative functions of all identified genes confirms the complexity of the plant response to drought stress.

Keywords: Transcription profiling

platform_organism:
Populus trichocarpa x Populus deltoides
Capsicum annuum
Solanum tuberosum
Ipomoea batatas
Triticum aestivum
Zea mays
Homo sapiens
Pinus pinaster
Populus euphratica
Populus tremula x Populus alba

overall_design: Two-condition experiment in roots and leaves, control leaves (CL) vs. drought-stressed leaves (DL) and control roots (CR) vs. drought-stressed roots (DR). Biological replicates: 4 control (1-4), drought-stressed (1-4), independently grown and harvested. One swap replicate per array.

first: A

last: Sziderics

first: M

last: Oufir

first: F

last: Trognitz

first: I

last: Matusikova

first: J

middle: F

last: Hausmann

first: E

last: Wilhelm


title: Murine C2C12 cells: GTP-stimulated cells vs. Control in SM

summary: To define the signalling of extracellular GTP as enhancer of myogenesis, we investigated if the gene expression profile of differentiated C2C12 cells (4 and 24 hours in SM) was affected by extracellular GTP.

overall_design: Two-condition experiment, GTP-treated C2C12 cells vs Control C2C12 cells at 4 h and 24 h of differentiation. Biological replicates: 1 control, 1 GTP-treated cells, independently grown and harvested. One replicate per array.


title: Fast Growth Increases the Selective Advantage of a Mutation Arising Recurrently during Evolution under Metal Limitation

city: Cambridge

first: HSIN-HUNG

last: CHOU

state: MA

address: 16 Divinity Avenue, Biolab 3079

department: Organismic and Evolutionary Biology

institute: Harvard University

summary: The goal was to use microarray to compare changes in the global transcription profiles between an engineered bacterial strain and one of its descendants subject to 600 generations of experimental evolution in batch culture. This comparison allowed us to identify a beneficial mutation that substantially increased expression of a novel cobalt transporter cassette in this descendant.

overall_design: The total RNA of the an engineered strain of Methylobacterium extorquens AM1 and an descendant derived from this engineered strain was extracted. RNA extractions were performed 3 times using 3 independent growth cultures of a given strain. The final RNA samples for the following microarray experiments consisted of an equal proportion of 3 independent extractions for each strain. The cDNA of the descendant was labeled with Alexa_555 while the engineered strain was labeled with Alexa_647 in the array dataset GSM371896.

first: Hsin-Hung

last: Chou

first: Julia

last: Berthet

first: Christopher

middle: J

last: Marx


title: Breaking the epigenetic barrier during reversion of post-implantation epiblast cells

first: Fuchou

last: Tang

state: Cambridgeshire

address: Tennis Court Road

department: Wellcome Trust/Cancer Research UK Gurdon Institute

country: United Kingdom

institute: University of Cambridge

summary: Primitive ectoderm cells (PE) in blastocysts represent the foundation of the pluripotent state, which is lost progressively during development.  For example, development of epiblast cells from PE in postimplantation embryos is accompanied by transcriptional and epigenetic changes, including DNA methylation and X inactivation (Refs); these changes alter the nature of epiblast cells fundamentally, affecting their responsiveness to signaling molecules, and constitute a robust boundary that prevents their reversion to a PE-like state.  Notably, epiblast cells unlike PE, are refractory to leukaemia inhibitory factor (LIF)/STAT3 signalling in vitro; instead, they respond to FGF/Activin to form self-renewing epiblast stem cells (EpiSCs) that are like human ES cells, which differ significantly from mouse embryonic stem cells (ES) derived from PE.  However, here we show that under appropriate conditions, epiblast cells from postimplantation embryos can respond to LIF/STAT3/fetal calf serum (FCS), and undergo reprogramming to form embryonic stem cell -like cells (repiES: reprogrammed epiblast ES-like cells).  Reprogramming of epiblast cells occurs progressively. First, they form colonies that retain key properties of epiblast cells (cEpi: cultured epiblast), which subsequently show erasure of epigenetic modifications, including DNA demethylation and X-reactivation to generate repiES.  Analysis also revealed that repiES progressively acquire a transcriptome profile of ES cells that is distinct from cEpi and EpiSCs.  In chimeras, repiES contributed to all the tissues, including germ cells.  Thus, we show for the first time that reversion of epiblast cells to repiES phenotype entails progressive loss of phenotypic and epigenetic memory of epiblast cells.  Our study provides insights into underlying mechanisms, and a tractable model for how signaling molecules induce epigenetic reprogramming of cells leading to an elemental pluripotent state.

