title: Whole transcriptome analyses of six thoroughbred horses before and after exercising using RNA-Seq

status: Public on May 01 2013

city: Seoul

first: HYEONGMIN

last: KIM

address: Seoul National Univ., Daehak-dong, Gwanak-gu,

department: College of Agriculture Sciences

country: South Korea

institute: Seoul National University

summary: We sequenced the whole mRNA of six thoroughbred horse (Equus caballus) blood and muscle tissues before and after exercising, generating a total of 1.3 billion short reads with 90-bp pair-end sequences from 24 samples. Comparing with current genome annotation, we identified 32,361 unigene clusters spanning 51.83 Mb that contained 11,933 (36.87%) annotated genes. More than 60% (20,428) unigene clusters did not match any current equine gene model. We identified 189,973 single nucleotide variations (SNVs) from the aligned sequences against the horse reference. Most SNVs (171,558 SNVs; 90.31%) were novel compared with over 1.1 million equine SNPs from two databases. Some genes have significantly different expression levels under different environment. We define those identical genes which have different expression levels are ‘differentially expressed’ and carried out differentially expressed gene analysis before and after exercise conditions. We discovered, 62 up- and 80 down-regulated genes in the blood and 878 up- and 285 down-regulated genes in the muscle from the 24 samples. Six out of 28 previously exercise-related known genes, HIF1A, ADRB2, PPARD, VEGF, TNC, and BDNF, were highly expressed in the muscle after exercise. We discovered 56 functionally unknown transcription factors that are probably associated with an early regulatory exercise mechanism from 91 differentially expressed transcription factors. We found interesting RNA expression patterns where different alternative splicing forms of the same gene showed reversed expressions before and after exercising.

overall_design: whole mRNA sequencing profiles of six thoroughbred horse (Equus caballus) blood and muscle tissues before and after exercising

first: Byung-Wook

last: Cho

first: Hee-Bal

last: Kim

first: Jong-Hwa

last: Park


title: Transcriptional characterization of a prospective series of primary plasma cell leukemia revealed genes associated with tumor progression and poorest outcome

city: MILAN

first: Luca

last: Agnelli

address: Venezian 1

department: Department of Advanced Diagnostics

country: Italy

institute: IRCCS Istituto Nazionale dei Tumori

summary: Plasma cell leukemia (PCL) is a rare form of plasma cell dyscrasia that presents either as a progression of previously diagnosed multiple myeloma (MM), namely secondary PCL (sPCL), or as the initial manifestation of disease, namely primary PCL (pPCL). Although presenting signs and symptoms include those seen in MM, pPCL is characterized by several aspects that clearly define more aggressive course. To provide insights into the biology of pPCL, we have investigated the transcriptional profiles of a cohort of 21 newly-diagnosed, homogeneously treated pPCL patients included in a multicenter prospective clinical trial. All but one pPCL had one of the main IGH translocations, whose associated transcriptional signatures resembled those observed in MM. A 503-gene signature was identified that distinguished pPCL from MM, from which emerged 28 genes whose trend in expression levels was found associated with the progressive stages of plasma cell dyscrasia in a large dataset of cases from multiple institutions, including samples from normal donors throughout PCL. The transcriptional pattern of the pPCL series was then evaluated in association with outcome. Three genes were identified having expression levels correlated with response to the first-line treatment with lenalidomide/dexamethasone, whereas a 27-gene signature was identified associated with overall survival independently of molecular alterations, hematological parameters and renal function. Overall, our data contribute to a fine dissection of pPCL and may provide novel insights into the molecular definition of a subgroup of high-risk pPCL.

overall_design:
This series of microarray experiments contains the gene expression profiles of purified plasma cells (PCs) obtained from 21 pPCL and 55 MM at diagnosis. PCs were purified from bone marrow samples using CD138 immunomagnetic microbeads according to the manufacturer's instructions (MidiMACS system, Miltenyi Biotec); the purity of the positively selected PCs was assessed by morphology and flow cytometry and was > 90% in all cases. 5.5 micrograms of single-stranded DNA target obtained from 100 ng of purified total RNA was fragmented and then labeled using the WT Terminal Labeling Kit according to the standard Affymetrix protocol (GeneChip¨ Whole Transcript (WT) Sense Target Labeling Assay Manual). The fragmented labeled single-stranded DNA target was hybridized for 16 hours and 30 minutes at 45¡C on GeneChip¨ Gene 1.0 ST array according to the standard Affymetrix protocol. Washing and scanning were performed using GeneChip System of Affymetrix (GeneChip Hybridization Oven 640, GeneChip Fluidics Station 450 and GeneChip Scanner 7G). Log2-transformed expression values were extracted from CEL files and normalized using NetAffx Transcript Cluster Annotations, Release 31 and robust multi-array average (RMA) procedure in Expression Console software (Affymetrix Inc.). Non-annotated transcript clusters were discarded. The expression values of transcript cluster ID specific for loci representing naturally occurring read-through transcriptions or mapped to more than one chromosomal location were summarized as median value for each sample.
gene expression analysis of 21 primary Plasma Cell Leukemia and 55 multiple myeloma tumors

first: Katia

last: Todoerti

first: Sonia

last: Fabris

first: Marta

last: Lionetti

first: Giacomo

last: Tuana

first: Luigia

last: Lombardi

first: Vitina

last: Grieco

first: Gabriella

last: Bianchino

first: Fiorella

last: D'Auria

first: Teodora

last: Statuto

first: Carmela

last: Mazzoccoli

first: Luciana

last: De Luca

first: Maria

middle: T

last: Petrucci

first: Fortunato

last: Morabito

first: Massimo

last: Offidani

first: Francesco

last: Di Raimondo

first: Antonietta

last: Falcone

first: Paola

last: Omed

first: Mario

last: Boccadoro

first: Antonio

last: Palumbo

first: Antonino

last: Neri

first: Pellegrino

last: Musto


title: Gene expression analysis of omental and mesenteric adipose tissue in Crohn's disease and ulcerative colitis

status: Public on May 09 2013

city: cusano milanino

first: Zulian

last: Alessandra

state: milano

address: via zucchi 18

institute: istituto auxologico italiano

summary: Our hypothesis is that in IBD patients intestinal bacteria translocation into the intra-abdominal fat depots affects adipocyte morphology and gene expression. The study aimed to study adipocyte gene expression  of omental (OM) and mesenteric (MES) adipose tissue of ulcerative colitis (UC) and crohn's disease (CD).

overall_design: Total RNA was extracted from isolated adipocytes from omental and mesenteric adipose tissue of CD and UC patients. Microarray experiments were performed in duplicates of 4 different pools of RNAs extracted from adipocytes isolated from OM and MES of UC patients (n=5) and CD patients (n=5) respectively.

first: A

last: Zulian


title: Analysis of genes overexpressed in hepatocellular carcinoma

status: Public on Apr 27 2013

city: Taipei

first: Yung-Ming

last: Jeng

address: 7 Chung-Shan South Road

department: Department of Pathology

country: Taiwan

institute: National Taiwan University Hospital

summary: Six pairs of hepatocellular carcinoma and their corresponding non-tumrous liver parenchymas were analyzed to identify the genes differentially expressed in hepatocellular carcinoma and nontumorous liver parenchyma.

overall_design: We analyzed mRNA expression pattern of 6 primary hepatocellular carcinomas and their corresponding nontumourous liver parenchyma. The specimens were obtained from a tissue bank in our hospital. All the clinical informations were de-linked from the specimens.

first: Yu-Ling

last: Chen


title: Placental gene-expression profiles of Intrahepatic cholestasis of pregnancy (ICP)

status: Public on Apr 18 2013

city: Shanghai

first: YouDong

last: Pan

state: Shanghai

address: 320 YueYang Road

department: Shanghai Institutes for Biological Sciences

country: China

institute: Chinese Academy of Sciences

summary: DNA microarray was applied to characterize the whole-genome expression profiles of placentas during ICP development.

overall_design: Pregnant women were categorized evenly into three groups: healthy group; mild ICP, with serum bile-acid concentration ranging from 10–40 uM; and severe ICP, with bile-acid concentration >40 uM.

first: JianFeng

first: QiaoLing

last: Du

first: Tao

last: Duan


title: Integrated microRNA and mRNA Signatures Associated with Survival in Triple Negative Breast Cancer

status: Public on Apr 19 2013

city: Bellinzona

first: Luciano

last: Cascione

state: Ticino

address: Via Chiesa 5

department: Lymphoma & Genomics Research Program

country: Switzerland

institute: IOR

type:
Non-coding RNA profiling by array
Expression profiling by array

summary: Triple negative breast cancer (TNBC) includes basal and non-basal subclasses. To further stratify TNBC we determined microRNA (miRNA) and mRNA expression profiles, linked specific miRNA signatures to patient survival and used miRNA/mRNA anti-correlations to identify TNBC subclasses associated with expression of canonical signal pathways

overall_design: RNA was isolated from formalin-fixed paraffin-embedded tissue cores of 165 primary tumors, 59 adjacent normal and 54 lymph node metastatic samples and expression of 664 miRNAs and 230 cancer-associated mRNAs was assessed for each sample using the nanoString nCounter platform. Kaplan-Meier distant-disease free and overall survival curves were compared using the log-rank test. Cox proportional hazard regression and risk score analysis were used to identify miRNAs for classification of patients with significantly different prognoses.

first: Pierluigi

last: Gasparini


title: Expression data from breast cancer cell lines

status: Public on Apr 22 2013

city: Rotterdam

first: Marcel

last: Smid

address: Dr. Molewaterplein 50

department: Medical Oncology

country: Netherlands

institute: Erasmus MC

summary: Breast cancer is a genetically and phenotypically complex disease. To understand the role of microRNAs in this molecular complexity, we performed miRNA expression analysis in a cohort of molecularly well-characterized human breast cancer (BC) cell lines to discover miRNAs associated with the most common molecular subtypes and the most frequent genetic aberrations.Using a microarray carrying LNA™ modified oligonucleotide capture probes (Exiqon), expression levels of 725 human miRNAs were measured in 51 BC cell lines. MiRNA expression was explored by unsupervised cluster analysis and then associated with the molecular subtypes and genetic aberrations commonly present in breast cancer. Unsupervised cluster analysis using the most variably expressed miRNAs divided the 51 BC cell lines into a major and a minor cluster predominantly mirroring the luminal and basal intrinsic subdivision of BC cell lines. One hundred and thirteen miRNAs were differentially expressed between these two main clusters of which half were related to the ER-status of the cell lines. Forty miRNAs were differentially expressed between basal-like and normal-like/claudin-low cell lines. Within the luminal-group of cell lines, 39 miRNAs were associated with ERBB2 overexpression and 24 miRNAs with E-cadherin gene mutations, which are frequent in this subtype of BC cell lines. In contrast, 31 different miRNAs were associated with E-cadherin promoter hypermethylation, which, contrary to E-cadherin mutation, is exclusively observed in BC cell lines that are not of luminal origin. The differential expression of 30 miRNAs were associated with p16INK4 status while only a few differentially expressed miRNAs were associated with BRCA1, or PIK3CA/PTEN, TP53 mutation status of the cell lines (P-value < 0.05). Twelve miRNAs were associated with DNA copy number variation of the respective locus. Luminal-basal and epithelial-mesenchymal associated miRNAs determine the overall subdivision of miRNA transcriptome of BC cell lines. Specific sets of miRNAs were associated with ERBB2 overexpression, p16INK4aor E-cadherin mutation or E-cadherin methylation status, which implies that these miRNAs may contribute to the driver role of the genetic aberrations. Additionally, miRNAs, which are located in a genomic region showing recurrent genetic aberrations, may themselves play a driver role in breast carcinogenesis or contribute to a driver gene in their vicinity. In short, our study provides detailed molecular miRNA portraits of BC cell lines, which can be exploited for functional studies of clinically important miRNAs.

