title: Gene expression profiles of human CD8+ T cells: IL-12 and IFN-a polarized in vitro stimulated and ex vivo sorted CCR7hi and CXCR3hi populations

status: Public on Sep 30 2011

city: Dallas

first: Fatema

middle: Zahra

last: Chowdhury

state: TX

address: 5323 Harry Hines Blvd

department: Immunology

institute: University of Texas Southwestern Medical Center

summary: CD8+ cytotoxic T lymphocytes (CTLs) play a major role in defense against intracellular pathogens, and their functions are specified by antigen recognition and innate cytokines. While effector CTLs eliminate the infection, a small population of memory cells are retained that yields more rapid and robust response upon re-infection. Antigen presenting cells secrete an array of innate cytokines including IL-12 and IFN-α after recognition of pathogens. Both IL-12 and IFN-α have been shown to act as the third signal regulating the development of CTLs. We have shown that these two cytokines have a non-redundant effect in generation of human effector CTL.  IL-12 alone is sufficient for effector CTL genesis marked by IFN-γ and TNF-α production, as well as increased cytolytic activity. Even in the presence of IFN-α, IL-12 programs CTLs that express the chemokine receptor CXCR3 and effector cytokines. Using microarray analysis we have investigated how IL-12 and IFN-α differentially regulate the genetic programming pathways that give rise to effector CTLs among multiple human donors. We have also analyzed the gene expression patterns of cells sorted from healthy human peripheral blood that display surface markers of effector memory CTL (designated as ex vivo) samples.

overall_design: 5 healthy human donor samples were used for the in vitro cultures. For each donor the CFSE labeled cells (CD8+CD45RA+) were cultured in the presence of neutralized, IL-12, IFN-a, and IL-12+IFN-a conditions and plate-bound anti-CD3+anti-CD28 for 3.5 days. Total RNA from CFSEhi (Undiv) and CFSElo (Div) sorted cells were used for Illumina Bead Array. 4 healthy human donor samples were used for the ex vivo samples. Total RNA was collected from FACS sorted CD8+CCR7hiCXCR3lo and CD8+CCR7loCXCR3hi cells without any stimulation.

middle: Z

first: J

middle: D

last: Farrar


title: Expression data from docetaxel-resistant prostate cancer cell lines

status: Public on Jan 01 2012

city: Barcelona

first: Mercedes

last: Marín-Aguilera

address: Villarroel 170

country: Spain

institute: Fundació Clínic per a la Recerca Biomèdica

summary:
Docetaxel-based chemotherapy is the standard first-line therapy in metastatic castration-resistant prostate cancer. However, most patients eventually develop resistance to this treatment.
The aim of the study was to identify key molecular genes and networks associated with docetaxel resistance in 2 models of docetaxel-resistant castration-resistant prostate cancer cell lines.

overall_design: DU-145 and PC-3 cells were converted to docetaxel-resistant cells, DU-145R and PC-3R, respectively. Whole-genome arrays were used to compare global gene expression between these 4 cell lines. Arrays were performed by triplicate for each cell line.

first: Susana

middle: G

last: Kalko

first: Begoña

last: Mellado


title: Host cells infected by various types of viruses

status: Public on Jan 03 2013

city: Singapore

first: Richard

middle: J

last: Sugrue

address: 60 Nanyang Drive

department: Divison of Molecular and Cell Biology

country: Singapore

institute: Nanyang Technological University

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

sample_organism:
Gallus gallus
Homo sapiens
Canis lupus familiaris
Mus musculus

overall_design: Refer to individual Series


title: Expression data from control and COUP-TFII siRNA treated PC3 cells

status: Public on Oct 25 2011

city: Houston

first: Jun

last: Qin

address: One Baylor Plaza

country: USA

institute: Baylor College of Medicine

summary: COUP-TFII, a member of the nuclear receptor superfamily plays a critical role in angiogenesis and organogenesis during embryonic development. Our results indicate that COUP-TFII expression is profoundly upregulated in prostate cancer patients and might serves as biomarker for recurrence prediction. Thus we conduct transcriptome comparison of control and COUP-TFII depleted PC3 cells to gain genomic insights on the biological processes that COUP-TFII is involved in prostate cancer cells. Ingenuity Pathway Analysis (IPA) shows that the most prominent altered pathways in the COUP-TFII depleted cells are related to cell growth; cell cycle progression and DNA damage response. Indeed many growth related genes including E2F1, p21, CDC25A, Cyclin A and Cyclin B are changed in COUP-TFII knockdown cells, suggesting that COUP-TFII might be an important regulator for prostate cancer cell growth. Further functional assays from cells and mice genetic studies confirm the hypothesis that COUP-TFII serve as the major regulator to control prostrate cancer growth. Together, results provide insight into the role of COUP-TFII in prostate tumorigenesis.

overall_design: PC3 Cells were transfected with siRNA  (Control or COUP-TFII siRNA) duplexes (40 nM) and total RNA was isolated 48 hours later.

first: San-Pin

last: Wu

first: Xin

last: Xie

first: Ming-Jer

last: Tsai

first: Sophia

middle: Y


title: The conjunctival transcriptome in scarring trachoma for Tanzanians

status: Public on Aug 01 2011

city: London

first: Matthew

last: Burton

address: Keppel Street

country: United Kingdom

institute: London School of Hygiene and Tropical Medicine

summary: Trachoma is a poorly understood immuno-fibrogenic disease process, initiated by Chlamydia trachomatis (Ct). Differences in conjunctival gene expression profiles between Tanzanians with trachomatous conjunctival scarring (with (TSI) and without (TS) inflammation) and controls (C) were investigated to identify relevant host responses. Tarsal conjunctival swab samples were collected for RNA isolation and Ct PCR. Transcriptome-wide microarray experiments were conducted on 41 samples (TSI 13, TS 15, C14). Cases had enrichment in proinflammatory cytokines and chemokines (IL1B, IL8, CXCL5, CCL20); Matrix metalloprotienases (MMP7, MMP9, MMP12); components of the cornified envelope / squamous metaplasia (SPRR2a, SPRR2F).

overall_design: Case - control study. Conjunctival swab samples were collected from people with trachomatious conjunctival scarring with (TSI) and without (TS) clinically visable inflammation compared to normal controls (C). Total RNA extracted and analysed on the Illumina WG-6 platrom. Additional extensive qRT-PCR work done on a large set of case-control pairs.

first: Victor

last: Hu

first: Julien

last: Bauer

first: David

last: Mabey

first: Martin

last: Holland

first: Robin

middle: L

last: Bailey


title: Gene expression after felodipine

status: Public on Aug 03 2011

city: Jacksonville

first: Tushar

last: Patel

state: FL

address: 4500 San Pablo Road

institute: Mayo Clinic

summary: Cholangiocarcinomac ells have bee treated with felodipine and gene expression analyzed. In order to identify genes with differential gene expression between the groups cb-f and cb-g we study 2 hybridizations on the HG-U133_Plus_2_IVT array using analysis of variance.  The analysis identified 7068 genes with significant gene expression differences between the groups (p<0.01).

overall_design: Cells were treated for 24 hrs with felodipine and collected for RNA extraction and hybridization on Affymetrix microarrays.

first: Chiara

last: Braconi


title: Study of gene expression of mouse cMET experiments

status: Public on Aug 15 2011

city: Boston

first: Chunsheng

last: Zhang

address: 33 Avenue Louis Pasteur

institute: Merck

summary: We analyzed the molecular changes in a mouse c-MET over-expression model of hepatocellular carcinoma (HCC).

overall_design: FVB mice overexpressing human c-MET carried one copy of the LAP-tTa transgene (the liver-specific LAP promoter driving the Tet-VP16 transactivator) and one copy of the TRE-c-MET transgene (Tet-operator regulated human c-MET gene).  Normal liver or liver tumor tissue (two per mouse) was collected and processed for gene expression profiling.


title: The conjunctival transcriptome in scarring trachoma

summary: Trachoma is a poorly understood immuno-fibrogenic disease process, initiated by Chlamydia trachomatis (Ct). Differences in conjunctival gene expression profiles between Ethiopians with trachomatous trichiasis (with (TTI) and without (TT) inflammation) and controls (NC) were investigated to identify relevant host responses. Tarsal conjunctival swab samples were collected for RNA isolation and Ct PCR. Transcriptome-wide microarray experiments were conducted on 42 samples (TTI 13, TT 15, NC14). Specific results were confirmed using multiplex quantitative RT-PCR for 16 mRNA targets in an independent collection of case-control samples: 386 case-control pairs (TTI 244, TT 142, NC 386). The gene expression profiles of cases were consistent with: squamous metaplasia (Keratins, SPRR), pro-inflammatory cytokine production (IL-1β, CXCL5, S100A7) and tissue remodelling (MMP7, MMP9, MMP12, HAS3). There was no difference in the level of IFNG between cases and controls. However, cases had increased INDO, NOS2A, and IL13RA2 and reduced IL13. Ct was detected in 1/772. Cases show evidence of ongoing inflammation and tissue remodelling, which were more marked where clinical inflammation was also present. Significantly, these processes appear to be active in the absence of current Ct infection. There was limited evidence of a TH1 response (INDO, NOS2A) and no association between a TH2 response and cases. The epithelium appears actively involved in late cicatricial stages of trachoma through production of pro-inflammatory factors (IL-1β, CXCL5, S100A7). Longitudinal studies are needed to investigate which aetiological factors and pathways are associated with progressive scarring and whether simply controlling chlamydial infection will halt progression in people with established cicatricial disease.

overall_design: Case - control study. Conjunctival swab samples were collected from people with trachomatous trichiasis with (TTI) and without (TT) clinically visable inflammation compared to normal controls (C). Total RNA extracted and analysed on the Illumina WG-6 platrom. Additional extensive qRT-PCR work done on a large set of case-control pairs.

first: Saul

middle: N

last: Rajak


title: An expression microarray approach for the identification of candidate metastable epialleles in the mouse genome

status: Public on Jul 15 2011

city: Ann Arbor

first: Jung

last: Kim

state: MI

address: 1400 E. Medical Center Drive

institute: University of Michigan

summary:
Genetic loci displaying environmentally responsive epigenetic marks, termed metastable epialleles, offer a solution to the paradox presented by genetically identical yet phenotypically distinct individuals.  The murine viable yellow agouti (Avy) locus is a well-described metastable epiallele that serves as a visual epigenetic biosensor. The Avy locus exhibits a high R-value or ratio of inter-individual (Vi) to inter-tissue (Vt) variance in gene expression, characteristic of what we term the ‘Agouti Expression Fingerprint.’ We propose a novel method for identification of candidate metastable epialleles based on the Agouti Expression Fingerprint, defining candidates as loci with R-values greater than 1.5 on expression microarray.
Using Expression data from tissues of the three germ layers (liver, kidney, brain), high variance in agouti RNA levels among isogenic animals coupled with low variance among tissue types in individual animals is demonstrated. Here, we provide proof of concept for the ‘Agouti Expression Fingerprint’; the characterization of epigenetically labile loci in humans will be crucial to the development of novel screening and therapeutic targets for human disease prevention.

