McArthur Lab Citation Tracker

Friday, July 17, 2009

The genome of the blood fluke Schistosoma mansoni.

Nature. 2009 Jul 16;460(7253):352-8

Authors: Berriman M, Haas BJ, Loverde PT, Wilson RA, Dillon GP, Cerqueira GC, Mashiyama ST, Al-Lazikani B, Andrade LF, Ashton PD, Aslett MA, Bartholomeu DC, Blandin G, Caffrey CR, Coghlan A, Coulson R, Day TA, Delcher A, Demarco R, Djikeng A, Eyre T, Gamble JA, Ghedin E, Gu Y, Hertz-Fowler C, Hirai H, Hirai Y, Houston R, Ivens A, Johnston DA, Lacerda D, Macedo CD, McVeigh P, Ning Z, Oliveira G, Overington JP, Parkhill J, Pertea M, Pierce RJ, Protasio AV, Quail MA, Rajandream MA, Rogers J, Sajid M, Salzberg SL, Stanke M, Tivey AR, White O, Williams DL, Wortman J, Wu W, Zamanian M, Zerlotini A, Fraser-Liggett CM, Barrell BG, El-Sayed NM

Schistosoma mansoni is responsible for the neglected tropical disease schistosomiasis that affects 210 million people in 76 countries. Here we present analysis of the 363 megabase nuclear genome of the blood fluke. It encodes at least 11,809 genes, with an unusual intron size distribution, and new families of micro-exon genes that undergo frequent alternative splicing. As the first sequenced flatworm, and a representative of the Lophotrochozoa, it offers insights into early events in the evolution of the animals, including the development of a body pattern with bilateral symmetry, and the development of tissues into organs. Our analysis has been informed by the need to find new drug targets. The deficits in lipid metabolism that make schistosomes dependent on the host are revealed, and the identification of membrane receptors, ion channels and more than 300 proteases provide new insights into the biology of the life cycle and new targets. Bioinformatics approaches have identified metabolic chokepoints, and a chemogenomic screen has pinpointed schistosome proteins for which existing drugs may be active. The information generated provides an invaluable resource for the research community to develop much needed new control tools for the treatment and eradication of this important and neglected disease.

Wednesday, April 29, 2009

Can subtle changes in gene expression be consistently detected with different microarray platforms?

BMC Genomics. 2008;9:124

Authors: Pedotti P, 't Hoen PA, Vreugdenhil E, Schenk GJ, Vossen RH, Ariyurek Y, de Hollander M, Kuiper R, van Ommen GJ, den Dunnen JT, Boer JM, de Menezes RX

BACKGROUND: The comparability of gene expression data generated with different microarray platforms is still a matter of concern. Here we address the performance and the overlap in the detection of differentially expressed genes for five different microarray platforms in a challenging biological context where differences in gene expression are few and subtle. RESULTS: Gene expression profiles in the hippocampus of five wild-type and five transgenic deltaC-doublecortin-like kinase mice were evaluated with five microarray platforms: Applied Biosystems, Affymetrix, Agilent, Illumina, LGTC home-spotted arrays. Using a fixed false discovery rate of 10% we detected surprising differences between the number of differentially expressed genes per platform. Four genes were selected by ABI, 130 by Affymetrix, 3,051 by Agilent, 54 by Illumina, and 13 by LGTC. Two genes were found significantly differentially expressed by all platforms and the four genes identified by the ABI platform were found by at least three other platforms. Quantitative RT-PCR analysis confirmed 20 out of 28 of the genes detected by two or more platforms and 8 out of 15 of the genes detected by Agilent only. We observed improved correlations between platforms when ranking the genes based on the significance level than with a fixed statistical cut-off. We demonstrate significant overlap in the affected gene sets identified by the different platforms, although biological processes were represented by only partially overlapping sets of genes. Aberrances in GABA-ergic signalling in the transgenic mice were consistently found by all platforms. CONCLUSION: The different microarray platforms give partially complementary views on biological processes affected. Our data indicate that when analyzing samples with only subtle differences in gene expression the use of two different platforms might be more attractive than increasing the number of replicates. Commercial two-color platforms seem to have higher power for finding differentially expressed genes between groups with small differences in expression.

