title: Ex vivo transcriptional profiling reveals a common set of genes important for the adaptation of Pseudomonas aeruginosa to chronically infected host sites

status: Public on Nov 14 2012

city: Braunschweig

first: Piotr

last: Bielecki

address: Inhoffenstr 7

department: Cell Biology

institute: Helmholtz Centre for Infection Research

summary: The opportunistic bacterium Pseudomonas aeruginosa is a major nosocomial pathogen causing both devastating acute and chronic persistent infections. During the course of an infection, P. aeruginosa rapidly adapts to the specific conditions within the host. In the present study, we aimed at the identification of genes that are highly expressed during biofilm infections such as in chronically infected lungs of patients with cystic fibrosis (CF), burn wounds and subcutaneous mouse tumours. We found a common subset of differentially regulated genes in all three in vivo habitats and evaluated whether their inactivation impacts on the bacterial capability to form biofilms in vitro and to establish biofilm-associated infections in a murine model. Additive effects on biofilm formation and host colonization were discovered by the combined inactivation of several highly expressed genes. However, even combined inactivation was not sufficient to abolish the establishment of an infection completely. These findings can be interpreted as evidence that either redundant traits encode functions that are essential for in vivo survival and chronic biofilm infections and/or bacterial adaptation is considerably achieved independently of transcription levels. Supplemental screens, will have to be applied in order to identify the minimal set of key genes essential for the establishment of chronic infectious diseases.

overall_design: Ex-vivo samples were collected from burn wound, cystic fibrosis patients mucous and mice tumor and stabilized for the RNA extraction. Recovered clinical strains were cultivated in rich medium control conditions untill the early stationary phase and RNA was extracted. The murine tumors were infected with burn wound isolated strains

first: Uliana

last: Komor

first: Agata

last: Bielecka

first: Mathias

last: Musken

first: Jacek

last: Puchalka

last: Pletz

first: Manfred

last: Ballmann

first: Vitor

last: Martins dos Santos

first: Siegfried

last: Weiss

first: Susanne

last: Häussler


title: Gene expression changes in response to aging compared to heat stress, oxidative stress and ionizing radiation in Drosophila melanogaster

status: Public on Nov 19 2012

city: Los Angeles

first: John

last: Tower

state: California

address: 1020 Childs Way, RRI213

department: Biological Sciences

country: USA

institute: University of Southern California

summary: Gene expression changes in response to aging, hyperoxia, hydrogen peroxide, ionizing radiation, and heat stress were compared using microarrays.  While aging shared features with each stress, aging was more similar to the stresses most associated with oxidative stress (hydrogen peroxide, hyperoxia, ionizing radiation) than to heat stress.  Aging is associated with down-regulation of numerous mitochondrial genes, including electron-transport-chain (ETC) genes and mitochondrial metabolism genes, and a sub-set of these changes was also observed upon hydrogen peroxide stress and ionizing radiation stress. Aging shared the largest number of gene expression changes with hyperoxia.  The extensive down-regulation of mitochondrial and ETC genes during aging is consistent with an aging-associated failure in mitochondrial maintenance, which may underlie the oxidative stress-like and proteotoxic stress-like responses observed during aging.

overall_design: Thirty five sample of RNA including Stress Controls (4 replicates), Heat Stress (3 replicates), Ionizing Radiation (4 replicates), Sugar (4 replicates), Hydrogen Peroxide (4 replicates), Young Controls (6 replicates) , Hyperoxia (6 replicates) and  Old (4 replicates) adult Drosophila were analysed by Affymetrix microarrays. Stress Controls were used as controls for Heat Stress, Ionizing Radiation, Sugar and Hydrogen Peroxide samples. Young Controls were used as controls for Hyperoxia and Old samples. All flies were male progeny of a cross between Oregon-R wildtype and transgenic strain w[1118];P{w+ rtTA}(3)[E2]/ TM3. Flies lacking the balancer but bearing the transgene were used.

first: Gary

middle: N

last: Landis

first: Jie

last: Shen


title: Expression data in C. elegans L2 larvae after nhr-23 inhibition and in controls

status: Public on Aug 29 2012

city: Bethesda

first: WeiPing

last: Chen

state: MD

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

department: GCL

institute: NIDDK/NIH

summary: NHR-23, a conserved member of the nuclear receptor family of transcription factors, is required for normal development in C. elegans where it plays a critical role in growth and molting. In a search for NHR-23 dependent genes, we performed whole genome comparative expression microarrays on both control and nhr-23 inhibited synchronized larvae. Genes that decreased in response to nhr-23 RNAi included several collagen genes. Unexpectedly, several hedgehog-related genes were also down-regulated after nhr-23 RNAi. A homozygous nhr-23 deletion allele was used to confirm the RNAi knockdown phenotypes and the changes in gene expression. Our results indicate that NHR-23 is a critical coregulator of functionally linked genes involved in growth and molting and reveal evolutionary parallels among the ecdysozoa.

overall_design: Synchronized populations of L1 larvae were plated with two sets of HT115 bacteria, one that had been transformed with the RNAi vector only (L4440 plasmid) and another that had been transformed with a vector targeting nhr-23 (clone 5174) [Proc Natl Acad Sci USA 98 (2001) 7360-7365].  Worms were kept on 2% agarose plates for 21 hr at 20C, collected, and approximately 200ml of worms resuspended in PBS were used in each individual experiment.  Total RNA was isolated from frozen pellets using a Mixer-Mill (Miller-Mill 300) following an RNeasy Mini Kit (Qiagen, Germantown, MD) according to manufacturer protocol.  Aliquots of cultures used for RNA isolation were kept on nhr-23 RNAi plates to confirm the knockdown phenotypic changes occurred during subsequent molts.

first: Nathaniel

last: Kouns

first: Johana

last: Nakielna

first: Frantisek

last: Behensky

first: Michael

middle: W

last: Krause

first: Zdenek

last: Kostrouch

first: Marta

last: Kostrouchova


title: Natural genetic variation in yeast longevity

status: Public on Jul 18 2012

city: New Orleans

first: jianliang

last: dai

address: 1430 tulane ave

department: Tulane Center for Aging

institute: Tulane University

summary: A quantitative genetic analysis of the yeast replicative life span was carried out by sampling the natural genetic variation

overall_design: Genomic DNA was extracted from 39 recombinant lines from a cross between strains S96 and YJM 789.

first: Stefan

last: Stumpferl

first: Sue

middle: E

last: Brand

first: James

middle: C

last: Jiang

first: Boguslawa

last: Korona

first: Anurag

last: Tiwari

first: Jianliang

last: Dai

first: Jae-Gu

last: Seo

first: S

middle: M

last: Jazwinski


title: ATFS-1 mediates a protective transcription program during mitochondrial stress

status: Public on Jul 12 2012

city: New York

first: Jeffrey

last: Zhao

state: NY

address: 1275 York Avenue

department: Genomics Core

institute: Memorial Sloan Kettering Cancer Center

summary:
ATFS-1 has been shown to regulate transcription of mitochondrial chaperone genes such as mtHsp70/hsp-6 and hsp-60 in response to mitocondrial stress. To identify the entire ATFS-1-mediated response, we compared the transcript profiles from wild-type and atfs-1(tm4525) worms raised in the absence and presence of mitochondrial stress.
We used microarrays to identify genes regulated by ATFS-1 during mitochondrial stress

overall_design: RNA samples were prepared from wild-type(wt) and atfs-1(tm4525)(mutant) worms fed either control(RNAi) or spg-7(RNAi). Worms were synchronized by bleaching, raised in liquid culture and harvested at the L4 stage. Control is denoted as C and spg-7 treatment is denoted T. Each experiment was performed in triplicate indicated as 1,2 and 3.

first: Amrita

last: Nargund

first: Cole

last: Haynes


title: Transcription data for Pseudomonas aeruginosa isolates TBCF10839 and TBCF121838 grown to early and late exponential growth phase

status: Public on Jun 30 2012

city: Hannover

first: Jens

last: Klockgether

address: Carl-Neuberg-Str. 1

department: Ped. Pneumology

country: Germany

institute: Hannover Medical School

summary: We used Affymetrix microarrays to monitor the gene expression of genomically related but phenotypically divergent strains TBCF10839 and TBCF121838

overall_design: Cultures were grown in LB medium 37°C. In mid-exp. phase (OD600 1.5) cell aliquots were harvested for subsequent RNA extraction. Culturing was continued until late exponential phase (OD600 3.0) when another aliquot was harvested for RNA extraction.


title: Expression data from wildtype and unc-37 mutant A-class motor neurons in C. elegans

status: Public on Sep 01 2012

city: Nashville

first: William

middle: Clay

last: Spencer

state: TN

address: 465 21st Ave S

department: Cell & Developmental Biology

institute: Vanderbilt University

summary: In Caenorhabditis elegans, VA and VB motor neurons arise as lineal sisters but synapse with different interneurons to regulate locomotion. VA-specific inputs are defined by the UNC-4 homeoprotein and its transcriptional corepressor, UNC-37/Groucho, which function in the VAs to block the creation of chemical synapses and gap junctions with interneurons normally reserved for VBs. To reveal downstream genes that control this choice, we have employed a cell-specific microarray strategy that has now identified unc-4-regulated transcripts. One of these genes, ceh-12, a member of the HB9 family of homeoproteins, is normally restricted to VBs. We show that expression of CEH-12/HB9 in VA motor neurons in unc-4 mutants imposes VB-type inputs. Thus, this work reveals a developmental switch in which motor neuron input is defined by differential expression of transcription factors that select alternative presynaptic partners. The conservation of UNC-4, HB9, and Groucho expression in the vertebrate motor circuit argues that similar mechanisms may regulate synaptic specificity in the spinal cord.

overall_design: We employ the mRNA-tagging method to isolate poly(A) RNA from wildtype and unc-37 mutant A-class motor neurons by expressing a 3X FLAG-tagged poly(A) binding protein PAB-1 in DA/VA neurons under control of the unc-4 promoter. A 2-round IVT protocol (modified from the Affymetrix small-sample protocol) was used to convert starting RNA into biotinylated aRNA.

first: Stephen

last: Von Stetina

first: David

last: Miller III

first: Rebecca

last: Fox


title: Gene Expression in Normal and Tumor Ovarian Epithelial Cells from Non-carriers and Heterozygous Carriers of a BRCA French Canadian Mutation.