overall_design: For expression microarray studies, total RNA was isolated from liver, kidney, and brain tissue from 10 male Avy/a mice (2 per each of the 5 coat color classes) at time of weaning and coat color determination (day 22). Using Affymetrix GeneChip Mouse Genome 2.0 arrays (Santa Clara, CA), we queried the entire mouse genome for candidate metastable epialleles that display the Agouti Fingerprint.  Approximately 100 of the greater than 40,000 transcripts on the mouse array displayed an expression pattern characterized as high inter-individual variation coupled with low inter-tissue variation (R-value > 1.5).

first: Caren

last: Weinhouse

first: Olivia

middle: S

last: Anderson

first: Tamara

middle: R

last: Jones

first: Shayna

last: Liberman

first: Muna

last: Nahar

first: Laura

last: Rozek

first: Randy

last: Jirtle

first: Dana

middle: C

last: Dolinoy


title: Global gene expression profiles and the progression of pro- and anti-inflammatory pathways in mouse models

status: Public on Aug 02 2011

city: Cincinnati

first: nupur

last: dasgupta

state: OH

address: 3333 Burnet Ave.

institute: Cincinnati Children's Hopital Medical Center

summary: Gaucher Disease (GD) is caused by defective glucocerebrosidase (GCase) activity and the consequent accumulation of its substrate, glucosylceramide (GC). This excess of accumulation of GC leads to broad functional impairments in multiple organs, but the pathogenic pathways leading to lipid laden macrophages (Gaucher cells) in visceral organs and their abnormal function is obscure.  To understand the molecular pathogenesis of GD, developmental global gene expression was conducted by microarray analyses of total mRNAs from lung and liver of two distinct GCase point-mutated mice (V394L/V394L and D409V/null) and genetic background matched wild-type controls.  INFg regulated pro-inflammatory and IL-4 regulated anti-inflammatory  cytokine/mediator network were constructed in the lung and liver of GCase mutant mice. Progressive alterations of the INFg and IL-4 pathways were similar, but to different degrees, in visceral tissues from the two different GCase mutated mice. These analyses implicate IFNg regulated pro-inflammatory and IL-4 regulated anti-inflammatory networks in the differential pathophysiological progression.

overall_design: In order to understand the molecular pathogenesis of GD, the disease progression in those models were inverstigated in two visceral tissues (lung and liver) at four time points according to the genotypes. 9V/null: 4 weeks (4w), 12 weeks (12w), 18 weeks (18w), 28 weeks (28w); 4L: weeks (4w), 12 weeks (12w), 18 weeks (18w), 28 weeks (28w). The data from those models were analyzed relative to the adult wild type at 28 weeks.


title: Transcriptome profiling and expression analyses of genes critical to wheat adaptation to low temperature

status: Public on Oct 05 2011

city: Albany

first: Debbie

last: Laudencia-Chingcuanco

state: CA

address: 800 Buchanan Street

department: Western Regional Research Center

institute: USDA-ARS

summary: Understanding the mechanism of low temperature (LT) adaptation is crucial to the development of cold-tolerant crops.  To identify the genes involved in the development of LT tolerance in the crown of hexaploid wheat we examined the global changes in genes expression during cold-treatment using the Affymetrix Wheat Genome Chip.

overall_design: Time-series experiment with 4 genotypes x 8 time-points X 3 biological replicates in random block design; 96 hybridizations


title: Gene expression profiling of CEBPA double-, single-mutant and CEBPA wild type AML

status: Public on Aug 04 2011

city: Rotterdam

first: Erdogan

last: Taskesen

address: dr. molewaterplein 50

country: Netherlands

institute: Erasmus University Medical Center (EMC)

summary: A previously predictive CEBPA double mutant (CEBPAdm) signature was hampered by the recently reported CEBPA silenced AML cases that carry a similar gene expression profile (GEP). Two independent AML cohorts were used to train and evaluate the predictive value of the CEBPAdm signature in terms of sensitivity and specificity. A predictive signature was created, containing 25-probe sets by using a logistic regression model with Lasso regularization, which selects discriminative probe sets between the classes, CEBPAdm and all other AML cases, CEBPA wild type (CEBPAwt) and CEBPA single mutant (CEBPAsm). Subsequently, a classifier was trained on the entire HOVON-SAKK cohort based on a two-class approach; CEBPAdm versus all other cases (CEBPAwt and CEBPAsm). This trained classifier subsequently classified 16 candidate CEBPAdm cases in the AMLSG-cohort out of 154 AML cases. This approach showed perfect sensitivity and specificity (both 100%). In addition, we have performed a classification between CEBPAdm ,CEBPAsm, and CEBPAwt to infer if we were able to accurately classify CEBPAsm cases. We observed that all CEBPAsm cases were classified as CEBPAwt, thus CEBPAsm cases do not have a consistent gene expression pattern and are different from the CEBPAdm group.

overall_design: All samples were obtained from untreated patients at the time of diagnosis. Cells used for microarray analysis were collected from the purified fraction of mononuclear cells after Ficoll density centrifugation. Routine diagnostic algorithms, including the characterization of molecular markers are performed.

first: Lars

last: Bullinger

first: Andrea

last: Corbacioglu

first: Mathijs

middle: A

last: Sanders

first: Claudia

last: Erpelinck

first: Bas

last: Wouters

first: Sonja

last: van der Poel-van de Luytgaarde

first: Frederik

last: Damm

first: Jurgen

last: Krauter

first: Arnold

last: Ganser

first: Richard

middle: F

last: Schlenk

first: Bob

last: Löwenberg

first: Ruud

last: Delwel

first: Hartmut

last: Döhner

first: Peter

last: Valk

first: Konstanze


title: Gene expression of EpH4 cells stably transfected with PR

status: Public on Jul 14 2011

city: Bethesda

first: Erika

last: Ginsburg

state: MD

address: 37 Convent Dr.

department: NCI

institute: NIH

summary: Whole mouse genome analysis to discover differentially expressed genes in EpH4 cells overexpressing PR

overall_design: Total RNA was extracted from stably transfected EpH4-PR mouse epithelial cell line

first: Goldhar

first: Plant

last: Vonderhaar


title: CTCF Binding Sites by ChIP-seq from ENCODE/University of Washington

status: Public on Jun 29 2011

city: Stanford

first: ENCODE

last: DCC

address: 300 Pasteur Dr

institute: ENCODE DCC

type: Genome binding/occupancy profiling by high throughput sequencing

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

This track is produced as part of the ENCODE Project. This track displays maps of genome-wide binding of the CTCF transcription factor in different cell lines using ChIP-seq high-throughput sequencing
For data usage terms and conditions, please refer to http://www.genome.gov/27528022  and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design:
Cells were grown according to the approved ENCODE cell culture protocols. Cells were crosslinked with 1% formaldehyde, and the reaction was quenched by the addition of glycine. Fixed cells were rinsed with PBS, lysed in nuclei lysis buffer, and the chromatin was sheared to 200-500 bp fragments using Fisher Dismembrator (model 500). Sheared chromatin fragments were immunoprecipitated with specific polyclonal antibodies at 4 degrees C with gentle rotation. Antibody-chromatin complexes were washed and eluted. The cross linking in immunoprecipitated DNA was reversed and treated with RNase-A. Following proteinase K treatment, the DNA fragments were purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. 20-50 ng of ChIP DNA was end-repaired, adenine ligated to Illumina adapters was added, and then a Solexa library was made for sequencing.
ChIP-seq affinity is directly reflected in raw tag density (Raw Signal), which is shown in the track as density of tags mapping within a 150 bp sliding window (at a 20 bp step across the genome). ChIP-seq affinity zones (HotSpots) were identified using the HotSpot algorithm described in Sabo et al. (2004). 1.0% false discovery rate thresholds (FDR 0.01) were computed for each cell type by applying the HotSpot algorithm to an equivalent number of random uniquely mapping 36mers. ChIP-seq affinity (Peaks) were identified as signal peaks within FDR 1.0% hypersensitive zones using a peak-finding algorithm.

last: Sandstrom


title: Comparison of the RNA expression profile between P-cadherin over-expressing BLM melanoma cells and empty-vector control cells

status: Public on Jun 01 2011

city: Leuven

first: Rekin's

last: Janky

address: Herestraat 49 Box 816

department: Nucleomics Core

country: Belgium

institute: VIB

summary: Van Marck et al. (Cancer Research, 2005) published that overexpression of P-cadherin in a melanoma cell line promoted homo- and heterotypic cell-cell adhesion, induced an epitheloïd morphology, and impaired cancer cell invasion. Therefore, we wanted to compare the RNA expression profile between the empty vector control cells and the P-cadherin overexpression variant. This experiment could provide us with effector molecules of P-cadherin that mediate its anti-invasive function.

overall_design: 2 samples were analyzed, each with 2 replicates

first: Koen

last: Jacobs

first: Marc

last: Bracke

first: Veerle

last: Van Marck


title: DNaseI Hypersensitivity by Digital DNaseI from ENCODE/University of Washington

status: Public on Jun 03 2011

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

This track is produced as part of the ENCODE Project. This track shows DNaseI sensitivity measured genome-wide in different cell lines using the Digital DNaseI methodology (see below), and DNaseI hypersensitive sites. DNaseI has long been used to map general chromatin accessibility and DNaseI hypersensitivity is a universal feature of active cis-regulatory sequences. The use of this method has led to the discovery of functional regulatory elements that include enhancers, insulators, promotors, locus control regions and novel elements. For each experiment (cell type) this track shows DNaseI sensitivity as a continuous function using sequencing tag density (Raw Signal), and discrete loci of DNaseI sensitive zones (HotSpots) and hypersensitive sites (Peaks)."
For data usage terms and conditions, please refer to http://www.genome.gov/27528022  and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: Cells were grown according to the approved ENCODE cell culture protocols. Digital DNaseI was performed by DNaseI digestion of intact nuclei, isolating DNaseI 'double-hit' fragments as described in Sabo et al. (2006), and direct sequencing of fragment ends (which correspond to in vivo DNaseI cleavage sites) using the Solexa platform (36 bp reads). Uniquely mapping high-quality reads were mapped to the genome. DNaseI sensitivity is directly reflected in raw tag density (Raw Signal), which is shown in the track as density of tags mapping within a 150 bp sliding window (at a 20 bp step across the genome). DNaseI sensitive zones (HotSpots) were identified using the HotSpot algorithm described in Sabo et al. (2004). 1.0% false discovery rate thresholds (FDR 0.01) were computed for each cell type by applying the HotSpot algorithm to an equivalent number of random uniquely mapping 36mers. DNaseI hypersensitive sites (DHSs or Peaks) were identified as signal peaks within FDR 1.0% hypersensitive zones using a peak-finding algorithm.


