title: Global post-transcriptional control of genes involved in amino acid metabolism by the Hfq-dependent GcvB RNA

first: Hans-Joachim

last: Mollenkopf

summary: The control of amino acid synthesis and transport in bacteria has been well-investigated at the transcriptional level. The discovery of a small Hfq-dependent regulatory RNA, GcvB, added another layer of gene expression control at the post-transcriptional level. GcvB RNA has been shown to directly regulate multiple ABC transporters for amino acids in E. coli and Salmonella using a highly conserved G/U-rich domain, R1. To identify additional GcvB targets, we have combined a sRNA pulse-expression and microarray analysis of whole transcriptome changes with biocomputational target searches for C/A- rich target sites in Salmonella. Moreover, we have included GcvB mutant RNAs in our microarray approach providing a new target search approach by inactivating conserved domains or target interaction sites. This dual approach revealed further amino acid transporters and, in addition, genes involved in amino acid metabolism as consensus R1-dependent GcvB targets. Moreover, GcvB RNA seems to bind with at least two binding sites to an R1-independent target, the glycine transporter cycA. Using GFP reporter gene fusions we have now validated 21 GcvB targets which is best to our knowledge with ~1% of all Salmonella protein coding genes, the largest bacterial sRNA-controlled regulon. Intriguingly, GcvB rewires many primary control circuits and, thus, constitutes an important metabolic knot.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

overall_design:
To predict direct GcvB targets with better confidence, we expanded the sRNA pulse-expression approach to assay effects of several versions of GcvB sRNA. We cloned GcvB wild-type and mutants RNAs deleted for the conserved regions R1 or R2 (Fig. 1A) under control of an arabinose-inducible PBAD promoter, yielding plasmid pBAD-GcvB (pKP1-1), pBAD-GcvB delta R1 (pKP2-6) and pBAD-GcvB delta R2 (pKP30-1). For confirmation of inducible expression, Salmonella wild-type carrying pBAD control vector (pKP8-35), and Salmonella ΔgcvB carrying either control vector (Ctr) or the above GcvB expression plasmids were grown to mid-exponential phase (OD600 of 1) and treated with L-arabinose for up to 15 min.
We used whole-genome S. typhimurium microarrays to determine relative mRNA expression changes at 10 min of induction, comparing the mRNA profiles of the pBAD-GcvB, pBAD-GcvB delta R1 or pBAD-GcvB delta R2 strains to that of the delta gcvB deletion mutant strain carrying the control vector. Microarrays used in this study were produced by in-situ synthesis as 8x15k multipack format from Agilent Technologies. Each microarray comprises 13268 60-mer S. typhimurium strain SL1344 specific oligonucleotides supplemented with 319 60-mer S. enterica subsp. serovar Typhimurium 14028S specific oligonucleotides, 360 60-mer S. typhimurium LT2 specific oligonucleotides and 360 60-mer oligonucleotides specific for 149 Salmonella sRNAs. The experimental design involves the use of Salmonella enterica serovar Typhimurium genomic DNA as the co-hybridized control for one channel on all microarrays. Two independent biological eperiments were analyzed.

first: Cynthia

middle: M

last: Sharma

first: Kai

last: Papenfort

first: Sandy

last: Pernitzsch

first: Jay

last: Hinton

first: Joerg

last: Vogel


title: A comprehensive assessment of array-based platforms and calling algorithms for detection of copy number variants (WGTP)

status: Public on Jun 30 2011

city: Toronto

first: Stephen

middle: W

last: Scherer

state: Ontario

address: 686 Bay Street

department: Genetics and Genomic Biology

country: Canada

institute: The Hospital for Sick Children

type: Genome variation profiling by genome tiling array

summary: Systematic evaluation of eleven array platforms commonly used for CNV detection to address parameters of quality and CNV calling.

overall_design: 18 Samples

first: Dalila

last: Pinto

first: Jeffrey

middle: R

last: MacDonald


title: Influence of TCDD onto HCC cell line

status: Public on Jul 01 2011

city: Rehovot

first: Einat

last: Sitbon

address: 10 Plaut St

country: Israel

institute: Rosetta Genomics

summary:
HepG2 cell lines were treated with TCDD, as an activator for the TF AhR/arnt since it  is a known ligand for AhR.
Hepatocellular carcinoma (HCC) is a fatal disease with currently very limited beneficial therapies. MicroRNAs (miRs), which are considered to be master regulators of gene expression, have significant influence on cellular pathways and phenotype, and are de-regulated in HCC, and hence are thought to be of great therapeutic potential as novel targets. We identified hsa-miR-191 as a potential target for HCC therapy. Inhibition of this miR causes decreased cell proliferation and induction of apoptosis in vitro as well as a significant reduction of tumor mass in vivo in an orthotopic liver xenograft model. This miR was also found to be up-regulated by a dioxin, a known liver carcinogen, and was found to be a key regulator of cancer related pathways.

overall_design: HCC cell lines  treated with TCDD and control. Although this experiment was done with dual channel, chanels are not compared, irrelevant samples were on the reciprocal channel. Sample data tables represent relevant, single channel data.

first: Noga

last: Yerushalmi

first: Eran

last: Elyakim

first: Avital

last: Dov


title: A comprehensive assessment of array-based platforms and calling algorithms for detection of copy number variants (2X244K)

overall_design: 72 Samples


title: Expression analysis of fully-grown oocyte collected from mouse wild-type, Hsf1 mutant or Hsf2 mutant

city: Toulouse

first: Florent

last: LE MASSON

address: 118 route de Narbonne Bat4R3b3

country: France

institute: CNRS

summary:
Investigation of whole genome gene expression level changes in oocytes Hsf1-/- or Hsf2-/- compared to wild-type.
The mutants analyzed in this study are further described in McMillan DR, Xiao X, Shao L, Graves K, Benjamin IJ. 1998. Targeted disruption of heat shock transcription factor 1 abolishes thermotolerance and protection against heat-inducible apoptosis. J Biol Chem. 273(13):7523-8 and in McMillan DR, Christians E, Forster M, Xiao X, Connell P, Plumier JC, Zuo X, Richardson J, Morgan S, Benjamin IJ. 2002. Heat shock transcription factor 2 is not essential for embryonic development, fertility, or adult cognitive and psychomotor function in mice. Mol Cell Biol. 22(22):8005-14.

overall_design: A microarray study using two technical replicates of total RNA recovered from wild-type fully-grown oocytes, Hsf1 mutant fully-grown oocytes and Hsf2 mutant fully-grown oocytes. Each microarray measures the expression level of 42,586 probe sets from Mus musculus with nine 60-mer probe pairs per gene.

last: Le Masson

first: Elisabeth

last: Christians

first: Timothy

middle: J

last: Westwood

first: Zak

last: Razak

first: Mo

last: Kaigo


title: A comprehensive assessment of array-based platforms and calling algorithms for detection of copy number variants (NimbleGen 720K)


title: Drought-responsive changes in poplar roots

status: Public on Jun 29 2011

city: Seibersdorf

first: Silvia

last: Fluch

address: Campus Seibersdorf

department: Biogenetics/Natural Resources

country: Austria

institute: Austrian Research Centers GmbH

summary: Drought-treated and corresponding control root tissue of poplar was subjected to array analyses

platform_organism:
Populus trichocarpa x Populus deltoides
Populus euphratica
Populus tremula x Populus alba

overall_design: Two-condition experiment, control (K) vs. Drought-stressed (S) leaves. Biological replicates: 3 control (1-3), drought-exposed (1-3), independently grown and harvested. One swap replicate per array.

first: S

first: M

last: Stierschneider

first: D

last: Kopecky

first: I

last: Matusikova


title: Differentially expressed genes in window trials are influenced by the wound healing process: lessons learned from a pilot study with Anastrozole

city: New York

first: Victor

middle: Piana

last: Andrade

state: NY

address: 408E 69th Street z427E

department: Breast Surgery

country: USA

institute: Memorial Sloan-Kettering Cancer Center

summary:
Introduction: Peri-operative window trials, using molecular biomarkers as surrogate endpoints for treatment response, offer investigators a uniqueopportunity to obtain intact tumor samples at 2 different time-points and thus evaluate potential biomarkers over a short period of time. Here we report results of a pilot trial designed to determine if treatment-mediated changes in gene expression can be detected after a 10-day exposure to anastrozole in estrogen receptor (ER)-positive breast cancer compared to untreated controls.
Methods: Paired tumor samples (biopsy and surgical) were obtained from 26 postmenopausal women with ER-positive breast cancer. Patients were assigned anastrozole (1mg/dy) for 10 days immediately prior to surgery (13 cases) or no treatment (13 controls). 502 cancer-related genes were examined by the DASL FFPE-based cDNA array (moderated t-test, p ≤ .005). Surrogate biomarkers reflecting changes in gene expression were examined by immunohistochemistry (IHC) (Wilcoxon rank-based test, p < .05).
Results: Sufficient RNA was available from 19 paired samples (8 controls, 11 cases). There was no difference in age, tumor size, grade, or baseline biomarker expression between groups. Within each group, 18 genes, reflecting roles in proliferation, angiogenesis, and apoptosis showed differential expression over time from biopsy to surgery (p < 0.005). Dysregulation of estrogen-related genes was present only in the treated group. Comparison between groups identified 5 genes that were significantly dysregulated between controls and treated cases (BAG1/ING1/ERBB4/IFNGR1/TFDP1). Reduction in Ki-67 index was observed in 7 (54%) treated cases in 1 (7.7%) control patient.
Conclusions:  Short-term (10-day) exposure to anastrozole resulted in significant dysregulation of 18 out of 502 cancer-related genes, and Ki-67 was reduced in 54% of cases. However, the local effects of wound healing may represent a confounding factor in the interpretation of peri-operative window trials as exposed by the changes in gene expression and the increased Ki-67 index in the control group.

overall_design: Prospective, paired tumor samples (biopsy and surgical) were obtained from 26 postmenopausal women with ER-positive breast cancer. Patients were assigned anastrozole (1mg/dy) for 10 days immediately prior to surgery (13 cases) or no treatment (13 controls). 502 cancer-related genes were examined by the DASL FFPE-based cDNA array. Sufficient RNA for gene expression analysis was obtained from 19/26 (73%) FFPE paired (biopsy and surgery) samples (8 controls, 11 anastrozole-treated patients

first: Tari

middle: A

last: King

first: Mary

last: Morrogh

middle: P

first: Asawari

last: Patil

first: Li-Xuan

last: Qin

first: Quianxing

last: Mo

first: Rita

last: Sakr

first: Adam

middle: B

last: Olshen

first: Crispinita

last: Arroyo

first: Edi

last: Brogi

first: Monica

last: Morrow


title: BBX32 Role in Early Light Signaling in Arabidopsis

city: Hayward

first: Don

last: Maszle

state: CA

address: 3935 Point Eden Way

institute: Mendel Biotechnology, Inc.

summary: The transcriptional profiles of several genotypes of Arabidopsis seedling shoots were compared in dark and after 1 hr of monochromatic red-light treatment.