Antioxidant responses and NRF2 in synergistic developmental toxicity of PAHs in zebrafish.

Toxicol Sci. 2009 Feb 20;

Authors: Timme-Laragy AR, Van Tiem LA, Linney EA, Di Giulio RT

Early piscine life-stages are sensitive to polycyclic aromatic hydrocarbon (PAH) exposure, which can cause pericardial effusion and craniofacial malformations. We previously reported that certain combinations of PAHs cause synergistic developmental toxicity, as observed with co-exposure to the aryl hydrocarbon receptor (AHR) agonist beta-naphthoflavone (BNF) and cytochrome P4501A inhibitor alpha-naphthoflavone (ANF). Herein, we hypothesized that oxidative stress is a component of this toxicity. We examined induction of antioxidant genes in zebrafish embryos (Danio rerio) exposed to BNF or ANF individually, a BNF+ANF combination, and a pro-oxidant positive control, tert-butylhydroperoxide (tBOOH). We measured total glutathione, and attempted to modulate deformities using the glutathione synthesis inhibitor buthionine sulfoxamine (BSO) and increase glutathione pools with N-acetyl cysteine (NAC). In addition, we used a morpholino to knockdown expression of the antioxidant response element transcription factor NRF2 to determine if this would alter gene expression or increase deformity severity. BNF+ANF co-exposure significantly increased expressions of superoxide dismutase1 and2, glutathione peroxidase 1, pi class glutathione-s-transferase, and glutamate cysteine-ligase to a greater extent than tBOOH, BNF, or ANF alone. BSO pretreatment decreased some glutathione levels, but did not worsen deformities, nor did NAC diminish toxicity. Knockdown of NRF2 increased mortality following tBOOH challenge, prevented significant upregulation of antioxidant genes following both tBOOH and BNF+ANF exposures, and exacerbated BNF+ANF-related deformities. Collectively, these findings demonstrate that antioxidant responses are a component of PAH synergistic developmental toxicity, and that NRF2 is protective against prooxidant and PAH challenges during development.

Gene expression analysis using agilent DNA microarrays.

Methods Mol Biol. 2009;529:133-45

Authors: Stangegaard M

Hybridization of labeled cDNA to microarrays is an intuitively simple and a vastly underestimated process. If it is not performed, optimized, and standardized with the same attention to detail as e.g., RNA amplification, information may be overlooked or even lost. Careful balancing of the amount of labeled cDNA added to each slide reduces dye-bias and slide to slide variation. Efficient mixing of the hybridization solution throughout the hybridization reaction increases signals several fold. The amount of near perfect target-probe hybrids may be reduced by efficient stringency washes of the hybridized microarray slides.

Proteomes and transcriptomes of Apicomplexa - Where's the message?

Int J Parasitol. 2008 Nov 1;

Authors: Wastling JM, Xia D, Sohal A, Chaussepied M, Pain A, Langsley G

The Apicomplexa now have some of the most comprehensive and integrated proteome datasets of all pathogenic micro-organisms. Coverage is currently at a level where these data can be used to help predict the potential biological function of proteins in these parasites, without having to defer to measurement of mRNA levels. Transcriptomic data for the Apicomplexa (microarrays, expressed sequence tag (EST) collections, serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS) tags) are also copious, enabling us to investigate the extent to which global mRNA levels correlate with proteomic data. Here, we present a proteomic and transcriptomic perspective of gene expression in key apicomplexan parasites, including Plasmodium spp., Toxoplasma gondii, Cryptosporidium parvum, Neospora caninum and Theileria spp., and discuss the alternative views of gene expression that they provide. Although proteomic evidence does not detect every protein for every gene, many examples of readily detected proteins whose corresponding genes display little or no detectable transcription, are detected across the Apicomplexa. These examples are not easily explained by the "guilt by association", or "stock and go" hypotheses of gene transcription. With the advent of ultra-high-throughput sequencing technologies there will be a quantum shift in transcriptional analysis which, combined with improving quantitative proteome datasets, will provide a core component of a systems-wide approach to studying the Apicomplexa.