status: Public on Jun 15 2012

city: Montréal

first: Diala

last: Abed-Rabbo

address: C.P. 6128, succursale Centre-ville

country: Canada

institute: Université de Montréal

summary: We hypothesized that the transcriptome of primary cultures of morphologically normal ovarian surface epithelial cells could be altered by the presence of a heterozygous BRCA1 or BRCA2 mutation. We aimed to discover early events associated to ovarian carcinogenesis, which could represent putative targets for preventive strategies of this silent killer tumor. We identified the first molecular signature associated with French Canadian BRCA1 or BRCA2  founder mutations in morphologically normal ovarian epithelial cells. We discovered that wild-type and mutated BRCA2 allelic transcripts were expressed not only in morphologically normal but also in tumor cells from 8765delAG BRCA2 carriers.  Further analysis of morphologically normal ovarian and tumor cells from C4446T BRCA1 carriers lead to the same observation. Our data support the idea that one single hit in BRCA1 or BRCA2 is sufficient to alter the transcriptome of phenotypically normal ovarian epithelial cell. The highest level of BRCA2 mutated allele transcript expression was measured in cells originating from the most aggressive ovarian tumor. The penetrance of the mutation and the aggressiveness of the related tumor could depend on a dosage effect of the mutated allele transcript.

overall_design: We studied the molecular profiles associated with 5 a priori classes of samples : 4 non-familial morphologically normal ovarian surface epithelium (NOSEs) : NM class, 2 BRCA1-mutated NOSEs : M1 class, 3 BRCA2-mutated NOSEs : M2 class, 3 BRCA1-mutated ovarian tumor cells (TOVs) : TM1 class and one BRCA2-mutated TOV :TM2 class. No technical replicates were included in this study. The following contrasts were computed : NM vs. M1, NM vs. M2, M1 vs. M2, M1 vs. TM1 and M (M1 union M2) vs. TM (TM1 union TM2).

last: Abd-Rabbo

first: Christine

last: Abaji

first: Guillaume

middle: B

last: Cardin

first: Ali

last: Filali-Mouhim

first: Caroline

last: Arous

first: L

last: Portelance

first: D

last: Escobar

first: Sofie

last: Cloutier

first: Patricia

last: Tonin

first: Diane

last: Provencher

first: Anne

last: Mes-Masson

last: Maugard


title: Use of Pleiotropy to Model Genetic Interactions in a Population

city: Bar Harbor

first: Gregory

last: Carter

state: ME

address: 600 Main Street

institute: The Jackson Laboratory

summary: Microarray data to analyze differential biology underlying pheromone response and overall mating efficiency in a yeast population

overall_design: One or more samples per strain, collected after 3 hr exposure to alpha-factor at 1uM.

first: G

first: T

last: Galitski


title: Effects of vitamin B12 on the gene expression of PAO1 under anaerobic growth conditions

status: Public on Jun 18 2012

city: Seoul

first: Sang Sun

last: Yoon

address: 250 Seongsanno, Seodaemun-gu

country: South Korea

institute: Yonsei University College of Medicine

summary:
Pseudomonas aeruginosa undergoes cell elongation and forms robust biofilms during anaerobic respiratory growth using nitrate (NO3-) as an alternative electron acceptor. Understanding the mechanism of cell shape change induced upon anaerobiosis is crucial to the development of effective treatments against P. aeruginosa biofilm infection. Anaerobic growth of PAO1 reached higher cell density in the presence of vitamin B12, an essential coenzyme of class II ribonucleotide reductase. In addition, cell morphology returned to a normal rod shape. These results suggest that vitamin B12, the production of which was suppressed during anaerobic growth, can restore cellular machineries for DNA replication and therefore facilitate better anaerobic growth of P. aeruginosa with normal cell division.
We used microarray to elucidate the global gene expression profiles underlying vitamin B12-induced changes in bacterial cell shape and growth-associated properties.

overall_design: Gene expression profiles of PAO1 grown in LBN (LB+NO3-) or LBN supplemented with 1 microM vitamin B12 are compared.

first: Sang-Sun


title: P. aeruginosa PA14 WT in LB medium vs in LB with 10 mM adenosine medium

status: Public on May 23 2012

city: State College

first: Thomas

middle: K.

last: Wood

state: PA

address: Room 161 Fenske Laboratory, University Park

department: Chemical Engineering & Biochemistry and Molecular Biology

institute: Pennsylvania State University

summary: To investigate the impact of adenosine on gene expression of wild-type PA14.

overall_design: To investigate the impact of adenosine on gene expression of wild-type PA14, cells were harvested after incubating for 7 h in LB medium and LB with 10 mM adenosine medium, and RNA was extracted with the RNeasy Mini Kit (Qiagen, Valencia, CA) using a bead beater (Biospec, Bartlesville, OK ) with RNAlater buffer (Applied Biosystems, Foster City, CA) to stabilize the RNA.


title: Effects of DHA-rich n-3 fatty acid supplementation on gene expression in blood mononuclear leukocytes: the OmegAD study

status: Public on May 24 2012

city: Stockholm

first: Inger

last: Vedin

address: Hälsovägen 7-9

department: Department of medicin

country: Sweden

institute: Karolinska Institutet

summary: The hypothesis was that 6 months of dietary n-3 FA supplementation to humans affected expression of genes that might be of significance for inflammatory processes and for Alzheimer´s disease.

overall_design:
Effects on global gene expression profiles (Affymetrix Human Focus array) in human blood mononuclear leukocytes obtained  from 11 patients with Alzheimer´s disease before and after 6 months of a DHA-rich n-3 fatty acid supplementation. Data analyses were performed using Gene chip Operating Software (GCOS) Version 1.4.

Two Samples, GSM735991 and GSM736014, are missing CEL files due to storage failure.

first: David

last: Brodin


title: microRNA expression profile in a murine model of hyperoxia-induced bronchopulmonary dysplasia

status: Public on May 01 2012

city: Rochester

first: Jie

last: Dong

state: MN

address: 200 1st SW

department: Thoracic Surgery

institute: Mayo Clinic

summary: We performed miRNA and mRNA profiling at postnatal day 14 and day 29 to compare hyperoxia-induced bronchopulmonary dysplasia and wild type. We built potential miRNA-mRNA interaction networks specific to brochopulmonary dysplasia.

overall_design: Replicated time course of mouse lung development at 2 time points (P14, P29). Three replicates per time point for bronchopulmonary dysplasia induced by hyperoxia mouse lung, and two replicates per time point for wild type mouse lung. This dataset represents the miRNA profiling component of the study.

first: Dennis

middle: A

last: Wigle


title: The yeast PP2A-CDC55 phosphatase regulates the transcriptional response to hyperosmolarity stress by regulating Msn2 and Msn4 [Time course 1]

status: Public on Jun 08 2012

city: Tulln

first: Christoph

last: Schueller

address: Konrad Lorenz Strasse 24

department: Department of Applied Genetics and Cell Biology

country: Austria

institute: University of Natural Resources and Life Sciences, Vienna (BOKU)

summary: The yeast PP2A-Cdc55 Serine/Threonine phosphatase regulates transcription under certain conditions. It is required for full activation of the environmental stress response mediated by the transcription factors Msn2 and Msn4. PP2A-Cdc55 contributes to sustained nuclear accumulation of Msn2 and Msn4 and extended chromatin recruitment under stress conditions such as hyperosmolarity stress. Transcript profiles of Msn2 and Msn4 double mutants are similar to cdc55 and the corresponding triple mutants. This argues for a Msn2/4 specific function of PP2A-Cdc55.

overall_design: Time course of 10 20 and 30 minutes hyperosmolarity treated yeast cells of wild type (W303), msn2msn4, cdc55, msn2msn4cdc55 genetic background.

last: Schüller

first: Andriy

last: Petryshyn

first: Eva

last: Klopf


title: Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa [LB]

status: Public on Apr 01 2012

city: Lausanne

first: Sylvain

last: Pradervand

address: Genopode

department: CIG

country: Switzerland

institute: UNI Lausanne

summary: The opportunistic human pathogen Pseudomonas aeruginosa can utilize several carbon and nitrogen compounds as energy sources, which allows the bacterium to grow on a variety of different environments. Nevertheless, the uptake and utilization of these compounds is organized in a hierarchical manner, which is guaranteed by a mechanism named catabolite repression. In P. aeruginosa catabolite repression is a post-transcriptional process with the translational repressor protein, Crc, as the main component. Crc recognizes CA-motifs (acronym for catabolite activity) present in the vicinity of the ribosome binfing site of corresponding target mRNAs and therefore compete with ribosome binding. Certain conditions, which are mainly related to changes in the carbon to nitrogen ratio, induce the two component system CbrAB, which activates the transcription of the sRNA CrcZ. The sRNA sequesters Crc and allows the translation of the target mRNAs. The main focus of this study was to identify novel direct targets of the CbrAB/Crc system with the use of a transcriptome analysis in combination with the search for CA-motifs. We were able to identify five novel targets (estA, acsA, dctP, bkdR and aroP2), which were involved in the uptake and utilization of less preferred carbon sources and amino acids. Direct interaction of Crc with these genes and the resulting regulation by CbrB and CrcZ were verified using mutational analysis and in vitro and in vivo experiments. Moreover, these targets were discussed in the light of growth and biofilm development in synthetic CF sputum medium which emphasised the importance of the CbrAB/Crc system as a regulator of chronic infection.

sample_organism:
Pseudomonas aeruginosa
Pseudomonas aeruginosa PAO1

overall_design: Comparative transcriptome analysis with the PAO1 wild type strain and PAO6673 (delta crc), PAO66711 (delta cbrB), PAO6679 (delta crcZ) strains grown in LB medium.

first: Sonnleitner

last: Elisabeth

first: Valentini

last: Martina

first: Wenner

last: Nicolas

first: Haichar

last: Feth El Zahare

first: Haas

last: Dieter

first: Lapouge

last: Karine


title: Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa.