overall_design: We validated our findings concerning reprogramming of epiblast cells by microarray analysis, which showed that repiES are clearly more similar to control ES cells and differ from the cEpi cells, Changes in the transcriptome must also account for the activation of the Oct4-deltaPE-GFP reporter via the distal enhancer. 12 samples

first: Kaiqin

last: Lao

first: Azim

last: Surani


title: Cholesterol's effect on M. tuberculosis

city: Newark

first: Saleena

last: Ghanny

address: 185 South Orange Ave

institute: Rutgers University

summary:
2 experiments:
(1) Mtb, Tween vs Cholesterol at 3 & 24hrs

(2) CDC1551 vs kstR mutant of CDC1551, with and w/o Cholesterol

overall_design: In triplicate, dye flips included.

first: Eugenie

last: Dubnau


title: Comparison of gene expression between wild-type and PTIP deficient chicken DT40 B cells

city: Kyoto

first: XIN

last: WANG

address: Yoshida Konoe, Sakyo-ku

department: Graduate School of Medicine

institute: Kyoto University

summary: PTIP (Pax2 transactivation domain-interacting protein) is a nuclear protein containing six BRCT domains. It has been shown that PTIP affects gene expression by controlling the activity of the transcription factor Pax2 and histone H3 lysine 4 methyltransferase complexes. In addition to its role in transcriptional regulation, PTIP has been implicated in DNA damage response. To ask if  the depletion of PTIP affects the expression level of genes encoding DNA damage response factors , we compared the whole transcripts between wild-type and PTIP deficient chicken DT40 B cell lines.

overall_design: The total RNAs were isolated from wild-type and PTIP deficient cells (PTIP-/-/-) using Sepasol®-RNA I (Nacalai tesque, Japan). The gene expression profiles were examined using Genechip® Chicken Genome Array (Affymetrix Cat #900590), by GeneticLab co. Ltd. Japan, following Genechip® protocol.

first: Xin

last: Wang

first: Katuya

last: Takenaka

first: Akira

last: Motegi

first: Shunichi

last: Takeda


title: Genome-Wide Microarray Analysis of Histone H2B Ubiquitylation mutant strains of Tetrahymena thermophila

status: Public on Oct 27 2009

city: Rochester

first: Martin

last: Gorovsky

state: NY

address: Hutchison Hall 432

department: Department of Biology

institute: University of Rochester

summary: Microarray analyses were performed to compare the gene expression profiles of wild-type and several mutant strains of the ciliated protozoan Tetrahymena thermophila. Elimination of H3K4 methylation (hht2-K4Q) and knockout of either of the ubiquitylation enzymes (delta-UBC2 and delta-BRE1) affects a broader spectrum of genes than elimination of H2B ubiquitylation (htb1-K115R).

overall_design: Cells at mid-logarithmic growing phase (cell density of 200,000 cells/ml) were collected. Then total RNAs were extracted and hybridized.