Clustering-based approaches to SAGE data mining.

BioData Min. 2008;1(1):5

Authors: Wang H, Zheng H, Azuaje F

ABSTRACT: Serial analysis of gene expression (SAGE) is one of the most powerful tools for global gene expression profiling. It has led to several biological discoveries and biomedical applications, such as the prediction of new gene functions and the identification of biomarkers in human cancer research. Clustering techniques have become fundamental approaches in these applications. This paper reviews relevant clustering techniques specifically designed for this type of data. It places an emphasis on current limitations and opportunities in this area for supporting biologically-meaningful data mining and visualisation.

A combination of LongSAGE with Solexa sequencing is well suited to explore the depth and the complexity of transcriptome.

BMC Genomics. 2008 Sep 16;9(1):418

Authors: Hanriot L, Keime C, Gay N, Faure C, Dossat C, Wincker P, Scote-Blachon C, Peyron C, Gandrillon O

ABSTRACT: BACKGROUND: "Open" transcriptome analysis methods allow to study gene expression without a priori knowledge of the transcript sequences. As of now, SAGE (Serial Analysis of Gene Expression), LongSAGE and MPSS (Massively Parallel Signature Sequencing) are the mostly used methods for "open" transcriptome analysis. Both LongSAGE and MPSS rely on the isolation of 21 pb tag sequences from each transcript. In contrast to LongSAGE, the high throughput sequencing method used in MPSS enables the rapid sequencing of very large libraries containing several millions of tags, allowing deep transcriptome analysis. However, a bias in the complexity of the transcriptome representation obtained by MPSS was recently uncovered. RESULTS: In order to make a deep analysis of mouse hypothalamus transcriptome avoiding the limitation introduced by MPSS, we combined LongSAGE with the Solexa sequencing technology and obtained a library of more than 11 millions of tags. We then compared it to a LongSAGE library of mouse hypothalamus sequenced with the Sanger method. CONCLUSION: We found that Solexa sequencing technology combined with LongSAGE is perfectly suited for deep transcriptome analysis. In contrast to MPSS, it gives a complex representation of transcriptome as reliable as a LongSAGE library sequenced by the Sanger method.

Comparison of the expression profiles of promastigotes and axenic amastigotes in Leishmania donovani using serial analysis of gene expression.

Parasitol Res. 2008 Jun 22;

Authors: Li Q, Zhao Y, Ni B, Yao C, Zhou Y, Xu W, Wang Z, Qiao Z

In this study, we examined the transcriptome of Leishmania donovani promastigotes and axenic amastigotes to identify differentially regulated mRNAs utilizing the serial analysis of gene expression (SAGE). The axenic culture of amastigotes was initiated from stationary phase promastigotes. Transformation from promastigote to amastigote occurred when cultures in Medium 199 (pH 5.5), supplemented with 20% (v/v) fetal bovine serum, were transferred from 26 degrees C to 37 degrees C. A total of 20,299 and 20,132 tags were generated from promastigote and amastigote libraries, respectively. The containing unique genes identified in these two SAGE libraries were 8,615 and 7,835, respectively. Characteristics of the expressed genes' frequency distribution were remarkably similar in both libraries: the most abundant tags (frequency >/=20), whose levels were equal to or >1.3% of the identified tags, constituted >23% of the total sequenced tags. Correspondingly, 75.72% or 71.65% of the genes accounted for those tags present at low abundance (frequency = 1) contributed only 32.13% or 27.89% of the total tags. A total of 968 genes (11.2% of the total genes in promastigotes and 12.4% in amastigotes) were recorded to have statistically different transcript levels between promastigotes and axenic amastigotes. Of the 968 distinct total genes, there are 326 promastigote-enriched transcripts and 642 amastigote-enriched mRNAs.

