title: Gene expression changes in mouse embryonic fibroblasts (MEF cells) deficient for Ing1 and Gadd45a

status: Public on Feb 04 2013

city: Mainz

first: Emil

last: Karaulanov

address: Ackermannweg 4

institute: Institute of Molecular Biology

summary: ING1b and GADD45a are nuclear proteins involved in the regulation of cell growth, apoptosis and DNA repair. We found that ING1b and GADD45a physically and functionally interact in the epigenetic regulation of specific target genes. In order to study this interaction further, we analysed the transcriptional changes in MEF cells from single and double Ing1/Gadd45 knockout mice via microarray profiling.

overall_design: Mouse embryonic fibroblasts (MEF cells) were isolated from embryonic day E15.5 male embryos, either wild-type (WT) or knockout for Ing1 (Ing1-/-), Gadd45a (Gadd45a-/-) or Ing1/Gadd45a (double knockout, DKO), and cultured for 3 passages. Samples were then collected in duplicates per MEF line for expression array profiling.

first: Andrea

last: Schäfer

first: Ulrike

last: Stapf

first: Gabi

last: Döderlein

first: Christof

last: Niehrs


title: Gene expression changes in embryonic stem cells in response to Tcf15-E47

status: Public on Feb 08 2013

city: Edinburgh

first: Sally

last: Lowell

address: 5 Little France Drive

department: MRC Centre for Regenerative Medicine

country: United Kingdom

institute: University of Edinburgh

summary: We set out to identify genes that respond rapidly to overexpression of a Tcf15-E47 heterodimeric transcription factor in mouse embryonic stem cells.

overall_design: Dox inducible Tcf15-E47 mouse embryonic stem cells were exposed to dox for 0, 8, 12, 24h and samples collected in duplicate.  Control parental cells were exposed to dox for 0 or 8 h and samples collected in duplicate. Total 12 samples.

first: Chia-Yi

last: Lin


title: Expression profiling of FACS-purified fetal germ cells

status: Public on Jan 25 2013

city: Singapore

first: Vibhor

last: Kumar

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

country: Singapore

institute: Genome Institute of Singapore

summary: We used microarrays to profile global gene expression changes of Pou5f1-GFP-positive germ cells between E11.5 to E15.5.

overall_design: Germ cells were FACS-purified from gonadal single cell suspension based on Pou5f1-GFP expression. Three timepoints were included in this study: E11.5 (male/female), E13.5 (male) and E15.5 (male). For each timepoint, three biological replicates were analyzed. The Pou5f1-GFP-negative (non-germ cell) fraction of E13.5 (male) gonads was also included as a control.

first: Jia-Hui

last: Ng

first: Shyam

last: Prabhakar

first: Huck-Hui


title: Identification of downstream targets of Ezh2 histone methyltransferase

status: Public on Feb 06 2013

city: Parkville

first: Matthew

last: Ritchie

state: Victoria

address: 1G Royal Parade

department: Epigenetics and Development Division

country: Australia

institute: The Walter and Eliza Hall Institute of Medical Research

summary: Genome-wide transcriptional profiles of the mammary stem cell (MaSC)-enriched and luminal populations were determined following targeted disruption of Ezh2 gene. Gene ontology analysis revealed changes in the expression of genes associated with cell cycle, DNA replication and DNA repair.

overall_design: To carry out ex vivo cre-mediated excision of Ezh2, freshly isolated MaSC-enriched (CD29hiCD24+) and luminal (CD29loCD24+) cells from 8-week old Ezh2T/+ F/F mice were infected with virus expressing cre recombinase or control virus (for infection methods refer to Toula et. al., Cell stem cell 2008 11; 3(4):429-41). The infected cells were purified by sorting for GFP+ cells. Total RNA was isolated from GFP+ cells and up to 250 ng of total RNA per sample was labelled and hybridized to Illumina Mouse WG6V2 expression BeadChip platform. Three independent samples were used for each population.

middle: E


title: Gadd45a and Ing1 interaction in epigenetic gene regulation

country: Germany

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Corticosterone regulation of mRNAs and and corresponding RU486 antagonism in astrocytes in vitro

status: Public on Jan 09 2013

city: Ann Arbor

first: Bradley

middle: S

last: Carter

state: MI

address: 109 Zina PItcher Place

department: Molecular and Behavioral Neuroscience Institute

country: USA

institute: University of Michigan

summary: Analysis of mRNA gene expression changes mediated by natural glucocorticoid ligand corticosterone in mouse cortical primary astrocyte cultures; also, parallel analysis of RU486 (mifepristone) antagonism of corticosterone-mediated mRNA changes.

overall_design: Total RNA obtained from astrocyte cultures subjected to 2, 4, or 6 hours of (1) 100nM corticosterone alone, (2) 500nM RU486 alone, or (3) 100nM corticosterone + 500nM RU486 exposure compared to vehicle control astrocyte cultures.

first: Robert

middle: C

last: Thompson


title: Genome-wide analysis of Xbp1s effect on gene expression in liver by Doxycycline induced TRE-Xbp1s overexpression in mouse

status: Public on Jan 08 2013

city: Dallas

first: Yingfeng

last: Deng

state: TX

address: 5323 Harry Hines Blvd

institute: UT Southwestern Medical Center at Dallas

summary: Analysis of Xbp1s overexpression in liver after 24 hr indution or 48 hr induction in LIXs mouse. As a control, WT mice after 2 hr refeed and 24 hr fast without refeed are used for analysis of postprandial gene expression. This microarray study was used to screen for target genes activated by Xbp1s in liver. Results provide important information for the role of Xbp1s during postprandial.

overall_design: Total RNA obtained from liver tissues from mice on Dox200 chow diet for 24 hr or 48 hr. Mice were fasted for 6 hr before sacrifice for liver.

first: Philipp

last: Scherer


title: The mouse cytosine-5 RNA methyltransferase NSun2 is a component of the chromatoid body and required for testis differentiation

status: Public on Feb 07 2013

city: Cambridge

first: Suraj

last: Menon

address: Li Ka Shing Center, Robinson Way

institute: Cambridge Research Institute, Cancer Research UK

summary: Post-transcriptional regulatory mechanisms are crucial for protein synthesis during spermatogenesis and often organized by the chromatoid body. Chromatoid bodies are large cytoplasmic ribonucleoprotein granules, whose precise function and composition remain unclear. Here, we identify NSun2 as a novel component of the chromatoid body, and further show that this RNA methylase is essential for germ cell differentiation in the mouse testis. Lack of NSun2 leads to down-regulation of genes controlling RNA processing and post-transcriptional repression pathways, including Ddx4, Mili, Piwil1 (Miwi) and Tudor domain containing (Tdrd) proteins. Germ cell differentiation was blocked specifically at the pachytene stage by lack of NSun2, as spermatogonial and Sertoli cells were unaffected in knockout mice. We observed the same phenotype when we simultaneously deleted NSun2 with Dnmt2, the only other characterized cytosine-5 RNA methyltransferase to date, indicating that Dnmt2 was not functionally redundant for NSun2 in spermatogonial stem cells or Sertoli cells. Thus, our data indicate that RNA methylation pathways play an essential role in male germ cell differentiation.

overall_design: Four sample groups: Testes from Wild-type and NSun2 knock out mice at 15 and 49 days; 6 samples in each group

first: Shobbir

last: Hussain

first: Michaela

last: Frye


title: Towards a comprehensive structural variation map of an individual human genome for personalized medicine

status: Public on Feb 12 2013

city: Toronto

first: Andy

last: Pang

state: ON

address: MaRS Centre - East Tower, 101 College Street, Room 14-701

country: Canada

institute: The Centre for Applied Genomics

type: Genome variation profiling by genome tiling array


title: Genes activated by 5-aza-dC (DAC) in pancreatic cancer

status: Public on Jan 01 2013

city: New York

first: Benjamin

last: Tycko

state: NY

address: 1130 St. Nicholas Ave.

department: ICG

institute: Columbia University


title: Therapeutic interference with mTorc1 restricts inflammation-associated and Stat3-dependent gastro-intestinal tumourigenesis in mice

status: Public on Jan 29 2013

first: Zhengdeng

last: Lei

address: 8 College Road

institute: Duke-NUS graduate medical school

summary: Gp130 receptor engagement on neoplastic cells provides a link by which an inflammatory microenvironment facilitates tumour promotion. Although hyperactivation of the gp130-dependent Stat3 signalling node is commonly observed in solid tumours, Stat3 remains a challenging therapeutic target. To mimic excessive Stat3 signalling, we molecularly validate the gp130FF mouse as a preclinical model for inflammation-associated intestinal-type gastric cancer (IGC), with aberrant mammalian target of rapamycin (mTOR) pathway activity as shared feature. Accordingly, administration of the mTorc1 inhibitor RAD001 reversibly reduced IGC burden in gp130FF mice and suppressed colitis-associated cancer in wild-type mice. Since the therapeutic effect of RAD001 occurs independently of Stat3 hyperactivation, which is also dispensable for gp130-dependent engagement of the PI3K/Akt/mTorc1 pathway, we conclude that mTorc1 signalling limits tumour promoting Stat3 activity

overall_design: The mouse whole-genome gene expression profiling was performed on Illumina's MouseWG-6 v2.0 Expression BeadChips for 24 mice, with 8 mice in each group (gp130WT antral tissue, gp130FF unaffected antral tissue and gp130FF tumour tissue).

first: Stefan

last: Thiem

first: Steve

last: Rozen

first: Patrick

last: Tan

first: Matthias

last: Ernst


title: 8 NSCLC cell lines treated with either 50nM or 100nM of AUY for 24 hours.

status: Public on Jan 18 2013

city: Santa Monica

first: Habib

last: Hamidi

state: CA

address: 2825 Santa Monica Blvd., Suite 200

institute: UCLA

summary: RNA from treated cell lines are compared to RNA from matched untreated samples on the same chip to assess differential gene expression.

first: Edward

last: Garon

first: Judy

last: Dering

first: Sharon

last: Pitts

first: Naeimeh

last: Kamranpour

first: Richard

last: Finn

first: Amrita

last: Desai

first: Wylie

last: Hosmer

first: Susan

last: Ide

first: Emin

last: Avsar

first: Michael

last: Jensen

first: Cornelia

last: Quadt

first: Manway

last: Liu

first: Steven

last: Dubinett

first: Dennis

last: Slamon


title: Gene expression profiles of monocytes derived from chronic lymphocytic leukemia patients and normal controls

status: Public on Jan 10 2013

city: Modena

first: Rossana

last: Maffei

state: Modena

address: Via del Pozzo 71

department: Dept of Hematology and Oncology

country: Italy

institute: University of Modena and Reggio Emilia

summary: We collected monocytes from peripheral blood of 5 chronic lymphocytic leukemia (CLL) patients and 5 healthy donors and we performed gene expression analysis by microarray. Comparison of gene expression profiles (GEPs) between CLL-derived and normal monocytes was used to discover molecular abnormalities in this nonmalignant immune cellular population in leukemia-bearing patients. Although analysed cells were not part of the malignant clone, in unsupervised hierarchical clustering analysis, GEPs of normal monocytes were clearly distinguishable from those of monocytes obtained from CLL patients. Supervised analysis identified 65 genes significantly up-regulated and 48 genes down-regulated in CLL monocytes compared with monocytes from normal controls (FC=2, p<0.05). Modification of gene expression profile would imply impairment of phagocytosis and production of immunosuppressive mediators in CLL-derived monocytes. The alterations described in our study further contribute to characterize the complexity of factors potentially involved in acquired immune deficiency of CLL patients.