first: Zhe

middle: A


title: Unamplified mRNA abundance in miR-124 and mock-transfected cells

city: Stanford

first: Patrick

middle: O

last: Brown

address: Beckman Center b439

department: Department of biochemistry

institute: Stanford university school of medicine

summary: To compare total RNA levels in miR-124 and mock-transfected cells (Figure S3), 5-10 ug of total RNA from miR-124-transfected cells or mock-transfected cells or universal reference RNA (Stratagene Cat# 740000) was reverse transcribed with Superscript III (Invitrogen Cat# 18080085) in the presence of aminoallul-dUTP 5-(3-aminoallyl)-dUTP (Ambion Cat# AM8439) and natural dNTPs (GE Healthsciences Cat# US77212) with 10 ug of N9 primer (Invitrogen). Subsequently, amino-allyl-containing cDNAs from miR-124 and mock-transfected cells were covalently linked to Cy5 NHS-monoesters, and universal reference cDNA was covalently linked to Cy3 NHS-monoesters (GE HealthSciences Cat# RPN5661). Cy5- and Cy3-labeled cDNAs were mixed and diluted into 50 ul of solution containing 3x SSC, 25 mM Hepes-NaOH (pH 7.0), 20 ug human Cot-1 DNA (Invitrogen Cat# 15279011), 20 ug of poly(A) RNA (Sigma Cat# P9403), 25 ug of yeast tRNA (Invitrogen Cat# 15401029), and 0.3% SDS. The sample was incubated at 95 C for 2 min, spun at 14,000 rpm for 10 mins in a microcentrifuge, then hybridized at 65 C

overall_design: An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.

first: Daniel

last: Hogan


title: Identification of StpA-binding sites on the Salmonella genome

city: Norwich

first: Sacha

last: Lucchini

address: Colney Lane

department: Molecular Microbiology

institute: Institute of Food Research

summary: We used ChIP-chip analysis to identify where the nucleoid associated protein StpA binds on the Salmonella Typhimurium genome. We identified 285 chromosomal regions bound by StpA, ranging in size from 400 to 3500 base pairs and containing 572 genes. Comparison of the ChIP data revealed that StpA displayed an identical binding profile to H-NS; all genomic regions associated with StpA were also bound by H-NS. Of the 88 StpA-dependent genes that are de-repressed in the absence of StpA, only 25 were bound by StpA, suggesting that StpA generally regulates gene expression indirectly.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

overall_design: To identify the direct StpA targets, we co-immunoprecipitated DNA from a strain expressing a 3xFLAG-tagged StpA-protein using an anti-3xFLAG antibody. As a negative control, we used the same antibody (anti-3xFLAG) to co-immunoprecipitate DNA from an isogenic strain expressing the non-tagged StpA-protein. Three independent biological replicates were generated. For comparative purposes we also performed the co-immunoprecipitation on wild type Salmonella using a monoclonal anti-H-NS antibody. In that case, the negative control was obtained by performing the co-immunoprecipitation protocol on wild type Salmonella without adding antibody.

first: Paul

last: McDermott

first: Arthur

last: Thompson

first: Jay

middle: C

last: Hinton


title: Global gene expression analysis during sporulation of the aquatic fungus Blastocladiella emersonii

city: São Paulo

first: André

last: Vieira

state: São Paulo

address: Av. Prof. Lineu Prestes 748

department: Bioquímica

country: Brazil

institute: Instituto de Química - USP

summary: Blastocladiella emersonii life cycle presents a number of drastic biochemical and morphological changes, mainly during two cell differentiation stages: the germination and the sporulation. To investigate the transcriptional changes taking place during the sporulation phase, which culminates with the production of the zoospores, motile cells responsible for the dispersal of the fungus, microarray experiments were performed.

overall_design:
Transcription comparisons between cell at the begining of the sporulation phase (zero min.) and at different time points throughout zoospore differentiation were performed in order to associate the gene expression profile with the morphologial changes which culminate with zoospore differentiation.
Microarray experiments along the sporulation phase were also realized in the presence of 1% glucose, once such carbohydrate is able to block zoospore differentiation.
The results obtained from both sets of experiments were compared in order to understand the glucose effect on sporulation.
There are at least 3 biological replicates (independent harvest) and 2 technical replicates of each array (L - left and R - right).

