title: Gene expression profiles in different proglottids of Moniezia expansa

first: Wenjuan

last: Zhao

summary:
We used complementary DNA microarray to analyze the gene expression profiles in different proglottids of Moniezia expansa. A total of 4056 sequences including full length and partial complementary DNAs representing novel, known, and control genes were analyzed. We utilized cDNA array for detection of gene expression profiles of different proglottids of M.expansa. The present study provides some interesting data to better understanding the mechanisms of procreation and may suggest some potential target molecules for a more effective treatment on verminosis.

1.Construction of microarray
By clustering the 2,642 sequences from the cDNA library, 1,078 unigenes of M.expansa, including full-length and partial cDNAs representing novel or known genes, were obtained for array construction. The negative control spots of non-tapeworm origin in the chip were the rice genes (48 spots). In addition, the array included 9 known genes(54 spots) as positive control genes that were provided by our library and 24 empty spots. Unigenes and all control genes were cloned into plasmid vector. The DZH (TC) chips were constructed by United Gene Holdings Limited of China (Shanghai, China) according to the method described by Li et al.
The cDNA inserts were amplified by use of the polymerase chain reaction (PCR) using universal primers to plasmid vector sequences and were then purified. All PCR products were examined by agarose gel electrophoresis to ensure the quality and the identity of the amplified clones as expected. Then the amplified PCR products were dissolved in a buffer containing 3×SSC solution. The solution with amplified PCR products were spotted onto glass slides (model DZH-TC) using a Cartesian PixSys 7500 motion control robot (Cartesian Technologies, Irvine, CA., USA) fitted with ChipMaker Micro-Spotting Technology (TeleChem International, Sunnyvale, CA., USA). The glass slides were then hydrated for 2 h in 70% humidity, dried for 0.5 h at room temperature, and UV crosslinked (65 mj/cm). They were further processed at room temperature by soaking in 0.2% sodium dodecyl sulfate (SDS) for 10 min, distilled H2O for 10 min, and 0.2% sodium borohydride (NaBH4) for 10 min. The slides were dried again and ready for use.
2.RNA preparation and probe labeling
Total RNAs were isolated from 6 M.expansas with scolex-neck proglottids, immature proglottids, mature proglottids and gravid proglottids. Tissue samples were ground into a fine powder in a 10 cm ceramic mortar (RNase-free) and were then homogenized in TRIzol (Biostar, Shanghai, China). After centrifugation, the supernatant was separated from the organic phase and was extracted in an equal volume of chloroform. The aqueous phase was then precipitated by an equal volume of isopropanol at 4°C, centrifuged to pellet the RNA and dissolved in Milli-Q H2O.
The fluorescent cDNA probes were prepared through reverse transcription with Cy3- or Cy5-deoxy UTP (Amersham Pharmacia Biotech, Piscataway, NJ, USA) as follows: 5 μg of oligo(dT18) was added and annealed to 3μg of mRNA by heating the mixture to 70˚C for 10 min, and then chilling it on ice. The final reaction buffer mixture contained dNTPs (200 μM dATP, dCTP and dGTP; 60 μM dTTP; and 60 μM Cy3- or Cy5-dUTP), 2 μl of Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and 1×reaction buffer 10μl. The reactions were carried out at 42 ºC for 2 h. RNA was hydrolyzed by adding 4 μl of 2.5 M NaOH, incubating the mixture at 65 ºC for 10 min and then neutralizing it with 4 μl of 2.5 M HCl. The RNA samples from the scolex-neck proglottids were labeled with Cy3-dUTP and those from immature proglottids, mature proglottids and gravid proglottids, respectively, were labeled with Cy5-dUTP. The two color probes were then mixed and diluted to 500 μl with TE, and concentrated using a Microcon YM-30 filter (Millipore, Bedford, MA, USA) to 10 μl. The sample was then vacuum dried.
3.Hybridization
The probe was dissolved in 20 ml of hybridization solution (5×SSC (0.75M NaCl and 0.075M sodium citrate), 0.4% SDS, 50% formamide). Microarrays were pre-hybridized with a hybridization solution containing 0.5 mg/ml of denatured salmon sperm DNA at 42°C for 6 h. Fluorescent probe mixtures were denatured at 95°C for 5 min and then applied onto the pre-hybridized chip under a cover glass. Chips were hybridized at 42°C for 15-17 h. Next, the hybridized chips were each washed at 60°C for 10 min in solutions of 2×SSC and 0.2% SDS, 0.1×SSC and 0.2% SDS, 0.1×SSC and then dried at room temperature.
4.Detection and analysis
The chips were scanned with a ScanArray 4000 (GSI Lumonics, Bellerica, MA, USA) at two wavelengths, 635 and 532 nm, to detect emission from both Cy5 and Cy3, respectively. The acquired images were analyzed using GenePix Pro 3.0 software. The intensities of each spot at the two wavelengths represent the quantity of Cy3-dUTP and Cy5-dUTP. Ratios of Cy5 to Cy3 were computed using the GenePix Pro 3.0 median of ratio method. Overall intensities were normalized using the corresponding GenePix default normalization factor. All spots flagged “Bad” or “Not Found” by GenePix software were removed from the final data. Only genes with raw intensity values for both Cy3 and Cy5 of >200 were chosen for differential analysis. Genes were identified as differentially expressed if the ratio was >2 or <0.5, or the absolute value of base 2 logarithm of the ratio was >1 or < -1.

overall_design: mature proglottids, immature proglottids, gravid proglottids vs common reference scolex-neck proglottids tissue. 2 biological replicates for each experiments.

first: Xinwen

last: Bo

first: Hui

last: Zhang

first: Xinhua

last: Wang

first: Lichao

last: Kang


title: KLF17 is a negative regulator of epithelial-mesenchymal transition and metastasis in breast cancer

status: Public on Nov 30 2009

city: Philadelphia

first: Louise

middle: C

last: Showe

state: PA

address: 3601 Spruce St

country: USA

institute: The Wistar Institute

summary: Metastasis is a complex multi-step process requiring the concerted action of many genes and is the primary cause of cancer deaths. Pathways that regulate metastasis enhancement and suppression both contribute to tumor dissemination process. In order to identify novel metastasis suppressors, we set up a forward genetic screen in a mouse model. We transduced a genome-wide RNAi library into the non-metastatic 168FARN breast cancer cell line, orthotopically transplanted the cells into mouse mammary fat pads, and then selected for cells that could metastasize to the lung and identified an RNAi for the KLF17 gene. Conversely, ectopic expression of KLF17 in highly metastatic 4T1 breast cancer cell line inhibited their ability to metastasize from the mammary fat pad to the lung. We showed that suppression of KLF17 expression promotes breast cancer cell invasion and epithelial-mesenchymal transition (EMT). We also showed that KLF17 functions by directly binding to the promoter of Id-1, a key metastasis regulator in breast cancer, to inhibit its transcription. Finally, we demonstrated that KLF17 expression is significantly down-regulated in primary human breast cancer samples and that the combined expression patterns of KLF17 and Id-1 can serve as a potential biomarker for lymph node metastasis in breast cancer.

overall_design: Illumina mouse array was used for 168FARN cells stably expressing a control vector, KLF17 cDNA or KLF17 shRNA. Triplicate samples were used to perform gene expression analysis on each cell line.

first: Kiranmai

last: Gumireddy

first: Anping

last: Li

first: Phyllis

middle: A

last: Gimotty

first: Andres

middle: J

last: Klein-Szanto

first: Dionyssios

last: Katsaros

first: George

last: Coukos

first: Lin

first: Qihong

last: Huang


title: Comparative Genomics Study of Clostridium botulinum

status: Public on Nov 26 2009

city: Rockville

first: Chun-Hua

last: Wan

state: MD

address: 9407 Medical Center Dr

department: PFGRC

institute: J Craig Venter Institute

summary:
The PFGRC has developed a cost effective alternative to complete genome sequencing in order to study the genetic differences between closely related species and/or strains. The comparative genomics approach combines Gene Discovery (GD) and Comparative Genomic Hybridization (CGH) techniques, resulting in the design and production of species microarrays that represent the diversity of a species beyond just the sequenced reference strain(s) used in the initial microarray design. These species arrays may then be used to interrogate hundreds of closely related strains in order to further unravel their evolutionary relationships.

Clostridium botulinum produces botulinum neurotoxin (BoNT)and is classified as a “Category A” select agent. BoNT can be classified into seven serotypes designated A-G. There is considerable genetic variation within these serotypes, as demonstrated by the recognition of at least 47 subtypes. The most studied serotype, BoNT/A, has been found in a large and diverse group of clostridia, most of which express the subtype BoNT/A1. The BoNT/A1 producing C. botulinum strain ATCC 3502, used to obtain an initial annotated genome sequence, is not representative of the diverse clostridia group producing BoNT. Nearly 50% of C. botulinum strains producing BoNT/A1 have been shown to also encode unexpressed variants of BoNT/B with a distinct cluster arrangement. This nucleotide cluster is completely absent from the published genome sequence. In addition, a recently identified novel BoNT/A1 strain lacks the gene cluster seen in the genome sequence of ATCC 3502. Furthermore, a strain designated Hall A Hyper differs greatly from the sequenced strain as indicated by its ability to produce higher quantities of BoNT/A1. The genetic and phenotypic basis for this difference in BoNT expression is currently unknown, and the sequences of the BoNT gene and the cluster are identical in both strains. This observation supports the hypothesis that genes outside the toxin cluster are involved in the regulation and maturation of BoNT.

The flow of genetic information within this group motivated us to identify novel genes for the purpose of creating a “species” DNA microarray to better understand the ancestral relationships among its members. Based on preliminary genotyping (MLST, and CGH using a single-genome-based array), 20 diverse C. botulinum strains were selected for sequencing. Sequence information obtained from this project, and from other publicly available sources, led to the development of a comprehensive species microarray for C. botulinum group members. The availability of the C. botulinum species DNA microarray has allowed us to carry out a collaborative CGH genotyping project to validate this microarray as well as understand the phylogenomic relationships among members of C. botulinum group.

overall_design: One hundred fifty six query strains were investigated in this study, with each query strain hybridized against the reference strain, ATCC3502.  Each strain has a single dye experiment.  Each oligo is spotted on the C. botulinum species microarray once.  Positive controls on the array consist of oligos designed from the sequenced reference genome, ATCC3502, and negative controls on the array consist of oligos designed from the thale cress plant, Arabidopsis thaliana.The microarrays also had Agilent internal controls.

first: L.

last: Papazisi

last: Appalla

first: E.

last: Wester

first: A.

last: Pineda

first: S.

last: Akkoush

last: Jin

last: Ratnayke

first: Q.

last: Sun

first: B.

middle: G

last: Remortel

first: C.

first: F.

last: Onmus-Leone

first: M.

last: Jacobson

first: G.

last: Lin

first: R.

