title: MG1655 temperature O2 response normalized data Tavazoie

status: Public on May 13 2008

city: Princeton

first: yirchung

last: Liu

state: NJ

address: 220 Carl Icahn Laboratory

department: Molecular Biology

institute: Princeton University

summary:
Microarray transcriptional profiling was utilized to observe the global cellular state correlates of the physiological responses to temperature and oxygen changes.  We provide evidence for the existence of correlations of Escherichia coli transcriptional responses to temperature and oxygen perturbations-precisely mirroring the co-occurrence of these parameters upon transitions between the outside world and the mammalian gastrointestinal-tract.
Keywords: time course

overall_design: Physiological perturbations were carried out under a controlled environment in the context of bioreactor  (Bioflo 110, New Brunswick Scientific) growth. Thermoelectric sensors and heaters were used to shift  temperature profiles between 25 C and 37 C, and polarographic dissolved oxygen sensors (Mettler  Toledo) and nitrogen gas was used to rapidly change oxygen saturation between anaerobic (0% dissolved  oxygen) and aerobic (16-21% dissolved oxygen) condition. The cultures were maintained in exponential  phase (O.D.600 0.2-0.4) through controlled dilution, where fresh media is pumped in and spent media is  pumped out at a controlled rate. Prior to the perturbations, cells were maintained in the pre-transition  environment for at least eight generations (8-24 hours). All experiments were performed in duplicate.

first: Saeed

last: Tavazoie

first: Y

middle: C


title: Whole Genome Microarray Analysis of the OmcF-deficient Mutant

status: Public on May 22 2008

city: Amherst

first: Derek

last: Lovley

state: MA

address: University of Massachusetts at Amherst, Dept. of Microbiology, Morrill IV north room 202

department: Microbiology

country: USA

institute: University of Massachusetts Amherst

summary:
OmcF is a monoheme c-type cytochrome in Geobacter sulfurreducens.  An OmcF-deficient mutant is impaired in iron reduction and electricity production.  Heme staining revealed altered expression of several c-type cytochromes in the OmcF-deficient mutant.  Whole genome DNA microarray analysis was performed in order to determine which genes demonstrate altered expression in the OmcF-deficient mutant compared to wildtype.
Keywords: cell type comparison

overall_design: RNA samples from three biological replicates were hybridized in duplicate on 12K Combimatrix sense-detecting arrays.  The experiment (omcF mutant) was labeled with Cy5, the control (wildtype) was labeled with Cy3.

first: Byoung-Chan

last: Kim

first: Bradley

middle: L

last: Postier

first: Raymond

middle: J

last: DiDonato

first: Swades

middle: K

last: Chaudhuri

first: Kelly

middle: P

last: Nevin

middle: R


title: Array CGH in congenital heart disease

status: Public on May 12 2008

city: Berlin

first: Fikret

last: Erdogan

address: Ihnestr. 73

country: Germany

institute: Max-Planck-Institute

type: Genome variation profiling by genome tiling array

summary:
Congenital heart disease (CHD) is the most frequent birth defect and affects nearly 1% of newborns. The etiology of CHD is largely unknown and only a small percentage can be assigned to environmental risk factors such as maternal diseases or exposure to mutagenic agents during pregnancy. Chromosomal imbalances have been identified in many forms of syndromic CHD, but next to nothing is known about the impact of DNA copy number changes in non-syndromic CHD. Here we present a sub-megabase resolution array CGH screen of a cohort with CHD as the sole abnormality at the time of diagnosis.
Keywords: array CGH

overall_design: In this BAC array CGH study 104 patients with congenital heart disease and some of their parents were screened for DNA copy number changes at submegabase resolution. No dye swap was performed.


title: Parasitization of Bemisia tabaci by Eretmocerus mundus

city: Bet Dagan

first: Murad

last: Ghanim

address: Hamacabim

department: Entomology

country: Israel

institute: Volcani Center

summary:
The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), and the viruses it transmits, are a major constraint to vegetable crops, worldwide. Although the whitefly is usually controlled using chemical pesticides, biological control agents constitute an important component in integrated pest management programs. One of these agents is the wasp Eretmocerus mundus (Mercet) (Hymenoptera: Aphelinidae). E. mundus lays its egg on the leaf underneath the pupa of B. tabaci. First instars of the wasp hatch and penetrate the whitefly larvae. Initiation of parasitization induces the host to form a cellular capsule around the parasitoid. Around this capsule, epidermal cells multiply and thick layers of cuticle are deposited. The physiological and molecular processes underlying B. tabaci-E. mundus interactions have not been investigated so far.
We have used a cDNA microarray containing 6,000 expressed sequence tags (ESTs) from the whitefly genome to study the parasitoid-whitefly interaction. We compared RNA samples collected across two time points of the parasitization process: when the parasitoid first instar started the penetration process and once it had fully penetrated the host. The results clearly indicated that genes known to be part of the defense pathways described in other insects are also involved in the response of B. tabaci to parasitization by E. mundus.  Some of the responses observed included the repression of a serine protease inhibitor (serpin) and the induction of a melanization cascade. A second set of genes that strongly responded to parasitization included bacterial genes encoded by whitefly symbionts. Quantitative real-time PCR and FISH analyses showed that proliferation of Rickettsia, a facultative secondary symbiont, was strongly induced following the initiation of the parasitization process, a result that supported previous reports suggesting that endosymbionts may be involved in the insect host resistance to various environmental stresses.
This is the first study examining the transcriptional response of a hemipteran insect to the attack of a biological control agent (Hymenopterous parasitoid), using a new genomic approach developed for this insect pest. The defense response in B. tabaci seems to resemble that of model organisms such as Drosophila melanogaster. Moreover, endosymbionts of B. tabaci seem to play a role in the response to parasitization, and this is supported by previously published results from aphids.
Keywords: time course

overall_design: 3 hybridizations were performed for RNA extracted from pre-penetrated B. tabaci nymphs by E. mundus, labeled with Cy5 and directly hybridized against 3 samples from non parasitized nymphs as a control and labeled with Cy3. Another 3 hybridizations were performed from nymphs fully penetrated by the parasitoid and hybridized against the same control.

first: Assaf

last: Mahadav

first: Dan

last: Gerling

first: Yuval

last: Gottlieb

first: Henryk

last: Czosnek


title: Microarray CGH (aCGH) analysis of a patient with Mental Retardation (MR)

status: Public on May 11 2008

city: Yokahama

first: Takeshi

last: Mizuguchi

address: Fukuura 3-9, Kanazawa-ku

department: Department of Human Genetics

country: Japan

institute: Yokohama City University

summary:
Background: Mental Retardation occurs with the prevalence of 2%-3% of general population. Molecular karyotyping by 1-Mb resolution microarray revealed that pathological genomic imbalances were found in 14%-20% of MR.
Aim: The aim of this study is to find the submicroscopic rearrangements in patient with MR using the custom BAC microarray (probe spacing at 0.75 Mb throughout the human genome).
Conclusion: We identified approximate 2.0-Mb deletion on 9q33.3-q34.11 in a female with MR (Patient 1).
Keywords: array CGH

overall_design:
Sample analyzed: a female with MR (Patient 1)
aCGH experiment: We developed and used custom 4.2K BAC microarray (probe spacing at 0.75 Mb throughout the human genome). To minimize the false positive result, we performed aCGH experiment in duplicate with dye-swaps.
Validation of aCGH: Results were validated by Fluorescence in situ hybridization (FISH).

first: Hirotomo

last: Saitsu

first: Naomichi

last: Matsumoto


title: Overexpression analysis of sigZ of B. cereus ATCC 14579.

city: Wageningen

first: Menno

middle: van der

last: Voort

address: Bomenweg 2

department: Agrotechnology and Food Sciences Group

country: Netherlands

institute: Wageningen UR

summary:
The Bacillus cereus ATCC 14579 alternative σ factor σZ and its putative regulon have been characterized. σZ shows overall similarity with ECF σ factors and sigZ constitutes an operon together with asfZ encoding its putative anti-σ factor. Expression analysis revealed sigZ to be induced by an array of stresses, including exposure to ethanol, alkaline pH and heat shock, and a typical promoter binding site for the sigZ-operon was identified by 5’RACE. Phenotypic characterization of B. cereus ATCC 14579 and its sigZ-deletion strain revealed diminished growth performance and sporulation capacity. The σZ-regulon was successfully established by transcriptome analysis of a nisin inducible sigZ-overexpression strain. Overexpression of sigZ was shown to affect expression of 42 genes, including 33 genes encoding proteins located in the extracytoplasm. The identified σZ regulon contained genes encoding proteins situated in the extracytoplasm involved in cell surface modifications and transport. The regulation of genes encoding cell surface modification proteins implies σZ to be involved in the regulation of interaction of B. cereus ATCC 14579 with its environments, which includes human intestinal cells, possibly influencing its virulence status.
Keywords: Comparative transcriptome study

overall_design: Both an empty vector strain and an sigZ overexpressing strain were grown to an OD600 of ~0.5, after which 0.2 ng/ml nisin was added to both cultures. After 90 minutes from both cultures a sample was taken for RNA isolation. Comparisons performed were in duplicate with dye swap, the empty vector strain after 90 min nisin with the overexpressing strain after 90 min nisin.

last: van der Voort


title: SAGE in Rhipicephalus (Boophilus) microplus following OP treatment of larvae from OP resistant/susceptible strains.

status: Public on May 14 2008

city: Kerrville

first: Leonel

middle: (Leo)

last: Saldivar

state: TX

address: 2700 Fredericksburg Rd.