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

overall_design: Refer to individual Series


title: DNA modification study of major depressive disorder: beyond locus-by-locus comparisons.

status: Public on Apr 28 2012

city: Toronto

first: Art

last: Petronis

state: Ontario

address: 250 College St

department: Campbell Family Mental Health Research Institute

institute: Centre for Addiction and Mental Health

type: Methylation profiling by genome tiling array

summary: Major depressive disorder (MDD) exhibits numerous clinical and molecular features that are consistent with putative epigenetic misregulation. Despite growing interest in epigenetic studies of psychiatric diseases, the methodologies guiding such studies have not been well defined. We performed DNA modification analysis in white blood cells from monozygotic twins discordant for MDD, in brain prefrontal cortex, and germline (sperm) samples from affected individuals and control subjects (total N = 304) using 8.1K CpG island microarrays and fine mapping. In addition to the traditional locus-by-locus comparisons, we explored the potential of new analytical approaches in epigenomic studies. In the microarray experiment, we detected a number of nominally significant DNA modification differences in MDD and validated selected targets using bisulfite pyrosequencing. Some MDD epigenetic changes, however, overlapped across brain, blood, and sperm more often than expected by chance. We also demonstrated that stratification for disease severity and age may increase the statistical power of epimutation detection. Finally, a series of new analytical approaches, such as DNA modification networks and machine-learning algorithms using binary and quantitative depression phenotypes, provided additional insights on the epigenetic contributions to MDD. Mapping epigenetic differences in MDD (and other psychiatric diseases) is a complex task. However, combining traditional and innovative analytical strategies may lead to identification of disease-specific etiopathogenic epimutations.

overall_design: We performed DNA methylome analysis on white blood cells from monozygotic twins discordant for depression (n=200), pre-frontal cortex (n=71), and germline samples (n=33) from affected individuals and controls (total n=304).  DNA samples were enriched for unmodified fraction of the genome using DNA-modification sensitive restriction enzyme digestion followed by adaptor-mediated PCR. The enriched fractions were labelled with a fluorescent dye (Cy5) and hybridized onto the array with a common reference pool (Cy3) generated from individuals unrelated to this study.

first: Gabriel

last: Oh

first: Sun-Chong

last: Wang

first: ZhengFei

last: Chen

first: Tarang

last: Khare

first: Mamoru

last: Tochigi

first: Catherine

last: Ng

first: Angela

last: Yang

first: Mrinal

last: Pal

first: Andrew

last: Kwan

first: Zachary

last: Kaminsky

first: Jonathan

last: Mill

last: Heath

first: Pamela

last: Madden

first: Cerisse

last: Gunasinghe

first: Jennifer

last: Tackett

first: Irving

last: Gottesman

first: Gustavo

last: Turecki

first: Naomi

last: Wray

first: Grant

last: Montgomery

first: Peter

last: McGuffin

first: Nicholas

last: Martin

first: Dorret

last: Boomsma

first: Rafal

last: Kustra


title: Protoanemonin: A new natural quorum sensing inhibitor that selectively activates iron starvation response

status: Public on Jun 13 2012

first: Piotr

last: Bielecki

summary: Many Gram-negative bacteria employ cell-to-cell communication mediated by N-acyl homoserine lactones (quorum sensing) to control expression of a wide range of genes including, but not limited to, genes encoding virulence factors. Outside the laboratory, the bacteria live in complex communities where signals may be perceived across species. We here present a newly found natural quorum sensing inhibitor, produced by the pseudomonads Pseudomonas sp. B13 and Pseudomonas reinekei MT1 as a blind end in the biodegradation of organochloride xenobiotics, which inhibits quorum sensing in P. aeruginosa in naturally occurring concentrations. This catabolite, 4-methylenebut-2-en-4-olide, also known as protoanemonin, has been reported to possess antibacterial properties, but seems to have dual functions. Using transcriptomics and proteomics, we found that protoanemonin significantly reduced expression of genes and secretion of proteins known to be under control of quorum sensing in P. aeruginosa. Moreover, we found activation of genes and gene products involved in iron starvation response. It is thus likely that inhibition of quorum sensing, as the production of antibiotics, is a phenomenon found in complex bacterial communities.

overall_design: Strain P. aeruginosa  MPAO1 was cultivated under defined ABT medium. The orginal culture was divided in the early exponential phase into two parts. One was treated with 125μM Protoanemonin and the other served as a control. The experiment was performed in duplicates.

first: Roberto

last: Bobadilla Fazzini

first: Mette

last: Skindersoe

first: Jacek

last: Puchałka

first: Michael

last: Givskov

first: Vitor

last: P Martins dos Santos


title: Identification of miRNA target genes in C. elegans by RIP-chip-SRM

status: Public on Mar 01 2012

city: Cambridge

first: Marko

last: Jovanovic

state: MA

address: 7 Cambridge Center

department: Core Group (Regev group) member

institute: Broad Institute of MIT and Harvard

type:
Other
Expression profiling by array

summary:
RIP-chip-SRM : a New Combinatorial Large Scale Approach Identifies a Set of Translationally Regulated bantam/miR 58 Targets in C. elegans

RNA binding protein immunopurification + microarray + targeted protein quantification via Selected Reaction Monitoring

This SuperSeries is composed of the SubSeries listed below.

first: Jovanovic

last: Marko

first: Hengartner

middle: O

last: Michael

first: Gerber

middle: P

last: Andre

first: Aebersold

last: Ruedi

first: Rehrauer

last: Hubert


title: Pseudomonas aeruginosa biofilm cells contain ribosome hibernation factors in the dormant antibiotic-tolerant subpopulation

status: Public on May 03 2012

city: Bozeman

first: Kate

last: McInnerney

state: MT

address: 109 Lewis Hall

department: Microbiology

institute: Montana State University

summary: Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptional responses of Pseudomonas aeruginosa growing in biofilms by using microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNAs for these housekeeping genes were low in cells at the bottom of the biofilms. Selective GFP labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNAs levels for genes regulated by the hypoxia induced regulator, Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-termanoxia. Transcripts for ribosomal proteins were primarily associated with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (rmf and PA4463) at the bottom of the biofilms. A P. aeruginosa ∆rmf strain had increased uptake of the membrane integrity stain, propidium iodide. Using selective GFP labeling and cell sorting, we showed that the dividing cells were more susceptible to tobramycin and ciprofloxacin than the dormant subpopulation. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state.

overall_design: 52-hour Pseudomonas aeruginosa TSA colony biofilms were cryoembedded, thin sectioned, and laser dissected (LCM) to obtain samples from the top and bottom 50 µm of the biofilms. 9 sections per biofilm were pooled. RNA was extracted with the RNeasy Micro kit, Turbo DNase treated, poly(A) tailed, and amplified using the Quantitect WTA kit. After clean up, the resulting product was fragmented and end labeled before hybridization.

first: Kerry

middle: S

last: Williamson

first: Lee

last: Richards

first: Ailyn

last: Perez-Osorio

first: Betsey

last: Pitts

first: Kathleen

first: Philip

last: Stewart

middle: J

last: Franklin


title: Genome wide transcriptional analysis of P. aeruginosa PAO1 response to kappa-opioid U-50,488 in poor nutrient medium

status: Public on May 21 2012

city: Chicago

first: Olga

middle: Y

last: ZABORINA

state: IL

address: 5841 S Maryland

department: Surgery

institute: University of Chicago

summary:
Here we examined virulence activation in Pseudomonas aeruginosa in response to the synthetic kappa opioid agonist U-50, 488 in nutrient poor media where growth conditions are limited and density dependent quorum sensing is not activated.
We used a microarray to define the virulence-related genes in P. aeruginosa grown in liquid poor nutrient medium to determine the effect of kappa-opioid receptor agonist on virulence/lethal phenotype

overall_design: All biological samples for gene expression analysis were prepared in triplicate. P. aeruginosa MPAO1 cells collected from liquid culture at 7 hrs of growth in 1.) 0.1xTY medium containing tryptone 1 g/L and yeast extract, 0.5 g/L, or 2.) 0.1xTY+25 mM potassium phosphate buffer, pH 6.0, or 3.) 0.1xTY+200 uM U-50,488, or 4.) 0.1xTY+  25 mM potassium phosphate buffer, pH 6.0+ 200 uM U-50,488 were used for RNA isolation. Microarray analysis was performed using Affymetrix P. aeruginosa GeneChips (Affymetrix, Santa Clara, CA) at the University of Chicago Functional Genomics Facility

first: John

last: Alverdy

last: Zaborina


title: Role of ndvB on gene expression in Pseudomonas aeruginosa

status: Public on Apr 26 2012

city: Ottawa

first: Trevor

last: Beaudoin

address: 451 Smyth Road

institute: University of Ottawa

summary:
ndvB is a gene expressed preferrentialy in biofilms of Pseudomonas aeruginosa and has been implicated in antibiotic resistance. This gene also has a role in signaling in some plant pathogens. A knockout ndvB strain was used to determine if it controlled any other gene expression related to antibiotic resistance
We used microarrays of wildtype and ndvB knockout P. aeruginosa grown in biofilms to identify the role of ndvB in gene expression

overall_design: Wildtype and ndvB knockout  PA14 biofilms were grown for 48 hours followed by RNA extraction. RNA was pooled from 2 wells for each condition to obtain a single biological replicate. Affymetrix microarray processing was performed on PA01 genechips and data analysis was carried out using R statistical framework with bio-conductor packages using RMA and MAS5.0 normalization procedures

first: Thien-Fah

last: Mah


title: Evidence of Dynamically Dysregulated Gene Expression Pathways in Hyper-responsive B Cells from African American Lupus Patients

city: Dallas

first: Igor

last: Dozmorov

state: TX

address: 5323 Harry Hines Boulevard

department: Immunology

institute: UT Southwestern Medical Center

summary: In this report we applied standard and original methods of analysis of gene expression microarray data to delineate differences in the molecular pathways impacted by stimulation of Epstein-Barr virus (EBV) transformed B cells derived from patients with systemic lupus erythematosus (SLE) and normal unrelated controls. In order to understand the dynamics and interconnections the B cell molecular pathways, the system was perturbed with a biologically relevant signal, co-crosslinking of the B cell antigen receptor (BCR) and FcγR2b and global gene expression changes were assessed at various timepoints post-stimulation. Both traditional analysis of differential gene expression and analysis of the dynamics of gene expression variations were performed. Combining these two methods in an analysis process we call Pathway Dysregulation Analysis allows us to establish model networks of functional gene expression important for B cell signaling responses and elucidate gene expression regulatory interconnections disrupted in B cells from individuals with lupus. Through this technique, we found two main groups of gene associations changed significantly with the disease phenotype, which included genes with established controlling function of the B cell activation, and genes involved in apoptosis initiation or prevention.

overall_design: Epstein-Barr virus (EBV) transformed B cells derived from two patients with systemic lupus erythematosus (SLE) and two normal unrelated controls were stimulated with a biologically relevant signal, co-crosslinking of the B cell antigen receptor (BCR) and FcγR2b.  Total RNA was isolated at various timepoints post-stimulation. Gene expression data were used for analysis of differential gene expression and analysis of the dynamics of gene expression variations.