middle: D

last: Fleischmann

last: Johnson

middle: N

last: Peterson


title: MLL and Pol2 Chip-chip in interphase and mitotic arrested cells

status: Public on Nov 25 2009

city: Cold Spring Harbor

first: Christopher

last: Vakoc

state: NY

address: 1 Bungtown Road

institute: Cold Spring Harbor Laboratory

type: Genome binding/occupancy profiling by genome tiling array

summary: Mixed Lineage Leukemia (MLL) and its metazoan Trithorax orthologs have been linked with the epigenetic maintenance of transcriptional activity. To identify mechanisms by which MLL perpetuates active transcription in dividing cells, we investigated its role during M-phase of the cell cycle. Unlike other chromatin modifying enzymes examined, we found that MLL associates with gene promoters packaged within condensed mitotic chromosomes. Genome-wide location analysis identified a globally rearranged pattern of MLL occupancy during mitosis in a manner favoring genes that were highly transcribed during interphase. Knockdown experiments revealed that MLL retention at gene promoters during mitosis accelerates transcription reactivation following mitotic exit. MLL tethers Menin, RbBP5, and ASH2L to its occupied sites during mitosis, but is dispensable for preserving histone H3K4 methylation. These findings implicate mitotic bookmarking as a component of Trithorax-based gene regulation which may facilitate inheritance of active gene expression states during cell division.

overall_design: anti-MLL ChIP (antibody 456) and anti-pol2 chip (sc-899) in chromatin prepared from interphase and mitotic HeLa cells

first: C

middle: R


title: The full cardiac G protein-coupled receptor repertoire reveals chamber-specific expression patterns

status: Public on Dec 01 2009

city: Montpellier

first: Laurent

last: Journot

address: 141 rue de la Cardonille

country: France

institute: MGX - Montpellier GenomiX

summary: Cardiac function is regulated by many hormones and neurotransmitters which exert their physiological effects through the activation of G protein-coupled receptors (GPCRs). Here, we quantified the expression of 395 endoGPCRs (all GPCRs excluding taste and odorant receptors) in male mouse right and left atria and ventricles by using high-throughput real-time RT-PCR and focused on the 135 most highly expressed transcripts. No cardiac functional data is available for almost half of these receptors. Cluster analysis allowed us to link GPCR expression patterns to cardiac function. Indeed, ventricles and atria are both contractile; however, the latter, and especially the right atrium, are central to the generation and regulation of cardiac rhythm. Accordingly, the right atrium exhibited the most specific signature whereas the vast majority of GPCRs found in ventricles were evenly expressed in both the right and left chambers. RT-PCR data were confirmed at the protein level for six selected transcripts (ETA, EP1, PAR1, Sfrp1, CCR2 and AT1a) and at the functional level in isolated mouse ventricular cardiomyocytes for the glutamate metabotropic receptor 1b.

overall_design:
Twenty hearts were dissected from 8-week-old C57BL/6 male mice.
Total RNAs were prepared from pooled cardiac chambers: right atria (RA) including the SAN, left atria (LA), right (RV) and left ventricles (LV) with 3 replicates for each.

first: Brigitte

last: Couette

first: Thomas

last: Moore-Morris

first: Annie

last: Varrault

first: Matteo

last: E. Mangoni

first: Anne

last: Le Digarcher

first: Vincent

last: Negre

first: Christelle

last: Dantec

first: Joel

last: Nargeot


title: Genes upregulated by cis-1,2-dichloroethene (cDCE) in Polaromonas sp. strain JS666

city: Ithaca

first: James

middle: M

last: Gossett

address: 220 Hollister Hall

department: Civil and Environmental Engineering

institute: Cornell University

type: Expression profiling by genome tiling array

summary:
Investigation of whole genome gene expression level changes in cultures of Polaromonas sp. JS666 grown on cDCE compared to the reference substrate glycolate.
JS666 is the first organism isolated capable of coupling growth to the aerobic oxidation of the chlorinated solvent, cis-1,2-dichloroethene (cDCE).

overall_design: A six chip study using total RNA extracted from cultures of JS666 grown on cDCE or glycolate in triplicate.

first: Laura

middle: K

last: Jennings

first: Michelle

last: Chartrand

first: Georges

last: Lacrampe-Couloume

first: Barbara

last: Sherwood Lollar

first: Jim

last: Spain


title: Ribose utilization by the human commensal Bifidobacterium breve UCC2003

city: Utrecht

first: Aldert

last: Zomer

address: Yalelaan 1

department: Department of Infectious Diseases and Immunology,Faculty of Veterinary Medicine

country: Netherlands

institute: Utrecht University

summary: Growth of B. breve UCC2003 on ribose leads to the transcriptional induction of the rbsACBDK gene cluster. Generation and phenotypic analysis of an rbsA insertion mutant established that the rbs gene cluster is essential for ribose utilization, and that its transcription is likely regulated by a LacI-type regulator encoded by rbsR, located immediately upstream of rbsA. Gel mobility shift assays using purified RbsRHis indicate that the promoter upstream of rbsABCDK is negatively controlled by RbsRHis binding to an 18-bp inverted repeat and that RbsRHis binding activity is modulated by D-ribose. The rbsK gene of the rbs operon of B. breve UCC2003 was shown to specify a ribokinase (EC 2.7.1.15), which specifically directs its phosphorylating activity towards D-ribose, converting this pentose sugar to ribose-5-phosphate.

overall_design: In order to investigate differences in global gene expression upon growth of B. breve UCC2003 on ribose, or a combination of ribose and glucose, as compared to glucose, DNA microarray experiments were performed. Total RNA was isolated from B. breve UCC2003 cultures grown on ribose, a combination of ribose and glucose, or glucose. All experiments were performed in duplicate. A dye swap was performed in one of the two biological replicates.

first: Karina

last: Pokusaeva

first: Ana-Rute

last: Neves

first: Mary

last: O'Connell-Motherway

first: John

last: MacSharry

first: Peter

last: Curley

first: Gerald

middle: F

last: Fitzgerald

first: Douwe

last: van Sinderen


title: Gene expression studies in normal, dysplastic and malignant cervical tissues

city: Chennai

first: Rajkumar

last: T

state: Tamilnadu

address: Adyar

department: Molecular Oncology

country: India

institute: Cancer Institute (WIA)

summary:
Gene expression profiling of normal cervix, CIN's and cancer cervix, with an intent to identify genes involved in the malignant transformation of normal cervix and to identify genes which can be used as biomarkers for early diagnosis.
Identification of genes for predicting radiation response using microarray gene expression studies

Keywords: Patient tissue samples

overall_design: Two dye experiments using Universal control RNA (Stratagene) and RNA from tissues. Biological replicates: Normal = 5; CIN1=4; CIN3=3; Cervical cancers = 28. One replicate per array.

first: T

last: Rajkumar


title: Genome-wide transcriptional profiling of NF1-haploinsufficiency in human: Coriell kindred

city: Bethesda

first: Alexander

last: Pemov

address: 49 Convent Drive

institute: National Institutes of Health

summary: Neurofibromatosis type 1 (NF1) is a common monogenic tumor-predisposition disorder that arises secondary to mutations in the tumor suppressor gene NF1. Haploinsufficiency of NF1 fosters a permissive tumorigenic environment through changes in signalling between cells; however, the intracellular mechanisms for this tumor-promoting effect are less clear. We hypothesized that the genetic effects of NF1-haploinsufficiency may be discerned by comparison of genome-wide transcriptional profiling in somatic, non-tumor cells (LCLs) from NF1-affected and –unaffected individuals.

overall_design:
Total RNA obtained from lymphoblastoid cell lines (LCLs) from NF1-affected individuals was compared to total RNA from LCLs of NF1-unaffected controls.

The supplementary file 'GSE18444_non-normalized_data.txt' contains non-normalized data for Samples GSM459535-GSM459552.

first: Douglas

last: Stewart


title: Oscillations in supercoiling drive circadian gene expression in cyanobacteria

city: Cambridge

first: Vikram

last: Vijayan

state: MA

address: 52 Oxford Street, Room 440

institute: Harvard University

summary: The cyanobacterium Synechococcus elongatus PCC 7942 exhibits oscillations in mRNA transcript abundance with 24-hour periodicity under continuous light conditions. The mechanism underlying these oscillations remains elusive – neither cis nor trans-factors controlling circadian gene expression phase have been identified. Here we show that the topological status of the chromosome is highly correlated with circadian gene expression state. We also demonstrate that DNA sequence characteristics of genes that appear monotonically activated and monotonically repressed by chromosomal relaxation during the circadian cycle are similar to those of supercoiling responsive genes in E. coli. Furthermore, perturbation of superhelical status within the physiological range elicits global changes in gene expression similar to those that occur during the normal circadian cycle.

sample_organism: Synechococcus elongatus PCC 7942 = FACHB-805

overall_design: Synechococcus elongatus PCC 7942 was subjected to two consecutive light/dark cycles and released into continuous light (T = 0). Cells were sampled every 4 hours from T = 24 to T = 84 hours for microarray analysis to characterize circadian gene expression. In a separate experiment, to characterize the response of S. elongatus to immediate chromosome relaxation, cells were sampled at T = 56 and T = 64 hours, immediately followed by novobiocin treatment (0.1 ug/ml), and the resulting response was measured by microarray after 5, 10, 30, 90, and 150 minutes. This experiment was designed to test whether chromosomal relaxation is sufficient to induce gene expression changes similar to those observed during the circadian cycle.

first: Rick

last: Zuzow

first: Erin

last: O'Shea


title: Comparative transcriptome and phenotype analysis of acid-stressed Bacillus cereus strain ATCC 10987

city: Wageningen

first: Maarten

last: Mols

address: Bomenweg 2

department: ATV

institute: Wageningen University

summary: The food-borne human pathogen Bacillus cereus is found in environments that often have a low pH, such as food and soil. The physiological response upon exposure to several levels of acidity were investigated of B. cereus model strain ATCC 10987, to elucidate the response of B. cereus to acid stress. pH 5.4, pH 5.0, pH 4.8 and pH 4.5 were selected to conduct microarray analyses, based on the differences in physiological response upon exposure to the acid conditions. The transcriptome data revealed response specific profiles. Showing mechanisms induced upon all the different acid down-shocks, such as nitrate reductase and energy production genes, and several genes specifically expressed differentially in mild or lethal levels of acidity, such as F1F0-ATPase and cydAB. Furthermore, mechanisms involved in oxidative stress response were found highly up-regulated in response to both mild and lethal acid stress. The induction of oxidative stress related genes may be a response to the formation of reactive oxygen species by a perturbation of the electron transport chain. Therefore, the formation of hydroxyl radicals and/ or peroxynitrite was monitored upon exposure to the different levels of acidity with a fluorescent probe in a flow cytometer. The formation of these oxidative compounds was shown to be specific for lethal pHs and a model to relate radical formation with the observed transcriptome profiles was proposed.

overall_design: Per acid down-shock three exposure times (i.e., 10, 30 and 60 min) were each compared with non-exposed cells (i.e., t0). In total 4 different acid down-shocks were applied, pH 5.4, pH 5.0, pH 4.8 and pH 4.5. The experiments were performed in duplicate and the duplicate samples were hybridised with a dye-swap.

first: Richard

last: van Kranenburg

first: Clint

last: van Melis

first: Roy

last: Moezelaar

first: Tjakko

last: Abee


title: Isolation and characterization of environmental Bacillus isolates which may harbor B. anthracis virulence genes

first: Chun-Hua

last: Wan

address: 9704 Medical Center Dr

department: Pathogen Functional Genomics Resources Center (PFGRC)

institute: The J. Craig Venter Institute

summary:
The goal of this project was to screen soil samples for bacteria that may harbor B. anthracis virulence-associated genes (VAGs). There is currently no information about the prevalence of these types of organisms in the environment. Due to increased environmental monitoring of select agents by programs such as BioWatch and biodetection systems in place at the United States Post Offices and Department of State locations, it has become critical that we not only better understand the natural range of B. anthracis but also how widespread B. anthracis virulence genes are in environmental communities. Naturally occurring isolates containing the B. anthracis virulence genes could generate false-positive results in tests that detect the anthrax toxins, capsule or their associated genes. Understanding the true diversity and pathogenic potential of Bacillus spp. and particularly the B. cereus group is crucial not only in terms of understanding data from environmental monitoring but also diagnosing patients with clinical presentations similar to anthrax in the future. Severe and fatal disease caused by strains similar to B. anthracis could unnecessarily initiate emergency responses if anthrax was incorrectly suspected. Conversely, these strains may be used as bioterror agents requiring science-based responses; presently our limited understanding of these organisms does not permit data-driven decision making.