department: Agriculture

institute: USDA

summary:
Organophosphate resistant and susceptible tick larvae from laboratory strains of the southern cattle tick, Rhipicephalus (Boophilus) microplus were exposed to low doses of the organophosphate (OP) acaricide, coumaphos.  Serial Analysis of Gene Expression (SAGE) was used to analyze differential gene expression in response to OP treatment and to compare the responses in OP-treated and untreated resistant and susceptible tick larvae.
** note: Contact person is Felix D. Guerrero.   email: felix.guerrero@ars.usda.gov
Keywords: SAGE, acaricide response, organophosphate.

overall_design:
four samples make up this series:
Munoz Treated: 6030 tags
Munoz Untreated: 7806 tags
SanRoman Treated: 7714 tags
SanRoman Untreated: 9065 tags

first: Felix

middle: D

last: Guerrero

first: Kylie

middle: G

last: Bendele

first: Erin

last: Pleasance

first: Richard

last: Varhol

first: Vishvanath

middle: M

last: Nene


title: Monitoring the ethanol stress response of a sigM deletion strain of B. cereus ATCC 14579.

summary:
Here, the role of σM and its regulon in stress response and survival of B. cereus ATCC 14579 was assessed by comparative transciptome and phenotypic analysis of this strain and its sigM deletion strain. Exposure of B. cereus ATCC 14579 to a wide range of stresses revealed expression of sigM, encoding σM, to be up-regulated mainly in the presence of ethanol and after alkaline pH-shock. Next to this, disc diffusion tests showed the sigM deletion strain to be more sensitive to oxidizing agents and to be more resistant to cell-wall targeting antibiotics than the wild-type strain. The σM regulon was subsequently determined by comparative transcriptional analyses of the wild-type and its sigM-deletion strain after exposure to ethanol. The putative σM-regulon was shown to consist of 29 genes, several of these genes are predicted to be involved in counteracting oxidative stress, such as an NADH oxidase, a ferredoxin, and a lysine decarboxylase or could encode enzymes involved in methionine metabolism, leading toward L-cysteine production, including luxS. Screening of promoter upstream regions allowed for the assessment of a B. cereus consensus promoter binding site for σM. Since the consensus promoter binding site for B. cereus ATCC 14579 σM, its regulon and the predicted functionalities are different from the corresponding features in B. subtilis, it can be concluded that σM plays a unique role in B. cereus stress response and survival.
Keywords: Stress response, comparative transcriptome study

overall_design: The sigM deletion strain of B. cereus ATCC 14579 was cultured to an OD600 of ~0.6. Here the first RNA sample was taken, 0 min. After sampling 4% of ethanol was added (v/v), and samples were taken after 10, 30 and 60 minutes of exposure. Comparisons performed were 0 min - 10 min, 0 min - 30 min, and 0 min - 60 min.


title: Gene induction by ethanol stress in B. cereus ATCC 14579

summary:
The stress response of B. cereus ATCC 14579 is monitored true time, showing an enormous response in gene expression.
Keywords: Stress response, comparative transcriptome analysis.

overall_design: The wild-type strain of B. cereus ATCC 14579 was cultured to an OD600 of ~0.6. Here the first RNA sample was taken, 0 min. After sampling 4% of ethanol was added (v/v), and samples were taken after 10, 30 and 60 minutes of exposure. Comparisons performed were 0 min - 10 min, 0 min - 30 min, and 0 min - 60 min.


title: Mouse Segmental Duplication and Copy-Number Variation

status: Public on May 10 2008

city: Seattle

first: Ze

last: Cheng

state: WA

address: 1905 NE Pacific St.

department: Genome Sciences

institute: University of Washington

summary:
Detailed analyses of the clone-based genome assembly reveal that the recent duplication content of mouse (4.94%) is now comparable to that of human (5.5%), in contrast to previous estimates from the whole-genome shotgun sequence assembly. The architecture of mouse and human genomes differ dramatically; most mouse duplications are organized into discrete clusters of tandem duplications that are depleted for genes/transcripts and enriched for LINE1 and LTR retroposons. We assessed copy-number variation of the C57BL/6J duplicated regions within 15 mouse strains used for genetic association studies, sequencing, and the mouse phenome project. We determined that over 60% of these basepairs are polymorphic between the strains (on average 20 Mbp of copy-number variable DNA between different mouse strains). Our data suggest that different mouse strains show comparable, if not greater, copy-number polymorphism when compared to human; however, such variation is more locally restricted. We show large and complex patterns of inter-strain copy-number variation restricted to large gene families associated with spermatogenesis, pregnancy, viviparity, phermone signaling, and immune response.
Keywords: comparative genomic hybridization

overall_design: Genomic DNA of 14 inbred mouse strains was tested against reference C57BL/6J sample. C57BL/6J DNA sample from another individual was tested as negative control.

first: Xinwei

last: She

first: Anne

last: Morrison

first: Sebastian

last: Zöllner

first: Deanna

last: Church

first: Evan

middle: E

last: Eichler


title: Transcript Analysis of Human Mammary Cell Subsets using SAGE

city: Vancouver

first: BCGSC

last: BC Cancer Agency

state: BC

address: 675 West 10th Avenue

department: Gene Expression

country: Canada

institute: Canada's Michael Smith Genome Sciences Centre

summary:
Highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue were isolated and compared their transcriptomes which were obtained using PCR-Long-SAGE technology.
Keywords: mammary progenitors, stem cells, Notch signaling, gene expression

overall_design: Four SAGE libraries were constructed on RNA samples extracted from highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells isolate from a normal human mammary tissue.

first: Afshin

last: Raouf

first: Yun

last: Zhao

first: Karen

last: To

first: John

last: Stingl

first: Allen

last: Delaney

first: Marry

last: Barbara

first: Norman

last: Iscove

first: Steven

last: Jones

last: McKinney

first: Joanne

last: Emerman

first: Samuel

last: Aparicio

first: Marco

last: Marra

first: Connie

last: Eaves


title: Transcript Analysis of Human Mammary Cell Subsets by Chip Technology

summary:
Highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue were isolated and compared their transcriptomes obtained using the Affymetrix GeneChip Human X3P Array.
Keywords: human mammary progenitors, stem cells, transcriptomes, Notch signaling, gene expression

overall_design: 12 Afffymetrix array hybridizations were performed on RNA samples extracted from highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells isolate from 3 different normal human mammary tissue.


title: Peroxide stress in Sigma54 mutant and Wt

status: Public on May 08 2008

city: Amsterdam

first: Douwe

last: Molenaar

address: De Boelelaan 1085

department: Systems Bioinformatics

institute: Vrije Universiteit Amsterdam

summary:
Analyses of the Wild type and the sigma 54 mutant strain NZ7306 with and without peroxide treatment: A Lactobacillus plantarum strain with a deletion in the alternative sigma factor 54 (?54) encoding gene rpoN, displayed a 100 fold higher sensitivity to peroxide as compared to its parental strain. This feature could be due to ?54-dependent regulation of genes involved in peroxide stress response. However, transcriptome analyses of the wild type and the mutant strain during peroxide exposure did not support such a role for ?54. Subsequent experiments revealed that the impaired expression of the mannose PTS operon in the rpoN mutant caused the observed increased peroxide sensitivity.
Keywords: genetic modification

overall_design: Two conditions were tested in the transcriptome comparison, the condition “peroxide stress exposure” and the condition “deletion of the rpoN-gene”. This resulted in 4 samples: “Wild type normal”, “rpoN-mutant normal”, “Wild type with peroxide”, and “rpoN-mutant with peroxide”. Samples were hybridised against a sample with only one variation, resulting in a total of 4 hybridisations (“Wild type vs Mutant”, “Wild type vs Wild type peroxide”, “Mutant vs Mutant peroxide”, and “Wild type peroxide vs Mutant peroxide”). The experiment was performed in duplicate (duplicates are designated A and B) and in order to discriminate the variation between the duplicates additional hybridisation were performed: “Wild type A vs Wild type B”  and “Mutant with peroxide A vs Mutant with peroxide B”. This resulted in a total of 10 hybridisations (2 x 4 and 2).

first: Marc

last: Stevens

first: Willem

last: De Vos

first: Anne

last: de Jong

first: Michiel

last: Kleerebezem


title: Monitoring the ethanol stress response of a sigZ deletion strain of B. cereus ATCC 14579.

first: Menno

last: Voort

summary:
The stress response of as sigZ deletion strain of B. cereus ATCC 14579 is monitored true time by use of microarrays. The sigZ regulon in ethanol stress response was determined and compared with the regulon determined by micorarray analysis of overexpression of sigZ.
Keywords: stress response, comparative transcriptome study

overall_design: The sigZ deletion strain of B. cereus ATCC 14579 was cultured to an OD600 of ~0.6. Here the first RNA sample was taken, 0 min. After sampling 4% of ethanol was added (v/v), and samples were taken after 10, 30 and 60 minutes of exposure. Comparisons performed were 0 min - 10 min, 0 min - 30 min, and 0 min - 60 min.