Validation of oligoarrays for quantitative exploration of the transcriptome.

BMC Genomics. 2008 May 30;9(1):258

Authors: Nygaard V, Liu F, Holden M, Kuo WP, Trimarchi J, Ohno-Machado L, Cepko CL, Frigessi A, Glad IK, van de Wiel MA, Hovig E, Lyng H

ABSTRACT: BACKGROUND: Oligoarrays have become an accessible technique for exploring the transcriptome, but it is presently unclear how absolute transcript data from this technique compare to the data achieved with tag-based quantitative techniques, such as massively parallel signature sequencing (MPSS) and serial analysis of gene expression (SAGE). By use of the TransCount method we calculated absolute transcript concentrations from spotted oligoarray intensities, enabling direct comparisons with tag counts obtained with MPSS and SAGE. The tag counts were converted to number of transcripts per cell by assuming that the sum of all transcripts in a single cell was 5 * 105. Our aim was to investigate whether the less resource demanding and more widespread oligoarray technique could provide data that were correlated to and had the same absolute scale as those obtained with MPSS and SAGE. RESULTS: A number of 1,777 unique transcripts were detected in common for the three technologies and served as the basis for our analyses. The correlations involving the oligoarray data were not weaker than, but, similar to the correlation between the MPSS and SAGE data, both when the entire concentration range was considered and at high concentrations. The data sets were more strongly correlated at high transcript concentrations than at low concentrations. On an absolute scale, the number of transcripts per cell and gene was generally higher based on oligoarrays than on MPSS and SAGE, and ranged from 1.6 to 9,705 for the 1,777 overlapping genes. The MPSS data were on same scale as the SAGE data, ranging from 0.5 to 3,180 (MPSS) and 9 to1,268 (SAGE) transcripts per cell and gene. The sum of all transcripts per cell for these genes was 3.8 * 105 (oligoarrays), 1.1 * 105 (MPSS) and 7.6 * 104 (SAGE), whereas the corresponding sum for all detected transcripts was 1.1 * 106 (oligoarrays), 2.8 * 105 (MPSS) and 3.8 * 105 (SAGE). CONCLUSION: The oligoarrays and TransCount provide quantitative transcript concentrations that are correlated to MPSS and SAGE data, but, the absolute scale of the measurements differs across the technologies. The discrepancy questions whether the sum of all transcripts within a single cell might be higher than the number of 5 * 105 suggested in the literature and used to convert tag counts to transcripts per cell. If so, this may explain the apparent higher transcript detection efficiency of the oligoarrays, and has to be clarified before absolute transcript concentrations can be interchanged across the technologies. The ability to obtain transcript concentrations from oligoarrays opens up the possibility of efficient generation of universal transcript databases with low resource demands.

Deep cap analysis gene expression (CAGE): genome-wide identification of promoters, quantification of their expression, and network inference.

