title: Response of the Pseudomonas host chromosomal transcriptome to carriage of the IncP-7 plasmid pCAR1.

status: Public on Dec 25 2009

first: Hideaki

last: Nojiri

summary: Plasmid carriage requires appropriate expression of the genes on the plasmid or host chromosome through cooperative transcriptional regulation. To clarify the impact of plasmid carriage on the host chromosome, we compared the chromosomal RNA maps of plasmid-free and plasmid-containing host strains using the incompatibility group P-7 archetype plasmid pCAR1, which is involved in carbazole degradation, and three distinct Pseudomonas strains. The possession of pCAR1 altered gene expression related to the iron acquisition systems in each host. Expression of the major siderophore pyoverdine was greater in plasmid-containing P. putida KT2440 and P. aeruginosa PAO1 than in the plasmid-free host strains, in part due to the expression of carbazole-degradative genes on pCAR1. The mexEFoprN operon encoding an efflux pump of the resistance-nodulation-cell division (RND) family was specifically upregulated by the carriage of pCAR1 in P. putida KT2440, whereas the expression of orthologous genes in the other species remained unaltered. Induction of the mexEFoprN genes increased the resistance of pCAR1-containing KT2440 to chloramphenicol compared to pCAR1-free KT2440. Our findings indicate that the possession of pCAR1 altered the growth rate of the host via the expression of genes on pCAR1 and the host chromosomes.

sample_organism:
Pseudomonas putida KT2440
Pseudomonas fluorescens Pf0-1
Pseudomonas aeruginosa PAO1

overall_design:
Samples GSM380019-GSM380021: 6 samples were compared including two biological replicates.
Samples GSM380025-GSM380026: 4 samples were compared including two biological replicates.
Samples GSM380027-GSM380028: 4 samples were compared including two biological replicates.
Samples GSM380029-GSM380030: 4 samples were compared including two biological replicates.

first: Masaki

last: Shintani

first: Yurika

last: Takahashi

first: Hiroki

last: Tokumaru

first: Kouji

last: Kadota

first: Hirohumi

last: Hara

first: Masatoshi

last: Miyakoshi

first: Kunihiko

last: Naito

first: Hisakazu

last: Yamane

first: Hiromi

last: Nishida


title: Transcriptome analysis of Rhizobium etli’s free-living and symbiotic non-growing state.

status: Public on Nov 04 2010

city: Leuven

first: Kristof

last: Engelen

address: Kasteelpark Arenberg 20

country: Belgium

institute: KULeuven

summary: Gene expression during stationary phase and symbiosis of R. etli CFN42 was compared to that of exponentially growing cells. This allowed us to better understand how R. etli adapts to a non-growing lifestyle, both the free-living and symbiotic state, as well as to determine to what extent this adaptation is similar in both states.

overall_design: R. etli CFN42 was grown at 30˚C in AMS medium supplied with 10 mM NH4Cl and 10 mM succinate while monitoring the optical density (OD) of the culture. Free-living samples were taken at OD600 = 0.3 and 6 hours after reaching the maximum OD, representing early exponential and stationary phase respectively. Bacteroid samples were obtained from nodules 3 weeks after inoculation of Common bean plants (Phaseolus vulgaris cv Limburgse vroege).

first: Maarten

last: Vercruysse

last: Fauvart

first: Jan

last: Michiels

first: Lore

last: Cloots


title: Ecdysone signaling and transcript signature in Drosophila cells resistant against methoxyfenozide