title: Genome-wide analysis of perinatal effect of nitric oxide (NO) donor molsidomine on the kidneys of Fawn-Hooded Hypertensive (FHH) rats at different ages

status: Public on Jul 01 2011

city: Utrecht

first: Sebastiaan

last: Wesseling

address: Heidelberglaan 100

department: Nephrology & Hypertension

institute: University Medical Center Utrecht

summary: Microarray was performed on kidneys in molsidomine-treated FHH rats in order to gain understanding why perinatal treatment with molsidomine in FHH was beneficial in FHH offspring (e.g. lower blood pressure and less renal damage)

overall_design: In total 62 samples were used. 6 different groups for each gender: 2d, 2w and adult rats with or without molsidomine treatment. Total 12 groups.


title: Functional genome analysis of Bifidobacterium breve UCC2003 reveals a major conserved host-colonization factor

first: Aldert

last: Zomer

address: Yalelaan 1

department: Department of Infectious Diseases and Immunology,Faculty of Veterinary Medicine

institute: Utrecht University

type:
Expression profiling by array
Genome variation profiling by array

summary: Development of the human gut microbiota commences at birth with bifidobacteria being among the first colonizers of the sterile newborn gastrointestinal tract. To date the genetic basis of bifidobacterial colonization and persistence remains poorly understood. Transcriptomic analysis of the 2,422,684 bp genome of Bifidobacterium breve UCC2003, a strain isolated from a nursling stool, during colonization of a mouse model revealed the differential expression of a type IVb or so-called Tad pilus-encoding cluster. Mutational analysis coupled with colonization experiments demonstrated that the UCC2003 tad gene cluster is essential for colonization. Immunogold transmission electron microscopy confirmed the presence of the Tad pili at the cell poles of B. breve UCC2003. Conservation of the Tad pilus-encoding locus among sequenced bifidobacterial genomes supports the notion of a ubiquitous and novel host colonization and persistence mechanism for bifidobacteria.

sample_organism:
Bifidobacterium breve
Mus musculus
Bifidobacterium breve UCC2003

overall_design:
Colonisation of BalbC mice:
Seven-week-old male, BalbC mice were housed in individually vented cages (Animal care systems, Colorado) under a strict 12 h light cycle. Mice (n = 5 per group) were fed a standard polysaccharide-rich mouse chow diet and water ad libidum. Mice were inoculated by oral gavage (109 cfu of B. breve UCC2003 pPKCM7 or B. breve UCC 2003-tadA pPKCM7  in 100 ul of PBS). Fecal pellets were collected at intervals over 25 days to enumerate bacteria. Twenty five days after inoculation, mice were sacrificed and their intestinal tracts quickly dissected. Aliquots of small intestine, caecum and large intestine were harvested for determination of colony forming units (cfu) (serial dilution plating on RCA agar plates with appropriate antibiotics). Caeca were placed in RNA later for immediate RNA isolation (see below).
Microarray procedures:
DNA microarrays containing oligonucleotide primers representing each of the 1926 annotated genes of B. breve UCC2003 were obtained from Agilent Technologies (Palo Alto, CA). An overnight culture of B. breve was used to inoculate (1 % inoculum) 50 ml of MRS broth. Cells were incubated at 37°C until an optical density at 600 nm (OD600) of 0.5 was reached. Cells were then harvested by centrifugation at 8,000 x g for 1 min at room temperature and immediately frozen at -80oC. Methods for cell disruption, RNA isolation,  RNA quality control were performed as described previously (van Hijum et al., 2005). Caeca, isolated from mice, were placed in RNAlater (Ambion) and immediately processed. Bacterial mRNA was extracted from caecal RNA preparations using the MicrobEnrich and MicrobExpress kits (Ambion) according to the manufacturer’s instructions. cDNA from bacterial mRNA was synthesized using the cDNA synthesis and labelling kit DSK-001 (Kreatech) according to the manufacturer’s instructions. 1 μl of each cDNA solution (10 ng) was amplified in triplicate using the GenomiPhi™ V2 DNA Amplification Kit (Amersham Biosciences) according to the manufacturer's protocol and labelled with Cy3 or Cy5 using Cy3-ULS and Cy5-ULS from the cDNA synthesis and labelling kit DSK-001 (Kreatech). Labelled and amplified cDNA was hybridized using the Agilent Gene Expression hybridization kit (part number 5188-5242) as described in the Agilent Two-Color Microarray-Based Gene Expression Analysis (v4.0) manual (publication number G4140-90050). Following hybridization, microarrays were washed as described in the manuals and scanned using Agilent's DNA microarray scanner G2565A. The scanning results were converted to data files with Agilent's Feature Extraction software (version 9.5). DNA microarray data was processed as previously described (Zomer et al. 2009). Differential expression tests were performed using a t test. A gene was considered differentially expressed between a test strain and control when an expression ratio of >5 or <0.2 relative to the result for the control was obtained with a corresponding P value that was <0.0001.

DNA microarrays containing oligonucleotide primers representing each of the 1926 annotated genes of B. breve UCC2003 were obtained from Agilent Technologies (Palo Alto, CA). An overnight culture of B. breve was used to inoculate (1 % inoculum) 50 ml of MRS broth. Cells were incubated at 37°C until an optical density at 600 nm (OD600) of 0.5 was reached. Cells were then harvested by centrifugation at 8,000 x g for 1 min at room temperature and immediately frozen at -80oC. Methods for cell disruption, DNA isolation were performed as described previously (O'Riordan et al. 1998). Genomic DNA (gDNA) labeling for comparative genome hybridizations (CGH) was undertaken according to the BµG@S standard protocols (Hinds et al. 2002). In brief 5 µg of bifidobacterial genomic DNA was labeled with dCTP Cy3 (UCC2003) or dCTP Cy5 (test strain) using random primers (Promega) and a DNA polymerase I large Klenow fragment (Invitrogen). Labelled gDNA was hybridized using the Agilent CGH kit (part number 5188-5220) as described in the Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis (v6.0) manual (publication number G4410-90010). Validation of the CGH was performed by comparing the hybridization efficiency of DNA of the B. breve type strain DSM 20213 to sequence identity of this strain with the probes on the microarray. A clear correlation was observed between sequence identity and hybridization efficiency, suggesting that an ln 2 fold change in signal intensity between target and control is sufficient to determine whether a gene is present or absent on a given genome. Therefore, a gene was considered absent when the ratio between a test strain and UCC2003 was < ln 2 with a corresponding P value that was <0.0001.

first: Douwe

last: van Sinderen


title: Identification of candidate genes affecting juvenile wood stiffness in Pinus radiata

city: Canberra

first: Xinguo

last: Li

state: ACT

address: Building 602, Black Mountain Laboratories

department: Plant Industry

country: Australia

institute: CSIRO

summary: Wood stiffness is the most important wood quality trait of forest trees for structural timber production. We investigated genes differentially transcribed in radiate pine trees with distinct wood stiffness using bulked segregant analysis (BSA) and cDNA microarrays. Transcript accumulation in earlywood (EW) and latewood (LW) of high (HS) and low stiffness (LS) trees in two progeny trials was compared.

overall_design: Radiata pine trees used for microarray experiment were selected from two progeny trials planted at Flynn and Kromelite, Australia. Based on the IML-based MOE measurement, five families with highest and lowest MOE each were selected from each trial, which represented two segregant populations with contrasting wood stiffness. Two individuals from each selected family were further sampled. Developing xylem tissues of selected trees in Flynn trial were sampled in spring (October) and autumn (April), representing earlywood (EW) and latewood (LW) of juvenile aged trees, respectively. Collection of xylem tissues from Kromelite trial was arranged in summer (late November) when latewood (LW) was formed. The xylem tissues were scraped at breast height with a sharp chisel after the bark was removed. In Flynn trial EW and LW tissues were collected from the same sampled trees on opposite sides of the trunk. Transcript accumulation was compared in trees with highest (HS) and lowest stiffness (LS) using xylem samples from Flynn collected in spring (EW) and autumn (LW), as well as Kromelite in summer (LW), respectively. Bulked segregant analysis (BSA) was used for the experiment design. Total RNA samples extracted from the five trees with HS were pooled at equal amount, and compared to the bulked five individuals with LS. This pooling strategy can partly minimize the genetic variation among different genotypes. Dye swaps were applied in each biological replicate.

first: Harry

middle: X

last: Wu

first: Simon

last: Southerton


title: Sensing prokaryotic mRNA signifies microbial viability and promotes immunity

status: Public on May 22 2011

city: Wageningen

first: Guido

last: Hooiveld

address: HELIX, Stippeneng 4

department: Div. Human Nutrition & Health

institute: Wageningen University

summary: MyD88-independent signal transduction associated with Toll-like receptors (TLRs) 3 and TLR4 is mediated through the adapter protein TRIF (TIR-domain-containing adapter-inducing interferon-beta). It has been proposed that TLR signalling is important for the transcription of crucial inflammasome components like NLRP3, a process that has been termed "priming". In order to test whether TRIF signalling was required for the priming of inflammasome components, we performed a genome wide transcriptional analysis on wild-type and Trif-knockout bone marrow derived macrophages (BMMs) before and 1, 3 and 6 hours after phagocytosis of E. coli. These results indicated that TRIF was involved in the activation and not transcriptional priming of the NLRP3 inflammasome.

overall_design: Bone marrow derived macrophages from WT and Trif knockout mice, stimulated with E.coli for up to 6hrs.

first: Leif

middle: E

last: Sander

first: Michael

last: Davis

first: Mark

middle: V

last: Boekschoten

first: Derk

last: Amsen

first: Christopher

last: Dasher

first: Bernard

last: Ryffel

first: Joel

last: Swanson

last: Müller

first: J

middle: M

last: Blander


title: MLL partner genes confer distinct biological and clinical signatures of pediatric AML, an AIEOP study

status: Public on Apr 21 2011

city: Padova

first: Andrea

last: Zangrando

address: Via Giustiniani, 3

department: Woman and Child's Health

country: Italy

institute: University of Padova

summary:
We retrospectively analyzed AML patients enrolled in the AIEOP since 2000, 42 patients with 11q23 rearrangement were analyzed by gene expression profile
Gene expression analyses were performed to compare AML MLL partner genes (AF9, AF10, AF6, ENL, ELL, Septin 6, and AF1q)

Keywords: Expression data

overall_design: Class comparison between different AML MLL partner genes

first: Martina

last: Pigazzi

first: Silvia

last: Bresolin


title: pcaGoPromoter - An R package for functional interpretation of principal component analysis of genome-wide gene expression data

status: Public on May 01 2011

city: Copenhagen

first: Morten

last: Hansen

address: Blegdamsvej 3B

department: Department of Cellular and Melocular Medicine

country: Denmark

institute: Faculty of Health Sciences

summary: Background and aim: Analysis of data obtained from genome wide gene expression experiments is challenging, due to the huge amount of variables, management of the data and the need for multivariate analysis. We here present the R package: pcaGoPromoter that facilitates the interpretation of genome wide expression data to overcome these problems. In a first step principal component analysis is applied to overview any differences between the observations and possible groupings. The next step is interpretation of the principal components with respect to both biological function and involvement of predicted transcription factor binding sites. The robustness of the results is evaluated using cross validation. Illustrative plots of PCA score plots and Gene Ontology terms are available. To illustrate the functionality of the R package, we designed a serum stimulation experiment, where the main biological outcome is well documented.  Results: Samples from the serum stimulation experiment were analyzed using the Affymetrix Human Genome U133 Plus 2.0 chip. The array data were analyzed by the tools of the pcaGoPromoter package, which resulted in a clear separation of the observations into the three experimental groups - controls, serum only and serum with inhibitor. The functional annotation of the axes in the PCA score plot showed the expected serum promoted biological processes such as cell cycle progression and the predicted involvement of the expected transcription factors including E2F. In addition unexpected results, e.g. the cholesterol synthesis in serum depleted cells and NF-κB activation in inhibitor treated cells were uncovered.  Conclusion:  The pcaGoPromoter R package provides a collection of tools for analyzing gene expression data. It works with any platform using gene symbols or Entrez Ids as probe identifiers. In addition support for several popular Affymetrix GeneChip platforms is provided. The tools give an overview of the data via principal component analysis, functional interpretation by Gene Ontology terms (biological processes), and indication of involvement of possible transcription factors. Thus, pcaGoPromoter structures the high-dimensional data of gene expression experiments and can be applied to generate hypotheses for further exploration.

