title: Adhesion GPCR, latrophilin-2, specifies cardiac lineage commitment through CDK5, Src, and P38MAPK

status: Public on Jan 06 2017

city: Seoul

first: Choon-Soo

last: Lee

address: 28 Yongon-Dong, Jongno-gu

institute: Seoul National University Hospital

summary: Identifying lineage-specific markers is pivotal for understanding developmental processes and developing cell therapies. Here, we report a new cardiomyogenic cell surface marker latrophilin-2 (Lphn2), an adhesion G protein-coupled receptor. When mouse and human pluripotent stem cells (PSCs) were stimulated with BMP4, Activin A, and bFGF, they differentiated into cardiac lineage cells. Lphn2 was selectively expressed on cardiac progenitor cells (CPCs) and cardiomyocytes (CMCs) during the differentiation of mouse PSCs, and cell sorting with an anti-Lphn2 antibody promoted the isolation of populations highly enriched in CPCs and CMCs. Lphn2 knock-down or knock-out PSCs did not express cardiac genes. To investigate the molecular mechanism underlying the induction of cardiac differentiation by Lphn2, we used the Phospho Explorer Antibody Array, which encompasses nearly all known signaling pathways. Lphn2-dependent phosphorylation was strongest for cyclin-dependent kinase 5 (CDK5) at Tyr15. We identified CDK5, Src, and P38MAPK as key downstream molecules of Lphn2. These findings provide a valuable tool for isolating cardiomyogenic progenitors and CMCs from PSCs and shed light on the still-unknown mechanisms of cardiac differentiation.

overall_design: 6 samples are analyzed by 3 groups.

first: Hyun-Jai

last: Cho

first: Jin-Woo

first: Jaewon

first: Yoo-Wook

last: Kwon

first: Hanseul

last: Park

first: Jongpil

last: Kim

first: Hyo-Soo


title: HNF4α transduction and 5'azacytidine treatment of naïve rat liver epithelial cells (rLEC)

status: Public on Jan 01 2017

city: Jette

first: jennifer

last: bolleyn

address: Laarbeeklaan 103

country: Belgium

institute: Vrije universiteit Brussel

type: Expression profiling by high throughput sequencing

summary: Neonatal liver-derived rat epithelial cells (rLEC) from biliary origin are liver progenitor cells that acquire a hepatocyte-like phenotype upon sequential exposure to hepatogenic growth factors and cytokines. Undifferentiated rLEC express several liver-enriched transcription factors including the hepatocyte nuclear factors (HNF) 3β and HNF6, but not the hepatic master regulator HNF4α. In this study, we investigated the impact of the ectopic expression of HNF4α in rLEC on both mRNA and microRNA level by means of microarray technology. In addition, we investigated the influence of subsequent DNA methyl transferase (DNMT) inhibition on the phenotype of undifferentiated rLEC by exposure to 5' azacytidine (AZA).

platform_organism:
Rattus norvegicus
synthetic construct

overall_design: rLEC were transduced with a lentiviral vector containing HNF4α and puromycin selected.  In addition, cells were exposed to 20 µM 5'azacytidine for 7 days

first: Jennifer

last: Bolleyn

first: Matthias

last: Rombaut

first: Nisha

last: Nair

first: Marinee

last: Chuah

first: Thierry

last: VandenDriessche

first: Vera

last: Rogiers

first: Joery

last: De Kock

first: Tamara

last: Vanhaecke


title: Resveratrol Supplementation Ameliorates Neuronal Insults in Cortical Brain Tissue of Nonhuman Primates Fed a High-Fat/Sucrose Diet

status: Public on Feb 14 2017

city: Baltimore

first: Supriyo

last: De

state: Maryland

address: 251 Bayview Blvd

department: Laboratory of Genetics and Genomics

country: USA

institute: NIA-IRP, NIH

summary: Chronic high-fat/high-sugar (HFS) feeding is linked to the development of insulin resistance as well as arterial wall and adipose tissue inflammation in nonhuman primates. These changes are significantly reduced when the animals are fed a HFS diet supplemented with resveratrol (RSV) for two years. Herein, we evaluated the occurrence of cerebral cortex injury in these HFS-fed middle-aged male rhesus monkeys and investigated the possibility of brain protection by RSV treatment. HFS caused a reduction in capillary density in the cerebral cortex that was preempted by RSV supplementation. The patterns of cDNA microarray analysis revealed upregulation of markers of oxidative stress, inflammation and apoptosis in the cortical cortex of HFS-fed monkeys, which was reversed by RSV. The underlying mechanism of RSV action included its ability to prevent the HFS-mediated NF-kappa-B activation and loss in mitochondrial aldehyde dehydrogenase 2 expression. We conclude that RSV may confer neuroprotection against HFS-mediated cerebral vascular dysfunction and activation of inflammatory pathways.

overall_design: Twenty-four adult (7-13 years old) male rhesus monkeys (Macaca mulatta) were maintained on standard NIH monkey chow (Purina Mills) during baseline assessment, and then quasi-randomized (such that average age and bodyweight did not differ significantly between groups) to one of three two-year dietary regimens: Four animals were fed a healthy standard diet (SD) with 13% Kcal in fat and less than 5% sucrose by weight, ten animals were fed a high fat and sugar (HFS) diet with 42% Kcal in fat and 27% sucrose by weight (Harlan, Teklad), and another ten animals (RESV) were fed the same HFS diet plus a flavored primate treat (Bioserve) containing 40 mg and 240 mg resveratrol (RSV) twice daily, during year 1 and 2, respectively. Monkeys in non-RSV groups received placebo treats.

first: Michel

last: Bernier

first: Devin

last: Wahl

first: Ahmed

last: Ali

first: Joanne

last: Allard

first: Shakeela

last: Faulkner

first: Artur

last: Wnorowski

first: Mitesh

last: Sanghvi

first: Ruin

last: Moaddel

first: Irene

last: Alfaras

first: Julie

last: Mattison

first: Zsuzsanna

last: Tucsek

first: Zoltan

last: Ungvari

first: Anna

last: Csiszar

first: Rafael

last: de Cabo


title: Identification of Senescence-Associated Circular RNAs (SAC-RNAs) reveals senescence suppressor CircPVT1

status: Public on Feb 13 2017

type: Non-coding RNA profiling by high throughput sequencing

summary: With advancing age, senescent cells accumulate in tissues and critically influence aging-associated diseases.  We report the first catalog of a novel class of regulatory RNAs, circular RNAs, differentially expressed in senescent (non-dividing) compared with dividing human fibroblasts.  Among them, we focused on the circular RNA CircPVT1, as it promoted proliferation by binding and neutralizing let-7, a microRNA capable of triggering senescence.  Interestingly, by suppressing let-7, CircPVT1 selectively elevated the expression of several proliferative proteins which were otherwise repressed by let-7.  In summary, we have uncovered a novel mechanism whereby circular RNAs control gene expression programs in senescent cells.

overall_design: Identification and validation of Senescence-Associated Circular RNAs (SAC-RNAs) in human diploid WI-38 fibroblasts

first: Amaresh

middle: C

last: Panda

first: Ioannis

last: Grammatikakis

first: Kyoung

middle: M

last: Kim

first: Jennifer

middle: L

last: Martindale

first: Rachel

last: Munk

first: Xiaoling

last: Yang

first: Kotb

last: Abdelmohsen

first: Myriam

last: Gorospe


title: Transcriptome States Reflect Imaging of Aging States

status: Public on Dec 19 2016

first: Supriyo

last: De

summary: We developed a morphological biomarker that detects multiple discrete sub-populations (or “age states”) at any given chronological age in a population of nematodes (C. elegans). Age-matched animals in different age states have distinct transcriptome profiles, one markedly older than the other as determined by comparison with other aging study microarray datasets. Here we characterized the frequencies of three healthy adult states and the transitions between them across the lifespan. Jaccard similarity showed expression profiles were more similar within states than between states. Chronologically identical individuals showed less similarity than morphologically identical individuals isolated on different days. We used short-lived and long-lived strains to confirm the general applicability of the state classifier and to monitor state progression. This exploration revealed healthy and unhealthy states, the former being favored in long-lived strains and the latter showing delayed onset. Short-lived strains rapidly transitioned through the putative healthy state. We expected state exit factors to be up regulated later in a given state. Several small heat shock protein encoding genes demonstrated oscillation within states. Oscillatory expression of sHSPs within states and proteasome components between states, supports an extension of a proteostasis collapse model to include periodic collapses and rebuilding phases.

overall_design: Six groups representing chronologically early and late stages for three aging states  (N=3-6 per state, total N=24 microarray samples)

first: D

last: Eckley

first: Christopher

middle: E

last: Coletta

first: Nikita

middle: V

last: Orlov

first: Mark

last: Wilson

first: Wendy

last: Iser

first: Paul

last: Bastian

first: Elin

last: Lehrmann

first: Yonqing

last: Zhang

first: Kevin

middle: G

last: Becker

first: Ilya

last: Goldberg


title: Racial differences in microRNA and gene expression in hypertensive women (Exiqon)

status: Public on Nov 16 2016

summary: Systemic arterial hypertension is an important cause of cardiovascular disease morbidity and mortality. African Americans are disproportionately affected by hypertension, in fact the incidence, prevalence, and severity of hypertension is highest among African American (AA) women. Previous data suggests that differential gene expression influences individual susceptibility to selected diseases and we hypothesized that this phenomena may affect health disparities in hypertension. Transcriptional profiling of peripheral blood mononuclear cells from AA or white, normotensive or hypertensive females identified thousands of mRNAs differentially-expressed by race and/or hypertension. Predominant gene expression differences were observed in AA hypertensive females compared to AA normotensives or white hypertensives. Since microRNAs play important roles in regulating gene expression, we profiled global microRNA expression and observed differentially-expressed microRNAs by race and/or hypertension. We identified novel mRNA-microRNA pairs potentially involved in hypertension-related pathways and differently-expressed, including MCL1/miR-20a-5p, APOL3/miR-4763-5p, PLD1/miR-4717-3p, and PLD1/miR-4709-3p. We validated gene expression levels via RT-qPCR and microRNA target validation was performed in primary endothelial cells. Altogether, we identified significant gene expression differences between AA and white female hypertensives and pinpointed novel mRNA-microRNA pairs differentially-expressed by hypertension and race. These differences may contribute to the known disparities in hypertension and may be potential targets for intervention.

