title: Expression profiles of Pseudomonas aeruginosa TBCF10839 wild type and nadk1 mutant under oxidative stress conditions

status: Public on May 06 2010

city: Hannover

first: Burkhard

last: Tuemmler

address: Carl-Neuberg-Str. 1

institute: Medizinische Hochschule Hannover

summary: Phagocytosis and killing of the opportunistic pathogen Pseudomonas aeruginosa by polymorphonuclear neutrophils (PMNs) is the major antipseudomonal host defense of vertebrates. By screening a transposon library of the clinical P. aeruginosa isolate TBCF10839 that can grow and replicate in PMNs, a mutant was identified that was most strongly sensitized to killing by PMNs. The inactivated gene PA1572 termed nadK1 was found to encode an ATP:NAD kinase. The transcriptomes of the TBCF10839 wild type and nadK1 mutant were investigated in the presence of PMNs or H2O2. Exposure to H2O2 led to diametrical mRNA expression profiles. H2O2-degrading enzymes were upregulated by wild type, but not by nadK1 mutant. This data demonstrates that NadK1 is crucial for the response of P. aeruginosa to reactive oxygen species.

overall_design:
The transcriptomes of Pseudomonas aeruginosa TBCF10839 wild type and nadK1 mutant were comparatively investigated in the presence of polymorphonuclear neutrophils or H2O2 in order to investigate the role of NadK1 in oxidative stress response. These samples represent test samples.
The expression profile of Pseudomonas aeruginosa strain TBCF10839 wild type cells, isolated from a Cystic Fibrosis patient, was investigated under standard growth conditions in batch culture in Luria broth (LB) with no treatment. These samples represent reference samples.
All samples were analyzed in duplicate.

first: Mario

last: Juhas

first: Prabhakar

last: Salunkhe


title: P. aeruginosa PA14 gene expression in artificial sputum medium (ASMDM)

status: Public on May 31 2010

city: Sydney

first: Jim

last: Manos

state: NSW

address: Camperdown

country: Australia

institute: University of Sydney

summary: Pseudomonas aeruginosa airway infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. In vitro models that closely mimic CF sputum are needed to improve understanding of the pathobiology of P. aeruginosa in the CF airway. We developed an artificial sputum medium (ASMDM) that more closely resembles the composition of CF sputum than current media. In order to validate the utility of ASMDM, we used GeneChip microarrays to compare expression data of P. aeruginosa UCBPP-PA14 (PA14) in ASMDM with published data for this strain grown under the same conditions in an artificial medium containing 10% (v/v) CF sputum. Thirty-seven of 39 nutrition-related genes were differentially expressed in the same manner in both media. However, 24 quorum-sensing (QS) genes, 23 Type III secretion system and several anaerobic respiration genes were more highly expressed in ASMDM than in sputum-containing medium. When grown to stationary phase in ASMDM, PA14 differentially expressed about 50 biologically significant genes compared to stationary phase growth in Luria Broth; genes involved in iron acquisition (pfeA, fepC) and in assimilatory nitrate reduction (nasC, nirD) were upregulated, while 24 QS genes, including the regulator rhlR, lasA, rsaL, aprADEI and phenazine genes phzC2DD2EG2 were downregulated. Downregulation of QS-regulated virulence genes has been noted in chronic P. aeruginosa infection. ASMDM thus appears highly suitable for studies on gene expression of (i) P. aeruginosa strains from acutely and chronically infected CF patients and (ii) established biofilms that are a hallmark of advanced CF lung disease.

overall_design:
PA14 was grown in four different ways:
1. Logarithmic growth for early gene expression
Cells were grown in MOPS-Glucose and separately in ASMDM. The average of two biological duplicates in each case was compared to the other to determine differential gene expression.
2. Stationary phase growth for gene expression
Cells were grown in Luria Broth and separately in ASMDM. The average of two biological duplicates in each case was compared to the other to determine differential gene expression.

first: Carina

last: Fung

first: Sharna

last: Naughton

first: Jonathan

last: Arthur

first: Lynne

last: Turnbull

first: Cynthia

last: Whitchurch

first: Barbara

last: Rose

first: Daniel

middle: J

last: Hassett

first: Colin

last: Harbour


title: Expression data in response to abiotic stresses in tomato at flowering stage

status: Public on Jun 12 2010

city: Varanasi

first: Ashutosh

last: Rai

state: Uttar Pradesh

address: Post Box-1. Jakhini Shahanshapur

department: Crop Improvment

country: India

institute: Indian Institute of Vegetable Research

summary:
A comparative study ware made to know the abiotic stress tolerance machanism between tolerant and susceptible plants at flowering stage.
The tolerance in response to abiotic stresses are sum of expression of thousands of genes at a particular stage.

overall_design: Tomato plants were exposed to drought and heat stress for RNA extraction and hybridization on Affymetrix microarrays. To study the molecular mechanism of abiotic stress tolerance to increase the tolerance in tomato plants, transcripts of tolerant and susceptible plants at flowering stage were compared in response to heat and water stress.

first: Upama

last: Mishra

first: Major

last: Singh

first: Rajesh

last: Kumar

first: Rama

middle: S

last: Dubey

first: Nagendra

middle: K

first: Neha

last: Jain

first: Hausila

middle: P

last: Pandey


title: Metabolically engineered urea degrading and urea importing Sake yeast strains K7 (WT), K7 Dur1,2 and K7 Dur3

status: Public on Apr 21 2010

city: Vancouver

first: Matthew

last: Dahabieh

address: 2205 East Mall

department: Wine Research Centre

country: Canada

institute: University of British Columbia

summary:
Excess/residual urea is a pervasion problem in wine and Sake fermentation. We sought to reduce residual urea levels (to reduce ethyl carbamate leves) by engineering the Sake yeast strain K7 to constitutively express either the urea amidolyase (Dur1,2) or urea importer (Dur3). We sought to then compare the gene expression profiles of the metabolically engineered yeast strains to the parental strain during fermentation.
Engineered strains would hopefully have gene expression profiles that were minimally different from the parental strain.

overall_design: Yeast strains were used to ferment Chardonnay grape must and total RNA harvested at 24 hrs into fermentation. 10 ug of total RNA was made into cDNA, and then labelled cRNA, with the Affymetrix GeneChip one cycle target amplification and labelling system. Fragmented cRNA was then hybridized to an Affymetrix YGS98 array in biological duplicate.

first: Zongli

last: Luo


title: Transcriptional changes in the absence of nth-1, xpa-1 and nth-1;xpa-1

status: Public on Apr 12 2010

city: Oslo

first: Jessica

middle: Margareta

last: Lindvall

address: Gaustadallee 24

country: Norway

institute: University of Oslo

summary:
Background: The ability of an organism to repair damages to DNA is   inextricably linked to aging and cancer.  We have characterized and   compared the transcriptome of C. elegans mutants deficient in DNA   base excision repair, nucleotide excision repair or both to elucidate   the transcriptional changes incurred by the reduction of these repair   pathways.

Results: The gene expression signatures from nth-1, xpa-1 and   nth-1;xpa-1 are deciphered. We find that the single mutants are more   similar in overall transcriptomic activity compared to the double mutant   and their corresponding wild-type (N2).  Interestingly, of the enriched   Gene Ontology terms for respective mutants are aging and   proteolysis for nth-1, aging, energy production and unfolded protein   response for xpa-1, and DNA repair and regulation of M-phase for   nth-1;xpa-1.  We also find striking similarities between the biological   processes found to be enriched in the single mutants, which show a shared   response in genes associated with aging and insulin signaling.  Loss of   either BER or NER induces an oxidative stress response, but does not   confer resistance to heat shock.

Conclusion: Taken together, our study provides a general overview about   the global transcriptional changes in the absence of nth-1, xpa-1 and   nth-1;xpa-1.  Our results also point to a two-tiered global response to   combat the effects of decreased DNA repair capacity.

overall_design: C. elegans RNAi mutants deficient in DNA excision repair, nth-1, xpa-1, nth-1;xpa-1 and the corresponding wild-type N2, were subjected to Affymetrix C. elegans microarrays. For nth-1, triplicates were run, whereas for xpa-1, nth-1;xpa-1 and N2, duplicates were run.

first: Oyvind

last: Fensgård

first: Henok

last: Kassahun

first: Torbjørn

last: Rognes

middle: M

first: Hilde

last: Nilsen


title: Caenorhabditis elegans infected with Staphylococcus aureus

city: Boston

first: Javier

middle: E

last: Irazoqui

state: MA

address: 55 Fruit St GRJ1416

department: Pediatrics

country: USA

institute: Massachusetts General Hospital

summary:
Young adult fer-15;fem-1 Caenorhabditis elegans were infected with Staphylococcus aureus for 8 h to determine the transcriptional host response to Staphylococcus aureus. Analysis of differential gene expression in C. elegans young adults exposed to two different bacteria: E. coli strain OP50 (control), wild-type Staphylococcus aureus RN6390. Samples were analyzed at 8 hours after exposure to the different bacteria. These studies identified C. elegans genes induced by pathogen infection.
Keywords: response to pathogen infection, innate immunity, host-pathogen interactions

overall_design: There are 6 samples total: 3 biological replicates of Staphylococcus aureus-infected animals, and 3 biological replicates of E. coli-fed controls. Three independent biological replicates were isolated for each treatment. All treatments were performed in parallel.

first: Emily

middle: R

last: Troemel

first: Frederick

last: Ausubel


title: BP1 Expression Promotes Tumorigenesis and Aggression in MCF-7 Breast Cancer Cells

status: Public on May 05 2010

city: Washington

first: Sidney

middle: W

last: Fu

state: DC

address: 2300 Eye St. NW, Ross 402C

department: Medicine

institute: George Washington University

summary: This study was designed to identify genes that are differentially expressed when BP1 homeobox gene is overexpressed in MCF-7 breast cancer cells. The goal is to understand the functional role of BP1 in breast tumorigenesis.

overall_design: MCF-7 overexpressing pcDNA3.2-BP1 clones, O1 and O4 as replicates vs. MCF-7 transfected with pcDNA3.2 vector clones, V1 and V2 as replicates

first: Erika

last: Ginsburg

first: Arnold

last: Schwartz

first: Yan-gao

last: Man

first: Joseph

last: Pinzone

first: Louis

last: Bivona

first: Holly

last: Stevenson

first: Samuel

last: Simmens

first: Christine

last: Teal

first: Kyung-Soon

last: Kim

first: Dowser

last: Alani

first: Weiwei

last: Wu

first: Barbara

last: Vonderhaar

first: Patricia

last: Berg


title: Expression data of fibroblasts from gingival/dermal tissues

status: Public on May 28 2010

city: Nagoya

first: Ryuji

last: Kato

state: Aichi

address: Furocho, Chikusa-ku

department: Graduate School of Engineering

country: Japan

institute: Nagoya University

summary: Fibroblasts are widely used cells for regenerative medicine in clinics, such as gingival or facial skin treatment. In fact, fibroblasts are considered as a mixture of various types of cells with "spindle shape" and there is no available clear marker.  Gingival and dermal fibroblasts are similar in their morphology and function; however it is considered that the cultured cells retain their original characteristics depending on the origin, which may contribute to the differential therapeutic effects.  For example, gingival wounds are known to heal relatively quickly with less scar formation compared with skin, which may imply their higher capability for regeneration as a therapeutic effect. The reason for this phenomenon may be partly due to characteristic differences between gingival and dermal fibroblasts including the expression of migration stimulating factor and matrix formation but these differences remain largely unknown. Recently, the characteristics of dermal fibroblasts have been reported to be different depending on body sites, such as face, trunk and plamoplantar skin. Although the expression of fibronectin and their splicing variants were known to be different between trunk and oral mucosal fibroblasts, there is still no detailed report on the functional differences between gingival and dermal fibroblasts. In this study, we investigated differential gene expression in normal gingival and dermal fibroblasts using DNA microarray to investigate the difference between the vague fibroblast-type cells from different tissue origin to achieve higher therapeutic effect in cell therapy.

