Molecular Targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) within the Zebrafish Ovary: Insights into TCDD-induced Endocrine Disruption and Reproductive Toxicity

TCDD is a reproductive toxicant and endocrine disruptor, yet the mechanisms by which it causes these reproductive alterations are not fully understood. In order to provide additional insight into the molecular mechanisms that underlie TCDD\u27s reproductive toxicity, we assessed TCDD-induced transcriptional changes in the ovary as they relate to previously described impacts on serum estradiol concentrations and altered follicular development in zebrafish. In silico computational approaches were used to correlate candidate regulatory motifs with observed changes in gene expression. Our data suggest that TCDD inhibits follicle maturation via attenuated gonadotropin responsiveness and/or depressed estradiol biosynthesis, and that interference of estrogen-regulated signal transduction may also contribute to TCDD\u27s impacts on follicular development. TCDD may also alter ovarian function by disrupting various signaling pathways such as glucose and lipid metabolism, and regulation of transcription. Furthermore, events downstream from initial TCDD molecular-targets likely contribute to ovarian toxicity following chronic exposure to TCDD. Data presented here provide further insight into the mechanisms by which TCDD disrupts follicular development and reproduction in fish, and can be used to formulate new hypotheses regarding previously documented ovarian toxicity

Immobilized probe and glass surface chemistry as variables in microarray fabrication

BACKGROUND: Global gene expression studies with microarrays can offer biological insights never before possible. However, the technology possesses many sources of technical variability that are an obstacle to obtaining high quality data sets. Since spotted microarrays offer design/content flexibility and potential cost savings over commercial systems, we have developed prehybridization quality control strategies for spotted cDNA and oligonucleotide arrays. These approaches utilize a third fluorescent dye (fluorescein) to monitor key fabrication variables, such as print/spot morphology, DNA retention, and background arising from probe redistributed during blocking. Here, our labeled cDNA array platform is used to study, 1) compression of array data using known input ratios of Arabidopsis in vitro transcripts and arrayed serial dilutions of homologous probes; 2) how curing time of in-house poly-L-lysine coated slides impacts probe retention capacity; and 3) the retention characteristics of 13 commercially available surfaces. RESULTS: When array element fluorescein intensity drops below 5,000 RFU/pixel, gene expression measurements become increasingly compressed, thereby validating this value as a prehybridization quality control threshold. We observe that the DNA retention capacity of in-house poly-L-lysine slides decreases rapidly over time (~50% reduction between 3 and 12 weeks post-coating; p < 0.0002) and that there are considerable differences in retention characteristics among commercially available poly-L-lysine and amino silane-coated slides. CONCLUSIONS: High DNA retention rates are necessary for accurate gene expression measurements. Therefore, an understanding of the characteristics and optimization of protocols to an array surface are prerequisites to fabrication of high quality arrays

Global analysis of phase locking in gene expression during cell cycle: the potential in network modeling

<p>Abstract</p> <p>Background</p> <p>In nonlinear dynamic systems, synchrony through oscillation and frequency modulation is a general control strategy to coordinate multiple modules in response to external signals. Conversely, the synchrony information can be utilized to infer interaction. Increasing evidence suggests that frequency modulation is also common in transcription regulation.</p> <p>Results</p> <p>In this study, we investigate the potential of phase locking analysis, a technique to study the synchrony patterns, in the transcription network modeling of time course gene expression data. Using the yeast cell cycle data, we show that significant phase locking exists between transcription factors and their targets, between gene pairs with prior evidence of physical or genetic interactions, and among cell cycle genes. When compared with simple correlation we found that the phase locking metric can identify gene pairs that interact with each other more efficiently. In addition, it can automatically address issues of arbitrary time lags or different dynamic time scales in different genes, without the need for alignment. Interestingly, many of the phase locked gene pairs exhibit higher order than 1:1 locking, and significant phase lags with respect to each other. Based on these findings we propose a new phase locking metric for network reconstruction using time course gene expression data. We show that it is efficient at identifying network modules of focused biological themes that are important to cell cycle regulation.</p> <p>Conclusions</p> <p>Our result demonstrates the potential of phase locking analysis in transcription network modeling. It also suggests the importance of understanding the dynamics underlying the gene expression patterns.</p

Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays

BACKGROUND: Spotted 70-mer oligonucleotide arrays offer potentially greater specificity and an alternative to expensive cDNA library maintenance and amplification. Since microarray fabrication is a considerable source of data variance, we previously directly tagged cDNA probes with a third fluorophore for prehybridization quality control. Fluorescently modifying oligonucleotide sets is cost prohibitive, therefore, a co-spotted Staphylococcus aureus-specific fluorescein-labeled "tracking" oligonucleotide is described to monitor fabrication variables of a Mycobacterium tuberculosis oligonucleotide microarray. RESULTS: Significantly (p < 0.01) improved DNA retention was achieved printing in 15% DMSO/1.5 M betaine compared to the vendor recommended buffers. Introduction of tracking oligonucleotide did not effect hybridization efficiency or introduce ratio measurement bias in hybridizations between M. tuberculosis H37Rv and M. tuberculosis mprA. Linearity between the mean log Cy3/Cy5 ratios of genes differentially expressed from arrays either possessing or lacking the tracking oligonucleotide was observed (R(2 )= 0.90, p < 0.05) and there were no significant differences in Pearson's correlation coefficients of ratio data between replicates possessing (0.72 ± 0.07), replicates lacking (0.74 ± 0.10), or replicates with and without (0.70 ± 0.04) the tracking oligonucleotide. ANOVA analysis confirmed the tracking oligonucleotide introduced no bias. Titrating target-specific oligonucleotide (40 μM to 0.78 μM) in the presence of 0.5 μM tracking oligonucleotide, revealed a fluorescein fluorescence inversely related to target-specific oligonucleotide molarity, making tracking oligonucleotide signal useful for quality control measurements and differentiating false negatives (synthesis failures and mechanical misses) from true negatives (no gene expression). CONCLUSIONS: This novel approach enables prehybridization array visualization for spotted oligonucleotide arrays and sets the stage for more sophisticated slide qualification and data filtering applications

Quantitative Measurement of Pathogen-Specific Human Memory T cell Repertoire Diversity Using a CDR3 beta-specific Microarray

BACKGROUND: Providing quantitative microarray data that is sensitive to very small differences in target sequence would be a useful tool in any number of venues where a sample can consist of a multiple related sequences present in various abundances. Examples of such applications would include measurement of pseudo species in viral infections and the measurement of species of antibodies or T cell receptors that constitute immune repertoires. Difficulties that must be overcome in such a method would be to account for cross-hybridization and for differences in hybridization efficiencies between the arrayed probes and their corresponding targets. We have used the memory T cell repertoire to an influenza-derived peptide as a test case for developing such a method. RESULTS: The arrayed probes were corresponded to a 17 nucleotide TCR-specific region that distinguished sequences differing by as little as a single nucleotide. Hybridization efficiency between highly related Cy5-labeled subject sequences was normalized by including an equimolar mixture of Cy3-labeled synthetic targets representing all 108 arrayed probes. The same synthetic targets were used to measure the degree of cross hybridization between probes. Reconstitution studies found the system sensitive to input ratios as low as 0.5% and accurate in measuring known input percentages (R2 = 0.81, R = 0.90, p \u3c 0.0001). A data handling protocol was developed to incorporate the differences in hybridization efficiency. To validate the array in T cell repertoire analysis, it was used to analyze human recall responses to influenza in three human subjects and compared to traditional cloning and sequencing. When evaluating the rank order of clonotype abundance determined by each method, the approaches were not found significantly different (Wilcoxon rank-sum test, p \u3e 0.05). CONCLUSION: This novel strategy appears to be robust and can be adapted to any situation where complex mixtures of highly similar sequences need to be quantitatively resolved