We have investigated 700 aerobic sporoform soil isolates obtained from two areas in the Southwest of the US. Soil samples from the first site had been taken from public access land approximately 50 meters across from the work site of a fatal pneumonia case in a welding factory. This took place in year 2003 when B. cereus was isolated from a metal worker. The second site was targeted because of a recent case involving a deceased mule suspected to have died of a B. anthracis infection. Soil samples were initially analyzed at the CDC. Isolates were obtained by heating the soil at 65 degrees Celcius for 30 minutes followed by plating on agar media. All isolates were screened by PCR for the presence of B. anthracis genomic traits such as toxin genes (cya, lef and pag) as well as chromosomal markers. All isolates were also tested for their hemolytic activity as well as phage sensitivity.

sample_organism:
Bacillus cereus
Bacillus anthracis str. Sterne

overall_design: Eighty-four query strains were investigated in this study, with each query strain hybridized against the reference strain, Sterne. Two dye-swap experiments were performed with seventeen strains, for a total of four hybridizations per query strain. The other strains have a single dye experiment, for a total of two hybridizations per query strain.  Each 70mer oligo spotted on the B. cereus species microarray is spotted once. Positive controls on the array consist of oligos designed from the sequenced reference genome, Sterne, and negative controls on the array consist of oligos designed from the thale cress plant, Arabidopsis thaliana.

first: L.

last: Papazisi

first: B.

last: Remortel

last: Appalla

first: F.

last: Onmus-Leone

first: H.

last: Sajadi

first: S.

last: Ratnayke

first: Q.

last: Sun

first: C.

first: N.

last: Podnecky

last: Beesley

first: R.

last: Fleischmann

first: A.

last: Hoffmaster

last: Peterson


title: Listeria monocytogenes SOS response determined after mitomycin C exposure

first: Stijn

last: van der Veen

summary:
The SOS response is a conserved pathway that is activated under certain stress conditions and is regulated by the repressor LexA and the activator RecA. The food-borne pathogen Listeria monocytogenes contains RecA and LexA homologs, but their roles in Listeria have not been established. In this study, we identified the SOS regulon in L. monocytogenes by comparing the transcription profiles of the wild-type strain and the ΔrecA mutant strain after exposure to the DNA damaging agent mitomycinC (MMC). The SOS response is an inducible pathway involved in DNA repair, restart of stalled replication forks, and in induction of genetic variation in stressed and stationary phase cells. It is regulated by LexA and RecA. LexA is an autoregulatory repressor which binds to a consensus sequence in the promoter region of the SOS response genes, thereby repressing transcription. A consensus LexA binding motif for L. monocytogenes has not been identified thus far. Generally, the SOS response is induced under circumstances in which single stranded DNA accumulates in the cell. This results in activation of  RecA, which in turn stimulates cleavage of LexA, and ultimately in the induction of the SOS response.

Keywords: stress response, loop design, SOS response, mitomycin c, listeria monocytogenes, RecA, LexA

overall_design: Triple loop design of 3 independent experiments (series A, B, and C) using Wt strain and recA mutant (activator of the SOS response).  Sampling was done before (t=0) and 1 hour after (t=60) exposure to mitomycin C (MMC) for both the wild-type strain and the recA mutant strain. One series therefore contains 4 samples and the complete experiments consists of 12 samples. Each of the 3 series was designed in a loop (wt, t=0 =>wt, t=60 => recA, t=60 => recA, t=0 => wt, t=0). These 3 loops were connected using series B as the central loop. Series B was connected to series A as follows: wt, t=0 (B) => recA, t=0 (A) and wt, t=60 (B) => recA, t=60 (A). Series B was connected to series C as follows: recA, t=0 (B) => wt, t=0 (C) and recA, t=60 (B) => wt, t=60 (C).

last: van der Veen'

first: Saskia

last: van Schalkwijk

first: Douwe

last: Molenaar

first: Willem

last: de Vos

first: 'Marjon

last: Wells-Bennik


title: Comparative Genomics of Bacillus cereus and Bacillus anthracis

last: Wan

summary: The aim of the study was to carry out a CGH study utilizing a set of 39 diverse Bacillus isolates. Thirty four B. cereus and five B. anthracis strains and isolates were chosen so as to represent different lineages based on previous characterizations, including MLEE and MLST (Helgason, Okstad et al. 2000; Helgason, Tourasse et al. 2004). They represent the spectrum of B. cereus phenotypic diversity by including soil, dairy and periodontal isolates in addition to virulent B. anthracis strains.

sample_organism:
Bacillus anthracis
Bacillus cereus

overall_design:
39 diverse Bacillus isolates were chosen for the study.Thirty four B. cereus and five B. anthracis strains. Dye swap experiments were performed yielding 2 hybridizations per query strain.
Each 70mer oligo spotted on the B. cereus species microarray is spotted once.  Positive controls on the array consist of oligos designed from the sequenced reference genome, STERNE, and negative controls on the array consist of oligos designed from the thale cress plant, Arabidopsis thaliana.

last: Papazisi

first: G.

last: Bock

last: Remortel

first: T.

last: Minogue

first: M.

last: Jones

last: Andreas

middle: B

last: Kolstø


title: Renal allograft dysfunction

city: Hangzhou

first: youying

last: mao

address: Yuhangtang Road,NO.388

country: China

institute: Zhejiang University

summary:
The study comprises various components:
We aim to screen out different gene expression profile in acute rejection on the kidney.
We aim to screen out different gene expression profile in acute tubular necrosis on the kidney.
We aim to screen out different gene expression profile in borderline change on the kidney.
We aim to screen out different gene expression profile in non-rejection on the kidney.
We aim to screen out different gene expression profile in presumed rejection on the kidney.
We aim to screen out different gene expression profile in renal recipients with stable function.
Results from the various study components can help to diagnose renal allograft dysfunction with different causes by distinct gene expression profile.
Keywords: acute rejection, acute tubular necrosis, borderline change, non-rejection, presumed rejection, renal recipients with stable function,

overall_design:
Samples GSM456771-GSM456800: This study has been accomplished with 15 patients of acute rejection on the kidney.Technical replicates: 2 replicates(except AR10 and AR13)
Samples GSM456801-GSM456810: This study has been accomplished with 5 patients of acute tubular necrosis on the kidney. Technical replicates: 2 replicates(except ATN4)
Samples GSM456811-GSM456831: This study has been accomplished with 11 patients of borderline change on the kidney.Tecnical replicates:2 replicates(except BL8 and BL11)
Samples GSM456833-GSM456850: This study has been accomplished with 9 patients of non-rejection on the kidney.Technical replicates: 2 replicates
Samples GSM456851-GSM456864: This study has been accomplished with 7 patients of presumed rejection on the kidney.Technical replicates: 2 replicates
Samples GSM456865-GSM456894: This study has been accomplished with 15 patients of renal recipients with stable function.Technical replicates: 2 replicates

first: Youying

last: Mao


title: Comparative Genomics Study of Vibrio cholerae

first: Chun-Hua

summary:
The PFGRC has developed a cost effective alternative to complete genome sequencing in order to study the genetic differences between closely related species and/or strains. The comparative genomics approach combines Gene Discovery (GD) and Comparative Genomic Hybridization (CGH) techniques, resulting in the design and production of species microarrays that represent the diversity of a species beyond just the sequenced reference strain(s) used in the initial microarray design. These species arrays may then be used to interrogate hundreds of closely related strains in order to further unravel their evolutionary relationships.

Many infectious agents that cause emerging and re-emerging diseases appear to evolve from non-virulent forms. We still lack a clear understanding about the natural history of various microbial agents that cause human infectious diseases and the events leading to acquisition of their pathogenic potential. There have been seven pandemics of V. cholerae throughout the history of the mankind. To date, the world population is still experiencing the seventh one which started in the early 1960s. From almost 200 recognized V. cholerae serotypes, the majority of these epidemics are associated with primarily O1 serotype. However there is evidence that this species is undergoing some phenotypic changes during the last decades. Such examples include shifts in some metabolic pathways used for biotyping, phage sensitivity profiling and the acquisition of plasmids that carry multiple genes conferring antimicrobial resistance. Furthermore, the recent emergence of a non-O1 serotype (‘Bengal strain’, classified serologically as O139) has prompted the experts to think that perhaps this genotype will be the predominant one in the upcoming (eighth) pandemic. Besides the O1 and O139, the non-O1 and non-O139 V. cholerae stains are occasionally associated with other severe forms of gastrointestinal disease in humans. Interestingly, many of these non-canonical strains lack the genes encoding the typical virulence factors for this species such as the Cholera-toxin (ctx) and toxin co-regulated pilus (tcpA). Therefore it has been hypothesized that this group of non-canonical V. cholerae pathogens consist of several sub-clones that elicit disease via unknown virulence determinants and underlying mechanisms.