overall_design:
Affymetrix plus2PM arrays were hybridized according to the manufacturer's procedure using RNA extracted from 52 cultured breast cancer cell lines. Most cellines were analyzed in triplicate. Gene expression data in log2 scale arrays were calculated and associated with diverse characteristics.
This submission represents the gene expression component of the study only

first: John

last: Martens


title: Expression analysis of Caenorhabditis elegans Bristol N2 prg-1 and Bristol N2 prg-1; daf-2 double mutant

status: Public on Jun 01 2013

city: Cambridge

first: Eric

middle: A.

last: Miska

address: Tennis Court Rd

department: Wellcome Trust/Cancer Research UK Gurdon Institute, The Henry We

country: United Kingdom

institute: University of Cambridge

type: Expression profiling by genome tiling array

summary: Investigation of whole genome gene expression level changes in early generation Caenorhabditis elegans Bristol N2 prg-1 and Bristol N2 prg-1; daf-2 double mutant, compared to late-generation strains.

overall_design: A seven chip study using total RNA recovered from three separate alleles of Caenorhabditis elegans Bristol N2 prg-1 and four separate allelic combinations of a double mutant strain, Bristol N2 prg-1; daf-2 , in which prg-1 (pk2298) and prg-1 (tm872) are combined with one of three daf-2 alleles (e1368, 1370, m41).


title: Deep sequencing of endogenous mRNA from Caenorhabditis elegans in the presence and absence of rotenone at 4 different time points

status: Public on Apr 16 2013

city: Jena

first: JenAge

last: Project

address: Beutenbergstr. 11

country: Germany

institute: Leibniz Institute for Age Research - Fritz Lipmann Institute

type: Expression profiling by high throughput sequencing

summary:
Background: We here show that inhibitors of mitochondrial complex I promote physical activity, stress resistance as well as lifespan of Caenorhabditis elegans despite normal food uptake, i.e. in the absence of DR. Dietary restriction (DR) extends lifespan and promotes metabolic health in evolutionary distinct species.
The RNA-seq data comprises 4 age groups (1, 5, 10 and 20 days  after L4) and 2 different conditions (rotenone and normal feeding (DMSO))

Jena Centre for Systems Biology of Ageing - JenAge (www.jenage.de)

overall_design: 22 samples: mRNA profiles of 1-day, 5-day and 10-day old worms as triplicates for each, rotenone and solvent control treatment; mRNA profiles of 20-day old worms as duplicates for each, rotenone and solvent control treatment

first: Sebastian

last: Schmeisser

first: Steffen

last: Priebe

first: Marco

last: Groth

first: Shamci

last: Monajembashi

first: Peter

last: Hemmerich

first: Reinhard

last: Guthke

first: Matthias

last: Platzer

first: Michael

last: Ristow


title: The MLH1 c.-27C>A and c.85G>T variants are borne on a European ancestral haplotype which underlies an autosomal dominant form of MLH1 epimutation.

status: Public on Apr 01 2013

city: Nijmegen

first: Ingrid

last: Vogelaar

address: Geert Grooteplein Zuid 10

department: Human Genetics

institute: Radboud University Medical Centre

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

summary:
Lynch syndrome, caused by germline heterozygous mutations of the DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2, or deletions affecting the EPCAM gene upstream of MSH2, is characterized by a predisposition to early-onset colorectal and additional extracolonic cancers. An alternative but rare cause of Lynch syndrome is a constitutional epimutation of MLH1, which is characterized by promoter methylation and transcriptional silencing of a single allele in normal tissues. Worldwide, five families with autosomal dominant transmission of a constitutional MLH1 epimutation linked to an MLH1 haplotype with two single nucleotide variants (c.-27C>A and c.85G>T) have been identified.
Array-based genotyping using Affymetrix SNP 6.0 data in four of these families revealed a shared haplotype extending across a ≤2.6 Mb region of chromosome 3p22 encompassing MLH1 and additional flanking genes, indicating common ancestry.

overall_design: Genomic DNA from 5 carriers of the c.-27C>A and c.85G>T variants was hybridized on Affymetrix SNP6.0 array according to manufacturer's procedures

first: Chau-To

last: Kwok

middle: P

first: Wendy

middle: A

last: van Zelst-Stams

first: Arjen

middle: R

last: Mensenkamp

first: Marjolijn

middle: J

last: Ligtenberg

first: Robert

middle: W

last: Rapkins

first: Robyn

middle: L

last: Ward

first: Nicolette

last: Chun

first: Uri

last: Ladabaum

middle: C

last: McKinnon

first: Marc

middle: S

last: Greenblatt

first: Megan

last: Hitchins


title: Gene expression analysis of growth hormone producing somatotroph pituitary adenomas

status: Public on Jun 10 2013

city: Oslo

first: Tove

last: Lekva

address: Sognsvannsveien 20

department: Section of Specialized Endocrinology and Institute for Internal Medicine

country: Norway

institute: OUS-HF, Rikshospitalet

summary: The epithelial marker E-cadherin plays a crucial role in epithelial mesenchymal transition (EMT). Decreased protein content in somatotroph adenomas has been associated with increased tumor size, invasion, and poor response to somatostatin analog (SA) treatment, but the potential mechanisms of EMT progression in these adenomas are lacking. Adenomas from sixteen acromegalic patients, eight with high (tertile 3)and eight with low (tertile 1) expression of E-cadherin where four adenomas treated with SA in each group, were screened for expressional candidate genes using Affymetrix human Gene Plus 2.0 Arrays. Analyses were performed to identify EMT-related transcripts.

overall_design: Expression levels were examined in adenomas from sixteen acromegalic patients, eight with high (tertile 3) and eight with low (tertile 1) expression of E-cadherin where four adenomas were treated with SA in each group.

first: Thor

last: Ueland

first: Jens

last: Berg

last: Bollerslev

first: Ole

middle: K

last: Olstad


title: Different response of WT and Ts keratinocyte to TPA treatment

city: Baltimore

first: Annan

last: Yang

state: MD

address: 725 N. Wolfe St, Biophysics 203

department: Physiology

country: USA

institute: Johns Hopkins University

summary: Ts keratinocyte has increased proliferation compare to WT keratinocyte upon TPA treatment. This result showed the different transcription regulation in Ts and WT keratinocyte upon TPA treatment.

overall_design: Total RNA extracted from WT or Ts keratinocytes, not treated or treated with TPA for 12 hours.

first: Duane

last: Currier

first: Roger

last: Reeves


title: Phenotypic characterization of retinoic acid differentiated SH-SY5Y cells by transcriptional profiling

city: Amsterdam

first: Joanna

last: Korecka

address: Meibergdreef 47

department: Neuroregeneration

institute: Netherlands Institute for Neuroscience

summary: Multiple genetic and environmental factors play a role in the development and progression of Parkinson’s disease (PD). The main neuropathological hallmark of PD is the degeneration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SN). To study genetic and molecular contributors to the disease process, there is a great need for readily accessible cells with prominent DAergic features that can be used for reproducible in vitro cellular screening. Here, we investigated the molecular phenotype of retinoic acid (RA) differentiated SH-SY5Y cells using genome wide transcriptional profiling combined with gene ontology, transcription factor and molecular pathway analysis. We demonstrated that RA induces a general neuronal differentiation program in SH-SY5Y cells and that these cells develop a predominantly mature DAergic-like neurotransmitter phenotype. This phenotype is characterized by increased dopamine levels together with substantial suppression of other neurotransmitter phenotypes, such as those for noradrenaline, acetylcholine, glutamate, serotonin and histamine. In addition, we show that RA differentiated SH-SY5Y cells express dopamine and noradrenalin neurotransmitter transporters that are responsible for uptake of MPP(+), a well known DAergic cell toxicant, and that MPP(+) treatment alters mitochondrial activity according to its proposed cytotoxic effect in DAergic neurons. Taken together, RA differentiated SH-SY5Y cells have a DAergic-like phenotype, and provide a good cellular screening tool to find novel genes or compounds that affect cytotoxic processes that are associated with PD.

overall_design: SH-SY5Y cell differentiation process was assessed by comparing RA differentiated and noRA differentiated cells in 8 days of culture.  Cells were compared at 6 different time points.

first: Ronald

middle: E

last: van Kesteren

first: Eva

last: Blaas

first: Sonia

middle: O

last: Spitzer

first: August

middle: B

last: Smit

first: Dick

middle: F

last: Swaab

first: Joost

last: Verhaagen

first: Koen

last: Bossers

first: Jorke

middle: H

last: Kamstra


title: The FLS (Fatty liver Shionogi) mouse, liver with non-alcoholic steatohepatitis (NASH)

status: Public on Mar 30 2013

city: Chiba

first: Semba

last: Toshihisa

state: Chiba

address: 1-8-1 Inohana, Chuo-ku,

department: Molecular Diagnosis

country: Japan

institute: Graduate School of Medicine, Chiba University

summary:
Comparison between livers of FLS mice and livers of DS (DD shionogi) mice
We used FLS mice as model animals of human NASH, while DS mice as control animals. FLS mice develops NASH spontaneously. DS mouse strain is a sister strain of the FLS mouse strain.

overall_design: We compared RNA from pooled livers of three FLS mice and three DS mice at 19 weeks. NASH in livers from FLS mice was confirmied pathologically while simple steatosis of DS mouse livers confirmed.

first: Toshihisa

last: Semba

first: Motoi

last: Nishimura

first: Osamu

last: Ohara

first: Takayuki

last: Ishige

first: Ken

last: Nonaka

first: Fumio

last: Imazeki

last: Nomura


title: Genome-wide analysis of Sox2-ablated optic cups compared with wild-type control optic cups at E16.5

status: Public on May 10 2013

city: Bethesda

first: Whitney

last: Heavner

address: Building 35 Rm 2B-203

department: NINDS

institute: National Institutes of Health

summary: To test the hypothesis that ablation of the SOX2 in the developing optic cup leads to expansion of peripheral optic cup signaling pathways, we screened whole-genome changes in gene expression in Sox2-mutant eyes compared with controls. Results confirm that ciliary epithelial (peripheral) identity is gained at the expense of neural retinal identity in Sox2-ablated optic cups.

