title: Toll-like receptor 2 (TLR2)-TLR9 crosstalk dictates IL-12 family cytokine production in microglia

summary: Microglia are the resident mononuclear phagocytes of the CNS parenchyma and represent an initial line of defense against invading microorganisms. Microglia utilize Toll-like receptors (TLRs) for pathogen recognition and TLR2 specifically senses conserved motifs of gram-positive bacteria including lipoproteins, lipoteichoic acids, and peptidoglycan (PGN) leading to cytokine/chemokine production. Interestingly, primary microglia derived from TLR2 knockout (KO) mice over-expressed numerous IL-12 family members, including IL-12p40, IL-12p70, and IL-27 in response to intact S. aureus, but not the less structurally complex TLR2 ligands Pam3CSK4 or PGN. The ability of intact bacteria to augment IL-12 family member expression was specific for gram-positive organisms, since numerous gram-negative strains were unable to elicit exaggerated responses in TLR2 KO microglia. Inhibition of SYK or IRAK4 signaling did not impact heightened IL-12 family member production in S. aureus-treated TLR2 KO microglia, whereas PI3K, MAPK, and JNK inhibitors were all capable of restoring exaggerated cytokine expression to wild type levels. Additionally, elevated IL-12 production in TLR2 KO microglia was ablated by a TLR9 antagonist, suggesting that TLR9 drives IL-12 family member production following exposure to intact bacteria that remains unchecked in the absence of TLR2 signaling. Collectively, these findings indicate crosstalk between TLR2 and TLR9 pathways to regulate IL-12 family member production by microglia. The summation of TLR signals must be tightly controlled to ensure the timely cessation and/or fine tuning of cytokine signaling to avoid nonspecific bystander damage due to sustained IL-12 release.

overall_design: TLR2 KO mice were backcrossed with C57BL/6 animals for a minimum of eight generations prior to use in these studies. Age- and sex-matched C57BL/6 mice were used as wild type (WT) controls. Primary mixed glial cultures were prepared from the cerebral corticies of neonatal mice (2-4 days of age) and microglia were harvested using a differential shaking technique with a purity of >98%. A USA300 community-acquired methicillin-resistant S. aureus (CA-MRSA) clinical isolate recovered from a patient with a fatal brain abscess was used to stimulate the microglia isolates.  Bacterial strains were heat-inactivated and used to stimulate microglia at 107 colony forming units (cfu)/well for 6 and 12 hours time points. Three replicates of each mouse type (WT, TLR2 KO) at both time points 6 and 12 hours were used for the microarray experiments.  Data was only usable for 2 replicates of the KO-12 hr group.

first: Monica

last: Holley

first: Elin

last: Lehrmann

first: William

last: Wood

first: Tammy

last: Kielian


title: A High-Fat Diet and NAD+ Rescue Premature Aging in Cockayne Syndrome

status: Public on Dec 22 2014

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: Cockayne syndrome (CS) is an accelerated aging disorder characterized by progressive neurodegeneration caused by mutations in the genes encoding the DNA repair proteins CSA or CSB. Csbm/m mice were given a high-fat, caloric-restricted or resveratrol-supplemented diet. The high-fat diet rescued the phenotype of Csbm/m mice at the metabolic, transcriptomic and behavioral levels. Additional analysis suggests that the premature aging seen in CS mice, nematodes and human cells results from aberrant PARP activation due to deficient DNA repair leading to decreased SIRT1 activity and mitochondrial dysfunction. Notably, β-hydroxybutyrate levels are increased by the high-fat diet; and β-hydroxybutyrate, PARP inhibition, or NAD+ supplementation can activate SIRT1 and rescue CS-associated phenotypes. Mechanistically, CSB is able to displace activated PARP1 from damaged DNA to limit its activity. This study connects two emerging longevity metabolites, β-hydroxybutyrate and NAD+, through the deacetylase SIRT1 and suggests possible interventions for CS.

overall_design: 4-month-old mice, WT and Csbm/m on a C57BL/6 background, were fed a standard AIN-93G diet (SD; carbohydrate:protein:fat ratio of 64:19:17 percent of kcal) ad libitum or at 40% CR, a SD supplemented with 100 mg/kgchow resveratrol ad libitum, or a high-fat diet ad libitum consisting of AIN-93G with 60% of calories from fat, primarily hydrogenated coconut oil (HFD; carbohydrate:protein:fat ratio of 16:23:61). Each group was on the specified diet for 8 months, after which the mice were sacrificed and the cerebellum was removed. For nicotinamide treatments, 4- or 18-month-old WT and Csbm/m mice were given daily injections of nicotinamide riboside (NR) (500 mg/kg/d, ip) or saline for one week, after which the mice were sacrificed and the cerebellum was removed. RNA was extracted from the cerebellums of all mice using Trizol, and RNA quality and quantity were tested using an Agilent 2100 BioAnalyzer with RNA 6000 nano chips. RNA was labeled using the standard Illumina protocol for Illumina TotalPrep RNA Amplification Kit. Labeled RNA was hybridized to Illumina's Sentrix MouseRef-8 v2 Expression BeadChips (Illumina, San Diego, CA) overnight and washed, stained and scanned the next day.

first: Morten

last: Scheibye-Knudsen

first: Sarah

middle: J

last: Mitchell

first: Evandro

middle: F

last: Fang

first: Teruaki

last: Iyama

first: Theresa

last: Ward

first: James

last: Wang

first: Christopher

middle: A

last: Dunn

first: Nagendra

last: Singh

first: Sebastian

last: Veith

first: M

middle: M

last: Hasan

first: Aswin

last: Mangerich

first: Mark

last: Wilson

middle: P

last: Mattson

first: Linda

middle: H

last: Bergersen

first: Victoria

middle: C

last: Cogger

first: Alessandra

last: Warren

first: David

middle: G

last: Le Couteur

first: Ruin

last: Moaddel

last: Wilson 3rd

first: Deborah

middle: L

last: Croteau

first: Rafael

last: de Cabo

first: Vilhelm

last: Bohr


title: Increased Human like Alzheimer features in a Novel Mouse Model with Oxidative DNA Repair Dysfunction

status: Public on Jan 05 2015

first: Supriyo

last: De

summary: The decline of cognitive function is a feature of normal human aging and is exacerbated in Alzheimer’s disease (AD).  DNA repair declines in brain cells during normal aging and even more so in AD.   Here we show that experimental reduction in levels of the base excision repair enzyme, DNA polymerase β (Polb) renders neurons vulnerable to age-related dysfunction and degeneration in a mouse model of AD.    Whereas 3xTgAD mice exhibit age-related extracellular amyloid b-peptide (Ab) accumulation and cognitive deficits, but no neuronal death, 3xTg/Polb+/- mice accumulates intracellular Ab and neurons die in the hippocampus and cerebral cortex.  The DNA repair-deficient 3xTgAD mice exhibited increased DNA strand breaks and apoptotic caspase activation with loss of hippocampal volume, and impaired synaptic plasticity and memory retention.  Molecular profiling revealed remarkable similarities in gene expression alterations in brain cells of AD patients and 3xTgAD/Polb+/- mice including multiple abnormalities suggestive of impaired cellular bioenergetics.  Our findings demonstrate that a modest decrement in oxidative DNA damage processing is sufficient to render neurons vulnerable to AD-related pathogenic molecular and cellular alterations that result in the dysfunction and death of neurons, and associated cognitive deficits.

overall_design: 4 mouse strains were used in these experiments,  the 3xTgAD and Pol β (+/-) mice were bred at the National Institute on Aging (Baltimore, Maryland). The original line 3xTgAD line was generated as described previously (Oddo, et. al 2003) and possess APPswe, PS1M146V, and tauP301L mutations. DNA polymerase beta heterozygous mice, Pol β (+/-), were crossed with the 3xTgAD mice to generate a 3xTgAD/Pol β (+/-) mouse. The Wt strain is C57Bl/6. At 20 months of age these mice were euthanized by cervical dislocation, the brain removed from the skull and dissected into regions of interest, the prefrontal cortex was used for the microarray studies.

first: P

last: Sykora

last: Misiak

first: Y

last: Wang

first: S

last: Ghosh

first: G

last: Leandro

first: D

last: Liu

first: J

last: Tian

first: B

last: Baptiste

first: W

last: Cong

last: Brenerman

first: E

last: Fang

first: K

last: Becker

first: R

last: Hamilton

last: Chigurupati

last: Zhang

last: Egan

last: Wilson III

first: V


title: The SIRT1 activator SRT1720 extends lifespan and improves health of mice fed a standard diet

status: Public on Dec 20 2014

last: De

summary: The prevention or delay of the onset of age-related diseases prolongs survival and improves quality of life while reducing the burden on the health care system. Activation of sirtuin 1 (SIRT1), an NAD+ deacetylase, improves metabolism and confers protection against physiological and cognitive disturbances in old age. SRT1720 is a specific SIRT1 activator that has health and lifespan benefits in adult mice fed a highfat diet. We found extension in lifespan, delayed onset of age-related metabolic diseases, and improved general health in mice fed a standard diet after SRT1720 supplementation. Inhibition of pro-inflammatory gene expression both in the liver and muscle of SRT1720-treated animals was noted. SRT1720 lowered phosphorylation of NF-κB pathway regulators in vitro only when SIRT1 was functionally present. Combined with our previous work, the current study further supports the beneficial effects of SRT1720 on health across the lifespan in mice.

overall_design: Groups of 28 week old male C57BL/6J mice were maintained on ad libitum AIN-93G SD diet, or an ad libitum AIN-93G diet supplemented with SRT1720 for the rest of their lives. SRT1720 was added at a dose of 1.33 g drug per kg of chow, formulated to provide daily doses of approximately 100 mg drug per kg bodyweight to the mice.  5 mice from each  group were selected and RNA was extracted from both muscle and liver tissue using 1.0mm glass beads in a Precellys 24 Tissue Homogenizer and Qiagen RNeasy Mini Kits for Fibrous Tissue according to manufacturer's specifications. Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips.  RNA samples were labeled using 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 and labeled by incorporation of biotin-16-UTP.  Arrays were hybridized using a total of 0.75ug of biotin-labeled cRNA at 58 degrees C for 16 hours to Illumina's Sentrix MouseRef-8 v2 Expression BeadChips. Each BeadChip has ~24,000 well-annotated RefSeq transcripts with approximately 30-fold redundancy. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.  Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina and the data was extracted using the Illumina GenomeStudio software(v1.6.0).  Any spots at or below the background were filtered out using an Illumina detection p value of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below.  Z-score = (raw value - avg)/std.

first: Sarah

last: Mitchell

first: Alejandro

last: Martin-Montalvo

first: Evi

last: Mercken

first: Hector

last: Palacios

first: Theresa

last: Ward

first: Robin

middle: K

last: Minor

first: George

last: Vlasuk

first: James

last: Ellis

first: David

last: Sinclair

first: Michel

last: Bernier

last: de Cabo


title: Methylome remodeling in regional breast cancer metastasis

status: Public on Dec 01 2014

city: New York

first: Marsha

last: Reyngold

state: NY

address: 1275 York Ave

department: Radiation Oncology

institute: Memorial Sloan-Kettering Cancer Center

type: Methylation profiling by genome tiling array

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

overall_design: Refer to individual Series


title: Methylome remodeling in regional breast cancer metastasis [expression]

summary: We sought to characterize methylome remodeling during regional metastasis.  We profiled the DNA methylome (n=44) and transcriptome (n=36) of matched primary breast tumors and regional metastases.

