title: Towards a PBMC "virogram assay" for precision medicine: concordance between ex vivo and in vivo viral infection transcriptomes

status: Public on Aug 07 2014

city: Tucson

first: Vincent

last: Gardeux

state: AZ

address: 1657 East Helen Street, PO Box 210240

department: Medicine

institute: University of Arizona

summary:
BACKGROUND: Understanding individual patient host response to viruses is key to designing optimal personalized therapy. Unsurprisingly, in-vivo human experimentation to understand individualized dynamic response of the transcriptome to viruses are rarely studied because of the obvious limitations stemming from ethical considerations of the clinical risk.

OBJECTIVE:
In this rhinovirus study, we first hypothesized that ex-vivo human cells response to virus can serve as a proxy for otherwise controversial in-vivo human experimentation. We further hypothesized that the N-of-1-pathways framework, previously validated in cancer, can be effective in understanding the more subtle individual transcriptomic response to viral infection.

METHOD:
N-of-1-pathways computes a significance score for a given list of gene sets at the patient level, using merely the 'omics profiles of two paired samples as input. We extracted the peripheral blood mononuclear cells (PBMC) of four human subjects, aliquoted in two paired samples, one subjected to ex-vivo rhinovirus infection. Their dysregulated genes and pathways were then compared to those of 9 human subjects prior and after intranasal inoculation in-vivo with rhinovirus. Additionally, we developed the Similarity Venn Diagram, a novel visualization method that goes beyond conventional overlap to show the similarity between two sets of qualitative measures.

RESULTS:
We evaluated the individual N-of-1-pathways results using two established cohort-based methods: GSEA and enrichment of differentially expressed genes. Similarity Venn Diagrams and individual patient ROC curves illustrate and quantify that the in-vivo dysregulation is recapitulated ex-vivo both at the gene and pathway level (p-values<0.004).

CONCLUSION:
We established the first evidence that an interpretable dynamic transcriptome metric, conducted as ex-vivo assays for a single subject, has the potential to predict individualized response to infectious disease without the clinical risks otherwise associated to in-vivo challenges. These results serve as a foundational work for personalized "virograms".

overall_design:
PBMC INCUBATED WITH VIRUSES:
The live PBMCs had been isolated from blood samples collected from four human subjects under a protocol approved by The University of Arizona Internal Review Board. Whole blood was obtained from donors and placed in Becton Dickenson’s CPT tubes that were centrifuged according to standard protocols to obtain PBMCs, and then each aliquoted in two paired samples. Each sample of the pair was subsequently exposed to and incubated with either (i) Human Rhinovirus serotype 16 (ex vivo infected sample) or to (ii) sterile medium (control ex vivo non-infected sample) and incubated at 37 °C in 5% CO2 for 24 h. This protocol resulted in 4 ex vivo infected + 4 ex vivo controls = 8 paired samples. RNA was extracted from these samples, amplified, tagged, and hybridized on Affymetrix Human Gene 1.0 ST microarrays according to standard operating procedures.

DATA NORMALIZATION:
Robust Multiple-array Average (RMA) normalization was applied on each patient data independently (2 paired samples at a time, to avoid bias in the single-patient experiments) using Affymetrix Power Tools (APT)

first: Anthony

last: Bosco

first: Jianrong

last: Li

first: Marilyn

last: Halonen

last: Jackson

first: Fernando

last: Martinez

first: Yves

last: Lussier


title: miRNA expression data from saliva, blood, semen and vaginal swap

status: Public on Aug 01 2014

city: daejeon

first: Jonglyul

last: Park

address: 52, Eoeun-dong, Yuseong-gu

department: Medical genomics

country: South Korea

institute: Korea Research Institute of Bioscience and Biotechnology

summary:
Body-fluid specific marker provide important information for crime scene, but some marker such as mRNA and protein can provide wrong information because cross-reaction.
We used microarrays to identify body-fluid specific miRNA marker for forensic use.

overall_design: Genome-wide miRNA profiling in 4 type of body fluid to identify each body fluid specific marker

first: Jong-Lyul

first: Seong-Min

first: Jeong-Hwan

last: Kim

first: Seon-Young

first: Seung-Hwan

last: Lee


title: Expression data with either EglN2 or control knockdown in T47D cells

status: Public on Jul 31 2014

city: Chapel Hill

first: Qing

last: Zhang

address: 450 West Drive

country: USA

institute: University of North Carolina at Chapel Hill

summary: In order to perform integrated analysis for EglN2 ChIP-Seq and microarray to identify EglN2 direct target genes, we performed the microarray analysis for T47D breast cancer cells with either control or EglN2 siRNA followed by hypoxia+DMOG treatment

overall_design: T47D cells were transfected with either control or EglN2 siRNA in duplicate with RNAi max. 36 hours following transfection, cells were treated with hypoxia (1%) and DMOG (1 mM) for 16 hours. Following treatment, total RNA was extracted using Rnaeasy kit from Qiagen.

first: Chengyang

last: Wang


title: Expression data from cultured human ovarian carcinoma cell lines with and without Cisplatin treatment

status: Public on Jul 29 2014

city: Singapore

first: Tuan Zea

last: Tan

address: 14 Medical Drive, #11-01, Singapore

department: Cancer Science Institute Singapore

country: Singapore

institute: National University of Singapore

summary: Chemo-resistance to platinum such as cisplatin is critical in the treatment of ovarian cancer. Recent evidences have linked epithelial-mesenchymal transition (EMT) with the drug resistance as a contributing mechanism. The current study explored the connection between cellular responses to cisplatin with EMT in ovarian cancer.

overall_design: 46 ovarian carcinoma cell lines expression data with and without Cisplatin treatment.

first: Tuan

middle: Z

middle: E

last: Miao

first: Jean

middle: P

last: Thiery

first: Seiichi

last: Mori


title: Activating c-KIT mutations confers oncogenic cooperativity and rescue RUNX1-ETO induced DNA-damage in human cells

city: Frankfurt

first: Claudia

last: Döring

address: Theodor Stern Kai 7

department: Medical School, Institite of Pathology

country: Germany

institute: Johann Wolfgang-Goethe University Frankfurt a.M.

summary: REtr causes genomic instability in U937 cells. Activated forms of c-KIT, like c-KIT(N822K), rescues the Retr induced genomic instability by increasing the rate of DNA repair by homologous recombination

overall_design: Human U937 cells were transduced with retroviral vectors encoding REtr or c-KIT(N822K), expanded in culture and FACS selected prior to molecular analysis

first: Manuel

last: Grez


title: Inflammation-induced chemokine expression in uveal melanoma cell lines stimulates monocyte chemotaxis

status: Public on Jul 30 2014

city: Copenhagen

first: Tina

last: Jehs

address: Blegdamsvej 3

country: Denmark

institute: University of Copenhagen

summary:
Purpose: Uveal melanoma (UM) is the most common primary intraocular tumor in adults and the presence of infiltrating leucocytes is associated with a poor prognosis. Little is known how infiltrating leucocytes influence the tumor cells. The purpose of this study was to investigate the effect of activated T cells on the expression of chemotactic cytokines in UM cells. Furthermore, we examined the ability of stimulated UM cells to attract monocytes.

Methods: We used an in vitro co-culture system in which UM cell lines and T cells were cultured together, but separated by a membrane. UM gene expression was quantified using a microarray. Protein expression in the supernatant was quantified with ELISA or cytometric bead array. For the monocyte migration assay, a trans-well plate was used.

Results: Gene-expression analysis of UM cell lines showed that co-culture with activated T cells resulted in an upregulation of chemokines such as CXCL8, CXCL9, CXCL10, CXCL11, CCL2, CCL5, vascular endothelial growth factor (VEGF), intracellular adhesion molecule 1 (ICAM1) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The upregulation of these molecules was confirmed at the protein level. This increase of chemokines coincided with an increased chemotactic capacity of the supernatant towards monocytes.

Conclusions: Cytokines derived from activated T cells shifted the UM cell-transcriptome towards a more inflammatory state, including upregulation of several chemokines, which led to an increased migration of monocytes. Therefore, UM cells might actively participate in generating a tumor- promoting inflammatory microenvironment.

overall_design: One replicate for each cell lines (92.1, Mel 270 and Mel 290) was analyzed before and after co-culture with activated T cells.

first: Carsten

last: Faber

first: Helene

middle: B

last: Juel

first: Inge

middle: H

last: Bronkhorst

first: Martine

middle: J

last: Jager

first: Mogens

last: Nissen


title: Precursor and mature microRNA expression in MCF7 cells in hypoxia

status: Public on Jul 25 2014

city: Bedford Park

first: Kanchana

middle: Veronika

last: Bandara

state: SA

address: Flinders Drive

department: Renal Department

country: Australia

institute: Flinders University

summary: Background: Cancers are commonly characterised by hypoxia and also by global reductions in the levels of mature microRNAs. We have examined the hypothesis that hypoxia might mediate this reduction through repressive effects on microRNA biogenesis proteins.  Methods: Breast cancer cell lines were exposed to hypoxia and manipulations of hypoxia inducible factor (HIF) and HIF hydroxylase activity. The effects of hypoxia on the mRNA and protein levels of enzymes involved in microRNA biogenesis (Dicer, Drosha, TARPB2, DCGR8, XPO5) was determined by RT PCR and immunoblotting. The effect of hypoxia on microRNAs was determined with microarray studies, RT PCR and reporter assays.  Results: In breast cancer lines there was significant reduction of Dicer mRNA and protein levels in cells exposed to hypoxia. This effect was independent of HIF but dependent on the HIF hydroxylase PHD2 and was partly mediated by feedback effects via microRNAs. Furthermore, several other proteins with critical roles in microRNA biogenesis (Drosha, TARBP2 and DCGR8) also showed significant and co-ordinated repression under hypoxic conditions. Despite these substantial alterations no, or modest, changes were observed in mature microRNA production  Conclusion: These observations provide further and important interfaces between oxygen availability and gene expression and a potential mechanistic explanation for the reduced levels of microRNAs observed in some cancers. They provide further support for the existence of feedback mechanisms in the regulation of the microRNA biogenesis pathway and the relative stability of microRNAs.

overall_design: MCF7 cells were treated with three different conditions.   Treatment-1: MCF7 cells were exposed to hypoxia (0.1% O2) for 48 h and harvested for RNA extraction (n=3).  Treatment-2: MCF7 cells were exposed to normoxia for 48 h and harvested for RNA extraction (n=3).  Treatment-3: Dicer inhibition in MCF7 cells by transient transfection of siRNAs targeting Dicer. Cells were transfected with 20 nM siRNA duplexes (Shanghai GenePharma Co., Ltd, China), using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s protocol. A second transfection was carried out after 24 h following the same protocol. Cells were harvested 24 h after the second transfection and used for RNA extraction (n=3).  RNA integrity was assessed using the Agilent 2100 Bioanalyzer. Affymetrix miRNA 3.1 Array Strip was used for RNA analysis. This array consisted probe sets unique to human mature and pre-miRNA hairpins. A detailed protocol can be found in the miRNA 3.1 Array Strips technical manual (Affymetrix). In summary, 100-300 ng of total RNA was used to synthesise double stranded cDNA using random hexamers. The cDNA was then amplified to produce antisense cRNA, which was then reverse transcribed in a second cycle of cDNA synthesis. The second cycle incorporates dUTP into the cDNA sequence, which allows it to be fragmented using uracil DNA glycosylase and apurinic/apyrimidic endonuclease I. Following biotinylation, these fragments were hybridised overnight to a Affymetrix miRNA 3.1 array. The arrays were then washed, stained using a fluorescently-labelled antibody, and scanned using a high-resolution scanner. Intensity data were analysed using Partek® software (Partek Inc.). Data were normalised by quantile normalisation and log 2 transformed. Differential expression was determined by ANOVA and corrected for false discovery.