Biotechniques. 2008 Apr;44(5):627-8, 630, 632

Authors: de Hoon M, Hayashizaki Y

In cap analysis gene expression (CAGE), short ( approximately 20 nucleotide) sequence tags originating from the 5' end of full-length mRNAs are sequenced to identify transcription events on a genome-wide scale. The rapid increase in the throughput of present-day sequencers provides much deeper CAGE tag sequencing, where CAGE tags can be found multiple times for each mRNA in a given experiment. CAGE tag counts can then be used to reliably estimate the cellular concentration of the corresponding mRNA. In contrast to microarray and SAGE expression profiling, CAGE identifies the location of each transcription start site in addition to its expression level. This makes it possible for us to infer a genome-wide network of transcriptional regulation by searching the promoter region surrounding each CAGE-defined transcription start site for potential transcription factor binding sites. Hence, deep CAGE is a unique tool for the construction of a promoter-based network of transcriptional regulation. CAGE-based expression profiling also allows us to identify dynamic promoter usage in time-course experiments and the specific promoter regulated by a given transcription factor in disruption experiments. The sheer size of the short-tag datasets produced by modern sequencers spurs a need for new software development to handle the amount of data generated by next-generation sequencers. In addition, new visualization methods will be needed to represent a promoter-based transcriptional network.

Tuesday, April 28, 2009

Variation of the genetic expression pattern after exposure to estradiol-17beta and 4-nonylphenol in male zebrafish (Danio rerio).

Gen Comp Endocrinol. 2008 May 29;

Authors: Ruggeri B, Ubaldi M, Lourdusamy A, Soverchia L, Ciccocioppo R, Hardiman G, Baker ME, Palermo F, Polzonetti-Magni AM

There is much concern about the increasing presence in the environment of synthetic chemicals that are able to disrupt the endocrine system. Among these compounds, 4-nonylphenol (4-NP) is one of the most studied xenoestrogens, due to its widespread accumulation in water sediment and consequent presence in fatty acid of aquatic organisms. Here, we have used a zebrafish microarray representing 16,399 genes to study the effects of 4-NP and estradiol-17beta (E2) in adult male zebrafish in order to elucidate the mechanism of action of 4-NP compared with that of E2. The microarray results showed that both 4-NP and E2 induced a strong expression of vitellogenin (VTG), the sex related precursor of the yolk proteins in oviparous vertebrates. Both treatments induced elevated protein turnover upregulating genes involved in proteolysis and those that are constituents of the ribosome. Many genes regulated by 4-NP and E2 are involved in energy metabolism, oxidative stress defense mechanisms, xenobiotic metabolism, and lipid metabolism. A different pattern of expression in the two treatments was found for genes involved in oxidative stress, since E2 seems to induce the mechanism of detoxification, while 4-NP seems to inhibit this protective mechanism of the cell. Overall, these findings demonstrate that the microarray approach can contribute significantly to the understanding of expression patterns induced by E2 and 4-NP in male zebrafish. The results also demonstrate that 4-NP is able to act through an alternative pattern to that of estradiol-17beta, modulating the expression of the same genes in a different manner.

Effect of starvation on the transcriptomes of the brain and liver in adult female zebrafish (Danio rerio).

Physiol Genomics. 2008 Aug 26;

Authors: Drew RE, Rodnick KJ, Settles ML, Wacyk J, Churchill EJ, Powell MS, Hardy RW, Murdoch GK, Hill RA, Robison BD

We used microarray and quantitative real-time PCR (qRT-PCR) analyses in adult female zebrafish (Danio rerio) to identify metabolic pathways regulated by starvation in the liver and brain. The transcriptome of whole zebrafish brain showed little response to 21 days of starvation. Only agouti-related protein 1 (agrp1) significantly responded, with increased expression in brains of starved fish. In contrast, a 21-day period of starvation significantly down-regulated 466 and up-regulated 108 transcripts in the liver, indicating an overall decrease in metabolic activity, reduced lipid metabolism, protein biosynthesis and proteolysis, and cellular respiration, and increased gluconeogenesis. Starvation also regulated expression of many components of the Unfolded Protein Response, the first such report in a species other than yeast (Saccharomyces cerevisiae) and mice (Mus musculus). The response of the zebrafish hepatic transcriptome to starvation was strikingly similar to that of rainbow trout (Oncorhynchus mykiss), and less similar to mouse, while the response of common carp (Cyprinus carpio) differed considerably from the other three species.

Molecular targets of TBBPA in zebrafish analysed through integration of genomic and proteomic approaches.