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: Effects of race and/or hypertensive status on gene expression was examined using microarrays to obtain transcriptional profiles from peripheral blood mononuclear cells (PBMCs) microRNA from African American or white, normotensive or hypertensive females (n=24).

first: Douglas

middle: F

last: Dluzen

first: Nicole

last: Noren Hooten

first: Yongqing

first: Frank

last: Glover

first: Salman

last: Tajuddin

first: Kimberly

middle: D

last: Jacob

first: Alan

middle: B

last: Zonderman

first: Michele

middle: K

last: Evans


title: Racial differences in microRNA and gene expression in hypertensive women. (miRNA transfection)

first: Supriyo

last: De

overall_design: The effect of transfection with pre-miRNA precursors hsa-miR-20a_5p, -30c_5p, -4763_5p and a scrambled negative control on global gene expression was examined in primary human aortic endothelial cells (HAECs) with three replicates per group.


title: Racial differences in microRNA and gene expression in hypertensive women (miRNA transfection, part 2)

overall_design: The effect of transfection with pre-miRNA precursors hsa-miR-4709_3p, -4717_3p and a scrambled negative control on global gene expression was examined in primary human umbilical vein endothelial cells (HUVECs) with three replicates per group.


title: Racial differences in microRNA and gene expression in hypertensive women (mRNA)

overall_design: Effects of race and/or hypertensive status on gene expression was examined using microarrays to obtain transcriptional profiles from peripheral blood mononuclear cells (PBMCs) mRNA from African American or white, normotensive or hypertensive females (n=24) and global microRNA (miRNA) expression in a larger cohort (n=40), with RT-qPCR quantification in an expanded cohort (n=80).


title: Racial differences in microRNA and gene expression in hypertensive women

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

summary:
Systemic arterial hypertension is an important cause of cardiovascular disease morbidity and mortality. African Americans are disproportionately affected by hypertension, in fact the incidence, prevalence, and severity of hypertension is highest among African American (AA) women. Previous data suggests that differential gene expression influences individual susceptibility to selected diseases and we hypothesized that this phenomena may affect health disparities in hypertension. Transcriptional profiling of peripheral blood mononuclear cells from AA or white, normotensive or hypertensive females identified thousands of mRNAs differentially-expressed by race and/or hypertension. Predominant gene expression differences were observed in AA hypertensive females compared to AA normotensives or white hypertensives. Since microRNAs play important roles in regulating gene expression, we profiled global microRNA expression and observed differentially-expressed microRNAs by race and/or hypertension. We identified novel mRNA-microRNA pairs potentially involved in hypertension-related pathways and differently-expressed, including MCL1/miR-20a-5p, APOL3/miR-4763-5p, PLD1/miR-4717-3p, and PLD1/miR-4709-3p. We validated gene expression levels via RT-qPCR and microRNA target validation was performed in primary endothelial cells. Altogether, we identified significant gene expression differences between AA and white female hypertensives and pinpointed novel mRNA-microRNA pairs differentially-expressed by hypertension and race. These differences may contribute to the known disparities in hypertension and may be potential targets for intervention. This SuperSeries is composed of the SubSeries listed below.

This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: The whole transcriptome analysis in a replicative senescence model.

status: Public on Sep 12 2016

summary: RNA-Seq was performed to screen transcripts in whole lyastes or mitoplast fraction prepared from young and senescent WI-38 human fibroblast

overall_design: A whole or mitoplast RNA was extracted from both young and senescent WI-38 cells, and used for RNA-Seq.

first: Ji

middle: H

last: Noh

first: Kyoung

last: Kim

first: Kotb

last: Abdelmohsen

first: Je-Hyun

last: Yoon

first: Amaresh

last: Panda

first: Rachel

last: Munk

first: Jiyoung

first: Jessica

last: Curtis

first: Christopher

middle: A

last: Moad

first: Fred

last: Indig

first: Wilson

last: de Paula

first: Dawood

last: Dudekula

first: Xiaoling

last: Yang

first: Jennifer

last: Martindale

first: Rafael

last: de Cabo

first: Myriam

last: Gorospe


title: Serum extracellular vesicle depletion processes affect release and infectivity of HIV-1 in culture

status: Public on Oct 23 2016

first: Supriyo

summary: Extracellular vesicles (EVs) are involved in intercellular communication in health and disease and affect processes including immune and antiviral responses. We have previously demonstrated that ultracentrifuged serum is depleted of EVs and, when used in cell culture media, is associated with declines in growth and viability of numerous cultured cell types. Although EVs had been reported to enhance or interfere with HIV-1 infection, depending on the setting, the effects of EVs on HIV-1 production and infectivity of released virions were unknown. In this study, we examined the effects of EV-depleted serum on HIV-1 replication in primary cells and cell lines, including two HIV-1 latency models. Cell culture media were prepared with EV-replete fetal bovine serum (FBS) or serum depleted of EVs via ultracentrifugation or a proprietary method (ThermoFisher/Gibco). T-cell and myeloid-lineage cell lines, including ACH-2 and U1 HIV-1 latency models, and primary cells were grown in 10% FBS-based culture media. Cell counts, viability, and proliferation were assessed throughout. HIV-1 production and infectivity were measured by p24 ELISA and luciferase reporter cell lines, respectively. Flow cytometry, Seahorse assays, and miRNA and mRNA expression arrays were done to assess cellular responses to EV-depleted conditions. Significant increases in HIV-1 production were observed in EV-depleted conditions, along with, in some cases, morphology changes and decreased cell viability. Add-back of pelleted EVs reduced HIV-1 production almost to baseline. Primary cells appeared to be less susceptible to EV depletion. ACH-2 and U1 latency models also produced more HIV-1 under EV-depleted conditions. Virus produced under EV-depleted conditions was more infectious. Changes in cellular metabolism and gene expression were associated with EV-depleted culture. The EV environment of HIV-1 infected cells appears to have a significant effect on virus production and infectivity. In cell lines of HIV-latency, significantly higher concentrations of p24 were observed in those cells cultured in EVD conditions. EV-dependence of cell cultures should be examined carefully prior to examining additional experimental variables. However, we also sound a cautionary note that direct actions of EVs may be accompanied by the effects of other, closely associated factors.

overall_design: HEK293T cells were incubated with FBS (CTR, N=4), extracellular vesicle-depleted serum (exosome-depleted, N=4), or extracellular vesicle-enriched (exosome-enriched, N=4)

first: Zhaohao

last: Liao

first: Dillon

last: Muth

first: Erez

last: Eitan

first: Meghan

last: Travers

first: Elin

last: Lehrmann

first: Roy

last: Cutler

first: Mark

middle: P

last: Mattson

first: Kenneth

middle: W

last: Witwer


title: A total RNA-Seq screen reveals that the splicing inhibitor Isoginkgetin blocks transcription elongation

status: Public on Sep 17 2016

city: boston

first: jesse

last: gray

state: MA

address: 77 avenue louis pasteur, NRB 356

department: Genetics

institute: Harvard Medical School

summary: Pharmacological perturbation is a powerful tool for understanding gene expression, but identification of the specific steps of this multi-step process targeted by small molecules remains challenging. Here we apply total RNA-Seq to distinguish specific pharmacological effects on transcription and pre-mRNA processing, revealing unexpectedly that the splicing inhibitor isoginkgetin blocks transcription elongation.An RNA-Seq screen reveals that the compounds TBBz and CYT387 mimic the transcriptional effects of isoginkgetin and are inhibitors of CSNK2A2, suggesting that isoginkgetin blocks transcription elongation via inhibition of CSNK2A2. Our results reveal RNA-Seq screening as a tool for disentangling complex pharmacological effects on gene expression.

overall_design: Total RNA sequencing of drug treated Hela cells for 6 or 18 hours.

first: Jesse

last: Gray

first: Michael

last: Springer

first: Sarah

last: Boswell


title: Gene expression profiling of cultured cells with serum depleted of extracellular vesicles

last: De

type:
Expression profiling by array
Expression profiling by RT-PCR

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


title: Novel RNA-binding activity of MYF5 enhances Ccnd1/Cyclin D1 mRNA translation during myogenesis

status: Public on Aug 08 2016

summary: Skeletal muscle contains long multinucleated and contractile structures known as muscle fibers, which arise from the fusion of myoblasts into nucleated myotubes during myogenesis.  The myogenic regulatory factor (MRF) MYF5 is the earliest to be expressed during myogenesis and functions as a transcription factor in muscle progenitor cells (satellite cells) and myocytes.  In mouse C2C12 myocytes, MYF5 is implicated in the initial steps of myoblast differentiation into myotubes.  Ribonucleoprotein immunoprecipitation (RIP) analysis showed that MYF5 bound a subset of myoblast mRNAs; prominent among them was Ccnd1 mRNA, which encodes the key cell cycle regulator CCND1 (Cyclin D1).  Biotin-RNA pulldown, UV-crosslinking, and gel shift experiments indicated that MYF5 was capable of binding the 3' untranslated region (UTR) and the coding region (CR) of Ccnd1 mRNA.  MYF5 silencing in proliferating growing myoblasts revealed that and MYF5 promoted CCND1 translation, and it also modestly increased transcription of Ccnd1 mRNA.  Importantly, silencing MYF5 reduced myoblast growth as well as differentiation of myoblasts into myotubes, while overexpressing MYF5 in C2C12 cells upregulated CCND1 expression.  We propose that MYF5 enhances early myogenesis in part by coordinately elevating Ccnd1 transcription and Ccnd1 mRNA translation.

overall_design: Four replicates were utilized from either Control (IgG) or MYF5-immunoprecipitated RNA samples from C2C12 cells growing in either growth medium (GM) or differentiation medium (DM) for a total of sixteen samples.

first: Amaresh

last: Panda

first: Kotb

last: Abdelmohsen

first: Xiaoling

last: Yang

first: Ioannis

last: Grammatikakis

first: Yongqing

last: Zhang

first: Dawood

last: Dudekula

first: Kevin

last: Becker

first: Elizabeth

middle: J

last: White

first: Gerald

last: Wilson


title: Gene expression profile of micropapillary bladder cancer

status: Public on Sep 03 2016

city: Houston

first: Li

state: TX

address: 1400 Pressler, unit 1410

department: Bioinformatics

institute: The University of Texas MD Anderson Cancer Center

summary:
Analyses of mRNA gene expression profiling performed on 43 micropapillary bladder samples and 89 conventional urothelial carcinoma samples.
Micropaillary tumors showed downregulation of miR-296 and activation of chromatin-remodeling complex RUVBL1. Micropapillary tumors evolve through luminal pathway.

overall_design: Total RNA samples obtained from 43 micropapillary bladder tumors and 89 conventional urothelial carcinomas were arrayed

first: Charles

last: Guo

first: Vipulkumar

last: Dadhania

first: Bogdan

last: Czerniak


title: Blast traumatic brain injury induced cognitive deficits are attenuated by pre- or post-injury treatment with the glucagon-like peptide-1 receptor agonist, exendin-4 [Day 3 dataset]

status: Public on Aug 05 2016

first: Supriyo

summary: Blast traumatic brain injury (B-TBI) affects military and civilian personnel. Presently there are no approved drugs for blast brain injury. Exendin-4, administered subcutaneously, was evaluated as a pre-treatment (48 hours) and post-injury treatment (2 hours) on neurodegeneration, behaviors and gene expressions in a murine open field model of blast injury. B-TBI induced neurodegeneration, changes in cognition and genes expressions linked to dementia disorders. Exendin-4, administered pre- or post-injury ameliorated B-TBI-induced neurodegeneration at 72 hours, memory deficits from days 7-14 and attenuated genes regulated by blast at day 14 post-injury.  The present data suggest shared pathological processes between concussive and B-TBI, with endpoints amenable to beneficial therapeutic manipulation by exendin-4. B-TBI-induced dementia-related gene pathways and cognitive deficits in mice somewhat parallel epidemiological studies of Barnes and co-workers who identified a greater risk in US military veterans who experienced diverse TBIs, for dementia in later life.