title: Gene expression profiling of a mouse model of metabolic syndrome

city: Rahway

first: Oscar

last: Puig

state: NJ

address: 126 East Lincoln Ave

department: Molecular Profiling Research Informatics

institute: Merck Research Laboratories

summary: Mice overexpressing reverse tetracycline-transactivator (rtTA) exhibited all four sequelae of metabolic syndrome (visceral obesity insulin resistance, dyslipidemia, and hypertension), a pro-inflammatory state and marked hepatic steatosis. Gene expression profiling of the adipose tissue, muscle and liver revealed changes in gene expression of key factors involved in lipid metabolism, insulin resistance, and inflammation.

overall_design:
Liver, epididymal White Adipose Tissue, kidney cortex, kidney medulla and muscle from wild type (FVB mice, Taconic Farms)  and rtTA overexpressing mice (line 6) were profiled in Merck/Agilent mouse 44k v1.0 arrays.
For each tissue, a reference pool was used composed of total RNA from that tissue obtained from FVB mice. Channel 1 is the reference pool, channel 2 is the sample.

first: Liwen

last: Zhang


title: A comprehensive assessment of array-based platforms and calling algorithms for detection of copy number variants (Human660W-Quad)

first: Stephen

last: Scherer

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

overall_design: 36 Samples

first: Dalila

last: Pinto

first: Jeffrey

last: MacDonald


title: Genome-wide methylation analysis of AOM/DSS-induced colon tumors

city: Chuo-ku

first: Satoshi

last: Yamashita

state: Tokyo

address: Tsukiji 5-1-1

country: Japan

institute: National Cancer Center Research Institute

type: Methylation profiling by genome tiling array

summary: Genome-wide methylation analysis was performed by methylated DNA immunoprecipitation (MeDIP)-CpG island (CGI) microarray analysis to identify candidate CGIs specifically methylated in mouse colon tumors associated with colitis.  We sucessfully identified 23 candidate CGIs methylated in tumors.

overall_design: Two samples were analyzed by MeDIP-CGI microarray.  One is a pool of two AOM/DSS-induced colon tumors in BALB/c mice and another is a pool of two normal colonic epithelial cell samples obtained from untreated BALB/c mice by the crypt isolation technique.  The pool of normal colonic epithelial cell samples was used as reference.  Dye-swaps were not perfromed.  The methylation statuses of CGIs identified by microarray were confirmed by another method, methylation-specific PCR.

first: Miki

last: Katsurano

first: Tohru

last: Niwa

first: Toshikazu

last: Ushijima


title: Ethylene and 1-MCP differentially regulate gene expression during onion (Allium cepa L.) sprout suppression

city: Crawley

first: John

last: Hammond

state: WA

address: 35 Stirling Highway

department: School of Plant Biology

country: Australia

institute: University of Western Australia

summary: Onion is regarded as non-climacteric. In onion, ethylene can suppress sprouting however, the ethylene binding inhibitor, 1-MCP can also suppress sprout growth although it is unknown how ethylene and 1-MCP elicit the same response.  In this study, onion bulbs were treated with 10 μL L-1 ethylene or 1 μL L-1 1-MCP individually or in combination for 24 h at 20°C before or after curing (six weeks) at 20 or 28°C then stored at 1°C.  Following curing, a subset of these same onion bulbs was stored separately under continuous air or ethylene (10 μL L-1) at 1°C

overall_design: Six treatments were chosen for microarray analysis; four samples were taken before curing immediately after treatment with ethylene or 1-MCP or ethylene and 1-MCP in combination for 24 h at 20°C.  The other two samples and two  were taken at the end of storage following 6 weeks curing at 28°C and after 29 weeks cold storage (1°C) in continuous air or continuous ethylene totalling 35 weeks storage.  The four pre-curing samples used were untreated (control) or treated with EB, MB or EMB and the two samples after storage were control bulbs stored in continuous ethylene or air at 1°C.  There were three biological replicates of each of the six treatments making 18 samples in total.

first: Katherine

last: Cools

first: Gemma

last: Chope

first: Andrew

last: Thompson

first: Leon

last: Terry


title: Transcriptome profiling reveals a novel role for Trichostatin A in antagonizing histone chaperone Chz1 mediated telomere anti-silencing.

status: Public on Jun 28 2011

city: Nanjing

first: Yakun

last: Wan

address: Sipailou #2

country: China

institute: Southeast University

summary: The histone chaperones play an important role in chromatin assembly and disassembly during replication and transcription. We assessed the global roles of histone chaperones in Saccharomyces cerevisiae. Microarray transcriptional analyses indicate that histone chaperones have their own specific target genes, and various histone chaperones have partially overlapping functions during transcriptional regulation. Histone deacetylase inhibitor TSA and histone chaperones Asf1, Vps75 and Rtt106 can function in parallel pathways to regulate transcription. Moreover, TSA can specifically antagonize histone chaperone Chz1-mediated telomere anti-silencing. This study demonstrates that a mutual cross-talk mechanism exists between histone chaperones and histone deacetylation in transcriptional regulation.

overall_design: All yeast strains used in this study are listed in the paper (Table S1). CHZ1, NAP1, ASF1, VPS75 and RTT106 genes were deleted individually by homologous recombination in BY4742 (WT) by using a previously described in (Wan, Y. et al. MCB, 2009) PCR-based procedure. The strains were cultured at 30°C in YPD (1% yeast extract, 2% peptone, 2% glucose) media. For TSA treatments, WT and deletion mutants were grown to mid-log phase (OD600=0.5), TSA (Sigma-Aldrich) was then added to the yeast cultures at a final concentration of 10 μM and cells were cultured for additional hour.Total RNA was isolated by hot acid phenol extraction protocol as previously described in Wan, Y. et al. MCB, 2009. Microarray labeling and hybridization reactions were performed as previously described in Wan, Y. et al. MCB, 2009 and Wan, Y. et al. NAR, 2010. Two color microarrays, comparing RNA from the experimental conditions (deletion mutations grown in YPD, WT and deletion mutations grown in YPD with TSA treatment) to RNA from the control WT cells grown in glucose-containing medium (YPD), were performed using Agilent whole-genome S. cerevisiae arrays.

first: Weiming

last: Chen


title: Gastric cancers of Western European and African patients

city: Amsterdam

first: Daoud

last: Sie

address: De Boelelaan 1117

department: Pathology

country: Netherlands

institute: Vrije Universiteit Medical Center

summary: Gastric cancer is the second most common cause of cancer death worldwide, but incidence and mortality rates show large variations across different countries. Variation in risk factors between different populations, including environmental and host factors influencing gastric cancer risk, have been reported but little is known about the biological differences between gastric cancers from different geographic locations. We set out to study genomic instability patterns of gastric cancers obtained from patients from United Kingdom (UK) and South Africa (SA).  DNA was isolated from 67 gastric adenocarcinomas, 33 UK patients, 9 Caucasian SA patients and 25 native SA patients. Microsatellite instability and chromosomal instability were analyzed by PCR and microarray comparative genomic hybridization, respectively. Data was analyzed by supervised univariate and multivariate analyses as well as unsupervised hierarchical cluster analysis.   Tumors from Caucasian and native SA patients showed significantly more microsatellite instable tumors (p<0.05). For the microsatellite stable tumors, geographical origin of the patients correlated with cluster membership, derived from unsupervised hierarchical cluster analysis (p=0.001). Several chromosomal alterations showed significantly different frequencies in tumors from UK patients and native SA patients, but not between UK patients and Caucasian SA patients and between native and Caucasian SA patients.  In conclusion, gastric cancers from South African and UK patients show differences in genetic instability patterns, indicating possible different biological mechanisms underlying the disease.

first: Tineke

middle: E

last: Buffart

first: Melanie

last: Louw

first: Nicole

middle: C

last: vanGrieken

first: Marianne

last: Tijssen

first: Beatriz

last: Carvalho

first: Bauke

last: Ylstra

first: Heike

last: Grabsch

first: Chris

last: Mulder

first: Cornelis

last: vandeVelde

first: Schalk

last: vanderMerwe

first: Gerrit

last: Meijer


title: A comprehensive assessment of array-based platforms and calling algorithms for detection of copy number variants (NimbleGen 2.1M)

first: Stephen

last: Scherer

overall_design: 33 Samples

first: Dalila

last: Pinto

first: Jeffrey

last: MacDonald


city: wuhan

first: Wei

last: Miao

state: hubei

address: donghu south road #7

institute: Institute of hydrobiology, CAS

summary: Five genome-wide microarrays were used to indentify gene expression in exposure to Tributyltin, Dichlorodiphenyltrichloroethane and 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin. The three environmental chemicals are persistent in environment and cause continuous toxical effects on human and aquatic life. Tetrahymena thermophila has the potential as a toxical model. The genome-wide microarray in Tetrahymena thermophila exposed to these toxicants was analyzed. First, genes differentially expressed under the treatment of each toxicant were identified and then their functions were categorized using GO enrichment analysis. The results may suggest that Tetrahymena thermophila is able to reflect different mechanism of different toxic-type toxicants which are similar to those in multicellular organisms. Regarding the complexity of TCDD effects, context likelihood of relatedness method (CLR) was applied to construct a TCDD-relevant network. General functions of the network are related to the epigenetic mechanism of TCDD. Based on the network analysis, a model of TCDD effect on T. thermophila was infered. Thus, Tetrahymena has the potential to be a good unicellular eukaryotic model for toxic mechanism research at genome level.

overall_design: Tetrahymena thermophila CU428 cells were cultured using 1XSPP, 30 degree, 100 rpm shaking, until 300,000 cells/ml. Cells at 300,000 cells/ml was incubated in SPP medium were treated. Two microarrays were used 4.02μl DMSO per ml SPP as the solvent and control; One microarray for 5 ppb TBT treatment 24 hours; One microarray for 1 ppb TCDD treatment 24 hours; One microarray for 4ppm DDT treatment for 24 hours.


title: Heavy metal resistance in Cupriavidus metallidurans CH34 is governed by an intricate transcriptional network

city: Mol

first: Rob

last: Van Houdt

address: Boeretang 200

department: Interdisciplinary Biosciences

country: Belgium

institute: SCK-CEN

summary:
Background: The high number of heavy metal resistance genes in the soil bacterium Cupriavidus metallidurans CH34 makes it an interesting model organism to study microbial responses to heavy metals. Results: In this study the transcriptional response of this bacterium was measured after challenging it to a wide range of sub-lethal concentrations of various essential or toxic metals. Considering the global transcriptional responses for each challenge as well as by identifying the overlap in upregulated genes between different metal responses, the sixteen metals could be clustered in three different groups. Additionally, next to the assessment of the transcriptional response of already known metal resistance genes, new metal response gene clusters were identified. The majority of the metal response loci showed similar expression profiles when cells were exposed to different metals, suggesting complex cross-talk at transcriptional level between the different metal responses. The highly redundant nature of these metal resistant regions – illustrated by the large number of paralogous genes – combined with the phylogenetic distribution of these metal response regions within evolutionary related and other metal resistant bacteria, provides important insights on the recent evolution of this naturally soil dwelling bacterium towards a highly metal-resistant strain found in harsh and anthropogenic environments. Conclusions: The metal-resistant soil bacterium Cupriavidus metallidurans CH34 displays myriads of gene expression patterns when exposed to a wide range of heavy metals at non-lethal concentrations. The interplay between the different gene expression clusters points towards a complex cross-regulated regulatory network governing heavy metal resistance in C. metallidurans CH34.
Keywords: Cupriavidus metallidurans CH34, transcriptional regulation, heavy metal resistance

overall_design: Two-condition experiments. Comparing samples after induction with heavy metals versus non-induced samples. Biological duplicate or triplicate. Each array contains 3 or 4 technical replicates.