overall_design: Gingival and dermal tissues were collected from healthy patients.  After successive stages primary culture and RNA extraction and hybridization on Affymetrix microarrays (Genome Focus Array). Dermal and gingival tissues were obtained from healthy volunteers (8 cases for dermal tissue; 6 females, 2 males , average age 48 and 8 cases for buccal gingival tissue; 6 females, 2 males, average age 43) whose informed consent was obtained according to a protocol approved by the ethics committee of Nagoya University Hospital.  After enzymatic digestion, tissues were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum at 37°C in the presence of 5% CO2 for about 4 weeks as reported previously. Total mRNAs were extracted from cells between passages 4-5 by Trizol reagent (Invitrogen, Carlsbad, CA, USA) and were applied to Human Focus Arrays (Affymetrix, Santa Clara, CA, USA) for microarray analysis according to the manufacturer's protocol (http://www.affymetrix.com/support/technical/manuals.affx). The gene expression data were analyzed by Arrayassist (Stratagene, La Jolla, CA, USA).  Briefly, 8,500 probes on the array, normalization and scaling (MAS5), flag-positive gene selection, unpaired t-test, and CV selection (<20) resulted in 5,284 genes to analyze. GO (Gene ontology) analysis was performed using the software default settings to find the gene group related to the same category of biological function by searching common key terms that were reported for each gene.

first: Katsumi

last: Ebisawa

first: Tomotaka

last: Sugimura

first: Mazlyzam

middle: A

last: Latif

first: Yusuke

last: Hori

first: Yuji

last: Narita

first: Minoru

last: Ueda

first: Hiroyuki

last: Honda

first: Hideaki

last: Kagami


title: Brief Hydrocortisone Infusion Differentially Influences Blood Leukocyte and Monocyte Gene Expression Before and After Endotoxin Administration in Humans (supported by NIH grant GM34695)

status: Public on May 21 2010

city: New Brunswick

first: Stephen

middle: F

last: Lowry

state: NJ

address: 125 Paterson St

department: Surgery

institute: UMDNJ

summary: Fourteen human subjects (18-40 years of age) were studied. The control group (saline + LPS [n = 7]) received a 12 hour infusion of saline and the experimental group (cortisol + LPS [n = 7]) received a 12 hour infusion of cortisol (3 µg/kg/min). After 6 hours of saline or cortisol infusion, intravenous lipopolysaccharide (2 ng/kg) was administered to all subjects. Blood samples were collected at -24 hours, 0 hours, 6 hours and 24 hours relative to LPS administration at 0 hours. Leukocytes and monocytes were isolated from these blood samples, total RNA extracted, and microarray analyses performed using Affymetrix Focus GeneChips®.

overall_design: Blood samples were collected at (-24)hr, 0hr, 6hr, and 24hr in relation to the LPS infusion. For both saline and cortisol treated subjects, infusion was started at (-6) hr and continued to (+6) hr.

first: Steve

last: Calvano


title: Loss of Caenorhabditis elegans UNG-1 uracil-DNA glycosylase affects apoptosis in response to DNA damaging agents.

status: Public on May 26 2010

first: Jessica

last: Lindvall

summary: The nematode Caenorhabditis elegans has been used extensively to study responses to DNA damage. In contrast, little is known about DNA repair in this organism. C. elegans is unusual in that it encodes few DNA glycosylases and the uracil-DNA glycosylase (UDG) encoded by the ung-1 gene is the only known UDG. C. elegans could therefore become a valuable model organism for studies of the genetic interaction networks involving base excision repair (BER). As a first step towards characterization of BER in C. elegans, we show that the UNG-1 protein is an active uracil-DNA glycosylase. We demonstrate that an ung-1 mutant has reduced ability to repair uracil-containing DNA but that an alternative Ugi-inhibited activity is present in ung-1 nuclear extracts. Finally, we demonstrate that ung-1 mutants show altered levels of apoptotic cell corpses formed in response to DNA damaging agents. Increased apoptosis in the ung-1 mutant in response to ionizing radiation (IR) suggests that UNG-1 contributes to repair of IR-induced DNA base damage in vivo. Following treatment with paraquat however, the apoptotic corpse-formation was reduced. Gene expression profiling suggests that this phenotype is a consequence of compensatory transcriptomic shifts that modulate oxidative stress responses in the mutant and not an effect of reduced DNA damage signaling.

overall_design: C. elegans RNAi mutants deficient in ung-1 and the corresponding wild-type N2, were subjected to Affymetrix whole C. elegans genome microarrays. Triplicates were run for each sample group.

first: Hanne

last: Skjeldam

first: Henok

last: Kassahun

first: Oyvind

last: Fensgard

first: Tanima

last: SenGupta

first: Eshrat

last: Babaie

first: Katarzyna

last: Arczewska

first: Hilde

last: Nilsen


title: Integration of the general amino acid control and nitrogen regulatory pathways in yeast nitrogen assimilation

status: Public on Mar 01 2010

city: Indianapolis

first: Ron

last: Wek

state: IN

address: 635 Barnhill Dr. MS4065

department: Biochemistry & Molecular Biology

institute: Indiana University School of Medicine

summary:
Two nutrient sensing and regulatory pathways, the general amino acid control (GAAC) and the target of rapamycin (TOR), control yeast growth and metabolism in response to changes in nutrient availability.  Starvation for amino acids activates the GAAC pathway, involving Gcn2p phosphorylation of eIF2 and preferential translation of GCN4, a transcription activator of genes involved in amino acid metabolism.  TOR senses nitrogen availability and regulates gene expression through transcription factors, such as Gln3p.  We used microarray analyses to address the integration of the GAAC and TOR pathways in directing the yeast transcriptome in response to amino acid starvation and rapamycin treatment.  Of the ~2500 genes whose expression was changed by 2-fold or greater, Gcn4p and Gln3p were required for 542 and 657 genes, respectively.  While Gcn4p activates a common core of 57 genes in response to amino acid starvation or rapamycin treatment, the different stress arrangements allow for variations in Gcn4p-directed transcription.  With few exceptions, genes requiring Gcn2p eIF2 kinase for induced expression also required Gcn4p, emphasizing the role of Gcn2p as an upstream activator of Gcn4p-directed transcription. There is also significant coordination between the GAAC and TOR pathways, with Gcn4p being required for activation of more genes during rapamycin treatment than Gln3p.  Importantly, TOR regulates the GAAC-directed transcription of genes required for assimilation of nitrogen sources, such as γ-amino-butyric acid.  Therefore, yeast has integrated gene expression responses to amino acid abundance and nitrogen source quality through the control of Gcn2p phosphorylation of eIF2 and GCN4 translation.

Keywords: gene expression

overall_design: In this study, we carried out microarray analyses in a collection of yeast strains deleted for GCN2, GCN4, and GLN3, individually or in combinations, to explore the importance of the TOR and GAAC pathways in directing the transcriptome in response to amino acid starvation and rapamycin treatment.

first: Kirk

last: Staschke

first: Souvik

last: Day

first: Jeanette

middle: N

last: McClintick

first: Howard

last: Edenberg

first: Ronald

middle: C


title: Transcriptional profiling of P. aeruginosa isolated from 3 individuals with cystic fibrosis over time

status: Public on May 25 2010

city: Ulsan

first: Taejoon

last: Kwon

address: 50 Unist-gil

department: Department of Biomedical Engineering

country: South Korea

institute: Ulsan National Institute of Science and Technology

summary: Pseudomonas aeruginosa chronically colonizes the lungs of individuals with CF, where it reaches high cell densities and produces a battery of virulence factors. Upon infection, a single strain of P. aeruginosa can colonize an individual’s lungs throughout his or her lifetime. To understand the evolution of P. aeruginosa during chronic lung infection, we conducted both genotypic and phenotypic analyses on clinical isogenic strains obtained from the lungs of three different individuals with CF. These strains were isolated over a period of approximately ten years and possess phenotypes that are commonly observed in isolates from the CF lung, such as the antibiotic resistant dwarf and mucoid phenotypes. Microarray analyses were carried out on isolates grown in a chemically defined medium that mimics the nutritional environment of the CF lung, synthetic CF sputum medium (SCFM).

overall_design: 17 clinically isolated P. aeruginosa strains from three individuals with CF (5 strains from individual P1, 7 strains from individual P2, 5 strains from individual P3). Two reference strains PAO1 and PA14. All experiments were biologically duplicated.

first: Holly

last: Huse

first: Marvin

last: Whiteley

first: James

last: Zlosnik

first: David

last: Speert

first: Edward

last: Marcotte


title: Human skeletal muscle - type 2 diabetes and family history positive individuals - Mexican American

status: Public on Apr 14 2010

first: Mary Elizabeth

last: Patti

address: 1 Joslin Place

department: Research Division

institute: Joslin Diabetes Center

summary: Type 2 diabetes mellitus (DM) is characterized by insulin resistance and pancreatic beta-cell dysfunction. In high-risk subjects, the earliest detectable abnormality is insulin resistance in skeletal muscle. Impaired insulin-mediated signaling, gene expression, and glycogen synthesis, and accumulation of intramyocellular triglycerides have all been linked with insulin resistance, but no specific defect responsible for insulin resistance and DM has been identified in humans. To identify genes potentially important in the pathogenesis of DM, we analyzed gene expression in skeletal muscle from healthy metabolically characterized nondiabetic (family history negative and positive for DM) and diabetic Mexican-American subjects. We demonstrate that insulin resistance and DM associate with reduced expression of multiple nuclear respiratory factor-1 (NRF-1)-dependent genes encoding key enzymes in oxidative metabolism and mitochondrial function. While NRF-1 expression is decreased only in diabetic subjects, expression of both PPARg coactivator 1-alpha and -beta (PGC1-a/PPARGC1, and PGC1-b/PERC), coactivators of NRF-1 and PPARg-dependent transcription, is decreased in both diabetic subjects and family history positive nondiabetic subjects. Decreased PGC1 expression may be responsible for decreased expression of NRFdependent genes, leading to the metabolic disturbances characteristic of insulin resistance and DM.

overall_design: Human muscle samples were obtained from five subjects with type 2 diabetes and ten subjects without diabetes, as well as 5 aliquots from a single subject without diabetes.  The subjects without diabetes were further classified as family history positive (four subjects) or family history negative (six subjects).