overall_design: A serum starved human monocyte cell line (ATCC® Number:  CRL-9853) was stimulated by serum in the presence or absence of the specific Erk-1/2 inhibitor U0126.

first: Thomas

last: Gerds

first: Ole

middle: H

last: Nielsen

first: Jacob

middle: B

last: Seidelin

first: Jesper

middle: T

last: Troelsen

first: Jørgen

last: Olsen


title: Transcriptomic characterization of the diabetic retinal response to chronic insulin treatment

status: Public on May 23 2011

city: Hershey

first: Penn State

last: COM

state: PA

address: 500 University Drive

department: Genome Sciences Facility

institute: Penn State College of Medicine

summary: The aim of this study was to identify alterations associated with diabetes-induced functional dysregulation of the retina and the effects of chronic insulin therapy.  Transcriptional profiling through microarray analysis was analyzed to find mRNA targets of interest in diabetic samples, as well as in the insulin treated group.   Verification of targets shown to be unrecovered in the insulin group was validated.

overall_design: Diabetes was induced in Sprague-Dawley male rats (Charles River Laboratories, Wilmington, MA) by intraperitoneal injection of 65 mg/kg streptozotocin (STZ)(Sigma-Aldrich, St. Louis, MO) in 10mM sodium citrate pH 4.5 vehicle. Control rats were injected with an equal dose of vehicle only.  Rats had free access to food and water, and were maintained on a 12 hour light/dark cycle. Blood glucose level and body weight were measured 6 days post-STZ or vehicle injection, and biweekly throughout the experiment. Only rats with blood glucose levels >250 mg/dL at the time of the original test and throughout the experiment were included in the diabetic groups.  The insulin treatment group received one 26mg subcutaneous pellet (LinShin Canada, Scarborough, Canada) delivered via trocar 6 weeks post-STZ injection. An additional 26 mg implant was introduced when body weight exceeded 300 g or when midday non-fasting blood glucose exceeded 250 mg/dL.  At the time of retina harvest, rats were given a lethal dose of pentobarbital, 100 mg/kg, (Ovation Pharmaceuticals Inc., Deerfield, IL) by intraperitoneal injection and sacrificed by decapitation. Retinas were rapidly excised snap- frozen in liquid nitrogen for subsequent experimentation.

first: Georgina

last: Bixler

first: Heather

middle: D

last: VanGuilder

first: Robert

last: Brucklacher

first: Wendy

middle: G

last: Dunton

first: Sarah

middle: K

last: Bronson

first: Willard

last: Freeman


status: Public on Apr 19 2011

city: Leiden

first: Seyed Yahya

last: Anvar

address: Einthovenweg 20

department: Center for Human and Clinical Genetics

institute: Leiden University Medical Center

summary: Oculopharyngeal muscular dystrophy (OPMD) is a late-onset progressive muscle disorder caused by a poly-alanine expansion mutation in PABPN1. The hallmark of OPMD is the accumulation of the mutant protein in insoluble nuclear inclusions. The molecular mechanisms associated with disease onset and progression are unknown. We performed a high-throughput cross-species transcriptome study of affected muscles from two OPMD animal models and from patients at pre-symptomatic and symptomatic stages. The most consistently and significantly OPMD-deregulated pathway across species is the ubiquitin-proteasome system (UPS). By analyzing expression profiles, we found that the majority of OPMD-deregulated genes are age-associated. Based on expression trends, disease onset can be separated from progression; the expression profiles of the proteasome-encoding genes are associated with onset but not with progression. In a muscle cell model, proteasome inhibition and the stimulation of immunoproteasome specifically affect the accumulation and aggregation of mutant PABPN1. We suggest that proteasome down-regulation during muscle aging triggers the accumulation of expPABPN1 that in turn enhances proteasome deregulation and leads to intranuclear inclusions (INI) formation.

overall_design:
Human quadriceps muscle samples were collected with the needle or by an open surgical procedure from OPMD patients and family members as well as from anonymous age-matching healthy individuals that gave informed consent. The presence of expansion mutation in PABPN1 in OPMD patients and pre-symptomatic was determined with sequencing. Bergstrom needle biopsies from the (pre)symptomatic patients were approved by the ethical committee. Total RNA was extracted from skeletal muscles using RNA Bee (Amsbio) according to the manufacturer's instructions. RNA integration number (RIN) was determined with RNA 6000 Nano (Agilent Technologies). RNA with RIN >7 were used for subsequent steps. RNA labeling was performed with the Illumina® TotalPrep RNA Amplification kit (Ambion) according to the manufacturer's protocol, and subsequently was hybridized to Illumina Human v3 Bead arrays.

SUBMITTER_CITATION: Anvar, S.Y., 't Hoen, P.A.C., Venema, A., van der Sluijs, B., van Engelen, B., Snoeck, M., Vissing, J., Trollet, C., Dickson, G., Chartier, A. et al. (2011). Deregulation of the ubiquitin-proteasome system is the predominant molecular pathology in OPMD animal models and patients. Skeletal Muscle 1:15

first: Seyed

middle: Y

first: Peter

last: 't Hoen

last: Venema

first: Barbara

last: van der Sluijs

first: Baziel

last: van Engelen

first: Marc

last: Snoeck

first: John

last: Vissing

first: Capucine

last: Trollet

first: George

last: Dickson

first: Aymeric

last: Chartier

first: Martine

last: Simonelig

first: Gert-Jan

last: van Ommen

first: Silvere

last: van der Maarel

first: Vered

last: Raz


title: Gene expression profiling in DBA/2J glaucoma

status: Public on Apr 15 2011

city: Mount Desert

first: Gareth

middle: Rhys

last: Howell

address: 600 Main Street

institute: The Jackson Laboratory

summary: In this study that was specifically designed to identify early stages of glaucoma in DBA/2J mice, we used genome-wide expression profiling and a series of computational methods. Our methods successfully subdivided eyes with no detectable glaucoma by conventional assays into molecularly defined stages of disease. These stages represent a temporally ordered sequence of glaucoma states. Using an array of tools, we then determined networks and biological processes that are altered at these early stages. Our strategy proved very sensitive, suggesting that similar approaches will be valuable for uncovering early processes in other complex, later-onset diseases. Early changes included upregulation of both the complement cascade and endothelin system, and so we tested the therapeutic value of separately inhibiting them. Mice with a mutation in the complement component 1a gene (C1qa) were robustly protected from glaucoma with the protection being among the greatest reported. Similarly, inhibition of the endothelin system was strongly protective. Since EDN2 is potently vasoconstrictive and was produced by microglial/macrophages, our data provide a novel link between these cell types and vascular dysfunction in glaucoma. Targeting early events such as the upregulation of the complement and endothelin pathways may provide effective new treatments for human glaucoma.

overall_design: Genome-wide assessment of gene expression changes was performed in DBA/2J mice. The optic nerve head and retina from 40 DBA/2J eyes at 10.5 months of age were separately profiled. These eyes were selected as they encompassed a range of glaucoma severity. Two control groups were also included; 10 eyes from 10.5 months old D2-Gpnmb+ mice (age and strain matched, no glaucoma control) and 10 eyes from 4.5 months old DBA/2J mice (young, pre-glaucoma).

first: G

first: S

middle: W

last: John


title: Analysis of gene expression regulated by Drosophila melanogaster Tis11

status: Public on Mar 25 2011

city: Heidelberg

first: Milan

last: Spasic

address: Im Neuenheimer Feld 280

country: Germany

institute: German Cancer Research Centre

summary: In mammalian cells, AU-rich elements (AREs) are well known regulatory sequences located in the 3' untranslated region (UTR) of many short-lived mRNAs that suppress gene expression at the posttranscriptional level. Tis11, a zinc finger RNA-binding protein homologous to mammalian tristetraprolin, targets ARE-containing reporter mRNAs for rapid degradation in SL2 cells. To identify Drosophila mRNA targets of Tis11, we performed genome-wide expression profiling after dsRNA-mediated depletion of endogenous Tis11 in SL2 cells. Furthermore, we studied the involvement of Tis11 in regulating the Drosophila immune response by profiling mRNA expression after LPS treatment, in the presence or absence of Tis11.

overall_design: SL2 cells were treated with either dsRNA against Tis11 or dsRNA against GFP (control). After 4 days of treatment with 12.5 microgram dsRNA / ml medium, total RNA was extracted and hybridized to Drosophila Genome 2.0 Arrays (Affymetrix, Cat. No. 900531). Where indicated, cells were treated with lipopolysaccharide (LPS, Sigma, O55:B5) at a concentration of 10 microgram / ml for 4 hours before lysis. All experiments were performed in 3 biological replicates.

first: Georg

last: Stoecklin


title: Linking phytoplankton community composition to seasonal changes in f ratio

status: Public on Mar 17 2011

city: Princeton

first: Bess

last: Ward

state: NJ

address: Guyot Hall

department: Geosciences

institute: Princeton University

summary: Seasonal changes in nitrogen assimilation have been studied in the western English Channel by sampling at approximately weekly intervals for 12 months. Nitrate concentrations showed strong seasonal variations. Available nitrogen in the winter was dominated by nitrate but this was close to limit of detection from May to September, after the spring phytoplankton bloom. 15N uptake experiments showed that nitrate was the nitrogen source for the spring phytoplankton bloom but regenerated nitrogen supported phytoplankton productivity throughout the summer. The average annual f ratio was 0.35, which demonstrated the importance of ammonia regeneration in this dynamic temperate region. Nitrogen uptake rate measurements were related to the phytoplankton responsible by assessing the relative abundance of nitrate reductase (NR) genes and the expression of NR among eukaryotic phytoplankton. Strong signals were detected from NR sequences that are not associated with known phylotypes or cultures. NR sequences from the diatom Phaeodactylum tricornutum were highly represented in gene abundance and expression, and were significantly correlated with f ratio. The results demonstrate that analysis of functional genes provides additional information, and may be able to give better indications of which phytoplankton species are responsible for the observed seasonal changes in f ratio than microscopic phytoplankton identification.

sample_organism:
synthetic construct
marine metagenome

overall_design:
NR gene diversity from seawater (two replicates of 16 blocks per array, 8 replicate features per probe, duplicate arrays for some samples)