Successful unrelated marrow transplantation for patients over the age of 40 with chronic myelogenous leukemia

AbstractSome older patients (> or =40 years) with chronic myelogenous leukemia (CML) who lack human leukocyte antigen (HLA)-identical sibling donors are not offered unrelated marrow transplantation because of concerns over excessive regimen-related toxicity, in particular due to graft-vs.-host disease (GVHD). The purpose of this study was to determine the efficacy and toxicity of unrelated marrow transplantation in older CML patients using a regimen designed to minimize the severity of GVHD. Thirty-one consecutive patients over the age of 40 with CML received unrelated marrow transplants between January 1988 and June 1997. Twenty-one patients were transplanted in chronic phase while ten were transplanted in the accelerated phase of their disease. Fifteen patients received transplants from phenotypically matched donors while 16 received marrow grafts from donors who were mismatched at one HLA locus. GVHD prophylaxis consisted of ex vivo T cell depletion of the donor marrow graft plus posttransplant cyclosporine administration. Durable engraftment was achieved in 29 of 31 patients (94%). The probability of developing grades II-IV or severe grades III-IV acute GVHD was 39.2 and 7.1%, respectively. There was no difference in the incidence of grades II-IV acute GVHD between patients transplanted with marrow grafts from phenotypically matched (38.1%) vs. those transplanted from mismatched unrelated donors (40%, p = 0.99). The 2-year probability of relapse for the entire population was 29.4%. Relapse was significantly higher for patients transplanted in accelerated phase (60%) than for those in chronic phase (13.8%, p = 0.027). The 2-year probability of overall survival and disease-free survival for the entire cohort was 56 and 45%, respectively. There was no significant difference in survival or disease-free survival for patients receiving phenotypically matched vs. mismatched marrow grafts. Immunological reconstitution for this cohort was compared with a younger (<40 years) patient population that had been similarly transplanted over the same time period. Immune function as assessed by total T cell, B cell, NK cell, and T cell subset reconstitution posttransplant was quantitatively equivalent in the two groups with most parameters normalizing within 18 months of transplant. We conclude that CML patients over the age of 40 who have either phenotypically matched or one antigen-mismatched unrelated donors can successfully undergo allogeneic marrow transplantation. T cell depletion of the marrow graft may be advantageous in these older patients by reducing GVHD severity, particularly in those patients transplanted with HLA-disparate marrow grafts.Biol Blood Marrow Transplant 1998;4(1):3-12

Development of a sensitive, highly controlled assay for molecular detection of the Philadelphia chromosome in patients with chronic myelogenous leukemia

The Philadelphia chromosome (Ph1), present in [ges]95% of chronic myelogenous leukemia (CML) patients, is a well-characterized translocation that results in a unique chimeric gene product (BCR/ABL) with transforming capability. Molecular methods utilizing the polymerase chain reaction (PCR) to detect BCR/ABL mRNA transcripts has been useful for detecting minimal residual disease (MRD) after treatment, as well as for establishing the diagnosis of CML. Amplification-based assays for the BCR/ABL transcript, however, have shown variable reproducibility and sensitivity. This variability may be largely due to technical differences and insufficient controls. In this report, we describe the development of a highly controlled, reproducible, and sensitive PCR assay to detect Ph1 that is well suited to clinical and research applications. A validation study of 82 samples was performed consisting of 25 dilutions of K562 cells (Ph1+) into normal cultured B cells, 26 pre- and post-transplant peripheral blood samples from CML patients, 16 peripheral blood samples for diagnosis of CML, and 15 peripheral blood samples from healthy individuals. RNA isolated from 3 to 5 million leukocytes was reverse transcribed (RT) and amplified by nested primer PCR. The products were characterized using agarose gel electrophoresis. Approximately 1000 Ph1-positive cells admixed with 106 normal cells were detectable after one round of amplification. In 60% of assays where one Ph1-positive cell was admixed with 106 normal cells, a BCR/ABL product was detectable after nested primer PCR. Specific measures to ensure accurate results in routine testing included (a) assessing RNA integrity and adequate cDNA preparation by detection of the constitutively expressed ABL mRNA, (b) monitoring sensitivity with the addition and detection of K562 RNA mixed with RNA from unknown samples (failure to detect the "spiked" K562 RNA indicates the presence of inhibitors or ribonucleases within the unknown RNA sample), (c) detection of nucleic acid contaminants by using negative controls in every assay, and (d) duplicate analysis of all samples and controls. Internally, this assay was 100% reproducible. Our results verify that nested primer RT-PCR is a fast, sensitive alternative to cytogenetic or Southern blot analysis for monitoring MRD after treatment and for diagnosis of CML. In addition, the highly controlled detection scheme presented here can be used as a general model for the development of other amplification-based detection assays.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31883/1/0000835.pd

