title: A role for the nucleoporin Nup170p in chromatin structure and gene silencing [Agilent microarrays]

city: Nanjing

first: Yakun

last: Wan

address: Sipailou #2

country: China

institute: Southeast University

summary: Embedded in the nuclear envelope, nuclear pore complexes (NPCs) not only regulate nuclear transport, but also interface with both transcriptionally active euchromatin and largely silenced heterochromatin, as well as the boundaries between these regions. It is unclear what functional role NPCs play in establishing or maintaining these distinct chromatin domains. Here we report that the yeast NPC protein Nup170p interacts with specific regions of the genome containing ribosomal protein and subtelomeric genes. At these locations, Nup170p functions to establish normal nucleosome positioning and as a repressor of transcription. We show that the function of Nup170p in subtelomeric gene silencing is linked to its association with the RSC chromatin-remodeling complex and the silencing factor Sir4p, and that the binding of Nup170p and Sir4p to subtelomeric chromatin is cooperative and necessary for the association of telomeres with the nuclear envelope. Our results establish the NPC as an active participant in the formation of peripheral heterochromatin.

overall_design: Two-color microarrays were performed using Agilent whole-genome Saccharomyces cerevisiae arrays (Agilent) to determine gene expression profiles in the nucleoporin mutant strains nup157∆, nup170∆, and nup188∆.  Similar analyses were performed on PMET3-HA3-STH1 cells depleted of Sth1p for either  2 hrs (sth1-deplete 2hrs) or depleted of Sth1p for 8 hrs followed by reinduction of Sth1p for an additional 4 hrs (sth1-reinduction 4hr), as well as WT cells grown in the presence or absence of methionine.  All experiments were performed with duplicate experimental and duplicate technical replicates of each condition as previously described (Wan et al., 2009).

last: Van de Vosse

first: Diego

last: Lapetina

first: Wei-Ming

last: Chen

first: Jung-Hsien

last: Chiang

first: John

last: Aitchison

first: Richard

last: Wozniak


title: Regulation of Anopheles gambiae male accessory gland genes influences post-mating response in female

status: Public on Feb 08 2013

city: Perugia

first: tania

last: dottorini

address: via del Giochetto

department: Dept. Exp.Medicine and Bichem. Sci.

country: Italy

institute: University of Perugia

summary: In Drosophila, the accessory gland proteins (Acps) secreted from the male accessory glands (MAGs) and transferred along with sperm into the female reproductive tract have been implicated in triggering postmating behavioral changes, including refractoriness to subsequent mating and propensity to egg laying. Recently, Acps have been found also in Anopheles, suggesting similar functions. Understanding the mechanisms underlying transcriptional regulation of Acps and their functional role in modulating Anopheles postmating behavior may lead to the identification of novel vector control strategies to reduce mosquito populations. We identified heat-shock factor (HSF) binding sites within the Acp promoters of male Anopheles gambiae and discovered three distinct Hsf isoforms; one being significantly up-regulated in the MAGs after mating. Through genome-wide transcription analysis of Hsf-silenced males, we observed significant down-regulation in 50% of the Acp genes if compared to control males treated with a construct directed against an unrelated bacterial sequence. Treated males retained normal life span and reproductive behavior compared to control males. However, mated wild-type females showed a ∼46% reduction of egg deposition rate and a ∼23% reduction of hatching rate (∼58% combined reduction of progeny). Our results highlight an unsuspected role of HSF in regulating Acp transcription in A. gambiae and provide evidence that Acp down-regulation in males leads a significant reduction of progeny, thus opening new avenues toward the development of novel vector control strategies.

overall_design: Total RNA from 4-d-old dsLacZ-treated controls and HSF-silenced males was extracted using TRIzol reagent (Life Technologies), following protocols set according to manufacturer’s instructions. RNA preparation, labelling, hybridization and data analysis were performed by the Oxford Gene Technology (OGT) company. In particular, RNA was further cleaned up using the RNeasy Mini Kit (Qiagen) followed by ethanol precipitation. Sample passing the purity metrics (260/280 and 260/230 ratio) were considered for labeling and hybridization procedures. Labeling was done with cyanine 3 (dsHSF1 and dsHSF123) and cyanine 5 (control dsLacZ). Samples and controls were hybridized in triplicates to the Agilent Mosquito Gene expression arrays (4x44,000, Agilent Technologies, Santa Clara, CA, USA).  Data analysis was performed by OGT according to a standardized procedure, as implemented by the software applications Feature Extraction (version 9.5.3.1.), and Genespring GX (version 11.0). Expression data was first normalised using linear and loess normalization in Feature Extraction, then it was imported into Genespring where it was converted into normalised log ratios. The distribution of the data between and within the treatments was checked via boxplots and hierarchical clustering to confirm consistency. Intensity measures from spots flagged as being of poor quality were excluded. Then, the control probes were removed for the statistical analysis: t-tests were carried out for each condition with null hypothesis of log ratio being equal to zero. This was done on the normalised log ratios from the 3 technical replicates. Probes meeting a cut-off of p<0.05 with Benjamini-Hochberg multiple testing correction and with a fold change cutoff greater than 2 were considered statistically and biologically relevant and were used to generate lists of differentially expressed targets.

first: Tania

last: Dottorini

last: Persampieri

first: Pietro

last: Palladino

first: Dean

last: Baker

first: Roberta

last: Spaccapelo

first: Nicola

last: Senin

first: Andrea

last: Crisanti


title: Hox-C9 activates the intrinsic pathway of apoptosis and is associated with spontaneous regression in neuroblastoma [cell line_genex_44k]

status: Public on Mar 28 2013

city: Heidelberg

first: Thomas

last: Wolf

address: Im Neuenheimer Feld 580

country: Germany

institute: DKFZ

summary:
Single-color gene expression profiles from 2 neuroblastoma cell lines were generated using 44K oligonucleotide microarrays. To gain insights into the molecular processes occurring upon HOXC9 re-expression, we analyzed gene expression profiles of IMR-32 and SK-N-AS cells after Hox-C9 induction using microarrays. We used gene ontology (GO) annotations to find classes of genes that are significantly over-represented in gene sets that were either up- or downregulated after HOXC9 re-expression.
One-color gene expression analysis

overall_design: Single-color gene expression profiles from 2 neuroblastoma cell lines were generated using 44K oligonucleotide microarrays. Total RNA of HOXC9- and GFP-expressing SK-N-AS and IMR-32 cells was isolated at 0, 6, 12, 24, 48 and 96 h using Trizol. To determine global differences in the expression profiles of HOXC9- and GFP-induced SK-N-AS and IMR-32 cells, mean expression levels of each gene between HOXC9-induced and control (GFP) cells were compared. Gene Ontology Tree Machine (GOTM) was used to identify functional categories associated with the condition of the respective cell line.

first: Hayriye

last: Kocak

first: Matthias

last: Fischer

first: Benedikt

last: Brors

first: André

last: Oberthuer


title: Gene expression analysis of SCG sympathetic neurons with loss of Egr3

status: Public on Mar 22 2013

city: Chicago

first: David

last: Quach

state: Illinois

address: 303 E. Chicago Ave.

department: Pathology

country: USA

institute: Northwestern University

summary: The transcription factor Egr3 has been shown to have a cell autonomous role in sympathetic nervous system (SNS) development. We utilized microarray analysis to identify potential downstream target genes deregulated with loss of Egr3. Both conditions were in the null Bax background to prevent apoptosis and therefore mitigate identification of apoptosis related genes. Our analysis identified genes involved in biological processes that were expected such as SNS development and axonogenesis as well as those that were unexpected such as dendritogenesis and axon guidance. This led us to investigate whether Egr3 is important in these unexpected biological processes within sympathetic neurons.

overall_design: Total RNA was obtained from superior cervical ganglion (SCG) dissected from P0 mice with the genotype of Egr3+/+; Bax-/- or Egr3-/-; Bax-/-. Each genotype had 3 samples each.

first: Warren

last: Tourtellotte


title: Analyses of transcriptomic responses generated by hepatocarcinogens in a battery of liver-based in vitro models

status: Public on Feb 20 2013

city: Brussels

first: Tatyana

last: Doktorova

state: Belgium

address: Laarbeeklaan 103, building G

department: FAFY

country: Belgium

institute: VUB

summary: For assessing the cancer-causing potential for humans of a chemical compound, the conventional approach is the use of the 2-year rodent carcinogenicity bioassay, thus alternatives such as in vitro toxicogenomics are highly desired. In the present study, the transcriptomics responses following exposure to genotoxic (GTX) and non-genotoxic (NGTX) hepatocarcinogens and non-carcinogens (NC) in five liver-based in vitro models, namely conventional and epigenetically-stabilized cultures of primary rat hepatocytes, the human hepatoma-derived HepaRG and HepG2 cell lines and the human embryonic stem cell-derived hepatocyte-like cells hES-Heps are examined and compared.

sample_organism:
Homo sapiens
Rattus norvegicus

platform_organism:
Homo sapiens
Rattus norvegicus

overall_design: The global gene expression profiles of five commonly used liver-based in vitro systems are investigated, namely conventional cultures of primary rat hepatocytes (HepsC), Trichostatin A (TSA)-stabilized cultures of primary rat hepatocytes (HepsT), human embryonic stem cell-derived hepatocyte-like cells (hES-Hep), HepG2 and HepaRG cells. These models are exposed to 15 prototypical compounds which have been carefully chosen and belong to 3 toxic classes i.e. (i) genotoxic (GTX) (aflatoxin B1, AFB1; 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK; 2-nitrofluorene, 2NF; benzo(a)pyrene, BaP; cyclophosphamide, CYCLO), (ii) non-genotoxic (NGTX) (methapyrilene hydrochloride, MPH; piperonylbutoxide , PIPB; Wy-14643, WYE; phenobarbital sodium, SPB; 12-O-tetradecanoylphorbol-13-acetate, TPA) carcinogens and (iii) non-carcinogens (NC) (nifedipine, NIF; clonidine, CND; D-mannitol, MAN; tolbutamide, TOL; diclofenac sodium, SDF). For the gene- and pathway- based analysis, the raw microarray data was re-annotated to Ensembl version 61 genome and Gene Chip Robust Multi-array Average (GC-RMA) normalized (Dai et al. 2005). This resulted in 11,187 and 18,919 probe sets for the rat and the human models, respectively.  For the classification analysis, the raw microarray data was annotated using the chip description files from Affymetrix and then GC-RMA normalized. In order to eliminate batch effects, data from the different studies were half-z normalized. Half-z-normalization adjusts the logarithmic expression values of transcripts within a group such that each transcript has zero mean.

