title: Hepatopancreatic multi-transcript expression patterns in the crayfish Cherax quadricarinatus during the molt cycle

city: Haifa

first: Moshe

last: Tom

department: Marine Biology and Biotechnology

country: Israel

institute: Israel Oceanographic and Limnological Research

summary:
Variations of hepatopancreatic transcript expression related to induced molt cycle were identified in male Cherax quadricarinatus using a cDNA microarray estimated to contain 2180 unique sequences. Molt induction was performed by X-organ sinus gland extirpation or by repeated 20-hydroxyecdysone injections. Manipulated males were sacrificed at premolt or early postmolt and a reference population at intermolt. Their isolated hepatopancreatic mRNA was hybridized onto the microarray and differentially-expressed genes were identified, sequenced, and annotated. Clusters of similarly expressed transcripts, in correlation with the four combinations of induction methods and molt stages were identified, containing both known and novel genes. Biologically interesting clusters were characterized by general shift of expression throughout premolt and early postmolt vs. intermolt, or by different premolt vs. postmolt expression. Several genes were differentially expressed in 20-hydroxyecdysone injected crayfish vs. X-organ sinus gland extirpated ones.
Keywords: Physiological state analysis

overall_design:
The experiment submitted in this series includes male Cherax quadricarinatus in five experimental conditions. Individual variability was integrated into the hybridization design, by using RNA populations of 3 individual crayfish males for each of four experimental conditions: injected-premolted, injected-postmolted, extirpated-premolted and extirpated-postmolted males. Three pools of reference intermolt individuals were used: Ref pool 1 contained RNA from 6 individuals whereas each of Ref pools 2 and 3 contained 3 individuals. Three hybridization sets which are biological replicates, each composed of 8 slides, were carried out. In each set, one individual from each treatment was compared to one of the Ref pools, and in addition the treated individuals were compared to each other in a loop design, totaling 8 slides. The design was balanced with respect to the dye labeling of the RNA populations, such that each individual RNA population was labeled with one dye on one slide and with the other dye in two other slides (see graphic presentation of the hybridization design in linked file GSE6947_Hybridization_design.pdf). The entire 24 slides set was analyzed as one integrative experiment, resulting with eight binary comparisons among experimental conditions as follows: extirpated-premolted vs. intermolt reference (Epre_Ref); extirpated-postmolted vs. intermolt reference (Epost_Ref); injected-premolted vs. intermolt reference (Ipre_Ref); injected-postmolted vs. intermolt reference (Ipost_Ref); extirpated-premolted vs. injected-premolted (Epre_Ipre); extirpated-premolted vs. extirpated-postmolted (Epre_Epost); injected-premolted vs. injected-postmolted (Ipre_Ipost); extirpated-postmolted vs. injected-postmolted (Epost_Ipost).
Data analysis was conducted under the R/BioConductor environment (Gentleman et al., 2005). Calculation and statistical analysis of log2 expression ratios (VALUE column of the sample data table) of each unique spot across a binary comparison was carried out using the LIMMA package (Linear Models for Microarray data Analysis; Smyth, 2004). GENEPIX quantified image files were imported into LIMMA, spots with quality flags < -49 were marked as unreliable, and the Cy5 and Cy3 intensities within each slide were normalized using the print-tip Loess method. No background correction was applied. LIMMA calculated for each spot on an individual slide an M-value [M=log2(Cy5)/Cy3); Cy5 / Cy3 are the normalized emission intensities of the spot] and an emission intensity A-value [A=(log2(Cy5)*Cy3))/2]. Subsequently, LIMMA fitted a linear model to the expression data followed by Empirical Bayes smoothing. Inter-duplicate correlation method was used at the linear modeling step. A meanA value, which is the average spot emission intensity across the analyzed experiment, was calculated for each unique spot and only spots with meanA > 8.5 were included in the analysis, avoiding faint emissions. The LIMMA summary statistics of each unique spot included an estimated average M-value across each binary comparison associated to two statistical parameters: B statistic, the log-odds that the spot is differentially expressed and p-value, derived from a moderated t-statistic after multiple testing correction according to Benjamini and Hochberg (1995). All these parameters are found in the sample data tables.

first: Yana

last: Yudkovski

first: Asaf

last: Shechter

first: Vered

last: Chalifa-Caspi

first: Meirav

last: Auslander

first: Ron

last: Ophir

first: Chantal

last: Dauphin-Villemant

first: Michael

middle: R

last: Waterman

first: Amir

last: Sagi


title: Sensitive expression profiling of T-cell subsets reveals parallel post-thymic differentiation for CD4+ and CD8+ lineages

status: Public on Jun 30 2007

city: COLOGNE

first: Frank

last: Huebel

state: NRW

address: Stoeckheimer Weg 1

department: MACS Molecular 9

country: Germany

institute: Miltenyi Biotec GmbH, MACS Molecular Business Unit

summary:
Gene expression profiling has opened new directions of investigation in scientific research, however, the need for substantial amount of biological material to perform these experiments limits significantly its application range. Here, we have developed procedures to perform microarray analysis on amplified cDNA from single cells, including primary T lymphocytes, and applied this technology to the study of CD4 and CD8 lineage differentiation. Gene expression profiling was performed on samples of 1000 cells from 10 different subpopulations, defining the major stages of post-thymic CD4+ or CD8+ T-cell differentiation. Surprisingly, our data revealed that while CD4+ and CD8+ T-cells evolve differently at early stages of differentiation, they become increasingly similar as they reach late differentiation stages. This suggests that the functional heterogeneity of antigen experienced CD4+ and CD8+ T-cells is located early during post-thymic differentiation, and that late stages of differentiation may represent a common end in the development of T-lymphocytes.
Keywords: Gene expression profiling

overall_design: Experiments to test the efficacy of our method applied to low cell numbers of human lymphocytes were performed using the immortalized Jurkat T-cell and Raji (Human Burkitt's lymphoma) B-cell line. First, a set of microarrays (duplicates representing a total of 12 microarrays) was obtained with amplified material from 1, 10, 100, 1000 or 10000 Jurkat T-cells hybridized against the same numbers of Raji B-cells (all titrated by dilution) and compared with profiles obtained with non amplified material from 10^7 Jurkat cells versus Raji cells. In a second set of experiments (duplicates representing a total of 22 microarrays), the influence of the FACS sorting procedure on gene expression profiling was tested. An increasing number of Jurkat cells (1, 3, 10, 30, 100, 300, 1000) were sorted by FACS and their amplified cDNA hybridized against amplified Raji cDNA (1000 cells). Gene expression profiles from these experiments were compared with the profile from 1000 Jurkat vs. Raji cells obtained by dilution. Another set of experiments (representing a total of 20 microarrays) was performed directly using primary CD8+ T lymphocytes obtained from two donor peripheral bloods. For this purpose, naïve (CD27+, CD28+, CD45RA+ and CCR7+) as well as highly differentiated (CD27-, CD28- and CCR7-) CD8+ T-cells were sorted by flow cytometry into 10, 100, 1000, 10000 cell samples. Naïve and highly differentiated cell probes were hybridized in a competitive manner on the microarrays (in duplicate) to provide differential gene expression profiles between these two subsets. In a last set of experiments a comparative study of 10 different subpopulations, defining major steps of both CD4+ or CD8+ T-cell differentiation processes (Fig. 3a and 3b), FACS sorted from a single blood sample of 20 ml was performed. Experiments were performed on two healthy donor blood samples (a total of 20 microarrays). For both CD4+ or CD8+ lineages, the probes obtained from the different subsets of antigen experienced cells were hybridized in a competitive manner against the probes obtained from respective naïve cell pool standards (composed of three independent FACS isolated and amplified 1000 CD4+ or CD8+ cell samples).


title: Characterization of human malaria parasite Plasmodium falciparum eIF4E homologue and mRNA 5′ cap status

status: Public on Jul 07 2007

city: Pathumthani

first: Philip

middle: James

last: Shaw

state: Pathumthani

address: 113 Phahonyothin Road

department: National Center for Genetic Engineering and Biotechnology

country: Thailand

institute: National Science and Technology Development Agency

summary:
The mRNA 5′ cap is normally essential for eukaryotic mRNA translation, stabilization and transport and both the cap and eIF4E are important elements of post-transcriptional gene regulation. To further our understanding of mRNA translation in the human malaria parasite Plasmodium falciparum, we have investigated the parasite translation initiation factor eIF4E and its interaction with 5′ capped mRNA. We have purified P. falciparum eIF4E as a recombinant protein and demonstrated that it has canonical mRNA 5′ cap binding activity. We used this protein to purify P. falciparum full-length 5′ capped mRNAs from total parasite RNA. Microarray analysis comparing total and eIF4E-purified 5′ capped mRNAs shows that a subset of 34 features were more than two-fold under-represented in the purified RNA sample, including 19 features representative of nuclear transcripts. The uncapped nuclear transcripts may represent a class of mRNAs targeted for storage and cap removal.
Keywords: total RNA vs purified capped mRNA

overall_design: The microarray data were obtained from four hybridizations using RNA from two independent GST-PfeIF4E purifications from separate malaria cultures.

middle: J

first: Napawan

last: Ponmee

first: Nitsara

last: Karoonuthaisiri

first: Sumalee

last: Kamchonwongpaisan

first: Yongyuth

last: Yuthavong


title: cDNA microarray analysis of gene expression in blood mononuclear cells of Yorkshire pigs with SLA-DRB1 defined genotypes

status: Public on May 30 2007

city: Guelph

first: Maria

middle: Isabel

last: Nino-Soto

state: Ontario

address: College Ave. West

department: Pathobiology

country: Canada

institute: University of Guelph

summary:
The major histocompatibility complex (SLA in pigs) encodes molecules for self-nonself discrimination, constitute a major transplantation barrier and is associated with a variety of diseases. Pigs with defined SLA genotypes are a resource for the study of immune response, disease resistance and production traits, as well as an important animal model for transplantation research. The aim of the present study was to explore the influence of defined SLA genotypes on gene expression patterns of immune-related genes in blood mononuclear cells (BMCs). Using a loop design, heterologous cDNA microarray hybridizations were performed to compare Yorkshire pigs representing three defined SLA-DRB1 genotypes (including a potentially new allele). Total RNA was treated with Dnase I treated and amplified. The amplified RNA was reverse transcribed using random primers and incorporating 5-(3-aminoallyl)-dUTP for post-synthesis labelling of the cDNA with the appropriate fluor dyes. Hybridization was performed on a bovine immune-endocrine in-house cDNA microarray printed on glass slides. Microarray fold-change analysis revealed that animals carrying confirmed SLA alleles (SLA-DRB1*070701 and SLA-DRB1*050502) showed consistent differences in gene expression when compared to the two other groups. On the other hand, the group carrying a potentially new allele showed differences that varied depending on the group it was being compared to. Genes that were identified as differentially expressed include macrophage inflammatory protein 1, interleukin 1, toll-like receptor 2 and caspase 1. A better understanding of SLA gene activity can facilitate the definition of new strategies to control animal health and optimize animal production.
Keywords: SLA-defined genotype comparative genomic cDNA microarray hybridizations

overall_design: Three groups (n = 2) were established, each representing a defined SLA-DRB1 genotype. A loop design was used to reciprocally compare the three groups. A total of 6 comparisons were performed with one animal per group being compared to two different animals from the two other groups. Each array was replicated twice with reciprocal labeling (dye-swap).