status: Public on Nov 08 2010

city: Ghent

first: Guy

last: Smagghe

address: Coupure Links

department: Crop Protection

institute: Ghent University

summary: Methoxyfenozide (RH-2485) represents the group of non-steroidal ecdysteroid agonists with a dibenzoylhydrazine structure, that are used as novel biorational insecticides in the control of insect pests. Here we report on the selection of Drosophila melanogaster S2 cells for resistance to inhibition of cell proliferation by methoxyfenozide by ~1000-fold over 4 months. Cells were exposed to gradually increasing concentrations of methoxyfenozide and selected out based on the ecdysteroid-sensitive response for cell proliferation. In the resistant cells, the ecdysteroid receptor (EcR/USP) complex was no longer active in the presence of methoxyfenozide. But when resistant cells were relaxed from pressure in methoxyfenozide-free medium, induction of the reporter construct was observed. In parallel, EcR/USP functionality was also restored when resistant cells were rescued by a Drosophila EcR plasmid. However, it was striking that in the resistant cells the ecdysteroid-sensitive response for cell proliferation was not restored upon methoxyfenozide withdrawal, indicating permanent changes in the physiology of the cells during selection. To investigate changes in gene expression caused by inactivation of the EcR/USP complex in resistant cells, Drosophila oligo 14kv1 microarrays were used and probed with cDNAs from resistant cells in the presence and absence of ecdysone agonist on one hand and from unselected sensitive cells on the other hand. A selection of 324 differentially expressed genes was assigned covering diverse functions as transport, enzyme activity, cytoskeleton organization, cell cycle machinery, transcription/translation and ecdysteroid signaling. Besides the identification of (primary and secondary) target genes of the EcR/USP signaling pathway, this analysis is also predicted to gain insights into the mechanism of resistance and the crosstalk between ecdysteroid signaling and cell proliferation-linked processes.

overall_design:
In this microarray experiment three biological conditions were under study: (i) untreated S2 cells (S2), (ii) a subpopulation of these untreated cells, which were grown under continuous pressure of methoxyfenozide and which were shown to become resistant to this compound (S2res) and (iii) a subpopulation of the resistant cells, which were again cultured without methoxyfenozide added to the medium (S2res-woMF). For each condition, two subpopulations (which were treated as biological replicates, although they are not exactly independent), were isolated and used for the RNA extractions.
The overall experimental design was an n+2 A-optimal design. When each hybridisation is respresented by an arrow (Cy5 → Cy3), the design can be respresented as follows: First loop = S2.1 → S2res.1 → S2.2 → S2res.2 → S2.1; Second loop = S2.1 → S2res-woMF.1 → S2.2 → S2res-woMF.2 → S2.1 (numbers following the condition name correspond to the biological replicate numbers). The two loops are thus connected by S2.1 and S2.2. Two analyses were performed: S2res compared to S2 and S2res-woMF compared to S2.

first: Hadi

last: Mosallanejad

first: Liesbeth

last: Badisco

first: Luc

last: Swevers

first: Thomas

last: Soin

first: Dries

last: Knapen

first: Jozef

last: Vanden Broeck


title: Ectopic activation of the immune response in the larval airway epithelium

status: Public on May 11 2009

city: Kiel

first: Christina

last: Wagner

address: Am Botanischen Garten 1-9

department: Zoophysiology

country: Germany

institute: Zoological Institute

summary:
The unsurpassed simplicity of the fruitfly’s airway epithelium, that is made of homogenous epithelial cells only, favours its use as a model to study general features and response characteristics of airway epithelia in general. All epithelial cells are able to launch an immune response as characterized by the expression of antimicrobial peptide genes. Infection induces a complex change in the expression profile of these epithelial cells. Outstanding are a priming of the immune system and the launch of a survival program, presumably to counteract infection induced apoptotic signals, which comprises the concurrent expression of known longevity genes such as dFoxo, and dThor. In regions of the airway epithelium with strong immune reactions, a complex remodelling of the airways can be observed, which is characterized by metaplasia and presumably also by hyperplasia of the affected epithelial cells. At the transcriptional level, this reorganization of the airway epithelium is mirrored by a recapitulation of genetic programs that are characteristic for early phases of airway development. Taken together, the response characteristics of the fly’s airway epithelium towards infections discloses features that are known from inflammatory diseases of the human lung, thus opening the opportunity to study fundamental aspects of these diseases in the fly.

