title: Minimal peroxide exposure of neuronal cells induces multifaceted adaptive responses.

first: Supriyo

last: De

summary: Oxidative exposure of cells occurs naturally and may be associated with cellular damage and dysfunction. Protracted low level oxidative exposure can induce accumulated cell disruption, affecting multiple cellular functions. Accumulated oxidative exposure has also been proposed as one of the potential hallmarks of the physiological/pathophysiological aging process. We investigated the multifactorial effects of long-term minimal peroxide exposure upon SH-SY5Y neural cells to understand how they respond to the continued presence of oxidative stressors. We show that minimal protracted oxidative stresses induce complex molecular and physiological alterations in cell functionality. Upon chronic exposure to minimal doses of hydrogen peroxide, SH-SY5Y cells displayed a multifactorial response to the stressor. To fully appreciate the peroxide-mediated cellular effects, we assessed these adaptive effects at the genomic, proteomic and cellular signal processing level. Combined analyses of these multiple levels of investigation revealed a complex cellular adaptive response to the protracted peroxide exposure. This adaptive response involved changes in cytoskeletal structure, energy metabolic shifts towards glycolysis and selective alterations in transmembrane receptor activity. Our analyses of the global responses to chronic stressor exposure, at multiple biological levels, revealed a viable neural phenotype in-part reminiscent of aged or damaged neural tissue. Our paradigm indicates how cellular physiology can subtly change in different contexts and potentially aid the appreciation of stress response adaptations.

overall_design: SH-SY5Y cells were maintained at 37oC in a humidified 5% CO2 incubator in AMEM/F12 supplemented with 10% FBS, 100 units/ml ?penicillin and 100 units/ml streptomycin.  Cells were treated with either phosphate-buffered saline (PBS) (CTR), or 10nM hydrogen peroxide(CMP) (H2O2: Sigma Aldrich, St Louis MO) for seven days.  Cell growth media and applied hydrogen peroxide treatments were changed daily.  Both control and hydrogen peroxide treated SH-SY5Y cell were serum-starved overnight prior to ligand stimulations with either, Beta-methylcholine(BMC) (Calbiochem, Gibbstown NJ) or 10ng/ml brain derived neurotrophic factor (BDNF) (Invitrogen, Carlsbad CA) for 2, 4, or 8 hours. Cells were then collected and used to generate total RNA.

first: Nicholas

last: Mitchell

first: Matthew

last: Stone


title: Euglycemic agent-mediated hypothalamic transcriptomic manipulation in the N171-82Q model of Huntington's disease is related to their physiological efficacy

status: Public on Oct 05 2012

city: Baltimore

state: Maryland

address: 251 Bayview Blvd

department: Laboratory of Genetics and Genomics

country: USA

institute: NIA-IRP, NIH

summary: Our aim was to find an effective treatment for the dysfunction in energy regulation and hypothalamic integrity occurring in many Huntington’s disease (HD) patients and mouse models. HD is a neurodegenerative disease caused by an expanded CAG repeat and is characterized by mutant huntingtin aggregates, cell death, progressive motor impairment, and cognitive alterations. Changes in neuroendocrine function, body weight, energy metabolism, euglycemia, appetite function, sleep, and gut function can also occur. It is likely that the locus of these alterations is the hypothalamus.

overall_design: Male B6C3-Tg(HD82Gln)81Dbo/J (N171-82Q) mice and age-matched wild-type (WT) mice were used. Transgenic mice were identified by PCR analysis of tail DNA. N171-82Q mice express 82 polyglutamine repeats and display multiple Huntington’s disease-like symptoms. Either Ex-4 (n=10, 300 µL of 0.1 µmol/L solution, Bachem, Torrance, CA), the GLP-1-transferrin hybrid molecule (n=10, GLP-1Tf, 1mg/kg), insulin (n=10, 5Units, Lantus) or a saline vehicle treatment (n=10, phosphate buffered saline, PBS; Sigma, St. Louis, MO) was administered by a once-daily subcutaneous injection. Injections were started when animals were pre-symptomatic (2 months of age) and continued for 5 weeks. 3 Mice from each treatment qroup were used for the microarray analysis. RNA was extracted from frozen hypothalamus tissue using 1.0mm glass beads (BioSpec Products, Inc.) in a Precellys 24 Tissue Homogenizer (Bertin Technologies) and Qiagen RNeasy Mini Kits according to manufacturer's specifications. Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips. The standard Illumina protocol using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) was used to biotin label the aRNA generated and then hybridized to Illumina's Sentrix MouseRef-8 v2 Expression BeadChips (Illumina, San Diego, CA). Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina.

first: Bronwen

last: Martin

first: Wayne

last: Chadwick

first: Wei-na

last: Cong

first: Nick

last: Pantaleo

first: Caitlin

middle: M

last: Daimon

first: Erin

middle: J

last: Golden

first: Kevin

middle: G

last: Becker

first: William

middle: H

last: Wood III

first: Olga

middle: D

last: Carlson

first: Josephine

last: Egan

first: Stuart

last: Maudsley


title: Hypoxia-induced gene expression in chemoradioresistant cervical cancer revealed by dynamic contrast enhanced MRI

status: Public on Aug 16 2012

city: Oslo

first: Heidi

last: Lyng

address: Montebello

department: Department of Radiation Biology

country: Norway

institute: Oslo University Hospital

summary: We explored the prognostic impact of the dynamic contrast enhanced MR imaging (DCE-MRI) parameter ABrix in cervical cancer combined with global gene expression data to reveal the underlying molecular phenotype of the parameter and construct a gene signature that reflected ABrix. Based on 78 cervical cancer patients subjected to curative chemoradiotherapy, we identified a prognostic ABrix parameter by pharmacokinetic analysis of DCE-MR images based on the Brix model, where tumors with low ABrix appeared to be most aggressive. Gene set enrichment analysis of 46 tumors with pairwise DCE-MRI and gene expression data showed a significant correlation between ABrix and the hypoxia gene sets, whereas gene sets related to proliferation, radioresistance, and wound healing were not significant. Hypoxia gene sets specific for cervical cancer created in cell culture experiments, including targets of the hypoxia inducible factor (HIF1α) and the unfolded protein response (UPR), were the most significant. In the remaining 32 tumors, low ABrix was associated with upregulation of HIF1α protein expression, as assessed by immunohistochemistry, consistent with increased hypoxia. Based on the hypoxia gene sets, a signature of 31 genes that were upregulated in tumors with low ABrix was constructed. This DCE-MRI hypoxia gene signature showed prognostic impact in an independent validation cohort of 109 patients.

overall_design:
Gene expression correlating with the DCE-MRI parameter ABrix were identified in 46 patients (DCE-MRI cohort) and used to find a hypoxia gene signature. The prognsotic impact of the gene signature was validated in an independent cohort of 109 patients (validation chort).
Cell culture experiments were performed to generate cervical cancer specific gene lists associated with hypoxia (GSM897799 - GSM897804).

first: Cathinka

last: Halle

first: Erlend

last: Andersen

first: Malin

last: Lando

first: Eva-Katrine

last: Aarnes

first: Grete

last: Hasvold

first: Marit

last: Holden

first: Randi

last: Syljuåsen

first: Kolbein

last: Sundfør

first: Gunnar

middle: B

last: Kristensen

first: Ruth

last: Holm

first: Eirik

last: Malinen


title: Claudin-7 is frequently overexpressed in ovarian cancer and promotes invasion

status: Public on Sep 19 2012

first: Supriyo

last: De

summary: Claudins are tight junction proteins that are involved in tight junction formation and function. Previous studies have shown that claudin-7 is frequently upregulated in epithelial ovarian cancer (EOC) along with claudin-3 and claudin-4. Here, we investigate in detail the expression patterns of claudin-7, as well as its possible functions in EOC.  Initially a total of 95 ovarian tissue samples (7 normal ovarian tissues, 65 serous carcinomas, 11 clear cell carcinomas, 8 endometrioid carcinomas and 4 mucinous carcinomas) were studied for claudin-7 expression using RT-PCR analysis. The gene for claudin-7, CLDN7, was found to be significantly upregulated in all the tumor tissue samples studied. Similarly, immunohistochemical analysis and western blotting showed that claudin-7 protein was significantly overexpressed in the vast majority of EOCs. Two cell lines(OVCAR-2 and OVCA420) were chosen for microarray based gene expression analysis using small interfering RNA-mediated(SiRNA) knockdown of claudin-7.  This treatment led to significant changes in gene expression that was validated by RT-PCR and immunoblotting.

overall_design: Ovarian cancer lines OVCA420 and OVCAR-2 were cultured in duplicate in McCoy’s 5A growth medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin).  Cells cultured in 6-well plates were transfected with either Claudin-7 specific siRNA oligos duplexes (Ambion, Inc.Austin, TX) or control siRNA #1 (Dharmacon) using LipofectAMINE 2000 (Invitrogen). Cells were treated for 72 hours to allow maximum knockdown, after which they were harvested for RNA preparation.  Total RNA, was extracted using Trizol, quality and quantity were checked using the Agilent 2100 Bioanalyzer with RNA 6000 Nano chip, (Agilent Technologies UK Ltd, West Lothian, UK). Biotinylated cRNA was generated using the Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX) starting with approximately 500 ng total RNA. Hybridization of the cRNA was to the Sentrix HumanRef-8 Expression BeadChip (Illumina, Inc., San Diego, CA).   Microarray data processing and analysis was performed using Illumina Bead Studio software. Hierarchical clustering analysis of the significant genes was done using the JMP 6.0.0 software. Validation of the expression patterns of selected genes was performed using RT-PCR.

first: Neetu

last: Dahiya

first: Kevin

last: Becker

first: Yongqing

last: Zhang

first: Patrice

last: Morin


title: Clinically significant copy number alterations and complex rearrangements of MYB and NFIB in adenoid cystic carcinoma of the head and neck

status: Public on Dec 31 2012

city: Gotenburg

first: Marta

last: Persson

address: Box 425

country: Sweden

institute: Sahlgrenska Cancer Center

type: Genome variation profiling by genome tiling array

summary: Using high-resolution, array-based comparative genomic hybridization (aCGH), we explored genomic alterations in 40 fresh-frozen ACC samples, the largest cohort to date, with the aims to: (1) identify recurrent CNAs in ACC; (2) identify novel candidate target genes; and (3) correlate recurrent CNAs with tumour grade and other clinical parameters to identify potential clinically useful biomarkers.

overall_design: High-resolution aCGH on fresh frozen tissue from 40 ACCs.

first: Ywonnes

last: Andrén

first: Christopher

middle: A

last: Moskaluk

first: Henry

middle: F

last: Frierson

first: Teresia

last: Kling

first: Sven

last: Nelander

first: Anders

last: Nordkvist

first: Fredrik

first: Göran

last: Stenman


title: Exendin-4, a glucagon-like peptide-1 receptor agonist prevents mTBI-induced changes in hippocampus gene expression and memory deficits in mice

status: Public on Oct 17 2012

summary: Traumatic brain injury (TBI) is a global problem reaching near epidemic numbers that manifests clinically with cognitive problems that decades later may result in dementias like Alzheimer’s disease (AD). Presently, little can be done to prevent ensuing neurological dysfunctions by pharmacological means. Recently, it has become apparent that several CNS diseases share common terminal features of neuronal cell death. The effects of exendin-4 (Ex-4), a neuroprotective agent delivered via a subcutaneous micro-osmotic pump, were examined in the setting of mild TBI (mTBI). Utilizing a model of mTBI, where cognitive disturbances occur over time, animals were subjected to four treatments: sham; Ex-4; mTBI and Ex-4/mTBI. mTBI mice displayed deficits in novel object recognition, while Ex-4/mTBI mice performed similar to sham. Hippocampal gene expression, assessed by gene array methods, showed significant differences with little overlap in co-regulated genes between groups. Importantly, changes in gene expression induced by mTBI, including genes associated with AD were largely prevented by Ex-4. These data suggest a strong beneficial action of Ex-4 in managing secondary events induced by a traumatic brain injury.