first: Mary

first: Atul

last: Butte

first: Sarah

last: Crunkhorn

first: Kenneth

last: Cusi

first: Reachel

last: Berria

first: Sangeeta

last: Kashyap

first: Yoshinori

last: Miyazaki

first: Isaac

last: Kohane

first: Maura

last: Costello

first: Robert

last: Saccone

first: Edwin

last: Landaker

first: Allison

middle: B

last: Goldfine

last: Mun

first: Ralph

last: DeFronzo

first: Jean

last: Finlayson

first: C.

last: Kahn

first: Lawrence

last: Mandarino


title: C. elegans gene expression in response to Y. pestis KIM5 infection

status: Public on Feb 05 2010

city: Durham

first: Devin

last: Bolz

state: NC

address: 270 Jones Building, Research Drive, Box 3580

department: Molecular Genetics and Microbiology

institute: Duke University Medical Center

summary: The response of the nematode C. elegans to Y. pestis infection was evaluated by gene expression profiling.  A synchronized population of nematodes were exposed to Y. pestis KIM5 for 24h.  Transcript levels from Y. pestis-treated animals were compared with animals maintained on relatively nonpathogenic E. coli OP50 for 24h.

overall_design: Three independent RNA isolations were performed following exposure to either Y. pestis KIM5 or E. coli OP50.  Exposures to the different pathogens were performed in parallel for each replicate isolation.

middle: D

first: Jennifer

middle: L

last: Tenor

first: Alejandro

last: Aballay


title: Gene Expression of MATa yeast segregants (YJM789 X S96) after alpha factor treatment

status: Public on Feb 18 2010

city: New Haven

first: Wei

last: Zheng

state: CT

address: Suite 503, 300 George Street

institute: Yale University

summary: In this study, we mapped for the first time differences in transcription binding among individuals and elucidated the genetic basis of such variation.  Whole-genome Ste12 binding profiles were determined using ChIP-Seq in pheromone-treated cells of 43 segregants of a cross between two highly diverged yeast strains, YJM789 and S288c, as well as the parental lines. We identified extensive Ste12 binding variation among individuals and mapped underlying cis- and trans- acting loci responsible for such variation. We showed that the majority of TF binding variation is cis-linked and that many variations are associated with polymorphisms residing in the binding motifs of Ste12 as well as those of several known and proposed Ste12 cofactors. We also identified two trans factors, AMN1 and FLO8, that modulate Ste12 binding to promoters of more than 10 genes under α-factor treatment.  Neither of these two genes was known to regulate Ste12 previously, and we suggest that they may be key mediators of gene activity and phenotypic diversity.  Ste12 binding strongly correlates with gene expression for more than 200 genes, indicating that binding variation is functional.  Many of the variable bound genes are involved in cell wall organization and biogenesis.  Overall, we identified key regulators of molecular diversity among individuals and provide novel insights into mechanisms of gene regulation.

overall_design: We measured gene expression levels after 30 minutes treatment with alpha factor for 43 MATa segregants from a YJM789 X S96 cross, as well as MATa parental lines. We also measured the gene expression levels without alpha factor treatment for parental lines (S96, HS959) and SEG8 as controls.

first: Hongyu

last: Zhao

first: Eugenio

last: Mancera

first: Lars

last: Steinmetz

first: Michael

last: Snyder


title: RIP-chip analysis of the C. elegans PUF protein FBF

status: Public on Jan 27 2010

city: Madison

first: Judith

last: Kimble

state: WI

address: 433 Babcock Drive

department: HHMI / Biochemistry

institute: University of Wisconsin-Madison

summary: The PUF family of RNA binding proteins has a conserved role in maintaining stem cell self-renewal.  FBF is a C. elegans PUF that is required to maintain germline stem cells (GSCs).  To understand how FBF controls GSCs, we sought to identify is target mRNAs.  Briefly, we immunoprecipitated FBF-mRNA complexes from worm extracts and then used microarrays to identify the FBF-associated mRNAs.  To focus on germline targets of FBF, we used a FBF-GFP transgene under the control of a germline promoter and we used an anti-GFP antibody to purify FBF-GFP from worm extracts.  In parallel, we also processed a strain expressing TUBULIN-GFP in the germline to control for mRNAs that non-specifically co-purify with GFP.  We found that FBF associates with >1,000 unique mRNAs and likely controls a broad network of key cellular and developmental regulators.

overall_design: Worm extracts were prepared from synchronized adult C. elegans (24 h after L4 stage) expressing either a rescuing FBF-1-GFP or TUB-GFP transgene under the control of a germline promoter (pie-1).  An immoblized anti-GFP antibody was then used to purify the GFP fusion proteins from extracts.  RNA was then extracted from the pellets and analyzed on Affymetrix microarrays.  Four biological replicates were performed, each consisting of a FBF-GFP and a TUB-GFP sample processed in parallel.

first: Aaron

last: Kershner


title: Genetic Analysis of Variation in Transcription Factor Binding in Yeast

type: Genome binding/occupancy profiling by high throughput sequencing

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: A genome-wide survey for a Juvenile hormone response element, DR4, in the fruit fly, Drosophila melanogaster

status: Public on Dec 31 2009

city: Lexington

first: Subba

middle: Reddy

last: Palli

state: KY

address: S-225 Agr. Sci. Ctr. N

department: Entomology

institute: University of Kentucky

summary:
Microarray analyses identified 713 genes whose expression increased or decrease by 1.5-fold with a P value <0.01 in response to exposure to JH for 3-12 hr. 497 were up-regulated, and 228 were down-regulated. 12 genes showed differentially regulated pattern that depended on the time of exposure to JH. 73 of these genes (57 genes up-regulated and 17 genes down-regulated) showed the presence of DR4 element in their promoter regions. We also screened the promoter regions of genes whose products are distributed in different cellular locations and various functional groups and found that the DR4 element is present in 275 out of 2850 genes screened. The distribution of DR4 element containing genes varied depending on both cellular location and gene function. The genes whose products are localized to the nucleus or cytoplasm showed higher proportion of DR4 element containing genes when compared to the genes coding for products localized to the membrane. Genes belonging to immune response and ligand activated receptor functional groups showed higher number of genes that contained DR4 elements when compared to the genes belonging to protein kinase or transmembrane receptor functional groups. The DR4 elements identified in the promoter regions of D. melanogaster genes bound to the nuclear proteins isolated from JH III-treated Drosophila L57 cells. In addition, the expression of DR4 element containing genes identified from various functional groups was induced by JH III in Drosophila mbn2 cells grown in the medium containing 1 µM JH III. Presence of DR4 element in JH-responsive genes is conserved across species. This study also demonstrates the feasibility of using genome-wide analysis for identification of genes regulated by various hormones and other signaling molecules.
Keywords: Time course

overall_design: We performed microarray analysis using RNA isolated form L57 cells that were exposed to 1 µM JH III for 0, 3, 6 and 12 hr and identified 497 genes that are induced by JH and 228 genes that are down-regulated by JH. Then, we searched the promoter regions of these genes and found that 57 out of these 497 JH induced genes, and 17 out of these 228 JH down-regulated genes had conserved DR4 element present in their promoters.

first: Yiping

last: Li


title: Comprehensive discovery of endogenous Argonaute binding sites in C. elegans

status: Public on Nov 24 2009

city: La Jolla

first: Gene

last: Yeo

state: CA

address: 2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab

institute: UCSD

overall_design: Total RNA were extracted from whole alg-1(gk214) (alg-1(-)) and N2 (wildtype) worms and used to compare the expression profiles of genes that were targeted by ALG-1 protein.

first: Amy

last: Pasquinelli


title: Microarray Analysis of mRNA Levels from RAW264.7 Macrophages Infected with Brucella abortus

status: Public on Jan 01 2010

first: Gary

last: Splitter

address: 1656 Linden Dr

department: Pathobiological Sciences

institute: University of Wisconsin - Madison

summary:
Identification of host responses at the gene transcription level provides a molecular profile of the events that occur following infection.  Brucella abortus is a facultative intracellular pathogen of macrophages that induces chronic infection in humans and domestic animals.  Using microarray technology, the response of macrophages 4 hours following B. abortus infection was analyzed to identify early intracellular infection events that occur in macrophages.  Of the more than 6,000 genes, we identified over 140 genes that were reproducibly differentially transcribed.  First, an increase in the transcription of a number of pro-inflammatory cytokines and chemokines, such as TNF-α, IL-1β, IL-1α, and members of the SCY family of proteins, was evident that may constitute a general host recruitment of antibacterial defenses.  Alternatively, Brucella may subvert newly arriving macrophages for additional intracellular infection.  Second, transcription of receptors and cytokines associated with antigen presentation, e.g., MHC class II and IL-12p40, were not evident at this 4 hour period of infection.  Third, Brucella inhibited transcription of various host genes involved in apoptosis, cell cycling, and intracellular vesicular trafficking.  Identification of macrophage genes whose transcription was inhibited suggests that Brucella utilizes specific mechanisms to target certain cell pathways.  In conclusion, these data suggest that B. abortus can alter macrophage pathways to recruit additional macrophages for future infection while simultaneously inhibiting apoptosis and innate immune mechanisms within the macrophage permitting intracellular survival of the bacterium.  These results provide insights into the pathogenic strategies used by Brucella to survive long-term within a hostile environment.
Keywords: Macrophage, intracellular pathogen, Brucella abortus, inflammatory immune response

overall_design: Five murine U74A gene chips (Affymetrix) were independently hybridized with cRNA from uninfected macrophage samples (2 independent samples) and macrophages infected 4 hrs with B. abortus (3 independent samples).  Washing and staining with streptavidin phycoerythrin was done using a GeneChip Fluidics station 400 (Affymetrix).  Scanning was performed with an Affymetrix GeneArray.  GeneChip Expression Analysis software (Affymetrix) was used to scan and analyze data.  Output was stored in an Excel spreadsheet.  Difference calls were assigned the following values: increased = 2, marginally increased = 1, no change = 0, marginally decrease = -1 and decreased = -2.  The sum of the six difference calls from the inter-group comparisons of 2 uninfected and 3 Brucella infected arrays (2 x 3) was calculated.  A sum of ≥8 or ≥ -8 was the cutoff values for increase/decrease, respectively.  Expressed sequence tag (EST) sequences were eliminated from the analysis.

first: Linda

last: Eskra

first: Angela

middle: J

last: Mathison


title: Expression data from wild type C. elegans and 5 osmotic stress resistant mutants exposed to hyper/isotonic environments

status: Public on Dec 04 2009

city: Philadelphia

first: Todd

last: Lamitina

state: PA

address: 3700 Hamilton Walk, A702

department: Physiology

institute: University of Pennsylvania

summary: Adaptation of C. elegans to hypertonic environments involves the accumulation of the organic osmolyte glycerol via transcriptional upregulation of the glycerol biosynthestic enzyme gpdh-1.  A number of mutants, termed osmotic stress resistant (osr) mutants, have been identified.  osr mutants cause constitutive upregulation of gpdh-1 and confer extreme resistance to hypertonicity.  We tested the hypothesis that osr mutants broadly activate a gene expression program normally activated by osmotic stress in wild type animals using Affymterix microarray analysis of the hypertonic stress response in wild type animals and of constituitive gene expression changes in five osr mutants.

overall_design: Young adult C. elegans were exposed to hypertonic growth plates for varying times prior to RNA extraction and hybridization on Affymetrix microarrays.  Since we wished to separate direct response from secondary responses to osmotic stress, we collected worms following short term exposures to hypertonic conditions (15 minutes and 1 hour) and after long term exposure to hypertonic conditions (6 hours or a full generation of growth under hypertonic conditions).  We also collected young adults from the osr mutants osm-7, osm-8, osm-11, dpy-9, and dpy-10 for microarray analysis.  These mutants were grown under isotonic conditions to determine whether that constitutively activate genes normally regulated by hypertonic stress in wild type animals.


title: The foraging gene in Drosophila mediates metabolic as well as behavioral plasticity

city: Mississauga

first: Clement

last: Kent

state: ON

address: 3359 Mississauga Rd N

department: Biology

institute: Univ. of Toronto Mississauga

summary:
The foraging gene in Drosophila, encoding PKG, is known for its importance as a natural variant affecting behavioral plasticity.  Here, we demonstrate that it also affects metabolic plasticity: Rovers show a greater response to changes in their food environment than do sitters. Using metabolic profiling, we define foraging- and food-dependent changes in metabolites indicating that Rover store energy predominantly as lipids, whereas sitter variants store it as carbohydrates, Such changes are reflected in gene expression patterns, indicating an influence of foraging on regulators of insulin signaling. Combining for alleles with mutants of positive insulin signaling regulators makes Rover responses sitter-like, but does not change sitter responses. Collectively these findings suggest that the effect on metabolic plasticity of foraging works through the insulin signaling pathway.