middle: Y

first: Reha

last: Yildirimman

first: Mathieu

last: Vinken

first: Mireia

last: Vilardell

first: Tamara

last: Vanhaecke

first: Hans

last: Gmuender

first: Roque

last: Bort

first: Gabriella

last: Brolen

first: Ruoya

last: Li

first: Christophe

last: Chesne

first: Joost

last: van Delft

first: Jos

last: Kleinjans

first: Jose

last: Castell

first: Petter

last: Bjorquist

first: Ralf

last: Herwig

first: Vera

last: Rogiers


title: Recurrent rearrangement during adaptive evolution in an interspecific yeast hybrid suggests a model for rapid introgression

status: Public on Apr 01 2013

city: Stanford

first: Barbara

last: Dunn

state: CA

department: Genetics

institute: Stanford University

summary:
Genome rearrangements are associated with eukaryotic evolutionary processes ranging from tumorigenesis to speciation.  Such rearrangements are especially common following the shock of interspecific hybridization, and some of these could be expected to have strong selective value.  To test this expectation we created de novo interspecific yeast hybrids between two diverged but largely syntenic species, Saccharomyces cerevisiae and S. uvarum, then experimentally evolved them under continuous ammonium limitation.  We discovered that a characteristic interspecific genome rearrangement arose multiple times in independently evolved populations.  We uncovered nine different breakpoints, all occurring in a ~1 kb region of chromosome 14, and all producing an “interspecific fusion junction” within the MEP2 gene coding sequence, such that the 5’ portion derives from S. cerevisiae and the 3’ portion derives from S. uvarum.  In most cases the rearrangements altered both chromosomes, resulting in what can be considered to be an introgression of a several-kb region of S. uvarum into an otherwise intact S. cerevisiae chromosome 14, while the S. uvarum chromosome 14 experienced an interspecific reciprocal translocation at the same breakpoint within MEP2, yielding a chimaeric chromosome.  The net result is the presence in the cell of two MEP2 fusion genes having identical breakpoints.  Given that MEP2 encodes for a high-affinity ammonium permease, that MEP2 fusion genes arise repeatedly under ammonium-limitation, and that three independent evolved isolates carrying MEP2 fusion genes are each more fit than their common ancestor, the novel MEP2 fusion genes are very likely adaptive under ammonium limitation.  Our results suggest that when homoploid hybrids form, the admixture of two genomes enables swift and otherwise unlikely evolutionary innovations.  Furthermore, the architecture of the MEP2 rearrangement suggests a model for rapid introgression, a phenomenon seen in numerous eukaryotic phyla, that does not invoke repeated backcrossing to one of the parental species.

Nomenclature: GSY86 TimeZeroInoculum = ancestral interspecific hybrid used to inoculate ammonium-limited chemostats into 3 replicate vessels A, B, C.
150gen = various single-clone isolates from 150 generations of evolutions from vessels A, B or C. 200gen = various isolates from 200 generations of evolutions from vessels A, B or C.

platform_organism:
Saccharomyces bayanus
Saccharomyces cerevisiae

overall_design: Logical Set: Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc.


title: Baseline serum anti-glycan antibodies of 113 subjects in a clinical trial of PROSTVAC-VF, a therapeutic cancer vaccine

city: Frederick

first: Christopher

last: Campbell

state: MD

address: 376 Boyles Street

institute: NCI

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

platform_organism:
Quercus alba
Gossypium hirsutum
Medicago sativa
Rhodotorula
Pandalus borealis
Gallus gallus
Homo sapiens
Bos taurus
Ovis aries
Mus musculus
Megathura crenulata
Bothrops moojeni
Frangula alnus
Senegalia senegal
Pachymenia carnosa
Dermestes lardarius

overall_design: Refer to individual Series


title: Gene expression profiling of rhesus macaques vaccinated with ALVAC-SIVgpe DNA + SIVgp120 protein subunit and unvaccinated controls after challenge with SIVmac251 - 11 wks post-infection

status: Public on Mar 01 2013

city: Atlanta

first: Gregory

middle: K

last: Tharp

state: GA

address: 954 Gatewood Dr

department: Developmental and Cognitive Neuroscience

institute: Yerkes National Primate Research Center

summary: The SIVmac251 macaque model has been used to evaluate the efficacy of vaccine for HIV. Exposure of macaques to a single high dose of SIVmac251 results in transmission of multiple viral variants, which contrasts the few HIV variants typically transmitted in humans. In here, we investigated whether the dose of SIVmac251 challenge affected vaccination efficacy and found that exposure of the immunized macaques to single high dose of SIVmac251 resulted in no vaccine efficacy, whereas exposure to a tenfold lower dose resulted in protection from SIVmac251 acquisition and protection from disease in animals that become infected. The dose of challenge did not affect the expression of inflammatory genes in the gut in acute infection, but at set point, a significant down regulation of interferon responsive genes and up regulation of genes involved in B and T-cell responses, was observed only in vaccinated animals exposed to a lower dose of SIVmac251. Accordingly, in these animals, we also found a significant correlation with vaccine induced T-cell responses and protection from disease. These data demonstrate that the evaluation of the efficacy of vaccine candidates for HIV relies on accurate modeling in macaques to better mimic HIV transmission to humans.

overall_design: A total of 34 RNA samples were hybridized on to Rhesus Affymetrix 3' Expression arrays. The study was composed of 9 vaccinated and 9 control animals subjected to a low dose challenge and 7 vaccinated and 9 control animals subjected to a high dose challenge

first: Monica

last: Vaccari

first: Brandon

middle: F

last: Keele

first: Steven

middle: E

last: Bosinger

first: Melvin

last: Doster

first: Zhong-Min

last: Ma

first: Justin

last: Pollara

first: Anna

last: Hryniewicz

first: Guido

last: Ferrari

first: Youngjun

last: Guan

first: Donald

last: Forthal

first: David

last: Venzon

first: Claudio

last: Fenizia

first: Tia

last: Morgan

last: Montefiori

first: Jeffrey

middle: D

last: Lifson

first: Chris

last: Miller

last: Silvestri

last: Felber

first: George

last: Pavlakis

first: James

last: Tartaglia

first: Genoveffa

last: Franchini


title: Hox-C9 activates the intrinsic pathway of apoptosis and is associated with spontaneous regression in neuroblastoma [tumor_genex_44k]

first: Thomas

last: Wolf

summary:
Single-color gene expression profiles from 649 neuroblastoma tumors were generated using 44K oligonucleotide microarrays. We aimed at determining the association of class I HOX gene expression patterns with prognostic markers in neuroblastoma.We investigated the functional consequences of HOXC9 re-expression on neuroblastoma growth and programmed cell death.
One-color gene expression analysis.

overall_design: Single-color gene expression profiles from 649 neuroblastoma tumors were generated using 44K oligonucleotide microarrays. Stages were classified according to the International Neuroblastoma Staging System. The expression patterns of the 39 class I HOX genes were analyzed in 649 neuroblastoma samples by microarrays, and the association with prognostic markers was determined.

first: Hayriye

last: Kocak

first: Matthias

last: Fischer

first: Benedikt

last: Brors

first: André

last: Oberthuer


title: Genome-wide Analysis Reveals SR Protein Cooperation and Competition in Regulated Splicing

city: Santa Cruz

first: Lily

last: Shiue

address: 1156 High Street

department: MCD Biology

institute: UCSC

summary: Global analyses of two prototypical SR proteins (SRFS1 and SFRS2 using splicing-sensitive arrays and CLIP-seq on mouse embryo fibroblasts carrying tet-repressible versions of the two genes.

overall_design: 4 biological replicate sets of SFRS1 or SFRS2 tet-repressible MEFs were grown for 5 days -/+ docycline. Total RNA was isolated and labeled targets hybridized to Affymetrix mouse splicing arrays.  This is the gene expression component of the study only.

first: Shatakshi

last: Pandit

first: Manuel

last: Ares

first: Xiang-Dong

last: Fu


title: Gene expression profiling of rhesus macaques vaccinated with ALVAC-SIVgpe DNA + SIVgp120 protein subunit and unvaccinated controls after challenge with SIVmac251

first: Gregory

last: Tharp


title: High resolution methylome analysis reveals widespread functional hypomethylation during adult human erythropoiesis

status: Public on Feb 05 2013

city: Bronx

first: Yiting

last: Yu

state: NY

address: 1300 Morris Park Ave

institute: Albert Einstein College of Medicine

type: Methylation profiling by genome tiling array

summary: Differentiation of hematopoietic stem cells to red cells requires coordinated expression of numerous erythroid genes. To understand the regulatory mechanisms governing lineage commitment we conducted a high resolution methylomic and transcriptomic analysis of six major stages of human erythroid differentiation. We observed widespread epigenetic differences between early and late stages of erythropoiesis with progressive loss of methylation being the dominant change during differentiation. Gene bodies, intergenic regions and CpG shores were preferentially demethylated during erythropoiesis. Epigenetic changes at transcription factor binding sites correlated significantly with changes in gene expression and were enriched for binding motifs for SCL, MYB, GATA and other factors not previously implicated in erythropoiesis. Demethylation at gene promoters was associated with increased expression of genes, while epigenetic changes at gene bodies correlated inversely with gene expression. Important gene networks encoding erythrocyte membrane proteins, surface receptors and heme synthesis proteins were found to be regulated by DNA methylation. Furthermore, integrative analysis enabled us to identify novel, potential regulatory areas of the genome that underwent differential methylation and correlated with corresponding changes in gene expression, as evident by epigenetic changes in a predicted PU.1 binding site in intron 1 of the GATA1 gene. This intronic site was found to be conserved across species and was validated to be a novel PU.1 binding site by qCHIP in erythroid cells. Altogether, our study provides a comprehensive analysis of methylomic and transcriptomic changes during erythroid differentiation and demonstrates that hypomethylation of the genome during erythroid differentiation has implications in modulating gene expression.

overall_design: We examined changes in methylation on days 0, 3,  7, 10, 13 and 16 of human erythroid culture. Two independent sets of experiments were performed.

first: Y

first: A

last: Verma


title: Identification of Biologically Relevant Enhancers in Human Erythroid Cells [Illumina BeadArray]

status: Public on Jan 28 2013

city: New Haven

first: Vince

last: Schulz

state: CT

address: 333 Cedar St. LMP 4085

department: Department of Pediatrics

institute: Yale University

summary: Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses and candidate enhancers identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the TSS of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or erythroid cell phenotypes. Candidate enhancers exhibited only moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a data set of erythroid-associated, biologically relevant, SNPs from the GWAS catalog of the NHGRI. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function, and provide insights into inherited and acquired hematologic disease.

overall_design: CD34+-selected stem and progenitor cells were expanded for three days in the absence of EPO, and total RNA was isolated from a portion of the cells.  The cells were further cultured in the presence of EPO, and RNA was isolated after cells differentiated into R3/R4 nucleated erythroid cells.  There were 3 replicates of CD34 cells and 9 replicates of R3/R4 erythroid cells.

first: Mack

last: Su

first: Laurie

middle: A

last: Steiner

first: Hannah

last: Bogardus

first: Tejaswini

last: Mishra

first: Vincent

middle: P

first: Ross

middle: C

last: Hardison

first: Patrick

middle: G

last: Gallagher


title: UVB irradiation does not directly induce detectable changes of DNA methylation in human keratinocytes

status: Public on Jan 30 2013

city: Duarte

first: Xiwei

last: Wu

address: 1500 E. Duarte Rd.