The arrays contain three sets of probes for different applications (rbcL and nitrate reductase (NR) from phytoplankton, and amoA from ammonia oxidizing bacteria). The paper to which this submission relates, and the experiments reported in it, used only the NR probe set.

first: Andrew

middle: P

last: Rees

first: Paul

middle: J

last: Somerfield

first: Ian

last: Joint


title: Zebrafish: Estrogen induced gene expression

status: Public on Mar 08 2011

city: Singapore

first: Justin

middle: Jeyakani

last: Joseph Gnanakkan

state: Singapore

address: 60, Biopolis street

department: Computational and Systems Biology

institute: Genome Institute of Singapore

summary: We performed microarray experiment on zebrafish exposed to estrogen (17β-estradiol; a classified carcinogen) and an anti-estrogen (ICI 182,780). Our transcriptome analysis captured multiple estrogen-responsive genes and signaling pathways that increased cell proliferation, promoted DNA damage and genome instability, and decreased tumor suppressing effects, suggesting a common mechanism for estrogen-induced carcinogenesis. Comparative analysis with human E2 responsive genes revealed a core set of conserved estrogen-responsive genes that demonstrate enrichment of ER binding sites and cell cycle signaling pathways. Knowledge-based and network analysis led us to propose that the mechanism involving estrogen-activated ER mediated down-regulation certain genes followed by up-regulation cell cycle-related genes is highly conserved. Our study provides first evidence of molecular conservation of estrogen-responsiveness between zebrafish and human cancer cell lines, hence demonstrating the potential of zebrafish for estrogen-related cancer research.

overall_design: 10 samples were analyzed in total. Three  samples in control, 3 samples treated with estrogen and 3 samples treated with estrogen in combination with ICI.

first: Sinnakaruppan

last: Mathavan

first: Serene

last: Lee

first: Siew

last: Lam

first: Karuturi

middle: R

last: Murthy

first: Edison

last: Liu

first: Zhiyuan

last: Gong


title: Transcription profiling of One month old Gossypium herbaceum plant leaf during drought and watered condition

status: Public on Jan 11 2011

city: Lucknow

first: Samir

last: Sawant

state: UP

address: Sikandrabad

department: Plant Molecular Biology Lab

country: India

institute: National Botanical Research Institute

summary: Comparative genomics has emerged as a promising means of unravelling the molecular networks underlying complex traits such as drought tolerance.We used Affymetrix microarray to detail the global programme of gene expression underlying in drought and watered condition and identified distinct classes of up-regulated genes during this process.leaf tissue were used for the expression analysis during watered and drought condition  with three biological replicates for each case.

platform_organism:
Gossypium barbadense
Gossypium hirsutum
Gossypium arboreum
Gossypium raimondii

overall_design: Here we assess the genotype-dependent component of the drought-induced transcriptome response in two contrasting cotton (Gossypium spp) genotypes differing in drought tolerance. Leaf tissue were used during drought and watered condtion for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain differential expression pattern during drought and wtered condition.

first: Alok

last: Ranjan

first: Deepti

last: Nigam


title: Expression data from Arabidopsis seedlings treated with Pseudomonas aeruginosa (strain PA14)

status: Public on Jan 22 2011

city: Boston

first: Suresh

last: Gopalan

state: MA

address: 185 Cambridge Street, CPZN 7250

department: Molecular Biology

institute: Massachusetts General Hosptal & Harvard Medical School

summary:
Early stages of host microbe adaptations involve 'system status changes' (rewiring of pre-existing cellular signaling networks and components) of the host and microbe. We posited that under certain environmental conditions these changes leads to maladaptations and favor emergence of new infectious diseases, and these adaptations will have characteristic signatures representative of the adaptation.
Here using Arabidopsis seedlings in a submerged environment treated with P. aerugionsa, we show one such rewired regulation where the master two-component regulator GacA (previously shown to act upstream of quorum sensing, including the regulator LasR, that in turn controls a subset of virulence factors) is completely dispensable. The gacA mutant behaves similar to wild type P. aeruginosa (strain PA14) by a number of read-outs. Consistent with that, the gene expression data here indicates that the transcriptome pattern of the host is identical when treated with wild type PA14 or PA14-gacA mutant.

overall_design: Single time point (10 day old Arabidopsis seedlings infected with wild type PA14 or mutant bacteria PA14DgacA, and analyzed 24h after infection) with two independent experimenal replicates per treatment

first: Frederick

last: Ausubel


title: Global transcriptome profiling after exposure to solute, matric, and sand desiccation stress in S. wittichii RW1

status: Public on Jan 19 2011

city: Dübendorf

first: David

last: Johnson

address: Uberlandstrasse 133

department: Environmental Microbiology

country: Switzerland

institute: Eawag

summary: Spingomonas wittichii strain RW1 can completely oxidize dibenzo-p-dioxins and dibenzofurans, which are persistent contaminants of soils and sediments. For successful application in soil bioremediation systems, strain RW1 must cope with fluctuations in water availability, or water potential. The objectives of this study were to characterize how strain RW1 responses to changes in different components of the total water potential (solute and matric potential) and to then connect these responses to more realistic scenarios of soil desiccation. To accomplish this task, transcriptome profiling was used to investigate the effects of decreasing the solute potential with sodium chloride (solute stress), decreasing the matric potential with high-molecular weight polyethylene glycol (matric stress), or inoculating cells directly into unsaturated sand (sand desiccation stress). Transcriptome profiling revealed a general response to solute, matric, and sand desiccation stress that involved synthesizing trehalose and modifying the composition of exopolysaccarides. Transcriptome profiling also revealed responses that were unique to each stress. Only solute and matric stress triggered the down-regulation of flagella genes. Only solute and sand desiccation stress triggered the up-regulation of two RNA polymerase ECF-type sigma factors along with several membrane proteins, mechanosensitive channels, and solute transporters. Finally, only matric stress triggered the up-regulation of the RNA polymerase sigma-32 factor along with several molecular chaperones. Together, this study revealed a general response to solute, matric and sand desiccation stress but also unique responses to only a subset of these stresses, suggesting that each stress affects strain RW1 in a fundamentally different way.

overall_design: Comparative transcriptome profiling was performed to assess the effects of acute (30 min) solute and matric stress (3 samples for acute solute stress, 3 samples for acute matric stress, 3 controls), the effects of chronic (24 hours) solute and matric stress (3 samples for chronic solute stress, 3 samples for chronic matric stress, 3 controls), and the effects of sand desiccation stress (4 samples for sand desiccation treatment, 3 controls).

first: Jan

last: van der Meer


title: Molecular and phenotypic characterization of a mouse model of oculopharyngeal muscular dystrophy reveals severe muscular atrophy restricted to fast glycolytic fibres

status: Public on Jan 14 2011

first: Seyed Yahya

last: Anvar

summary: Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by ptosis, dysphagia and proximal limb weakness. Autosomal-dominant OPMD is caused by a short (GCG)8–13 expansions within the first exon of the poly(A)-binding protein nuclear 1 gene (PABPN1), leading to an expanded polyalanine tract in the mutated protein. Expanded PABPN1 forms insoluble aggregates in the nuclei of skeletal muscle fibres. In order to gain insight into the different physiological processes affected in OPMD muscles, we have used a transgenic mouse model of OPMD (A17.1) and performed transcriptomic studies combined with a detailed phenotypic characterization of this model at three time points. The transcriptomic analysis revealed a massive gene deregulation in the A17.1 mice, among which we identified a significant deregulation of pathways associated with muscle atrophy. Using a mathematical model for progression, we have identified that one-third of the progressive genes were also associated with muscle atrophy. Functional and histological analysis of the skeletal muscle of this mouse model confirmed a severe and progressive muscular atrophy associated with a reduction in muscle strength. Moreover, muscle atrophy in the A17.1 mice was restricted to fast glycolytic fibres, containing a large number of intranuclear inclusions (INIs). The soleus muscle and, in particular, oxidative fibres were spared, even though they contained INIs albeit to a lesser degree. These results demonstrate a fibre-type specificity of muscle atrophy in this OPMD model. This study improves our understanding of the biological pathways modified in OPMD to identify potential biomarkers and new therapeutic targets.

overall_design:
A17.1 transgenic mice have previously been described. Male A17.1 mice and WT controls were generated by crossing the heterozygous carrier strain A17.1 obtained from Rubinsztein's group with the FvB background mice. The mice were genotyped by PCR 3–4 weeks after birth. Wild type FvB and A17.1 mice were housed in minimal disease facilities (Royal Holloway, University of London) with food and water ad libitum.
Total RNA was extracted from skeletal muscles using RNA Bee (Amsbio) according to the manufacturer's instructions. RNA integration number (RIN) was determined with RNA 6000 Nano (Agilent Technologies). RNA with RIN >7 were used for subsequent steps. RNA labelling was performed with the Illumina® TotalPrep RNA Amplification kit (Ambion) according to the manufacturer's protocol, and subsequently was hybridized to Illumina Mouse v1.1 Bead arrays.

first: Capucine

last: Trollet

first: Seyed

first: Andrea

last: Venema

first: Iain

last: Hargreaves

first: Keith

last: Foster

first: Alban

last: Vignaud

first: Arnaud

last: Ferry

first: Elisa

last: Negroni

first: Christophe

last: Hourde

first: Martin

middle: A

last: Baraibar

first: Peter

last: 't Hoen

first: Janet

last: Davies

middle: C

last: Rubinsztein

first: Simon

last: Heales

first: Vincent

last: Mouly

first: Vered

last: Raz

first: Gillian

last: Butler-Browne

first: Silvere

last: van der Maarel

first: George

last: Dickson


title: Shewanella oneidensis MR-1 etrA deletion mutant strain expression profiling determines fine-tuning regulatory role in anaerobic metabolism

status: Public on Jan 29 2011

city: Atlanta

first: Claribel

last: Cruz-Garcia

state: Georgia

address: 311 Ferst Dr

department: Civil and Environmental Engineering

institute: Georgia Institute of Technology

summary:
Comparisson of expression profiling of a etrA deletion mutant strain (experimental sample) with that of the wild type Shewanella oneidensis MR-1 strain to assess global direct/indirect genetic regulation
EtrA in Shewanella oneidensis MR-1 shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulator Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is complex and not well understood. Whole-genome expression profiling using a etrA gene deletion mutant as the experimental sample and the wild type strain as the reference, determine that EtrA fine-tunes the expression of genes involved in various anaerobic metabolic pathways, including nitrate, fumarate and dimethyl sulfoxide reduction. Moreover, genes involved in prophage activation and and genes implicated in aerobic metabolism were also differentially expressed. In contrast to previous studies that attributed a minor regulatory role to EtrA in Shewanella spp., this study demonstrates that EtrA acts as a global transcriptional regulator and cofers physiological advantages to the strain under certain growth conditions.

