title: Chronic Pentylenetetrazol withdrawal induced successive gene expression changes in Drosophila head

status: Public on Oct 02 2007

first: Abhay

last: Sharma

summary:
Kindling induced by Pentylenetetrazol is an established rodent model of epileptogenesis. The molecular basis of the long-term plasticity involved is however not clear. In addition, rodent models of kindling plasticity are not useful for large-scale screening of compounds to identify antiepileptogenic drugs. Given this, we have developed a fly model of chronic PTZ- and withdrawal-induced behavioral plasticity. In our fly model, the chronic PTZ treatment is given for 7 days. This is then followed by 7 day long withdrawal. Expression profiling of fly heads at three time points in the 7 day long withdrawal period – 8th day, 10th day, and 14th day from the beginning of the treatment - showed a dynamic and widespread alteration of various functional categories of genes.
Keywords: time series

overall_design: D. melanogaster Oregon-R wild type cultures were grown at 24 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old adult males, unmated, were grown in either normal food (NF) or food containing 8 mg/ml of PTZ for seven days. Following this, the control and PTZ treated individuals were shifted to vials containing NF and maintained further for seven days. Each vial contained 30 individuals in the beginning of the treatment. Heads were harvested at three time points during withdrawal - 8th day, 10th day, and 14th day from the beginning of the treatment. Heads of flies frozen in liquid nitrogen were collected, after vigorous shaking of the vial containing the flies, using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four PTZ exposed individuals, during treatment or withdrawal period. For isolation, TRI REAGENT (Sigma) was used as per manufacturer’s recommendation. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of control and treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and PTZ treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The preprocessing and quantification of the 16 bit TIFF images were carried out using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 2.21, Excel Add-In, Stanford) under the conditions of one class response and 100 permutations.

first: Sharma

last: Abhay

first: Mohammad

last: Farhan

first: Singh

last: Priyanka


title: Array CGH data of 64 prostate cancer specimens

status: Public on Oct 03 2007

city: Stanford

first: Jonathan

last: Pollack

state: CA

address: 269 Campus Drive, CCSR3245A

department: Pathology

country: USA

institute: Stanford University

summary:
The dataset comprises array CGH data of 64 prostate cancer specimens performed on Stanford cDNA microarrays, to accompany the study of J Lapointe et al (2007).  For each array, Channel 2 represents Cy5-labeled prostate genomic DNA, and Channel 1 Cy3-labeled normal male genomic DNA.
Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc.
Keywords: Logical Set, array CGH

overall_design: Using regression correlation

first: Jacques

last: Lapointe


title: Chronic Pentylenetetrazol treatment induced successive gene expression changes in Drosophila head

city: Delhi

state: Delhi

address: Delhi University campus, Mall Road

department: Functional Genomics Unit

country: India

institute: CSIR-Institute of Genomics and Integrative Biology (IGIB)

summary:
Pentylenetetrazol (PTZ)-induced kindling in rodents is an established model of epileptogenesis. The molecular basis of the associated long-term plasticity is however not clear. Further, rodent models of kindling plasticity are not amenable to large-scale screening of compounds for identifying antiepileptogenic drugs. In this context, we have developed a fly model of chronic PTZ- and withdrawal-induced behavioral plasticity. In our model, the chronic treatment is given for 7 days, followed by 7 day long withdrawal. Gene expression profiles of fly heads secondary to chronic PTZ were examined to identify molecular correlates of behavior in our model. Expression profiling of fly heads at three time points in the 7 day long chronic PTZ - 12 hrs, 2nd day, and 7th day - showed a dynamic and widespread alteration of various functional categories of genes.
Keywords: Time Series

overall_design: D. melanogaster Oregon-R wild type flies were grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin.  The cultures were grown at 24 +/- 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Three to four days old unmated adult males were grown in either normal food (NF) or food containing 8 mg/ml of PTZ for seven days. Following this, the control and PTZ treated flies were shifted to vials containing normal food and maintained further for seven days. Each vial contained 30 flies in the beginning of the treatment. Heads were harvested at three time points during PTZ treatement - 12hrs, 2nd day, and 7th day. Flies frozen in liquid nitrogen were shaken and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four PTZ exposed individuals, during treatment or withdrawal period, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of control and treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and PTZ treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The preprocessing and quantification of the 16 bit TIFF images were carried out using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 3.0, Excel Add-In, Stanford) under the conditions of one class response and 100 permutations.


city: Umea

first: Jan

last: Larsson

address: Umea University

department: Molecular Biology

country: Sweden

institute: Umea University

type: Expression profiling by genome tiling array

summary:
Two chromosome-targeting and gene regulatory systems are present in Drosophila melanogaster. The male X-chromosome is targeted by the male-specific lethal complex believed to mediate the two-fold up-regulation of the X-linked genes. The highly heterochromatic 4th chromosome is specifically targeted by the Painting of Fourth (POF) protein which together with heterochromatin protein 1 (HP1) modulate the expression level of genes on the 4th chromosome. Here we report high-resolution mapping of POF and HP1 on the 4th chromosome using chromatin immunoprecipitation followed by tiling microarrays (ChIP-chip) in S2 cells and salivary glands. The enrichments are compared to transcript profiles of the two cell types used. POF specifically binds to genes with a strong preference for exons. The POF binding profile correlates to the binding profile of HP1 which in addition displays a typical “peak” in the promoter regions of bound genes. POF and HP1 binds to active genes and the binding correlates with levels of transcription and changes in binding levels are paralleled by a comparable change in transcription. Our results provide a high resolution description of the chromosome 4 specific gene regulatory system provided by the combinatorial effects of POF and HP1.
Keywords: Transcript profiling

overall_design: Transcript profiling in S2 cells and salivary glands. Two biological replicates, one replicate per array.

first: Per

last: Stenberg

first: Anna-Mia

last: Johansson


title: Round up hi-load herbicide treatment for 2 h

status: Public on Nov 01 2007

city: Tsukuba

first: Emiko

last: Kitagawa

state: Ibaraki

address: Onogawa 16-1

country: Japan

institute: National Institute of Advanced Industrial Science and Technology

summary:
The number and type of synthetic chemicals that are being produced worldwide continues to increase significantly. While these industrial chemicals provide numerous benefits, there is no doubt that some have potential to damage the environment and health. Toxicity must be evaluated and use must be carefully controlled and monitored in order to minimize potential damage. DNA microarray technology has become an important new technique in toxicology. We are using the yeast Saccharomyces cerevisiae as a model organism for toxicological study because it is a simple, fast-growing eukaryote that has been thoroughly characterized. In order to evaluate toxicity by newly synthesized or mixture chemicals, toxicity-induced gene expression alteration profiles by known chemicals should be collected. In our study, cells need to be exposed with same experimental cellular condition, semi lethal (IC50), respectively. In the case of round up (CAS; 40465-66-5), the exposure dose was decided as 1500 times dilution by growth curve with continuously diluted exposure. Roundup is the brand name of a systemic, broad-spectrum herbicide contains the active ingredient glyphosate.
Glyphosate is classed as a moderately toxic herbicide and in EPA toxicity class III.
//
Genomic profile of roundup treatment of yeast using DNA microarray analysis:
The herbicide Roundup, which contains glyphosate as the active ingredient, was first introduced in 1974 and has enjoyed widespread use in Japan and elsewhere in the world. Roundup-induced reactions occurring in the yeast Saccharomyces cerevisiae may have a predictive value for understanding responses in higher eukaryotes, and we applied yeast DNA microarray analysis for this purpose. Functional characterization of up-regulated open reading frames (ORFs) following Roundup treatment suggests that Roundup affects membrane structures and cellular organelles. Expression profiles induced by treatments with detergents, oils and hydrostatic pressure were similar to those following Roundup treatment based on cluster analysis. Glyphosate alone was not found to inhibit yeast growth at the concentration contained in the Roundup treatment used for microarray analysis. The toxicity of Roundup appeared to be due to detergent in the product.
Keywords: stress response

overall_design: Series contains triplicate hybridization results from independent biological samples respectively.

first: Sophon

last: Sirisattha

first: Hitoshi

last: Iwahashi


title: Control cDNA microarray profiles of Drosophila head

summary:
In the process of developing a biological systems model of kindling-like plasticity in the fruit fly, we performed a control gene expression profiling analysis in which RNA isolated from different pools of heads of wild-type flies grown on normal food (NF) were compared between themselves using cDNA microarrays. Significance analysis of microarray (SAM) did not reveal any gene as differentially expressed below a false discovery rate (FDR) of 96.88%.
Keywords: comparative

overall_design: D. melanogaster Oregon-R wild type flie were grown in standard fly medium, i.e., NF, consisting of agar-agar, maize powder, brown sugar, dried yeast and nipagin, at 24 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. To obtain flies used in the experiments, individuals from identical cultures were first allowed to lay eggs in milk bottles containing normal fly media. Flies were shifted to fresh bottle every 12 hr. First 4 sets of bottles were discarded. Flies that emerged in subsequent bottles were only used. Those that emerged in the beginning were first discarded and then flies were collected within 1 day, at 4 hr interval. Flies were anesthetized with diethyl ether, and males and females separated immediately after collection using a stereomicroscope. Male flies were thereafter maintained for another 3-4 days in standard culture vials containing NF in batches of 30 individuals. Flies were frozen in liquid nitrogen, shaken, and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, each representing flies collected from four vials two times of which comprised a single parallel set of treatment, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche). The cDNA was purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. The eight batches of purified cDNA was used for labeling with Cy3 and Cy5 dyes (Amersham Biosciences), four each, using Microarray RNA Target Synthesis Kit T7 (Roche) and labeled products were purified by Microarray Target Purification Kit (Roche). The two cRNA samples of each biological replicate, one labled with Cy3 and another with Cy5, were pooled together, precipitated, washed and air-dried. The dried pellet was dissolved in 18MΩ RNAase free water (Sigma). Hybridization solution was prepared by mixing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma) and added to the labeled product. This mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto). The slides were covered by lowering down a 24X60 mm coverslip (ESCO, Portsmouth, USA). Hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Coverslips were removed by submerging the slides in a solution containing 1X SSC and 0.1% SDS at 50ºC. Slides were washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Finally, slides were washed in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Microarray slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices), using both green and red lasers. The 16 bit TIFF images were preprocessed and quantified using Gene Pix Pro 6.0 software (Molecular Devices). Data normalization was performed using Acuity 4.0 software (Molecular Devices). Ratio based normalization was used for all slides. All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 2.21, Excel Add-In, Stanford) under the conditions of one class response and 100 permutations. SAM however did not reveal any gene as differentially expressed in NF versus NF below a false discovery rate (FDR) of 96.88%. It was therefore concluded that our experimental design is robust enough to reliably compare control versus treated samples next.