middle: I

first: Razi

last: Jafari

first: Bonnie

middle: A

last: Mallard


title: Small RNAs from the lycopod Selaginella moellendorffii

city: University Park

last: Axtell

state: PA

address: 280 Mueller Lab

department: Biology

country: USA

institute: Pennsylvania State University

type: Non-coding RNA profiling by high throughput sequencing

summary:
The small RNAs of Selaginella moellendorffii were sampled by direct pyrosequencing (454 Life Sciences).  These data represent microRNAs, siRNAs, and other expressed small RNAs of ~18-~28 nts. in length.
Keywords: Small RNA sequencing - high throughput

overall_design: One sample of above-ground tissues of greenhouse-grown S. moellendorffii was used to extract and sequence small RNAs.

first: David

middle: P

last: Bartel


title: Gene expression in undifferentiated human ES cells - Illumina

status: Public on Jun 27 2007

city: Cleveland

first: Paul

last: Tesar

state: Ohio

address: 10900 Euclid Ave.

department: Genetics & Genome Sciences

institute: Case Western Reserve University

summary:
The application of human embryonic stem (ES) cells in medicine
and biology has an inherent reliance on understanding the starting
cell population. Human ES cells differ from mouse ES cells and the
specific embryonic origin of both cell types is unclear. Previous
work suggested that mouse ES cells could only be obtained from
the embryo before implantation in the uterus1–5. Here we show
that cell lines can be derived from the epiblast, a tissue of the postimplantation
embryo that generates the embryo proper. These
cells, which we refer to as EpiSCs (post-implantation epiblastderived
ES cells), express transcription factors known to regulate
pluripotency, maintain their genomic integrity, and robustly differentiate
into the major somatic cell types as well as primordial
germ cells. The EpiSC lines are distinct from mouse ES cells in
their epigenetic state and the signals controlling their differentiation.
Furthermore, EpiSC and human ES cells share patterns of
gene expression and signalling responses that normally function
in the epiblast. These results show that epiblast cells can be maintained
as stable cell lines and interrogated to understand how
pluripotent cells generate distinct fates during early development.
*Note: EpiSCs were previously referred to as post-ES cells
Keywords: cell type comparison

overall_design: 3 biological replicates of human ES cells were compared to mouse ES cells and EpiScs.

first: Josh

middle: G

last: Chenoweth

last: McKay


title: SNP array analysis of chromosomal instability patterns discriminates rectal adenomas from carcinomas

city: Amsterdam

first: Esther

last: Lips

address: Plesmanlaan 121

department: Molecular Pathology

country: Netherlands

institute: NKI-AVL

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

summary:
Total mesorectal excision (TME) is the standard treatment for rectal cancer, while transanal  endoscopic microsurgery (TEM) is a recently introduced surgical approach for the treatment  of rectal adenomas. Incorrect preoperative staging before TEM is a problem. To identify  genetic changes that might correlate with tumour stage and could lead to optimized  treatment selection we performed a genome-wide chromosomal instability search in a  homogeneous, clinical cohort of rectal tumours. 78 rectal tumours during different clinical  stages were analysed with 10K single nucleotide polymorphism (SNP) arrays. Logistic  regression was performed to build a quantitative model of specific chromosomal aberrations.  Overall, most cases (95%) had one or more chromosomal aberrations. We observed a  clear correlation between the total number of aberrations and the different tumour stages.  Specifically, the chromosomal events: gain of 8q22–24, 13q and 20q, and loss of 17p and  18q12–22, were far more abundant in carcinoma than in adenoma. In adenoma fractions  from cases with a carcinoma (infiltrating at least in the submucosa), twice the amount of such  ‘malignant aberrations’ was observed, compared to pure adenomas. Furthermore, combined  aberrations such as gain of 13q and loss of 18q were only found in adenomatous fractions  of carcinomas and not in benign lesions. Based on these five genomic events associated with  carcinoma, a clear distinction between adenoma and carcinoma tissue could be made. These  data should be validated further in order that they may be used in preoperative staging of  rectal tumours.
Keywords: SNP array copy number and LOH study

overall_design: 78 tumor samples and 19 reference normal samples

middle: H

first: Eelco

last: de Graaf

first: Rob

last: Tollenaar

first: Ronald

last: van Eijk

first: Karoly

last: Szuhai

first: Tom

last: Karsten

first: Yasuhito

last: Nanya

first: Seishi

last: Ogawa

first: Cornelis

last: van de Velde

last: Eilers

last: van Wezel

first: Hans

last: Morreau


title: S. mansoni gene expression 21-27d post-infection in wt and RAG-/- mice

status: Public on Jun 28 2007

city: San Francisco

first: Steve

last: Miller

state: CA

address: 1600 Divisadero St, Room B-232, Box 1613

department: Laboratory Medicine

institute: University of California, San Francisco

summary:
RNA isolated from S. mansoni worms grown in C57BL/6 or RAG-/- mice at 21, 23, 25 and 27 days post-infection was hybridized to a S. mansoni custom microarray.  Expression was compared to a reference pool containing equal amounts of RNA from each condition and time point assayed.
Keywords: Time course with two growth conditions (wt and RAG-/- mice)

overall_design: Wild-type (C57BL/6) or RAG-/- mice were infected with S. mansoni cercariae.  Worms were isolated by liver perfusion at 21, 23, 25 and 27 days post-infection.  RNA was isolated and amplified.  A reference pool was made from equal amounts of amplified RNA from each of 8 samples.  Sample RNA was labeled with Cy5 and reference RNA was labeled with Cy3 dyes.  Labeled RNAs were hybridized to a S. mansoni microarray and analyzed for relative signal.

first: Emmitt

last: Jolly

first: Rebecca

last: Blank

first: James

last: McKerrow


title: Voluntary exercise, rat, colon mucosa

city: Munich

first: Katja

last: Buehlmeyer

state: Bavarian

address: Connollystreet 32

department: Institute of Public Health Research

institute: TU Munich

summary:
Colon cancer is the second most cancer type in Europe. Its development is highly influenced by life style factors such as nutrition and physical inactivity. Detailed biological mechanisms are so far unclear. The purpose of this study was to investigate the effects of chronic wheel running on gene expression in rat colon mucosa. Therefore six-week-old male Wistar rats (exercise (EX) group, n=20) completed a stress free voluntary running exercise period of 12 weeks. Sedentary rats served as a control (CO, n=9) group. In the colon mucosa, steady-state mRNA expression levels of approximately 10,000 genes were compared between both groups by micro-array analysis (MWG rat 10k array). 8,846 mRNAs were detected above background level. Chronic exercise led to a decreased expression of 47 genes at a threshold-factor of 2.0. 3 genes were found to be up-regulated in the EX group. The identified genes encode proteins involved in signal transduction (n=11), transport (n=8), immune system (n=7), cytoskeleton (n=6), protein targeting (n=6) and metabolism (n=5). Among the genes regulated by chronic exercise, the betaine-homocysteine methyltransferase 2 (BHMT2) seems to be of particular interest. Physical activity may protect against aberrant methylation by repressing the BHMT2 gene and thus contribute to a decreased risk of developing colon cancer. In summary, our experiment presents the first gene expression pattern in rat colon mucosa following chronic wheel running and therefore represents an important step in understanding the molecular mechanisms responsible for the preventive effect of physical activity on the development of colon cancer.
Keywords: voluntary running exercise, animal model

overall_design: Pooled and high-concentrated total RNA-samples of the EX and CO group were obtained. Reverse transcription, labelling and hybridization were performed according to the manufacturer’s manual (MWG Biotech AG; Ebersberg, Germany). For the fluorescence detection Cy3- and Cy5-labeled dCTP (Amersham Bioscience Europe, Freiburg, Germany) were used to produce fluorescence labelled first-strand cDNAs. Arrays were scanned (Affimetrix 428 Array Scanner, Santa Clara, CA) under dried conditions. Data were normalized and analyzed via ImaGene 4.2 software (BioDiscovery, Los Angeles, CA). Three independent hybridizations were carried out. Genes were considered as regulated if: a) the expression ratio was more than 2-fold in at least two hybridizations and  b) signal intensity values were at least 2-fold above overall background signal intensity in every group. Criteria for the inclusion into the EX pool sample were an adequate RNA-amount, RNA purity and the running distance per night of the respective rat: rats with extremely low or extremely high running distances were excluded to standardize the influence of physical activity on the colon mucosa. Ten rats from the EX group ran between 5747±2888 m and 8446±3434 m per night. Out of these specimen eight samples (animal number 1, 2, 3, 6, 8, 17, 19 and 20) had the right amount (more than 60 µg total RNA) and purity (checked by optical density (OD) 260 280-1 nm absorption) of total RNA. Eight samples were randomly taken for the CO pool.

first: Horst

last: Michna

first: Thorsten

last: Schulz

first: Birgit

last: Kindermann

first: Frank

last: Doering

first: Hannelore

last: Daniel


title: F plasmid induced changes in expression levels in E. coli

status: Public on May 29 2007

city: Koeln

first: Bettina

last: Harr

address: Weyertal 121Dept of Genetics

institute: Institut für Genetik

summary:
We measured expression level changes associated with the introduction of the F plasmid in 2 bacterial host strains (MG1655 and DH5alpha)
Keywords: parallel sample

first: Christian

last: Schloetterer


title: High throughput mapping of origins of replicationand histone H3 actylation in human cells

status: Public on May 26 2007

city: Chicago

first: Isabelle

last: Lucas

state: IL

address: 5841 S Maryland

institute: University of Chicago

type: Genome binding/occupancy profiling by genome tiling array

summary:
Mapping origins of replication has been challenging in higher eukaryotes. We have developed a rapid, genome-wide method to map origins in asynchronous human cells by combining the nascent strand abundance assay with a highly-tiled microarray platform, and we validated the technique by two independent assays.  We applied this method to analyze the enrichment of nascent DNA in three 50 kb regions containing known origins of replication in the MYC, LMNB2, and HBB genes, a 200 kb region containing the rare fragile site, FRAXA, and a 1075 kb region on chromosome 22, and detected most of the known origins, as well as 28 new origins.  Surprisingly, the 28 new origins were small in size, and predominantly located within genes.  Our study also revealed a strong correlation between origin replication timing and chromatin acetylation.
Keywords: origin mapping, nascent strand DNA, histone acetylaction mapping, ChIP-chip

overall_design:
Goal #1:  Map the origins of DNA replication in human cells.  This experiment is based on the fact that during S phase, short newly replicated DNA fragments (300bp-1kb) are generated only at the origins of replication. By combining the nascent strand assay with a tiled microarray platform, we have developed a rapid, non-PCR based, high-throughput approach to map active origins in asynchronous human cells.  Two independent biological samples were analyzed.  On the array design, four control regions, MYC (chr8), LMNB2 (chr19), HBB (chr11), and FRAXA (chrX), containing known origins were used as positive controls.