middle: V

first: Michael

last: Michael

first: Jonathan

middle: M

last: Gleadle


title: Human XCR1+ Dendritic Cells Derived In Vitro from CD34+ Progenitors Closely Resemble Blood Dendritic Cells, Including Their Adjuvant Responsiveness, Contrary to Monocyte-Derived Dendritic Cells

status: Public on Jul 23 2014

city: marseille

first: Thien-Phong

last: Vu Manh

address: 163 avenue de Luminy

country: France

institute: CIML

summary: Functional characterization of human dendritic cell subsets is limited due to the very low frequency of these cells in vivo. We developed an in vitro culture system for the simultaneous generation of XCR1+ DCs and MoDCs from cord blood CD34+ cells. Their global gene expression profiles at steady state and under activation, phenotypes, morphologies and responses to different TLR ligands where characterized and compared with those of their in vivo counterparts. The study demonstrated that the XCR1+ DCs generated in vitro from cord blood CD34+ cells are equivalent to blood XCR1+ DCs and also allowed a rigorous comparison of this DC subset with MoDC which are often considered as the reference model for DCs. Altogether, our results showed that in vitro generated XCR1+ DCs are a better model for the study of blood DC than the conventionally used MoDCs.

overall_design: The different dendritic cell subsets used for the study were either generated in vitro from cord blood CD34+ cells with recombinant human cytokines or isolated from  peripheral blood. The subsets were purified by fluorescence-activated cell sorting and their responses to PolyI:C, LPS or R848 were studied including by gene expression profiling.

first: S

last: Balan

first: V

last: Ollion

first: N

last: Colletti

first: R

last: Chelbi

first: F

last: Montanana-Sanchis

first: H

last: Liu

first: Tp

first: C

last: Sanchez

first: J

last: Savoret

first: I

last: Perrot

first: A

last: Doffin

first: E

last: Fossum

first: D

last: Bechlian

last: Chabannon

first: B

last: Bogen

last: Asselin-Paturel

first: M

last: Shaw

first: T

last: Soos

last: Caux

last: Valladeau-Guilemond

last: Dalod


title: Variation in primary CD4+ T cell response to activation across time and people

status: Public on Aug 28 2014

city: Boston

first: Liang

last: Yang

state: MA

address: 77 Avenue Louis Pasteur

department: Pathology

institute: Harvard Medical School

type:
Expression profiling by array
Other

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

Variation in individuals' adaptive immune response is believed to influence susceptibility to complex diseases in humans. The genetic basis of such variation is poorly understood. We analyzed the response of human CD4+ T cells from a cohort of 348 healthy humans, representing three major ancestries, to in vitro stimulation, alone or in Th17 conditions, or with IFNb stimulation.

overall_design: The SuperSeries includes three SubSeries. The first is a pilot microarray time-course analysis (45' to 72 hrs) of CD4+ T cells from a single pool of donors, performed to define the key timepoints of the response. The second is a microarray analysis of responses, in 5 defined conditions (unbiased stimulation at 4 and 48 hrs, Th17 conditions, IFNb supplementation) of CD4+ T cells from 15 healthy individuals. The third is a focused signature analysis (Nanostring) of responses of  236 transcripts (also in the same 5 conditions) in CD4+ T cells of 348 healthy donors.


title: Human perirenal adipose tissue

status: Public on Jul 11 2014

city: Gothenburg

first: Per-Arne

last: Svensson

department: Department of molecular and clinical medicine

country: Sweden

institute: institute of medicine

summary:
Analysis of perirenal adipose tissue from healthy kidney donors (age 44±9 years, BMI 25.8±3.3 kg/m2, mean±SD).
The samples were taken bylaparoscopic technique beneath the incision plane that was created in the renal fascia (Greotas fascia) close to the renal vein.

overall_design: Ten samples of human perirenal adipose tissue

first: Maria

middle: K

last: Pereira


title: SM5-1-conjugated gold nanoparticles for antibody therapy in hepatocellular carcinoma

city: Beijing

first: ma

middle: xi

last: bo

address: No.95,Zhongguancun East Road

country: China

institute: Chinese Academy of Sciences

summary:
As a humanized mouse antibody, SM5-1 can target a membrane protein of about 230kDa over-expressed in hepatocellular carcinoma (HCC), melanoma and breast cancer and it has been found to inhibit the progress of tumor cells. In this study, SM5-1 conjugated gold nanoparticles were prepared to study the antitumor efficacy in the treatment of HCC-LM3-fLuc tumor. The results showed that AU-SM5-1 could inhibit HCC-LM3 hepatocellular carcinoma cell proliferation up to 71.26% at the concentration of 0.5mg/ml contrast with SM5-1 and gold nanoparticles.
In order to address the mechanism of the antiproliferative effects of AU-SM5-1, we examined the gene expression in HCC-LM3-fLuc  tumor cells based on gene-chip screening.

overall_design: The gene chip results showed that some cycle-related and reactive oxygen species (ROS) related genes including up-regulated P21 and down-regulated duox2 and nox1 genes which were validated by real-time quantitative polymerase chain reaction (PCR).

first: Xibo

last: Ma

first: Yushen

last: Jin


title: Effects of Lipocalin 2 (Lcn2), iron, and the bacterial siderophore Enterobactin on A549 respiratory cell gene expression

status: Public on Jul 03 2014

city: Ann Arbor

first: Rork

last: Kuick

state: MI

address: University of Michigan, SPH II, 1415 Washington Heights, Room M2533

institute: University of Michigan

summary: Iron is essential for many cellular processes and is required by bacteria for replication. To acquire iron from the host, pathogenic Gram-negative bacteria secrete siderophores, including Enterobactin (Ent). However, Ent is bound by the host protein Lipocalin 2 (Lcn2), preventing bacterial reuptake of aferric or ferric Ent.  In two experiments we treated A549 (lung cancer cell line) cells with Lcn2, Ent, and iron, alone and in combination.  In experiment 1, biological duplicates of 4 conditions were used: PBS control, Lcn2, Lcn2+Ent, and Lcn2+Ent+iron. In experiment 2, 4 biological replicates of 4 conditions were used: PBS control, Ent, iron, and Ent+iron.  Targets made from the samples were hybridized to Affymetrix Human Gene 1.0 ST arrays to measure transcript abundances.  The RMA algorithm was used to estimate transcript levels. Replicate samples were exchangeable, so we fit one-way ANOVA models to log2-transformed data separately to each experiment, and tested for pairwise differences between groups in each experiment, as well as asking if the Ent vs. PBS differences were larger or smaller than the Ent+iron vs. iron differences (Ent by iron interactions).  We report results for 29096 probe-sets that were not annotated as positive or negative controls on the array.  A supplementary Excel workbook is provided that contains the estimated expression level, some probe-set annotation, and simple statistical analysis for each probe-set.  It may be convenient for some users, however obtaining newer probe-set annotation may be advisable.

overall_design: A549 (ATCC CCL-185) cells, a human type II pneumocyte cell line, were cultured in F12K (Invitrogen, Carlsbad, CA) media supplemented with 10% fetal bovine serum (Invitrogen) and 1:100 penicillin streptomycin (Invitrogen).  24-well plates were seeded with A549 cells at a concentration of 35000 cells/well. After two days, cells were weaned from serum and antibiotics overnight. Cells were then stimulated overnight with the indicated combinations of 50 uM Ferric ammonium citrate (FAC) (Sigma, St. Louis, MO), 50 um Ent (Sigma or EMC Microcollections, Tubingen, Germany), and/or 25 uM lipocalin 2 (Lcn2) in F12K media lacking serum or antibiotics. Prior to incubation with cells, siderophore-Lcn2 complexes were prepared by sequential incubation at room temperature of FAC and Ent for 30 minutes followed by addition of Lcn2 and incubation for an additional 30 minutes. In experiment 1, 2 biological replicates of each of these 4 conditions were grown: PBS,   25 uM lipocalin 2 (Lcn2), a combination of 25 uM Lcn2 with 50 uM Enterobactin (Ent),  and a combination of Lcn2, Ent and 50 uM Ferric Ammonium Citrate (FAC). In experiment 2, 4 biological replicates of each of these 4 conditions were grown: PBS, 50 uM Ent, 50 uM FAC, and a combination of 50 uM Ent and 50 uM FAC. Cells were harvested and RNA was extracted using an miRNeasy Mini Kit (Qiagen). Biotinylated cDNA targets were prepared according to the Ambion WT expression kit protocol from 150 ng total RNA.  There are no batches within each experiment - the samples in each group are exchangeable.  Targets were hybridized to Affymetrix Human Gene 1.0 ST arrays.  Arrays were scanned and quantified by usual procedures, and transcript abundances for 29096 non-control probe-sets estimated by an RMA algorithm.

first: Victoria

middle: I

last: Holden

first: Steven

last: Lenio

first: Michael

middle: A

last: Bachman


title: Gene expression in normal and tumor samples from patients with HNSCC

status: Public on Jul 07 2014

city: Jackson

first: Sharon

last: Lobert

state: MS

address: 2500 N. State St.

department: School of Nursing

institute: University of Mississippi Medical Center

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

summary:
Tissue samples were collected from patients diagnosed with HNSCC (oropharynx, hypopharynx, larynx). Samples were taken from the tumor site (tumor samples) and from a site distant to the tumor (normal samples) prior to therapy.
The study goals were to 1) compare the expression of microRNAs in tumor and normal samples from individual patients and 2) determine whether the differences in expression of microRNAs is associated with patient survival outcomes.

overall_design: Fifteen paired normal and tumor samples from individual patients were analyzed (a total of 30 samples).


title: Aromatic inhibitors derived from ammonia-pretreated lignocellulose hinder bacterial ethanologenesis by activating regulatory circuits controlling inhibitor efflux and detoxification (I)

status: Public on Jul 18 2014

city: Madison

first: Robert

middle: C

last: Landick

state: WI

address: 1550 Linden Dr

department: Great Lakes Bioenergy Research Center

institute: University of Wisconsin - Madison

summary: Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5‑hydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts.