overall_design: Large-scale gene expression profiling (GEP) was performed on total RNA extracted from purified CD14+ monocytes (RNeasy Mini kit Plus, QIAGEN, Valencia, CA, USA) isolated from 5 individual CLL patients and 5 healthy controls by hybridization on 4X44K Whole Human Genome Microarray (Agilent Technologies, Palo alto, CA). Fluorescence data were analysed with Feature Extraction Software v.10.5 (Agilent Technologies) an QC Chart tool v.1.3. Agglomerative two-dimensional clustering analysis and supervised analyses based on t-test were performed using Gene Spring GX (Agilent) software. Genes were defined as differentially expressed between groups at a significant level of p<0.05 and with a fold change cut off ± 2 in all the pair wise comparisons.

first: Roberto

last: Marasca


title: Gene expression profiling of γ34.5 deleted HSV1 infected Mouse Embryonic Fibroblasts

status: Public on Jan 15 2013

city: Miami

first: Tianli

last: Xia

state: FL

address: 1550 NW 10th Ave

institute: University of Miami

summary: Gene expression analysis of wild type and STING knock-out Mouse Embryonic Fibroblasts (MEFs) infected with γ34.5 deleted HSV1. Genes whose expression that are affected by HSV1 in a STING dependent manner will be identified and signaling pathways regulated by STING will be elucidated.

overall_design: Primary MEFs were mock treated or infected with γ34.5 deleted HSV1 at M.O.I. 1. Total RNA was extracted 3 hours post infection for array analysis.

first: Glen

last: Barber


title: Gene expression profiling of macrophages: Implications for an immunosuppressive effect of dissolucytotic gold ions

status: Public on Dec 31 2012

city: Braunschweig

first: Robert

last: Geffers

address: Inhoffenstr. 7

department: Dep. Molecular Bacteriology

institute: HCI - Helmholtz Centre for Infection Research

summary: Gold salts has been used in the treatment of rheumatoid arthritis but has been replaced by biologicals such as TNF-alpha inhibitors. The mechanisms behind the anti-inflammatory effect of metallic gold ions are still unknown, however, recent data showed that charged gold atoms are released from pure metallic gold implants by macrophages via a dissolucytosis membrane, and that gold ions are taken up by local macrophages, mast cells and to some extent fibroblasts. These current findings offer new treatment options for metallic gold and deeper understanding of the effect of metallic gold on key inflammatory cells as macrophages are essential. In the present study the impact of phagocytised gold ions on the global gene expression profile of the human monocytic cell line THP-1 was investigated, using microarray analysis comprising approximately 20,000 genes. The gene expression data was confirmed by measurement of three secreted proteins. A unique gene expression signature of dissolucytotic macrophages that had taken up gold ions was demonstrated. A large number of regulated genes were functionally related to immunomodulation/protection. Gold ion uptake into macrophages induced downregulation of central inflammatory cytokines as TNF-alpha, IL-32 and CD28. The data obtained in this study offer new insights into the mode of action of gold ions and suggest a future role of metallic gold as implants or topical applications in treating inflammation.

overall_design: To determine the effect of gold phagocytosis on global gene expression

first: O

last: Seifert

first: A

last: Matussek

first: C

last: Andersson

first: R


title: Effect of infection with Aggregatibacter actinomycetemcomitans or Porphyromonas gingivalis on primary murine bone marrow-derived dendritic cells

status: Public on Jan 11 2013

city: Bonn

first: Moritz

last: Kebschull

address: Welschnonnenstr. 17

department: Department of Periodontology

institute: University of Bonn

summary: To determine the biological mechanisms underlying a dampened immune response to Porphyromonas gingivalis, as compared to Aggregatibacter actinomycetemcomitans challenge, we infected primary BMDCs with either pathogen or left uninfected

overall_design: Total RNA from uninfected BMDCs compared to BMDCs infected with either  Aggregatibacter actinomycetemcomitans or Porphyromonas gingivalis

last: Nowak

first: Justus


title: Gene expression signatures following nerve injury in a rat model (0d-14d)

city: Shanghai

first: Guohui

last: Ding

address: 320 Yueyang Road

country: China

institute: Shanghai Institutes for Biological Sciences

summary: We used microarrays to distinguish the gene expression differences among different time points after injury.

overall_design: We generated L4-6 dorsal root ganglia (DRG) tissues and proximal sciatic nerve (SN) tissues (0.5cm) at 0d, 1d, 4d, 7d and 14d after sciatic nerve resection.

first: Bin

last: Yu

first: Xiaosong

last: Gu


title: ICL0012 - Host response in human Calu-3 cells to infection with NS1trunc124: A/Vietnam/1203-CIP048_RG4/2004 (H5N1) or WT: A/Vietnam/1203/2004 (H5N1)

status: Public on Jan 05 2013

city: Seattle

first: Michael

last: Katze

state: WA

address: Rosen Building 960 Republican St.

department: Microbiology

institute: University of Washington

summary: Differential expression was determined in Calu-3 cells between mock infected and infection with NS1trunc124: A/Vietnam/1203-CIP048_RG4/2004 (H5N1) or WT: A/Vietnam/1203/2004 (H5N1) at different times post infection.

overall_design:
Purpose: To obtain samples for transcriptional analysis in triplicate using the VN1203 pathogenicity mutant NS1-trunc124.

Overview of Experiment:
. Time Points: 0, 3, 7, 12, 18 and 24 hrs post infection.
. Done in triplicate.
. Triplicates are defined as 3 different wells, plated at the same time using the same cell stock for all replicates.
. Time-matched mocks were done in triplicate from the same cell stock as the rest of the samples.
. Culture medium (the same as what the virus stock is in) was used for the mock infections.

Calu-3 cells were infected with NS1trunc124: A/Vietnam/1203-CIP048_RG4/2004 (H5N1) or mock infected and samples were collected at 0, 3, 7, 12, 18 and 24 hpi.
Calu-3 cells were infected with WT: A/Vietnam/1203/2004 (H5N1) and samples were collected at 7 and 24 hpi.
There are 3 mock (2 included here) and 3 infected replicates for each time point. Expression profiles were determined.

middle: G

first: Amie

middle: J

last: Eisfeld

first: Yoshihiro

last: Kawaoka

first: Shufang

last: Fan

first: G

middle: L

last: Law

first: Jean

last: Chang


title: Expression data from the human cerebellum brain

city: Changsha

first: Chao

last: Chen

address: 110 Xiangya Road

institute: Central South University

summary:
Schizophrenia (SCZ) and bipolar disorder (BD) are highly heritable psychiatric disorders. Associated genetic and gene expression
changes have been identified, but many have not been replicated and have unknown functions. We identified groups of genes
whose expressions varied together, that is co-expression modules, then tested them for association with SCZ. Using weighted gene
co-expression network analysis, we show that two modules were differentially expressed in patients versus controls. One,
upregulated in cerebral cortex, was enriched with neuron differentiation and neuron development genes, as well as disease
genome-wide association study genetic signals; the second, altered in cerebral cortex and cerebellum, was enriched with genes
involved in neuron protection functions. The findings were preserved in five expression data sets, including sets from three brain
regions, from a different microarray platform, and from BD patients. From those observations, we propose neuron differentiation
and development pathways may be involved in etiologies of both SCZ and BD, and neuron protection function participates in
pathological process of the diseases.

overall_design: We use microarray technology to understand the etiology and pathology of psychiatric diseases at the transcriptomic level. Postmortem human brain samples came from the Stanley Medical Research Institute’s Neuropathology Consortium and Array collections, including schizophrenia, bipolar disorder and control samples.

first: Chunyu

last: Liu


title: Global gene expression analysis of peripheral blood cells, pluripotent cell lines, and iPS-derived T cells

status: Public on Dec 25 2012

city: Tokyo

first: Toshinobu

last: Nishimura

address: 4-6-1 Shirokanedai, Minato-ku

department: Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine

country: Japan

institute: The Institute of Medical Science, The University of Tokyo

summary: Exhaustion of antigen-specific T cells represents a major challenge to adoptive immunotherapy against many types of cancer and viral infection. In an effort to overcome this problem, we reprogrammed clonally expanded antigen-specific CD8+ T cells from an HIV-1-infected patient to pluripotency. The T cell-derived induced pluripotent stem cells (T-iPSCs) were then redifferentiated into CD8+ T cells that had high proliferative capacity and elongated telomeres. To confirm that T-iPSCs were compatible to other embryonic stem cells (ESCs) but different from T cells, and that redifferentiated T cells were compatible to T cells but different from natural killer (NK) cells, we analyzed and compared the gene expression profiles of the samples by cDNA microarray. These analyses revealed that our T-iPSCs and redifferentiated T cells were essentially the same as their counterpart pluripotent stem cells and T cells, respectively.

overall_design: We generated human T-iPSC lines from peripheral blood T cells of healthy donors. To confirm that established T-iPSCs were typical iPSCs, the gene expression profile of one T-iPSC clone derived from a healthy person (TkT3V1-7) was compared to those of peripheral blood T cells (CD4+ T cell and CD8+ T cell) and ESCs (KhES3). We also established T-iPSCs (H254SeVT-3) from a T-cell clone (H25-#4) which was derived from an HIV-1-infected patient. H254SeVT-3 was redifferentitated into functional T cells (reT-1 and reT-2.1) in vitro. The gene expression profiles of reT-1 and reT-2.1 were compared to those of H25-#4 and peripheral blood NK cells to clarify that the redifferentiated T cells carried the same characteristics as the original T-cell clone.

first: Shin

last: Kaneko

first: Hiromitsu

last: Nakauchi


title: Human lymphoblastoid cell lines: controls vs. patients with autism

status: Public on Dec 21 2012

city: Greenwood

first: Chin-Fu

state: South Carolina

address: 113 Gregor Mendel Circle

department: Center for Molecuar Studies

institute: Greenwood Genetic Center

summary: Autism spectrum disorders (ASDs) are relatively common neurodevelopmental conditions whose biological basis has been incompletely determined. We analyzed the metabolic profile of lymphoblastoid cell lines from patients with ASDs and normal individuals, using the Biolog Phenotype plates. To validate our metabolic findings, we utilized the Agilent Whole Human Genome Oligo Microarray to evaluate the level of gene expression in the tested cell lines. As a comparison for gene expression profiles in cells of patients with ASDs, we also performed microarray analysis for lymphoblastoid cell lines from patients with intellectual disability (ID). Two independent experiments were performed for each sample. To maximize the contrast between samples, we implemented a loop experimental design.

overall_design: Loop design for maximal contrast.

first: Cheng

middle: Y

last: Kuan

first: Chin

middle: F

first: Luigi

last: Boccuto

first: Chales

last: Schwartz


title: Novel dedifferentiated liposarcoma xenograft models reveal PTEN down regulation as a malignant signature and response to PI3K pathway inhibition

status: Public on Dec 19 2012

city: Los Angeles

first: Linh

middle: My

last: Tran

state: California

department: Medicine - Pulmonary & Critical Care

institute: University of Los Angeles

summary: Liposarcoma is a type of soft tissue sarcoma, exhibiting poor survival and a high recurrence rate.  Treatment is generally limited to surgery and radiation, emphasizing the need to understand this disease.  Because very few in vivo and in vitro models can reproducibly recapitulate the human disease, we generated several xenograft models from surgically resected human dedifferentiated liposarcoma.  Our study demonstrates that all xenografts recapitulate morphologic and gene expression characteristics of the patient tumors after continuous in vivo passages.  Importantly, xenograftability is directly correlated with disease specific survival of liposarcoma patients.  When treated with the PI3K/AKT/mTOR pathway inhibitor rapamycin alone or in combination with the multi-kinase inhibitor sorafenib, all xenografts responded with increased lipid content and a more differentiated gene expression profile.

overall_design:
One-color arrays:
Vehicle vs combination (rapamycin & sorafenib) treatment for 2 separate dedifferentiated liposarcoma xenografts.

