Thursday, June 26, 2014
Human pharmaceuticals have been well documented in receiving waters yet their impacts on aquatic species are not clear. We have exposed adult zebrafish for 6 weeks to waterborne acetaminophen, gemfibrozil, venlafaxine, and carbamazepine at two doses (0.5 and 10 μg L-1). Fish were then exposed to a mixture of all four pharmaceuticals or wastewater effluent (5 and 25%) to assess whether transcriptional responses are similar with mixtures.. For all exposures, reproduction was significantly reduced and histopathological changes induced in kidney with at least the high dose exposure. Livers were pooled to provide sufficient RNA for microarray analyses. Hepatic transcriptional responses were determined with a modified Agilent 44K zebrafish microarry using a single channel approach. Significantly different probes were identified with a 2-way ANOVA (sex and treatment) and rank product analyses with a 10% false discovery rate. Transcriptional responses were particularly marked with acetaminophen exposure and there was broad overlap in the significant probes found between doses and across gender for this compound. 52 probes were at least 20 fold up- or down- regulated in acetaminophen exposed fish; 3 probes were 100 fold up-regulated (apolipoprotein Eb precursor, cdc73, and a hypothetical protein). Unique probes were identified for all exposures suggesting a unique transcriptional response may occur for each pharmaceutical, the pharmaceutical mixture, and wastewater effluent. Interestingly, there was almost no overlap in the transcriptional response found with single pharmaceutical exposure and either the mixture or wastewater effluent exposure. Indeed, the large transcriptional response from acetaminophen exposure was largely absent in fish exposed to the pharmaceutical mixture and wastewater effluent. This suggests that identifying individual or clusters of genes that may be useful in effects based monitoring may be difficult for pharmaceutical compounds.
Posted by Andrew G. McArthur at 11:11 AM
Inhibition of endogenous MTF-1 signaling in zebrafish embryos identifies novel roles for MTF-1 in development
Biochim Biophys Acta. 2014 Apr 18;1843(9):1818-1833
The metal responsive element-binding transcription factor-1 (MTF-1) responds to changes in cellular zinc levels caused by zinc exposure or disruption of endogenous zinc homeostasis by heavy metals or oxygen-related stress. Here we report the functional characterization of a complete zebrafish MTF-1 in comparison with the previously identified isoform lacking the highly conserved cysteine-rich motif (Cys-X-Cys-Cys-X-Cys) found in all other vertebrate MTF-1 orthologs. In an effort to develop novel molecular tools, a constitutively nuclear dominant-negative MTF-1 (dnMTF-1) was generated as tool for inhibiting endogenous MTF-1 signaling. The in vivo efficacy of the dnMTF-1 was determined by microinjecting in vitro transcribed dnMTF-1 mRNA into zebrafish embryos (1-2 cell stage) followed by transcriptomic profiling using an Agilent 4x44K array on 28- and 36-hpf embryos. A total of 594 and 560 probes were identified as differentially expressed at 28 hpf and 36 hpf, respectively, with interesting overlaps between timepoints. The main categories of genes affected by the inhibition of MTF-1 signaling were: nuclear receptors and genes involved in stress signaling, neurogenesis, muscle development and contraction, eye development, and metal homeostasis, including novel observations in iron and heme homeostasis. Finally, we investigate both the transcriptional activator and transcriptional repressor role of MTF-1 in potential novel target genes identified by transcriptomic profiling during early zebrafish development.
Posted by Andrew G. McArthur at 11:06 AM
Friday, May 16, 2014
Zittermann, S.I., A.G. McArthur, N.V. Fittipaldi, V. Braun, L. Vrbova, D. Middleton, G. Mallo, R. Ahmed, P. Huk, M. Lombos, V.G. Allen. 2014. Whole genome sequencing of Salmonella Enteritidis for public health investigation. Presentation at the 114th General Meeting of the American Society for Microbiology, Boston, Massachusetts.
Posted by Andrew G. McArthur at 10:14 AM
Thursday, December 05, 2013
Developmental expression of the Nfe2-related factor (Nrf) transcription factor family in the zebrafish, Danio rerio
PLoS One. 2013 Oct 24;8(10):e79574
Transcription factors in the CNC-bZIP family (NFE2, NRF1, NRF2 and NRF3) regulate genes with a wide range of functions in response to both physiological and exogenous signals, including those indicating changes in cellular redox status. Given their role in helping to maintain cellular homeostasis, it is imperative to understand the expression, regulation, and function of CNC-bZIP genes during embryonic development. We explored the expression and function of six nrf genes (nfe2, nrf1a, nrf1b, nrf2a, nrf2b, and nrf3) using zebrafish embryos as a model system. Analysis by microarray and quantitative RT-PCR showed that genes in the nrf family were expressed throughout development from oocytes to larvae. The spatial expression of nrf3 suggested a role in regulating the development of the brain, brachia and pectoral fins. Knock-down by morpholino anti-sense oligonucleotides suggested that none of the genes were necessary for embryonic viability, but nfe2 was required for proper cellular organization in the pneumatic duct and subsequent swim bladder function, as well as for proper formation of the otic vesicles. nrf genes were induced by the oxidant tert-butylhydroperoxide, and some of this response was regulated through family members Nrf2a and Nrf2b. Our results provide a foundation for understanding the role of nrf genes in normal development and in regulating the response to oxidative stress in vertebrate embryos.