first: Pieter

last: Monsieurs

first: Hugo

last: Moors

first: Paul

last: Janssen

first: Ann

first: Ilse

last: Coninx

first: Max

last: Mergeay

first: Natalie

last: Leys


title: Pyrococcus furiosus DSM 3638 growth curve, tiling arrays

status: Public on Jun 27 2011

city: Seoul

first: Sung Ho

last: Yoon

address: 120 Neungdong-ro, Gwangjin-gu

department: Department of Bioscience and Biotechnology

country: South Korea

institute: Konkuk University

type: Expression profiling by genome tiling array

summary: Experimentally mapped transcriptome structure of Pyrococcus furiosus DSM 3638 by hybridizing total RNA (including RNA species <200 nt) to genome-wide high-density tiling arrays (60 mer probes tiled every 16 nt).

overall_design:
Pyrococcus furiosus DSM 3638 growth curve experiments were conducted in batch culture. Reference samples were cultured at mid-log phase (OD600 = 0.096).
Seven samples were collected that spanned the key phases of the growth curve. Total RNA from samples of growth curve and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. Dye-flip experiments were done for each sample. Log ratios were calculated for each probe (growth curve sample/reference). Transcriptome browser is available at http://baliga.systemsbiology.net/enigma/.

first: Sung-Ho

first: David

last: Reiss

first: J

last: Bare

first: Min

last: Pan

first: Angeli

middle: L

last: Menon

first: Michael

last: Adams

first: Nitin

middle: S

last: Baliga


title: Common and overlapping oncogenic pathways contribute to the evolution of acute myeloid leukemias.

city: Berlin

first: Lars

last: Bullinger

address: Augustenburger Platz 1

department: Hematology, Oncology and Tumorimmunology

country: Germany

institute: Charité

summary: In this report we demonstrate that the ability to alter self-renewal in vitro and in vivo is a more generalized property of leukemia-associated oncogenes. We further demonstrate that disparate leukemia-associated oncogenes initiate early common and overlapping transformation and self-renewal gene expression programs to mediate these effects. Furthermore, elements of these programs can be detected in established leukemia stem cells from an animal model and across a large cohort of patients with differing acute myeloid leukemia (AML) subtypes, where they strongly predict for disease biology. Finally, individual genes from the programs are demonstrated to partially phenocopy the leukemia-associated oncogenes and themselves alter self-renewal in committed murine progenitors and generate AML when expressed in murine bone marrow.

overall_design:
A total of 253 RNA samples derived from adult AML patients were provided by the German Austrian AML Study Group (AMLSG) [AMLSG trials AML HD98A (ClinicalTrials.gov Identifier: NCT00146120), and AML HD98B (Schlenk et al., 2009)].
Conventional cytogenetic banding, and FLT3, CEBPA and NPM1 mutational analysis were performed as previously described (Schlenk et al., N Engl J Med 2008). Detailed clinical, cytogenetic and molecular cytogenetic information are also provided in Table S3 along with the publication.
Following enrichment, all samples contained at least 80% leukemic cells. Gene expression profiling (GEP) was performed as previously described (Bullinger et al., N Engl J Med 2004).

Separate filtering and batch centering were performed for the group of 253 Samples (relative to GSE16432).  Filtered data presented as a supplementary file at the foot of this record.


title: A microarray approach to identify genes involved in seed and mesocarp development in peach

city: Legnaro

first: claudio

last: Bonghi

state: Padova

address: Viale dell'UNiversità, 16

country: Italy

institute: University of Padova

summary:
Background
Field observations and a few physiological studies pointed out that peach embryogenesis and fruit development are strictly related. In fact, attempts to stimulate parthenocarpic fruit development by means of external tools failed. Moreover, physiological disturbances during the early embryo development lead to seed abortion and fruitlet abscission. Later on, the interactions between seed and fruit development become less stringent. Genetic and molecular information about seed and fruit development in peach is limited.

Results
The isolation of 455 genes differentially expressed in seed and fruit was done by means of a comparative analysis of the transcription profiles carried out in peach (Prunus persica, cv Fantasia) seed and mesocarp throughout development by means of µPEACH 1.0, the first peach microarray. Genes differentially expressed in the two organs and specific of developmental stages had been identified, and some were validated as markers. Genes representative of the main functional categories are present, among which several transcription factors such as MADS-box, bZIP, Aux/IAA, AP2, WRKY, and HD. Some of these showed a similar transcription profile in the two organs, while others displayed an opposite pattern, being more expressed in embryo at early development and in mesocarp at ripening.

Conclusions
The µPEACH1.0, although developed from ripe fruit ESTs, resulted in being suitable for studying seed/mesocarp interactions. Among the differentially expressed genes, marker genes specific for organ and stage of development have been selected.

overall_design: Comparisons were carried out by pooling stage 1 and 2 (named early development, e) and stage 3 and 4 (named late development, l), separately for mesocarp (M) and seed (S) of cultivar Fantasia, and using a simple loop experimental design. RNA has been extracted from fruit harvest at above-mentioned stages of development. At least four hybridizations have been conducted for a total of four technical replicates (with dye-swap).

first: Claudio

first: Fiorenza

last: Ziliotto

first: Angela

last: Rasori

first: Angelo

last: Ramina

first: Alice

last: Tadiello

first: Giorgio

last: Casadoro

first: Livio

last: Trainotti


title: The RPB2 flap loop of human RNA polymerase II is dispensable for transcription initiation and elongation.

status: Public on Jun 24 2011

city: madison

first: jeff

last: grass

state: wi

address: 1550 linden dr

department: biochemistry

institute: uw-madison

type: Genome binding/occupancy profiling by genome tiling array

summary:
Deletion of the flap loop in the Rp2 subunit of human RNAPII did not alter the ability of human RNAPII to initiate transcription or elongate the initiated transcripts in vivo and in vitro, and was also dispensable for elongation factor-mediated enhancement and inhibition of transcript elongation in vitro.
ChIP:chip of wild-type and flap deletion human RNAPII revealed that the mutant RNAPII was not defective for promoter binding, initiation and elongation in vivo.

overall_design: Eight ChIP datasets were generated as log2(IP/input) fluorescence intensities using anti-FLAG IP of wild-type RPB2-FLAG cells in biological triplicate, anti-FLAG IP of delta-FL RPB2 FLAG cells in biological triplicate, and anti-RPB1 CTD, 8WG16 of both wild-type and delta-FL cells in biological duplicate. The two 8WG16- IP datasets were combined to measure the distribution of RPB1 signal.

first: Murali

last: Palangat

first: Jeffrey

last: Grass

first: Marie-France

last: Langelier

first: Benoit

last: Coulombe

first: Robert

last: Landick


title: Assessment of chemical mixtures and ground water effects on Daphnia magna transcriptomics

status: Public on Jun 23 2011

city: Vicksburg

first: Natalia

last: Vinas

state: MS

address: 3909 Halls Ferry Rd

institute: Mississippi State University

summary: Small organisms can be used as biomonitoring tools to assess chemicals in the environment. Chemical stressors are especially hard to assess and monitor when present as complex mixtures. Here, Daphnia magna were exposed for 24 hours to five different munitions constituents 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT), trinitrobenzene (TNB), dinitrobenzene (DNB), or 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX) as well as to 8 different munitions mixtures and ground water contaminated with munitions constituents. To better understand possible mixture effects, gene expression changes from all treatments were compared using high-density microarrays. While mixtures and ground water exposures had genes and gene functions in common with single chemical exposures, unique functions were also affected, which was consistent with the non-additivity of chemical effects in these mixtures.

overall_design: The study consisted of three different experiments: (1) exposure to a concentration corresponding to 70% of 1/10th of the LC50 value of six individual MCs (TNT, 2,4-DNT, 2,6-DNT, DNB, TNB, RDX) and a control; (2) exposure to eight different laboratory mixtures of the previously mentioned MCs. Different combinations of MCs including four mixtures (Mixtures 5, 6, 7 and 8) representative of field collected groundwater from LAAP (Louisiana Army Ammunition Plant) were created; and (3) exposure to MC-contaminated ground water field-collected from 3 different wells (85, 108, and 141) at the LAAP. All exposures were conducted for 24h.

first: N

last: Garcia-Reyero

first: L

last: Escalon

first: Po-Ru

last: Loh

first: Jennifer

last: Laird

first: Alan

last: Kenendy

first: Bonnie

last: Berger

first: Edward

last: Perkins


title: Sulfolobus solfataricus P2 growth curve, tiling arrays

first: Sung Ho

last: Yoon

summary: Experimentally mapped transcriptome structure of Sulfolobus solfataricus P2 by hybridizing total RNA (including RNA species <200 nt) to genome-wide high-density tiling arrays (60 mer probes tiled every 25 nt).

overall_design:
Sulfolobus solfataricus P2 growth curve experiments were conducted in batch culture. Reference samples were cultured at mid-log phase (OD600 = 0.312).
Eight samples were collected that spanned the key phases of the growth curve. Total RNA from samples of growth curve and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. Dye-flip experiments were done for each sample. Log ratios were calculated for each probe (growth curve sample/reference). Transcriptome browser is available at http://baliga.systemsbiology.net/enigma/.

first: Sung-Ho

first: David

last: Reiss

first: J

last: Bare

first: Min

last: Pan

first: Adam

last: Barnebey

first: Steven

last: Yannone

first: Nitin

last: Baliga


title: A flucytosine-responsive APSES protein, Mbs1, plays pleiotropic roles in stress response, differentiation, and virulence of Cryptococcus neoformans

first: Yong-Sun

last: Bahn

address: 134 Shinchon-dong Seodaemun-gu

department: Biotechnology

institute: Yonsei University

summary: A family of APSES transcription factor is known to be fungal-specific transcriptional regulators and play important roles in governing growth, differentiation, and virulence of diverse fungal pathogens. Yet none of APSES-like transcription factors have been identified and investigated in a basidiomycetous fungal pathogen, Cryptococcus neoformans. In the present study we discovered an APSES-like transcription factor, mbs1 (Mbp1/Swi4-like APSES protein 1), as one of novel flucytosine-responsive genes (total 194 genes) identified through comparative transcriptome analysis of C. neoformans hybrid sensor kinase mutants, tco1 and tco2 mutants, which displayed differential flucytosine-susceptibility. Supporting the microarray data, Northern blot and quantitative RT-PCR analysis confirmed that expression of mbs1 is rapidly induced in response to flucytosine in the wild-type strain, but not in the tco1 and tco2 mutants. Furthermore, C. neoformans with deletion of the mbs1 gene exhibited increased susceptibility to flucytosine. Intriguingly, mbs1 plays pleiotropic roles in diverse cellular process of C. neoformans. mbs1 positively regulates ergosterol biosynthesis and thereby its inhibition confers increased susceptibility and resistance to amphotericin B and azole drugs, respectively. mbs1 is also involved in DNA damage repair counteracting genotoxic stresses. During sexual differentiation mbs1 represses pheromone production, but promotes cell-cell fusion. Furthermore mbs1 is required for production of antioxidant melanin pigment and full virulence of C. neoformans. Finally we also performed DNA microarrray analysis to identify mbs1-regulated genes in C. neoformans. A majority of them were found to be involved in cell cycle regulation and DNA repair. Therefore, this study provides a novel antifungal therapeutic method for treatment of cryptococcosis.