Two-color arrays:
Comparison of patient tumor, several passages of xenograft generated from that tumor, and cells cultured from xenografted tumors.

first: Kathleen

middle: B

last: Smith

middle: M

first: Brenna

last: Tam

first: Elizabeth

last: Shurell

first: Yunfeng

last: Li

first: Daniel

last: Braas

first: William

middle: D

last: Tap

first: Heather

middle: R

last: Christofk

first: Sarah

last: Dry

first: Fritz

last: Eilber

first: Hong

last: Wu


title: Molecular Phenotyping of Immune Cells from Young NOD Mice

status: Public on Dec 30 2012

city: Memphis

first: Dorothy

middle: N.

last: Kakoola

state: TN

address: 1030 Jefferson Avenue

department: Medicine

institute: University of Tennessee Health Science Center

summary:
Islet leukocytic infiltration (insulitis) is first obvious at around 4 weeks of age in the NOD mouse – a model for human type 1 diabetes (T1DM). The molecular events leading to insulitis are poorly understood. Since TIDM is caused by numerous genes, we hypothesized that multiple molecular pathways are altered and interact to initiate this disease.
Analysis of the global gene expression profiles using microarrays followed by hierarchical clustering revealed that the majority (~90%) of the differentially expressed genes in NOD mice relative to control mice were repressed. Further analysis of these genes using a modern suite of multiple bioinformatics approaches identified abnormal molecular pathways that can be divided broadly into 2 categories: metabolic pathways, which were predominant at 2 weeks, and immune response pathways, which were predominant at 4 weeks. Network analysis by Ingenuity pathway analysis (IPA) identified several putative key genes/molecules that may play a role in regulating these pathways, including five that were common to both ages (TNF, HNF4A, IL15, Progesterone, and YWHAZ), and others that were unique to 2 weeks (e.g. MYC/MYCN, TGFB1, and IL2) and to 4 weeks (e.g. IFNG, beta-estradiol, p53, NFKB, AKT, PRKCA, IL12, and HLA-C).

overall_design: Spleen leukocytes were collected from NOD mice and two control strains, NON, and C57BL/6, at 2 and 4 weeks of age (n=5 for each strain and each age group). NON is the original diabetes-resistant control strain closely related genetically to the NOD strain, whereas C57BL/6 is a more distantly related strain with some immunogenetic similarities to NOD. RNA expression was analyzed on Affymetrix expression arrays MOE 430 A and MOE430B. The two arrays were combined prior to statistical analysis of the data giving a total of ~45,000 probe sets per sample.

first: Jian

middle: N

first: Nataliya

middle: I

last: Lenchik

first: Dominic

last: Desiderio

first: Dana

last: Marshall

first: Ivan

last: Gerling


title: RIP:  miR-202 mimic-transfected HeLa cells vs. negative control

city: New Haven

first: Alan

last: Fu

state: CT

address: 60 College St.

institute: Yale University

summary: Potential miR-202 targets were identified using a targetome-wide RIP-based microarray.  HeLa cells were transfected with either a miR-202 mimic or a scrambled single-stranded RNA negative control.  RNA was isolated from total cell lysate prior to IP and from antibody-immobilized Protein G agarose beads-RNP complexes (post-IP).

overall_design: Ribonucleoprotein IP was performed using the RIP-Assay kit for microRNA (MBL) according to the protocol described by the manufacturer. Briefly, anti-EIF2C2/Ago2 monoclonal antibody (Novus Biologicals, LLC) was incubated with Protein G plus agarose beads (Pierce) at 4°C overnight to prepare antibody-immobilized beads. 20 million cells were harvested and washed four times with ice-cold DEPC-treated PBS. The cell pellet was lysed with 500 μl of lysis buffer and the supernatant was incubated with Protein G agarose beads without antibody to reduce nonspecific adsorption. The cell lysate was then transferred into a tube containing antibody-immobilized Protein G agarose beads and incubated for 3 hours at 4°C.  Genome-wide expression levels were analyzed in total RNA samples from total cell lysate and antibody-immobilized Protein G agarose beads-RNP complexes from cells transfected with miR-202 mimic or negative control using the Agilent 44K 60-mer human whole-genome microarray. Signal hybridization and scans were performed by MOGene, LC (St Louis, MO) (run in biological duplicate). The normalized signal intensities of probes from RNP-bead complexes (post-IP fraction) were divided by the signal intensities from total cell lysate (pre-IP fraction) in miR-202 mimic-transfected cells. Transcripts were identified as members of the global miRNA targetome if they exhibited an enrichment fold change > 2.0. From this list, post-IP to pre-IP signal intensity ratios in miR-202 mimic-transfected cells were divided by post-IP to pre-IP signal intensity ratios in NC cells.  Transcripts exhibiting normalized signal ratios of > 1.5 were considered to be bound with miR-202 in the RNA-induced silencing complex (RISC), and therefore potential direct miR-202 targets.

first: Yong

last: Zhu

first: Aaron

last: Hoffman

first: Ran

last: Liu

first: Tongzhang

last: Zheng

first: Frank

last: Slack


title: LV-Induced Dendritic Cells for AML Immunotherapy

first: Robert

last: Geffers

summary: Wilms’ tumor 1 antigen (WT1) is overexpressed in acute myeloid leukemia (AML). Unfortunately, clinical immunotherapeutic use of WT1 peptides against AML has been inconclusive. A tricistronic lentiviral vector co-expressing a truncated form of WT1 (lacking the DNA-binding domain), GM-CSF and IL-4 was used to transduce human monocytes. The overnight transduction procedure induced the self-differentiation of monocytes into highly viable and immunophenotypically stable “SmartDC/tWT1” (GM-CSF+, IL-4+, WT1+, IL-6+, IL-8+, TNF-?+, MCP-1+, HLA-DR+, CD86+, CCR2+, CCR5+). RNA expression pattern analyses revealed up-regulation of several  toll-like receptors, interferons and interferon-stimulated genes in lentivirally-reprogrammed SmartDC/tWT1 in comparison with conventional DCs. Remission samples from a FLT3-ITD+ AML patient and surplus material from the donor lymphocyte infusion (DLI) obtained from the matched related donor were used to produce SmartDC/tWT1. In our donor-patient model, donor T cells expanded with SmartDC/tWT1 showed high reactivity against WT1 and against primary AML. Intracellular processing of WT1 peptides by SmartDC/tWT1 resulted in superior WT1-specific CD8+ cytotoxicity against the primary leukemia blasts than exogenous peptide pulsing. Therefore, SmartDC/tWT1 produced in a single day of ex vivo culture with broad homeostatic, innate immunity, antigenic presentation and cytotoxic T cell stimulation properties can be clinically developed in combination with DLI for boosting anti-leukemia responses.

overall_design: Seeking to better characterize possible effects of truncated WT1 over-expression in the transcriptional activity of dendritic cells, we compared the gene expression pattern of SmartDC/tWT1 with those of SmartDC and Conventional DC.

first: R

first: B

last: Sundarasetty

last: Stripecke


title: An integrative genomic and transcriptomic analysis reveals potential targets associated with cell proliferation in uterine leiomyomas

status: Public on Dec 16 2012

city: Botucatu

first: Priscila

middle: Daniele Ramos

last: Cirilo

state: SP

address: Distrito de Rubião Junior S/N

department: Genética

country: Brazil

institute: Instituto de Biociências de Botucatu

summary: Transcriptional profiling of uterine leiomyoma tumor samples for integrative analysis with copy number alterations data. Goal was to identify up- and/or down-expressed genes associated with genomic gains and/or losses, respectively. After, it was applied to GSEA and CONEXIC algorithm to integrate the data. Protein-protein interactions also was building and the data were validated using qRT-PCR and IHC techniques.

overall_design: Experiment condition, 51 uterine leiomyoma samples from patients at the secretory or proliferative menstrual cycle phases were labeled with Cy3 and adjacent normal myometrium reference samples were labeled with Cy5. Each uterine leiomyoma and adjacent normal myometrium sample were obtained from the same patient and were mixed and hybridized on a glass slide Agilent 4x44 platform.

first: Fabio

middle: A

last: Marchi


title: Overexpression of CD157 contributes to epithelial ovarian cancer progression by promoting mesenchymal differentiation.

city: Biella

first: giovanna

last: chiorino

address: via malta 3

department: Cancer Genomics

institute: Fondo Edo Tempia

summary: Epithelial ovarian carcinoma (EOC) is an aggressive tumor often diagnosed at an advanced stage, when there is little prospect for cure. Despite some advances in surgical and chemotherapeutic strategies, only marginal improvements in patient outcome have been obtained. Hence, understanding the biological mechanisms underpinning EOC progression is critical for its treatment and to ameliorate patients survival. Recently, we reported that CD157 is expressed in EOC and controls tumor cell migration and invasion. Using stable overexpression and knockdown in OVCAR-3 and OV-90 ovarian cancer cell lines, we demonstrated that CD157 overexpression promotes morphological and functional changes, characterized by downregulation of epithelial marker and upregulation of mesenchymal ones. These are mediated at the transcriptional level by altering the expression of Snail and Zeb1 transcriptional repressors. The effects of CD157 overexpression on ovarian cancer phenotype translate into increased tumor cell motility and mesotelial invasion, while its knockdown significantly reduces the migratory potential, implying a direct correlation between CD157 expression levels and EOC aggressiveness.  The analysis of the transcriptomic profiling highlighted 378 significantly differentially expressed genes, representing the signature of CD157-overexpressing EOC cells. The overall picture deduced from the analysis of these modulated transcripts indicated that high CD157 expression results in strengthening of a number of biological functions that favour tumor progression (including cell differentiation, cell motility and migration), and weakening of selected biological processes that hinder the tumor progression (such as apoptosis, cell death and response to stress). Collectively, these data support a causal role of CD157 in the control of ovarian cancer progression motivating the existence of a direct correlation between the expression levels of CD157 and the adverse clinical outcome in EOC patients, and suggest that CD157 may represent a valuable therapeutic target.

overall_design: Gene expression analysisi of control cell lines (OVCAR-3/mock and OV-90/mock) and testing cell lines (OVCAR-3/CD157 and OV-90/CD157), with two replicates, with dye swap, performed for each sample.