The flow of genetic information within this group motivated us to identify novel genes for the purpose of creating a "species" DNA microarray to better understand the ancestral relationships among its members. Based on preliminary genotyping (MLST, and CGH using a single-genome-based array), 10 diverse V. cholerae and one V. mimicus were selected for sequencing. Sequence information obtained from this project, and from other publicly available sources, led to the development of a comprehensive species microarray for V. cholerae group members. The availability of the V. cholerae species DNA microarray has allowed us to carry out a collaborative CGH genotyping project to validate this microarray as well as understand the phylogenomic relationships among members of V. cholerae group.

overall_design: One hundred thirteen query strains were investigated in this study, with each query strain hybridized against the reference strain, El Tor. Each strain has a single dye experiment. Each oligo is spotted on the V. cholerae species microarray once. Positive controls on the array consist of oligos designed from the sequenced reference genome, El Tor, and negative controls on the array consist of oligos designed from the thale cress plant, Arabidopsis thaliana. The microarrays also had Agilent internal controls.

first: L.

last: Appalla

first: E.

last: Wester

last: Pineda

last: Akkoush

last: Jin

last: Ratnayke

last: Sun

first: B.

last: Fleischmann

first: O

last: Stine

first: J.

last: Johnson

last: Peterson


title: Comparative transcriptome and phenotype analysis of acid-stressed Bacillus cereus strain ATCC 14579

first: Maarten

last: Mols

summary: The food-borne human pathogen Bacillus cereus is found in environments that often have a low pH, such as food and soil. The physiological response upon exposure to several levels of acidity were investigated of B. cereus model strain ATCC 14579, to elucidate the response of B. cereus to acid stress. pH 5.4, pH 5.0, pH 4.8 and pH 4.5 were selected to conduct microarray analyses, based on the differences in physiological response upon exposure to the acid conditions. The transcriptome data revealed response specific profiles. Showing mechanisms induced upon all the different acid down-shocks, such as nitrate reductase and energy production genes, and several genes specifically expressed differentially in mild or lethal levels of acidity, such as F1F0-ATPase and cydAB. Furthermore, mechanisms involved in oxidative stress response were found highly up-regulated in response to both mild and lethal acid stress. The induction of oxidative stress related genes may be a response to the formation of reactive oxygen species by a perturbation of the electron transport chain. Therefore, the formation of hydroxyl radicals and/ or peroxynitrite was monitored upon exposure to the different levels of acidity with a fluorescent probe in a flow cytometer. The formation of these oxidative compounds was shown to be specific for lethal pHs and a model to relate radical formation with the observed transcriptome profiles was proposed.

first: Richard

last: van Kranenburg

first: Clint

last: van Melis

first: Roy

last: Moezelaar

first: Tjakko

last: Abee


title: Trichoderma harzianum CECT 2413 expression in liquid basal medium or glucose-, chitin-, or tomato plant-containing media

city: Villamayor

first: M Belen

last: Suárez

state: Salamanca

address: Rio Duero

department: CIALE

country: Spain

institute: Universidad de Salamanca

summary: Trichoderma harzianum CECT 2413 expression in liquid basal medium and in the presence of glucose, chitin or tomato plants.

overall_design: Four different experimental conditions were carried out: basal (MS), glucose (MS-G), chitin (MS-Q) and tomato plant (MS-P). Two biological replicates were analyzed by microarray for each experimental condition. Three independent cultures of mycelium were pooled for each biological replicate.

first: Ilanit

last: Samolski

first: Alberto

last: de Luis

first: Enrique

last: Monte

first: Belen

last: Suarez


title: microRNA profiling in human macrophages under immunogenic treatments in vitro

status: Public on Nov 24 2009

city: Jerusalem

first: Hermona

last: Soreq

address: Safra Campus, Givat Ram

department: Department of Biological Chemistry

country: Israel

institute: The Hebrew University of Jerusalem

summary: MicroRNAs (miRs) contribute to both neuronal and immune cell fate, but their involvement in inter-tissue communication remained unexplored. The brain, via vagal secretion of acetylcholine (ACh), suppresses peripheral inflammation by intercepting cytokine production; therefore, we predicted that microRNAs targeting acetylcholinesterase (AChE) can attenuate inflammation. Here, we report that inflammatory stimuli induce leukocyte over-expression of the AChE-targeting miR-132. Injected LNA-modified anti-miR-132 oligonucleotide depleted miR-132 levels while elevating AChE in mouse circulation and tissues.  In transfected cells, a mutated 3’UTR miR-132 binding site increased AChE mRNA expression, whereas cells infected with a lentivirus expressing pre-miR-132 showed suppressed AChE. Transgenic mice over-expressing 3'UTR-null AChE showed excessive inflammatory mediators and impaired cholinergic anti-inflammatory regulation, in spite of substantial miR-132 up-regulation in brain and bone marrow. Our findings identify the AChE mRNA-targeting miR-132 as a functional regulator of the brain-to-body resolution of inflammation, opening new avenues for study and therapeutic manipulations of the neuro-immune dialogue.

platform_organism:
Homo sapiens
Mus musculus

overall_design: Pathogen-negative leukopacks (Buffy coat) obtained from the Blood Bank at the Hadassah Medical Center, Ein Kerem were transferred into 50 ml conical tubes (~10 ml/tube) and diluted 1:2 with Dulbecco's phosphate buffered saline (PBS, Biological Industries, Beit Haemek, Israel). The diluted blood was gently layered on top of 20 ml of lymphocyte separation medium (LSM)/Ficoll (MP Biomedicals, Solon, OH) without mixing the layers. Following centrifugation (500g, 30 min, 20°C, without brake), three layers were obtained (plasma & platelets, lymphocytes, erythrocytes & granulocytes). The upper phase was drawn with a Pasteur pipette until reaching 2 mm above the interphase cloud, and then 5 to 10 ml interphase lymphocytes were drawn gently with a 1ml pipette, washed once with 45 ml PBS, and centrifuged (400g , 10 min, room temp.). Pellets from all tubes were resuspended in a total of 40 ml PBS. An aliquot of cells was counted in a hemacytometer using 0.5% Trypan Blue Solution (Biological Industries) to determine cell number and viability. Cells were then centrifuged (200g, 5 min, room temp) and resuspended at a concentration of ~ 5 X 106 cells/ml in RPMI-1640 (Sigma, St. Louis, MO) supplemented with 2.5% heat-inactivated (56°C, 30 min) human serum (Sigma). 10 ml of cell suspension were plated in 75 ml flasks and incubated (2 hrs, 37°C, 5% CO¬2). Non-adherent cells were then removed and the adherent monocytes washed 3 times with warm PBS. Cells were incubated for 2hr in 10 ml per dish of RPMI-1640 supplemented with LPS-free 10% foetal bovine serum (FBS), 2 mM L-Glutamine and 0.5% penicillin/streptomycin (GIBCO, Carlsbad, CA) and washed 3 times with warm PBS and 10 ml of complete RPMI supplemented with 10 U/ml granulocyte-monocyte colony stimulating factor (GM-CSF, Sigma, G5035) added every 2-3 days. By ~day 7 monocytes were differentiated to macrophages. Escherichia coli LPS (Sigma) was added to the growth medium to a final concentration of 1μg/ml, which was found optimal to induce innate immune reaction. For comparison, 1 μM of the TLR9 ligand CpG 2006 (type B; Microsynth GmbH) were added for 24 hr. Cells were washed 2-3 times with cold PBS, then incubated in 5 ml of cold filtered PBS + EDTA 2 mM (pH = 7.2) for 10-15 min on ice. Flasks were then tapped firmly to loosen cell attachment, and cells were gently scraped and collected by centrifugation (4°C, 800 rpm, 10 min).  Short RNAs isolated from LPS-treated, CpG-treated and control cells were labeled with CyDyes 3 and 5  (each with a dye-swap), and hybridized with slides with spotted miRvana probe set (Ambion) as described for each sample.

first: Iftach

last: Shaked

first: Ari

last: Meerson

first: Yochai

last: Wolf

first: Ran

last: Avni

first: David

last: Greenberg

first: Adi

last: Gilboa-Geffen


title: Transcriptional gene silencing mediated by interplay of MOM1 and Pol V

city: Beaucouzé

first: Etienne

last: Bucher

address: 42 rue Georges Morel

institute: IRHS

summary: In plants, multiple levels of epigenetic control suppress transposon movement and permit somatic as well as transgenerational transmission of gene expression patterns. A variety of factors are involved that establish and/or maintain epigenetic marks, such as covalent modification of DNA and histones. Alteration of epigenetic marks leads to the enhancement or release of transcriptional gene silencing (TGS). TGS regulator MOM1 is special in this respect since it silences transcription by an unknown mechanism operating without obvious changes in epigenetic marks. We have isolated an enhancer of the mom1 mutation that points towards regulatory interplay between MOM1 and the plant-specific RNA Polymerase V (Pol V). Pol V transcribes heterochromatic loci and silences them. Although its biochemical properties have been studied; it is still not clear how Pol V is targeted to heterochromatin. We now provide evidence that Pol V is required for MOM1-mediated suppression of transcription at a subset of its chromosomal targets. Thus, Pol V interacts with MOM1 in the control of gene silencing. Interestingly, functional relationships between mutations in MOM1 and Pol V genes range from enhancement to independence or even suppression at different target loci.

overall_design: 4 samples: wt, mom1, nrpe1, mom/nrpe1 with 3 replicates each

first: Chotika

last: Yokthongwattana

last: Buche

first: Marian

last: Čaikovski

first: Isabelle

last: Vaillant

first: Joël

last: Nicolet

first: Regula

last: Bänniger

first: Ortrun

last: Scheid

first: Jerzy

last: Paszkowski


title: miRNA degradation in frozen tissue

city: Heidelberg

first: Mirco

last: Castoldi

address: Im Neuenheimer Feld 156

department: Department of Pediatric Oncology, Hematology and Immunology

country: Germany

institute: University of Heidelberg

summary: To determine the effect of RNA degradation on miRNA expression profiling.

overall_design:
To mimic total RNA degradation in snap-frozen livers and duodenums from mice (wild type female, C57/Bl6) tissues were stored in a -80°C freezer and then transferred to dry ice. Each frozen tissue was sliced into five identical pieces which then were transferred into eppendorf tubes. At time point zero (T0), all samples were placed on ice and total RNA was extracted immediately from (T0) and at later time points [30 min (T30), 60 min (T60), 120 min (T120) and 240 min (T240)]. RNA integrities were assessed using the Agilent Bioanalyzer 2100 which calculates RIN values of assayed RNAs. We found that at T0, the RNA integrity for both liver and duodenum was above 7, indicating good quality total RNA. However, 30 min on ice was sufficient to reduce RNA integrity, indicated by the decrease in RINs. These findings indicate that RNA degradation can take place in defrosted tissue at low temperature (i.e. on ice). For comparison, we assessed the extent to which RNA integrity is preserved in freshly harvested tissues when tissue processing is delayed. Liver and duodenum samples were collected from mice and either processed immediately or maintained on ice, as described above. Bioanalyzer electropherograms of the liver samples did not indicate any significant total RNA degradation even when samples remained on ice for up to 4 hrs. By contrast, duodenal samples (which are rich in RNases) do show high susceptibility to degradation similar to the snap-frozen defrosted material. These findings suggest that tissues such as duodenum or pancreas should either be processed immediately or snap-frozen and processed individually.
To assess to which extent total RNA degradation affects miRNA expression profiles, we hybridized to the miCHIP microarray total RNAs extracted from freshly harvested livers and duodenums liver and from snap frozen livers and duodenums. Hierarchical clustering (HCL), by Pearson correlation, was used to cluster samples based on their miRNA expression profiles. Our analysis shows that miRNAs extracted from freshly harvested liver is less degraded (matrix plot analyses). Consistent with high level of RNases present in duodenum, miRNAs from freshly harvested duodenum are extensively degraded. Based on these data, we conclude that samples with low RIN values (less than 7) do not merit analysis on miRNA arrays.