Keywords: Ectopic activation of IMD, larval airways, third instar larvae, Gal4/UAS-system, two-colour microarrays

overall_design:
Ectopic activiation of the Immune deficiency pathway in the larval airway epithelium using the Gal4/UAS-system of Brand and Perrimon. The activation was induced by the overexpression of the pattern recognition receptor PGRP-LE using a driver line which was specific for the tracheal system of the larvae. In general four replicates were performed including dye-swaps in two-colour arrays.
A table of significantly-regulated genes from the SAM-output and GeneTraffic-output has been linked as a supplementary file at the foot of this record.

last: Roeder


title: Pmr, a histone-like protein H1 (H-NS) family protein encoded by the IncP-7 plasmid pCAR1, is a key global regulator that alters host function

status: Public on May 25 2010

city: Tokyo

first: Hideaki

last: NOJIRI

address: 1-1-1 Yayoi, Bunkyo-ku

department: Biotechnology Research Center

country: Japan

institute: The University of Tokyo

type: Genome binding/occupancy profiling by genome tiling array

type: Expression profiling by genome tiling array

summary: H-NS family proteins are known as a member of nucleoid associated proteins (NAPs) conserved in genus Pseudomonas bacteria. Incompatibility group (Inc) P-7 archetype plasmid pCAR1 is transmissible among various Pseudomonas strains and carries genes encoding H-NS family protein, Pmr. P. putida KT2440 is one of pCAR1 hosts, which possesses five genes encoding H-NS family proteins, PP_1366 (TurA), PP_3765 (TurB), PP_0017 (TurC), PP_3693 (TurD), and PP_2947 (TurE) on its chromosome. Quantitative RT-PCR showed that the presence of Pmr did not affect on the transcriptions of genes encoding H-NS family proteins, and only Pmr, TurA and TurB were majorly transcribed. In vitro Pull down assay revealed that Pmr strongly interacted with Pmr itself and TurA, TurB, and TurE. Transcriptome comparisons of pmr disruptant, with KT2440 and KT2440(pCAR1) showed that the pmr disruption had larger effects on the host transcriptome than those by pCAR1 carriage. The transcriptional levels of some genes increased by pCAR1 carriage were reverted to those of pCAR1-free KT2440 by disruption of pmr, such as mexEFoprN genes for efflux pump, and parI. Transcriptional levels of many genes on the putative foreign DNA regions were not altered by carriage of pCAR1 but by pmr disruption. Identification of genome-wide Pmr binding sites by ChAP-chip analysis showed that Pmr preferentially binds to the foreign DNA region. Pmr binding sites were well overlapping with the location of the differentially transcribed genes by disruption of pmr. Our findings indicate that Pmr is a key factor to optimize the gene transcription onpCAR1 and host chromosome.

platform_organism:
Pseudomonas
Pseudomonas putida KT2440

overall_design:
ChIP-chip: Pseudomonas putida KT2440 harboring pCAR1 cells ChIPed with His-tag ( C-terminus of pmr) vs Input in Pseudomonas putida KT2440 harboring pCAR1 cells
RNA mapping: 4 samples were compared including two biological replicates.

first: Choong-Soo

last: Yun

first: Chiho

last: Suzuki

last: Naito

first: Toshiharu

last: Takeda

first: Yurika

last: Takahashi

first: Fumiya

last: Sai

first: Tsuguno

last: Terabayashi

last: Miyakoshi

first: Masaki

last: Shintani

last: Nojiri


title: Menin links the stress response to genome stability in Drosophila melanogaster

status: Public on Oct 25 2010

city: Mississauga

first: Zak

last: Razak

state: Ontario

address: 3359 Mississauga Rd

country: Canada

institute: Canadian Drosophila Microarray Centre

summary: Mutants of the Mnn1 gene are hyper-sensitive to several stresses and display increased genome instability when subjected to conditions, such as heat shock, generally regarded as non-genotoxic.  We describe a role for Menin as a global regulator of heat shock gene expression and critical factor in the maintenance of genome integrity

overall_design: 2-colour microarray design using 36 microarrays testing shock vs non shock in Drosophila melanogaster. Using a test of loss-of-heterozygosity, we show that Drosophila strains lacking a functional Mnn1 gene or expressing a Mnn1 dsRNAi are characterized by increased genome instability in response to short, repeated but non-lethal heat shock or hypoxia treatments.  The same was true for strains lacking all Hsp70 genes.