first: Nicolas

last: Dominguez

first: Andrea

middle: L

last: Sestak

last: Harley

first: Judith

last: James

first: Joel

last: Guthridge


title: Stimulation of Host Immune Defenses by a Small Molecule Protects C. elegans from Bacterial Infection

status: Public on Apr 14 2012

city: Boston

first: Read

last: Pukkila-Worley

address: 55 Fruit Street

department: Molecular Biology and Medicine

institute: Massachusetts General Hospital

summary: The nematode Caenorhabditis elegans offers currently untapped potential for carrying out high-throughput, live-animal screens of low molecular weight compound libraries to identify molecules that target a variety of cellular processes.  We previously used a bacterial infection assay in C. elegans to identify 119 compounds that affect host-microbe interactions among 37,214 tested.  We subsequently found that one of these small molecules, RPW-24, protects C. elegans from bacterial infection by stimulating the host immune response of the nematode.  Using transcriptome profiling, epistasis pathway analyses with C. elegans mutants, and an RNAi screen, we showed that RPW-24 promotes resistance to Pseudomonas aeruginosa infection by inducing the transcription of a remarkably small number of C. elegans genes (~1.3% of all genes) in a manner that partially depends on the evolutionarily-conserved p38 MAP kinase pathway and the transcription factor ATF-7.  These data demonstrated that the immunostimulatory activity of RPW-24 is required for its efficacy and define a novel C. elegans-based strategy to identify compounds with activity against antibiotic-resistant bacterial pathogens.  Here we present the microarray data that were used to define the genes that are differentially regulated in wild-type nematodes following exposure to RPW-24.

overall_design: There are six samples total that comprise three biological replicates of wild-type animals exposed to either 70 μM RPW-24 or DMSO for 15 hours at 15°C.  For a given biological replicate, N2 C. elegans animals in the late L4 larval stage were exposed to RPW-24 or DMSO in parallel to each other.


title: Toxicogenomic responses of C. elegans to gold nanoparticles

status: Public on Apr 16 2012

city: Lexington

last: Tsyusko

state: KY

address: 1100 S. Limestone St

department: Plant and Soil Sciences

institute: University of Kentucky

summary: We used Au nanoparticles (Au-NPs) as a model for studying particle specific effects of manufactured nanomaterials (MNMs) by examining the toxicogenomic responses in a model soil organism, free living nematode Caenorhabditis elegans. Global genome expression for nematodes exposed to 4-nm citrate-coated Au-NPs at the LC10 (5.9 mg L-1) revealed significant differential expression of 797 genes. The levels of expression for five genes (apl-1, dyn-1, act-5, abu-11, and hsp-4) were confirmed independently with qRT-PCR.  Seven common biological pathways associated with 38 of these genes were identified.  Activation of 26 pqn/abu genes from noncanonical Unfolded Protein Response (UPR) pathway and up-regulation of molecular chaperones (hsp-16.1, hsp-70, hsp-3 and hsp-4) were observed and are likely indicative of endoplasmic reticulum stress.  Inhibition of abu-11 with RNAi showed increase in mortality in Au-NP exposed nematodes suggesting possible involvement of abu-11 (a gene associated with specific to C. elegans UPR) in a protective mechanism against Au-NPs.  Exposure to Au-NPs also caused activation of genes involved in apoptosis and necrosis and resulted ultimately in 10% mortality.  These results demonstrate that Au-NPs are bioavailable and cause adverse effects to a model ecoreceptor which activate both general and specific biological pathways.

overall_design: Age-synchronized L3 nematodes (about 2000 nematodes per replicate with 3 replicates per treatment) were exposed to LC10 (5.9 mgL-1 ) of 4-nm citrate-coated Au-NP using 6 cm petri-dishes with 4 ml of exposure solution per dish. The N2 nematodes at L3 stageused were used and they were exposed in 50% K-Medium (31.68 mM KCl, 51.37 mM NaCl) for 12h.  At 12 h all solutions were replaced with 50% K-Medium and left for another 12 h.  After 24 h RNA was extracted from each of the replicates .

first: Jason

last: Unrine

first: David

last: Spurgeon

first: Daniel

last: Starnes

first: Michael

last: Tseng

first: Greg

last: Joice

first: Paul

last: Bertsch


title: Expression data from Pseudomonas aeruginosa PAO1 and its isogenic ampR mutant in the presence and absence of sub-MIC ß-lactam exposure.

status: Public on Mar 30 2012

city: Miami

first: Deepak

last: Balasubramanian

state: Fl

institute: FLORIDA INTERNATIONAL UNIVERSITY

summary: The transcriptional regulator AmpR controls expression of the AmpC ß-lactamase in P. aeruginosa and other bacteria. Studies have demonstrated that in addition to regulating ampC expression, AmpR also regulates the expression of the sigma factor AlgT/U and the production of some quorum-sensing regulated virulence factors. In order to understand the ampR regulon, we compared the expression profiles of PAO1 and its isogenic ampR mutant, PAO∆ampR in the presence and absence of sub-MIC ß-lactam stress. The analysis demonstrates that the ampR regulon is much more extensive than previously thought, with the deletion of ampR affecting the expression of over 300 genes. Expression of an additional 207 genes are affected by AmpR when the cells are exposed to sub-MIC ß-lactam stress, indicating that the ampR regulon in P. aeruginosa is much more extensive than previously thought.

overall_design: An inframe deletion of ampR was generated in P. aeruginosa PAO1. The wild type and ampR mutant strains were grown to mid-log phase and subjected to sub-MIC ß-lactam exposure. Total RNA was isolated from 2-hour ß-lactam exposed and unexposed cells to monitor changes in gene expression arising due to loss of ampR in the presence and absence of ß-lactam exposure.

first: Kalai

last: Mathee


title: Effects of (E)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8) on P. aeruginosa PAO1 persister cells (three biological replicates)

status: Public on Mar 24 2012

city: Syracuse

first: Jiachuan

last: Pan

address: 121 Link Hall

institute: Syracuse Unievrsity

summary: Twenty eight genes in PAO1 persister cells were consistently induced by treatment with 1 ug/mL BF8 for 1 h.

overall_design: Persister cells were isolated from PAO1 overnight cultures and treated with and without BF8 for microarray analysis.

first: J

first: D

last: Ren


title: Expression Profiling of Caenorabditis elegans tdp-1 loss-of-function mutant

status: Public on Feb 21 2012

city: Baltimore

first: Jiou

last: Wang

address: 615 N. Wolfe St.

institute: Johns Hopkins University

summary:
Transactive response DNA-binding protein of 43 kDa (TDP-43), a heterogeneous nuclear ribonucleoprotein (hnRNP) with diverse activities, is a common denominator in several neurodegenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Orthologs of TDP-43 exist from mammals to invertebrates, but their functions in lower organisms remain poorly understood. Here we systematically studied mutant Caenorhabditis elegans lacking the nematode TDP-43 ortholog, TDP-1. To understand the global gene expression regulation induced by the loss of tdp-1, the C. elegans transcriptomes were compared between the N2 WT animals and the tdp-1(ok803lf) mutant. Transcriptional profiling demonstrated that the loss of TDP-1 altered expression of genes functioning in RNA processing and protein folding. These results suggest that the C. elegans TDP-1 as an RNA-processing protein may have a role in the regulation of protein homeostasis and aging.
Global gene expression profiling was performed to compare the transcriptome of wild-type (N2) Caenorabditis elegans and that of tdp-1(ok803) loss-of-function mutant.

overall_design: We analyzed mixed stages of Caenorabditis elegans, wild-type N2 versus tdp-1(ok803), using the Affymetrix C. elegans genome array. Three biological replicates were performed.

first: Tao

last: Zhang

first: C

last: Talbot

first: Haiping

last: Hao


title: Analysis of the Pseudomonas aeruginosa regulon controlled by the sensor kinase KinB and sigma factor RpoN

city: Aurora

last: Schurr

state: CO

institute: University of Colorado School of Medicine

summary: Alginate overproduction by P. aeruginosa, also known as mucoidy is associated with chronic endobronchial infections in cystic fibrosis (CF).  Alginate biosynthesis in this bacterium is initiated by the extracytoplasmic function sigma factor (σ22, AlgU/T).  In the wild type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered by the anti-sigma factor MucA that inhibits alginate production.  However, the degradation of MucA by activated intramembrane proteases AlgW and/or MucP can lead to the conversion from nonmucoid strains to mucoid.  Previously we reported that the absence of the sensor kinase KinB in PAO1 causes the initiation of AlgW-dependent proteolysis of MucA resulting in alginate overproduction.  In the kinB mutant this activation requires alternate sigma factor RpoN (σ54).  To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid kinB mutant and an isogenic nonmucoid kinB rpoN double mutant.  In the kinB mutant, RpoN controlled the expression of approximately 20% of the genome.  Besides alginate biosynthesis and regulator genes such as AlgW, KinB, in concert with RpoN, also control a large number of genes including: those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility.  In an acute pneumonia murine infection model, mice exhibited better survival when challenged with the kinB mutant than wt PAO1.  Together, these data strongly suggest that KinB controls virulence factors important for acute pneumonia and conversion to mucoidy.

overall_design: 6 Samples total. Three with kinB mutant and three kinB wild type.

first: F

middle: H

last: Damron

first: Joshua

last: Owings

first: Yuta

last: Okkotsu

first: John

last: Varga

first: Jill

middle: R

first: Joanna

last: Goldberg

first: Hongwei

middle: D

last: Yu


title: Microarray analysis of TAP::ALG-1 associated RNAs isolated from synchronized 'wild-type' animals and 'mir-58' mutants

first: Marko

last: Jovanovic

summary:
To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein (C. elegans strain WS4303). We crossed the TAP::ALG-1 transgene into the mir-58(n4640) mutant background to generate the strain WS5041. For simplicity, we will hereafter term the TAP::ALG-1 transgenic animals as wild typeand the transgenic WS5041 animals as mir-58.
We compared the mRNA population that coimmunopurified with TAP::ALG-1 from synchronized L4 stage wild-type animals with that from synchronized L4 stage mir-58 mutant animals by one-color Affymetrix gene arrays. miR-58 target mRNAs should be specifically underrepresented in the latter samples.

overall_design: Strains WS4303 (wt) and WS5041 (mir-58) were used for TAP::ALG-1 IPs. All experiments were conducted in three independent replicates. For each replicate, WS4303 and WS5041 were grown in parallel. 150 ng of TAP::ALG-1 associated RNA isolated from synchronized late L4 animals were sent to the GeneCore facilty in Heidelberg, Germany (http://www.genecore.embl.de/index.cfm), and the microarray data were generated according to their standard protocol (Weinmann et al. 2009).