overall_design: Total RNA was isolated from whole eyes enucleated from six wild-type controls and six Sox2-mutant embryos for a total of twelve eyes per genotype. Each embryo (pair of eyes) was analyzed as a single sample.


title: Expression of miR-196a in Huntington's disease transgenic (HD) mice

status: Public on May 30 2013

city: Tainan

first: Shang-Hsun

last: Yang

department: Department of Physiology, College of Medicine; Institute of Basic Medical Sciences

institute: National Cheng-Kung University

summary: We intend to screen altered genes after overexpression of miR-196a in  HD transgenic mice. Two transgenic mouse lines were used in this study, including HD transgenic mice and HD transgenic mice overexpressing miR-196a. The mice were all at approximate 12 months of age. At this point, HD transgenic mice showed severve motor dysfunctions, whereas HD transgenic mice overexpressing miR-196a displayed mild motor dysfunctions.

overall_design: We used the striatum tissues from 2 HD transgenic mice and 3 HD transgenic mice overexpressing miR-196a. The mice were all at approximate 12 months of age. Two technique repeats were performed for each sample.

first: Pei-Hsun

last: Cheng

first: Chia-Ling

last: Li

first: Yu-Fan

last: Chang

first: Shaw-Jeng

last: Tsai

first: Yen-Yu

last: Lai

first: Anthony

last: Chan

first: Chuan-Mu

last: Chen


title: Characterization of the transcriptomes of genetically diverse Listeria monocytogenes exposed to hyperosmotic and low temperature conditions

status: Public on May 04 2013

city: Berkeley

first: Juliana

last: Durack

state: CA

address: Barker Hall

department: MCB

institute: University of California

summary:
A screening process was used to determine hyperosmotic as well as cold stress tolerance of a diverse set of Listeria monocytogenes strains and was based on the capacity to grow at sub lethal levels of NaCl and 4°C. Analysis of gene expression in cells adapted to cold and hyperosmotic stress revealed significant strain specificity in terms of individual gene expression profiles though general patterns of expression within functional gene subsets were largely similar. .  Strain ATCC19115 revealed 150 significantly up- and 146 down-regulated genes in cells adapted to both hyperosmotic and cold stress, 82 up- and 56 down-regulated genes were matching in response to both stresses in strain 70-1700, whereas ScottA, a relatively stress-tolerant strain, showed up-regulation of 77 genes whilst 106 genes were significantly down-regulated. Among these homologously expressed genes only 22 were found to be significantly up-regulated and only 7 were down-regulated in all three strains adapted to hyperosmotic stress and cold temperature, suggesting a dominating strain specific response.   Overall adaptation to hyperosmotic and low temperature growth conditions in three L. monocytogenes strains were found to have many parallels, especially when examined using a statistically-based ontological approach. Evidence was presented for strong activation of genes associated with protein synthesis, in particular those coding proteins of the ribosome and involved directly in transcription. Genes associated with DNA maintenance; modification to cell envelope, and cell division were also up-regulated. On the other hand strong suppression of genes associated with carbohydrate metabolism and transport as well as flagella assembly was evident in stress adapted cells most likely due to energy preservation via CodY regulon. This suggests that adaptation to these adverse conditions engages similar mechanisms to cope with the induced stressful environments.
The microarray component of the experiments had the aim of determining:  i) To examine the gene expression responses of three L. monocytogenes strains that had different intrinsic salt resistances following adaptation to hyperosmotic stress.  ii) To examine the gene expression responses of three L. monocytogenes strains that had different intrinsic cold tolerances following adaptation to cold stress.  iii) To evaluate common pathways of adaptation to hyperosmotic and cold stresses

overall_design: Control cultures  (four biological replicates; exception strain ATCC19115 for the 10%NaCl stress which had three biological replicates) were labeled with Cy3 (exception strain ATCC19115 for the 10%NaCl-3 dyeswapped, where the control was labeled with Cy5 and sample with Cy3) for each treatment sample for three different L. monocytogenes strains. Sample cultures (four biological replicates; exception strain ATCC19115 for the 10%NaCl which had three biological replicates) were labeled with Cy5 (exception strain ATCC19115 for the 10%NaCl-3 dyeswapped, where the control was labeled with Cy5 and sample with Cy3) for each treatment sample for three different L. monocytogenes strains.    Condition one –sub-lethal hyperosmotic stress:  Control culture (Cy3-labelled RNA, except ATCC19115 for the 10%NaCl-3 dyeswapped , where the control was labeled with Cy5 and sample with Cy3) - strains were grown to mid exponential growth phase at 25°C (in a shaking water bath) in brain-heart infusion broth.   Test cultures (Cy5-labelled RNA, except ATCC19115 for the 10%NaCl-3 dyeswapped , where the control was labeled with Cy5 and sample with Cy3) – strains grown to mid exponential growth at 25°C phase (in a shaking water bath) in BHI broth supplemented with 8% additional NaCl (strain 701700), 10% additional NaCl (strain ATCC19115) or 12% additional NaCl (ScottA).    Condition two – cold stress:  Control culture (Cy3-labelled RNA) - strains were grown to mid exponential growth phase at 25°C (in a shaking water bath) in brain-heart infusion broth.  Test cultures (Cy5-labelled RNA) – strains grown to exponential growth phase at 4°C in BHI broth.

first: John

last: Bowman


title: Gene expression profiling of HIV-1 latently infected memory CD4+ T cells

status: Public on Mar 15 2013

first: Luigi

last: Marchionni

address: 1550 Orleans St., CRB2, Room 1M52

department: Oncology

summary:
Suppressive HAART does not eradicate HIV-1 and viral DNA persists as a stably integrated form in the absence of viral particle production. As a consequence, latent reservoirs are refractory to antiretroviral drugs and invisible to immune surveillance. The largest latent reservoir consists of resting memory CD4+ T cells. These cells can resume viral infection when activated through antigen recognition, causing bursts of viremia (blips).

Current therapies targeting latent HIV-1 have focused primarily on the “shock and kill” approach, which employs “anti-latency” drugs – most notably histone deacetylase (HDAC) inhibitors – to reactivate and flush latent provirus from its cellular reservoirs in the absence of global T cell activation. This approach is predicated on the notions that viral reactivation will lead to the demise of the infected cell, and that HAART will prevent spreading of the infection. On the contrary, recent evidence indicates that latently infected CD4+ T cells of HIV-1 patients on HAART survive in vitro viral reactivation with the HDAC inhibitor, SAHA, even when co-cultured with autologous CD8+ cytotoxic T lymphocytes (CTL). Moreover, it remains to be addressed the impact of anti-latency drugs on viral reservoirs undergoing low-level ongoing replication, inherently more resistant to the cytopathic effects of HIV-1 and residing in anatomical sites hard to reach for some antiretroviral drugs (e.g. macrophages).

As a consequence, there is a need to develop alternative therapeutic approaches aimed at eliminating or decreasing the latent reservoir. Progress in that direction has been hindered by the lack of biomarkers uniquely or differentially expressed on latently infected compared to their uninfected counterparts.

To gain insight into the cellular mechanisms that take place in the context of latency, and with the goal of identifying distinctive markers that distinguish latently infected CD4+ T cells, we have used an in vitro model developed in our laboratory to study the expression profile of latently infected CD4+ T cells by microarray analysis.

overall_design:
We have used a culture system, previously established in our laboratory, to generate and isolate quiescent latently infected CD4+ T cells in vitro. In this in vitro HIV-1 latency model, CD4+ T cells are activated, infected with full length, replication competent HIV-1, and then returned to quiescence in the presence of IL-7, yielding a culture of quiescent latently infected and uninfected cells.

We showed that HIV-1 p24gag expressed during viral replication persists in the cytoplasm of latently infected cells for several days before being degraded. Therefore, we exploited the presence of cytoplasmic p24gag to sort latently infected from uninfected cells by FACS from the same initial cell culture.

Total RNA was isolated from sorted latently infected and uninfected cells generated from CD4+ T cells of four different donors. Paired RNA samples from infected and uninfected cells were labeled with Cy3 and Cy5 to allow dual-color competitive hybridization. Moreover, to control for the dye bias in our experiments, we implemented a dye swap protocol (reciprocal labeling) for paired RNA samples from 2 donors.

Samples were analyzed by dual-color competitive hybridization on the Agilent whole human genome microarrays (41,000 unique probes).

This is the first comparative genomic profiling of primary latently infected resting memory CD4+ T cells versus their uninfected counterparts sorted from the same culture. Microarray analyses performed in this study revealed profound differences between latently infected and uninfected cells. Of relevance are genes involved, not only in previously described pathways related with transcriptional and post-transcriptional regulation, but affecting proliferation, survival, cell cycle progression and cell metabolism. This could explain why latently infected cells have been resistant to reactivation with current anti-latency approaches. Thus, targeting of more downstream steps, such as the ones identified in this study, may be able to enhance viral flushing from refractory latent reservoirs. In addition, we identified a panel of surface makers differentially expressed in latently infected cells, which seem worth investigating for their potential use as biomarkers. Indeed, they might allow the enrichment of this latent reservoir for molecular in depth studies, for monitoring the size of the latent reservoir in the clinical setting, as well as for the development of new therapeutic strategies aimed at eradicating this reservoir.

first: Maria

last: Iglesias-Ussel

first: Fabio

last: Romerio


title: Patterns of miRNA expression in primary plasma cell leukemias: implications for tumor progression in multiple myeloma

first: Luca

last: Agnelli

summary: Primary plasma cell leukemia (pPCL) is a rare and very aggressive form of plasma cell dyscrasias. To date, no information of microRNA expression in pPCL has been reported. To investigate the role of miRNAs in pPCL, we analyzed global miRNA expression profiles of highly purified malignant plasma cells from 18 previously untreated patients included in a prospective clinical trial. MiRNA expression patterns were evaluated in the context of the molecular abnormalities of the disease and in comparison with a representative cohort of multiple myeloma (MM) patients. We identified a series of deregulated miRNAs in pPCL (42 up-regulated and 41 down-regulated) which may have a putative role in contributing to tumor progression in MM. Furthermore, we integrated miRNA and gene expression data with computational prediction of miRNA targets, finding that miRNAs differentially expressed between MM and pPCL could regulate genes with important functions in cancer. Overall, our study represents the first attempt to investigate the involvement of miRNAs in pPCL and may contribute to develop functional approaches to investigate the activity of deregulated miRNAs in aggressive forms of plasma cell dyscarsias and their possible role as novel therapeutic targets.