Comprehensive quality control utilizing the prehybridization third-dye image leads to accurate gene expression measurements by cDNA microarrays

BACKGROUND: Gene expression profiling using microarrays has become an important genetic tool. Spotted arrays prepared in academic labs have the advantage of low cost and high design and content flexibility, but are often limited by their susceptibility to quality control (QC) issues. Previously, we have reported a novel 3-color microarray technology that enabled array fabrication QC. In this report we further investigated its advantage in spot-level data QC. RESULTS: We found that inadequate amount of bound probes available for hybridization led to significant, gene-specific compression in ratio measurements, increased data variability, and printing pin dependent heterogeneities. The impact of such problems can be captured through the definition of quality scores, and efficiently controlled through quality-dependent filtering and normalization. We compared gene expression measurements derived using our data processing pipeline with the known input ratios of spiked in control clones, and with the measurements by quantitative real time RT-PCR. In each case, highly linear relationships (R(2)>0.94) were observed, with modest compression in the microarray measurements (correction factor<1.17). CONCLUSION: Our microarray analytical and technical advancements enabled a better dissection of the sources of data variability and hence a more efficient QC. With that highly accurate gene expression measurements can be achieved using the cDNA microarray technology

Cross-species global and subset gene expression profiling identifies genes involved in prostate cancer response to selenium

BACKGROUND: Gene expression technologies have the ability to generate vast amounts of data, yet there often resides only limited resources for subsequent validation studies. This necessitates the ability to perform sorting and prioritization of the output data. Previously described methodologies have used functional pathways or transcriptional regulatory grouping to sort genes for further study. In this paper we demonstrate a comparative genomics based method to leverage data from animal models to prioritize genes for validation. This approach allows one to develop a disease-based focus for the prioritization of gene data, a process that is essential for systems that lack significant functional pathway data yet have defined animal models. This method is made possible through the use of highly controlled spotted cDNA slide production and the use of comparative bioinformatics databases without the use of cross-species slide hybridizations. RESULTS: Using gene expression profiling we have demonstrated a similar whole transcriptome gene expression patterns in prostate cancer cells from human and rat prostate cancer cell lines both at baseline expression levels and after treatment with physiologic concentrations of the proposed chemopreventive agent Selenium. Using both the human PC3 and rat PAII prostate cancer cell lines have gone on to identify a subset of one hundred and fifty-four genes that demonstrate a similar level of differential expression to Selenium treatment in both species. Further analysis and data mining for two genes, the Insulin like Growth Factor Binding protein 3, and Retinoic X Receptor alpha, demonstrates an association with prostate cancer, functional pathway links, and protein-protein interactions that make these genes prime candidates for explaining the mechanism of Selenium's chemopreventive effect in prostate cancer. These genes are subsequently validated by western blots showing Selenium based induction and using tissue microarrays to demonstrate a significant association between downregulated protein expression and tumorigenesis, a process that is the reverse of what is seen in the presence of Selenium. CONCLUSIONS: Thus the outlined process demonstrates similar baseline and selenium induced gene expression profiles between rat and human prostate cancers, and provides a method for identifying testable functional pathways for the action of Selenium's chemopreventive properties in prostate cancer