first: Suely

last: Comes


title: The effect of energy balance on bovine mammary tissue gene expression during Streptococcus uberis mastitis challenge

status: Public on Oct 26 2009

city: Tjele

first: Kasey

middle: M

last: Moyes

address: Blichers Allé 20, P.O. BOX 50

department: Animal Health, Welfare, and Disease

institute: University of Aarhus

summary:
Background
Our objective was to compare mammary tissue gene expression profiles during a Streptococcus uberis (S. uberis) mastitis challenge between lactating cows subjected to dietary-induced negative energy balance (NEB; n = 5) and cows fed ad libitum to maintain positive energy balance (PEB; n = 5).  The NEB cows were feed-restricted to 60% of calculated net energy for lactation requirements for 7 d, and cows assigned to PEB were fed the same diet for ad libitum intake.  Five days after feed restriction, one rear mammary quarter of each cow was inoculated with 5,000 cfu of S. uberis (O140J).  At 20 h post-inoculation, both the S. uberis-infected mammary quarters (i.e., YES) and non-infected rear quarters (i.e., NO) from all cows were biopsied for RNA extraction.

Effect of S. uberis Intramammary Infection (IMI; YES vs NO)
S. uberis IMI resulted in 2,102 oligos (1,939 annotated genes) differentially expressed genes (DEG; YES versus NO).  Of these, 1,082 genes were up-regulated during IMI and were primarily involved with the immune response, e.g., IL6, TNF, IL8, IL10, SELL, LYZ, and SAA1.  Genes down-regulated (1,020) included those associated with milk fat synthesis, e.g., LPIN1, LPL, CD36, and BTN1A1.  Pathway analysis of all DEG indicated that IL-10 Signaling, IL-6 Signaling, and Glucocorticoid Receptor Signaling were among the top canonical pathways affected by infection.  Interestingly, LXR/RXR Signaling was also a primary affected pathway, indicating a relationship between immune system response and metabolism during an IMI.  Network analysis indicated that TNF had positive relationships (i.e. up-regulation) with genes involved with immune system function (e.g., CD14, IL8, IL1B, and TLR2) and negative relationships (i.e., down-regulation) with genes involved with lipid metabolism (e.g., GPAM, SCD, FABP4, CD36, and LPL) and antioxidant activity (SOD1).  Results provided information into the early response factors associated with the innate immune response to S. uberis infection.  Our study indicated that IMI challenge with S. uberis (strain O140J) elicited a strong transcriptomic response, leading to an overall up-regulation of genes involved in the innate immune response and a down-regulation of genes involved with lipid metabolism.

Effect of NEB Within Infected Quarters (YES)
Energy balance (NEB vs. PEB) resulted in 285 DEG (FDR ≤ 0.05), with 85 DEG up-regulated and 200 DEG down-regulated.  Canonical pathways most affected by NEB were IL-8 Signaling (10 genes), Glucocorticoid Receptor Signaling (13), and NRF2-mediated Oxidative Stress Response (10).  Among genes differentially expressed by NEB, Cell Growth and Proliferation (48) and Cellular Development (36) were the most enriched functions.  Up-regulated genes included ANXA1, HMOX1, and HLAA; down-regulated genes included NOTCH1 and CCND1.  Regarding immune response, HLAA was up-regulated due to NEB, whereas the majority of genes involved in immune response were down-regulated (e.g., AKT1, IRAK1, MAPK9, and TRAF6).  Results helped identify mechanisms affected by dietary-induced NEB that may be associated with the impairment of immune function and increased risk of mastitis in cows experiencing postpartal NEB.

Effect of NEB Within Non-Infected Quarters (NO)
Energy balance (NEB vs. PEB) resulted in 278 DEG within non-infected rear mammary quarters.  Up-regulated DEG most affected by NEB (genes = 180) included genes involved in lipid metabolism and amino acid metabolism.  The down-regulated DEG most affected by NEB  (n = 98) included genes involved in carbohydrate metabolism, anti-inflammatory response, and apoptosis.  Among genes up-regulated, Ingenuity Pathway Analysis® identified Lipid Metabolism (8), Molecular Transport (14), and Small Molecule Biochemistry (15) as some of the most enriched molecular functions. Genes down-regulated by NEB were associated with Cell Growth and Proliferation (21) and Cell Death (18).  Results indicate that dietary induced-NEB alters genes primarily associated with cellular metabolism, proliferation, and tumorigenesis but no candidate genes were identified as being associated with risk of disease.

overall_design:
10 Holstein cows in mid-lactation (~90 days in milk) were subjected to either dietary-induced negative energy balance (n=5) or fed ad libitum to maintain positive energy balance (n=5). The cows were inoculated with 5,000 cfu of Streptococcus uberis strain O140J into the mammary quarter. Both the infected and non-infected rear quarters of all cows were biopsied at 20 hours post-inoculation for gene expression profiling. Mammary tissue was cohybridzed with RNA from a pool of bovine tissues. All samples were hybridized to duplicate slides with reverse labeling (40 microarrays total).