overall_design: This series of microarray experiments contains the microRNA profiles of purified plasma cells (PCs) obtained from 39 multiple myeloma (MM) and 18 primary plasma cell leukemia (pPCL) at diagnosis. PCs were purified from bone marrow specimens, after red blood cell lysis with 0.86% ammonium chloride, using CD138 immunomagnetic microbeads. The purity of the positively selected PCs was assessed by morphology and flow cytometry and was > 90% in all cases. 500 nanograms of total RNA was processed in accordance with the manufacturer's protocols (Agilent Technologies) to generate Cy3-labelled RNA, which were purified on chromatography columns (Micro Biospin 6, Bio-Rad, Hercules, CA) and then hybridized on an Agilent microarray (G4470B) at 55¡C for 17 hr in a rotating oven. Images at 5 um resolution were generated using an Agilent scanner G2505B. The Feature Extraction 10.7.3.1 software (Agilent Technologies) was used to obtain the microarray raw-data. The raw gTotalGeneSignal has been recalculated using the procedures described in Agilent Feature Extraction Software version 10.1 manual. Non-human probes, miRNAs flagged as ÒabsentÓ (i.e. expressed below background levels) throughout the whole dataset and miRNAs expired according to Sanger miRBase Release 15 (April 2010) were discarded, and a quantile normalization was applied on raw data using the aroma.light package for Bioconducor. The data were then converted to obtain positive values throughout the dataset, at a minimum value of 1, and log2 transformed.

first: Lionetti

last: Marta

first: Musto

last: Pellegrino

first: Fabris

last: Sonia

first: Agnelli

last: Luca

first: Todoerti

last: Katia

first: Mosca

last: Laura

first: Tuana

last: Giacomo

first: Grieco

last: Vitina

first: Bianchino

last: Gabriella

first: D'Auria

last: Fiorella

first: Statuto

last: Teodora

first: Mazzoccoli

last: Carmela

first: DeLuca

last: Luciana

first: Petrucci

last: Maria Teresa

first: Morabito

last: Fortunato

first: Offidani

last: Massimo

first: DiRaimondo

last: Francesco

first: Falcone

last: Antonietta

first: Omede

last: Paola

first: Boccadoro

last: Mario

first: Palumbo

last: Antonio

first: Neri

last: Antonino


title: Expression data from undifferentiated and differentiated mouse female ES cells PGK12.1

status: Public on Feb 15 2013

city: Seattle

first: Xinxian

last: Deng

state: WA

address: 1959 NE Pacific St.

department: Laboratory Medicine and Pathology

institute: University of Washington

summary:
Affymetrix 430 2.0 mouse arrays were used for expression analyses in undifferentiated and differentiated PGK12.1 ES cells. We found that the X:autosome expression ratios calculated from the mean expression values of X-linked and autosomal genes from microarrays was ~1.4 in undifferentiated female ES cells and then decreased to 1.2 in PGK12.1 cells after 15-day embryoid body differentiation. Thus, a substantial level of X upregulation is already evident in these ES cells prior to differentiation.
Our findings based on Affymetrix expression arrays are consistent with microarray analysis from other labs and our RNA-seq analysis of mouse female PGK12.1 ES cells.

overall_design: Mouse female ES cells PGK12.1 were differentiated by the EB (embryoid body) differentiation protocol. The presence of two active X chromosomes in undifferentiated female ES cells and one active X in 15-day differentiated cells was verified by Xist RNA FISH. Total RNA was prepared from undifferentiated and 15-day differentiated PGK12.1 and was used for expression array analyses to examine exprssion of the X chromosome during differentiation.

first: Joel

last: Berletch

first: Christine

middle: M

last: Disteche


title: Gene expression profiling of cidofovir treatment and resistance in SiHa cells

status: Public on Jan 31 2013

city: Leuven

first: Rekin's

last: Janky

address: Herestraat 49 Box 816

department: Nucleomics Core

country: Belgium

institute: VIB

summary: Cidofovir is an acyclic nucleoside phosphonate with strong antiviral activity against a broad spectrum of DNA viruses. Although it has previously been shown that cidofovir exerts an antiproliferative effect on HPV positive cells by the induction of apoptosis, the exact mechanism of action remains to be unraveled. In order to study the activity of cidofovir against HPV, gene expression profiling was performed in cidofovir-treated and cidofovir-resistant SiHa cells by means of microarrays (HG-U133 Plus 2, Affymetrix).

overall_design: Controls (untreated SiHa wild-type cells): 24h, 48h and 72h of growth. Cidofovir treated (50 µg/ml) SiHa cells: 24h, 48h and 72h of growth. Cidofovir resistant cells (#55 and #230): 72h of growth. All samples were performed in triplicates.

first: Tim

last: De Schutter

first: Dimitrios

last: Topalis

first: Graciela

last: Andrei

first: Robert

last: Snoeck


title: Upregulation of the mammalian X chromosome is associated with enhanced transcription initiation, MOF-mediated H4K16 acetylation, and longer RNA half-life

status: Public on Mar 05 2013

type: Genome binding/occupancy profiling by genome tiling array

summary:
Many animal species employ a chromosome-based mechanism of sex determination, which has led to coordinate evolution of dosage compensation systems. Dosage compensation not only corrects the imbalance in the number of X-chromosomes between the sexes, but is also hypothesized to correct dosage imbalance within cells due to mono-allelic X expression and bi-allelic autosomal expression, by upregulating X-linked genes (termed ‘Ohno’s hypothesis’).
To identify molecular mechanisms of X upregulation in mammals we established genome-wide profiles for the initiation and elongation forms of RNA polymerase II (PolII), PolII-S5p (phosphorylated at serine 5) and PolII-S2p (phosphorylated at serine 2), and for histone modifications in mouse cell lines and tissues. We found that in addition to being enriched in PolII-S5p but not in PolII-S2p, X-linked promoters were also enriched in two epigenetic marks, H4K16ac and H2AZ, dependent on expression levels. To address the function of the H4K16 acetyltransferase MOF occupancy profiles were established and knockdowns of MOF and MSL1 were done in mouse ES cells. Our results support a conserved role for the MSL complex to enhance transcription initiation of X-linked genes.

sample_organism:
Mus musculus
Mus musculus x Mus spretus

overall_design: Comparison of the profiles of RNA PolII, the H4K16ac acetyltransferase MOF and histone active marks on the X versus autosomes in mouse


title: Allele-specific maps of RNA polymerase II phosphorylated at serine 5 in mouse cultured hybrid cells and mouse hybrid brain

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We report the application of single-molecule-based sequencing technology for high-throughput profiling of RNA polymerase II phosphorylated at serine 5 (PolII-S5p; the transcription initiation form) in female mouse cultured hybrid cells and female hybrid brain derived from mouse systems with skewed X inactivation based on crosses between C57BL/6J (BL6) and M. spretus. In these systems, alleles can be differentiated by frequent SNPs between mouse species, and the active X (Xa) compared to the haploid set of autosomes from the same species. To examine PolII-S5p occupancy in vivo, ChIP-seq was done in brain from an adult female F1 mouse in which the BL6 X is always active and the spretus X inactive. Uniquely mapped reads containing informative SNPs were assigned to each haploid chromosome set (BL6 or spretus) and were counted to establish allele-specific PolII-S5p occupancy profiles. We found that PolII-S5p allele-specific occupancy with or without normalization by input genomic DNA sequencing data showed that expressed genes on the Xa (>1RPKM) had 30% higher PolII-S5p peak levels at their promoters compared to autosomal genes from the same species (BL6). This result was confirmed by performing an independent allele-specific ChIP-seq analysis on fibroblasts derived from embryonic kidney (Patski cell line) that have the opposite X inactivation pattern from the brain sample, i.e. an Xa from M. spretus and an Xi from BL6. These findings suggest that transcription initiation of X-linked genes is enhanced to contribute to X upregulation in cell lines and in vivo.

overall_design: Examination of allele-specific PolII-S5p occupancy in mouse hybrid cells and brain.

first: Wenxiu

last: Ma

first: William

last: Noble

first: Jay

last: Shendure


title: Expression data from mouse ES cells after control RNAi (scramble siRNAs) or specific RNAi (siRNAs for specific genes) treatment

summary:
To address the functional role of MOF in mammalian X upregulation, male and female mouse ES cells were transfected with a mixture of three small interfering RNA duplexes, each of which targets a different region of Mof mRNA. We found that MOF knockdown in mouse ES cells caused a greater drop in expression of X-linked genes compared to autosomal genes, as measured by expression array analyses. The strongest effect was observed on medium-expressed X-linked genes.
We next examined components of the two known MOF protein complexes, MSL1 (male-specific lethal1) and NSL1 (nonspecific lethal1). Knockdown of MSL1 but not NSL1 in undifferentiated female ES cells PGK12.1 specifically caused a decrease in expression levels of X-linked genes. Cells co-transfected with both MOF and MSL1 siRNAs had similar expression changes to MSL1 knockdown alone, indicating that these components probably operate within the same complex but are not additive. Our findings that key components of the MSL but not NSL complex play a role in upregulation of mammalian X-linked genes in ES cells.

overall_design: Mouse ES cells were treated by Invitrogen scramble siRNA duplexes or specific siRNA duplexes and used for RNA extraction and hybridization on Affymetrix microarrays. Six RNA samples from two independent double-RNAi treatments and one single-RNAi treatment in undifferentiated female ES cells PGK12.1, and RNA samples from two single-RNAi treatments in undifferentiated male ES cells WD44 or E14 were assayed for expression changes by arrays. RNA samples from three MSL1RNAi treatments, two MOF/MSL1RNAi treatments and three NSL1RNAi treatments in undifferentiated female ES cells PGK12.1 were assayed by arrays.


title: Expression data from Spdef +/+ and Spdef -/- mice

status: Public on Feb 08 2013

city: Boston

first: Ilene

last: Gipson

state: MA

address: 20 Staniford ST

institute: Schepens Eye Research Institute

summary:
Goblet cell numbers decrease within the conjunctival epithelium in drying and cicatrizing ocular surface diseases. Factors regulating goblet cell differentiation in conjunctival epithelium are unknown. Recent data indicate that the transcription factor SAM-pointed domain epithelial-specific transcription factor (Spdef) is essential for goblet cell differentiation in tracheobronchial and gastrointestinal epithelium of mice. Using Spdef -/- mice, we determined that Spdef is required for conjunctival goblet cell differentiation and that Spdef -/- mice, which lack conjunctival goblet cells, have significantly increased corneal surface fluorescein staining and tear volume, a phenotype consistent with dry eye.
Microarray analysis of conjunctival epithelium in Spdef -/- mice identified 43 signficantly upregulated genes and 110 signficantly downregulated genes in the conjunctival epithelium of Spdef -/- mice compared to that of Spdef +/+ control mice (3 fold change, p<0.01). Downregulated genes of particular interested included goblet cell-specific genes (Muc5ac, Tff1, Gcnt3). Upregulated genes included epithelial cell differentiation/keratinization genes (Sprr2h, Tgm1) and pro-inflammatory genes (Il1-α, Il-1β, Tnf-α), all of which are upregulated in dry eye. Interestingly, four Wnt pathway genes were downregulated.

overall_design: Conjunctival epithelium of Spdef +/+ and Spdef -/- mice was collected by laser capture microdissection for RNA extraction and hybridization on Affymetrix microarrays to determine if gene expression patterns in the conjunctival epithelium of Spdef -/- mice is altered compared to that of Spdef +/+ mice.