overall_design: There are more than 95% of genome homology between JEC21 and H99. Therefore 9 slides of JEC21 (cryptococcus neoformans var. neoformans serotype D) 70-mer oligo are used in this analysis, 3 biological replicate experiments are performed, total RNAs are extracted  3 strains from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), mbs1Δ, mbs2Δ), We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy5 as Sample dye and Cy3 as a control dye.

first: Min-Hee

last: Song

last: Kim

first: Ja-Kyung

first: Jang-Won

last: Lee

first: Theodore

last: White

first: Anna

last: Floyd

first: Joseph

last: Heitman

first: Kirsten

last: Nielsen


title: Expression analysis of Shigella flexneri 2a strain 301

city: Beijing

first: Junping

last: Peng

address: No 6, Rongjing Eastern Street, BDA

institute: Chinese center for disease control and prevention

summary:
Investigation of whole genome gene expression to identify overlooked sRNAs and sORFs.
Background
The completion of numerous genome sequences has introduced an era of whole-genome study. However, many real genes, including small RNAs (sRNAs) and small ORFs (sORFs), are missed in genome annotation. In order to improve genome annotation, we sought to identify novel sRNAs and sORFs in Shigella, the principal etiologic agents of bacillary dysentery or shigellosis.
Results
Firstly, we identified 64 sRNAs in Shigella which is experimentally validated in other bacteria based on sequence conservation. Secondly, among possible approaches to search for sRNAs, we employed computer-based and tiling array based methods, followed by RT-PCR and northern blots. This allowed us to identify 12 sRNAs in Shigella flexneri strain 301. We also find 29 candidate sORFs.
Conclusions
This investigation provides an updated and comprehensive annotation of the Shigella genome, increases the expected numbers of sORFs and sRNAs with the corresponding impact on future functional genomics and proteomics studies.  Our method can be used for the large scale reannotation of sRNAs and sORFs in any microbe whose genome sequence is available.

overall_design: Study using total RNA recovered from five conditions.

first: Peng

last: Junping


title: Toc1, Toc2, and Skn7 null mutants treated with Flucytosine

summary: A family of APSES transcription factor is known to be fungal-specific transcriptional regulators and play important roles in governing growth, differentiation, and virulence of diverse fungal pathogens. Yet none of APSES-like transcription factors have been identified and investigated in a basidiomycetous fungal pathogen, Cryptococcus neoformans. In the present study we discovered an APSES-like transcription factor, Msa1 (Mbp1/Swi4-like APSES protein 1), as one of novel flucytosine-responsive genes (total 194 genes) identified through comparative transcriptome analysis of C. neoformans hybrid sensor kinase mutants, tco1 and tco2 mutants, which displayed differential flucytosine-susceptibility. Supporting the microarray data, Northern blot and quantitative RT-PCR analysis confirmed that expression of MSA1 is rapidly induced in response to flucytosine in the wild-type strain, but not in the tco1 and tco2 mutants. Furthermore, C. neoformans with deletion of the MSA1 gene exhibited increased susceptibility to flucytosine. Intriguingly, Msa1 plays pleiotropic roles in diverse cellular process of C. neoformans. Msa1 positively regulates ergosterol biosynthesis and thereby its inhibition confers increased susceptibility and resistance to amphotericin B and azole drugs, respectively. Msa1 is also involved in DNA damage repair counteracting genotoxic stresses. During sexual differentiation Msa1 represses pheromone production, but promotes cell-cell fusion. Furthermore Msa1 is required for production of antioxidant melanin pigment and full virulence of C. neoformans. Finally we also performed DNA microarrray analysis to identify Msa1-regulated genes in C. neoformans. A majority of them were found to be involved in cell cycle regulation and DNA repair. Therefore, this study provides a novel antifungal therapeutic method for treatment of cryptococcosis.

overall_design: There are more than 95% of genome homology between JEC21 and H99. Therefore 24 slides of JEC21 (cryptococcus neoformans var. neoformans serotype D) 70-mer oligo are used in this analysis, 3 biological replicate experiments are performed, total RNAs are extracted under 2 conditions (with or without treatment of Flucytosine) with 4 strains from H99 (H99 Wild type strain (Cryptococcus neoformans var. grubii serotype A), tco1Δ , tco2Δ , skn7Δ), We use the mixed all of total RNAs from this experiment as a control RNA. We use Cy5 as Sample dye and Cy3 as a control dye.

last: Yoon


title: Genome-wide expression quantitative trait loci (eQTL) analysis in maize

status: Public on Jun 22 2011

city: Johnston

first: Kevin

middle: Robert

last: Hayes

state: IA

address: 7200 NW 62nd Ave

institute: Pioneer Hi-Bred International

summary: Background: Expression QTL analyses have shed light on transcriptional regulation in numerous species of plants, animals, and yeasts.  These microarray-based analyses identify regulators of gene expression as either cis-acting factors that regulate proximal genes, or trans-acting factors that function through a variety of mechanisms to affect transcript abundance of unlinked genes.  Results: A hydroponics-based genetical genomics study in roots of a Zea mays IBM2 Syn10 double haploid population identified tens of thousands of cis-acting and trans-acting eQTL.  Cases of false-positive eQTL, which results from the lack of complete genomic sequences from both parental genomes, were described.  A candidate gene for a trans-acting regulatory factor was identified through positional cloning.  The unexpected regulatory function of a class I glutamine amidotransferase controls the expression of an ABA 8’-hydroxylase pseudogene.

overall_design:
For each of two biological replicates, tissue was collected from B73 and Mo17 parental controls and the IBM2 Syn10 doubled haploid mapping population at 4 weeks after germination (V5 stage).  One hundred thirty-five IBM2 Syn10 lines were used in this study.  The tissue sampled was the 1.5 cm tip of all crown roots that had developed at the time of harvest, representing the most metabolically active region based on whole-tissue visualization of triphenyl tetrazolium chloride staining. Tissues were flash frozen in liquid nitrogen and stored at -80º C.   They were manually ground into a fine powder using a mortar and pestle on dry ice.
Two-channel hybridizations were considered as single channel. Therefore, the same raw data file is linked to two Samples appended as _Cy3 and _Cy5.

first: Beth

last: Holloway

first: Stanley

last: Luck

first: Mary

last: Beatty

first: J-Antoni

last: Rafalski

first: Bailin

last: Li


title: Protein expression in primary murine dermal fibroblasts grown on fibronectin vs. plastic

city: Toronto

first: Benjamin

last: Alman

address: TMDT, 101 College Street

department: Developmental and Stem Cell Biology

institute: Hospital for Sick Children

summary: We were interested in identifying proteins that are regulated in response to the extracellular matrix molecule, fibronectin, in dermal fibroblasts.  To determine proteins regulated by exposure to fibronectin, primary murine dermal fibroblasts were cultured on tissue culture plastic or on tissue culture plates coated with a thin layer of fibronectin (2 μg/cm2).  Fibroblasts were seeded at 35,000 cells/10 cm2 and were harvested 48 hours later. Cell pellets from these fibroblasts were shipped to Kinexus Bioinformatics Corp. (Vancouver, B.C.) where they were lysed and analyzed using the Kinex TM Antibody Microarray (KAM 1.2), which contains 800 (500 pan-specific and 300 phosphorylation site-specific) antibodies. This enabled us to identify candidate proteins regulated by exposure to fibronectin.

overall_design: Primary murine dermal fibroblasts were grown on fibronectin and tissue culture plastic for 48 hours. Cell pellets from these fibroblasts were shipped to Kinexus Bioinformatics Corp. (Vancouver, B.C.) where they were lysed and analyzed using the Kinexus Antibody Microarray (KAM 1.2), which contains 800 (500 pan-specific and 300 phosphorylation-site specific) antibodies.

last: Bielefeld


title: VirF gene from Shigella spp is able to induce chromosomal genes in E. coli

status: Public on Jun 25 2011

city: Rome

first: Gianni

last: Prosseda

address: via dei sardi 70

department: Biology and Biotechology C. Darwin

institute: Saoienza , University of Rome

summary: In order to understand whether the evolutionary acquisition of virF gene may have altered the transcriptional program in Shigella's harmless ancestor (E. coli) promoting the inactivation of genes potetially detrimental to the full expression of the invasivity phenotype, we performed a global transcriptional analysis of E. coli cells expressing or lacking the virF gene. To this end we used the E. coli K12 MG1655 strain, carrying the virF-encoding plasmid pMYSH6504, or its virF depleted derivative pMY6504R.

overall_design: Two condition experiment: the E. coli K12 MG1655 strain, carrying the virF-encoding plasmid pMYSH6504, and its virF depleted derivative pMY6504R.  Two replicate for each condition


title: Genomic prevalence of heterochromatic H3K9me2 and transcription do not discriminate pluripotent from terminally differentiated cells (ChIP-chip)

status: Public on Jun 21 2011

city: Basel

first: Dirk

last: Schuebeler

address: Fabrikstrasse 24

country: Switzerland

institute: Friedrich Miescher Institute for Biomedical Research

summary: Cellular differentiation entails reprogramming of the transcriptome from a pluripotent to a unipotent fate. This process was suggested to coincide with a global increase of repressive heterochromatin, which results in a reduction of transcriptional plasticity and potential. Here we report the dynamics of the transcriptome and an abundant heterochromatic histone modification, dimethylation of histone H3 at lysine 9 (H3K9me2), during neuronal differentiation of embryonic stem cells. In contrast to the prevailing model, we find H3K9me2 to occupy over 50% of chromosomal regions already in stem cells. Marked are most genomic regions that are devoid of transcription and a subgroup of histone modifications. Importantly, no global increase occurs during differentiation, but discrete local changes of H3K9me2 particularly at genic regions can be detected. Mirroring the cell fate change many genes show altered expression upon differentiation. Quantitative sequencing of transcripts reveals however that the total number of active genes is equal between stem cells and several tested differentiated cell types. Together, these findings reveal high prevalence of a heterochromatic mark in stem cells and challenge the model of low abundance of epigenetic repression and resulting global transcription in stem cells.  Instead cellular differentiation entails local rather than global changes in epigenetic repression and transcriptional activity.

overall_design: Our dataset comprises of 2 ChIP-seq samples using chromatin from embryonic stem (ES) cells and derived terminal neurons (TN)  which was immunoprecipitated, using antibodies against H3K27me3 and H3K4me2. For the same cells we generated H3K9me2 ChIP-chip samples in biological replicates. For ES cells, neurons and mouse embryonic fibroblasts (MEFs) we generated biological replicates of RNA-seq data.

first: Florian

last: Lienert

first: Fabio

last: Mohn

first: Vijay

middle: K

last: Tiwari

first: Tuncay

last: Baubec

first: Tim

last: Roloff

first: Dimos

last: Gaidatzis

first: Michael

last: Stadler

last: Schübeler


title: Methanococcus maripaludis S2 growth curve, tiling arrays

first: Sung Ho

last: Yoon

summary: Experimentally mapped transcriptome structure of Methanococcus maripaludis S2 by hybridizing total RNA (including RNA species <200 nt) to genome-wide high-density tiling arrays (60 mer probes tiled every 14 nt).