department: Computational and Quantitative Medicine

institute: City of Hope National Medical Center

summary: Unprotected exposure to UVB radiation from the sun and the resulting DNA damage are thought to be responsible for physiological changes in the skin and for a variety of skin cancers, including basal cell and squamous cell carcinoma and malignant melanoma. Although the mutagenic effects of UVB have been well documented and studied mechanistically, there is only limited information as to whether UV light may also be responsible for inducing epigenetic changes in the genome of exposed cells. DNA methylation is a stable epigenetic modification involved in gene control. To study the effects of UVB radiation on DNA methylation, we repeatedly exposed normal human keratinocytes to a UVB light source. After a recovery period, we analyzed global DNA methylation patterns in the irradiated and control cells using the methylated-CpG island recovery assay (MIRA) method in combination with high-resolution microarrays. Bioinformatics analysis revealed only a limited number of possible differences between UVB-exposed and control cells. However, these minor apparent changes could not be independently confirmed by bisulfite sequencing-based approaches. This study reveals that UVB irradiation of keratinocytes has no recognizable global effect on DNA methylation patterns and suggests that changes in DNA methylation, as observed in skin cancers, are not immediate consequences of human exposure to solar UVB irradiation.

overall_design: DNA methylation analysis of control and UVB irradiated keratinocytes. The MIRA assay was used for enrichment of methylated DNA. NimbleGen CpG island plus promoter arrats were used.

first: Christoph

last: Lahtz

first: Sang-In

last: Kim

first: Steven

last: Bates

first: Arthur

middle: X

last: Li

first: Gerd

last: Pfeifer


title: Widespread splicing changes in human brain development and aging

status: Public on Jan 22 2013

city: Moscow

first: Pavel

last: Mazin

address: Skolkovo Innovation Center, Building 3

department: Biomed

country: Russia

institute: Skolkovo Institute of Science and Technology

summary: Differences in transcript splicing are well documented among tissues and between sexes in humans and other organisms. Further, splicing changes in selected genes have been reported in human and mouse brain development. Using high-throughput RNA sequencing we characterized the transcriptome-wide splicing changes occurring over the lifespan in the human brain. We found that in two brain regions, prefrontal cortex and cerebellum, as many as 37% of all expressed protein-coding multi-exon genes undergo significant splicing changes between birth and 98 years of age. Approximately 40% of these changes take place in aging. We confirmed our findings using exon arrays and PCRs and detected identified splicing changes at the protein level. We further associated distinct patterns of age-related splicing changes with expression of the corresponding splicing factors. Thus, splicing plays an important role in shaping the human brain transcriptome in both development and aging.

overall_design: Human post-mortem brain samples from prefrontal cortex and cerebellum from 30 individuals with ages from birth to 100 years were collected. For each five individuals of similar age RNA, extracted from dissected tissue, was pooled resulting in 5 pooled samples. Obtained RNA was hybridized with Affymetrix Human Exon 1.0 ST arrays.

first: Jieyi

last: Xiong

first: Xiling

last: Liu

first: Zheng

last: Yan

first: Xiaoyu

last: Zhang

first: Liu

last: He

first: Mehmet

last: Somel

first: Yuan

last: Yuan

first: Yi-Ping

last: Chen

first: Yuhui

last: Hu

first: Ning

last: Fu

first: Zhibin

last: Ning

first: Rong

last: Zeng

first: Wei

first: Mikhail

last: Gelfand

first: Philipp

last: Khaitovich


title: The gene expression profiling array of mRNAs and LncRNA between portal vein tumor thrombus tissues and paired tumor tissues in hapatocellular carcinoma

status: Public on Jan 19 2013

city: shanghai

first: WEIXING

last: GUO

address: CHANGHAI ROAD

institute: ehbh

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

summary: Investigation of whole genome gene expression level changes of mRNAs and LncRNAs in portal vein tumor thrombus tissues and paired tumor tissues in hapatocellular carcinoma. The different expression genes were further analyzed.

overall_design:
The human mRNA and LncRNA microarray analysis of the 6 samples (3  portal vein tumor thrombus tissues and 3 paired tumor tissue) were completed. Total RNA from each sample was quantified using the NanoDrop ND-1000 and RNA integrity was assessed using standard denaturing agarose gel electrophoresis. Total RNA of each sample was used for labeling and array hybridization as the following steps: 1) Reverse transcription with by Invitrogen Superscript ds-cDNA synthesis kit; 2) ds-cDNA labeling with NimbleGen one-color DNA labeling kit; 3) Array hybridization using the NimbleGen Hybridization System and followed by washing with the NimbleGen wash buffer kit; 4) Array scanning using the Agilent Scanner G2505C.

Scanned images (TIFF format) were then imported into NimbleScan software (version 2.5) for grid alignment and expression data analysis. Expression data were normalized through quantile normalization and the Robust Multichip Average (RMA) algorithm included in the NimbleScan software. The Probe level (*_norm_RMA.pair) files and mRNA level (*_RMA.calls) files were generated after normalization. All mRNAs level files were imported into Agilent GeneSpring GX software (version 11.5.1) for further analysis.mRNAs that at least 3 out of 6 samples have values greater than or equal to lower cut-off: 50.0 (All Targets Value) were chosen for further data analysis. Differentially expressed mRNAs were identified through Volcano Plot filtering. Pathway analysis and GO analysis were applied to determine the roles of these differentially expressed mRNAs played in these biological pathways or GO terms. Finally, Hierarchical Clustering was performed to show the distinguishable mRNAs expression pattern among samples.

This array contains ~19000 RefSeq coding genes and ~21000 long non-coding RNAs (lncRNA), and each sample data table contains both mRNAs and LncRNAs data.

first: Wei-Xing

last: Guo

first: Yu-Qiang

last: Cheng


title: aCGH was used to identify the overall genomic imbalances of SCC of the vulva in 14 patients

status: Public on Jan 16 2013

city: oslo

first: Francesca

last: Micci

address: Ullernchausseen 70

department: Institute for medical informatics

country: Norway

institute: Oslo Universitetssykehus

type: Genome variation profiling by genome tiling array

summary: The imbalances scored by arrayCGH mapped to different chromosomes with losses being more common than gains. Frequent losses of large chromosomal segments were scored from 3p and 8p whereas same-sized gains were frequent from 3q and 8q. This is the first study of vulvar tumors using arrayCGH, and some frequent imbalances could be defined precisely.

overall_design: 14 patients we analyzed without replicates against a commercial common reference.

first: Sverre

last: Heim


title: Gene expression profiling of primary canine insulinomas and their metastases

status: Public on Jan 13 2013

city: Utrecht

first: Floryne

last: Buishand

address: Yalelaan 108

department: Clinical Sciences of Companion Animals

country: Netherlands

institute: Faculty of Veterinary Medicine, Utrecht University

summary: Insulinomas (INS) are rare pancreatic endocrine tumours (PETs), arising from tumoral beta-cells from the pancreatic islets of Langerhans. Canine INS are malignant in >95% of cases. This study aims to provide further insights on molecular pathways involved in INS pathogenesis and to identify prognostic genes. Canine specific cDNA microarray representing 20,313 genes was used to analyze expression profiles in a panel of ten primary INS and ten INS metastases. Eighty-four genes were found to be significantly down-regulated in the group of metastases in comparison to the group of primary INS. From the microarray analysis it also followed that primary INS, based on their gene expression profiles, can be divided into two groups, which differ in their malignancy potential. It was also demonstrated that canine INS have both endocrine and exocrine cell features. Furthermore, the present study has revealed pancreatic lipase and chymotrypsinogen B1 as candidate genes that can be followed up in prospective studies as prognostic markers for INS. Our findings add to the current knowledge of INS pathogenesis and tumor progression, which can be used for future therapeutic and prognostic strategies, for both dogs and humans.

overall_design: From 1993 to 2009, ten histologically confirmed primary insulinomas (INS) and their accompanying metastases were obtained from dogs that were presented at the Department of Clinical Sciences of Companion Animals of the Faculty of Veterinary Medicine of Utrecht University. Tumors were resected by experienced surgeons, and fragments (5-15 mm diameter) for RNA extraction were carefully removed from the central portion of the tumors to avoid contamination with normal adjacent tissue. Tissue specimens collected for RNA isolation were snap-frozen in liquid nitrogen immediately after surgical removal and stored at  70oC. Various clinical and pathological parameters were evaluated retrospectively for the dogs included in this study. Half of both the primary insulinoma samples, as well as half of the metastasis samples was labelled with Cy3, while the other half of both groups was labelled with Cy5. A common reference cRNA pool consisting of equal quantity of cRNA from all 20 tumor samples was used to hybridize against each sample on the array.

middle: O

first: Jolle

last: Kirpensteijn

first: Alexandra

last: Jaarsma

first: Ernst-Jan

middle: M

last: Speel

first: Marja

last: Kik

first: Jan

last: Mol


title: Increase in chromatin accessibility to MNase at sites of DNA replication

status: Public on Jan 17 2013

city: Seattle

first: Jairo

last: Rodriguez

state: WA

address: 1100 Fairview Ave N

department: Basic Sciences

institute: Fred Hutchinson Cancer Research Center

type: Genome binding/occupancy profiling by genome tiling array

summary: Faithful DNA replication is essential for normal cell division and differentiation. In eukaryotic cells, DNA replication takes place on chromatin. This poses the critical question as to how DNA replication can progress through chromatin, which is inhibitory to all DNA-dependent processes. Here, we have developed a novel genome-wide method to measure chromatin accessibility to micrococcal nuclease that is normalized for nucleosome density, NCAM (Normalized Chromatin Accessibility to MNase) assay.  This method enabled us to discover that chromatin accessibility increases specifically at and ahead of DNA replication forks in normal S phase and during replication stress.   We further found that Mec1, a key regulatory ATR-like kinase in the S-phase checkpoint, is required for both normal chromatin accessibility around replication forks and replication fork rate during replication stress.