overall_design: Male ICR mice weighing 30–40 g were kept five per cage under a constant 12-h light/dark cycle, at room temperature (23°C). Food (Purina rodent chow) and water were available ad libitum. Each mouse was used for one experiment and for one time point only. The Ethics Committee of the Sackler Faculty of Medicine approved the experimental protocol (M-09-055), in compliance with the guidelines for animal experimentation of the National Institutes of Health (DHEW publication 85–23, revised, 1995). A minimal number of mice were used for the study and all efforts were made to minimize suffering. All experimental manipulations were conducted during the light phase of the cycle.  Experimental mTBI was induced using the concussive head trauma device described previously (Milman et al. 2005; Zohar et al. 2003). Briefly, mice were lightly anesthetized (Isoflurane) and placed under the weight-drop concussive head trauma instrument. The device consists of a metal tube (inner diameter 13 mm), placed vertically over the mouse head. A metal weight (30 g) was dropped from the top of the tube (80 cm) and struck the skull at the temporal right side between the corner of the eye and the ear. A sponge supported the head, allowing some antero-posterior motion without any rotational head movement at the moment of the impact. Immediately after the injury, mice were placed back in their cages for recovery. The effect of the injury upon behavior and cognition was studied from 7 days following the trauma. Sham treated mouse groups were treated identically only the weight was not dropped.  The peptide Ex-4 was obtained from Bachem (Torrance, CA). In order to obtain a constant steady-state concentration of Ex-4 in mice prior to injury; Ex-4 was delivered by use of micro-osmotic pumps (7 day infusion duration), 2 days prior to the induction of mTBI. For the Ex-4 treated animals the peptide was dissolved in an equal volume of saline and dimethyl sulfoxide (DMSO) mixture and delivered from a subcutaneously implanted ALZET Micro-osmotic pump (Model 1007D, Alzet, Cupertino, CA) at a rate of 3.5 pM/kg/min.  In drug treatment control groups, saline and DMSO pumps were implanted in each animal following the same surgical procedure as used for the Ex-4 treatment animals. In all animals, pumps were placed, posterior to the scapulae. Pump implantation was performed under anesthesia (ketamine/xylazine) utilizing sterile procedures. Mini pumps were implanted 48 hours prior to the weight drop injury. The number of animals from each treatment group that went on to microarray analysis were as follows, Sham, n = 5; mTBI, n = 4; Ex-4, n = 5; Ex-4/ mTBI, n = 4.  Mouse cognition was assessed using the novel object recognition paradigm.  After the completion of the novel object behavioral assessment, animals were euthanized and the hippocampus dissected for total RNA isolation. Total RNA was prepared using the Qiagen RNeasy Mini Kit (Qiagen, Inc. Valencia CA) following the manufacturer's specifications. Quantity and quality of the RNA was assessed using the Agilent 2100 Bioanalyzer with RNA 6000 Nano Chips. The total RNA (500 ng) was used to generate biotin-labeled cRNA using the Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791). In short, 500 ng of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP. This cRNA was used in the hybridization reaction. Briefly, a total of 750 ng of biotin-labeled cRNA was hybridized at 58oC for 16 hours to Illumina's SentrixMouse Ref-8, v2 Expression BeadChips (Illumina, San Diego, CA). Each BeadChip has ~24,000 well-annotated RefSeq transcripts with an approximately 30-fold redundancy. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3. Arrays were scanned at a resolution of 0.8 m using the Beadstation 500 X from Illumina, data was extracted from the image using Illumina BeadStudio software, v3.4.0.

first: David

last: Tweedie

first: Lital

last: Rachmany

first: Vardit

last: Rubovitch

first: Elin

last: Lehrmann

first: Evelyn

last: Perez

first: Jonathan

last: Miller

first: Barry

last: Hoffer

first: Nigel

last: Greig

first: Chaim

last: Pick


title: Stromal factors SDF1alpha, sFRP1 and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells

status: Public on Jun 25 2012

first: Supriyo

last: De

summary:
The identified stromal factors SDF1alpha, sFRP1 and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells.
Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons are potentially useful for treating Parkinson’s disease (PD) through cell replacement therapy.  Generation of DA neurons from hESCs has been achieved by co-culture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA).  However, the factor(s) produced by stromal cells that constitute SDIA is unknown.  We previously reported that medium conditioned by PA6 cells can generate functional DA neurons in the human embryonal carcinoma stem cell line, NTera2.  Here we further examined the effects of PA6-conditioned medium and found that it can induce DA neuronal differentiation in both the NTera2 cell line and the hESC line, I6.  To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with proteomic analysis of PA6-conditioned medium.  Four candidate factors (hepatocyte growth factor (HGF), stromal cell-derived factor-1 alpha (SDF1alpha), secreted frizzled-related protein 1 (sFRP1) and vascular endothelial growth factor D (VEGFD)) were identified and immunoaffinity capillary electrophoresis (ICE) was used to establish the protein concentration of these factors in conditioned medium.  Upon addition of SDF1alpha, sFRP1, and VEGFD, we observed an increase in the number of tyrosine hydroxylase- and TuJ1- positive cells in both the NTera2 and I6 cell lines.  These results indicate that SDF1alpha, sFRP1 and VEGF-D are major components of SDIA, and suggest the potential use of these defined factors to elicit dopaminergic differentiation of pluripotent stem cells as a therapeutic intervention in PD.

overall_design: Mouse embryonic fibroblasts were grown in DMEM medium supplemented with 10% FBS; this conditioned media was used as the control.  PA6, mouse stromal cells, were grown in MEM-alpha supplemented with 10% FBS; this conditioned media is known to induce differentiation in hES cells.

first: Catherine

last: Schwartz

first: Tara

last: Tavakoli

first: Charmaine

last: Jamais

first: Sung

last: Park

first: Terry

middle: E

last: Phillips

first: Pamela

last: Yao

first: Stuart

last: Maudsley

first: Mahendra

middle: S

last: Rao

first: Wu

last: Ma

first: Ernest

last: Arenas

first: Mark

middle: P

last: Mattson


title: IL-10 transcription is negatively regulated by BAF180, a component of the SWI/SNF chromatin remodeling enzyme

status: Public on Mar 27 2012

summary: Several versions of SWI/SNF complexes, BAF and PBAF, have been described based on unique subunit composition. We find that T cell development in the thymus and lymphoid periphery is largely normal in the absence of the PBAF-specific component BAF180.  However, BAF180-deficient Th2 cells express high levels of the immunoregulatory cytokine IL-10.  BAF180 binds directly to regulatory elements in the Il-10 locus but is replaced by BAF250 BAF complexes in the absence of BAF180, resulting in increased histone acetylation and CBP recruitment to the IL-10 locus.  These results demonstrate that BAF180 is a repressor of IL-10 transcription in Th2 cells and suggest that the differential recruitment of different SWI/SNF subtypes can have direct consequences on chromatin structure and gene transcription.

overall_design:
mouse primary T helper lymphocytes, naïve and Th2 cells, resting and stimulated, comparison of wild-type and BAF180/Pbrm1 deficient cells
RNA from T helper cells was compared in the presence (WT) and absence (KO) of the Pbrm1/BAF180 gene.  Comparison was made in 2 T helper subsets (Naive and Th2), each under 2 condtions (resting and stimulated), in triplicate (A,B,C).  Resting naïve CD4+ T cells (N) were purified to 95% purity from the spleens and lymph nodes of 4-6 week old Balb/c mice (WT, or BAF180 cKO) using CD4+CD62L+ T cell Purification Kit II per manufacturer’s instructions (Miltenyi).  Stimulated Naive cells (S) were prepared from resting naive cells by stimulation for 24 hours on anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml.  Resting Th2 cells (2U) were prepared from resting naive cells by plating onto anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml in the presence of 10ng/ml IL-4, 10ug/ml anti-IFN-gamma. IL-2 (100U/ml) was added 24 hours later. Cultures were expanded in IL-2 (100U/ml) three days after initial culture.  Stimulated Th2 cells (2S) were prepared from resting Th2 cells, stimulated with PMA (50ng/ml) and Ionomycin (500ng/ml) for 1.5 hours.  RNA was harvested from 3 replicates of the primary Th cells under each condition. RNA was isolated using RNAeasy Kit (Qiagen), Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips.  RNA was labeled using the standard Illumina protocol and Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791)  Biotin labeled cRNA was hybridized to Illumina’s Sentrix MouseRef-8,v2 Expression BeadChips.

first: Andrea

middle: L

last: Wurster

first: Patricia

last: Precht

first: Kevin

last: Becker

first: William

last: Wood

first: Yongqing

last: Zhang

first: Zhong

last: Wang

first: Michael

last: Pazin


title: Beta-microseminoprotein (MSMB) downregulation is associated with increased drug resistance in ovarian cancer

status: Public on Mar 12 2012

first: Supriyo

last: De

summary: Resistance to current chemotherapeutic agents is major cause of therapy failure in ovarian cancer patients. To better understand mechanisms of drug resistance, and possibly identify novel targets for therapy, we generated a series of ovarian cancer cell lines that are resistant to various chemotherapeutic drugs (cisplatin, doxorubicin, and paclitaxel), and identified key resistance genes and pathways using whole-genome expression analysis. Our data identify a number of genes altered in the drug resistant lines compared to drug-sensitive cells, and further validation finds an interesting candidate MSMB to be consistently decreased at both the mRNA and protein levels in all the drug-resistant ovarian cancer cells. Through knockdown and overexpression experiments in cell culture models, we show that MSMB has a functional role in drug resistance. Using a mouse xenograft model, we show that re-expression of MSMB in drug-resistant cells can partially reverse the drug resistant phenotype. MSMB-expressing cells have increased caspase-3 activity and known downstream targets, including the PAK2-MLCK-actin pathway, are found activated, providing a possible molecular mechanism for the roles of MSMB in drug resistance. Thus, our study identifies a novel gene in ovarian cancer drug resistance and elucidates a portion of the signaling pathway that may be crucial in its function. Our data suggest a new mechanism for the development of drug resistance in ovarian cancer and identify possible new targets for therapy.

overall_design: To study the mechanisms involved in the development of resistance to anti-cancer agents, the drug-sensitive OV90 ovarian cancer cell line was used to generate a series of cell lines, each individually resistant to different drugs. Repeated cycles of drug exposure followed by recovery periods led to the development drug resistance in these cells. Using this approach, we generated OV90 sublines resistant to cisplatin, doxorubicin and paclitaxel. The lines resistant to cisplatin (OV90C-A, OV90C-D), doxorubicin (OV90D-6, OV90D-7) and paclitaxel (OV90P-3, OV90P-7) all exhibited significant resistance to their corresponding drugs compared to the parental OV90 cell. Cross resistance was also investigated and we found that the cisplatin resistant lines (OV90C-A and OV90C-D) were not cross-resistant to doxorubicin or paclitaxel.  In contrast, the doxorubicin resistant cells (OV90D-6 and OV90D-7) were cross-resistant to paclitaxel, and the paclitaxel resistant cells (OV90P-3 and OV90P-7) were also resistant to both cisplatin and doxorubicin. To identify genes and pathways important in the development of drug resistance, we performed gene expression profiling analysis on the OV90 drug sensitive cell line and on the resistant cell lines using Illumina Sentrix microarrays. For each of the resistance types (cisplatin, doxorubicin, and paclitaxel) two independent sublines were profiled in duplicate (two different cultures). Multidimensional scaling (MDS) analysis based on the genes expression data showed that the cell lines were grouped according to their resistance phenotype, demonstrating  that the selection for resistance to different drugs led to overall different patterns of gene expression changes. This suggested different mechanisms of resistance for the different drugs.