first: Jovanovic

last: Marko

first: Hengartner

last: Michael

first: Gerber

last: Andre

first: Aebersold

last: Ruedi

first: Rehrauer

last: Hubert


title: Novel targets of the CbrAB/Crc carbon catabolite control system revealed by transcript abundance in Pseudomonas aeruginosa [BSM]

first: Sylvain

last: Pradervand

summary:
The opportunistic human pathogen Pseudomonas aeruginosa can utilize several carbon and nitrogen compounds as energy sources, which allows the bacterium to grow on a variety of different environments. Nevertheless, the uptake and utilization of these compounds is organized in a hierarchical manner, which is guaranteed by a mechanism named catabolite repression. In P. aeruginosa catabolite repression is a post-transcriptional process with the translational repressor protein, Crc, as the main component. Crc recognizes CA-motifs (acronym for catabolite activity) present in the vicinity of the ribosome binfing site of corresponding target mRNAs and therefore compete with ribosome binding. Certain conditions, which are mainly related to changes in the carbon to nitrogen ratio, induce the two component system CbrAB, which activates the transcription of the sRNA CrcZ. The sRNA sequesters Crc and allows the translation of the target mRNAs. The main focus of this study was to identify novel direct targets of the CbrAB/Crc system with the use of a transcriptome analysis in combination with the search for CA-motifs. We were able to identify five novel targets (estA, acsA, dctP, bkdR and aroP2), which were involved in the uptake and utilization of less preferred carbon sources and amino acids. Direct interaction of Crc with these genes and the resulting regulation by CbrB and CrcZ were verified using mutational analysis and in vitro and in vivo experiments. Moreover, these targets were discussed in the light of growth and biofilm development in synthetic CF sputum medium which emphasised the importance of the CbrAB/Crc system as a regulator of chronic infection.
keyword(s): genetic modification

overall_design: Comparative transcriptome analysis with the PAO1 wild type strain and PAO6673 (delta crc), PAO66711 (delta cbrB), PAO6679 (delta crcZ) strains grown in BSM-Succinate medium.

first: Sonnleitner

last: Elisabeth

first: Valentini

last: Martina

first: Wenner

last: Nicolas

first: Haichar

last: Feth El Zahare

first: Haas

last: Dieter

first: Lapouge

last: Karine


title: The complex interplay of genetic pathways in C.elegans following the treatment with humic substances

status: Public on Feb 03 2012

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: Low concentrations of the dissolved leonardite humic acid HuminFeed® (HF) prolonged the lifespan and enhanced the thermal stress resistance of the model organism Caenorhabditis elegans. Furthermore growth was impaired and reproduction delayed, effects which have also been identified in other polyphenolic monomers, including tannic acid, rosmarinic acid, and caffeic acid. Moreover, a chemical modification of HF (HF-HQ), which increases its phenolic/quinonoid moieties, magnified the biological impact on C. elegans. To gain a deep insight into the molecular basis of these effects, we performed global transcriptomics on young adult (3 d) and old adult (11 d) nematodes exposed to two concentrations of HF and young adults (3 d) exposed to two concentrations of HF-HQ.

overall_design: The global transcriptome was compared in Caenorhabditis elegans mutant strain GE24, pha-1(e2123) exposed to 0, 0.2 and 2.0 mM HuminFeed® (HF) or Huminfeed-Hydroquinone (HF-HQ). Nematodes were harvested as 3 or 11 day old adults (for HF) or 3 day old adults (for HF-HQ).

first: Ralph

last: Menzel

first: Suresh

last: Swain

first: Stefanie

first: Kerstin

last: Pietsch

first: Christian

last: Steinberg

first: Matt

last: Arno

first: Estibaliz

last: Astarloa


title: Microarray analysis to identify Egfr-responsive genes

status: Public on Jan 06 2012

city: Columbus

first: Amanda

last: Simcox

state: OH

address: 484 W 12th Ave

department: Molecular Genetics

institute: Ohio State University

summary: The highly conserved Epidermal Growth Factor-receptor (Egfr) pathway is required in all animals for normal development and homeostasis; consequently, aberrant Egfr signaling is implicated in a number of diseases. Genetic analysis of Drosophila melanogaster Egfr has contributed significantly to understanding this conserved pathway and has led to the discovery of new components and targets.  Here we used microarray analysis of Drosophila third instar wing discs, in which Egfr signaling was perturbed, to identify new Egfr-responsive genes. Upregulated transcripts included five known targets suggesting the approach was valid. We investigated the function of 29 previously uncharacterized genes, which had pronounced responses. The Egfr pathway is important for wing-vein patterning and using reverse genetic analysis we identified five genes that showed venation defects. Three of these genes are expressed in vein primordia and all showed transcriptional changes in response to altered Egfr activity consistent with being targets of the pathway. Genetic interactions with Egfr further linked two of the genes, Sulfated (Sulf1), an endosulfatase gene, and CG4096, an ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) gene, to the pathway. Sulf1 showed a strong genetic interaction with the neuregulin-like ligand vein (vn) and may influence binding of Vn to heparan-sulfated proteoglycans (HSPGs). Genetic evidence also shows that CG4096 functions by modulating activity of the Egfr ligands. The substrate(s) and how ligand activity is affected are unknown, but interestingly vertebrate EGF ligands are regulated by a related ADAMTS protein. We conclude Sulf1 and CG4096 are negative feedback regulators of Egfr signaling that function in the extracellular space to influence ligand activity.

overall_design: 3 replicates each of wing disc samples in which EGFR dominant negative (EGFR-DN) or EGFR activated (EGFR-TOP) transgenes were expressed with the 71B-Gal4 driver.


title: Saccharomyces cerevisiae and propolis sensitivity

status: Public on Nov 29 2011

city: Ribeirão Preto

first: Patricia

middle: Alves

last: de Castro

state: São Paulo

address: Av. do Café

department: Pharmaceuticals Sciences

country: Brazil

institute: USP

summary: The main objective was to identify genes regulated after the BY4742 yeast cells were exposed to 0.125% for 5 or 10 min. The experiment was further valited by microbiological assays.

overall_design: The control for this experiment that used 0.125% propolis as a treatment have 0.68% ethanol for 5 or 10 min (what corresponds to 60% alcoholic extract for 0.125% propolis). For analysis, the cells were exposed to 0.125% for 5 or 10 min were compared to the control.


title: mRNA microarray analysis on young adult Caenorhabditis elegans exposed to MWCNT

status: Public on Dec 31 2011

city: seoul

first: jinhee

last: choi

address: 90 Jeonnong-dong, Dongdaemun-gu

department: College of Urban Science

institute: University of Seoul

summary: In order to evaluate the identification of genes and pathways, the global gene expression profiles were assessed in response to multiwall carbon nanotube (MWCNT) on the soil nematode, Caenorhabditis elegans. We performed whole genome DNA microarray experiments with subsequent quantitative analysis conducted on selected genes.

overall_design: We used synchronized C. elegans populations exposed to MWCNT for 4 and 24h, and whole genome microarrays to screen for global changes in C. elegans transcription profiles. Young adults of C.elegans were selected  for RNA extraction and hybridization on Affymetrix microarrays.

first: Jinhee

last: Choi

first: Ji-Yeon

last: Roh


title: Meta-analysis of global transcriptomics of Quercetin and Tannic acid exposed C. elegans

summary: Recent research has highlighted that the polyphenols Quercetin (Q) and Tannic acid (TA) are capable of extending the lifespan of C. elegans. To gain a deep understanding of the underlying molecular genetics, we analyzed the global transcriptional patterns of nematodes exposed to Quercetin or Tannic acid concentrations that are non-effective (in lifespan extension), lifespan extending or toxic.

overall_design: The global transcriptome was compared in wild type nematodes raised in the presence of 0, 50, 100, and 200 µM Quercetin (Q) or 0, 100, 200, and 300 µM Tannic acid (TA).

first: Nadine

last: Saul


title: Time course expression data in wild-type and TF-deletion yeast

city: singapore

first: Xiaowei

last: XUE

address: 60 Biopolis Street, #02-01, Genome

department: systems biology 1

country: Singapore

institute: Genome Institute of Singapore

summary: To investigate the role of transcriptional factors Gcn4, Leu3, Gat1 and Met31 in the response to 3AT-induced amino acid starvation, we performed time-course microarray studies of wild-type, single deletion and double deletion strains. The analyses provide insight into a complex regulatory response involving at least four transcription factors.

overall_design: Yeast Saccharomyces Cerevisiae BY4741 wild-type and deletion strains were collected at various time points after 3AT induced amino acid starvation.

last: Xue

first: Aili

last: Yeo


title: Fitness Landscape of Antibiotic Tolerance in Pseudomonas aeruginosa Biofilms

status: Public on Nov 21 2011

city: Princeton

first: Alison

last: Hottes

state: NJ

address: 220 Carl Icahn Lab Washington Road

department: Lewis-Sigler Institute for Integrative Genomics

institute: Princeton University

summary:
Bacteria in biofilms have higher antibiotic tolerance than their planktonic counterparts.  A major outstanding question is the degree to which the biofilm-specific cellular state and its constituent genetic determinants contribute to this hyper-tolerant phenotype.  Here, using genome-wide functional profiling of a complex, heterogeneous mutant population of Pseudomonas aeruginosa MPAO1, we identified large sets of mutations that contribute to antibiotic tolerance predominantly in the biofilm or planktonic setting only.  Our mixed population-based experimental design recapitulated the complexity of natural biofilms and, unlike previous studies, revealed clinically observed behaviors including the emergence of quorum sensing-deficient mutants.  Our study revealed a substantial contribution of the cellular state to the antibiotic tolerance of biofilms, providing a rational foundation for the development of novel therapeutics against P. aeruginosa biofilm-associated infections.
This dataset compares the expression of SAH108, a strain with enhanced antibiotic tolerance in the biofilm state, to expression in wild-type strains.

overall_design: We compared the expression of two biological replicates from strain SAH108 to samples from three wild-type, reference strains.   All samples were collected from exponentially-growing planktonic cultures.

first: Sasan

last: Amini

middle: K

first: Lincoln

middle: E

last: Smith

first: Saeed

last: Tavazoie


title: mRNA microarray analysis on young adult Caenorhabditis elegans exposed to benzene, toluene, formaldehyde and a BTF mix

summary: In order to evaluate the identification of genes and pathways, the global gene expression profiles were assessed in response to three chemicals (benzene (B), toluene (T), formaldehyde (F)) individually and as a BTF mix on the soil nematode, Caenorhabditis elegans. We performed whole genome DNA microarray experiments with subsequent quantitative analysis conducted on selected genes.

overall_design: We used synchronized C. elegans populations exposed to B, T, F or a BTF mix, and whole genome microarrays to screen for global changes in C. elegans transcription profiles. Young adults of C. elegans were selected for RNA extraction and hybridization on Affymetrix microarrays.