first: Priyanka

last: Singh


title: Aflatoxin formation and gene expression in response to carbon source media shift in Aspergillus parasiticus

status: Public on Oct 01 2007

city: Mississippi State

first: Jeff

last: Wilkinson

state: MS

department: Biochemistry and Molecular Biology

institute: Mississippi State University

summary:
Aflatoxins are toxic and carcinogenic polyketide metabolites produced by fungal species, including Aspergillus flavus and A. parasiticus.  The biosynthesis of aflatoxins is modulated by many environmental factors, including the availability of a carbon source.  The gene expression profile of A. parasiticus was evaluated during a shift from a medium with low concentration of simple sugars, Yeast Extract (YE), to a similar medium with sucrose, Yeast Extract Sucrose (YES).  Total RNA and aflatoxins (B1, B2, G1, and G2) were quantified from fungal mycelia harvested pre- and post-shifting.  When compared to YE media, YES caused temporary reduction of the aflatoxin levels detected at 3 hours post-shifting and they remained low well past 12 hours post-shift.  Aflatoxin levels did not exceed the levels in YE until 24 hrs post shift, at which time point a 10-fold increase was observed over YE.  Microarray analysis comparing the RNA samples from the 48 hr YE culture to the YES samples identified a total of 2120 genes that were expressed across all experiments, including most of the aflatoxin biosynthesis genes.  One-way Analysis of Variance (ANOVA) identified 56 genes that were expressed with significant variation across all time points.  Three genes responsible for converting norsolorinic acid to averantin were identified among these significantly expressed genes.  The potential role of these genes involvement in the regulation of aflatoxin biosynthesis is discussed.
Keywords: time course, media shift, Aspergillus, aflatoxin

overall_design: Each experiment consisted of one 48 hour YE sample coupled to either a 3, 6, 12, 24, or 48 hour sample from the YE to YES shift.  The 48 hour YE to 3, 6, 12 and 24 hour YES comparisons were repeated with duplicate biological replicates and with duplicate dye-flip; the 0 hour to 48 hour comparisons were done in triplicate each with a dye-flip.

first: Jeffery

middle: R

first: Jiujiang

last: Yu

first: Hamed

middle: K

last: Abbas

first: Brian

middle: E

last: Scheffler

first: Henam

middle: S

last: Kim

first: William

middle: C

last: Nierman

first: Deepak

last: Bhatnagar

first: Thomas

last: Cleveland


title: A transcript profile of Medicago truncatula 2HA tissue culture with auxin treatment

city: Canberra

first: Peta

last: Holmes

state: Australian Capital Territory

address: Building 46, Australian National University

department: Reseach School of Biological Sciences

country: Australia

institute: Genomic Interactions Group

summary:
Leaf explants of the superembryogenic Medicago truncatula line 2HA were treated with auxin (1-naphthaleneacetic acid) for one week to induce the formation of roots (Imin et al J Exp Bot 58:439-451). Gene expression in the leaves and the NAA treated tissue cultures was compared to identify transcripts expressed during the commitment to root formation in tissue culture.
We have used the Affymetrix Medicago Genome Array GeneChip to compared gene expression in Medicago truncatula leaves and leaf explants that have been cultured for one week on NAA, to identify genes expressed during the commitment to root formation in tissue culture.
Keywords: Cell type comparison

platform_organism:
Sinorhizobium meliloti
Medicago sativa
Medicago truncatula

overall_design: Medicago truncatula 2HA leaves and leaf explants treated with NAA for one week were collected; total RNA was extracted and used for hybridization to Affymetrix arrays

first: Nijat

last: Imin

first: Nicolas

last: Goffard


title: Fine mapping of a de novo interstitial 10q22-q23 duplication in a patient with congenital heart disease and microcephaly

city: Berlin

first: Fikret

last: Erdogan

address: Ihnestr. 73

country: Germany

institute: Max-Planck-Institute

type: Genome variation profiling by genome tiling array

summary:
In this study we report a female patient with an interstitial duplication of a region (10q22-q23) which is rarely reported in the literature. We fine mapped the duplication with array CGH, which revealed an 18.6 Mb duplication at 10q22.2-q23.33. There were no other deletions or duplication at another chromosome region. The duplicated region includes 89 annotated genes.
The main clinical features of the patient are microcephaly and congenital heart disease. These are likely to be caused by dosage effect of one or several genes in the duplicated region. Finding of similar phenotypes in the other patients with 10q11-q22 duplications and in two out of three patients with 10q22-q23 duplications, suggest presence of genes involved in the development of these features at 10q22. Most of the duplication cases were investigated only by conventional chromosome analyses and fine mapping of these duplications will help identifying candidate genes/regions.
Keywords: Array CGH

overall_design: One patient with congenital heart defect and microcephaly was analyzed with submegabase resolution array CGH. No dye-swops no replicates was done


title: Secretion stress caused by overexpression of heterologous α-amylase in Bacillus subtilis

city: Haren

first: Andrzej

last: Lulko

address: Kerklaan 30

country: Netherlands

institute: University of Groningen

summary:
Transcriptome analysis was used to investigate the global stress response of the Gram-positive bacterium Bacillus subtilis caused by overproduction of the well-secreted AmyQ α-amylase from Bacillus amyloliquefaciens. Analyses of the control and overproducing strains were carried out at the end of exponential growth and in stationary phase, when protein secretion from B. subtilis is optimal. Among the genes that showed increased expression were htrA and htrB, which are part of the CssRS regulon that responds to high-level protein secretion and heat stress. The analysis of the transcriptome profiles of a cssS mutant compared to the wild-type, under identical secretion stress conditions, revealed several genes with altered transcription in a CssRS-dependent manner, for example citM, ylxF, yloA, ykoJ and several genes of the flgB operon. However, a high affinity CssR-binding was only observed for htrA and htrB, and possibly for citM. In addition, the DNA macroarray approach reveal that several genes of the sporulation pathway are downregulated by AmyQ overexpression, and a group of motility-specific (σD-dependent) transcripts were clearly upregulated. Subsequent flow cytometric analyses demonstrate that upon overproduction of AmyQ as well as a non-secretable variant of the α-amylase, the process of sporulation is severely inhibited. The same experiments were implemented to investigate the expression levels of the hag promoter, a well-established reporter for σD-dependent gene expression. This approach confirmed the observations based on our DNA macroarray analyses and led us to conclude that expression levels of several genes involved in motility are maintained at high levels under all conditions of α-amylase overproduction.
Keywords: secretion stress response

sample_organism: Bacillus subtilis subsp. subtilis str. 168

overall_design: Secretion stress was applied by overproducing the well-secreted AmyQ α-amylase (pKTH10 vector) from B. amyloliquefaciens. Besides examining secretion stress in wild-type cells, we compared transcriptome profiles of a cssS mutant strain under conditions of high-level AmyQ production. Samples for transcriptome analyses were collected at the late exponential growth stage (one hour before the transition point) and 3 hours upon entry in the stationary growth phase. Three independent cultures of each strain were used and cells were sampled for macroarray experiments. Duplicate spots were averaged in Array-Pro software (Media Cybernetics, Inc.) and the signal was normalized after background subtraction by calculation of the percentage of total signal per gene using Microsoft Excel.

middle: T

first: Jan-Willem

last: Veening

first: Girbe

last: Buist

first: Wiep-Klaas

last: Smits

first: Evert-Jan

last: Blom

first: Aaron

last: Beekman

first: Sierd

last: Bron

first: Oscar

middle: P

last: Kuipers


title: Effect of gabapentin on gene expression in Drosophila head

status: Public on Oct 05 2007

first: Abhay

last: Sharma

summary:
We recently developed a fly model of pentylenetetrazol (PTZ)-induced long-term behavioral plasticity. Pharmacological validation suggests this model to be kindling-like. Hence, our model is of relevance in understanding the molecular pathogenesis of epileptogenesis as well as in screening potential antiepileptogenic agents. Gabapentin (GBP) is an antiepileptic drug, and in the process of developing our fly model we generated gene expression profile of flies’ head secondary to treatment of the insects with GBP. Our results provide novel insights in to the drug’s mode of action.
Keywords: Dose response

overall_design: Oregon-R wild type D. melanogaster was grown in standard fly medium consisting of agar-agar, maize powder, brown sugar, dried yeast, and nipagin.  Flies were cultured at 24 + 1oC, 60% RH, and 12 hrs light (9 AM to 9 PM) and 12 hours dark cycle. Ten to eleven days old unmated adult males were grown in either normal food (NF) or food containing 16 mg/ml of GBP for three days. Each culture vials contained 30 flies. Flies frozen in liquid nitrogen were agitated and the heads collected using cooled sieves. Total RNA was isolated from eight pools of frozen heads, every two of which represented a single parallel set of treatment in which four vials contained NF treated control flies, and four GBP treated individuals, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from 10 µg of total RNA using Microarray cDNA Synthesis Kit (Roche), and the cDNA purified using Micorarray Target Purification Kit (Roche), according to the manufacturer’s protocol. Each of the four sets of NF control and GBP treated cDNA samples, belonging to the four biological replicates, was used for labeling with either Cy3 or Cy5 dyes (Amersham Biosciences) using Microarray RNA Target Synthesis Kit T7 (Roche). The labeled products were purified by Microarray Target Purification Kit (Roche). The Cy3 and Cy5 labeled two cRNA samples of each biological replicate were pooled together, precipitated, washed, air-dried, and dissolved in 18MΩ RNAase free water (Sigma). Dye swapping was accomplished by hybridizing two arrays with NF control as Cy3- and GBP treated as Cy5- labeled sample, and the rest two as the opposite, NF as Cy5- and drug treated as Cy3- labeled sample. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D12Kv1, CDMC, Toronto). The slides were covered by a coverslip (ESCO, Portsmouth, USA) and hybridization was allowed to take place in hybridization chamber (Corning) at 37ºC for 16 hrs. Following hybridization, the coverslips were removed in a solution containing 1X SSC and 0.1% SDS at 50ºC, and the slides washed in 1X SSC and 0.1% SDS (three times for 15 minutes each) in a coplin jar at 50ºC with occasional swirling and then transferred to 1X SSC and washed with gentle swirling at room temperature (twice for 15 minutes each). Slides were given a final wash in 0.1X SSC for 15 minutes and then liquid was quickly removed from the slide surface by spinning at 600 rpm for 5 minutes. Slides were scanned at 10µm resolution in GenePix 4000A Microarray Scanner (Molecular Devices). The 16 bit TIFF images were preprocessed and quantified using Gene Pix Pro 6.0 software (Molecular Devices). Ratio based normalization was performed using Acuity 4.0 software (Molecular Devices). All Spots with raw intensity less then 100U and less then twice the average background was ignored during normalization. Normalized data was filtered for the selection of features before further analysis. Only those spot were selected which contained only a small percentage (<3) of saturated pixels, were not flagged bad or found absent (flags >= 0), had relatively uniform intensity and uniform background (Rgn R2 (635/532) >= 0.6) and were detectable above background (SNR >= 3). Analyzable spots in at least three of the four biological replicates performed were retrieved for downstream analysis using Significance Analysis of Microarrays (SAM 2.21, Excel Add-In, Stanford) under the conditions of one class response and 100 permutations.