last: van der Voort


title: Candidate prostate cancer genes through comparative expression-profiling of seminal vesicle

city: Stanford

first: Jonathan

last: Pollack

state: CA

address: 269 Campus Drive, CCSR3245A

department: Pathology

institute: Stanford University

summary:
BACKGROUND: Prostate cancer is the most frequently diagnosed cancer among men in the United States.  In contrast, cancer of the seminal vesicle is exceedingly rare, despite that the prostate and seminal vesicle share similar histology, secretory function, androgen dependency, blood supply, and (in part) embryonic origin.  We hypothesized that gene-expression differences between prostate and seminal vesicle might inform mechanisms underlying the higher incidence of prostate cancer.
METHODS: Whole-genome DNA microarrays were used to profile gene expression of 11 normal prostate and 7 seminal vesicle specimens (including 6 matched pairs) obtained from radical prostatectomy.  Supervised analysis was used to identify genes differentially expressed between normal prostate and seminal vesicle, and this list was then cross-referenced to genes differentially expressed between normal and cancerous prostate.  Expression patterns of selected genes were confirmed by immunohistochemistry using a tissue microarray.
We identified 32 genes that displayed a highly statistically-significant expression pattern with highest levels in seminal vesicle, lower levels in normal prostate, and lowest levels in prostate cancer.  Among these genes was the known candidate prostate tumor suppressor GSTP1 (involved in xenobiotic detoxification).  The expression pattern of GSTP1 and four other genes, ABCG2 (xenobiotic transport), CRABP2 (retinoic acid signaling), GATA3 (lineage-specific transcription) and SLPI (immune response), was confirmed by immunohistochemistry.
CONCLUSIONS: Our findings identify candidate prostate cancer genes whose reduced expression in prostate (compared to seminal vesicle) may be permissive to prostate cancer initiation.  Such genes and their pathways may inform mechanisms of prostate carcinogenesis, and suggest new opportunities for prostate cancer prevention.
Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc.
Disease State: normal prostate vs normal seminal vesicle sample
Individual
Keywords: Logical Set

overall_design: cDNA microarrays from the Stanford Functional Genomics Facility were used for expression profiling of 11 normal prostate and 7 seminal vesicle specimens (6 of which were matched pairs), against a universal RNA reference.  Extracted expression ratios were normalized by array then mean centered by gene, and expression differences between normal prostate and seminal vesicle identified using Significance Analysis of Microarrays (SAM).

first: Max

last: Thompson


title: Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions

status: Public on May 07 2008

first: Bas

last: van Steensel

address: Plesmanlaan 121

department: division of Molecular Biology

institute: Netherlands Cancer Institute

type: Genome binding/occupancy profiling by genome tiling array

summary:
Proper genome functionality is underpinned by the non-random, spatial or ganisation of chromatin. At the periphery of the nucleus, the association of chr omatin with the nuclear lamina is thought to facilitate both structural organisa tion and regulation of gene expression. Except for a small number of individual loci, the regions of the human genome that locate at the nuclear lamina have not  been identified. Here we used DamID to map the interaction sites of lamin B1 at  high resolution in human fibroblasts. We show that almost half of the human gen ome preferentially associates with the nuclear lamina. Chromatin-lamina interact ions occur via sharply demarcated domains of up to several megabases in size, wh ich are bordered by similar sized domains that do not associate with the lamina.  Lamina associated domains (LADs) are typified by low gene density and low gene expression levels. This is reflected by low levels of PolII binding and histone modification H3K4me2, in combination with enrichment of H3K27me3 (and H3K9?). A considerable subset of LADs is flanked by active promoters pointing outwards, or  by the insulator protein CTCF, suggesting possible mechanisms of LAD confinemen t. Finally, we found that chromosome re-arrangements occur more frequently betwe en loci located either within or outside LADs. The identification of enormous, s harply defined stretches of the human genome that associate with the nuclear lam ina provides unprecedented insights into the 3D organisation of chromatin within  the nucleus. Our results reveal the division of the human genome into periphera l and internal domains, which is echoed in genome functionality.
Keywords: DamID

overall_design: DamID profiling of LaminB1 and Emerin in human fibroblasts. Two biological replicate samples of LaminB1 and of Emerin were hybridized in dye swap orientation on high density array covering human chromosome HSA4. Two biological replicate samples of LaminB1 were hybridized in dye swap orientation on a series of 8 high density arrays covering the whole human genome.

first: Lars

last: Guelen

first: Ludo

last: Pagie


title: Anaerobic induction in B. cereus ATCC 14579

summary:
A comparative transcriptome approach was used to assess genes involved in metabolism and pathogenesis that are specifically activated during anaerobic growth of the spore-forming food-borne human pathogen Bacillus cereus ATCC 14579. Growth under anaerobic conditions in Brain Heart Infusion broth revealed a reduced growth rate and a lower yield as compared to that under aerobic conditions. Comparative transcriptome analysis of cells harvested at early- and mid-exponential growth phase, transition phase and stationary phase, subsequently showed hundreds of genes to be induced under anaerobic condition. These included novel genes identified for anaerobic growth of B. cereus, encoding metabolic pathways, such as the arginine deiminase pathway (ArcABDC), a formate dehydrogenase (FdhF) and a pyruvate fomate lyase (Pfl), and alternative respiratory proteins, such as arsenate reductases. Furthermore, the nitrosative stress response was induced in the anaerobic transition phase of growth, conceivably due to the production of nitric oxide as a by-product of nitrite and nitrate respiration. Notably, both hemolytic enzyme and enterotoxin encoding genes were activated in different oxygen limiting conditions, i.e. hemolytic enzyme encoding genes were induced during anaerobic growth, whereas enterotoxin encoding genes were induced in the transition and stationary phase of aerobic cultures reaching a high cell density. These data point to metabolic rearrangements, stress adaptation and activation of the virulent status of B. cereus under anaerobic conditions, such as encountered in the human GI-tract.
Keywords: time course, anaerobic growth

overall_design:
B. cereus ATCC 14579 was grown in BHI in 50 ml. Aerobic in a Erlenmeyer flask, shaking at 200 rpm. Anaerobic in a closed flask, flushed with Nitrogen-gas for 30 min, also shaking at 200 rpm.

Transcriptome analyses	Phase compared to mid-exponential phase
Anaerobic (OD600)
0.2 compared to 0.4	Early-exponential
1.0 compared to 0.4	Transition
1.1 compared to 0.4	Stationary

Aerobic (OD600)
0.2 compared to 0.8	Early-exponential
4.0 compared to 0.8	Transition
8.0 compared to 0.8	Stationary

Aerobic to anaerobic (OD600)
Anaerobic 0.6 to aerobic 0.6

first: Tjakko

last: Abee


title: Drosophila endogenous small RNAs bind to Argonaute2 in somatic cells

city: Koto-ku

first: Toutai

last: MITUYAMA

state: Tokyo

department: Artificial Intelligence Research Team

institute: National Institute of Advanced Industrial Science and Technology (AIST)

summary:
RNA silencing is a conserved mechanism in which small RNAs trigger various forms of sequence specific gene silencing by guiding Argonaute complexes to target RNAs via base-pairing. RNA silencing is thought to have evolved as a form of nucleic-acid-based immunity to inactivate viruses and transposable elements (TEs). Although the activity of TEs in animals has been thought to be largely restricted to the germline, recent studies have shown that they may also actively transpose in somatic cells, creating somatic mosaicism in animals3. In the Drosophila germline, piRNAs arise from repetitive intergenic elements including retrotransposons by a Dicer-independent pathway and function through the PIWI subfamily of Argonautes to ensure silencing of retrotransposons. Here we show that in Drosophila cultured S2 cells, Argonaute2 (AGO2), an AGO subfamily member of Argonautes, associates with endogenous small RNAs of 20-22 nucleotides in length, which we have collectively named esiRNAs (endogenous siRNAs). esiRNAs can be divided into two groups: one that mainly corresponds to a subset of retrotransposons, and the other that arises from stem-loop structures. esiRNAs are produced in a Dicer-2-dependent manner from distinctive genomic loci, are modified at their 3’ ends and can direct AGO2 to cleave target RNAs. Depletion of Dicer2 or AGO2 in S2 cells coincided with higher levels of retrotransposon transcripts. Together, our findings indicate that in Drosophila, different types of small RNAs and Argonautes are utilized to repress retrotransposons in germline and somatic cells.
Keywords: AGO2, small RNA 454 sequencing - high throughput

overall_design: Cloning of small RNAs associated with AGO2 in S2 cells and deep sequence analysis. Presented here is a list of non-redundant AGO2-associated small RNAs.

first: Yoshinori

last: Kawamura

first: Kuniaki

last: Saito

first: Taishin

last: Kin

first: Kiyoshi

last: Asai

first: Takafumi

last: Sunohara

first: Tomoko

middle: N

last: Okada

first: Mikiko

last: Siomi

first: Haruhiko


title: Transcriptome analyses of ragged seedling2 shoot apical meristem (SAM) vs non-mutant SAM

status: Public on May 03 2008

city: Ithaca

first: Michael

last: Scanlon

state: NY

address: 140 Emerson Hall

department: Department of Plant Biology

institute: Cornell University

summary:
The recessive mutation ragged seedling2-R (rgd2-R) conditions defective lateral development of maize leaves.  Although dorsiventral patterning is established in rgd2-R mutant leaves, some swapping of adaxial/abaxial epidermal identity may occur.  Molecular analyses indicate that RGD2 is required for the coordination of dorsiventral and lateral patterning during maize leaf development, yet the RGD2 gene product and the mechanisms of RGD2 function are unknown. In order to investigate the genetic mechanisms downstream of RGD2 gene function, SAM-specific transcriptional profiling of rgd2-R is compared to that of nonmutant using laser microdissection microarray. Whole SAMs were laser-microdissected from rgd2-R mutant and non-mutant seedlings grown under the same controlled conditions. Microarray profiles were generated for 28,671 maize cDNAs contained on gene chips SAM 1.1and SAM 3.0 (http://www.plantgenomics.iastate.edu/maizechip/).
Keywords: Genetic modification

overall_design: Two-condition experiment, rgd2 SAM vs non-mutant SAM cells. 6 Biological replicates with dye swap. One replicate per array.

