Human macrophage response to L. (Viannia) panamensis: microarray evidence for an early inflammatory response.

BACKGROUND: Previous findings indicate that susceptibility to Leishmania (Viannia) panamensis infection of monocyte-derived macrophages from patients and asymptomatically infected individuals were associated with the adaptive immune response and clinical outcome. METHODOLOGY/PRINCIPAL FINDINGS: To understand the basis for this difference we examined differential gene expression of human monocyte-derived macrophages following exposure to L. (V.) panamensis. Gene activation profiles were determined using macrophages from healthy volunteers cultured with or without stationary phase promastigotes of L. (V.) panamensis. Significant changes in expression (>1.5-fold change; p<0.05; up- or down-regulated) were identified at 0.5, 4 and 24 hours. mRNA abundance profiles varied over time, with the highest level of activation occurring at earlier time points (0.5 and 4 hrs). In contrast to observations for other Leishmania species, most significantly changed mRNAs were up- rather than down-regulated, especially at early time points. Up-regulated transcripts over the first 24 hours belonged to pathways involving eicosanoid metabolism, oxidative stress, activation of PKC through G protein coupled receptors, or mechanism of gene regulation by peroxisome proliferators via PPARα. Additionally, a marked activation of Toll-receptor mediated pathways was observed. Comparison with published microarray data from macrophages infected with L. (Leishmania) chagasi indicate differences in the regulation of genes involved in signaling, motility and the immune response. CONCLUSIONS: Results show that the early (0.5 to 24 hours) human monocyte-derived macrophage response to L. (Viannia) panamensis is not quiescent, in contrast to published reports examining later response times (48-96 hours). Early macrophage responses are important for the developing cellular response at the site of infection. The kinetics and the mRNA abundance profiles induced by L. (Viannia) panamensis illustrate the dynamics of these interactions and the distinct biologic responses to different Leishmania species from the outset of infection within their primary host cell.

Novel role of NADPH oxidase in ischemic myocardium: a study with Nox2 knockout mice.

Several potential sources of reactive oxygen species (ROS) in cells exist. One source is NADPH oxidase, which is especially important for superoxide radical production. Nox2 is a primary regulatory subunit of NADPH oxidase. In the present study, we examined the role of ROS and NADPH oxidase in ischemic preconditioning (IP)-mediated cardioprotection by using Nox2(-/-) mice. Both wild-type (WT) and Nox2(-/-) mice were subjected to either 30 min of ischemia followed by 2 h of reperfusion (IR) or IP prior to 30 min ischemia and 2 h of reperfusion. Reduction in left ventricular developed pressure (60.1 versus 63 mmHg), dp/dt (max) (893 versus 1,027 mmHg/s), and aortic flow (0.9 versus 1.8 ml/min) was observed in Nox2(-/-)IPIR compared to WTIPIR along with increased infarct size (33% versus 22%) and apoptosis after 120 min of reperfusion. Differentially regulated genes were demonstrated by comparing gene expression in WTIPIR versus Nox2(-/-) IPIR hearts. Selected differentially regulated genes such as ß-catenin, SRPK3, ERDR1, ACIN1, Syntaxin-8, and STC1 were validated by real-time PCR. Taken together, this is the first report identifying important, differentially expressed genes during ischemic preconditioning in Nox2(-/-) mice by using microarray analysis.

Performance comparison of one-color and two-color platforms within the MicroArray Quality Control (MAQC) project.

Microarray-based expression profiling experiments typically use either a one-color or a two-color design to measure mRNA abundance. The validity of each approach has been amply demonstrated. Here we provide a simultaneous comparison of results from one- and two-color labeling designs, using two independent RNA samples from the Microarray Quality Control (MAQC) project, tested on each of three different microarray platforms. The data were evaluated in terms of reproducibility, specificity, sensitivity and accuracy to determine if the two approaches provide comparable results. For each of the three microarray platforms tested, the results show good agreement with high correlation coefficients and high concordance of differentially expressed gene lists within each platform. Cumulatively, these comparisons indicate that data quality is essentially equivalent between the one- and two-color approaches and strongly suggest that this variable need not be a primary factor in decisions regarding experimental microarray design.

Making and using spotted DNA microarrays in an academic core laboratory.