first: S

last: Morone

first: N

last: Lo Buono

last: Parrotta

first: A

last: Giacomino

first: G

last: Nacci

last: Brusco

last: Larionov

first: P

last: Ostano

first: M

last: Mello Grand

last: Chiorino

first: E

last: Ortolan

last: Funaro


title: Genes activated by 5-aza-dC (DAC) in pancreatic cancer (KPC-Brca1) epithelial cells

first: Benjamin

last: Tycko

institute: Columbia University

summary: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that resists current treatments.  To test epigenetic therapy against this cancer we used the DNA demethylating drug 5-aza-2’-deoxycytidine (DAC) in a KrasLSL-G12D; p53LSL-R270H/+; Pdx1-cre; Brca1flex2/flex2 (KPC-Brca1) mouse model of aggressive stroma-rich PDAC.  In untreated tumors, we found globally decreased 5-methyl-cytosine (5mC) in malignant epithelial cells and in cancer-associated myofibroblasts (CAFs), and increased amounts of 5-hydroxymethyl-cytosine (5HmC) in CAFs, in progression from pancreatic intraepithelial neoplasia (PanIN) to PDAC.  DAC further reduced DNA methylation and slowed PDAC progression, markedly extending survival in an early treatment protocol and significantly though transiently inhibiting tumor growth when initiated later, without adverse side effects.  Escaping tumors contained areas of sarcomatoid transformation with disappearance of CAFs.  Mixing-allografting experiments and proliferation indices showed that DAC efficacy was due to inhibition of both the malignant epithelial cells and the stromal CAFs.  Expression profiling and immunohistochemistry highlighted DAC-induction of STAT1 in the tumors, and DAC plus gamma-interferon produced an additive anti-proliferative effect on PDAC cells.  DAC induced strong expression of the testis antigen DAZL in CAFs.  These data show that DAC is effective against PDAC in vivo and provide a rationale for future studies combining hypomethylating agents with cytokines and immunotherapy.

overall_design: Treatment of a short-term explant culture of malignant epithelial cells from a KPC-Brca1 mouse pancreatic carcinoma, with 0.5 micromolar 5-aza-dC (decitabine; DAC) for 48 hours. The experiment includes 3 replicate plates untreated and 3 replicates treated.

first: Reena

last: Shakya

first: Tamas

last: Gonda


title: ICL0011 - Host response in human Calu-3 cells to infection with A/Vietnam/1203-CIP048_RG3/2004 (H5N1) (PB1-F2 deletion), A/Vietnam/1203-CIP048_RG3/2004 (H5N1) (PB2-627E mutant) or WT: A/Vietnam/1203/2004 (H5N1)

first: Michael

last: Katze

summary: Differential expression was determined in Calu-3 cells between mock infected and infection with one of 3 Influenza viruses (wild-type VN1203, VN1203 mutant PB1-F2del, VN1203 mutant PB2-627E) at different times post infection.

overall_design:
Purpose: To obtain samples for transcriptional analysis in triplicate using the VN1203 pathogenicity mutants PB1-F2del and PB2-627E.

Overview of Experiment:
. Time Points: 0, 3, 7, 12, 18 and 24 hrs post infection.
. There are two time points for wild type VN1203.
. Done in triplicate.
. Triplicates are defined as 3 different wells, plated at the same time using the same cell stock for all replicates.
. Time-matched mocks were done in triplicate from the same cell stock as the rest of the samples.
. Culture medium (the same as what the virus stock is in) was used for the mock infections.

Calu-3 cells were infected with A/Vietnam/1203-CIP048_RG3/2004 (H5N1) (PB1-F2 deletion), A/Vietnam/1203-CIP048_RG3/2004 (H5N1) (PB2-627E mutant) or mock infected and samples were collected at 0, 3, 7, 12, 18 and 24 hpi.
Calu-3 cells were infected with WT: A/Vietnam/1203/2004 (H5N1) and samples were collected at 7 and 24 hpi.
There are 3 mock and 3 infected replicates for each time point. Expression profiles were determined.

first: Amie

last: Eisfeld

first: Yoshihiro

last: Kawaoka

first: Shufang

last: Fan

last: Law

first: Jean

last: Chang


title: Genes activated by 5-aza-dC (DAC) in pancreatic (KPC-Brca1) cancer-associated fibroblasts (CAFs)

overall_design: Treatment of a short-term explant culture of cancer-associated fibroblasts (CAFs) from a KPC-Brca1 mouse pancreatic carcinoma, with 2 micromolar 5-aza-dC (decitabine; DAC) for 48 hours. The experiment includes 3 replicate plates untreated and 3 replicates treated.


title: Altered CD45 isoform combination in C77G carriers influences the function of CD4+CD25high FoxP3+ regulatory T cells

first: Robert

last: Geffers

summary: Disorders in regulatory T cell (Treg) function can result in the break-down of immunological self-tolerance. Thus, the identification of mechanisms controlling the activity of Treg is of great relevance. We used Treg from individuals carrying the C77G polymorphism as human models to study the role of CD45 molecules. The C77G mutation occurs with low frequency in healthy individuals and prevents splicing of CD45 exon A thereby leading to an aberrant expression pattern of CD45 isoforms in affected individuals. Resting and in vitro expanded/activated CD4+CD25highFoxp3+ Treg from carriers of C77G strongly expressed CD45RA isoforms whereas they were almost absent in cells from individuals with wild-type CD45. The expression patterns of CD45RB and CD45RC did not differ between C77G and control Treg but CD45R0 molecules were reduced in C77G cells. Resting C77G Treg showed diminished upregulation of activation markers and a reduced proliferative potential when stimulated with anti-CD3/TcR or anti-CD3/CD28 mAb suggesting decreased responsiveness to activating stimuli. This is in line with the observation that CD3/CD28-mediated activation of in vitro expanded Treg resulted in lower levels of TGF-? and IL-10 transcripts in C77G cells compared to controls. Furthermore, microarray studies revealed distinct gene expression patterns in Treg from C77G carriers and individuals with wild-type CD45. In addition, the capacity to suppress proliferation of conventional CD4+ T cells was reduced in C77G Treg. These data suggest that the changes in CD45 isoform combination resulting from C77G alter the responsiveness of Treg to CD3/TcR- and/or CD28-mediated signalling. Targeting of CD45 isoforms might be an approach to modulate Treg function.

overall_design:
Total RNA was isolated from expanded and stimulated (2h with 1:5 CD3/CD28 beads) Treg of wild-type and C77G carriers using RNeasy Plus Micro Kit (Qiagen). Two pools of three wild-type individuals and three C77G individuals were prepared using equal amounts of RNA from each single donors. The two samples were each hybridised twice on an Agilent human Gene Expression Microarray 4x44K.
cell type comparison

first: R

first: C

last: Pokoyski

last: Schwinzer


title: Chronic Caloric Restriction Preserves Mitochondrial Function in Senescence Without Increasing Mitochondrial Biogenesis

city: Rochester

first: Zhifu

last: Sun

state: MN

address: 200 1st ST SW

department: HSR-BSI

institute: Mayo Clinic

summary: Caloric restriction (CR) without malnutrition appears to mitigate many detrimental effects of aging, in particular the age-related decline in skeletal muscle mitochondrial function.  Although the mechanisms responsible for this protective effect remain unclear, CR is commonly believed to increase mitochondrial biogenesis; a concept that is now demanding closer scrutiny.  Here we show that lifelong CR in mice prevents age-related loss of mitochondrial function, measured in isolated mitochondria and permeabilized muscle fibers.  We find that these beneficial effects of CR occur without increasing mitochondrial abundance.  Furthermore, whole-genome expression profiling and large-scale proteomic surveys revealed expression patterns inconsistent with increased mitochondrial biogenesis.  These observations, combined with lower protein synthesis rates support an alternative hypothesis that CR preserves mitochondrial function not by increasing mitochondrial biogenesis, but rather by decreasing mitochondrial oxidant emission, increasing antioxidant scavenging, thereby minimizing oxidative damage to cellular components.

overall_design: Cross-sectional comparison of skeletal muscle from young (8mo), old (24mo) and old caloric restricted mice, obtained from the colony maintained on behalf of the National Institute on Aging.

first: Ian

last: Lanza

first: Piotrek

last: Zabielski

first: Katherine

last: Klaus

first: Dawn

last: Morse

first: Carrie

middle: H

last: Heppelmann

first: H

last: Bergen

first: Stephane

last: Walrand

first: Kevin

last: Short

first: Matthew

last: Johnson

first: Jill

last: Schimke

first: Daniel

last: Jakaitis

first: Yan

middle: W

last: Asmann

first: K

middle: S

last: Nair


title: Effects of far-infrared rays on prostate cancer cells and normal prostate epithelial cells

city: Osaka

first: Hiroki

last: Shima

address: 3-7-41 Nakatsu YAMAMOTO  Bild. 4F. Kitaku

institute: SHIMA Institution for Quantum Medicine

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

overall_design: Refer to individual Series

first: Jun

last: Qiu

first: Yoshie

last: Yoshikawa


title: Vitamin D3 affects TNFa mediated activation of dendritic cells

city: Aachen

first: Mykola

last: Goncharenko

address: Pauwelstrasse 30

department: Institute for Biomedical Technology - Cell Biology

institute: IBMT - Cell Biology, University Hospital, RWTH Aachen

summary:
The ability of dendritic cells (DC) to initiate immunity and induce antigen-specific tolerance makes DC ideal targets for pharmacological intervention into immune responses. NF-kB factors are a family of transcriptional regulators important for DC development and function. However, the identity of NF-kB target genes that are central to DC biology is largely unknown. In the present study inhibition of the NF-kB activation by the IkBa super repressor (IkBa-SR) and DNA microarray analysis were used to determine the repertoire of NF-kB responsive genes in DC.

A number of immunomodulatory compounds have been suggested to target the NF-kB signalling pathway and/or NF-kB-mediated transcription of pro-inflammatory target genes. 1a,25-dihydroxyvitamin D3 (VD3) exerts its effects through the vitamin D3 receptor (VDR), a member of the nuclear hormone receptor superfamily. Microarray analysis was also selected as an approach of choice for the analysis of the effects of VD3 on the activation of DC, and to survey the involvement of VDR in repression of NF-kB regulated genes. These genes can be potentially useful targets for the development of more specific anti-inflammatory agents for clinical applications.
Keywords: drug treatment, TNFa treatment, VD3 treatment

overall_design: DC were generated in vitro from bone marrow cells of VDRmut and VDRwt control mice as described previously (Hieronymus, T., T. C. Gust, R. D. Kirsch, T. Jorgas, G. Blendinger, M. Goncharenko, K. Supplitt, S. Rose-John, A. M. Muller, and M. Zenke. 2005. Progressive and controlled development of mouse dendritic cells from Flt3+CD11b+ progenitors in vitro. J Immunol 174:2552-2562). Immature DC were pre-treated with 1a,25-dihydroxyvitamin D3 (VD3), or left untreated. TNFa was added where appropriate, and cells incubated further for 4 h. Total RNA was amplified, labelled and hybridised to Affymetrix MOE430A arrays. TNFa up- and down-regulated genes, as well as genes affected by VD3 were analysed.

