Biomedical subjects

Jean-Marie Rouillard

Publications and source records attributed to Jean-Marie Rouillard.

9 recordsLinked to original sources

PubMed2006

An open-access long oligonucleotide microarray resource for analysis of the human and mouse transcriptomes.

Two collections of oligonucleotides have been designed for preparing pangenomic human and mouse microarrays. A total of 148,993 and 121,703 oligonucleotides were designed against human and mouse transcripts. Quality scores were created in order to select 25,342 human and 24,109 mouse oligonucleotides. They correspond to: (i) a BLAST-specificity score; (ii) the number of expressed sequence tags matching each probe; (iii) the distance to the 3' end of the target mRNA. Scores were also used to compare in silico the two microarrays with commercial microarrays. The sets described here, called RNG/MRC collections, appear at least as specific and sensitive as those from the commercial platforms. The RNG/MRC collections have now been used by an Anglo-French consortium to distribute more than 3500 microarrays to the academic community. Ad hoc identification of tissue-specific transcripts and a approximately 80% correlation with hybridizations performed on Affymetrix GeneChiptrade mark suggest that the RNG/MRC microarrays perform well. This work provides a comprehensive open resource for investigators working on human and mouse transcriptomes, as well as a generic method to generate new microarray collections in other organisms. All information related to these probes, as well as additional information about commercial microarrays have been stored in a freely-accessible database called MEDIANTE.

Animals↗

PubMed2005

Layer-by-layer assembly of nacre-like nanostructured composites with antimicrobial properties.

In a recent report, we have presented the layer-by-layer (LBL) assembly of a biomimetic nanostructured composite from Na(+)-montmorillonite clay nanosheets and poly(diallylmethylammonium chloride) (Tang, Z.; Kotov, N.; Magonov, S.; Ozturk, B. Nat. Mater. 2003, 2, 413). The structure, deformation mechanism, and mechanical properties of the material are very similar to those of natural nacre and lamellar bones. This fact prompts further investigation of these composites as potential bone implants. LBL assembly affords preparation of multifunctional composites, and here we demonstrate that not only mechanical strength, but also antibacterial activity, can be introduced in these implantable materials by alternating clay layers with starch-stabilized silver nanoparticles. The resulting composite showed excellent structural stability with no detectable levels of silver lost over a 1 month period. Evaluation of the antibacterial properties showed almost complete growth inhibition of E. coli over an 18 h period. The amount of silver eluted from the LBL composite over a 1 month period was determined to be only 0.5-3.0 microg/L. This concentration of silver did not prevent the growth of the mammalian tissue cultures. The LBL composite has shown biocompatibility with the human osteoblast cell line.

Animals↗

PubMed2004

Potential of DNA microarrays for developing parallel detection tools (PDTs) for microorganisms relevant to biodefense and related research needs.

Development of parallel detection tools using microarrays is critically reviewed in view of the need for screening multiple microorganisms in a single test. Potential research needs with respect to probe design and specificity, validation, sample concentration, selective target enrichment and amplification, and data analysis are discussed. Data illustrating selected probe design issues for detecting multiple targets in mixed microbial systems is presented. Challenges with respect to cost, time, and ease of use compared to other methods are also summarized.

Animals↗

PubMed2004

Gene2Oligo: oligonucleotide design for in vitro gene synthesis.

There is substantial interest in implementing a bioinformatics tool that allows the design of oligonucleotides to support the development of in vitro gene synthesis. Current protocols to make long synthetic DNA molecules rely on the in vitro assembly of a set of short oligonucleotides, either by ligase chain reaction (LCR) or by assembly PCR. Ideally, such oligonucleotides should represent both strands of the final DNA molecule. They should be adjacent on the same strand and overlap the complementary oligonucleotides from the second strand to ensure good hybridization during assembly. This implies that the thermodynamic properties of each oligonucleotide have to be consistent across the set. Furthermore, any given oligonucleotide has to be totally specific to its target to avoid the creation of incorrectly assembled sequences. We have developed Gene2Oligo (http://berry.engin.umich.edu/gene2oligo/), a web-based tool that divides a long input DNA sequence into a set of adjacent oligonucleotides representing both DNA strands. The length of the oligonucleotides is dynamically optimized to ensure both the specificity and the uniform melting temperatures necessary for in vitro gene synthesis. We have successfully designed and used a set of oligonucleotides to synthesize the Saccharomyces cerevisiae cytochrome b5 by using both LCR and assembly PCR.

Algorithms↗

PubMed2004

Amplification and overexpression of the IGF2 regulator PLAG1 in hepatoblastoma.

There is evidence that 8q amplification is associated with poor prognosis in hepatoblastoma. A previous comparative genomic hybridization analysis identified a critical region in chromosomal bands 8q11.2-q13. Using restriction landmark genomic scanning in combination with a virtual genome scan, we showed that this region is delineated by sequences within contig NT_008183 of chromosomal subbands 8q11.22-q11.23. A real-time PCR-based genomic copy number assay of 20 hepatoblastomas revealed gain or amplification in this critical chromosomal region in eight tumors. The expression of four genes and expressed sequence tags (ESTs) within this newly defined region was assayed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) in four tumors with and six tumors without gain or amplification. The PLAG1 oncogene was found to be highly expressed in all but one tumor compared to normal liver tissue. Furthermore, quantitative RT-PCR revealed that the expression level of the developmentally regulated transcription factor PLAG1 was 3-12 times greater in hepatoblastoma tumors and cell lines compared to age-matched normal liver and comparable to the expression in fetal liver tissue. PLAG1 has been shown be a transcriptional activator of IGF2 in other tumor types. Using luciferase reporter assays, we demonstrated that PLAG1 transactivates transcription from the embryonic IGF2 promoter P3, also in hepatoblastoma cell lines. Thus, our results provide evidence that PLAG1 overexpression may be responsible for the frequently observed up-regulation of IGF2 in hepatoblastoma and therefore may be implicated in the molecular pathogenesis of this childhood neoplasia.