first: Xiaolan

last: Zhang


title: BCG vaccine strains lack narK2 and narX induction and exhibit altered phenotypes during dormancy

city: Aurora

first: Ryan

middle: William

last: Honaker

state: CO

institute: University of Colorado Denver

summary:
Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease that affects one-third of the world’s population. The sole extant vaccine for tuberculosis is the live attenuated Mycobacterium bovis bacille Calmette-Guerin (BCG). We examined 13 representative BCG strains from around the world to ascertain their ability to express DosR-regulated dormancy antigens. These are known to be recognized by T-cells of M. tuberculosis infected individuals, especially those harboring latent infections. Differences in expression of these antigens could be valuable for use as diagnostic markers to distinguish BCG vaccination from latent tuberculosis. We determined that all BCG strains were defective for induction of two dormancy genes, narK2 (Rv1737c) and narX (Rv1736c). NarK2 is known to be necessary for nitrate respiration during anaerobic dormancy. Analysis of the narK2/X promoter region revealed a base substitution mutation in all tested BCG strains and M. bovis in comparison to the M. tuberculosis sequence. We also show that nitrate reduction by BCG strains during dormancy was greatly reduced compared to M. tuberculosis and varied between tested strains. Several dormancy regulon transcriptional differences were also identified among the strains, as well as variation in their growth and survival. These findings demonstrate defects in DosR regulon expression during dormancy and phenotypic variation between commonly used BCG vaccine strains.
Keywords: Comparison of induction of a subset of genes between various mycobacterial strains.

sample_organism: Mycobacterium tuberculosis variant bovis BCG

platform_organism:
Mycobacterium tuberculosis CDC1551
Mycobacterium tuberculosis H37Rv

overall_design: 12 different BCG strains were examined as well as M. tuberculosis H37Rv and M. bovis. Two arrays per strain were analyzed, one with the addition of nitric oxide and the other utilizing hypoxia treatment, both conditions shown to induce expression of the dormancy regulon. The reference sample for each array was log phase M. tuberculosis H37Rv.

middle: W

first: Amanda

last: Stewart

first: Stephanie

last: Schittone

first: Angelo

last: Izzo

first: Michèl

last: Klein

first: Martin

middle: I

last: Voskuil


title: Characterization of anaerobic catabolism of p-coumarate in Rhodopseudomonas palustris

first: Yasuhiro

last: Oda

address: Box 357242, 1705 N.E. Pacific Street, HSB K-354

department: Department of Microbiology

summary:
To address the question of how photosynthetic bacterium Rhodopseudomonas palustris metabolize lignin derived compound p-coumarate, transcriptomics and quantitative proteomics were combined to characterize gene expression profiles at both the mRNA level and protein level in Rhodopseudomonas palustris grown with succinate, benzoate, and p-coumarate as the carbon source.
Keywords: Comparison of transcriptome profiles

overall_design: Transcriptome profiles among Rhodopseudomonas palustris cells grown with succinate, benzoate, and p-coumarate as the carbon source were compared.

first: Chongle

last: Pan

first: Dale

middle: A

last: Pelletier

first: Bing

first: Patricia

last: Lankford

first: Nagiza

middle: F

last: Samatova

first: Caroline

middle: S

last: Harwood

first: Robert

last: Hettich


title: Gene expression in melanocytes and metastases melanoma cells from the pig

city: Maisons-Alfort

first: Giorgia

last: Egidy

address: 7, ave Gral de Gaulle

country: France

institute: INRA-ENVA

summary:
Melanoblastoma-bearing Libechov minipigs (MeLiM) provide an animal model for the study of spontaneous cutaneous melanoma. We compared the serial analysis of gene expression (SAGE) profile between normal skin melanocytes and melanoma cells from a pulmonary metastasis of MeLiM.
Keywords: disease state study

overall_design: To minimise the contribution of cells other than melanocytes, we constructed SAGE libraries from a primary culture of melanoma cells derived from a pulmonary melanoma metastasis of a young MeLiM and from a pigmented melanocyte cell line, PigMel, derived from the skin of a healthy Meishan pig.

first: Sophia

last: Julé

first: Jean-Jacques

last: Panthier


title: Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome

city: University Park

first: Michael

middle: J

last: Axtell

state: PA

address: 280 Mueller Lab

department: Biology

institute: Pennsylvania State University

type: Expression profiling by high throughput sequencing

summary:
MicroRNAs (miRNAs) regulate the expression of target mRNAs in plants and animals. Plant miRNA targets have been predicted based on their extensive and often conserved complementarity to the miRNAs, as well as from miRNA over-expression experiments; many of these target predictions have been confirmed by isolating the products of miRNA-directed cleavage. Here, we present a transcriptome-wide experimental method, called “degradome sequencing,” to directly detect cleaved miRNA targets without relying on predictions or over-expression. The 5' ends of poly-adenylated, uncapped mRNAs from Arabidopsis were directly sampled resulting in an empirical snapshot of the degradome. miRNA-mediated cleavage products were easily discerned from an extensive background of degraded mRNAs, which collectively covered the majority of the annotated transcriptome. Many previously known Arabidopsis miRNA targets were confirmed and several novel targets were also discovered. Quantification of cleavage fragments revealed that those derived from TAS transcripts, which are unusual in their production of abundant secondary siRNAs, accumulated to very high levels. A subset of secondary siRNAs are also known to direct cleavage of targets in trans; degradome sequencing revealed many cleaved targets of these trans-acting siRNAs (tasiRNAs). This empirical method is broadly applicable to discover and quantify cleaved targets of small RNAs without a priori predictions.
Keywords: Degradome sequencing

overall_design:
20 or 21 nt tags from the 5' ends of uncapped, polyA+ RNAs were sequenced by next generation sequencing technologies in order to empirically detect cleaved microRNA targets.
4 libraries using different methodologies and samples were used.  See Addo-Quaye et al. (2008) Current Biology citation below for details.

first: Charles

last: Addo-Quaye

first: Tifani

last: Eshoo

first: David

last: Bartel


title: Transcriptome profiling of feeding-to-fasting transition in chicken liver using a chicken 20K oligo microarray

status: Public on May 06 2008

city: Rennes

first: Sandrine

last: Lagarrigue

address: 65 rue de St Brieuc

institute: Agrocampus Rennes/ INRA

summary:
Four weeks old male broilers, fed ad libitum or fasted for 16h or 48h were used to describe the evolution of global gene expression profiles in chicken liver during a 48h fasting period using a chicken 20K oligo array. Among the 20460 oligos on the microarray, 13057 were identified as aligning with a unique coding region of the 2.1 Washington University assembly of the chicken sequence genome. So the statistical analyses were performed on this 13057 gene set. A total number of 2062 differentially expressed genes were identified. The number of genes differentially expressed after 48h of fasting compared to the Fed state was 4-fold higher than after 16h of fasting. Analysis was focused on 1162 genes selected among these 2062 genes for which a human ortholog could be identified, thus allowing functional information collect. Quantitative real-time polymerase chain reaction (qRT-PCR) validated our results.
Keywords: Chicken fasted, transcriptional profiling, differentially expressed genes

overall_design: Biological replicates were dispatch as follow: 6 for fed chickens, 5 for fasted 16h and 7 for fasted 48h. 5 micrograms of total RNA from each sample were labeled with Cy5 and then hybridized with an aliquot of the reference (Cy3).

first: Colette

last: Desert

first: Michel

last: Duclos


title: Role of DksA in the pathogenesis and physiology of Campylobacter jejuni.

city: Ames

first: Byeonghwa

last: Jeon

state: IA

address: 1113 Vet Med

institute: ISU

summary:
DksA is well-known for its regulatory role in the transcription of ribosomal RNA and genes involved in amino acid synthesis in many bacteria. DksA is also reported to control expression of virulence genes in pathogenic bacteria. Here, we elucidated the roles of the DksA-like protein (CJJ81176_0160, Cj0125c) in the pathogenesis of Campylobacter jejuni. Like in other bacteria, transcription of stable RNA was repressed by DksA under stressful conditions in C. jejuni. Transcriptomic and proteomic analyses of C. jejuni 81-176 and its isogenic dksA mutant showed differential expression of many genes involved in iron-related metabolism, flagellar synthesis and amino acid metabolism. Also the dksA mutant of C. jejuni demonstrated a decreased ability to invade into intestinal cells and to induce release of interleukin-8 from intestinal cells. These results suggest the DksA-like protein plays an important regulatory role in the physiology and virulence of C. jejuni.
Keywords: dksA mutation of Campylobacter jejuni

sample_organism: Campylobacter jejuni subsp. jejuni 81-176

overall_design: The design utilized a commercially available two color microarray slide for the enture transcriptome of Campylobacter jejuni.  Six hybridizations were performed each with independently extracted samples of either C. jejuni wildtype of C. jejuni dksA mutant cDNA samples.  A dye swap was utilized to help minimize dye dependent bias.  Thus there were six biological replicates of each sample.