Since its major launch into academia in the mid-1990s, spotted DNA microarray technology has expanded and matured into an important mainstream tool for genomic-scale gene expression studies across many species with many applications. Based on the principles of enzymatic nucleic acid labeling and DNA hybridization, the basic techniques were initially developed and disseminated by Patrick Brown's laboratory at Stanford and by others using "open source" approaches to techniques and instrumentation. Accessibility of microarrays has now become an important component of institutional research support. Indeed, the challenge facing many investigators when designing genome-scale experiments is to choose an appropriate platform and method from among the many microarray options available to them, both commercial and academic. The combination of microarray instrumentation and methods used for gene expression studies vary tremendously at different institutions and yet together function equally well as a whole. Instead of presenting a definitive set of instrumentation and methods, this chapter describes one such functional solution. It describes the specific implementation of instrumentation, standard operating procedures, and approaches for microarray fabrication and gene expression studies that are used routinely at the Microarray Resource within the W. M. Keck Biotechnology Resource Laboratory at Yale. The procedures have evolved through 6 years of operation and have resulted in at least 50 publications acknowledging the use of microarray slides and/or services provided by the Resource. The protocols that are presented for array fabrication, quality control, labeling, and hybridization utilize both "home-brew" and commercially available products to achieve an optimized set of cost-effective tools. The aim is to provide a compendium of approaches and protocols to aid those starting out on the core laboratory path and to provide insight into the types of microarray services and studies that are undertaken in this particular academic core laboratory.

Crystal structure of the human LRH-1 DBD-DNA complex reveals Ftz-F1 domain positioning is required for receptor activity.

The DNA-binding and ligand-binding functions of nuclear receptors are localized to independent domains separated by a flexible hinge. The DNA-binding domain (DBD) of the human liver receptor homologue-1 (hLRH-1), which controls genes central to development and metabolic homeostasis, interacts with monomeric DNA response elements and contains an Ftz-F1 motif that is unique to the NR5A nuclear receptor subfamily. Here, we present the 2.2A resolution crystal structure of the hLRH-1 DBD in complex with duplex DNA, and elucidate the sequence-specific DNA contacts essential for the ability of LRH-1 to bind to DNA as a monomer. We show that the unique Ftz-F1 domain folds into a novel helix that packs against the DBD but does not contact DNA. Mutations expected to disrupt the positioning of the Ftz-F1 helix do not eliminate DNA binding but reduce the transcriptional activity of full-length LRH-1 significantly. Moreover, we find that altering the Ftz-F1 helix positioning eliminates the enhancement of LRH-1-mediated transcription by the coactivator GRIP1, an action that is associated primarily with the distantly located ligand-binding domain (LBD). Taken together, these results indicate that subtle structural changes in a nuclear receptor DBD can exert long-range functional effects on the LBD of a receptor, and significantly impact transcriptional regulation.

Modulation of human nuclear receptor LRH-1 activity by phospholipids and SHP.

The human nuclear receptor liver receptor homolog 1 (hLRH-1) plays an important role in the development of breast carcinomas. This orphan receptor is efficiently downregulated by the unusual co-repressor SHP and has been thought to be ligand-independent. We present the crystal structure at a resolution of 1.9 A of the ligand-binding domain of hLRH-1 in complex with the NR box 1 motif of human SHP, which we find contacts the AF-2 region of hLRH-1 using selective structural motifs. Electron density indicates phospholipid bound within the ligand-binding pocket, which we confirm using mass spectrometry of solvent-extracted samples. We further show that pocket mutations reduce phospholipid binding and receptor activity in vivo. Our results indicate that hLRH-1's control of gene expression is mediated by phospholipid binding, and establish hLRH-1 as a novel target for compounds designed to slow breast cancer development.

KARMA: a web server application for comparing and annotating heterogeneous microarray platforms.