last: Ibberson

first: Vladimir

last: Benes

first: Matthias

middle: W

last: Hentze

first: Martina

middle: U

last: Muckenthaler


title: Transcriptional profiling at different sites in lungs of pigs during acute bacterial respiratory infection

city: Tjele

first: Jakob

last: Hedegaard

address: PO-box 50

department: Department of Genetics and Biotechnology

country: Denmark

institute: Aarhus University, Faculty of Agricultural Sciences

summary: The local transcriptional response was studied in different locations of lungs from pigs experimentally infected with the respiratory pathogen Actinobacillus pleuropneumoniae serotype 5B, using porcine cDNA microarrays. This infection gives rise to well-demarcated infection loci in the lung, characterized by necrotic and haemorrhagic lesions. Lung tissue was sampled from necrotic areas, from visually unaffected areas and from areas bordering on necrotic areas. Expression pattern of these areas from infected pigs was compared to healthy lung tissue from un-infected pigs. Transcription of selected genes important in the innate defence response were further analysed by quantitative real-time reverse-transcriptase PCR. A clear correlation was observed between the number of differentially expressed genes as well as the magnitude of their induction and the sampling location in the infected lung, with the highest number of differentially expressed genes, and the most highly induced genes found in necrotic areas. Interestingly, a group of differentially regulated genes was represented in all three areas, comprising genes encoding cytokines, acute phase proteins, and factors related to regulation of apoptosis and the complement system. Interferon-gamma was down-regulated in both necrotic and bordering areas. Evidence of neutrophil recruitment was seen by the up-regulation of chemotactic factors for neutrophils. In conclusion, we found subsets of genes expressed at different levels in the three selected areas of the infected lung as compared to the control group. Thus it is demonstrated that an infection with clearly defined infected loci leads to a rapid disseminated intra-organ response in neighbouring seemingly unaffected tissue areas of the infected organ. Within the lung, we found a clear division of induced genes as, in unaffected areas a large part of differently expressed genes were involved in systemic reactions to infections, while differently expressed genes in necrotic areas were mainly concerned with homeostasis regulation.

overall_design: Expression profiling was conducted by hybridising each sample against a common reference consisting of a pool of equal amount of total-RNA from all samples.

first: Shila

last: Mortensen

first: Kerstin

last: Skovgaard

first: Christian

last: Bendixen

first: Peter

last: Heegaard


title: Transcriptional profiling of the rat hypothalamus response to TCDD

city: Toronto

first: Paul

last: Boutros

state: Ontario

address: 101 College Street, Suite 800

country: Canada

institute: Ontario Institute for Cancer Research

summary: Rodents exposed to the environmental contaminant, TCDD, suffer from a number of acute and chronic toxicities, including lethality and a wasting syndrome. Hypothesizing that the wasting syndrome may be caused by changes in neural control of energy flux and metabolism, we profiled the transcriptional response of rat hypothalamus to TCDD. We employed two separate rat strains: the Long-Evans strain is sensitive to TCDD toxicities while the Han/Wistar strain is over four orders of magnitude more resistant. Surprisingly, few transcriptional changes were induced by TCDD in either strain. Only four genes were altered in Long-Evans rats, including three classic TCDD-responsive genes: Cyp1a1, Cyp1b1, and Nqo1. These three genes were also altered in Han/Wistar rats, along with 133 additional genes. However, the magnitudes of alteration of these additional genes was very modest, with most changes well below two-fold in magnitude. We therefore concluded that rat hypothalamus is mostly refractory to TCDD exposure, at least at the doses and time-points surveyed here.

overall_design: Two strains, each with drug-treated vs. vehicle-control

first: Allan

last: Okey

first: Raimo

last: Pohjanvirta


title: 48-h stretched human periodontal ligament cells

status: Public on Nov 21 2009

city: Suita

first: Chiharu

last: Fujihara

state: Osaka

address: 1-8, Yamadaoka

department: Periodontology

country: Japan

institute: Osaka University Graduate School of Dentistry

summary: Human periodontal ligament (HPDL) is continuously exposed to mechanical stress in vivo. In this study, in utilizing DNA chips, we analyzed the influences of mechanical stress on the gene expression profile of HPDL cells in vitro. HPDL cells were obtained from extracted first premolars of individuals undergoing tooth extraction for orthodontic treatment. Then, HPDL cells were applied to a stretch apparatus. They were constantly stretched and relaxed at 0.5 Hz for 48hr with 110％ force elongation. After the application of the cyclic tension force, total RNA was extracted. Then, in utilizing the DNA chips (Human Oligo 30K DNA Chip containing almost all human genes in the genome). We analyzed the differences of gene expression between the stretched and the non-stretched control HPDL cells The DNA chip analysis identified 17 up-regulated genes that showed at least 2-fold difference in their relative intensities between the stretched and the control. This result included the genes for the glutamate receptor binding protein: HOMER1, for the growth factor receptor: CNTFR, for the ECM remodeling protein: MMP15, for the protein interacting with calcineurin A: DSCR1, for the cytoskeletal protein: LRRFIP1, for the glutamate receptor: GRIN3A and some novel genes. On the basis of these data, we suggest that mechanical stress, in other words in vivo occlusal force, may affect the functions of HPDL cells

overall_design: Gene expression profiles were constructed using Human Oligo DNA Chip 30K (Hitachi software engineering) in human periodontal ligament cells (The 48-h stretched and the 48-h non-stretched, control).

first: Satoru

last: Yamada


title: Determining lovastatin-induced differences in expression between statin sensitive and insensitive MM cells

summary:
The goals of this study were to determine global differences in transcript expression and regulation between MM cells that are sensitive or insensitive to lovastatin-induced apoptosis.  To this end, two sensitive (KMS11 and H929) and two insensitive (LP1 and SKMM1) MM cell lines treated with 20uM lovastatin or an ethanol vehicle control for 16 hours.  mRNA was extracted and prepared for mRNA expression microarrays (HG-U133 Plus 2) in triplicate.

Keywords: drug-response across cell-lines

overall_design: Four cell-lines, each treated with lovastatin or vehicle control, with three replicates per condition

first: James

last: Clendening

first: Igor

last: Jurisica

first: Linda

middle: Z

last: Penn


title: Profiling uterine fibroid specimens with an in-house array representing all 90 human tyrosine kinases and their ligands

city: La Jolla

first: Yong

last: Jiang

state: CA

address: 3855 Health Science Dr.

department: Medicine

institute: UCSD-Moores Cancer Center

summary: The aim of this study is to compare the expression patterns of tyrosine kinases and their ligand activators between matched myometrium and leiomyoma tissues. For this, total RNA samples extracted from 42 pairs of matched fibroid and adjacent myometrial tissues were hybridized to a set of 840 customized oligonucleotide microarrays to compare the expression profiles of 244 selected human genes including all 90 tyrosine kinases and 103 ligands. Among the 244 genes surveyed, 38 were found to be differentially expressed between pairs of myometrium and fibroid tissues. Clustering analysis of the expression ratios of these 38 genes from 42 pairs of samples has led to the subdivision of fibroid samples into three groups, based on the expression ratios of two peptide ligands, CYR61 and EFNA4. Real-time quantitative RT-PCR measurements of additional 32 pairs of samples further confirmed the existence of these three groups of fibroid samples. In conclusion, peptide ligand activators of protein tyrosine kinases are differentially expressed between myometrium and fibroid tissues. This finding supports the notion that tyrosine kinases may be exploited as medical targets to treat symptomatic fibroids without surgical intervention.

overall_design: This study has been accomplished with 42 patients fibroid specimen and their matched myometrium, each pair of specimen has been repeated at least 3 times.

first: Guangli

last: Suo

first: Xin

last: Lu

first: Anil

last: Sadarangani

first: Brian

last: Cowan

first: Jean

middle: Y

last: Wang


title: Persistent Organic Pollutants cause insulin resistance and ruin the health effects of salmon oil

city: Pierre Bénite

first: emmanuelle

last: meugnier

address: Cens Eli Bat 2 D- Hopital Lyon Sud

summary:
Transcriptional profiling of Rat liver submitted to high fat diets made with either crude (HFC) or refined salmon oil (HFR).

Keywords: transcriptomic analysis

overall_design: Two groups of rats were submitted  to high fat diet containing either crude salmon oil (HFC) or refined salmon oil (HFR). After 28 days, livers were retrieved and RNA extracted. A pool of RNA from HFR group was used as the reference group.  Each HFC was compared to reference group (pool of HFR).  Five biological replicates were processed.

first: Emmanuelle

last: Meugnier

first: Sandra

last: Pesenti

first: Hubert

last: Vidal

first: Jerome

last: Ruzzin


title: A systematic evaluation of pattern discovery algorithms

first: Paul

last: Boutros

summary: Pattern discovery algorithms are methods for discovering recurrent, non-random motifs widely used in the analysis of biological sequences. Many algorithms exist but few comparisons have been made amongst them. We systematically profile eight representative methods at multiple parameter settings across 174 diverse experimental datasets, including ten novel ChIP-on-chip datasets. We executed 16,777 pattern discovery analyses to assess prediction accuracy, CPU usage and memory consumption. For 144 datasets we developed a gold-standard using machine-learning algorithms; cross-validation was used for the remaining datasets. Performance was highly disparate, with median accuracy ranging from 32% to 96%. Importantly we were unable to replicate previously reported algorithm-rankings, emphasizing the need to use many and diverse experimental datasets. We found deterministic algorithms like Projection and Oligo/Dyad had the highest prediction accuracy. Computational efficiency was not linearly related to dataset size and becomes critical: some algorithms are intractably slow on large datasets. This work provides the first combined assessment of the CPU, memory, and prediction accuracies of pattern discovery algorithms on real experimental datasets.

overall_design:
HL60-Mnt-ChIP: ChIP-Chip with 10 biological replicates
HL60-Trrap-ChIP: ChIP-Chip with 13 biological replicates

first: Adam

middle: P

last: Hanley


title: Effect of rosuvastatin and atorvastatin in combination with high-cholesterol diet on cholesterol homeostasis

status: Public on Nov 20 2009

city: Ljubljana

first: Peter

last: Juvan

address: Zaloska 4

department: Institute of Biochemistry

country: Slovenia

institute: Faculty of Medicine, University of Ljubljana

summary: The aim of the study was to see any effect of rosuvastatina or atorvastatin on cholesterol homeostasis after 24 hours in C57BL/6 hyperlipidemic mice (induced by diet). Experiments were perfomed using a custom Steroltalk v2 microarray. Effect of both statins on drug metabolism was also evaluated. All experiments were done within the European sixth Framework program “Steroltalk” (www.steroltalk.net).

overall_design: Animals were given one peroral application of vehicle (tap water), 20 mg/kg rosuvastatin or 40 mg/kg atorvastatin after one week of 1% cholesterol diet. After 24h they were sacrificed and total RNA was isolated from the livers. Each sample was hybridized with a reference sample to Steroltalk v2 microarrays.

first: Tadeja

last: Režen

first: Damjana

last: Rozman


title: MicroRNA Expression in Rat Myocardial Infarction intervened by propranolol

status: Public on Nov 19 2009

city: Harbin

first: Wenliang

last: Zhu

state: Heilongjiang

address: Xuefu road 194

department: 1Institute of Clinical Pharmacology

institute: The Second Affiliated Hospital of Harbin Medical University

summary: We established a rat model of myocardial Infarction by performing a surgical operation of left anterior descending coronary artery occlusion under sterie conditions. Wistar rats were previously randomly divided into 4 groups: control, ischemia, ischemia-propranolol, and non-ischemia-propranolol. Our data suggested that propranolol could reverse many microRNAs with too high or too low expression in the ischemia group versus control group.