overall_design: E.coli ethanologen strain GLBRCE1 was grown in 3 media, AFEX corn stover hydrolysate (ACSH), synthetic hydrolysate (SynH) and syntetic hydrolysate with added lignotoxins (SynH_LT).  Fermentations were carried out in 3 L bioreactors (Applikon Biotechnology) containing 2.45 L of ACSH or SynH media, and cultures were diluted into ACSH or SynH with initial OD at 0.2, grown anaerobically overnight, and then inoculated into bioreactors to a starting OD600 of 0.2.  3 biological replicates (independent cultures) were grown in each medium.  RNA samples were obtained at 4 time points, corresponding to exponential (Exp), transitional (Trans), stationary (Stat1) and late stationary (Stat2) growth phases.

first: David

last: Keating

first: Yaoping

last: Zhang

first: Irene

last: Ong

first: Sean

last: McIlwain

first: Eduardo

last: Morales

first: Jeffrey

last: Grass

first: Mary

last: Tremaine

first: William

last: Bothfeld

first: Alan

last: Higbee

first: Arne

last: Ulbrich

first: Allison

last: Balloon

middle: S

last: Westphall

first: Josh

last: Aldrich

last: Lipton

first: Joonhoon

last: Kim

first: Oleg

last: Moskvin

first: Yury

last: Bukhman

first: Joshua

last: Coon

first: Patricia

last: Kiley

first: Donna

last: Bates


status: Public on Jul 01 2014

city: Tokyo

first: Jun-ichi

last: Satoh

address: 2-522-1 Noshio, Kiyose

department: Bioinformatics

country: Japan

institute: Meiji Pharmaceutical University

summary: Inflammasome, activated by pathogen-derived and host-derived danger signals, constitutes a multimolecular signaling complex that serves as a platform for caspase-1 (CASP1) activation and interleukin-1beta (IL1B) maturation. The activation of NLRP3 inflammasome requires two-step signals. The first “priming” signal (Signal 1) enhances gene expression of inflammasome components. The second “activation” signal (Signal 2) promotes the assembly of inflammasome components. Deregulated activation of NLRP3 inflammasome contributes to the pathological processes of Alzheimer’s disease (AD) and multiple sclerosis (MS). However, at present, the precise mechanism regulating NLRP3 inflammasome activation and deactivation remains largely unknown. By genome-wide gene expression profiling, we studied the molecular network of NLRP3 inflammasome activation-responsive genes in a human monocyte cell line THP-1 sequentially given two-step signals. We identified the set of 83 NLRP3 inflammasome activation-responsive genes. Among them, we found the NR4A nuclear receptor family NR4A1, NR4A2, and NR4A3, the EGR family EGR1, EGR2, and EGR3, the IkappaB family NFKBIZ, NFKBID, and NFKBIA as a key group of the genes that possibly constitute a negative feedback loop for shutting down inflammation following NLRP3 inflammasome activation. By molecular network analysis, we identified a complex network of NLRP3 inflammasome activation-responsive genes involved in cellular development and death, and immune and inflammatory responses, where transcription factors AP-1, NR4A, and EGR serve as a hub. Thus, NLRP3 inflammasome activation-responsive genes constitute the molecular network composed of a set of negative feedback regulators for prompt resolution of inflammation.

overall_design: To load the Signal 1 (S1), THP-1 cells were incubated for 3 hours in the culture medium with or without inclusion of 0.2 microgram/ml lipopolysaccharide (LPS). To load the Signal 2 (S2), they were incubated further for 2 hours in the culture medium with inclusion of 10 microM nigericin sodium salt dissolved in ethanol or the equal v/v% concentration of ethanol (vehicle), followed by processing for microarray analysis on Human Gene 1.0 ST Array (Affymetrix).


title: Cerebrovascular blood microRNA

city: Sacramento

first: Glen

last: Jickling

state: CA

institute: University of California Davis

summary: blood miRNA expression in ischemic stroke compared to controls

overall_design: miRNA expression in blood cells from patients with ischemic stroke were compared to controls with vascular risk factors


title: RNA profiling from ovarian FFPE specimens

first: Svitlana

last: Tyekucheva

address: 450 Brookline Ave

institute: Dana-Farber Cancer Institute

summary: To study feasibility of gene expression profiling from FFPE tissues using NuGen amplified mRNA hybridized on Affymetrix GeneChip Human Gene 1.0 ST arrays, we designed a pilot study utilizing samples from prostate cancer cohort. We selected samples from large-scale epidemiologic studies and clinical trials representative of a wide variety of fixation times, block ages and block storage conditions.

overall_design: Five serous carcinoma and six clear cell carcinoma samples with technical replicates


title: A High-Resolution Copy Number Variation Resource for Clinical Genetics

status: Public on Jul 24 2014

city: Toronto

first: Stephen

middle: W

last: Scherer

state: Ontario

address: 686 Bay Street

department: Genetics and Genomic Biology

country: Canada

institute: The Hospital for Sick Children

type: Genome variation profiling by genome tiling array

summary:
Purpose: Chromosomal microarray analysis (CMA) to assess copy number variation (CNV) content is now used as a first tier genetic diagnostic test for individuals with unexplained neurodevelopmental disorders (NDD) or multiple congenital anomalies (MCA). Over 100 cytogenetic labs worldwide are using the Affymetrix CytoScan HD 2.7M array to genotype >15,000 clinical samples per month. The aim of this study is to develop a CNV resource from a population control cohort that can be used as a community resource for interpretation of clinical and research samples.
Methods: We have genotyped a large population control set (1,000 individuals from our Ontario Population Genomics Platform (OPGP)) using the Affymetrix CytoScan HD microarray comprising 2.7 million probes. Four independent algorithms were applied to detect and assess high confidence CNVs. Reproducibility and validations were quantified using sample replicates and Quantitative-PCR (QPCR), respectively.
Results: DNA from 873 individuals from the OPGP cohort passed quality control and we have identified 71,178 CNVs (81 CNVs/individual) distributed across 796 different cytogenetic regions in the genome; 9.8% of the CNVs were previously unreported. After applying three layers of filtering criteria, from our high confidence CNVs dataset, we obtained a >95% reproducibility and >90% validation rate. Due to the array's high probe density within genic regions, our high confidence CNV data set show 73% of the detected CNVs overlapped at least one gene.
Conclusion: The genotype data and annotated CNVs presented in this study will represent a valuable public resource enabling clinical genetics research and diagnostics.

overall_design:
For array quality control, CEL files were processed using modules from the Affymetrix power tools and genotypes were extracted from the CHP file. Samples passing the median of the absolute pairwise differences (MAPD) < 0.20 and waviness-sd < 0.11 were retained for further analysis. After multiple checks, we excluded 52 samples that do not meet quality control (QC) cutoffs. To confirm the sample's self-reported gender, we have matched the sex chromosome information from the array and identified six samples with gender mismatch, which were excluded from the analysis. We also excluded 47 samples due to excessive CNV calls. A final set of 895 samples were used for further analysis. This number included 22 sample replicates (indicated by _1 following the Sample title), which were used to determine reproducibility of the array calls.

The CNV data for this study is available from dbVar (NCBI), DGVa (EBI) accession number estd212, and DGV.

first: Mohammed

last: Uddin


title: RNA profiling from prostate FFPE specimens

overall_design: We profiled seven paired tumor and adjacent normal prostate tissue samples from three patients with Gleason score 8, one with Gleason score 7 and three with Gleason 6 disease. 11 samples had two or three technical replicates.


title: RNA profiling from ovarian and prostate FFPE specimens

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

overall_design: Refer to individual Series


title: Effect of HPV 16 E6, E7 oncoproteins on the expression level of cellular genes

status: Public on Jun 26 2014

city: Debrecen

first: György

last: Veress

address: Nagyerdei krt. 98

department: Department of Medical Microbiology

country: Hungary

institute: University of Debrecen

summary: The life cycle of human papillomaviruses (HPV) is strictly linked to the differentiation of their natural host cells. The HPV E6 and E7 oncoproteins can delay the normal differentiation program of keratinocytes, however, the exact mechanisms responsible for this have not yet been identified. The goal of this study was to investigate the effects of HPV16 oncoproteins on the expression of genes involved in keratinocyte differentiation. Primary human keratinocytes transduced by LXSN (control) retroviruses or virus vectors expressing HPV16 E6, E7 or E6/E7 genes were subjected to gene expression profiling. The results of microarray analysis showed that HPV 16 E6 and E7 have the capacity to down-regulate the expression of several genes involved in keratinocyte differentiation. Quantitative real-time polymerase chain reaction (qRT-PCR) assays were performed to confirm microarray data. To investigate the effects of the HPV oncoproteins on the promoters of selected keratinocyte differentiation genes, luciferase reporter assays were performed. Our results suggest that the HPV 16 E6 and/or E7 oncogenes are able to down-regulate the expression of several genes involved in keratinocyte differentiation, at least partially by down-regulating their promoter activity. This activity of the HPV oncoproteins may have a role in the productive virus life cycle, and also in virus induced carcinogenesis.

overall_design: Primary human foreskin keratinocytes were transduced by retrovirus vectors containing HPV 16 E6, E7, E6/E7 or the control vector LXSN. The global gene expression patterns of transduced keratinocytes were analyzed on Affymetrix microarrays

first: Eszter

last: Gyöngyösi

first: Anita

last: Szalmás

first: Annamária

last: Ferenczi

first: Szilárd

last: Póliska

first: József

last: Kónya


title: Transcriptional signature of Th17 cells expressing ICOS-based CARs

city: Philadelphia

first: Carl

last: June

state: Pennsylvania

address: 3400 Civic Center Blvd, TRC 8th Floor 8-110E

department: Pathology and laboratory medicine

institute: University of Pennsylvania

summary:
Analysis of TH17 cells redirected with chimeric antigen receptors (CAR) expressing various signaling domains (including CD28, 4-1BB and ICOS) after surrogate antigen stimulation.
Our results showed that T cells redirected with an ICOS-based CAR specifically retained a genotype of TH17 cells with expression of Il17a, Il17f, Il1r1, Ccl20, Rorc, and in the absence of Foxp3

overall_design: CAR-redirected TH17 cells from three different human normal donors were stimulated with immobilized recombinant mesothelin. Gene expression levels were determined prior to stimulation (day 0) and 4, 8, 24 and 96 hours upon antigen recognition.

first: Sonia

last: Guedan


title: Genes regulated by TAZ in lung cancer cell lines

status: Public on Jun 25 2014

first: Akira

last: Saito

address: 7-31-, Hongo, Bunkyo

institute: Tokyo University

summary: To investigate the roles of TAZ in lung cancer cell proliferation, we compared the expression profiles of A549 and H441 lung adenocarcinoma cell lines transfected with control siRNA and siTAZ.

overall_design: We collected RNA from A549 and H441 cell lines transfected with control siRNA and siTAZ.

first: Satoshi

last: Noguchi

first: Asayo

last: Imaoka

first: Mitsuhiro

last: Ohshima

first: Yoshimitsu

last: Abiko

first: Patrick

last: Micke

first: Takahide

last: Nagase


title: MicroRNA (miRNA) expression of IMCD tubule suspension

status: Public on Jun 21 2014

city: Daegu

first: Jae-Eun

last: Kim

address: Dongin-dong 101

department: Department of Biochemistry and Cell Biology

institute: School of Medicine, Kyungpook National University

summary:
miRNA plays a role as post-transcriptional regulator. However, miRNAs in the kidney collecting duct cell have not been well understood. So we aimed to profile miRNAs in the kidney inner medullary collecting duct (IMCD) cells, and to identify the vasopressin-responsive miRNAs in the kidney IMCD cells.
The microarray assay revealed that relative expression of miRNAs in the kidney IMCD cells was changed by desmopressin (dDAVP) stimulation.