Goal #2: Map the hyperacetylated histone H3 (K9/K14) regions in human cells for comparison with the origin mapping data.  For this purpose, we combined the ChIP assay with the microarray technology.  Two independent biological samples were analyzed.  On the array design, two control regions, the hyperacetylated region, TUBA1 (chr2), and the hypoacetylated region, HET405 (chr9), were used as positive and negative controls, respectively, for our ChIP-on chip experiments.

Note: For statistical purposes, each probe was duplicated on the array, creating four data sets, FWD1, FWD2, REV1, and REV2.  To summarize our array design, 93% of the five regions on chromosomes 8, 11, 19, 22 and X were covered at the highest density with a 20 nt overlap between probes; for the remaining 7%, the gaps were 507±864 nt in length (range 31-9817 nt).

first: Aparna

last: Palakodeti

first: Yanwen

last: Jiang

first: David

last: Young

first: Anthony

last: Fernald

first: Michelle

middle: M

last: Le Beau


title: GAF zinc

status: Public on Jun 26 2007

city: Groningen

first: Anne

middle: de

last: Jong

address: Nijenborgh 7

department: Molecular Genetics

institute: university of Groningen

summary:
Streptococcus pneumoniae is a major cause of serious infections such as pneumonia and meningitis in both children and adults worldwide. Here, we describe the development of a high-throughput genome-wide technique, Genomic Array Footprinting (GAF), for the identification of genes essential for this bacterium at various stages during infection. GAF enables negative screens by means of a combination of transposon mutagenesis and microarray technology for the detection of transposon insertion sites. We tested several methods for the identification of transposon insertion sites and found that amplification of DNA adjacent to the insertion site by PCR resulted in non-reproducible results, even when combined with an adapter. However, restriction of genomic DNA followed directly by in vitro transcription circumvented these problems. Analysis of parallel reactions generated with this method on a large mariner transposon library, showed that it was highly reproducible and correctly identified essential genes. Comparison of a mariner library to one generated with the in vivo transposition plasmid pGh:ISS1, showed that both have an equal degree of saturation, but that 9% of the genome is preferentially mutated by either one. The usefulness of GAF was demonstrated in a screen for genes essential for survival of zinc stress. This identified a gene encoding a putative cation efflux transporter, and its deletion resulted in an inability to grow under high zinc conditions. In conclusion, we developed a fast, versatile, specific, and high-throughput method for the identification of conditionally essential genes in S. pneumoniae.
Keywords: GAF zinc

overall_design:
Selection for genes essential for survival of high zinc conditions.
Aliquots of a marinerT7 transposon library generated in S. pneumoniae R6 containing approximately 20,000 independent mutants were diluted 1 to 20 in GM17 broth or in GM17 broth containing 0.5 mM ZnSO4. Both cultures were grown for 10 generations as determined by OD600 after which the bacteria were spun down (9,000g, 4 min) and used for the extraction of genomic DNA.

first: Jetta

last: Bijlsma


title: Type I interferon signaling is required for activation of the inflammasome during Francisella infection

status: Public on May 23 2007

first: Sajeev

last: Batra

institute: UCSF

summary:
Francisella are pathogenic bacteria whose virulence is linked to their ability to replicate within the host cell cytosol. Entry into the macrophage cytosol activates a host protective multimolecular complex called the inflammasome to release the proinflammatory cytokines IL-1 and IL-18 and trigger caspase-1 dependent cell death. Here we show that cytosolic Francisella induce a type I interferon (IFN) response that is essential for caspase-1 activation, inflammasome mediated cell death, and release of IL-1 and IL-18.  Extensive type I IFN dependent cell death resulting in macrophage depletion occurs in vivo during Francisella infection. Type I IFN is also necessary for inflammasome activation in response to cytosolic Listeria but not vacuole localized Salmonella or extracellular ATP. These results show the specific connection between type I IFN signaling and inflammasome activation, two sequential events triggered by recognition of cytosolic bacteria. To our knowledge, this is the first example of positive regulation of inflammasome activation. This connection underscores the importance of cytosolic recognition of pathogens and highlights how multiple innate immunity pathways interact before commitment to critical host responses.
Keywords: murine macrophage response to Francisella tularensis subspecies novicida infection

overall_design: We analyzed a series of 18 MEEBO arrays on which were hybed RNA randomly amplified from bone marrow derived macrophages infected or not with WT Francisella tularensis subspecies novicida or a the mglA mutant strain GB2.

first: Thomas

last: Henry

first: Anna

last: Brotcke

middle: S

last: Weiss

first: Lucinda

last: Thompson

first: Denise

last: Monack


title: Gene expression in response to stresses in the Chytridiomycete Blastocladiella emersonii.

status: Public on Jun 22 2007

city: São Paulo

first: Raphaela

middle: Castro

last: Georg

state: São Paulo

address: Av. Prof Lineu Prestes, 748

department: Bioquímica

country: Brazil

institute: Universidade de São Paulo

summary:
We explored transcriptional responses of the chytridiomycete Blastocladiella emersonii to some stress environmental conditions. Three distinct cDNA libraries constructed from mRNAs extracted from cells submitted to heat shock and different cadmium concentrations (50 and 100 mM) were sequenced and generated 6,350 high-quality EST sequences. These ESTs were assembled into 2,326 unigenes, being 51% of them not previously described in B. emersonii and consequently represent a worthy contribution of this work. We assigned to approximately 59% of the unigenes an orthologue in other organism, whereas 41% remained without a putative identification. Using the transcriptome data of B. emersonii we constructed a microarray slide containing 3,773 different cDNAs which comprise many biological processes of this fungus and analyzed its expression pattern in response to cadmium and heat shock. Through cDNA microarray assays we observed that 3,3% and 5,1% of the genes present in B. emersonii microarray slide were up-regulated in response to heat shock and cadmium, respectively. The main categories to which belong these genes were protein folding and proteolysis, proteins with antioxidant properties, cellular transport, carbohydrate and amino acid metabolism. Interestingly, in response to cadmium stress, B. emersonii induced six different genes codifying Glutathione S-transferases and six Metacaspases, as well as several proteins from sulfur amino acid metabolism.
Keywords: stress response; heavy metal stress response; heat shock response

overall_design: Direct comparison between cadmium treated cells and control cells(without cadmium) or heat shock treated cells (38oC) and control cells(27oC). Some hybridizations are dye-swaped. There are at least 3 biological replicates (independent harvest) and 2 technical replicates of each array (L - left and R - right).

middle: C

first: Suely

middle: L

last: Gomes


title: Comparison of gene expression in human embyronic  stem cells grown in MEF-CM or defined media conditions

status: Public on Jun 24 2007

city: Athens

first: Tom

last: Schulz

state: GA

address: 111 Riverbend Rd

institute: Novocell

summary:
A fully defined media for human embryonic stem cells (hESCs) was designed after examining the phosphorylation status of 42 receptor tyrosine kinases (RTKs) on hESCs exposed to mouse embryonic fibroblast conditioned medium (CM).  Activation of the insulin and insulin-like growth factor-1 receptors (IR, IGF1R) as well as EGFR family members including ERBB2 and ERBB3 led to the design of a simple defined media which supported robust long-term growth of multiple hESC lines and allowed massive expansion of undifferentiated cells. Illumina bead arrays were used to demonstrate the maintenance of gene expression profiles characteristic of pluripotent cells in cultures grown in CM or defined media.
Keywords: Comparison of cells in different culture conditions

overall_design:
Human embyronic stem cells were maintained in standard MEF-CM conditions, or in a defined medium: DC-HAIF. The samples analyzed were:

BG02 cells grown in MEF-CM for 64 passages,
BG02 cells grown in defined media for 10 passages,
BG02 cells grown in defined media for 32 passages,
BG03 cells grown in MEF-CM for 35 passages.

Single analyses of the samples were performed using the Illumina bead array system. Comparable labeling, hybridization, detection and data analysis was used with previous analyses of these cell lines (Liu et al 2006 BMC Dev Biol 6:20).

first: Linlin

last: Wang

first: Eric

last: Sherrer

first: Derek

last: Dauphin

first: Soojung

last: Shin

first: Angelique

last: Nelson

first: Carol

middle: B

last: Ware

first: Mei

last: Zhan

first: Chao-Zhong

last: Song

first: Sandii

middle: N

last: Brimble

first: Amanda

last: McLean

first: Elizabeth

middle: W

last: Uhl

first: Jonathan

middle: D

last: Chesnut

first: Mahendra

last: Rao

last: Blau

first: Allan

last: Robins


title: Quantitative Genomics study of sex dimorphism in D. Melanogaster

city: Gainesville

first: Lauren

middle: M.

last: McIntyre

state: FL

department: Dept. of Molecular Genetics and Microbiology

institute: University of Florida

summary:
A diallel including reciprocals but not selfs experiment.
Keywords: Agilent microarray, sexual dimorphism, genetical genomics, genetic variation

overall_design: Nine randomly selected D. melanogaster lines were used as parents for this diallel. Male and female intact adult progeny were collected.  Crosses were repeated in four independent sub blocks such that every cross appeared with every other cross at least once.  Two sub blocks were pooled for each replicate.  Each array was a pair of male and females from the same cross with the dye flipped between replicates

first: Sergey

middle: V

last: Nuzhdin

first: Marta

last: Wayne

first: Lawrence

last: Harshman

first: Artyom

last: Kopp

first: Marina

last: Telonis-Scott

first: Lisa

last: Bono


title: A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing

status: Public on Jun 17 2007

first: Frank

last: Huebel

summary:
MicroRNAs (miRNAs) are small non-coding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents, enriched in euronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide novel computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the difference in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.
Keywords: miRNA microarray; expression profiling

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design:
To validate the results of the miRNA profiling achieved by sequence  analysis of small RNA clone libraries we performed additional dual color microarray hybridizations using a synthetic miRNA pool as a common reference. 5 μg total RNA of the samples hsa_Hepatoma-PLC (array
# 517063), hsa_Hepatocell-carcinoma-Huh7-mock (array #517064), hsa_Gliobl-SNB19 (array #517065), hsa_Hippocamp-adult (array #517066), and hsa_T-cell-CD4-A301 (array #517067), were hybridized on miRNA arrays carrying probes for all miRNA described in the present study. Sample hsa_T-cell-CD4-A301 was also analysed using 10 μg of total RNA to check for reproducibility and potential bias due to varying amounts of input RNA (array #517068).