title: High-resolution mapping of the pCAR1 plasmid transcriptomes in Pseudomonas

status: Public on Oct 18 2008

city: Sendai

first: Masatoshi

state: Miyagi

address: Aoba-ku, Katahira, 2-1-1

department: Department of Environmental Life Sciences

institute: Tohoku University

summary:
High-resolution mapping of the pCAR1 plasmid transcriptomes in the original host Pseudomonas resinovorans CA10 and the transconjugant Pseudomonas putida KT2440(pCAR1)
While plasmids are replicated autonomously in their hosts, the transcription of plasmid genes can be switched through horizontal transfer by the change in the transcriptional networks.  To examine whether and how the plasmid genome is differentially expressed, we analyzed the transcriptomes of the 199,035-bp IncP-7 carbazole catabolic and conjugative plasmid pCAR1 in the original host Pseudomonas resinovorans CA10 and the transconjugant Pseudomonas putida KT2440(pCAR1) during growth on carbazole or succinate using the high-resolution tiling array.  The tiling array successfully detected the relatively large catabolic operons, for which transcription was induced during growth on carbazole regardless of the host.  Compared between the hosts, nearly identical regions of pCAR1 were transcribed, but two hypothetical operons, i.e., ORF100-108 and ORF145-146, were transcribed at higher levels in KT2440(pCAR1) than in CA10.  We verified the differential expression in heterologous hosts using quantitative RT-PCR.  The tiling array analysis clearly revealed the transcription start sites, for which the positions and extents agreed with the primer extension experiments.  Our data demonstrate that the transcriptome of the transmissible plasmid is altered through horizontal transfer, and we identified probable genes that are involved in plasmid functions in various hosts.  This approach can be used to visualize flexible prokaryotic transcriptomes comprehensively.
Keywords: high-resolution RNA mapping

sample_organism:
Pseudomonas putida
Metapseudomonas resinovorans

overall_design: We analyzed the plasmid transcriptomes in two strains, Pseudomonas resinovorans CA10 and Pseudomonas putida KT2440(pCAR1), during growth on succinate and carbazole as the sole sources of carbon using the pCAR1 tiling array. We conducted two independent biological replicates for each strain and growth condition.

first: Hiromi

last: Nishida


title: Expression profiling of mammary stem cell enriched fractions and other fractions from fetal and adult mouse.

status: Public on Feb 08 2012

city: La Jolla

first: Benjamin

middle: T

last: Spike

state: California

address: 10010 N Torrey Pines Rd

department: Gene Expression Laboratory

country: USA

institute: The Salk Institute

summary: Examination of genome-wide, gene expression level differences among adult and fetal mammary sub-populations including fetal CD24high cells and adult CD49fhighCD24medium cells which harbor enriched mammary stem cell activity.

overall_design: A genome wide 12-plex expression array using linear-amplified (Eberwine) mRNA from dissociated and FACS sorted  fetal and adult mammary gland populations. Samples represent independent pooled biological replicates.


title: miR-309-dependent unstable maternal mRNAs are stabilized in smaug mutants

status: Public on Apr 23 2009

summary: The behavior of 410 miR-309-dependent maternal mRNAs (from Bushati et al., 2008) in embryos from wild type and smg-mutant females relative to wild-type stage 14 oocyte reference RNA. All are unstable in wild-type while almost 85% are stabilized in smg mutants