overall_design: All paired samples (n = 88) were obtained following surgical resection at the MSKCC as part of routine clinical care, and snap frozen. Tumors were obtained in accordance with Institutional Review Board policies at the MSKCC. Each sample was examined histologically by a breast pathologist and microdissected to achieve >70% tumor cell content. Genomic DNA or RNA was extracted using the DNeasy kit (Qiagen) or RNeasy Kit (Qiagen) as per the manufacturer’s instructions. Expression analysis of tumors was performed using the Affymetrics U133 2.0 microarray (Affymetrix). Affymetrix CEL files were imported into the Partek Genomics Suite 6.1 (Partek), using RMA normalization.


title: Methylome remodeling in regional breast cancer metastasis [DNA methylation]

summary:
Genome-wide DNA methylation profiling of of 44 matched primary breast tumors and metastases.
We sought to characterize methylome remodeling during regional metastasis.  We profiled the DNA methylome (n=44) and transcriptome (n=36) of matched primary breast tumors and regional metastases.

overall_design: All paired samples (n = 88) were obtained following surgical resection at the MSKCC as part of routine clinical care. Tumors were obtained in accordance with Institutional Review Board policies at the MSKCC. Each sample was examined histologically by a breast pathologist and microdissected to achieve >70% tumor cell content. Genomic DNA was extracted using the DNeasy kit (Qiagen)  as per the manufacturer’s instructions. Bisulphite converted DNA from the 44 primary breast tumors and matching metastases were hybridised to the Illumina Infinium 450k Human Methylation Beadchip.

first: Timothy

last: Chan


title: Exosomes secreted by a nematode parasite transfer small RNAs to mammalian cells and regulate genes of the innate immune system

status: Public on Nov 25 2014

city: Edinburgh

first: Alasdair

last: Ivens

address: Kings Buildings

country: United Kingdom

institute: Centre for Immunity, Infection and Evolution

type: Non-coding RNA profiling by high throughput sequencing

sample_organism:
Heligmosomoides polygyrus
Litomosoides sigmodontis


title: MNKs act as a regulatory switch for eIF4E1 and eIF4E3 driven mRNA translation in DLBCL

status: Public on Nov 18 2014

first: Supriyo

summary: The phosphorylation of eIF4E1 at serine 209 by MNK1 or MNK2 has been shown to initiate oncogenic mRNA translation, a process that favours cancer development and maintenance. Here, we interrogate the MNK-eIF4E axis in diffuse large B-cell lymphoma (DLBCL) and show a distinct distribution of MNK1 and MNK2 in germinal centre B-cell (GCB) and activated B-cell (ABC) DLBCL. Despite displaying a differential distribution in GCB and ABC, both MNKs functionally complement each other to sustain cell survival. MNK inhibition ablates eIF4E1 phosphorylation and concurrently enhances eIF4E3 expression. Loss of MNK protein itself down-regulates total eIF4E1 protein level by reducing eIF4E1 mRNA polysomal loading without affecting total mRNA level or stability. Enhanced eIF4E3 expression marginally suppresses eIF4E1-driven translation but exhibits a unique translatome that unveils a novel role for eIF4E3 in translation initiation. Together, we propose that MNKs can modulate oncogenic translation by regulating eIF4E1-eIF4E3 levels and activity in DLBCL.

overall_design: As the knockdown of eIF4E1 or eIF4E3 caused significant cell death, we overexpressed either protein in HLY-1 cells and performed sucrose density gradient fractionation. RNA was extracted from polysome fractions #9-10, containing highly translated polysome-bound mRNAs, from total RNA and from an empty vector control. These samples, in triplicate, were used for either translatome or transcriptome analysis using Illumina HumanHT-12 v4 BeadChips for gene expression analysis.

first: Ari

last: Landon

first: Parameswary

last: Muniandy

first: Amol

last: Shetty

first: Elin

last: Lehrmann

first: Laurent

last: Volpone

first: Simone

last: Houng

first: Yongqing

last: Zhang

first: Bojie

last: Daia

first: Raymond

last: Peroutka

first: Krystyna

last: Mazan-Mamczarz

last: Steinhardt

first: Anup

last: Mahurkar

first: Kevin

last: Becker

first: Katherine

last: Borden

first: Ronald

middle: B

last: Gartenhaus


title: Effects of nicastrin (NCSTN) siRNA knockdown in HEK293 and HEK001 cells.

status: Public on Nov 26 2014

city: St. Louis

first: Elisha

last: Roberson

state: MO

address: 660 South Euclid Ave., CB 8045

department: Internal Medicine & Genetics

institute: Washington University in St. Louis

summary: The goal of this study was to determine if knockdown of nicastrin induced a proinflammatory phenotype in HEK001 and HEK293 cells. Nicastrin (NCSTN) is a member of the gamma-secretase complex, and has been identified as the most frequently mutated gene in familial hidradenitis suppurativa. While much research has been done into the effects of PSEN1 and PSEN2 loss, less is known about isolated NCSTN haploinsufficiency. Two cell lines were knocked down with either NCSTN siRNA or an siRNA to luciferase in triplicate. RNA was extracted from drug selected knockdowns and profiled on the Illumina Human HT-12 v4 beadarray. Gene ontology analysis of differentially expressed genes revealed a proinflammatory and decreased proliferation signature in keratinocytes. HEK293 cells demonstrated expression signatures for decreased cholesterol synthesis and interferon-alpha signaling, as well as increased p53 signaling and caspase mediated cytoskeletal cleavage.

overall_design: 12 total samples. Two cell lines (HEK001 & HEK293) each with two treatments (NCSTN siRNA knockdown or pLKO luciferase knockdown) in three parallel 3 replicates each. For each line gives the changes specific to knockdown of gamma-secretase component nicastrin (NCSTN).

middle: D

first: Li

last: Cao


title: Expression data from low- and high-pathogenicity avian influenza-infected chicken and duck cells

status: Public on Dec 04 2014

city: Sutton Bonington

first: Suresh

middle: Varma

last: Kuchipudi

address: College Road

department: School of Veterinary Medicine and Science

institute: University of Nottingham

summary:
While infection of chickens with highly pathogenic avian influenza (HPAI) H5N1 subtypes often leads to complete mortality within 24 to 48 h, infection of ducks in contrast causes mild or no clinical signs. Rapid onsets of fatal disease in chickens, but with no evidence of severe clinical symptoms in ducks, suggest underlying differences in their innate immune mechanisms.  To understand the molecular basis for such difference, chicken and duck primary lung cells, infected with a low-pathogenicity avian influenza (LPAI) and two HPAI H5N1 viruses, were subjected to RNA expression profiling using Affymetrix Chicken GeneChip arrays.
We used microarrays to analyze the gene expression profiles of primary chicken and duck lung cells infected with H2N3 LPAI and two H5N1 influenza virus subtypes to understand the molecular basis of host susceptibility and resistance. We have identified a set of key genes and pathways that could play an important role in mediating innate host resistance to avian influenza in chickens and ducks.

sample_organism:
Anas platyrhynchos
Gallus gallus

overall_design: 24 hours following infection, total RNA from cells was extracted. Replicate RNA samples from each of the virus-infected (H2N3, H5N1 50-92, or H5N1 ty-Ty) or mock-infected chicken and duck cells (4 treatment groups for each species) were used for microarray analysis. Each of the RNA samples was hybridized to one GeneChip® Chicken Genome Array (Affymetrix), and a total of 16 array chips were used.

middle: V

first: Stephen

last: Dunham

first: Kin-Chow

last: Chang


title: Exosomes secreted by a nematode parasite transfer small RNAs to mammalian cells and regulate genes of the innate immune system [Heligmosomoides polygyrus]

first: Alasdair

last: Ivens

summary: In mammalian systems, extracellular small RNAs can operate in a paracrine manner to communicate information between cells, relying on transport within vesicles. “Foreign” small RNAs derived from bacteria, plants and parasites have also been detected in mammalian body fluids, sparking interest in whether these could mediate inter-species communication. However, there is no mechanistic framework for RNA-mediated interspecies communication and the active movement of RNA via vesicles has not been shown outside of mammals. Here we demonstrate that specific microRNAs and Y RNAs are packaged into vesicles secreted by a gastrointestinal nematode, Heligmosomoides polygyrus, which naturally infects mice.

overall_design: Total RNA was extracted from the secretion product of adult worms and compared to the profile of small RNAs in adult worms, eggs and infective larvae.

first: Amy

last: Buck


title: Integrative AUF1 PAR-CLIP analysis uncovers AUF1 roles in translation and genome integrity (PAR-CLIP)

status: Public on Nov 05 2014

first: Supriyo

last: De

summary: We report that AUF1 modulates global mRNA stability and translation, in turn promoting the maintenance of DNA integrity.

overall_design: Please see individual series. In short, for AUF1 PAR-CLIP, the four isoforms of AUF1 (p37, p40, p42, and p45) tagged with a Flag epitope were expressed in HEK293 cells. For total RNA-Seq HEK293 cells were transfected with Control siRNA, AUF1 siRNA, Empty Vector, Flag-AUF1 p37, p40, p42, or p45 as well as WI-38 cells were collected at PDL 15 and 55 and also transfected with Control siRNA, AUF1 siRNA, HuR siRNA. For Ribo-Seq HeLa cells were transfected with Control siRNA, AUF1 siRNA, or HuR siRNA.

first: Je-Hyun

last: Yoon

first: Markus

last: Hafner

first: Subramanya

last: Srikantan

first: Kotb

last: Abdelmohsen

first: Ioannis

last: Grammatikakis

first: Jiyoung

last: Kim

first: Jennifer

last: Martindale

first: Xiaoling

last: Yang

first: Min-Ju

last: Kang

first: William

last: Wood

first: Janice

last: Barnes

first: Nicole

middle: N

last: Hooten

first: Michele

last: Evans

first: Vidisha

last: Tripathi

first: Kannanganattu

last: Prasanth

first: Thomas

last: Tuschl

first: Nicholas

middle: T

last: Ingolia

first: Myriam

last: Gorospe


title: Integrative AUF1 PAR-CLIP analysis uncovers AUF1 roles in translation and genome integrity (RNA-Seq 2)

status: Public on Nov 04 2014

first: Supriyo

last: De

type: Expression profiling by high throughput sequencing

first: Je-Hyun

last: Yoon

first: Markus

last: Hafner

first: Kevin

last: Becker


title: Integrative AUF1 PAR-CLIP analysis uncovers AUF1 roles in translation and genome integrity (Ribosome)

overall_design: Please see individual series. In short, For AUF1 PAR-CLIP, the four isoforms of AUF1 (p37, p40, p42, and p45) tagged with a Flag epitope were expressed in HEK293 cells. For total RNA-Seq HEK293 cells were transfected with Control siRNA, AUF1 siRNA, Empty Vector, Flag-AUF1 p37, p40, p42, or p45 as well as WI-38 cells were collected at PDL 15 and 55 and also transfected with Control siRNA, AUF1 siRNA, HuR siRNA. For Ribo-Seq HeLa cells were transfected with Control siRNA, AUF1 siRNA, or HuR siRNA.