first: Pablo

last: Landgraf

first: Alain

last: Sewer

first: Ute

last: Bissels

first: Andreas

last: Bosio

first: Mihaela

last: Zavolan

first: Thomas

last: Tuschl


title: Cis-transcriptional variation in maize inbred lines B73 and Mo17 leads to additive expression - Immature ear data

status: Public on Jun 21 2007

city: Saint Paul

first: Nathan

last: Springer

state: MN

address: 1445 Gortner Ave

department: Plant Biology

institute: University of Minnesota

summary:
Microarray analysis of gene expression patterns in immature ear, seedling, and embryo tissues from the maize inbred lines B73 and Mo17 identified numerous genes with variable expression. Some genes had detectable expression in only one of the two inbreds; most of these genes were detected in the genomic DNA of both inbreds, indicating that the expression differences are likely caused by differential regulation rather than by differences in gene content. Gene expression was also monitored in the reciprocal F1 hybrids B73xMo17 and Mo17xB73. The reciprocal F1 hybrid lines did not display parental effects on gene expression levels. Approximately 80% of the differentially expressed genes displayed additive expression patterns in the hybrids relative to the inbred parents. The approximately 20% of genes that display nonadditive expression patterns tend to be expressed at levels within the parental range, with minimal evidence for novel expression levels greater than the high parent or less than the low parent.  Analysis of allele-specific expression patterns in the hybrid suggested that intraspecific variation in gene expression levels is largely attributable to cis-regulatory variation in maize. Collectively, our data suggest that allelic cis-regulatory variation between B73 and Mo17 dictates maintenance of inbred allelic expression levels in the F1 hybrid, resulting in additive expression patterns.
Keywords: genotype comparison

overall_design: Affymetrix expression profiling was used to study gene expression in immature ear tissue from maize. Three biological replicates were performed for four different genotypes; B73, Mo17, B73xMo17 and Mo17xB73.

first: Robert

last: Stupar


title: Mycoplasma gallisepticum Rlow attached to MRC-5 cells microarray

status: Public on May 20 2007

city: Storrs

first: Steven

last: Geary

state: CT

address: 61 North Eagleville Rd

institute: University of Connecticut

summary:
The goal of this study was to identify genes important to the interaction of Mycoplasma gallisepticum with host cells in an in vitro system.  An interaction time of one hour was chosen since it is less than a generation time and thus all changes can be attributed to transcriptional changes.  A total of 60 transcripts were determined to be significantly up- or down-regulated under these conditions, including several hypothetical genes.  Also several metabolism-related genes and ATP synthase genes were significantly down-regulated.
Keywords: transcriptional response to host cells

overall_design:
Five separate biological experiments and extractions were performed yielding five total replicate hybridizations.  The microarray slide contains 760 oligonucleotide spots in duplicate including several Arabidopsis controls and blank spots.  Individual spots were analyzed using BAGEL and a significantly different data set determined by including spots that were significantly (p<.05) up- or down-regulated by greater than 1.5-fold reproducibly (both duplicate spots).

"Broth-grown and attached M. gallisepticum BAGEL analysis" output file provided as a Series data table.

first: Katharine

middle: R

last: Cecchini

first: Timothy

last: Gorton

middle: J


title: Sugarcane transcriptome - Response to herbivory by Diatraea saccharalis

first: Milton

middle: Yutaka

last: Nishiyama Jr

address: Av. Professor Lineu Prestes, 748

department: Biochemistry

institute: Universidade de São Paulo - USP

summary:
Sugarcane stalk borer larvae were grown on artificial diet and maintained at 25°C and 60±10% relative humidity with a 14 h/10 h light/dark cycle. Second instar larvae were maintained under fasting conditions for 18 h and transferred to two-month old plants (genotype SP80-3280, CTC, Brazil). Leaves were collected after 30 min and 24 h of exposure to herbivory for the control and experimental groups. Two plantlets were used for each time point. Extraction of total RNA was performed separately on each sample pool.
Keywords: time course of stress response

overall_design: RNA from experimental sample and from its respective control sample (untreated plants) was used for hybridization to dual channel arrays. Two biological replicates were used for each experimental point. The quantification of each hybridization was submitted in two files, one for each slide side (technical replicates).

first: Carla

last: Barsalobres

first: Ane

last: Medeiros

first: Marcio

last: Silva Filho

first: Flávia

last: Rocha

last: Papini-Terzi

middle: Y

last: Nishiyama-Jr

first: Ricardo

middle: Z

last: Vêncio

first: Renato

last: Vicentini

first: Gláucia

last: Souza


title: Global transcriptional response of Escherichia coli O157:H7 to growth transitions in glucose minimal medium

status: Public on Jun 15 2007

city: East Lansing

last: Whittam

state: MI

address: 165 Food Safety & Tox

department: Microbiology

institute: Michigan State University

summary:
Background: Global patterns of gene expression of Escherichia coli K-12 during growth transitions have been deeply investigated, however, comparable studies of E. coli O157:H7 have not been explored, particularly with respect to factors regulating virulence genes and genomic islands specific to this pathogen. To examine the impact of growth phase on the dynamics of the transcriptome, O157:H7 Sakai strain was cultured in MOPS minimal media (0.1% glucose), RNA harvested at 10 time points from early exponential to full stationary phase, and relative gene expression was measured by co-hybridization on high-density DNA microarrays.
Results: Analysis of variance (R/MAANOVA, Fs test) identified 442 (36%) of 1239 O157-specific ORFs and 2110 (59%) of 3647 backbone ORFs that changed in expression significantly over time.  QT cluster analysis placed 2468 of the 2552 significant ORFs into 12 groups; each group representing a distinct expression pattern.  ORFs from the largest cluster (n=1078) decreased in expression from late exponential to early stationary phase: most of these ORFs are involved in functions associated with steady state growth.  Also represented in this cluster are ORFs of the TAI island, encoding tellurite resistance and urease activity, which decreased ~4-fold and most ORFs of the LEE island, which decreased ~2-fold by early stationary phase.  ORFs encoding proteins secreted via the LEE encoded type III secretion system, such as tccP and espJ, also decreased in expression from exponential to stationary phase. Three of the clusters (n=154) comprised genes that are transiently upregulated at the transition into stationary phase and included genes involved in nutrient scavenging.  Upregulated genes with an increase in mRNA levels from late exponential to early stationary phase belonged to one cluster (n=923) which includes genes involved in stress responses (e.g. gadAB, osmBC, and dps).  These transcript levels remained relatively high for >3h in stationary phase.  The Shiga toxin genes (stx1AB and stx2B) were significantly induced after transition into stationary phase.
Conclusions:  Expression of 307 O157-specific ORFs was modulated in a growth dependent manner.  These results provide a baseline transcriptional profile that can be compared to patterns of gene expression of this important foodborne pathogen under adverse environmental conditions.
Keywords: time course

overall_design: The results are based on O157:H7 Sakai cultures collected from 10 time points of growth in minimal media with 4 replicates at each point for a total of 40 samples. The samples collected from the mid-exponential phase 3-hour time point were used as a common reference for hybridization and analysis for all subsequent time points in a blocked reference design.  This design allowed for indirect comparison between all time points via the 3-hour samples.  We incorporated the dye-swaps among the four biological replicates.

first: Teresa

middle: M

last: Bergholz

first: Lukas

last: Wick

first: Weihong

last: Qi


title: S. coelicolor YSK4425 R5- Medium Time-course Study (Culture #1)

status: Public on Jun 14 2007

city: Minneapolis

first: Karthik

middle: P

last: Jayapal

address: 421 Washington Ave SE

department: Chemical Engineering and Materials Science

summary:
Antibiotic biosynthesis in Streptomyces species is controlled by a complex genetic and biochemical network of global and pathway specific regulators.  Details of their precise interactions in mediating temporal and spatial expression of secondary metabolite genes remain poorly defined.  In this study, we employed whole-genome microarray methods to investigate the temporal transcriptome profiles of S. coelicolor A3(2) ∆afsS::apr mutant strain and compared with the M145 Wild-type strain (series submitted separately).
Keywords: Time course

overall_design: Time-course study involved analysis of 8 samples (16h, 20h, 22h, 24h, 26h,  31h, 37h and 40h). All arrays used cDNA labeled with Alexa647 and gDNA labeled with Cy3.

first: Wei

last: Lian

first: Wei-Shou

last: Hu


title: The NsrR regulon of Escherichia coli K-12

status: Public on May 18 2007

city: Birmingham

first: Chrystala

last: Constantinidou

department: School of Biosciences

country: United Kingdom

institute: University of Birmingham

summary:
Successful pathogens must be able to protect themselves against reactive nitrogen species generated either as part of host defence mechanisms, or as products of their own metabolism. The regulatory protein, NsrR (a member of the Rrf2 family of transcription factors), plays key roles in this stress response. Microarray analysis was carried out to reveal the regulon of NsrR.
Keywords: Response to repressor titration and different growth conditions

overall_design: The nsrR gene of E. coli MG1655 is located immediately upstream of, and is co-transcribed with, the rnr gene encoding RNase R. Expression of this transcription unit is cold-shock inducible, which may well involve secondary structures in the mRNA in the vicinity of the nsrR coding region. Deletion of the nsrR gene might therefore perturb rnr expression by a simple consequence of polarity, or by disruption of mRNA structural elements involved in post-transcriptional regulation. The consequence would likely be secondary effects on global RNA stability, hence comparison of the transcriptomes of an nsrR mutant and its parent would be difficult to interpret. To avoid such potential artifacts, we exploited the observation that regulation by NsrR is extremely sensitive to repressor titration by NsrR-binding sites provided on a multicopy plasmid. Transformation of an nsrR+ strain with plasmid pGIT1, which carries the ytfE promoter in multicopy, titrates out NsrR to phenocopy an NsrR- mutation. As a control, pGIT8 carries the same promoter fragment, but a 1 bp deletion mutation in the NsrR binding site eliminates repressor titration in vivo. Strain MG1655 transformed with either pGIT1 or pGIT8 was grown anaerobically under the carefully controlled conditions described previously, so that growth rates were almost identical, and RNA was isolated from early exponential phase cultures grown in the presence or absence of nitrite. For each growth condition, four independent biological replicates were completed, and to validate comparisons between different plasmids and growth conditions, a pool of RNA from the control strain (MG1655 transformed with pGIT8 and grown in the absence of nitrite) was included in every microarray to provide a common reference.