overall_design: In this study we sought to analyze the chromatin structural changes that take place at sites of DNA replication. To this end, we obtained yeast cell populations synchronously undergoing DNA replication by α-factor G1 arrest and release. In order to analyze chromatin structure at sites of DNA replication we first mapped the genomic locations undergoing DNA replication to high-resolution, strand-specific microarrays tiling chromosomes III, VI, and XII, covering ~14% of the genome. Two complementary approaches were taken: (1) we mapped fork positions by chromatin immunoprecipitation (ChIP) of a FLAG-tagged DNA polymerase 1 (Pol1), a replication fork component and (2) we mapped the sites of active DNA synthesis by generating DNA copy number profiles. We then analyzed chromatin structure at the sites of DNA replication by Micrococcal nuclease mononucleosome mapping and by generating histone H3 density maps. We further generated a Normalized Chromatin Accessibility to MNase (NCAM) signal by normalizing MNase mononucleosome signal for histone H3 density. NCAM signal represents a measure of nucleosome accessibility to MNase that is normalized for nucleosome content. These three approaches allowed us to assess potential changes in nucleosome positioning, nucleosome density, and nucleosome accessibility during DNA replication. We searched for changes in chromatin structure by comparing, during S phase, regions undergoing DNA replication to those not yet replicated, and also by comparing the same region before replication (not replicated, G1 arrested control) and during DNA replication in S phase. We typically harvested S phase cells at 30 or 60 minutes after release from G1 arrest. Experimental conditions included releasing cells into rich media at 24°C or into rich media containing 200 mM Hydroxyurea (HU). Both conditions slowed down replication fork rate and made these experiments feasible. For each strain, samples for the different experiments (Chromatin for Pol1 and H3 ChIP, in vivo MNase digestion, and DNA for DNA copy number profiles) were harvested simultaneously for each time point. Therefore, comparisons between time points for each strain must be made using samples from the same replicate experiment. Our analysis included WT cells, as well as S phase checkpoint mutants (Δmec1 Δsml1 and mec1-100 Δsml1), as well as control strains (Δsml1).

first: Toshio

last: Tsukiyama


title: Expression profiling in miR-290 6dt1 mammary tumors

status: Public on Jan 15 2013

city: Bethesda

first: Natalie

last: Goldberger

address: 9000 Rockville Pike

summary: mRNA expression data from mammary tumors extracted 30 days after orthotopic injection of miR-290-expressing and negative control 6dt1 cells into female FVB/N mice.

overall_design: At 6 weeks of age, FVB/N female mice were orthotopically injected with 1.0x10E5 6dt1 cells, either expressing miR-290 or a negative control RNA construct. 4 negative control tumors and 3 miR-290 tumors were analyzed.

first: Renard

last: Walker

first: Chang-Hee

last: Kim

first: Scott

last: Winter

first: Kent

last: Hunter


title: Discovery of a splicing regulator required for cell cycle progression

status: Public on Jan 08 2013

city: Tampa

first: Elena

middle: S

last: Suvorova

address: 3720 Spectrum Blvd

institute: University of South Florida

summary:
Toxoplasma gondii temperature sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1).  The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature.  Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome.  Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators.
We describe transcriptome of the temperature sensitive mutant 12-109C6. Transcriptome studies demonstrated that gene expression is largely downregulated in the mutant at the restrictive temperature 40oC and is accompanied by a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs.

overall_design: RNA samples were isolated in duplicate from mutant 12-109C6 parasites grown for 24h at permissive (34oC) and 6h and 24h at non-permissive temperatures (40oC). Samples were hybridized to the Toxoplasma gondii Affymetrix microarray (ToxoGeneChip: http://ancillary.toxodb.org/docs/Array-Tutorial.html). Hybridization data was preprocessed with Robust Multi-array Average (RMA) and normalized using per chip and per gene median polishing and analyzed using the software package GeneSpring GX (Agilent Technologies, Santa Clara CA).

first: Michael

middle: W

last: White


title: Gene expression data of micro RNA from skeletal muscle sample of pig DL and Pi breed

city: Dummerstorf

first: Klaus

last: Wimmers

address: Wilhelm-Stahl-Allee 2

institute: Leibniz Institute for Farm Animal Biology (FBN)

summary:
Total of 675 porcine miRNA candidates datasets revealed by 100% aligment homologs and review literatures publication.
Probe-sets were screened, analysed for differential expression between different breeds (german landrace and pietrain) at different stages of development (63, 91 dpc and adult stage)

overall_design: microRNAs of skeletal muscle from german landrace and pietrain at 63, 91 dpc and adult stages were hybridized to Affymetrix GeneChip miRNA arrays.

first: Siriluck

last: Ponsuksili


title: miRNA expression analysis in AKXD RI strain mammary tumors

summary: Transcriptional profiling of miRNA levels in mammary tumors from 18 [PyMT x AKXD]F1 sublines. The PyMT strain was FVB/N-TgN(MMTV-PyVT)634Mul.

overall_design: Mammary tumor total RNA from mice representing one of 18 AKXD RI strains were pooled to represent each strain and expression profiled using a custom miRNA microarray.


title: Identification of DNA-replication domains in single S-phase cells: part 4 whole genome amplified single-cell DNA

city: Leuven

first: Niels

last: Van der Aa

address: Herestraat 49 bus 602

institute: KU Leuven

summary: DNA-replication is a key process in life and can lead to disease when disturbed. Cell-type specific early and late replication domains have been discovered throughout genomes by analysis of DNA from populations of cells. However, cell to cell differences and the association of these differences with other cellular processes remain largely elusive. Here we demonstrate for the first time that consecutive domains of early and late DNA-replication can be detected in single S-phase cells using array comparative genomic hybridization, providing proof-of-concept for a novel tool to investigate DNA-replication genome wide at the single-cell level. Furthermore, methods to profile the genome of a single cell for DNA-copy number aberrations are revolutionizing both basic genome research and clinical genetic diagnosis. It is thus important to apprehend not only technical but also biological reasons for false positive copy number detection. None of the current single-cell copy number calling methods distinguishes between a cell in G1-, S- or G2/M-phase of the cell cycle and mostly use cells isolated randomly from populations. We demonstrate that the oscillating pattern between early and late replicating loci instigates significantly more false-positive DNA copy-number calls in a diploid cell in S-phase cells than in G1- or G2/M-phase, depending on the specific aCGH-signal normalization method used. We propose a work-flow to detect single cells in S-phase and to correct for DNA-replication bias before copy number profiling.

overall_design: The genome of 7 S-phase, 2 M-phase and 2 G-phase single cells was amplified using the Sureplex amplification system. These test samples were hybridized comparatively to commercial male reference DNA.

first: Jiqiu

last: Cheng

first: Mateiu

last: Ligia

first: Masoud

last: Zamani Esteki

first: Parveen

last: Kumar

first: Evelyne

last: Vanneste

first: Yves

last: Moreau

first: Joris

middle: R

last: Vermeesch

first: Thierry

last: Voet


title: Identification of DNA-replication domains in single S-phase cells: part 3 multi-cell controls

overall_design: DNA was extracted from many cells from 1 S-phase, 1 G1-phase and 1 G2/Mphase enriched cell population from an EBV-transformed lymphoblastoid cell line from a male carrying a 750kb amplification on the short arm of chromosome 4. These test samples were hybridized comparatively to commercial male reference DNA.


title: Identification of DNA-replication domains in single S-phase cells: part 2 whole genome amplified single-cell

overall_design: The genome of 2 S-phase, 1 M-phase and 1 G-phase single cells were amplified using the Sureplex amplification system. These test samples were hybridized comparatively to commercial male reference DNA,


title: Developmental Changes in the Transcriptome of Human Cerebral Cortex Tissue: Long Noncoding RNA Transcripts

city: Detroit

first: Hui

last: JIA

state: MI

address: 275 E. Hancock

institute: wsu

summary: To examine the hypothesis that lncRNA expression varies with age, potentially paralleling 25 known developmental trends in synaptogenesis, myelination, and energetics, we quantified levels of nearly 6000 lncRNAs in 36 surgically resected human neocortical samples (primarily derived from temporal cortex) spanning infancy to adulthood.

overall_design: One-color (Alexa 555 green) microarray experiment was performed separately on each of the 36 surgically-resected human neocortical samples (primarily derived from temporal cortex) spanning infancy to adulthood. 8 probes per gene and 5 on-chip replicates for each probe.

first: Leonard

last: Lipovich

first: Adi

middle: L

last: Tarca

first: Juan

last: Cai

last: Jia

first: Harry

middle: T

last: Chugani

first: Kirstin

middle: N

last: Sterner

first: Lawrence

middle: I

last: Grossman

first: Monica

last: Uddin

first: Patrick

last: Hof

first: Chet

last: Sherwood

first: Christopher

last: Kuzawa

first: Morris

last: Goodman

first: Derek

last: Wildman


title: Identification of DNA-replication domains in single S-phase cells: part 1 whole genome amplified single-cell DNA

first: Niels

last: Van der Aa

overall_design: The genome of 4 S-phase, 3 M-phase and 4 G-phase single cells was amplified using the Sureplex amplification system. These test samples were hybridized comparatively to commercial male reference DNA,

first: Jiqiu

last: Cheng

first: Mateiu

last: Ligia

first: Masoud

last: Zamani Esteki

first: Parveen

last: Kumar

first: Evelyne

last: Vanneste

last: Moreau


title: Identification of DNA-replication domains in single S-phase cells: part 2 multi-cell controls

first: Yves

first: Joris

last: Vermeesch

first: Thierry

last: Voet


title: Mammary tumors

first: Natalie

last: Goldberger

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

overall_design: Refer to individual Series


title: Identification of DNA-replication domains in single S-phase cells: part 1 multi-cell controls

overall_design: DNA was extracted from many cells from 1 S-phase, 1 G1-phase and 1 G2/Mphase enriched cell population for two female normal control EBV-transformed lymphoblastoid cell lines. These test samples were hybridized comparatively to commercial male reference DNA.


title: Dosage dependent tumor suppression by histone deacetylase 1 and 2 by regulation of Myc collaborating genes and p53 function

status: Public on Jan 11 2013

city: Amsterdam

first: Jan-Hermen

last: Dannenberg

state: N-Holland

address: Plesmanlaan 121

institute: Antoni van Leeuwenhoek hospital

summary:
comparative genome hybridisation of Hdac1/2 cKO lymphomas and matched normal tissue
Histone deacetylases (HDACs) are epigenetic erasers of lysine-acetyl marks. Inhibition of HDACs using small molecule inhibitors (HDACi) is a potential strategy in the treatment of various diseases and is approved for treating hematological malignancies. Harnessing the therapeutic potential of HDACi requires knowledge of HDAC-function in vivo. Here, we generated a thymocyte-specific gradient of HDAC-activity using compound conditional knockout mice for Hdac1 and Hdac2. Unexpectedly, gradual loss of HDAC-activity engendered a dosage dependent accumulation of immature thymocytes and correlated with the incidence and latency of monoclonal lymphoblastic thymic lymphomas. Strikingly, complete ablation of Hdac1 and Hdac2 abrogated lymphomagenesis due to a block in early thymic development. Genomic, biochemical and functional analyses of pre-leukemic thymocytes and tumors revealed a critical role for Hdac1/Hdac2-governed HDAC-activity in regulating a p53-dependent barrier to constrain Myc-overexpressing thymocytes from progressing into lymphomas by regulating Myc-collaborating genes. One Myc-collaborating and p53-suppressing gene, Jdp2, was derepressed in an Hdac1/2-dependent manner and critical for the survival of Jdp2-overexpressing lymphoma cells.  Although reduced HDAC-activity facilitates oncogenic transformation in normal cells, resulting tumor cells remain highly dependent on HDAC-activity, indicating that a critical level of Hdac1 and Hdac2 governed HDAC-activity is required for tumor maintenance.