Posted by Andrew G. McArthur at 12:11 PM
Thursday, June 20, 2013
Antimicrobial Agents & Chemotherapy, 2013, 57(7): 3348-57.
The field of antibiotic drug discovery and the monitoring of new antibiotic resistance elements have yet to fully exploit the power of the genome revolution. Despite the fact that the first genomes sequenced of free living organisms were those of bacteria, there have been few specialized bioinformatic tools developed to mine the growing amount of genomic data associated with pathogens. In particular, there are few tools to study the genetics and genomics of antibiotic resistance and how it impacts bacterial populations, ecology, and the clinic. We have initiated development of such tools in the form of the Comprehensive Antibiotic Research Database (CARD; http://arpcard.mcmaster.ca). The CARD integrates disparate molecular and sequence data, provides a unique organizing principle in the form of the Antibiotic Resistance Ontology (ARO), and can quickly identify putative antibiotic resistance genes in new unannotated genome sequences. This unique platform provides an informatic tool that bridges antibiotic resistance concerns in health care, agriculture, and the environment.
Posted by Andrew G. McArthur at 4:11 AM
Wednesday, April 24, 2013
Exposure to natural and anthropogenic stressors often leads to oxidative stress—a disruption in the regulation of intracellular redox conditions. Animals have evolved protective responses to mitigate damage caused by oxidative stress. However, the mechanisms by which the oxidative stress response is regulated during development are poorly understood. Oxidants, electrophiles, and some phenolic anti-oxidants initiate this response by activating NF-E2-related factor 2 (NRF2) and related cap’n’collar (CNC)- basic-leucine zipper (bZIP) family proteins, which bind to the anti-oxidant response element (ARE) and activate transcription of genes such as glutathione S-transferases (GST), NAD(P)H-quinone oxidoreductase (NQO1), glutamate-cysteine ligase (GCL), and superoxide dismutase (SOD). In order to determine the genes that are induced or repressed in response to oxidative stress during development, and whether there is a “core” set of oxidant responsive genes that is induced by structurally distinct activators of NRF2, zebrafish (Danio rerio) larvae (96 hours post-fertilization) were exposed to model oxidants (tert-butylhydroquinone (tBHQ), tert-butylhydroperoxide (tBOOH), diquat (DQ) or sulforaphane (SFN)) and gene expression was measured 6 hr later by microarray and Q-RT-PCR. There was a robust response to oxidative stress by all chemicals, with a total of 1281 probes significantly altered in expression. The compounds caused overlapping but distinct patterns of altered gene expression. A core set of genes responded to all oxidants. However, other genes exhibited oxidant-specific changes in expression. Principal components analysis revealed that the changes in gene expression caused by SFN, a sulfhydryl-reactive agent, were distinct from those produced by the other oxidants. The results demonstrate that the oxidative stress response in developing animals is dependent upon the nature of the oxidative stress.
Posted by Andrew G. McArthur at 4:06 AM
Monday, March 04, 2013
Although cadmium (Cd) exerts its toxic effects through the generation of oxidative stress, many of the cellular mechanisms involved in Cd toxicity are not fully established. Utilizing zebrafish embryos as a model, we undertook a transcriptomic-based approach to further investigate the role of Cd as a developmental toxicant and to assist in the identification of novel mechanisms of cellular dysfunction. Zebrafish embryos (24, 48, 72 and 96 hpf) were continuously exposed to Cd (25, 50, 100, 150 µM) for 24 hours prior to mortality assessment. No significant mortality was observed at any of the Cd concentrations prior to 72 hpf. However, at 96 and 120 hpf the 50 µM Cd dose resulted in 20% and 40% mortality, respectively, while the 100 and 150 µM Cd doses resulted in an average of 75% and >95% mortality, respectively. To investigate global changes in gene expression, zebrafish embryos (72, 96 and 120 hpf) were exposed to 50 µM Cd for 4 or 8 hours prior to sampling. Global gene expression profiling was performed using the Agilent 4x44K Zebrafish Oligo Microarray. Overall, 788 probes were significantly upregulated, while 679 probes were downregulated in one or more treatments. Principle component analysis was employed to find trends among the various treatments. The first principle component separated out the 96 and 120 hpf Cd-treated embryos, consistent with the mortality curves suggesting that zebrafish embryos become most sensitive to Cd toxicity around 96 hpf. The majority of genes altered by Cd exposure resulted in the upregulation of a large subset of genes responsive to oxidative stress, genes involved in glutathione synthesis and heme/iron homeostasis, mitochondrial uncoupling proteins, and various solute carriers with roles in zinc transport/homeostasis or mitochondrial oxidative phosphorylation. The majority of genes downregulated by Cd were involved in cell cycle control and DNA replication. A thorough analysis of genes differentially regulated by Cd during zebrafish development will be presented.