first: JIAE

last: YUN

first: BYEONGHWA

last: JEON

first: YI-WEN

last: BARTON

first: PAUL

last: PLUMMER

first: QIJING

last: ZHANG

first: SANGRYEOL

last: RYU


title: Novel regions of acquired uniparental disomy discovered in acute myeloid leukemia

city: London

first: Manu

last: Gupta

address: Charterhouse Sq.

department: Medical Oncology

country: United Kingdom

institute: Queen Mary University of London

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

summary:
The acquisition of uniparental disomy (aUPD) in acute myeloid leukemia (AML) results in homozygosity for known gene mutations. Uncovering novel regions of aUPD has the potential to identify previously unknown mutational targets, therefore, we aimed to develop a comprehensive map of the regions of aUPD in AML. Here, we have analyzed a large set of diagnostic AML samples (n=455) using genotype arrays. Acquired UPD was found in 17% of the samples with a non-random distribution particularly affecting chromosomes 13q, 11p and 11q. Novel recurrent regions of aUPD were uncovered at 2p, 17p, 2q, 17q, 1p and Xq. Overall, aUPDs were observed across all cytogenetic risk groups, although samples with aUPD13q (5.4% of samples) belonged exclusively to the intermediate-risk group. All cases with a high FLT3-ITD level, measured previously, had aUPD13q covering the FLT3 gene. Of the 120 aUPDs observed, the majority (87%) were due to mitotic recombination while only 13% were due to non-disjunction. This study demonstrates aUPD is a frequent and significant finding in AML and pinpoints regions that may contain novel mutational targets.
Keywords: Genomic analysis of AML samples

overall_design: Genomic DNA from 459 diagnostic AML samples were analysed using Affymetrix 10K 2.0 SNP arrays.  Genomic DNA from the blood of ten unrelated controls was used as reference for all 459 AML samples. Remission DNA from 8 AML samples was also studied. Prevalence and regions of homozygosity were identified.

first: Manoj

last: Raghavan

first: Rosemary

last: Gale

first: Claude

last: Chelala

first: Christopher

last: Allen

first: Gael

last: Molloy

first: Tracy

last: Chaplin

middle: C

last: Linch

first: Jean-Baptiste

last: Cazier

first: Bryan

last: Young


title: NSF 20K Oligo Arrays to Dissect Rice Defense Response Pathways

city: Davis

first: Ki-hong

last: Jung

address: one shields

department: Plant pathology

institute: UC DAVIS

summary:
With the availability of a high quality draft rice genome sequence, large mutant collections, and gene expression oligo arrays for rice, we are now well positioned to dissect rice defense pathways. To do this, we performed global expression analyses to identify genes that are differentially expressed in 10 mutant lines (i.e., Xa21, NH1ox, NRR1ox, GR978, spl11, spl17, NBS2-PI9ox, MPK5ox, OsCoi1ox, and OsNahG1ox), exhibiting altered defense responses. As controls, we used wild typle varieties same with these mutants.
Keywords: disease response

overall_design: Two-condition experiment, 10 mutants vs wild type control with treatment or without treatment. Biological replicates: 2-4 control, independently grown and harvested. Technical replicates: 1-2 control. One replicate per array.

first: Guo-liang

last: Wang

first: Pamela

last: Ronald

first: Jean

last: Leach

first: Ki

middle: H

first: Chan

last: Park


title: Gene expression changes in Drosophila head secondary to ethosuximide treatment during Pentylenetetrazole withdrawal.

city: Delhi

first: Abhay

last: Sharma

state: Delhi

address: Delhi University campus, Mall Road

department: Functional Genomics Unit

country: India

institute: CSIR-Institute of Genomics and Integrative Biology (IGIB)

summary:
Drosophila melanogaster adult flies fed on normal food (NF) containing 16 mg/ml of pentylenetetrazole (PTZ) in the food show a hyperkinetic behavior within 24 hours. Half of that concentration, i.e., 8 mg/ml, of the PTZ, if fed for seven days, though doesn’t cause seizure-like behavior, results in a decreased climbing speed in flies. This change in locomotor behavior is progressive and becomes significant only on seventh day of the treatment. This climbing deficit is ameliorated when flies are treated concomitantly with PTZ and either of the two antiepileptic drugs (AEDs), sodium valproate (NaVP) or levetiracetam (LEV). Further experiments based on different regimes of combination and singular PTZ treatment demonstrated that whereas NaVP show a weaker prophylactic and stronger symptomatic effect, LEV exhibit an opposite effect, i.e., stronger prophylactic and weaker symptomatic action. These observations are consistent with the therapeutic effect of antiepileptic drugs. Time series of microarray gene expression profiling (earlier GEO submission) during chronic PTZ provided evidence of underlying downregulation of three main categories of biological processes, synaptic remodeling, energy metabolism and transport, in that order, in fly head. Transcriptomic analysis secondary to NaVP and LEV (earlier GEO submission) was found to cause statistically significant upregulation of two biological process categories each, energy metabolism and transport in case of NaVP and synaptic remodeling and energy metabolism in case of LEV. Time series of genome wide expression profiling secondary to PTZ and LEV combination treatment (earlier GEO submission) showed neutralizing effect of LEV on PTZ induced expression changes. Mining of published transcriptomic and proteomic data pertaining to epilepsy or established rodent animal models of epileptogenesis supported the relevance of the fly model in understanding the underlying brain pathophysiology.  Systems modeling using RNA interference and small molecules demonstrated the robustness of the fly model. In brief, the above results clearly showed that the fly model is valuable not only in screening of antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents, but also in advancing our knowledge of mechanisms of action of drugs used in treating human patients suffering from various neurological and neuropsychiatric conditions, pharmacogenomics of these drugs, identification of potential drug targets, and selection of potential candidate genes for association analysis in epilepsy. Considering the above fly locomotor behavior model as relevant in kindling attainment, it was of obvious interest to study the behavioral and transcriptomic changes post-kindling, i.e., after withdrawing PTZ following seven day long chronic PTZ administration. It was observed that when PTZ is discontinued and flies climbing speed is monitored for next seven days of PTZ discontinuation, climbing speed deficit initially disappears and then a progressive increase in climbing speed is detected. Behavioral pharmacology of AEDs in post-kindling regime, like fly kindling described above, showed its usefulness in screening of potential antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents. The present submission relates to transcriptomic changes in fly head secondary to ethosuximide (ETH) treatment following discontinuation of PTZ for seven days (after seven days of chronic PTZ treatment). To delineate possible prophylactic or symptomatic effect of ETH at behavioral level, flies were treated with ETH for first three days and then shifted to NF for next four days (3 + 4 day regime) or flies treated with NF for first four days and then drug treated for next three days (4 + 3 day regime), in that order. At transcriptomic level, we have now examined the effect of ETH in 3 + 4 day regime. Gene expression profiles have been generated at both time points, 3rd and 7th day, i.e., on 10th and 14th day from the beginning of PTZ treatment. Effect of three day long chronic ETH treatment on fly head microarray gene expression profile, in otherwise normally grown flies, has already been determined (earlier GEO submission). Similarly, transcriptomic changes after one day, three days and seven days of PTZ discontinuation (i.e., 8th, 10th and 14th day of the beginning of chronic PTZ treatment) have already been deciphered (earlier GEO submission). However, unlike the previous experiment in which flies were maintained in the same container during the post-kindling period of seven days, flies needed to be shifted in fresh vials once in the present experiment because of the demand of 3 + 4 day regime. Since change in housing conditions has the potential to influence head transcriptome, we are also submitting here microarray expression profile of fly head after treating flies with PTZ for seven days, maintaining the flies to NF containing vials for three days and then shifting and maintaining them in fresh NF containing vials for next four days. Repetition of expression profiling on 3rd day of withdrawal (i.e., 10th day from beginning of PTZ treatment) was not needed because both the previous and the present experiment were similar with respect to housing conditions. Repetition of expression profiling on 7th day (i.e., 14th day from the beginning of PTZ treatment) was however required in view of change in housing conditions. The present submission therefore includes this later expression profile also.
Keywords: Drug response

overall_design: D. melanogaster Oregon-R wild type flies were grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin.  The cultures were grown at 24 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 8 mg/ml PTZ for seven days. Each treatment vial contained 30 flies in the beginning. Next, flies were treated in either of the following manner: (1) shifted to vials containing 3.48 mg/ml ETH media for three days, (2) shifted to ETH media for three days and then again shifted to fresh vials containing NF for next four days, and (3) shifted to NF vials for three days and then again shifted to fresh NF vial for the next four days. Heads were harvested at the end of either of the three treatment conditions. For this, flies frozen in liquid nitrogen were shaken and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four individuals treated in either one of the three ways described earlier, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of control and treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and drug treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10 mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto, Canada). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10 µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The preprocessing and quantification of the 16 bit TIFF images were carried out using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 3, Excel Add-In, Stanford) under the conditions of one class response, imputation and 100 permutations.

first: A

first: P

last: Singh

first: M

last: Farhan


title: Gene expression changes in Drosophila head secondary to gabapentin treatment during Pentylenetetrazole withdrawal.