first: Subramanya

last: Srikantan

first: Kotb

last: Abdelmohsen

first: Ioannis

last: Grammatikakis

first: Jiyoung

last: Kim


title: Integrative AUF1 PAR-CLIP analysis uncovers AUF1 roles in translation and genome integrity (RNA-Seq 1)

overall_design: Please see individual series. For AUF1 PAR-CLIP, the four isoforms of AUF1 (p37, p40, p42, and p45) tagged with a Flag epitope were expressed in HEK293 cells. For total RNA-Seq HEK293 cells were transfected with Control siRNA, AUF1 siRNA, Empty Vector, Flag-AUF1 p37, p40, p42, or p45 as well as WI-38 cells were collected at PDL 15 and 55 and also transfected with Control siRNA, AUF1 siRNA, HuR siRNA. For Ribo-Seq HeLa cells were transfected with Control siRNA, AUF1 siRNA, or HuR siRNA.

first: Jennifer

last: Martindale

first: Xiaoling

last: Yang

first: Min-Ju

last: Kang

first: William

last: Wood


title: Integrative AUF1 PAR-CLIP analysis uncovers AUF1 roles in translation and genome integrity


title: Long non-coding RNA expression profiles  for 7 human samples derived from patients with laryngeal cancer

status: Public on Jul 23 2014

city: Ningbo

first: Zhisen

last: Shen

address: No.57 Xingning Road

department: Department of Otorhinolaryngology (Head and Neck Surgery)

country: China

institute: Lihuili Hospital of Ningbo University

summary: Investigation of lncRNA expression profile of laryngeal cancer

overall_design: A seven chip study using total RNA extracted from 7 squamous cell carcinoma tissues and paired adjacent normal tissues


title: Resveratrol prevents diet-induced arterial degeneration and stiffening in nonhuman primates

status: Public on Jul 21 2014

summary: Cardiovascular (CV) disease is a leading cause of morbidity and mortality in Western societies. Even after accounting for traditional CV risk factors (e.g. obesity, smoking and hypertension), the inflammation-driven thickening and stiffening of central arteries is a strong predictor of adverse outcomes. Arterial wall changes are universally associated with advancing age and show unparalleled worsening in metabolic syndrome. In mice, resveratrol ameliorates a high-fat diet induced arterial wall inflammation and slows age-associated physiologic deteriorations within the arterial wall. Here we tested resveratrol in adult male rhesus monkeys, an experimental model relevant to humans. A diet rich in fat and sucrose (HFS) led to an increase in body weight as well as thickening and stiffening of the aortic wall, marked by diffuse inflammation, fibrosis and fat infiltration. Dietary resveratrol supplementation prevented diet-induced structural and functional alterations within the aortic wall, and abrogated the deleterious vascular endothelial and smooth muscle responses. Integrative genomic and proteomic analyses of aortic tissues revealed molecular signatures consistent with improved vascular functions. Thus, resveratrol conferred protection against the initiation of diet-induced inflammatory events that progress to pathological thickening and stiffening of large arteries. Dietary resveratrol may therefore hold promise as a novel therapy to ameliorate metabolic stress-induced CV disease.

overall_design: After baseline assessment, four male rhesus monkeys remained on the healthy standard diet  (SD), 10 male rhesus monkeys were begun on a high fat/high sucrose (HFS) diet and 10 male rhesus monkeys were begun on a high fat/high sucrose (HFS) diet plus Resveratrol, 80mg/day.  After one year of dietary intervention, the amount of resveratrol was increased to 240mg/day for one additional year.  Tissues were then harvested for the array experiments.

first: Julie

last: Mattison

first: Mingyi

last: Wang

first: Michel

last: Bernier

first: Jing

last: Zhang

first: Sung-Soo

last: Park

first: Stuart

last: Maudsley

first: Steven

middle: S

last: An

first: Lakshmi

last: Santhanam

first: Bronwen

last: Martin

first: Shakeela

last: Faulkner

first: Christopher

last: Morrell

first: Joseph

last: Baur

last: Pearson

first: Edward

last: Lakatta

first: Rafael

last: de Cabo


title: RNA-seq of the brain of Nothobranchius furzeri at different time points

status: Public on Jul 29 2014

city: Jena

first: JenAge

last: Project

address: Beutenbergstr. 11

country: Germany

institute: Leibniz Institute for Age Research - Fritz Lipmann Institute

summary:
Background: Comparison of temporal gene expression profiles
The RNA-seq data comprises 5 age groups: 5,12,20,27,39 weeks

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

overall_design: 25 samples: 5 biological replicates for each age group

first: Mario

last: Baumgart

first: Marco

last: Groth

first: Steffen

last: Priebe

first: Aurora

last: Savino

first: Giovanna

last: Testa

first: Andreas

last: Dix

first: Roberto

last: Ripa

first: Michela

last: Ori

first: Eva

last: Tozzini

first: Reinhard

last: Guthke

first: Matthias

last: Platzer

first: Alessandro

last: Cellerino


title: Genome-wide maps of Runx3 bound regions in CD4 splenic dendritic cells.

status: Public on Jun 22 2014

city: Rehovot

first: Yoram

last: Groner

state: istael

department: Molecular genetics

country: Israel

institute: Weizmann institute of science

type: Genome binding/occupancy profiling by high throughput sequencing

summary: ChIP-seq was conducted using FACS-isolated CD11chiMHCII+CD4+ splenic WT DC and anti-Runx3 antibodies (Ab).

overall_design: Two biological Runx3 IP repeats and two input controls from sorted CD4 DC

last: Dicken

first: Dena

last: Leshkowitz


title: Gene expression analysis of human keratinocytes (HaCaTs) after non-thermal plasma treatment

status: Public on Jun 12 2014

city: Greifswald

first: Anke

last: Schmidt

state: MV

address: Felix-Hausdorff-Str. 2

department: Plasma Life Science

institute: INP Greifswald

summary:
Investigation of gene expression in human skin keratinocytes (HaCaT) following non-thermal plasma treatment for 20 s, 60 s, and 180 s compared to untreated and H2O2-treated controls.
Microarrays were used to analyze and investigate the biological effects of non-thermal plasma on human keratinocyte cells. Using an argon plasma jet kinpen, regulated transcripts were analyzed and further described in Schmidt et al. (2014): “Transcript profiling identifies an important role for Nrf2/Keap1-pathway after non-thermal plasma treatment in human keratinocytes”.

overall_design: A study using total RNA recovered from at non-thermal plasma treated-probes (n>6), as well as H2O2-treated, argon-gas treated, and untreated HaCaT controls.

first: Thomas

last: von Woedtke


title: SRT2104 extends survival of male mice on a standard diet and preserves bone and muscle mass

status: Public on Sep 22 2014

first: Supriyo

last: De

summary:
Increased expression of SIRT1 extends the lifespan of lower organisms and delays the onset of age-related diseases in mammals. Here, we show that SRT2104, a synthetic small molecule activator of SIRT1, extends both mean and maximal lifespan of mice fed a standard diet. This is accompanied by improvements in health, including enhanced motor coordination, performance, bone mineral density and insulin sensitivity associated with higher mitochondrial content and decreased inflammation. Short-term SRT2104 treatment preserves bone and muscle mass in an experimental model of atrophy. These results demonstrate it is possible to design a small molecule that can slow aging and delay multiple age-related diseases in mammals, supporting the therapeutic potential of SIRT1 activators in humans.
Key words: Sirtuins, lifespan, healthspan, osteoporosis, muscle wasting, inflammation

overall_design: Groups of 28 week old male C57BL6/J mice were maintained for the rest of their lives or until sacrifice on either an ad libitum AIN-93G SD diet, an ad libitum AIN-93G diet supplemented with SRT2104 or a caloric restricted AIN-93G diet consisting of 60% of what the ad lib animals ate. SRT2104 was added at a dose of 1.33 g drug per kg of chow, formulated to provide daily doses of approximately 100 mg drug per kg bodyweight to the mice.  5 mice from each  group were selected after 41 weeks and RNA was extracted from both muscle and liver tissue using 1.0mm glass beads in a Precellys 24 Tissue Homogenizer and Qiagen RNeasy Mini Kits for Fibrous Tissue according to manufacturer's specifications. Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips.  RNA samples were labeled using 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 and labeled by incorporation of biotin-16-UTP.  Arrays were hybridized using a total of 0.75ug of biotin-labeled cRNA at 58 degrees C for 16 hours to Illumina's Sentrix MouseRef-8 v2 Expression BeadChips. Each BeadChip has ~24,000 well-annotated RefSeq transcripts with approximately 30-fold redundancy. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.  Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina and the data was extracted using the Illumina GenomeStudio software(v1.6.0).  Any spots at or below the background were filtered out using an Illumina detection p value of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below.  Z-score = (raw value - avg)/std.

first: Evi

last: Mercken

first: Sarah

middle: J

last: Mitchell

first: Alejandro

last: Martin-Montalvo

first: Robin

last: Minor

first: Maria

last: Almeida

first: Ana

last: Gomes

first: Morten

last: Scheibye-Knudsen

first: Hector

last: Palacios

first: Jordan

last: Licata

first: Yongqing

last: Zhang

first: Kevin

last: Becker

first: Husam

last: Khraiwesh

first: José

last: Gonzalez-Reyes

last: Villalba

first: Joseph

last: Baur

first: George

last: Vlasuk

first: James

last: Ellis

first: David

last: Sinclair

first: Michel

last: Bernier

first: Rafael

last: de Cabo


title: Non-steroidal Anti-inflammatory Drugs Decrease E2F1 Expression and Inhibit Cell Growth in Ovarian Cancer Cells

status: Public on Jun 20 2014

first: Supriyo

last: De

summary: Epidemiological studies have shown that the regular use of non-steroidal anti-inflammatory (NSAIDs) drugs is associated with a reduced risk of various cancers. In addition, in vitro and experiments in mouse models have demonstrated that NSAIDs decrease tumor initiation and/or progression of several cancers. However, there are limited preclinical studies investigating the effects of NSAIDs in ovarian cancer. Here, we have studied the effects of two NSAIDs, diclofenac and indomethacin, in ovarian cancer cell lines and in a xenograft mouse model. Diclofenac and indomethacin treatment decreased cell growth by inducing cell cycle arrest and apoptosis. In addition, diclofenac and indomethacin reduced tumor volume in a xenograft model of ovarian cancer. To identify possible molecular pathways mediating the effects of NSAID treatment in ovarian cancer, we performed microarray analysis of ovarian cancer cells treated with indomethacin or diclofenac. Interestingly, several of the genes found downregulated following diclofenac or indomethacin treatment are transcriptional target genes of E2F1. E2F1 was downregulated at the mRNA and protein level upon treatment with diclofenac and indomethacin, and overexpression of E2F1 rescued cells from the growth inhibitory effects of diclofenac and indomethacin. In conclusion, NSAIDs diclofenac and indomethacin exert an anti-proliferative effect in ovarian cancer in vitro and in vivo and the effects of NSAIDs may be mediated, in part, by downregulation of E2F1.