first: Eva

last: Kaergel

first: Verena

last: Schroeder

first: Tatjana

last: Gust

first: Claus

last: Scheidereit

first: Martin

last: Zenke

first: Thomas

last: Hieronymus


title: Inducing and Predicting Immune Tolerance After Kidney Transplantation

status: Public on Dec 15 2012

city: Stanford

first: Li

last: Li

address: 300 pastuer dr.,

department: Pediatric

institute: Stanford University

summary:
Background
Immune tolerance and persistent mixed chimerism can be achieved reproducibly after combined organ and hematopoietic cell transplantation in mice conditioned with total lymphoid irradiation plus anti-thymocyte globulin. We studied the safety and reproducibility of this approach in a cohort of kidney transplant patients, and tried to identify immune monitoring procedures that can predict tolerance and guide complete immunosuppressive drug withdrawal.
Methods
Ten patients conditioned with 10 doses of total lymphoid irradiation and 5 doses of anti-thymocyte globulin were given kidney transplants and an injection of CD34+ hematopoietic progenitor cells and T cells from HLA matched donors. Blood cell monitoring included changes in chimerism, balance of T cell subsets, gene expression, and responses to donor alloantigens.
Results
Nine of 10 patients developed multi-lineage chimerism without graft versus host disease (GVHD), and all had excellent graft function at the last observation point. Five of these with chimerism persisting for at least 12 months were completely withdrawn from immunosuppressive drugs for 6 to 35 months. Blood cells from patients off drugs showed development of specific unresponsiveness to donor alloantigens, “tolerance” profiles on gene microarrays, early high ratios of regulatory versus conventional naïve T cells, and early high levels of chimerism among NK cells.
Conclusions
Total lymphoid irradiation, and anti-thymocyte globulin promoted the development of persistent chimerism and tolerance in a cohort of patients given kidney transplants and donor cell injections. Assays were identified that can assist in the safe withdrawal of immunosuppressive drugs.

overall_design: 45 Agilent Microarray samples were conducted, including 16 tolerance patients, 10 chronic rejection patients, 5 healthy normal controls, and 7 paired pre and post-transplant induced tolerance patients. The aim is to see whether induced tolerance patients show operational tolerance gene expression signature and can withdraw or minimize the immunosuppression regimens.

first: John

last: Scandling

first: Stephan

last: Busque

first: Sussan

last: Dejbakhsh-Jones

first: Claudia

last: Benike

first: Szu-chuan

last: Hsieh

first: Tara

last: Sigdel

first: Aditi

last: Mukhopadhyay

first: Tyson

last: Holmes

first: Judith

last: Shizuru

first: Robert

last: Lowsky

first: Edgar

middle: G

last: Engleman

first: Minnie

last: Sarwal

first: Samuel

last: Strober


title: Genome-wide transcriptional analysis of flagellin induced reprogramming in mouse corneal epithelial cells in response to Pseudomonas aeruginosa

city: Detroit

first: Fu-Shin

middle: X

last: Yu

state: Michigan

address: 4717 St. Antoine, Room K417

department: Ophthalmology

institute: Wayne State University School of Medicine

summary: We previously showed that pre-exposure of the cornea to TLR5 ligand flagellin induces profound mucosal innate protection against pathogenic microbes by reprogramming gene expression.   To date, there was no genome-wide cDNA array to detect full scale of flagellin mediated reprogramming of gene expression in mucosal surface epithelial cells. Taking advantage of readily accessible, easily procurable epithelial cell population, this study is the first report to use genome-wide cDNA microarray approach to document genes associated with flagellin-induced protection against Pseudomonas aeruginosa infection in corneal epithelial cells (CECs).

overall_design: Total RNA obtained from isolated mouse corneal epithelial cells of the control (cells scrapped off from the corneas without infection), Pseudomonas aeruginosa infected (6 h post infection) and flagellin pretreated (24 h), followed by Pseudomonas aeruginosa infection (6 h).

last: Gao

first: Gi

last: Yoon


title: miR148b is a major coordinator of breast cancer progression in a relapse-associated microRNA signature by targeting ITGA5, ROCK1, PIK3CA, NRAS and CSF1

city: Padova

first: Gerolamo

last: Lanfranchi

address: Via U. Bassi, 58/B

department: CRIBI - Biotechnology Center and Biology Department

institute: University of Padova

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

summary:
Breast cancer is often fatal during its metastatic dissemination. To unravel the role of microRNAs (miRs) during malignancy, we analyzed miR expression in 77 primary breast carcinomas and identified 16 relapse-associated miRs that correlate with survival and/or distinguish tumor subtypes in different datasets. Among them, miR-148b, downregulated in aggressive breast tumors, was found to be a major coordinator of malignancy. In fact, it is able to oppose various steps of tumor progression when overexpressed in cell lines by influencing invasion, survival to anoikis, extravasation, lung metastasis formation and chemotherapy response. miR-148b controls malignancy by coordinating a novel pathway involving over 130 genes and, in particular, it directly targets players of the integrin signaling, such as ITGA5, ROCK1,

PIK3CA/p110α, NRAS as well as CSF1, a growth factor for stroma cells. Our findings reveal the importance of the identified 16 miRs for disease outcome predictions and suggest a critical role for miR-148b in the control of breast cancer progression.

This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series.

first: Daniela

last: Cimino

first: Cristiano

last: De Pittà

first: Francesca

last: Orso

first: Matteo

last: Zampini

first: Silvia

last: Casara

first: Elisa

last: Penna

first: Elena

last: Quaglino

first: Marco

last: Forni

first: Christian

last: Damasco

first: Eva

last: Pinatel

first: Riccardo

last: Ponzone

first: Chiara

last: Romualdi

first: Cathrin

last: Brisken

first: Michele

last: De Bortoli

first: Nicola

last: Biglia

first: Paolo

last: Provero

last: Taverna


title: Gene expression analysis of wild type and KAP1 KO mouse T cell progenitors

city: Milan

middle: R.

last: Santoni de Sio

address: via Olgettina 58

department: SR-TIGET

institute: San Raffaele Scientific Institute

summary: The modulation of chromatin status at specific genomic loci controls lymphoid differentiation. Here, we investigated the role played in this process by KAP1, the universal cofactor of KRAB-containing Zinc Finger Proteins (KRAB-ZFPs), a tetrapod-restricted family of transcriptional repressors. T cell-specific Kap1 knockout mice displayed a significant expansion of immature thymocytes and imbalances in the ratios of mature T cells in the thymus and the spleen, with impaired responses to TCR stimulation. Transcriptome and chromatin studies revealed that KAP1 directly controls the expression of a number of genes involved in TCR and cytokine signalling, among which Traf1 and FoxO1, and is strongly associated with cis-acting regulatory elements marked by the H3K9me3 repressive mark on the genome of thymic T cells. Likely responsible for tethering KAP1 to at least part of its genomic targets, a small number of KRAB/ZFPs are selectively expressed in T lymphoid cells. These results reveal the so far unsuspected yet important role of KRAB/KAP1-mediated epigenetic regulation in T lymphocyte differentiation and activation.

overall_design: Cells were harvested form the thymus of 3 CD4-Cre/Kap1flox (KAP1 KO in the T lineage) mice and 3 wt/Kap1flox littermate controls

first: Adamandia

last: Kapopoulou

first: Didier

last: Trono


title: Characteristics of Cross-Hybridization and Cross-Alignment in Pseudo-Xenograft samples by RNA-Seq and Microarrays [Illumina MouseWG-6 v2.0]

status: Public on Dec 14 2012

city: Miami

first: Camilo

last: Valdes

address: Locator C-213.  1120 NW 14 Street

department: Center for Computational Science

institute: University of Miami

summary: In order to study molecular changes in the stroma from tissue samples it is recommended to separate tumor tissue from stromal tissue. This is particularly relevant to mouse tumor xenograft models where tumor, particularly metastatic tumors, can be small and difficult to separate from the host tissue. In our research we compared qualitatively the ability of high-throughput mRNA sequencing, RNA-Seq, and microarrays to detect tumor (human) and stromal (mouse) expression from mixed tumor-stromal samples in terms of the genes and pathways that are involved in cross-alignment (RNA-Seq) and cross-hybridization (microarrays).

overall_design: Human samples consisted of total RNA obtained from MDA-MB-231 human breast carcinoma cell line and isolated from three independent cultures of sub-confluent MDA-MB-231 cell lines in exponential phase of growth.  Mouse samples were obtained from NOD scid gamma mice, and normal lung tissue was harvested from three independent age-matched mice.

first: Jennifer

last: Clarke


title: Prediction of Breast Cancer Estrogen Receptor Status using Machine Learning

city: Edmonton

first: Kathryn

last: Graham

state: Alberta

address: 11560 University Ave

department: Oncology

institute: University of Alberta

summary: Gene expression profiles were generated from 199 primary breast cancer patients. Samples 1-176 were used in another study, GEO Series GSE22820, and form the training data set in this study. Sample numbers 200-222 form a validation set. This data is used to model a machine learning classifier for Estrogen Receptor Status.

overall_design: RNA was isolated from 199 primary breast cancer patients. A machine learning classifier was built to predict ER status using only three gene features.

first: John

last: Mackey


title: miR-148b modulates the expression of multiple genes

first: Gerolamo

last: Lanfranchi

summary: To identify which miR-148b targets were involved in tumorigenesis, a microarray analysis was performed for miR-148b over-expressing cells versus controls and 129 (49 up and 80 down) modulated genes were revealed.

overall_design:
The effects of miR-148b on cancer progression depend on the direct and indirect regulation of multiple target genes. To identify miR-148b modulated genes, MDA-MB-231 cells were transfected with miR-148b precursors or negative controls (pre-miR-148b or control) and used 48h later for microarray and western blot (WB) analyses. When a “Whole Human Genome Oligo Microarray” (Agilent) platform was employed, 129 differentially expressed genes (49 upregulated, 80 downmodulated) were found at 48h, considering a fold change (FC) cut of 1.5 and a false discovery rate (FDR) of 16% (Table S4).
Crossing these results with the list of putative miR-148b targets (3642) obtained by the miRecords System, we observed that 33 of the modulated genes were also miR-148b predicted targets and interestingly, 26 out of these 33 genes were downmodulated.

first: Daniela

last: Cimino

first: Cristiano

last: De Pittà

first: Matteo

last: Zampini

first: Silvia

last: Casara

first: Francesca

last: Orso

first: Piero

last: Sismondi


title: Whole heart transcriptional response to complete and epiblast-specfic deletion of Zic3 in 15.5 dpc mouse embryos

status: Public on Dec 12 2012

city: HOUSTON

first: Zhengxin

last: Jiang

state: Texas

address: ONE BAYLOR PLAZA

department: MOLECULAR AND HUMAN GENETICS

institute: BAYLOR COLLEGE OF MEDICINE

summary: In order to better understand the molecular basis for the heart defects seen in Zic3 null and epiblast CKO embryos, we investigated whether complete or epiblast-specific deletion of Zic3 would impact later embryonic heart development at the transcriptional level by whole genome expression microarray.

overall_design: The whole heart was carefully dissected out from 15.5 dpc Zic3 +/y, Zic3 flox/y, Zic3 flox/y; Sox2-cre, and Zic3 -/y embryos, total RNAs were extracted and purified using RNeasy Mini Kit (QIAGEN). Spectrophotometry (NanoDrop-1000 Spectrophotometer, Thermo Fisher Scientific) and microfluidic electrophoresis (Experion Automated Electrophoresis System, Bio-Rad Laboratories) were used for RNA quality control. In vitro transcription was performed using Illumina TotalPrep RNA Amplification Kit (Applied Biosystems/Ambion). cRNAs were hybridized onto Illumina MouseWG-6 v2.0 Expression BeadChips (Illumina) per manufacturer’s instructions.