summary:
Drosophila melanogaster adult flies fed on normal food (NF) containing 16 mg/ml of pentylenetetrazole (PTZ) in the food show a hyperkinetic behavior within 24 hours. Half of that concentration, i.e., 8 mg/ml, of the PTZ, if fed for seven days, though doesn’t cause seizure-like behavior, results in a decreased climbing speed in flies. This change in locomotor behavior is progressive and becomes significant only on seventh day of the treatment. This climbing deficit is ameliorated when flies are treated concomitantly with PTZ and either of the two antiepileptic drugs (AEDs), sodium valproate (NaVP) or levetiracetam (LEV). Further experiments based on different regimes of combination and singular PTZ treatment demonstrated that whereas NaVP show a weaker prophylactic and stronger symptomatic effect, LEV exhibit an opposite effect, i.e., stronger prophylactic and weaker symptomatic action. These observations are consistent with the therapeutic effect of antiepileptic drugs. Time series of microarray gene expression profiling (earlier GEO submission) during chronic PTZ provided evidence of underlying downregulation of three main categories of biological processes, synaptic remodeling, energy metabolism and transport, in that order, in fly head. Transcriptomic analysis secondary to NaVP and LEV (earlier GEO submission) was found to cause statistically significant upregulation of two biological process categories each, energy metabolism and transport in case of NaVP and synaptic remodeling and energy metabolism in case of LEV. Time series of genome wide expression profiling secondary to PTZ and LEV combination treatment (earlier GEO submission) showed neutralizing effect of LEV on PTZ induced expression changes. Mining of published transcriptomic and proteomic data pertaining to epilepsy or established rodent animal models of epileptogenesis supported the relevance of the fly model in understanding the underlying brain pathophysiology.  Systems modeling using RNA interference and small molecules demonstrated the robustness of the fly model. In brief, the above results clearly showed that the fly model is valuable not only in screening of antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents, but also in advancing our knowledge of mechanisms of action of drugs used in treating human patients suffering from various neurological and neuropsychiatric conditions, pharmacogenomics of these drugs, identification of potential drug targets, and selection of potential candidate genes for association analysis in epilepsy. Considering the above fly locomotor behavior model as relevant in kindling attainment, it was of obvious interest to study the behavioral and transcriptomic changes post-kindling, i.e., after withdrawing PTZ following seven day long chronic PTZ administration. It was observed that when PTZ is discontinued and flies climbing speed is monitored for next seven days of PTZ discontinuation, climbing speed deficit initially disappears and then a progressive increase in climbing speed is detected. Behavioral pharmacology of AEDs in post-kindling regime, like fly kindling described above, showed its usefulness in screening of potential antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents. The present submission relates to transcriptomic changes in fly head secondary to gabapentin (GBP) treatment following discontinuation of PTZ for seven days (after seven days of chronic PTZ treatment). To delineate possible prophylactic or symptomatic effect of GBP at behavioral level, flies were treated with GBP for first three days and then shifted to NF for next four days (3 + 4 day regime) or flies treated with NF for first four days and then drug treated for next three days (4 + 3 day regime), in that order. At transcriptomic level, we have now examined the effect of GBP in 3 + 4 day regime. Gene expression profiles have been generated at both time points, 3rd and 7th day, i.e., on 10th and 14th day from the beginning of PTZ treatment. Effect of three day long chronic GBP treatment on fly head microarray gene expression profile, in otherwise normally grown flies, has already been determined (earlier GEO submission). Similarly, transcriptomic changes after one day, three days and seven days of PTZ discontinuation (i.e., 8th, 10th and 14th day of the beginning of chronic PTZ treatment) have already been deciphered (earlier GEO submission). Unlike the previous experiment in which flies were maintained in the same container during the post-kindling period of seven days, flies needed to be shifted in fresh vials once in the present experiment because of the demand of 3 + 4 day regime. Since change in housing conditions has the potential to influence head transcriptome, we have also generated microarray expression profile of fly head after treating flies with PTZ for seven days, maintaining the flies to NF containing vials for three days and then shifting and maintaining them in fresh NF containing vials for next four days (submitted in GEO previously).
Keywords: Drug Response

overall_design: D. melanogaster Oregon-R wild type flies were grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin.  The cultures were grown at 24 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 8 mg/ml PTZ for seven days. Each treatment vial contained 30 flies in the beginning. Next, flies were treated in either of the following two manners: (1) shifted to vials containing 16 mg/ml GBP media for three days, and (2) shifted to GBP media for three days and then again shifted to fresh vials containing NF for next four days. Heads were harvested at the end of either of the two treatment conditions. For this, flies frozen in liquid nitrogen were shaken and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four individuals treated in either one of the two ways described earlier, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of control and treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and GBP treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10 mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto, Canada). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10 µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The preprocessing and quantification of the 16 bit TIFF images were carried out using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 3, Excel Add-In, Stanford) under the conditions of one class response, imputation and 100 permutations.


title: Transcriptional Response of 0.1%YE-grown M. sedula cells to ferrous sulfate

status: Public on May 02 2008

city: Raleigh

first: Kathryne

middle: Sherlock

last: Auernik

state: NC

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

department: Chemical and Biomolecular Engineering

institute: North Carolina State University

summary:
(from abstract): Iron oxidation is a desirable trait of biomining microorganisms, although the mechanism is not well-understood in extreme thermoacidophiles. The complete genome sequence of the extremely thermoacidophilic archaeon Metallosphaera sedula DSM 5348 (2.2 Mb, ~2300 ORFs) provides insights into biologically catalyzed metal sulfide oxidation. Comparative genomics was used to identify pathways and proteins (in)directly involved with bioleaching. As expected, the M. sedula genome encodes genes related to autotrophic carbon fixation, metal tolerance, and adhesion. Also, terminal oxidase cluster organization indicates the presence of hybrid quinol-cytochrome oxidase complexes. Comparisons with the mesophilic biomining bacterium Acidithiobacillus ferrooxidans ATCC 23270 indicate that the M. sedula genome encodes at least one putative rusticyanin, involved in iron oxidation. The fox gene cluster, involved in iron oxidation in the thermoacidophilic archaeon Sulfolobus metallicus, could also be identified. These iron-oxidizing components are missing from genomes of non-leaching Sulfolobales like Sulfolobus solfataricus P2 and Sulfolobus acidocaldarius DSM 639. Whole genome transcriptional response analysis showed that 88 ORFs were up-regulated 2-fold or more in M. sedula upon addition of ferrous sulfate to yeast extract-based medium; these included components of terminal oxidase clusters predicted to be involved with iron oxidation, as well as genes predicted to be involved with sulfur metabolism. Many hypothetical proteins were also differentially transcribed, indicating that aspects of the iron and sulfur metabolism of M. sedula remain to be identified and characterized.
Keywords: substrate response

overall_design: Dye flip of Mse cells includes two samples, yeast exact (YE) and yeast extract + ferrous sulfate (YEF). The first slide, Y3F5, has YE RNA labeled with cy3 and YEF RNA labeled with cy5, while the second slide, F3Y5, has YEF RNA labeled with cy3 and YE RNA labeled with cy5. YE serves as the reference condition, with the expectation that ORFs involved with Fe2+ oxidation (and SO4 metabolism) will be upregulated on YEF (log2 fold change of YE-YEF < -1).

first: Robert

last: Kelly


title: Estrogen insensitivity in a model of estrogen receptor positive breast cancer lymph node metastasis.

first: Wendy

last: Dye

institute: UCDHSC

summary:
The lymphatic system is a common avenue for the spread of breast cancer cells and dissemination through it occurs at least as frequently as hematogenous metastasis. Approximately 75% of primary breast cancers are estrogen receptor (ER) positive and the majority of these maintain receptor expression as lymph node (LN) metastases. However, it is unknown if ER function is equivalent in cancer cells growing in the breast and in the LNs. We have developed a model to assess estrogen responsiveness in ER(+) breast tumors and LN metastases. Fluorescent ER(+) MCF-7 tumors were grown in ovariectomized nude mice supplemented with estradiol. Once axillary LN metastasis arose, estradiol was withdrawn (EWD), for 1 or 4 weeks, or continued, to assess estradiol responsiveness. On EWD, proliferation rates fell similarly in tumors and LN metastases. However, estradiol-dependent ER down-regulation and progesterone receptor induction were deficient in LN metastases, indicating that ER-dependent transcriptional function was altered in the LN. Cancer cells from estradiol-treated and EWD primary tumors and matched LN metastases were isolated by laser capture microdissection. Global gene expression profiling identified transcripts that were regulated by the tissue microenvironment, by hormones, or by both. Interestingly, numerous genes that were estradiol regulated in tumors lost estradiol sensitivity or were regulated in the opposite direction by estradiol in LN metastases. We propose that the LN microenvironment alters estradiol signaling and may contribute to local antiestrogen resistance.
Keywords: Breast cancer lymph node metastasis, ER positive, MCF7, xenograft

overall_design: 10 samples, including 3 each of estrogen and estrogen withdrawn axillary lymph nodes and 2 each of estrogen and estrogen withdrawn primary mammary gland tumors.

first: J

last: Harrell

first: D

last: Harvell

first: Mauricio

last: Pinto

first: Paul

last: Jedlicka

first: Carol

last: Sartorius

first: Kathryn

middle: B

last: Horwitz


title: Dilated Cardiomyopathy in Dobermans and Boxers

city: Rensselaer

first: Sridar

middle: V

last: Chittur

address: One Discovery Drive, CRC 342G

department: Center for Functional Genomics

institute: SUNY-University at Albany

summary:
This study attempts at investigating the changes in cardiac gene expression that occur in Dilated Cardiomyopathy (DCM). DCM in Dobermans and Boxers are the focus of this study. Control heart tissue as well as Pacing tissue used is from mongrel dogs.