first: Bingxue

last: Yan

first: Cheryl

last: Sherman-Baust

first: Patrice

last: Morin


title: DNA microarray analysis of the anti-inflammatory effects of PDTC on IL-6 signaling in HepG2 cells

status: Public on Jan 05 2012

summary: Interleukin-6 (IL-6) is a proinflammatory cytokine that exerts a wide range of cellular, physiological and pathophysiological responses. Pyrrolidine dithiocarbamate (PDTC) antagonizes the cellular responsiveness to IL-6 through impairment in STAT3 activation and downstream signaling. Here, a transcriptional profiling was conducted as a basis for understanding the biological properties of PDTC in human HepG2 hepatocarcinoma cells. A global comparison of mRNA identified a highly significant difference of dysregulated gene expression transduced by PDTC versus IL-6 in HepG2 cells. Through an unbiased pathway analysis method, we have uncovered the mammalian target of rapamycin (mTOR) pathway together with rapid and dynamic alterations in REDD1 (regulated in development and DNA damage response 1) expression as one of the underlying molecular mechanisms responsible for IL-6 resistance to PDTC. Quantitative PCR and Western blot analyses validated the microarray data by showing the reciprocal pattern of REDD1 expression and subsequent mTOR inhibition after stimulation with PDTC relative to IL-6.

overall_design: The HepG2 human hepatocarcinoma cell line (American Type Culture Collection) was routinely grown and maintained in Minimum Eagle's medium (MEM)/F12 (1:1) supplemented with 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin and 50 ug/ml streptomycin under standard cell culture conditions at 37°C and 5% CO2 in a humid environment. Cells were seeded in 35- or 60-mm dishes and grown to ~90% confluence after which they were serum starved for 4 h followed by the addition of either vehicle, PDTC (50 µM) or  human recombinant IL-6 (20 ng/ml) for 2 replicates, labeled a and b, of the 5 time points: 0hr, 1hr, 2hr, 4hr and 8hr.

first: Shaoming

last: Song

first: Rajib

middle: K

last: Paul

last: Wood III

first: Sutapa

last: Kole

first: Michel

last: Bernier


title: Amitriptyline-mediated cognitive enhancement in aged 3xTg Alzheimer’s disease mice is associated with neurogenesis and neurotrophic activity

status: Public on Dec 31 2011

summary: Approximately 35 million people worldwide suffer from Alzheimer’s disease (AD). Existing therapeutics, while moderately effective, are currently unable to stem the widespread rise in AD prevalence. AD is associated with an increase in amyloid beta (A) oligomers and hyperphosphorylated tau, along with cognitive impairment and neurodegeneration. Several antidepressants have shown promise in improving cognition and alleviating oxidative stress in AD but have failed as long-term therapeutics. In this study, amitriptyline, an FDA-approved tricyclic antidepressant, was administered orally to aged and cognitively impaired transgenic AD mice (3xTgAD).  After amitriptyline treatment, cognitive behavior testing demonstrated that there was a significant improvement in both long- and short-term memory retention.  Amitriptyline treatment also caused a significant potentiation of non-toxic A monomer with a concomitant decrease in toxic oligomer A load, compared to vehicle-treated 3xTgAD controls. In addition, amitriptyline administration caused a significant increase in dentate gyrus neurogenesis as well as increases in expression of neurosynaptic marker proteins.  Amitriptyline treatment resulted in increases in hippocampal brain-derived neurotrophic factor protein as well as increased tyrosine phosphorylation of its cognate receptor (TrkB).  These results indicate that amitriptyline has significant beneficial actions in aged and damaged AD brains and that it shows promise as a tolerable novel therapeutic for the treatment of AD.

overall_design: Male 3xTgAD mice, 14 months of age, were maintained on a 12hr light dark cycle in pathogen free conditions. Animals received food and water ad libitum. The test group received 100ug/g body weight amitriptyline-hydrochloride per os in their drinking water (Sigma-Aldrich, St. Louis MO) for 4 months (n = 15), and the control group received water (n = 15). The Hippocampus and Cortex were removed from 3 replicates of each group and RNA purification was done using glass bead disruption followed by the RNEasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. The RNA was examined for quantity and quality using an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).  The samples were hybridized to Illumina's SentrixMouse Ref-8 v2. Expression BeadChips (Illumina, San Diego, CA).

first: Wayne

last: Chadwick

first: Nick

last: Mitchell

first: Jenna

last: Caroll

first: Yu

last: Zhou

first: Sung-Soo

last: Park

first: Liyun

first: Elin

last: Lehrmann

first: Bronwen

last: Martin

first: Stuart

last: Maudsley


title: Genetic recombination is directed away from functional genetic sites in mice.

status: Public on May 22 2012

city: Bethesda

first: Kevin

last: Brick

state: MD

address: 5/205 Memorial Drive

department: GBB

institute: NIDDK

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Genetic recombination occurs during meiosis, the key developmental program of gametogenesis. Recombination in mammals has been recently linked to the activity of a histone H3 methyl-transferase, PRDM9, the product of the only known speciation gene in mammals. PRDM9 is thought to determine the preferred recombination sites - recombination hotspots - through sequence-specific binding of its highly polymorphic multi-Zn-finger domain. Nevertheless, Prdm9 knockout mice are proficient at initiating recombination. Here we map and analyze the genome-wide distribution of recombination initiation sites in Prdm9 knockout mice and in two mouse strains with different Prdm9 alleles and their F1 hybrid. We show that PRDM9 determines the positions of practically all hotspots in the mouse genome, with the remarkable exception of the pseudoautosomal region – the only area of the genome that undergoes recombination in 100% of cells. Surprisingly, hotspots are still observed in Prdm9 knockout mice and as in wild-type, these hotspots are found at H3K4 trimethylation marks. However, in the absence of PRDM9, the majority of recombination is initiated at promoters and at other sites of PRDM9-independent H3K4 trimethylation. Such sites are rarely targeted in wild-type mice indicating an unexpected role of the PRDM9 protein in sequestering the recombination machinery away from gene promoter regions and other functional genomic elements.

overall_design: Detection of meiotic double strand breaks in mouse testis and comparison with H3K4me3 marks.

first: Fatima

last: Smagulova

first: Pavel

last: Khil

first: Rafael-Daniel

last: Camerini-Otero

first: Galina

last: Petukhova


title: Expression profiling of lung cancer cell lines

status: Public on Jun 18 2012

city: Dallas

first: Luc

last: Girard

state: TX

address: 5323 Harry Hines Blvd

department: Hamon Center for Therapeutic Oncology Research

institute: UT Southwestern Medical 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, ECOG performance status (PS) 0-2, consented to biopsies to test for biomarker assessment. Sorafenib was given at 400 mg orally twice daily until tumor progression or an unacceptable toxicity. Tumor evaluations were performed at baseline and every 8 weeks. Correlations of outcomes by biomarker groups were analyzed.
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 PS of 0-1. Median prior chemotherapies for stage IV NSCLC were two. Median follow-up was 9.4 (range: 1.3-32.2) months. Eight-week DCRs by histology were 59.1%, 57.1%, and 55.6% for adenocarcinomas, squamous cell carcinomas and other histologies respectively. Patients with tumors harboring 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 tumors harboring 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%).
Conclusion: 8-week DCR was better in patients treated with sorafenib who had EGFR wild-type tumors (including those with K-RAS mutations) when compared to those who had EGFR mutations. Additional analyses are warranted to further explore the biology of these tumors and related patient outcomes with sorafenib treatment. ClinicalTrials.gov number, NCT00411671.

overall_design:
To develop gene expression signatures for in vitro drug response and other phenotypes. To characterize human bronchial epithelial cells (HBECs) and human small airway epithelial cells (HSAECs), which are normal lung cells derived from the bronchus and peripheral areas resp. and were immortalized with CDK4 and hTERT.
Expression profiling was done on 124 NSCLC and 39 SCLC cell lines, using either Illumina HumanWG-6 V3 or HumanHT-12 V4. In addition, 30 HBEC-KTs, 15 HSAEC-KTs and 14 HSAEC-UIs were profiled with Illumina HumanWG-6 V3. (KT: immortalized with CDK4 and hTERT, UI: unimmortalized)

first: John

last: Minna

first: Boning

last: Gao


title: Krüppel-like factor 4 regulates T cell function

status: Public on Jun 24 2011

first: Supriyo

last: De

summary: Krüppel-like factor 4 (Klf4), a transcription factor mediating context-dependent activation or repression, plays a key role in maintaining pluripotency of stem cells and in regulating cell differentiation. However, the precise function of Klf4 in T cell development and differentiation is largely unknown. Here we showed that Klf4 was highly expressed in thymocyte subsets and mature T cells, and was rapidly down-regulated in mature T cells after activation. In T cell-specific Klf4 conditional knockout mice, we observed a modest reduction of thymocytes (27%). This was due partly to the loss of Klf4 repression of Cdkn1b expression in thymocytes resulting in decreased proliferation of DN thymocytes. Together, these findings identify Klf4 as a critical regulator in T cell development.

overall_design: Klf4fl/fl mice (Katz et al., 2002) were crossed with CD4-Cre mice (Lee et al., 2001) to generate Klf4fl/fl-CD4-Cre+ (Klf4 conditional knockout in T cells) and Klf4fl/fl-CD4-Cre- (control) mice. Mice used for experiments were between 8 and 10 weeks old.  Total RNA was extracted from freshly isolated DP+SP4 thymocytes from 3 KO mice and 3 control mice. RNA samples were labeled and hybridized to Illumina Sentrix MouseRef-8 v2 bead arrays.

first: Jie

last: An

first: Susanne

last: Golech

first: Jettanong

last: Klaewsongkram

first: Yongqing

last: Zhang

first: Gail

last: Huston

first: William

last: Wood III

first: Robert

last: Wersto

last: Becker

first: Susan

last: Swain

first: Nan-ping

last: Weng


title: Transcriptome-wide mapping of RNA:protein interactions of seven SR proteins and NXF1 in P19 cells by iCLIP

status: Public on Mar 07 2016

city: Frankfurt am Main

first: Michaela

last: Müller-McNicoll

address: Max-von-Laue-Str. 13

department: Institute for Cell Biology and Neuroscience

country: Germany

institute: Goethe University Frankfurt

type: Expression profiling by high throughput sequencing

summary: We used GFP-tagged SR proteins expressed at endogenous levels and iCLIP to identify and compare endogenous RNA targets of individual SR proteins, map the preferential sites of binding, compare binding pattern and binding motifs between family members and to NXF1 and quantify binding of SR proteins and NXF1 to spliced versus unspliced RNAs to study the role of SR proteins in mRNA export via NXF1.

overall_design: For each GFP-tagged SR proteins and NXF1, 3 to 6 biological replicates were subjected to the iCLIP procedure with UV-crosslinking at 254 nm. An anti-GFP antibody as used for IPs. As negative control served samples prepared from a P19 cell line expressing only GFP-NLS fusion protein.