overall_design: Experimental conditions used to create a blast traumatic brain injury (B-TBI) and the subsequent model characterization have been described in detail previously. In short, male ICR mice (30-40 g) were anaesthetized with a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg). Once fully anaesthetized, the animals were positioned ‘side-on’ to the blast overpressure on an elevated platform in a circle, 7 meters from the source of the detonation generated by the detonation of 500 g of trinitrotoluene (TNT). The maximum overpressure generated by the detonation was 2.5 PSI (17.23 kPa). The following animal treatment groups were utilized: sham operated no blast (Sham); blast traumatic brain injury (B-TBI); Ex-4 as a pre-treatment to the blast injury (Ex-4/B-TBI); Ex-4 as a post-treatment to the blast injury (B-TBI/Ex-4) and Ex-4 but no injury (Ex-4). Hippocampal brain gene expression was examined on day 3 and day 14 (Experiment GSE44625) after the blast traumatic brain injury (B-TBI) or sham procedure. Animals received 3.5 pM/kg/min (21ug/kg/day) Exendin-4 using Alzet micro-osmotic pumps either as a pre-treatment to the blast injury 48 hours before (Ex-4 pre-TBI) or as a post-treatment 2 hours after the blast injury (Ex-4 post-TBI). Animals in a previous data set on Day 14 (GSE44625) were administered Ex-4 as a pre-treatment.

first: David

last: Tweedie

first: Lital

last: Rachmany

first: Vardit

last: Rubovitch

first: Yazhou

last: Li

first: Harold

last: Holloway

first: Elin

last: Lehrmann

first: Evelyn

last: Perez

first: Barry

last: Hoffer

first: Chaim

last: Pick

first: Nigel

last: Greig


title: TT-Seq captures the human transient transcriptome

status: Public on Jun 03 2016

city: Göttingen

first: Björn

last: Schwalb

address: Fassberg 11

country: Germany

institute: MPI

type: Genome binding/occupancy profiling by high throughput sequencing

summary: A large portion of the genome is transcribed but many of the resulting RNAs live only transiently and can generally not be mapped. Here we develop transient transcriptome sequencing (TT-Seq), a protocol that maps transcriptionally active regions in a nearly uniform manner and allows for unbiased monitoring of cellular RNA synthesis activity. Application of TT-Seq to human K562 cells recovers stable mRNAs and long intergenic non-coding RNAs, and additionally maps over 10,000 transient RNAs including enhancer RNAs, antisense RNAs, promoter-associated upstream antisense and convergent RNAs. TT-Seq also provides RNA half-lives, and reveals that transient RNAs are short and lack U1 motifs and secondary structure. TT-Seq further uncovers transcription termination sites and reveals a universal DNA motif for RNA polymerase II release.

overall_design: TT-Seq is an adaptation of 4sU-Seq for human cells. The labeled RNA is fragmented prior to 4sU-purification, which leads to an overall enrichment of transient transcripts. 8 RNA-Seq (TT-Seq, 4sU-Seq, total RNA-Frag-Seq and total RNA-Seq; in biological duplicates) were sequenced on a HiSeq 1500

first: Margaux

last: Michel

first: Benedikt

last: Zacher

first: Katja

last: Frühauf

first: Carina

last: Demel

first: Achim

last: Tresch

first: Julien

last: Gagneur

first: Patrick

last: Cramer


title: Selection of reference genes for gene expression studies related to hypoxia in cervical cancer

status: Public on Mar 29 2016

city: Oslo

first: Heidi

last: Lyng

address: Montebello

department: Department of Radiation Biology

country: Norway

institute: Oslo University Hospital

summary: The present work aimed to identify reference genes for RT-qPCR studies of hypoxia in cervical cancer. From 422 candidate reference genes selected from the literature, we used Illumina array-based expression profiles to identify 182 genes not affected by hypoxia treatment in eight cervical cancer cell lines or correlated with the hypoxia-associated dynamic contrast-enhanced magnetic resonance imaging parameter ABrix in 42 patients. Among these genes, we selected nine candidates (CHCHD1, GNB2L1, IPO8, LASP1, RPL27A, RPS12, SOD1, SRSF9, TMBIM6) that were not associated with tumor volume, stage, lymph node involvement or disease progression in array data of 150 patients, for further testing by RT-qPCR. geNorm and NormFinder analyses of RT-qPCR data of 74 patients identified CHCHD1, SRSF9 and TMBIM6 as the most suitable set of reference genes, with stable expression both overall and across patient subgroups with different hypoxia status (ABrix) and clinical parameters. The suitability of the three candidates as reference genes were validated in studies of the hypoxia-induced genes DDIT3, ERO1A, and STC2. After normalizing with CHCHD1, SRSF9 and TMBIM6, the RT-qPCR data of these genes showed a significant correlation with Illumina expression (P<0.001, n=74) and ABrix (P<0.05, n=32), and the STC2 data were associated with clinical outcome, in accordance with the Illumina data. Thus, CHCHD1, SRSF9 and TMBIM6 seem to be suitable reference genes for studying hypoxia-related gene expression in cervical cancer samples by RT-qPCR. STC2 might be a useful prognostic hypoxia biomarker in cervical cancer that warrants further investigation.

overall_design: Totally 150 tumor samples and 12 cell line samples were analysed with Illumina WG-6 and 4 cell line samples were analysed with Illumina HT-12

first: Christina

middle: S

last: Fjeldbo

first: Eva

last: Aarnes

first: Erik

last: Malinen

first: Gunnar

last: Kristensen


title: SR protein family in P19 cells

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

institute: Goethe University Frankfurt


title: Integrative analysis of DCE-MRI and gene expression profiles in construction of a gene classifier for assessment of hypoxia-related risk of chemoradiotherapy failure in cervical cancer

summary:
We have previously identified a prognostic 31-gene expression signature in locally advanced cervical cancer that is associated with tumor hypoxia and reflected by the dynamic contrast enhanced magnetic resonance (DCE-MR) image parameter ABrix. To bring the signature closer to clinical use, we here aimed to construct a classifier with key signature genes that retained an association to ABrix and separated the patients into groups with different hypoxia status and chemoradiotherapy outcome.
A training cohort of 42 patients and two independent cohorts of 108 and 131 patients were included. Gene expression data were generated from tumor biopsies by two Illumina array generations (WG6, HT12), and for a subset of 74 patients, by the RT-qPCR platform. The amplitude ABrix in the Brix pharmacokinetic model was extracted from DCE-MR images of 64 patients and used as a measure of hypoxia.
Different classifiers were constructed in the training cohort. By evaluating their ability to separate the patients correctly according to ABrix, a 6-gene classifier with strong correlation to ABrix was identified. In validation analyses, the classifier separated the patients into two groups with 5-years progression-free survival probabilities of 85% and 60%. Both the prognostic value and ABrix-association were confirmed in an independent cohort, and robustness across array generations and assay platforms was demonstrated. The prognostic value was independent of existing clinical markers.
A robust, prognostic hypoxia classifier has been constructed, and might be used alone or combined with DCE-MR imaging to achieve an early indication of a patient’s risk of failure and allocate high risk patients to adjuvant hypoxia-targeted therapy.

overall_design: Total 150 patient and 2 cell line samples were analysed with Illumina WG-6, while 136 patient and 6 cell line samples were analyzied with Illumina HT-12.

first: Cathinka

last: Julin

first: Malin

last: Lando

last: Forsberg

first: Eva-Katrine

first: Jan

last: Alsner

first: Eirk


title: miRNA expression of lymphoma cell lines with or without HDAC inhibitor treatment

status: Public on May 07 2016

city: Akita

first: Akihiro

last: Kitadate

address: Hondo 1-1-1

department: Department of Hematology, Nephrology, and Rheumatology

country: Japan

institute: Akita University Graduate School of Medicine

summary: Various types of HDAC inhibitor are now planned or on going to use against non-Hodgkin’s lymphoma including CTCL. However there is a lack of molecular based evidence not only for which HDACi might be effective to use against advanced CTCL but also for which genes might be pivotal target gene(s) of HDACi. To investigate expression changes of miRNAs by HDACis, we conducted miRNA microarray by using CTCL cell lines.

overall_design: CTCL cell lines (My-La, HH, HUT78) and  CD4+ T-cells of healthy controls were treated with SAHA (5 µM),  panobinostat (80 nM), or DMSO for 24hrs. After treatment, total RNA was extracted from cells, and miRNA microarray analysis was performed.

first: Hiroyuki

last: Tagawa


title: Effects of Sex, Strain, and Energy Intake on Hallmarks of Aging in Mice

status: Public on Jun 27 2016

first: Supriyo

last: De

summary: Calorie restriction (CR) is the most robust non-genetic intervention to universally delay the onset of age-related diseases and extend mean and maximum lifespan. However, species, strain, sex, diet, age of onset, and level of CR are emerging as important variables to consider for a successful CR response. Here, we investigated the role of strain, sex and level of CR on outcomes of health and survival in mice. Response to CR varied from lifespan extension to no effect on survival, while consistently delaying the onset and impact of diseases independently of strain, sex and level of dietary restriction. CR led to transcriptional and metabolomics changes in the liver indicating anaplerotic filling of the Krebs cycle together with fatty acid fueling of mitochondria. Additionally, CR prevented the age-associated decline in the proteostasis network. Further, CR increased mitochondrial number and preserved their ultrastructure and function with age. Abrogation of mitochondrial function by deletion of fumarate hydratase or malate dehydrogenase 2 negated the life-prolonging effects of CR in yeast and worms. In F1 hybrid strains of mice, the lifespan response to CR tracked with the dam, indicating that the mitochondrial haplotype is an important regulator of CR. Our data illustrate the complexity of the CR responses within a single animal species in the context of aging, with a clear separation of outcomes related to health and survival, highlighting the complexities of translation of CR into human interventions.

overall_design: The study examines the effects of sex (male/female), mouse strain (DBA2/J versus C57BL/J), and diet (Ad libitum, 20% caloric restriction (20%CR), or 40%CR) using six biological replicate samples per group.

first: Sarah

last: Mitchell

first: Julio

last: Matute

first: Morten

last: Scheibye-Knudsen

first: Evandro

last: Fang

first: Miguel

last: Aon

first: Jose

last: Gonzalez

first: Sonia

last: Cortassa

first: Susmita

last: Kaushik

first: Marta

last: Gonzalez-Freire

first: Devin

last: Wahl

first: Ahmed

last: Ali

last: Calvo-Rubio

first: María

middle: I

last: Burón

first: Vincent

last: Guiterrez

first: Theresa

last: Ward

first: Hector

last: Palacios

first: Huan

last: Cai

first: David

last: Frederick

first: Christopher

last: Hine

first: Filomena

last: Broeskamp

first: Lukas

last: Habering

first: John

last: Dawson

first: T

last: Beasley

first: Junxiang

last: Wan

first: Yuji

last: Ikeno

first: Gene

last: Hubbard

first: Kevin

last: Becker

first: Yongqing

last: Zhang

first: Vilhelm

last: Bohr

first: Dan

last: Longo

first: Placido

last: Navas

first: Luigi

last: Ferrucci

last: Sinclair

first: Pinchas

last: Cohen

first: Josephine

last: Egan

first: James

middle: R

first: Joseph

last: Baur

last: Allison

first: José

last: Villalba

first: Frank

last: Madeo


title: Age-related changes in microRNA levels in serum

status: Public on Mar 03 2016

first: Supriyo

last: De


title: Exercise-mimetic AICAR transiently benefits brain function

status: Public on Dec 21 2015

summary: Exercise enhances learning and memory in animals and humans. The role of peripheral factors that may trigger the beneficial effects of running on brain function has been sparsely examined. In particular, it is unknown whether AMP-kinase (AMPK) activation in muscle can predict enhancement of brain plasticity. Here we compare the effects of running and administration of AMPK agonist 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR, 500 mg/kg), for 3, 7 or 14 days in one-month-old male C57BL/6J mice, on muscle AMPK signaling. At the time-points where we observed equivalent running- and AICAR-induced muscle pAMPK levels (7 and 14 days), cell proliferation, synaptic plasticity and gene expression, as well as markers of oxidative stress and inflammation in the dentate gyrus (DG) of the hippocampus and lateral entorhinal cortex (LEC) were evaluated. At the 7-day time-point, both regimens increased new DG cell number and brain-derived neurotrophic factor (BDNF) protein levels. Furthermore, microarray analysis of DG and LEC tissue showed a remarkable overlap between running and AICAR in the regulation of neuronal, mitochondrial and metabolism related gene classes. Interestingly, while similar outcomes for both treatments were stable over time in muscle, in the brain an inversion occurred at fourteen days. The compound no longer increased DG cell proliferation or neurotrophin levels, and upregulated expression of apoptotic genes and inflammatory cytokine interleukin-1β. Thus, an exercise mimetic that produces changes in muscle consistent with those of exercise does not have the same sustainable positive effects on the brain, indicating that only running consistently benefits brain function.