first: Nina

last: Filenko

first: Stephen

last: Spiro

first: Douglas

middle: F

last: Browning

first: Derrick

last: Squire

first: Tim

last: Overton

first: Jeff

middle: A

last: Cole


title: High-throughput sequencing of small RNAs from Drosophila

status: Public on Jun 10 2007

first: James

middle: Graham

last: Ruby

address: 1700 4th Street QB3 s404

summary:
High-throughput sequencing of Drosophila melanogaster small RNAs.
total RNA, ~18-26nt RNAs isolated using PAGE, ligation to adapters requires 5' monophosphate and 3' OH
Keywords: high-throughput 454 sequencing

overall_design: Small RNAs were sequenced from D. melanogaster at various developmental stages.


title: Specific inhibition of p300-HAT alters Global Gene Expression and Repress HIV replication

city: Bangalore

first: madavan

last: vasudevan

state: Karnataka

address: Kasturi Nagar

country: India

institute: Theomics International Private Limited

summary:
Reversible acetylation of histone and nonhistone proteins plays pivotal role in cellular homeostasis. Dysfunction of histone acetyltransferases (HATs) leads to several diseases including cancer, neurodegenaration, asthma, diabetes, AIDS and cardiac hypertrophy. We describe the synthesis and characterization of a set of p300-HAT specific small molecule inhibitors from a natural nonspecific HAT inhibitor, garcinol, which is highly toxic to cells. We show that the specific inhibitor selectively represses the p300-mediated acetylation of p53 in vivo. Furthermore, inhibition of p300-HAT down regulates several genes but significantly a few important genes are also upregulated. Remarkably, these inhibitors were found to be nontoxic to T cells, inhibit histone acetylation of HIV infected cells and consequently inhibit the multiplication of HIV.
Keywords: Response to Inhibition of p300-HAT

overall_design: The total RNA was isolated from control and treated cells using TRIZOL (Invitrogen) method. The Micromax direct labeling kit, MPS502 (PerkinElmer) was used to synthesize the labeled cDNA from 70 ug of total RNA and further process the hybridized cDNA on the array. All the steps were carried out according to manufacturer’s instructions (www.perkinelmer.com/lifesciences). The array slides were scanned immediately by PerkinElmer Scan array Gx Microarray scanner. The Scan array software (PerkinElmer) was used for grid wise normalization of array images. Four arrays were used with at least two biological treatments of cells and dye swap experiments were included in the final analysis. The data was analysed by GeneSpring GX and Biointerpreter software from Genotypic Technology, Bangalore. The differential expression was considered if the Log 2 mean of at least -1 for the down regulated genes and +1 for the upregulated genes. We considered only the genes that were reproducible from all four replicates.

first: Tapas

last: Kundu

first: Parag

last: Sadhale

first: Ashok

last: Reddy


title: Sugarcane transcriptome - Phosphate starvation

first: Milton

last: Nishiyama Jr

summary:
Rooted sugarcane plantlets, originated from in vitro meristem culture (genotype SP80-3280, CTC, Brazil), were greenhouse acclimatized by initial cultivation on 1/20th strength Hoagland and Arnon (1950) nutrient solution. Nutrient solutions were aired from an oil-less compressor and replaced every 7 days, increasing nutrient concentration to ¼ strength in 3 weeks. Plants were then individually transferred to 2.8 L pots filled with fresh ¼ strength nutrient solution.  After one week, half of the plants were transferred to fresh solution containing 250 µM Pi, while the other half was transferred to nutrient solution deprived of phosphate (Pi), with H2PO4 being replaced by H2SO4 (Muchhal et al., 1996). Roots from six plants from each treatment (0 and 250 µM Pi) were harvested 6, 12, 24 and 48 h after exposure to phosphate starvation and immediately frozen in liquid nitrogen. For each time point and each treatment, root samples were aggregated in three pools of two samples each. Extraction of total RNA was performed separately on each sample pool.
Keywords: time course of stress response

first: Rodrigo

last: Drummond

first: Marcelo

last: Menossi

first: Raul

middle: S

last: Almeida

first: Antonio

last: Figueira

first: Flávia

last: Rocha

last: Papini-Terzi

last: Nishiyama-Jr

first: Ricardo

last: Vêncio

first: Renato

last: Vicentini

first: Gláucia

last: Souza


title: Ratios for Htz1D cells (Agilent array)

status: Public on May 16 2007

city: Jerusalem

first: Tommy

last: Kaplan

address: Givat Ram Campus

department: School of Computer Science and Engineering

institute: Hebrew University

summary:
Chromatin plays roles in processes governed by different time scales. To assay the dynamic behaviour of chromatin in living cells, we used genomic tiling arrays to measure histone H3 turnover in G1-arrested S. cerevisiae at single-nucleosome resolution over 4% of the genome, and over the entire genome at lower (~265 bp) resolution. We find that nucleosomes at promoters are replaced more rapidly than at coding regions, and that replacement rates over coding regions correlate with polymerase density. In addition, rapid histone turnover is found at known chromatin boundary elements. These results suggest that rapid histone turnover serves to functionally separate chromatin domains and prevent spread of histone states.
Keywords: Chip-chip, htz1, histone turnover

overall_design: Ratios between Gal-induced H3-Flag and constitutive H3-Myc at Htz1-deleted cells. Hybridization to 44K Agilent arrays covering the yeast genome at ~265bp-resolution.

first: Michael

last: Dion

first: Minkyu

last: Kim

first: Stephen

last: Buratowski

first: Nir

last: Friedman

first: Oliver

last: Rando


title: Adapted tolerance to Benzalkonium chloride in E. coli K12

city: Aas

first: Solveig

last: Langsrud

address: Osloveien 1

country: Norway

institute: Matforsk, Norwegian Food Research Institute

summary:
Benzalkonium chloride (BC) is a commonly used disinfectant and preservative. This study describes changes in expression level on the transcriptomic and proteomic level for Escherichia coli K12 gradually adapted to a tolerance level towards BC of 7-8 times the initial MIC. Results from DNA arrays and two-dimensional (2-DE) gel electrophoresis for global gene and protein expression studies were confirmed by real time quantitative PCR. Peptide mass fingerprinting by MALDI-TOF MS was used to identify differentially expressed proteins. Changes in expression level in adapted cells were shown for porins, drug transporters, glycolytic enzymes, ribosomal subunits and several genes and proteins involved in protection against oxidative stress and antibiotics. Adapted strains showed increased tolerance to several antibiotics.  In conclusion, E. coli K12 adapted to higher tolerance to BC, acquired several general resistance mechanisms including responses normally related to the multiple antibiotic resistance (Mar) regulon and protection against oxidative stress. The results revealed that BC treatment might result in superoxide stress in E. coli.
Keywords: Study of resistance mechanism

overall_design: E. coli K12 was adapted to grow in the presence of benzalkonium chloride in three independen experiments resulting in three adapted strains (BCadapted1, BCadapted2 and BCadapted3). Control strains were cultivated in TSB before harvesting and adapted strains were cultivated either in TSB with or without 50 ppm benzalkonium chloride before harvesting. Cultures for transcriptional analysis were harvested three times at different days resulting in three replicate experiments. Samples from the fist day is termed replicate1, day 2 is termed replicate2 and day three replicate3. For the controls, samples from two cultures were sampled for replicate one and two.

first: Erlend

last: Bore

first: Michel

last: Hebraud

first: Ingrid

last: Chafsey

first: Christophe

last: Chambon

first: Camilla

last: Skjæret

first: Birgitte

last: Moen

first: Trond

last: Møretrø

first: Ayvind

first: Knut

last: Rudi


title: Regulation of mRNA transcript expression by hypoxia in human peripheral blood lymphocytes

city: Oxford

first: Jerome

middle: Tremblay

last: Brooks

state: Oxon

address: Parks Road

department: Department of Physiology Anatomy and Genetics

institute: University of Oxford

summary:
The aim of the study is to evaluate oxygen regulated gene expression in human peripheral blood lymphocytes using microarray analysis.
The results of this study are expected to provide useful marker transcripts whose expression is highly affected by hypoxia for further hypoxic gene expression studies using peripheral blood lymphocytes.
Keywords: peripheral blood lymphocytes, hypoxia-regulated transcripts

overall_design: The lymphocyte isolation and incubation at 1% or 20% Oxygen tensions are explained in detail in the methods section of Smith TG, Brooks JT et al., (2006) Mutation of von Hippel-Lindau Tumour Suppressor and Human Cardiopulmonary Physiology PLoS Medicine 3, e290. Briefly, following venesection, 40 ml of blood was drawn from a human volunteer. Peripheral blood lymphocytes were isolated from the blood sample by centrifugation over density gradient medium (Ficoll-Paque plus, Amersham Biosciences UK) followed by incubation at 37 degrees Celsius to allow for monocyte adhesion. The isolated lymphocytes were then divided into two aliquots which incubated at either 20% or 1% ambient oxygen tensions at 37 degrees Celsius for 20 hours. Lymphocytes from each aliquot were then harvested by centrifugation and RNA was extracted from the lymphocytes using a column based extraction method (RNAqueous-4PCR, Ambion US). The entire procedure was repeated three times on the same volunteer to give 6 RNA samples from the three paired sets of hypoxic/normoxic incubations. Following in vitro transcription and incorporation of biotin-16 UTP into the amplified transcripts, the six samples were then analysed for transcript expression on a Sentrix Human 6-Expression BeadChip (Illumina California US) platform. This platform contains six individual microarrays allowing all six samples to be analysed simultaneously on a single platform. The results from the three paired sets of hypoxic/normoxic exposures were used to statistically determine any transcripts showing hypoxic regulation in human peripheral blood lymphocytes.

first: Gareth

last: Elvidge

first: Lousie

last: Glenny


title: Ratios for G1 arrested cells (Printed array)

first: Tommy

last: Kaplan

summary:
Chromatin plays roles in processes governed by different time scales. To assay the dynamic behaviour of chromatin in living cells, we used genomic tiling arrays to measure histone H3 turnover in G1-arrested S. cerevisiae at single-nucleosome resolution over 4% of the genome, and over the entire genome at lower (~265 bp) resolution. We find that nucleosomes at promoters are replaced more rapidly than at coding regions, and that replacement rates over coding regions correlate with polymerase density. In addition, rapid histone turnover is found at known chromatin boundary elements. These results suggest that rapid histone turnover serves to functionally separate chromatin domains and prevent spread of histone states.
Keywords: Chip-chip, time course, histone turnover

overall_design: Ratios between Gal-induced H3-Flag and constitutive H3-Myc at 5 time points for G1-arrested yeast. Hybridization to high-resolution printed arrays of ~4% of the yeast genome.

