Michael D. Kane

Effects of estrogens and antiestrogens on gene expression of fathead minnow (Pimephales promelas) early life stages.

Endocrine disrupting chemicals (EDCs) are known to contaminate aquatic environments and alter the growth and reproduction of organisms. The objective of this study was to evaluate the sensitivity and utility of fathead minnow (Pimephales promelas) early life‐stages as a model to measure effects of estrogenic and antiestrogenic EDCs on physiological and gene expression endpoints relative to growth and reproduction. Embryos (<24‐h postfertilization, hpf) were exposed to a potent estrogen (17α‐ethinyl estradiol, EE2, 2, 10, and 50 ng L−1); a weak estrogen (mycotoxin zearalenone, ZEAR, same concentrations as above); an antiestrogen (ZM 189, 154; 40, 250, and 1000 ng L−1); and to mixtures of EE2 and ZM until swim‐up stage (∼170 hpf). Exposure to all concentrations of ZEAR and to the lowest concentration of ZM resulted in increased body sizes, whereas high concentrations of EE2 decreased body sizes. There was a significant increase in the frequency of abnormalities (mostly edema) in larvae exposed to all concentrations of EE2, and high ZEAR, and EE2 + ZM mixture groups. Expression of growth hormone was upregulated by most of the conditions tested. Exposure to 50 ng L−1 ZEAR caused an induction of insulin‐like growth factor 1, whereas exposure to 40 ng L−1 ZM caused a downregulation of this gene. Expression of steroidogenic acute regulatory protein gene was significantly upregulated after exposure to all concentrations of EE2 and luteinizing hormone expression increased significantly in response to all treatments tested. As expected, EE2 induced vitellogenin expression; however, ZEAR also induced expression of this gene to similar levels compared to EE2. Overall, exposure to EE2 + ZM mixture resulted in a different expression pattern compared to single exposures. The results of this study suggest that an early life stage 7‐day exposure is sufficient to recognize and evaluate effects of estrogenic compounds on gene expression in this fish model.

Activation of Protein Kinase C by Trimethyltin: Relevance to Neurotoxicity

Abstract: The differentiated PC12 cell neuronal model was used to determine the effect of trimethyltin (TMT) on protein kinase C (PKC). Cells treated with 5–20 µM TMT showed a partial and sustained PKC translocation within 30 min and persisted over a 24‐h period. TMT treatment was accompanied by a low level of PKC down‐regulation over 24 h, which was small compared with that produced by phorbol esters. Confocal imaging of differentiated PC12 cells showed that PKC translocates to the plasma membrane and the translocation is blocked by the PKC inhibitor chelerythrine (1 µM). Phorbol myristate‐induced PKC down‐regulation or inhibition with chelerythrine provided protection against TMT‐induced cytotoxicity. It was concluded that TMT‐induced PKC translocation and activation contribute to the cytotoxicity of TMT in differentiated PC12 cells.

A Survey of Scholarly Literature Describing the Field of Bioinformatics Education and Bioinformatics Educational Research

This article provides an overview of the state of research in bioinformatics education in the years 1998 through 2013. It identifies current curricular approaches for integrating bioinformatics education, concepts and skills being taught, pedagogical approaches and methods of delivery, and educational research and evaluation results.

Characterization of ontogenetic changes in gene expression in the fathead minnow (pimephales promelas)

Recently, researchers have begun looking at changes in gene expression in the fathead minnow (Pimephales promelas) after contaminant exposure as a way to develop biomarkers of exposure and effects. However, the bulk of this research has been conducted on adults, with few studies focusing on early life stages. Expression of selected genes important in growth, development, and reproduction in teleosts was quantified by quantitative polymerase chain reaction during different developmental time periods (from 0 to 28 d postfertilization [dpf]). Over the developmental period studied, there was a significant up-regulation of growth hormone mRNA and no significant changes in the expression of insulin-like growth factor 1. Thyroid hormone receptors A and B were detected in 4 dpf embryos and their expression stayed relatively constant. The variation in cytochrome P45019A mRNA expression was large during the first week of development, returning to 0 dpf expression levels thereafter. Estrogen receptor 2B was up-regulated during the first three weeks postfertilization, returning to prehatch values by 28 dpf. Expression of hydroxysteroid dehydrogenase 3B and steroidogenic acute regulatory protein increased after the third or fourth week postfertilization, respectively. Vitellogenin exhibited a large degree of variation within time points, especially after day 15, and a significant up-regulation for this gene was observed at 7 and 10 dpf. Knowledge of the normal changes in gene expression during embryo and larval development will allow for better experimental design and selection of suitable biomarkers when testing the potential toxicological effects of contaminants in this model fish species.