overall_design: The serous ovarian adenocarcinoma cell lines HEY, OVCAR5 and UCI-101 were grown in culture then seeded in 60 mm dishes and treated for 24 hours with 300 mM diclofenac, indomethacin or no treatment (Control).  RNA was isolated and one sample from each group was labeled and hybridized to Illumina Sentrix bead arrays.

first: Blanca

last: Valle

first: Theresa

last: D’Souza

first: William

last: Wood

first: Robert

last: Wersto

first: Patrice

last: Morin


title: Erythropoietin promotes breast tumorigenesis through tumor initiating cell self-renewal

status: Public on May 31 2014

city: Chapel Hill

middle: Y.

last: Kim

state: NC

address: 450 West Dr.

department: Medicine and Genetics

institute: University of North Carolina at Chapel Hill

summary: The erythropoietin (EPO) hormone induces red blood cell production and its recombinant form is the most prescribed drug for the treatment of anemia, including that arising in cancer patients. Based on randomized trials showing that EPO administration to cancer patients result in a decreased survival, we investigated the impact of EPO modulation on tumorigenesis. Using genetically engineered mouse models of breast cancer we found that EPO promoted tumorigenesis by activating JAK/STAT signaling specifically in breast tumor initiating cells (TICs) and promoting their self-renewal.  Moreover, we define an active role for endogenous EPO in breast cancer progression and breast TIC self-renewal and demonstrate a potential application of EPO pathway inhibition in breast cancer therapy.

overall_design: reference x sample

first: Bing

last: Zhou

middle: Y


title: SIRT1 but not its increased expression is essential for lifespan extension in caloric restricted mice.

status: Public on Apr 25 2014

summary:
The SIRT1 deacetylase is one of the best-studied potential mediators of some of the anti-aging effects of calorie restriction (CR); but its role in CR-dependent lifespan extension has not been demonstrated. We previously found that mice lacking both copies of SIRT1 displayed a shorter median lifespan than wild type mice on an ad libitum diet. Here we report that median lifespan extension in CR heterozygote SIRT1+/- mice was identical (51%) to that observed in wild type mice but SIRT1+/- mice displayed a higher frequency of some certain pathologies. Although larger studies in different genetic backgrounds are necessary , these results provide strong initial evidence for the requirement of SIRT1 for the anti-aging effects of CR, but suggest that its high expression is not required for CR-induced lifespan extension.
Key words: SIRT1, caloric restriction, lifespan, anti-aging

overall_design: 2-5 month old male mice of 3 different genotypes (SIRT1+/+, SIRT1+/-, and SIRT1-/-) that had normal, reduced or no expression of SIRT1 were treated with either a 40% caloric restricted diet (CR) or an ad libitum diet (AL). 2-4 replicates of each experimental condition were used in the analysis.

first: Evi

middle: M

last: Mercken

first: Jia

last: Hu

first: Susan

last: Krzysik-Walker

first: Min

last: Wei

first: Ying

last: Li

first: Michael

middle: W

last: McBurney

first: Valter

last: Longo


title: Synergistic and independent actions of multiple terminal nucleotidyl transferases in 3’ tailing of small RNAs in Arabidopsis

status: Public on Jul 01 2015

city: Lincoln

first: Chi

last: Zhang

state: NE

address: 1901 Vine Street

institute: University of Nebraska - Lincoln

summary: Small silencing RNAs are key regulators of gene expression in both plants and animals. HEN1-mediated 3’ terminal 2’-O-methylation plays a crucial role in small RNA stability control. In the absence of HEN1, small RNAs are frequently uridylated (untemplated uridine addition) and trimmed, a phenomenon that is conserved across species. However, the underlying molecular mechanism is largely unknown. In this study, we have discovered UTP: RNA uridylyltransferase (URT1) acts redundantly with HESO1 in the uridylation of miRNAs, in addition to its role in oligo-adenylated mRNA uridylation. We show both common and distinctive features of URT1 and HESO1 in catalyzing miRNA uridylation.

overall_design: non coding RNA deep sequencing

first: Guodong

last: Ren

first: Bin

last: Yu


title: Runx3 regulated genes in splenic CD4+ dendritic cells

first: Yoram

last: Groner

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

overall_design: Refer to individual Series


title: Resveratrol improves adipose insulin signaling and reduces the inflammatory response in adipose tissue of rhesus monkeys on a high-fat, high-sugar diet.

status: Public on Apr 21 2014

first: Supriyo

last: De

summary: Obesity is associated with a chronic, low-grade, systemic inflammation that may contribute to the development of insulin resistance and type 2 diabetes. Resveratrol, a natural compound with anti-inflammatory properties, is shown to improve glucose tolerance and insulin sensitivity in obese mice and humans. Here we tested the effect of a 2-year resveratrol administration on the pro-inflammatory profile and insulin resistance caused by a high-fat, high-sugar (HFS) diet in white adipose tissue (WAT) from rhesus monkeys. Eighty mg/day of resveratrol for 12-month followed by 480 mg/day for the second year decreased adipocyte size, increased sirtuin 1 expression, decreased NF-kB activation and improved insulin sensitivity in visceral but not subcutaneous WAT from HFS-fed animals. These effects were reproduced in 3T3-L1 adipocytes cultured in media supplemented with serum from monkeys fed HFS +/- resveratrol diets. In conclusion, chronic administration of resveratrol exerts beneficial metabolic and inflammatory adaptations in visceral WAT from diet-induced obese monkeys.

overall_design: Twenty-four adult (7-13 years old) male rhesus monkeys (Macaca mulatta) were housed individually in standard nonhuman primate caging on a 12h light/12h dark cycle, room temperature (78 +/- 2 degrees F), and humidity at 60 +/- 20%. One pairing was maintained throughout the study; all other monkeys had extensive visual, auditory, and olfactory but limited tactile contact with monkeys housed in the same room. Monkeys received 2 meals per day at estimated ad libitum levels throughout the study. Water was always available ad libitum. Monkeys were monitored minimally 3 times daily by trained animal care staff.  During baseline assessments, all monkeys were maintained on a commercially available closed formula monkey chow.  After baseline assessment, four male rhesus monkeys remained on the healthy standard diet  (SD), 10 male rhesus monkeys were begun on a high fat/high sucrose (HFS) diet and 10 male rhesus monkeys were begun on a high fat/high sucrose (HFS) diet plus Resveratrol, 80mg/day.  After one year of dietary intervention, the amount of resveratrol was increased to 480mg/day for one additional year.  Tissues were then harvested for the array experiments.

first: Yolanda

last: Jimenez-Gomez

first: Julie

middle: A

last: Mattison

first: Kevin

last: Pearson

first: Alejandro

last: Martin-Montalvo

first: Hector

middle: H

last: Palacios

first: Alex

last: Sossong

last: Ward

first: Caitlin

last: Younts

first: Kaitlyn

last: Lewis

first: Joanne

last: Allard

first: Dan

first: Jonathan

middle: P

last: Belman

first: Maria

last: Malagon

first: Placido

last: Navas

first: Mitesh

last: Sanghvi

first: Ruin

last: Moaddel

first: Edward

last: Tilmont

first: Richard

last: Herbert

first: Christopher

last: Morrell

first: Josephine

last: Egan

first: Joseph

last: Baur

first: Luigi

last: Ferrucci

last: Bogan

first: Michel

last: Bernier

first: Rafael

last: de Cabo


title: ChIP-Sequencing of AL5 over-expressing Arabidopsis and its knock out mutant

status: Public on Apr 12 2014

city: Beijing

first: Wei

last: Wei

address: Chaoyang District, Beichen West Road, Campus #1, No.2

department: State Key Lab of Plant Genomics

institute: Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

summary: Plant homeodomain (PHD) finger proteins affect growth and development by regulating transcription and reading epigenetic modifications of histones, but their functions in abiotic stress responses remain largely unclear. Here we characterize seven Arabidopsis thaliana Alfin1-like PHD finger proteins (ALs) in the response to abiotic stresses. ALs localize to the nucleus and repress transcription. Except AL6, all the ALs bind to G-box element. Changes of the amino acids at positions 34 and 35 in AL6 cause the loss of G-box binding ability. Expression of the ALs responded differently to osmotic stress, salt, cold and abscisic acid treatments. AL5 was induced by multiple stresses, and AL5-overexpressing plants showed higher tolerance to salt, drought and freezing stress than Col-0. Also, al5 mutants showed reduced stress tolerance. ChIP-Seq assay helps find the direct targets of AL5.

overall_design: Polyclonal antibody of AL5 protein was used to perform ChIP experiment. Two samples were analyzed, AL5 OE sample and its knock out mutant. Data was analyzed as OE sample Vs mutant sample and help find targets of AL5 protein.

first: Yu-Qin

last: Zhang

first: Jian-Jun

last: Tao

first: Hao-Wei

last: Chen

first: Qing-Tian

last: Li

first: Wan-Ke

first: Biao

last: Ma

first: Qing

last: Lin

first: Jin-Song

first: Shou-Yi


title: Reconstructing and reprogramming the tumor propagating potential of glioblastoma stem-like cells: RNA-seq

status: Public on Apr 14 2014

city: Cambridge

first: Esther

last: Rheinbay

state: MA

address: 7 Cambridge Center

institute: Broad Institute

summary: Developmental fate decisions are dictated by master transcription factors (TFs) that interact with cis-regulatory elements to direct transcriptional programs. Certain malignant tumors may also depend on a cellular hierarchy reminiscent of normal development but superimposed on underlying genetic aberrations. In glioblastoma (GBM), a subset of stem-like tumor- propagating cells (TPCs) appears to drive tumor progression and underlie therapeutic resistance, yet remain poorly understood. Here, we identify a core set of neurodevelopmental TFs (POU3F2, SOX2, SALL2, OLIG2) essential for GBM propagation. These TFs coordinately bind and activate TPC-specific regulatory elements, and are sufficient to fully reprogram differentiated GBM cells to ‘induced’ TPCs that recapitulate the epigenetic landscape and phenotype of native TPCs. We reconstruct a TF network model that highlights critical interactions and identifies novel therapeutic targets for eliminating TPCs. Our study establishes the epigenetic basis of a developmental hierarchy in a devastating malignancy, provides detailed insight into the underlying gene regulatory programs, and suggests attendant therapeutic strategies.

overall_design:
3'end RNA sequencing

Submitter declares that the raw data are being deposited in dbGaP.