overall_design: Fresh IMCD tubules were prepared from rat kidney and incubated in the medium in the absence or presence of dDAVP (1 nM, 2 h). Total RNA purified from IMCD tubules was used to analyze miRNA expression by GeneChip® miRNA 3.0 Array (Affymetrix).

first: Hyun

last: Jung

first: Tae

last: Kwon


title: Expression data from 13 week human fetal scalp epidermis sorted for expression of alpha 6 integrin and CD133

city: New York

first: Denise

last: Gay

address: 550 1st avenue

institute: NYU langone

summary:
CD133 is expressed by a subpopulation of human fetal hair follicle placode cells during early hair development. Its expression, which is gradually lost as the placode matures, correlates with early morphogenesis.
We used microarrays to analyze differences in gene expression between alpha-6 integrin+CD133+ basal cells and other human fetal epidermal populations to provide candidates defining this unique hair follicle placode subpopulation.

overall_design: 13 week human fetal scalp epidermis was sorted into 3 populations based upon expression of alpha 6 integrin and CD133 and RNA analyzed for gene expression.

first: George

last: Cotsarelis


title: Expression data for Control and SMRT-Depleted MCF-7 Cells

city: Houston

first: Carolyn

middle: L

last: Smith

state: Texas

address: One Baylor Plaza

department: Molecular and Cellular Biology

institute: Baylor College of Medicine

summary:
Estrogens are an important regulator of breast cancer disease progression, and they function by binding the estrogen receptor-α (ERα) to regulate changes in gene expression. ERα is able to both activate and inhibit gene transcription in a gene-specific manner and do so by binding target DNA sequences and recruiting coactivators and corepressors which can modulate the chromatin environment. Silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) is known to act as coactivator and corepressor of ERα in a gene-specific manner.
We used a microarray analysis to examine the gene expression changes that occur when the coregulator SMRT is depleted from the ERα positive MCF-7 breast cancer cell line.

overall_design: We sought to determine the genes that are regulated by depletion of the coregulator SMRT using Affymetrix Human Gene 1.0 ST Array. To this end, we transfected MCF-7 cells with control siRNA  or SMRT-targeting siRNA for 48 h and treated for an additional 4 or 24 h with vehicle (0.1% EtOH) or 1 nM estradiol (E2). A total of 24 samples were analyzed, separated into eight groups each with three experimental replicates in each group, siControl-Veh 4 h, siControl -E2 4 h, siSMRT-Veh 4 h, siSMRT-E2 4 h, siControl-Veh 24 h, siControl-E2 24 h, siSMRT-Veh 24 h, siSMRT-E2 24 h.

first: Sudipan

last: Karmakar

first: Julia

last: Blackmore

first: Liguo

last: Wang

first: Wei

last: Li


title: TCAB1：A Potential Target for Diagnosis and Therapy in Head and Neck Cancers

status: Public on Jun 15 2014

city: Chengdu

first: chongkui

last: sun

state: Sichuan

address: No.14, Sec. 3, Renmin Road South

department: State key laboratory of oral diseases

institute: Sichuan University

summary: Previous studies indicated TCAB1, also known as WRAP53, had oncogenic feature in a certain extend. However, there is none direct study on the function of TCAB1 in tumorigenesis and development of head and neck cancers. First of all, we verified the function of TCAB1 in head and neck cancers. Knockdown TCAB1 would inhibit cell proliferation in Vitro as well as in Vivo, meanwhile, depletion TCAB1 would decrease the invasion potential of OSCC Cal-27 cells. cDNA microarray analysis showed many pathways and factors associated with occurrence and development of carcinomas were implicated in this process. Our study indicated TCAB1 might be a potential target for prognosis and therapy in head and neck cancers.

overall_design: Immunohistochemistry assay and western blot showed TCAB1 was overexpressed in clinical nasopharyngeal carcinoma and head and neck cell lines. Depletion endogenous TCAB1 by shRNA lentivirus in Cal-27 would verify the function of TCAB1 in cells proliferation and invasion. Furthermore, cDNA microarray was performed to detect some pathways or factors involved in the process.

first: Chongkui

last: Sun

first: Ling

last: Hu

first: Yaping

last: Gou

first: Kun

first: Ping

last: Zhang

first: Chaoliang

first: Xiangli

last: Kong

first: Liying

last: Xiao

first: Qin

last: Yang

first: Qianming

last: Chen

first: Yan


title: Sex dependent gene expression in human pluripotent stem cells

status: Public on Jun 18 2014

city: Jerusalem

first: Daniel

last: Ronen

address: Edmond J. Safra Campus, Givat Ram

department: Genetics

country: Israel

institute: The Hebrew University of Jerusalem

summary:
During early development before gonadal differentiation, sex chromosomes are the main difference  between males and females. We examined any genetically  driven sex dimorphisms  in human pluripotent stem cells focusing on Y chromosome contribution.
In order to understand the consequence of these differences, human ES cells were differentiated  into EBs under the influence of Estrone (E1) and the global expression diffrences between male and females were examined.

overall_design: Male and female ES cells were differentiated by EB formation under E1 for RNA extraction and hybridization on Affymetrix GENE ST 1.0 microarrays (GPL6244). RNA was extracted  at 0d 3d and 11d

first: Nissim

last: Benvenisty


title: Affymetrix CytoScan HD array data for patient with Intellectual Disabilities

city: México.

first: Ruth

last: Ruiz-Esparza

address: Centro Médico Nacional Siglo XXI, IMSS, México,

department: Pediatría

country: Mexico

institute: Instituto Mexicano del Seguro Social

summary: Standard cytogenetic GTG-banding analysis (550 bands) revealed an interstitial deletion on 15q21q22 chromosomal region. Refinement of the 15q21.2 deletion intervals was conducted using aCGH (Cytoscan HD, Affymetrix) on DNA samples from the patient. This assay revealed the presence of submicroscopic alterations on chromosomes 1, 9 and 15. The result was arr[hg19] 9p24.1(6,619,823-6,749,335)x3, 1q44(248,688,586-248,795,277)x1, 15q21.2q22.2(50,848,301-61,298,006)x1.

overall_design: Affymetrix CytoScan HD arrays were performed according to the manufacturer's directions on DNA extracted from peripheral blood samples.

first: Ana

last: Velazquez-Wong

middle: R

last: Esparza-Garrido


title: Expression data from biopsies of TGP patients

city: Bronx

first: Pilib

last: Ó Broin

state: NY

address: 1301 Morris Park Ave

institute: Albert Einstein College of Medicine

summary:
We study the global gene expression profiles of TGP patients with or without graft loss to determine if a clinical and/or gene expression profile can predict allograft survival.
Transplant glomerulopathy (TGP) carries a poor prognosis and is associated with decreased allograft survival. In a large series of kidney transplant recipients, graft loss was observed in 38% of TGP patients at 5 years compared to 5% in patients without TGP. Among patients with TGP, predictors of poor outcome included C4d deposition in the peritubular capillaries, higher serum creatinine and proteinuria at diagnosis, the severity of interstitial fibrosis, and GBM duplication (higher Banff ‘chronic glomerulopathy [cg]’ score), and the presence of DSA. However, the importance of microvascular inflammation determined by peritubular capillaritis (ptc) and glomerulitis (g) scores and the strength of DSA has not been explored thoroughly. We have recently reported the intragraft gene expression profiles of TGP patients, with or without DSA. However, the association between gene expression profiles of biopsies and the allograft survival has not been investigated. In this study, in addition to known clinical and demographic factors associated with poor outcomes in TGP, we specifically aimed to investigate if intragraft gene expression profiles by microarrays, microvascular inflammation scores, and the strength of DSA determined by mean fluorescence intensity (MFI) values could predict allograft loss in TGP patients.

overall_design: The study population was derived from renal allograft biopsies diagnosed with TGP at Montefiore Medical Center, Bronx, NY from January 2009 to December 2012. All biopsies were clinically indicated for elevated creatinine or proteinuria. A retrospective chart review was conducted to record clinical, demographic, immunological, and histopathological parameters and graft outcomes.  Graft loss was defined as requiring dialysis and/or retransplantation during follow-up. Of the 525 patients who underwent clinically indicated transplant kidney biopsies, 52 patients (10%) had diagnosis of TGP. Median follow up time after the biopsy was 23 months (range, 1-46). Seventeen patients (32%) lost their allografts (defined by return to dialysis or re-transplantation) at a median time of 16 months (range, 1-44). A subgroup of patients who were enrolled in the IRB-approved “Immune Monitoring Study” and had clinically indicated biopsy samples were included in the analysis. A total of 28 biopsy samples (8 graft loss and 20 functioning allograft) were used for gene expression profiling.

first: Enver

last: Akalin


title: Interferon-gamma critically determines dendritic cell function

status: Public on Jun 01 2014

city: Doha

first: Francesco

middle: Maria

last: Marincola

address: Al Nasr Tower, AL Corniche Street, PO Box 26999

country: Qatar

institute: Sidra Medical and Research Center

summary: A growing body of evidence suggests that inflammatory cytokines have a dualistic role in immunity. In this study, we sought to determine the direct effects IFN-gamma on the differentiation and maturation of human peripheral blood monocyte-derived dendritic cells (moDC). Here, we report that following differentiation of human peripheral-blood monocytes into moDCs with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4, interferon-gamma (IFN-gamma) induces moDC maturation and up-regulates the co-stimulatory markers CD80, CD86, CD95, and MHC Class I, enabling moDCs to effectively generate antigen-specific CD4+ and CD8+ T cell responses for multiple viral and tumor antigens. Interestingly, early exposure of monocytes to high concentrations of IFN-gamma promotes monocyte differentiation into macrophages, despite the presence of GM-CSF and IL-4. However, under low concentrations of IFN-gamma, monocytes continue to differentiate into dendritic cells possessing a unique gene-expression profile, resulting in impairments in subsequent maturation by IFN-gamma and an inability to generate effective antigen-specific CD4+ and CD8+ T cell responses compared to standard moDCs. Monocytes differentiated in the presence of low levels of IFN-gamma downregulate IFN-gamma receptor expression, impairing their response to an inflammatory rechallenge. These findings demonstrate the ability of IFN-gamma to impart differential programs on human moDCs which shape the antigen-specific T cell responses they induce. Timing and intensity of exposure to IFN-gamma can thus determine whether moDCs are tolerogenic or immunostimulating.

overall_design: Human monocyte-derived dendritic cells from 4 healthy donors were differentiated with either GM-CSF and IL-4 (n=4) or GM-CSF, IL-4, and IFN-gamma (n=4). These samples were subsequently hybridized to arrays as 4 biological repeats for each of the two treatment conditions.