Keywords: stress response, comparative genomic hybridization, genotype by environment

overall_design: Design: 3 genotypes x 2 treatments x 3 replicates. Treatments were leaving flies on lab food (control) or food-depriving them overnight with access to water. Three genotypes differed in foraging alleles: Rovers are homozygous for[R] (strain: BB), sitters homozygous for[s] (strain: ee, mutant sitters homozygous for[s2] (strain:s2) . Adult flies were 6-7 days post emergence. Three samples of equal numbers of male and females were collected per combination, frozen in liquid nitrogen, and heads were sieved for RNA extraction. This Gene by Environment design tests how presence or absence of food for a short period (overnight) affects gene expression in different foraging genotypes.

first: Marla

last: Sokolowski

first: Ralph

last: Greenspan


title: Molecular mechanisms involved in adipose tissue-derived stromal cell hepatic conversion

status: Public on Dec 15 2009

city: Roma

first: Nathalie

last: Saulnier

address: Largo Francesco Vito,1

country: Italy

institute: Università Cattolica del Sacro Cuore di Roma

summary:
Background: Adipose tissue-derived stromal cells (ATSCs) hold great promises in regenerative medicine, due to their easy retrieval, high proliferative capacity, and multi-lineage differentiation potential. In the last decade, several studies have reported the plasticity of ATSCs toward a hepatic fate. Nonetheless, the molecular mechanisms underlying the conversion from a mesenchymal to an epithelial phenotype remain poorly understood.
Aim: In this study, we compared the full genome expression profiles of ATSCs cultured for 4 weeks under pro-hepatogenic conditions to undifferentiated ATSCs, in order to depict the molecular events involved in ATSC hepatic transdifferentiation.
Methods: Molecular analysis was performed using the Affymetrix human focus arrays. Sets of differentially expressed genes were functionally categorized in order to understand which pathways drive the hepatic conversion and interesting target genes were validated by Q-PCR.
Results: We showed that ATSC-derived hepatocyte-like cells activate several genes associated with specific liver functions, including protein metabolism, innate immune response regulation, and biodegradation of toxic compounds. Furthermore, microarray analysis highlighted the downregulation of several transcripts involved in stemness maintenance along with genes associated with a mesenchymal phenotype.
Conclusion: Taken together, our data suggest that the in vitro system used in this study drove ATSCs toward a hepatic conversion through a subtle regulation of molecular pathways controlling stem cell properties and lineage commitment that promote mesenchymal-epithelial-transition.

overall_design: Adipose tissue was obtained from 3 patients undergoing partial abdominoplasty. Adipose tissue-derived stromal cells (ATSC) were isolated according to standard procedures, using the in vitro adherence property of these cells. At passage culture 4, ATSC were submitted to an in vitro hepatogenic regimen, consisting of the sequential addition of growth factors. After 1 month of in vitro differentiation, cells were harvested and their transcriptome was compared to control ATSC.

first: Anna

last: Piscaglia

first: Marta

last: Barba

first: Maria

last: Puglisi

first: Giovambattista

last: Pani

first: Sergio

last: Alfieri

first: Antonio

last: Gasbarrini


title: Gene expression regulation in response to heat stress in different yeast strains

status: Public on Nov 09 2009

city: Pittsburgh

first: Xinghua

last: Lu

address: 5607 Baum Blvd

department: Dept Biomedical Informatics

institute: University of Pittsburgh

summary: Different yeast strains were subjected to heat stresss; samples were collected at 0, 5, 10, 15, 20, 25 and 30 minutes after heat stress; microarray analysis were performed using the Affymetrix Y-GS98 microarray.

overall_design: 7 time points, heat stress, 3 strains

first: L

last: Cowart

first: Yusuf

last: Hannun


title: Exon arrays of ENCODE tier 1, tier 2 and tier 3 cell types

status: Public on Nov 19 2009

city: Stanford

first: ENCODE

last: DCC

address: 300 Pasteur Dr

institute: ENCODE DCC

summary:
This data was generated by ENCODE. If you have questions about the data, contact the submitting laboratory directly (Richard Sandstrom mailto:sull@u.washington.edu). If you have questions about the Genome Browser track associated with this data, contact ENCODE (mailto:genome@soe.ucsc.edu).

At cell harvest, a subset of cells was stored at -20oC in RNALater.  Total RNA from 5 X 106 cells were purified using Ribopure (Ambion) according to vendor recommended protocols.  Total RNA quality was assessed on RNA 6000 Nano Chips (Agilent) using a Bioanalyzer (Agilent).  Approximately 3ug of total RNA for each cell type was sent to the Center for Array Technology (CAT) for labeling and hybridization to Affymetrix Human Exon 1.0 ST arrays.  Briefly, a Whole Transcript Sense Target Labeling Assay (Affymetrix) was used by the CAT to reduce the rRNA, perform in vitro transcription (IVT) and create labeled sense strand DNA for hybridization to the arrays.  Hybridizations were carried out according the manufacturers protocol.  Intensity files from the scanned exon arrays were sent to our lab for analysis at the exon level (Affymetrix ExACT 1.2.1 software). Samples were quantile normalized with PM-GCBG background correction and PLIER (Probe Logarithmic Intensity Error) summarized.

For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: Continuing exon profiling of ENCODE approved cell types, these coincide with DNaseI hypersensitivity sequencing assays, ChIP-seq (histone modifications, transcription factors), and other ENCODE assay types.

first: J

last: Stamatoyannopoulos


title: The Ess1 prolyl isomerase is required for the transcription termination of small non-coding RNAs via Nrd1 pathway

status: Public on Oct 16 2009

city: Albany

first: Steven

last: Hanes

state: NY

address: 150 New Scotland Ave

department: Developmental Genetics

institute: Wadsworth Center

summary: Genome-wide studies have identified abundant small, non-coding RNAs including snRNAs, snoRNAs, cryptic unstable transcripts (CUTs), and upstream regulatory RNAs (uRNAs) that are transcribed by RNA polymerase II (pol II) and terminated by a Nrd1-dependent pathway.  Here, we show that the prolyl isomerase, Ess1, is required for Nrd1-dependent termination of ncRNAs.  Ess1 binds the carboxy terminal domain (CTD) of pol II and is thought to regulate transcription by conformational isomerization of Ser-Pro bonds within the CTD.  In ess1 mutants, expression of ~10% of the genome was altered, due primarily to defects in termination of snoRNAs, CUTs, SUTs and uRNAs.  Ess1 promoted dephosphorylation of Ser5 (but not Ser2) within the CTD, most likely by the Ssu72 phosphatase, and we provide evidence for a competition between Nrd1 and Pcf11 for CTD-binding that is regulated by Ess1-dependent isomerization.  This is the first example of a prolyl isomerase required for interpreting the “CTD code.”

overall_design:
Two separate experiments were done: a standard Expression Array, and a Tiling Array.

For the Expression Array, two independent methods were used.  First, we compared wild-type and ess1(H164R) ts-mutants, where the mutant allele is integrated at the normal genomic locus.  Cells were shifted to 34°C for 2 hr and RNA was prepared.  In the second method, we used ESS1 deletion mutants that carried wild-type or H164R on a centromeric plasmid driven by the GAL1 promoter.  These constructs were activated by a GAL4-ER-VP16 driver, which even in the absence of beta-estradiol (“0 hormone”), drives low level protein expression.

For the Tiling Array we compared wild-type and ess1(H164R) ts-mutants after a shift to 34°C for 2 hr.

first: Navjot

last: Singh

first: Zhuo

last: Ma

first: Trent

last: Gemill

first: Xiaoyun

last: Wu

first: Holland

last: DeFiglio

first: Anne

last: Rossettini

first: Christina

last: Rabeler

first: Olivia

last: Beane

first: Randall

last: Morse

first: Michael

last: Palumbo


title: Microarray for suspension cells of PA14 WT with and without NE (50 uM and 500 uM) for 7 h

status: Public on Aug 27 2009

city: College Station

first: Manjunath

last: Hegde

state: TX

address: 524 Jack E. Brown Building TAMU

department: Chemical Engineering

institute: Texas A&M University

summary: It has been proposed that the gastro-intestinal tract environment containing high levels of neuroendocrine hormones is important for gut-derived P. aeruginosa infections. In this study, we report that the hormone norepinephrine increases P. aeruginosa PA14 growth, virulence factor production, invasion of HCT-8 epithelial cells, and swimming motility in a concentration-dependent manner. Transcriptome analysis of P. aeruginosa exposed to 500 µM, but not 50 µM, norepinephrine for 7 h showed that genes involved in the regulation of the virulence determinants pyocyanin, elastase, and Pseudomonas quinolone signal (PQS, 2-heptyl-3-hydroxy-4-quinolone) were up-regulated. The production of rhamnolipids, which are also important in P. aeruginosa infections, was significantly decreased on semi-solid surfaces, but not in planktonic cultures, upon exposure to norepinephrine. Swarming motility, a phenotype that is directly influenced by rhamnolipids, was also decreased upon 500 µM norepinephrine exposure. The increase in the transcriptional activation of lasR, but not that of rhlR, suggest that the effects of norepinephrine are mediated primarily through the las quorum sensing pathway. Together, our data strongly suggests that norepinephrine can play an important role in gut-derived infections by increasing the pathogenicity of P. aeruginosa PA14.