title: Gene expression in  P. aeruginosa cystic fibrosis isolates grown on Nematode Growth Medium

city: Sydney

first: Jim

last: Manos

state: NSW

address: Camperdown

country: Australia

institute: University of Sydney

summary:
Pseudomonas aeruginosa (P. aeruginosa) lung infection is a significant cause of mortality in patients with cystic fibrosis (CF). Existing experimental data in our lab showed significantly different levels of virulence of "early" and "late" P. aeruginosa infection isolates in a C. elegans slow killing model. We wished to examine the expression profile of these isolates in order to explore genes that may be responsible for the observed differences. The expression profiles of two pairs of isolates (four isolates in total) were compared to each other  using the Affymetrix P. aeruginosa PAO1 genome array, to gain insight into properties mediating virulence in these isolates. Data analysis was carried out using BIOCONDUCTOR software.
Keywords: Comparative strain hybridization

overall_design: Two pairs of isolates (four isolates in total) were compared to each other when grown on Nematode Growth Medium (NGM).

first: Christopher

last: Harmer

first: Barbara

last: Rose

first: Jonathan

last: Arthur

first: Jamie

last: Triccas

first: Mark

last: Elkins

first: Honghua

last: Hu

first: Peter

last: Bye


title: Gene expression profile of Tra1 dependent genes

status: Public on Dec 30 2011

city: Worcester

first: Michael

middle: R.

last: Green

address: 364 Plantation Street

department: Molecular, Cell and Cancer Biology

institute: University of Massachusetts Medical School

summary: Promoter-specific transcriptional activators (activators) stimulate transcription through direct interactions with one or more components of the transcription machinery, termed the “target”. Previous studies have provided evidence that the Tra1 subunit of the yeast SAGA (Spt-Ada-Gcn5-acetyltransferase) complex is the target of the yeast activator Gal4. However, several other general transcription factors, in particular the mediator complex, have also been implicated as Gal4 targets. To investigate the essentiality of Tra1 as a target of Gal4, here we derive Tra1 mutants that are selectively defective for interaction with Gal4 in vivo (Gal4 Interaction Defective (GID) mutants). In contrast to wild-type Tra1, Tra1 GID mutants are not recruited by Gal4 to the promoter and cannot support Gal4-directed transcription activation, demonstrating that the Gal4–Tra1 interaction is required for Gal4 function. In yeast strains expressing a Tra1 GID mutant, Gal4 promoter binding is unexpectedly also diminished indicating that Gal4 and Tra1 bind cooperatively. Consistent with cooperative binding, we demonstrate that the interaction between Gal4 and Tra1 occurs predominantly on the promoter and not off DNA. Finally, we show that although Tra1 is also targeted by other activators, these interaction are unaffected by GID mutations, revealing an unanticipated specificity of the Gal4-Tra1 interaction.

overall_design: Strain MDC1(TRA1 WT) and MDC3 (Tra1 ts) were grown at 30˚C and then shifted to 37˚C for 60, 90 and 120 mins.

first: Ling

last: Lin


title: Analysis of Gal4-directed transcription activation using Tra1 mutants selectively defective for interaction with Gal4

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

overall_design: Refer to individual Series


title: Gene expression profile of Tra1 GID (Gal4 interaction defective) mutants

overall_design: 3 samples were analyzed in duplicate with completely randomized design


title: Laboratory evolution of Jen1p-independent lactate transport in Saccharomyces cerevisiae: identification of ADY2 alleles by whole genome resequencing and mRNA expression analysis

status: Public on Dec 21 2011

city: Delft

first: Jean-Marc

last: Daran

address: Julianalaan 67

department: Department of Biotechnology

country: Netherlands

institute: Delft University of Technology

summary: Background: Evolutionary engineering is a powerful approach to isolate suppressor mutants and industrially relevant genotypes. Until recently, DNA microarray analysis was the only affordable genome-wide approach to identify the responsible mutations. This situation has changed due to the rapidly decreasing costs of whole genome (re)sequencing. DNA microarray-based mRNA expression analysis and whole genome resequencing were combined in a study on lactate transport in Saccharomyces cerevisiae. Jen1p is the only S. cerevisiae lactate transporter reported in literature. To identify alternative lactate transporters, a jen1Δ strain was evolved for growth on lactate.    Results: Two independent evolution experiments yielded Jen1p-independent growth on lactate (μmax 0.14 and 0.18 h-1 for single-cell lines IMW004 and IMW005, respectively). Whereas mRNA expression analysis did not provide leads, whole-genome resequencing showed different single nucleotide changes (C755G/Leu219Val and C655G/Ala252Gly) in the acetate transporter gene ADY2. Analysis of mRNA levels and depth of coverage of DNA sequencing combined with karyotyping, gene deletions and diagnostic PCR showed that in IMW004 an isochromosome III (~475 kb), which contains two additional copies of ADY2C755G, was formed via crossover between YCLWΔ15 and YCRCΔ6. Introduction of the ADY2 alleles in a jen1 ady2 strain resulted in growth on lactate (μmax 0.14 h-1 for Ady2pLeu219Val and 0.12 h-1 for Ady2pAla252Gly).     Conclusions: Whole-genome resequencing of yeast strains obtained from independent evolution experiments enabled rapid identification of a key gene that was not identified by mRNA expression analysis of the same strains. Reverse metabolic engineering showed that mutated alleles of ADY2 (C655G and C755G) encode efficient lactate transporters.

overall_design: The goal of the present study was to investigate whether evolutionary engineering and subsequent elucidation of the underlying mutations can lead to the identification of alternative lactate transporters in S. cerevisiae. Transport of carboxylic acids plays a key role in weak organic acids stress and the responsible membrane transporters are often poorly studied and encoded by multiple redundant genes . Additionally, import of lactate is an essential step in the reutilization of lactate produced during intense exercise in mammals [8]. In S. cerevisiae, Jen1p was previously identified as the only efficient lactate importer by generation of a UV-mutant unable to grow on lactate and subsequent functional complementation with a genomic library . The lactate uptake rate of the resulting jen1 knockout strain was close to the detection limit. Interestingly, export of lactate in engineered lactate producing S. cerevisiae is unaffected by deletion of JEN1 (our unpublished results). These observations suggest the presence of at least one alternative lactate transporter. To test this, a jen1 knockout strain was constructed and evolved for faster growth on lactate as the sole carbon and energy source. The evolved strains were subjected to a combination of mRNA expression analysis and whole genome DNA (re)sequencing to identify the relevant mutations. The resulting leads were tested for lactate transport activity via knockout studies in the evolved strains and reverse engineering of the portable genetic elements into non-evolved strains.

first: Marinka

last: Almering

first: Stefan

last: de Kok


title: Coordinated increase in cellular RNA and protein content induced by overexpression of Far1, a cyclin dependent kinase inhibitor, involves large transcriptional reprogramming and requires the Sfp1 protein.

status: Public on Sep 29 2011

city: Milan

first: Chiara

last: Balestrieri

address: Via Olgettina 58

department: Center for Omics Sciences

country: Italy

institute: IRCCS San Raffaele Scientific Institute

summary: The FAR1 gene encodes a large protein, whose major function is inhibition of cyclin-dependent kinase complexes involved in the G1/S transition. It has been proposed that Far1, together with the G1 cyclin Cln3, may be part of a cell sizer mechanism that controls the entry into S phase. A genome-wide transcriptional analysis of FAR1-overexpressing and far1 deleted cells grown in ethanol- or glucose-supplemented minimal media indicates that FAR1 overexpression induces strong transcriptional remodelling, metabolism being the most affected cellular property. These data suggest that the Far1/Cln3 sizer regulates cell growth either directly or indirectly by affecting metabolism and pathways known to modulate ribosome biogenesis. A crucial role in mediating the effect of Far1 overexpression is played by the Sfp1 protein, a key transcriptional regulator of ribosome biogenesis, whose presence is mandatory to allow a coordinated increase in both RNA and protein levels in ethanol-grown cells.

overall_design: The experimental plan was designed to study the effect of alteration of dosage of the FAR1 gene on the transcriptional pattern of cells exponentially growing in media supplemented with either glucose or ethanol as a carbon source. To this end wild type cells (strain W303-1A), far1 deleted mutant (background W303-1A, relevant genotype far1::his3) and strain overexpressing FAR1 (background W303-1A, transformed with plasmid pTet-FAR1-15Myc (Alberghina et al., 2004)) were collected in independent triplicates in exponential growth as detailed in growth protocol. Microarray experiments were performed using a yeast S98 chip oligonucleotide array (Affymetrix, Inc.) according to the manufacturer's instructions (http://www.affymetrix.com/support/technical/manuals.affx).

first: Marco

last: Vanoni

first: Lorenzo

last: Querin


title: Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant of Pseudomonas aeruginosa

status: Public on Dec 06 2011

city: Nottingham

first: victoria

last: wright

state: Notts

address: University Park

department: MOL

institute: University of Nottingham

summary:
Background  Small colony variants (SCVs) are slow-growing bacteria, which often show increased  resistance to antibiotics and cause latent or recurrent infections. It is therefore  important to understand the mechanisms at the basis of this phenotypic switch.  Methodology/Principal findings  One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and  to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by  emergence of large colonies with a frequency of 10-5 on media without antibiotics  while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed  growth, defective motility, and strongly reduced levels of the quorum sensing  Pseudomonas quinolone signal (PQS). Whole genome expression analysis further  suggested a multi-layered antibiotic resistance mechanism, including simultaneous  over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS  modification operon arnBCADTEF, and the PhoP-PhoQ two-component system.  Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAOSCV.  Finally, genomic analysis revealed the presence of mutations in phoP and phoQ  genes as well as in the mexZ gene encoding a repressor of the mexXY and mexABoprM  genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA  gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of  the rarely used aspartic acid codon GAU to the more common GAC, possibly causing  an increase of tufA mRNA translation. High expression of phoP and phoQ was  confirmed for the SCV variant while the revertant showed expression levels reduced to  wild-type levels.  Conclusions  By combining data coming from phenotypic, gene expression and proteome analysis,  we could demonstrate that resistance to aminoglycosides in one SCV mutant is  multifactorial including overexpression of efflux mechanisms, LPS modification and is  accompanied by a drastic down-regulation of the Pseudomonas quinolone signal  quorum sensing system.
We used microarrays to study changes in gene expression during early and late stationary phase of SCV and WT strains.

overall_design: SCV and WT cultures were grown in triplicate until early (24h) and late (48h) stationary phase.  RNA was extracted, labelled and hybridised on Affymetrix P. aeruginosa expression microarrays.