title: LMKB/MARF1 localizes to mRNA processing bodies, interacts with Ge-1, and regulates IFI44L gene expression

status: Public on Jun 09 2014

city: Boston

first: Rajeev

last: Malhotra

address: Yawkey 5700, 55 Fruit St

department: Medicine

institute: MGH

summary: The mRNA processing body is a cellular structure that regulates the stability of cytoplasmic mRNA.  MARF1/LMKB is an RNA-binding protein that is associated with maintenance of mRNA homeostasis and genomic stability.  To investigate the function of LMKB in a human B lymphocyte cell line, BJAB cells were treated with either control lentivirus or a lentivirus containing LMKB siRNA.

overall_design: 3 controls and 3 LMKB-KD samples

first: Donald

last: Bloch

first: Pingcheng

middle: P

last: Li

first: Emily

middle: G

middle: F

last: Berenson

first: Rita

last: Galdos

first: Pankaj

last: Arora

first: Connie

last: Wu

first: Weihong


title: Translational validation of personalized treatment strategy based on genetic characteristics of glioblastoma [xenograft]

status: Public on Jun 12 2014

city: Seoul

first: Young Taek

last: Oh

address: 50 Irwon-dong, Gangnam-gu

institute: Samsung Medical Center

summary: Glioblastoma (GBM) heterogeneity in the genomic and phenotypic properties has potentiated personalized approach against specific therapeutic targets of each GBM patient. The Cancer Genome Atlas (TCGA) Research Network has been established the comprehensive genomic abnormalities of GBM, which sub-classified GBMs into 4 different molecular subtypes. The molecular subtypes could be utilized to develop personalized treatment strategy for each subtype. We applied a classifying method, NTP (Nearest Template Prediction) method to determine molecular subtype of each GBM patient and corresponding orthotopic xenograft animal model. The models were derived from GBM cells dissociated from patient's surgical sample. Specific drug candidates for each subtype were selected using an integrated pharmacological network database (PharmDB), which link drugs with subtype specific genes. Treatment effects of the drug candidates were determined by in vitro limiting dilution assay using patient-derived GBM cells primarily cultured from orthotopic xenograft tumors. The consistent identification of molecular subtype by the NTP method was validated using TCGA database. When subtypes were determined by the NTP method, orthotopic xenograft animal models faithfully maintained the molecular subtypes of parental tumors. Subtype specific drugs not only showed significant inhibition effects on the in vitro clonogenicity of patient-derived GBM cells but also synergistically reversed temozolomide resistance of MGMT-unmethylated patient-derived GBM cells. However, inhibitory effects on the clonogenicity were not totally subtype-specific. Personalized treatment approach based on genetic characteristics of each GBM could make better treatment outcomes of GBMs, although more sophisticated classifying techniques and subtype specific drugs need to be further elucidated.

overall_design: Gene expression profiling experiments were conducted for 25 patient-derived xenograft glioblastoma samples using Affymetrix Human Gene 1.0 ST arrays according to manufacturer's protocol.

first: Kyeung

last: Joo

first: Jinkuk

last: Kim

first: Juyoun

last: Jin

first: Misuk

first: Ho

middle: J

last: Seol

first: Do-Hyun

last: Nam


title: Gene expression changes in a breast cancer stem cell model.

status: Public on Jun 22 2014

city: Bruce

first: Kristine

last: Hardy

state: ACT

address: University of Canberra

institute: University of Canberra

summary:
Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. The epithelial cell line MCF7, can be induced to undergo EMT with the induction of PKC by PMA. 5-10% of the resulting cells have a CSC phenotype. This study looks at the transcriptome of these cells and how it differs from cells with a non-CSC phenotype.
Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. The epithelial cell line MCF7, can be induced to undergo EMT with the induction of PKC by PMA. 5-10% of the resulting cells have a CSC phenotype. This study looks at the transcriptome of these cells and how it differs from cells with a non-CSC phenotype.

overall_design: MCF7 cells were stimulated with PMA and FACS sorted. 4 samples, no replicates

first: Anjum

last: Zafar

first: Sudha

last: Rao


title: Translational validation of personalized treatment strategy based on genetic characteristics of glioblastoma [surgical samples]


title: Translational validation of personalized treatment strategy based on genetic characteristics of glioblastoma


title: Comparison of gene expression between mandibular and iliac bone-derived cells

status: Public on Jun 14 2014

first: Tae-Geon

last: Kwon

address: Samduck 2 Ga, Jung Gu

department: Department of Oral & Maxillofacial Surgery

institute: School of Dentistry Kyungpook National University

summary:
Recently, there is increasing report of bone necrosis exceptionally in jaw bone areas after long term administration of anti-resorptive agent, bisphosphonate (BP). It is still unknown that why BP does not result in osteonecrosis but potentiate fracture repair in axial skeleton such as long bones. Many reports had shown that mandibular bone or calvarial bone exhibits lesser resoption than iliac bone graft for maxillofacial bone defect.(Jackson et al 1986, Koole et al 1989),(Crespi et al 2007) These might be attributed to the embryological difference between the two different type of the bone; intramembranous versus endochondral bone formation. Maxilla and mandibular alveolar and basal bone are originated from neural crest cells and exhibit intramembranous bone formation.(Chai and Maxson 2006) However, axial skeleton such as iliac or tibial bones are originated from mesoderm and undergo endochondral bone formation.(Helms and Schneider 2003)    It had been reported that there is distinct phenotype difference in human bone cells from different skeletal origin.(Akintoye et al 2006, Kasperk et al 1997),(Matsubara et al 2005) Akintoye et al.(Akintoye et al 2006) reported that mandibular bone marrow stromal cells (BMSCs) displayed a fibroblast-like morphology similar to iliac BMSCs in histological appearance. However, mandibular bone contained less red marrow and hematopoietic cells, whereas the iliac bone contained more red marrow thereby contributing more to hematopoiesis. This differential capacity of mandibular BMSCs with less hematopoietic stem cells is better than that of iliac BMSCs because mitosis in hematopoietic stem cells is usually stopped. They also reported that maxillofacial BMSCs shows higher proliferation rate and alkaline phosphotase activity.(Akintoye et al 2006, Akintoye et al 2008) However, in vivo response of human BMSCs to osteogenic induction was higher in iliac bone than maxillomandibular bone.(Akintoye et al 2006) Other reports had shown that BMSCs from mandible showed better in vitro and in vivo bone formation capacity compared to tibia from rats.(Aghaloo et al 2010) Matsubara et al. reported that osteogenic potential of human or canine alveolar bone and iliac bone-derived cells (BC) are similar but showed difference in chondrogenic and adipogenic potential.(Matsubara et al 2005) From the previous studies, the site-related difference of mandibular and iliac BC are clear. However, the pattern of the site-specificity are not exactly same among above mentioned studies. These might be attributed to the difference in the experimental subjects (species, age) and study design (in-patient or random comparison, etc). Moreover, underlying genetic mechanism of these skeletal-site dependent difference had not been fully investigated.   The investigation of site-specific differences in the gene expression would be help to understand the mechanism of bisphosphonate-related osteonecrosis of the jaw (BRONJ), which recently has become an major issue in dentistry. Considering that bisphosphonate is frequently prescribed to elderly patients who have a higher risk of osteoporosis, it is necessary to conduct a experimental analysis of cells from older-aged donors. It had been suggested that anatomical site-specific difference can be influenced by 1) difference in the regional blood supply, 2) difference in bone composition (lamellar bone versus trabecular bone), 3) difference in physiological strain exerted to bone, 4) difference in gene expression according to skeletal site.(Kasperk et al 1997)   In this study, bone cells derived from elderly donors went through microarray analysis under the hypothesis that there would be the differences in gene expressions involved in bone formation or cell proliferation of bone cells from iliac and mandible. The purpose of this study was to investigate the difference in gene expression between the human mandible and iliac bone so that we can understand the genetic difference in the two different type of bones.            <<<References>>>    1. Jackson IT, Helden G, Marx R. Skull bone grafts in maxillofacial and craniofacial surgery. J Oral Maxillofac Surg 1986; 44:949-955.  2. Koole R, Bosker H, van der Dussen FN. Late secondary autogenous bone grafting in cleft patients comparing mandibular (ectomesenchymal) and iliac crest (mesenchymal) grafts. J Craniomaxillofac Surg 1989; 17 Suppl 1:28-30.  3. Crespi R, Vinci R, Cappare P, Gherlone E, Romanos GE. Calvarial versus iliac crest for autologous bone graft material for a sinus lift procedure: a histomorphometric study. Int J Oral Maxillofac Implants 2007; 22:527-532.  4. Chai Y, Maxson RE, Jr. Recent advances in craniofacial morphogenesis. Dev Dyn 2006; 235:2353-2375.  5. Helms JA, Schneider RA. Cranial skeletal biology. Nature 2003; 423:326-331.  6. Kasperk C, Helmboldt A, Borcsok Iet al. Skeletal site-dependent expression of the androgen receptor in human osteoblastic cell populations. Calcif Tissue Int 1997; 61:464-473.      <<< Important Abstract of Associated reference>>>     Kingsmill VJ1, McKay IJ, Ryan P, Ogden MR, Rawlinson SC. Gene expression profiles of mandible reveal features of both calvarial and ulnar bones in the adult rat.J Dent. 2013 Mar;41(3):258-64. doi: 10.1016/j.jdent.2012.11.010. Epub 2012 Nov 23.    OBJECTIVES:  Limb and mandibular alveolar bone of the mandible are susceptible to disuse osteopenia, whilst skull and mandibular basal bone appear to resist excessive generalised bone loss. We wanted to compare the site-specific transcriptome of anatomically and functionally distinct bones to confirm the composite nature of the mandible at the molecular level.  METHODS: Gene expression profiles were obtained for the mandible, ulna, and calvaria of adult male rats using Affymetrix Rat Genome 230 2.0 GeneChips. Ingenuity Pathways Assist generated association maps, and RGD database software identified site-specific pathways.  RESULTS: The majority of expressed transcripts (84%) are common to all three sites. The mandible expressed 873 transcripts in common with ulna but not calvaria, and 1014 transcripts in common with calvaria but not ulna. Transcripts in these groups were excluded if they showed significant differential expression (>2-fold) and the remaining mapped genes were filtered for those related to modulation of gene transcription. Analysis of these genes revealed common pathways shared by the mandible and ulna, or mandible and calvaria, which were not shared by the calvaria and ulna.  CONCLUSIONS: There were relatively few differences in the expression of genes responsible for the bone formation process per se in different functional skeletal sites. Differential transcription factor expression suggests that it is the regulation of bone formation and not the mechanism of bone formation itself that differs between the skeletal sites.  CLINICAL SIGNIFICAN
Objectives: The exact genetic difference between the jaw and long bone had not been clearly understood. The purpose of this study was to investigate the difference in gene expression between the human mandible and iliac bone-derived cells.
Methods: Primary cells were obtained from mandibular and iliac bone from the 6 healthy, elderly donors (average age 60.2 years) during the reconstruction of mandibular alveolar bone defects. To investigate site-specific difference, within-patient comparison were carried out with cell proliferation and osteoblastic differentiation assay. Gene expression profile of mandible and iliac BC (bone-derived cell) were compared using cDNA microarray analysis using Affymetrix GeneChip®.
Results: The mandibular BC showed stronger proliferative capacity but weaker osteoblastic differentiation. The comparison of the gene expression profile identified that 82 genes were significantly up-regulated and 66 genes were down-regulated 1.5 fold or greater in mandible compared to iliac BC. The most significantly differentially regulated genes were associated with skeletal system development and morphogenesis (SIX1, MSX1, MSX2, HAND2, PRRX1, OSR2, HOX gene family, PITX2). Microarray analysis revealed that Msx1 was 2.03 fold and Msx2 was 1.99 fold up-regulated in mandible compared to iliac BC (both p<0.01). HOX gene group in mandibular BC was down-regulated (p<0.01). Osteopontin was also 2.84 fold down-regulated in mandible BC (p<0.01). We found that the results of the microarray were reproducible with qRT-PCR.
Conclusions: Site-specific difference between jaw and long bone can be explained by the different gene expression pattern, which is primarily related with embryological origin of these two different bones.