first: M

middle: L

last: Suvà

first: E

first: S

last: Gillespie

first: B

middle: E

last: Bernstein


title: Long Noncoding RNA MALAT1 Controls Cell Cycle Progression by Regulating the Expression of Oncogenic Transcription Factor B-MYB

status: Public on May 16 2014

first: Supriyo

last: De

summary:
The long noncoding MALAT1 RNA is upregulated in cancer tissues and its elevated expression is associated with hyper-proliferation, but the underlying mechanism is poorly understood. We demonstrate that MALAT1 levels are regulated during normal cell cycle progression. Genome-wide transcriptome analyses in normal human diploid fibroblasts reveal that MALAT1 modulates the expression of cell cycle genes, and is required for G1/S and mitotic progression. Depletion of MALAT1 leads to activation of p53 and its target genes. The cell cycle defects observed in MALAT1-depleted cells are sensitive to p53 levels, indicating that p53 is a major downstream mediator of MALAT1 activity. Furthermore, MALAT1-depleted cells display reduced expression of B-MYB (Mybl2), an oncogenic transcription factor involved in G2/M progression, due to altered binding of splicing factors on B-MYB pre-mRNA and aberrant alternative splicing. In human cells, MALAT1 promotes cellular proliferation by modulating the expression and/or premRNA processing of cell cycle-regulated transcription factors. These findings provide mechanistic insights on the role of MALAT1 in regulating cellular proliferation.
Keywords: MALAT1; MALAT-1, NEAT2, ncRNA; E2F, alternative splicing; pre-mRNA splicing factors

overall_design: WI38 cells (normal human diploid fibroblasts) were transfected with a control oligo (CTR) or antisense oligos to MALAT1 and RNA was isolated after 48 hr. Two antisense oligos were use for MALAT1 (AS-1 and AS-2).  Arrays were done for 3 sets of samples in triplicate (control, AS-1 and AS-2).

first: Vidisha

last: Tripathi

first: Zhen

last: Shen

first: Arindam

last: Chakraborty

first: Sumanprava

last: Giri

first: Susan

last: Freier

first: Xiaolin

last: Wu

first: Yongqing

first: Myriam

last: Gorospe

first: Supriya

middle: G

last: Prasanth

first: Ashish

last: Lal

first: Kannanganattu


title: MicroRNA-520c-3p targets eIF4GII and acts as a tumor suppressor in diffuse large B cell lymphoma

status: Public on Mar 19 2014

summary: Deregulation of the translational machinery is emerging as a critical contributor to lymphomagenesis. Various miRNA alterations have been identified in lymphoma, but their role in disrupting the cap-dependent translation regulation complex remains poorly understood. Here, we demonstrate the translation initiation factor, eIF4GII, as a direct target and major mediator of miR-520c-3p function through 3’UTR of eIF4GII mRNA. We established that elevated miR-520c-3p represses translation, initiates premature senescence and blocks cell proliferation in diffuse large B-cell lymphoma (DLBCL). Moreover, miR-520c-3p overexpression diminishes DLBCL cells colony formation and reduces tumor growth in a lymphoma xenograft mouse model. miR-520c-3p overexpressing cells display lowered eIF4GII levels. Consequently, downregulation of eIF4GII by siRNA induces cellular senescence, decreases cell proliferation and ability to form colonies. Our in vitro and in vivo findings we further validated in patient samples; DLBCL primary cells demonstrated low miR-520c-3p levels with reciprocally highly up-regulated eIF4GII protein expression. In contrast, normal donor B-cell lymphocytes had low levels of eIF4GII protein and elevated miR-520c-3p levels. Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of cap-dependent translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity. These findings may have implications for therapeutic interventions in patients with DLBCL.

overall_design: Human cervical carcinoma HeLa cells were transfected by using Oligofectamine (Invitrogen) with 50 nM small RNAs Pre-miR-520c-3p or control miRNA Pre-miR-Ctrl (Ambion). Linear sucrose gradient fractionation was performed on the transfected HeLa cells. Briefly, ~ 6 × 106 cells were incubated for 15 min with 100 mg/ml cycloheximide. Cells were lysed in cytoplasmic lysis buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl2, 0.3% IGEPAL CA-630, RNaseOUT (Invitrogen) and, protease inhibitor cocktail for 5 min on ice and centrifuged (10,000 × g, 10 min, 4°C). Cytoplasmic extracts were loaded onto the 10–50% sucrose gradients and after centrifugation (Beckman SW41, 35,000 rpm, 3 h, 4°C) the material was fractionated into eleven 1 ml aliquots labeled FR1-FR11 using a gradient fractionator (Brandel) and monitored by optical density measurement (A254).   Total RNA was extracted from three biological replicates of each fraction numbered 1-11 as well as unfractionated cell pellets using Trizol (Invitrogen).  Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips. Total RNA samples were labeled using Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX) 0.5g of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.  A total of 0.75ug of this biotin-labeled cRNA was hybridized at 58 degrees C for 16 hours to Illumina's Sentrix Human HT-12, v3 Expression BeadChips (Illumina, San Diego, CA). Each BeadChip targets more than 25,000 annotated genes with more than 48,000 probes. Probes were designed using the RefSeq (Build 36.2, Rel 22) and the UniGene (Build 199) databases with an average of 15-fold redundancy. The arrays were washed, blocked and the biotin labeled cRNA detected by staining with streptavidin-Cy3.  Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina. Data was extracted using the Illumina GenomeStudio software(v1.6.0). Any spots at or below the background were filtered out using an Illumina detection p-value of 0.02 and above. The natural log of all remaining scores was used to find the avg and std of each array and the z-score normalization was calculated and presented below.  Z-score = (raw value - avg)/std.

first: Krystyna

last: Mazan-Mamczarz

first: X

middle: F

last: Zhao

first: Bojie

last: Dai

first: James

last: Steinhardt

first: Kimberly

last: Berk

first: Ari

last: Landon

first: Mariola

last: Sadowska

first: Sharon

last: Corl

last: Zhang

first: Elin

last: Lehrmann

first: Kevin

last: Becker

first: Zhenqiu

last: Liu

first: Ronald

middle: B

last: Gartenhaus


title: A genetically engineered ovarian cancer mouse model based on fallopian tube transformation mimics human high-grade serous carcinoma development

status: Public on Apr 11 2014

first: Supriyo

last: De

summary:
Recent evidence suggests that ovarian high-grade serous carcinoma (HGSC) originates from the epithelium of the fallopian tube. However, most mouse models are based on the previous prevailing view that ovarian cancer develops from the transformation of the ovarian surface epithelium. Here, we report the extensive histological and molecular characterization of the mogp-TAg transgenic mouse, which expresses the SV40 large T-antigen (TAg) under the control of the mouse müllerian-specific Ovgp-1 promoter. Histologic analysis of the fallopian tubes of mogp-TAg mice identified a variety of neoplastic lesions analogous to those described as precursors to ovarian HGSC. We identified areas of normal appearing p53-positive epithelium that are similar to “p53 signatures” in the human fallopian tube. More advanced proliferative lesions with nuclear atypia and epithelial stratification were also identified that were morphologically and immunohistochemically reminiscent of human serous tubal intraepithelial carcinoma (STIC), a potential precursor of ovarian HGSC. Beside these noninvasive precursor lesions, we also identified invasive adenocarcinoma in the ovary of 56% of the mice. Microarray analysis revealed several genes differentially expressed between the fallopian tube of mogp-TAg and WT C57BL/6. One of these genes, Top2a, which encodes topoisomerase II-alpha, was shown by immunohistochemistry to be concurrently expressed with elevated p53 and specifically elevated in mouse STICs, but not in surrounding tissues. TOP2A protein was also found elevated in human STICs, low-grade, and high-grade serous carcinoma. The mouse model reported here displays a progression from normal tubal epithelium to invasive HGSC in the ovary, and therefore closely simulates the current emerging model of human ovarian HGSC pathogenesis. This mouse therefore has the potential to be a very useful new model for elucidating the mechanisms of serous ovarian tumorigenesis, as well as for developing novel approaches for the prevention, diagnosis, and therapy of this disease.
Keywords: transgenic mouse model, ovarian cancer, fallopian tube, intraepithelial carcinoma

overall_design: 6 mouse fallopian tubes (FT) were analyzed with experimental repeats; 3 wildtype C57BL6 mice (FT) and 3 transgenic mogp-TAg (FT), with one set of each at 7, 8 and 9 weeks of age.

first: Cheryl

last: Sherman-Baust

first: Elisabetta

last: Kuhn

first: Blanca

last: Valle

first: Ie-Ming

last: Shih

first: Robert

last: Kurman

first: Tian-Li

last: Wang

first: Tomokazu

last: Amano

first: Minoru

last: Ko

first: Ichiro

last: Miyoshi

first: Yoshihiko

last: Araki

first: Yongqing

first: Patrice

last: Morin


title: AMPK agonist AICAR improves cognition and motor coordination in young and aged mice

status: Public on Jan 17 2014

summary:
Normal aging can result in a decline of memory and muscle function.  Exercise may prevent or delay these changes. However, aging associated frailty may preclude physical activity. AMP-activated protein kinase (AMPK) is a transcriptional regulator important for muscle physiology. In the present study we investigated effects of AMPK agonist 5-aminoimidazole-4-carboxamide riboside (AICAR) on memory and motor function in young (2-month-old), aged (23-month-old) C57Bl/6 mice, and transgenic mice with muscle-specific mutated AMPK alpha-subunit (AMPK-DN). Mice were injected with AICAR (500 mg/kg) for 3 to 14 days and tested in the Morris water maze, rotarod and open field.  Improved water maze performance and motor function was observed, albeit at longer duration of administration, in aged (14 days AICAR) than young (3 days AICAR) mice. In the AMPKDN mice, the compound did not enhance behavior, providing support for a muscle mediated mechanism. In addition, microarray analysis of muscle and hippocampal tissue derived from aged mice treated with AICAR revealed changes in gene expression in both tissues, which correlated with behavioral effects in a dose-dependent manner. Pronounced up-regulation of mitochondrial genes in muscle was observed. In the hippocampus genes relevant to neuronal development and plasticity were enriched. Altogether, endurance related factors may mediate both muscle and brain health in aging, and could play a role in new therapeutic interventions.
Key words: Skeletal Muscle, Brain, Exercise, AMPK, Learning and Memory, Morris water maze

overall_design: 23-month old female C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were housed under standard conditions, 3 mice per cage, with food and water ad libitum.  Mice were injected intraperitoneally with 5-Aminoimidazole-4-carboxamide-1-a-D-ribofuranoside (AICAR, Toronto Research Chemicals Inc., Canada) dissolved in saline, 500 mg/kg/day for 3 (ACR3), 7 (ACR7) or 14 (ACR14) days or saline vehicle, for 3 (CTR) days.   Thirty-three days after the final injection animals were deeply anesthetized with isofluorane and perfused transcardially with 0.9% NaCl solution.  The gastrocnemius muscle and Hippocampus were quickly removed, frozen on dry ice and stored at −80 °C for RNA isolation and microarray analysis.