overall_design: genomic DNA from LckCre+;Hdac1/2 cKO lymphomas and matched normal genomic DNA was hybridized onto a Nimblegen whole genome array

first: Marinus

last: Heideman

first: Roel

middle: H

last: Wilting

first: Eva

last: Yanover

first: Arno

last: Velds

first: Johann

last: de Jong

first: Ron

last: Kerkhoven

first: Heinz

last: Jacobs

first: Lodewyk

middle: F

last: Wessels


title: Real-time quantitative PCR analysis of 88 microRNAs involved in human cell differentiation and development in involved skin of systemic scleroderma (SSc), localized scleroderma (LSc) and Keloid.

city: Kumamoto

first: Katsunari

last: Makino

state: Kumamoto

address: 1-1-1 Honjo

department: Dermatology and Plastic Surgery

country: Japan

institute: Kumamoto University

summary: Skin specimens were derived from involved skin of 3 systemic scleroderma (SSc), 3 localized scleroderma (LSc) and 3 keloid patients. These skin samples and 3 control skins were collected and fixed in formaldehyde immediately after resections. The microRNA (miRNA) isolation from human skin tissue was performed using miRNeasy FFPE kit (Qiagen). For PCR array, miRNAs were reverse-transcribed into first strand cDNA using RT2 miRNA First Strand Kit (SABiosciences). A mixture of equal amounts of miRNAs from 3 normal skins, 3 SSc, 3 LSc or 3 keloid were prepared, and miRNA expression profile in each disease in vivo was evaluated using RT2 Profiler PCR Array. The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added into 96-well RT2 miRNA PCR Array that includes primer pairs for 88 human miRNAs (SABiosciences).

overall_design: Skin specimens were derived from involved skin of 3 systemic scleroderma (SSc), 3 localized scleroderma (LSc) and 3 keloid patients. These skin samples and 3 control skins were collected and fixed in formaldehyde immediately after resections. Control donors were each matched with diseases for age, sex, and biopsy site.


title: miR-29c Targets Tropomyosin-1α and Is Down-regulated in Rat Remnant Kidneys and IgA Nephropathy Patients with Interstitial Fibrosis

city: Shanghai

first: Yi

last: Fang

address: No 180 Fenglin Road

department: nephrology

institute: Zhongshan Hospital

summary: MicroRNAs (miRNAs) are small regulatory RNA molecules that modulate the activity of specific mRNA targets and play important roles in a wide range of physiologic and pathologic processes.  We hypothesized that miRNAs might be involved in the progression of CKD.  In our previous studies we found chronic renal damages developed progressively in rats with 5/6 nephrectomy.  L-mimosine(L-Mim) intervention from wk 5 to wk 12 improved renal function and resulted in additional accumulation of HIF-1 α and -2 α at wk 12.  In the current study we found miR-29c was up-regulated in the L-Mim treated group compared with the control using Agilent miRNA microarrays.  Of the microRNAs and proteins that exhibited reciprocal changes in expression following the L-Mim treatment, miR-29c and tropomyosin 1α (TPM1), which is involved in stress fiber function, met the sequence criteria for microRNA-target interaction,  were later confirmed by 3'-untranslated region reporter analysis.  TGFβ1 treatment (3 ng/ml, 24 hours) decreased miR-29c expression and up-regulated protein expression of TPM1 in human renal epithelial cells. Overexpression of miR-29c significantly attenuated TGF-β1 induced increase in TPM1 in vitro. Moreover,  intrarenal expression of miR-29c was decreased in IgAN patients with moderate to severe tubulointerstital fibrosis (TIF), compared with IgAN patients without TIF, and intrarenal protein expression of TMP1 was significantly increased in IgAN patients with TIF.  The results suggest that intrarenal expression of miR-29c was down-regulated while its predicted target, TPM1 was up-regulated in the progression of CKD.  Short term stabilizing of HIF up-regulates miR-29c and attenuates CKD in the remnant kidney model.

overall_design: Four weeks after 5/6 nephrectomy, rats were treated with intraperitoneal injections of vehicle or L-mimosine (L-Mim, Calbiochem), a prolyl 4-hydroxylase inhibitor (PHD), at a dosage of 50 mg/kg every other day.  At the end of wk 12 after 5/6 nephrectomy, all rats (n=4, for each group) were sacrificed and blood samples were collected via cardiac puncture.  Renal tissue were harvested, one piece of which was fixed in neutral formalin and then embedded in paraffin.  The remaining kidney tissue was dissected in ice-cold PBS to, remove medulla, and then snap-frozen in liquid nitrogen before transferring to storage at -80°C until further analysis.

first: Xiaoqiang

last: Ding

first: Mingyu

last: Liang

first: Xiaofang

last: Yu

first: JiaMing

last: Zhu

first: Yong

last: Liu

first: Alison

middle: J

last: Kriegel


title: Identification of DNA-replication domains in single S-phase cells: part 3 whole genome amplified single-cell DNA

first: Niels

last: Van der Aa

overall_design: The genome of 1 S-phase, 2 M-phase and 1 G-phase single cells was amplified using the Sureplex amplification system. These test samples were hybridized comparatively to commercial male reference DNA,

first: Jiqiu

last: Cheng

first: Mateiu

last: Ligia

first: Masoud

last: Zamani Esteki

first: Parveen

last: Kumar

first: Evelyne

last: Vanneste

first: Yves

last: Moreau

first: Joris

last: Vermeesch

first: Thierry

last: Voet


title: Identification of DNA-replication domains in single S-phase cells


title: Interaction effects of knockdown of PML and TNFalpha treatment in HUVEC cells

status: Public on Jan 10 2013

city: Cleveland

first: Hung-Ying

last: Kao

state: OH

address: 2123 Adelbert Road, RT400-L

department: Biochemistry

institute: Case Western Reserve University

summary: To study the interaction effects between promyelocytic leukemia (PML) gene knockdown and tumor necrosis factor-alpha (TNFalpha) signaling in human umbilical vein endothelial cells (HUVECs), we transfected control or two independent PML siRNAs into HUVEC cells without or with 20 ng/mL TNFalpha treatment for 20 h. The total RNA was extracted for gene expression microarray analyses.

overall_design: The experiment is a 3x2 two-factor design. One factor is siRNA and it has three levels (siCtrl, siPML1, siPML2). siCtrl is RISC-inducing non-targeting control siRNA. siPML1 and siPML2 designate two independent siRNAs against the PML gene. Two independent siRNAs were used to eliminate the off-target effects of siRNA. The other factor is TNFalpha treatment (20 ng/mL for 20 h) and it has two levels: Untreated (U) or Treated (T). Each sample has technical duplicates.

first: Xiwen


title: Dysregulation of REST-regulated coding and non-coding RNAs in a cellular model of Huntington’s disease

status: Public on Jan 03 2013

city: London

first: Caroline

middle: Emma

last: Johnston

address: 125 Coldharbour Lane

country: United Kingdom

institute: Kings College London

summary: Abstract - Huntingtin (Htt) protein interacts with many transcriptional regulators, with widespread disruption to the transcriptome in Huntington’s disease (HD) brought about by altered interactions with the mutant Htt (muHtt) protein. Repressor Element-1 Silencing Transcription Factor (REST) is a repressor whose association with Htt in the cytoplasm is disrupted in HD, leading to increased nuclear REST and concomitant repression of several neuronal-specific genes, including brain-derived neurotrophic factor (Bdnf). Here, we explored a wide set of HD dysregulated genes to identify direct REST targets whose expression is altered in a cellular model of HD but that can be rescued by knock-down of REST activity.  We found many direct REST target genes encoding proteins important for nervous system development, including a cohort involved in synaptic transmission, at least two of which can be rescued at the protein level by REST knock-down.  We also identified several microRNAs (miRNAs) whose aberrant repression is directly mediated by REST, including miR-137, which has not previously been shown to be a direct REST target in mouse.  These data provide evidence of the contribution of inappropriate REST-mediated transcriptional repression to the widespread changes in coding and non-coding gene expression in a cellular model of HD that may affect normal neuronal function and survival.

overall_design: Biological replicates total RNA samples from 2 cell types (Hdh7/7 and Hdh109/109) cells were used. Expression in 'HD' cells (Hdh109/109) was compared with expression in wildtype (Hdh7/7) cells.

first: Chiara

last: Soldati

first: Angela

last: Bithell

first: Kee-Yew

last: Wong

first: Lawrence

last: Stanton

first: Noel

last: Buckley


title: Detection of mutant NPM1 mRNA in acute myeloid leukemia (AML) using custom gene expression arrays [Validation cohort]

status: Public on Jan 01 2013

city: Rotterdam

first: Martin

middle: van

last: Vliet

address: Lichtenauerlaan 40

department: Bioinformatics

institute: SkylineDx

summary: Abstract Mutations in the gene encoding nucleophosmin (NPM1) carry prognostic value for patients with acute myeloid leukemia (AML). Various techniques are currently being used to detect these mutations in routine molecular diagnostics. Incorporation of accurate NPM1 mutation detection on a gene expression platform would enable simultaneous detection with various other expression biomarkers. Here we present an array based mutation detection using custom probes for NPM1 WT mRNA and NPM1 type A, B, and D mutant mRNA. This method was 100% accurate on a training cohort of 505 newly diagnosed unselected AML cases. Validation on an independent cohort of 143 normal karyotype AML cases revealed no false negative results, and one false positive (sensitivity 100.0%, and specificity 98.7%). Based on this, we conclude that this method provides a reliable method for NPM1 mutation detection. The method can be applied to other genes/mutations as long as the mutant alleles are sufficiently high expressed.

overall_design: Validation cohort of 143 AML cases analyzed using the AMLprofiler

last: van Vliet

first: Erik

last: van Beers


title: Feed restriction, but not L-carnitine infusion, affects the liver transcriptome with an evident and inhibition of energy production and sterol synthesis and increase of gluconeogenesis  in mid-lactating dairy cows

status: Public on Jan 07 2013

city: Corvallis

first: Massimo

last: Bionaz

state: OR

address: 316A Withycombe Hall

department: Animal and Rangeland Sciences

institute: Oregon State University

summary: Feed restriction and L-carnitine infusion are known to affect the liver metabolism of dairy cows. In the present experiment the effects on liver transcriptome of feed restriction and L-carnitine abomasal infusion and the interaction of the two in mid-lactation Holstein dairy cows was assessed. Data clearly indicated a lack of transcriptomics effect by L-carnitine but a strong effect due to feed restriction. The functional analysis identified a overall reduction of cholesterol synthesis and oxidative phosphorylation and data suggested an increase flux toward gluconeogenesis and fatty acid oxidation.

overall_design: The liver biopsy was performed after 14 days of treatment in 8 Holstein dairy cows in a 2 x 2 factorial arrangment with 5 days washout between treatments. A dye-swap reference design (reference =  mixture of RNA from several bovine tissues) was used.

