Project description:Using a range of copper we have utilized Metal-induced Protein Precipitaiton (MiPP) technique to elucidate the copper midpoints of the proteins found in an E. coli proteome. Using bottem-up proteomics techniques and TMT-10 plex isobaric mass-tags we were able to use MiPP to classify proteins based off of their sensitivity to copper.
Project description:Metal oxide nanoparticles can exert adverse effects on humans and aquatic organisms. However, the toxic effects and mechanisms of MO-NPs are not clearly understood.We investigated the toxic effects and mechanisms of copper oxide, zinc oxide, and nickel oxide nanoparticles in Danio rerio using microarray analysis.
Project description:Multidrug-resistant (MDR; resistance to >3 antimicrobial classes) Salmonella enterica serovar I 4,[5],12:i:- strains were linked to a 2015 foodborne outbreak from pork. Strain USDA15WA-1, associated with the outbreak, harbors an MDR module and the metal tolerance element Salmonella Genomic Island 4 (SGI-4). Characterization of SGI-4 revealed that conjugational transfer of SGI-4 resulted in the mobile genetic element (MGE) replicating as a plasmid or integrating into the chromosome. Tolerance to copper, arsenic, and antimony compounds was increased in Salmonella strains containing SGI-4 compared to strains lacking the MGE. Following Salmonella exposure to copper, RNA-seq transcriptional analysis demonstrated significant differential expression of diverse genes and pathways, including induction of numerous metal tolerance genes (copper, arsenic, silver, and mercury). Evaluation of swine administered elevated concentrations of zinc oxide (2,000 mg/kg) and copper sulfate (200 mg/kg) as an antimicrobial feed additive (Zn+Cu) in their diet for 4 weeks prior to and 3 weeks post-inoculation with serovar I 4,[5],12:i:- indicated that Salmonella shedding levels declined at a slower rate in pigs receiving in-feed Zn+Cu compared to control pigs (no Zn+Cu). The presence of metal tolerance genes in MDR Salmonella serovar I 4,[5],12:i:- may provide benefits for environmental survival or swine colonization in metal-containing settings.
Project description:<p>Increasing studies associating glycerophospholipids with various pathological conditions highlight the need for their thorough characterization. However, the intricate composition of the lipidome due to the presence of lipid isomers poses significant challenges to structural lipidomics. This study uses the anodic corrosion of two metals in a single theta nESI emitter as a tool to simultaneously characterize lipids at multiple isomer levels. Anodic corrosion of cobalt and copper in the positive ion mode generates the metal-adducted lipid complexes, [M+Co]2+ and [M+Cu]+, respectively. Optimization of parameters such as the distances of the electrodes from the nESI tip allowed the achievement of the formation of one metal-adducted lipid product at a time. Collision-induced dissociation (CID) of [M+Co]2+ results in preferential loss of the fatty acyl (FA) chain at the sn-2 position, thus generating singly charged sn-specific fragment ions. Whereas, multistage fragmentation of [M+Cu]+ via CID generated a C=C bond position-specific characteristic ion pattern induced by the π-Cu+ interaction. The feasibility of the method was tested on PC lipid extract from egg yolk to identify lipids on multiple isomer levels. Thus, the application of dual metal anodic corrosion allows lipid isomer identification with reduced sample preparation time, no signal suppression by counter anions, low sample consumption and no need for an extra apparatus.</p>
Project description:DHPM-thiones rescue Ab-mediated toxicity in a metal-dependent manner that strongly synergizes with clioquinol, a known metal-binding and cytoprotective compound. RNA-seq experiments reveal a modest, yet specific effect on metal-responsive genes that do not change with the inactive control compound.