overall_design: For growth rate measurements, a single colony of PA14 was grown overnight in Luria Bertini (LB) broth, and re-inoculated in serum-RPMI medium in the presence of 50 µM and 500 µM NE to an initial turbidity of ~ 0.05 at 600 nm. The turbidity of the cultures at 600 nm was monitored every hour and growth rate of the exponentially growing cultures was calculated. Growth curves were obtained in triplicate using three independent cultures, and the statistical significance of specific growth rate was determined using the unpaired Student’s t-test. Total RNA isolation and microarray analysis PA14 was grown overnight in LB to turbidity at 600 nm of ~5.0 and diluted in 100 mL of serum-RPMI medium to an initial turbidity at 600 nm of 0.05. Different concentrations of NE (50 µM or 500 µM) were added and the cultures grown for 7 hours till late exponential phase (turbidity at 600 nm of ~0.5, 1.1 and 1.5 with 0 µM, 50 µM and 500 µM NE, respectively). Cell pellets were prepared and RNA was isolated as described previously (Ren et al. 2004a). The P. aeruginosa Genome Array (Affymetrix, P/N 510596) containing 5,500 of the 5,570 open reading frames of P. aeruginosa PA01, was used to analyze changes in the PA14 transcriptome. cDNA synthesis, fragmentation and hybridizations were as described previously (Gonzalez Barrios et al. 2006). Hybridization was performed for 16 h at 50°C, and the total cell intensity was scaled to an average value of 500. The probe array images were inspected for any image artifact. Background values, noise values and scaling factors of all the arrays were examined and were comparable. The intensities of polyadenosine RNA control were used to monitor the labelling process. For each binary microarray comparison of differential genes expression, if the gene with the larger transcription rate did not have a consistent transcription rate based on the 13 probe pairs (P-value less than 0.05), these genes were discarded. A gene was considered differentially expressed when the P-value for comparing two chips was lower than 0.05 (to assure that the change in gene expression was statistically significant and that false positives arise less than 5%), and the expression ratio (between cells treated with NE and control) was greater than 4.0 for the 50 µM NE array and 2.0 for 500 µM NE array (based on the standard deviation of fold-change values) (Ren et al. 2004b). Gene functions and locus tag numbers were obtained from the Pseudomonas Genome Database (http://www.pseudomonas.com/download.jsp).

first: Thomas

last: Wood

first: Arul

last: Jayaraman


title: Expression data from early response of Saccharomyces cerevisiae to the antimalarial drug quinine

status: Public on Sep 11 2009

city: Lisbon

first: Sandra

last: Santos

address: Av Rovisco Pais

department: BSRG

country: Portugal

institute: IBB-IST

summary:
In this study, we used the eukaryotic model Saccharomyces cerevisiae to better understand quinine’s mode of action and the mechanisms underlying the cell response to the drug.
We performed a transcriptional profiling of the yeast response to a quinine concentration that exerted a very slight effect over cellular growth.

overall_design: Four arrays, corresponding to two replicates of each condition under study, were analysed.

last: dos Santos

last: Tenreiro

first: Margarida

last: Palma

first: Jorg

last: Becker

first: Isabel

last: Sá-Correia


title: Genes responsive to the addition of spermidine or spermine to a polyamine-deficient Saccharomyces cerevisiae mutant

status: Public on Aug 20 2009

city: Bethesda

first: WeiPing

last: Chen

state: MD

address: Bldg 8, Room 1A11, NIDDK/NIH

department: GCL

institute: NIDDK/NIH

summary: The naturally occurring polyamines putrescine, spermidine or spermine are ubiquitous in all cells. Although polyamines have prominent regulatory roles in cell division and growth, precise molecular and cellular functions are not well established in vivo. In this work we have performed a microarray experiment in a polyamine mutant (delta-spe3 delta-fms1) strain to investigate the responsiveness of yeast genes to supplementation with spermidine and spermine. Expression analysis identified genes responsive to the addition of either excess spermidine (10-5 M) or spermine (10-5 M) compared to a control culture containing 10-8 M spermidine. 247 genes were up-regulated >2-fold, and 11 genes were up-regulated more than 10-fold after spermidine addition. Functional categorization of the genes showed induction of transport related genes, and genes involved in methionine, arginine, lysine, NAD and biotin biosynthesis. 268 genes were down-regulated more than 2-fold, and 6 genes were down-regulated more than 8-fold after spermidine addition. A majority of the down-regulated genes are involved in nucleic acid metabolism and various stress responses. In contrast, only few genes (18) were significantly responsive to spermine. Thus, results from global gene expression profiling demonstrate a more major role for spermidine in modulating gene expression in yeast than spermine.

overall_design: 5 control replicates vs. 3 spermine (SP)-treated or 5 spermidine (SPD)-treated samples.

first: Manas

last: Chattopadhyay

first: Margaret

last: Cam

first: Weiping

first: George

last: Poy

first: Herbert

last: Tabor

first: David

last: Stiles


title: Resistance to hop iso-α-acids in yeast involves active export and vacuolar sequestration

status: Public on Nov 13 2009

city: Delft

first: Jean-Marc

last: Daran

address: Julianalaan 67

department: Department of Biotechnology

country: Netherlands

institute: Delft University of Technology

summary: The hop plant, Humulus lupulus L., contains an exceptionally high content of secondary metabolites, the hop iso-α-acids, which possess a range of beneficial properties including antiseptic action. Studies performed on the mode of action of hop iso-α-acids have hitherto been restricted to lactic acid bacteria. The present study investigates molecular mechanisms of hop iso-α-acid resistance in the model eukaryote Saccharomyces cerevisiae. Growth inhibition occurred at concentrations of hop iso-α-acids that were an order of magnitude higher than those found with hop-tolerant prokaryotes. Chemostat-based transcriptome analysis and phenotype screening of the S. cerevisiae haploid gene deletion collection were used as complementary methods to screen for genes involved in hop iso-α-acids detoxification and tolerance. Further analysis of deletion mutants confirmed that yeast tolerance to hop iso-α-acids involves two major processes: active export of iso-α-acids across the plasma membrane and active proton pumping into the vacuole by the V-ATPase to enable vacuolar sequestration of iso-α-acids. Furthermore, iso-α-acids were shown to affect cellular metal homeostasis by acting as strong zinc and iron chelator.

overall_design: Two complementary genome-wide approaches were employed to investigate cellular responses of S. cerevisiae to hop extracts enriched in iso-α-acids. Microarray transcriptome analysis was performed on chemostat cultures of an S. cerevisiae reference strain grown in the presence and absence of iso-α-acids. In addition, screening of the nearly complete set of yeast open reading frame (ORF) haploid knock-outs generated by the Saccharomyces Genome Deletion Project (SGDP) (Open Biosystems) identified the mutants with increased hop sensitivity. Subsequently, involvement of selected genes and cellular processes in hop acid sensitivity and tolerance was analyzed by construction and detailed analysis of selected mutant strains.

first: Lucie

last: Hazelwood

first: Jack

middle: T

last: Pronk

first: JM


title: Involvement of Snf7 and Rim101 in regulation of TIR1 and anaerobically up-regulated genes in yeast

status: Public on Sep 25 2009

summary: Despite the scientific and applied interest in anaerobic metabolism of Saccharomyces cerevisiae, not all genes whose transcription is up-regulated under anaerobic conditions have yet been linked to known transcription factors. Experiments with a reporter construct in which the promoter of the anaerobically up-regulated TIR1 gene was fused to LacZ revealed a complete loss of anaerobic up-regulation in a snf7Δ mutant. Anaerobic up-regulation was restored by expression of a truncated allele of RIM101 that encodes for a constitutively active Rim101p transcription factor. Analysis of LacZ expression in several deletion mutants confirmed that the effect of Snf7p on anaerobic up-regulation of TIR1 involved Rim101p and did not require a functional multi-vesicular body sorting pathway (in which Snf7p also participates). Transcriptome analysis in anaerobic chemostat cultures revealed that 26 additional genes exhibited a Snf7p/Rim101p dependent anaerobic up-regulation. Since, in its activated form, Rim101p is generally known as a transcriptional repressor, its role in anaerobic up regulation of TIR1 and other ‘anaerobic’ yeast genes must involve additional factors. Further studies with deletion mutants in NRG1, NRG2 and SMP1, which were previously shown to be regulated by Rim101p, showed that these genes were not involved in the regulation of TIR1. However, the aerobic repression mechanism of TIR1 involved the general repressor Ssn6p-Tup1p complex. The physiological relevance of Snf7p/Rim101p-mediated transcriptional up-regulation of several genes in anaerobic yeast cultures was evident from reduced growth of a snf7Δ under anaerobic conditions.

overall_design:
The aim of the present study is to investigate the role of SNF7 in the regulation of TIR1, to assess whether this role involves the Rim101p pathway, and to investigate whether Snf7p and/or Rim101p are also involved in regulation of other ‘anaerobic’ S. cerevisiae genes. After analyzing transcriptional regulation of TIR1 in several genetic backgrounds, a chemostat-based transcriptome analysis was performed on snf7Δ and rim101Δ strains as well as on the isogenic reference strain. Sets of genes that were differentially expressed in the snf7 and rim101 deletion strains were then compared to sets of genes that were previously shown to be transcriptionally up-regulated in anaerobic chemostat cultures of S. cerevisiae (Piper et al., 2002; Tai et al., 2005)
Piper MD, Daran-Lapujade P, Bro C, Regenberg B, Knudsen S, Nielsen J & Pronk JT (2002) Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem 277: 37001-37008.
Tai SL, Boer VM, Daran-Lapujade P, Walsh MC, de Winde JH, Daran JM & Pronk JT (2005) Two-dimensional transcriptome analysis in chemostat cultures. Combinatorial effects of oxygen availability and macronutrient limitation in Saccharomyces cerevisiae. J Biol Chem 280: 437-447.

first: Ishtar

middle: S

last: Snoek

first: Yde

middle: H

last: Steensma

first: jean-Marc


title: Pseduomonas aeruginosa PAO1 exposed to subinhibitory colistin

status: Public on Sep 01 2009

city: Cork

first: Fergal

last: O'Gara

address: UCC, Western Rd

department: Microbiology Dept, Univerisity College Cork

country: Ireland

institute: BIOMERIT Research Centre

summary: Colistin is an important cationic antimicrobial peptide (CAMP) in the fight against Pseudomonas aeruginosa infection within the cystic fibrosis (CF) lungs. The effects of sub-inhibitory colistin on gene expression in P. aeruginosa were investigated by transcriptome microarray and functional analysis.  Analysis revealed an alteration in the expression of 60 genes in total from a variety of pathways.  Genes associated with bacterial chronic colonisation and virulence such as response to osmotic stress, motility, and biofilm formation, as well as those associated with LPS modification and quorum sensing are the most highly represented. Most striking among these is the upregulation of the PQS biosynthesis operon including pqsH, pqsE, and the anthranilate biosynthetic genes phnAB. Early activation of this central component of the QS-network may represent a switch to a more robust population, with increased fitness in the competitive environment of the CF-lung.

overall_design: Three independent cultures of the P. aeruginosa strain PAO1 were exposed to 0.15 µg colistin ml–1.  The untreated and treated samples were grown from OD600 0.05 to 0.8 and subsequently total RNA was extracted using the Ambion RiboPureTm- Bacteria kit according to the manufacturer’s instructions.