last: Belmont


title: Gene expression profiling of endothelial cells isolated from wildtype and Crim1KST264/KST264 adult mice kidneys

city: Brisbane

first: Melissa

middle: Helen

last: Little

state: Queensland

address: Carmody Rd, St. Lucia

department: Institute for Molecular Bioscience

institute: The University of Queensland

summary: Previous analysis has shown that the Crim1KST264/KST264 mice develop progressive renal fibrosis as well as vascular abnormalities characterised by increased vascular leakiness and loss of endothelium integrity. To further define this endothelium abnormality, CD31+ endothelial cells were MACS sorted from both wildtype and Crim1KST264/KST264 kidneys for expression profiling to determine the biological processes that are perturbed.

overall_design: Total RNA was extracted from CD31+ MACS sorted endothelial cells from 4 pairs of mixed gender wildtype and Crim1KST264/KST264 adult mice kidneys

first: Yu

middle: L

last: Phua


title: ERβ activation by genistein promotes metastatic progression in prostate cancer

status: Public on Dec 27 2012

city: Vancouver

first: Hisae

last: Nakamura

address: 675 West 10th Ave

institute: BC Cancer Agency

summary: Androgen signalling through the androgen receptor (AR) plays a critical role in prostate cancer (PCa) initiation and progression. Estrogen and the estrogen receptor, in synergy with androgen, are essential for cell growth of the normal and malignant prostate. However, the exact role that estrogen plays in prostate carcinogenesis, and the precise mechanisms involved, remain unclear.   We have previously demonstrated the metastasis-promoting effect of an estrogen receptor Beta (ERβ) agonist (genistein) and the inhibitory action of an anti-estrogen (ICI 182, 780) in patient-derived PCa xenograft models mimicking localized and metastatic disease. In this study, we compared the gene expression profiles of treated and untreated PCa xenografts using microarrays to identify a unique set of genes that were both up-regulated by genistein treatment and down-regulated by the anti-estrogen, ICI 182,780. Five of the six genes identified from this comparison belonged to the metallothionein (MT) gene family. Knock-down of ERβ led to a reduction in MT gene expression, confirming its role in regulating these genes. Using qRT-PCR, the differences in expression levels were validated in the metastatic and non-metastatic LTL313 PCa xenograft tumour lines, both of which were originally derived from the same PCa patient. Together our data provides evidence for the role of ERβ signalling in PCa metastasis and implicates estrogen-stimulated MT gene expression in this process.

overall_design: Three samples each (total RNA extracted from three tumours of three different animals from each group) for control, genistein and ICI were used for the array.

first: Yuwei

last: Wang

first: Hui

last: Xue

first: Mark

middle: T

last: Romanish

first: Dixie

last: Mager

first: Cheryl

last: Helgason

first: Yuzhuo


title: Identification of genes upregulated by MeTA-array in human pancreatic cancers

status: Public on Dec 11 2012

city: Sendai

first: Shinichi

last: Fukushige

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

department: Department of Diabetes, Metabolism and Endocrinology

institute: Tohoku University Graduate School of Medicine

summary: Identification and characterization of epigenetically silenced genes is very important for cancer research. Particularly, information of hypermethylated genes provides clues to understand roles of epigenetics in tumorigeneses, and genes frequently methylated in a tumor-specific manner can be used as tumor markers. DNA methylation inhibitors such as 5-aza-cytidine or 5-aza-2’-deoxycytidine were widely used to search epigenetically silenced genes. However, these inhibitors frequently upregulate genes whose promoters remain unmethylated. We tried to improve the specificity and sensitivity in detecting such methylation-mediated silenced genes in cancer and successfully developed a new method termed “methyl-CpG targeted transcriptional activation (MeTA)” by using a transcriptional activating fragment with a methyl-CpG binding domain (MBD) that specifically recognizes and binds to methylated DNAs. Because MBD proteins in fact mediate transcriptional repression of tumor suppressor genes associated with promoter hypermethylation in cancer, MeTA is thought to be one of the ideal methods to search such genes. In the present study, we applied this method to three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2, and PANC-1, with a normal pancreatic ductal epithelial cell line HPDE (as the control). All of these cell lines have already been analyzed their expression profiles by 5-aza-2’-deoxycytidine. We first analyzed the expression of five genes by RT-PCR with Southern hybridization, NEFH, NPTX2, SFRP1, TIMP3, and UCHL1; these genes are known to be methylated in at least any one of these cancer cell lines. Upregulation by “MeTA” was confirmed in all of these genes. Then we searched for upregulated-genes, by two-folds or more, in all the three cancer cell lines after MeTA; nineteen such upregulated genes were identified. Among these, sixteen genes except NEFH, HOXA9, and CLDN5 have not been reported previously using the conventional DNA methylation inhibitors. Methylation status of two genes, SLC32A1 and CSMD2, were further analyzed by methylation-specific PCR and found that SLC32A1 and CSMD2 were methylated in 100% (21/21) and 83% (15/18) pancreatic cancer cell lines analyzed, respectively. Our results suggest that “MeTA” is a highly efficient method to isolate methylation-mediated transcriptionally silenced genes in human pancreatic cancer and that this method can be applied to other types of human cancer.

overall_design: Three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2, and PANC-1, with a normal pancreatic ductal epithelial cell line HPDE (as the control) were transfected with pcDNA6/myc-His vector or pcDNA6-3xFLAG-NFkB (AD)-MBD and were harvested 48 h after transfection.

first: Hideyuki

last: Shimizu


title: Gene array experiment to identify genes that are upregulated by saturating creatine levels in vitro and potentially inhibiting creatine transporter (CrT) activity

status: Public on Dec 07 2012

city: Oxford

first: Sevasti

last: Zervou

address: Roosevelt Drive

institute: University of Oxford

summary: We sought to investigate the mechanisms of CrT modulation by saturating extracellular Cr. For this purpose, we used an in vitro system of 3T3 fibroblasts stably overexpressing CrT and followed a global gene array approach to test the hypothesis that saturating intracellular [Cr] increases the expression of candidate molecules that inhibit CrT activity. In this way, we would elucidate mechanisms of redox regulation in cases of fluctuating energy demands.

overall_design: 3T3-CrT cells were grown on 6-well plates to 70% confluency. n=3 replicate wells were treated with 5mM creatine monohydrate in growth media and n=3 wells remained untreated (controls).

first: Tanmoy

last: Ray

first: Natasha

last: Sahgal

first: Liam

last: Sebag-Montefiore

first: Debra

last: Medway

first: Philip

last: Ostrowski

first: Stefan

last: Neubauer

first: Craig

last: Lygate


title: NF-kB target genes in dendritic cells

first: Mykola

last: Goncharenko

summary:
The ability of dendritic cells (DC) to initiate immunity and induce antigen-specific tolerance makes DC ideal targets for pharmacological intervention into immune responses. NF-kB factors are a family of transcriptional regulators important for DC development and function. However, the identity of NF-kB target genes that are central to DC biology is largely unknown. In the present study IkBa super repressor (IkBa-SR) and DNA microarray analysis were used to determine the repertoire of NF-kB responsive genes in DC. In DC these genes regulate vital DC functions of antigen uptake and presentation, motility, survival, etc.

Taking in account limitations of the genome-wide microarray analysis, generated transcription factor data were confirmed by the independent means of RT-PCR and chromatin immunoprecipitation. Kinetics of NF-kB induction by well-known DC activatory agents TNFa and LPS were further analysed. NF-kB regulated genes can be potentially useful targets for the development of more specific anti-inflammatory agents for clinical applications.
Keywords: drug treatment, adenovirus transduction

overall_design: DC were generated in vitro from bone marrow of CAR transgenic mice as described previously (Hieronymus, T., T. C. Gust, R. D. Kirsch, T. Jorgas, G. Blendinger, M. Goncharenko, K. Supplitt, S. Rose-John, A. M. Muller, and M. Zenke. 2005. Progressive and controlled development of mouse dendritic cells from Flt3+CD11b+ progenitors in vitro. J Immunol 174:2552-2562). Immature DC were transduced with AdIkBa-SR adenovirus, AdOVA control virus, or left untreated. Cells were then stimulated with TNFa, or left unstimulated. Total RNA was amplified, labelled and hybridised to Affymetrix MOE430A arrays. TNFa up- and down-regulated genes, as well as genes regulated by AdOVA control virus, and suppressed by AdIkBa-SR were analysed.

first: Eva

last: Kaergel

first: Thomas

last: Hieronymus

first: Tatjana

last: Gust

first: Matt

last: Cotton

first: Claus

last: Scheidereit

first: Martin

last: Zenke


title: Coxiella burnetii infects JEG trophoblastic cell line, a model of placenta infection

city: marseille

first: adil

last: ef

address: 27 bd jean Moulin

country: France

institute: URMITE

summary: Q fever is a zoonosis caused by Coxiella burnetii, an obligate intracellular bacterium usually found in myeloid cells. The infection is a source of severe obstetrical complications in humans and cattle, and of chronic evolution in pregnant women. As C. burnetii is found in the placenta of aborted foetuses in humans and ruminants, we wondered if it may infect trophoblasts. In this work, we showed that C. burnetii, infected JEG trophoblastic cells without replication and was localized within phagolysosomes. We analyzed gene expression programs induced by C. burnetii in JEG trophoblastic cell line and compared it with transcriptomic program of BeWo trophoblasts in which C. burnetii replicates. These transcriptomic programs induced by C. burnetii in JEG trophoblasts was poor and markedly different from that induced by C. burnetii in BeWo trophoblasts. Hence, the differences in transcriptomic programs may explain the different intracellular fate of C. burnetii in JEG and BeWo cells. Our results suggest that C. burnetii may use trophoblastic cells as a reservoir by interfering with gene expression.

overall_design: Comparaison between unstimulated JEG cell line and Coxiella burnetii stimulated JEG cell line (bacterial ratio 200:1) for 6 hours

first: Amira

middle: B

last: Amara

first: Abdoulaye

middle: O

last: Barry

first: Christian

last: Capo

first: Eric

last: Ghigo

first: Jean-Louis

last: Mege


title: Evaluation of gene expression between CD44+ and CD44- cells in colonospheres

city: Chuo-ku

first: Hirokazu

last: Ohata

state: Tokyo

address: Tsukiji 5-1-1

institute: National Cancer Center Research Institute

summary: To identify genes that are differentially expressed between CD44+ and CD44- cells in colonospheres, we have employed whole genome microarray expression profiling as a discovery platform. Colonospheres from four colon cancer patients were sorted into CD44+ and CD44- cells using the FACS Aria II Cell Sorter, and total RNA extracted from the cells were labeled with Cy3 and used for microarray analyses with Agilent Whole Human Genome Oligo Microarrays.

overall_design: Gene expression in fluorescence-activated cell-sorted CD44+ and CD44- cells derived from four colon cancer patients was measured.