first: Christina

last: Marko

first: Balaraj

last: Menon


title: Expression data from inflorescence (unopened flower buds) of Arabidopsis thaliana - comparison between Ler and ask1 mutant

status: Public on Feb 01 2013

city: University Park

first: Dihong

last: Lu

state: PA

address: 416 Life Sciences Building

institute: Penn state university

summary:
ARABIDOPSIS SKP1-LIKE1 (ASK1) protein is involved in regulating flower development. We have compared ask1 mutant floral transcriptome with wild-type Ler to identify the role of ASK1-containing E3 ubiquitin ligases in regulating flower transcriptome.
In this dataset, we include the expression data obtained from Arabidopsis thaliana Ler and ask1 mutant flower buds. We identified 42 genes and 74 genes that are down-regulated and up-regulated, respectively.

overall_design: Totally 5 samples were analyzed: 3 samples of ask1 and 2 samples of Ler. The average values were compared between ask1 and Ler.

first: Weimin

last: Ni


title: Genome-wide microarray analysis of normal human fibroblasts in response to kaemferol, daidzein, kaemferol/genistein, and daidzein/genistein

status: Public on Jan 24 2013

city: Gdańsk

first: Magdalena

last: Gabig-Cimińska

address: Wita Stwosza 59

department: of Medical Biology and Genetics

country: Poland

institute: University of Gdańsk

summary: Establishment of a transcriptomic profile of human cells treated with kaemferol, daidzein, kaemferol/genistein, or daidzein/genistein with particular emphasis on signature of genes coding for enzymes involved in glycosaminoglycan synthesis stands for the present study. The hypothesis tested was that kaemferol, daidzein, kaemferol/genistein, and daidzein/genistein influence expression of some genes, among which are those coding for enzymes required for the synthesis of different GAGs being pathologically accumulated in mucopolysaccharidoses. Results provide important information concerning the extent of action of kaemferol, daidzein, kaemferol/genistein, and daidzein/genistein at the molecular level in terms of modulation of gene expression.

overall_design: Total RNA was obtained from human dermal fibroblasts (HDFa) subjected to 24 or 48 hours in vitro exposure to 30, 60 or 100 µM kaemferol; 60 or 100 µM daidzein; 30 µM + 30 µM kaemferol/genistein; 30 µM + 30 µM daidzein/genistein; or 0.05%DMSO. Three replicates each.


title: Transcriptomic Analysis of PNN- and ESRP1-regulation in Human Corneal Epithelial Cells

city: Durham

first: Jian-Liang

last: Li

state: NC

address: 111 TW Alexander Dr

institute: NIEHS

summary: Pnn depletion in developing mouse corneal epithelium led to disrupted alternative splicing of multiple ESRP-regulated epithelial-type exons. In human corneal epithelial cells (HCET), ESRP1 and PNN displayed close localization in and around nuclear speckles and their physical association in protein complexes was identified.  In this study, gene expression profiling was performed to identify PNN- and ESRP1-regulated alternative pre-mRNA splicing in human corneal epithelial cells. Immortalized human corneal epithelial cells harboring doxycycline-inducible shRNA against PNN or ESRP1 were created.  Whole transcriptome array analysis on ESRP1 or PNN knockdown HCET cells revealed clear alterations in transcript level and splicing pattern of specific subsets of genes with significant overlaps in their candidate targets. Our data suggest that ESRP1 and PNN modulate alternative splicing of a specific subset of exons, but not general splicing events. ESRP1 and PNN may together participate in the regulation of epithelial-specific splicing program in a genome-wide fashion.

overall_design: Parental HCET, shRNA-PNN HCET, and shRNA-ESRP1 HCET cells were cultured for 3 days with/without doxycycline. Total RNA was isolated from four biological replicates of each sample group and then subjected to hGlue3_0 transcriptome array analysis.

first: Jeong

last: Joo

first: Creg

last: Correia

first: Jian

first: Maria

last: Lopez

first: Henry

middle: V

last: Baker

first: Stephen

last: Sugrue


title: Expression data from Arabidopsis wt and ga1-3, brm-1, ga1-3/brm-1 mutants

status: Public on Mar 08 2013

city: Warsaw

first: Maciej

last: Kotliński

state: Mazowieckie

address: Pawinskiego 5a

department: Laboratory of Microarray Analysis

institute: University of Warsaw / Institute of Biochemistry and Biophysics, PAS

summary: Arabidopsis brm plants depleted in a SWI/SNF-type ATPase BRM have decreased level of endogenous gibberellins and a phenotype that in many respects resembles the phenotype of mutants with repressed GA signaling or biosynthesis, like ga1-3. ga1-3/brm double mutant showed several additive and synergistic effects. To examine whether the phenotypic traits of brm, ga1-3 and ga1-3/brm lines are reflected at the gene expression level, we compared the expression profiles of brm, ga1-3, ga1-3/brm and wild-type plants using microarray analysis.

overall_design: Microarray analysis was performed on total RNA isolated from shoots of 18-d-old wt, brm, ga1-3, and ga1-3/brm seedlings. Three biological replicates were examined for each genotype. Plants were grown simultaneously under the same conditions.

first: Rafal

last: Archacki

last: Kotlinski

first: Marta

last: Koblowska

first: Roksana

last: Iwanicka-Nowicka

first: Andrzej

last: Jerzmanowski


title: Evaluation of gene expression profile in postmortem brain with Alzheimer´s disease-type neuropathological changes

status: Public on Jan 11 2013

city: São Paulo

first: Renato

middle: David

last: Puga

state: São Paulo

address: Dr Ovidio Pires de Campos,785

department: Instituto de Psiquiatria

country: Brazil

institute: Universidade de São Paulo

summary: Unravel the mechanisms underlying brain aging and Alzheimer´s disease (AD) has been difficult because of complexity of the networks that drive these aging-related changes. Analysis of the gene expression in the brain is a valuable tool to study the function of the brain under normal and pathological conditions. Gene microarray technology allows massively parallel analysis of most genes expressed in a tissue, and therefore is an important research tool that potentially can provide the investigative power needed to address the complexity of brain aging and neurodegenerative processes. One of the reasons that account for the resistance of AD pathogenesis to analysis is that clinically normal subjects may exhibit considerable AD pathology, blurring criteria for distinguishing subjects with normal aging or AD. Here, we analyzed hippocampal and cortex frontal gene expression from 32 subjects separated in individuals presenting, 1) both pathologic and clinical AD (definitive AD); 2) AD pathology and normal clinic (pathologic AD); 3) cognitive impairment, without AD pathology (others dementias); and 4) no cognitive impairment, without AD pathology (normal individuals). Our results show that based on gene expression profile these individuals we could verify similarity between the definitive AD group and the group that only had AD-type pathology (pathologic AD).

overall_design:
Specimens of hippocampus and cortex frontal used in this study were obtained at autopsy from 32 subjects. Neuropathology, specifically NFT and NP, was assessed in accordance to the Braak staging and CERAD scores, respectively. Based on pathologic and clinic criteria, subjects were categorized into four groups: 1) nine subjects with AD neuropathologic (Braak = IV / V / VI and CERAD ≠ 0) presenting cognitive impairment (CDR ≥ 1), termed “definitive AD” (dAD); 2) five subjects with AD neuropathologic (Braak = IV / V / VI and CERAD ≠ 0) and without cognitive impairment (CDR = 0), termed “pathologic AD” (pAD); 3) nine subjects without AD neuropathologic (Braak = 0 / I / II and CERAD ≠ C) presenting cognitive impairment (CDR ≥ 1), termed “other dementias” (OD); 4) - nine subjects without AD neuropathologic (Braak = 0 / I / II and CERAD ≠ C) and without cognitive impairment (CDR = 0), termed “normal” (N).  RNA isolation and Amplification. From total RNA, a two-round linear amplification procedure (T7-based protocol) was carried out for all samples and for a pool of RNAs
obtained from 15 distinct human cell lines used as reference. Labeled cDNA was generated in a reverse transcriptase reaction using amplified RNA (aRNA). Equal amounts of test and reference cDNA reverse color Cy-labeled aRNA targets were competitively hybridized against the cDNA probes in a customized cDNA platform with 4,608 ORESTES representing human genes. Dye-swap was performed for each sample as control for dye bias and used as replicate.

first: Aderbal

last: Silva

first: Helena

last: Brentani


title: High fidelity patient-derived xenografts for accelerating prostate cancer discovery and drug development

status: Public on Jan 12 2013

city: Vancouver

first: Shawn

last: Anderson

state: BC

address: 2660 Oak Street

country: Canada

institute: Vancouver Prostate Centre

type: Genome variation profiling by genome tiling array

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

overall_design: Refer to individual Series


title: Phenotypic changes in PMN from production to site of infection

city: Toronto

first: Flavia

last: Lakschevitz

state: ON

address: 150 College st room 221

department: Faculty of Dentistry

institute: University of Toronto

summary: Neutrophils, key cells of the innate immune system, are responsible for preventing bacterial infections. It has recently been established that neutrophils are capable of complex changes in gene expression during inflammation. The concept that neutrophils merely respond to external signals has been replaced by the concept that activated neutrophils can secrete a variety of cytokines and also have an active regulatory role in angiogenesis and tumoural fate. Currently, neutrophil research relies mostly in animal models that reproduce human physiology and pathology. Although, in many respects, there is a conservation of functions in mice and humans, little is known about genomic conservation of neutrophils between mice and humans.   We hypothesize that neutrophils are transcriptionally active cells that alters their gene expression profile as they migrate into different compartments. To identify changes in neutrophil gene expression in the circulation and inflamed tissue we used recent advances in neutrophil isolation, RNA amplification and microarray technologies that allowed us to compare the transcriptome of neutrophils. Additionally, using bioinformatics, we investigate the genomic conservation of neutrophils between mice and humans.

overall_design: Total RNA obtained from isolated neutrophils from Bone Marrow, Blood and Peritoneal Exudate from Black 6 mice.

first: Michael

last: Glogauer


title: High fidelity patient-derived xenografts for accelerating prostate cancer discovery and drug development (expression)

summary: Prostate cancer research is hampered by a lack of preclinical models which accurately reproduce clinical heterogeneity. We have established a bank of transplantable patient-derived prostate tumor xenograft lines, using subrenal capsule grafting of human tumor tissue into immuno-deficient mice. This panel includes the first lines generated from prostate cancer biopsy tissue, and also new lines from metastatic tissue. The lines retained salient features of the original patient tumors, including histopathological and molecular characteristics. Furthermore, they span major subtypes of prostate cancer, capturing diverse inter- and intra-tumoral heterogeneity. Host castration led to the development of castrate-resistant tumors, including the first model of neuroendocrine transdifferentiation. This publicly-available resource provides novel tools for the next-generation of prostate cancer research and therapy development.