first: Karla

last: Neugebauer


title: Global Analysis of Glucocorticoid Receptor-Associated Transcripts and Identification of a Target RNA Motif

summary: Posttranscriptional regulation is emerging as a key factor in glucocorticoid (GC)-mediated gene regulation.  We investigated the role of the human glucocorticoid receptor (GR) as an RNA-binding protein and its effect on mRNA turnover in human airway epithelial cells.  Cell treatment with the potent GC budesonide accelerated the decay of CCL2 mRNA (t1/2=8±1 min vs. 62±17 min in DMSO-treated cells) and CCL7 mRNA (t1/2=15±4 min vs. 114±37 min), but not that of CCL5 mRNA (t1/2=231±8 min vs. 266±5 min) in the BEAS-2B cell line.   This effect was inhibited by pre-incubation with an anti-GR antibody, indicating that GR itself plays a role in the turnover of these transcripts.  Co-immunoprecipitation and biotin pulldown experiments showed that GR associates with CCL2 and CCL7 mRNAs, but not CCL5 mRNA. These methods confirmed CCL2 mRNA targeting by GR in human primary airway epithelial cells.  Association of the GR was localized to the 5’UTR of CCL2 mRNA, and further mapped to nucleotides 44-60.  The collection of transcripts associated with GR, identified by immunoprecipitation of GR-mRNA complexes followed by microarray analysis, revealed 479 transcripts that associated with GR.  Computational analysis of the primary sequence and secondary structures of these transcripts yielded a GC-rich motif, which was shown to bind to GR in vitro.  This motif was used to predict binding of GR to an additional 7889 transcripts. These results indicate that cytoplasmic GR interacts with a subset of mRNA through specific sequences and can regulate turnover rates, suggesting a novel posttranscriptional role for GR as an RNA-binding protein.

overall_design: The BEAS-2B cell line is derived from human tracheal epithelium transformed by an adenovirus 12-SV40 hybrid virus.  For isolation of glucocorticoid receptor-associated transcripts, cytoplasmic lysates obtained from 1.6x107 cells/condition, 4 replicates per condition, were used to generate a collection of transcripts associated with GR by immunoprecipitation of the GR-mRNA complexes.  The RNA from these IP's were extracted and labeled with the Illumina TotalPrep RNA Amplification Kit which generates biotin-labeled cRNA.   This was hybridized to Illumina's Sentrix HumanRef-8,v3 Expression BeadChips.  The data from 3 replicates of GR-IP and 4 replicates of Control-IP were used for global analysis of GR-mRNA interactions.

first: Faoud

middle: T

last: Ishmael

first: Xi

last: Fang

first: Kenneth

middle: R

last: Houser

last: Pearce

first: Kotb

last: Abdelmohsen

first: Ming

last: Zhan

first: Myriam

last: Gorospe

first: Cristiana

last: Stellato


title: Dynamic BRG1 Recruitment During T Helper Differentiation And Activation Reveals Distal Regulatory Elements

status: Public on Mar 01 2011

first: Supriyo

last: De

summary: We found binding of the remodeling protein BRG1 was programmed by lineage and activation signals.  BRG1 binding was positively correlated with gene activity at protein-coding and miRNA genes.  BRG1 binding was found at promoters and distal regions, including known and novel distal regulatory elements.  Distal BRG1 binding correlated with expression, and novel distal sites possessed enhancer activity, suggesting a general role for BRG1 in long-distance gene regulation.  Together, these findings suggest BRG1 interprets differentiation and activation signals and plays a causal role in gene regulation, chromatin structure, and cell fate.  Our findings indicate BRG1 binding is a useful marker for identifying cis-regulatory regions in protein-coding and miRNA genes.

overall_design: Compare BRG1 binding in T helper subsets genome wide; Naïve, resting Th1, resting Th2, Stimulated Th1, Stimulated Th2, Stimulated Th17, compared to input DNA as negative control

first: Michael

last: Pazin

first: Andrea

last: Wurster

first: Kevin

first: Patricia

last: Precht


title: Expression data for non-small-cell lung cancer

status: Public on Jan 30 2013

first: Hao

last: Tang

state: Texas

department: Clinical Sciences

summary: Purpose  Prospectively identifying who will benefit from adjuvant chemotherapy (ACT) would improve clinical decisions for individual non-small-cell lung cancer (NSCLC) patients. Most current molecular signatures for lung cancer are prognostic only and provide limited information with regard to the functional importance of the genes selected. In this study, we aim to develop and validate a functional gene set that predicts the clinical benefit of ACT in NSCLC.  Experimental Design  An 18-hub-gene prognosis signature was developed through a systems biology approach using a large NSCLC dataset from the Director’s Challenge Consortium. The prognostic value of this signature was tested in NSCLC patients from UT Lung SPORE cohort and additional five public datasets. The 18-hub-gene set was then integrated with genome-wide functional (RNAi) data and genetic aberration data to derive a 12-gene predictive signature for ACT benefit in NSCLC.   Results  We showed that the 18-hub-gene set can robustly predict the prognosis of patients with adenocarcinoma in all validation datasets across four microarray platforms. The refined 12-gene functional set was successfully validated in two independent datasets. The predicted benefit group showed significant improvement in survival after ACT (JBR.10 clinical trial data: hazard ratio=0.36, p=0.038; UT Lung SPORE data: hazard ratio=0.34, p=0.017), while the predicted non-benefit group showed no survival improvement.  Conclusions  This is the first study to integrate genetic aberration, genome-wide RNAi functional data, and mRNA expression data to identify a functional gene set that is predictive for ACT benefits. This 12-gene predictive signature has been validated in two independent NSCLC cohorts.

overall_design: Patients were eligible to enter the study if they underwent curative resection for NSCLC at MD Anderson Cancer Center between December 1996 and June 2007, and patients with radiation therapy were excluded from the study. All tissue samples were obtained by surgical resection from patients who had provided written informed consent. Tissues were stored at −140°C after being snap frozen in liquid nitrogen. Serial sectioning of each sample was used to histologically evaluate tumor and malignant cells content before RNA extraction. The primary tumor tissues from 176 patients were selected randomly from similar samples in the UT Lung SPORE tumor collection based on stringent, predefined quality control procedures before any data analysis, including the presence of ≥70% tumor tissue and ≥50% malignant cells in the frozen tissue used for RNA extraction. In this cohort, 133 patients are adenocarcinomas (ADCs) and 43 patients are squamous cell carcinomas (SCCs); 49 patients received ACT (mainly Carboplatin plus Taxanes) and 127 patients did not receive ACT.

first: Guanghua

last: Xiao

first: Carmen

last: Behrens

first: Joan

last: Schiller

first: Jeffrey

last: Allen

first: Chi-Wan

last: Chow

first: Milind

last: Suraokar

first: Alejandro

last: Corvalan

last: White

first: Ignacio

last: Wistuba

first: John

last: Minna

first: Yang

last: Xie


title: Membranous Expression of Ectodomain Isoforms of the Epidermal Growth Factor Receptor (EGFR) Predicts Outcome after Chemoradiotherapy of Lymph Node Negative Cervical Cancer

status: Public on Jul 19 2011

first: Heidi

last: Lyng

summary:
We compared the prognostic significance of ectodomain isoforms of the epidermal growth factor receptor (EGFR), which lack the tyrosine kinase (TK) domain, with that of the full length receptor and its autophosphorylation status in cervical cancers treated with conventional chemoradiotherapy.
We further explored signaling pathways that were correlated with high expression of ectodomain isoforms by gene expression analysis of 110 of the tumors, and validated the results in an independent cohort of 41 tumors.

overall_design: Pathways associated with the expression of ectodomain isoforms were studied by gene expression analysis with Illumina beadarrays in 110 patients and validated in an independent cohort of 41 patients.

first: Cathinka

last: Halle

first: Malin

last: Lando

first: Debbie

last: Svendsrud

first: Trevor

last: Clancy

first: Marit

last: Holden

first: Kolbein

last: Sundfør

first: Gunnar

last: Kristensen

first: Ruth

last: Holm


title: Deletion of the Mammalian INDY Homologue in Mice Mimics Aspects of Dietary Restriction and Protects Against Diet and Age-Induced Adiposity and Insulin Resistance

status: Public on Aug 04 2011

first: Supriyo

last: De

summary: Reduction in expression of the INDY gene in Drosophila melanogaster and Caenorhabditis elegans prolongs life span, and in D. melanogaster augments mitochondrial biogenesis in a manner akin to caloric restriction. However, the cellular mechanism by which INDY does this is unknown. In order to examine the effect that INDY might have on energy metabolism and insulin sensitivity in mammals we created the first knock out mouse model of the mammalian INDY homologue SLC13A5. Here, we show that deletion of the mammalian homologue of INDY, SLC13A5 (mINDY) in mice (mINDY-/- mice) have increased mitochondrial biogenesis, hepatic lipid oxidation, and energy expenditure, but decreased hepatic de novo lipogenesis. Loss of mIndy activates hepatic AMPK, which induces PGC-1?, inhibits ACC-2, and reduces SREBP-1c levels.  In contrast to wild- type mice, mINDY-/- mice have reduced body weight and are protected from insulin resistance that evolves with high-fat feeding and aging. These studies demonstrate that mIndy functions as a novel regulator of mammalian energy metabolism and suggest that mIndy might be a novel therapeutic target for the treatment of obesity and type 2 diabetes.

overall_design: An SLC13A5 knockout mouse model (mINDY-/-) was generated.  The litters  produced when heterozygous (KO/+) mINDY mice (mINDY+/-) were bred, contained the expected ratio of Illumina Mouse-ref8Indy wild-type (mINDY+/+), mINDY+/- mice, and homozygous mIndy (mINDY-/-) pups, indicating no substantial embryonic lethality. In wild-type mice, Indy mRNA expression was highest in liver, and very low in white adipose tissue, brown adipose tissue, skeletal muscle, small and large intestines and the pancreas confirming previous mINDY expression levels in rodents, and humans. In the liver, mINDY mRNA expression was completely abolished in the INDY-/- mice and ~50% reduced in heterozygous mINDY+/- mice.  Total RNA from the Liver tissue of 5 replicate Wt and 5 replicate Knockout mice were labeled and hybridized to Illumina Mouse-ref8  v2 bead arrays.  Analysis of gene expression was carried out by Hierarchy clustering/K-means clustering and Principal Components Analysis (PCA), as well as The Parameterized Analysis of Gene Enrichment (PAGE) and Ingenuity Pathways Analysis IPA (Ingenuity Systems, CA, USA).

first: Andreas

last: Birkenfeld

first: Hui-Young

last: Lee

first: Fitsum

last: Guebre-Egziabher

first: Michael

last: Jurczak

first: Francois

last: Jornayvaz

first: Tiago

last: Alves

first: Dongyang

last: Zhang

first: Jennifer

last: Hsiao

last: Martin-Montalvo

first: Antje

last: Fischer-Rosinsky

first: Joachim

last: Spranger

first: Lothar

last: Willmitzer

last: Pfeiffe

first: Christopher

last: Carmean

first: Dirk

last: Weismann

first: Felix

last: Knauf

first: Pablo

last: Irusta

first: Rafael

last: De Cabo

first: Stephen

last: Helfand

first: Varman

last: Samue

first: Gerald

middle: I

last: Shulman


title: A single-round of antigen receptor signaling programs naïve B cells to receive T cell help

status: Public on May 24 2010

first: Supriyo

last: De

summary: To simulate transient B cell activation that is the likely initiator of T-dependent responses, we examined the molecular and functional consequences of a single-round of immunoglobulin M (IgM) signaling. This form of activation triggered early cytosolic signaling and transcription factor NF-kB activation indistinguishably from conventional continuous IgM cross-linking, but did not induce G1 progression. However, single-round IgM signaling changed the expression of chemokine and chemokine receptor genes implicated in initiating T-dependent responses, as well as accentuated responsiveness to CD40 signaling. Several features of single-round IgM signaling in vitro were recapitulated in B cells after short-term exposure to antigen in vivo. We propose that transient BCR signals prime B cells to receive T cell help by increasing the probability of B-T encounter and creating a cellular environment that is hyper-responsive to CD40 signaling.