Antagonism of cyanide toxicity by isosorbide dinitrate: possible role of nitric oxide

In a search for improved cyanide antidotes, the efficacy of isosorbide dinitrate (ISDN), was compared with that of the known cyanide antidote, NaNO2. ISDN was as effective as an optimal dose of NaNO2 in protecting mice against cyanide lethality. To study the mechanism involved, the extent of formation of the cyanide scavenger, methemoglobin, in the action of ISDN was determined. ISDN (300 mg/kg, p.o.) increased methemoglobin from 5 to 10% of total hemoglobin, while, in contrast, NaNO2 (100 mg/kg, i.p.) increased methemoglobin levels to 50% of total hemoglobin. Lowering the dose of NaNO2 to 30 mg/kg reduced methemoglobin levels to approximately 10% of total hemoglobin and in turn nearly abolished its antidotal effect. Decreasing methemoglobin to less than control levels using methylene blue failed to abolish cyanide antagonism by ISDN. Thus, methemoglobin formation by ISDN does not account for its antidotal action. Further studies comparing the respiratory depressant effects of cyanide in the presence of ISDN or NaNO2 also indicated that these two antidotes have different mechanisms of action. Efforts to produce tolerance to the antidotal effect of ISDN against cyanide toxicity were unsuccessful. It is suggested that the well-known ability of ISDN to generate nitric oxide may account for the noted cyanide antagonism.

An information technology emphasis in biomedical informatics education

Unprecedented growth in the interdisciplinary domain of biomedical informatics reflects the recent advancements in genomic sequence availability, high-content biotechnology screening systems, as well as the expectations of computational biology to command a leading role in drug discovery and disease characterization. These forces have moved much of life sciences research almost completely into the computational domain. Importantly, educational training in biomedical informatics has been limited to students enrolled in the life sciences curricula, yet much of the skills needed to succeed in biomedical informatics involve or augment training in information technology curricula. This manuscript describes the methods and rationale for training students enrolled in information technology curricula in the field of biomedical informatics, which augments the existing information technology curriculum and provides training on specific subjects in Biomedical Informatics not emphasized in bioinformatics courses offered in life science programs, and does not require prerequisite courses in the life sciences.

Pharmacogenomics Training Using an Instructional Software System

Objectives. To implement an elective course in pharmacogenomics designed to teach pharmacy students about the fundamentals of pharmacogenomics and the anticipated changes it will bring to the profession. Design. The 8 sessions of the course covered the basics of pharmacogenomics, genomic biotechnology, implementation of pharmacogenetics in pharmacy, information security and privacy, ethical issues related to the use of genomic data, pharmacoepidemiology, and use and promotion of GeneScription, a software program designed to mimic the professional pharmacy environment. Assessment. Student grades were based on completion of a patient education pamphlet, a 2-page paper on pharmacogenomics, and precourse and postcourse survey instruments. In the postcourse survey, all students strongly agreed that genomic data could be used to determine the optimal dose of a drug and genomic data for metabolizing enzymes could be stored in a safe place. Students also were more willing to submit deoxyribonucleic acid (DNA) data for genetic profiling and better understood how DNA analysis is performed after completing the course. Conclusions. An elective course in pharmacogenomics equipped pharmacy students with the basic knowledge necessary to make clinical decisions based on pharmacogenomic data and to teach other healthcare professionals and patients about pharmacogenomics. For personalized medicine to become a reality, all pharmacists and pharmacy students must learn this knowledge and these skills.