overall_design: One-month-old C57BL/6J male mice (Jackson Laboratory, Bar Harbor, ME) were individually housed in standard conditions with food and water ad libitum, and were maintained in accordance with the National Institutes of Health guidelines. All protocols for procedures were approved by the NIA’s Institutional Animal Care and Use Committee. Animals were divided into 3 groups - control (CTR), AICAR-treated (ACR) or voluntary running (RUN). ACR mice received a daily intraperitoneal injection (IP) of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR, Toronto Research Chemicals Inc., Canada) of 500 mg/kg/day, dissolved in saline; CTR and RUN animals received IP saline vehicle; RUN animals had free access to running wheels. Animals were sacrificed after deep anesthesia with isofluorane (Henry Schein Animal Health, OH) after 7, 14 or 28 days, and the dentate gyrus (DG) and lateral entorhinal cortex (LEC) were collected and immediately frozen and stored at -80ºC.

first: Davide

last: Guerrieri

first: Henriette

last: van Praag


title: Senescence-associated lncRNAs: senescence-associated long noncoding RNAs.

status: Public on Feb 08 2016

summary: Noncoding RNAs include small transcripts, such as microRNAs and piwi-interacting RNAs, and a wide range of long noncoding RNAs (lncRNAs). Although many lncRNAs have been identified, only a small number of lncRNAs have been characterized functionally. Here, we sought to identify lncRNAs differentially expressed during replicative senescence. We compared lncRNAs expressed in proliferating, early-passage, 'young' human diploid WI-38 fibroblasts [population doubling (PDL) 20] with those expressed in senescent, late-passage, 'old' fibroblasts (PDL 52) by RNA sequencing (RNA-Seq). Numerous transcripts in all lncRNA groups (antisense lncRNAs, pseudogene-encoded lncRNAs, previously described lncRNAs and novel lncRNAs) were validated using reverse transcription (RT) and real-time, quantitative (q)PCR. Among the novel senescence-associated lncRNAs (SAL-RNAs) showing lower abundance in senescent cells, SAL-RNA1 (XLOC_023166) was found to delay senescence, because reducing SAL-RNA1 levels enhanced the appearance of phenotypic traits of senescence, including an enlarged morphology, positive β-galactosidase activity, and heightened p53 levels. Our results reveal that the expression of known and novel lncRNAs changes with senescence and suggests that SAL-RNAs play direct regulatory roles in this important cellular process.

overall_design: RNA was extracted from both young and senescent WI-38 cells and used for total RNA-Seq.

first: Kotb

last: Abdelmohsen

first: Amaresh

last: Panda

first: Min-Ju

last: Kang

first: Jason

last: Xu

first: Roza

last: Selimyan

first: Je-Hyun

last: Yoon

first: Jennifer

last: Martindale

first: William

last: Wood 3rd

first: Myriam

last: Gorospe


title: RNA-seq of the killifish N.furzeri during normal aging (cross-sectional and longitudinal study) and treatment with rotenone

status: Public on Dec 03 2015

city: Jena

first: JenAge

last: Project

address: Beutenbergstr. 11

institute: Leibniz Institute for Age Research - Fritz Lipmann Institute

summary:
The study consists of three parts: 1) normal aging in liver and skin (cross-sectional); 2) treatment with rotenone in brain, liver and skin; 3) longitudinal study of 45 fish with different ages at their death measured at two different time points by fin clipping

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

overall_design: 185 samples: 1) 50 normal aging samples: 2 tissues (liver and skin) * 5 age groups (5,12,20,27,39 weeks) * 5 replicates; 2) 45 samples: 3 tissues (brain, liver and skin) * 2 age groups (9,27 weeks) for controls and 1 age group for treatment group * 5 replicates; 3) 90 samples: 45 fish, each measured at 10 and 20 weeks (fin clipping)

first: Mario

last: Baumgart

first: Uwe

last: Menzel

first: Steffen

last: Priebe

first: Marco

last: Groth

first: Matthias

last: Platzer

first: Alessandro

last: Cellerino


title: Hypsibius dujardini high resolution developmental transcriptomic time-course

status: Public on Feb 18 2016

city: Haifa

first: Itai

last: Yanai

address: Technion City

department: Biology

country: Israel

institute: Technion - Israel Institute of Technology

summary: Classical embryological studies revealed that during mid-embryogenesis vertebrates show similar morphologies. This “phylotypic stage” has recently received support from transcriptome analyses, which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a ‘zootypic’ stage, however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum, including a segmented worm, a flatworm, a roundworm, a water bear, a fruitfly, a sea urchin, a zebrafish, a sea anemone, a sponge, and a comb jelly. For each species, we collected the embryonic transcriptomes at ~100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly, we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation, and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes, also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility.

overall_design: 126 single embryo samples.

first: Michal

last: Levin

first: Leon

last: Anavy


title: Wnt5A promotes an adaptive, senescent-like stress response, while continuing to drive invasion in melanoma cells.

status: Public on Aug 07 2015

first: Supriyo

last: De

summary: We have previously shown that Wnt5A drives invasion in melanoma. We have also shown that Wnt5A promotes resistance to therapy designed to target the BRAF(V600E) mutation in melanoma. Here, we show that melanomas characterized by high levels of Wnt5A respond to therapeutic stress by increasing p21 and expressing classical markers of senescence, including positivity for senescence-associated β-galactosidase (SA-β-gal), senescence-associated heterochromatic foci (SAHF), H3K9Me chromatin marks, and PML bodies. We find that despite this, these cells retain their ability to migrate and invade. Further, despite the expression of classic markers of senescence such as SA-β-gal and SAHF, these Wnt5A-high cells are able to colonize the lungs in in vivo tail vein colony-forming assays. This clearly underscores the fact that these markers do not indicate true senescence in these cells, but instead an adaptive stress response that allows the cells to evade therapy and invade. Notably, silencing Wnt5A reduces expression of these markers and decreases invasiveness. The combined data point to Wnt5A as a master regulator of an adaptive stress response in melanoma, which may contribute to therapy resistance.

overall_design: To better understand the molecular mechanisms governing the response of highly invasive cells to IR as compared to that of poorly invasive cells, we performed microarray analysis of both poorly and highly invasive cells at early and late timepoints after irradiation. Cells were treated with y-irradiation, and RNA was taken at 1 hour, 24 hours and 5 days after irradiation. Microarray analysis was performed using Illumina Human HT-12 ver3 expression arrays, and each time point was compared to RNA from untreated cells.

first: Marie

last: Webster

first: Mai

first: Kathryn

last: Kinzler

first: Amanpreet

last: Kaur

first: Jessica

last: Appleton

first: Michael

last: O’Connell

first: Katie

last: Marchbank

first: Alexander

last: Valiga

first: Vanessa

middle: M

last: Dang

first: Michela

last: Perego

first: Gao

last: Zhang

first: Ana

last: Slipicevic

first: Frederick

last: Keeney

first: Elin

last: Lehrmann

last: Wood III

first: Kevin

last: Becker

first: Andrew

last: Kossenkov

first: Dennie

middle: T

last: Frederick

first: Keith

last: Flaherty

first: Xiaowei

first: Meenhard

last: Herlyn

first: Maureen

last: Murphy

first: Ashani

last: Weeraratna


title: Multi-layered population structure in Island Southeast Asians

status: Public on Feb 04 2016

city: Cambridge

first: Luca

last: Pagani

state: UK

address: Henry Wellcome Building, Fitzwilliam Street

department: Biological Anthropology

country: United Kingdom

institute: University of Cambridge

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

summary: The history of human settlement in Southeast Asia has been complex and involved several distinct dispersal events. Here we report the analyses of 1825 individuals from Southeast Asia including new genome-wide genotype data for 146 individuals from three Mainland Southeast Asian (Burmese, Malay and Vietnamese) and four Island Southeast Asian (Dusun, Filipino, Kankanaey and Murut) populations. While confirming the presence of previously recognized major ancestry components in the Southeast Asian population structure, we highlight the Kankanaey Igorots from the highlands of the Philippine Mountain Province as likely the closest living representatives of the source population that may have given rise to the Austronesian expansion. This conclusion rests on independent evidence from various analyses of autosomal data and uniparental markers.

overall_design: 196 samples were analysed with the Illumina platform OmniExpress BeadChips  and are described herein.

last: Mörseburg

first: Francois-Xavier

last: Ricaut

first: Bryndis

last: Yngvadottir

first: Eadaoin

last: Harney

first: Cristina

last: Castillo

first: Tom

last: Hoogervorst

first: Tiago

last: Antao

first: Pradiptajati

last: Kusuma

first: Harilanto

last: Razafindrazaka

first: Alexia

last: Cardona

first: Denis

last: Pierron

first: Thierry

last: Letellier

first: Joseph

last: Wee

first: Syafiq

last: Abdullah

first: Mait

last: Metspalu

first: Toomas

last: Kivisild


title: Circular RNAs in monkey muscle: age-dependent changes

status: Public on Nov 16 2015

first: Supriyo

last: De

summary: Here, we describe the identification of circRNA in monkey (Rhesus macaque) skeletal muscle.  RNA sequencing analysis was employed to identify and annotate ~12,000 circRNAs, including numerous circular intronic RNAs (ciRNAs), from monkey skeletal muscle tissue of  various ages.

overall_design: Identification and validation of circular RNAs (circRNAs) in skeletal muscle tissues (vastus lateralis) were obtained from young (Y), middle-aged (MA), and old (O) monkeys.