overall_design: Primary B lymphocytes were isolated using Auto-MACS (Miltenyi Biotec) by negative selection. B cell purity was 90-95% based on flow cytometric analysis with CD19 staining.  Purified B cells (2x10^6/ml) were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 55nM beta-mercaptoethanol, 2mM L-glutamine and 100IU penicillin and 100ug/ml streptomycin at 37degrees C. For pulsed anti-IgM treatment experiments, B cells were incubated with 10ug/ml goat anti-mouse IgM F(ab’)2 (Jackson ImmunoResearch Laboratories) at 4 degrees C for 30 min. Unbound anti-IgM was removed from the medium by washing and centrifuging the cells at 4 degrees C. The cells were resuspended in chilled complete medium and shifted to 37 degrees C by placing in an incubator or in water-bath. For continuous anti-IgM treatment experiments, B cells were stimulated with 10ug/ml anti-IgM at 4 degrees C for 30 min, then incubated at 37 degrees C.

first: Bazarragchaa

last: Damdinsuren

first: Yongqing

first: Ashraf

last: Khalil

first: William

last: Wood III

first: Kevin

last: Becker

first: Mark

last: Shlomchik

first: Ranjan

last: Sen


title: Transcriptomic reprogramming of Ethanol adapted vs Ethanol non adapted culture

status: Public on Sep 01 2009

city: Toulouse

first: Laurent

last: Benbadis

address: 135 Av de Rangueil

country: France

institute: LISBP INSA CNRS UMR5504

summary: We have previously shown that fed-batch processes with the longest uncoupling phase (ethanol adapted) were characterized by induction of storage carbohydrates, a metabolic event typical of yeast cells experiencing nutrient limitation, at the onset of this phase, whereas this metabolic event was not seen in processes with a short uncoupling phase (ethanol non adapted culture). Taken together, our results suggested that reproducible high bioethanol performance in aerated fed-batch process may be linked to the ability of yeast cells to impede ethanol toxicity by triggering a metabolic remodelling reminiscent to that of cells entering a quiescent G0/G1 state. The aim of this study was to search for genes implicated in the induction an ethanol adapted culture vs ethanol non-adapted culture.

overall_design: We measure the changes in the gene expression of Ethanol adapted culture (Test : fermentation I in ref 17005001[PMID]) and Ethanol non-adapted (reference : Fermentation II in ref 17005001[PMID]) at the same ethanol concentration of 60 g/l and the same growth rate of the cells (0,14 h-1 :Test) and (0,13 h-1 : reference) to reduce the risk of observing secondary effects due to growth and ethanol stress. For each sample, total RNAs from one yeast culture (no biological replicate) were extracted four times (technical replicates -extract). For labelling, we employed a dye-switch (dCTP-Cy3 and dCTP-Cy5) repeated 2 times and hybridized cDNA on four independent microarrays, given rise to eight data value per gene (each gene is duplicate on the slide).

first: Marlene

last: Cot

first: Jean

last: François


title: Fasting Cycles Retard Growth of Tumors and Sensitize a Range of Cancer Cell Types to Chemotherapy

status: Public on Mar 21 2012

summary: Short-term starvation (STS or fasting) provides protection to normal cells, mice, and possibly patients from a variety of chemotherapy drugs, but the possibility that it may also protect tumor cells renders its translational potential uncertain. Here we show that fasting cycles can be as effective as toxic chemotherapy drugs, and increase chemotherapy efficacy in the treatment of melanoma, glioma, breast cancer, and neuroblastoma. In vitro, STS sensitizes to chemotherapy 15 of the 17 cancer cell lines tested. In combination with chemotherapy STS results in a synergistic 20-fold increase in DNA damage, increased phosphorylation of pro-aging genes AKT and S6 kinase, reduced expression of stress resistance transcription factors FOXO3a and NFkB, elevated superoxide, and activated caspase-3; all changes not observed in normal tissues. Several of these effects are linked to the activity of heme oxygenase 1 (HO-1), whose modulation was sufficient to regulate chemotherapy-dependent cell death in breast cancer cells. These studies suggest that multiple fasting cycles have the potential to replace certain toxic chemotherapy drugs and to sensitize a wide range of tumors to chemotherapy.

overall_design: To obtain an unbiased view of the gene expression changes occurring in cancer cells in response to fasting, we performed genome-wide microarray analyses, using  Illumina's Sentrix MouseRef-8 v2 Expression BeadChips (Illumina, San Diego, CA), on the subcutaneous 4T1 breast cancer tumor mass, heart, muscle and liver tissues from balb-c mice that were either fasted for 48 hours or fed an ad lib diet. Three mice from each of the starvation and the ad lib fed groups were used for the array studies.

first: Changhan

last: Lee

first: Lizzia

last: Raffaghello

first: Fernando

last: Safdie

first: Giovanna

last: Bianchi

first: Alejandro

last: Martin-Montalvo

first: Vito

last: Pistoia

first: Saewon

last: Hwang

first: Laura

last: Emionite

last: de Cabo

first: Valter

last: Longo

first: Sebastian

last: Brandhorst

first: Min

last: Wei

first: Annalisa

last: Merlino


title: Histone methylations and gene expression in naïve, central (TCM) and effector (TEM) memory CD8 T cells.

status: Public on Mar 07 2009

first: Supriyo

last: De

summary:
Histone methylations play a major role in regulating the chromatin state and gene expression, yet little is known about their involvement in differential gene expression and function of memory CD8 T cells.  Here, we report a genome-wide analysis of two histone H3 methylations (H3K4me3 and H3K27me3) and gene expression in naïve, central (TCM) and effector (TEM) memory CD8 T cells.  Analysis of 16,314 annotated genes in CD8 T cell subsets revealed that gene expression were positively correlated with the levels of H3K4me3 and negatively correlated with the levels of H3K27me3 in these gene loci.  The correlation between differential H3K4me3 orH3K27me3 levels with gene expressions in memory CD8 T cells displayed four distinct modes: repressive, active, poised, and bivalent, reflecting their complex regulation and different function of these genes.  Furthermore, accessible chromatin states of different gene loci were preferentially influenced by different histone modifications as demonstrated here high levels of H3K9ac found in active gene loci without high levels of H3K4me3. These findings reveal a histone methylation based complex regulation of differential gene expression in memory CD8 T cells.  Thus, change of chromatin structure mediated by histone methylation may serve a fundamental basis for the rapid transcriptional response of memory CD8 T cells.

Keywords: Histone methylations, chromatin state, CD8 memory T cells

overall_design: Enriched naïve and memory CD8 T cells were purified into CD8+CD45RA+CD62L+ naïve T cells, CD8+CD45RA-CD62L+ central memory T cells (TCM), and CD8+CD45RA-CD62L- effector memory T cells (TEM) by a cell sorter (MoFlo; Dako Cytomation, Carpentaria, CA).  Triplicates of each cell type were either used right away or incubated with anti-CD3 and anti-CD28 Ab (anti-CD3/CD28) coupled magnetic beads (Invitrogen) at the cell:bead ratio of 1:1 for 16 hours in RPMI-1640 with 10% Fetal bovine serum and penicillin (10 U/ml)/streptomycin (10 ug/ml) (Invitrogen).  The freshly isolated and 16 hr stimulated cells from several donors as a pool were used for gene expression microarray analysis.  All sample data was normalized to a standard control RNA labeled and hybed along with each sample and all data from the 3 replicates was averaged.

first: Yasuto

last: Araki

first: Zhibin

last: Wang

first: Chongzhi

last: Zang

first: William

last: Wood

first: Dustin

last: Schones

first: Kairong

last: Cui

first: Tae-Young

last: Roh

first: Brad

last: Lhotsky

first: Robert

last: Wersto

first: Weiqun

last: Peng

first: Kevin

last: Becker

first: Keji

last: Zhao

first: Nan-ping

last: Weng


title: Extension of lifespan in C. elegans by naphthoquinones that act through stress hormesis mechanisms

summary: Hormesis occurs when a low level stress elicits adaptive beneficial responses that protect against subsequent exposure to severe stress.  Recent findings suggest that mild oxidative and thermal stress can extend lifespan by hormetic mechanisms.  Here we show that the botanical pesticide plumbagin, while toxic to C. elegans nematodes at high doses, extends lifespan at low doses.  Because plumbagin is a naphthoquinone that generates free radicals in vivo, we investigated whether it extends lifespan by activating an adaptive cellular stress response pathway.  Mammalian NF-E2-related factor 2 (Nrf2) and its C. elegans ortholog SKN-1, mediate protective responses to oxidative stress by promoting target gene expression via antioxidant response elements (ARE).  Genetic analyses showed that skn-1 mediates plumbagin’s lifespan-extending effect in C. elegans.  Further screening of a series of plumbagin analogs identified three additional naphthoquinones that could induce SKN-1 targets in C. elegans.  Naphthazarin showed skn-1-dependent lifespan extension, over an extended dose range compared to plumbagin, while the other naphthoquinones, oxoline and menadione, had differing effects on C. elegans survival and failed to activate ARE reporter expression in cultured mammalian cells.  Our findings reveal the potential for low doses of naturally occurring naphthoquinones to extend lifespan by engaging a specific adaptive cellular stress response pathway.

overall_design: Approximately 1,000 age-synchronized young adults were treated for two days with either 100 µM plumbagin or DMSO as a vehicle control and then washed three times in M9 and flash frozen with dry ice.  RNA was isolated with the Absolutely RNA Miniprep Kit (Agilent Technologies, USA).  RNA concentration and quality was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, USA).  Microarray hybridizations were performed with the C. elegans 4x44K Oligo Microarray (Agilent Technologies, USA).  Raw microarray hybridization intensity data from four separate experiments were log-transformed and normalized to generate z-scores and subsequent z-ratios (Cheadle et al. 2003).

first: Piper

last: Hunt

first: Tae

last: Son

first: Mark

last: Wilson

first: Quian-Sheng

last: Yu

first: Yongqing

last: Zhang

first: Nigel

last: Greig

last: Mattson

first: Simonetta

last: Camandola

first: Catherine

last: Wolkow


title: Identification of transformation-related pathways in a breast epithelial cell model using a ribonomics approach.

status: Public on Jul 29 2008

first: Supriyo

last: De

summary:
Employing MCT-1 oncogene mediated transformation of immortalized breast epithelial MCF10A cells; we characterized the largely reciprocal association of these two RBPs with target mRNAs and their influence on protein expression vis-à-vis cellular transformation.  Using a ribonomics approach, we identified mRNAs from cancer-related pathways whose association with AUF1 and/or HuR were altered when comparing immortalized with transformed MCF10A cells.
Significantly, we were able to demonstrate that knockdown of HuR expression using RNA interference, reduced anchorage-independent growth capacity in transformed MCF10A cells as well as decreased protein expression of a number of validated target genes. Our data demonstrate that the global alterations in binding of HuR and AUF1 with target transcripts have a critical role in post-transcriptional regulation of genes encoding proteins involved in breast epithelial cell transformation.

overall_design: In this study, using a microarray approach we demonstrate that the dynamic global changes in association of two RBPs, HuR and AUF1, with cancer-related mRNAs have important influence on cell transformation in a MCT-1-mediated breast epithelial transformation model.