first: Priyanka

last: Singh

first: Mohd

last: Farhan


title: Saccharomyces cerevisiae sorted and untreated

city: Leipzig

first: Jeannette

last: Achilles

address: Permoserstraße 15

department: Environmental Microbiology

institute: Helmholtz-Centre for Environmental Research - UFZ

summary:
Gene expression of a vital, stained and sorted subpopulation of Saccharomyces cerevisiae with high affinity to glucose, harvested at a dilution rate of D=0.160 h-1, and of cells from the whole population without further treatment grown at the same dilution rate were analysed.
The isolation of RNA was accomplished by using lyticase to lyse the cells and the Qiagen RNeasy Mini Kit with some modifications within the manufacturers´ protocol.
6 µg of the total RNA per sample was used for each microarray experiments. The indirect labelling by the tyramide-signal-amplification method (MicromaxTM TSATM labelling and detection Kit from Perkin Elmer life sciences) was used to increase the Cy3 and Cy5 signals of microarray detection.
Each cDNA containing Biotin- and Fluorescein-nucleotides respectively was purified with a QIAquick PCR purification kit and suspended in 11 µl of the formamide containing hybridization buffer. The slides were hybridized at 42°C over night under a cover slip.
The microarrays were scanned by the Axon 4000B scanner; image intensity data were extracted and analysed with GenePix® Pro 6.0 software.
Data from different scans of Dye-swap experiment were extracted by GenePix Pro 6.0 software, normalized and united. An outliertest has been applied in order to find outliers amongst the gene replicats.  Subsequently, a t-test (1% and 5% probability of error) has been used in order to find regulated genes.
Keywords: sorted yeast cells

overall_design: Microarray experiments were realised with dye swap and cDNA labelling was proceeded in loop design.

first: Susann

last: Müller

first: Frank

last: Stahl

first: Hauke

last: Harms

first: Cornelia

last: Repenning


title: FDCPmix, ES cell line

city: Neuherberg

first: Martin

last: Irmler

state: Bayern

address: Ingolstaedter Landstrasse 1

department: Institute of Experimental Genetics

institute: Helmholtz Zentrum München GmbH

summary:
Hemotopoietical FDCPmix cell line stays as pluripotent cell line depend of IL3 treatment. Cytokine cocktails initiate terminal differentiation in myeloid cell. The cell line was transfected with MLL (leukaemia factor) causing in reaction with several transcription factors diverse tumors. Expression profiling analysis to get an insight into the mechanism of MML leukaemia factor. The factor is stimulated by Tamoxifen (OHT). On several time points after stimulation expression profiles were performed
Keywords: FDCPmix, ES cells, murine, tumorigenesis, cDNA arrays

overall_design: FDCPmix 4 time points (3h, 7h, 12h, 23h)after Tamoxifen treatment versus cells 0h after treatment, including 50% dye sway experiments

first: Johannes

last: Beckers

first: Marion

last: Horsch


title: Gene expression non-additivity in immature ears of a heterotic F1 maize hybrid

city: Milan

first: Giorgio

last: Pea

address: via Celoria 26

department: Department of Biomolecular Sciences and Biotechnology

country: Italy

institute: University of Milan

summary: Non-additive gene regulation has been recently suggested as an important factor promoting phenotypic variation and plasticity. In order to obtain a description of gene expression status at an early stage of ear development in a maize (Zea mays L.) F1 hybrid as relative to its parental inbreds, we compared gene expression profiles in immature ears of elite inbred lines B73 and H99 to one of their F1 hybrids (B73xH99) using cDNA microarray technology. Results show several genes expressed at a significantly different level between both inbred lines and their hybrid. In addition, gene expression non-additivity in the hybrid was detected on a broad scale, consisting of both dominance and over-dominance components, indicating that complex non-additive interactions at the molecular level exist in the developing ear of the studied maize hybrid. Non-additively regulated genes belong to a wide range of molecular functions, indicating that several regulatory and metabolic patterns are possibly affected during ear development in the investigated hybrid. We discuss the possibility that observed gene expression non-additivity in immature ear might be an early molecular manifestation of hybrid vigor, the most exploited factor for maize agronomic improvement.

overall_design:
We directly contrasted both B73 and H99 inbred lines vs. their F1 heterotic hybrid transcriptomes in immature ear. Our experimental design consisted of 10 cDNA microarray hybridizations, 5 for each combination of hybrid vs. inbred genotypes, involving 20 separate labeling reactions. Labeling dyes were swapped in two of the five replicates for each combination. In each hybridization, control channel was assigned to the F1 hybrid, and differences in transcriptional levels between B73 and H99 were inferred using the hybrid as a common reference sample in an indirect experimental design. To take in account variability in transcript population among individuals, total RNA coming from different isolations, each collected on multiple individuals, were mixed before poly(A+) RNA purification. All hybridizations on microarray slides were then performed using cDNA independently labeled from the poly(A+) RNA purification product for each genotype.
Base-two logarithms of expression ratios were subjected to one-class response significance analysis in SAM v. 2.20 software [Tusher et al. 2001]. For each EST the estimates of additive parameter "a" and dominance parameter "d" (middle-parent heterosis) were obtained as
a = (L2 – L1)/2
d = (L1 + L2)/2
(where L1 and L2 are mean base-two logarithms of expression ratios of F1 vs. B73 and F1 vs. H99, respectively). Positive values of "a" indicate expression values bigger in B73 than in H99. The dominance/additivity ratio (d/|a|) was also calculated [Falconer 1989]. Evaluation of statistical significance of parameters was done by calculating standard errors of the estimates "a" and "d" as standard errors of linear functions of the means. Significance testing was done correspondingly, using an F-test for linear contrasts. P-values for the families of tests corresponding to each parameter were subjected to global error analyses using a method based on fitting mixture distribution [Allison et al. 2002], allowing to estimate the false discovery rates (FDR) and false negative rates (FNR). Confidence intervals for d/|a| ratios were obtained by Fieller’s method [Piepho and Emrich 2005], allowing to classify the genes into different dominance type classes.

first: Simona

last: Ferron

first: Luca

last: Gianfranceschi

first: Pawel

last: Krajewski

first: Mario

last: Pe'


title: Displacement and re-accumulation of centromeric cohesin during transient pre-anaphase centromere splitting

status: Public on Sep 30 2007

city: London

first: Maria

last: Ocampo-Hafalla

address: 44 Lincoln's Inn Fields

country: United Kingdom

institute: Cancer Research UK London Research Institute

type: Genome binding/occupancy profiling by genome tiling array

summary:
The ring-shaped cohesin complex links sister chromatids until their timely segregation during mitosis. Cohesin is enriched at centromeres, where it provides the cohesive counter-force to bi-polar tension produced by the mitotic spindle. As a consequence of spindle tension, centromeric sequences transiently split in pre-anaphase cells, in some organisms up to several micrometeres. This ‘centromere breathing’ presents a paradox, how sister sequences separate where cohesin is most enriched. We now show that in the budding yeast S. cerevisiae, cohesin binding diminishes over centromeric sequences that split during breathing. We see no evidence for cohesin translocation to surrounding sequences, suggesting that cohesin is removed from centromeres during breathing. Two pools of cohesin can be distinguished. Cohesin loaded before DNA replication, that has established sister chromatid cohesion, disappears during breathing. In contrast, cohesin loaded after DNA replication is partly retained. As sister centromeres re-associate after transient separation, cohesin is re-loaded in a manner independent of the canonical cohesin loader Scc2/Scc4. Efficient centromere re-association requires the cohesion establishment factor Eco1, suggesting that re-establishment of sister chromatid cohesion contributes to the dynamic behaviour of centromeres in mitosis. These findings provide new insights into cohesin behaviour at centromeres.
Keywords: ChIP-chip

overall_design:
SAMPLES

Origin of each biological sample: Saccharomyces cerevisiae (W303background)

Growth conditions: Cells containing the MET3-CDC20 allele were grown at 25°C in SC medium lacking methionine with 2% raffinose or 2% glucose as the carbon source (Uhlmann et al. 2000). Cultures were synchronized in G1 with α-factor (5 μg/ml) and released into medium containing 100 mM hydroxyurea (HU) for arrest in early S-phase. For arrest in metaphase, cells were released from α-factor or HU arrest by filtration and re-suspension in YP medium supplemented with 5 mM methionine to deplete Cdc20. To inactivate cohesin loading, cells harbouring the temperature sensitive scc2-4 allele (Ciosk et al. 2000) were shifted to the restrictive temperature of 35°C for 1 hour before release. To prevent metaphase spindle assembly, or to disassemble already formed spindles, 7.5 μg/ml nocodazole was added to the medium for 1 hour. To wash out nocodazole, cells were filtered again, washed, and re-suspended in fresh medium lacking nocodazole for 1 hour. Expression of Scc1 to induce differentially epitope-tagged cohesin under control of the GAL1 promoter, or of Cdc14, was achieved by addition of 2% galactose for 1 hour to cultures grown in raffinose-containing medium.

EXPERIMENTAL DESIGN

Experiment type: ChIP-chip = Chromatin immunoprecipitation (ChIP) followed by hybridization to a high-density oligonucleotide microarray (chip).