first: Haji

last: Akbar

first: David

middle: B

last: Carlson

first: Sandra

last: Rodriguez-Zas

first: Robin

middle: E

last: Everts

first: Harris

middle: A

last: Lewin

first: James

middle: K

last: Drackley

first: Juan

last: Loor


title: affy_med_2011_09-Analysis of Medicago truncatula root transcriptome in response to mycorrhization by Glomus intraradices under phosphate and nitrogen limitation

city: evry

first: stephanie

last: huguet

address: 2 rue gaston cremieux

country: France

institute: inra

summary: affy_med_2011_09: In natural ecosystems most vascular plants develop symbiosis with arbuscular mycorrhizal (AM) fungi which help them acquire nutrients such as phosphorus (P) and nitrogen (N). P has long been known to control AM symbiosis which takes place only when P is limiting. For N, however, its role in controlling mycorrhization is less clear. We have chosen the model plant Medicago truncatula to analyze the impact of P limitation and both P and N limitation on Medicago root transcriptome in response to the AM fungus Rhizophagus irregularis (formerly Glomus intraradices (BEG141)). These analyses may help us uncover signaling events involved in the interaction between these symbionts as well as genes encoding transporters potentially important for nutrient exchanges in these conditions. --We will compare the root transcriptome of Medicago truncatula plants inoculated with Rhizophagus irregularis to that of non-inoculated plants grown under P limitation (or both P and N limitation) after 4 weeks of culture

platform_organism:
Sinorhizobium meliloti
Medicago sativa
Medicago truncatula

overall_design: 12 arrays - Medicago; wt vs mutant comparison

first: Hoai-Nam

last: Truong

first: Stephanie

last: Huguet

first: Sandrine

last: Balzergue

first: Marie-Laure

last: Martin-Magniette


title: Comparative Transcriptomic Analysis of Salmonella in Desiccation

city: Bedford Park

first: Haiping

last: Li

state: IL

address: 6502 S. Archer Rd

department: CFSAN/OFS/Moffett Center

institute: Food and Drug Administration

summary: To explore the mechanisms of Salmonella desiccation resistance, we studied the transcriptomic responses in Salmonella Tennessee (Tennessee), using S. Typhimurium LT2 (LT2), a strain weakly resistant to desiccation, as a reference strain. In response to 2 h air-drying at 11% equilibrated relative humidity, approximately one-fourth of the ORFs in the Tennessee genome and one-fifth in LT2 were differentially expressed (> 2-fold). Among all differentially expressed functional groups (>5-fold) in both strains, the expression fold change associated with fatty acid metabolism was the highest, and constituted 51 and 35% of the total expression fold change in Tennessee and LT2, respectively. Tennessee showed greater changes in expression of genes associated with stress response and envelope modification than LT2, while showing lesser changes in protein biosynthesis expression. Expression of flagella genes was significantly more inhibited in stationary phase cells of Tennessee than LT2 both before and after desiccation.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2

sample_organism: Salmonella enterica subsp. enterica serovar Tennessee

overall_design: Salmonella Typhimurium LT2 ATCC strain 19585 (LT2) and Tennessee strain K4643 (Tennessee) were grown in TSB at 37 ºC with shaking for 20 h. Cells (~8 log CFU) for each strain were added on the discs and either stored at -80ºC directly, as control samples; or kept at 11% ERH at 25°C for 2 h and then transferred into -80 ºC, as desiccation treated samples. Three independent experiments were carried out for each condition.


title: Detection of mutant NPM1 mRNA in acute myeloid leukemia (AML) using custom gene expression arrays [Training cohort]

last: Vliet

overall_design: Training cohort of 505 AML cases analyzed using the AMLprofiler


title: Synergistic effects of temperature and pCO2 on growth, development and gene expression of Strongylocentrotus purpuratus larvae

city: Santa Barbara

first: Tyler

middle: G

last: Evans

state: California

address: Marine Science Institute

department: Ecology, Evolution and Marine Biology

institute: University of California Santa Barbara

summary: Transcriptional profiling of purple sea urchin (Strongylocentrotus purpuratus) larvae cultured under four different seawater conditions: (i)13°C/400 µatm pCO2, (ii)13°C/1100 µatm pCO2, (iii)18°C/400 µatm pCO2 (iv)18°C/1100 µatm pCO2. The goal was to determine the effects of temperature and CO2, both important climate change variables, on gene expression

overall_design: Larvae were cultured under four different seawater conditions (ii)13°C/1100 µatm pCO2, (iii)18°C/400 µatm pCO2 (iv)18°C/1100 µatm pCO2, (i)13°C/400 µatm each with three replicate cultures for each condition and sampled at pluteus stage


title: The AmyR regulon in Erwinia amylovora

city: Vernon Hills

first: DONGPING

last: WANG

address: 777 Forest Edge Dr

department: Amlan

institute: OilDri Corporation of America

summary: Microarray analysis was used to identify genes that were controlled by AmyR in minimal and on immature pear fruits. Consistent with amylovoran production, an inverse correlation was observed between amyR expression and the expression level of amylovoran biosynthetic genes in liquid media. Interestingly, over-expression of AmyR suppressed the expression of type III secretion system genes including hrpA, hrpN and dspEF after pear fruit infection. Consistent with levan production and swarming motility, levasucrase and flagellar genes were both down-regulated both in the amyR mutant and over-expression strains in liquid media. Together, our results suggest that AmyR plays an important role in regulating bacterial exopolysaccharide production and virulence in E. amylovora.

overall_design: A total of 14 samples were analyzed in two conditions: For the in vitro condition (MBMA medium + 1% sorbitol), Erwinia amylovora wild type strain (2 replicates); E. amylovora amyR mutant strain (3 replicates); E. amylovora amyR over-expression strain (3 replicates); For the in vivo condition (on wounded immature pear fruits), Erwinia amylovora wild type strain (2 replicates); E. amylovora amyR mutant strain (2 replicates); E. amylovora amyR over-expression strain (2 replicates).

first: Dongping

last: Wang

first: Youfu

last: Zhao


title: Effect of AP2IX-9 overexpression on CTG transcriptome upon alkaline treatment

first: Olivier

last: Lucas

state: FL

address: 3720 spectrum blvd

summary: Overexpression of DDHA-AP2IX-9 under a tubulin promoter in type III CTG strain. Differential expression analysis under addition of shield-1 and comparison to the non-bradyzoite induced parental culture revealed a global downregulation of the bradyzoite gene expression program including canonical markers like BAG1.

overall_design: 11 samples total. Each conditions in duplicates except wildtype CTG under tachyzoite condition in triplicates.

first: Michael

last: White


title: The frequency of antigen-specific T cells is regulated by self-specific non-clonal neighbors

city: Baltimore

first: Nevil

middle: John

last: Singh

address: 685 W Baltimore Street

department: Microbiology & Immunology

institute: UMB

summary: In order to repeatedly mount successful immune responses against a wide range of pathogens, vertebrates must maintain sufficient numbers of diversely specific T cells over a long time. The cellular principles ensuring this are not clear. We show that the density of an antigen-specific T cell population is regulated by neighboring T cells not specific for the same antigen. Their activity is nevertheless T cell specific, due to the shared recognition of a self-peptide. Such regulation was sufficient to prevent autoimmune arthritis even in a lymphopenic model system without other regulatory T cells. This homeostatic strategy, based on the specific recognition of unique sub-threshold ligands, avoids excessive loss of bystander specificities during multiple immune responses, preserving the valuable diversity of the T cell repertoire.

overall_design: Ten of the centi-pools selected from screening that showed very high or no "deletor" activity against 5C.C7 T cells were analyzed for gene expression patterns that may correlate with their activity. The reference sample consisted of RNA from Mouse T cells (Naïve, Memory, and Tolerant) that were mixed in equal proportion.


title: Insecticide resistance mechanisms Myzus persicae: Genotypes Insecticide vs. Genotypes Control

city: Valdivia

first: Andrea

last: Silva

state: Valdivia

address: Isla teja s/n

department: Ciencias Ambientales y Evolutivas

country: Chile

institute: Universidad Austral de Chile

summary: Transcripcional responses in three genotypes of Muzus persicae, each exhibiting different resistance mechanisms, in response to an anti.cholinesterase insecticide

overall_design: Two-condition experiment in threee different genotypes: Insecticide vs. acetone plus water. Genotypes S (exhibiting no resistance mechanism) Biological replicates: 3. Genotypes RS (resistant through a kdr sodium channel mutation) Biological replicates: 2.  Genotypes MR (carrying kdr and MACE mutations) Biological replicates: 2.

middle: X

first: Georg

last: Jander

first: John

last: Ramsey

first: Christian

middle: C

last: Figueroa


title: Mouse and zebrafish genes in vitro and in vivo

city: Leiden

first: Alex

last: Mohseny

address: Albinusdreef 2

department: Pathology

institute: LUMC

summary: Gene expression of mouse cells was studied by extracting total RNA from cultured mouse mesenchymal stem cells (MSCs) and zebrafish injected with mouse MSCs, in addition the host response was studied by hybridizing the zebrafish RNA to zebrafish arrays.

sample_organism:
Danio rerio
Mus musculus

platform_organism:
Danio rerio
Mus musculus

overall_design: Two-condition experiment, low passage MSCs vs high passage MSCs and tumorigenic MSCs, in vitro and inside zebrafish embryos.

first: Alexander

first: Anne-Marie

last: Cleton-Jansen


title: Inhibition of the mitochondrial TCA cycle by monofluoroacetate in Arabidopsis thaliana

city: Tucson

first: David

middle: M.

last: Rhoads

state: Arizona

address: 1140 East S. Campus Dr.

department: Plant Sciences

institute: University of Arizona

summary: Plant mitochondria signal to the nucleus leading to altered transcription of nuclear genes by a process called mitochondrial retrograde regulation (MRR). MRR is implicated in metabolic homeostasis and responses to stress conditions. Transcriptional consequences on nuclear gene expression of mitochondrial perturbations were examined by a microarray analyses. Expression of 606 genes was altered by monofluoroacetate (MFA) inhibition of the TCA cycle in leaves of soil grown Arabidopsis plants in the dark for 10 hours. Functional gene category (MapMan) and cluster analyses showed that genes with expression levels affected by perturbation from MFA inhibition were most similarly affected by biotic stresses such as pathogens, not oxidative stresses. Overall, the data provide further evidence for the presence of mtROS-independent MRR signaling, and support the proposed involvement of MRR and mitochondrial function in plant responses to biotic stress.

overall_design: Three independent (bio-replicate) experiments were done using three independent plant samples in the treatments. For each, approximately 30 plants were used for the treated sample and about 30 plants were used as the control sample. Plants were treated with 5 mM monofluoroacetate in 0.01% Tween 20 and incubated in the dark at room temperature (25C) for 6 hours. RNA was isolated from the inhibitor treated and control plants and used for microarray experiments. For each independent (bio-replicate) experiment, two microarrays were utilized using Cy3 and Cy5 dye-labeled samples and dye swapping was incorporated- 2 microarrays for each of 3 independent experiments (6 microarrays total).