overall_design: Human mandible and iliac bone chip was were collected from 6 donors (average 60.2 years, ranged from 56 to 69 years; 3 males, 3 females).   Primary cells from this bone chips were utilized to analysis. Therefore 6 sets of iliac and mandibular bone-derived cells were obtained.     <<detailed method>>  The experimental protocol using human tissue was approved by the Institutional Review Board of Kyungpook National University Hospital (KNUH_10-1093). Informed consent was obtained from each donors who were undergoing iliac bone graft to mandibular alveolar bone defect. To collect osteoblast-like primary cells migrated from bone chips cortical or cortico-cancellous bone was harvested with the similar method described previously.   Human mandible and iliac bone chip was were collected from 6 donors (average 60.2 years, ranged from 56 to 69 years; 3 males, 3 females), of healthy general condition without clinical inflammatory lesion in oral cavity. After complete reflection of periosteal layer, the mandibular bone and iliac crest was visualized. Mandibular cortical or cortico-cancellous bone chip was collected by a ronguer during the host bone trimming and decortication procedure for iliac bone graft to edentulous mandible.   Similarly, small amount of iliac cortico-cancellous bone chip was collected seperately during the iliac bone harvesting procedure from the same patient. The harvested bone were broke into small pieces and the size of the bone particle became to be 2~4mm in length. The collected bone samples from the two sites were treated with the same method. The bone sample were gently rinsed with Dulbecco's modified Eagle's Medium (DMEM, Lonza Group Ltd., Basel, Switzerland) containing heparin to exclude fat and blood.   The particulated bone were then transferred into culture flasks and cultured with DMEM supplemented with 20% fetal bovine serum (FBS, GIBCO-Invetrogen, Carlsbad, CA), 100 U/ml penicillin, 100 mg/ml streptomycin sulphate and 2 mM glutamine incubated at 37°C in a humidified atmosphere of 5% CO2 in air. The medium was first changed 2 days after seeding to remove non-adherent cells and the media (DMEM - 20% FBS) was changed every 2 days until the cells migrated out from the explants and reached confluency.   Subconfluent primary cells were trypsinized and replated to 75 Cm2 flask. At passage 2, the cells were harvested to extract RNA for cDNA microarray.

last: KWON

first: Jung-Tae

last: Lee


title: Posterior Amorphous Corneal Dystrophy Is Associated with a Deletion of Small Leucine-rich Proteoglycans on Chromosome 12

status: Public on Jun 04 2014

city: Los Angeles

first: Anthony

middle: J.

last: Aldave

address: 200 Stein Plaza DSERC 3-143

department: Ophthalmology

institute: Stein Eye Institute, UCLA

summary: Posterior amorphous corneal dystrophy (PACD) is a rare, autosomal dominant disorder affecting the cornea and iris. After next-generation sequencing of the family in which linkage analysis identified a chromosomal locus for PACD on 12q21.33 failed to yield a pathogenic mutation, array-based copy number analysis and qPCR detected a deletion on 12q21.33 containing four genes encoding small leucine-rich proteoglycans (SLRPs): KERA, LUM, DCN, and EPYC.  Two other families with PACD also demonstrated deletion of these SLRPs, which together with murine models of KERA, LUM, and DCN deficiency provide convincing evidence that PACD is caused by haploinsufficiency of these SLRPs.

overall_design: Copy number analysis of 750K SNP and 1.9 non-polymorphic probes present on the Affymetrix CytoScan HD array. Samples were separated by affection status. Eight unaffected and 5 affected samples from across 3 families were analyzed. Analysis was performed using the Affymetrix Chromosome Analysis Suite (ChAS) v.2.0 software.

first: Michelle

first: Ricardo

last: Frausto


title: Final efficacy and biomarker analysis of the sorafenib arm of the BATTLE (Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination) trial

first: Pierre

last: Saintigny

address: 1515 Holcombe

department: Thoracic / Head and Neck Medical Oncology

institute: The University of Texas M.D. Anderson Cancer Center

summary:
Purpose: To determine the 8-week disease control rate (DCR) of sorafenib monotherapy in patients with advanced non-small-cell lung cancer (NSCLC) in the BATTLE trial.
Methods: Patients with pre-treated NSCLC consented to baseline biopsies for pre-specified biomarkers assessment and biomarker discovery. Sorafenib was given at 400 mg orally twice daily until tumor progression or unacceptable toxicity. Outcomes by pre-specified biomarkers were analyzed and a Sorafenib sensitivity signature developed using high-throughput gene expression profiles of NSCLC cell lines and baseline biopsies.
Results: 105 patients were eligible and 98 patients were evaluable. Median age was 62 (range 34-81) years, 51% of patients were male, 75% were former/current smokers, and 89% had an ECOG performance status of 0-1.  Median prior chemotherapies for stage IV NSCLC were two. Median follow-up was 9.4 (range: 1.3-32.2) months.  Overall, 8-week DCR was 58.2%. Patients with EGFR mutations had significantly lower 8-week DCR compared to patients with wild-type tumors (23.1% vs. 64.2%, P=0.0119), and patients with K-RAS mutations had the highest 8-week DCR (67%). Most commonly reported treatment-related adverse events include hand-foot syndrome (59.6%), fatigue (42.3%), rash (40.4%), diarrhea (38.5%), and weight loss (38.5%). Sorafenib sensitivity signature developed in cell lines was associated with an improved outcome.
Conclusion: Patients with wild-type EGFR, including those with K-RAS mutation, may benefit from sorafenib as opposed to patients with EGFR mutation. We identify a gene expression signature associated with an improved outcome in patients with wild-type EGFR treated with sorafenib. BATTLE-2 trial is ongoing to validate those results. ClinicalTrials.gov number, NCT00411671.

overall_design: Gene expression profiles were measured in 37 core biopsies from patients with refractory non-small cell lung cancer treated with sorafenib in the  Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial. We report a Sorafenib sensitivity gene expression signature trained in vitro (separate GEO submission), and tested in baseline biopsies collected in the BATTLE trial.

first: Ignacio

last: Wistuba

first: John

middle: V

last: Heymach

first: George

last: Blumenschein Jr.

first: Edward

first: Scott

last: Lippman

first: Roy

last: Herbst

first: Waun

last: Hong

first: J

first: Kevin

last: Coombes

first: Li

last: Mao


title: An EGFR-mutation signature reveals features of the EGFR-dependent phenotype and identifies MACC1 as an EGFR-associated regulator of MET.

summary:
EGFR-mutated non-small cell lung cancers bear hallmarks including sensitivity to EGFR inhibitors,  and low proliferation, and increased MET.  However, the biology of EGFR dependence is still poorly understood. Using a training cohort of chemo-naive lung adenocarcinomas, we have developed a 72-gene signature that predicts (i) EGFR mutation status in four independent datasets; (ii) sensitivity to erlotinib in vitro; and (iii) improved survival, even in the wild-type EGFR subgroup. The signature includes differences associated with enhanced receptor tyrosine kinase (RTK) signaling, such as increased expression of endocytosis-related genes, decreased phosphatase levels, decreased expression of proliferation-related genes, increased folate receptor-1 (FOLR1) (a determinant of pemetrexed response), and higher levels of MACC1 (which we identify as a regulator of MET in EGFR-mutant NSCLC).  Those observations provide evidence that the EGFR-mutant phenotype is associated with alterations in the cellular machinery that links the EGFR and MET pathways and create a permissive environment for RTK signaling.
We have developed a gene expression signature that predicts (i) EGFR mutation in chemo-naive and, to a lesser extent, in chemo-refractory NSCLC patients; (ii) EGFR TKI response in vitro; and (iii) survival in wild-type EGFR patients. The signature also identifies novel features of EGFR mutant NSCLC including increased levels of endocytosis-related genes and MACC1, which appears be an EGFR mutant associated regulator of MET.

overall_design: Gene expression profiles were measured in 124 core biopsies from patients with refractory non-small cell lung cancer in the  Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial. We used the BATTLE dataset to test an EGFR-mutation gene expression signature trained in chemo-naive lung adenocarcinoma. The signature was computed as an index, called EGFR index.

last: Kim

middle: M


title: Microarray analysis of colon carcinoma cell lines

status: Public on May 31 2014

city: Berlin

first: Angela

last: Relogio

address: Invalidenstraße 43

department: Institute for Theoretical Biology (ITB)

institute: Humboldt University of Berlin and Charité Medical University of Berlin

summary: To investigate potential differences between strong and weak oscillators at the gene expression level we carried out a transcriptome analysis for each cell line.   Our results indicate that phenotypic circadian clock differences are reflected by gene expression differences both in genes of the core network, but also in additional genes not directly associated with circadian clock functions.

overall_design: We carried out a gene expression analysis of 6 colon cancer cell lines (HCT116, HT-29, RKO, SW480, LIM1512 and CaCo2) for each the RNA was collected at two time points (0hr and 48 hr after syncronization). As a reference the osteosarcoma U2OS cell line was used. No treatment was applied.

last: Relógio

first: Philippe

last: Thomas

first: Bert

last: Maier

first: Reinhold

last: Schaefer

first: Ulf

last: Leser

first: Achim

last: Kramer

first: Christine

last: Sers


title: Expression data from baseline and post-endurance training in human PBMCs

status: Public on May 30 2014

city: Sao Paulo

first: Rodrigo

middle: Gonçalves

last: Dias

state: Sao Paulo

address: Av. Dr. Eneas Carvalho de Aguiar, 44

country: Brazil

institute: Heart Institute (InCor)

summary:
There is an association between transcriptome and the exercise-related phenotype. Peripheral blood cells suffer alterations in the gene expression pattern in response to perturbations caused by exercise. The acute response to endurance activates stress and inflammation, as well as growth and tissue repair responses.
Peripheral blood mononuclear cell (PBMC) samples were obtained at baseline and after an endurance training protocol from healthy male untrained police recruits. Samples were used to identify differentially expressed transcripts in response to training and further infer about activated metabolic and signalling pathways. Additionally, baseline expression was associated with the oxygen uptake change and stratified individuals according to their percent change in oxygen uptake.