Number of hybridizations performed: 13 ChIP-chip samples; 2 SUPernatant samples

Analysis: ChIP samples were normalized against SUPernatant fractions

Quality control: Confirmation of results by duplication of experiments, by usage of different epitope tags for the same protein, and by usage of different subunits of the same protein complex. Also, whole cell extract, SUPernatant, and ChIP fractions were checked by Western blotting.

Protocols: ChIP-chip and hybridization protocols were performed as previously described (Katou et al. 2003; Lengronne et al. 2004; Lengronne et al. 2006). A summary is provided below:

ChIP. Cells were grown under the conditions described above and cross-linked with 1% formaldehyde. Cells were disrupted using a Multi-Beads Shocker, and whole cell extracts were sonicated to obtain 400-600 bp genomic DNA fragments. Chromatin immunoprecipitation (ChIP) was performed with anti-HA antibody or anti-PK monoclonal antibody coupled to protein A Dynabeads. Immunprecipates were eluted and incubated overnight at 65ºC to reverse the cross-link. The immunoprecipitated genomic DNA was then incubated with proteinase K, extracted twice with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RNaseA. The DNA was purified, concentrated by ethanol precipitation and amplified by PCR after random priming. 10ug of the amplified DNA was digested with DNase I and end-labelled using TdT Terminal Transferase and Biotin-N6-ddATP.

Hybridization and scanning. Samples were hybridized in buffer containing SSPE, TritonX-100, denatured salmon sperm DNA and biotin-labelled control oligonucleotide, oligo B2. Samples were denatured at 95ºC for 10 minutes before being hybridized to the arrays for 16 hours at 42ºC in a GeneChip Hybridization Oven 640. The washing and scanning protocol (FlexMidi_euk2v3_450), provided by Affymetrix, was performed automatically on a GeneChip Fluidics Station 450. Arrays were scanned using the GeneChip Scanner 3000 7G, and primary analysis of tiling chip data was performed using the Affymetrix GeneChip Operating Software (GCOS).

ARRAY DESIGN

General array design: in situ synthesized arrays from Affymetrix

Location and ID of each spot on arrays: available upon request from Affymetrix

Probe type: oligonucleotide

Availability of arrays: commercially available from Affymetrix
S. cerevisiae (Chromosome VI) rikDACF, P/N# 510636
S. cerevisiae T	iling Array (Forward), P/N# 520286

REFERENCES

Ciosk R, Shirayama M, Shevchenko A, Tanaka T, Toth A, Shevchenko A, Nasmyth K (2000) Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins. Mol Cell 5: 1-20

Katou Y, Kanoh Y, Bandoh M, Noguchi H, Tanaka H, Ashikari T, Sugimoto K, Shirahige K (2003) S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424: 1078-1083

Lengronne A, Katou Y, Mori S, Yokobayashi S, Kelly GP, Itoh T, Watanabe Y, Shirahige K, Uhlmann F (2004) Cohesin relocation from sites of chromosomal loading to places of convergent transcription. Nature 430: 573-578

Lengronne A, McIntyre J, Katou Y, Kanoh Y, Hopfner K-P, Shirahige K, Uhlmann F (2006) Establishment of sister chromatid cohesion at the S. cerevisiae replication fork. Mol Cell 23: 787-799

Uhlmann F, Wernic D, Poupart M-A, Koonin EV, Nasmyth K (2000) Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103: 375-386

first: Yuki

last: Katou

first: Katsuhiko

last: Shirahige

last: Uhlmann


title: Broad, ectopic expression of the sperm protein, PLCZ1 induces parthenogenesis and ovarian tumours in mice

city: Kobe

first: Anthony

middle: CF

last: Perry

state: Hyogo-ken

address: 2-2-3 Minatojima-minamimachi

department: Center for Developmental Biology

institute: RIKEN

summary:
Mammalian metaphase II (mII) exit and embryogenesis are induced at fertilization by a signal thought to come from the sperm protein, phospholipase C-zeta (PLCZ1).  Meiotic progression can also be triggered without sperm, as in parthenogenesis, although the classic mouse in vivo parthenogenetic model, LT/Sv, fails in meiosis I due to an unknown molecular etiology.  We here dissect PLCZ1 specificity and function in vivo and address its ability to interfere with maternal meiotic exit.  Wild-type mouse Plcz1 expression was restricted to post-pubertal testes, and the brains of both sexes, with region-specifying elements mapping to a 4.1 kb Plcz1 promoter fragment.  When broad ectopic PLCZ1 expression was forced in independent transgenic lines, they initially appeared healthy.  Their oocytes underwent unperturbed meiotic maturation to mII but subsequently exhibited autonomous intracellular free calcium oscillations, second polar body extrusion, pronucleus formation and parthenogenetic development.  Transfer of transgenic cumulus cell nuclei into wild-type oocytes induced activation and development, demonstrating a direct effect of PLCZ1 analogous to fertilization.  Whilst Plcz1 transgenic males remained largely asymptomatic, females developed abdominal swellings caused by benign ovarian teratomas that were under-represented for paternally- and placentally-expressed transcripts.  Plcz1 was not over-expressed in the ovaries of LT/Sv or in human germline ovarian tumours.  The narrow spectrum of PLCZ1 activity indicates that it is modulated by tissue-restricted accessory factors.  This work characterizes a novel model in which parthenogenesis and tumourigenesis follow full meiotic maturation and are linked to fertilization by PLCZ1.
Keywords: miRNA profiling; expression profiling of mouse miRNAs_SampleB

overall_design: Fourteen samples were analyzed for the study.


title: Analysis of on-chip hybridization kinetics for optimization of gene expression experiments.

city: Paris

first: Luce

last: Dauphinot

address: 47 boulevard de l'hôpital

department: ICM

country: France

institute: INSERM U1127-CNRS UMR7225-UPMC

summary:
DNA microarray technology is a powerful tool for getting the overview of gene expression in biological samples. Although the successful application of microarray-based expression analysis was demonstrated in a number of applications, the main problem with this approach is the fact that expression levels deduced from hybridization experiments do not necessarily correlate with RNA concentrations. Moreover, oligonucleotide probes corresponding to the same gene can give different hybridization signals. Apart from cross-hybridizations and differential splicing, this could be due to secondary structures of probes or targets. In addition, for low copy genes, hybridization equilibrium may be reached after hybridization times much longer than the one commonly used (overnight, 15 hours). Thus, hybridization signals could depend on kinetic properties of the probe, which may vary between different oligonucleotide probes immobilized on the same microarray.
To validate these hypothesis, on-chip hybridization kinetics and duplexes thermostability analysis were performed using oligonucleotide microarrays containing 50-mer probes corresponding to mouse genes.
We demonstrate that differences in hybridization kinetics between the probes can influence the interpretation of expression data. Ways to improve the reliability of microarray-based expression analysis are discussed.
Keywords: kinetics, gene expression, microarrays

overall_design:
Total RNA were extracted from 5 thymus and 6 cortex of B10D2 mice.
For each tissue, equal amounts of each RNA were pooled and 15µg were labeled by reverse-transcription and hybridized on neurotransmission microarrays.

Three replicates of each experiment have been performed.

first: E

last: Khomyakova

first: M

middle: A

last: Livshits

first: M-C

last: Durand

first: L

first: S

last: Cohen-Kaminsky

first: J

last: Rossier

first: F

last: Soussaline

last: Potier


title: Interlocking Transcriptional Feedback Loops Control White-Opaque Switching in Candida albicans

city: San Francisco

first: David

middle: J

last: Galgoczy

address: 600 16th Street

department: Biochemistry and Biophysics

institute: UCSF

summary:
The human pathogen Candida albicans can assume either of two distinct cell types, designated ‘‘white’’ and ‘‘opaque.’’  Each cell type is maintained for many generations; switching between them is rare and stochastic, and occurs without any known changes in the nucleotide sequence of the genome.  The two cell types differ dramatically in cell shape, colony appearance, mating competence, and virulence properties.  In this work, we investigate the transcriptional circuitry that specifies the two cell types and controls the switching between them.  First, we identify two new transcriptional regulators of white-opaque switching, Czf1 and white-opaque regulator 2 (Wor2). Analysis of a large set of double mutants and ectopic expression strains revealed genetic relationships between CZF1, WOR2, and two previously identified regulators of white-opaque switching, WOR1 and EFG1. Using chromatin immunoprecipitation, we show that Wor1 binds the intergenic regions upstream of the genes encoding three additional transcriptional regulators of white-opaque switching (CZF1, EFG1, and WOR2), and also occupies the promoters of numerous white- and opaque-enriched genes.  Based on these interactions, we have placed these four genes in a circuit controlling white-opaque switching whose topology is a network of positive feedback loops, with the master regulator gene WOR1 occupying a central position. Our observations indicate that a key role of the interlocking feedback loop network is to stably maintain each epigenetic state through many cell divisions.
Keywords: ChIP-chip

overall_design: Chromatin-immunoprecipitation (ChIP) was performed using one of two different affinity purified antibodies (α-Wor1Nterm or α-Wor1Cterm ), raised against a peptides near the N- and C- termini of Wor1.  Precipitated DNA was amplified, fluorescently labeled, and competitively hybridized against genomic DNA (input DNA) on custom DNA tiling microarrays containing 60-mer oligonucleotides tiled at 80bp intervals across the entire C. albicans genome (ChIP-chip).  For each antibody, two microarrays were hybridized with DNA from two separate IPs of an opaque wild type a strain; a single ChIP was performed in a wor1Delete/wor1Delete (white) a strain as a control for the N-terminal antibody experiments and two ChIPs were performed in the deletion strain for the C-terminal antibody experiments.

first: Rebecca

last: Zordan

first: Mathew

middle: G

last: Miller

first: Brian

middle: B

last: Tuch

first: Alexander

middle: D

last: Johnson


title: CGH Analysis of C. jejuni isolates

city: Ottawa

first: John

middle: H

last: Nash

state: Ontario

address: 100 Sussex Drive

department: Institute for Biological Sciences

country: Canada

institute: National Research Council

summary:
We performed microarray CGH analysis of 104 neuropathogenic and enteritis only stains of C. jejuni.
Keywords: Individual hybridizations, CGH

overall_design: We measured signal intensity for tester strain versus control strain Cj NCTC 11168 for 104 isolates of C. jejuni.