overall_design:
mRNA was extracted from staged embryos as well as stage 14 oocytes as described previously (Tadros et al., 2007a). To assay mRNA quality, known stable (rpA1) and unstable (Hsp83) transcripts were probed on Northern blots. Total RNA was then reverse transcribed with random primers (Tadros et al., 2007a) and labeled asÊdescribed in the Indirect Labeling of Total RNA for Microarray Hybridization protocol at http://www.flyarrays.com.  The fluorescently labeled cDNA probes were hybridized to 14Kv1 microarrays obtained from the Canadian Drosophila Microarray Centre (http://www.flyarrays.com;
GPL3603: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL3603).  Hybridization and scanning were performed using a PerkinElmer/GSI ScanArray 4000 scanner and the ScanArray software as previously described (Neal et al., 2003). The 16 bit TIFF image files were quantified using QuantArray Version 3 (PerkinElmer), using the adaptive quantification algorithm and analyzed using GeneTraffic Duo3.2 (Iobion Informatics/Stratagene). . The 14Kv1 arrays were normalized to a set of known stable transcripts: Rpl1, RpL32, Rps5a, Rps3, RpL22, mRpS30, mRpS22, CG6764, bonsai, RpS29, RpL11, mRpL1, CG6764, CG317, RpL37A, and RpL40.

first: Sarah

middle: M

last: Votruba

first: Chun

middle: H

last: He

first: Linan

last: Chen

first: J

last: Westwood

first: Howard

middle: D

last: Lipshitz


title: Gene expression changes in Drosophila head secondary to levetiracetam treatment during Pentylenetetrazole withdrawal.

status: Public on May 03 2008

city: Delhi

first: Abhay

last: Sharma

state: Delhi

address: Delhi University campus, Mall Road

department: Functional Genomics Unit

country: India

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

summary:
Drosophila melanogaster adult flies fed on normal food (NF) containing 16 mg/ml of pentylenetetrazole (PTZ) in the food show a hyperkinetic behavior within 24 hours. Half of that concentration, i.e., 8 mg/ml, of the PTZ, if fed for seven days, though doesn’t cause seizure-like behavior, results in a decreased climbing speed in flies. This change in locomotor behavior is progressive and becomes significant only on seventh day of the treatment. This climbing deficit is ameliorated when flies are treated concomitantly with PTZ and either of the two antiepileptic drugs (AEDs), sodium valproate (NaVP) or levetiracetam (LEV). Further experiments based on different regimes of combination and singular PTZ treatment demonstrated that whereas NaVP show a weaker prophylactic and stronger symptomatic effect, LEV exhibit an opposite effect, i.e., stronger prophylactic and weaker symptomatic action. These observations are consistent with the therapeutic effect of antiepileptic drugs. Time series of microarray gene expression profiling (earlier GEO submission) during chronic PTZ provided evidence of underlying downregulation of three main categories of biological processes, synaptic remodeling, energy metabolism and transport, in that order, in fly head. Transcriptomic analysis secondary to NaVP and LEV (earlier GEO submission) was found to cause statistically significant upregulation of two biological process categories each, energy metabolism and transport in case of NaVP and synaptic remodeling and energy metabolism in case of LEV. Time series of genome wide expression profiling secondary to PTZ and LEV combination treatment (earlier GEO submission) showed neutralizing effect of LEV on PTZ induced expression changes. Mining of published transcriptomic and proteomic data pertaining to epilepsy or established rodent animal models of epileptogenesis supported the relevance of the fly model in understanding the underlying brain pathophysiology.  Systems modeling using RNA interference and small molecules demonstrated the robustness of the fly model. In brief, the above results clearly showed that the fly model is valuable not only in screening of antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents, but also in advancing our knowledge of mechanisms of action of drugs used in treating human patients suffering from various neurological and neuropsychiatric conditions, pharmacogenomics of these drugs, identification of potential drug targets, and selection of potential candidate genes for association analysis in epilepsy. Considering the above fly locomotor behavior model as relevant in kindling attainment, it was of obvious interest to study the behavioral and transcriptomic changes post-kindling, i.e., after withdrawing PTZ following seven day long chronic PTZ administration. It was observed that when PTZ is discontinued and flies climbing speed is monitored for next seven days of PTZ discontinuation, climbing speed deficit initially disappears and then a progressive increase in climbing speed is detected. Behavioral pharmacology of AEDs in post-kindling regime, like fly kindling described above, showed its usefulness in screening of potential antiepileptic, antiepileptogenic, disease-modifying and neuroprotective agents. The present submission relates to transcriptomic changes in fly head secondary to levetiracetam (LEV) treatment following discontinuation of PTZ for seven days (after seven days of chronic PTZ treatment). To delineate possible prophylactic or symptomatic effect of LEV at behavioral level, flies were treated with LEV for first three days and then shifted to NF for next four days (3 + 4 day regime) or flies treated with NF for first four days and then drug treated for next three days (4 + 3 day regime), in that order. At transcriptomic level, we have now examined the effect of LEV in 3 + 4 day regime. Gene expression profiles have been generated at both time points, 3rd and 7th day, i.e., on 10th and 14th day from the beginning of PTZ treatment. Effect of three day long chronic LEV treatment on fly head microarray gene expression profile, in otherwise normally grown flies, has already been determined (earlier GEO submission). Similarly, transcriptomic changes after one day, three days and seven days of PTZ discontinuation (i.e., 8th, 10th and 14th day of the beginning of chronic PTZ treatment) have already been deciphered (earlier GEO submission). Unlike the previous experiment in which flies were maintained in the same container during the post-kindling period of seven days, flies needed to be shifted in fresh vials once in the present experiment because of the demand of 3 + 4 day regime. Since change in housing conditions has the potential to influence head transcriptome, we have also generated microarray expression profile of fly head after treating flies with PTZ for seven days, maintaining the flies to NF containing vials for three days and then shifting and maintaining them in fresh NF containing vials for next four days (submitted in GEO previously).
Keywords: Drug Response