first: Michael

last: Dion

first: Minkyu


title: Gene Expression of a Hybrid Rice Strain(LYP9) and Its Parental Cultivars

status: Public on Jun 08 2007

city: Beijing

first: shuhui

last: song

address: Airport Industrial Zone B-10

department: Genomics & Bioinformatics

country: China

institute: Beijing Institute of Genomics, Chinese Academy of Sciences, China

summary:
Based our serial analysis of gene expression (SAGE) data from an elite Chinese super-hybrid rice (LYP9) and its parental cultivars (93-11 and PA64s) in three major tissue types at three different developmental stages, we obtained a much more comprehensive view of genes that related to rice heterosis and analyzed the potential effects of gene-expression difference on the heterosis of rice.These heterotic expression genes among different genotypes provided new avenues for exploring the molecular mechanisms underlying heterosis, including variable gene expression patterns.
Keywords: Heterosis study by SAGE

overall_design: We constructed nine SAGE libraries parallelly, including root at the first tillering stage, leaf at the milky stage of rice grain maturation, and panicle at the pollen-maturing stage of hybrid rice (LYP9) and its paternal lines (9311, PA64s).

first: Jingyue

last: Bao

first: Shuhui

last: Song

first: Jun

last: Yu


title: A gene expression signature of confinement in peripheral blood of red wolves (Canis rufus)

status: Public on Jun 13 2007

city: Raleigh

first: Erin

last: Kennerly

state: NC

institute: NCSU

summary:
Background: The idea of using whole genome microarrays to detect peripheral blood biomarkers for physiological status a fairly new and unexplored method. Identifying biomarkers that can be linked to stress and immune response would be of great importance not only in animal management practices but in humans as well. The main objective of this research was to explore this concept using the North American Red Wolf (Canis rufus) which are exposed to a wide variety of environments from free ranging to confinement.
Results: Transcription profiling of peripheral blood samples from 13 red wolf individuals in the Alligator River region of North Carolina revealed a strong signal of differentiation between confined and free-range animals.  482 out of 2,980 transcripts detected on the human Illumina Ref8 oligonucleotide bead arrays were found to differentiate these groups at a false discovery rate of 5 percent.  Over-representation of genes in focal adhesion, insulin signaling, proteasomal, and tryptophan metabolism pathways suggests the activation of proinflammatory and stress responses in confined animals.
Conclusions:  Integration of immunological and physiological signals may leave a signature of lifestyle in the patterns of gene expression in the blood and suggest the possible development of biomarkers for disease and normal conditions.
Keywords: Stress Response/Disease State

sample_organism:
Canis latrans
Canis rufus

overall_design: 14 whole blood samples were collect from captive and free ranging red wolves. Information pertaining to sex and current holding status was obtained for each individual. In total we had 6 captive wolves (2 females and 4 males), 7 free range wolves (2 females and 5 males) and 1 free range female coyote. Total RNA was extracted from whole blood samples stored in RNAlater at -20C. Total RNA was amplified and biotin labeled using Ambion's Illumina RNA Amplification Kit (#I1755). Each labeled sample was hybridized on 4 independent arrays, giving a total of 56 arrays (4/sample).  Hybridization was done at 42C overnight. Arrays were washed and stained with strepavidin linked Cy3. Arrays were scanned on Illumina's BeadStation Scanner and raw intensity data was extracted using Illumina Beadstudio. Raw intensities were log base 2 transformed and then median center standardized in SAS using the proc stdize.

first: Anne

last: Acton

first: Simon

last: Gregory

last: Stoskopf

first: Greg

last: Gibson


title: A global gene expression analysis in the shoot apical meristem of maize (Zea mays L.)

status: Public on Jun 07 2007

city: Ames

first: Patrick

middle: S.

last: Schnable

state: IA

address: 2035B Roy J Carver Co-Lab

institute: Iowa State University

summary:
All above ground organs of higher plants are ultimately derived from specialized organogenic structures called shoot apical meristems (SAMs). It is comprised of pluripotent stem cells, which divide to regenerate themselves as well as to provide cells to form other organs such as leaves and stems. To study global gene expression in maize SAM and very young primordia (P0 and P1), RNA was extracted from both SAMs (SAMs per se plus P0 and P1) and above-ground portions of seedlings collected from 14-day old B73 seedlings. These RNA samples were labeled and hybridized with cDNA microarrays that have over 37,000 informative spots from maize. Statistical analyses showed that approximately 7.4% and 6.0% of the tested genes were significantly (p <0.0001) up- and down-regulated, respectively. Several control genes, whose expressions were confirmed in the maize SAM in the previous studies, were also up-regulated (p <0.01). The significantly up-regulated genes involved many novel transcription factor genes and regulatory genes as well as some kinds of enzymes, suggesting these genes play important roles in meristem maintenance and the early stage of leaf development in maize. Interestingly, several retrotransposon-related genes were greatly up-regulated in the SAM. This finding raised the possibility that retrotransposons are involved in regulatory mechanisms of maize SAM.
Keywords: cell type comparison design

overall_design: An experimental aim is to identify genes preferentially expressed in maize SAM by comparing the transcript abundant between SAMs and seedlings using cDNA microarrays that have over 37,000 informative spots from maize.


title: The Chromatin-Remodeling Factor FACT Contributes to Centromeric Heterochromatin Independently of RNAi

status: Public on May 10 2007

city: Stockholm

first: Karl

last: Ekwall

address: Alfred Nobels Alle 7

country: Sweden

institute: Karolinska Inst

summary:
Centromeres exert vital cellular functions in mitosis and meiosis. A specialized histone and other chromatin-bound factors nucleate a dynamic protein assembly that is required for the proper segregation of sister chromatids. In several organisms including the fission yeast S. pombe, a RNAi-related pathway further strengthens the epigenetic maintenance of centromere location, primarily through repressing transcription near centromeres. Little is known  how accessory factors such as histone chaperones and chromatin remodelling factors might be required to establish a functioning centromere. Here we show that the chaperone and remodelling complex FACT is required for transcriptional gene silencing at centromeres and for accurate chromosome segregation during mitosis. We show that Spt16 and Pob3 are two subunits of the S. pombe FACT complex. Surprisingly, yeast strains deleted for Pob3 are viable and show a marked loss of gene silencing at centromeric repeats and at the mating locus. Importantly, Pob3-null strains fail to undergo mitosis at high accuracy and phenocopy histone methylation and RNAi mutants. Processing of centromeric RNA transcripts into siRNAs is maintained in Pob3 mutants, but Swi6-association at the centromere is affected. Our studies provide the first experimental evidence for a role of the RNA polymerase II cofactor FACT in centromere function.
Keywords: Expression profiling of pob3 vs wt

overall_design: cDNA expression profiling was carried out according to (Xue et al., 2004). We used the S. pombe ORF spotted microarrays (Eurogentec, Belgium custom DNA microarray services).GeneSpring software was used for all data analysis. Expression profiling mutant vs wt data sets were normalized using Lowess (per spot per chip) intensity-dependent normalization, which corrects nonlinear rates for dye incorporation. Cut off values of 2,0 were used to generate ‘high’ and ‘low’ gene lists for ORF and IGR regions.

first: Erwan

last: Lejeune

first: Miriam

last: Bortfeld

first: Sharon

last: White

first: Robin

last: Allshire

first: Andreas

middle: G

last: Ladurner


title: Clostridium acetobutylicum ATCC 824 60-mer array protocol setup

city: Newark

first: Eleftherios

middle: Terry

last: Papoutsakis

state: DE

address: 15 Innovation Way

department: Chemical Engineering

institute: University of Delaware

summary:
Set up and modification of Agilent standard protocols for a new custom 60-mer array for C. acetobutylicum.
Keywords: Test of platform and protocols

overall_design: RNA samples were taken from both C. acetobutylicum ATCC and M5 strains at several points during the exponential phase of growth and the samples for each strain were mixed. Several amounts of labeled material and PMT settings were tested. For the ATCC824 strain the samples were taken at OD (600 nm) = {1.09, 1.8, 2.6, 2.0} whereas for the the M5 strain samples were taken at OD (600 nm) = {0.868, 1.36, 2.40, 3.20,  4.20}. The different amount of materials loaded into the slides were {5.0, 3.0, 1.0, 0.5} micrograms and the PMT values were {(60,10), (60,20), (80,10), (80,20), (80,40), (100,100)}.

first: Carlos

last: Paredes

first: Jacob

last: Borden

first: Ryan

last: Sillers

first: Shawn

last: Jones

first: Nathan

last: Cheng

middle: T


title: E. coli Transcriptome Dynamics during the Transition from Aerobic to Micro-aerobic Conditions

city: Sheffield

first: Jonathan

last: Partridge

address: Firth Court, Western Bank

department: Molecular Biology and Biotechnology

institute: The University of Sheffield

summary:
Escherichia coli is a metabolically versatile bacterium that is able to grow in the presence and absence of oxygen. Here, the process of adaptation was investigated by determining changes in transcript profiles when aerobic steady-state cultures were depleted of air.
Escherichia coli strain MG1655 was grown in a New Brunswick Scientific Bioflow 1000 fermentation vessels (1.8 l capacity) with culture agitation speed constant at 300 rpm and the temperature maintained at 37 °C. Oxygen levels were monitored using galvanic oxygen electrodes while the pH was maintained at 7.2 ±0.2 by automatic titration with sterile KOH. Evans defined medium was used as the growth medium with glucose (15 mM) as the carbon source with the dilution rate being 0.2 h-1. Aerobic cultures were maintained by sparging the chemostat with air (0.4 l min-1). The switch to micro-aerobic conditions was achieved by switching off the air sparging the culture. After a period of 5, 10, 15 and 60 min exposure to air, cells were harvested directly into RNA Protect (Qiagen) to stabilize RNA before total RNA purification using Qiagen’s Rneasy Midi kit as recommended by manufacturer’s instructions.
Keywords: time-course, oxygen-depletion

overall_design:
Analysis used aerobic steady state RNA as control samples for comparison to the experimental samples taken at 5, 10, 15 and 60 min. Indirect comparisons were made across multiple arrays with raw data pulled from different channels for data analysis and comparison to the control data. Data analysis was carried out using Imagene, version 5.1 and Genesight version 4 (Biodiscovery Inc). The mean values from each channel were log2 transformed and normalized using the LOWESS algorithm to remove intensity dependent effects within the calculated values. Normalised values were used to calculate the Cy3/Cy5 fluorescence ratios from experimental and biological repeats (seperate chemostat runs)with all replicates combined. Data was compiled from two aerobic and two anaerobic cultures each with a dye-swap replicate, thus providing two biological repeats within four technical repeats.
Presented is the LOG2 normalised data comparing the control (aerobic) with the identified experimental (5, 10, 15, 60 min air depletion) with supplementary material provided in the form of Raw array files.

middle: D

first: Jeffrey

last: Green


title: Gene expression profiling of Streptococcus mutans in biofilm and planktonic conditions

status: Public on May 07 2007

first: Moshe

last: Shemesh

address: Hebrew University-Hadassah POB 12272

department: Institute of Dental Sciences

institute: Hebrew University-Hadassah

summary:
In vitro comparative transcriptome analysis was carried to determine genes that are differentially expressed in S. mutans biofilm compared with free-living cells. DNA-microarray analyses indicated that about 12 % of genes showed significant differential expression: 139 were activated and 104 were repressed in biofilm vs. planktonic environment.
Keywords: cell type comparison

overall_design: S. mutans UA159 was grown to stationary phase planktonic bacteria  and the biofilm generated on polystyrene multidishes. the total RNA was extracted and, after Reverse transcription and probe labeling, Hybridized to DNA-microarray.