overall_design: 3 primary tumors and 29 xenograft tumors

first: Dong

last: Lin

first: Alexander

last: Wyatt

first: Sonal

last: Brahmbhatt

first: Anne

last: Haegert


title: High fidelity patient-derived xenografts for accelerating prostate cancer discovery and drug development (aCGH)

overall_design: 3 primary tumors and 21 xenograft tumors


title: Effects of dsRNA-GFP as exogenous control on honey bee RNAi assays: gene expression changes in workers

status: Public on Jan 10 2013

city: Piracicaba

first: Ana

middle: Durvalina

last: Bomtorin

address: R Ajudante Albano 847

institute: Universidade de Sao Paulo

summary: A green fluorescence protein (GFP)-derived dsRNA (dsRNA-GFP) has been used as an exogenous control for Apis mellifera RNAi assays by multiple research groups. Its sequence does not share any significant homology with any known honey bee genes. Although dsRNA-GFP is not expected to trigger an RNAi response in treated bees, undesirable effects on gene expression, pupal pigmentation or developmental timing have been routinely observed. To better understand the multiple molecular and phenotypic effects of dsRNA-GFP in honey bees and to evaluate its use as a control for RNAi studies, we examined the impact of dsRNA-GFP on global gene expression patterns in developing workers. We found that dsRNA-GFP causes large-scale changes in gene expression associated with multiple biological processes. Furthermore, dsRNA-GFP exposure tended to preferentially decrease, rather than increase, expression of genes compared to controls.

overall_design: Gene expression differences were analyzed using a dye-swap design. Second instar worker larvae received 0.5 μg of dsRNA-GFP. Non-treated and dsRNA-GFP treated 7 day-old workers (W7d) were sampled.

middle: D

first: Francis

last: Nunes

first: Angel

last: Barchuk

first: Zila

last: Simoes


title: dsGFP as exogenous control on RNAi assays and its effects on gene expression in Apis mellifera

summary: Double stranded RNAs are used to induce gene silencing in functional studies. In Apis mellifera green fluorescent protein (GFP) dsRNA (dsGFP) has been used as an exogenous control as its sequence has no homology in honeybee genome. However, some undesirable effects are observed after dsGFP treatment. A microarray approach comparing gene expression differences between untreated and dsGFP treated groups, containing honeybees workers in two different developmental stages, pre-pupae and light-brown eyed pupae, were used to test the reliability of dsGFP as a control for RNAi experiments. According to these microarrays results dsGFP can be used as a control in RNAi assays as long as the affected genes are taken into account in the analysis.

overall_design: Analysis used loop-design to compare dsGFP treated and noon-treated workers in pre-pupae and light-brown-eyed pupae stages.

first: Aline

last: Aleixo

first: Christina

last: Grozinger

middle: P


title: Epigenome analysis of smear cells from the uterine cervix consisting of 24 normals and 24 cervical intraepithelial neoplasias (all HPV+)

city: London

first: Andrew

last: Teschendorff

address: 72 Huntley Street

department: UCL Cancer Institute/Institute for Women's Health

institute: University College London

summary: The Illumina Infinium 27k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 27,000 CpGs in smear cells from the uterine cervix (liquid based cytology samples), obtained from 48 women. All women tested positive for the human papilloma virus (HPV+). Of the 48 samples, 24 were cytologically normal while the other 24 exhibited morphological transformation (cervical intraepithelial neoplasia of grade 2 or higher - CIN2+).

overall_design: Bisulphite converted DNA from the 48 samples were hybridised to the Illumina Infinium 27k Human Methylation Beadchip v1.2

first: Martin

last: Widschwendter


title: Dual Pten/p53 suppression enhances Notch signaling and sarcoma progression

status: Public on Jan 01 2013

city: LYON

first: Jiri

last: ZAVADIL

state: Other

address: 150 COURS ALBERT THOMAS

country: France

institute: IARC WHO

summary:
Soft tissue sarcomas (STS) are a heterogeneous group of tumors associated with poor clinical outcome. While a subset of STS are characterized by simple karyotypes and recurrent chromosomal translocations, the mechanisms driving cytogenetically complex sarcomas are largely unknown. Clinical evidence led us to partially inactivate Pten and p53 in the smooth muscle lineage of mice, which developed high-grade undifferentiated pleomorphic sarcomas (HGUPS), leiomyosarcomas (LMS) and carcinosarcomas (CS) that widely recapitulate the human disease, including the aberrant karyotype and metastatic behavior. Pten was found haploinsufficient whereas the wild-type allele of p53 invariably gained point mutations. Gene expression profile showed upregulated Notch signaling in Pten∆/+p53∆/+ tumors compared to Pten+/+p53∆/+. Consistently, Pten silencing exacerbated the clonogenic and invasive potential of p53-deficient bone marrow-derived mouse mesenchymal stem cells and tumor cells, while activating the Notch pathway. Moreover, the increased oncogenic behavior of Pten∆/+p53∆/+ and shPten-transduced Pten+/+p53∆/+ tumor cells was counteracted by treatment with a gamma secretase inhibitor (GSI), suggesting that the aggressiveness of those tumors can be attributed, at least in part, to enhanced Notch signaling. This study demonstrates a cooperative role for Pten and p53 suppression in complex karyotype sarcomas while establishing Notch as an important functional player in the crosstalk of these pathways during tumor progression. Our results highlight the importance of molecularly subclassifying high-grade sarcoma patients for targeted treatments.
Compare Pten∆/+p53∆/+  to Pten+/+p53∆/+ high-grade undifferentiated pleomorphic sarcomas (HGUPS)

overall_design:
4 Pten∆/+p53∆/+ were compared to 5 Pten+/+p53∆/+
Keywords: Differential gene expression.

first: Maria

last: Guijarro

first: Sonika

last: Dahiya

first: Laura

last: Danielson

first: Miguel

last: Segura

first: Frances

last: Vales-Lara

first: Silvia

last: Menendez

first: Dorota

last: Popiolek

first: Kush

last: Mittal

first: Jian-Jun

last: Wei

last: Zavadil

first: Carlos

last: Cordón-Cardó

first: Pier

last: Pandolfi

first: Eva

last: Hernando


title: Gene expression in  nonagenarian (Vitality 90+)

city: Tampere

first: Saara

last: Marttila

address: Biokatu 6

department: School of Medicine

country: Finland

institute: University of Tampere


title: Expression data from human colonic biopsy samples  (adenoma-carcinoma)

city: Budapest

first: Wichmann

last: Barna

address: Szentkirály

country: Hungary

institute: SOTE

summary: Whole genomic microarray analysis was performed in order to identify gene expression profile alterations focusing on the dysplastic adenoma-carcinoma transition. Our aims were to determinate characteristic transcript sets for developing diagnostic mRNA expression patterns for objective classification of benign and malignant colorectal diseases and to test the classificatory power of these markers on an independent sample set.

overall_design: Total RNA was extracted from colonic biopsy samples of histologically negative patients and of patients with adenoma or colorectal cancer and were hybridized on Affymetrix HGU133 Plus 2.0 microarrays

first: Orsolya

last: Galamb

first: Barna

last: Wichmann

first: Bela

last: Molnar


title: Expression data from human colonic biopsy samples

summary: Age-related transcriptome changes can contribute to better understanding of gastrointestinal disorders such as colorectal cancer in older population

overall_design: Total RNA was extracted from colonic biopsy samples from histologically normal children and the samples were hybridized on Affymetrix HGU133 Plus 2.0 microarrays

first: Katalin

last: Leiszter

first: Kinga

last: Toth


title: Genome-wide microarray analysis of normal human fibroblasts in response to genistein

first: Magdalena

last: Gabig-Cimińska

summary: Establishment of a transcriptomic profile of human cells treated with genistein with particular emphasis on signature of genes coding for enzymes involved in glycosaminoglycan synthesis stands for the present study. The hypothesis tested was that genistein influences expression of some genes among which are those coding for enzymes required for synthesis of different GAGs being pathologically accumulated in mucopolysaccharidoses. Results provide important information concerning the extent of action of genistein at the molecular level in terms of modulation of gene expression by this substance.

overall_design: Total RNA obtained from Human Dermal Fibroblasts (HDFa) subjected to 1, 24 or 48 hours in vitro exposure to 30, 60 or 100 µM genistein, or 0.05%DMSO (K1), compared to untreated control (K2).


title: DNA methylation profiling in colon cancer

status: Public on Jan 22 2013

city: Leiden

first: Eddy

last: van Roon

address: Albinusdreef 2

department: Pathology

institute: LUMC

type: Methylation profiling by genome tiling array

summary: Profiling of CpG island methylation in 19 primary colon cancer and paired normal samples

overall_design: Agilent 244k CpG island microarray


title: miR-182 is Overexpressed in Head and Neck Squamous Cell Carcinomas Leading to Downregulation of Specific Transcripts

city: Baltimore

first: Michael

last: Ochs

address: 550 North Broadway, Ste 1103

department: Oncology Biostatistics

institute: Johns Hopkins University

summary: This study correlated miR expression with predicted targets to determine those miRs that both downregulated a subset of their predicted targets and showed differential expression in cancer samples compared to controls

overall_design: Case: Control with HNSCC tumors and uvulopalatopharyngoplasty (UPPP) samples

first: Joseph

middle: A

last: Califano


title: Microarray analysis of an RXR agonist HX630 regulated gene expression in murine pituitary corticotroph tumor cells (AtT20 cell)

status: Public on Jan 27 2013

city: Sendai

first: Akiko

last: Saito-Hakoda

address: 2-1, Seiryo-machi, Aoba-ku

department: Molecular Endocrinology

institute: Tohoku University Graduate School of Medicine

summary: To gain insight into the functional mechanisms of an RXR agonist HX630 in murine pituitary corticotroph tumor cells (AtT20 cell), especially concerning cell proliferation and apoptosis, we conducted whole genome microarray analysis to identify gene expression patterns, networks, and pathways in AtT20 cells treated with HX630 at 10 μM for 48 hr. 2,173 up-regulated and 3,169 down-regulated genes were identified that demonstrated fold-changes of at least 1.5 and a P-value <0.01 in AtT20 cells with HX630 at 10 uM compared with control.  Both the up- and down-regulated differentially expressed genes were submitted to IPA, which groups significant genes according to biological processes in which they function, and identifies various functions, networks, and pathways. These analyses showed that the categories of the biological functions and diseases significantly regulated by HX630 were associated with cell death, growth and cancer. Furthermore, it was identified that a gene network associated with caspase 3 was significantly induced by HX630.These data indicated that HX630 induced cell death and suppressed cell growth in AtT20 cells, consistent with the results of in vitro studies.

overall_design: AtT20 cells grown to 50 % confluence in regular medium in 12-multiwell plates were incubated either without or with HX630 at 10 μM in DMEM supplemented with 1 % stripped FBS media for 48 hr. The cells were then lysed and their total RNAs were isolated for microarray analysis.