overall_design: Total RNA was extracted from three biological replicate cultures of both S. oneidensis etrA deletion mutant and the wild type strain, grown individually on anaerobic minimal medium supplemented with 2 mM nitrate as the electron acceptor and lactate as the electron donor. Total RNA was collected from mid-log phase cultures. cDNA from each sample was prepared and labeled with Cy-dyes. Three biological replicates per treatment were used for the hybridization of six microarray slides including technical replicates (dye-swap) per experiment.

last: Cruz-García

first: Alison

middle: E

last: Murray

first: Jorge

middle: L

last: Rodrigues

first: Jeffrey

last: Gralnick

first: Lee-Ann

last: McCue

first: Margaret

middle: F

last: Romine

first: Frank

last: Loeffler

first: James

last: Tiedje


title: An Invasive Gene Expression Signature in Pancreas Cancer.

status: Public on Jan 01 2011

city: Dublin

first: Annamarie

last: Rogers

address: AMNCH

department: Surgery

country: Ireland

institute: Trinity College Dublin

summary:
Gene expression profiling has demonstrated clinical utility as a predictive tool in clinical oncology. We have identified genes associated with invasion of pancreatic cancer, and with potential for identifying early recurrence.
We used Affymetrix Human U133 Plus 2.0 microarrays to identifiy specific predictive profiles in pancreatic cancer, and the evolution of gene expression.  We identified distinct classes of up-regulated genes during this process.

overall_design: Primary and metastatic pancreatic cancer cell lines (BxPC-3 and AsPC-1), were stimulated with with phorbol-12-myristate 13-acetate (PMA), a known inducer of invasion.  Affymetrix gene expression microarray analysis was performed, comparing PMA stimulated BxPC-3 and AsPC-1 gene expression to unstimulated controls, and also PMA stimulated BxPC-3 verses stimulated AsPC-1 cell lines.  Differential gene expression was identified using ArrayAssist bioinformatics software. Gene expression changes were confirmed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) (Assays-on-demand, Taqman, ABI systems).  Pathway Assist and GOstat were used to identify pathway and gene ontology changes.

first: AnnaMarie

first: Myles

last: Smith

first: Padraig

last: Doolan

first: Colin

last: Clarke

first: Martin

last: Clynes

first: Joseph

last: Murphy

first: Aoife

last: McDermott

first: Niall

last: Swan

first: Paul

last: Crotty

last: Fridgway

first: Kevin

last: Conlon


title: DNaseI Digital Genomic Footprinting from ENCODE/University of Washington [Human]

status: Public on Dec 29 2010

city: Stanford

first: ENCODE

last: DCC

address: 300 Pasteur Dr

institute: ENCODE DCC

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

This track, produced as part of the ENCODE Project, contains deep sequencing DNase data that will be used to identify sites where regulatory factors bind to the genome (footprints).
Footprinting is a technique used to define the DNA sequences that interact with and bind DNA-binding proteins, such as transcription factors, zinc-finger proteins, hormone-receptor complexes, and other chromatin-modulating factors like CTCF. The technique depends upon the strength and tight nature of protein-DNA interactions. In their native chromatin state, DNA sequences that interact directly with DNA-binding proteins are relatively protected from DNA degrading endonucleases, while the exposed/unbound portions are readily degraded by such endonucleases. A massively parallel next-generation sequencing technique to define the DNase hypersensitive sites in the genome was adopted. Sequencing these next-generation-sequencing DNase samples to significantly higher depths of 300-fold or greater produces a base-pair level resolution of the DNase susceptibility maps of the native chromatin state. These base-pair resolution maps represent and are dependent upon the nature and the specificity of interaction of the DNA with the regulatory/modulatory proteins binding at specific loci in the genome; thus they represent the native chromatin state of the genome under investigation. The deep sequencing approach has been used to define the footprint landscape of the genome by identifying DNA motifs that interact with known or novel DNA binding proteins.
For data usage terms and conditions, please refer to http://www.genome.gov/27528022  and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

overall_design: Cells were grown according to the approved ENCODE cell culture protocols. Digital DNaseI was performed by DNaseI digestion of intact nuclei, followed by isolating DNaseI 'double-hit' fragments as described in Sabo et al. (2006), and direct sequencing of fragment ends (which correspond to in vivo DNaseI cleavage sites) using the Solexa platform (27 bp reads). High-quality reads were mapped to the GRCh37/hg19 human genome using Bowtie 0.12.5 (Eland was used to map to NCBI36/hg18); only unique mappings were kept. DNaseI sensitivity is directly reflected in raw tag density (Signal), which is shown in the track as density of tags mapping within a 150 bp sliding window (at a 20 bp step across the genome). DNaseI hypersensitive zones (HotSpots) were identified using the HotSpot algorithm described in Sabo et al. (2004). False discovery rate thresholds of 1.0% (FDR 0.01) were computed for each cell type by applying the HotSpot algorithm to an equivalent number of random uniquely mapping 36-mers. DNaseI hypersensitive sites (DHSs or Peaks) were identified as signal peaks within 1.0% (FDR 0.01) hypersensitive zones using a peak-finding algorithm. Only DNase Solexa libraries from unique cell types producing the highest quality data, as defined by Percent Tags in Hotspots (PTIH ~40%) were designated for deep sequencing to a depth of over 200 million tags.

first: John

last: Stamatoyannopoulos


title: Dendritic cells and stress translation

status: Public on Dec 31 2010

city: Marseille

first: Philippe

last: Pierre

address: Campus Luminy - case 906

country: France

institute: CIML - CNRS - INSERM

summary: In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation that exhibits specific mechanisms to control the immune response. Here we show that in response to polyriboinosinic:polyribocytidylic acid (poly I:C), DCs mount a specific transcription program during which the growth arrest and DNA damage-inducible protein 34 (GADD34/MyD116), a phosphatase 1 (PP1) cofactor, is expressed. Together with its constitutively active counterpart CReP, GADD34 promotes an extensive dephosphorylation of the translation initiation factor eIF2-alfa in activated DCs. In turn, dephosphorylation of eIF2-alfa prevents the translation inhibition normally associated with cellular stress or detection of cytoplasmic double-stranded RNA. These observations have important implications in linking pathogen detection with the integrated stress responses molecular machinery. The importance of this regulation for DC function is exemplified by the alteration of IFN-beta production or the induction of caspase-3 cleavage upon inhibition of PP1 activity.

overall_design: LPS-activated murine bone-marrow derived dendritic cells were transfected or pulsed with pIC and incubated for 4h or 16h. Cells were harvested, total RNA was extracted and analysed by Affymetrix microarrays analysis.

first: Giovanna

last: Clavarino

first: Maurizio

last: Ceppi

first: Nuno

last: Claudio

first: Till

last: Wenger

first: Enrico

last: Schmidt

first: Evelina

last: Gatti


title: Analysis of Gene Expression in the Chicken Otocyst

status: Public on Mar 01 2011

city: Palo Alto

first: Samuel

last: Myllykangas

address: 269 Campus Drive, CCSR 3240

department: Oncology

institute: Stanford University

summary: The inner ear arises from multipotent placodal precursors that are gradually committed to the otic fate and further differentiate into all inner ear cell types, with the exception of a few neural crest-derived cells.  The otocyst has a pivotal role during inner ear development: otic progenitor cells sub-compartmentalize into non-sensory regions and regions giving rise to prosensory domains where subsequently also hair cells differentiate.  The genes and pathways underlying this progressive subdivision and differentiation process are not entirely known.  The goal of this study was to identify a comprehensive set of genes expressed in the chicken otocyst using the serial analysis of gene expression (SAGE) method.  Our analysis revealed several hundred transcriptional regulators, potential signaling proteins, and receptors.  We identified a substantial collection of genes that were previously known in the context of inner ear development, but we also found many new candidate genes, such as Sox4, Sox5, Sox7, Sox8, Sox11, and Sox18, which previously were not known to be expressed in the developing inner ear.  Despite its obvious limitation of not being all-inclusive, the generated otocyst SAGE library is a practical bioinformatics tool to study otocyst gene expression and to identify candidate genes for developmental studies.

overall_design: Otocysts were dissected from HH stage 18-19 chicken embryos, total RNA was extracted, and subjected to a commercial long-SAGE protocol, resulting in a library of concatemerized tags.  3,512 individual clones of SAGE concatemers were sequenced resulting in 39,326 17-bp tags with tag counts up to 718 for the most abundant tag; 3,292 tags that were represented between two and five times, whereas the majority of tags (11,717) were only found once.  Overall, we identified 16,008 unique sequence tags.

first: Saku

last: Sinkkonen

first: Veronika

last: Starlinger

first: Deepa

last: Galaiya

first: Roman

last: Laske

first: Kazuo

last: Oshima

first: Stefan

last: Heller


title: Towards an understanding of the mechanism of hypoevolution of Schistosoma japonicum schistosomula from Microtus fortis

status: Public on Dec 02 2010

city: Brisbane

first: Geoffrey

last: Gobert

address: 300 Herston Rd

institute: QIMR

summary: More than 40 species of mammal have been reported to be infected naturally with Schistosoma japonicum (Chinese mainland strain) in China. The reed vole, Microtus fortis, is the only known mammalian host in which the schistosomes are unable to mature and cause significant pathogenic changes. Gene expression profiling of the 10 day old schistosomula was performed.

overall_design: Microarray analysis was also used to identify differences in gene expression between Schistosoma japonicum schistosomula from BALB/c mice and from Microtus fortis. 10 day old schistosomula were isolated, total RNA obtained and Agilent one colour labeling used. A custom designed Agilent microarray was used to determine what differential gene expression occurs between parasites maintained in either a permissive (mouse) or non-permissive (vole) hosts.

first: Jinbiao

last: Peng

middle: N

first: Jiaojiao

last: Lin


title: Legionella pneumophila transcriptome during intracellular multiplication in human macrophages

city: St-Anne-de-Bellevue

first: Sebastien

last: Faucher

state: QC

address: 21,111 Lakeshore

department: Natural Resource Sciences

country: Canada

institute: McGill University

summary: Legionella pneumophila is the causative agent of Legionnaires’ disease, an acute pulmonary infection. L. pneumophila is able to infect and multiply in both phagocytic protozoan, such as Acanthamoeba castellanii, and mammalian professional phagocytes. The best-known virulence determinant used by L. pneumophila to infect host cells is a Type IVb translocation system named Icm/Dot, which is used to modify the host cell functions to the benefit of the bacteria. To date the Icm/Dot systeme is known to translocate more than 100 effectors. While the transcriptional response of Legionella to the intracellular environement of A. castelannii as already been investigated, much less is known of how Legionella reacts transcriptionnally inside human macrophages. In this study, the transcriptome of L. pneumophila was monitored during exponential and post-exponential phase in rich AYE broth and during infection of human cultured macrophages by using microarray and a RNA amplification procedure called SCOTS to allow for the study of conditions of low bacterial loads. Among the genes induced intracellularly are those involved in amino acid synthesis pathway leading to L-arginine, L-histidne and L-proline as well as many transport system involved in amino acid and iron uptake. The Icm/Dot systems is not differentially expressed inside cells compare to the E phase control but the effectors are strongly induced. The intracellular transcriptome was further used to identify putative new Icm/Dot effectors and translocation was show to occur for 3 of them. This study provides a comprehensive view of how L. pneumophila react to the human macrophages intracellular environment.