Chemosphere. 2008 Oct 29;

Authors: De Wit M, Keil D, Remmerie N, Ven KV, Brandhof EJ, Knapen D, Witters E, Coen WD

Tetrabromobisphenol-A (TBBPA) is nowadays one of the most frequently used brominated flame retardants (BFRs) and can be considered as a high production volume chemical. Over the last decade, numerous reports of increasing concentrations of BFRs in the environment and humans have been published. However, the toxicological knowledge on TBBPA, and more specifically its molecular mode of action, is rather fragmentary. In this study two populations of adult zebrafish (Danio rerio) were exposed for 14 days to 0.75muM and 1.5muM TBBPA. Subsequently, we employed a combined transcriptomic and proteomic approach to evaluate the molecular effects of TBBPA in zebrafish liver. Oligonucleotide microarrays were used to study the effects on gene expression levels. These results were validated through real-time PCR. The proteome of the liver was analysed by means of differential in-gel electrophoresis (DiGE), an innovative application of traditional 2D-PAGE. Combination of the extracted datasets allowed reassembling of individual molecular responses into a comprehensive overview of affected molecular pathways. Interpretation of the results depicted an interference of thyroid and Vitamin A homeostasis in the exposed zebrafish, TBBPA also elicited responses indicating onset of oxidative stress and general stress responses. Additionally, numerous differentially expressed transcripts could be associated with defence mechanisms or corresponded to metabolizing enzymes. Furthermore, cellular metabolism was clearly affected, illustrated as disturbance of e.g. lipid, carbohydrate, and organic acid metabolic processes. Summarizing, these results enabled us to hypothesize several working mechanisms of TBBPA and demonstrated the potential of a combined genome and proteome approach to generate detailed mechanistic toxicological information.

Positive correlation between gene coexpression and positional clustering in the zebrafish genome.

BMC Genomics. 2009 Jan 22;10(1):42

Authors: Ng YK, Wu W, Zhang L

ABSTRACT: BACKGROUND: Co-expressing genes tend to cluster in eukaryotic genomes. This paper analyzes correlation between the proximity of eukaryotic genes and their transcriptional expression pattern in the zebrafish (Danio rerio) genome using available microarray data and gene annotation. RESULTS: The analyses show that neighbouring genes are significantly coexpressed in the zebrafish genome, and the coexpression level is influenced by the intergenic distance and transcription orientation. This fact is further supported by examining the coexpression level of genes within positional clusters in the neighbourhood model. There is a positive correlation between gene coexpression and positional clustering in the zebrafish genome. CONCLUSIONS: The study provides another piece of evidence for the hypothesis that coexpressed genes do cluster in the eukaryotic genomes.

Discovery and characterization of novel vascular and hematopoietic genes downstream of etsrp in zebrafish.

PLoS ONE. 2009;4(3):e4994

Authors: Gomez GA, Veldman MB, Zhao Y, Burgess S, Lin S

The transcription factor Etsrp is required for vasculogenesis and primitive myelopoiesis in zebrafish. When ectopically expressed, etsrp is sufficient to induce the expression of many vascular and myeloid genes in zebrafish. The mammalian homolog of etsrp, ER71/Etv2, is also essential for vascular and hematopoietic development. To identify genes downstream of etsrp, gain-of-function experiments were performed for etsrp in zebrafish embryos followed by transcription profile analysis by microarray. Subsequent in vivo expression studies resulted in the identification of fourteen genes with blood and/or vascular expression, six of these being completely novel. Regulation of these genes by etsrp was confirmed by ectopic induction in etsrp overexpressing embryos and decreased expression in etsrp deficient embryos. Additional functional analysis of two newly discovered genes, hapln1b and sh3gl3, demonstrates their importance in embryonic vascular development. The results described here identify a group of genes downstream of etsrp likely to be critical for vascular and/or myeloid development.

Revealing genes associated with vitellogenesis in the liver of the zebrafish (Danio rerio) by transcriptome profiling.