overall_design:
Methanococcus maripaludis MM901, a wild type Methanococcus maripaludis S2 with an in frame deletion of the uracil phosphoribosyltransferase gene (Proc. Natl. Acad. Sci. U S A 107: 11050-11055) growth curve experiments were conducted in batch culture. Reference samples were cultured at mid-log phase (OD660 = 0.804).
Eight samples were collected that spanned the key phases of the growth curve. Total RNA from samples of growth curve and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. Dye-flip experiments were done for each sample. Log ratios were calculated for each probe (growth curve sample/reference). Transcriptome browser is available at http://baliga.systemsbiology.net/enigma/.

first: Sung-Ho

first: David

last: Reiss

first: J

last: Bare

first: Min

last: Pan

first: Sujung

last: Lim

first: John

last: Leigh

first: Nitin

last: Baliga


title: Changes in glycogene expression during maturation of dendritic cells

status: Public on Jun 14 2011

city: La Jolla

first: Steven

last: Head

address: 10550 N Torrey Pines Rd

institute: The Scripps Research Institute

summary: To investigate the expression of glycosyltransferases in dendritic cells and the changes in expression associated with maturation.

overall_design:
RNA preparations from 5 different immature and mature dendritic cells subsets, with and without LPS from 2 different donors (20 different samples); (iDC, IL-10 iDC, Dexamethason iDC, Vitamin D3 iDC, im. macrophage), (mDC, IL-10 mDC, Dexamethason mDC, Vitamin D3 mDC, m. macrophage) were sent to both Microarray Core (E) and Core(C).
The RNA was purified on an RNeasy Column, amplified, labeled, and hybridized to the Glycov3 microarrays.

first: Yvette

last: Van Kooyk


title: Transcriptome analysis of Escherichia coli mutants involved in inorganic polyphosphate metabolism

status: Public on Jun 15 2011

city: Santiago

first: Francisco

last: Chavez

address: 3425, Las Palmeras

country: Chile

institute: Universidad de Chile

summary:
Inorganic polyphosphate (Poly P) is a polymer of various phosphate residues linked by phosphoanhydride bonds as in ATP. It is found in all cells in nature with roles in the origin and survival of species, particularly in bacteria.
To study the role of the inorganic polyphosphate in bacteria, we obtained knockout mutants of polyP metabolism genes in Escherichia coli K12. We performed DNA microarray experiments of single mutants in polyphosphate kinase 1 (PPK1), exopolyphosphatase (PPX) and also with the double mutant (PPK1 and PPX).
The mutant strains growth normally in LB medium but have different colony morphology phenotypes. All mutants have flagellation problems and a detail description of all gain and lost phenotypes o these strains will be published soon because we performed a complete phenotypic microarray study of all three mutant strains.

overall_design:
In the study presented here, the expression of all the genes of Escherichia coli K12 was measured in three mutant strains lacking the enzymes related to the inorganic polyphosphate metabolism. We used reverse dye labelling for all co-hybridizations.

Supplementary files: There are two raw data file versions -- a 'full version' including Gene Symbols, ECK numbers, Platform GPL534 IDs, and the expression values -and- an 'abbreviated version'with only the Gene Symbol name and the expression values.

first: Cecilia

last: Mauriaca

first: Camilo

last: Valdivieso

first: Alex

last: Vergara

first: Carlos

last: Jerez

last: Chávez


title: Cultured human amniotic fluid-derived mesenchymal stromal cells [PIQOR data]

status: Public on Jun 18 2011

city: Malaga

first: Jose Maria

last: Miranda-Sayago

state: Malaga

address: Campus Teatinos, SN

department: Biochemistry, Molecular Biology and Immunology

country: Spain

institute: University of Malaga

summary: Human amniotic fluid-derived mesenchymal stromal cells (hAMSC) have become one of the main cell populations used in regenerative medicine and for the study of various clinical disorders. These cells have a great capacity for proliferation and differentiation, do not form teratomas when transplanted into animal models and their stemness seems to be between embryonic cells and adult mesenchymal cells. Prior to their use in cell therapy they must be cultured and expanded in vitro, but the effect this process has on their fitness, a determining factor for the success or failure of cell therapy, is unknown. We undertook a follow-up of gene and microRNAs (miRNAs) expression using microarray of a hAMSC population kept under in vitro culture conditions for the first 15 passages. Significant changes were noted in the expression of various mRNAs and miRNAs, particularly down-regulation of TP53, increased expression of hsa-miR-125a and up-regulation of CDKN2D. The variations in TP53 and hsa-miR-125a may act as an indicator of the stemness of the hAMSC, whereas CDKN2D may indicate the reduction in the proliferative capacity of these cells in a TP53-independent mechanism. The genes described in this study will help evaluate the fitness of hAMSC, thus guaranteeing their biological quality for use in regenerative medicine.

overall_design: The variations in the expression patterns of the mRNAs were studied using topic-defined PIQOR Stem Cell microarrays. Human amniotic fluid-derived mesenchymal stromal cells pellets of 5 x 10E5 cells were cryopreserved in liquid nitrogen and send to Miltenyi Biotec for their analysis. A pool of non-stimulated lymphocytes (non-mitotic cell cycle) obtained by Ficoll isolation in the Immunology Service of Carlos Haya hospital was used as a control of the expression.

first: Jose

first: Nieves

last: Fernandez-Arcas


title: Continued studies the glycosylation changes in a cell line model of embryo implantation of steroid-exposed and control Ichikawa cells

summary: This experiment continues our investigation of glycosylation in endometrial epithelium.  During the menstrual cycle, estrogen and progesterone sensitise the uterus to the implanting embryo. There is a major expansion of Golgi and secretory function with increases in mucin (MUC1) and the appearance of specific glycans.  We are using Ishikawa cells as a model for in vitro implantation, and have shown the presence of steroid receptors and alterations in glycosylation after hormone exposure.

overall_design:
We are using Ishikawa cells as a model for in vitro implantation, and have shown the presence of steroid receptors and alterations in glycosylation after hormone exposure.  We now wish to profile glycogenes in steroid (E+P) exposed and control cells.
We now wish to profile glycogenes in steroid (E+P) exposed and control cells. Steroid conditions are: estradiol (E2 at 10exp -8M) and medroxyprogesterone acetate (MPA at 10 exp -7M)): 24h with E2, followed by E2+MPA for 72h. Cells were grown on plastic or glass.

last: Aplin


summary: The main objective of my laboratory is to identify the molecular mechanisms of HIV-1 entry and infection of the CNS, focusing on PG-species as the primary attachment receptors, and their interactions with certain HIV-1 proteins.

overall_design:
Microarray analysis to examine glycan-related gene expression in primary human cells infected with HIV-1 to confirm that distinct cell-associated PGs play differential role(s) in viral capture and transfer via BMVECs and astrocytes
To examine glycan-related gene expression in primary human cells (CNS-based cells such as BMVECS, astrocytes and microglia, as well as primary human control peripheral blood mononuclear cells, such as CD4+ T-cells and macrophages)  infected with HIV-1 at different time-points (6 hours post-infection, 24-hours post-infection and 7-days post-infection) as compared to uninfected cells (all of the above cells, uninfected, in triplicates, with matching time-points).

first: Elias

last: Argyris


title: Cultured human amniotic fluid-derived mesenchymal stromal cells [miRXplore data]

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
Mus musculus
Rattus norvegicus

overall_design: miRNA patterns were studied in in vitro culture samples of human amniotic fluid-derived mesenchymal stromal cells at passages P1, P3, P5 and P10 with miRXplorer microarrays using a synthetic pool of miRNAs. Cell pellets of 5 x 10E5 cells were cryopreserved in liquid nitrogen and sent to Miltenyi Biotec for their analysis.


title: Bacillus subtilis CU1065 ramoplanin stimulon

status: Public on Jun 17 2011

city: Ithaca

middle: D.

last: Helmann

address: 372 Wing Hall

department: Microbiology

institute: Cornell University

summary: Transcriptional response of Bacillus subtilis to ramoplanin in wild-type CU1065.

overall_design: Bacillus subtilis CU1065, WT (-RAM) vs. (+RAM) and liaR (yvqC) deletion (-RAM) vs. (+RAM). The experiment was conducted in triplicate using three independent total RNA preparations. Combined reference RNA was labeled with Cy3 and ramoplanin treated/untreated samples were labeled with Cy5.

first: Anna-Barbara

last: Hachmann

middle: D


title: The effect on cancer cells of the glycodendrimers believed to decrease tumor growth, prolong survival time, and enhance immune response

summary: The potential therapeutic relevance of synthetic glycodendrimers was shown on animal subjects bearing colorectal carcinoma and melanoma. The application of glycodendrimers led to decreased tumor growth and prolonged survival time of the animals, accompanied by an enhancement of immune response. Comparison of mass spectra and biochemical analyses have indicated that the uptake of glycodendrimers by target cells resulted in changes of surface glycosylation. Moreover our research shows changes in membrane constitution. Recently our lab found changes in proliferation potential and expression of GnT-III and GnT-V in cancer cells in contrast to the peripheral blood mononuclear cells from healthy donors. As there are a sizable amount of enzymes involved in glycan metabolism and signaling pathways, the glyco-chip would be a very effective tool to determine which genes are involved in metabolism of these glycodendrimers. Our lab would like to develop expression profiles of cancer cells treated by glycodendrimers and a corresponding untreated control.

overall_design:
For chip analyses we have chosen cell lines derived from chronic myeloid leukemia (K562), multiple myeloma (U266) and colon carcinoma (HT-29). There are untreated control samples and post glycodendrimers treatment samples from each cell line.
All samples were produced and developed in triplicate for a total of 18 samples.  The RNA was labeled and hybridized onto the GLYCOv3 array.  .

first: Anna

last: Fiserova


title: Expression data from patients with advanced ovarian cancer

city: boston

first: panagiotis

last: konstantinopoulos

state: MA

address: 330 brookline av

department: hem/onc

institute: bidmc

summary:
Integration of several ovarian cancer datasets to identify a reproducible predictors of survival
Four microarray datasets from different institutions were reprocessed in a uniform manner, into a single training dataset. Survival analysis was performed and a validation cohort (61 patients from 3 institutions) was profiled using a custom array to confirm the prognostic value of the predictors.