first: Qing

last: Wei

first: Tarighi

last: Saeed

first: Andreas

last: Dotsch

first: Susanne

last: Haussler

first: Mathias

last: Muesken

first: Victoria

last: Wright

first: Miguel

last: Camara

first: Paul

last: Williams

first: Steven

last: Haenen

first: Bart

last: Boerjan

first: Annelies

last: Bogaerts

first: Evy

last: Vierstraete

last: Verleyen

first: Liliane

last: Schoofs

first: Ronnie

last: Willaert

first: Valerie

last: De

first: Jan

last: Michiels

first: Ken

last: Vercammen

first: Aurelie

last: Crabbe

first: Pierre

last: Cornelis


title: Analysis of Pseudomonas aeruginosa evolving in the cystic fibrosis lung uncovers limited evidence that mutator lineages are more genetically variable than non-mutator lineages

status: Public on Oct 31 2011

city: Bozeman

first: Kate

last: McInnerney

address: 109 Lewis Hall

institute: Montana State University

summary: Pseudomonas aeruginosa evolving in the cystic fibrosis (CF) lung encounters selection via iron-limitation, antibiotics, immune system effectors and other microbes. Standing genetic variation, which depends on rates of horizontal gene transfer (HGT) and mutation supply, controls response to challenges.  HGT may increase if new clones successfully invade, while mutator strains increase new mutations.  We sought to ascertain genomic signatures of invasion and whether, in the absence of novel invasion, mutator-containing P. aeruginosa populations from chronically infected CF lung are more genetically variable than nonmutator populations. Forty-nine strains from 14 patients treated over three years at Necker Children’s Hospital in Paris were phenotyped for antibiotic resistance, mucoidy and mutator status, and then genotyped by rep-PCR, PFGE and MLST analysis. Overall, strains exhibited greater genetic similarity within patients than among patients, with initial and terminal clones differing markedly between series, indicating unrelated clones independently established infections and resisted invasions that might enlarge genetic variation by sexual recombination. Mutator series were more likely to be multiply antibiotic-resistant, but were no more genetically variable at the single nucleotide level. DNA microarray analyses of bacterial genomes in two longitudinal series of equal duration, one containing and one lacking mutators, revealed both series conspicuously lack genes encoding proteins involved in attachment, motility and amino acid biosynthesis.  The mutator series also contained fewer genes hybridizing to canonical PAO1  genome sequences. These data suggest genetic variation arising from mutators may be limited in scope, transient in nature or not easily resolved by fingerprinting, MLST or comparative genomic analyses.

overall_design: Clinical P. aeruginosa hypermutator and nonhypermutator lineages from CF lung. Array-based genome hybridization.

first: Ashley

last: Warren

first: Christine

last: Chandler

first: Carla

last: Boulianne

first: Kami

last: Chiotti

first: Michael

last: Franklin

first: Francois

last: Taddei

first: Isabelle

last: Sermet-Gaudelus

first: Agnes

last: Ferroni

first: Scott

last: Miller

first: Frank

last: Rosenzweig


title: topA mutant of P. aeruginosa PA14

city: Boston

first: Laurence

middle: G

last: Rahme

address: 50 Blosson St, Thier 340

institute: Massachusetts General Hospital

summary: We have tested the effect of deletion of Topoisomerase I on genes transcription of the bacteria Pseudomonas. aeruginosa. Topoisomerase I is a conserved protein that modulate the topology state of the DNA from super-coiled to relaxed. To this end we have compared the transcription profile of a topA::GentR mutant to that of the wild-type strain. The topA mutant contains in topA gene a Mariner transposon with a gentamicin resistance cassette. It is part of the non-redundant mutant library of P. aeruginosa PA14 strains.

overall_design: WT and mutant cells were grown to OD 2.0 in LB 200rpm shaking at 37C.

first: Ronen

middle: N

last: Hazan

first: Jianxin

last: He


title: Expression data from C. elegans

status: Public on Oct 14 2011

city: Bandar Tun Razak,

first: Sue-Mian

last: Then

state: Kuala Lumpur

address: Jalan Yaacob Latif,

department: UKM Medical Molecular Biology Institute (UMBI)

country: Malaysia

institute: Universiti Kebangsaan Malaysia (UKM)

summary: We used microarrays to investigate the effects of microgravity and space radiation on the genome-wide expression of the C. elegans.

overall_design: Three technical replicates of wild type C. elegans (CC1 strain) which exposed to space radiation are analyzed along with ground control.

first: N-F

last: Jusoh

first: S

middle: M


title: PA14_mexR vs. wildtype planktonic cells in minimal medium with C-30

status: Public on Sep 20 2011

city: College Station

first: Mingming

last: Pu

address: 3122 TAMU

department: Chemical Engineering

institute: Texas A&M University

summary: Mutations that made the cells insensitive to the QS inhibition by C-30 were identified in mexR, a multi-drug resistance operon repressor. Gene expression of mexR mutant relative to wild-type at the presence of C-30 was examined.

overall_design:
Strains: PA14_mexR and wildtype.
Medium: OS minimal medium + 0.1% Adenosine as carbon source.
Compounds: 50 µM C-30 added at OD600=0.25.
Time: 2 hr.
Temp: 37 ºC.
Cell type: Planktonic Cells.

first: Toshinari

last: Maeda

first: Rodolfo

last: García-Contreras

first: Yunho

last: Lee

first: Thomas

last: Wood


title: Expression data from the T84 human intestinal epithelial cell line

status: Public on Nov 23 2011

first: Douglas

last: Kominsky

department: Medicine

institute: University of Colorado Denver

summary: Epithelial cells of the intestine respond to IFN-gamma, which is a critical regulator of the inflammatory response.  Here we sought to determine the impact that this cytokine has on epithelial cell gene expression in an in vitro inflammation model.

overall_design: T84 cells were treated with IFN-gamma for 0, 6, or 18 hrs for RNA extraction and hybridization on Affymetrix microarrays.

first: Sean

last: Colgan

first: Robert

last: Hershberg


title: Expression data from C. elegans

status: Public on Dec 02 2011

city: Laurel

first: Jada

last: Lewis

state: MD

address: 8301 Muirkirk Road

institute: FDA

summary: We exposed wild-type Vibrio cholerae E7496, multiple Vibrio cholerae virulence factor deleted genes with intact hemolysin A gene [CVD109] and without hemolysin A gene [CVD110] in E7946, and E.coli OP50 to wild-type C.elegans N2 for 18 hours. We used microarrays to detail the global gene expression and identified distinct classes of up-regulated and down-regulated genes during this process.

overall_design: C. elegans were exposed to Vibrio cholerae and E.coli then hybridization on Affymetrix microarray chips.

first: Surasri

last: Sahu

first: Isha

last: Patel

first: Hediye

last: Cinar


title: Comparison between Caenorhabditis elegans wild-type and Nep-1 mutant strains

status: Public on Sep 24 2011

city: Bologna

first: Elena

last: Bonora

address: via Massarenti )

department: Dept of Gynaecology

institute: University of Bologna

summary:
RET/GDNF and ET3/EDNRB regulate cell survival, differentiation and migration of neural crest-derived cells. Many signalling mediators of RET have been characterized but the target genes at the end of the signalling cascade are largely unknown. Since the RET/EDNRB crosstalk has been previously shown, we used a Caenorhabditis elegans knockout strain of Nep-1, a homologue of human ECE1 (endothelin-converting enzyme-1), to identify new target genes. Transcriptome comparison between wild-type and Nep-1 strains at different stages identified vit-3 as a differentially expressed gene. Molecular studies of the vit3 mammalian homologue, Apoliporotein B (APOB), were performed in the murine Neuro2a cell line, a model of ENS development. Apob expression in Neuro2a is specifically activated by the RET/GDNF signalling pathway, since Ret silencing abolished Apob increase, and this effect is induced by MAPK P38 kinase activation. Mouse Apob promoter study revealed that there is a p53-dependent repressor element in the promoter region which blocks Apob expression and we show that actually p53 binds to this region. We demonstrated that RET/GDNF and EDNRB/endothelin 3 (ET-3) cooperate in inducing neuronal differentiation resulting in Apob activation.  We also show that Apob expression is downregulated in mouse embryos homozygous for the mutation RetC620R and presenting a severe HSCR phenotype, whereas heterozygous mice, phenotypically normal, present a significant increase in Apob expression. These data suggest that Apob has an important role in RET-mediated neuronal development and APOB decrease may have an impact in human disorders where RET absence has been already identified, such as HSCR and Parkinson disease.
Gene expression analysis using Affymetrix GeneChip C. elegans arrays in order to identify genes up- or down-regulated in nep-1 strains, homologue of human ECE1 (endothelin-converting enzyme 1).

overall_design: Comparison of the transcriptomes between wt and nep-1 strains in larval stage L3 and adult C. elegans.

first: Cecilia

last: Evangelisti

first: Alberto

last: Goldoni

first: Giovanni

last: Romeo


title: Genome wide transcriptional analysis of P. aeruginosa PAO1 response to pH at 25 mM phosphate background

status: Public on Oct 06 2011

city: Chicago

first: Olga

last: ZABORINA

address: 5841 S Maryland

department: Surgery

institute: University of Chicago

summary: During extreme physiological stress, the intestinal tract can be transformed into a harsh environment characterized by regio- spatial alterations in oxygen, pH, and phosphate concentration. When the human intestine is exposed to extreme medical interventions, the normal flora becomes replaced by pathogenic species whose virulence can be triggered by various physico-chemical cues leading to lethal sepsis. We previously demonstrated that phosphate depletion develops in the mouse intestine following surgical injury and triggers intestinal P. aeruginosa to express a lethal phenotype that can be prevented by oral phosphate ([Pi]) supplementation.

overall_design:
We used a microarray to define the virulence-related genes in P. aeruginosa grown as lawns on NGM at pH7.5 vs pH6.0
All samples for gene expression analysis were prepared in triplicate. P. aeruginosa MPAO1 cells collected from lawns grown on NGM/[Pi]25 mM, pH 6.0 or  NGM/[Pi]25, pH 7.5 were used for RNA isolation as previously described. Microarray analysis was performed using Affymetrix P. aeruginosa GeneChips (Affymetrix, Santa Clara, CA) at the University of Chicago Functional Genomics Facility

first: John

last: Alverdy

last: Zaborina


title: Extreme calorie restriction and energy source starvation in Saccharomyces cerevisiae represent distinct physiological states

status: Public on Aug 16 2011

first: Jean-Marc

last: Daran

summary: Cultivation methods used to investigate microbial calorie restriction often result in carbon and energy starvation. This study aims to dissect cellular responses to calorie restriction and starvation in Saccharomyces cerevisiae by using retentostat cultivation. In retentostats, cells are continuously supplied with a small, constant carbon and energy supply, sufficient for maintenance of cellular viability and integrity but insufficient for growth. When glucose-limited retentostats cultivated under extreme calorie restriction were subjected to glucose starvation, calorie-restricted and glucose-starved cells were found to share characteristics such as increased heat-shock tolerance and expression of quiescence-related genes. However, they also displayed strikingly different features. While calorie-restricted yeast cultures remained metabolically active and viable for prolonged periods of time, glucose starvation resulted in rapid consumption of reserve carbohydrates, population heterogeneity due to appearance of senescent cells and, ultimately, loss of viability. Moreover, during starvation, calculated rates of ATP synthesis from storage carbohydrates were 2-3 orders of magnitude lower than steady-state ATP-turnover rates calculated under extreme calorie restriction in retentostats. Stringent reduction of ATP turnover during glucose starvation was accompanied by a strong down-regulation of genes involved in protein synthesis. These results demonstrate that extreme calorie restriction and carbon starvation represent different physiological states in S. cerevisiae.