BMC Genomics. 2009 Mar 31;10(1):141

Authors: Levi L, Pekarski I, Gutman E, Fortina P, Hyslop T, Biran J, Levavi-Sivan B, Lubzens E

ABSTRACT: BACKGROUND: : In oviparous vertebrates, including fish, vitellogenesis consists of highly regulated pathways involving 17 beta-estradiol (E2). Previous studies focused on a relatively small number of hepatic expressed genes during vitellogenesis. This study aims to identify hepatic genes involved in vitellogenesis and regulated by E2, by using zebrafish microarray gene expression profiling, and to provide information on functional distinctive genes expressed in the liver of a vitellogenic female, using zebrafish as a model fish. RESULTS: : Genes associated with vitellogenesis were revealed by the following paired t-tests (SAM) comparisons: a) two-month old vitellogenic (Vit2) females were compared with non-vitellogenic (NV) females, showing 825 differentially expressed transcripts during early stages of vitellogenesis, b) four-month old vitellogenic (Vit4) females were compared with NV females, showing 1,046 differentially expressed transcripts during vitellogenesis and c) E2-treated males were compared with control males, showing 1,828 differentially expressed transcripts regulated by E2. A Venn diagram revealed 822 common transcripts in the three groups, indicating that these transcripts were involved in vitellogenesis and putatively regulated by E2. In addition, 431 transcripts were differentially expressed in Vit2 and Vit4 females but not in E2-treated males, indicating that they were putatively not up-regulated by E2. Correspondence analysis showed high similarity in expression profiles of Vit2 with Vit4 and of NV females with control males. The E2-treated males differed from the other groups. The repertoire of genes putatively regulated by E2 in vitellogenic females included genes associated with protein synthesis and reproduction. Genes associated with the immune system processes and biological adhesion, were among the genes that were putatively not regulated by E2. E2-treated males expressed a large array of transcripts that were not associated with vitellogenesis. The study revealed several genes that were not reported before as being regulated by E2. Also, the hepatic expression of several genes was reported here for the first time. CONCLUSION: Gene expression profiling of liver samples revealed 1,046 differentially expressed transcripts during vitellogenesis of which at least ~64% were regulated by E2. The results raise the question on the regulation pattern and temporal pleiotropic expression of hepatic genes in vitellogenic females.

Analysis of endocrine disruption in southern california coastal fish using an aquatic multispecies microarray.

Environ Health Perspect. 2009 Feb;117(2):223-30

Authors: Baker ME, Ruggeri B, Sprague LJ, Eckhardt-Ludka C, Lapira J, Wick I, Soverchia L, Ubaldi M, Polzonetti-Magni AM, Vidal-Dorsch D, Bay S, Gully JR, Reyes JA, Kelley KM, Schlenk D, Breen EC, Sásik R, Hardiman G

BACKGROUND: Endocrine disruptors include plasticizers, pesticides, detergents, and pharmaceuticals. Turbot and other flatfish are used to characterize the presence of chemicals in the marine environment. Unfortunately, there are relatively few genes of turbot and other flatfish in GenBank, which limits the use of molecular tools such as microarrays and quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to study disruption of endocrine responses in sentinel fish captured by regulatory agencies. OBJECTIVES: We fabricated a multigene cross-species microarray as a diagnostic tool to screen the effects of environmental chemicals in fish, for which there is minimal genomic information. The array included genes that are involved in the actions of adrenal and sex steroids, thyroid hormone, and xenobiotic responses. This microarray will provide a sensitive tool for screening for the presence of chemicals with adverse effects on endocrine responses in coastal fish species. METHODS: We used a custom multispecies microarray to study gene expression in wild hornyhead turbot (Pleuronichthys verticalis) collected from polluted and clean coastal waters and in laboratory male zebrafish (Danio rerio) after exposure to estradiol and 4-nonylphenol. We measured gene-specific expression in turbot liver by qRT-PCR and correlated it to microarray data. RESULTS: Microarray and qRT-PCR analyses of livers from turbot collected from polluted areas revealed altered gene expression profiles compared with those from nonaffected areas. CONCLUSIONS: The agreement between the array data and qRT-PCR analyses validates this multispecies microarray. The microarray measurement of gene expression in zebrafish, which are phylogenetically distant from turbot, indicates that this multispecies microarray will be useful for measuring endocrine responses in other fish.