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 PTZ for seven days. Each treatment vial contained 30 flies in the beginning. Next, flies were treated in either of the following two manners: (1) shifted to vials containing 5 mg/ml LEV media for three days, and (2) shifted to LEV media for three days and then again shifted to fresh vials containing NF for next four days. Heads were harvested at the end of either of the two treatment conditions. For this, 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 individuals treated in either one of the two ways described earlier, 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 LEV 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), 10 mg/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, Canada). 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 10 µm 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, Excel Add-In, Stanford) under the conditions of one class response, imputation and 100 permutations.

first: A

first: P

last: Singh

first: M

last: Farhan


title: A systematic search for transgenerational effect of drug exposure to adult males using a novel Drosophila model

status: Public on Mar 26 2009

summary: Drosophila melanogaster adult flies fed on food containing 16 mg/ml of pentylenetetrazole (PTZ) in the food show a hyperkinetic behavior within 24 hours. Half of that concentration, i.e., 8 mg/ml, of PTZ, if fed for seven days, though doesn’t cause seizure-like behavior, results in a decreased climbing speed in flies. This change in locomotor behavior is progressive and becomes significant only on seventh day of the treatment. We also examined flies’ locomotor behavior secondary to PTZ withdrawal. Interestingly, an increased climbing speed was found to develop seven days after withdrawal. Importantly, antiepileptic drugs showed effectiveness in the above fly model. Earlier, we submitted in GEO time series of fly head microarray gene expression profiling during chronic PTZ and PTZ withdrawal phase. We also submitted previously expression profiles associated with antiepileptic drug treatment. Here, we have undertaken a different line of work. Having developed a well characterized acquired model of behavioral and gene expression plasticity, we found an opportunity here to investigate if drug exposure to adult males could cause transgenerational effect. To probe this, we carried out a systematic study at both behavioral and microarray gene expression levels. In the latter, we asked the question that do F0 testis, F1 males’ head, F1 females’ head, F1 testis, F2 males’ head and F2 females’ head show gene expression changes if F0 male parents had a history of PTZ exposure? A total of 28 microarray slides were used in this study.