title: A microRNA network regulates proliferative timing and extracellular matrix synthesis during cellular quiescence in fibroblasts [mRNA]

status: Public on Dec 05 2012

city: Princeton

middle: Jungwoo

last: Suh

state: NJ

address: 14 Washington Rd

department: Molecular Biology

institute: Princeton University

summary:
Background: Although quiescence—reversible cell-cycle arrest—is a key part in the life history and fate of many mammalian cell types, the mechanisms of gene regulation in quiescent cells are poorly understood. We sought to clarify the role of microRNAs as regulators of the cellular functions of quiescent human fibroblasts.
Results: Using microarrays, we discovered that the expression of the majority of profiled microRNAs differed between proliferating and quiescent fibroblasts. Fibroblasts induced into quiescence by contact inhibition or serum starvation had similar microRNA profiles, indicating common changes induced by distinct quiescence signals. By analyzing the gene expression patterns of microRNA target genes with quiescence, we discovered a strong regulatory function for miR-29, which is downregulated with quiescence. Using microarrays and immunoblotting, we confirmed that miR-29 targets genes encoding collagen and other extracellular matrix proteins and that those target genes are induced in quiescence. In addition, overexpression of miR-29 resulted in more rapid cell cycle re-entry from quiescence. We also found that let-7 and miR-125 were upregulated in quiescent cells. Overexpression of either one alone resulted in slower cell cycle re-entry from quiescence, while the combination of both together slowed cell cycle re-entry even further.
Conclusions: microRNAs regulate key aspects of fibroblast quiescence including the proliferative state of the cells as well as their gene expression profiles, in particular, the induction of extracellular matrix proteins in quiescent fibroblasts.

overall_design: mRNAs were analyzed by two color microarray from three separate human neonatal dermal fibroblasts cell lines transfected either with a miR-29b mimic or a control short dsRNA. One sample was labeled and analyzed in duplicate to ensure consistency in labeling and hybridization technique.

middle: J

first: Matthew

middle: Y

last: Remillard

first: Aster

last: Legesse-Miller

first: Elizabeth

last: Johnson

first: Johanna

middle: M

last: Lemons

first: Talia

middle: R

last: Chapman

first: Joshua

last: Forman

first: Mina

last: Kojima

last: Silberman

first: Hilary

last: Coller


title: EID1 knochdown by small RNA induces the large-scale conversion of adipose-derived mesenchymal stem cells into hematopoietic stem cells

status: Public on Dec 01 2012

city: Beijing

first: James

last: Yin

address: Dongcheng District, 5 Nanmengcang

institute: The military general hospital of Beijing PLA

summary: Comparison of expression profiles in adipose derived MSCs (AD-MSCs) without or with transfection of siR-EID1 and shR-EID1 and cord blood-derived HSCs (CB-HSCs). After MSCs were transfected with sRNA-EID1, they could be converted into HSCs. The goal was to determine possible molecular mechanisms of MSC transdetermination.

overall_design: Two-condition experiments: AD-MSCs vs CB-HSCs, and AD-MSCs transfected with the combination of siR-EID1 and shR-EID1 vs AD-MSCs transfected with shR-EID1.

middle: Q

first: Shanshan

last: Wang

first: Hong

first: Ruxiang

last: Xu


title: Gene expression data from Guthrie blood-spot cards

city: Grand Rapids

first: Karl

last: Dykema

state: MI

address: 333 Bostwick Ave. NE

institute: Van Andel Institute

summary:
Background: Standard Guthrie cards have been widely used to collect blood samples from neonates for newborn screening programs, and to a lesser extent, from normal controls and patients in research studies.  Ease of blood collection (small quantity and less pain), transportation, and storage are the advantages of using these cards.  It is believed that RNA obtained from these samples is of low quantity and degraded quality.  However, we recently discovered that approximately 3,500 expressed genes can be detected from blood spot samples using in-house made, low resolution cDNA microarrays.  Here, we established a new and improved methodology to acquire gene expression profiles from blood spot cards using commercially-available high resolution microarrays.  We determined the optimal number of blood spot punches required for maximal RNA extraction, eliminated uses of trizol and chloroform for RNA extraction by using a modified protocol of the illustra Mini Spin Kit from GE-Whatm  an, concentrated the quantity of RNA templates before amplification, improved amplification efficiency using the new Ribo-SPIA technology in WT-Ovation Pico System (WT-Pico) by NuGEN, before the samples were hybridized onto 4x44K whole human genome gene expression microarrays from Agilent.  Nine dried blood spot samples were collected from a control population and stored at ~ -80 °C between 6 months to 2 years.  High quality RNA was extracted from the buffy coat of the same individuals as a reference and processed using the standard Agilent microarray procedure.  Commercially-available brain RNA was used as a positive control in both standard and new procedures for microarrays.
Results: Three 3-mm punches produced the highest yield of total RNA using the non-trizol extraction method.  Three to six nanogram per microliter of RNA can be concentrated and is sufficient to be amplified using the WT-Pico.  Approximately 9,000 expressed genes can be detected after normalization and background correction of the microarray data.
Conclusion: Genome-wide gene expression profile can be obtained from archived dried blood spot samples.  Our new and improved methodology will add value to the perception of utilizing archival Guthrie cards eg. neonatal blood spot cards as unique biospecimens for molecular genomics and diagnostic studies of perinatal diseases such as pediatric cancers.

Keywords: Gene Expression experiment

overall_design:
Archival guthrie blood-spot cards may contain valuable data for epidemiological or other studies.
Showing microarray data from guthrie blood-spot cards


title: Genome-wide analysis of gene expression in Krt1+/+ versus Krt1-/- epidermis and full-thickness skin.

city: Bonn

first: Joachim

last: Schultze

state: NRW

address: Carl-Troll-Strasse 31

department: Genomics and Immunoregulation

institute: LIMES (Life and Medical Sciences Center Genomics and Immunoregulation)

summary: Keratin 1 (KRT1) and its heterodimer partner keratin 10 (KRT10) constitute the intermediate filament cytoskeleton of suprabasal skin keratinocytes. They participate in formation of the epidermal barrier, which protects against dehydration and inflammation. Mutations in KRT1 cause keratinopathic ichthyosis with erythema, recurrent inflammation, and barrier defects. Here, we show that genetic deletion of Krt1 in mice causes a defective inside-out epidermal barrier, pre- and postnatal increases in Mrp8/Mrp14, interleukin (IL) 18, IL-33, and thymic stromal lymphopoietin (TSLP) in skin extracts, and systemic release of IL-18 into newborn serum. Perinatal lethality was partially rescued by treatment with glucocorticoids to promote barrier repair or with IL- 18-blocking antibodies in utero. In human keratinocytes, IL-18 release was cellautonomous and caspase-1-dependent, indicating KRT1-dependent inflammasome activation. Our data reveal a novl function of KRT1 in controlling inflammasome activity and stimulating barrier formation, thereby integrating the keratin cytoskeleton into the epidermal immune response. In view of their widespread expression, keratins merit investigation of their functions in inflammatory conditions, including asthma and inflammatory bowel disorders.

overall_design: Total RNA was obtained from epidermis or full-thickness skin of Krt1+/+ and Krt1-/- mice (C57BL/6 background) at P0 (newborn).

first: Wera

last: Roth

first: Thomas

last: Margin

first: Andrea

last: Staratschek-Jox


title: Transcriptomic profiling of murine ES and iPS cells, embryoid bodies, and ES and IPS cell-derived cardiomyocytes

summary: In this study we have compared functional and molecular properties of highly purified murine induced pluripotent stem (iPS) cell- and embryonic stem (ES) cell-derived cardiomyocytes (CM). In order to obtain large amounts of purified CM, we have generated a transgenic murine iPS cell line, which expresses puromycin resistance protein N-acetyltransferase and EGFP under the control of the cardiomyocyte-specific α-myosin heavy chain promoter (alphaMHC-Puro-IRES-GFP, aPiG). We demonstrate that murine aPIG-iPS and aPIG-ES cells differentiate into spontaneously beating CM at comparable efficiencies. When selected with puromycin both cell types yielded more than 97% pure population of CMs. Both aPIG-iPS and aPIG-ES cell-derived CM express typical cardiac transcripts and structural proteins and possess similar sarcomeric organization. Action potential recordings revealed that iPS- and ES cell-derived CM respond to beta-adrenergic and muscarinic receptor modulation, express functional voltage-gated sodium, calcium and potassium channels and possess comparable current densities. Comparison of global gene expression profiles of CM generated from iPS and ES cells revealed that both cell types cluster close to each other but are highly distant to undifferentiated ES or iPS cells as well as unpurified iPS and ES cell-derived embryoid bodies (EB). Both iPS and ES cell-derived CMs express genes and functional categories typical for CM. They are enriched in genes involved in transcription and genes coding for structural proteins involved in cardiac muscle contraction and relaxation. They also express genes involved in heart and muscle developmental processes, ion export and ion binding processes and various metabolic processes for ATP synthesis. These CMs downregulate genes involved in immune response, cell cycle and cell division, thus demonstrating the CMs population is mitotically inactive. Most surface signaling pathways are also downregulated. Thus, a transgenic aPiG-iPS cell line can provide a robust supply of highly purified and functional CMs for future in vitro and in vivo studies.

overall_design: Seven different experimental groups were included into analysis: undifferentiated murine ES cells (1) and undifferentiated murine iPS cells (2), murine ES cell-derived embyroid bodies (3) and murine iPS cell-derived embryoid bodies at day 16 of differentiation (4), murine ES cell-derived cardiomyocytes (5) and murine iPS cell-derived cardiomyocytes (6) at day 16 of differentiation (they were generated by puromycin selection for 7 days prior to RNA isolation). Adult mouse tail tip fibroblasts (7) were used as a control for iPS cells. Total RNA samples were prepared from three independent biological replicates in groups 1-6. In group 7, single RNA probes were analyzed as three technical replicates.

first: Tomo

last: Saric

first: Azra

last: Fatima

last: Gaarz

first: Jürgen

last: Hescheler


title: Expression profiling of human peripheral blood before and after omega 3 dietary supplement.

city: Huntington

last: Denvir

state: WV

address: One John Marshall Drive

department: Biochemistry and Microbiology

institute: Marshall University School of Medicine

summary: Patients with early stage CLL were recruited into the study and administered a course of omega 3 dietary supplements. Expression profiling of each patient was performed at baseline (before administering the supplement) and after consumption of supplements. Patients were divided into two groups according to the level of NFkB activation at baseline, and then within each of the two groups significant changes in gene expression between baseline and the highest dose of supplements consumed were sought.

overall_design: Patients were divided into two groups based on initial NFkB levels being below (low expressers) or above (high expressers) the median NFkB Activity level (2.14x10^6 NFkB Luminescence units/µg protein). Within each group, paired two class SAM was performed using the maximum supplement dose for each patient versus the baseline.

first: W

middle: E

last: Hardman

first: Johannes

middle: F

last: Fahrmann

first: Oscar

last: Ballester

first: Gabriella

first: Theodore

last: Witte

first: Alexander

last: Salazar

first: Gabriela

last: Ion

first: Donald

middle: A

last: Primerano

first: Goran

last: Boskovic


title: Transcriptome Analysis in Patients with Progressive Coronary Artery Disease

status: Public on Nov 30 2012

city: Bad Krozingen

middle: G

last: Nuehrenberg

state: Baden-Württemberg

address: Südring 15

department: Klinik für Kardiologie II

institute: Universitäts-Herzzentrum Freiburg • Bad Krozingen

summary:
The identification of classic risk factors for coronary artery disease unveiled pathophysiologic mechanisms of atherosclerosis. Among them, inflammation as a systemic process measurable in peripheral blood plays a central role in plaque progression. However, other mechanisms of plaque progression remain to be fully understood. Therefore, this study sought to further investigate systemic correlates of plaque progression by global gene expression in peripheral blood.
Microrarray gene expression analysis revealed 93 genes differentially expressed between the groups, of which 23 genes have no known function. Among the remaining 70 genes, 10 (14%) were identified to be associated with progenitor and pluripotent cells whereas only 3 genes (4%) had been associated with atherosclerosis. A risk prediction gene signature was developed by a multivariable statistical approach model integrating comprehensive laboratory and clinical patient data. This signature identified plaque progression with 81% sensitivity and 80% specificity (AUC: 0.86) for new patients, as estimated by resampling techniques. Array results were validated by qPCR for the genes ankyrin-2 (ANK2) and glutathione S-transferase theta 1 (GSTT1). In conclusion, patients with pogressive coronary artery disease despite good risk factor control exhibit particular gene expression patterns in peripheral blood. Understanding the functional implications of the observed changes might help to design new approaches to control atherosclerosis progression.