The supplementary file 'GSE15344_raw_data_readme.txt' contains a description of the contents of the raw data files.

first: James

last: Drackley

first: Dawn

middle: E

last: Morin

first: Massimo

last: Bionaz

first: Sandra

last: Rodriguez-Zas

first: Robin

last: Everts

first: Harris

last: Lewin

first: Juan

last: Loor


title: Global gene expression differences between GT1, RH-ERP2009 and RH-JSR

city: St Louis

first: Michael

middle: Sean

last: Behnke

address: 660 S. Euclid Ave

institute: Washington University School of Medicine

summary: Previous studies have shown that continuous in vitro passage can result in changes to the global gene expression profiles in mammalian cells.  To examine the extent of gene expression differences between highly homologous type I strains, we took advantage of the T. gondii Affymetrix microarray containing probes to more than 8,000 genes.  We compared the RH-JSR and GT1 strains, two isolates that share the small plaque phenotype, to the large plaque isolate RH-ERP2009.  Following hybridization and normalization of data, we used two independent methods to generate lists of genes with expression differences that were statistically significant  and 2-fold or greater in at least one sample.  The 1-way ANOVA (P ≤ 0.05) identified 520 genes that were significantly differentially expressed between GT1, RH-ERP2009 and RH-JSR.  Statistical Analysis of Microarray (SAM) (FDR = .05%) analysis identified 475 transcripts that were differentially expressed.  Both lists were combined (610 genes) and used to perform pairwise fold comparisons between the three samples.  This process identified 113 genes that were similarly expressed in GT1 and RH-JSR and differentially expressed in RH-ERP2009.  Differentially expressed genes were located across all chromosomes and showed no pattern of clustering to one particular region of the genome.  Interestingly, three out of 34 ABC transporters (ToxoDB) were significantly upregulated in RH-ERP2009 (hypergeometric distribution, P = 0.01)

overall_design: Three type I strains of Toxoplasma gondii, GT1, RH-ERP2009 and RH-JSR, were compared for global gene expression differences.  Three biological replicates were obtained for each strain.

first: Asis

last: Khan

first: L

middle: D

last: Sibley

middle: W

last: White


title: Response of gastric epithelial progenitors to H. pylori isolates from Swedish patients with chronic atrophic gastritis 2

city: Saint Louis

first: Jeremiah

last: Faith

address: Campus Box 8510

institute: Washington University in St. Louis

summary: Helicobacter pylori infection is associated with development of gastric adenocarcinoma in a subset of infected humans, especially those that develop an antecedent condition, chronic atrophic gastritis (ChAG) characterized by loss of acid-producing parietal cells. Studies in a gnotobiotic transgenic mouse model of ChAG, with an engineered ablation of parietal cells and an associated expansion of gastric epithelial progenitors (GEPs), have shown that a subset of GEPs is able to harbor intracellular collections of H. pylori.  To better understand H. pylori’s adaptation to ChAG, we sequenced the genomes of 24 isolates, obtained from 6 individuals, each sampled over a 4-year interval, as they maintained normal gastric histology, or progressed from normal histology to ChAG, or experienced worsening ChAG, or proceeded from ChAG to cancer. Analyses of gene content and single nucleotide polymorphisms (SNPs) demonstrated that H. pylori populations within study participants were largely clonal, and remarkably stable over the 4-year interval, regardless of disease state. Because they exhibited such broad inter-host variation (38.6±4.7 SNPs/1000bp of genome), and did not cluster according to host pathology, we sought to identify common functional properties by performing GeneChip studies of the responses of a cultured mouse gastric stem cell-like line (mGEPs) to infection with sequenced strains. The results yielded a shared 695-member set of genes differentially expressed after infection with ChAG-associated, but not normal or heat killed strains: 434 of these genes were also represented in dataset of responses to the cancer-associated strain.  Ingenuity Pathway Analysis revealed that ChAG- and ChAG/cancer- associated responses were  significantly enriched in genes associated with tumorigenesis in general, and gastric carcinogenesis in specific cases. Whole genome transcriptional profiling of a sequenced ChAG strain during mGEP infection disclosed a set of responses that included upregulation of hopZ, an adhesin belonging to a family of outer membrane proteins. Expression profiles of wild-type and hopZ? strains revealed a number of pH-regulated genes affected by loss of HopZ, including HopP which binds sialylated glycans produced by GEPs in vivo. Genetic inactivation of hopZ produces a fitness defect in gnotobiotic transgenic mice but not their wild-type littermates. This study illustrates an approach for identifying GEP responses specific to ChAG, and bacterial genes important for survival in a gastric ecosystem that lacks parietal cells.