Adult↗

PubMed2003

Profiling of pathway-specific changes in gene expression following growth of human cancer cell lines transplanted into mice.

BACKGROUND: Tumor cells cultured in vitro are widely used to investigate the molecular biology of cancers and to evaluate responses to drugs and other agents. The full extent to which gene expression in cancer cells is modulated by extrinsic factors and by the microenvironment in which the cancer cells reside remains to be determined. Two cancer cell lines (A549 lung adenocarcinoma and U118 glioblastoma) were transplanted subcutaneously into immunodeficient mice to form tumors. Global gene-expression profiles of the tumors were determined, based on analysis of expression of human genes, and compared with expression profiles of the cell lines grown in culture. RESULTS: A bioinformatics approach associated genes that showed changes in their expression levels with functional classes as defined by either the GO gene annotations or MeSH terms in the literature. The classes of genes expressed at higher levels in cells grown in vitro indicated increased cell division and metabolism, reflecting the more favorable environment for cell proliferation. In contrast, in vivo tumor growth resulted in upregulation of a significant number of genes involved in the extracellular matrix (ECM), cell adhesion, cytokine and metalloendopeptidase activity, and neovascularization. When placed in comparable tissue environments, the U118 cells and the A549 cells expressed different sets of ECM and cell adhesion-related genes, suggesting different mechanisms of extracellular interaction at work in the different cancers. CONCLUSIONS: Studies of this type allow us to examine the specific contribution of cancer cells to gene expression patterns within an in vivo tumor mixed with non-cancerous tissue.

Animals↗

PubMed2003

OligoArray 2.0: design of oligonucleotide probes for DNA microarrays using a thermodynamic approach.

There is a substantial interest in implementing bioinformatics technologies that allow the design of oligonucleotides to support the development of microarrays made from short synthetic DNA fragments spotted or in situ synthesized on slides. Ideally, such oligonucleotides should be totally specific to their respective targets to avoid any cross-hybridization and should not form stable secondary structures that may interfere with the labeled probes during hybridization. We have developed OligoArray 2.0, a program that designs specific oligonucleotides at the genomic scale. It uses a thermodynamic approach to predict secondary structures and to calculate the specificity of targets on chips for a unique probe in a mixture of labeled probes. Furthermore, OligoArray 2.0 can adjust the oligonucleotide length, according to user input, to fit a narrow T(m) range compatible with hybridization requirements. Combined with on chip oligonucleotide synthesis, this program makes it feasible to perform expression analysis on a genomic scale for any organism for which the genome sequence is known. This is without relying on cDNA or oligonucleotide libraries. OligoArray 2.0 was used to design 75 764 oligonucleotides representing 26 140 transcripts from Arabidopsis thaliana. Among this set, we provide at least one specific oligonucleotide for 93% of these transcripts.

Algorithms↗

PubMed2003

Amplification and overexpression of the L-MYC proto-oncogene in ovarian carcinomas.

Gene amplification is an important mechanism of oncogene activation in various human cancers, including ovarian carcinomas (OvCas). We used restriction landmark genomic scanning (RLGS) to detect amplified DNA fragments in the genomes of 47 primary OvCas. Visual analysis of the RLGS gel images revealed several OvCa samples with spots of greater intensity than corresponding spots from normal tissues, indicating possible DNA amplification in specific tumors. Two primary tumors (E1 and S12) shared four high-intensity spots. A recently developed informatics tool termed Virtual Genome Scans was used to compare the RLGS patterns in these tumors with patterns predicted from the human genome sequence. Virtual Genome Scans determined that three of the four fragments localized to chromosome 1p34-35, a region containing the proto-oncogene L-MYC. Sixty-eight primary OvCas, including 40 analyzed by RLGS, were screened by quantitative polymerase chain reaction (PCR) for possible amplification of L-MYC. Ten tumors with increased L-MYC copy number were identified, including tumor E1, which showed an approximately 24-fold increase in copy number compared to normal DNA. Southern analysis of several tumors confirmed the quantitative PCR results. Using sequence tagged site (STS) markers flanking L-MYC, increased DNA copy number in tumor E1 was found to span the region flanking L-MYC between D1S432 and D1S463 ( approximately 3.1 Mb). Other tumors showed amplification only at the L-MYC locus. Using oligonucleotide microarrays, L-MYC was found to be more frequently overexpressed in OvCas than either c-MYC or N-MYC relative to ovarian surface epithelium. Quantitative reverse transcriptase-PCR analysis confirmed elevated L-MYC expression in a substantial fraction of OvCas, including nine of nine tumors with increased L-MYC copy number. The data implicate L-MYC gene amplification and/or overexpression in human OvCa pathogenesis.

Base Sequence↗

PubMed2002

OligoArray: genome-scale oligonucleotide design for microarrays.

SUMMARY: OligoArray is a program that computes gene specific and secondary structure free oligonucleotides for genome-scale oligonucleotide microarray construction or other applications. AVAILABILITY: The program code is distributed under the GNU General Public License and is freely available for non-profit use via request from the authors.

Computer-Aided Design↗