The four datasets were obtained from the following reports:

Spentzos D, Levine DA, Ramoni MF, et al. Gene expression signature with independent prognostic significance in epithelial ovarian cancer. J Clin Oncol 2004;22:4700-10
Bild AH, Yao G, Chang JT, et al. Oncogenic pathway signatures in human cancers as a guide to targeted therapies. Nature 2006;439:353-7
Marquez RT, Baggerly KA, Patterson AP, et al. Patterns of gene expression in different histotypes of epithelial ovarian cancer correlate with those in normal fallopian tube, endometrium, and colon. Clin Cancer Res 2005;11:6116-26
Zhang L, Volinia S, Bonome T, et al. Genomic and epigenetic alterations deregulate microRNA expression in human epithelial ovarian cancer. Proc Natl Acad Sci U S A 2008;105:7004-9

overall_design: 61 samples

first: Dimitrios

last: Spentzos


title: Microarray of RNA of TCR transgenic CD8 T cells to assess the role of protein glycosylation in the decision between deletion vs. anergy in immune tolerance

last: Head

summary: Activation of peripheral T lymphocytes in the absence of pathogen induced inflammation or costimulatory signals results in tolerance. Two different tolerance mechanisms have been described, deletion and anergy.  We recently demonstrated that an important variable which is responsible for the decision between anergy and deletion for CD8 T cells is the strength of signaling through the T cell receptor(Redmond and Sherman, Immunity 22:275,2005).  However, it is not know what the downstream signals are that result in death(deletion) vs. survival(anergy)of the activated cells.  Marth and co-workers have previously shown that CD8 T cell apoptosis under conditions similar to those that occur during tolerance in vivo may involve a change in protein glycosylation (Van Dyken et.al., Mol.Cell.Bio.27:1096,2007).In order to assess the role of protein glycosylation in the decision between deletion vs.anergy in immune tolerance, we have prepared purified TCR transgenic CD8 T cells stimulated in vivo using a strong antigenic signal (anergy conditions) or a weak antigenic signal (deletion conditions) with cognate antigen.  We wish to assess transcriptional differences in genes involved in protein glycosylation in these 2 cell populations.  The control population will be naive trangenic CD8 cells.

overall_design:
Dr. Sherman's lab aims to assess the role of protein glycosylation in the decision between deletion vs. anergy in immune tolerance, they have prepared purified TCR transgenic CD8 T cells stimulated in vivo using a strong antigenic signal (anergy conditions) or a weak antigenic signal (deletion conditions) with cognate antigen.  Dr. Sherman's lab wishes to assess transcriptional differences in genes involved in protein glycosylation in these 2 cell populations.  Then control population will be naïve transgenic CD8 cells.
RNA preparations of mouse CD8 T cells from the B10D2 strain with three different conditions (Naïve, Deleted, and Anergic) were sent to the Microarray Core (E). The RNA was amplified, labeled, and hybridized to both the GLYCOv3 and Mouse 430A_2.0 microarrays.

first: Linda

last: Sherman


title: To understand which glycan-related genes are involved in the recognition, signaling, and binding of nutrients (HMOs)

summary: Dr. Lebrilla's lab is working on another set of transcriptomics experiments which will measure whether HMO (human milk oligosacharides) elicit a differential response on Caco2 in the presence of B.infantis. They wish to supplement the data obtained with the Illumina platform with glycan-related expression data obtained with the Gene-chips to achieve a more complete understanding on how Caco2 cells respond to the presence of HMOs and B.infantis. The transcriptomics data is being complemented by qPCR based bacterial-epithelial cells binding assays, and glycan arrays assays for which they have initiated a collaboration with the CFG Core H (David Smith).

overall_design:
The influence, signaling and adhesion between Bifidobacterium infantis, a probiotic microorganism abundant in infant feces, and Caco2 epithelial cells. Our hypothesis is that HMOs act as signaling molecules for both Caco2 cells and B. infantis to indicate the presence in the gut, of a nutrient niche adapted for the nourishment and recruitment of HMO-consuming probiotic microorganism. HMOs therefore could play a role in the expression and binding to GBPs both on epithelial cells and in probiotic gut microorganisms. Preliminary transcriptomics data analysis obtained by using the Illumina Human Ref-8 Expression Bead Chip, has revealed that co-incubation of Caco2 cells with HMO upregulates genes belonging to chemokines, adhesion molecules, growth factors and glycan degradation, thus indicating that HMOs are actively recognized by these cells.  We are working on another set of transcriptomics experiments which will measure whether HMO elicit a differential response on Caco2 in the presence of B.infantis. We wish to supplement the data obtained with the Illumina platform with glycan-related expression data obtained with the Gene-chips to achieve a more complete understanding on how Caco2 cells respond to the presence of HMOs and B.infantis. The transcriptomics data is being complemented by qPCR based bacterial-epithelial cells binding assays, and glycan arrays assays for which we have initiated a collaboration with the CFG Core H (David Smith). We are requesting the analysis of 16 RNA samples from Caco2 cells on the Glyco-gene chips, for testing respectively 4 independent biological replicates for each of the 4 different co-incubation conditions.
RNA preparations from Caco2 cells were sent to Microarray Core (E).  The RNA was amplified, labeled, and hybridized to GLYCO_v3 microarrays.

first: Carlito

last: Lebrilla


title: Transcriptional responses of Fusarium graminearum to plant cell wall substrates

status: Public on Jun 16 2011

city: Illkirch

first: Raphael

last: Carapito

address: Bd Sébastien Brandt

institute: Ecole Supérieure de Biotechnologie de Strasbourg

summary: We report a complete transcriptomic study of Fusarium graminearum in response to glucose, cellulose, xylan and cell wall fragments with a whole genome microarray from febit.

overall_design: Fusarium graminearum was cultured at 25 °C on minimal M3 medium with glucose, birch wood xylan, carboxy methyl cellulose or hop cell wall as sole carbon source at a concentration of 10 g/L. Microarray experiments were achieved with a Geniom device (febit biomed, Germany).  Each of the four tested condition was tested with two biological and two technical replicates.


title: Glycan analysis of promary human lymphatic endothlial cells (LEX) in their resting and activated states, in normal and tumor tissues

first: Steven

summary: Trafficking of leukocytes (dendritic cells, memory T cells, neutrophils) and tumor cells through the lymphatic network is a key process in inflammation and immunity and an important mechanism in metastatic spread of human cancers (1-3). Such trafficking involves both communication with and passage across lymphatic endothelium, the distinct endothelium that lines lymphatic vessels within the peripheral tissues and forms the lymphatic sinuses within lymph nodes. However, in comparison with blood vascular endothelium, there is only a rudimentary understanding of the molecular phenotype of lymphatic endothelium, and only a basic knowledge of the glycoconjugates that regulate leukocyte-endothelial and tumor cell-endothelial interactions in the lymphatic compartment.

overall_design:
We would anticipate that changes in glycosylation of LEC cell surface proteins following activation might affect important functions associated withy LEC including eg. interactions with leukocytes and/or sequestration of GAG-binding chemokines. We already have shown that ligand binding to the lymphatic endothelial hyaluronan receptor LYVE-1 is reversibly masked by terminal sialation in LEC and that functional regulation of LYVE-1 is likely to be important in inflammation. An advantage of our proposal is that we can routinely isolate primary LEC in relatively large numbers, and have the necessary ethical approval to do so from human tissue.
Glycan analysis of primary human lymphatic endothelial cells (LEC) in their resting and activated states, in normal and tumour tissues and a comparison of the LEC glycan structures with those from blood vessel endothelial cells

first: David

last: Jackson


overall_design:
RNA preparations from monocytes (Mo), immature dendritic cells (iDC) and mature dendritic cells (mDC), from different healthy human donors were sent to both Microarray Core (E) and Core(C).  The RNA was put on an RNeasy Column, amplified, labeled, and hybridized to the Glycov3 microarrays.  Data was sent to Dr. van Kooyk's lab for analysis.
The RNA was put on an RNeasy Column, amplified, labeled, and hybridized to the Glycov3 microarrays.  Data was sent to Dr. van Kooyk's lab for analysis.

first: Yvette

last: Van Kooyk


title: To identify specific glycosylated structures on brain endothelial cells that mediate rolling, adhesion, and diapedesis of leukocytes in the brain

summary: The blood-brain barrier (BBB) dynamically controls and maintains a precisely balanced brain microenvironment necessary for reliable functioning. It is made up of highly specialized endothelial cells (ECs) in the lining of the vascular wall. These ECs are surrounded by the basal lamina, astrocytic perivascular endfeet, pericytes, microglia and neuronal processes that have been shown to contribute to barrier function1. In essence, the brain endothelium limits both transcellular and paracellular passage of cells and molecules into the central nervous system (CNS). Transcellular passage of hydrophilic molecules is limited due to a low rate of transcytotic vesicles, an extremely low pinocytotic activity, expression of active efflux transporters, and high metabolic activity. Paracellular diffusion of hydrophilic molecules and trafficking of immune cells is restricted by a network of tight junctions (TJs)2-5. Due to these characteristics, the BBB is able to protect the CNS from sudden changes in blood composition and uncontrolled influx of immune cells. In a large number of neuro-inflammatory diseases, an impaired function and an opening of the BBB are observed. In diseases of the CNS like multiple sclerosis or stroke or after brain trauma, the BBB becomes inflamed (mediated by for example reactive oxygen species (ROS) and hypoxia) and its function severely impaired which gives rise to enhanced cellular infiltration, thus contributing to tissue damage and neurological deficits.   Due to the specialized nature of brain ECs, transmigration of leukocytes through cerebrovascular endothelium is likely to differ from that in other vascular beds. Generally, the transmigration process is closely controlled by the interaction of leukocytes with the endothelial surface followed by low velocity rolling, arrest, firm adhesion, and finally transmigration. For the diapedesis of immune cells across the cerebral vasculature a re-arrangement of the cytoskeleton and opening of the TJ complexes is required to allow cells to pass into the sub endothelial space. In the initial step of the adhesive cascade leukocytes adhere to the endothelium with low affinity. This adhesion is mediated by several members of the selectin family and their corresponding ligands. Despite the low affinity of these interactions, resulting contact of leukocytes with the endothelium leads to further activation of both cell types and finally transmigration of the leukocytes through the BBB6,7.

overall_design:
The glycosylation of endothelial cells of different vascular bed origins
To date it is unknown which specific carbohydrate structures on the BBB endothelium mediate leukocyte capture and rolling, and whether these structures are differentially expressed onto inflamed brain EC compared to normal brain ECs and vascular beds of other organs. Initial studies using qPCR and FACS analysis within our group reveal that brain endothelial cells have a different profile in their glycosylation-related genes compared to microvascular ECs upon inflammation, which may result in a different glycosylation profile of adhesive structures and may underlie rolling, adhesion and diapedesis of leukocytes. In this project we wish to identify specific, glycosylated structures on brain endothelial cells that mediate capture, rolling and diapedesis of leukocytes in the brain. To investigate the expression of glycosyltransferases in dendritic cells and the changes in expression associated with maturation. RNA preparations from stimulated and non-stimulated hcMEC/D3 (human brain endothelial cells line) and FMVEC (human promary microvascular endothelial cells isolated from foreskins) were sent to both Microarray Core (E) and Core(C). The RNA was put on an RNeasy Column, amplified, labeled, and hybridized to the Glycov3 microarrays. Data was sent to Dr. van Kooyk's lab for analysis.


title: Analysis of the enzymes involved in PEN5 carbohydrate metabolism

summary: Dr. Eric Vivier's lab would like to tackle the enzymes involved in PEN5 carbohydrate metabolism. We have generated these cell lines by repeated cell sorting and cell cloning of the parental HSB-2 T cell line and the parental JY B cell line. H+ are PEN5+ HSB-2 cells, H- are PEN5- HSB-2 cells; JY+ are PEN5+ HSB-2 cells, JY- are PEN5- HSB-2 cells.