first: Joanne

last: Cummins

first: Jerry

last: Reen

first: Christine

last: Baysse

first: Marleis

last: Mooji

last: O' Gara


title: Air-liquid interfacial biofilm vs planktonic S. cerevisiae cells

status: Public on Nov 25 2009

city: Corvallis

first: Alan

last: Bakalinsky

state: OR

address: Wiegand Hall

department: Food Science & Technology

institute: Alan Bakalinsky

summary: Goal was to identify yeast genes whose expression changed as a function of the shift from growth in bulk culture to growth in an air-liquid interfacial biofilm.

overall_design: Cells were grown 24 h at 30°C in YEPD to a density of about 500,000,000 cells/ml. At harvest, sugar was found to be depleted as measured by an enzymatic dip stick (Diastic, Bayer). Cells were pelleted and washed twice in sterile distilled water by centrifugation and then diluted 10-fold into 100 ml of Flor medium (YNB + 4% ethanol + leucine + histidine + uracil) in triplicate 250 ml beakers. Cultures were then grown statically in Flor medium at 27°C. Within a few hours following inoculation, the bulk liquid appeared to be clear, no biofilm was evident by visual observation, and a layer of settled cells was evident at the bottom of the beakers. After 48 h, a visible air-liquid interfacial biofilm covered the entire surface while the thickness of the layer of settled cells appeared unchanged. After 48 h, biofilm cells (“floaters”) were collected by aseptic aspiration. Once the biofilm cells were removed, cells at the bottom of the beaker (“sinkers”) were collected similarly. Cells from both populations were washed once in sterile distilled water by centrifugation prior to RNA isolation. Significant cell clumping was evident in both populations of cells by microscopic observation. While cell viability was estimated by plating on YEPD, the resultant cfu/ml values could not be directly correlated with cell counts in a hemacytometer because clumps of cells containing at least one viable cell presumably produced only a single colony. Further, counting individual cells accurately in the numerous clumps containing large numbers of cells was not possible. Nonetheless, when cells were counted (clumps were counted as single cells) and compared to cfu/ml for the same suspension, the cfu/ml values were about 2-fold higher than the corresponding values for cell counts using the hemacytometer for both biofilm and bottom layer cells.

first: Severino

last: Zara

first: Michael

middle: K

last: Gross

first: Giacomo

first: Marilena

last: Budroni


title: Expression data from Pseudomonas aeruginosa (wild type and lasRrhlR mutant strains) exposed to human neutrophils

status: Public on Aug 04 2009

city: Copenhagen

first: Morten

last: Alhede

address: Blegdamsvej 3B

department: International Health, Immunology and Microbiology

country: Denmark

institute: University of Copenhagen

summary:
In the present in vitro study, interactions between P. aeruginosa (sessile biofilms as well as planktonic cells) and PMNs were analyzed by means of DNA microarray based transcriptomics. We found that the P. aeruginosa wild type biofilms, in contrast to planktonic cultures and quorum sensing (QS) deficient strains, respond to PMN exposure in a rather aggressive manner. The response does not involve protective mechanisms such as those involved in oxidative stress. Rather it is dominated by QS controlled virulence determinants such as those encoded by pqs, phz, rhlAB, all of which are designed to cripple Eukaryotic cells including PMNs and macrophages. Our comparative analysis supports the view that QS plays a major role in mechanisms by which P. aeruginosa evades host defense systems.
Keywords: Stress response

sample_organism:
Pseudomonas aeruginosa
Pseudomonas aeruginosa PAO1

overall_design: The biofilms were allowed to grow and develop in the biofilm flow chambers for 3 days before challenge with PMNs. Fresh PMNs from human volunteers were isolated and resuspended in RPMI-1640 medium (BIOCHROM AG) as previously described by Bjarnsholt et al. 2005 (Bjarnsholt et al., 2005). Prior to PMN injection the flow was halted. Ten million PMNs were resuspended in 6 ml of RPMI media and injected into the flow chamber. The concentration of PMNs was found by microscopy to be approximately 1 PMN per 1000 bacterial cells. PMNs and biofilm were incubated for 2 hours. The flow chambers were rolled every 15 minutes to ensure that the entire biofilm was exposed to the PMNs. After incubation the fluid inside the chamber was discarded and the attached biofilm cells were loosened by rolling. The cells were then mechanically removed and collected in 6 ml of RNAlater®

first: Thomas

last: Bjarnsholt

first: Peter

middle: O

last: Jensen

first: Matt

last: Parsek

first: Niels

last: Høiby

last: Bovbjerg Rasmussen

last: Givskov


title: Molecular mechanisms of freeze injuries in commercial baker’s yeast

status: Public on Jul 31 2009

city: Tsukuba

first: Satomi

middle: Mizukami

last: Murata

address: Kannondai 2-1-12

institute: National Food Research Institute

summary:
Frozen dough baking is useful method in the modern bread-making industry.  However, the fermentation activity of baker’s yeast dramatically decreased after thawing due to freeze injuries, because baker’s yeast cells contained in dough experience freeze injuries during freeze-thaw processes.  Here, we performed genome-wide expression analysis to determine genetic response in baker’s yeasts under freeze-thaw condition using a DNA microarray analysis.  Functional and clustering analyses in gene expression reveal that genes could be characterized by the term of freeze-thaw stress.  Under short-term freeze stress (freeze treatment for 3 day), genes involved in ribosomal protein were up-regulated.  Under long-term freeze stress (freeze treatment for longer than 7 day), genes involved in energy synthesis were up-regulated.  In each phase, genes involved in protein damage, several stresses and trehalose and glycogen metabolism were also up-regulated.  Through these freeze stress, yeast cells may improve reduced efficiency of translation and enhanced cell protection mechanism to survive under freeze stress condition.  These regulations of these genes would be controlled by the cAMP-protein kinase A pathway.
Keywords: baker’s yeast, freeze-thaw stress, gene expression, freezing period

overall_design: All experiments were done in duplicate from two independent samples.

first: Jun

last: Shima


title: Gene expression profiling of patients with allergy to latex and/or vegetable food

status: Public on Jul 30 2009

first: Nathalie

last: Saulnier

summary:
Background: The prevalence of individuals allergic to latex, exhibiting cross-hypersensitivity with plant-derived food has been frequently reported as the so-called latex-fruit syndrome. Nonetheless, molecular mechanisms underlying allergy to latex and/or fruit are poorly understood.

Objective: The aims of this study were to identify candidate genes that may be associated with the pathogenesis of allergy to latex and /or vegetable food, and to assess if similar molecular pathways  are involved in both types of hypersensitivity.

Methods: DNA microarray analysis was performed to screen the molecular profiles of peripheral blood mononuclear cells isolated from patients with allergy to latex, to fruit, or with latex-fruit syndrome, and from control healthy subjects.

Results: Gene expression profiling identified an overlapping dataset of genes commonly regulated in all the atopic patients enrolled in this study, suggesting that similar molecular mechanisms are involved in the pathogenesis of allergy to the fruit and /or latex. Several regulators of the innate and acquired immunity reported to polarize the immunological response towards a Th2-mediated immune response were overexpressed in patients. Furthermore, evidences suggested that T regulatory cell expression might be defective in allergic patients, as a consequence of a dysregulation of some inflammatory cytokines. Finally, several  transcription factors that may be responsible for the Th1/Th2 imbalance were modulated in allergic patients.

Conclusion and clinical implications: This study identified  relevant genes that may help to elucidate the molecular mechanisms underlying allergic disease. Knowledges of critical targets, along with  transcription factors regulating gene activity may facilitate the development of new therapies.

overall_design:
This study aimed at the understanding of the molecular pathways involved in allergy to latex and fruit. As latex and vegetable food allergens cross-react, it could be hypothesized that similar pathways are involved in both types of hypersensitivity.

For this purpose, patients allergic to latex (n=6), allergic to vegetable food (n=5), or suffering from latex-fruit syndrome (n=6) were enrolled from the Unit of Allergy of the Gemelli Hospital of Rome. Moreover, 4 healthy volunteers were added to this study. Five ml of blood were harvested from each individual, and PBMCs were isolated on ficoll gradient. Following sample preparation (as recommended by the manufacturer), each sample was hybridized on Affymetrix human focus array. Data were processed with Affymetrix MAS 5.0 software and averaged intensity of signals from biological replicates were calculated for further analysis.

The CEL files for this study were lost in a computer crash.

first: Eleonora

last: Nucera

first: Giorgia

last: Altomonte

first: Antonio

last: Gasbarrini

first: Giampiero

last: Patriarca


title: Gene expression from CEPH-Utah,CHB+JPT, and YRI individuals to examine gene expression correlation

status: Public on Aug 13 2009

first: Renuka

last: Nayak

address: 3516 Civic Center Blvd

institute: HHMI, University of Pennsylvania

summary: We used microarrays to examine gene expression levels in individuals from different populations (cephgp, chb, jpt, yri). The chb and jpt populations are combined in the analysis.

overall_design: We measured gene expression levels in 295 lymphoblastoid cell lines representing 4  ethnically-distinct populations.  For some cell lines, RNA extracts were hybridized twice, representing technical replicates. For these cell lines, we take the average signal intensity as a measure of relative abundance of a gene.

last: Kearns

first: Richard

last: Spielman

first: Vivian

middle: G

last: Cheung


title: Bio-electrospraying the nematode Caenorhabditis elegans

status: Public on Jul 02 2009

city: London

first: Stephen

last: Sturzenbaum

address: 150 Stamford Street

department: Analytical, Environmental and Forensic Sciences

country: United Kingdom

institute: King's College London

summary: Bio-electrospray, the direct jet-based cell handling apporach, is able to handle a wide range of cells. Studies at the genomic, genetic, and the physiological level have shown that, post-treatment, cellular integrity is unperturbed and a high percentage (>70%, compared to control) of cells remain viable. Although, these results are impressive, it may be argued that cell based systems are oversimplistic. This study utilizing  a well characterised multicellular model organism, the non-parasitic nematode Caenorhabditis elegans. Nematodes were subjected to bio-electrosprays  to demonstrate that bio-electrosprays can be safely applied to nematodes.

overall_design: The global  transcriptomic response was compared  in control nematodes (negative control),  heat shocked nematodes (positive control). and nematodes subjected to bio-electrospray.

first: Napachanok

last: Mongkoldhumrongkul

first: Suresh

last: Swain

first: Suwan

last: Jayasinghe

middle: R


title: Comparison of gene expression between wild type (N2) and hlh-30(tm1978) mutant worms

status: Public on Jul 24 2009

city: Worcester

first: Marian

last: Walhout

address: 364 Plantation St - LRB#605

institute: UMASS Medical School

summary: The hlh-30 gene encodes a C. elegans basic-helix-loop-helix (bHLH) transcription factor; We compared RNA from wild type worms and worms mutant for the hlh-30 gene to identify putative target genes of the HLH-30 transcription factor.

overall_design: Total RNA from three biological replicates were isolated from embryos, L1 larvae, and L2 larvae from both wild type and hlh-30(tm1978) mutant worms

first: Christian

middle: A

last: Grove

first: Inmaculada

last: Barrasa

first: Federico

last: De Masi


title: Transcriptome analysis of the roxSR and anr mutant strains of Pseudomonas aeruginosa under aerobic conditions

city: Bunkyo-ku

first: Hiroyuki

last: Arai

state: Tokyo

address: Yayoi 1-1-1

institute: The University of Tokyo

summary: To assess the role of two redox-sensitive transcriptional regulators, RoxSR and ANR, in Pseudomonas aeruginosa under aerobic conditions, microarray analysis was performed. Transcriptome profiles of roxSR mutant and anr mutant aerobically grown in LB medium were determined by Affymetrix GeneChip at both the exponential phase and early stationary phase and compared to that of the wild type strain.