overall_design: H. pylori strains that had been grown to log phase were used to infect mGEP cells.. After 24h, media and non-attached bacteria were washed off and the cells harvested by trypsinization. RNA was prepared from bacteria infecting mGEP cells or bacterial cultures  grown in cell media for 24h in the absence of mGEP cells.

first: M

last: Giannakis

first: J

middle: I

last: Gordon


title: The effect of stpA deletion on S. Typhimurium gene expression during growth in rich medium

first: Sacha

last: Lucchini

summary: StpA is a paralogue of the nucleoid associated protein H-NS that is conserved in a range of enteric bacteria and had no known function in Salmonella enterica serovar Typhimurium. Here, we show that 5% of the Salmonella genome is regulated by StpA, which contrasts with the situation in Escherichia coli where deletion of stpA only had minor effects on gene expression. The StpA-dependent genes of S. Typhimurium are a specific subset of the H-NS regulon that are predominantly under the positive control of sigma38 (RpoS), CRP-cAMP and PhoP. The regulatory role of StpA varied at different growth phases; StpA only controlled sigma38 levels at mid-exponential phase when it prevented inappropriate activation of sigma38 during rapid bacterial growth. In contrast, StpA only activated the CRP-cAMP regulon during late exponential phase.

overall_design: The effect of stpA deletion on S. Typhimurium gene expression during growth in LB was analysed at 4 different time points (early-log, mid-log, late-log, and stationary phase) where the gene expression profile of the stpA-deletion strain was compared to that of the parental strain. Between two and three biological replicates were performed for each strain and time point. For this study, we used Salmonella genomic DNA as the comparator which also acted as the control for spot quality.

first: Paul

last: McDermott

first: Arthur

last: Thompson

first: Jay

last: Hinton


title: Gold induction of C. metallidurans CH34

city: Mol

first: Rob

last: Van Houdt

address: Boeretang 200

department: Interdisciplinary Biosciences

country: Belgium

institute: SCK-CEN

summary: Whole-genome expression microarray experiments were conducted to assess the response of C. metallidurans CH34 to aqueous Au(III)-chloride and identify possible biochemical pathways for Au detoxification. Four microarray experiments were conducted to assess gold response at low, medium and high Au(III)-chloride concentrations (i.e., 10, 50 and 100 µM Au(III)-chloride) and different induction times (10 and 30 minutes with 50 µM Au(III)-chloride). Based on these arrays, the differentially expressed genes upon gold-exposure can be identified.

overall_design: The four microarray experiments (10, 50 and 100 µM Au(III)-chloride at 10 minutes and 50 µM Au(III)-chloride at 30 minutes) were all performed in biological triplicate and containing three (in-slide) technical repeats. For all conditions, the metal-induced sample (Cy5) was compared with the non-induced sample (Cy5) to identify those genes that were differentially expressed upon gold exposure.

first: Frank

last: Reith

first: Pieter

last: Monsieurs

first: Hugo

last: Moors

first: Abderafi

last: Benotmane

first: Cornelia

last: Grosse