first: Avshalom

last: Tam

first: Doron

last: Steinberg


title: Mutation accumulation and the yeast transcriptome

status: Public on Jun 01 2007

city: Cambridge

first: Christian

last: Landry

state: MA

address: 16 Divinity avenue

department: OEB

institute: Harvard University

summary:
Spontaneous mutations were accumulated in haploid strains of yeast. Expression profiling was performed on four MA lines and the ancestor in a complete loop design.
Keywords: Gene expression profiling

overall_design: Every line is compared to every other line, for a total of four replicates per line.

middle: R

first: Bernardo

last: Lemos


title: Expression changes in mouse embryo fibroblasts associated with the loss of menin (menin-null)

city: Bethesda

first: Christopher

last: Pan

state: MD

address: Rm 5228, Bldg 50, South Dr.

department: NHGRI

institute: NIH

summary:
Multiple endocrine neoplasia, type 1 (MEN1) is an autosomal dominant familial cancer syndrome characterized primarily by endocrine tumors of the parathyroids, anterior pituitary and enteropancreatic endocrine tissues. Affected individuals carry a germline loss-of-function mutation of the MEN1 gene, and tumors arise after loss of the second allele. Homozygous loss of Men1 in the germline of mice results in early embryonic lethality, with defective development of neural tube, heart, liver, and craniofacial structures. We generated immortalized Wild type (Wt) and menin-null mouse embryo fibroblast (MEF) cell lines and evaluated their characteristics, including global expression patterns. The Wt and menin-null cell lines were aneuploid, and the nulls did not display tumorigenic characteristics in soft-agar assay. Expression arrays in menin-null MEFs revealed altered expression of several extracellular matrix proteins that are critical in organogenesis. Specifically, transcripts for fibulin-2 (Fbln2), periostin (Postn) and versican (Cspg2, chondroitin sulfate proteoglycan), genes critical for the developing heart and known to be induced by TGF-beta, were decreased in their expression in menin-null MEFs. Fbln2 expression was the most affected, and the reduction in menin-null MEFs for Fbln2, Postn and Cspg2 was 16.18, 5.37 and 2.15-fold respectively. Menin-null MEFs also showed poor response to TGF-beta-induced Smad3-mediated transcription in a reporter assay, supporting a role for menin in this pathway. The expression changes associated with the loss of the tumor suppressor menin provide insights into the defective organogenesis observed during early embryonic development in Men1-null mouse embryos.
Keywords: Wild type and menin-null mouse embryo fibroblasts

overall_design: Expression arrays were generated by printing PCR amplified inserts for clones from from mouse embryo cDNA libraries. Fibroblst cell lines were established from wild type (WT) and menin-null (Null) mouse embryos. RNA isolated from 4 each of WT and Null mouse embryo fibroblast cell lines were labeled with Cy3 and Cy5 fluorochromes and each WT/Null pairs were hybridized to the arrays, and duplicate hybridizations were also carried out with the same WT/Null pairs by reversing the dye used to label the RNA- a total of 32 hybridizations.  Self vs Self RNA hybridizations were also one for RNA samples.

first: Settara

middle: C

last: Chandrasekharappa

first: Youngmi

last: Ji

first: Yidong

last: Chen

first: Abdel

last: Elkahloun


title: H9 hES cells vs. universal RNAs

city: Münster

first: Boris

last: Greber

address: Röntgenstraße 20

department: Cell and Developmental Biology

institute: Max Planck Institute for Molecular Biomedicine

summary:
Comparison of human embryonic stem cell transcriptome with universal RNA pool (10 human cell lines) to reveal hES cell-specific gene expression.
Keywords: cell type comparison

overall_design: RNA was isolated from three replicate exponentially growing cultures of undifferentiated human embryonic stem cells (feeder-free, in MEF-conditioned medium). Human universal RNA was purchased from Stratagene.

first: Hans

last: Lehrach

first: James

last: Adjaye


title: Differential Gene Expression of Enriched Germ Cells Responding to a Short Heat Stimulus

status: Public on Jun 02 2007

city: Chunan, Miaoli

first: Wen-chuan

last: Lee

address: 52 Ke-dung 2nd Rd.

department: Division of Biotechnology

country: Taiwan

institute: Animal Technology Institute Taiwan

summary:
It is well recognized that the quality of boar sperm is severely influenced by higher temperature in summer, thereby reducing the productivity of semen.  cDNA microarray is a high throughput technique frequently used in genome-wide screening for specific genes related a known phenotype or disease.  A testis cDNA microarray was established for the investigation of the differentially expressed genes, responding to a short heat stimulus, in enriched germ cells isolated from immature pigs where the first wave of spermatogenesis was just initiated.  Two cDNA libraries were constructed from the testis tissues of two mature Duroc boars with or without whole body heat shock treatment.  About 27,000 single colonies were randomly picked for one-direction sequencing.  After the trimming of vector derived sequences, 22,969 high quality ESTs were obtained, which assembled into 3,207 contigs and 9,541 singletons, representing 12,748 tentative unique transcripts.  A porcine testis cDNA microarray was established with single spot of the amplified cDNA plus 20 spots of positive and negative controls.  We conducted a cell model experiment to screen the differentially expressed genes responding to a short heat shock.  Small population of genes was up- or down-regulated with at least 1.5-fold of differences, including transcriptional factors, kinases, phosphatases, binding proteins and some transcripts with unknown functions.  Further examination on these genes might contribute to a clear picture on the heat-tolerance of spermatogenesis in boar and benefit the breeding of boars with elite semen quality in all reasons.
Keywords: stress response

overall_design: An enriched germ cells population isolated from the testis of 3-month-old immature porcine were cultured in DMEM/F12 medium with 10% FBS for 1 day and then treated with or without a 1-hour heat shock (39C).  Total RNA were extracted after 2-hour recovery at 34C and amplified to antisense RNA(aRNA).  The cDNA microarray experiments were carried out with 3 biological repeats and dye swap technical repeats each pair.

first: B-H

last: Gau

first: I-M

last: Chu

first: M-C

last: Huang

first: K-T

last: Yang

first: S-H

last: Chiou

first: Y-H

last: Fan

first: M-Y

first: J-H

last: Lin

first: C-K

last: Chuang

first: S-Y

first: W-C


title: Knock-down of three core transcription factors in hEC cells

summary:
Three transcription factors were silenced in human embryonic carcinoma cells, OCT4, NANOG, and SOX2. Downstream effects were analysed by means of microarrays to obtain insights into the regulation of self-renewal in these cells.
Keywords: RNAi-chip

overall_design: Cells were transfected with esiRNA against the respective transcription factor transcripts. Knock-down efficiencies were high as monitored by real-time RT-PCR and Western blotting. Samples for chip hybridisations were harvested 2.5 days after transfection.


title: Hepatic transcriptome response to glucocorticoid receptor activation in rainbow trout.

status: Public on May 02 2007

city: Woods Hole

first: Neelakanteswar

last: Aluru

address: 45 Water Street

department: Biology

institute: Woods Hole Oceanographic Institution

summary:
Stress induced elevation in plasma cortisol levels is shown to be involved in the metabolic re-adjustments to stress but the genetic basis of this response is still unclear. Cortisol signaling in teleosts is thought to be mediated predominantly by the glucocorticoid receptor (GR), a ligand-bound transcription factor and previous studies have shown that RU486, a GR antagonist offsets corticosteroid signaling in teleosts. In order to elucidate the genetic basis of GR-mediated metabolic re-adjustments, we exposed in vitro primary culture of trout hepatocytes to cortisol (100 ng/ml; stressed levels) or RU486 (1000 ng/ml) or a combination of both (RU486 + Cortisol), and analyzed the transcriptional responses using a low density custom-made targeted trout cDNA array. These results were further validated using quantitative real-time PCR. Cortisol treatment significantly increased glucose production in hepatocytes and this response is blocked by RU486 suggesting the activation of GR-mediated corticosteroid signaling. Cortisol treatment significantly modulated the transcript abundance of several genes, previously shown to be involved in molecular and biochemical responses to stress and most of these effects are abolished by RU486, suggesting GR-mediated effects. In addition, cortisol treatment affected key genes involved in reproduction supporting previous findings on inhibitory effects of cortisol on reproduction. Taken together, our results provide evidence on the transcriptional changes involved in cortisol signaling in response to stress.
Keywords: cDNA microarray, RU486, Cortisol, GR signaling, rainbow trout, hepatocytes

overall_design: Microarray experiments were designed to comply with minimum information about microarray experiment (MIAME) guidelines. The experimental design include treatment of primary culture of trout hepatocytes with either control (0.01% ethanol; vehicle), Cortisol (100 ng/ml), RU486 (GR antagonist; 1000 ng/ml) or a combination of both RU486 (1000 ng/ml) and cortisol (100  ng/ml). Hepatocytes were sampled 24 h after treatment and used for microarray analyses. RNA isolated from these samples was co-hydridized with a common reference standard. A common reference standard was made by pooling equal amounts of RNA from untreated hepatocytes isolated from the experimental fish. A total of 16 hybridizations were performed (4 treatments X 4 biological replicates per treatment) in this experiment. Dye swapping was carried out so that each treatment had two samples labeled with either Cy3 or Cy5.

first: Mathilakath

last: Vijayan


title: Reactive oxygen species trigger a regulatory module involved in the early responses of rice seedlings to cold stress

status: Public on May 31 2007

city: Orono

first: Benildo

last: de los Reyes

state: ME

address: 5735 Hitchner Hall

department: School of Biology and Ecology

institute: University of Maine

summary:
Plants respond to low temperature through an intricately coordinated transcriptional network controlled by specific groups of transcription factors. Major regulatory pathways in plants that evolved to withstand freezing by cold acclimation have been elucidated in Arabidopsis. A prominent pathway is the CBF/DREB regulon, which was also shown to be evolutionarily conserved between temperate and warm-season plants. This study exploited the wide contrast in chilling tolerance between indica and japonica rice as model to dissect the hierarchical organization of early response regulatory networks by integrative analysis of promoter architecture and gene expression profiles.

Analysis of the transcriptome of japonica rice identified a group of genes that were upregulated during the initial 24 hours at 10oC. Included among the 120 ‘early response’ genes were two transcription factors (ROS-bZIP1, OsMyb4) and another larger sub-group with a common denominator of having the as1/ocs element in their promoters. ROS-bZIP1, OsMyb4 and the as1/ocs element-containing genes were also induced by exogenous H2O2 at ambient temperature, thus are likely components of a regulatory module (ROS-bZIP1-as1/ocs regulatory module) that is activated by elevated intracellular ROS induced by cold stress. Comparative analysis of the expression of ROS-bZIP1-as1/ocs regulatory module between tolerant (CT6748-8-CA-17) and intolerant (INIAP12) rice cultivars showed positive correlation of the activity of the regulon with genotypic differences in chilling tolerance.