first: Kotb

last: Abdelmohsen

first: Amaresh

middle: C

last: Panda

first: Ioannis

last: Grammatikakis

first: Jiyoung

last: Kim

first: Jun

last: Ding

first: Ji

last: Noh

first: Kyoung

first: Julie

last: Mattison

first: Rafael

last: de Cabo

first: Myriam

last: Gorospe


title: Caloric restriction induces heat shock response and inhibits B16F10 cell tumorigenesis both in vitro and in vivo

status: Public on Aug 06 2015

summary:
Caloric restriction (CR) without malnutrition is one of the most consistent strategies for increasing mean and maximal lifespan and delaying the onset of age-associated diseases. Stress resistance is a common trait of many long-lived mutants and life-extending interventions, including CR. Indeed, better protection against heat shock and other genotoxic insults have helped explain the pro-survival properties of CR. In this study, both in vitro and in vivo responses to heat shock were investigated using two different models of CR. Murine B16F10 melanoma cells treated with serum from CR-fed rats showed lower proliferation, increased tolerance to heat shock and enhanced HSP-70 expression, compared to serum from ad libitum-fed animals. Similar effects were observed in B16F10 cells implanted subcutaneously in male C57BL/6 mice subjected to CR. Microarray analysis identified a number of genes and pathways whose expression profile were similar in both models. These results suggest that the use of an in vitro model could be a good alternative to study the mechanisms by which CR exerts its anti-tumorigenic effects.
KEY WORDS: caloric restriction, heat shock, stress response, tumorigenesis, aging

overall_design: In Vivo:  Male C57BL/6 mice (3 month old) were single-housed in duplex caging in a room maintained at a constant temperature (20-22 °C) and humidity (30-70%) in a light:dark 12:12-h schedule, according to established animal protocols and NIH guidelines.  They were fed either a standard purified mouse diet (NIH-31) ad libitum (AL; n=10) or maintained on a 40% calorie restriction regimen (CR; n=10) for the six week study. B16F10 melanoma cells (ATCC® CRL-6475™) were purchased from American Type Culture Collection (Manassas, VA); they were cultured in Dulbecco's Modified Essential Medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin (Gibco, Gaithersburg, MD) under standard cell culture conditions.   One month into the study, 5 mice from each diet group were injected with 1x106 B16F10 melanoma cells in the periscapular region.  Fourteen days later, animals were euthanized and tumors were excised and frozen for RNA isolation.  In Vitro:  B16F10 melanoma cells were incubated in media with 10% serum from either AL- or CR-fed rats, serum was obtained from overnight fasted, anesthetized 6-month-old male Fisher 344 rats.  Cells were grown for 96 hours before being harvested, washed and collected for RNA isolation.  The RNA was extracted using the RNeasy Mini Kit from Qiagen using standard protocols and labeled with Biotin using the standard Illumina protocol. Samples were hybed overnight to Illumina's Sentrix MouseRef-8 v1.1 Expression BeadChips.  Arrays were washed, stained with Cy-3 and scanned at 0.8 micron resolution in a 500 GX Illumina Bead Array Reader.

first: Marta

last: Novelle

first: Ashley

last: Davis

first: Nathan

middle: L

last: Price

first: Ahmed

last: Ali

first: Stefanie

last: Fürer-Galvan

first: Yongqing

last: Zhang

first: Kevin

last: Becker

first: Michel

last: Bernier


title: Novel RNA- and FMRP-binding protein TRF2-S regulates axonal mRNA transport and presynaptic plasticity

status: Public on Nov 06 2015

first: Supriyo

last: De

summary: Despite considerable evidence that RNA-binding proteins (RBPs) regulate mRNA transport and local translation in dendrites, roles for axonal RBPs are poorly understood.  Here we demonstrate that a nontelomeric isoform of telomere repeat-binding factor 2 (TRF2-S) is a novel RBP that regulates axonal plasticity.  TRF2-S interacts directly with target mRNAs to facilitate their axonal delivery.  The process is antagonized by fragile X mental retardation protein (FMRP).  Distinct from the current RNA-binding model of FMRP, we show that FMRP occupies the GAR domain of TRF2-S protein to block the assembly of TRF2-S-mRNA complexes.  Overexpressing TRF2-S and silencing FMRP promotes mRNA entry to axons, and enhances axonal outgrowth and neurotransmitter release from presynaptic terminals.  Our findings suggest a pivotal role for TRF2-S in an axonal mRNA localization pathway that enhances axon outgrowth and neurotransmitter release.

overall_design: RNA immunoprecipitated samples using either anti-rat TRF2-S or rabbit IgG antibodies were used to generate biotin-labeled RNA using the Illumina Total Prep RNA Amplification Kit (Ambion), which was hybridized to Illumina's rat Ref-12 Expression BeadChips (Illumina, San Diego, CA), containing 22,523 well-annotated RefSeq transcripts with ~30-fold redundancy.  The arrays were scanned using an Illumina BeadStation 500X Genetic Analysis Systems scanner and the image data extracted using Illumina BeadStudio software, version 1.5, normalized by Z-score transformation and used to calculate differences in signal intensities.  Significant values were calculated from two groups of independent experiments, using a two-tailed Z-test with P < 0.05, a false discovery rate <0.30, a z ratio absolute value not less than 1.5, and an average signal intensity not less than zero.  The results also had to pass the filtering and one-way independent ANOVA test by sample groups less than 0.05, and detection p-value for any probe in the comparison group less than 0.02.

first: Peisu

first: Kotb

last: Abdelmohsen

first: Yong

last: Liu

first: Kumiko

last: Tominaga-Yamanaka

first: Je-Hyun

last: Yoon

first: Grammatikakis

last: Ioannis

first: Jennifer

last: Martindale

middle: G

first: In

last: Yang

first: Mark

last: Mattson


title: Hepatitis C virus up-regulates B-cell receptor signaling: a novel mechanism for HCV-associated B-cell lymphoproliferative disorders

status: Public on Oct 14 2015

summary: B-cell receptor (BCR) signaling is essential for the development of B-cells and plays a critical role in B-cell neoplasia. Increasing evidence indicates an association between chronic hepatitis C virus (HCV) infection and B-cell lymphoma, however, the mechanisms by which HCV causes B-cell lymphoproliferative disorder are still unclear. Herein, we demonstrate the expression of HCV viral proteins in B-cells of HCV-infected patients and show that HCV up-regulates BCR signaling in human primary B-cells. HCV nonstructural protein NS3/4A interacts with CHK2 and down-regulates its activity, modulating HuR posttranscriptional regulation of a network of target mRNAs associated with B-cell lymphoproliferative disorders. Interestingly, the BCR signaling pathway was found to have the largest number of transcripts with increased association with HuR and was up-regulated by NS3/4A. Our study reveals a previously unidentified role of NS3/4A in regulation of host BCR signaling during HCV infection, contributing to a better understanding of the molecular mechanisms underlying HCV-associated B-cell lymphoproliferative disorders.

overall_design: A series of FLAG epitope-tagged NS3/4A truncation and deletion mutants were cloned into a lentiviral-vector CD532, and stable cell lines of SUDHL6-overexpressing FLAG epitope-tagged NS3/4A truncation and deletion mutants generated by lentiviral transduction, selection and maintenance with 0.5 mg/ml puromycin.  Ribonucleoprotein immunoprecipitation (RNP IP) using an anti-HuR antibody of control (CTR_S6_532_2_x) and NS3/4A-overexpressing (NS3_S6_8_2_x) SUDHL6-cells generated HuR-IP RNA samples for microarray analysis of differences in HuR binding activity caused by NS3/4A-overexpression. Three independent experiments are reported for each condition.

first: Bojie

last: Dai

first: Annie

middle: Y

last: Chen

first: Christopher

last: Corkum

first: Raymond

last: Peroutka

first: Ari

last: Landon

first: Simone

last: Houng

first: Parameswary

last: Muniandy

first: Elin

last: Lehrmann

first: Krystyna

last: Mazan-Mamczarz

first: James

last: Steinhardt

first: Mikhail

last: Shlyak

first: Qing

first: Ferenc

last: Livak

first: Tomasz

last: Michalak

first: Rohit

last: Talwani

first: Ronald

last: Gartenhaus


title: Human umbilical cord matrix mesenchymal stem cells suppress the growth of breast cancer by expression of tumor suppressor genes

first: Supriyo

last: De

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

sample_organism:
Homo sapiens
Rattus norvegicus

platform_organism:
Homo sapiens
Rattus norvegicus

overall_design: Refer to individual Series


title: The mid-developmental transition and the evolution of animal body plans

first: Itai

last: Yanai

sample_organism:
Caenorhabditis elegans
Platynereis dumerilii
Drosophila melanogaster
Strongylocentrotus purpuratus
Danio rerio
Mnemiopsis leidyi
Nematostella vectensis
Schmidtea polychroa
Hypsibius dujardini
Amphimedon queenslandica


title: The aging microenvironment promotes melanoma cell invasion and therapy resistance

status: Public on Apr 08 2016

summary: Cancer is a disease of aging, and incidence and mortality from all cancers increase logarithmically after the age of 45. Older patients have a much poorer prognosis for melanomas of equal stage, compared to younger individuals. The role of the tumor microenvironment in modulating cancer behavior is widely recognized. We hypothesized that age-related changes in the tumor microenvironment could affect the progression of melanoma. We demonstrate that the aging microenvironment increases the invasion of melanoma cells. Using skin fibroblasts isolated from healthy donors, we have built artificial skin and demonstrate that melanoma cells invade more rapidly when exposed to aged fibroblasts. In a mouse model of melanoma (YUMM1.7, BrafV600E/Cdkn2a-/-/Pten-/-), congeneic to C57/BL6 mice, melanomas invade faster in aged mice. Gene expression analyses suggest that melanoma cells are undergoing DNA damage when exposed to an aged microenvironment, and we confirm this in cellular assays. Proteomics analysis of aging fibroblasts suggest that fibroblasts secrete molecules (laminin b2, Wnt inhibitors) associated with increased resistance to targeted therapy. Indeed, melanoma cells injected into aged mice respond less well to PLX4720 than those injected into young mice.  This suggests that  exciting new drugs targeting the BRAF pathway may be less effective in aging patients.

overall_design: To assess the effects of the aging microenvironment on melanoma cell gene expression, we co-cultured dermal fibroblasts from young (<35 years), medium or aged ( >55 years) individuals with three different melanoma cell lines (UACC1273, M93-047 and UACC903) in triplicate using transwell chambers. After 96 h of co-culture, fibroblasts in the upper well were removed, and melanoma cells (lower chamber) were subjected to microarray analysis.