first: Akira

last: Sugawara

last: Hakoda


title: Cancer Outlier Gene Profile Sets Elucidate Pathways and Patient-Specific Targets in Head and Neck Squamous Cell Carcinoma [Affymetrix SNP6]

summary: This study integrated Affymetrix SNPchip data for CNV estimation, Affymetrix HuEx1.0 data for gene expression estimation, and Illumina HumanMethylation27k BeadChip data for promoter methylation to estimate pathway activity.

overall_design: Case:Control with HNSCC tumors and uvulopalatopharyngoplasty (UPPP) samples


title: Cancer Outlier Gene Profile Sets Elucidate Pathways and Patient-Specific Targets in Head and Neck Squamous Cell Carcinoma [Affymetrix HuEx1.0]

summary: This study integrated Affymetrix SNPchip data for CNV estimation, Affymetrix HuEx1.0 data for gene expression estimation, and Illumina HumanMethylation27k BeadChip data for promoter methylation to estimate pathway activity


title: Effect of BAY 87-2243 on gene expression in hypoxic H460 cells

status: Public on Dec 08 2012

city: Wuppertal

first: Peter

last: Ellinghaus

address: Aprather Weg 18a

department: GDD-CS-GBM

institute: Bayer Pharma AG

summary:
The activation of the transcription factor Hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small-molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high throughput screen led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations but did not affect expression levels of genes outside of the HIF-1 pathway. Lead structure BAY 87-2243 was found to inhibit HIF-1α protein accumulation under hypoxic conditions in NSCLC cell line H460 but had no effect on HIF-1α protein accumulation and HIF target gene expression in RCC4 cells lacking VHL activity or in H460 cells after inhibition of HIF prolyl hydroxylase activity. BAY 87-2243 had no effect on HIF-α-mRNA levels. Antitumor activity of BAY 87-2243 and suppression of HIF-1 target gene expression in vivo was demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial production of reactive oxygen species (ROS) by blocking complex I activity but has no effect on complex III activity. Lowering of mitochondrial ROS production to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors.
We used microarrays to detail the global programme of gene expression that is induced in NSCLC cell line H460 upon hypoxia (16 h incubation at 1 % pO2) and evaluated a dose-dependent effect of our HIF-1-pathway inhibitor BAY 87-2243 on genes tthat are affected by hypoxia.

overall_design: Specificity of BAY 87-2243 for the suppression of HIF-1-mediated gene transcription on a genome-wide scale was evaluated by microarray hybridizations using Affymetrix GeneChip Human Gene 1.0 ST arrays. RNA from normoxic H460 cells and from hypoxic H460 cells incubated with 1, 10 and 100 nM BAY 87-2243 respectively was subjected to array hybridization. Of those 30 genes that were most strongly suppressed by 100 nM BAY 87-2243 in hypoxic H460 cells compared to DMSO-treated hypoxic H460 cells, virtually all of them are induced by prior hypoxia and most of these genes have been described in the literature as HIF-1 target genes

first: Holger

last: Summer


title: Targeting the Phosphoinositide 3-Kinase p110α Isoform Impairs Cell Proliferation, Survival and Tumor Growth in Small Cell Lung Cancer

status: Public on Dec 01 2012

city: Zurich

first: Hubert

last: Rehrauer

address: Winterthurerstr. 190

department: FGCZ

institute: ETH Zurich / University of Zurich

summary: Purpose: The phosphoinositide 3-kinase (PI3K) pathway is fundamental for cell proliferation and survival and is frequently altered and activated in neoplasia, including carcinomas of the lung. In this study we investigated the potential of targeting the catalytic class IA PI3K isoforms in small cell lung cancer (SCLC), which is the most aggressive of all lung cancer types.  Experimental Design: The expression of PI3K isoforms in patient specimens was analyzed. The effects on SCLC cell survival and downstream signaling were determined following PI3K isoform inhibition by selective inhibitors or down-regulation by small interfering RNA.  Results: Over-expression of the PI3K isoforms p110α and p110β was shown by immunohistochemistry in primary SCLC tissue samples. Targeting the PI3K p110α with RNA interference (RNAi) or selective pharmacological inhibitors resulted in strongly affected cell proliferation of SCLC cells in vitro and in vivo, while targeting p110β was less effective. Inhibition of p110α also resulted in increased apoptosis and autophagy, which was accompanied by decreased phosphorylation of Akt and components of the mammalian target of rapamycin (mTOR) pathway, such as the ribosomal S6 protein, and the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). A DNA microarray analysis revealed that p110α inhibition profoundly affected the balance of pro- and anti-apoptotic Bcl-2 family proteins. Finally, p110α inhibition led to impaired SCLC tumor formation and vascularization in vivo.  Conclusion: Together our data demonstrate the key involvement of the PI3K isoform p110α in multiple tumor-promoting processes in SCLC.

overall_design: 3 control samples, 3 samples for two treaments

first: Anna

last: Wojtalla

first: Alexandre

last: Arcaro


title: Cancer Outlier Gene Profile Sets Elucidate Pathways and Patient-Specific Targets in Head and Neck Squamous Cell Carcinoma

type:
Methylation profiling by array
Expression profiling by array
Genome variation profiling by SNP array
Non-coding RNA profiling by array

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

platform_organism:
Homo sapiens
synthetic construct

overall_design: Refer to individual Series


title: RNA-seq of Taxus chinensis cells

status: Public on Nov 16 2012

city: Wuhan

first: Longjiang

last: Yu

address: 1037 Luoyu Road

department: College of life science and technology

institute: Huazhong University of Science & Technology

summary: We reported the application of high-throughput sequencing technology (RNA-seq) for the transcriptome of T. chinensis cells and the transcriptional alternatives of that responded to MeJA were comprehensively and quantitatively assessed with high-throughput sequencing technology (RNA-seq). By sequencing > 29 million reads (200 bp in length) of cDNA from each of MeJA-treated T. chinensis cells at 16 h (T16) and the control (T0), we identified 46,581 transcripts and uncovered 13,469 genes differentially expressed in response to MeJA. We provided functional clues for understanding the regulation mechanisms of MeJA-mediated defense responses and taxol biosynthesis.

overall_design: Examination of 2 different time treated with MeJA in Taxus chinensis cells.

first: Long-jiang

first: Peng

last: Zhang

first: Shu-tao

last: Li

first: Meng


title: Cancer Outlier Gene Profile Sets Elucidate Pathways and Patient-Specific Targets in Head and Neck Squamous Cell Carcinoma [Illumina HumanMethylation]


title: MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system

status: Public on Jan 14 2013

city: New York

first: Marian

last: Mellen

address: 1230 York Avenue

institute: The Rockefeller University

type: Methylation profiling by high throughput sequencing

summary: The high level of 5-hydroxymethylcytosine (5hmC) present in neuronal genomes suggests  that mechanisms interpreting 5hmC in the central nervous system (CNS) may differ from those present  in embryonic stem cells. Here we present quantitative, genome-wide analysis of 5hmC, 5-  methylcytosine (5mC) and gene expression in differentiated CNS cell types in vivo. We report that  5hmC is enriched in active genes, and that surprisingly strong depletion of 5mC is observed over these  regions. The contribution of these epigenetic marks to gene expression depends critically on cell type.  We identify methyl-CpG binding protein 2 (MeCP2) as the major 5hmC binding protein in the brain,  and demonstrate that MeCP2 binds 5hmC and 5mC containing DNA with similar high affinities. The  Rett Syndromecausing mutation R133C preferentially inhibits 5hmC binding. These findings support a  model in which 5hmC and MeCP2 constitute a cell specific epigenetic mechanism for regulation of  chromatin structure and gene expression.