overall_design:
We have originally described a cell surface molecule called PEN5, as a sulfated lactosamine, that is selectively expressed on a mature subset of human NK cells (Vivier, J. Exp. Med. 1993). Later, we have shown that the PEN5 carbohydrate decorates PSGL-1, confering L-selectin binding properties (André et al. PNAS, 2000). We have recently performed a Glycan Array Screening via Core H (Glycomics 917). These studies indeed reveal a very nice binding of the anti-PEN5 mAb to a subset of sulfated lactosamines. We would like to tackle the enzymes involved in PEN5 carbohydrate metabolism. We thus requesting a Gene microarray analysis performed using RNA extracted from PEN5+ and PEN5- NK cells, as well from the H+, H-, JY+ and JY- stable cell lines. We have generated these cell lines by repeated cell sorting and cell cloning of the parental HSB-2 T cell line and the parental JY B cell line. H+ are PEN5+ HSB-2 cells, H- are PEN5- HSB-2 cells; JY+ are PEN5+ HSB-2 cells, JY- are PEN5- HSB-2 cells.
RNA preparations from variants of the HSB-2 (human T leukemia) and JY (human B lymphoma) cell lines, which were express or not PEN5 were sent to Microarray Core (E).  These stable variants are referred to as H+ (HSB-2 cells expressing PEN5), H- (HSB-2 cells NOT expressing PEN5), JY+ (JY cells expressing PEN5), and JY- (JY cells NOT expressing PEN5).  Samples were prepared at 3 different time points.  The RNA was amplified, labeled, and hybridized to the GLYCOv3 microarrays.  Data was sent to Dr. Eric Vivier for analysis.

first: Eric

last: Vivier


title: A glycoanalysis of WEHI 231 B cell line to determine if there is a different profile of carbohydrates following apoptosis, especially if there is an exposure of fucose residues

summary: We have determined that there is a carbohydrate change on CD44 on leukocytes during apoptosis.  This change is important for phagocytosis of the apoptotic cells and is inhibitable by fucoidin but not mannan or monosaccharides.  In addition, this occurs without new protein synthesis.

overall_design:
We want to immunoprecipitate CD44 from apoptotic and control cells and determine whether there is a different profile of carbohydrates, especially exposure of fucose residues.  We have used a lectin to isolate a protein consistent with the molecular weights of CD44. We would also like to do a profile of the apoptotic versus control membranes from the B cells as a comparison.
RNA preparations were sent to the Microarray Core (E). The RNA was amplified, labeled, and hybridized to the GLYCOv3 microarrays.  Data was provided to Dr. Mills' laboratory for analysis.

first: Joan

last: Cook-Mills


title: Microarray analysis for differential gene expression in M1-SDHHbtail

city: Columbus

first: Vijay

last: Pancholi

state: OH

address: 420W 12th Ave, TMRF-288

institute: Ohio State University

summary: A total of 215 gene were found to be differentially expressed; 90 of them were up regulated and 125 genes were down regulated. Most notably the functional categories that were affected include Virulence genes, carbohydrate genes and lipid synthesis genes. Some of the virulence genes were down regulated by more than 16-32 fold. These results indicate that SDH plays an important role in the regulation of virulence.

overall_design:
S.pyogenes strain SF370 (wild-type) was procured from ATCC (ATCC 700294). M1-SDHHbtail (SDH cytoplasmically retained) mutant strain PGB-1 was obtained using pFW5 vector as described before ( Boel G. Jin, H. and Pancholi, V. (2005). Inhibition of cell surface export of groupA streptococcal anchorless surface dehydrogenase (SDH) affects bacterial adherence and antiphagocytic properties. Infect. Immun. 73:6237-6248).

Both strains  (M1-Wild-type and PGB1(M1-SDHHbtail) were grown in Todd-Hewitt broth to their late log phase. Bacteria  were harvested by centrifugation and washed twice with sterile PBS. Total RNA was isolated from  these washed streptococci using Qiagen RNeasy Mini kit.

For synthesis and labeling of cDNA, 20 ug of total RNA, 1.5 ug of random hexamer, 0.5mM dNTPs (except that 0.2mM of dTTP was replaced by the same amount of amino-allyl dUTP to incorporate dUTP into first-strand cDNA) were used in each reverse transcriptase reaction (Superscript II Reverse Transcriptase, Invitrogen).

Purified cDNA preparations from the wild-type and PGB1 mutant strains were labeled with either Alexafluor-555 or Alexafluor-647 depending on the experimental design ( i.e. dye swap experiment). Differentially labeled probes were then combined and purified.  Using three independently isolated RNA preparations (biological replicates), a total of 8 experiments (incorporating  dye swaps) were performed. Accordingly eight hybridization measurements for this mutant were performed. Thus
Exp-1 and -2 (GSM380310, GSM380320) are the technical replicates of the biological sample-1, Exp-3,-4 and -5 (GSM380321, GSM380322, GSM380331) are technical replicates of biological sample-2 and Exp-6,-7, and -8 (GSM380332,GSM380333, GSM380334) are technical replicates of the biological sample-3.

Signals of the bound reagents on the microarray spots in terms of relative fluorescence values were measured and quantified by a laser scanner (GenePix 4100 ) at 10um/pixel resolution. The resulting images were processed using Gene Pix Pro  software (version 4.0, Axon instruments). The raw data were obtained in the form of GenPix  *.gpr output files.

The web application CARMAweb (Comprehensive R based microarray analysis web service) was used for the normalization and analysis of microarray data.  All raw data (in the form of  *. GPR files) were uploaded to the web application in the data directory. Using appropriate navigation tree, background correction from the foreground signal was applied and within microarray normalization was achieved using the Lowess method.  Genes flagged as bad spots by the scanning software were excluded from the analysis and all flagged spots were given a weight of zero. The normalized data were then subjected to fold-change analysis and t-statistics using Bioconductor multtest package. CARMAweb allows to set Log2 cut-off values for both the M (regulation) and the A (average expression) values. Differentially expressed genes (Log2 values of Mutant647-red vs. wild-type555-green or Log2 values Mutant-555(green) vs Wild-type 647-red) were defined based on cut-off value >1 Log2< i.e. all genes that show a two or more-fold up- or down-regulation.  Bioconductors Multtest package provided suitable method to adjust P values according to multiple hypothesis testing problems.

first: Hong

last: Jin


title: Diagnostic marker for lung cancer through microarray analysis

summary: Dr. Kate Olson's Lab is interested to expand these analysis to comparisons between human normal lung tissue and human lung tumor tissue. In hopes that this will help in finding a diagnostic marker for lung cancer. Since there will be more variability between these samples, we would like to get preliminary data on ten normal lung tissue and ten lung tumor tissues from the same patient.

overall_design:
We have already analyzed 4 cell lines and we saw gene expression variability related to how metastatic the cell lines were. This has been written and submitted for publication.  We would like to expand these analysis to comparisons between human normal lung tissue and human lung tumor tissue.  We hope that this will help in finding a diagnostic marker for lung cancer. Since there will be more variability between these samples, we would like to get preliminary data on ten normal lung tissue and ten lung tumor tissues from the same patient. The samples were dissected by a pathologist prior to extraction to maximize the amount of normal tissue included in the tumor samples.
RNA samples from non tumor and tumor lung cancer patients were received by Core E.  The RNA was amplified, labeled, and hybridized to the GLYCOv3 microarrays.

first: Kate

last: Rittenhouse-Olson


title: Glyco-gene expression in naive and activated murine regulatory T-cells vs. conventional T-cells

summary: In collaboration with Professor Anne Dell at the South Kensington Campus of Imperial College London, we have conducted preliminary glycomic surveys of C57BL/6 CD25+ and CD25- CD4+ T cells. Cells selected in serum-containing culture medium demonstrated unusual N-glycan profiles compared to those prepared in phosphate buffered saline (PBS). These profiles were subsequently shown to result from cellular sequestration of glycoproteins present in fetal calf serum (FCS) used to supplement the medium (1).   Analysis of the N-glycan profile of murine CD4+CD25+ Tregs prepared in PBS revealed distinct differences from CD4+CD25- T cells – namely, a reduction in sialic acid capping and loss of core fucose.

overall_design:
Preliminary studies are underway to ascertain the minimum medium required to sustain general T cell survival, whilst maintaining ‘clean’ glycomic profiles. The effect of glycoprotein withdrawal on CD25+ and CD25- CD4+ T cells in culture was investigated by selecting the cells in FCS-free medium and incubating them for six hours with either FCS-containing medium, or PBS supplemented with FCS, fetuin or fetuin-derived glycans. Early apoptosis was measured using Annexin-V (BD Biosciences, Heidelberg, Germany) and late apoptosis using 7-aminoactinomycin D (7-AAD) (BD Biosciences), in parallel with a complementary apoptosis detection method – measurement of the mitochondrial membrane potential using the BD™ Mitoscreen Kit (BD Biosciences).
This apoptosis staining study demonstrated that there was a remarkably higher percentage of death in both CD25+ and CD25- CD4+ T cells cultured in PBS versus cells cultured in RPMI-1640 or medium containing FCS. Both CD25+ and CD25- CD4+ T cells were highly susceptible to apoptosis in glycoprotein-free medium, demonstrating their exquisite sensitivity to the lack of glycoprotein survival factors. Continuing studies are addressing the minimum medium required to sustain cells undergoing polyclonal activation in vitro and the impact of activation on the N-glycan profiles of Tregs versus conventional T cells. To complement this work, we used Core E resources to investigate glycosyltransferase gene expression in freshly isolated versus activated C57BL/6 CD25+ and CD25- CD4+ T cells.

first: Oliver

last: Garden


title: Investigate the anti-tumor mechanisms of But4ManNAc and butyrate in two breast cancer cell lines

summary: But4ManNAc - an analog of ManNAc, the committed precursor of sialic acid - decreases metastatic potential, inhibits growth, and triggers apoptosis in many cancer cells. The anti-tumor effects of But4ManNAc may result from a synergistic interaction between butyrate-induced changes in gene expression and ManNAc-induced altered sialylation.  This experiment provides insight into synergistic interactions by identifying changes in gene expression specific to But4ManNAc and not shared with butyrate.

overall_design:
Specifically, microarrays will compare gene expression of metastatic breast cancer cells (MDA-MB-231) treated with But4ManNAc to that of cells treated with sodium butyrate at concentrations which inhibit invasiveness but do not trigger apoptosis.  Differences in gene expression  demonstrate a synergistic interaction between butyrate and sialylation as well as suggest mechanisms to account for this synergy.
RNA preparations of treated and control metastatic breast cancer MDA-MB-231 cells were sent to Microarray Core (E). Five conditions where sent: cells treated with 50uM But4ManNAc, 125uM But4ManNAc, 50uM But5Man, 125uM But5Man, and Ethanol (control). Three replicate samples from each condition were used in the study.  The RNA was amplified, labeled, and hybridized to the GLYCOv3 microarrays. Data was analyzed to identify differences in gene expression that demonstrate a synergistic interaction between butyrate and sialylation, as well as suggest mechanisms to account for this synergy.

first: Kevin

last: Yarema


title: Gene expression in B cells from mice with the autoimmune disease nephrotoxic nephritis

summary: Studying the differences in glycosylation in B cells of normal mice and mice with an autoimmune disease (nephrotoxic nephritis).

overall_design:
Differences in glycosylation in B cells of normal mice and mice with an autoimmune disease (nephrotoxic nephritis) were studied via gene expression analysis.
RNA from mouse resting spleen B cells and B cells stimulated with antiCD40, CpG, and IL4 for 5 days, was isolated and prepared. One sample for each class was prepared.  RNA was labeled and hybridized to the GLYCOv3 array. Resulting Gene expression patterns were analyzed.