A.R Timme-Laragy, S.I. Karchner, R.C. Harbeitner, A.G. McArthur, M.E. Hahn. 2013. Nrf2 gene regulation during oxidative stress in embryonic development. Presentation at the Society of Toxicology 52cd Annual Meeting, San Antonio, Texas.
Nrf2 is a transcription factor that regulates antioxidant defenses in response to oxidative stress. Embryonic development is highly susceptible to disruption by exposure to chemicals, including those that alter redox balance. The role of Nrf2 in the oxidative stress response (OSR) during embryonic development remains unclear. Our previous work identified a novel Nrf2 paralog, nrf2b, in zebrafish (Danio rerio). This study builds upon that work to elucidate the roles of nrf2a and nrf2b in regulating the OSR during vertebrate embryonic development. Zebrafish embryos were micro-injected with antisense morpholino oligonucleotides (MO) to knock down translation of either nrf2a or nrf2b, or a standard morpholino control (co-MO). At 48 hours post fertilization, embryos were exposed to the pro-oxidant and Nrf2 activator, tertbutylhydroquinone (tBHQ) or vehicle (DMSO) for 4 hours, and preserved for RNA isolation. Microarrays were conducted using Agilent’s V3 44k array, and selected genes validated by QPCR. In response to tBHQ, 71 probes were up-regulated in the co-MO group, including gstp1, gclc, ferritin, peroxiredoxin1, hsp70, sod1, and other genes typically found as part of the OSR. Interestingly, we found that an important and often overlooked part of the OSR is the down-regulation of genes, including cathespin, various complement components, and apolipoprotein Ea. Knockdown of Nrf2a or Nrf2b blocked some but not all of the tBHQ-induced changes in OSR gene expression and the effects of Nrf2a-MO and Nrf2b-MO were distinct. The results show that Nrf2 paralogs primarily regulate distinct gene sets, with some overlapping targets, in response to oxidative stress in embryos. This study also highlights the importance of gene down-regulation as a component of the OSR during embryonic development.
Posted by Andrew G. McArthur at 3:23 AM
Wednesday, October 24, 2012
Molecular typing of Mycobacterium tuberculosis isolates from Yaoundé reveals RIF resistance markers, clonal relatedness and mutation patterns
Asian Pacific Journal of Tropical Disease, 2012, 2(5): 342-347.
Objective: To understand the spread of drug resistance and identifying diagnostic probes among the local tuberculosis (TB) strains in order to design rational control tools for tuberculosis controls. Methods: TA cloning and sequencing were used to characterize mutation associated with RIF resistance in 69 bp region of the gene, rpoB. The analysis identified two regions of mutations but no unusual insertion and deletion. No mutation was observed in RIF sensitive strains. Results: We employed Random Amplified Polymorphic DNA (RAPD) analysis for typing strains of M. tuberculosis to determine whether new strains were present among M. tuberculosis isolates circulating in Yaounde. Three groups (I to III) of M. tuberculosis were identified among 93 isolates randomly selected. RAPD analysis provided a rapid and easy means of identifying polymorphism in M. tuberculosis isolates, and it was found to be a valuable alternative epidemiological tool. RAPD was used to select the new site of diagnostic by PCR. Also single nucleotide polymorphisms between M. tuberculosis and M. bovis were found, suggesting that RAPD can be a useful technique for distinguishing between species. Conclusions: Molecular typing is defined as the integration of conventional epidemiological approach to track specific strains of pathogens in order to understand the distribution of disease in populations.
Posted by Andrew G. McArthur at 3:57 AM
Monday, July 30, 2012
During the course, I will be providing both lectures and computer labs related to an "Introduction to Microarray Approaches in Ecotoxicogenomics".
During the course, I will be providing both lectures and computer labs related to an "Introduction to Microarray Approaches in Ecotoxicogenomics".
Posted by Andrew G. McArthur at 3:52 AM
Sunday, June 24, 2012
Posted by Andrew G. McArthur at 12:57 PM