first: Krystyna

last: Mazan-Mamczarz

first: Patrick

last: Hagner

first: Bojie

last: Dai

first: William

last: Wood III

first: Zhenqui

last: Liu

first: Ronald

last: Gartenhaus


title: Resveratrol Diet Aging (Heart, Liver, Muscle and Fat Tissue)

status: Public on Aug 05 2008

summary:
Resveratrol delays age-related deterioration and mimics transcriptional aspects of dietary restriction without extending lifespan

A small molecule that safely mimics the ability of dietary restriction (DR) to delay age-related diseases in laboratory animals is greatly sought after. We and others have shown that resveratrol mimics effects of DR in lower organisms. In mice, we find that resveratrol induces gene expression patterns in multiple tissues that parallel those induced by DR and every-other-day feeding. Moreover, resveratrol-fed elderly mice shows a marked reduction in signs of aging including reduced albuminuria, decreased inflammation and apoptosis in the vascular endothelium, increased aortic elasticity, greater motor coordination, reduced cataract formation, and preserved bone mineral density. However, mice fed a standard diet did not live longer when treated with resveratrol beginning at mid-life. Our findings indicate that resveratrol treatment has a range of beneficial effects in mice but does not increase the longevity of ad libitum-fed animals when started mid-life.

overall_design: Male C57BL/6NIA mice at 11 months of age were maintained on a standard purified mouse diet (AIN-93G) for one month prior to the start of the experiment. Beginning at one year of age, SD and EOD groups were fed a standard AIN-93G diet or AIN-93G plus 0.01% or 0.04% resveratrol for the duration of the study. Three separate groups were placed on an HC diet (AIN-93G modified by the addition of hydrogenated coconut oil to provide 60% of calories from fat) or HC + 0.01% or 0.04% resveratrol 6 weeks later and remained on those diets throughout the study. SD and HC mice were fed ad libitum. EOD mice were fed ad libitum on alternate days then moved to a separate cage without food for 24 h. Food intake and body weight were measured on a weekly basis for the duration of the study.

first: Kevin

last: Pearson

first: Joseph

last: Baur

first: Kaitlyn

middle: N

last: Lewis

first: Leonid

last: Peshkin

first: Nathan

last: Price

first: Nazar

last: Labinskyy

last: Swindell

first: Davida

last: Kamara

first: Robin

last: Minor

first: Evelyn

last: Perez

first: Hamish

last: Jamieson

first: Stephen

last: Dunn

first: Kumar

last: Sharma

first: Nancy

last: Pleshko

first: Laura

last: Woollett

first: Anna

last: Csiszar

first: Yuji

last: Ikeno

first: David

last: Le Couteur

first: Peter

last: Elliott

last: Becker

first: Placido

last: Navas

first: Donald

last: Ingram

first: Norman

last: Wolf

first: Zoltan

last: Ungvari

last: Sinclair

first: Rafael

last: de Cabo


title: Ovarian Cancer Cell Lines pre-microRNA Transfection

status: Public on Aug 29 2008

first: Supriyo

last: De

summary:
MicroRNAs (miRNAs) represent a class of small non-coding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Emerging evidence suggests the potential involvement of altered regulation of miRNA in the pathogenesis of cancers, and these genes are thought to function as both tumor suppressors and oncogenes. Using microRNA microarrays, we identify several miRNAs aberrantly expressed in human ovarian cancer tissues and cell lines. miR-221 stands out as a highly elevated miRNA in ovarian cancer, while miR-21 and several members of the let-7 family are found downregulated. Public databases were used to reveal potential targets for the highly differentially expressed miRNAs. In order to experimentally identify transcripts whose stability may be affected by the differentially expressed miRNAs, we transfected precursor miRNAs into human cancer cell lines and used oligonucleotide microarrays to examine changes in the mRNA levels.

Keywords: Expression data from various ovarian cancer cell lines transfected with pre-microRNA.

overall_design: 2 Cell lines, UCI101 and BG1,  were tranfected with various miRNA precursors, 4 different miRNA candidates (miR-98, miR-424, miR-34c and let-7f), (Ambion) and control siRNA (Dharmacon) in 24 well plates using siPORT neo-FX (Ambion) according to the manufacturer’s protocol. Three days post-transfection, RNA was prepared using RNeasy kit (Qiagen) and changes in RNA levels analyzed using Illumina microarrays.  In Short, biotinylated cRNA was prepared using the Illumina RNA Amplification Kit (Ambion, Inc., Austin, TX) according to the manufacturer’s directions starting with approximately 500 ng total RNA. Samples were purified using the RNeasy kit (Qiagen, Valencia, CA). Hybridization to the Sentrix HumanRef-8 Expression BeadChip (Illumina, Inc., San Diego, CA), washing and scanning were performed according to the Illumina BeadStation 5006manual (revision C). Array data processing and analysis was performed using Illumina Bead Studio software.  The changes in miRNA levels were monitored using N-code cDNA synthesis and qPCR.

first: Neetu

last: Dahiya

first: Cheryl

last: Sherman-Baust

first: Tian-Li

last: Wang

first: Ben

last: Davidson

first: Ie-Ming

last: Shih

first: Yongqing

last: Zhang

first: William

last: Wood III

first: Kevin

first: Patrice

last: Morin


title: CHD5, A Brain-Specific Chromatin Remodeling Enzyme, Regulates Expression Of Neuronal Genes.

status: Public on Sep 22 2011

summary: CHD5 is frequently deleted in neuroblastoma, and appears to be a tumor suppressor gene; however, little is known about the role of CHD5.  We found CHD5 mRNA was restricted to brain; by contrast most other remodeling ATPases were broadly expressed.  CHD5 protein isolated from mouse brain was associated with HDAC2, p66, MTA3 and RbAp46 in a megadalton complex.  CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons.  CHD5 protein was predominantly nuclear in primary rat neurons and brain sections.  Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons.  CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme.  Aging and Alzheimers gene sets were strongly affected by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct.  Together, these results indicate that CHD5 is found in a NuRD-like multi-protein complex.  CHD5 is restricted to the brain, unlike the closely related family members CHD3 and CHD4.  CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes.  CHD5 is linked to regulation of aging and Alzheimer’s genes.

overall_design: CHD5 KD shRNA sequences were designed according to the instructions for the pLKO.1 system (Addgene). Control was as described (Sci307-1098,2005) Scramble, clone 1864 from Addgene). Virus was packaged using HEK-293T cells, pLKO.1 vector with shRNA inserts for CHD5, and the control.   48 hours after transfection of 293 cells, medium containing virus was filtered (0.45 micron), then applied for 6 hours to primary cortical neurons one day after the neurons were plated (Day 1). Medium was removed, and replaced with Neural Basal Medium, and the cells were cultured until Day 5, 9 or 12.  RNA was harvested from 3 replicates of the treated primary cortical neurons at each time point. RNA was isolated using RNAeasy Kit (Qiagen), Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips.  RNA was labeled using the standard Illumina protocol and Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791)  Biotin labeled cRNA was hybridized to Illumina's Sentrix Rat Ref-12 v1 Expression BeadChips.

first: Rebecca

last: Casaday Potts

first: Peisu

first: Andrea

last: Wurster

first: Patricia

last: Precht

first: Mohamed

last: Mughal

first: Mark

last: Mattson

first: Michael

last: Pazin


title: CXCL12 Mediated T-Cell Migration Via Activation of Wnt5A

status: Public on Jan 01 2009

first: Supriyo

summary:
The chemokine CXCL12 and its receptor CXCR4 play important roles in signaling and migration of T-cells, but little is known about the transcriptional events involved in CXCL12-mediated T-cell migration. In this study we performed microarray analysis on CXCL12- treated T-cells, and found that the Wnt family of proteins was significantly upregulated during CXCL12 treatment. Confirmation of these results by real-time PCR and Western analysis indicated that the non-canonical Wnt pathway was specifically upregulated during CXCL12 treatment. In vitro and in vivo knockdown studies confirm that b-catenin (the key mediator of canonical Wnt signaling) is not involved in the CXCL12-mediated migration of T-cells. However, Wnt5A, a non-canonical Wnt protein, increases signaling through the CXCL12/ CXCR4 axis via Protein Kinase C (PKC). Our results demonstrated that CXCL12 required Wnt5A to mediate T-cell migration, and the treatment of T-cells with recombinant Wnt5A sensitized T-cells to CXCL12 induced migration. Additionally, Wnt5A expression was required for the sustained expression of CXCR4, both transcriptionally and translationally. These results could be translated in vivo, using EL4 thymoma metastasis as a model of T-cell migration. Taken together our data indicate, for the first time, that Wnt5A is a critical mediator of the CXCL12/ CXCR4 signaling axis.

Keywords: Wnt5A, CXCL12, CXCL12, CXCR4, T-cell Migration

overall_design: Primary T cells were treated with human CXCL12 (Peprotech, Rocky Hill, NJ) at 100 ng/ml  per 10 million cells overnight in a humidified incubator at 37ºC with 5% CO2. Control cells were incubated in media only. Cells were harvested and washed with ice cold PBS for 2 times followed by the addition of ice cold TRIzol (Invitrogen, Carlsbad, CA) and frozen at -80ºC overnight. Total RNA was isolated using the RNA isolation kit manufactured by Qiagen (Valencia, CA). The cDNA was prepared from equal amount of RNA using a cDNA preparation kit (Bio-Rad, Hercules, CA) followed by preparation of cRNA according to manufacturer’s instructions (Agilent, Santa Clara, CA).  The cRNA was amplified and labeled with either Cy-3 or Cy-5, using the Agilent low-input linear amplification kit, according to manufacturer’s protocols. Labeled cRNA were applied to the Human 44K whole genome oligo array slides (Agilent, Santa Clara, CA).  Slides were hybridized in a rotating chamber overnight at 60ºC in 6X SSC. Next day, slides were washed with 0.005% Triton X-102 for 10 minutes, and then in 0.1X SSC, 0.005% Triton X-102 for 5 minutes on ice. Slides were dried using a nitrogen-filled air gun, and scanned using an Agilent scanner. Images were analyzed using the Agilent Feature Extractor Software, Version A.7.5.1 and ratios for each spot were calculated.

first: Manik

middle: C

last: Ghosh

first: Gary

last: Collins

first: Bolormaa

last: Vandanmagsar

first: Kalpesh

last: Patel

first: Margaret

last: Brill

first: Arnell

last: Carter

first: Ana

last: Lustig

last: Becker

first: Chineye

last: Emeche

first: Amanda

last: French

last: O’Connell

first: Mai

last: Xu

first: Ashani

last: Weeraratna

first: Dennis

last: Taub


title: Ovarian Cancer MicroRNA

status: Public on Jun 30 2008

last: De

summary:
Comparison of various ovarian tumors and ovarian cell lines.
Keywords: Various ovarian tumors and cell lines.