first: Amanpreet

last: Kaur

first: Katie

last: Marchbank

first: Vanessa

last: Dang

first: Michael

last: O’Connell

first: Marie

last: Webster

first: Phil

last: Cheng

first: Alex

last: Valiga

first: Rachel

last: Morissette

first: Nazli

last: McDonnell

first: Luigi

last: Ferrucci

first: Andrew

last: Kossenkov

first: Katrina

last: Meeth

first: Marcus

last: Bosenberg

first: Hsin-Yao

last: Tang

first: Xianfang

last: Yin

first: William

last: Wood

first: Kevin

last: Becker

first: Keith

last: Flaherty

first: Dennie

last: Frederick

first: Jennifer

last: Wargo

first: Katherine

last: Aird

first: Rugang

last: Zhang

first: Xiaowei

last: Xu

first: Qin

last: Liu

first: David

last: Speicher

first: Ashani

last: Weeraratna


title: Antitumor Activity of (R,R’)-4-methoxy-1-naphthylfenoterol in a rat C6 glioma xenograft model in the mouse

first: Supriyo

last: De

summary:
(R,R’)-4-methoxy-1-naphthylfenoterol (MNF) inhibits in vitro proliferation of several types of cancer cell lines. In this study, the in vivo antitumor effects of MNF were evaluated using rat C6 glioma cells implanted subcutaneously into the lower flank of 5 week-old NMRI/Nude female Swiss mice. Three days after the inoculation, the mice were subjected to intraperitoneal injections of saline or MNF (2mg/kg) for five days per week for16 days. Tumor volumes were measured everyday using slide calipers. Significant reductions in mean tumor volumes were observed in mice receiving MNF when compared with the saline-treated group (p<0.001, n=17-19). At the end of the study, animals were sacrificed and tumors were collected for cDNA microarray, quantitative RT-PCR and immunoblot analyses. Significant decrease in expression of genes involved in cellular proliferation, including mitotic checkpoint kinase MAD3L (Bub1b), cyclin-dependent kinase inhibitor 3 (Cdkn3) and cyclin A2 (Ccna2), as well as molecular markers for glioblastoma, such as oligodendrocyte transcription factor 1 (Olig1) and SRY-box 4 (Sox4) was observed in tumors of MNF-treated mice as compared to saline-injected controls. The efficacy of MNF against C6 glioma cancer in vivo was accompanied by marked reduction in the expression of cell cycle regulator proteins, including cyclin A and cyclin D1, in tumor extracts. This study is the first demonstration of MNF-dependent chemoprevention in a glioblastoma xenograft model in the mouse and may offer a potential mechanism for its anticancer action in vivo.
Keywords: fenoterol derivative; C6 glioma; tumor xenografts; microarray analysis;

overall_design: Total RNA was isolated from rat C6 glioma xenografts harvested from vehicle and MNF-treated nude mice (n = 3 per group, cohort 1). This analysis was repeated in a second cohort of animals (n = 3 per group, cohort 2). Total cellular RNA was extracted using an RNeasy plus mini kit (QIAGEN, Valencia, CA), and its quality was assessed using an Agilent BioAnalyzer using RNA 6000 Nano Chips (Agilent Technologies, Santa Clara, CA). Transcriptional profiling was determined using Illumina Sentrix BeadChips (Illumina, San Diego, CA). Total RNA was used to generate biotin-labeled cRNA with the Illumina TotalPrep RNA Amplification Kit.  In short, 0.5ug 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 incorporating biotin-16-UTP.  A total of 0.75µg of biotin-labeled cRNA was hybridized at 58 °C for 16 h to Illumina's Sentrix Rat Ref-12 Expression BeadChips.  The arrays were washed, blocked and the labeled cRNA was detected by staining with streptavidin-Cy3. Hybridized arrays were scanned using an Illumina BeadStation 500X Genetic Analysis Systems scanner and the image data extracted using Illumina’s GenomeStudio software, version 1.6.1.  For statistical analysis, the expression data were filtered to include only probes with a consistent signal on each chip and an Illumina detection  p value < 0.02.

first: Michel

last: Bernier

first: Rajib

last: Paul

first: Katina

last: Dossou

first: Artur

last: Wnorowski

first: Ruin

last: Moaddel

first: Anuradha

last: Ramamoorthy

first: Arnaud

last: Paris

first: Jean

middle: F

last: Cloix

first: Irving

last: Wainer


title: Human umbilical cord matrix mesenchymal stem cells suppress the growth of breast cancer by expression of tumor suppressor genes [rat]

summary: Human and rat umbilical cord matrix mesenchymal stem cells (UCMSC) possess the ability to control the growth of breast carcinoma cells. Comparative analyses of two types of UCMSC suggest that rat UCMSC-dependent growth regulation is significantly stronger than that of human UCMSC. Their different tumoricidal abilities were clarified by analyzing gene expression profiles in the two types of UCMSC. Microarray analysis revealed differential gene expression between untreated naïve UCMSC and those co-cultured with species-matched breast carcinoma cells. The analyses screened 17 differentially expressed genes that are commonly detected in both human and rat UCMSC. The comparison between the two sets of gene expression profiles identified two tumor suppressor genes, adipose-differentiation related protein (ADRP) and follistatin (FST), that were specifically up-regulated in rat UCMSC, but down-regulated in human UCMSC when they were co-cultured with the corresponding species' breast carcinoma cells. Over-expression of FST, but not ADRP, in human UCMSC enhanced their ability to suppress the growth of MDA-231 cells. The growth of MDA-231 cells was also significantly lower when they were cultured in medium conditioned with FST, but not ADRP over-expressing human UCMSC. In the breast carcinoma lung metastasis model generated with MDA-231 cells, systemic treatment with FST-overexpressing human UCMSC significantly attenuated the tumor burden. These results suggest that FST may play an important role in exhibiting stronger tumoricidal ability in rat UCMSC than human UCMSC and also implies that human UCMSC can be transformed into stronger tumoricidal cells by enhancing tumor suppressor gene expression.

overall_design: Rat UCMSC and Mat B III breast carcinoma cells were cultured indirectly for 48 hours using Transwell culture dish. Naïve rat UCMSC were cultured under same condition without the addition of carcinoma cells.

first: Naomi

last: Ohta

first: Susumu

last: Ishiguro

first: Atsushi

last: Kawabata

first: Deepthi

last: Uppalapati

first: Marla

last: Pyle

first: Deryl

last: Troyer

first: Yongqing

first: Kevin

last: Becker

first: Masaaki

last: Tamura


title: In vitro caloric restriction induces protective genes and functional rejuvenation in senescent SAMP8 astrocytes

status: Public on Jun 23 2015

first: Supriyo

last: De

summary:
Astrocytes are key cells in brain aging, helping neurons to undertake healthy aging or otherwise letting them enter into a spiral of neurodegeneration. We aimed to characterize astrocytes cultured from senescence-accelerated prone 8 (SAMP8) mice, a mouse model of brain pathological aging, along with the effects of caloric restriction, the most effective rejuvenating treatment known so far. Analysis of the transcriptomic profiles of SAMP8 astrocytes cultured in control conditions and treated with caloric restriction serum was performed using mRNA microarrays. A decrease in mitochondrial and ribosome mRNA, which was restored by caloric restriction, confirmed the age-related profile of SAMP8 astrocytes and the benefits of caloric restriction. An amelioration of antioxidant and neurodegeneration-related path- ways confirmed the brain benefits of caloric restriction. Studies of oxidative stress and mitochondrial function demonstrated a reduction of oxidative damage and partial improvement of mito- chondria after caloric restriction. In summary, caloric restriction showed a significant tendency to normalize pathologically aged astrocytes through the activation of pathways that are protective against the age-related deterioration of brain physiology.
Key words: astrocytes; caloric restriction; mitochondria; oxidative stress; RNA microarrays; SAMP8.

overall_design:
Primary cultures enriched in astrocytes were obtained from cerebral cortical tissue from 2-day-old SAMP8 and SAMR1 mice.  Astrocyte cultures were established and experiments were routinely carried out after 21 days in culture.  Established astrocyte cultures of both SAMR1 and SAMP8 consisted of 85-90% astrocytes, 10-15% microglia and 0.1-1% oligodendroglia.  Sera from rats subjected to ad libitum (AL) diet and to CR were obtained as described for the establishment of the CR in vitro model (de Cabo et al., 2003). Serum was heat inactivated at 56°C prior to use in astrocyte culture experiments. Treatment in vitro was performed by adding 10% volume CR or AL serum onto the astrocyte culture medium for 48 h, the cells were harvested and RNA was extracted for the microarray studies. Three biological replicates for each condition were done and RNA was extracted for the microarray studies.

Please note that SAM models were developed from AKR/J by Kyoto University. Five litters with severe senescence were selected to further propagate and examine these characteristics. Litters that showed normal aging were selected as a senescence-resistant series (R-series). The genetic background of the SAM mice became suspect after the pathological findings were different from the AKR/J mouse. Each SAM model is genetically different. Each SAM colony was acquired by Harlan by Takeda Chemical Ltd. in 2002.
And here is the link to the company site.
http://www.harlan.com/products_and_services/research_models_and_services/research_models/sam_inbred_mice/samp8tahsd.hl

first: Silvia

last: Garcıa-Matas

first: Rajib

first: Patricia

last: Molina-Martınez

first: Hector

last: Palacios

first: Vincent

last: Gutierrez

first: Ruben

last: Corpas

first: Merce

last: Pallas

first: Rosa

last: Cristofol

first: Rafael

last: de Cabo

first: Coral

last: Sanfeliu


title: Blast traumatic brain injury induced cognitive deficits are attenuated by pre- or post-injury treatment with the glucagon-like peptide-1 receptor agonist, exendin-4


title: Copy number variations distinguish lung adenocarcinomas from squamous cell carcinomas

status: Public on Nov 13 2015

city: Dallas

first: Luc

last: Girard

address: 5323 Harry Hines Blvd

department: Hamon Center for Therapeutic Oncology Research

institute: UT Southwestern Medical Center

type: Genome variation profiling by genome tiling array

summary: For a number of clinical and biological reasons, the accurate classification of non-small cell lung carcinoma (NSCLC) into adenocarcinoma (ADC) and squamous cell carcinoma (SCC) is essential. DNA-based tests, which are not currently used, are more robust when applied to formalin-fixed paraffin-embedded tissues. To develop a molecular-based classification of NSCLC based on genome wide copy number variations (CNVs), the corresponding TCGA, SPORE and CANARY patient datasets were used as training and independent validation data. The signature genes were selected by advanced supervised classification algorithms and restricted to known important oncogenes/tumor suppressors, resulting in a final 27-gene signature that was able to classify ADC from SCC with 0.85-0.87 accuracies of SPORE validation sets and 0.96-0.98 accuracy of CANARY validation sets. Even by using the top 7 genes in this signature, the accuracies of the validation sets were still as high as 0.80 and 0.97, respectively. These signature genes also classified adenocarcinoma and squamous cell carcinomas from the non-malignant lung samples with accuracies of 91-97%.

overall_design: Copy number analysis of Agilent 244K CGH arrays was performed for 162 early stage Non-Small Cell Lung Cancer (NSCLC) patients

first: Ignacio

last: Wistuba

first: Milind

last: Suraokar

first: Kai

last: Song


title: Human umbilical cord matrix mesenchymal stem cells suppress the growth of breast cancer by expression of tumor suppressor genes [human]

overall_design: Human UCMSC and MDA-231 breast carcinoma cells were cultured indirectly for 48 hours using Transwell culture dish. Naïve human UCMSC were cultured under same condition without the addition of carcinoma cells.

first: Naomi

last: Ohta

first: Susumu

last: Ishiguro

first: Atsushi

last: Kawabata

first: Deepthi

last: Uppalapati

first: Marla

last: Pyle

first: Deryl

last: Troyer

first: Yongqing

last: Zhang

first: Kevin


title: Defective Mitophagy in XPA via PARP1 activation and NAD+/SIRT1-depletion:  Implications for neurodegeneration (mouse)

status: Public on Jun 04 2015

first: Supriyo

last: De

summary: Mitochondrial dysfunction is a common feature in neurodegeneration and aging. We identify mitochondrial dysfunction in xeroderma pigmentosum group A (XPA), a nucleotide excision DNA repair disorder with severe neurodegeneration, in silico and in vivo. XPA deficient cells show defective mitophagy with excessive cleavage of PINK1 and increased mitochondrial membrane potential. The mitochondrial abnormalities appear to be caused by decreased activation of the NAD+-SIRT1-PGC-1α axis triggered by hyperactivation of the DNA damage sensor PARP1. This phenotype is rescued by PARP1 inhibition or by supplementation with NAD+ precursors that also rescue the lifespan defect in xpa-1 nematodes. Importantly, this pathogenesis appears common to ataxia-telangiectasia and Cockayne syndrome, two other DNA repair disorders with neurodegeneration, but absent in XPC, a DNA repair disorder without neurodegeneration. Our findings reveal a novel nuclear-mitochondrial cross-talk that is critical for the maintenance of mitochondrial health.