overall_design: Fasting blood samples were withdrawn at baseline and after the exercise training protocol, 48 hours after the last exercise session. RNA was extracted from peripheral blood mononuclear cells, and hybridized into Affymetrix microarrays.

first: Michelle

last: Silva

first: Alexandre

last: Pereira

first: José

middle: E

last: Krieger


title: Whole Adult Organism Transcriptional Profiling of Metal Exposures in Zebrafish

status: Public on Jun 10 2014

city: Ft. Detrick

last: Baer

state: MD

address: 568 Doughten Drive

department: Environmental Health Program

institute: Excet, Inc. / USACEHR

summary: A convergence of technological breakthroughs in the past decade has facilitated the development of rapid screening tools for biomarkers of toxicant exposure and effect. Platforms using the whole adult organism to evaluate the genome-wide response to toxicants are especially attractive. Recent work demonstrates the feasibility of this approach in vertebrates using the experimentally robust zebrafish model. In the present study, we evaluated gene expression changes in whole adult zebrafish following an acute 24 hour exposure to three metals with known human health risks. Male adult zebrafish were exposed to nickel chloride, cobalt chloride, and sodium dichromate concentrations corresponding to their respective 96 hr LC20, LC40 and LC60. Histopathology was performed on a subset of metal-exposed zebrafish to phenotypically anchor transcriptional changes associated with each metal. Comparative analysis identified subsets of differentially expressed transcripts both overlapping and unique to each metal. Application of gene ontology (GO) and transcription factor enrichment algorithms revealed a number of key biological processes perturbed by metal exposures and the master transcriptional regulators mediating gene expression changes. Metal exposures differentially activated biological processes associated with ribosome biogenesis, proteosomal degradation and p53 signaling cascades, while repressing oxygen-generating pathways associated with amino acid and lipid metabolism. Despite appreciable effects on gene regulation, nickel exposures did not induce any morphological alterations in zebrafish organs and tissues. Histopathological effects of cobalt remained confined to the olfactory system, while chromium targeted the gills, pharynx and intestinal mucosa. A number of enriched transcription factors mediate the observed gene response to metal exposure, including known targets such as p53, HIF1 and the myc oncogene and novel regulatory factors such as XBP1, GATA6 and HNF3. This work uses an experimentally innovative approach to capture global responses to metal exposures and provides mechanistic insights into metal toxicity mechanisms.

overall_design: Metal induced changes in gene expression in zebrafish were measured after 24 h exposures to each of three metals (nickel, chromium or cobalt). A total of 48 arrays were processed - 16 arrays per metal with 4 replicates for each exposure condition (control, low, mid, and high).

first: Naissan

last: Hussainzada

first: John

last: Lewis

first: Danielle

last: Ippolito

first: David

last: Jackson

first: Jonathan

middle: D

last: Stallings


title: Differential expression of microRNA in Muc2-/- mouse colonic epithelial cells

city: Chicago

first: Wancai

last: Yang

state: IL

address: 840 South Wood Street

department: Pathology

institute: University of Illinois at Chicago

summary:
Intestinal epithelia are protected by a layer of mucin secreted by goblet cells against mechanical and chemical injuries, potent causes of inflammation, and the most abundant secreted intestinal mucin is encoded by the Muc2 gene. Genetic deletion of Muc2 causes intestinal inflammation in early stage and tumors after 3 months. The underlying mechanisms are not clear, but epigenetic alterations, particularly, up- and down-regulated microRNAs are involved in the malignant transformation from colitis to cancer.
We used miRNA array to profile the differential expression of the miRNAs in Muc2-/- mouse colonic epithelial lin comparison with those in wild-type mice.

overall_design: Total RNA were extracted from mouse colonic epithelial cells and Muc2-/- and +/+, and the RNA were hybridized on Affymetrix miRNA microarray to determine the alterations of miRNAs during colitis development and its malignant transformation from colitis to cancer. To the end, we found miRNA were differential expressed in the Muc2-/- mice, among them 20 miRNAs were significantly downregulated and 71 miRNAs were significantly upregulated  in Muc2-/- mice, in comparison with Muc2+/+ mice (change fold >2 or <0.5; T<0.01, p value< 0.05, q value< 0.05).

first: Yonghua

last: Bao


title: Global impact of Salmonella type III secretion effector SteA on host cells

status: Public on May 28 2014

city: Sevilla

first: Gabriel

last: Gutierrez

address: PO BOX 1095

country: Spain

institute: University of Seville

summary: Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems (T3SSs), encoded in pathogenicity islands 1 (SPI1) and 2 (SPI2), respectively. These systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with certain host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both T3SSs. Nothing is known about the function of this protein inside the host cells. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA.  We found that constitutive synthesis of SteA in epithelial cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also represses genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. Consisted with this analysis a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, reduction of cytotoxicity, cell-cell adhesion and migration capability, and increase in endocytosis.

overall_design: Three experiments. Each experiment replicated three times. Control: HeLa cell transfected with plasmid pBABE without steA, Experiment 1:HeLa line 2 transfected with plasmid pBABE with steA. Experiment 2: HeLa line 4 transfected with plasmid pBABE with steA

first: Francisco

last: Ramos-Morales

first: Elena

last: Cardenal-Muñoz

last: Gutiérrez


title: Gene expression in FIH-1 silenced human nucleus pulposus cells

status: Public on May 27 2014

city: Philadelphia

first: Makarand

last: Risbud

state: PA

address: 1015 Walnut St.

department: Orthopaedic Surgery

institute: Thomas Jefferson University

summary: To evaluate the effect of Hypoxia-inducible factor 1-alpha inhibitor (HIF-1) in nucleus pulposus (NP) cells, human NP cells were lentivirally transduced with either control or FIH-1 targeted shRNA. Gene expression changes between samples from control and FIH-1 silenced cells were evaluated using a microarray.

overall_design: Primary NP cells were isolated from human discs and grown in culture. Cells were transduced with either control or FIH-1 targeting shRNA constructs. Silencing of FIH-1 was confirmed for each set using qRT-PCR and Western blot. Total RNA from NP cells was used for RNA extraction and hybridization on the Affymetrix microarray. Control samples: C1, C2, and C3 and FIH-1-silenced samples: E1, E2, and E3.

first: Y

last: Hirose

first: Z

last: Johnson

last: Schoepflin

first: D

last: Markova

first: K

last: Chiba

last: Toyama

first: I

last: Shapiro

first: M


title: Regulatory interplay of Cockayne syndrome B ATPase and stress-response gene ATF3 following genotoxic stress

status: Public on May 23 2014

city: Illkirch

first: Egly

last: Jean-Marc

address: 1 rue Laurent Fries

institute: IGBMC

summary:
Gene expression profile in CS1AN deficient and CSBwt restored cell lines after 24 hours of UV or alphe-amanitin treatment (only for restored). The comaprison of expression profile between 0 and 24 hours revealed
continouse suppresion of transcription upon UV treatment in CS1AN cell line and alpha-amanitin treated CS1AN CSBwt restored cells.

overall_design: Gene expression profiles were obtained in duplicates for un-treated (assigned as 0h time point) and treated (assigned as 24h time point) cells.

first: Jean-Marc

last: Egly

first: Alexey

last: Epanchintsev


title: Gene profile in fetal human heart and brain

status: Public on May 21 2014

city: madison

first: jesus

last: Iruretagoyena

address: 1010 Mound street

institute: University of wisconsin

summary: To describe normal cardiac and brain development during late first and early second trimester in human fetuses using microarray and pathways analysis and the creation of a corresponding “normal” database.  RNA  from recovered tissues was used for transcriptome analysis with Affymetrix 1.0 ST microarray chip. From the amassed data we investigated differences in cardiac and brain development within the 10-18 GA period dividing the sample by GA in three groups: 10-12 (H1), 13-15(H2) and 16-18(H3) weeks. A fold change of 2 or above adjusted for a false discovery rate of 5% was used as initial cut-off to determine differential gene expression for individual genes. Test for enrichment to identify functional groups were carried out using the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Array analysis correctly identified the cardiac specific genes, and transcripts reported to be differentially expressed were confirmed by qRT-PCR.

overall_design: 14 hearts-10 brains


title: Transcriptome analysis reveals molecular profiles associated with evolving steps of monoclonal gammopathies

status: Public on May 22 2014

city: Salamanca

first: Norma C.

state: Salamanca

address: Campus Miguel de Unamuno

department: Hematologia

institute: Centro de Investigación del Cáncer de Salamanca

summary: To gain further insights into the role of the transcriptome deregulation in the transition from a normal plasma cell (NPC) to a clonal PC and from an indolent clonal PC to a malignant PC, we performed gene expression profiling in 20 patients with MGUS, 33 with high-risk SMM and 41 with MM. The analysis showed that 126 genes were differentially expressed in MGUS, SMM and MM as compared to NPC. Interestingly, 17 and 9 out of the 126 significant differentially expressed genes were small nucleolar RNA molecules (snoRNA) and zinc finger proteins. GADD45A was the most significant up-regulated gene in clonal PC compared to NPC. Several proapoptotic genes (AKT1 and AKT2) were downregulated and antiapoptotic genes (APAF1 and BCL2L1) were upregulated in MM, both symptomatic and asymptomatic, compared to MGUS. Myc mediated apoptosis signaling is one of the top canonical pathways differentiating the asymptomatic and symptomatic myeloma. When we looked for those genes progressively modulated through the evolving stages of monoclonal gammopathies, eight snoRNA showed a progressive increase while APAF1, VCAN and MEGF9 exhibited a progressive downregulation in the transition from MGUS to SMM and to MM. In conclusion, our data show that although MGUS, SMM and MM are not clearly distinguishable groups according to their GEP, several signaling pathways and genes were significant deregulated in the different steps of transformation process.

overall_design: Bone marrow (BM) samples were obtained from 20 patients with MGUS, 33 with high-risk SMM and 41 with MM. All samples corresponded to newly diagnosed untreated patients. To avoid misclassification or overlapping entities we decided to focus on MGUS patients with more than two years of stable follow-up. Samples were classified according to the International Myeloma Working Group criteria. The criteria for defining high-risk SMM has been described previously. In addition, five healthy donors were also included in order to relate the deregulation of gene expression profiling of clonal populations to normal condition. The study was approved by the research ethic committees of all participating centers and written informed consent was obtained from all patients in accordance with the Helsinki Declaration. The main clinical and laboratory characteristics of these patients are shown in Supplementary Table 1.