first: Tali

last: Kobilo

last: Zhang

first: Davide

last: Guerrieri

first: Sarah

last: Collica

first: Henriette

last: van Praag


title: Identification of subsets of urothelial carcinoma

status: Public on Feb 10 2014

city: Houston

first: Woonyoung

last: Choi

state: TX

address: 1515 Holcombe

institute: Md Anderson Cancer center

summary: Identification of bladder cancer subsets

overall_design: 142 primary bladder tumors including superficial and invasive tumors were arrayed. 73 invasive tumors out of 142 tumors were used for muscle invasive baldder cancer classification

first: I-ling

last: Lee

first: David

last: McConkey


title: Genome wide evidence of Austronesian-Bantu admixture and cultural reversion in a hunter-gatherer group of Madagascar

status: Public on Dec 19 2013

first: Luca

last: Pagani

state: UK

address: Henry Wellcome Building, Fitzwilliam Street

department: Biological Anthropology

institute: University of Cambridge

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

summary: Linguistic and cultural evidence suggests that Madagascar was the final point of two major dispersals of Austronesian and Bantu speaking populations. Today, Mikea are described as the last known Malagasy population reported to be still practising a hunter-gatherer life-style. It is unclear, however, whether Mikea descend from a remnant population that existed before the arrival of Austronesian and Bantu agriculturalists or whether it is only their life-style that separates them from the other contemporary populations of South Madagascar. In order to address these questions we have performed a genome wide analysis of >700,000 SNP markers on 21 Mikea, 24 Vezo and 24 Temoro individuals together with 50 individuals from Bajo and Lebbo populations from Indonesia. Our analyses of these data in the context of data available from other Southeast Asian and African populations reveal that all three Malagasy populations are derived from the same admixture event involving Austronesian and Bantu sources. In contrast to the fact that most of the vocabulary of the Malagasy speakers is derived from the Barito group of the Austronesian language family we observe that only one third of their genetic ancestry is related to the populations of Java-Kalimantan-Sulawesi area. Because no additional ancestry components distinctive to for the Mikea were found, it is likely that they have adopted their hunter-gatherer way of life by through cultural reversion.

overall_design: 69 samples were analysed with the Illumina platform OmniExpress BeadChips  and are described herein.

first: Denis

last: Pierron

first: Harilanto

last: Razafindrazaka

first: Francois-Xavier

last: Ricaut

first: Tiago

last: Antao

first: Mélanie

last: Capredon

first: Clement

last: Sambo

first: Chantal

last: Radimilahy

first: Jean-Aimé

last: Rakotoarisoa

first: Roger

last: Blench

first: Thierry

last: Letellier

first: Toomas

last: Kivisild


title: Age-related changes in microRNA levels in serum [miRNA]

status: Public on Dec 18 2013

first: Supriyo

last: De

summary: microRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression by targeting specific mRNAs. Altered expression of circulating miRNAs have been associated with age-related diseases including cancer and cardiovascular disease. Although we and others have found an age-dependent decrease in miRNA expression in peripheral blood mononuclear cells (PBMCs), little is known about the role of circulating miRNAs in human aging. Here, we examined miRNA expression in human serum from young (mean age 30 years) and old (mean age 64 years) individuals using next generation sequencing technology and real-time quantitative PCR. Of the miRNAs that we found to be present in serum, three were significantly decreased in 20 older individuals compared to 20 younger individuals: miR-151a-5p, miR-181a-5p and miR-1248. Consistent with our data in humans, these miRNAs are also present at lower levels in the serum of elderly rhesus monkeys. In humans, miR-1248 was found to regulate the expression of mRNAs involved in inflammatory pathways and miR-181a was found to correlate negatively with the pro-inflammatory cytokines IL-6 and TNFa and to correlate positively with the anti-inflammatory cytokines TGFb and IL-10.  These results suggest that circulating miRNAs may be a biological marker of aging and could also be important for regulating longevity. Identification of stable miRNA biomarkers in serum could have great potential as a noninvasive diagnostic tool as well as enhance our understanding of physiological changes that occur with age.

overall_design: Examination of microRNAs isolated from human serum from 11 young (mean age 30 yrs) and 11 old (mean age 64 yrs) individuals and from peripheral blood mononuclear cells from one young (30 yr) and one old (64 yr) individual.

first: Nicole

last: Hooten

first: Megan

last: Fitzpatrick

first: William

last: Wood 3rd

first: Ngozi

last: Ejiogu

first: Yongqing

last: Zhang

first: Julie

last: Mattison

first: Kevin

last: Becker

first: Alan

last: Zonderman

first: Michele

middle: K

last: Evans


title: Comparison of the long-term effects of calorie restriction without malnutrition on global gene expression profiles of rat and human skeletal muscle

status: Public on Aug 01 2013

sample_organism:
Homo sapiens
Rattus norvegicus

platform_organism:
Homo sapiens
Rattus norvegicus


title: GTL1 regulates ploidy-dependent cell growth through direct transcriptional repression of APC/CCS52A1 in Arabidopsis

city: Columbus

first: Kengo

last: Morohashi

state: OH

address: 1060 Carmack Rd

department: Molecular Genetics

institute: The Ohio State University

type: Genome binding/occupancy profiling by genome tiling array


title: Identification of distinct basal and luminal subsets of human bladder cancers with different sensitivities to frontline chemotherapy

first: Woonyoung

last: Choi


title: Comparison of the long-term effects of calorie restriction without malnutrition on global gene expression profiles of rat and human skeletal muscle [Rat]

summary: With the population of older and overweight individuals on the rise in the Western world, there is an ever greater need to slow the aging processes and reduce the burden of age-associated chronic disease that would significantly improve the quality of human life and reduce economic costs. Caloric restriction (CR), is the most robust and reproducible intervention known to delay aging and to improve healthspan and lifespan across species (1); however, whether this intervention can extend lifespan in humans is still unknown. Here we report that rats and humans exhibit similar responses to long-term CR at both the physiological and molecular levels. CR induced broad phenotypic similarities in both species such as reduced body weight, reduced fat mass and increased the ratio of muscle to fat. Likewise, CR evoked similar species-independent responses in the transcriptional profiles of skeletal muscle. This common signature consisted of three key pathways typically associated with improved health and survival: IGF-1/insulin signaling, mitochondrial biogenesis and inflammation. To our knowledge, these are the first results to demonstrate that long-term CR induces a similar transcriptional profile in two very divergent species, suggesting that such similarities may also translate to lifespan-extending effects in humans as is known to occur in rodents. These findings provide insight into the shared molecular mechanisms elicited by CR and highlight promising pathways for therapeutic targets to combat age-related diseases and promote longevity in humans.

overall_design: Male Fisher 344 rats (n=54) were randomly assigned to two groups at 2 months of age. One group was kept ad libitum (AL) fed throughout their lifespan while the calorie restriction (CR) group was progressively brought down to a 40% CR. All animals were fed a NIH-31 standard chow (Harlan Teklad, Indianapolis, IN, USA). Rats were singly housed in an environmentally controlled vivarium with unlimited access to water and a controlled photoperiod (12 hr. light;12 hr. dark). Body weights and food intake were recorded biweekly. All rats were maintained between 68-72°F according to animal protocols and NIH guidelines.   Total RNA was extracted from the vastus lateralis skeletal muscle using Trizol Reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions, n=5 from each group. Total RNA samples were biotin labeled and hybridized to RatRef-12 v1 Gene Expression beadchips (Illumina, San Diego, CA) following Illumina protocols. Arrays were washed and scanned using an Illumina BeadArray 500GX reader.  Microarray florescent signals were extracted using the Illumina GenomeStudio Gene Expression software(v1.6.0) and any spots at or below the background were filtered using an Illumina detection p-value of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated . Correlation analysis, sample clustering analysis and principal component analysis include all of probes are performed to identify/exclude any possible outliners. The resulting dataset was next analyzed with DIANE 6.0, a spreadsheet based microarray analysis program.  Gene set enrichment analysis use gene expression values or gene expression change values for all of the genes in the microarray. Parametric analysis of gene set enrichment (PAGE) was used [pubmed 20682848] for gene set analysis. Gene Sets include the MSIG database [Link], Gene Ontology Database [Link], GAD human disease and mouse phenotype gene sets [pubmed: 20092628] were used to explore functional level changes.

first: Evi

last: Mercken

first: Seth

last: Crosby

first: Lellean

last: JeBailey

first: Susan

last: Krzysik-Walker

first: Dennis

last: Villareal

first: Rafael

last: de Cabo

first: Luigi

last: Fontana


title: Comparison of the long-term effects of calorie restriction without malnutrition on global gene expression profiles of rat and human skeletal muscle [Human]

first: Supriyo

last: De

overall_design: Percutaneous biopsy specimens of vastus lateralis muscle were taken from 15 Human subjects from a group of middle-aged (58.7±7.4 yrs.), weight-stable members of the Calorie Restriction Society who have been practicing ~30% CR with adequate nutrition (at least 100% of RDI for each nutrient) for an average of 9.6 years and 10 age matched controls that were eating a typical Western diet. Total RNA was extracted from the skeletal muscle using Trizol Reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions, RNA quality and quantity was checked using an Agilent 2100 Bio-analyzer and the RNA 6000 nano-chips (Agilent Technologies, Palo Alto, CA).  Total RNA samples were biotin labeled and hybridized to Sentrix Human HT-12, v3 Expression BeadChips (Illumina, San Diego, CA), following Illumina protocols. Arrays were washed, stained and scanned using an Illumina BeadArray 500GX reader.  Microarray florescent signals were extracted using the Illumina GenomeStudio Gene Expression software(v1.6.0) and any spots at or below the background were filtered using an Illumina detection p-value of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated . Correlation analysis, sample clustering analysis and principal component analysis include all of probes are performed to identify/exclude any possible outliners. The resulting dataset was next analyzed with DIANE 6.0, a spreadsheet based microarray analysis program.  Gene set enrichment analysis use gene expression values or gene expression change values for all of the genes in the microarray. Parametric analysis of gene set enrichment (PAGE) was used [pubmed 20682848] for gene set analysis.


title: Gene expression analysis of HaCaTs after non-thermal plasma treatment

status: Public on Apr 26 2013

first: Anke

last: Schmidt

summary:
Investigation of gene expression in cultured human skin epithelial keratinocytes (HaCaT) following non-thermal plasma treatment for 60 s, compared to untreated control. Non-thermal atmospheric pressure plasma has recently gained attention in the field of biomedical and clinical applications. In the area of plasma medicine research one promising approach is to promote wound healing by stimulation of cells involved. To understand basic molecular and cellular mechanisms triggered by plasma treatment we investigated biological effects of an argon plasma jet (kinpen) on human epithelial skin cells. Consequently, whole-genome microarrays were used to analyze this interaction in detail and identified a statistically significant modification of 3,274 genes including 1,828 up- and 1,446 down-regulated genes. Particularly, plasma-treated cells are characterized by differential expression of a considerable number of genes involved in the response to stress. In this regard, we found a plasma-dependent regulation of oxidative stress answer and increased expression of enzymes of the antioxidative defense system (e.g. 91 oxidoreductases). Our results demonstrate that plasma induces cell reactions of stress-sensing but also of proliferative nature. Consistent with gene expression changes as well as Ingenuity Pathway Analysis prediction, we propose that stimulating doses of plasma may protect epithelial skin cells in wound healing by promoting proliferation and differentiation. In conclusion, gene expression profiling may become an important tool in identifying plasma-related changes of gene expression. Our results underline the enormous clinical potential of plasma as a biomedical tool for stimulation of epithelial skin cells
We investigated biological effects of an argon plasma jet on HaCaTs. Microarray were used to analyzed this interaction in detail. The transcripts analyzed in this study are further described in Schmidt et al. (2013): Non-thermal plasma treatment is associated with changes in transcriptome of human epithelial skin cells. Accepted in Journal Free Radical Research

overall_design: A study using total RNA recovered from at least 8 non-thermal plasma treated samples (300 ms*µl/cell) and untreated HaCaT controls.