first: Ann

middle: L

last: Umbach

first: Jelena

last: Zarkovic

first: Jianping

last: Yu

last: Ruckle

first: Lee

last: McIntosh

first: Jeffery

last: Hock

first: Scott

last: Bingham

first: Samuel

first: Rajani

middle: M

last: George

first: Chalivendra

last: Subbaiah


title: Inhibition of the mitochondrial electron transport chain by antimycin A in Arabidopsis thaliana

summary: Plant mitochondria signal to the nucleus leading to altered transcription of nuclear genes by a process called mitochondrial retrograde regulation (MRR). MRR is implicated in metabolic homeostasis and responses to stress conditions. Transcriptional consequences on nuclear gene expression of mitochondrial perturbations were examined by a microarray analyses.  Expression of 1316 was altered by antimycin A (AA) inhibition of the cytochrome respiratory pathway in leaves of soil grown Arabidopsis plants in the dark for 6 hours. Functional gene category (MapMan) and cluster analyses showed that genes with expression levels affected by perturbation from AA or MFA inhibition were most similarly affected by biotic stresses such as pathogens, not oxidative stresses. Overall, the data provide further evidence for the presence of mtROS-independent MRR signaling, and support the proposed involvement of MRR and mitochondrial function in plant responses to biotic stress.

overall_design: Three independent (bio-replicate) experiments were done using three independent plant samples and independent chemicals in the treatments. For each, approximately 30 plants were used for the treated sample and about 30 plants were used as the control treated sample. Plants were treated with 20 uM antimycin A in 0.01% Tween 20 and incubated in the dark at room temperature (25C) for 6 hours. RNA was isolated from the inhibitor treated and control treated plants and used for microarray experiments. For each independent (bio-replicate) experiment, two microarrays were utilized using Cy3 and Cy5 dye-labeled samples and dye swapping was incorporated- so, minimum of 2 microarrays for each of 3 independent experiments (6 microarrays). In addition, two of the microarrays were duplicated so that a total of 8 slides were used in the data analyses.

first: Michael

last: White


title: Arabidopsis transcriptome responses to mitochondrial perturbations

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

overall_design: Refer to individual Series


title: Integration of molecular biology tools to obtain information about abundantly expressed genes in flowers of Oncidium Gower Ramsey

city: Taipei

first: Choun Sea

last: Lin

address: 128 Academia Road, Section 2

department: Agriculture Biotechnology Research Center

country: Taiwan

institute: Academia Sinica

summary: Orchids are commercially important flowers, comprising one of the largest families of plants. Model plants such as Arabidopsis do not contain all plant genes. In this study, several molecular biology tools were integrated to isolate the flower-specific gene promoters of Oncidium Gower Ramsey (GR). A cDNA library of reproductive tissues was constructed and a microarray was established and used to compare gene expression in flowers and leaves. Five genes were highly expressed in flower tissues and the subcellular locations of the corresponding proteins were identified using fluorescent protein-fusion protein lip transient transformation, and related to their putative functions. BAC clones of the five genes, together with nine previously published flower and reproductive growth specific genes in Onc. GR, were identified and promoter regions were cloned by screening an Onc. GR BAC library. Interestingly, three of the five novel flower-abundant genes were putative trypsin inhibitor (TI) genes, OnTI1, OnTI2 and OnTI3, which were tandemly duplicated in the same BAC clone. Their promoters were identified using transient GUS reporter gene transformation and stable Arabidopsis transformation analyses. In conclusion, a combination of tools including a cDNA microarray, a BAC library, and a bombardment assay can be successfully employed to identify orchid genes and promoters.

overall_design: A total of 960 cDNAs derived from the Onc. GR flower cDNA library were amplified using PCR incorporating the T3 and M13 reverse universal primers. The PCR products were purified using the QIAquick PCR Purification kit (Qiagen, Germantown, MD) and eluted into 100 µl of 0.1× Tris-EDTA buffer, pH 8.0. Purified PCR products were printed on GAPS II-coated slides (Corning, New York, NY) using the OmniGrid 100 microarray (Genomic Solutions, Ann Arbor, MI), and arranged into 1.8 × 1.8-cm arrays (spot size: 100 μm) twice. These DNAs were cross-linked to the slide by baking the array at 80°C for 2 h. After printing, the slides were left to dry overnight. The total RNA from leaves and flowers (25 μg) was used for cDNA synthesis with either Cy3 or Cy5 dye molecules, using the 3DNA Expression Array Detection kit for microarrays (Array 50, version 2, Genisphere, Hatfield, PA). cDNA hybridization and washing procedures were performed according to the manufacturer's instructions. All experiments were carried out in triplicate, with repeats of each dye combination (n = 3).

first: Chen-Tran

last: Hsu

first: De-Chih

last: Liao

first: Fu-Hui

last: Wu

first: Shu-Chen

last: Shen

first: Shu-Jen

last: Chou

first: Shu-Yun

last: Tung

first: Chang-Hsien

last: Yang

first: Ming-Tsair

last: Chan

first: Choun-Sea


title: Crp induces switching of the CsrB and CsrC RNAs in Yersinia pseudotuberculosis and links nutritional status to virulence

status: Public on Dec 31 2012

city: Braunschweig

first: Ann

middle: Kathrin

last: Heroven

state: Niedersachsen

address: Inhoffenstr. 7

department: Molecular Infection Biology

institute: Helmholtz Centre for Infection Research

summary: Colonization of the intestinal tract and dissemination into deeper tissues by the enteric pathogen Yersinia pseudotuberculosis demands expression of a special set of virulence factors important for the initiation and the persistence of the infection. In this study we demonstrate that many virulence-associated functions are coregulated with the carbohydrate metabolism. This link is mediated by the carbon storage regulator (Csr) system, including the regulatory RNAs CsrB and CsrC, and the cAMP receptor protein (Crp), which both control virulence gene expression in response to the nutrient composition of the medium. Here, we show that Crp regulates the synthesis of both Csr RNAs in an opposite manner. A loss of the crp gene resulted in a strong upregulation of CsrB synthesis, whereas CsrC levels were strongly reduced leading to downregulation of the virulence regulator RovA. Switching of the Csr RNA involves Crp-mediated repression of the response regulator UvrY which activates csrB transcription. To elucidate the regulatory links between virulence and carbon metabolism, we performed comparative metabolome, transcriptome and phenotypic microarray analyses and found that Crp promotes oxidative catabolism of many different carbon sources, whereas fermentative patterns of metabolism are favoured when crp is deleted. Mouse infection experiments further demonstrated that Crp is pivotal for a successful Y. pseudotuberculosis infection. In summary, placement of the Csr system and important virulence factors under control of Crp enables this pathogen to link its nutritional status to virulence in order to optimize biological fitness and infection efficiency through the infectious life cycle.

overall_design: Y. pseudotuberculosis YPIII or the isogenic crp mutant strain were grown to late stationary phase at 25°C. Four biological replicates were employed for each experiment consisting of two pooled individual cultures and two pooled RNA preparation samples, respectively. Total RNA was extracted using SV Total RNA Isolation System (Promega). The samples were treated with RNase-free DNase (Roche Applied Science) and the quality of the RNA was confirmed by the lack of PCR amplification of the hns gene and by using an Agilent 2100 Bioanalyzer.

first: Johannes

last: Klein


title: Granule-exocytosis of granulysin and granzyme B as a potential key mechanism in vaccine-induced immunity in cattle against the nematode Ostertagia ostertagi

city: Beltsville

first: Robert

middle: W

last: Li

address: Barc East

department: Animal & Natural Resources Institute

institute: USDA

summary: Ostertagia ostertagi is considered one of the most economically important bovine parasites. As an alternative for anthelmintic treatment, an experimental host-protective vaccine was previously developed based on ASP-proteins derived from the adult worms. Intramuscular injection of this vaccine, combined with QuilA as adjuvant, significantly reduced the faecal egg counts by 59 %. However, the immunological mechanisms triggered by the vaccine are still unclear. Therefore, in this study, the differences in immune responses at the site of infection, i.e. the abomasal mucosa, between ASP/QuilA-vaccinated animals and QuilA-vaccinated control animals were investigated on a transcriptomic level, using a whole genome bovine micro-array, combined with histological analysis. Sixty nine genes were significantly impacted in animals protected by the vaccine, 48 of which were upregulated. A correlation study between the parasitological parameters and gene transcription levels showed that the transcription levels of two of the upregulated genes, granulysin (GNLY) and granzyme B (GZMB) negatively correlated to cumulative faecal egg counts and total worm counts, respectively. Both genes also positively correlated to each other, and to another upregulated gene, the IgE receptor subunit FCER1A. Surprisingly, these three genes also correlated significantly to CMA1, a mast cell marker, and to cell counts for mast cells and cells previously described as globule leukocytes. Furthermore, immunohistochemical data showed that GNLY was present in the granules of globule leukocytes and that it was secreted in the mucus. Overall, the results suggest a potential role of granule exocytosis by globule leucocytes, potentially IgE-mediated, in the vaccine induced protection against O. ostertagi

overall_design: 12 helminth‐free Holstein crossbred calves at 7‐10 months of age were randomly divided in 2 groups of 6 animals. All animals were immunized three times intramuscularly in the neck with a three‐week interval. One group received 750 μg of QuilA with Tris‐buffer instead of antigen (negative control). The other group received ~30 μg of the native ASP fraction per immunization in combination with 750 μg of QuilA adjuvant. After the final immunization, the animals were challenged with a trickle infection of 30,000 infective L3 larvae (1,000 L3/day for 30 days, 5 days a week during a period of 6 weeks). 3 weeks after the last challenge infection, all animals were sacrified and total RNA from fundic abomasa was extracted. A total of 12 microarrays were used. Only biological replicates were used.

first: F

last: Van Meulder

first: S

last: Van Coppernolle

first: J

last: Borloo

first: M

last: Rinaldi

first: K

last: Chiers

first: W

last: Van den Broeck

last: Vercruysse

first: E

last: Claerebout

first: Peter

last: Geldhof


title: Dynamics of 5-hydroxymethylcytosine and chromatin marks in mammalian neurogenesis

city: Duarte

first: Xiwei

last: Wu

state: CA

address: 1500 E. Duarte Rd.

department: Computational and Quantitative Medicine

institute: City of Hope National Medical Center

summary:
H3K36me3 (ChIp-ChIp), H3K4me3 (ChIp-ChIp), H3K27me3 (ChIp-ChIp), 5mC (MIRA) and 5hmC (hMeDIP)  profiles were analyzed in neural progenitor cells (NPC) and neurons by using Nimblegen Mouse ChIP-chip 2.1M Economy Whole-Genome Tiling - 4 Array Set.
In order to compare two different techniques of 5hmC profiling, we performed 5hmC profiling with Hydroxymethyl Collector™ Kit (Active Motif) method and hybridized it on mouse Chr7 fragment (Nimblegen). As an independent experiment, 5hmC profiling was performed by using hMeDIP method and hybridized on mouse Chr7 fragment (Nimblegen).  After MIRA enrichment and genome amplification, DNA was hybridized on mouse Chr7 fragment (Nimblegen).