title: XBP-1 transduction of PEL

status: Public on Sep 28 2007

first: Paul

last: Kellam

state: London

address: 46 Cleveland Street

department: Infection

institute: UCL

summary:
Transcription of human and KSHV genes in PEL following the transduction of spliced and unspliced XBP-1
Keywords: Cell type comparison

platform_organism:
Homo sapiens
Human gammaherpesvirus 8

overall_design: Two colour sample Cy5, reference Cy3, reference is a mixture of mRNA from uninfected, HeLa cell and KSHV lytic cycle induced PEL.

first: Sam

last: Wilson


title: 8-chip high-resolution aCGH experiment NA15510 vs. NA18505

city: New Haven

first: Jan

middle: Oliver

last: Korbel

state: CT

address: 266 Whitney Avenue

institute: Yale University

summary:
We introduce high-throughput and massive paired-end mapping (PEM), a large-scale genome sequencing method to identify SVs 3 kb or larger that combines the rescue and capture of paired-ends of 3 kb fragments, massive 454 Sequencing, and a computational approach to map DNA reads onto a reference genome. PEM was used to map SVs in an African and putatively European individual and identified shared and divergent SVs relative to the reference genome. Overall, we fine-mapped more than 1000 SVs and documented that the number of SVs among humans is much larger than initially hypothesized; many of the SVs potentially affect gene function. The breakpoint junction sequences of more than 200 SVs were deduced with a novel pooling strategy and computational analysis. Array-CGH was used for validation.
Keywords: array CGH

overall_design: 2 samples were analyzed with 8 different Nimblegen chips (385k); thus ~30M probes were used to interrogate copy number variants in NA15510 (using NA18505 as control) at high resolution.

middle: O

last: Urban

first: Jason

last: Affourtit

last: Godwin

first: Fabian

last: Grubert

middle: F

last: Simons

first: Philip

middle: M

last: Kim

first: Dean

last: Palejev

first: Nicholas

last: Carriero

first: Lei

last: Du

first: Bruce

last: Taillon

first: Andrea

last: Tanzer

first: Eugenia

last: Saunders

first: Jianxiang

last: Chi

first: Fengtang

last: Yang

first: Nigel

last: Carter

first: Matthew

last: Hurles

first: Sherman

last: Weissman

first: Timothy

last: Harkins

first: Mark

last: Gerstein

first: Michael

last: Egholm

last: Snyder

first: Zhoutao

last: Chen


title: Smooth Muscle Cells

city: Durham

first: Jen-Tsan Ashley

state: NC

address: 101 Science Drive

department: Duke Institute for Genome Sciences & Policy

institute: Duke University

summary:
This set contains 68 arrays of two series of experiments. 60 arrays are used to profile the heterogeneity of SMCs from different anatomic locations. 8 arrays are used to profile the change of gene expression of vascular smooth muscle cells upon serum exposure.
An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.
Keywords: all_pairs

overall_design: Using regression correlation

first: Jen-Tsan


title: Differential gene expression of Listeria monocytogenes during high hydrostatic pressure processing

status: Public on Sep 29 2007

city: Hobart

first: John

middle: Phillip

last: Bowman

state: Tasmania

address: College Road, Sandy Bay

department: School of Agricultural Science

institute: University of Tasmania

summary:
High hydrostatic pressure processing (HPP) is currently being used as a treatment for certain foods to control the presence of food-borne pathogens, such as Listeria monocytogenes. Genomic microarray analysis was performed to determine the effects of HPP on L. monocytogenes in order to understand how it responds to mechanical stress injury. Reverse transcriptase PCR analysis of tufB and rpoC indicated that the reduction of mRNA expression in HPP treated cells was dependent on intensity and time of the treatment. Treatments of 400 and 600 MPa for 5 min. on cells in the exponential growth phase though leading to partial or complete cellular inactivation still resulted in measurable relative differential gene expression. Gene set enrichment analysis indicated HPP induced increased expression of genes associated with DNA repair mechanisms, transcription and translation protein complexes, the septal ring, the general protein translocase system, flagella assemblage and chemotaxis, and lipid and peptidoglycan biosynthetic pathways. HPP on the other hand appears to suppress a wide range of energy production and conversion, carbohydrate metabolism, and virulence associated genes accompanied by strong suppression of the SigB and PrfA regulons. HPP also induced repression of genes negatively controlled by pleotrophic regulator CodY. HPP-induced cellular damage appears to lead to increased expression of genes linked to sections of the cell previously shown in bacteria to be damaged or altered during HPP exposure and suppression of gene expression associated with cellular growth processes and virulence.
Keywords: Stress response

overall_design: Two separate treatments were conducted one at 400 MPa (5 min) treatment with 3 biological replicates and a seond at 600 MPa (5 min) with 3 biological replicates compared. In the case of the 600 MPa (5 min) treatment two technical replicates per biological replicate were also included in the downstream data processing as mRNA levels were quite variable after this treatment as compared to the milder high pressure treatment.

first: Claudio

last: Bittencourt

first: Tom

last: Ross


title: Genome-wide Analysis of KAP1 Binding Suggests Auto-regulation of KRAB-ZNFs

city: Davis

first: Kimberly

middle: Rose

last: Blahnik

address: GBSF 1 Shields Ave

department: Genome and Biomedical Sciences

institute: University of California Davis

summary:
We performed a genome-scale ChIP-chip comparison of two modifications (trimethylation of lysine 9 and trimethylation of lysine 27) of histone H3 in Ntera2 testicular carcinoma cells and in three different anatomical sources of primary human fibroblasts. We found that in each of the cell types the two modifications were differentially enriched at the promoters of the two largest classes of transcription factors. Specifically, zinc finger genes were bound by H3me3K9 and homeobox genes were bound by H3me3K27. We have previously shown that the PRC2 complex is responsible for mediating trimethylation of lysine 27 of histone H3 in human cancer cells. In contrast, there is little overlap between H3me3K9 targets and components of the PRC2 complex, suggesting that a different histone methyltransferase is responsible for the H3me3K9 modification.  Previous studies have shown that SETDB1 can trimethylate H3 on lysine 9, using in vitro or artificial tethering assays.  SETDB1 is thought to be recruited to chromatin by complexes containing the KAP1 co-repressor. To determine if a KAP1-containing complex mediates trimethylation of the identified H3me3K9 targets, we performed ChIP-chip assays and identified KAP1 target genes using human 5kb promoter arrays. We found that a large number of promoters of zinc finger transcription factors were bound by both KAP1 and H3me3K9 in normal and cancer cells. To expand our studies of KAP1, we next performed a complete genomic analysis of KAP1 binding using a 38 array tiling set, identifying ~7000 KAP1 binding sites. The identified KAP1 targets were highly enriched for C2H2 zinc fingers, especially those containing KRAB domains. Interestingly, although most KAP1 binding sites were within core promoter regions, the binding sites near ZNF genes were greatly enriched within transcribed regions of the target genes. Because KAP1 is recruited to the DNA via interaction with KRAB-ZNF proteins, we suggest that expression of KRAB-ZNF genes may be controlled via an auto-regulatory mechanism involving KAP1.
Keywords: Human whole genome tiling array

overall_design:
Amplicons were applied either to ENCODE arrays, 5 kb promoter arrays, or to the human genome tiling array set consisting of 38 arrays (see www.nimblegen.com for details). The labeling and hybridization of DNA samples for ChIP-chip analysis was performed by NimbleGen Systems, Inc, except that ENCODE arrays were hybridized at UC Davis. Briefly, each DNA sample (1 μg) was denatured in the presence of 5'-Cy3- or Cy5-labeled random nonamers (TriLink Biotechnologies) and incubated with 100 units (exo-) Klenow fragment (NEB) and dNTP mix [6 mM each in TE buffer (10 mM Tris/1 mM EDTA, pH 7.4; Invitrogen)] for 2 h at 37°C. Reactions were terminated by addition of 0.5 M EDTA (pH 8.0), precipitated with isopropanol, and resuspended in water. Then, 13ug of the Cy5-labeled ChIP sample and 13ug of the Cy3- labeled total sample were mixed, dried down, and resuspended in 40 μl of NimbleGen Hybridization Buffer (NimbleGen Systems) plus 1.5 ug of human COT1 DNA. After denaturation, hybridization was carried out in a MAUI Hybridization System (BioMicro Systems) for 18 h at 42°C. The arrays were washed using NimbleGen Wash Buffer System (NimbleGen Systems), dried by centrifugation, and scanned at 5-μm resolution using the GenePix 4000B scanner (Axon Instruments). Fluorescence intensity raw data were obtained from scanned images of the oligonucleotide tiling arrays using NIMBLESCAN 2.0 extraction software (NimbleGen Systems). For each spot on the array, log2-ratios of the Cy5-labeled test sample versus the Cy3-labeled reference sample were calculated. Then, the biweight mean of this log2 ratio was subtracted from each point; this procedure is approximately equivalent to mean-normalization of each channel.
Total RNA was prepared from 5x106 Ntera cells using RNAeasy Kit (Qiagen) following the manufacturer’s instructions. RNA quality was ensured using the Agilent Systems Bioanalyzer. The RNA was hybridized to human whole genome expression microarrays from Nimblegen, which contain probes for every human gene based on genome build HG18 from the UCSC database (more details at http://www.nimblegen.com). 10 ug total RNA were used to synthesize cDNA using the SuperScript Double-Stranded cDNA synthesis kit (Invitrogen).  The labeling of RNA samples, array hybridization and preliminary RNA expression analysis (data normalization) was performed by NimbleGen Systems, Inc.


title: Genome-wide gene expression in HDAC5 wildtype and knockout mice treated with chronic cocaine or saline

status: Public on Sep 27 2007

city: Boston

first: Will

last: Renthal

state: MA

address: 60 Fenwood Rd.