sample_organism: Legionella pneumophila subsp. pneumophila str. Philadelphia 1

overall_design:
The aim of this work was to study the gene expression profile of Legionella during infection of macrophages infected at a low multiplicity of infection (MOI). SCOTS is a method that allows amplification of small amounts of bacterial RNA from infected host cells, while discarding host cell transcripts and ribosomal RNA.
Before infection, macrophages derived from the THP-1 monocyte cell line were pre-treated with antibodies against the L. pneumophila major outer membrane protein, which increases the efficiency of bacterial entry into host cells . After 2 hours of infection, the macrophages were washed and treated with gentamicin for 1 hour, to synchronize the infection and kill extracellular bacteria, and cells were washed 3 times and fresh medium was added. Samples for the first time point (T0) were collected after the gentamicin treatment. Samples were also collected after 6h (T6) and 18h (T18).
Samples from all conditions, including growth in AYE broth to exponential (E) or post exponential (PE) phase, were treated with three consecutive rounds of SCOTS and the resulting cDNA was labeled and hybridized to the microarray slides. As a reference channel, labeled gDNA was also hybridized.  For each condition studied, three independent biological replicates and two technical replicates were analyzed, resulting in six replicates for each condition. The data was background subtracted and normalized by calculating the contribution of each spot to the total intensity and the ratio to gDNA was recorded. A one tailed student's T test was used for statistical analysis and the ratio between test conditions (PE, T0, T6 and T18) and the control conditions (E phase or T0) was calculated.

first: Catherine

last: Mueller

first: Howard

last: Shuman


title: Klf4 gene disruption is linked to enhanced cellular proliferation and tumor formation

status: Public on Nov 25 2010

first: Engda

last: Hagos

state: GA

address: 615 Michael Street

department: Digestive Diseases

institute: Emory University

summary: Kruppel-like factor 4 (KLF4) is involved in diverse biological processes such as cell cycle control, differentiation, transcriptional regulation, DNA repair and apoptosis. Furthermore, KLF4 is a tumor suppressor and play a role in regulating genomic stability. In order to investigate the changes in gene expression in the klf4 null MEFs, we compared the gene expression patterns of the wild type and klf4 null by microarray.

overall_design: Total RNA isolated from Klf4 -/- MEFs compared to Klf4 +/+ MEFs. RNA was processed from cells that had reached 80-90 confluency in triplicate using Trizol reagent as recommended by the manufacturer (Invitrogen, Carlsbad, CA). RNA was subjected to DNase I treatment in order to remove any contaminating genomic DNA. Final purification was performed on RNAeasy columns (Qiagen, Valencia, CA), according to the manufacturer's recommendations.


title: Gene expression changes in mouse forebrain deficient in Dnmt1 and Dnmt3a

status: Public on Dec 03 2010

city: Los Angeles

first: jian

last: feng

address: 695 Charles E Young Dr

department: Human Genetics

institute: UCLA

summary: Two major DNA methylation-catalyzing enzymes, Dnmt1 and Dnmt3a, are expressed in postmitotic neurons, but their function in the adult central nervous system is unclear.  We generated conditional mutant mice (CamKIIa Cre; Dnmt loxP) that lack either Dnmt1 or Dnmt3a, or both, exclusively in forebrain excitatory neurons and found only double-knockout (DKO) mice exhibited abnormal hippocampal CA1 long-term plasticity and deficits of learning and memory.  DKO neurons also exhibit a significant up-regulation of immune genes, such as class I MHC and Stat1, which are implicated in synaptic plasticity.

overall_design: Four pairs of 2-3 month-old DKO and litter mate control were used.  RNA samples were extracted from the cortex and hippocampus for gene expression array analysis.

first: Jian

last: Feng

first: Guoping

last: Fan


title: Comparison of SCOTS and standard microarray protocol

platform_organism: Legionella pneumophila subsp. pneumophila str. Philadelphia 1

overall_design: To validate the use of SCOTS, we compare the expression patterns obtained by SCOTS to those obtained when a standard microarray protocol was used. RNA from bacteria in E phase was treated using SCOTS or a standard microarray protocol where the cDNA is labeled during the reverse transcription reaction as previously published. The resulting cDNA was hybridized to the microarray slides as described previously and data analysis was carried out the same way as for the SCOTS treated samples.


title: Expression data from Arabidopsis thaliana - Pseudomonas interaction

status: Public on Oct 07 2010

city: Potsdam

first: Jens

last: Schwachtje

address: Am Mühlenberg 1

department: Energy Metabolism

institute: Max-Planck-Institute for Molecular Plant Physiology

summary:
Some soil bacteria promote plant growth, including Pseudomonas species.
With this approach we detected significant changes in Arabidopsis genes related to primary metabolism that were induced by the bacteria.

overall_design: Pseudomonas G62 was applied to roots of 18 day-old Arabidopsis seedlings and the transcriptional profile of whole seedlings after 6 hours of treatment was analyzed.

first: Ina

last: Thormählen

first: Joost

middle: T

last: van Dongen


title: Expression data collected using a custom Affymetrix array (Adenoma Biomarker Gene Chip) designed to differentiate neoplastic colorectal from non-neoplastic

status: Public on Nov 13 2010

city: North Ryde

first: Iain

last: Beeston

state: NSW

address: 11 Julius Avenue

institute: Clinical Genomics

summary:
This array was designed to verify a number of previously identified markers for colo-rectal adenoma.
It contains probesets from the Affymetrix Hu133, HuGene and other novel probesets not included on other arrays.

overall_design: 68 samples of colon tissues were analysed, covering the classes normal, adenoma and carcinoma.

first: Lawrence

last: LaPointe

first: Peter

last: Molloy

first: Graeme

last: Young


title: Hippocampus of APPV717I-CT100 Tg mice brain: Control vs. Triple hippocampus

status: Public on Sep 28 2010

city: Seoul

first: Yoo-Hun

last: Suh

address: 28 Yongon-dong

country: South Korea

institute: Seoul National University

summary: To investigate the regulatory genes responsible for the neuropathology in AD, we performed microarray analysis with APPV717I-CT100 transgenic mice, an animal model of AD. We extracted total RNA of hippocampus in three brains of 11 month-old APPV717I-CT100 Tg mice. Total RNA was prepared from using Trizol reagent.

overall_design: Control vs APPV717I-CT100 Tg mice.  Three hippocampus per array were used.

first: Keun-A

last: Chang

first: TAE-YOUNG

last: HA


title: UV effects in mouse melanocytes

city: Bethesda

first: Sean

last: Davis

address: 37 Convent Drive, Room 6138

institute: National Cancer Institute

summary: Ultraviolet (UV) radiation is a major melanoma risk factor, yet underlying mechanisms remain poorly understood. Here we  introduce a mouse model permitting fluorescence-aided melanocyte imaging and isolation following in vivo UV irradiation. We use expression profiling to show that activated neonatal skin melanocytes isolated following a melanomagenic UVB dose bear a distinct, persistent interferon-response signature, including genes associated with immunoevasion. UVB-induced melanocyte activation, characterized by aberrant growth and migration, was abolished by antibody-mediated systemic blockade of interferon-gamma (IFN-gamma), but not type-I interferons. IFN-gamma was produced by macrophages recruited to neonatal skin by UVB-induced chemokine receptor Ccr2 ligands. Admixed recruited skin macrophages enhanced transplanted melanoma growth by inhibiting apoptosis; notably, IFN-gamma blockade abolished macrophage-associated melanoma growth and survival. IFN-gamma-producing macrophages were identified in 70% of human melanomas examined. Our data reveal an unanticipated role for IFN-gamma in promoting melanocytic cell survival/immunoevasion, and suggest IFN-gamma-R signaling represents a novel therapeutic melanoma target.

overall_design: Biologic replicates of UVA- and UVB-treated mouse melanocytes, as well as untreated mouse melanocytes and mouse keratinocytes, were used to define melanocyte expression signatures associated with UV treatment.

first: M

last: Zaidi

first: Paul

middle: S

last: Meltzer

first: Glenn

last: Merlino


title: Transcriptome-wide signature of hybrid breakdown associated with intrinsic reproductive isolation in lake whitefish species pairs (Coregonus spp. Salmonidae)

status: Public on Aug 01 2010

city: Quebec

first: Sébastien

last: Renaut

state: qc

address: 1030 Avenue de la Médecine

department: Biology

institute: Université Laval

summary: Genetic analyses of speciation have focused nearly exclusively on retrospective analyses of reproductive isolation between highly divergent species. Yet, a full understanding of the speciation process must encompass analysis of the consequences of genomic divergence in young lineages still capable of exchanging genes under natural conditions. The accumulation of conditionally neutral genetic variation may lead to the evolution of divergent gene networks. In a hybrid background, such mutations may no longer compensate one another, resulting in the appearance of selectively disadvantageous traits, including disruption of gene expression regulation. Here, we documented genome-wide patterns of gene expression divergence between young lineages of normal and dwarf lake whitefish and their backcross hybrids for which strong, yet incomplete post-zygotic isolation barriers exist. A significant proportion (33%) of backcross hybrids showed developmental abnormalities not seen in parental forms and eventually leading to death. While the transcriptome of parental forms was nearly identical during embryonic development, suggesting a role for stabilizing selection, all hybrids displayed strongly divergent patterns of gene expression. By comparing healthy, surviving hybrids against moribund ones, we observed that over 2000 genes were misregulated in these abnormal embryos. In particular, misregulation was significantly biased towards essential developmental genes which were strongly underexpressed. Furthermore, genes previously documented to be highly transgressive (exaggerated inter-individual variance) were almost invariably underexpressed in hybrids. Our results thus clearly showed a transcriptome-wide signature of hybrid breakdown in young, incipient species and demonstrated a persuasive link between misexpression of essential developmental genes and post zygotic isolation.

platform_organism:
Osmerus mordax
Oncorhynchus mykiss
Salmo salar
Coregonus clupeaformis
Oncorhynchus tshawytscha

overall_design: Samples of dwarf, normal, backcross-healthy and backcross-moribund were hybridized in a loop design, involving eight biological replicates for the backcross-healthy and backcross-moribund comparison and six for the others. Dye swap was performed between each replicate. As a result, we obtained a final set of 32 microarray slides.

first: Louis

last: Bernatchez


title: EXPRESSION OF VAV1 IN GLIOBLASTOMA MULTIFORME

status: Public on Sep 30 2010

city: salamanca

first: Juan

middle: Luis

last: Garcia

address: Campus Miguel de Unamuno

institute: Center Cancer Research

summary:
Background: Even though much progress has been made in the understanding of the molecular nature of glioma, the survival rates of patients affected of this tumour have not changed significantly during these years. Thus, a deeper understanding of this malignancy is still needed in order to predict its outcome and improve patient treatment. Here, we report that VAV1, a GDP/GTP exchange factor for Rho/Rac proteins with oncogenic potential that is involved in the regulation of cytoskeletal dynamics and cell migration.
Methodology/Principal Findings: VAV1 is overexpressed in 32 patients diagnosed with high-grade glioma. Such overexpression is linked to the parallel upregulation of a number of genes coding for proteins also involved in cell invasion- and migration-related processes. Unexpectedly, immunohistochemical experiments revealed that VAV1 is not expressed in glioma cells. Instead, VAV1 is found in non-tumoral astrocyte-like cells that are located either peritumoraly or perivascularly, suggesting that its expression is linked to synergistic signalling cross-talk between cancer and infiltrating cells.
Conclusions/Significance: Interestingly, we show that the pattern of expression of VAV1 is a good prognostic factor to unveil populations of high-grade glioma patients with different survival and progression free survival rates.