Project description:Manipulation of zinc at the host-pathogen interface depends on both the identity of the pathogen and the nature of the host cell. In this study, we examine the response of bone marrow derived macrophages from 129S6/SvEvTac mice to infection by Salmonella Typhimurium. Unlike Balb/c and C57BL/6 mice, 129S6/SvEvTac mice possess a functional Slc11a1 (Nramp-1), a phagosomal transporter of divalent cations. We examine the changes in gene expression upon treatment with live or heat killed Salmonella at 2 Hrs and 18 Hrs post infection, and observed widespread changes in metal transport, metal-dependent, and metal homeostasis genes, suggesting significant remodeling of iron, copper, and zinc availability by host cells.
Project description:MTF1 is a highly conserved metal-binding transcription factor in eukaryotes. MTF1 binds to DNA sequence motifs, termed metal response elements (MREs) to induce the expression of genes involved in metal and oxidative stress homeostasis. MTF1 is responsive to both metal excess and deprivation, and can also protect cells from oxidative and hypoxic stresses. Disruption of metal homeostasis leads to the development of several pathological states. Despite its roles in these processes , MTF1 has been shown to be required for developmental processes such as embryonic liver formation. In this study, we used multiple strategies to understand the mechanism by which MTF1 functions in skeletal muscle differentiation and to determine the role cellular copper (Cu) status plays in this process. We provide the functional relationships between MTF1, Cu, myogenic gene promoters, and MyoD, an specific component of the myogenic transcriptional machinery in differentiating primary myoblasts derived from mouse satellite cells. We found that MTF1 is induced and translocated to the nucleus upon initiation of myogenesis. Consistent with previous studies from our laboratory , addition of non-toxic concentrations of Cu promote myogenesis and enhanced MTF1 expression. CRISPR/Cas9-mediated depletion of MTF1 demonstrated that MTF1 is essential for proper development of skeletal muscle, as partial Mtf1 knockdown leads to apoptosis to differentiating myoblasts. MTF1 was also found to bind Cu at a carboxy-terminal tetra-cysteine cluster, which may contribute to the mobilization of Cu to the nucleus during myogenesis. ChIP-seq and ChIP-qPCR analyses showed that MTF1 binds at the promoter regions of myogenic genes as part of a complex with MyoD, the master transcriptional regulator of the myogenic lineage. These results have the potential to initiate a new area of research in MTF1 function and in the regulation of myogenesis. Furthermore, these studies set the basis to understand the role of Cu at the transcriptional level, affects growth and development and will contribute to the largely unexplored are of muscular phenotypes observed in human pathologies associated to Cu misbalance, such as Menkes’ and Wilson’s diseases.
Project description:Background: Toxicogenomics provides new opportunities for innovative and proactive approaches to chemical screening, risk assessment, and predictive toxicology. If applied to ecotoxicology, genomics tools could greatly enhance the ability to detect toxicants and understand the modes of toxicity in an environmental setting. However, few studies have yet to illustrate the potential of genomic techniques in ecotoxicology. Objective: Therefore, our objective was to demonstrate the potential utility of gene expression profiling in ecotoxicology using Daphnia magna, a standard aquatic ecotoxicity test organism. Methods: D. magna were exposed to copper, cadmium, and zinc at the 1/10 LC50 for 24 hours. Following each exposure, RNA was isolated, reverse transcribed, and the cDNA was hybridized to a 5000 clone cDNA microarray for D. magna. Differentially expressed cDNAs were sequenced and homology searches revealed each gene product's potential function. Real time PCR was used to verify the differential expression of several genes, and enzyme assays were used to assess the significance of these changes. Results: We identified distinct expression profiles in response to acute copper, cadmium, and zinc exposures and discovered specific biomarkers of exposure including two probable metallothioneins, and a ferritin mRNA with a functional IRE. The gene expression patterns support known mechanisms of metal toxicity and reveal novel modes of action including zinc inhibition of chitinase activity. Conclusions: Using a cDNA microarray for traditional ecotoxicology organism, D. magna, we have identified novel biomarkers of exposure and revealed possible modes of toxicity, providing experimental support for the utility of ecotoxicogenomics. Keywords: comparative toxicant exposure