overall_design: Pseudomonas aeruginosa wild type (PAO1ut), roxSR mutant (ROX1), and anr mutant (PAO6261) strains were cultivated aerobically in LB in Erlenmeyer flasks, and total RNAs were extracted at both the exponential phase (OD600 = 0.3) and early stationary phase (OD600 = 1.4). The experiment was performed in duplicate independent cultures.

first: Takuro

last: Kawakami

first: Miho

last: Kuroki

first: Masaharu

last: Ishii

first: Yasuo

last: Igarashi


title: The regulation of reserve carbohydrate metabolism in S cerevisiae in response to nutrient availability

first: Jean-Marc

last: Daran

summary: In Saccharomyces cerevisiae, glycogen and trehalose are important reserve carbohydrates that accumulate under nutrient limitation in batch cultures. An inherent draw-back of batch studies is that specific growth rate and substrate and product concentrations are variable over time and between cultures. The aim of this present study was to identify the nutritional requirements associated with high accumulation of reserve carbohydrates at a fixed specific growth rate (0.10 h-1) in anaerobic chemostat cultures that were limited by one of five different nutrients (carbon, nitrogen, sulfur, phosphorus or zinc). Reserve carbohydrates accumulation is not a general response to nutrient limitation. Over the conditions tested, accumulation occurs essentially under nitrogen (and to a lesser extent carbon) limited conditions. This was confirmed by the transcriptional profile of the genes involved in trehalose biosynthesis. We show that the transcriptional induction of both glycogen and trehalose biosynthesis genes was to a large extent driven by the regulator Msn2/4. However, the main regulatory control of glycogen biosynthesis was post-translational. Under nitrogen limitation, the ratio of glycogen synthase over glycogen phosphorylase increased up to eight-fold, thus enabling an increased flux towards glycogen biosynthesis.

overall_design: We studied this in anaerobic chemostat cultures at a dilution rate of 0.10 h-1 where growth was limited by five different nutrients (carbon, nitrogen, sulfur, phosphorus or zinc limitations). In addition, we studied the expression of these pathways at transcriptional and post-transcriptional levels and assessed the role of Msn2/4 in mediating transcriptional induction of glycogen and trehalose genes in the absence of stress.

first: Lucie

last: Hazelwood

first: Jack

last: Pronk


title: C. elegans fer-1 mutant gene expression profile

status: Public on Jun 25 2009

city: Philadelphia

first: Predrag

last: Krajacic

address: 3700 Hamilton Walk A700 Richards

institute: University of Pennsylvania

summary:
In vertebrate muscle, loss of Dysferlin results in the activation of compensatory muscle gene  expression, even at pre-pathological stages. We hypothesized that if C. elegans  fer-1 is also expressed in muscle, then fer-1 mutant worms might also exhibit  compensatory muscle gene expression.
To test this hypothesis, we used Affymetrix microarrays to profile gene expression from synchronized wild type and fer-1 mutant  adults. To improve the specificity of this approach, we profiled two well characterized loss-of-function fer-1 mutants (hc1ts and hc24ts) and considered genes that changed similarly in both mutants as fer-1-regulated transcripts.

overall_design: For each genotype, six individual replicates were performed and were hybridized to Affymetrix C. elegans Genechips using the manufacturer’s  recommended protocols. The best four preparations (as determined by the overall Pearson Correlation within a  genotype) were used for RNA labeling and hybridization.

first: Tejvir

last: Khurana

first: Todd

last: Lamitina


title: Monocytic U937 cells and receptor cross-linking

status: Public on Jul 20 2009

city: Singapore

first: Jayapal

last: Manikandan

country: Singapore

institute: NUS

summary:
Monocytic U937 cell lines and crosslinked CD32 & CD64 at different time points
Keywords: time-course

first: Manikandan

last: Jayapal

first: Pushparaj

last: Peter Natesan


title: Transcriptomic analysis of the sulfate starvation response in Pseudomonas aeruginosa

status: Public on Jun 22 2009

city: Manchester

first: Michael

middle: A.

last: Kertesz

address: Oxford Rd

department: Faculty of Life Sciences

institute: University of Manchester

summary:
Gene expression in response to changes in sulfur supply was studied in P. aeruginosa E601, a cystic fibrosis isolate that displays mucin sulfatase activity, and in P. aeruginosa PAO1. A large family of genes was found to be upregulated by sulfate limitation in both isolates, encoding sulfatases and sulfonatases, transport systems, oxidative stress proteins, and a sulfate-regulated TonB/ExbBD complex. These genes were localized in five distinct islands on the genome, and encoded proteins with a significantly reduced content of cysteine and methionine. Growth of P. aeruginosa E601 with mucin as sulfur source led to a sulfate starvation response, but also to induction of genes involved with type III secretion systems.
Keywords: starvation response, strain comparison

overall_design: Gene expression in exponential-phase cells was analysed using Affymetrix arrays. Sulfur was supplied either as sulfate, or as the non-sulfate S sources cyclamate (sodium cyclohexylsulfamate) and mucin (human colon cell line mucin LS174T). Control experiments were also done with combinations of these sulfur sources. Studies with mucin as sulfur source were carried out with P. aeruginosa E601 (a CF isolate with mucin sulfatase activity) and comparative studies were performed with P. aeruginosa PAO1. Experiments with strain E601 were done in 3-4 fold replication, experiments with strain PAO1 with 2-3 fold replication.

first: Tewes

last: Tralau

first: Stephane

last: Vuilleumier

first: Christelle

last: Thibault

first: Barry

last: Campbell

first: Anthony

last: Hart


title: Transcriptional changes due to the [GAR+] prion

status: Public on Jul 01 2009

city: Cambridge

first: Jessica

middle: C. S.

last: Brown

state: MA

address: 9 Cambridge Center

department: biology

institute: Whitehead/MIT

summary: Comparison between [gar-] and [GAR+] samples of the W303 background grown in 2% glucose.

overall_design: Three separate [gar-] and [GAR+] cultures were grown to late exponential phase (OD600~0.8) prior to phenol-chloroform extraction.  Biological replicates are [gar-] samples #1, 2, and 3 and [GAR+] samples #1, 2, and 3.  [gar-] and [GAR+] sample sets were compared to determine transcriptional differences.

first: Susan

last: Lindquist


title: Caenorhabditis elegans as a Genomic Model for Toxicology

status: Public on Jun 03 2009

city: Ft. Detrick

first: John

last: Lewis

address: 568 Doughten Drive

department: Biomarkers Program

institute: U.S. Army Center for Environmental Health Research

summary:
Effective toxicological testing of the vast number of new and existing chemicals currently in use will require efficient and cost effective methods. We evaluated the utility of a simple, low cost toxicity testing system employing the nematode Caenorhabditis elegans to identify toxicologically relevant changes in gene expression.
Dichlorvos and fenamiphos, which are organophosphorous pesticides that inhibit acetylcholinesterase were chosen as model toxicants to test the usefulness of the C. elegans toxicity testing system, and mefloquine, which appears to perturb neuronal Ca++ homeostasis, provided an out-group for analysis.

Keywords: gene expression array-based (RNA / in situ oligonucleotide)

overall_design: Concentrations of dichlorvos, fenamiphos, or mefloquine sufficient to prevent the development of 10%, 50%, or 90% of mid-vulval L4 worms to the early gravid adult stage at 24 h after exposure were determined in range finding experiments. For definitive experiments, mid-vulval L4 worms were exposed to one of the foregoing concentrations of dichlorvos, fenamiphos, or mefloquine for 8h. Exposures were performed in triplicate.

first: Maria

last: Szilagyi

first: Elizabeth

last: Gehman

first: William

middle: E

last: Dennis

first: David

last: Jackson


title: Expression data from sequential P. aeruginosa cystic fibrosis (CF) isolates

status: Public on May 26 2009

city: Munich

first: michael

last: Hogardt

state: Bavaria

address: Marchioninistreet 17

department: Bacteriology

country: Germany

institute: Max von Pettenkofer-Institut

summary:
To provide a more detailed survey of adaptive changes in the physiology of P. aeruginosa (PA) during long-term infection of the cystic fibrosis (CF) lung, we performed a comparative proteome and transcriptome analysis of a set of isogenic sequential non-mutator and mutator isolates from three selected CF patients. Recently, we showed that during CF lung persistence PA mutators converge to a virulence-attenuated phenotype. In this study, we demonstrate that besides virulence-associated traits (VATs) the adaptation process of PA predominantly comprises metabolic pathways. In end-stage mutator strains, transcripts of genes encoding VATs, chemotaxis, degradation of aromatic compounds and several two-component regulatory systems were decreased. In contrast, several transcripts of genes or proteins involved in metabolism of fatty acids, nucleotides, amino acids and the generation of energy were increased. Of particular interest is the increased expression level of genes involved in (i) the anaerobic arginine-deiminase pathway, (ii) the anaerobic respiration such as nitrate-uptake protein OprF, redox-active azurin and cytchrome c551 peroxidase, (iii) the micro-aerobic respiration such as high oxygen-affinity cytochrome oxidase cbb3 (iv) the tricarboxylic acid cycle (TCA), glyoxylate shunt and anaplerotic carboxylation reactions to oxaloacetate. Strikingly, an increased transcription of the anaerobic regulator gene anr correlates with the up-regulation of ANR-dependent genes. In conclusion, these changes in transcriptome and proteome indicate an adaptive shift towards constitutive expression of genes of metabolic pathways obviously required for growth under micro-aerobic and nutritional conditions of suppurative CF lung tissue. Finally, these results provide us with new targets for antimicrobial agents and biomarkers reflecting adaptation of PA towards progressive CF lung disease.
Keywords: in vitro study/interstrain comparison/clinical isolates/early nonmutator vs. late mutator; variable time point of isolation from cf respiratory secretions

overall_design:
P. aeruginosa isolates recovered from different time points of chronic cystic fibrosis lung disease were cultered in vitro, harvested for RNA extraction and hybridization on Affymetrix microarrays. We compared the transcriptome (triplicate microarrays) of early non-mutator P. aeruginosa isolates with late mutator isolates with high mutation frequency probably the driving force of an efficient adaptation to changing environements to conclude from differences in gene expression to the requirements of CF lung environment.
Second publication of array data to be added later

first: Christina

last: Hoboth

first: Reinhard

last: Hoffmann

first: Anja

last: Eichner


title: DNA microarray analyses for HepG2 cells exposed to silver nanoparticles

status: Public on Jun 01 2009

city: Sapporo

first: Akinobu

last: Ito

address: North-13, West-8, Kitaku

department: Graduate School of Engineering

institute: Hokkaido University

summary: From the result of the gene expression analyses of human hepatoma cell line, HepG2, a number of genes associated with cell proliferation and DNA repair were distinctively up-regulated by Ag-nanoparticle exposure, suggesting that Ag-nanoparticles might stimulate cell proliferation and DNA damage, which are considered to be mechanisms playing an important role for carcinogenesis and tumor progression.  The inductions of these genes involved in cell proliferation were also observed in PS-nanoparticles and Ag2CO3-exposed cells.  In addition, the inductions of DNA repair-associated genes were also observed in Ag2CO3-exposure.  These results suggest that both “nanoshape” and “silver” can cause the inductions of these gene expression patterns.  Furthermore, cysteine, a strong ionic silver ligand partially abolished these gene expressions induced by silver nanoparticles.  Ionic silver sourced from Ag-nanoparticles could not fully explain these gene expressions.