overall_design: Mice carrying WT, or CX (Csa-/-/Xpa-/-) alleles in a C57BL/6 background were maintained under standard laboratory conditions and allowed free access to water and control casein pelleted diet (Research Diets D12450B). At 3 months of age, 3 replicates of each of the CX and WT mice were given subcutaneous interscapular injections of 500 mg of Nicotinamide riboside/kg body weight/day or the equivalent volume of saline for 14 consecutive days at 4:00 pm.  On day 15, the mice were sacrificed and half of the cerebellum was harvested for purification of mitochondria, with the left half snap-frozen, homogenized, and aliquoted for RNA isolation.  Total RNA extraction was done using a TRIzol Plus RNA purification kit as per manufacturer’s protocol.  Quality and quantity of the total RNA was tested using the Agilent 2100 Bio-Analyzer and RNA 6000 nano kits.   The RNA was labeled using the standard Illumina protocol and hybed overnight to Mouse Ref-8 Illumina arrays.  The arrays were scanned using the Beadstation 500 X from Illumina.

first: Evandro

last: Fang

first: Morten

last: Scheibye-Knudsen

first: Lear

middle: E

last: Brace

first: Henok

last: Kassahun

first: Tanima

last: SenGupta

first: Hilde

last: Nilsen

first: James

last: Mitchel

first: Deborah

last: Croteau

first: Vilhelm

last: Bohr


title: EnD-Seq and AppEnD: Sequencing 3' ends to identify non-templated tails and degradation intermediates

status: Public on Jun 01 2015

city: Chapel Hill

first: William

last: Marzluff

state: NC

address: 207 Fordham Hall

institute: The University of North Carolina at Chapel Hill

summary: Existing methods for detecting RNA intermediates resulting from exonuclease degradation are low-throughput and laborious. In addition, mapping the 3’ ends of RNA molecules to the genome after high-throughput sequencing is challenging, particularly if the 3’ ends contain posttranscriptional modifications. To address these problems, we developed EnD-Seq, a high-throughput sequencing protocol that preserves the 3’ end of RNA molecules, and AppEnD, a computational method for analyzing high-throughput sequencing data. Together these allow determination of the 3’ ends of RNA molecules, including nontemplated additions. Applying EnD-Seq and AppEnD to histone mRNAs revealed that a significant fraction of cytoplasmic histone mRNAs end in one or two uridines, which have replaced the 1-2 nts at the 3’ end of mature histone mRNA maintaining the length of the histone transcripts. Histone genes in fly embryos and ovaries show the same pattern, but with different tail nucleotide compositions. We increase the sensitivity of EnD-Seq by using cDNA priming to specifically enrich low-abundance tails of known sequence composition allowing identification of degradation intermediates. In addition, we show the broad applicability of our computational approach by using AppEnD to gain insight into 3’ additions from diverse types of sequencing data, including data from small capped RNA sequencing and some alternative polyadenylation protocols.

sample_organism:
Drosophila melanogaster
Homo sapiens

platform_organism:
Drosophila melanogaster
Homo sapiens

overall_design: Libraries were prepared essentially as described in Slevin et al. (Mol. Cell  53: 1020-1030, 2014)

first: Joshua

middle: D

last: Welch


title: Defective Mitophagy in XPA via PARP1 activation and NAD+/SIRT1-depletion:  Implications for neurodegeneration (human)

overall_design: Four replicates of XPA-deficient (GM04312C) and the matched complemented (GM15876A) control cells as well as primary fibroblasts, transfected with either the shRNA to XPA or a scrambled version of the shRNA, as a control, were grown and total RNA was extracted.  The RNA was labeled using the standard Illumina protocol and hybed overnight to Human-HT12 arrays.  The arrays were scanned using the Beadstation 500 X from Illumina.


title: Defective Mitophagy in XPA via PARP1 activation and NAD+/SIRT1-depletion:  Implications for neurodegeneration

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Roles and Programming of Arabidopsis ARGONAUTE Proteins During Turnip Mosaic Virus Infection

status: Public on Apr 06 2015

city: Lincoln

first: Hernan

last: Garcia-Ruiz

state: NE

address: 4240 Fair Street

department: Plant Pathology

institute: University of Nebraska-Lincoln

summary: AGO protein immunoprecipitation was combined with high-throughput sequencing of associated small RNAs. AGO2, AGO10, and to a lesser extent AGO1 were shown to associate with siRNAs derived from silencing suppressor (HC-Pro)-deficient TuMV-AS9, but not with siRNAs derived from wild-type TuMV. Co-immunoprecipitation and small RNA sequencing revealed that viral siRNAs broadly associated with wild-type HC-Pro during TuMV infection. These results support the hypothesis that suppression of antiviral silencing during TuMV infection, at least in part, occurs through sequestration of virus-derived siRNAs away from antiviral AGO proteins by HC-Pro.

overall_design: Catalytic mutant HA-AGO1-DAH, HA-AGO2-DAD and HA-AGO10-DAH or catalytically active HA-AGO10-DDH were immunoprecipitated from buffer (mock) or inoculated rosette leaves (at 7 dpi) or noninoculated cauline leaves at 10 dpi with wt TuMV or suppressor-deficient TuMV-AS9. Inflorescence from TuMV infected plants was collected at 10 dpi with wt TuMV. HC-Pro was tagged with 6xHIS in TuMV-HIS and suppressor-deficient TuMV-HIS-AS9. HC-Pro  was immunoprecipitated from noninoculated cauline leaves of inflorescence at 10 dpi. HC-Pro AS9 was immunoprecipitated from noninoculated cauline leaves at 15 dpi. Total RNA was extracted from input fraction and small RNAs separated by size fractionation. Small RNAs in input and HA-AGO or HC-Pro immunoprecipitation fractions were converted to DNA Amplicons by 5' (GUUCAGAGUUCUACAGUCCGACGAUC) or  3’ (CTGTAGGCACCATCAAT) adaptor ligation followed by RT-PCR. DNA Amplicons were sequenced using the Illumina HiSeq2000 platform. Duplicate libraries were made per treatment. For each library, hits to TuMV, to TuMV-HIS and to Arabidopsis thaliana were included in separate files.

first: Noah

last: Fahlgren

last: Carrington


title: Delineation of a conserved arrestin-biased signaling repertoire in vivo

status: Public on Mar 05 2015

summary: Biased G protein-coupled receptor agonists engender a restricted repertoire of downstream events from their cognate receptors, permitting them to produce mixed agonist-antagonist effects in vivo. While this opens the possibility of novel therapeutics, it complicates rational drug design, since the in vivo response to a biased agonist cannot be reliably predicted from its in vitro efficacy. We have employed novel informatic approaches to characterize the in vivo transcriptomic signature of the arrestin pathway-selective parathyroid hormone analog [D-Trp12, Tyr34]-bPTH(7-34) in six different murine tissues after chronic drug exposure. We find that [D-Trp12, Tyr34]-bPTH(7-34) elicits a distinctive arrestin-signaling focused transcriptomic response that is more coherently regulated across tissues than that of the pluripotent agonist, hPTH(1-34). This arrestin-focused network is closely associated with transcriptional control of cell growth and development. Our demonstration of a conserved arrestin-dependent transcriptomic signature suggests a framework within which the in vivo outcomes of arrestin-biased signaling may be generalized.

overall_design: Eleven-week-old male wild type C57BL/6J or congenic arrestin3-null mice were infused with human 40 µg/kg d PTH(1-34), 40 µg/kg d bovine (D-Trp12, Tyr34)-PTH(7-34), or PBS vehicle via Alzet osmotic minipumps (Model #1004, Durect Corp., Cupertino, CA) for 28 days. Minipumps were implanted subcutaneously in the upper back of anesthetized mice. The pump released 0.25 uL/hr in vehicle of 1 mM acetic acid in sterile PBS. At the end of the infusion period, animals were sacrificed and target tissues: calvarial bone, heart, lung, liver, kidney and aorta, were harvested and stored at -80 ˚C until mRNA isolation. Animal protocols were approved by the institutional animal care and use committee at Duke University School of Medicine and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

first: Stuart

last: Maudsley

first: Bronwen

last: Martin

first: Diane

last: Gesty-Palmer

first: Huey

last: Cheung

first: Calvin

last: Johnson

first: Shamit

last: Patel

last: Becker

middle: H

last: Wood III

first: Elin

last: Lehrmann

first: Louis

last: Luttrell


title: MOTS-c: A Novel Peptide Encoded by a Short Mitochondrial Open Reading Frame Regulates Metabolic Homeostasis

status: Public on Mar 07 2015

first: Supriyo

last: De

summary: Mitochondria are known to be functional organelles, but their role as a signaling unit is increasingly being appreciated. The recent identification of a short open reading frame (sORF) in the mitochondrial DNA (mtDNA) that encodes a signaling peptide, humanin, suggests the possible existence of additional sORFs in the mtDNA that yield bioactive peptides. Here we report the identification of a sORF within the mitochondrial 12S rRNA encoding a 16-amino-acid peptide named MOTS-c (mitochondrial open-reading-frame of the twelve S rRNA -c) that regulates insulin sensitivity and metabolic homeostasis. MOTS-c is detected in various tissues and in circulation in an age-dependent manner. Its primary target organ appears to be the skeletal muscle and its cellular actions inhibit the folate cycle and its tethered de novo purine biosynthesis, causing a significant accumulation of AICAR levels concomitantly with AMPK activation. MOTS-c treatment in mice prevented age-dependent and high-fat diet-induced insulin resistance, as well as diet-induced obesity. These results suggest that mitochondria may be more actively engaged in regulating metabolic homeostasis than previously recognized, through the production of peptides encoded within its genome that act at the cellular and organismal level.

overall_design: Human embryonic kidney cells (HEK293 cell line) were cultured in 10-cm dishes in 7 mL of phenol-free DMEM supplemented with 10% FBS and incubated with water (controls) or the 16-amino-acid peptide mitochondrial open-reading-frame of the twelve S rRNA-c (MOTS-c, 10 uM) for 4 or 72 hours prior to RNA extraction.

first: Changhan

last: Lee

first: Jennifer

last: Zeng

first: Brian

last: Drew

first: Tamer

last: Sallam

first: Alejandro

last: Martin-Montalvo

first: Junxiang

last: Wan

first: Su

last: Kim

first: Hemal

last: Mehta

first: Andrea

last: Hevener

first: Rafael

last: de Cabo

first: Pinchas

last: Cohen


title: Distinct inhibitory effects on mTOR signaling by ethanol and INK128 in diffuse large B-cell lymphoma

status: Public on Mar 03 2015

summary: The mechanistic target of rapamycin, (mTOR) kinase plays a pivotal role in controlling critical cellular growth and survival pathways, and its aberrant induction is implicated in cancer pathogenesis. Therefore, suppression of active mTOR signaling has been of great interest to researchers; several mTOR inhibitors have been discovered to date. Ethanol (EtOH), similar to pharmacologic mTOR inhibitors, has been shown to suppress the mTOR signaling pathway, though in a non-catalytic manner. Despite population studies showing that the consumption of EtOH has a protective effect against hematological malignancies, the mechanisms behind EtOH’s modulation of mTOR activity in cells and its downstream consequences are largely unknown. Here we evaluated the effects of EtOH on the mTOR pathway, in comparison to the active-site mTOR inhibitor INK128, and compared translatome analysis of their downstream effects in diffuse large B-cell lymphoma (DLBCL).