institute: Brigham and Women's Hospital

summary:
Some individuals are more vulnerable than others to chronic psychiatric illnesses, such as drug addiction or depression, but the neural and molecular mechanisms responsible for this are poorly understood.  Although addiction and depression are known to have a strong genetic component, recent evidence has suggested that epigenetic mechanisms, such as histone acetylation, may also contribute.  However, the mechanisms which integrate drugs of abuse or stress with changes in chromatin structure remain unclear.  Here, we identify the activity-dependent class II histone deacetylase, HDAC5, as a central integrator of these stimuli with changes in chromatin structure and gene expression.  We found that chronic, but not acute, exposure to cocaine or stress decreases HDAC5 function in the nucleus accumbens (NAc), a major brain reward region, which allows for increased histone acetylation and transcription of HDAC5 target genes.  This regulation is behaviorally important, as loss of HDAC5 causes hypersensitive responses to chronic, not acute, cocaine or stress.  Coupling gene expression microarrays and several bioinformatic approaches, we have gone on to identify the most likely HDAC5 target genes responsible for the behavioral maladaptations.  One HDAC5 target, the Substance P Receptor, is upregulated in HDAC5 knockout mice, has increased histone acetylation on its promoter, and modulates cocaine reward, as measured by conditioned place preference.  These findings suggest that proper balance of histone acetylation is a crucial factor in the saliency of a given emotional stimulus, and that disruption of this balance is involved in the transition from normal adaptive responses to chronic psychiatric illness.
Keywords: genetic modification and its impact on cocaine responses in the nucleus accumbens

overall_design: HDAC5 knockout mice and their wildtype littermate controls were injected i.p. with saline or 20 mg/kg cocaine for 7 days.  Twenty-four hours after the last injection, mice were sacrificed and their NAc were dissected.  NAc from four mice were pooled.  Total RNA was extracted from each pool, labeled, and hybridized to Illumina Mouse 6 V1.1 full genome microarrays.  For the first 12 microarrays, 8 mice/condition (WT saline, WT cocaine, KO saline, KO cocaine), were pooled into 2 groups, resulting in 4 mice per array and a total of two biological replicates and four technical replicates.  For the second 18 microarrays, a newly treated set of 16 mice/condition were pooled into 4 groups for a total of 4 mice per array.   This resulted in 4 more biological replicates per condition and 2 more technical replicates.  In total, 30 microarrays yielded 6 biological replicates and 6 technical replicates (1-2 technical replicates per condition).  The goal of this experiment was to identify gene expression differences between HDAC5 knockout and wildtype mice that may underlie their behavioral differences in response to cocaine.


title: Foxm1 Transcription Factor Accelerates Formation of Lung Tumors

city: Chicago

first: Tanya

last: Kalin

state: IL

address: 5841 S. Maryland Ave.

department: Medicine

institute: University of Chicago

summary:
The Forkhead Box m1 (Foxm1 or Foxm1b) protein (previously called HFH-11B, Trident, Win, or MPP2) is abundantly expressed in human non-small cell lung cancers where it transcriptionally induces expression of genes essential for proliferation of tumor cells. In this study, we used Rosa26-Foxm1 transgenic mice, in which the Rosa26 promoter drives ubiquitous expression of Foxm1 transgene, to identify new signaling pathways regulated by Foxm1. Lung tumors in Rosa26-Foxm1 mice were induced using the 3-methylcholanthrene (MCA)/ butylated hydroxytoluene (BHT) lung tumor initiation/ promotion protocol. MCA/BHT-treated Rosa26-Foxm1 mice displayed a significant increase in the proliferation of lung tumor cells as well as in the number and size of lung adenomas. Elevated tumor formation in Rosa26-Foxm1 transgenic lungs was associated with persistent pulmonary inflammation, macrophage infiltration and increased expression of Cdc25C phosphatase, cyclin E2, chemokine ligands Cxcl5, Cxcl1 and Ccl3, cathepsins, and Matrix metalloprotease 12, all of which stimulate signaling pathways essential for cellular proliferation, pulmonary inflammation and remodeling. Lung tumors from Rosa26-Foxm1 mice displayed increased expression of Cyclooxygenase-2 (Cox-2), whereas diminished Cox-2 levels were observed in Foxm1-deficient lung tumors from Mx-Cre Foxm1 fl/fl mice. Cell culture experiments with A549 human lung adenocarcinoma cells demonstrated that depletion of Foxm1 by either siRNA transfection or treatment with Foxm1-inhibiting ARF 26-44 peptide significantly reduced Cox-2 expression. Foxm1 protein bound to the –3479/–3461 and –7826/–7795 bp regions of the human Cox-2 promoter, indicating that the human Cox-2 gene is a direct transcriptional target of Foxm1 in lung tumor cells.
Keywords: Influence of genetic modification to the tumor development

overall_design: Rosa26-Foxm1 TG mice and wild type FVB/N mice were injected intraperitoneally (i.p.) with one dose of 3-methylcholanthrene (MCA; 15 µg/g mouse weight), followed by six weekly i.p. injections of butylated hydroxytoluene (BHT; the first dose was 150 µg/g mouse weight, and subsequent doses were 200 µg/g mouse weight). Seven mice from each group were sacrificed at 12 weeks and 24 weeks after MCA injection and examined for lung tumors using a dissecting microscope. Lung tissues were used to prepare total RNA with RNA-STAT-60 (Tel-Test "B" Inc. Friendswood, TX).

first: Lucille

last: Meliton

first: Vladimir

last: Kalinichenko

first: Maria

last: Tretiakova


title: Expression profiling of human adipose tissue in obese subjects

status: Public on Sep 26 2007

first: Corneliu

last: Henegar

state: Ile de France

address: 15 Rue de l'Ecole de Medecine

institute: Institut national de la santé et de la recherche médicale

summary:
The molecular mechanisms by which obesity increases the risk of cardiovascular diseases are poorly understood. The purpose of this study was to identify candidate biomarkers overexpressed in adipose tissue of obese subjects that could link expanded fat mass to atherosclerosis. We compared gene expression profile in subcutaneous adipose tissue (scWAT) of 28 obese and 11 lean subjects using microarray technology. This analysis identified 240 genes significantly overexpressed in scWAT of obese subjects. The genes were then ranked according to the correlation between gene expression and body mass index (BMI). In this list, the elastolytic cysteine protease cathepsin S was among the highly correlated genes. RT-PCR and Western blotting confirmed the increase in cathepsin S mRNA (P=0.006) and protein (P<0.05) in obese scWAT. The circulating concentrations of cathepsin S were also significantly higher in obese than in nonobese subjects (P<0.0001). Both cathepsin S mRNA in scWAT and circulating levels were positively correlated with BMI, body fat, and plasma triglyceride levels. In addition, we show that the proinflammatory factors, lipopolysaccharide, interleukin-1β, and tumor necrosis factor-α increase cathepsin S secretion in human scWAT explants. This study identifies cathepsin S as a novel marker of adiposity. Since this enzyme has been implicated in the development of atherosclerotic lesions, we propose that cathepsin S represents a molecular link between obesity and atherosclerosis.
Keywords: disease state analysis

overall_design:
scWAT samples were obtained, after an overnight fast, by needle biopsy from the peri-umbilical area while the female subjects were under local anesthesia (1% xylocaïne). In a first exploratory phase, differential gene expression of scWAT was compared between 11 nonobese [mean body mass index (BMI) 23.3±0.56 range 19-26 kg/m2] and 28 obese women (mean BMI 40.3±1.41 range 32-60 kg/m2).

To analyze adipose tissue gene expression in the first set of lean and obese subjects, we used a common reference pool that was generated by mixing equal amounts of total RNA extracted from adipose tissue samples of patients undergoing plastic surgery. Amplified RNA (aRNA) from the reference pool was labeled with Cy3, and aRNA from the 39
subjects was labeled with Cy5. A total of 39 pangenomic microarray hybridizations were performed for this differential comparison. After scanning the 39 arrays, the images were analyzed and data were normalized using the Stanford Microarray Database Online resources (http://genomewww5.
stanford.edu/MicroArray/SMD/). The log2 Cy5/Cy3 ratios of the 39 subjects were extracted. Spots with an average intensity <2.5-fold above the background were eliminated. We filtered genes with signal recovered in at least 8/11 lean and 20/28 obese subject ratios.

first: Karine

last: Clement

first: Soraya

last: Taleb

first: Christine

last: Rouault

first: Veronique

last: Pelloux


title: Transcriptional response of wild type ABA-treated Arabidopsis seedlings

city: Hayward

first: Don

middle: R

last: Maszle

address: 3935 Point Eden Way

institute: Mendel Biotechnology, Inc.

summary:
The transcriptional profile of wild-type Col-0 Arabidopsis seedling 4 hr after treatment with ABA was compared with mock-treated controls.
Keywords: aba_treatment

overall_design: Wild-type Col-0 Arabidopsis seedlings were grown axenically on vertically-oriented square tissue culture plates under 24 hr light on standard MS media for 14 days.  Treatments were applied by pouring a solution of either 100 uM ABA (in solution of MS basal salts + 0.1% Tween 20) or mock solution over seedlings until all tissue was submerged. The seedlings remained submerged in the solution for 5 minutes, the solution was poured off, and then plates were returned to the growth chamber for 4 hr before collection and processing for microarrays.  Two biological replicates per treatment (each containing 20-24 seedling shoots, separated from roots) were hybridized to microarrays.


title: Cell cycle of yeast deleted for yox1

city: Seattle

first: Tata

last: Pramila

state: WA

address: 1100, Fairview Avenue N

department: Basic Sciences

institute: FHCRC

summary:
Transcription profiles across the cell cycle of yox1 deleted yeast cells. BY2399 cells (yox1 delete cells isogenic with W303a) were arrested with alpha factor in YEPD media. After release from arrest cells were sampled every 5 min for 2 hr.
Keywords: cell cycle time course

overall_design: Cells were synchronized with alpha factor and sampled every 5 min across 2 cell cycles. A total of 25 samples were analyzed. Technical replicates were included. cDNA of the cell cycle samples were labelled with Cy5. For Cy3 labelling, asynchronous yeast population was used.