overall_design:
1. Oligonucleotide microarray analyses
Total RNAs were extracted using the Triazol reagent (Life Technologies, Gaithersburg, MD, USA) and purified with the RNeasy Mini kit (Qiagen, Valencia, CA, USA). The integrity of RNA samples obtained was assessed using the 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). Double-stranded cDNAs and biotinylated cRNAs were synthesized using a T7-polyT primer and the BioArray RNA labelling Kit (Enzo Farningdale, NY, USA), respectively.  Labelled RNAs were then fragmented and hybridised to HU-133A oligonucleotide arrays (Affymetrix, Santa Clara, CA, USA) according to standard Affymetrix protocols. After hybridization and washes, arrays were scanned using the Gene Array Scanner (Affymetrix), and the expression value for each probe set calculated using the MAS 5.0 software (Affymetrix). All examples had a scaling factor lower than threefold and 3’/5’ of GAPDH probe set <2.5. Gene levels were transformed to base two logarithms. A median normalization approach was applied. Only genes with al least three “present” calls across all samples were selected. All these steps were done at the Genomics and Proteomics Unit of the “Centro de Investigación del Cáncer, Salamanca”.
2. Microarray data analyses
To visualize clusters of genes with similar expression patterns, we used a hierarchical clustering method (Cluster and TreeView software) based on the average-linkage method with the centred correlation metric [26]. A multidimensional scaling method (BRB Arrays Tools version 3.0) was also utilized by using Euclidean distance criteria [27]. Supervised learning was used to identify genes with statistically significant changes in expression among different classes by using the Significant Analysis of Microarrays (SAM) algorithm [28]. All data were permuted over 100 cycles by using the two-class (unpaired) and multi-class response format. Significant genes were selected based on the lowest false discovery ratio (between 0.6 and 0.9). In addition, nonparametric tests such as Wilcoxon rank sum test and Kruskal-Wallis test to compare more than two unpaired group were also used (SPSS 18, SPSS Inc).
3. Functional annotation of microarray data
Probe sets showing significant expression change were functionally annotated and grouped according to biological function criteria using GeneOntology biological process descriptions. The functional analysis to identify the most relevant biological mechanism, pathways and functional categories in gene dada sets was generated using the Ingenuity Pathway software (Ingenuity Systems, Mountain View, CA, USA) available in the web (www.ingenuity.com) [29]. A functional network was considered significant when it fulfilled the following criteria: i) to have a minimal score of 15; ii) to have a minimum of 20 direct functional interactions among the network members.
4. Quantitative reverse transcription-PCR
Total RNA was quantified in a RNA 6000 Nano Chip (Agilent Technologies) and quantitative PCR performed using the QuantiTect SYBR Green RT–PCR kit (Qiagen). To quantify VAV1 mRNA levels, we used two different sets of probes: PAIR A (5’-AAC AAC GGG AGG TTC ACC CT-3’ and 5’-GGT CCC TCA TGG CAT CCA-3’) and PAIR B (5’-AGC CAT TGG ACC CTT TCT ACG-3’ and 5’-GCC ATG GAC ATA GGG CTT CA-3’). Amplifications were performed using the iCycler apparatus (Bio-Rad Laboratories, California, USA). Analyses of data were done using the iCycler iQ Optical System Software, version 3.0a (Bio-Rad Laboratories). Primers to GAPDH were used as intersample normalizing controls. Variations in expression of VAV1 mRNA were represented as the mean value of the fold change respect the VAV1 expression levels detected in sample #19209 with both pairs of oligonucleotide primers.
5. Immunohistochemical analyses
The VAV1 antibody was generated in rabbits using a synthetic peptide and purified by affinity chromatography in Bustelo’s laboratory. This antibody recognizes VAV1 proteins from humans and mice but it does not recognize other VAV family members (unpublished data). For immunostaining, tissue sections were washed thrice with Xylene and once with 100% ethanol, rehydrated by sequential changes in 80%, 70%, and 50% ethanol and a final incubation in phosphate-buffered saline (PBS). Each rehydrating step involved 3 min incubations with the indicated solutions. Endogenous peroxidases were quenched by the addition of a 3% H2O2 solution in methanol for 30 min at room temperature (RT). Tissue sections were subsequently washed twice with PBS. Antigen retrieval was performed by incubation in 1 mM EDTA for 30 min at 37°C. The slides were washed twice in PBS and blocked in blocking buffer (Zymed, CA, USA) for 30 min at RT. Specimens were then incubated with the primary antibody (1:250 dilution) in blocking buffer. After an 1 hr incubation at 37°C, slides were washed three times in PBS, incubated with a biotinylated secondary antibody for 30 min at 37°C, washed thrice in PBS, incubated with horseradish peroxidase-streptavidin for 30 min at 37°C, washed three times in PBS, and developed using the AEC substrate (Zymed). Slides were then washed twice in water, counterstained with hematoxilin (Zymed), washed again in water, and mounted with GVA (Zymed). Samples were analyzed by light microscopy and images acquired suing an Axiophot imaging system (Zeiss, Munich, Germany).
6. Fluorescence in situ hybridization analyses
FISH experiments were carried out in 40 cases of glioblastoma multiforme (grade IV) positive for VAV1 expression. For this purpose, we performed dual-colour FISH analyses with locus-specific probes for centromere 7 (Abbott Molecualr, Des Plaines) exactly as previously described.[30] Polysomies were defined when more than 10% of the nuclei surveyed contained three or more CEP signals (chromosome-specific FISH probes that hybridize to highly repetitive human satellite DNA sequences, usually located near centromeres).
7. Immunohistochemistry and fluorescence in situ hybridization (FISH) in paraffin-embedded tumours
Four um sections were cut from routinely processed paraffin blocks and mounted onto glass slides with a charged coating. Sections were dewaxed in Xylene and then rehydrated using increasing concentrations of alcohol before being rinsed briefly in water. Slides were heated 2 min in 1 mM EDTA (pH 9.0) in a microwavable pressure cooker. After antigen retrieval, slides were incubated 1 h at RT in a moist chamber with a primary antibody diluted in PBS supplemented with 10% foetal calf serum. Slides were incubated for 1 h with fluorochrome-conjugated antibodies to the appropriate IgG isotypes in a moist chamber in the dark. Finally, slides were washed thrice in PBS containing 0.5% Tween 20 three before FISH analysis.
8. Degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) analyses
After the staining of tissue sections with VAV1 antibodies (see above), the regions of the tumour were identified, microdissected, and collected using the PALM® microscope system (P.A.L.M. Microlaser Technologies, Munich, Germany). The genomic DNA was extracted as indicated by Isola et al [31] with modifications to small DNA amounts. Those included the resuspension of the microdissected sections in extraction buffer followed by a digestion with proteinase K (0.6 mg/ml). All samples were resuspended in 10 ul of 10 mM Tris-HCl (pH 7.4) and 0.1 mM EDTA. DOP-PCR amplification was performed in two steps. For the first, low-stringency step, 1 ul of sample was added to 4 ul of buffer A (2.5 ul of 600 uM dNTPs (Roche, Pleasanton, CA), 0.5 ul of 10 uM DOP primer 5’-CCGACTCGAGNNNNNNNATGTGG-3’, where N= A, C, G, or T) [32] and 1 ul of 5x Sequenase Reaction Buffer (Amersham, Cleveland, OH). Reactions were performed using 5 cycles of 30ºC for 5 min, 37ºC for 2 min, and 96ºC for 2 min, adding 0.65 units of Sequenase in each 30ºC step. The first phase product was then subjected to the second step usin

first: Jose

last: Coucero

last: Gomez-Moreta

first: Angel

last: Santos-Briz

first: Vincent

last: Sauzeau

first: Eva

last: Lumbreras

first: Manuel

last: Delgado

first: Cristina

last: Robledo

first: Monica

last: Almunia

first: Xose

last: Bustelo

first: Jesus

middle: M

last: Hernández


title: Identification of genes differentially expressed between control and SMILE siRNA transfected HeLa cells

status: Public on May 18 2010

city: Nantes

first: RACAPE

last: MAUD

address: 30 Bd Jean Monnet

institute: INSERM UMR 643

summary: SMILE, a molecule for which no information is available in the literature, is overexpressed in the blood of patients tolerating a kidney graft without treatment. We performed microarray analysis to determine cellular functions affected by SMILE silencing.

overall_design: Each condition of control and SMILE siRNA transfected cells was made in triplicates.

first: Maud

last: Racapé

first: Sophie

last: Brouard

first: Richard

last: Danger


title: Multi-modal proteomic target discovery and orthogonol confirmation of preclinical diabetic retinopathy drug development biomarkers

status: Public on Jun 01 2010

city: Hershey

first: Penn State

last: COM

address: 500 University Drive

institute: Penn State College of Medicine

summary: The aim of this study was to identify proteomic alterations associated with functional dysregulation of the retina with diabetes that could eventually be used as surrogate endpoints in preclinical drug testing studies. A multi-modal approach of antibody (Luminex)-, electrophoresis (2-DIGE)-, and LC-MS (iTRAQ)-based quantitation methods was used to provide broad coverage of the retinal proteome. Transcriptional profiling through microarray analysis was also included to increase coverage and provide insight into potential regulation of protein expression changes at the mRNA level.  The different technologies proved complementary, with limited coverage overlap between methods.

overall_design: Diabetes was induced in Sprague-Dawley male rats (Charles River Laboratories, Wilmington, MA) by intraperitoneal injection of 65 mg/kg streptozotocin (STZ) (Sigma-Aldrich, St. Louis, MO) in 10mM sodium citrate pH 4.5 vehicle. Control rats were injected with an equal dose of vehicle only.  Rats had free access to food and water, and were maintained on a 12 hour light/dark cycle. Blood glucose level and body weight were measured 6 days post-STZ or vehicle injection, and biweekly throughout the experiment. Only rats with blood glucose levels >250 mg/dL at the time of the original test and throughout the experiment were included in the diabetic groups. At the time of retina harvest, rats were given a lethal dose of pentobarbital, 100 mg/kg, (Ovation Pharmaceuticals Inc., Deerfield, IL) by intraperitoneal injection and sacrificed by decapitation. Retinas were rapidly excised snap- frozen in liquid nitrogen for subsequent experimentation.

first: Willard

last: Freeman

first: Georgina

last: Bixler

first: Heather

middle: D

last: VanGuilder

first: Lydia

last: Kutzler

first: Robert

last: Brucklacher

first: Sarah

middle: K

last: Bronson

first: Scot

last: Kimball