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: Wistar rats were initially anesthetized with pentobarbital (40 mg/kg, i.v.), which were previously randomly divided into 4 groups: control, ischemia (MI), ischemia-propranolol (MI-PRO), and non-ischemia-propranolol (NMI-PRO). The rat model of MI was established by performing a surgical operation of left anterior descending coronary artery occlusion under sterie conditions. Successful occlusion was confirmed by an increase in the amplitude of R wave of lead I during the first few seconds of each occlusion and elevation of S-T segment of lead II, and a 20-30% decline in the arterial blood pressure compared to the pre-ischemic values mesured by a previously installed ECG recorder (BL 420, ChengDu TME Technology Co, Ltd, ChengDu, China). Propranolol was administrated 2 months before the experiment with daily oral doses of 50 mg/kg. Hearts were quickly isolated after the rats were sacrificed and the ischemic zone of the left ventricle was prepared for the miRNA microarray experiment. Control and NMI-PRO animals underwent open chest procedures without coronary artery occlusion.


title: Genome-wide characterization of the transcriptional program of Myc-dependent transformation, expression study

summary:
We profiled Myc binding in a normal breast cell-line (MCF10A) under basal conditions after ectopic expression of Myc. We showed that ectopic Myc expression increases tumour formation in vivo and in vitro. We then profiled genome-wide Myc binding using Agilent promoter arrays in MCF10A cells expressing wild-type and ectopic Myc. We show that Myc binds to a greater number of spots in wild-type than in Myc cells, but that some targets are unique to each condition.

Keywords: genetic-modification and stress-response

overall_design: We compared ectopic Myc to wild-type cells under wild-type and serum-deprived conditions with 4 replicates per condition

first: Amanda

last: Wasylishen

first: Romina

last: Ponzielli

first: Igor

last: Jurisica

first: Linda

last: Penn


title: bre1 vs WT transcriptional profile

city: San Francisco

first: William

last: Hwang

address: Lim Lab - Box 0525, 35 Medical Center Way

department: Neurosurgery

institute: UCSF

summary: Relative mRNA abundance of all S. cerevisiae genes was measured in bre1 compared to wild-type.

overall_design: Arrays done in quadruplicate with dye-swaps.

first: Hiten

last: Madhani


title: CGH profiling of Bacillus cereus isolates from the CDC historical strain collection

city: Rockville

first: Chun-Hua

last: Wan

summary:
The goal of this study is the discovery of (a) meaningful phylogenomic relationships among members of this B. cereus/B. anthracis group, and (b) reliable gene-phenotype associations, e.g. recognition of links between genomic traits and the ability of certain strains to cause various forms of disease. We also tried to elucidate genome evolution aspects that may lead to the emergence of variants that are capable (or have the potential) of causing anthrax-like disease. This large-scale comparative genomics approach is unprecedented for this taxonomic group.

Dr. A. Hoffmaster (CDC) provided the PFGRC with 73 B. cereus and B. anthracis isolates from the CDC culture collection. Of these, 27 were isolated from patients with severe or systemic disease; ten isolates of this group were obtained from patients (welding factory workers) with anthrax-like disease or from the environment near their workplace. Another set of 26 represented isolates from food-born illnesses. Of the 26 gastrointestinal disease isolates (GIDI), 10 were obtained from patients with diarrhea, whereas another set of 10 had been shown to harbor the emetic (vomit) toxin gene by PCR. The rest of the group consisted of 20 isolates with various phenotypes. All strains were screened for their genomic content using the B. cereus/B. anthracis species microarray.

sample_organism:
Bacillus anthracis
Bacillus cereus
Bacillus anthracis str. Sterne
Bacillus cereus G9842

overall_design: Seventy-three query strains were investigated in this study, with each query strain hybridized against the reference strain, Sterne. Dye-swap experiments were performed with all the 73 strains on both chipA and chipB of the microarray, for a total of four or more hybridizations per query strain. Each 70mer oligo spotted on the B. cereus species microarray is spotted once. Positive controls on the array consist of oligos designed from the sequenced reference genome, Sterne, and negative controls on the array consist of oligos designed from the thale cress plant, Arabidopsis thaliana.

first: L

last: Papazisi

first: B

last: Remortel

last: Appalla

first: F

last: Onmus-Leone

first: H

last: Sajadi

first: S

last: Ratnayke

first: Q

last: Sun

first: C

first: C.

last: Beesley

first: R

last: Fleischmann

first: A

last: Hoffmaster

last: Peterson


title: Whole-Genome Transcriptional analysis of Sulfate and Sulfonate assimilation by Bradyrhizobium japonicum USDA 110

status: Public on Nov 17 2009

city: Saint Paul

first: Masayuki

last: Sugawara

state: MN

address: 250 Borloug Hall, 1991 Upper Buford Circle

department: Soil, Water, and Climate

institute: University of Minnesota

summary: The ability of Bradyrhizobium japonicum and B. elkanii strains to utilize alkane and aromatic sulfonates as sole sources of sulfur for growth was investigated. All of the strains tested were able to utilize alkane sulfonates, but not aromatic sulfonates for growth. Whole-genome transcriptional profiling was used to assess B. japonicum USDA 110 genes involved in growth on alkane sulfonates, as compared to growth on sulfate and cysteine. Two sets of genes, bll7007 to bll7011 and bll6449 to 6456 were highly expressed during growth with sulfate and sulfonates. These genes were predicted to encode alkanesulfonate monooxygenases and ABC transporter components. Reverse transcription-PCR (RT-PCR) analyses showed that these genes were organized in two operon-like structures and expressed as polycistronic messages. The sulfonate monooxygenase encoded by bll7010 (ssuD) complemented an E. coli mutant defective in utilization of sulfonates. The expression of many genes that were induced during growth on cysteine and taurine were under the control of the FixLJ-FixK2-FixK1 symbiotic nitrogen fixation cascade, indicating there is a novel linkage between sulfur metabolism and nitrogen fixation. Taken together, results of this study indicate that Bradyrhizobium sp. strains are metabolically diverse and likely use organosulfur compounds for growth and survival, and for legume nodulation and nitrogen fixation in soil systems.

overall_design: Three independent biological materials were prepared for sulfate or sulfonate supplemented cells. Total 12 arrays including dye swap were analyzed.

first: Gopit

last: Shah

first: Michael

last: Sadowsky

first: Oleg

last: Paliy

first: Prasad

last: Gyaneshwar


title: A Region of Human HoxD that Confers Polycomb-Group Responsiveness

city: Boston

first: Peter

last: Kharchenko

address: 10 Shattuck St.

department: DBMI

institute: Harvard Medical School

summary: Polycomb group (PcG) proteins are essential for accurate axial body patterning during embryonic development. PcG-mediated repression is conserved in metazoans and is targeted in Drosophila by Polycomb response elements (PREs). Targeting sequences in humans have not been described.  While analyzing chromatin architecture in the context of human embryonic stem cell (hESC) differentiation, we discovered a 1.8kb region between HOXD11 and HOXD12 (D11.12) that is associated with PcG proteins, is nuclease hypersensitive, and shows alteration as hESCs differentiate. D11.12 repressed luciferase expression from a reporter construct both before and after differentiation of mesenchymal stem cells into adipocytes.  Full repression by D11.12 required a highly conserved region and YY1 binding sites.  Repression relied upon PcG proteins Bmi1 and Eed and a YY1-interacting partner, RYBP. We conclude that D11.12 is a Polycomb-dependent regulatory region with similarities to Drosophila PREs, indicating conservation in the mechanisms that target PcG function in mammals and flies.

overall_design: Data contains microarray measurements of the MNase-digested mononucleosomal fragments in hES cells, derived MSCs, osteoblasts and adipocytes. The ChIP-chip data includes Suz12, Bmi1 and H3K27me3 measurements in MSCs and adipocytes.

first: Caroline

last: Woo

middle: V

first: Laurence

last: Daheron

last: Park

first: Robert

middle: E

last: Kingston


title: Molecular evolution of sex-biased genes in the Drosophila ananassae subgroup

city: Planegg-Martinsried

first: Sonja

last: Grath

address: Grosshaderner Str. 2

department: Department Biology II

institute: LMU

summary: Genes with sex-biased expression often show rapid molecular evolution between species. Previous population genetic and comparative genomic studies of Drosophila melanogaster and D. simulans revealed that male-biased genes have especially high rates of adaptive evolution. To test if this is also the case for other lineages within the melanogaster group, we investigated gene expression in D. ananassae, a species that occurs in structured populations in tropical and subtropical regions. We used custom-made microarrays and published microarray data to characterize the sex-biased expression of 129 D. ananassae genes whose D. melanogaster orthologs had been classified previously as male-biased, female-biased, or unbiased in their expression and had been studied extensively at the population-genetic level.

overall_design: In total, 12 arrays were analyzed. These arrays include 4 biological replicates for strain KK1 (Kota Kinabalu, Malaysia) + 4 dye-swaps, and 2 biological replicates for strain KK2 (Kota Kinabalu, Malaysia) + 2 dye-swaps.

first: John

last: Baines

last: Parsch


title: Transcriptional signatures associated with species divergence in New Zealand Pachycladon (Brassicaceae)

city: Palmerston North

first: Claudia

last: Voelckel

address: Riddet Road

department: Allan Wilson Centre for Molecular Ecology and Evolution

country: New Zealand

institute: Massey University

summary: Comparative gene expression profiling of a) above-ground tissues of three fruiting species (P. cheesemanii, ch, P. exile, ex, P. novae-zelandiae, nz), b) above-ground tissues of two rosette stage species (P. fastigiatum, fa, P. enysii, en) and c) roots of all five species using heterologous A. thaliana microarrays

sample_organism:
Pachycladon cheesemanii
Pachycladon fastigiatum
Pachycladon novaezelandiae
Pachycladon enysii
Pachycladon exile

overall_design: 8-12 specimens per species grown in common garden, RNA from roots and aboveground tissues extracted separately from each specimen, individual samples pooled in equal amounts resulting in five above ground samples (ch_l, ex_l, nz_l, fa_l, en_l) and five root samples (ch_r, ex_r, nz_r, fa_r, en_r), 36 two channel microarrays used in total: five above-ground samples hybridized in a loop-like fashion using 14 arrays, five root samples hybridized in a loop-wise fashion using 10 arrays; five root and three leaf samples hybridized in a loop-like fashion using 12 arrays, thus each sample hybridized multiple times (4-12) while being labelled either with cy3 or cy5

first: B

last: Janssen

first: L

last: Luo

first: P

last: Lockhart


title: Effect of TCPOBOP and PCN in combination with high-cholesterol diet on genes involved in cholesterol homeostasis

last: Juvan

summary: TCPOBOP (1,4-bis[2-(3,5-dichloropyridyloxy)]benzene) and PCN (pregnenolone 16α-carbonitrile) are inducers of drug metabolism through activation of nuclear receptors CAR (constitutive androstane receptor) and PXR (pregnane X receptor), respectively. Mouse experiment was designed to study the effect of CAR and PXR activation on cholesterol homeostasis genes and other genes, which are present on the Steroltalk v2 microarray. Treatments were combined with standard and high-cholesterol diet to observe the interference of high liver cholesterol on nuclear receptor transcription regulation. All experiments were done within the European sixth Framework program “Steroltalk” (www.steroltalk.net). Results form these experiments give new knowledge about involvement of ‘xenosensors’ CAR and PXR in regulation of endogenous liver metabolism.

overall_design: Animals were injected i.p. 3mg/kg TCPOBOP, 40 mg/kg PCN or vehicle (corn oil) in combination of one week of standard or 1% cholesterol diet prior tretament. After 24h they were sacrificed and total RNA was isolated from the livers. Each sample was hybridized with a reference sample to Steroltalk v2 microarrays.