first: Linda

middle: L

last: Breeden


title: Expression profile of human preadipocytes cultured with activated macrophages medium

status: Public on Sep 24 2007

summary:
Extracellular matrix (ECM) remodelling occurs during tissue repair and inflammation-related pathologies with deposition of specific proteins.  White adipose tissue (WAT) was recently shown to be the site of substantial interstitial fibrosis. ECM components, such as fibronectin, and their receptors integrins control cell migration, proliferation and differentiation. Adipocyte differentiation which is under the control of a specific transcriptional network is associated with decrease of fibronectin-rich matrix. Considering macrophage accumulation in white adipose tissues from obese subjects, we recently demonstrated that macrophage-secreted factors provoke an inhibition of differentiation with a proinflammatory state of human preadipocytes. The functional analysis of gene expression measurements obtained from cDNA microarray experiments reveals that themes related to the “extracellular matrix” are among the most significantly enriched in overexpressed genes in inflammatory preadipocytes. ECM remodelling, characterized by high deposition of fibronectin, collagen I and tenascin-C and important clustering of ECM receptors integrin alpha-5, is associated with increased proliferation and migration of preadipocytes. siRNA deletion of NF-kappaB in inflammatory preadipocytes decreased fibronectin network formation and their proliferation. Cyclin D1 and FAK constitute pivotal molecular targets in the synergistic promotion of migration and proliferation of the inflammatory preadipocytes. Importantly, interactions between preadipocytes and macrophages are favoured in 3D collagen I, a microenvironment mimicking fibrosis context of obese WATs.
These data suggest that inflammatory preadipocytes could contribute to the production of fibrosis components and, in the context of WAT repair, these altered properties of preadipocytes could further lead to reconstitution of new fat clusters.
Keywords: cell type comparison

overall_design:
Isolation of human preadipocytes:
Subcutaneous adipose tissues biopsies were obtained from non obese (BMI < 30 kg/m2) young female patients undergoing elective surgery. None of the patients had diabetes or metabolic disorders and taking medications. This study was approved by the Ethical Committees of Hôtel-Dieu (Paris, France). Minced adipose tissue was digested by collagenase treatment. The digested material was filtered and centrifuged. The resulting pellet (stroma-vascular fraction, SVF) was resuspended in erythrocyte lysis buffer (154 mM NH4Cl, 5.7 mM K2HPO4 and 0.1 mM EDTA, pH 7.0) at 250g for 10 min. After washing in PBS, the SVF cells were suspended in DMEM-10% FBS and used for cell culture at passage 2 in order to eliminate non preadipocyte cell contamination as confirmed by negative staining for macrophage markers (Ham 56 and Mac-1).
Preparation of human blood monocyte-derived macrophages and conditioned medium:
Blood from overweight (BMI > 25 kg/m2) female patients was immediately processed for plasma blood mononuclear cells (PBMC) isolation. Blood sample was layered on PBMC isolation medium (GE healthcare, little Chaford, UK). PBMCs were suspended in PBS/2% FBS/1mmol/L EDTA were incubated at room temperature for 15 min with CD14-positive selection cocktail, followed by 10 min incubation period with magnetic nanoparticles (Stemcell Technologies, Grenoble, France). The bead-coupled CD14+ cells were maintained for 24h in 2 ml of RPMI-10 % FBS at a cell density of 4x105 cells. After 8 days of differentiation, the cells were incubated in RPMI-1 % FBS for 24 h with 100 ng/ml lipopolysaccharides (LPS from E. Coli 0127:B8, Sigma St Louis, Mi, USA) prior collecting the medium (Ac CM). Control medium was RPMI-1 % FBS kept at 37°C for 24h in the absence of macrophages. The conditioned media of the macrophages obtained from 3-5 individuals were pooled and stored at –80°C until used. Distinct pools were used for each culture experiment.
Preparation of Adipose Tissue Macrophages (ATM) and conditioned medium:
Isolated human SVF cells were obtained from subcutaneous adipose tissue biopsies, as described above. SVF cells were suspended in PBS/2% FBS/1mmol/L EDTA were incubated at room temperature for 15 min with CD34-positive selection cocktail, followed by 10 min incubation period with magnetic nanoparticles (Stemcell Technologies, Grenoble, France). The CD34-negative cell fraction was incubated with CD14 positive selection cocktail. The bead-coupled CD14+ cells were maintained for 24h in 1 ml of RPMI-1 % FBS at cell density of 4x105 cells in 12-well plates to obtain macrophage conditioned media (ATM CM), which was stored at - 80°C before use.
Differentiation of human preadipocytes:
Preadipocytes were cultured for 24h in DMEM-10 % FBS at a cell density of 105 cells per well in 12-well plates. The preadipocytes were then incubated with control RPMI or Ac CM (corresponding to 1x105 activated macrophages) and DMEM/F12 induction medium (final concentration of 50 nM insulin, 100 nM dexamethasone, 0.25 mM IBMX and 100 nM rosiglitazone) for 4 days, as described in nous. Next, this medium was replaced by control RPMI or Ac CM and DMEM/F12 culture medium (final concentration of 50nM insulin and 100 nM rosiglitazone). The medium was changed every 2 days until 10-12 days. For ATM CM, the experimental conditions were as described above except that the proportion of ATM CM corresponds to 2x105 AT macrophages.

first: Daniele

last: Lacasa


title: Gene expression specificity in relation to the main cellular fractions of the adipose tissue

summary:
In human obesity, the stroma vascular fraction (SVF) of white adipose tissue (WAT) is enriched in macrophages. These cells may contribute to low-grade inflammation and to its metabolic complications. Little is known about the effect of weight loss on macrophages and genes involved in macrophage attraction. We examined subcutaneous WAT (scWAT) of 7 lean and 17 morbidly obese subjects before and 3 months after bypass surgery. Immunomorphological changes of the number of scWAT-infiltrating macrophages were evaluated, along with concomitant changes in expression of SVF-overexpressed genes. The number of scWAT-infiltrating macrophages before surgery was higher in obese than in lean subjects (HAM56+/CD68+; 22.6 ± 4.3 vs. 1.4 ± 0.6%, P < 0.001). Typical "crowns" of macrophages were observed around adipocytes. Drastic weight loss resulted in a significant decrease in macrophage number (–11.63 ± 2.3%, P < 0.001), and remaining macrophages stained positive for the anti-inflammatory protein interleukin 10. Genes involved in macrophage attraction (monocyte chemotactic protein [MCP]-1, plasminogen activator urokinase receptor [PLAUR], and colony-stimulating factor [CSF]-3) and hypoxia (hypoxia-inducible factor-1{alpha} [HIF-1{alpha}]), expression of which increases in obesity and decreases after surgery, were predominantly expressed in the SVF. We show that improvement of the inflammatory profile after weight loss is related to a reduced number of macrophages in scWAT. MCP-1, PLAUR, CSF-3, and HIF-1{alpha} may play roles in the attraction of macrophages in scWAT.
Keywords: cell type comparison

overall_design:
Subcutaneous abdominal white adipose tissue was obtained from nine healthy female subjects (mean body mass index, BMI 28±7 kg/m2) undergoing plastic surgery in agreement with French laws on biomedical research. Mature adipocytes were collected after collagenase (type II, Gibco, Cergy pontoise, France) cell dissociation at 37°C, filtration, decantation, and centrifugation. The floating cellular layer was kept free of any detectable stromavascular element and contained only mature adipocytes filled with triglyceride droplets (tested by light microscopy). SVF cells were recovered at the bottom of the tubes after centrifugation. For measurements of mRNA levels, adipocytes and SVF cells were lysed with denaturing buffer from RNeasy kit (Qiagen, Courtaboeuf, France), then stored at –80°C until RNA preparation.
Total RNA was prepared using the RNeasy total RNA Mini kit (Qiagen). RNA concentration and integrity were assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Massy, France). For microarray experiments, 1 µg of total RNA from each total RNA sample preparation was amplified by MessageAmp RNA Kit (Ambion, Austin, TX, USA) and 3 µg of amplified RNA was labeled with cyanin dyes (Cy) using the CyScribe First-Strand cDNA labeling kit (Amersham Biosciences, Orsay, France).
We compared total RNA isolated from adipocytes and from SVF cells. The microarray experiments were performed after pooling an equal amount of total RNA from adipocyte and from SVF cell preparations and repeated six times. Amplified RNA from SVF cells was labeled with Cy3 whereas aRNA from adipocytes was labeled with Cy5. The labeled cDNA mixtures were hybridized according to the protocol described at http://cmgm.stanford.edu/pbrown/protocols/index

first: Raffaella

last: Cancello

first: Nathalie

last: Viguerie


title: Purification of neuronal precursors from the adult mouse brain: a comprehensive gene expression analysis

status: Public on Sep 22 2007

city: Bergisch Gladbach

first: Sandra

middle: Juliane

last: Pennartz

address: Friedrich-Ebert-Strasse 68

department: Research & Development

institute: Miltenyi Biotec GmbH

summary:
The progeny of neural stem cells in the subventricular zone (SVZ) of the adult mammalian brain consists in polysialylated NCAM-expressing immature neurons (PSA(+) cells), which migrate to the olfactory bulb (OB) to differentiate into GABAergic interneurons. We purified murine PSA(+) cells directly from the adult brain by FACS and analyzed their gene expression profile by SAGE. Comparative analyses led to the identification of precursor-enriched genes, including Survivin, Sox-4, Meis2, Dishevelled-2, C3aR1 and Riken 3110003A17, and many so far uncharacterized transcripts. Cluster analysis showed that groups of genes involved in axon guidance and gene clusters implicated in chemotaxis are strongly upregulated, indicating a role of both cues in the control of cell migration in the adult brain. Furthermore, genes involved in apoptosis and cell proliferation are co-expressed, suggesting that the amount of precursors that is present in the adult brain is a result of an equilibrium of these processes.
Keywords: neuronal precursors, adult SVZ, mouse, gene expression analysis

overall_design:
Please see Pennartz et al., Mol Cell Neurosci. 2004 Apr;25(4):692-706 and PhD thesis "Gene expression analysis of neuronal precursors
from adult mouse brain and differential screen for
neural stem cell markers"  http://deposit.ddb.de/cgi-bin/dokserv?idn=973392754

first: Richard

last: Belvindrah

first: Harold

last: Cremer

first: Marcus

last: Conradt

first: Andreas

last: Bosio

first: Stefan

last: Tomiuk


title: Acid1 overexpression fibroblasts

first: Martin

last: Irmler

address: Ingolstaedter Landstrasse 1

institute: Helmholtz Zentrum München GmbH

summary:
NIH3T3 mouse fibroblasts with over-expression of the transcription cofactor Acid1. An induced/non-induced system regulated by treatment with Tamoxifen (OHT) stimulates the transport of Acid1 from the cytoplasm into the nucleus that leads to an overexpression of the protein in the nucleus (induced situation). 3 and 6 hours after induction of Acid1 expression profiles were performed
Keywords: murine, fibroblast, Acid1, cDNA arrays

overall_design: NIH3T3 fibroblast overexpressing Acid1 3h and 10h after treatment including 50% dyeswap experiments