Keywords: control vs pacing vs disease; strain specific disease

overall_design: 3 Dobermans-DCM, 4 Boxers-DCM, 3 mongrels-control and 3 mongrels-pacing

first: Marcy

middle: L

last: Kuentzel

first: Mark

last: Oyama


title: Gene expression changes in Drosophila head secondary to sodium valproate treatment during Pentylenetetrazole withdrawal.

summary:
Drosophila melanogaster adult flies fed on normal food (NF) containing 16 mg/ml of pentylenetetrazole (PTZ) in the food show a hyperkinetic behavior within 24 hours. Half of that concentration, i.e., 8 mg/ml, of the PTZ, if fed for seven days, though doesn’t cause seizure-like behavior, results in a decreased climbing speed in flies. This change in locomotor behavior is progressive and becomes significant only on seventh day of the treatment. This climbing deficit is ameliorated when flies are treated concomitantly with PTZ and either of the two antiepileptic drugs (AEDs), sodium valproate (NaVP) or levetiracetam (LEV). Further experiments based on different regimes of combination and singular PTZ treatment demonstrated that whereas NaVP show a weaker prophylactic and stronger symptomatic effect, LEV exhibit an opposite effect, i.e., stronger prophylactic and weaker symptomatic action. These observations are consistent with the therapeutic effect of antiepileptic drugs. Time series of microarray gene expression profiling (earlier GEO submission) during chronic PTZ provided evidence of underlying downregulation of three main categories of biological processes, synaptic remodeling, energy metabolism and transport, in that order, in fly head. Transcriptomic analysis secondary to NaVP and LEV (earlier GEO submission) was found to cause statistically significant upregulation of two biological process categories each, energy metabolism and transport in case of NaVP and synaptic remodeling and energy metabolism in case of LEV. Time series of genome wide expression profiling secondary to PTZ and LEV combination treatment (earlier GEO submission) showed neutralizing effect of LEV on PTZ induced expression changes. Mining of published transcriptomic and proteomic data pertaining to epilepsy or established rodent animal models of epileptogenesis supported the relevance of the fly model in understanding the underlying brain pathophysiology.  Systems modeling using RNA interference and small molecules demonstrated the robustness of the fly model. In brief, the above results clearly showed that the fly model is valuable not only in screening of antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents, but also in advancing our knowledge of mechanisms of action of drugs used in treating human patients suffering from various neurological and neuropsychiatric conditions, pharmacogenomics of these drugs, identification of potential drug targets, and selection of potential candidate genes for association analysis in epilepsy. Considering the above fly locomotor behavior model as relevant in kindling attainment, it was of obvious interest to study the behavioral and transcriptomic changes post-kindling, i.e., after withdrawing PTZ following seven day long chronic PTZ administration. It was observed that when PTZ is discontinued and flies climbing speed is monitored for next seven days of PTZ discontinuation, climbing speed deficit initially disappears and then a progressive increase in climbing speed is detected. Behavioral pharmacology of AEDs in post-kindling regime, like fly kindling described above, showed its usefulness in screening of potential antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents. The present submission relates to transcriptomic changes in fly head secondary to NaVP treatment following discontinuation of PTZ for seven days (after seven days of chronic PTZ treatment). To delineate possible prophylactic or symptomatic effect of NaVP at behavioral level, flies were treated with NaVP for first three days and then shifted to NF for next four days (3 + 4 day regime) or flies treated with NF for first four days and then drug treated for next three days (4 + 3 day regime), in that order. At transcriptomic level, we have now examined the effect of NaVP in 3 + 4 day regime. Gene expression profiles have been generated at both time points, 3rd and 7th day, i.e., on 10th and 14th day from the beginning of PTZ treatment. Effect of three day long chronic NaVP treatment on fly head microarray gene expression profile, in otherwise normally grown flies, has already been determined (earlier GEO submission). Similarly, transcriptomic changes after one day, three days and seven days of PTZ discontinuation (i.e., 8th, 10th and 14th day of the beginning of chronic PTZ treatment) have already been deciphered (earlier GEO submission). Unlike the previous experiment in which flies were maintained in the same container during the post-kindling period of seven days, flies needed to be shifted in fresh vials once in the present experiment because of the demand of 3 + 4 day regime. Since change in housing conditions has the potential to influence head transcriptome, we have also generated microarray expression profile of fly head after treating flies with PTZ for seven days, maintaining the flies to NF containing vials for three days and then shifting and maintaining them in fresh NF containing vials for next four days (submitted in GEO previously).
Keywords: Drug Response

overall_design: D. melanogaster Oregon-R wild type flies were grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin.  The cultures were grown at 24 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 0.33 mg/ml PTZ for seven days. Each treatment vial contained 30 flies in the beginning. Next, flies were treated in either of the following two manners: (1) shifted to vials containing 24 mg/ml NaVP media for three days, and (2) shifted to NaVP media for three days and then again shifted to fresh vials containing NF for next four days. Heads were harvested at the end of either of the two treatment conditions. For this, flies frozen in liquid nitrogen were shaken and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four individuals treated in either one of the two ways described earlier, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of control and treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and NaVP treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10 mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto, Canada). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10 µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The preprocessing and quantification of the 16 bit TIFF images were carried out using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 3, Excel Add-In, Stanford) under the conditions of one class response, imputation and 100 permutations.


title: Gene expression changes in Drosophila head secondary to vigabatrin treatment during Pentylenetetrazole withdrawal.

summary:
Drosophila melanogaster adult flies fed on normal food (NF) containing 16 mg/ml of pentylenetetrazole (PTZ) in the food show a hyperkinetic behavior within 24 hours. Half of that concentration, i.e., 8 mg/ml, of the PTZ, if fed for seven days, though doesn’t cause seizure-like behavior, results in a decreased climbing speed in flies. This change in locomotor behavior is progressive and becomes significant only on seventh day of the treatment. This climbing deficit is ameliorated when flies are treated concomitantly with PTZ and either of the two antiepileptic drugs (AEDs), sodium valproate (NaVP) or levetiracetam (LEV). Further experiments based on different regimes of combination and singular PTZ treatment demonstrated that whereas NaVP show a weaker prophylactic and stronger symptomatic effect, LEV exhibit an opposite effect, i.e., stronger prophylactic and weaker symptomatic action. These observations are consistent with the therapeutic effect of antiepileptic drugs. Time series of microarray gene expression profiling (earlier GEO submission) during chronic PTZ provided evidence of underlying downregulation of three main categories of biological processes, synaptic remodeling, energy metabolism and transport, in that order, in fly head. Transcriptomic analysis secondary to NaVP and LEV (earlier GEO submission) was found to cause statistically significant upregulation of two biological process categories each, energy metabolism and transport in case of NaVP and synaptic remodeling and energy metabolism in case of LEV. Time series of genome wide expression profiling secondary to PTZ and LEV combination treatment (earlier GEO submission) showed neutralizing effect of LEV on PTZ induced expression changes. Mining of published transcriptomic and proteomic data pertaining to epilepsy or established rodent animal models of epileptogenesis supported the relevance of the fly model in understanding the underlying brain pathophysiology.  Systems modeling using RNA interference and small molecules demonstrated the robustness of the fly model. In brief, the above results clearly showed that the fly model is valuable not only in screening of antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents, but also in advancing our knowledge of mechanisms of action of drugs used in treating human patients suffering from various neurological and neuropsychiatric conditions, pharmacogenomics of these drugs, identification of potential drug targets, and selection of potential candidate genes for association analysis in epilepsy. Considering the above fly locomotor behavior model as relevant in kindling attainment, it was of obvious interest to study the behavioral and transcriptomic changes post-kindling, i.e., after withdrawing PTZ following seven day long chronic PTZ administration. It was observed that when PTZ is discontinued and flies climbing speed is monitored for next seven days of PTZ discontinuation, climbing speed deficit initially disappears and then a progressive increase in climbing speed is detected. Behavioral pharmacology of AEDs in post-kindling regime, like fly kindling described above, showed its usefulness in screening of potential antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents. The present submission relates to transcriptomic changes in fly head secondary to vigabatrin (VGB) treatment following discontinuation of PTZ for seven days (after seven days of chronic PTZ treatment). To delineate possible prophylactic or symptomatic effect of VGB at behavioral level, flies were treated with VGB for first three days and then shifted to NF for next four days (3 + 4 day regime) or flies treated with NF for first four days and then drug treated for next three days (4 + 3 day regime), in that order. At transcriptomic level, we have now examined the effect of VGB in 3 + 4 day regime. Gene expression profiles have been generated at both time points, 3rd and 7th day, i.e., on 10th and 14th day from the beginning of PTZ treatment. Effect of three day long chronic VGB treatment on fly head microarray gene expression profile, in otherwise normally grown flies, has already been determined (earlier GEO submission). Similarly, transcriptomic changes after one day, three days and seven days of PTZ discontinuation (i.e., 8th, 10th and 14th day of the beginning of chronic PTZ treatment) have already been deciphered (earlier GEO submission). Unlike the previous experiment in which flies were maintained in the same container during the post-kindling period of seven days, flies needed to be shifted in fresh vials once in the present experiment because of the demand of 3 + 4 day regime. Since change in housing conditions has the potential to influence head transcriptome, we have also generated microarray expression profile of fly head after treating flies with PTZ for seven days, maintaining the flies to NF containing vials for three days and then shifting and maintaining them in fresh NF containing vials for next four days (submitted in GEO previously).
Keywords: Drug Response

overall_design: D. melanogaster Oregon-R wild type flies were grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin.  The cultures were grown at 24 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 8 mg/ml PTZ for seven days. Each treatment vial contained 30 flies in the beginning. Next, flies were treated in either of the following two manners: (1) shifted to vials containing 24 mg/ml VGB media for three days, and (2) shifted to VGB media for three days and then again shifted to fresh vials containing NF for next four days. Heads were harvested at the end of either of the two treatment conditions. For this, flies frozen in liquid nitrogen were shaken and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four individuals treated in either one of the two ways described earlier, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of control and treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and VGB treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10 mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto, Canada). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10 µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The preprocessing and quantification of the 16 bit TIFF images were carried out using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 3, Excel Add-In, Stanford) under the conditions of one class response, imputation and 100 permutations.