first: Jeffrey

last: Ravetch


title: Gene microarray screening of RNA from bruce 4 ES cells

summary: The sample is wild type male bruce 4 ES cells which has been used in core f to generate knockout mice.

overall_design:
RNA samples from wild type male bruce 4 ES cells, which has been used in core f to generate knockout mice, were screened.  Scraped cells, pre-plated and non-pre-plated trypsinized cells were compared to determine the effects of trypsin and fibroblasts on gene expression of the ES cells.
Mouse ES cell pellets were delivered to Core E for RNA Extraction. *Concurrent analysis of glycan profiles in these cells was performed in Core C*. RNA from scraped ES cells, trypsinized cells, and pre-plated trypsinized cells was isolated and prepared in triplicate.  RNA was labeled and hybridized to the GLYCOv3 array. Resulting Gene expression patterns were analyzed by Core F.

first: James

last: Paulson


title: miRNA profiling of 29 cases of diffuse large B cell lymphoma

status: Public on Aug 02 2011

city: Madrid

first: Santiago

last: Montes-Moreno

state: Madrid

address: C/ Melchor Fernández Almagro, 3

department: Molecular Pathology

institute: CNIO

summary: 29 cases of RCHOP treated DLBCL were profiled for miRNA expression. Different integrated annalysis were performed, including mirNA differential expression between cases classified according to the COO signature (see later) and correlation tests between gene and miRNA expression.

overall_design: Cases classified according to the cell of origin classsifer (Wright G, Tan B, Rosenwald A, Hurt EH, Wiestner A, Staudt LM. A gene expression-based method to diagnose clinically distinct subgroups of diffuse large B cell lymphoma. Proc Natl Acad Sci U S A 2003;100:9991-6.) based on gene expression were subjected to miRNA expression profiling.

first: S

last: Montes Moreno

first: N

last: Martinez


title: The role of glycans and glycosylation in cell-type specific effects on viral infectivity

last: Head

summary: Our lab has previously used the Glyco v3 gene-chip to analyze RNA from human macrophages and T-cells, as part of a project examining the effect of cellular origin on the viral infectivity of HIV/SIV. Our experiments have led us to hypothesize that the different glycosylation pathways present in macrophages and T-cells result in the production of virions with differently glycosylated viral proteins and different glycans present on the virion surface. Furthermore, studies with glycosidases using the Glyco v3 gene-chip have indicated that these differences in the glycosylation profiles of virions derived from different cell types may influence viral infectivity.

overall_design:
Here we used glyco v3 chips for identifying the enzymes active in the glycosylation pathways of 174xCEM and 293 cells, which are cell types commonly used in HIV and SIV research. These analyses will allow us a better understanding of the role of glycans and glycosylation in cell-type specific effects on viral infectivity because there is a much larger amount of data on virus derived from these cell types and we will be able to relate our studies more directly to previous work in our lab and other labs.
RNA from 174xCEM and HEK293 cells, cell types commonly used in HIV and SIV research, was isolated and sent to Microarray Core (E).  RNA was prepared in duplicate, totaling 4 samples.  Samples were labeled and hybridized to the GLYCOv3 array and gene expression patterns were used to identify enzymes active in glycosylation pathways.

first: Howard

last: Fox


title: Cardiac microRNA profiling in mice treated with lipopolysacharide

city: New York

first: Konstantinos

last: Drosatos

department: Medicine

institute: Columbia University

summary: To identify the microRNA (miR) profile in the hearts of mice that were treated with lipopolysaccharide (LPS) and compare this profile with respective profiles that have been published in studies with mice that underwent pressure overload heart failure, 8 mouse samples were hybridized to Sanger 14 Multi-Species miR microarrays. As a result, we observed that particular cardiac miRs that are modulated during pressure overload heart failure are not affected significantly by treatment with LPS

overall_design: Total RNA from pieces of hearts obtained from 8 C57BL/6 mice that were treated either with saline (4 mice) or with lipopolysacharide (4 mice).

first: Ira

last: Goldberg


title: Expression analysis of Salmonella enterica serovar Typhimurium 14028 delta GidA mutant

status: Public on Jul 20 2011

city: Madison

first: Daniel

last: Shippy

address: 1675 Observatory Dr

institute: University of Wisconsin-Madison

summary:
Investigation of whole genome gene expression level changes in a Salmonella enterica serovar Typhimurium 14028 delta GidA mutant
The mutant described in this study is further analyzed in Shippy, D. C., N. M. Eakley, P. N. Bochsler, and A. A. Fadl. 2011. Biological and virulence characteristics of Salmonella enterica serovar Typhimurium following deletion of glucose-inhibited division (gidA) gene. Microb Pathog.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S

overall_design: A single chip study using three separate cultures of wild-type Salmonella enterica serovar Typhimurium 14028 and three separate cultures of a single mutant, delta GidA Salmonella enterica serovar Typhimurium 14028.

first: A

last: Fadl

first: D


title: qRT-PCR of ex vivo Salmonella enterica in NRAMP-1 minus/plus macrophage background

status: Public on Jun 13 2011

city: Rockville

first: John

last: Braisted

state: MD

address: 9704 Medical Center Dr

department: PFGRC

institute: J Craig Venter Institute

summary: A major virulence factor in S. enterica is the pathogenicity island Spi2. The goal of the experiment was to determine the impact of the NRAMP-1 (natural resistance-associated macrophage protein-1) on expression of Spi2 in S. enterica wild type (WT) and triple mutant strain (TMS) with deletions in SPI-1 TTSS, SPI-2 TTSS, flagella. The stain 14028 was used for the experiment. Cells were grown in a NRAMP-1 wildtype (plus) or deleted (minus) macrophage background. Total RNA was extracted and processed by qRT-PCR.

platform_organism: Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S

overall_design:
Two strains ( WT and TMS) grown under 2 conditions (NRAMP-1 wildtype (plus) or NRAMP-1 deleted (minus)) - total 4 samples were analyzed in the study.
All PCRs were done in duplicates, located on the same PCR plate. For each duplicate the mean value was calculated and used as a data point for further normalization. We averaged the Cp values for all the data points in a sample and then subtracted this sample average Cp from all the data points of the sample. Only 192 primer pairs from 288 on the platform GPL11111 were used.

first: Fred

last: Heffron

first: Marcus

last: Jones

first: Tatiana

last: Dracheva


title: qRT-PCR of ex vivo Salmonella enterica in TLR4 minus/plus macrophage background

summary: A major virulence factor in S. enterica is the pathogenicity island Spi2.  The goal of the experiment was to determine the impact of the toll-like receptor 4 (TLR-4) on expression of Spi2 in S. enterica wild type (WT) and triple mutant strain (TMS) with deletions in SPI-1 TTSS, SPI-2 TTSS, flagella. The stain 14028 was used for the experiment.  Cells were grown in a TLR-4 wildtype (plus) or deleted (minus) macrophage background.  Total RNA was extracted and processed by qRT-PCR.

overall_design:
Two strains (WT and TMS) grown under 2 conditions (TLR-4 wildtype (plus) or  TLR-4 deleted (minus)) - total 4 samples were analyzed in the study.
All PCRs were done in quadruplicates, located on the same PCR plate. For each quadruplicates the median value was calculated and used as a data point for further normalization.  A set of controls was selected for all data sets using the following criteria: the data points for the controls should have Cp <40 and be present in all the samples.


title: Glyco gene profiling of two Hela cell lines to help identify NKp30L expressed on tumoral cells

first: Steven

summary:
NKp30 is one of the first NK-specific triggering receptor involved in tumour cell lysis to be identified. It is part of the so-called Natural Cytotoxicity Receptors (NCRs) also including NKp44, NKp46 and NKp80 (Bottino, 2005). Human NKp30 is a 190 amino-acids transmembrane protein with an extracellular V-type Ig-like domain containing two putative N-glycosylation sites (Pende, 1999). Three alternative splices can yield three different intracellular domains. An additional alternative splice can induce the deletion of 25 AA in the extracellular domain leading to the formation of a predicted C2-type Ig-like domain instead of a V-type Ig-like domain (Hollyoake, 2005; Neville, 1999).
Dr. Romagne lab's aim is to identify NKp30L expressed on tumoral cells. Preliminary Results: Two in vitro systems allow us to detect the NKp30L on the surface of target cells. The first reporter system is a reporter cell line expressing a NKp30 chimeric protein. Upon engagement of this chimeric protein with the NKp30L, cell activation specific for this interaction is measured. The second reporter systems uses the NKp30Fc recombinant protein as a detection tool or eventually as a blocking reagent. It is made with the sequence encoding the V-type Ig-like domain of NKp30. Work to qualify and define cell lines for the expression of NKp30L was carried out in order to identify in our two reporter systems the best cells lines to use. We finally identified the Hela EV2 and Hela PF cell lines as a positive and negative for NKp30L respectively whereas other NK receptor ligands expression is identical in these two cells lines. The NKp30L is sensitive to trypsin digestion and to PNGase F digestion in the NKp30Fc FACS staining assay. These results indicate that at least one NKp30L is probably a N-Glycan carried by a trypsin-sensitive protein at the cell surface. We performed a glycan gene expression profiling of these two Hela cell lines as this will be of major importance to help characterizing the differences in glycans between these two variants of Hela cells and the nature of the NKp30L.

overall_design: RNA preparations of Hela EV2 and Hela cells from EV23, EV2B, EV2III(positive) and PF4, PFB, PFIII (negative) cells were sent to the Microarray Core (E). The RNA was amplified, labeled, and hybridized to the GLYCOv3 microarrays.

first: François

last: Romagne


title: Genome-Wide gene expression of Tetrahymena thermophila conjugation

first: Ronald

last: Pearlman

address: 4700 Keele St

department: Core Molecular Biology Facility

institute: York University

summary: Eleven genome wide microarrays containing the predicted coding sequences (putative genes) for the ciliated protozoan Tetrahymena thermophila used to study gene expression in conjugation cells (C-0, C-15m, C-2, C-4, C-6, C-8, C-10, C-12, C-14, C-16, C-18). Combined these eleven microarrays with 50 microarrays described in Miao et al (2009) and other 6 microarrays, we constructed the Tetrahymena gene network (TGN) using three methods: the Pearson correlation coefficient, the Spearman correlation coefficient and the context likelihood of relatedness (CLR) algorithm. The accuracy and coverage of the three networks were evaluated using four conserved protein complexes in yeast, and the CLR network was found to be the best network, with a Z-score threshold 3.49. Then the TGN was partitioned, and 55 modules were found. In addition, analysis for the arbitrarily determined 1200 hubs showed that these hubs could be sorted into six groups according to expression profiles. We also investigated human disease orthologs in Tetrahymena that are missing in yeast and found evidence indicating that some of these were involved in the same process in Tetrahymena as in human.

overall_design: For conjugation, B2086 and CU428 cells that had been starved for 18 hours were resuspended in 10 mM Tris (pH 7.5) at 200,000 cells/ml, mixed, and samples were collected at 15 min, 2, 4, 6, 8, 10, 12, 14, 16, 18 hours after the mixture （referred to as C-0, C-15m, C-2, C-4, C-6, C-8, C-10, C-12, C-14, C-16 and C-18).