overall_design: The yeast was first grown for 14 days under extreme calorie restriction in anaerobic, glucose-limited retentostats (Boender et al., 2009, Appl.Environ.Microbiol., 75: 5607-5614.). Subsequently, starvation was started by terminating the glucose feed. Yeast transcriptional reprogramming in response to calorie restriction and starvation was monitored by microarray analysis. Independent duplicate retentostat cultures, and subsequently starvation, were sampled for transcriptome analysis using Affymetrix microarrays. One time-point was sampled during calorie restriction (T0) and four time points were sampled during the starvation phase 10, 30, 60 and 120 minutes after switching of the feed, resulting in a dataset of 10 arrays.

first: Leonie

last: Boender

first: Marinka

last: Almering

first: Madelon

last: Dijk

first: Antonius

last: van Maris

first: Johannes

last: de Winde

first: Jack

middle: T

last: Pronk

first: Pascale

last: Daran-Lapujade


title: In-Vivo Expression Profiling of Pseudomonas aeruginosa Infections Reveals Niche-Specific and Strain-Independent Transcriptional Programs

city: Braunschweig

first: Piotr

last: Bielecki

address: Inhoffenstr 7

department: Cell Biology

institute: Helmholtz Centre for Infection Research

summary: Pseudomonas aeruginosa is a threatening, opportunistic pathogen causing disease in immunocompromised individuals. The hallmark of P. aeruginosa virulence is its multi-factorial and combinatorial nature. It renders such bacteria infectious for many organisms and it is often resistant to antibiotics. To gain insights into the physiology of P. aeruginosa during infection, we assessed the transcriptional programs of three different P. aeruginosa strains directly after isolation from burn wounds of humans. We compared the programs to those of the same strains using two infection models: a plant model, which consisted of the infection of the midrib of lettuce leaves, and a murine tumor model, which was obtained by infection of mice with an induced tumor in the abdomen. All control conditions of P. aeruginosa cells growing in suspension and as a biofilm were added to the analysis. We found that these different P. aeruginosa strains express a pool of distinct genetic traits that are activated under particular infection conditions regardless of their genetic variability. The knowledge herein generated will advance our understanding of P. aeruginosa virulence and provide valuable cues for the definition of prospective targets to develop novel intervention strategies.

overall_design: Samples collected from human burn wounds were stabilized and used for P. aeruginosa RNA extraction. The strains from burn wound infection were later used for infection models namely plant and murine tumor infection as well as rich medium controls with planctonic and biofilm growth. The RNA was treated for bacterial RNA enrichment and amplification. All samples were treated in the same way.

first: Jacek

last: Puchalka

first: Melissa

last: Wos

first: Holger

last: Loessner

first: Justyna

last: Glik

first: Marek

last: Kawecki

first: Mariusz

last: Kowalski

first: Burkhard

last: Tummler

first: Siegfried

last: Weiss

first: Vitor

middle: A

last: Martins dos Santos


title: Engineering topology and kinetics of sucrose metabolism in Saccharomyces cerevisiae for improved ethanol yield

status: Public on Sep 06 2011

first: Jean-Marc

last: Daran

summary: Sucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-H+ symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9 % higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport.  This prediction was tested by engineering the promoter and 5’ coding sequences of SUC2, resulting in relocation of invertase to the cytosol. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 95 generations of anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11 % higher ethanol yield on sucrose in chemostat and batch cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes.  Deletion of AGT1, which had been duplicated during laboratory evolution, restored the growth characteristics of the unevolved iSUC2 strain. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes.

overall_design: The goal of the present study was to investigate whether a relocation of sucrose hydrolysis to the cytosol can be used to improve ethanol yields on sucrose and which additional steps may be required to improve sucrose utilization by strains that only express intracellular invertase. To this end, the SUC2 gene was modified to cause an exclusive intracellular localization. Growth and product formation by the engineered strain were compared with that of the parental strain in anaerobic sucrose-limited chemostat cultures. Subsequently, evolutionary engineering was used to improve sucrose uptake kinetics and an evolved strain was characterized for growth and product formation in chemostat cultures. Transcriptome analysis and gene deletion studies were used to identify genetic changes in the evolved strain that contribute to its improved sucrose-uptake kinetics.

first: Thiago

last: Basso

first: Stephan

last: de Kok


title: Expression pattern of Pseudomonas aeruginosa biofilm-residing bacteria exposed to gentamicin or surface acoustic waves or gentamicin and surface acoustic waves combined

status: Public on Sep 01 2011

city: Ramat-Gan

first: Ehud

last: Banin

address: Bldg 206

country: Israel

institute: Bar-Ilan University

summary:
Bacteria are known to respond to various environmental stimuli including nutrient deprivation, osmotic stress, and exposure to antibiotics. Our experimental data showed that P. aeruginosa biofilm formed in urinary catheters is very suseptible to gentamicin treatment when combined with exposure to surface acoustic waves
Our goal was to establish whether P. aeruginosa is capable of specifically sensing the surface acoustic waves as well as to try and decipher the molecular mechanism underlying the increased biofilm suseptibility to antibiotics treatment

overall_design: To fully assess the bacterial response we subjected the P. aeruginosa biofilm to four different treatments: control (no treatment); gentamicin treatment alone; surface acoustic waves treatment alone and combined treatment of gentamicin with surface acoustic waves.

first: Moran

last: Kopel


title: PHO4 target expression for yeast S. cerevisiae under phosphate perturbation

status: Public on Aug 01 2011

first: Xiaowei

last: XUE

institute: Genome Institute of Singapore

summary: In this study, we determined the expression profiles of Pho4 and Cbf1 targeted genes in phosphate perturbation.

overall_design: Yeast S. cerevisiae in log phase was grown in PNB medium with no phosphate for 3 hours and then shift to various phosphate concentration (from 0 to 10 mM) for 80 minutes.

first: Jin-Quan

last: Run

last: Xue

first: Neil

last: Clarke


title: Cellular responses of Saccharomyces cerevisiae at near-zero growth rates: transcriptome analysis of anaerobic retentostat cultures

status: Public on Aug 23 2011

summary: Extremely low specific growth rates (below 0.01 h-1) represent a largely unexplored area of microbial physiology. Retentostats enable controlled, energy-limited cultivation at near-zero specific growth rates while avoiding starvation. In this study, anaerobic, glucose-limited retentostats were used to analyze physiological and genome-wide transcriptional responses of Saccharomyces cerevisiae to cultivation at near-zero specific growth rates. Cultures at near-zero specific growth rates exhibited several characteristics previously associated with quiescence, including accumulation of storage polymers and an increased expression of genes involved in storage metabolism, autophagy and exit from the replicative cell cycle into G0. Analysis of transcriptome data from glucose-limited retentostat and chemostat cultures showed, as specific growth rate was decreased, quiescence-related transcriptional responses already set in at specific growth rates above 0.025 h-1. Many genes involved in mitochondrial processes were specifically upregulated at near-zero specific growth rates, possibly reflecting an increased turn-over of organelles under these conditions. Prolonged (> 2 weeks) cultivation in retentostat cultures led to induction of several genes that were previously implicated in chronological ageing. These observations stress the need for systematic dissection of physiological responses to slow growth, quiescence, ageing and starvation and indicate that controlled cultivation systems such as retentostats can contribute to this goal.

overall_design: Independent duplicate retentostat cultures were subjected to microarray analysis at four time points after switching the effluent line to the filter unit (2, 9, 16 and 22 d). Microarray analysis of independent, triplicate anaerobic glucose-limited chemostat cultures grown at a specific growth rate of 0.025 h-1 (t = 0) were also performed as part of this study, resulting in a dataset of 11 arrays.

first: Leonie

last: Boender

first: Antonius

last: van Maris

first: Erik

last: de Hulster

first: Marinka

last: Almering

first: Ida

last: van der Klei

first: Marten

last: Veenhuis

first: Johannes

last: de Winde

first: Jack

last: Pronk

first: Pascale

last: Daran-Lapujade


title: Genomic Characterization of Adaptive Mutations that Effect Minimal Fitness Trade-offs in Evolved Clones of Saccharomyces cerevisiae

status: Public on Nov 02 2011

city: Stanford

first: Jared

last: Wenger

state: CA

address: 300 Pasteur Drive

institute: Stanford University Medical School

summary:
In some of the earliest uses of genome-wide gene-expression microarrays and array-based Comparative Genomic Hybridization (aCGH), a set of diploid yeasts that had undergone experimental evolution under aerobic glucose limitation was used to explore how gene expression and genome structure had responded to this selection pressure. To more deeply understand how adaptation to one environment might constrain or enhance performance in another we have now identified the adaptive mutations in this set of clones using whole-genome sequencing, and have assessed whether the evolved clones had become generalists or specialists by assaying their fitness under three contrasting growth environments: aerobic and anaerobic glucose limitation and aerobic acetate limitation. Additionally, evolved clones and their common ancestor were assayed for gene expression, biomass estimates and residual substrate levels under the alternative growth conditions. Relative fitnesses were evaluated by competing each clone against a common reference strain in each environment. Unexpectedly, we found that the evolved clones also outperformed their ancestor under strictly fermentative and strictly oxidative growth conditions. We conclude that yeasts evolving under aerobic glucose limitation become generalists for carbon limitation, as the mutations selected for in one environment are advantageous in others. High-throughput sequencing of the evolved clones uncovered mutations in genes involved in glucose sensing, signaling, and transport that in part explain these physiological phenotypes, with different sets of mutations found in independently-evolved clones. Earlier gene expression data from aerobic glucose-limited cultures had revealed a shift from fermentation towards respiration in all evolved clones explaining increased fitness in that condition. However, because the evolved clones also show higher fitness under strictly anaerobic conditions and under conditions requiring strictly respirative growth, this switch cannot be the sole source of adaptive benefit. Furthermore, because independently evolved clones are genetically distinct we conclude that there are multiple mutational paths leading to the generalist phenotype.
Strain Name: Parental strain (CP1AB) or evolved clones (E1 - E5)
Media: aerobic / anaerobic

overall_design: 36 hybridizations