Development and evaluation of a PCR and mass spectroscopy (PCR–MS)-based method for quantitative, type-specific detection of human papillomavirus

Knowledge of the central role of high-risk human papillomavirus (HPV) in cervical carcinogenesis, coupled with an emerging need to monitor the efficacy of newly introduced HPV vaccines, warrant development and evaluation of type-specific, quantitative HPV detection methods. In the present study, a prototype PCR and mass spectroscopy (PCR-MS)-based method to detect and quantitate 13 high-risk HPV types is compared to the Hybrid Capture 2 High-Risk HPV DNA test (HC2; Digene Corp., Gaithersburg, MD) in 199 cervical scraping samples and to DNA sequencing in 77 cervical tumor samples. High-risk HPV types were detected in 76/77 (98.7%) cervical tumor samples by PCR-MS. Degenerate and type-specific sequencing confirmed the types detected by PCR-MS. In 199 cervical scraping samples, all 13 HPV types were detected by PCR-MS. Eighteen (14.5%) of 124 cervical scraping samples that were positive for high-risk HPV by HC2 were negative by PCR-MS. In all these cases, degenerate DNA sequencing failed to detect any of the 13 high-risk HPV types. Nearly half (46.7%) of the 75 cervical scraping samples that were negative for high-risk HPV by the HC2 assay were positive by PCR-MS. Type-specific sequencing in a subset of these samples confirmed the HPV type detected by PCR-MS. Quantitative PCR-MS results demonstrated that 11/75 (14.7%) samples contained as much HPV copies/cell as HC2-positive samples. These findings suggest that this prototype PCR-MS assay performs at least as well as HC2 for HPV detection, while offering the additional, unique advantages of type-specific identification and quantitation. Further validation work is underway to define clinically meaningful HPV detection thresholds and to evaluate the potential clinical application of future generations of the PCR-MS assay.

Interactive analysis of systems biology molecular expression data

BackgroundSystems biology aims to understand biological systems on a comprehensive scale, such that the components that make up the whole are connected to one another and work through dependent interactions. Molecular correlations and comparative studies of molecular expression are crucial to establishing interdependent connections in systems biology. The existing software packages provide limited data mining capability. The user must first generate visualization data with a preferred data mining algorithm and then upload the resulting data into the visualization package for graphic visualization of molecular relations.ResultsPresented is a novel interactive visual data mining application, SysNet that provides an interactive environment for the analysis of high data volume molecular expression information of most any type from biological systems. It integrates interactive graphic visualization and statistical data mining into a single package. SysNet interactively presents intermolecular correlation information with circular and heatmap layouts. It is also applicable to comparative analysis of molecular expression data, such as time course data.ConclusionThe SysNet program has been utilized to analyze elemental profile changes in response to an increasing concentration of iron (Fe) in growth media (an ionomics dataset). This study case demonstrates that the SysNet software is an effective platform for interactive analysis of molecular expression information in systems biology.

Trimethyltin Stimulates Protein Kinase C Translocation Through Receptor‐Mediated Phospholipase C Activation in PC12 Cells

Abstract: Trimethyltin (TMT) is a potent neurotoxic compound that initiates a delayed neuronal cell death. Previously we have shown that TMT‐induced cytotoxicity is associated with protein kinase C (PKC) translocation and activation. The present study investigates the mechanism underlying TMT‐stimulated PKC translocation in PC12 cells. TMT exposure led to a rapid increase in intracellular levels of inositol 1,4,5‐trisphosphate (IP3), a product of phospholipase C (PLC). This was significantly decreased by pretreating cells with antagonists to either the cholinergic muscarinic receptor (atropine) or the glutamatergic metabotropic receptor [(+)‐α‐methyl‐4‐carboxyphenylglycine; (+)‐MCPG]. Furthermore, the rise in IP3 level was blocked by pretreating cells with a PLC inhibitor (U‐73122) or by a combination of atropine and (+)‐MCPG. This pretreatment also significantly decreased TMT‐stimulated PKC translocation, indicating that TMT‐mediated PKC translocation was related to PLC activation, presumably through formation of diacylglycerol, an endogenous activator of PKC and product of PLC. It is interesting that atropine and (+)‐MCPG did not provide protection against TMT‐induced cytotoxicity in these cells. However, these data suggest that TMT causes the release of cellular constituents that activate G protein‐coupled receptors, ultimately leading to PKC translocation.