overall_design: SUDHL-2 and SUDHL-4 diffuse large B-cell lymphoma (DLBCL) cell lines (ATCC) were cultured in RPMI Medium 1640 (Gibco BRL) supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO2. EtOH-treated cells were cultured in sealed flasks to maintain EtOH concentrations, 20mM in the culture medium, water was added to EtOH control wells. INK128-treated cells were treated with INK128 at 40nM (Selleckchem) dissolved in dimethyl sulfoxide (DMSO) or 0.01% DMSO was added for the control wells. To create polysomal fractions, cells were lysed in cytoplasmic lysis buffer (5mM Tris, 2.5mM MgCl2, 1.5mM KCl, 1% Triton X-100, 0.5% Sodium Deoxycholate and 2 mM DTT), loaded on 10 - 50% linear sucrose gradients and fractionated. The RNA in each fraction was monitored by optical density measurement (A254) and eleven fractions were collected with a fraction collector (Brandel). The RNA from each fraction was isolated by Trizol (Invitrogen) and used for RT-qPCR analysis. RNA from high molecular weight polysomal fractions, with actively translated mRNAs (fractions 9 - 11) were pooled and used for microarray analysis. RNA was isolated using Qiagen RNeasy protocols and quality and quantity were checked with an Agilent 2100 Bio-Analyzer using RNA 6000 Nano chips. Labeling and amplification were done with the standard Illumina protocol and the biotin-labeled cRNA was hybridized to Illumina's HumanHT-12 V4.0 Expression BeadChip. The arrays were washed, blocked and the biotin-labeled cRNA was detected by staining with streptavidin-Cy3.

first: Krystyna

last: Mazan-Mamczarz

first: Raymond

last: Peroutka

first: James

last: Steinhardt

first: Moriah

last: Gidoni

first: Yongqing

last: Zhang

first: Ari

last: Landon

first: Bojie

last: Dai

first: Simone

last: Houng

first: Parameswary

middle: A

last: Muniandy

first: Efroni

last: Sol

first: Kevin

first: Ronald

middle: B

last: Gartenhaus


title: Sunitinib treatment exacerbates intratumoral heterogeneity in metastatic renal cancer

status: Public on May 25 2015

city: Edinburgh

first: Fiach

last: O'Mahony

address: Crewe Road South

department: Pathology

institute: University of Edinburgh

summary: The aim of this study was to investigate the effect of VEGF targeted therapy (sunitinib) on intratumoral heterogeneity (ITH) in metastatic clear cell renal cancer (mRCC).

overall_design: 138 samples from patients with clear cell renal cell carcinoma, including biological replicates of nephrectomy samples.  RNA extracted  fresh frozen tissue samples.

first: Grant

last: Stewart

first: Thomas

last: Powles

first: Alan

last: MacKay


title: Gene expression profiling of patient's DCIS-IDC tandem lesions by RNA sequencing analysis

status: Public on Feb 26 2015

city: Kansas City

first: Hanan

last: Elsarraj

state: KS

address: 3901 Rainbow Blvd.

department: Pathology and Laboratory Medicine

institute: University of Kansas Medical Center

summary: Tandem DCIS/IDC are defined as ductal carcicnoma in situ (DCIS) lesions that have concurrent invasive ductal carcinoma (IDC) within the same breast. These are identified radiologically by an area of clustered microcalcifications adjacent to (contiguous with) an invasive mass. Our radiologist (Dr. William P. Smith) has provided us with biopsy cores from each region. One core from each region (DCIS and IDC) has bas been collected and subjected to RNA sequencing for our studies to compare changes from DCIS to IDC in each individual patient.

overall_design: 6 pairs of DCIS-IDC samples were collected, and analysed by RNA sequencing

first: William

middle: P

last: Smith

first: Kelli

last: Valdez

first: Whitney

last: Michaels

first: Markus

last: Hook

first: Jeremy

last: Chein

first: Fariba

last: Behbod


title: Gene expression analysis of Influenza vaccine response in Young and Old

status: Public on Jan 27 2015

city: New Haven

first: Hailong

last: Meng

state: CT

address: 300 George Str

institute: Yale University


title: Gene expression analysis of Influenza vaccine response in Young and Old - Year 2 - PBMC

status: Public on Jan 26 2015

summary: We profiled gene expression from a stratified cohort of subjects to define influenza vaccine response in Young and Old

overall_design: Differential gene expression by human PBMCs from Healthy Adults receiving Influenza Vaccination (Fluvirin, Novartis). Healthy adults (older >65, younger 21-30 years) were recruited at seasonal Influenza Vaccination clinics organized by Yale University Health Services during October to December of 2011-2012 seasons. With informed consent, healthy individuals were recruited as per a protocol approved by Human Investigations Committee of the Yale University School of Medicine. Each subject was evaluated by a screening questionnaire determining self-reported demographic information, height, weight, medications and comorbid conditions. Participants with acute illness two weeks prior to vaccination were excluded from study. Blood samples were collected into BD Vacutainer Sodium Heparin tube at four different time points, once prior to administration of vaccine and three time points after vaccination on days 2/4, 7 and 28. Peripheral Blood Mononuclear Cells (PBMC) were isolated from heparinized blood using Histopaque 1077 in gradient centrifugation. About 1.0x10^7 freshly isolated PBMC were lysed in Triso and immediately stored in -800C. Total RNA in aqueous phase of Trisol - Chloroform was isolated in an automated QiaCube instrument using miRNeasy according to manufacturer’s instructions. Integrity of RNA samples were assessed by Agilent 2100 BioAnalyser Samples were processed for cRNA generation using Illumina TotalPrep cRNA Amplification Kit and subsequently hybridized to Human HT12-V4.0 BeadChip at Yale Center for Genomic Analysis (YGCA).

first: Albert

last: Shaw

first: Subhasis

last: Mohanty

first: Samit

last: Joshi

first: Ikuyo

last: Ueda

first: Jean

last: Wilson

first: Juilee

last: Thakar

first: Steven

last: Kleinstein

first: Sui

last: Tsang


title: Gene expression analysis of Influenza vaccine response in Young and Old - Year 1 - PBMC

overall_design: Differential gene expression by human PBMCs from Healthy Adults receiving Influenza Vaccination (Fluvirin, Novartis). Healthy adults (older >65, younger 21-30 years) were recruited at seasonal Influenza Vaccination clinics organized by Yale University Health Services during  2010 – 2011 season. With informed consent, healthy individuals were recruited as per a protocol approved by Human Investigations Committee of the Yale University School of Medicine. Each subject was evaluated by a screening questionnaire determining self-reported demographic information, height, weight, medications and comorbid conditions. Participants with acute illness two weeks prior to vaccination were excluded from study. Blood samples were collected into BD Vacutainer Sodium Heparin tube at four different time points, once prior to administration of vaccine and three time points after vaccination on days 2/4, 7 and 28. Peripheral Blood Mononuclear Cells (PBMC) were isolated from heparinized blood using Histopaque 1077 in gradient centrifugation. About 1.0x10^7 freshly isolated PBMC were lysed in Triso and immediately stored in -800C. Total RNA in aqueous phase of Trisol - Chloroform was isolated in an automated QiaCube instrument using miRNeasy according to manufacturer’s instructions. Integrity of RNA samples were assessed by Agilent 2100 BioAnalyser Samples were processed for cRNA generation using Illumina TotalPrep cRNA Amplification Kit and subsequently hybridized to Human HT12-V4.0 BeadChip at Yale Center for Genomic Analysis (YGCA).


title: RNA-binding protein AUF1 promotes myogenesis by regulating MEF2C expression levels

status: Public on Dec 22 2014

first: Supriyo

last: De

summary:
The mammalian RNA-binding protein AUF1 (AU-binding factor 1, also known as heterogeneous nuclear ribonucleoprotein D, hnRNP D) binds to numerous mRNAs and influences their post-transcriptional fate.  Given that many AUF1 target mRNAs encode muscle-specific factors, we investigated the function of AUF1 in skeletal muscle differentiation.  In mouse C2C12 myocytes, where AUF1 levels rise at the onset of myogenesis and remain elevated throughout myocyte differentiation into myotubes, RIP (RNP immunoprecipitation) analysis indicated that AUF1 binds prominently to Mef2c (myocyte enhancer factor 2c) mRNA, which encodes the key myogenic transcription factor Mef2c.  By performing mRNA half-life measurements and polysome distribution analysis, we found that AUF1 associated with the 3’UTR of Mef2c mRNA and promoted Mef2c translation without affecting Mef2c mRNA stability.  In addition, AUF1 promoted Mef2c gene transcription via a lesser-known role of AUF1 in transcriptional regulation.  Importantly, lowering AUF1 delayed myogenesis, while ectopically restoring Mef2c expression levels partially rescued the impairment of myogenesis seen after reducing AUF1 levels.  We propose that Mef2c is a key effector of the myogenesis program promoted by AUF1.
Keywords: ribonucleoprotein complex; post-transcriptional gene regulation; muscle cell differentiation; myocytes; mRNA translation; mRNA stability; post-transcriptional gene regulation; transcriptome

overall_design: C2C12 mouse myoblasts were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% serum (Invitrogen) and antibiotics (Invitrogen).  Differentiation was induced on sub-confluent cultures by replacing the growth media (GM, DMEM with 10% FBS) with differentiation media (DM, DMEM with 2% horse serum).  At various time points after differentiation, cells were harvested and RNA was extracted with phenol-chloroform and either saved as input samples for microarrys or subjected to AUF1 or IgG ribonucleoprotein immunoprecipitation.  C2C12 cells cultured in GM and DM were lysed in 20 mM Tris-HCl at pH 7.5, 100 mM KCl, 5 mM MgCl2, and 0.5% NP-40 for 10 min on ice and centrifuged at 15,000 × g for 10 min at 4°C.  The supernatants were incubated with protein-A Dynabeads beads coated with anti-AUF1 (Millipore) or with control IgG (Santa Cruz Biotechnology) antibodies for 2 hr at 4°C.  The beads were washed with NT2 buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40), followed by incubation with 20 units of RNase-free DNase I for 15 min at 37°C to remove the DNA.  The samples were then incubated for 15 min at 55°C with 0.1% SDS/0.5 mg/ml Proteinase K to digest proteins.  The RNA from the IP samples was extracted using phenol-chloroform, precipitated, and used along with the  RNA from the input samples for cDNA microarrays.

first: Amaresh

last: Panda

first: Kotb

last: Abdelmohsen

first: Je-Hyun

last: Yoon

first: Jennifer

last: Martindale

first: Xiaoling

last: Yang

first: Jessica

last: Curtis

first: Evi

last: Mercken

first: Devon

last: Chenette

first: Yongqing

last: Zhang

first: Robert

middle: J

last: Schneider

first: Kevin

last: Becker

first: Rafael

last: de Cabo

first: Myriam

last: Gorospe