Quantification of Giardia transcripts during in vitro excystation: Interest for the estimation of cyst viability.

Water Res. 2009 Mar 25;

Authors: Bertrand I, Maux M, Helmi K, Hoffmann L, Schwartzbrod J, Cauchie HM

The aim of the present study was to evaluate the potential of transcript quantification as an indicator of Giardia cyst viability. The variations of beta-giardin, EF1A and ADHE mRNAs were quantified during excystation by real-time RT-PCR assays and compared with the percentages of viability estimated using propidium iodide staining and in vitro excystation. The first experiments were performed with purified G. duodenalis assemblage B cysts. When 55% of excysting protozoa were observed, the increase of the selected transcripts ranged from 0.40+/-0.13 to 0.97+/-0.11log(10) after 1h of incubation in excystation medium. Purified cysts were also stored at 4 degrees C for up to 56days and analysed at several sampling times. Significant correlations were observed between the variations of the selected mRNAs and the percentages of viability estimated with staining and excystation methods. Among the three transcripts, beta-giardin appeared to be the most appropriate to study the viability of Giardia cysts concentrated from wastewater samples.

Lipidomic analysis reveals that phosphatidylglycerol and phosphatidylethanolamine are newly generated phospholipids in an early-divergent protozoan, Giardia lamblia.

Mol Biochem Parasitol. 2009 May;165(1):67-78

Authors: Yichoy M, Nakayasu ES, Shpak M, Aguilar C, Aley SB, Almeida IC, Das S

The pathogenic protozoan Giardia lamblia is known to not synthesize membrane lipids de novo. Therefore, it is possible that lipids in the small intestine, where trophozoites colonize, play key roles in regulating the growth and differentiation of this important pathogen. The focus of the current study is to conduct a complete lipidomic analysis and to test the hypothesis that Giardia has some ability to generate new phospholipids (PLs). Using mass spectrometry, now we show that phosphatidylglycerols (PGs) are major PLs followed by phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) in non-encysting and encysting trophozoites, as well in cysts. The fatty acids attached to these PLs consist mostly of palmitate, palmitoleate, oleate, and linoleate. Results also indicate that PGs and PEs, unlike PCs, are not present in bovine bile and serum, the major sources of lipids of the culture medium, and that they could therefore be produced by fatty acid and headgroup remodeling reactions, circumventing the synthesis of entirely new PLs via de novo pathways. Genomic and transcriptional analyses show the presence of giardial phosphatidylglycerolphosphate synthase (gpgps) and phosphatidylserine decarboxylase (gpsd) genes, which are expressed throughout the life cycle. Bioinformatic and phylogenetic analyses further indicated that both genes are of prokaryotic origin and that they have undergone duplication in the course of evolution. Our studies suggest that the abundance of PG in Giardia is unique among eukaryotes and that its synthesis thus could serve as a potential target for developing new therapies against this waterborne parasite.

Using morpholinos for gene knockdown in Giardia intestinalis.

Eukaryot Cell. 2009 Apr 17;

Authors: Carpenter ML, Cande WZ

We used translation-blocking morpholinos to reduce protein levels in Giardia intestinalis. Twenty-four hours after electroporation with morpholinos targeting either GFP or kinesin-2b, levels of these proteins were reduced by 60%. An epitope-tagged transgene can also be used as a reporter of morpholino efficacy for targets lacking specific antibodies.