first: Tadeja

last: Režen

first: Damjana

last: Rozman


title: Changes in the Pattern of DNA Methylation Associate with Twin Discordance in SLE

city: Badalona, Barcelona

first: Esteban

last: Ballestar

state: N/A = Not Applicable

address: Ctra de Can Ruti, Camí de les Escoles s/n

institute: Josep Carreras Research Institute (IJC)

summary: This is the first high-throughput analysis of DNA methylation in autoimmune diseases. We have used a cohort of MZ twins discordant for three diseases whose clinical signs often overlap: systemic lupus erythematosus (SLE), rheumatoid arthritis and dermatomyositis. Only MZ twins discordant for SLE featured widespread changes in the DNA methylation status of a significant number of genes. Individual analysis confirmed the existence of DNA methylation and expression changes in genes relevant to SLE pathogenesis. Our findings not only identify potentially relevant DNA methylation markers for the clinical characterization of SLE patients but also support the notion that epigenetic changes may be critical in the clinical manifestations of autoimmune disease.

overall_design: Total DNA isolated by standard procedures from 59 White Blood Cell (WBC) samples corresponding to monozygotic twins discordant for three different autoimmune diseases: systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and dermatomyositis (DM) and two additional controls for each MZ twin pair.

first: Biola

last: Javierre

first: Agustin

last: Fernandez

first: Julia

last: Richter

first: Fatima

last: Al-Shahrour

first: J

middle: I

last: Martin-Subero

first: Javier

last: Rodriguez-Ubreva

first: Maria

last: Berdasco

first: Mario

last: Fraga

first: Terrance

last: O'Hanlon

first: Lisa

last: Rider

first: Filipe

last: Jacinto

first: F

last: Lopez-Longo

first: Joaquin

last: Dopazo

first: Marta

last: Forn

first: Miguel

middle: A

last: Peinado

first: Luis

last: Carreño

first: Amr

middle: H

last: Sawalha

last: Harley

first: Reiner

last: Siebert

first: Manel

last: Esteller

first: Frederick

last: Miller


title: ChIP-chip of HA-tagged Lsr2 in Mycobacterium tuberculosis and Mycobacterium smegmatis

status: Public on Nov 15 2009

first: Blair

last: Gordon

address: 1 King's College Circle

department: Molecular Genetics

institute: University of Toronto

summary: Bacterial nucleoid-associated proteins play important roles in chromosome organization and global gene regulation. We find that Lsr2 of Mycobacterium tuberculosis is a novel nucleoid-associated protein that specifically binds AT-rich regions of the genome, including regions encoding major virulence factors, such as the ESX secretion systems, the lipid virulence factors PDIM/PGL, and the PE/PPE families of antigenic proteins. Comparison of genome-wide binding data with expression data indicates that Lsr2 binding results in transcriptional repression. Domain swamping experiments demonstrate that Lsr2 has an N-terminal dimerization domain and a C-terminal DNA binding domain. NMR analysis of the DNA binding domain of Lsr2 and its interaction with DNA reveals a novel structure and a unique mechanism that enables Lsr2 to discriminately target AT-rich sequences through interactions with the minor groove of DNA. Taken together, we provide evidence that mycobacteria have employed a structurally distinct molecule with an apparently different DNA recognition mechanism to achieve an equivalent function as the Enterobacteriaceae H-NS, coordinating global gene regulation and virulence in this group of medically important bacteria.

sample_organism:
Mycolicibacterium smegmatis
Mycobacterium tuberculosis H37Rv

platform_organism:
Mycobacterium tuberculosis H37Rv
Mycolicibacterium smegmatis MC2 155

overall_design: Comparison of Lsr2 chromatin-immunoprecipitated DNA sequences to total reference DNA

first: Yifei

last: Li

first: Linru

last: Wang

first: Anna

last: Sintsova

first: Harm

last: van Bakel

first: Songhai

last: Tian

first: William

last: Navarre

first: Bin

last: Xia

first: Jun

last: Liu


title: Pichia angusta cells glucose- vs methanol-grown

city: Haren

first: Tim

last: van Zutphen

state: Groningen

address: Kerklaan 30

department: Molecular Cell Biology

institute: University of Groningen

summary: Transcriptional profiling of Pichia angusta NCYC 495 leu1.1 cells growing exponentially on mineral medium containing 0.5% glucose versus a shift for 2 hours to mineral medium containing 0.5% methanol.

overall_design: Two-conditions experiment, 4 individual glucose-grown cultures were shifted at OD660nm 2.3 to fresh mineral medium OD 0.2 containing 0.5% methanol for 2 hours. Transcriptional profiling was performed using total RNA from the glucose-grown cells at the moment of the shift versus cells adapted to methanol for 2 hours.

first: Richard

middle: J

last: Baerends

first: Kim

last: Susanna

first: Anne

last: de Jong

first: Oscar

last: Kuipers

first: Marten

last: Veenhuis

first: Ida

last: van der Klei


title: Nucleases Encoded by Integrated Elements CJIE2 and CJIE4 Inhibit Natural Transformation of Campylobacter jejuni

status: Public on Nov 14 2009

city: Albany

first: Craig

middle: T

last: Parker

address: 800 Buchanan St

department: PSM

institute: USDA ARS

summary: The species Campylobacter jejuni is naturally competent for DNA uptake; nevertheless, nonnaturally transformable strains do exist. For a subset of strains we previously showed that a periplasmic DNase, encoded by dns, inhibits natural transformation in C. jejuni. In the present study, genetic factors coding for DNase activity in absence of dns were identified. DNA arrays indicated that nonnaturally transformable dns-negative strains contain putative DNA/RNA non-specific endonucleases encoded by CJE0566 and CJE1441 of strain RM1221. These genes are located on C. jejuni integrated element 2 and 4. Expression of CJE0566 and CJE1441 from strain RM1221 and a homologous gene from strain 07479 in DNase-negative Escherichia coli and C. jejuni strains indicated that these genes code for DNases. Genetic transfer of the genes to a naturally transformable C. jejuni strain resulted in a decreased efficiency of natural transformation. Modelling suggests that the C. jejuni DNases belong to the Serratia nuclease family. Overall, the data indicate that the acquisition of prophage encoded DNA/RNA non-specific endonucleases inhibits the natural transformability of C. jejuni through hydrolysis of DNA.

overall_design: The genomic diversity of 15 naturally competent or nonnaturally transformable  Campylobacter jejuni strains  were examined by microarray-based comparative genomic indexing (CGI) analysis. The CGI analysis allowed the assessment of CDS content for each C. jejuni strain relative to the C. jejuni DNA microarray, which comprises ORFs from strains NCTC 11168, RM1221. ORFs were spotted in duplicate. Genomic DNA from strains NCTC 11168/RM1221 were used as a reference DNA and competitively hybridized with genomic DNA from each of the other C. jejuni strains. Two replicates for each strain were performed. Data normalization was performed as in Parker et al. J Clin Microbiol 2006, 44(11):4125-4135.

first: Esther

last: Gaasbeek

first: Jaap

last: Wagenaar

first: Magalie

last: Guilhabert

first: Jos

last: van Putten

first: Fimme

last: van der Wal


title: Aerobic transcriptome of Porphyromonas gingivalis

status: Public on Nov 13 2009

city: Richmond

first: Janina

last: Lewis

state: VA

address: 521 North 11th Street

department: Philips Institute of OCMB

institute: Virginia Commonwealth University

summary: Transcriptional profiling of P. gingivalis cells comparing control cells grown in anaerobic conditions with cells grown in the presence of 6% of oxygen

overall_design: Two-condition experiment, anaerobically vs. aerobically grown cells. Biological replicates: 3 independently grown and harvested bacterial cells. Two technical replicates for two experiments.  Four replicates per array.


title: MacroH2A1 ChIP-chip from IMR90 cells

city: Dallas

first: W. Lee

last: Kraus

state: TX

address: 5323 Harry Hines Blvd.

institute: UT Southwestern Medical Center

summary: MacroH2A1 is a histone variant that is enriched on the inactive X chromosome (Xi) in mammals and is postulated to play an important, but unknown, role in the repression of gene expression.  Here we show that although macroH2A1 marks repressed autosomal chromatin, it positively regulates transcription when located in the transcribed regions of many target genes.  We used chromatin immunoprecipitation coupled with tiling microarrays (ChIP-chip) to determine the genomic localization of macroH2A1 in IMR90 human primary lung fibroblasts and MCF-7 breast cancer cells.  The patterns of macroH2A1 deposition are largely similar across the autosomes of both cell lines.  Our studies revealed a genomic localization pattern unique among histone variants, namely the occupation by macroH2A1 of large chromatin domains (>500 kb in some cases) that contain repressive chromatin marks (e.g., histone H3 lysine 27 trimethylation).  The boundaries of macroH2A1-containing domains tend to occur in promoter proximal regions.  Not all promoters, however, serve as macroH2A1 boundaries; many macroH2A1-containing chromatin domains invade the transcribed regions of genes whose products play key roles in development and cell-cell signaling.  Surprisingly, the expression of many of these genes is positively regulated by macroH2A1.  MacroH2A1 also plays a role in augmenting signal-regulated transcription, specifically for genes responsive to serum starvation.  Collectively, our results document an unexpected role for macroH2A1 in the escape from heterochromatin-associated silencing and the enhancement of autosomal gene transcription.

overall_design: Two macroH2A1 ChIP-chip biological replicates from IMR90 human embryonic lung fibroblasts are included.

first: Matthew

last: Gamble

first: Lee


title: Expression of phosphofructokinase in Neisseria meningitidis

status: Public on Nov 11 2009

city: Gent

first: Gino

last: Baart

state: East Flanders

address: Technologiepark 927

department: Plant Systems Biology

country: Belgium

institute: Ghent University

summary: Neisseria meningitidis serogroup B is a pathogen that can infect diverse sites within the human host. According to the N. meningitidis genomic information and experimental observations glucose can be completely catabolized through the Entner-Doudoroff pathway and the pentose phosphate pathway. The Embden-Meyerhof-Parnas pathway is not functional, because the gene for phosphofructokinase is not present. The phylogenetic distribution of phosphofructokinase indicates that in most obligate aerobic organisms PFK is lacking. We conclude that this is because of the limited contribution of PFK to the energy supply in aerobically grown organisms in comparison with the energy generated through oxidative phosphorylation. Under anaerobic or microaerobic conditions the available energy is limiting and PFK provides an advantage, which explains the presence of PFK in many (facultative) anaerobic organisms. In accordance with this, in silico flux balance analysis predicted an increase of biomass yield as a result of PFK expression. However, analysis of a genetically engineered N. meningitidis strain that expresses a heterologous phosphofructokinase showed that the yield of biomass on substrate decreased in comparison with a pfkA deficient control strain, which was associated mainly with an increase in CO2 production, whereas production of by-products was comparable between the two strains. This might explain why the pfkA gene has not been obtained by horizontal gene transfer, since it is initially unfavourable for biomass yield. No large effects related to heterologous expression of pfkA were observed in the transcriptome. Although our results suggest that introduction of PFK does not contribute to a more efficient strain in terms of biomass yield, achievement of a robust, optimal metabolic network that enables a higher growth rate or a higher biomass yield, might be possible after adaptive evolution of the strain, which remains to be investigated.