overall_design: Analysis of epigenetic changes during neural differentiation due to comparisson of  epigenetic patterns in neural progenitor cells versus neurons.

first: Maria

last: Hahn

first: Runxiang

last: Qiu

first: Arthur

first: Jun

last: Wang

first: Julie

last: Zhang

first: Jonathan

last: Jui

first: Seung-Gi

last: Jin

first: Yong

last: Jiang

first: Gerd

middle: P

last: Pfeifer

first: Qiang

last: Lu


title: Gene expression profiling in the ovary of Queen conch (Strombus gigas) exposed to tributyltin-polluted environments in the British Virgin Islands

city: Providence

first: Daniel

middle: James

last: Spade

state: RI

address: Box G-E5

department: Pathology and Laboratory Medicine

institute: Brown University

summary: Ecotoxicogenomics in field experiments have yielded valuable mechanistic information for organisms present in polluted environments. The Queen conch (Strombus gigas) is a threatened species and populations are declining due to anthropogenic impact that includes pollution from boating activities.  In the British Virgin Islands (BVI), local Queen conch populations have exhibited imposex, a condition in which both male and female gonadal characteristics are present and studies in the BVI suggest that tributyl tin (TBT), a chemical used in boat paint, is correlated to increased incidence imposex.  This present study utilized a previously validated 8 x 15K Queen conch microarray to characterize the response of the ovarian transcriptome in conch found in polluted environments with high TBT in the BVIs.  There polluted sites, Road Harbour (RH) and Trellis Bay (TB), are harbours with high boating activity while the reference sites, Guana Island (GI) and Anegada (AN), are areas with low boating activity.  Microarray analysis revealed that there were 17 transcripts with high homology to known genes that were differentially expressed in the environments with high TBT and these included 6 induced and 11 down-regulated transcripts (p<0.01). These differentially expressed transcripts included phosphoenolpyruvate carboxylase, transposase, and high-affinity phosphate transporter PT1. When considering both RH and TB together in comparison to GI, functional enrichment showed that the biological processes and molecular functions of calcium ion binding, immune response, and negative regulation of cell proliferation were over represented in the polluted sites.  Gene set enrichment analysis revealed that transcripts involved in the biological processes of general metabolism, immune, lipid metabolism, and stress were affected in polluted environments. Although difficult to directly link changes at the transcriptomics level to TBT in the harbour, this analysis provides novel insight into pathways impacted in regions that experience heavy boating activity in the BVIs.

overall_design: Adult female queen conchs were collected from field sites in the British Virgin Islands with varying levels of TBT contamination and imposex incidence.  Contaminated sites included Rhode Harbour (RH, n=3) and Trellis Bay (TB, n=3), and the low-pollution site was Guana Island (GA, n=5).  Ovaries were preserved in RNAlater, transported to the laboratory, and RNA was prepared using a phenol-chloroform extraction and a CsCl gradient.  RNA was labeled with Cy3 for a single-dye design, per the Agilent protocol.

first: Cassander

last: O'Neil

middle: J

first: Yanping

first: Rosalinda

last: Kan

first: Christopher

last: Martyniuk

first: Nancy

middle: D

last: Denslow

first: Bruce

last: McDonald


title: Manipulation of Temperature and Photoperiod Identifies Molecular Networks Associated with the Para- to Endo-dormant Transition in Leafy Spurge Crown Buds

city: Fargo

first: Munevver

last: Dogramaci

state: ND

address: 1605 Albrecht Blvd N

institute: USDA-ARS

summary: Leafy spurge is a model for studying well-defined phases of dormancy in underground adventitious buds (UABs) of herbaceous perennial weeds, which is a primary factor allowing many invasive perennial weeds to escape conventional control measures. A 12-week ramp down in both temperature (27°C → 10°C) and photoperiod (16 h → 8 h light) is required to induce a transition from para- to endo-dormancy in UABs of leafy spurge. To evaluate the effects of photoperiod and temperature on molecular networks associated with this transition, we compared global transcriptome data-sets obtained from UABs of leafy spurge exposed to a ramp down in both temperature and photoperiod (RDtp) vs. a ramp down in temperature (RDt) alone. Analysis of transcriptome data-sets indicated that numerous genes associated with circadian clock, photoperiodism, flowering, and hormone responses (CCA1, COP1, HY5, MAF3, MAX2) were preferentially expressed during the transition from para- to endo-dormancy. Gene-set enrichment analyses highlighted metabolic pathways associated with ethylene, auxin, flavonoids, and carbohydrate metabolism; whereas, sub-network enrichment analyses identified hubs (CCA1, CO, FRI, mir172A, EINs, DREBs) of gene networks associated with carbohydrate metabolism, circadian clock, flowering, and stress and hormone responses during the transition to endodormancy. These results helped refine existing models for the transition to endodormancy in UABs of leafy spurge, which strengthened the roles of circadian clock associated genes, DREBs, COP1-HY5, carbohydrate metabolism, and involvement of hormones (ABA, ethylene, and strigolactones). Further, we propose that the RDtp treatment ultimately leads to a chain effect, responsive to photoperiod and temperature signaling, to synchronize molecular processes associated with the transition from para- to endo-dormancy.

platform_organism:
Manihot esculenta
Euphorbia esula

overall_design:
Plant material, environmental treatments, and vegetative growth
Leafy spurge plants were propagated from the uniform biotype (1984-ND001) and maintained in a greenhouse as described by Anderson and Davis (2004). Prior to the start of each experiment, plants were acclimated in a Conviron growth chamber (Model PGR15) for one week at 27°C, 16:8 h light:dark photoperiod. Each experiment was replicated four times, and each replicate contained 30 plants. Six plants from each replicate were used to determine vegetative growth rate, and crown buds from the remaining 24 plants were collected for transcriptome studies. All samples were collected between 11:00 and 13:00 a.m. to avoid diurnal variation.

 We previously established conditions for induction and release of dormancy phases under controlled environments (Doğramacı et al. 2010; Foley et al. 2009). To induce endodormancy, paradormant plants were subjected to a ramp-down in temperature (27°C → 10°C) and photoperiod (16 h → 8 h light), i.e., RDtp, for 12 weeks (Fig. 1, Exp-1). To distinguish the individual effects of temperature and photoperiod on molecular networks involved in endodormancy induction (see Fig. 1, Exp-2), we compared three-month old paradormant plants subjected to a ramp-down in temperature (27°C → 10°C) under constant photoperiod (16 h light) for 12 weeks (i.e., RDt) to RDtp plants. Additionally, a set of paradormant plants were kept under constant temperature and light (27°C, 16 h light) as a control.

At the end of each treatment, the aerial portion of the plant was decapitated to determine the dormancy status of the crown buds by their vegetative growth rate in the greenhouse, as described by Foley et al. (2009). New vegetative shoot growth was recorded weekly; results were analyzed using the generalized linear mixed model (PROC GLIMMIX) procedure of SAS 9.2, and 95% confidence intervals for treatment by week means were generated.

Transcriptome analyses
At the end of each treatment, crown buds were collected from intact plants to study transcriptome profiles and were stored at -80°C. RNA extraction, cDNA synthesis and fluorescent labeling, microarray hybridization using ~23K element arrays, and spot intensity analyses were performed as previously described (Doğramacı et al. 2010). Transcriptome data obtained from Exp-1 and -2 (Fig. 1) was used for various bioinformatics analysis. GeneMaths XT 2.1 software was used to normalize the arrays, to conduct statistical analyses, and to generate Venn diagrams as previously described by Doğramacı et al. (2010). Expression data for both Exp-1 and -2 are deposited at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) as GEO dataset query GSE19217 and GSE??, respectively.

The entire data set was further analyzed using Ariadne Pathway Studio 7 Software-Resnet Plant Version 2.1 (Ariadne Genomics Inc., Rockville, MD, USA) to obtain Gene Set Enrichment Analysis (GSEA) and Sub Network Enrichment Analysis (SNEA). GSEA is used to determine if predefined sets of genes are over-represented (P<0.05) between treatments; focusing on gene sets based on AraCyc metabolic pathways http://pmn.plantcyc.org/ARA/class-instances?object=Pathways, and gene ontology (GO) (http://www.geneontology.org/). SNEA algorithm was used to identify the networks by highlighting over-represented ontologies based on published gene regulation hierarchies, protein:protein interactions, or protein modification targets. Furthermore, these networks were visualized using the Union Selected Pathway function of the software for expression targets, binding partners, protein modification targets, and also custom advanced function to generate sub-networks as neighbors of proteins based on expression, regulation, molecular transport, protein modification, promoter binding, molecular synthesis, chemical reaction, and direct regulation.

first: James

middle: V

last: Anderson


title: QTL-region-specific microrrays reveal differential expression of signaling pathways associated with the liability for the inverted teat defect

city: Dummerstorf

first: Klaus

last: Wimmers

address: Wilhelm-Stahl-Allee 2

institute: Leibniz Institute for Farm Animal Biology (FBN)

type: Expression profiling by genome tiling array

summary:
The inverted teat defect is an inherited disorder characterised by the failure of teats to protrude from the udder surface. The number and identity of relevant genes are unknown.
Simultaneously, 1,587 probe-sets were screened, of which some 1,000 had present calls and were analysed for differential expression between mesenchymal and epithelial tissue of three categories of teats, i.e. normal teats of non-affected (NN) and affected animals (AN) and inverted teats (AI) of the later.

overall_design: RNAs of mesenchymal and epithelial teat tissues of 5 non-affected and 5 affected animals were hybridized to custom-designed application-specific array.

first: Siriluck

last: Ponsuksili

first: Kunsuda

last: Chomwisarutkun

first: Eduard

last: Murani

first: Silvio

last: Tetzlaff


title: PrlR regulon in Brucella melitensis 16M

city: NAMUR

first: LETESSON

middle: Jean-Jacques

last: marie

address: 61 rue de Bruxelles

institute: University of Namur

summary:
Investigation of whole genome gene expression level changes in a Brucella melitensis delta prlr mutant compared to the wild type strain.
The mutants analyzed in this study are further described in A. Mirabella, R-M Yanez, R.M. Delrue, S. Uzureau, M.S. Zygmunt, A. Cloeckaert, X. De Bolle, J.J. Letesson (2012). The two component system PrlS/PrlR of Brucella melitensis is required for persistence in mice and appears to respond to ionic strength. Microbiology

overall_design: A six chip study using total RNA recovered from three separate wild-type cultures of Brucella melitensis 16M and three separate cultures of a prlR mutant strain.  Each chip measures the expression level of 3,198 genes from Brucella melitensis 16M with nineteen 60 mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

first: Aurélie

last: Mirabella

first: Rosse-Mary

last: Yanez Villanueva

first: Julien

last: Lemaire

first: Xavier

last: De Bolle

first: Jean-Jacques

last: Letesson