overall_design: From a large database of 45,727 coronary angiograms, peripheral blood was drawn from two patient groups with good risk factor control, but different clinical evolution: First, 16 patients with significant lesion progression leading to repeated coronary interventions and second, 16 patients with angiographically documented stable courses.

first: T

first: F

last: Neumann


title: Quiescent Fibroblasts Exhibit High Metabolic Activity

first: Eric

last: Suh

summary: Many cells in mammals exist in the state of quiescence, which is characterized by reversible exit from the cell cycle. Quiescent cells are widely reported to exhibit reduced size, nucleotide synthesis, and metabolic activity. Much lower glycolytic rates have been reported in quiescent compared with proliferating lymphocytes. In contrast, we show here that primary human fibroblasts continue to exhibit high metabolic rates when induced into quiescence via contact inhibition. By monitoring isotope labeling through metabolic pathways and quantitatively identifying fluxes from the data, we show that contact-inhibited fibroblasts utilize glucose in all branches of central carbon metabolism at rates similar to those of proliferating cells, with greater overflow flux from the pentose phosphate pathway back to glycolysis. Inhibition of the pentose phosphate pathway resulted in apoptosis preferentially in quiescent fibroblasts. By feeding the cells labeled glutamine, we also detected a “backwards” flux in the tricarboxylic acid cycle from α-ketoglutarate to citrate that was enhanced in contact-inhibited fibroblasts; this flux likely contributes to shuttling of NADPH from the mitochondrion to cytosol for redox defense or fatty acid synthesis. The high metabolic activity of the fibroblasts was directed in part toward breakdown and resynthesis of protein and lipid, and in part toward excretion of extracellular matrix proteins. Thus, reduced metabolic activity is not a hallmark of the quiescent state. Quiescent fibroblasts, relieved of the biosynthetic requirements associated with generating progeny, direct their metabolic activity to preservation of self integrity and alternative functions beneficial to the organism as a whole.

overall_design: mRNAs were analyzed by two color microarray from two separate human neonatal dermal fibroblasts cell lines in proliferating, 7 days contact inhibition, or 14 days contact inhibition. Contact inhibited samples were co-hybridized to proliferating samples as a control, while an additional array co-hybridized the two proliferating samples to analyze reproducibility.

first: Johanna

last: Lemons

first: Xiao-Jiang

last: Feng

first: Bryson

middle: D

last: Bennet

first: Aster

last: Legesse-Miller

first: Elizabeth

last: Johnson

first: Irene

last: Raitman

last: Pollina

first: Herschel

last: Rabitz

first: Joshua

last: Rabinowitz

first: Hilary

last: Coller


title: A microRNA network regulates proliferative timing and extracellular matrix synthesis during cellular quiescence in fibroblasts

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

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

overall_design: Refer to individual Series


title: Intralesional host response within eschars in Mediterranean spotted fever

first: adil

last: ef

summary: The host response within the eschar of inoculation during Mediterranean spotted fever (MSF) has been poorly studied. Our objective was to evaluate the host response by comparing transcriptional profiles of eschars to controls using a whole-genome microarray. Hierarchical clustering revealed a signature of eschars consisting of 698 genes. The genes included in this signature were mainly up-regulated and were predominantly associated with immune response and signalling. New molecules involved notably in microbicidal and innate immunity response have also been found up-regulated in eschars such as MMP1, Defensin β4, the proinflammatory S100A9, and the T cell attracting CCL-18. Genes down-regulated were mainly associated with biological regulation. We also observed that eschars from severe cases of MSF displayed a specific signature with notably difference in degree of modulation compared to eschars from non severe MSF cases. Some parameters identified in this work should be tested as biomarkers for prognostic assessment in future studies.

overall_design: To evaluate the host response by comparing transcriptional profiles of eschars to controls

first: E

last: Botelho-Nevers

first: A

last: Ben Amara

last: El Filali

first: N

last: Mouffok

last: Boudebouch

first: V

last: Merhaj

first: D

last: Raoult

first: J

last: Mège


title: transcriptional profiling of NUP98-CDX1, NUP98-CDX4 and EGFP overexpressing primitive mouse bone marrow cells

city: Singapore

first: jinmiao

last: chen

address: Biopolis

institute: Singapore Immunology Network

summary: The first genetic hits towards leukemia are thought often to confer to the mutant clone a slight proliferative/survial advantage. These pre-malignant clones can then be maintained under physiological cues in normal pool for long periods. We have searched for this type of first genetic hits and found that ectopic expression of novel nucleoporin 98 (NUP98)-homeodomain fusion proteins in primitive bone marrow cells can induce long-lasting hematopoietic stem cell (HSC) activity in these cells. More specifically, NUP98-CDX1/2 was able to sustain HSC activity with high frequencies despite massive in vitro expansions and in vivo residence. Continual expression of NUP98-CDX1/2 did not perturb lymphoid, myeloid or erythroid/platelet differentiation and NUP98-CDX1/2 was able to reprogram committed progenitors into cells with attributes of HSC. Switching off of NUP98-CDX1/2 expression did not lead to “return” to normal HSC but acute differentiation. Related NUP98-CDX4 induced leukemia with the shortest latency ever reported for HOX transcription factors.  In this case switching off of NUP98-CDX4 expression cured the full-blown leukemia and again the cured cells showed only transient repopulating activity in vivo. These two types of pre-leukemic cells generated here will provide useful models for elucidating the self-renewal mechanisms of HSC as well as leukemic stem cells.

overall_design: Total RNA from NUP98-CDX lines was compared to EGFP overexpressing lines. The experiment was done in triplicate.

first: Zhi-hao

last: Wu

first: Shuzk-Cheng

last: Loh

first: Jin-miao

last: Chen

first: Christiane

last: Ruedl

first: Klaus

last: Karjalainen


title: An eQTL study in the Japanese population [gene expression_1]

status: Public on Nov 27 2012

city: Kodaira

first: Daimei

last: Sasayama

address: 4-1-1 Ogawahigashi-machi

summary: A genome-wide eQTL analysis was performed in whole blood samples collected from 76 Japanese subjects. RNA microarray analysis was performed for 3 independent samples that were genotyped in a genome-wide scan. The correlations between the genotypes of 534,404 autosomal single nucleotide polymorphisms (SNPs) and the expression levels of 30,465 probes were examined for each sample. The SNP-probe pairs with combined correlation coefficients of all 3 samples corresponding to P < 3.10 × 10-12 (i.e., Bonferroni-corrected P < 0.05) were considered significant. SNP-probe pairs with a high likelihood of cross-hybridization and SNP-in-probe effects were excluded to exclude false positive results. We identified 102 cis-acting and 5 trans-acting eQTL regions. The cis-eQTL regions were widely distributed both upstream and downstream of the gene, as well as within the gene.

overall_design:
RNA microarray data obtained from 3 independent samples originally recruited for other studies investigating the gene expression levels in psychiatric disorders were used in the present study. For the purpose of the present analyses, genomic DNA was collected from 24 subjects (13 men and 11 women, mean age [SD] = 39.9 [7.6] years) in sample 1, 24 subjects in sample 2 (12 men and 12 women, 34.1 [11.5] years), and 28 subjects (14 men and 14 women, 41.4 [11.8] years) in sample 3. Some of the subjects had depressive symptoms, but all were physically healthy and without clinically significant systemic disease (e.g., malignant disease, diabetes mellitus, hypertension, renal failure, or endocrine disorders). Subjects were recruited from the outpatient clinic of the National Center of Neurology and Psychiatry Hospital, Tokyo, Japan, through advertisements in free local information magazines or through our website announcement. All the subjects were biologically unrelated Japanese individuals who resided in the same geographical area (western Tokyo). The study protocol was approved by the ethics committee at the National Center of Neurology and Psychiatry, Japan. Written informed consent was obtained from every subject after the study was explained to them. Venous blood was collected between 1100 and 1200 h in PAXgene tubes (Qiagen, Valencia) from each subject and was incubated at room temperature for 24 h for RNA stabilization. RNA was extracted from whole blood according to the manufacturer’s guidelines by using the PAXgene Blood RNA System Kit (PreAnalytix GmbH, Hombrechtikon, Switzerland). The RNA was quantified by optical density readings at A260nm by using the NanoDrop ND-1000 (Thermo Scientific, Rockford). Gene expression analysis was performed using Agilent Human Genome 4 × 44 K arrays (Agilent Technologies, Santa Clara). Raw signal data for each of the 3 independent samples were analyzed separately by the GeneSpring GX software (Agilent Technologies). Data were filtered according to the expression level for quality control to eliminate genes that were below the 20th percentile threshold. The expression value of each gene was normalized to the median expression value of all genes in each chip. A total of 30,465 probes were included in the analysis.
Genomic DNA was obtained from venous blood samples. Genotyping was performed by Riken Genesis (Yokohama, Japan) using the Illumina HumanOmni1-Quad BeadChip (Illumina, Inc., San Diego). A total of 713,495 autosomal SNPs were assessed for quality using the PLINK v1.07 software. All SNPs with a call rate below 95%, a deviation from Hardy-Weinberg equilibrium at an error level of P < 0.001, or a minor allele frequency of less than 10% were excluded. The remaining 534,404 SNPs were used for further analysis.

first: Hiroaki

last: Hori

first: Seiji

last: Nakamura

first: Ryo

last: Miyata

first: Toshiya

last: Teraishi

first: Kotaro

last: Hattori

first: Miho

last: Ota

first: Noriko

last: Yamamoto

first: Teruhiko

last: Higuchi

first: Naoji

last: Amano

first: Hiroshi

last: Kunugi


title: Gene expression in human prostate cancer cell lines regulated by HIC1 gene

status: Public on Nov 17 2012

city: Shanghai

first: Jianhua

last: Wang

address: No.280 Chongqing Road

department: School of Medicine

institute: Shanghai Jiaotong University

summary: This study aimed to further our understanding of the role that hypermethylatioted in cancer 1 (HIC1) plays in prostate cancer (PCa) development. Microarrays were searched for some genes that had correlated expression with HIC1 mRNA. Our data showed that HIC1 promoter hypermethylation was presented in cell lines, tissues and plasma of PCa patients. According to fold-change screening between restoring expression of HIC1 and its respective control cells, both up-regulated and down-regulated genes were commonly observed in PC3 and C4-2B cells.

overall_design: The restoring expression HIC1 in PCa lines were respectively noted as PC3-HIC1 and C4-2B-HIC1 cells, and the respective controls were noted as C4-2B-GFP and PC3-GFP cells.

first: Jianghua

last: Zheng

first: Jinglong

first: Xueqing

last: Sun


title: An eQTL study in the Japanese population [gene expression_3]

institute: National Center of Neurology and Psychiatry