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: Samples from ovarian tumors and ovarian cell lines were examined for their microRNA expression patterns.

first: Neetu

last: Dahiya

first: Cheryl

last: Sherman-Baust

first: Tian-Li

last: Wang

first: Ben

last: Davidson

first: Ie-Ming

last: Shih

first: Yongqing

last: Zhang

first: William

last: Wood

first: Kevin

first: Patrice

middle: J

last: Morin


title: SRT1720 extends healthspan and lifespan in diet-induced obese mice

status: Public on Aug 18 2011

first: Supriyo

summary: Activation of Sirt1, the mammalian homolog of an NAD+-dependent deacetylase known to modulate lifespan in lower organisms, is thought to hold promise as a strategy for delaying aging in mammals. SRT1720, a novel compound developed as a specific and potent activator of Sirt1, has shown promising effects to glucose homeostasis in short-term studies of rats and mice. Here we show SRT1720 extends both mean and maximum lifespan of mice fed a high-fat diet and has concrete benefits to health including reduced liver steatosis and increased insulin sensitivity and locomotor activity. Gene expression profiles and markers of inflammation and apoptosis were also restored to levels more reflective of standard diet controls. Furthermore, the benefits incurred by SRT1720 occurred in the absence of any observable toxicity. The current findings provide hope that safe and effective treatments may be developed to mitigate age-related diseases and enhance lifespan in humans.

overall_design: Male C57BL/6J mice obtained at 12 weeks of age were maintained on a standard purified mouse diet (AIN-93G) until 56 weeks of age prior to the start of the experiment. Beginning at 56 weeks of age, the SD group was fed a standard AIN-93G diet for the duration of the study. Three separate groups were placed on a high-fat diet (HFD) (AIN-93G modified by the addition of hydrogenated coconut oil to provide 60% of calories from fat) or HFD + 30mg/kg body weight SRT1720 (HFD-L) or 100mg/kg body weight SRT1720 (HFD-H) and remained on those diets throughout the study. All mice were fed ad libitum. Food intake and body weight were measured biweekly for the duration of the study.

first: Robin

last: Minor

first: Joseph

last: Baur

first: Theresa

last: Ward

first: Anna

last: Csiszar

first: Evi

middle: M

last: Mercken

first: Kotb

last: Abdelmohsen

first: Yu-Kyong

last: Shin

first: Carles

last: Canto

first: Melissa

last: Krawczyk

first: Basil

last: Hubbard

first: Elin

last: Lehrmann

last: Swindell

last: Pearson

middle: G

first: Myriam

last: Gorospe

first: Josephine

last: Egan

first: Mark

last: Talan

first: Johan

last: Auwerx

first: Christoph

last: Westphal

first: James

last: Ellis

first: Zoltan

last: Ungvari

first: David

last: Sinclair

first: Peter

last: Elliott

first: Rafael

last: de Cabo


title: AGEMAP_Lung

status: Public on Dec 28 2007

first: Supriyo

last: De

summary:
To assess the molecular changes associated with aging in different tissues of mice using cDNA microarrays to examine the transcriptomes of young, middle aged and old mice.
Mice of both genders were examined in relation to the effects of age in multiple tissues.
Keywords: age- and gender comparison.

overall_design: Up to 40 mice were compared according to age and gender.

first: Jacob

last: Zahn

first: Poosala

last: Suresh

first: Art

last: Owen

first: Donald

last: Ingram

first: Ana

last: Lustig

first: Arnell

last: Carter

first: Ashani

last: Weeraratna

first: Dennis

last: Taub

first: Krystyna

last: Mazan-Mamczarz

first: Edward

last: Lakatta

first: Kenneth

last: Boheler

first: Xiangru

last: Xu

last: Mattson

first: Geppino

last: Falco

first: Minoru

last: Ko

last: Schlessinger

first: Jeffery

last: Firman

first: Sarah

last: Kummerfeld

first: William

last: Wood

first: Alan

last: Zonderman

first: Stuart

last: Kim

first: Kevin

last: Becker


title: AGEMAP_Skeletal_Muscle

first: Supriyo

last: De

first: Jacob

last: Zahn

first: Poosala

last: Suresh

first: Art

last: Owen

first: Donald

last: Ingram

last: Lustig

last: Carter

first: Myriam

last: Gorospe

first: Mark

first: David


title: Transcriptome analysis of murine thymocytes reveals age-associated changes in thymic gene expression

status: Public on May 05 2009

summary:
The decline in adaptive immunity, naïve T-cell output and a contraction in the peripheral T cell receptor (TCR) repertoire with age are largely attributable to thymic involution and the loss of critical cytokines and hormones within the thymic microenvironment. To assess the molecular changes associated with this loss of thymic function, we used cDNA microarray analyses to examine the transcriptomes of thymocytes from mice of various ages ranging from very young (1 month) to very old (24 months). Genes associated with various biological and molecular processes including oxidative phosphorylation, T- and B- cell receptor signaling and antigen presentation were observed to significantly change with thymocyte age.  These include several immunoglobulin chains, chemokine and ribosomal proteins, annexin A2, vav 1 and several S100 signaling proteins. The increased expression of immunoglobulin genes in aged thymocytes could be attributed to the thymic B cells which were found to be actively producing IgG and IgM antibodies. Upon further examination, we found that purified thymic T cells derived from aged but not young thymi also exhibited IgM on their cell surface suggesting the possible presence of auto-antibodies on the surface thymocytes with advancing age. These studies provide valuable insight into the cellular and molecular mechanisms associated with thymic aging.

Keywords: Thymocytes,  thymic involution, aging, time course

overall_design: 3 replicates each from the 6, 16 and 24 month old animals were labeled with Cy-3 and hybridized with a Cy-5 labeled pool from 1 month old mice.  The data from the 2 channels are being reported here separately.

first: Ana

first: Arnell

first: Dorothy

last: Bertak

first: Divya

last: Enika

first: Bolormaa

last: Vandanmagsar

first: Ashani

last: Weeraratna

first: Dennis

middle: D

last: Taub


title: Survival effect of PDGF-CC rescues neurons from apoptosis in both brain and retina by regulating GSK3b phosphorylation

status: Public on Feb 26 2010

summary: Platelet-derived growth factor-CC (PDGF-CC) is the third member of the PDGF family discovered after more than two decades of studies on the original members of the family, PDGF-AA and PDGF-BB. The biological function of PDGF-CC remains largely to be explored. We report here a novel finding that PDGF-CC is a potent neuroprotective factor that acts by modulating glycogen synthase kinase (GSK)3beta activity. In several different animal models of neuronal injury, such as axotomy-induced neuronal death, neurotoxin-induced neuronal injury, 6-OHDA-induced Parkinson's dopaminergic neuronal death and ischemia-induced stroke, PDGF-CC protein or gene delivery protected different types of neurons from apoptosis in both the retina and brain. On the other hand, loss-of-function assays using PDGF-C null mice, neutralizing antibody or shRNA showed that PDGF-CC deficiency/inhibition exacerbated neuronal death in different neuronal tissues in vivo.  Mechanistically, we revealed that the neuroprotective effect of PDGF-CC was achieved by regulating GSK3beta phosphorylation and expression. Our data demonstrate that PDGF-CC is critically required for neuronal survival, and may potentially be used to treat neurodegenerative diseases. Inhibition of the PDGF-CC/receptor pathway for different clinical purposes should be conducted with caution to preserve normal neuronal functions.

overall_design:
PDGF-C deficient mice were bred onto C57BL/6 background for more than six generations and littermates were used. After optic nerve crush (ONC), PDGF-CC protein × 1 µl of active recombinant human PDGF-CC protein 0.5 µg/µl (rhPDGF-CC) was injected into mouse vitreous once a week for two weeks. Seven days after the ONC injury and PDGF-CC protein treatment, retinae were harvested and total RNA isolated using the TRIzol reagent (Invitrogen) followed by the RNeasy Mini kit (Qiagen) purification according to the manufacturer's instructions. Microarray assay was performed using the Mouse-6 Expression BeadChips containing approximately 24,000 annotated mouse transcripts (Illumina Inc). Three biological repeats were included in the microarray assay. Two tailed Student's t-test was used for statistical analysis of gene expression data. Functional grouping of the differentially expressed genes was performed using several different tools including the WebGestalt (http://bioinfo.vanderbilt.edu/webgestalt) and the Ingenuity Pathways Analysis (https://analysis.ingenuity.com/pa/login/login.jsp).

The supplementary file 'GSE19207_non-normalized.txt' contains raw data for Samples GSM476021-GSM476026.

first: Zhongshu

last: Tang

first: Pachiappan

last: Arjunan

first: Chunsik

last: Lee

first: Yang

last: Li

first: Anil

last: Kumar

first: Xu

last: Hou

first: Bin

last: Wang

first: Piotr

last: Wardega

first: Fan

last: Zhang

first: Lijin

last: Dong

first: Yongqing

first: Shi-Zhuang

first: Hao

last: Ding

first: Johan

last: Lennartsson

first: Nobuo

last: Nagai

first: Yihai

last: Cao

first: Xuri


title: ATM regulates a DNA damage response post-transcriptional RNA operon in lymphocytes

status: Public on Jan 05 2011

first: Supriyo

last: De

summary: Maintenance of genomic stability depends on the DNA damage response (DDR), a biological barrier in early stages of cancer development. Failure of this response results in genomic instability and high predisposition toward lymphoma, as seen in patients with ataxia-telangiectasia mutated (ATM) dysfunction. ATM activates multiple cell cycle checkpoints and DNA repair following DNA damage, but its influence on posttranscriptional gene expression has not been examined on a global level. We show that ionizing radiation (IR) modulates the dynamic association of the RNA-binding protein HuR with target mRNAs in an ATM-dependent manner, potentially coordinating the genotoxic response as an RNA operon. Pharmacologic ATM inhibition and use of ATM-null cells revealed a critical role for ATM in this process. Numerous mRNAs encoding cancer-related proteins were differentially associated with HuR depending on the functional state of ATM, in turn affecting expression of encoded proteins. The findings presented here reveal a previously unidentified role of ATM in controlling gene expression post-transcriptionally.  Dysregulation of this DDR RNA operon is likely relevant to lymphoma development in ataxia-telangiectasia individuals. These novel RNA regulatory modules and genetic networks provide critical insight into the function of ATM in oncogenesis.

overall_design: B-lymphocyte cell lines GM02184 (wild type, ATM +/+) and GM03332 (AT, ATM -/-) were either untreated or exposed to 1 Gy of IR. 6 h later cells were harvested and used for immunoprecipitation (IP) in the presence of HuR antibody (Santa Cruz Biotech.). RNA from IP material was extracted and used for microarray analysis.