first: Lucía

last: López Corral

first: Luis

middle: A

last: Corchete

first: Norma


title: aCGH/SNP data from Hepatosplenic T cell lymphoma patient samples

status: Public on May 24 2014

city: Leuven

first: Julio

last: Finalet

address: Herestraat 49

country: Belgium

institute: KULeuven

summary: Hepatosplenic T-cell lymphoma (HSTL) is an aggressive lymphoma cytogenetically characterized by isochromosome 7q   [i(7)(q10)], of which the molecular consequences remain unknown. We report here results of an integrative genomic and transcriptomic (expression microarray and RNA-sequencing) study of six HSTL cases with i(7)(q10) and three cases with ring 7   [r(7)], a rare variant aberration. Using high resolution array CGH, we prove that HSTL is characterized by the common loss of a 34.88 Mb region at 7p22.1p14.1 (3506316-38406226 bp) and duplication/amplification of a 38.77 Mb region at 7q22.11q31.1 (86259620-124892276 bp). Our data indicate that i(7)(q10)/r(7)-associated loss of 7p22.1p14.1 is a critical event in the development of HSTL, while gain of 7q sequences drives progression of the disease and underlies its intrinsic chemoresistance. Loss of 7p22.1p14.1 does not target a postulated tumor suppressor gene but unexpectedly enhances the expression of CHN2 from the remaining 7p allele, resulting in overexpression of β2-chimerin and dysregulation of a pathway involving RAC1 and NFATC2 with a cell proliferation response. Gain of 7q leads to increased expression of critical genes, including RUNDC3B, PPP1R9A and ABCB1, a known multidrug resistance gene. RNA-sequencing did not identify any additional recurrent mutations or gene fusions, suggesting that i(7)(q10) is the only driver event in this tumor. Our study confirms the previously described gene expression profile of HSTL and identifies a set of 24 genes, including three located on chromosome 7 (CHN2, ABCB1 and PPP1R9A), distinguishing HSTL from other malignancies

overall_design: Total genomic DNA was isolated from fresh frozen lymphoma samples using standard procedures. Genomic profiling, following the manufacturer’s protocols, was performed using the Affymetrix CytoScan HD arrays (www.affymetrix.com)

last: Ferreiro

first: Leila

last: Rouhigharabaei

first: Helena

last: Urbankova

first: Jo-Anne

last: van der Krogt

first: Lucienne

last: Michaux

first: Shashirekha

last: Shetty

first: Laszlo

last: Krenacs

first: Thomas

last: Tousseyn

first: Pascale

last: De Paepe

first: Anne

last: Uyttebroeck

first: Gregor

last: Verhoef

first: Tom

last: Taghon

first: Peter

last: Vandenberghe

first: Jan

last: Cools

first: Iwona

last: Wlodarska


title: Expression data from BRAFV600E A375 melanoma cells treated with vehicle or vemurafenib

status: Public on May 20 2014

city: East Melbourne

first: Tiffany

middle: Joy

last: Parmenter

address: 1 St Andrews Pl

institute: Peter MacCallum Cancer Centre

summary:
Vemurafenib is a BRAF inhibitor with specificity for the most common BRAF mutant encountered in melanomas (BRAFV600E). Vemurafenib suppresses the proliferation of BRAF mutant human melanoma cells by suppressing downstream activation of the MEK/ERK mitogen activated protein kinases.
We used microarrays to examine the transcriptional response of a vemurafenib-sensitive BRAFV600E human melanoma cell line (A375) to vemurafenib in order to further delineate the mechanisms by which BRAFV600E drives cell proliferation and energy metabolism in human melanoma.

overall_design: BRAFV600E A375 human melanoma cells were treated with vehicle (0.1% DMSO) or 10 μM vemurafenib for 24 h after which total RNA was extracted. Cells were prepared and RNA was extracted in 3 separate batches (three different cell stocks on three separate days) providing three independent replicates (n=3). Paired replicates (prepared from the same stock of cells on the same day) are denoted by A, B and C.

first: Grant

last: McArthur


title: Metabolic and gene expression changes induced by the naturally occurring ΔNp53 isoform link mTOR pathway and mitochondrial alterations to the progeroid phenotype.

status: Public on May 08 2014

city: Boulder

first: Shih-chieh

last: Lin

state: CO

address: 215 UCB

department: Chemistry and Biochemistry

country: USA

institute: University of Colorado at Boulder

summary: This study is to find the cellular and molecular mechanisms by which a naturally-occurring ΔNp53 isoform causes accelerated aging in humans. The biological function of ΔNp53, which lacks only 40 N-terminal amino acids, represents an example of p53 as a regulator of mammalian aging. When expressed together with WTp53 in mice, ΔNp53 causes an aging phenotype such as shorter life span, reduced body mass, organ atrophy and osteoporosis. Because p53 must form a tetramer to regulate transcription, we generated p53 clones (based upon the structure of the native p53 tetramer) containing one ΔNp53 linked with one WTp53 to form a functional ΔNp53:WTp53 tetramer with 1:1 stoichiometry. Thus, our strategy ensured each p53 tetramer contained 2 ΔNp53 and 2 WTp53 proteins. Importantly, ΔNp53:WTp53 form stable tetramers, based upon gel filtration chromatography and structural analysis using electron microscopy. Furthermore, the ΔNp53:WTp53 tetramer activates transcription equally well compared with WTp53 tetramers in an in vitro reconstituted transcription system. Having verified the stoichiometry, stability, structure, and activity of these ΔNp53:WTp53 tetramers, here we used microarray analysis to compare global gene expression patterns in p53-null H1299 cells expressing either WTp53 or ΔNp53:WTp53. As expected, global gene expression was largely similar, since the differences between ΔNp53:WTp53 tetramers and WTp53 tetramers are slight: only 2 of 4 p53 proteins will be different in the ΔNp53:WTp53 tetramer. Among only several dozen genes that were selectively up- or down-regulated 2-fold or greater, many genes known to regulate mammalian aging were altered in cells expressing ΔNp53:WTp53, including insulin signaling pathway members (IRS1, INPP5D, PLK3, MAP3K1, FGF5) and regulators of glucose metabolism (SLC2A2, CRYAB, LRCH1).  Expression of other key metabolic genes were also altered in cells expressing ΔNp53:WTp53 tetramers, suggesting that global me tabolic changes might contribute to ΔNp53:WTp53 pathology.  In collaboration with Metabolon (Durham, NC), we identified approximately one hundred metabolites that were significantly up- or down-regulated in H1299 cells expressing ΔNp53:WTp53. The metabolome analysis was a powerful complement to the gene expression data, and further suggested that the mTOR pathway (e.g. across-the-board up-regulation of amino acid levels) and mitochondrial function (e.g. up-regulation of carnitine, important for a-oxidation of fatty acids) was altered in cells expressing ΔNp53:WTp53.  These findings were subsequently validated using biochemical and cell-based approaches. Furthermore, whereas equal expression of ΔNp53 and WTp53 cause accelerated aging in mammals, due to alternative splicing and translation initiation ΔNp53 is a naturally-occurring isoform whose expression levels can change throughout the lifetime. Thus, the cellular and molecular mechanisms identified from this work will likely reflect changes common to normal, physiological aging.

overall_design:
Comparison of global gene expression profiles in WTp53 or ΔNp53:WTp53 expressed H1299
RNA from FACS sorted GFP positive hybrid to Affymatrix Gene
The exp 0, exp 4 and exp 5  are independent replicate/transfections/biological experiments.
The exp d, exp e and exp f  are control experiments.

first: Dylan

last: Taatjes


title: Phenotypic, genomic and functional characterization reveals no differences between CD138++ and CD138low subpopulations in multiple myeloma cell lines

status: Public on May 19 2014

first: Teresa

last: Paíno

institute: Centro de investigación del cáncer

summary: Despite recent advances in the treatment of multiple myeloma (MM), it remains an incurable disease potentially due to the presence of resistant myeloma cancer stem cells (MM-CSC). Although the presence of clonogenic cells in MM was described more than 30 years ago, the phenotype of MM-CSC is still a matter of debate, especially with respect to the expression of syndecan- 1 (CD138). Here, we demonstrate the presence of two subpopulations - CD138++ (95-99%) and CD138low (1-5%) - in eight MM cell lines. To find out possible stem-cell-like features, we have phenotypically, genomic and functionally characterized the two subpopulations. Our results show that the minor CD138low subpopulation is morphologically identical to the CD138++ fraction and does not represent a more immature B-cell compartment (with lack of CD19, CD20 and CD27 surface expression). Moreover, both subpopulations have similar gene expression and genomic profiles. Importantly, both CD138++ and CD138low subpopulations have similar sensitivity to bortezomib, melphalan and doxorubicin. Finally, serial engraftment in SCID mice shows that CD138++ as well as CD138low cells have self-renewal potential and they are also phenotypically interconvertible. Overall, our results differ from previously published data which attribute a B-cell phenotype to MM-CSC and urge the need to explore more reliable markers to discriminate true clonogenic myeloma cells.

overall_design: To compare the gene expression profile of the subpopulations CD138++ and CD138low within the MM cell line, RPMI-8226. RNA was isolated from CD138++ (n=3) and CD138low (n=3) RPMI-8226 cells. RNA was hybridized to Human Gene 1.0 ST array (Affymetrix) according to Affymetrix protocols [Gutierrez, 2005, 402]. Microarray data were normalized using the Robust Multichip Analysis (RMA) algorithm implemented in the Affymetrix Expression Console. Data analysis was carried out using DNA-Chip Analyzer software (DChip). The comparison criteria used in DChip analysis were fold change E/B≥2 or B/E≥2, mean difference E-B>100 or B-E>100 and the lower 90% confidence bound of fold-change was used

first: María

last: Sarasquete

first: Bruno

last: Paiva

first: Patryk

last: Krzeminski

first: Laura

last: San-Segundo

first: Luis

last: Corchete

first: Alba

last: Redondo

first: Mercedes

last: Garayoa

first: Ramón

last: García-Sanz

first: Norma

middle: C

last: Gutiérrez

first: Enrique

last: Ocio

first: Jesús

last: San-Miguel


title: Phenotypic, genomic and functional characterization reveals no differences between CD138++ and CD138low subpopulations in multiple myeloma cell lines (Copy Number)

overall_design: To compare the Copy Number Abnormalities of the subpopulations CD138++ and CD138low within the MM cell line, RPMI-8226. DNA was isolated from CD138++ (n=3) and CD138low (n=3) RPMI-8226 cells. The detection of CNA was investigated using the CytoScan HD array (Affymetrix, Santa Clara, CA, USA) which includes more than 2.6 million copy number markers and 750.000 SNPs. Briefly, genomic DNA was digested with Nsp I restriction endonuclease, ligated to adaptors that recognize the cohesive four base pair overhangs and amplified by PCR. PCR product was purified and fragmented with DNAse I and then end-labeled using terminal deoxynucleotidyl transferase and hybridized to the CytoScan HD array. The arrays were processed using the Fluidics Station 450, GeneChip Scanner 3000 7 G and AGCC (AffymetrixGeneChip Command Console Software).   For the analysis we employed the Affymetrix® Chromosome Analysis Suite (ChAS) Software v2.0 and Nexus Copy Number Software Version 7.0 (BioDiscovery, El Segundo, CA, USA).  The complete data set was analyzed by visual inspection using ChAS software. CNA were reported when the three following criteria were achieved: minimum of 50 markers per segment, 200 Kb minimum genomic sizes and 50% overlap with known copy number variants (Database of Genomic Variants).