first: Johannes

last: Beckers

first: Marion

last: Horsch


title: Quantitative proteomics of anaerobic and aerobic yeast cultures

city: Delft

first: Marco

middle: J.L de

last: Groot

state: Zuid Holland

address: Mekelweg 4

department: Mediamatics

institute: Delft University of Technology

summary:
Saccharomyces cerevisiae is unique among yeasts for its ability to grow rapidly in the complete absence of oxygen. S. cerevisiae is therefore an ideal eukaryotic model to study physiological adaptation to anaerobiosis. Recent transcriptome analyses have identified hundreds of genes that are transcriptionally regulated by oxygen availability but the relevance of this cellular response has not been systematically investigated at the key control level of the proteome. Therefore, the proteomic response of the S. cerevisiae to anaerobiosis was investigated using metabolic stable isotope labeling in aerobic and anaerobic glucose-limited chemostat cultures, followed by proteome analysis to relatively quantify protein expression. Using independent replicate cultures and stringent statistical filtering, a robust dataset of 474 quantified proteins was generated, of which 249 showed differential expression levels. While some of these changes were consistent with previous transcriptome studies, many responses of S. cerevisiae to oxygen availability were hitherto unreported. Comparison of transcriptome and proteome from identical cultivations yielded strong evidence for post-transcriptional regulation of key cellular processes, including glycolysis, amino-acyl tRNA synthesis, purine-nucleotide synthesis and amino-acid biosynthesis. The use of chemostat cultures provided well-controlled and reproducible culture conditions, which are essential for generating robust datasets at different cellular information levels. Integration of transcriptome and proteome data led to new insights in the physiology of anaerobically growing yeast that would not have been apparent from differential analyses at either the messenger RNA or protein level alone, thus illustrating the power of multi-level studies in yeast systems biology.
Protein levels versus transcript level: Systematic analysis of the control levels at which the yeast response to anaerobiosis takes place was performed using previously published transcript data obtained from yeast cultures grown under strictly identical conditions as described for the current proteome analysis. Affymetrix microarrays from five aerobic and four anaerobic independent culture replicates were used for this analysis. These comparison data are summarized in the table below. These array data are publicly available at the gene expression repository Gene Expression Omnibus under accession number GSE4804.
Keywords: proteomic, nanoflow-LC-MS/MS

overall_design: The relative protein levels of anaerobically versus aerobically grown S. cerevisiae were investigated using metabolic stable isotope labeling. The biomass was produced using carbon limited chemostat cultures. Each biomass was produced twice, once using 14N labeled nitrogen source (ammonium sulfate) and once using 98% 15N nitrogen source. Subsequently, biomass was harvested, and protein extracts were prepared. Two mixed samples were prepared from these four protein samples, containing equal protein content of 14N aerobic and 15N anaerobic cultures for the first sample and equal protein content of 15N aerobic and 14N anaerobic cultures for the second sample. The two resulting samples were separated on an SDS-PAGE gel, and each lane was equally fragmented into 40 slices resulting in sub-samples based on protein size. This was followed by an in-gel protein digestion using trypsin, and analysis in triplicate by nanoflow-LC-MS/MS. Proteins were identified using MS/MS, and the relative protein levels were obtained by comparing the extracted ion chromatograms of the co-eluting 14N and 15N labeled peptide pairs

last: de Groot

first: Pascale

last: Daran-Lapujade

first: Bas

last: van Breukelen

first: Theo

last: Knijnenburg

first: Erik

last: de Hulster

first: Marcel

last: Reinders

first: Jack

middle: T

last: Pronk

first: Albert

last: Heck

first: Monique

last: Slijper


title: AIN-1 and AIN-2::GFP IP of C.elegans miRNA targets

status: Public on Sep 21 2007

city: Boulder

first: liang

last: zhang

state: CO

address: University of Colorado at Boulder, Han lab, Department of MCD Biology, Campus box 0347

institute: university of colorado at boulder

summary:
MicroRNAs (miRNAs) regulate gene expression for diverse functions, but only a limited number of mRNA targets have been experimentally identified. We show that GW182 family proteins AIN-1 and AIN-2 act redundantly to regulate the expression of miRNA targets, but not miRNA biogenesis. Immunoprecipitation (IP) and mass spectrometry indicate that AIN-1 and AIN-2 interact only with miRNA-specific Argonaute proteins ALG-1 and ALG-2 and with components of the core translational initiation complex. Known miRNA targets are enriched in AIN-2 complexes, correlating with the expression of corresponding miRNAs. Combining IP with pyrosequencing and microarray analysis of RNAs associated with AIN-1/AIN-2, we identified 106 previously annotated miRNAs plus 9 new candidate miRNAs, but nearly no siRNAs, and more than 3500 potential miRNA targets including nearly all known ones. Our results demonstrate an effective biochemical approach to systematically identify miRNA targets and provide valuable insights regarding the properties of miRNA effector complexes.
Keywords: IP microarray of miRNA targets

overall_design: RNAs were isolated from each immunocomplex after IP, or from total worm lysate, using the Trizol reagents (Invitrogen) according to vendor’s protocol. Three independent biological replicates were generated for each IP sample and for the total worm lysate samples from strains expressing AIN-2::GFP or GFP alone driven by the ain-2 promoter. Two biological replicates were generated for wt total lysates. After being resuspended into equal volumes of water, the samples were shipped to the Washington University Microarray Center for microarray analysis. The samples were amplified once using the MessageAmp aRNA kit (Ambion) and labeled with cy3 or cy5 using the MicroMax ASAP kit (Perkin Elmer). The hybridizations were performed according to the standard method. Dye label flipping was included to control the dye label bias.Signals from replicates of total worm lysates from wt and strains containing the ain-2::gfp or the ain-2 promoter::gfp transgene were mean normalized and averaged respectively to generate standard profiles of gene expression in these worm strains. We then calculated the ratio of signal of each gene from each IP sample to the standard gene expression profile of the corresponding worm strain. Based on this ratio, a percentile rank of each gene relative to all genes in each IP replicate was calculated. The percentile ranks in the three replicates of each IP were averaged. Student t test was utilized to determine if the average percentile ranks of enrichment of individual genes were significantly higher (p value) than the mean enrichment of all genes in the IP samples. To determine the AIN-1 or AIN-2 associated genes, we used the following criteria: (1) average percentile ranks of enrichment is greater than the mean enrichment of all genes in AIN-1 or AIN-2 IP with p<0.01; (2) average signal in AIN-1 or AIN-2 IP replicates is greater than the background signal (including 2X standard deviation (SD)) (Background signal and SD were calculated based on signals from empty spots on each microarray); (3) criteria 1 is not be satisfied for the same gene in the corresponding control IP.

first: Liang

last: Zhang

first: Lei

last: Ding

first: Tom

last: Cheung

first: Meng-Qiu

last: Dong

first: Jun

last: Chen

first: Aileen

middle: K

last: Sewell

first: Xuedong

last: Liu

first: John

last: Yates III

first: Min

last: Han


title: Jmjd1a / Jmjd2c RNAi expression profile

city: Singapore

first: Huck

middle: Hui

last: Ng

state: Singapore

address: Biopolis street

department: Stem Cell and Development biology

country: Singapore

institute: Genone Institute of Singapore

summary:
Global gene expression effects of silencing Jmjd1a and Jmjd2c genes
We used microarrays to detail the global programme of gene expression after silencing Jmjd1a gene and Jmjd2c gene separately
Keywords: dose response

overall_design: three biological replicates each for Jmjd1a and Jmjd2c RNAi transcript levels were compared with six biological replicated of LUC RNAi

first: Yuin-Han

last: Loh

first: Weiwei

first: Xi

first: Joshy

last: George

first: Huck-Hui


title: Transcriptional and Physiological Responses of Bradyrhizobium japonicum to Desiccation-Induced Stress

status: Public on Sep 25 2007

city: St. Paul

first: Eddie

middle: J

last: Cytryn

state: MN

address: 1991 Upper Buford Circle

department: Soil Water and Climate

institute: University of Minnesota

summary:
The growth and persistence of rhizobia and bradyrhizobia in soils are negatively impacted by drought conditions. In this study, we used genome-wide transcriptional analyses to obtain a comprehensive understanding of the response of Bradyrhizobium japonicum to drought. Desiccation of cells resulted in the differential expression of 15 to 20% of the 8,480 B. japonicum open reading frames, with considerable differentiation between early (after 4 h) and late (after 24 and 72 h) expressed genes. While 225 genes were universally up-regulated at all three incubation times in response to desiccation, an additional 43 and 403 up-regulated genes were common to the 4/24- and 24/72-h incubation times, respectively. Desiccating conditions resulted in the significant induction (>2.0-fold) of the trehalose-6-phosphate synthetase (otsA), trehalose-6-phosphate phosphatase (otsB), and trehalose synthase (treS) genes, which encode two of the three trehalose synthesis pathways found in B. japonicum. Gene induction was correlated with an elevated intracellular concentration of trehalose and increased activity of trehalose-6-phosphate synthetase, collectively supporting the hypothesis that this disaccharide plays a prominent and important role in promoting desiccation tolerance in B. japonicum. Microarray data also indicated that sigma(54)- and sigma(24)-associated transcriptional regulators and genes encoding isocitrate lyase, oxidative stress responses, the synthesis and transport of exopolysaccharides, heat shock response proteins, enzymes for the modification and repair of nucleic acids, and the synthesis of pili and flagella are also involved in the response of B. japonicum to desiccation. Polyethylene glycol-generated osmotic stress induced significantly fewer genes than those transcriptionally activated by desiccation. However, 67 genes were commonly induced under both conditions. Taken together, these results suggest that B. japonicum directly responds to desiccation by adapting to changes imparted by reduced water activity, such as the synthesis of trehalose and polysaccharides and, secondarily, by the induction of a wide variety of proteins involved in protection of the cell membrane, repair of DNA damage, stability and integrity of proteins, and oxidative stress responses.

Keywords: stress response; time course

overall_design: B. japonicum cells were grown in minimal medium supplemented with yeast extract and glycerol as a carbon source. At mid-log phase cells were filtered onto nitrocellulose membranes and incubated in desiccators for 4, 24 and 72 hours under severe desiccation [27% relative humidity (RH)], and full hydration (100%RH).  Transcriptional profiling of B. japonicum was compared between cells grown under desiccating (27%) and hydrating (100%) conditions

first: Woo-Suk

last: Chang

first: Michael

last: Sadowsky