first: Krystyna

last: Mazan-Mamczarz

first: Patrick

last: Hagner

first: Bojie

last: Dai

first: Elin

last: Lehrmann

first: Kevin

last: Becker

first: Jack

last: Keene

first: Myriam

last: Gorospe

first: Zhenqui

last: Liu

first: Ronald

middle: B

last: Gartenhaus


title: AGEMAP_Heart

first: Jacob

last: Zahn

first: Poosala

last: Suresh

first: Art

last: Owen

first: Donald

last: Ingram

first: Ana

last: Lustig

first: Arnell

last: Carter

first: Ashani

last: Weeraratna

first: Dennis

last: Taub

first: Edward

last: Lakatta

first: Kenneth

last: Boheler

first: Xiangru

last: Xu

first: Mark

last: Mattson

first: Geppino

last: Falco

first: Minoru

last: Ko

first: David

last: Schlessinger

first: Jeffery

last: Firman

first: Sarah

last: Kummerfeld

first: William

last: Wood

first: Alan

last: Zonderman

first: Stuart

last: Kim


title: AGEMAP_Bone_Marrow

first: Supriyo

last: De

first: Jacob

last: Zahn

first: Poosala

last: Suresh

first: Art

last: Owen

first: Donald

last: Ingram

first: Myriam

last: Gorospe

first: Krystyna

last: Mazan-Mamczarz

first: Kevin

last: Becker


title: Age Map, Cerebellum (A)

status: Public on Jun 22 2009

summary:
To assess the transcriptional changes in different brain regions and spinal cord associated with aging and either a caloric restricted (CR) or ad libitum (AL) diet. cDNA microarray and quantitative PCR analyses were used to examine the transcriptomes of various neuronal tissues in young, middle aged and old mice. Mice of both genders were examined as well as the effects of their placement on either caloric restricted (CR) or ad libitum (AL) diets.
Keywords: age-, gender-, and diet- comparison

overall_design: 67 mice were compared according to age, gender, and diet

first: Xiangru

last: Xu

first: Ming

last: Zhan

first: Wenzhen

last: Duan

first: Vinayakumar

last: Prabhu

first: Randall

last: Brenneman

last: Wood III

first: Jeff

first: Huai

last: Li

first: Peisu

last: Zhang

first: Carol

last: Ibe

first: Dan

last: Longo

first: Suresh

last: Poosala

first: Mark

last: Mattson


title: Growth hormone receptor deficiency is associated with a major reduction in pro-aging signaling, cancer, and diabetes in humans

status: Public on Dec 25 2010

first: Supriyo

last: De

summary: Mutations in growth signaling pathways extend life span, as well as protect against age-dependent DNA damage in yeast and decrease insulin resistance and cancer in mice. To test their effect in humans, we monitored for 22 years Ecuadorian individuals who carry mutations in the growth hormone receptor (GHR) gene that lead to severe GHR and IGF-1 (insulin-like growth factor-1) deficiencies. We combined this information with surveys to identify the cause and age of death for individuals in this community who died before this period. The individuals with GHR deficiency exhibited only one nonlethal malignancy and no cases of diabetes, in contrast to a prevalence of 17% for cancer and 5% for diabetes in control subjects. A possible explanation for the very low incidence of cancer was suggested by in vitro studies: Serum from subjects with GHR deficiency reduced DNA breaks but increased apoptosis in human mammary epithelial cells treated with hydrogen peroxide. Serum from GHR-deficient subjects also caused reduced expression of RAS, PKA (protein kinase A), and TOR (target of rapamycin) and up-regulation of SOD2 (superoxide dismutase 2) in treated cells, changes that promote cellular protection and life-span extension in model organisms. We also observed reduced insulin concentrations (1.4 μU/ml versus 4.4 μU/ml in unaffected relatives) and a very low HOMA-IR (homeostatic model assessment-insulin resistance) index (0.34 versus 0.96 in unaffected relatives) in individuals with GHR deficiency, indicating higher insulin sensitivity, which could explain the absence of diabetes in these subjects. These results provide evidence for a role of evolutionarily conserved pathways in the control of aging and disease burden in humans

overall_design: Primary Human Mammalian Epithelial Cells (HMECs), were cultured in HMEC medium (ScienCell) at 37oC and 5% CO2 in Poly-L-Lysine coated culture dishes (Sigma). Treatment consisted of cells being stimulated with HMEC basal medium containing either 15% GHRD serum or 15% control serum for 6 hours. Cells were then harvested and processed for RNA extraction.

first: Jaime

last: Guevara-Aguirre

first: Priya

last: Balasubramanian

first: Marco

first: Min

last: Wei

first: Federica

last: Madia

first: Chia-Wei

last: Cheng

first: David

last: Hwang

first: Alejandro

last: Martin-Montalvo

first: Jannette

last: Saavedra

first: Sue

last: Ingles

first: Rafael

last: de Cabo

first: Pinchas

last: Cohen

first: Valter


title: AGEMAP_Gonads

first: Jacob

last: Zahn

first: Poosala

last: Suresh

first: Art

last: Owen

first: Donald

last: Ingram

first: Ana

last: Lustig

first: Arnell

last: Carter

first: Ashani

last: Weeraratna

first: Dennis

last: Taub

first: Myriam

last: Gorospe

first: Krystyna

last: Mazan-Mamczarz

first: Edward

last: Lakatta

first: Kenneth

last: Boheler

first: Xiangru

last: Xu

first: Geppino

last: Falco

first: Minoru

last: Ko

last: Schlessinger

first: Jeffery

last: Firman

first: Sarah

last: Kummerfeld

first: William

last: Wood

first: Alan

last: Zonderman

first: Stuart

last: Kim

first: Kevin

last: Becker


title: AGEMAP_Adrenals

first: Supriyo

last: De

first: Jacob

last: Zahn

first: Poosala

last: Suresh

first: Art

last: Owen

first: Donald

last: Ingram

first: Ana

last: Lustig

last: Carter

first: Mark

last: Mattson

first: David


title: Age Map, Striatum (B)

first: Xiangru

last: Xu

first: Ming

last: Zhan

first: Wenzhen

last: Duan

first: Vinayakumar

last: Prabhu

first: Randall

last: Brenneman

last: Wood III

first: Jeff

first: Huai

last: Li

first: Peisu

last: Zhang

first: Carol

last: Ibe

first: Dan

last: Longo

first: Suresh

last: Poosala


title: AGEMAP_Kidney

first: Supriyo

last: De

first: Jacob

last: Zahn

first: Poosala

last: Suresh

first: Arnell

first: Ashani

last: Weeraratna

first: Dennis

last: Taub

first: Myriam

last: Gorospe

first: Krystyna

last: Mazan-Mamczarz

first: Edward

last: Lakatta

first: Kenneth

last: Boheler

first: Geppino

last: Falco

first: Minoru

last: Ko

last: Schlessinger

first: Jeffery

last: Firman

first: Sarah

last: Kummerfeld

first: William

last: Wood

first: Alan

last: Zonderman

first: Stuart

last: Kim

first: Kevin

last: Becker


title: Age Map, Cerebral Cortex (B)

first: Supriyo

last: De

first: Xiangru

last: Xu

first: Ming

last: Zhan

first: Wenzhen

last: Duan

first: Vinayakumar

last: Prabhu

first: Randall

last: Brenneman

last: Wood III

first: Jeff

first: Huai

last: Li

first: Peisu

last: Zhang

first: Carol

last: Ibe

first: Dan

last: Longo

first: Suresh

last: Poosala

first: Mark

last: Mattson


title: AGEMAP_Spleen

first: Jacob

last: Zahn

first: Poosala

last: Suresh

first: Art

last: Owen

first: Donald

last: Ingram

first: Ana

last: Lustig

first: Arnell

last: Carter

first: Ashani

last: Weeraratna

first: Dennis

last: Taub

first: Myriam

last: Gorospe

first: Krystyna

last: Mazan-Mamczarz

first: Edward

last: Lakatta

first: Kenneth

last: Boheler

first: Geppino

last: Falco

first: Minoru

last: Ko

first: David

last: Schlessinger

first: Jeffery

last: Firman

first: Sarah

last: Kummerfeld

first: William

last: Wood


title: Age Map, Spinal Cord (B)

first: Supriyo

last: De

summary:
To assess the transcriptional changes in different brain regions and spinal cord associated with aging and either a caloric restricted (CR) or ad libitum (AL) diet. cDNA microarray and quantitative PCR analyses were used to examine the transcriptomes of various neuronal tissues in young, middle aged and old mice. Mice of both genders were examined as well as the effects of their placement on either caloric restricted (CR) or ad libitum (AL) diets.
To assess the transcriptional changes in different brain regions and spinal cord associated with aging and either a caloric restricted (CR) or ad libitum (AL) diet. cDNA microarray and quantitative PCR analyses were used to examine the transcriptomes of various neuronal tissues in young, middle aged and old mice. Mice of both genders were examined as well as the effects of their placement on either caloric restricted (CR) or ad libitum (AL) diets.
Keywords: age-, gender-, and diet- comparison

first: Xiangru

last: Xu

first: Ming

last: Zhan

first: Wenzhen

last: Duan

first: Vinayakumar

last: Prabhu

first: Randall

last: Brenneman

last: Wood III

first: Jeff

first: Huai

last: Li

first: Peisu

last: Zhang

first: Carol

last: Ibe

first: Alan

last: Zonderman

first: Dan

last: Longo

first: Suresh

last: Poosala

first: Kevin

last: Becker

first: Mark

last: Mattson


title: Age Map, Hippocampus (B)

last: Firman


title: Age Map, Spinal Cord (A)


title: Age Map, Striatum (A)


status: Public on Jul 07 2007

summary:
Naïve CD4+ T cells were isolated from spleen of AND TcR transgenic/green fluorescence protein (GFP) transgenic mice (Kaye et al., Nature 1989;341:746, Wright et al, Blood 2001;97:2278) that recognize a peptide of pigeon cytochrome C in the context of I-Ek and express CD44lo, CD62Lhi, CD45RBhi, and CD25-.  After 4 days in vitro stimulation with antigen presenting cells (APC) under either Th1 or Th2 condition, naïve cells become Th1 or Th2 effector cells expressing CD44hi, CD62L lo, CD45RBhi, and CD25+.  Additional 3 days culture in the absence of APC, those effector cells become rested expressing a phenotype similar to memory cells (CD44 hi, CD62L lo, CD45RB lo and CD25-).  These rested effector cells were adaptively transferred into thymectomized, lethally irradiated, and T cell depleted bone marrow reconstituted mice and memory cells were isolated after 4-12 weeks by flow sort.
Keywords: Microarray analysis of Th1 and Th2 effector and memory CD4+ T cells

overall_design: Generation and purification of Th1 and Th2 effector and memory CD4+ T cells of 42 samples.

first: K

last: McKinstry

first: Susanne

last: Golech

first: W

middle: H

last: Lee

first: Gail

last: Huston

first: N

last: Weng

first: Susan

last: Swain


title: Expression patterns in liver after resveratrol treatment of mice on a high-calorie diet

status: Public on Nov 03 2006

first: Supriyo

last: De

summary:
Resveratrol (3,5,4'-trihydroxystilbene) extends the lifespan of diverse species including yeast, worms, and flies. In these organisms, lifespan extension is dependent on Sir2, a conserved deacetylase proposed to underlie the beneficial effects of caloric restriction (CR). Here we show that resveratrol shifts the physiology of middle-aged mice on a high-calorie diet towards that of mice on a standard diet and significantly increases their survival. Resveratrol produces changes associated with longer lifespan including increased insulin sensitivity, reduced IGF-1, increased AMPK and PGC-1α activity, increased mitochondrial number, and improved motor function. Parametric analysis of gene set enrichment (PAGE) revealed that resveratrol opposed the effects of the high-calorie diet in 144 out of 153 significantly altered pathways. These data show that improving general health in mammals using small molecules is an attainable goal and point to new approaches for treating obesity-related disorders and diseases of ageing.
Keywords: Drug treatment

overall_design: One-year-old male C57BL/6 mice were maintained on AIN-93G standard diet (SD), AIN-93G modified to provide 60% of calories from fat (HC), or HC diet with the addition of 0.04% resveratrol (HCR).  Total RNA from the livers of 5 replicate animals from the first 2 groups and 4 from the 3rd group were labeled and hybridized to Agilent 44K whole genome microarrays.

first: Joseph

middle: A

last: Baur

last: Pearson

first: Nathan

last: Price

first: Hamish

last: Jamieson

first: Carles

last: Lerin

first: Avash

last: Kalra

first: Vinayakumar

middle: V

last: Prabhu

first: Joanne

last: Allard

first: Guillermo

last: Lopez-Lluch

first: Kaitlyn

last: Lewis

first: Paul

last: Pistell

first: Olivier

last: Boss

first: Dana

last: Gwinn

first: Mingyi

last: Wang

first: Sharan

last: Ramaswamy

first: Kenneth

middle: W

last: Fishbein

first: Richard

last: Spencer

first: Edward

last: Lakatta

first: David

last: Le Couteur

first: Reuben

last: Shaw

first: Placido

last: Navas

first: Pere

last: Puigserver

first: Donald

last: Ingram

first: Rafael

last: de Cabo

last: Sinclair