overall_design:
Isolation and sorting was performed as described in (Kriaucionis and  Heintz, 2009). Briefly, cerebella were dissected as described above and homogenized in homogenization  medium (0.25 M sucrose, 150 mM KCl, 5 mM MgCl2, 20 mM Tricine pH 7.8, 0.15 mM spermine,  0.5 mM spermidine, EDTA-free protease inhibitor cocktail (Roche) using loose (A) and tight (B) glassglass  dounce.Homogenate was supplemented with 50% iodixanol (OptiPrep, Axis-Shield,  Scotland), 150 mM KCl, 5 mM MgCl2, 20 mM Tricine pH 7.8; and laid on a 29% iodixanol cushion. GC nuclei from WT and Mecp2-null mice were sorted by selecting a specific FSC/SSC  population identified from GC EGFP+ mice. We observed that this FSC/SSC gated population in GC EGFP+  animals resulted >92% of the sorted nuclei being EGFP positive. Thus, the gate selected allows us  to enrich the sample from 65-70% to 92-96% on GC cells. 5hmC was pulled down as described (Song et al., 2010). After  purification DNA was amplified as described in TruSeq DNA Sample kit. 1-0.5 μg of DNA was used for each experiment.  Sonicated DNA was end-repaired following by ligation to Illumina paired end sequencing adapters (Illumina,  PE‐ 102‐ 1003).), following  by amplification with Illumina primers. Input samples were produced for  each cell types in both procedures  5hmC enriched were then sequenced using Illumina platform obtaining more than 50 x10-6, 36-  bp single-end reads per sample. Reads were aligned to mm9 mouse genome assembly using Bowtie v0.12.7  (-m1 --best).  Two biological replicas were done for each of the cell  types
Single animals per replica were euthanized  with CO2 and cerebella were dissected. 4 cerebella was immediately homogenized in ice-cold  homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100  μg/ml cycloheximide, Complete-EDTA-free protease inhibitors (Roche) and RNasin RNase inhibitors  (Promega, Madison, WI) using a Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at  2,000 × g, 4 °C, to pellet cell debris, and incubated with 1% IGEPAL CA-630 (NP-40, Sigma) and 30 mM  DHPC (Avanti Polar Lipids, Alabaster, AL) for 5 min. Lysates were centrifuged for 15 minutes at 20,000 × g to  pellet insoluble material. At this point, 30 μL of supernatant was saved as input sample. Two custom made  mouse anti-GFP (clones 19C8 and 19F7; see Heiman et al., 2008) antibodies were captured on Dynal  magnetic beads (Invitrogen Corporation, Carlsbad, CA) coupled with protein L (Pierce, Rockford, IL) . The  homogenate was incubated with antibody coupled beads at 4°C with end-over-end rotation for approximately  16 hours. Beads were subsequently collected on a magnetic rack, washed three times with high-salt wash  buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM DTT, 100 μg/ml cycloheximide)  and RNA was released and purified using Rneasy Micro Kit (Qiagen, Valencia, CA) with in-column DNase  digestion. RNA quantity and quality were determined with a Nanodrop 1000 spectrophotometer (Wilmington,  DE) and Agilent 2100 Bioanalyzer using RNA 6000 Pico Chip. 4 biological replicas was produced per each cell  type. 10 ng of total RNA from cerebellum were converted to cDNA using the NuGEN Ovation RNA-Seq  (NuGEN,San Carlos,CA,USA) following manufacturers’ instructions. The Single Primer Isothermal  Amplification (SPIA) method used in this protocol allows the amplification of RNA target in double stranded  cDNA under standardized conditions that markedly deplete rRNA without preselecting mRNA. cDNA obtained  was quality scored by RNA 6000 PicoChip for Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). 1 μg  of cDNA per sample was sonicated using Covaris-S2 system (Covaris Inc., Woburn, MA, USA), cDNA  fragments of 200 bp were end-repaired and adapters were ligated for HiSeq 2000 (Ilumina Inc., San Diego,  CA, USA) technology using TruSeq DNA Sample kit (Illumina) and following manufacturer`s instructions.  Quality of libraries was assesed using HT DNA High Sensitivity Chip (Agilent) for 2100 Bioanalyzer.
Single animals per replica were euthanized  with CO2 and cerebella were dissected. 4 cerebella was immediately homogenized in ice-cold  homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100  μg/ml cycloheximide, Complete-EDTA-free protease inhibitors (Roche) and RNasin RNase inhibitors  (Promega, Madison, WI) using a Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at  2,000 × g, 4 °C, to pellet cell debris, and incubated with 1% IGEPAL CA-630 (NP-40, Sigma) and 30 mM  DHPC (Avanti Polar Lipids, Alabaster, AL) for 5 min. Lysates were centrifuged for 15 minutes at 20,000 × g to  pellet insoluble material. At this point, 30 μL of supernatant was saved as input sample. Two custom made  mouse anti-GFP (clones 19C8 and 19F7; see Heiman et al., 2008) antibodies were captured on Dynal  magnetic beads (Invitrogen Corporation, Carlsbad, CA) coupled with protein L (Pierce, Rockford, IL) . The  homogenate was incubated with antibody coupled beads at 4°C with end-over-end rotation for approximately  16 hours. Beads were subsequently collected on a magnetic rack, washed three times with high-salt wash  buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM DTT, 100 μg/ml cycloheximide)  and RNA was released and purified using Rneasy Micro Kit (Qiagen, Valencia, CA) with in-column DNase  digestion. RNA quantity and quality were determined with a Nanodrop 1000 spectrophotometer (Wilmington,  DE) and Agilent 2100 Bioanalyzer using RNA 6000 Pico Chip. 4 biological replicas was produced per each cell  type. 10 ng of total RNA per IP or input sample were converted to cDNA using the NuGEN Ovation RNA-Seq  (NuGEN,San Carlos,CA,USA) following manufacturers’ instructions. The Single Primer Isothermal  Amplification (SPIA) method used in this protocol allows the amplification of RNA target in double stranded  cDNA under standardized conditions that markedly deplete rRNA without preselecting mRNA. cDNA obtained  was quality scored by RNA 6000 PicoChip for Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). 1 μg  of cDNA per sample was sonicated using Covaris-S2 system (Covaris Inc., Woburn, MA, USA), cDNA  fragments of 200 bp were end-repaired and adapters were ligated for HiSeq 2000 (Ilumina Inc., San Diego,  CA, USA) technology using TruSeq DNA Sample kit (Illumina) and following manufacturer`s instructions.  Quality of libraries was assesed using HT DNA High Sensitivity Chip (Agilent) for 2100 Bioanalyzer.
Single animals per replica were euthanized  with CO2 and cerebella were dissected. 4 cerebella was immediately homogenized in ice-cold  homogenization buffer (10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol (DTT), 100  μg/ml cycloheximide, Complete-EDTA-free protease inhibitors (Roche) and RNasin RNase inhibitors  (Promega, Madison, WI) using a Teflon-glass homogenizer. Homogenates were centrifuged for 10 minutes at  2,000 × g, 4 °C, to pellet cell debris, and incubated with 1% IGEPAL CA-630 (NP-40, Sigma) and 30 mM  DHPC (Avanti Polar Lipids, Alabaster, AL) for 5 min. Lysates were centrifuged for 15 minutes at 20,000 × g to  pellet insoluble material. At this point, 30 μL of supernatant was saved as input sample. Two custom made  mouse anti-GFP (clones 19C8 and 19F7; see Heiman et al., 2008) antibodies were captured on Dynal  magnetic beads (Invitrogen Corporation, Carlsbad, CA) coupled with protein L (Pierce, Rockford, IL) . The  homogenate was incubated with antibody coupled beads at 4°C with end-over-end rotation for approximately  16 hours. Beads were subsequently collected on a magnetic rack, washed three times with high-salt wash  buffer (10 mM HEPES [pH 7.4], 350 mM KCl, 5 mM MgCl2, 1% NP-40, 0.5mM DTT, 100 μg/ml cycloheximide)  and RNA was released and purified using Rneasy Micro Kit (Qiagen, Valencia, CA) wit

first: Nathaniel

last: Heintz


title: Transcriptomics Analysis of Lungs and Peripheral Blood of Crystalline Silica Exposed Rats

status: Public on Dec 31 2012

city: Morgantown

first: Rajendran

last: Sellamuthu

state: WV

address: 1095 Willowdale Rd

department: HELD

institute: National Institute for Occupational Safety and Health

summary: Non-invasive or minimally invasive surrogate approaches to detect/predict target organ toxicity have significant practical applications in occupational toxicology. Presently, using a rat model, we have investigated the potential application of peripheral blood transcriptomics as a practical approach to study the mechanisms of silica-induced pulmonary toxicity. Rats were exposed by inhalation to crystalline silica for one week (15 mg/m3, 6-hours/day, 5 days/week). Pulmonary toxicity and global gene expression profiles of lungs and peripheral blood were determined in the control and silica exposed rats at 32-weeks following termination of silica exposure. A significant elevation in bronchoalveolar lavage fluid (BALF) lactate dehydrogenase (LDH) activity and moderate histological changes in the lungs, including type II pneumocyte hyperplasia and fibrosis, indicated silica-induced pulmonary toxicity in the rats. Similarly, significant infiltration of neutrophils and elevated monocyte chemotactic protein-1 (MCP1) level in the lungs suggested silica-induced pulmonary inflammation in the rats. Microarray analysis of global gene expression profiles identified significant differential expression (>1.5 fold change and FDR p<0.01) of 520 and 537 genes, respectively, in the lungs and blood of the silica exposed rats. Bioinformatics analysis of the differentially expressed genes demonstrated significant similarity in the biological processes, molecular networks, and canonical pathways enriched by silica exposure in the lungs and blood of the rats. Several genes involved in functions relevant to silica-induced pulmonary toxicity such as inflammation, respiratory diseases, cancer, cellular movement, fibrosis, etc, were found significantly differentially expressed in the lungs and blood of the silica exposed rats. The results of this study, in addition to providing molecular insights into the mechanisms underlying silica-induced pulmonary toxicity, suggested the potential application of peripheral blood gene expression profiling as a toxicologically relevant and minimally invasive surrogate approach to study the mechanisms underlying silica-induced pulmonary toxicity.  Non-invasive or minimally invasive surrogate approaches to detect/predict target organ toxicity have significant practical applications in occupational toxicology. Presently, using a rat model, we have investigated the potential application of peripheral blood transcriptomics as a practical approach to study the mechanisms of silica-induced pulmonary toxicity. Rats were exposed by inhalation to crystalline silica for one week (15 mg/m3, 6-hours/day, 5 days/week). Pulmonary toxicity and global gene expression profiles of lungs and peripheral blood were determined in the control and silica exposed rats at 32-weeks following termination of silica exposure. A significant elevation in bronchoalveolar lavage fluid (BALF) lactate dehydrogenase (LDH) activity and moderate histological changes in the lungs, including type II pneumocyte hyperplasia and fibrosis, indicated silica-induced pulmonary toxicity in the rats. Similarly, significant infiltration of neutrophils and elevated monocyte chemotactic protein-1 (MCP1) level  in the lungs suggested silica-induced pulmonary inflammation in the rats. Microarray analysis of  global gene expression profiles identified significant differential expression (>1.5 fold change  and FDR p<0.01) of 520 and 537 genes, respectively, in the lungs and blood of the silica exposed rats. Bioinformatics analysis of the differentially expressed genes demonstrated significant similarity in the biological processes, molecular networks, and canonical pathways  enriched by silica exposure in the lungs and blood of the rats. Several genes involved in functions relevant to silica-induced pulmonary toxicity such as inflammation, respiratory diseases, cancer, cellular movement, fibrosis, etc, were found significantly differentially expressed in the lungs and blood of the silica exposed rats. The results of this study, in addition  to providing molecular insights into the mechanisms underlying silica-induced pulmonary toxicity, suggested the potential application of peripheral blood gene expression profiling as a  toxicologically relevant and minimally invasive surrogate approach to study the mechanisms underlying silica-induced pulmonary toxicity.

overall_design: A total of 12 rat blood samples were analyzed in this gene expression experiment. Rats were exposed to crystalline silica at a concentration of 15 mg/m3, 6-hours/day for 5 consecutive days. Rats exposed simultaneously to filtered air served as the controls. The control (n=6) and silica exposed (n=6) rats were sacrificed at post-exposure time interval of 32 weeks following termination of silica exposure and blood gene expression profiles were determined.

first: Christina

last: Umbright

first: Jenny

middle: R

last: Roberts

first: Amy

last: Cumpston

first: Walter

last: McKinney

first: Bean

last: Chen

first: David

last: Frazer

first: Shengqiao

last: Kashon

first: J

last: Pius


title: Defining Gene Signatures for Protective Immunity to Tuberculosis in the Chinese Cynomolgus Macaque Model

status: Public on Nov 15 2012

city: Salisbury

first: Alice

last: Hicks

address: Porton Down

department: TB research

institute: Health Protection Agency

summary: Gene expression profiles in PPD-stimulated and unstimulated peripherhal blood mononuclear cells isolated from chinese cynomolgus macaques before and after BCG vaccination and before and after M. tuberculosis challenge were compared in animals able to control TB infection and those that developed TB disease in order to identify potential correlates of protection and/or biomarkers of disease.

overall_design: 6 macaques received BCG vaccination prior to challenge and were able to control TB infection (vaccinated controllers). 3 unvaccinated macaques were also able to control TB infection after challenge (unvaccinated controllers) and 3 other unvaccinated macaques developed TB disease and reached humane endpoint criteria (unvaccinated progressors). PBMCs isolated at 8 and 18 weeks post BCG-vaccination from the vaccinated controllers and at 6 weeks post M.tb challenge and at 2 time-points when the animals were naive in all animals were stimulated with PPD. RNA was extracted from the cells and hybridised to and Agilent rhesus macaque GE microarray in a one-colour hybridisation. Gene expression post-vaccination and/or post-challenge was compared with expression before vaccination/challenge when the animals were naive.


title: Genome-wide analysis of primary plasma cell leukemia identifies recurrent imbalances associated with transcriptional Profile alterations

status: Public on Oct 12 2012

city: MILAN

first: Luca

last: Agnelli

address: Venezian 1

department: Department of Advanced Diagnostics

country: Italy

institute: IRCCS Istituto Nazionale dei Tumori

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