overall_design: Two-condition experiment, 3 replicates per condition

middle: G

first: Marc

last: Langenhof

first: Bas

last: van de Waterbeemd

first: Hendrik-Jan

last: Hamstra

first: Bert

last: Zomer

first: Leo

middle: L

last: van der Pol

first: Coen

last: Beuvery

first: Johannes

last: Tramper

first: Dirk

middle: D

last: Martens


title: Genomic hybridization of several Actinobacillus pleuropneumoniae serotypes' reference strains compared to strain JL03

city: Wuhan

first: Lu

last: Li

email: sakura.tree@163.com, xuzf@mail.hzau.edu.cn, xuzhuofei@sohu.com, kobe2071@tom.com

state: Hubei

address: Shizishan Street 1

institute: Huazhong Agricultural University

type: Genome variation profiling by genome tiling array

summary:
To make clear the genetic diversity and virulence evolution at the genome-wide level of A. pleuropneumoniae

Keywords: comparative genomic hybridization

overall_design: Gene content in various Actinobacillus pleuropneumoniae strains compared to A. pleuropneumoniae JL03 (GenBank Accession number: CP000687). Strains have been compared to the JL03 strain by comparative genomic hybridization using genome-wide 60-mer oligonucleotides microarrays representing the JL03 genome. Genes have been deemed present or divergent in the various strains.

first: Huanchun

last: Chen

first: Rui

last: Zhou

first: Zhuofei

last: Xu


title: A proteomic view on the cell biology of Staphylococcus aureus proteome

status: Public on Nov 12 2009

city: Greifswald

first: Jan

last: Pané-Farré

address: F.-L.-Jahn-Str. 15

department: Department of Microbial Physiology

summary:
The genome sequence is the “blue-print of life”, but proteomics is required to relate this blue-print of life to the actual physiology of living cells. Because of their low complexity – only about 2.000 proteins make a Staphylococcus aureus cell viable – bacteria are excellent model systems to identify the entire protein assembly of a living organism. Many of the studies on physiological proteomics of bacteria, however, still rely on 2D gel-based technologies that visualize only a minor part of the proteome. In this study it will be shown that the majority of proteins expressed in the Gram-positive human pathogen S. aureus can be identified and even quantified by a combination of gel-based and gel-free approaches, the latter having undergone formidable improvements over the last 10 years. S. aureus has been the model of choice for our proteomic studies, because it combines high pathogenicity with increasing antibiotic resistance, which calls for new leads in the development of anti-staphylococcal therapy.
On the way towards the entire proteome of S. aureus, we analysed four subproteomic fractions: cytosolic proteins, membrane-bound proteins, cell-surface associated and extracellular proteins, the two latter fractions containing most of the virulence factors. To obtain quantitative results, we compared growing cells and non-growing/stationary phase cells using the 15N metabolic labelling approach. With almost 2.000 proteins, 80 % of the expressed proteome was identified and even quantified in growing and non-growing cells. A comprehensive inventory of the entire protein assembly during the two physiologically distinct states is presented, which integrates data ranging from gene expression to subcellular localization. Three major protein classes were distinguished: (i) Proteins induced in the stationary phase only (ii) Vegetative proteins, no longer synthesized but stable in the stationary phase (iii) Vegetative proteins, no longer required in non-growing cells and finally degraded. This quantitative proteomics approach for growing/non-growing cells, which represents a proof-of-principle for whole-cell physiological proteomics, can now be extended to address particular infection-relevant physiological states. Importantly, the present model study represents the highest coverage of a bacterial proteome so far (except low complexity bacteria). It thus paves the way towards a new understanding of cell physiology and pathophysiology of S. aureus and related pathogenic bacteria, opening a new era in infection-related research on this crucial pathogen.

platform_organism: Staphylococcus aureus subsp. aureus NCTC 8325

platform_organism: Staphylococcus aureus subsp. aureus Mu50

platform_organism: Staphylococcus aureus subsp. aureus N315

platform_organism: Staphylococcus aureus subsp. aureus MRSA252

platform_organism: Staphylococcus aureus subsp. aureus MSSA476

platform_organism: Staphylococcus aureus subsp. aureus USA300

overall_design: For the DNA-microarray experiment RNA was extracted from two independently grown cultures. One was grown in 14N labelled BioExpress medium and the other in 15N labelled BioExpress medium. Samples of exponentially growing cells (OD600=0.5) and of cell at 5h after entry into stationary phase were harvested from every culture. Equal amounts of all four RNAs were pooled and the pool was used as common reference (Cy3-labeld) for the four sample RNAs (Cy3). Thus, in total four hybridizations, one for each sample versus the common reference, were performed.

first: Dörte

last: Becher

first: Michael

last: Hecker


title: mRNA abundance in HEK293T cells mock or miR-124 transfected

status: Public on Nov 10 2009

city: Stanford

first: Patrick

middle: O

last: Brown

address: Beckman Center b439

department: Department of biochemistry

institute: Stanford university school of medicine

summary:
HEK293T cells were either mock or miR-124 transfected. Cells were lysed 12 hours after transfection with Trizol reagent. RNA was isolated, amplified, labeled (cy5), and comparatively hybridized with a universal reference (cy3) on HEEBO arrays in biological triplicate. In addition, each biological replicate was measured twice (#2's). Data from the technical replicates was averaged before analysis. SS=steady state
Compound Based Treatment: miR-124

overall_design: Set of arrays that are part of repeated experiments

first: Daniel

last: Hogan


title: Human Argonaute Immunoaffinity Purification-associated RNA

summary: Immunoaffinity Purifications of Human Argonaute  Twelve hours after transfections with mock or with miR-124, we washed each 15-cm plate once with phosphate-buffered saline (usually two plates were used per IP), then added 1 ml of 4 C lysis buffer (150 mM KCl, 25 mM Tris-HCl [pH 7.4], 5 mM Na-EDTA [pH 8.0], 0.5% Nonidet P-40, 0.5 mM DTT, 10 ul protease inhibitor cocktail [Pierce Cat# 78437], 100 U/ml SUPERaseIn [Ambion Cat# AM2694]). Following a 30-min incubation at 4 C, we scraped the plates, combined the lysates, and then spun them at 4 C for 30 min at 14,000 RPM in a microcentrifuge. We collected the supernatant and filtered it through a 0.45-um syringe filter. We froze an aliquot of lysate in liquid nitrogen for reference RNA isolation. We then added 0.22 mg/ml heparin to the lysate. We mixed the lysate with 2.5 mg of Dynal m-280 streptavidin beads (250 ul from original storage solution) coupled to biotinylated 4F9 ago antibody (~12.5 ug), which we equilibrated immediately prior to use by washing twice with 1 ml of lysis buffer. We incubated the beads with the lysate for 2 h at 4 C and then washed the beads twice with 1.25 ml of ice-cold lysis buffer for 5-min each. Five percent of the beads were frozen for SDS PAGE analysis after the second wash. RNA was extracted directly from the remaining beads using lysis buffer from Invitrogen's Micro-to-Midi kit (Invitrogen Cat# 12183-018). We purified RNA from the lysate and RNA extracted from the beads with the Micro-to-Midi kit as per vender's instructions, except that the percentage isopropanol used for binding to the column was 70%, instead of 33%, to promote the binding of small RNAs. RNA was amplified with ambion kit 1755. RNA from total cell lysate was labeled with cy3 and IPd RNA was labeled with cy5

overall_design: An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.


title: Correlating global gene regulation to morphogenesis and maturation in the chick extra-embryonic vascular system

city: Montpellier

first: John

last: De Vos

address: 80 av. Augustin Fliche

department: Institute for Research in Biotherapy

institute: University Hospital of Montpellier

summary: Formation of blood vessels requires the concerted regulation of an unknown number of genes in a spatial-, time- and dosage-dependent manner. We investigated vascular development in vivo by determining global gene regulation throughout the formation of the chick chorio-allantoic membrane (CAM).  Our study provides a comprehensive molecular map of vascular maturation during developmental angiogenesis and might thus be a valuable resource to streamline further research of candidates susceptible to mediate pathological angiogenesis.

overall_design: The developmental stage of the embryos was determined after isolation of the CAM according to Hamburger & Hamilton (HH) (1992). CAMs were isolated from embryos at developmental day E5 (HH26), E7 (HH30), E10 (HH>35) and E14 (HH40), (n=3 embryos/day). mRNA was isolated and hybridized to Affymetrix chicken GeneChips using the Affymetrix standard protocol. We compared one embryo at a lower HH stage to three embryos at a more advanced HH stage and repeated the comparison with two more embryos. Overall, we compared expression between E7 and E5, E10 and E5, E14 and E5, E10 and E7 and E14 and E10.

first: Sophie

last: Javerzat

first: Anne-Lise

last: Peille

first: Natalia

last: Platonova

first: Julie

last: Millour

last: Herbert

first: Laurent

last: Gross

first: Véronique

last: Pantesco

first: Ahlame

last: Saidi

first: Roy

last: Bicknell

first: Andreas

last: Bikfalvi

first: Martin

last: Hagedorn


title: Induction of target genes of AP2-Sp in AP2-O::Sp parasites

status: Public on Nov 09 2009

city: tsu

first: masao

last: yuda

state: mie

address: edobashi 2-174

institute: mie university

summary: Sporozoite-specific genes were induced in parasites that express AP2-O whose AP2 domain was swapped with that of AP2-Sp.

overall_design: DNA construct encoding AP2 domain of AP2-Sp  was introduced into P. bergei schizonts. It was integrated into the locus of the endogenious AP2-O gene, resulting in swap of the AP2-domain (AP2-O::Sp parasites). Gene expression was analyzed in ookinetes cultured for 20h.

first: Masao

last: Yuda

first: Shuji

last: Shigenobu


title: Translation Profiling +/- miR-124

last: Brown

summary:
Ribosome Occupancy Experiments: for these experiments Channel 1 is amplified RNA (aRNA) from mRNA unbound to any ribosomes. Channel 2 is aRNA from mRNA associated with ribosomes.

Ribosome Density Experiments: for these experiments Channel 1 is amplified RNA (aRNA) from mRNA present in the light fractions of the polysome. Channel 2 is aRNA from mRNA associated with ribosomes deep in  the polysome.

overall_design: Experiments are biological replicates of ribosome occupancy and density 12 hours post mock transfection or miR-124 transfection.


title: CBP60g and CBP60h play partially redundant and critical roles in salicylic acid signaling

status: Public on Nov 04 2009

city: St Paul

first: lin

last: wang

address: 350 Cargill building, 1500 Gortner Ave

department: Plant Biology

summary: Our previous study on CBP60g, a calmodulin binding protein that is important for disease resistance and microbe-associated molecular pattern (MAMP)-induced SA accumulation, led to our discovery of a closely related family member CBP60h. CBP60h is also important for defense against P. syringae but is induced differently by pathogen and MAMP stimulus. Transcriptome profiling of cbp60h mutants suggested that CBP60h might be primarily functioning in response against P. syringae. We constructed a double mutant of cbp60g and cbp60h, which demonstrated severely defective defense against P. syringae and SA accumulation. Profiling of the cbp60g/h showed that its expression pattern is very similar to that of pad4. Transient expression in Tobacco showed that both CBP60g and CBP60h localized to nucleus. Our observation suggest that CBP60g and CBP60h share partially redundant but critical role in defense response and SA signaling.

overall_design: This experiment consists of three biological replicates. For each genotype, two leaves per plant were pooled from three pots to prepare total RNA.

first: Lin

last: Wang

first: Jane

last: Glazebrook