title: Dual roles of the nuclear cap binding complex and SERRATE in pre-mRNA splicing and microRNA processing in Arabidopsis

status: Public on May 01 2008

city: Oldenburg

first: Sascha

last: Laubinger

address: Carl-von-Ossietzky-Str. 9-11

institute: University of Oldenburg

type: Expression profiling by genome tiling array

summary:
The processing of Arabidopsis thaliana microRNAs (miRNAs) from longer primary transcripts (pri-miRNAs) requires the activity of several proteins, including DICER-LIKE1 (DCL1), the double stranded RNA binding protein HYPONASTIC LEAVES1 (HYL1), and the zinc finger protein SERRATE (SE). It has been noted before that the morphological appearance of weak se mutants is reminiscent of plants with mutations in ABH1/CBP80 and CBP20, which encode the two subunits of the nuclear cap-binding complex. We report that, like SE, the cap-binding complex is necessary for proper processing of pri-miRNAs. Inactivation of either ABH1/CBP80 or CBP20 results in decreased levels of mature miRNAs accompanied by increased levels of pri-miRNAs. Whole genome tiling array analyses reveal that se, abh1/cbp80 and cbp20 mutants also share similar pre-mRNA splicing defects, leading to the accumulation of many partially spliced transcripts. This is unlikely to be an indirect consequence of improper miRNA processing or other mRNA turnover pathways, since introns retained in se, abh1/cbp80 and cbp20 mutants are not affected by mutations in other genes required for miRNA processing or for non-sense-mediated mRNA decay. Taken together, our results uncover dual roles in splicing and miRNA processing that distinguish SE and the cap-binding complex from specialized miRNA processing factors such as DCL1 and HYL1.
Keywords: Tiling array analysis of RNA populations from wild type, se, abh1 and cbp20 mutants

overall_design: Wild type, se-1, abh1-285 and cbp20-1 mutant plants were grown under continuous light at 23°C on soil for 14 days. RNA from total plants was isolated using the RNeasy Plant Mini Kit (Qiagen). Targets (hybridization probes) for tiling array hybridization were generated from 1 µg total RNA (three biologigal replicates A, B and C) using the MessageAmp II-Biotin Enhanced Kit (Ambion) using unmodified NTPs instead of biotinylated NTPs, the GeneChip® WT Double-Stranded cDNA Synthesis Kit and the GeneChip® WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix). Targets were hybridized to Affymetrix Arabidopsis Tiling 1.0R arrays. For washing and scanning, the Affymetrix protocol was used.

first: Timo

last: Sachsenberg

first: Georg

last: Zeller

first: Wolfgang

last: Busch

first: Jan

middle: U

last: Lohmann

first: Gunnar

last: Rätsch

first: Detlef

last: Weigel


title: GNF mouse atlas_macrophages

status: Public on May 05 2008

city: San Diego

first: John

middle: R

last: Walker

address: 10675 John Jay Hopkins

institute: Genomics Institute of the Novartis Research Foundation

summary:
Monocytes and macrophages express an extensive repertoire of G Protein-Coupled Receptors (GPCRs) that regulate inflammation and immunity. In this study we performed a systematic microarray analysis of GPCR expression in primary mouse tissues to identify family members that are either enriched in macrophages compared to a panel of other cell types, or are regulated by an inflammatory stimulus, the bacterial product lipopolysaccharide (LPS).
Keywords: macrophage

overall_design: Macrophage, osteoblast, osteoclast, and mast cell samples were profiled on our GNF1M array.

first: Kate

last: Schroder

first: Allison

last: Pettit

first: Liza

last: Raggatt


title: DK5653/DK1622 Vegetative

first: Mitchell

last: Singer

address: 1 Shields Ave

department: Microbiology

institute: The University of California, Davis

summary:
Isovaleryl-CoA (IV-CoA) is usually derived from the degradation of leucine using the Bkd (branched-chain ketoacid dehydrogenase) complex. In myxobacteria we have previously identified an alternative pathway for IV-CoA formation that branches from the well-known mevalonate dependent isoprenoid biosynthesis pathway and we could already identify the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (MvaS) to be involved in this pathway in Myxococcus xanthus which is induced under leucine limiting conditions like in mutants impaired in leucine degradation or during myxobacterial fruiting body formation. Here we show that proteins involved in leucine degradation are also involved in the alternative IV-CoA biosynthesis by catalyzing the reverse reactions of that used in leucine degradation. Moreover, we conducted a global gene expression experiment comparing vegetative cells of wild type and a bkd mutant. Thus we could identify a five-gene operon which was highly upregulated in a bkd mutant and that contains the mvaS gene and other genes which might be involved in a mevalonate-shunt pathway leading from mevalonate to 3-methylglutaconyl-CoA. Additionally, several genes involved in outer membrane biosynthesis and a plethora of genes encoding regulatory proteins are decreased explaining the complex phenotype of a bkd mutant that includes a lack of adhesion in developmental submers culture.
Keywords: Vegetative growth mutant vs. wild-type comparison

overall_design: 6 independent biological replicates. Normalized ratios to Cy3.

first: Ivy

last: Jose

first: Rolf

last: Müller

first: Helge

last: Bode

first: Michael

last: Ring

first: Gertrud

last: Schwär


title: Microarray of Genes affected by BlxR deletion

city: Madison

first: Gary

last: Splitter

state: WI

address: 1656 Linden Dr

department: Pathobiological Sciences

institute: University of Wisconsin - Madison

summary:
A mini-microarray containing spotted oligos corresponding to 289 B. melitensis genes was used to identify candidate genes whose transcription is influenced by BlxR.  This microarray contains genes encoding the type IV secretion system, flagella, transcriptional regulators, transporters, and proteins involved in outer membrane biogenesis, iron acquisition, and denitrification.  RNA was prepared from both B. melitensis 16M, and the blxR deletion strain, 16M(delta)blxR, grown to late log phase in TSB.
Keywords: genetic modification

overall_design:
Bacteria were grown in TSB until mid/late-log phase.  Before RNA isolation, phenol-ethanol (1:19) was added, cells were pelleted by centrifugation and stored at -80°C.  Total RNA was extracted from cell pellets using the MasterPure RNA Purification Kit (Epicentre), and DNA was removed using TURBO DNA-free (Ambion).  cDNA was synthesized from 7 ug of total RNA in the presence of CyDye-3-dCTP (Amersham Biosciences).  The labeled cDNA was purified using the ChipShot cDNA System (Promega).  For each sample, RNA was extracted from two independently grown cultures and cDNA was generated three times for each RNA sample.
For the mini-microarray, 289 potential open reading frames (ORF’s) of the B. melitensis 16M genome were selected based on their hypothesized involvement in virulence.  These included genes for proteins involved in: type IV secretion, flagella, outer membrane biogenesis, transcriptional regulation, denitrification, iron uptake, and peptide and small molecule transport.  Oligonucleotides (70-mers) representing these 289 genes were purchased from Qiagen, re-suspended in 3x SSC, and spotted onto Ultra GAPS coated slides (Corning) using the MicroGrid Microarrayer (Apogent Discoveries).  Slides were desiccated in a vacuum oven with Drierite for 48 h and UV cross linked using a Stratalinker at 600 mJ.  The printing of each ORF spot was verified by hybridizing with Cy3 labeled 9 mer (Qiagen).  The slides were stored under vacuum at room temp with “Drierite” pellets until further use.
16M genomic DNA was labeled by incorporation of CyDye-5-dCTP (Amersham Biosciences) using High Prime DNA Labeling (Roche) according to manufacturer’s instructions.  Unincorporated nucleotides were removed using Micron YM-30 Columns (Millipore).
Cy3-labeled cDNA was mixed with 0.75 ug cy5-labeled gDNA, and hybridization buffer (Pronto Plus Kit, Promega) was added to a final volume of 35 ul.  The entire sample was loaded onto the printed array and covered with a HybriSlip coverslip (Molecular Probes).  Arrays were placed in specialized hybridization chambers (Telechem Inc., Sunnyvale, CA), and incubated at 42°C overnight.  Slides were washed using the Pronto Plus Hybridization System (Promega) and dried by centrifugation at 1000 rpm for 5 min.
Slides were scanned using the GenePix4000B scanner (Axon Instruments) with independent excitation of the fluorophores Cy3 and Cy5.  Background intensities for each spot were calculated using GenPixPro 3.0 software (Axon Instruments) via the segmentation method.  The signal intensity for each spot was calculated as the difference between the average signal intensity and the average local background intensity.  Data was normalized to gDNA levels by calculating the ratios of cDNA to gDNA for each spot.  Data was then log2 transformed, and the signal ratio for each spot was divided by the mean signal ratio of the entire array to validate comparisons between different arrays.  Data normalization and statistical analysis were performed using TIGR MeV.

GPR files were not submitted because they cannot be located.

first: Gireesh

last: Rajashekara

first: Amy

last: Rambow-Larsen