overall_design: In this study, we examined the gene expression alteration in human hepatoma cell line, HepG2 following exposures of silver nanoparticles (7-10 nm), polysthylene nanoparticles (15 nm) and silver carbonate using DNA microarray with 8795 human genes. Furthermore, we also performed the DNA microarray analyses for the cells exposed to silver nanoparticle in the presence of 5mM N-acetyl-L-cysteine to examine the contribution of silver ion to silver nanoparticle exposure.

first: Koji

last: Kawata

first: Masato

last: Osawa

first: Satoshi

last: Okabe


status: Public on May 23 2009

city: College Station

first: Akihiro

last: Ueda

address: 3122 TAMU

institute: Texas A&M University

overall_design: Microarray analysis for the biofilm cells of Pseudomonas aeruginosa PA14 wild-type vs the tpbA (PA14_13660) mutant in LB medium at 4 and 7 h at 37C.

first: Thomas

last: Wood


title: TheRole of Rad23/Rad4 protein complex in transcription and DNA repair in yeast

city: Cardiff

first: Julia

last: Smirnova

address: Tenovus building Ground Floor

department: Cardiff School of Medicine

institute: University of Cardiff

summary: The Rad23/Rad4 protein complex plays a major role in DNA damage recognition during nucleotide excision repair (NER) in yeast. We recently showed that two distinct pathways contribute to efficient NER in yeast. The first operates independently of de novo protein synthesis and requires a nonproteolytic function of the 19S regulatory complex of the 26S proteasome and Rad23. The second pathway requires de novo protein synthesis, and relies on the activity of a newly identified E3 ubiquitin ligase that ubiquitinates Rad4 in response to UV. Surprisingly, we found that cells deleted of either Rad23 or Rad4 caused reduced Rad4 and Rad23 mRNA levels respectively. We considered the possibility of an unexpected role of Rad23 and Rad4 in regulating the expression of genes involved in the transcriptional response to DNA damage. Gene expression profiling has suggested that Rad23 and Rad4 may function as a complex to affect transcription of a small subset of genes in response to UV damage. To determine how Rad4 and Rad23 contribute to the regulation of these genes, we have examined the occupancy of Rad4/Rad23 in their promoter regions by chromatin immunoprecipitation (ChIP), both in the presence and absence of UV damage. Our preliminary ChIP data suggests that the Rad4/Rad23 complex regulates a set of genes in response to UV light.

overall_design: We perform arrays on Rad4 single, Rad23 single and Rad4/23 double mutants. Each set has 3 biological repeats. All samples are untreated, mRNA prepared from logarithmically growing cultures.

first: Simon

last: Reed

first: Zheng

last: Zhou


title: Ecotoxicogenomic analysis on the soil nematode Caenorhabditis elegans exposed to BPA, DEHP and NP

status: Public on Feb 28 2009

city: seoul

first: jinhee

last: choi

address: 90 Jeonnong-dong, Dongdaemun-gu

department: College of Urban Science

institute: University of Seoul

summary:
Gene expression analysis was conducted on the wildtype Caenorhabditis elegans exposed to bisphenol A (BPA), di(2-ethylhexyl) phthalate (DEHP) and nonylphenol (NP) using whole genome microarray. The microarray study was conducted in an ecotoxicological context, by investigating the  response of global gene expression with that of classical toxicological endpoints, such as, mortality, growth, reproduction and development. Results provide insight into global transcription response of C.elegans to these endocrine disrupting chemicals exposure and also contribute to enhance the potential of C.elegans microarray in ecotoxicology (ecotoxicogenomics).
key word : ecotoxicogenomics

overall_design: Young adults of wildtype C.elegans from age-synchronized population were exposed to NP, DEHP and BPA at the concentrations equivalent to 1/100 of 24h LC50 of each chemical, for 24 h and RNA extraction and hybridization were subsequently conducted using C.elegans Genome array (Affymetrix).

first: Jinhee

last: Choi


title: A novel extracytoplasmic function (ECF) sigma factor regulates virulence in Pseudomonas aeruginosa

status: Public on Apr 21 2009

city: Amsterdam

first: Marian

last: Llamas

address: van der Boechorststraat 7

department: Medical Microbiology

institute: VU Medical Center

summary: Next to the two-component and quorum sensing systems, cell-surface signaling (CSS) has been recently identified as an important regulatory system in Pseudomonas aeruginosa. CSS senses signals from outside the cell and transmits them into the cytoplasm. It consists of a TonB-dependent outer membrane receptor, a cytoplasmic membrane-localized sigma factor regulator (or anti-sigma factor), and an extracytoplasmic function (ECF) sigma factor. Upon perception of the extracellular signal by the receptor the ECF sigma factor is activated and promotes the transcription of a specific set of gene(s). Although most P. aeruginosa ECF sigma factors are involved in the regulation of iron uptake, we have identified a novel ECF sigma factor (PA0675) involved in the regulation of virulence. By microarray analysis of cells overexpressing PA0675 from the pMUM3 plasmid we have identified the genes regulated by this sigma factor.

overall_design: Two different samples are analyzed namely P. aeruginosa (pMMB67EH) (control/reference sample) and P. aeruginosa (pMUM3) (overexpressing the PA0675 ECF sigma factor). Two different replicates per sample are included.

first: M

first: W

last: Bitter


title: mRNA expression data in Δrsf1mutant during growth on, and transition to growth on glycerol as sole carbon source

status: Public on May 29 2009

city: Detroit

first: George

middle: Gordon

last: Roberts III

state: MI

address: 540 E. Canfield

department: Immunology/Microbiology

institute: Wayne State University

summary:
Rsf1p is a putative transcription factor required for efficient growth using glycerol as sole carbon source but not for growth on the alternative respiratory carbon source ethanol.
We use microarrays to determine the differences in the transcriptional program between the Δrsf1 mutant and the wild type during respiratory growth on glycerol as well as the transition to growth on glycerol as sole carbon source.
Keywords: Mutant analysis during timecourse following switching carbon source from dextrose to glycerol

overall_design: Δrsf1or the isogenic parent strain were grown to early log (A600=0.6) in YPD and then washed twice in prewarmed YPG (30ºC) and returned to the air shaker in YPG for 15, 30 or 60 minutes. "Limit" conditions were provided by harvesting cells grown in YPD to early log phase without shifting to YPG and by harvesting cells grown in YPG to early log phase. Since the Δrsf1 mutant and its isogenic parent strain grow equally well on the respiratory carbon source ethanol, cells were also harvested after being grown in ethanol to early log phase.

last: Roberts

first: Alan

middle: P

last: Hudson


title: DCC binding and function (Expression Analysis)

status: Public on Mar 01 2009

city: Berkeley

first: Barbara

middle: J.

last: Meyer

address: 16 Barker Hall #3204

department: MCB

institute: HHMI/UCB

summary:
In many species, a dosage compensation complex (DCC) is targeted to X chromosomes of one sex to equalize levels of X gene products between males (1X) and females (2X).  Here we identify cis-acting regulatory elements that target the C. elegans X chromosome for repression by the DCC.  The DCC binds to discrete, dispersed sites on X of two types.  rex sites recruit the DCC in an autonomous, DNA sequence-dependent manner using a 12 bp consensus motif that is enriched on X.  This motif is critical for DCC binding, is clustered in rex sites, and confers much of X-chromosome specificity.  Motif variants enriched on X by 3.8-fold or more are highly predictive (95%) for rex sites.  In contrast, dox sites lack the X-enriched variants and cannot bind the DCC when detached from X.  dox sites are more prevalent than rex sites and, unlike rex sites, reside preferentially in promoters of some expressed genes.  These findings fulfill predictions for a targeting model in which the DCC binds to recruitment sites on X and disperses to discrete sites lacking autonomous recruitment ability.  To relate DCC binding to function, we identified dosage-compensated and non-compensated genes on X.  Unexpectedly, many genes of both types have bound DCC, but many do not, suggesting the DCC acts over long distances to repress X gene expression.  Remarkably, the DCC binds to autosomes, but at far fewer sites and rarely at consensus motifs.  DCC disruption causes opposite effects on expression of X and autosomal genes.  The DCC thus acts at a distance to impact expression throughout the genome.

Keywords: dosage compensation, condensin, X chromosome, gene expression, epigenetics C. elegans

overall_design: For microarray analysis, the nematode strains and number of experiments was as follows: TY2222, her-1(hv1y101); xol-1(y9) sdc-2(y74) unc-9(e101), XO embryos (8 biological replicas and 6 wild-type XX embryos controls ), dpy-27(y57) XX embryos (3 biological replicas and 3 wild-type XX embryos controls), and sdc-2(y93, RNAi) XX embryos (3 biological replicas and 3 wild-type XX embryos controls).

first: Judith

last: Jans

first: John

middle: M

last: Gladden

first: Edward

last: Ralston

first: Catherine

last: Pickle

first: A

last: Michel

first: Rebecca

last: Pferdehirt

first: Michael

middle: B

last: Eisen


title: Ecotoxicity of silver nanoparticles on the soil nematode Caenorhabditis elegans using functional ecotoxicogenomics

summary:
Gene expression analysis was conducted on the wildtype Caenorhabditis elegans exposed to silver nanoparticles (AgNPs) using whole genome microarray. Differentially expressed genes from the microarray were selected for the quantitative analysis. The microarray study was conducted in an ecotoxicological context: the integration of gene expression with organism and population level endpoints (survival, growth, reproduction) was investigated, to test the ecotoxicological relevance of AgNPs-induced gene expression. Results provide insight into the global transcription response of C.elegans to AgNPs exposure and also contribute to enhance the potential of C.elegans microarray in ecotoxicology (ecotoxicogenomics).

key word: ecotoxicogenomics

overall_design: Young adults of wildtype C.elegans from age-synchronized population were exposed to 0.1mg/L of AgNPs for 24 h and RNA extraction and hybridization were subsequently conducted using C.elegans Genome array (Affymetrix).


title: Transcriptional response of Drosophila cells to FHV infection (infection experiment)

status: Public on Apr 01 2009

city: Ann Arbor

first: David

last: Miller

address: 3560B MSRB II, 1150 W. Medical Center Dr.

department: Internal Medicine

institute: University of Michigan

summary:
Virus infections induce cellular gene up and down regulation, and these changes often provide clues to cellular pathways utilized by viruses.
We used microarrays to examine the transcriptional responses of cultured Drosophila S2 cells to infection with Flock House virus (FHV).

overall_design: Cultured S2 cells were infected with FHV at an MOI of 10 and we measured global transcript levels at 12 h after infection compared to control mock infected cells using Affymetrix Drosophila Genome 1.0 microarray chips.

middle: J