We have developed a universal web server application (KARMA) that allows comparison and annotation of user-defined pairs of microarray platforms based on diverse types of genome annotation data (across different species) collected from multiple sources. The application is an effective tool for diverse microarray platforms, including arrays that are provided by (i) the Keck Microarray Resource at Yale, (ii) commercially available Affymetrix GeneChips and spotted arrays and (iii) custom arrays made by individual academics. The tool provides a web interface that allows users to input pairs of test files that represent diverse array platforms for either single or multiple species. The program dynamically identifies analogous DNA fragments spotted or synthesized on multiple microarray platforms based on the following types of information: (i) NCBI-Unigene identifiers, if the platforms being compared are within the same species or (ii) NCBI-Homologene data, if they are cross-species. The single-species comparison is implemented based on set operations: intersection, union and difference. Other forms of retrievable annotation data, including LocusLink, SwissProt and Gene Ontology (GO), are collected from multiple remote sites and stored in an integrated fashion using an Oracle database. The KARMA database, which is updated periodically, is available on line at the following URL: http://ymd.med.yale.edu/karma/cgi-bin/karma.pl.

Biosphere: the interoperation of web services in microarray cluster analysis.

UNLABELLED: The growing use of DNA microarrays in biomedical research has led to the proliferation of analysis tools. These software programs address different aspects of analysis (e.g. normalisation and clustering within and across individual arrays) as well as extended analysis methods (e.g. clustering, annotation and mining of multiple datasets). Therefore, microarray data analysis typically requires the interoperability of multiple software programs involving different analysis types and methods. Such interoperation is often hampered by the heterogeneity inherent in the software tools (which may function by implementing different interfaces and using different programming languages). To address this problem, we employed the simple object access protocol (SOAP)-based web service approach that provides a uniform programmatic interface to these heterogeneous software components. To demonstrate this approach in the microarray context, we created a web server application, Biosphere, which interoperates a number of web services that are geographically widely distributed. These web services include a clustering web service, which is a suite of different clustering algorithms for analysing microarray data; XEMBL, developed at the European Bioinformatics Institute (EBI) for retrieving EMBL Nucleotide Sequence Database sequence data; and three gene annotation web services: GetGO, GetHAPI and GetUMLS. GetGO allows retrieval of Gene Ontology (GO) annotation, and the other two web services retrieve annotation from the biomedical literature that is indexed based on the Medical Subject Headings (MeSH) terms. With these web services, Biosphere allows the users to do the following: (i) cluster gene expression data using seven different algorithms; (ii) visualise the clustering results that are grouped statistically in colour; and (iii) retrieve sequence, annotation and citation data for the genes of interest. AVAILABILITY: Biosphere and its web services described in Web Service Description Language (WSDL) can be accessed at http://rook.cecid.hku.hk:8280/BiosphereServer.

YMD: a microarray database for large-scale gene expression analysis.

The use of microarray technology to perform parallel analysis of the expression pattern of a large number of genes in a single experiment has created a new frontier of medical research. The vast amount of gene expression data generated from multiple microarray experiments requires a robust database system that allows efficient data storage, retrieval, secure access, data dissemination, and integrated data analyses. To address the growing needs of microarray researchers at Yale and their collaborators, we have built the Yale Microarray Database (YMD). YMD is Web-accessible with the following features: (i) a Web program that tracks DNA samples between source plates and arrays, (ii) the capability of finding common genes/clones across different array platforms, (iii) an image file server, (iv) laboratory-based user management and access privileges, (v) project management, (vi) template data entry, (vii) linking gene expression data to annotation databases for functional analysis. YMD is currently being used on a pilot basis by several laboratories for different organisms and array platforms.

A dynamic approach to mapping coordinates between microplates and microarrays.

The retrieval of useful data from spotted microarray slides requires keeping track of which microplate wells and DNA sample corresponds to each spot on each array slide. Existing approaches are closely coupled with the type of arrayer in use and are computer operating-system-specific. To support the microarray researcher community at large who use different arrayers and computer platforms, increased flexibility, generality, and portability of these approaches are required. In this paper, we describe a general algorithm that correlates the well positions of DNA samples in each microplate to the positions of the spots on each array slide. Based on this algorithm, we have implemented a flexible and platform-independent program named MicroArray Convolutor (MAC) that provides a Web solution allowing the user to: (a) import a text file that identifies the DNA samples and their well locations, (b) select a transformation method that converts data in 96-well plate format into 384-well plate format, and (c) specify the output format of the array lists dependant on the configuration of the array platform as well as the downstream analysis software chosen for the array. MAC and its source code can be accessed via the following Web address: http://ymd.med.yale.edu/kei-cgi/kc_mac_dev8.pl.