first: Thomas

last: von Woedtke


title: Facilitates chromatin transcription complex is a marker and target of aggressive cancers with lower survival rates

status: Public on Mar 22 2013

city: Buffalo

first: Robert

middle: William

last: Leach

address: 701 Ellicott Street

department: Center for Computational Research

institute: Center of Excellence in Bioinformatics

summary: The Facilitates Chromatin Transcription (FACT) is involved in chromatin remodeling during transcription, replication and DNA repair and is considered to be an ubiquitously expressed complex that has no known associations with any disease. However, we discovered that FACT is expressed in very limited number of cells of adult mammalian organism, mostly presented by stem and undifferentiated cells. Here, we show that FACT is present in poorly differentiated aggressive cancers with an overall low survival rate. FACT acts as an "accelerator" of tumor transformation. It cannot drive transformation like an oncogene, but it increases the efficiency of oncogene-induced transformation. FACT expressing cancer cells cannot grow in the absence of FACT possibly due to the FACT involvement in selective chromatin remodeling of genes that stimulate proliferation, inhibit cell death and differentiation and that are induced in response to cell stress.  We propose that FACT is a marker of an aggressive cancer phenotype and Patients with FACT-positive tumors are noted for an overall poorer survival rate. FACT expression in primary tumors may be used as a predictor of metastatic disease. FACT may also be used as a target of anti-cancer therapy because tumor cells expressing FACT are dependent on FACT function, while normal cells either do not express FACT or are not sensitive to FACT inhibition.

overall_design: Examination of SSRP1 binding sites in HT1080 cells, 3 replicates of untreated cells and 3 replicates of curaxin-treated cells, each with 3 replicate input.

first: Henry

last: Garcia

first: Jeffrey

middle: C

last: Miecznikowski

first: Alfiya

last: Safina

first: Mairead

last: Commane

first: Anja

last: Ruusulehto

first: Sami

last: Kilpinen

first: Yan

last: Li

first: Seamus

last: Degan

first: Angela

middle: R

last: Omilian

first: Olga

last: Guryanova

first: Olympia

last: Papantonopoulou

first: Jianmin

last: Wang

first: Michael

last: Buck

first: Song

last: Liu

first: Carl

last: Morrison

first: Katerina

last: Gurova


title: Changes in mouse cognition and hippocampal gene expression observed in a mild physical- and blast-traumatic brain injury

status: Public on Mar 18 2013

first: Supriyo

last: De

institute: NIA-IRP, NIH

summary:
Warfare has long been associated with traumatic brain injury (TBI) in militarized zones. Common forms of TBI can be caused by a physical insult to the head-brain or by the effects of a high velocity blast shock wave generated by the detonation of an explosive device. While both forms of trauma are distinctly different regarding the mechanism of trauma induction, there are striking similarities in the cognitive and emotional status of survivors. Presently, proven effective therapeutics for the treatment of either form of TBI are unavailable. To be able to develop efficacious therapies, studies involving animal models of physical- and blast-TBI are required to identify possible novel or existing medicines that may be of value in the management of clinical events. We examined indices of cognition and anxiety-like behavior and the hippocampal gene transcriptome of mice subjected to both forms of TBI. We identified common behavioral deficits and gene expression regulations, in addition to unique injury-specific forms of gene regulation. Molecular pathways presented a pattern similar to that seen in gene expression. Interestingly, pathways connected to Alzheimer’s disease displayed a markedly different form of regulation depending on the type of TBI. While these data highlight similarities in behavioral outcomes after trauma, the divergence in hippocampal transcriptome observed between models suggests that, at the molecular level, the TBIs are quite different. These models may provide tools to help define therapeutic approaches for the treatment of physical- and blast-TBIs. Based upon observations of increasing numbers of personnel displaying TBI related emotional and behavioral changes in militarized zones, the development of efficacious therapies will become a national if not a global priority.
Keywords: Physical-traumatic brain injury; Blast-traumatic brain injury; Cognitive dysfunction; Gene expression; Molecular pathway(s); Neurodegeneration; Stem cells; Alzheimer’s disease

overall_design: A mild physical-TBI was induced using a concussive head trauma device described previously (Milman et al., 2005; Zohar et al., 2003). Briefly, mice were lightly anesthetized (Isoflurane) and placed under the weight-drop concussive head trauma instrument. The device consisted of a metal tube (inner diameter 13 mm), placed vertically over the mouse head. A metal weight (30 g) was dropped from the top of the tube (80 cm) and struck the skull at the right side temporal area between the corner of the eye and the ear. A sponge supported the head, allowing some antero-posterior motion without any rotational head movement at the moment of the impact.  Experimental conditions used to create a mild low-level blast-TBI and the subsequent model characterization, have been described in detail elsewhere (Rubovitch et al., 2011). In brief, mice were anaesthetized with a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg). Once the animals were fully anaesthetized they were placed at a defined distance from a detonation source, in this case 7 meters. Pressure sensors were used to measure the explosion shock wave pressure (PSI) generated by the detonation (Free-Field ICP® Blast Pressure Sensor; PCB Piezoelectronics, Depew, NY, USA, Model 137). At 7 meters from the source of the detonation, the animals were exposed to a maximum of a 2.5 PSI (17.2 kPa) pressure shock wave. Immediately after the induction of the injury, mice were placed back in their cages. Once the animals had recovered from the anesthesia, basic neurological assessments were undertaken to identify any acute neurological dysfunction. Only animals exhibiting no evidence of acute neurological damage post injury were subsequently used in further experiments. Sham treated mouse groups were treated identically; however, they were not exposed to physical- or blast-TBI.  Mouse hippocampus tissues were randomly selected from the larger library of samples generated from the behavioral experiments and the numbers utilized in the gene expression study were as follows: sham, n = 5: physical-TBI, n = 4; blast-TBI, n = 7.

first: David

last: Tweedie

first: Lital

last: Rachmany

first: Vardit

last: Rubovitch

first: Yongqing

last: Zhang

first: Kevin

last: Becker

first: Evelyn

last: Perez

first: Barry

middle: J

last: Hoffer

first: Chaim

last: Pick

first: Nigel

last: Greig


title: Metformin mild supplementation improves health and survival of mice

status: Public on Jul 31 2013

summary: Metformin, a commonly used drug prescribed to treat type-2 diabetes, has been found to extend health span and delay cancer incidence and progression. Here, we report that starting chronic treatment with low dose of metformin (0.1% w/w in diet) at one year of age extends health and lifespan in male mice, while a higher dose (1% w/w) was toxic. Treatment with low dose metformin mimicked some of the benefits of calorie restriction, such as improved physical performance, increased insulin sensitivity, and reduced LDL and cholesterol levels. At a molecular level, metformin increased AMP-activated protein kinase activity without attenuation of the mitochondrial electron transport chain activities. Metformin treatment resulted in lower chronic inflammation and increased antioxidant protection, suggesting that the ability of metformin to improve health of laboratory animals may stem from these factors. Our results support that metformin supplementation could be beneficial in extending health and lifespan in humans.

overall_design: Groups of one-year old male B6C57/J mice were maintained on ad libitum AIN-93G SD diet, or a 40% caloric restriction of the AIN-93G SD diet, or an ad libitum AIN-93G diet supplemented with 0.1% w/w metformin for the rest of their lives.  5 mice from each of these 3 groups were selected and RNA was extracted from both muscle and liver tissue using 1.0mm glass beads in a Precellys 24 Tissue Homogenizer  and Qiagen RNeasy Mini Kits for Fibrous Tissue according to manufacturer's specifications. Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips.  RNA samples were labeled using the Illumina TotalPrep RNA Amplification Kit.  In short, 0.5g of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.  Arrays were hybridized using a total of 0.75ug of biotin-labeled cRNA at 58 degrees C for 16 hours to Illumina's Sentrix MouseRef-8 v2 Expression BeadChips. Each BeadChip has ~24,000 well-annotated RefSeq transcripts with approximately 30-fold redundancy. The arrays were washed, blocked and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.  Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina and the data was extracted using the Illumina GenomeStudio software(v1.6.0).  Any spots at or below the background were filtered out using an Illumina detection p value of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented below.  Z-score = (raw value - avg)/std.

first: Alejandro

last: Martin-Montalvo

first: Evi

last: Mercken

first: Sarah

last: Mitchell

first: Hector

last: Palacios

first: Patricia

last: Mote

first: Morten

last: Scheibye-Knudsen

first: Ana

last: Gomes

first: Theresa

last: Ward

first: Robin

last: Minor

first: Marie-José

last: Blouin

first: Matthias

last: Schwab

last: Pollak

first: Vilhelm

last: Bohr

first: Donald

last: Ingram

last: Sinclair

first: Norman

middle: S

last: Wolf

first: Stephen

last: Spindler

first: Michel

last: Bernier

first: Rafael

last: De Cabo


title: Cancer-produced metabolites of 5-lipoxygenase induce tumor-evoked Bregs via peroxisome proliferator-activated receptor alpha

status: Public on Mar 13 2013

first: Supriyo

last: De

summary: Breast cancer cells facilitate distant metastasis through the induction of immunosuppressive regulatory B cells, designated tBregs. We report here that, to do this, breast cancer cells produce metabolites of the 5-lipoxygenase (5-LO) pathway such as leukotriene B4 (LTB4) to activate the proliferator-activated receptor alpha (PPARalpha) in B cells. Inactivation of LTB4 signaling or genetic deficiency of PPARalpha in B cells blocks the generation of tBregs and thereby abrogates lung metastasis in mice with established breast cancer. Thus, in addition to eliciting fatty acid oxidation and metabolic signals, PPARalpha initiates programs required for differentiation of tBregs. We propose that PPARalpha in B cells or/and tumor 5-LO pathways represents new targets for pharmacological control of tBreg-mediated cancer escape.