overall_design: Unless mentioned otherwise, standard method of fly handling and manipulation was used. Freshly generated isogenic 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 food containing 8 mg/ml PTZ for seven days. Flies were then shifted to vials containing NF. Seven days after shifting, F0 males were mated to same age group females maintained on NF throughout, without any drug exposure. Males and females were housed in groups in vials containing NF. Eggs collected on NF were used to generate F1 individuals as unmated males and virgin females. F1 males were mated to same age group females which were independently collected and maintained on NF. These females had no history of drug exposure in prior generations. Males and females were housed in groups in vials containing NF. Eggs collected on NF were used to generate F2 individuals as unmated males and virgin females. F0 testis, F1 males’ head, F1 females’ head, F1 testis, F2 males’ head and F2 females’ head were used for RNA isolation. Total RNA was isolated from pools of heads or testis, using TRI REAGENT (Sigma) according to the manufacturer’s protocol. Double stranded cDNA was synthesized from extracted 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 (no PTZ exposure of F0 males) 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 control as Cy3- and experimental as Cy5- labeled sample, and the rest two as the opposite, control as Cy5- and experimental as Cy3- labeled sample. Two sets of microarrays were generated for F1 males. The labeled product was mixed with hybridization solution containing hybridization buffer (DIG Easy Hyb; Roche), 10 mg/ml salmon testis DNA (0.05 mg/ml final concentration, Sigma) and 10 mg/ml yeast tRNA (0.05 mg/ml final concentration, Sigma). The hybridization mixture was denatured at 65ºC and applied onto cDNA microarray slides (D14Kv1, CDMC, Toronto, Canada). 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 10 µm 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, Excel Add-In, Stanford) under the conditions of one class response, imputation and 100 permutations. Analysis was also carried out using fold-change criterion in Acuity 4.0 itself.

first: Priyanka


title: Early pig response to experimental infection with Actinobacillus pleuropneomuniae

status: Public on May 04 2007

city: Tjele

first: Jakob

last: Hedegaard

address: PO-box 50

department: Department of Genetics and Biotechnology

country: Denmark

institute: Aarhus University, Faculty of Agricultural Sciences

summary:
The objective of this study was to identify porcine genes which expression was affected by experimental infection with A. pleuropneumoniae within 24 hours after experimental challenge. Microarray experiments were conducted to reveal genes being significantly differentially expressed in infected versus non-infected lung tissue and in liver and lung lymph node tissue from three challenged versus two non-challenged animals.
Keywords: Host response

overall_design:
Three comparisons were done:
- infected versus non-infected lung tissue sampled from three challenged pigs
- liver tissue from three challenged versus two non-challenged animals
- lung lymph node tissue from three challenged versus two non-challenged animals

All experiments were conducted as common reference design.

first: Kerstin

last: Skovgaard

first: Shila

last: Mortensen

first: Peter

last: Sørensen

first: Tim

middle: K

last: Jensen

first: Henrik

last: Hornshøj

first: Christian

last: Bendixen

last: Heegaard


title: Chronic potassium chloride (KCl) treatment induced gene expression changes in Drosophila head

status: Public on Mar 18 2008

summary:
We have recently developed a Drosophila behavioral- and transcriptomic- based (systems) model of chronic pentylenetetrazole induced locomotor plasticity. Pharmacological validation at behavioral level and at the level of fly head microarray gene expression shows that the model is of potential value in screening of compounds for detecting antiepileptic, antiepileptogenic, disease-modifying and neuroprotective activities. While screening compounds using this model at behavioral level, potassium chloride (KCl) was identified as having therapeutic potential. Examination of transcriptomic changes induced by KCl in fly head further supports its potential use as a therapeutic agent in neurological and neuropsychiatric conditions.
Keywords: Drug Response

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 + 1 C, 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 100 mM KCl for three days. Each treatment vial contained 30 flies. Heads were harvested at the end of seven day treatment. For this, 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 KCl 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). 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 KCl 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 (D14Kv1, CDMC, Toronto, Canada). 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, imputation and 100 permutations.

first: Mohammad