A hypothetical model of an ROS-mediate gene regulon was developed. Based on this model, it was hypothesized that the putative ROSbZIP1-as1/ocs regulatory module has a prominent role in configuring early or rapid responses to chilling in rice seedlings and that such pathway is independent of the CBF/DREB-mediated and ABA-mediated regulons.
Background
Keywords: Response to temperature at different time points

overall_design:
This experiment involves the analysis of changes in gene expression in response to cold stress in a total of five time points (0.5, 2, 6, 12 and 24 hrs). Control (29C) and cold stress (10C) samples were labeled with Cy5 and Cy3, respectively. No dye swap replicate was performed in this experiment.

Gene expression analysis was performed with two independent biological replicates (R1 and R2) for each time point. In each biological replicate, there were two technical replicates (T1 and T2). These technical replicates were represented by duplicate set of spots in each microarray slide.

first: Chen

last: Cheng

first: Kil-Young

last: Yun

first: Habtom

middle: W

last: Ressom

first: Bijayalaxmi

last: Mohanty

first: Vladimir

middle: B

last: Bajic

first: Yulin

last: Jia

first: Song-Joong


title: Differentially expressed genes in developing queens and workers - effects of juvenile hormone (JH)

city: Alfenas

first: Angel

middle: Roberto

last: Barchuk

state: Minas Gerais

address: Rua Gabriel Monteiro Silva 700, Campus Unifal

institute: Federal University of Alfenas

summary:
In honeybees, differential feeding of female larvae promotes the occurrence of two different phenotypes, a queen and a worker, from identical genotypes.  Nutritional factors affect both endocrine responses and gene expression and, thus, change the larval developmental pattern.  It is widely accepted that two types of alterations occur during this process: incremental alterations, which affect general growth, and character state alterations that result in the presence or absence of specific structures. Previous studies revealed a link between incremental alterations and differential expression of physiometabolic genes. Here, we report evidence for molecular changes that affect character states, controlling the expression of worker-like characteristics. By using cDNA microarray analyses of more than 6,000 Apis mellifera ESTs we found 240 differentially expressed genes (DEG) between developing queens and workers. Comparative analyses showed that many genes recorded as up-regulated in prospective workers appear to be unique to A. mellifera suggesting that the workers developmental pathway involves the participation of novel genes. We also show that workers up-regulate more developmental genes than queens, whereas queens up-regulate a greater predominance of physiometabolic genes. The latter include genes coding for metabolic enzymes and genes whose products are known to regulate the rate of mass-transforming processes and the general growth of the organism (e.g., tor).  Many DEG are likely to be involved in processes favoring the development of caste-specific structures, like brain, legs and ovaries, as well as genes that code for cytoskeleton constituents.  Treatment of developing worker larvae with juvenile hormone (JH) revealed 52 JH responsive genes, specifically during the critical period of castes development. Using Gibbs sampling and Expectation Maximization algorithms, we discovered eight overrepresented cis-elements from 4 gene groups. Graph theory and complex networks concepts were adopted to attain powerful graphical representations of the interrelation between cis-elements and genes and objectively quantify the degree of relationship between these entities. We suggest that clusters of functionally related DEGs are co-regulated during caste development in honeybees.  This network of interactions is activated by nutrition-driven stimuli in early larval stages. Our data are consistent with the hypothesis that JH is a key component of the developmental determination of queen-like characters. We propose a conceptual model of caste differentiation in A. mellifera based on gene-regulatory networks.
Keywords: caste differentiation during larval development; JH effect

overall_design: Slides were hybridized with samples from queens and workers (larval instars 3, 4 and 5S2), and from JH-treated (1h and 24h) and -Control L4 workers

first: Alexandre

last: Cristino

first: Robert

last: Kucharski

first: Luciano

last: Costa

first: Zilá

middle: L

last: Paulino Simões

first: Ryszard

last: Maleszka


title: Mesenchymal stem Cells vs. Fibroblasts

city: Cologne

first: Sebastian

last: Knoebel

address: Nattermannallee 1

institute: MiltenyiBiotec GmbH

summary:
PMID
Keywords: cell type comparison

overall_design:
RNA was prepared from 4 independent samples of cultivated human foreskin fibroblasts and human MSC, respectively using the Nucleospin-II-RNA-Extraction-Kit (Macherey&Nagel). MSC were isolated via their plastic adherence. Briefly, mononuclear cells were isolated from bone marrow by a Ficoll/ Paque Plus (Amersham Biosciences AB, Uppsala, Sweden) gradient separation. RNA samples were quantified by spectrophotometry and RNA integrity was checked by gel electrophoresis (Bioanalyser 2001, Agilent). A RNA pool prepared from equal amounts of all RNA samples was used as common reference for all hybridizations.
Linear amplification of RNA was done using a modified protocol of a method described previously (1). Amplified RNA (aRNA) samples were quantified by spectrophotometry and quality was checked by gel electrophoresis (Bioanalyser 2001, Agilent). One microgram of aRNA from each sample was labelled by reverse transcription with Cy5 (individual samples) and Cy3 (common reference) fluorescence, respectively. Labelled samples were then hybridised on PIQOR(TM) Stem Cell Microarrays (Miltenyi Biotec), which contain cDNAs of 942 genes spotted as four-fold replicates on different positions of the array. As a qualitative measurement for the validity of the data and to check for the uniformity of the hybridisation process, the coefficient of variation (cv) of the four ratios for the respective gene was calculated. Hybridisation, scanning and data analysis were performed as described in detail (2). Briefly, image capture and signal quantification of hybridised PIQORTM microarrays were done with the ScanArrayLite4000 Microarray Scanner (PackardBiosciences) and ImaGene software Version 5.0 (BioDiscovery, Los Angeles, CA, USA). Local background was subtracted from the signal to obtain the net signal intensity and the ratio of Cy5/Cy3. Subsequently, the mean of the ratios of 4 corresponding spots representing the same cDNA was computed. The mean ratios were normalised using the Median method. For normalisation only those spots were used for which the fluorescent intensity in one of the two channels was twice the mean background of all unflagged spots. Only genes displaying a net signal intensity 2-fold higher than the mean background were used for further analysis. Two class unpaired significance analysis of microarray (SAM) was performed using the 2-fold change criterion and a false discovery rate of 0,00000%. Only those genes present in all experiments were subjected to SAM.

first: Jeannette

middle: M

last: Moebius

first: Edith

middle: E

last: Straach

last: Ma

first: Andreas

last: Bosio

first: Christoph

last: Piechaczek


title: Hepatic transcriptional profiling of juvenile broiler chickens divergently selected for high or low body weight

status: Public on May 30 2007

first: Larry

middle: Albert

last: Cogburn

address: 531 South College Ave.

department: Animal and Food Sciences

summary:
Divergently selected chickens for either high growth (HG genotype) or low growth (LG genotype) developed at SRA-INRA, France were used to profile hepatic gene expression during juvenile development (1 to 11 weeks of age) and to identify differentially expressed genes associated with genotype and age. The HG and LG chickens are different in various phenotypic and metabolic measurements, including growth rate, abdominal fat, plasma glycemia, insulinemia, T4, T3, triglyceride and NEFA. The HG and LG chickens are valuable as a model for biomedical and agricultural traits. The Del-Mar 14K Chicken Integrated Systems microarrays were used for a transcriptional scan in liver during juvenile development using a balanced block hybridization design. Log2-transformed fluorescence intensities were analyzed with a two-stage mixed model. A total number of 531 differentially expressed genes were identified. The greatest number of genes was detected at 7 weeks of age where 178 genes were up-regulated in the HG and 162 genes up-regulated in the LG. The differentially expressed genes include metabolic enzymes, acute phase proteins, immune factors and transcription factors involved in various pathways (i.e.,  fatty acid and amino acid metabolism, glycolysis, oxidative phosphorylation, growth factor signaling and immune defense). Several of these functional genes are also identified as positional candidate genes within QTLs in an F2 population established from an intercross between the HG and LG lines.
Keywords: divergently selected chickens, growth, transcriptional profiling, differentially expressed genes

overall_design: A balance block design was used for microarray hybridizations, where half of the birds of each genotype and age were labeled with Alexa Flour 647 (red) and the other half with Alexa Flour 555 (green). Four biological replicates were used for each genotype (HG or LG) at six different ages (1, 3, 5, 7, 9 and 11 wk). An additional hybridization of eight replicates for each age and genotype was performed to verify the small number of differentially expressed genes detected at 1 and 3 weeks in the first hybridization.  The second hybridization included four technical replicates (same birds that were originally used for the first hybridization) and four additional biological replicates with different birds from each genotype at 1 and 3 weeks of age.

first: Nares

last: Trakooljul

first: Jean

last: Simon

middle: A


title: profiling of midbrain from mice s.c. injected with 4T-1 mammary carcinoma cells vs. PBS

city: Ciudad de Buenos Aires

first: Mariano

middle: Cesar

last: Salibe

address: Av. Patricias Argentinas 435

country: Argentina

institute: Gentron LLC

summary:
The main function of the nervous system is to maintain homeostasis by sensing and reacting to signals that reach a certain threshold. For example, the brain can sense immune peripheral events through soluble compounds or the vagus nerve and can react through activation of the hypothalamus-pituitary-adrenal axis, resulting in the modulation of an ongoing immune response. Cancer progression is characterized by high mutation rates, with each mutation potentially promoting alarm signals during the 10 to 15 years of cancer development before clinical detection. It is not known, however, whether the brain can recognize the presence of a peripheral tumour, and if this recognition can be molecularly assessed. Using genome-wide expression analysis in a model of mammary carcinoma, we show that a tumor growing at the periphery can indeed promote changes in the expression levels of defined sets of genes in the midbrain. These changes occur as early as 18 h after tumour cell injection and involve specific signalling pathways. These findings prove that cancer-derived signals are effective in eliciting specific changes in gene expression in discrete brain regions and open a question regarding the potential role of brain genes in cancer outcomes.
Keywords: gene expression profile of the midbrain in response to peripheral tumor cells

overall_design: We used two-color oligonucleotides in-house printed microarrays in a case-control direct design with two technical replicates in a dye swap design. We analyzed 3 different time points: 18, 72 and 192 h after tumor cells or PBS injection. We performed 5 samples (biological replicates) for 18 h and 4 samples for 72 h and 192 h time points.

middle: J

last: Alvarez

first: Cintia

last: Serra

first: Daniela

last: Celi

first: Guillermo

last: Vila-Ortiz

first: Alejandra

last: Ramirez

first: Osvaldo

last: Podhajcer

first: Fernando

last: Pitossi