overall_design: Both 4T1 cells and a subset of them designated 4T1.2 are 6-thioguanine resistant cell lines selected from the 410.4 tumor without mutagen treatment.  When injected into BALB/c mice, they spontaneously produces highly metastatic tumors that can metastasize to the lung, liver, lymph nodes and brain while the primary tumor is growing in situ. These cell lines can be rendered non-metastatic (4T1-PE and 4T1.2-PE) by treatment with TARC-PE38 chemotoxin.  RNA was extracted from both of these cell lines as well as the TARC-PE38 treated cell lines using Qiagen RNeasy kits.  The total RNA from 3 replicates of each group was labeled using Illumina TotalPrep RNA Amplification Kits from Ambion.  0.75ug of  this biotin-labeled cRNA was hybridized at 58 degrees C for 16 hours to Illumina's Sentrix MouseRef-8 v1.1 Expression BeadChips, washed, and the biotin labeled cRNA was detected by staining with streptavidin-Cy3.  Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina. Data was extracted using the Illumina BeadStudio software(v3.1.7).  Any spots at or below the background were filtered out using an Illumina detection p value of 0.02 and above. The natural log of all remaining scores were used to find the avgerage and standard deviation of each array and the z-score normalization was calculated and presented below.  Z-score = (raw value - avg)/std.

first: Katarzyna

last: Wejksza

first: Catalina

last: Lee-Chang

first: Monica

last: Bodogai

first: Jessica

last: Bonzo

first: Frank

last: Gonzalez

first: Elin

last: Lehrmann

first: Kevin

last: Becker

first: Arya

last: Biragyn


title: Drivers of gene expression in cervical cancer

status: Public on Jan 31 2013

city: Oslo

first: Heidi

last: Lyng

address: Montebello

department: Department of Radiation Biology

country: Norway

institute: Oslo University Hospital

summary: Gene expression profiling of 82 patients with cervical cancer was performed. The expression data were correlated with copy number alterations of the same patients, as assessed with array CGH in a separate study, in order to identify drivers of cervical cancer carcinogenesis.

overall_design: In total, 82 patients with cervical cancer, stage 1b bulky through 4a, were included. Pretreatment biopsies were taken from the tumors and analyzed with Illumina gene expression BeadArrays (Human WG-6 v3).

first: H

first: M

last: Lando


title: Identification of eight candidate target genes of the prognsotic 3p loss in cervical cancer by integrative genomic profiling

status: Public on Feb 28 2013

summary:
We performed integrative gene dosage and expression profiling to identify candidate target genes of the prognostic 3p loss in cervical cancer.
Candidate target genes were proposed from the correlation between gene dosage and gene expression. Combined network, gene set, and gene ontology analysis of the gene expression profiles depicted interaction partners of the candidate targets and proposed biological processes that were affected by the 3p loss.

overall_design: Gene dosages from array CGH data (previously submiited to ArrayExpress) were correlated with Illumina gene expression data in the integrative patient cohort. The prognostic significance of the candidate target genes was validated based on the Illumina gene expression data of the validation cohort. Changes in gene expressions after knockdown of three candidate targets, RYBP, TMF1 and PSMD6, were studied by Illumina beadarrays in the cervical cancer cell lines HeLa, SiHa and CaSki.

first: Malin

first: Kristin

last: Snipstad

first: Trevor

last: Clancy

first: Mariska

last: Bierkens

first: Marit

last: Holden

first: Trond

last: Stokke

first: Kolbein

last: Sundfør

first: Ruth

last: Holm

first: Gunnar

last: Kristensen

first: Renske

middle: D

last: Steenbergen


title: beta-Arrestin Pathway-Selective G Protein-Coupled Receptor Agonists Engender Unique Biological Efficacy In Vivo

status: Public on Mar 07 2013

city: Baltimore

first: Supriyo

last: De

address: 251 Bayview Blvd

summary: Biased GPCR agonists are orthosteric ligands that possess pathway-selective efficacy, activating or inhibiting only a subset of the signaling repertoire of their cognate receptors.  In vitro, D-Trp12,Tyr34-bPTH(7-34) (PTH-{beta}arr), a biased agonist for the type 1 parathyroid hormone receptor, antagonizes receptor-G protein coupling but activates arrestin-dependent signaling.  In vivo, both PTH-{beta}arr and the conventional agonist PTH(1-34) stimulate anabolic bone formation. To understand how two PTH1R ligands with markedly different in vitro efficacy could elicit similar in vivo responses, we analyzed transcriptional profiles from calvarial bone of mice treated for 8 weeks with vehicle, PTH-{beta}arr or PTH(1-34).  Treatment of wild type mice with PTH-{beta}arr primarily affected pathways that promote expansion of the osteoblast pool, notably cell cycle regulation, cell survival and migration.  These responses were absent in beta-arrestin2 null mice, identifying them as downstream targets of beta-arrestin2-mediated signaling.  In contrast, PTH(1-34) primarily affected pathways classically associated with enhanced bone formation, including collagen synthesis and matrix mineralization.  PTH(1-34) actions were less dependent on beta-arrestin2, as might be expected of a ligand capable of G protein activation.  These results illustrate the uniqueness of biased agonism in vivo and demonstrate that functional selectivity can be exploited to change the quality of GPCR efficacy.

overall_design: The derivation of beta-arrestin2 null mice was previously described.  Mice were backcrossed for greater than nine generations onto a C57BL/6J background.  Either Human PTH(1–34) (40 µg/kg•d), bovine (D-Trp12, Tyr34)-PTH(7-34) (40 µg/kg•d) or PBS vehicle was administered to nine week-old male mice via intraperitoneal injection, daily for 8 weeks. Total cellular RNA was isolated from the calvaria of the treated mice and RNA was isolated according to the TRIzol manufacturer’s protocol. The total RNA was analyzed for concentration (ng/uL) and purity (ratios of 260/280 nm and 260/230 nm) using a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE). RNA integrity was analyzed using the Experion RNA HighSens Analysis Kit (Bio-Rad Laboratories, Inc., Hercules CA).  Hybridization targets were prepared from this total RNA using the Affymetrix one cycle kit (Cat#900431) which creates biotinylated cRNA.  The labeled cRNA samples were hybridized to GeneChip Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA) in the Duke Microarray Facility of the Duke Institute for Genome Sciences & Policy. Three replicates were performed for each condition. Raw hybridization intensity data were log transformed and normalized to yield Z-scores, which in turn were used to calculate a Z-ratio value for each gene with respect to the control geneset. The Z-ratio was calculated as the difference between the observed gene Z-scores for the experimental and the control comparisons divided by the standard deviation associated with the distribution of these differences.

first: Louis

middle: M

last: Luttrell

first: Shaoxi

last: Liao

first: Ling

last: Yuan

first: Diane

last: Gesty-Palmer


title: Expression data from exponentially proliferating ovarian cancer cell lines

status: Public on Jan 26 2013

city: Melbourne

first: Karen

middle: Elizabeth

last: Sheppard

state: Victoria

address: St Andrews Place

department: Research

country: Australia

institute: Perter MacCallum Cancer Centre

summary: We used microarrays to assess gene expression in proliferating ovarian cancer cell lines

overall_design: Ovarian cancer cell lines were plated and harvested when 50-80% confluent.    RNA was extracted and hybridized on Affymetrix microarrays. At least 3 biological repeats were performed for each cell line.

first: Jason

last: Ellul


title: Age-related changes in microRNA levels in serum [mRNA]

status: Public on Oct 09 2013

overall_design: HeLa cells were transfected with 20 nM biotinylated miRIDIAN  miR-1248 mimic (Thermo Scientific) or a negative control, biotinylated miRIDIAN C. elegans miR-67 (here named Con-miR) using Lipofectamine 2000.  Forty eight hours post transfection, total RNA was isolated using the mirVana kit (Invitrogen) according to manufacturer’hree replicate experiments were performed and a technical repeat was included for each sample.  Samples were analyzed using Illumina's Sentrix Human HT-12 ver4 Expression BeadChips (Illumina, San Diego, CA).

first: Nicole

middle: N

last: Hooten

first: Megan

last: Fitzpatrick

first: William

last: Wood

first: Ngozi

last: Ejiogu

first: Yongqing

last: Zhang

first: Julie

middle: A

last: Mattison

first: Kevin

last: Becker

first: Alan

last: Zonderman

first: Michele

last: Evans


title: Multiple oxygen tension environments reveal diverse patterns of transcriptional regulation in primary astrocytes

status: Public on Sep 20 2012

summary: The central nervous system normally functions at O2 levels which would be regarded as hypoxic by most other tissues.  However, most in vitro studies of neurons and astrocytes are conducted under hyperoxic conditions without consideration of O2-dependent cellular adaptation.  We analyzed the reactivity of astrocytes to 1, 4 and 9% O2 tensions compared to the cell culture standard of 20% O2, to investigate their ability to sense and translate this O2 information to transcriptional activity.  Variance of ambient O2 tension for rat astrocytes resulted in profound changes in ribosomal activity, cytoskeletal and energy-regulatory mechanisms and cytokine-related signaling.  Clustering of transcriptional regulation patterns revealed four distinct response pattern groups that directionally pivoted around the 4% O2 tension, or demonstrated coherent ascending/decreasing gene expression patterns in response to diverse oxygen tensions. Immune response and cell cycle/cancer-related signaling pathway transcriptomic subsets were significantly activated with increasing hypoxia, whilst hemostatic and cardiovascular signaling mechanisms were attenuated with increasing hypoxia.  Our data indicate that variant O2 tensions induce specific and physiologically-focused transcript regulation patterns that may underpin important physiological mechanisms that connect higher neurological activity to astrocytic function and ambient oxygen environments.  These strongly defined patterns demonstrate a strong bias for physiological transcript programs to pivot around the 4% O2 tension, while uni-modal programs that do not, appear more related to pathological actions.  The functional interaction of these transcriptional ‘programs’ may serve to regulate the dynamic vascular responsivity of the central nervous system during periods of stress or heightened activity.

overall_design: Cerebral cortices were removed from 6-8-day-old Wistar rats,  cortical astrocytes were isolated and these cells were maintained in a humidified incubator at 37°C (95% air; 5% CO2).   This resulted in a culture of cortical astrocytes, as confirmed by visual inspection the following day and later by glial fibrillary acidic protein immunohistochemistry.  Any cortical astrocyte culture that was not homogenous was disposed of and not used in this study.  Culture medium was exchanged every 7 days and cells were grown in culture for up to 14 days.  24hr prior to experimentation cells were transferred to an hypoxic workstation equilibrated with either 1%, 4%, 9% or 21% O2 and 5% CO2, with the remaining percentage gas being N2. Once cortical astrocytes had reached approximately 90% confluence (75cm2 flask) they were subjected to hypoxia as above, washed with PBS, removed from the flask base with 0.05% trypsin-EDTA (Gibco) and then gently centrifuged (500xg).  The cell pellet was re-suspended in PBS, centrifuged twice more to remove any traces of media,  then triturated in 8-10 volumes of RNAlater (Applied Biosystems), frozen and stored at -80oC.  Messenger RNA was amplified and labeled with biotin using the standard Illumina protocol (Illumina TotalPrep RNA Amplification Kit Ambion; Austin, TX, cat # IL1791), and hybridized to Illumina's Sentrix Rat Ref-12,v1 Expression BeadChips (Illumina, San Diego, CA).  Three replicates from each treatment group (1%, 21%, 4%, and 9% O2) were hybridized and the data was extracted using the Illumina BeadStudio software(v3.4).

first: Wayne

last: Chadwick

first: John

middle: P

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last: Zhou

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last: Wang

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last: Peers

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