Project description:A shotgun metagenome microarray was created and used to investigate gene transcription during vinyl chloride (VC) dechlorination by a microbial enrichment culture called KB1. The array was constructed by spotting genomic fragments amplified from short-insert libraries of KB1 metagenomic DNA. Subsequently, the microarrays were interrogated with RNA extracted from KB1 during VC dechlorination (VC+methanol), and in the absence of VC (methanol-only). The most differentially expressed spots, and spots with the highest intensities, were then chosen to be sequenced. Sequencing revealed that Dehalococcoides (Dhc) genes involved in transcription, translation and energy generation were up-regulated during VC degradation. Furthermore, the results indicated that the reductive dehalogenase homologous (RDH) gene KB1rdhA14 is the only RDH gene up-regulated upon VC degradation, and that multiple RDH genes were more highly transcribed in the absence of VC. Numerous hypothetical genes from Dehalococcoides were also more highly transcribed in methanol only treatments and indicate that many uncharacterized proteins are involved in cell maintenance in the absence of chlorinated substrates. Spots with genes from Spirochaetes, Chloroflexi, Geobacter, Methanogens and phage organisms were differentially expressed and sequencing provided information from these uncultivated organisms that can be used to design primers for more targeted studies. This array format is powerful, as it does not require a priori sequence knowledge. This study provides the first report of such arrays being used to investigate transcription in a mixed community, and shows that this array format can be used to screen metagenomic libraries for functionally important genes. 2 Biological replicate experimens conducted 1 month apart. In the first there were 2 dye-swapped duplicates (total 4) of VC+MeOH versus MeOH only. In the second experiment there was one set of dye swapped arrays. Thus 6 arrays were performed including biological replicates, dye swapped replicates and technical duplicates.

Project description:Methylmercury (MeHg) toxicity in humans manifests deficits in neurological function. Cases of prenatal exposure to mercury have established that the developing nervous system is most highly susceptible to perturbation by MeHg. At a cellular level, MeHg-induced defects result from altered neuronal proliferation, migration and pathfinding. However, the molecular targets of MeHg that give rise to these outcomes are not fully understood. In an overall effort to identify the fundamental molecular targets of MeHg in neural development, we are investigating the effects of MeHg on gene expression and protein function in the Drosophila model. Since the fundamental signaling pathways in development of multicellular animals have been extensively characterized in Drosophila, and demonstrate high degree of conservation in vertebrates, we believe these data will lead us to the most pertinent pathways affected by MeHg and begin to elucidate the mechanism of MeHg neural toxicity relevant to cases of human exposure to this prevalent environmental toxin. Our aim is to identify fundamental signaling pathways in neural development that are targets for MeHg poisoning. Our hypothesis is that MeHg, by direct interaction with cysteine thiol groups, alters the function signaling pathway proteins and subsequently alters transcription of target genes in the respective pathways. Our hypothesis is supported by our recent data demonstrating a direct action of MeHg in activating the Notch receptor pathway and upregulating target gene expression. We anticipate a microarray analysis will elucidate additional fundamental signaling pathways where transcription is affected by this toxin. Fertilized female flies are fed on food containing methylmercury (1-20 micromolar) or solvent control (DMSO) for a period of five days to allow for MeHg penetration in to eggs. Flies are then transfered to a cage for embryo collection for a discrete window of time (e.g. 1hr). Embryos are aged 16-24 hours and collected for total RNA extraction using the Trizol reagent. Total RNA is quantitated by spectrophotometry with a Nanodrop reader. While concentration dependent effects of MeHg are of interest, initial experiments will be conducted on samples exposed to a single concetration known to illicit effect in other bioassays (e.g. 10 micromolar). The goal is to see which genes are up- or down-regulated with MeHg exposure, as compared to control embryos that are not exposed. Emphasis in the analysis stage will be in identifying target genes of known signaling pathways.

Project description:The developing human brain is uniquely vulnerable to methylmercury (MeHg) intoxication. Given the limited experimental access to MeHg-intoxicated human fetal brain, neither the threshold levels for neurotoxic effects nor the mechanisms underlying neurodevelopmental MeHg toxicity are clear. The observed persistent and latent effects in human and animal exposures and the relatively nonspecific neurodevelopmental changes suggest that MeHg impacts fundamental neurodevelopmental processes. Human cortical development is characterized by the sequential emergence of different neural stem cells, precursors, intermediate progenitors and postmitotic neurons, a sequence that can be replicated in human-induced pluripotent stem cell (hiPSC)-derived differentiating neuronal cultures. As the developing human cortex has been shown to be particularly vulnerable to fetal MeHg exposure, we assessed here by means of single cell RNA sequencing (scRNAseq) MeHg’s effects on different stages of differentiation and cell types present in early cortical cultures differentiated from hiPSC cells. Cortical cultures differentiating from hiPSC were exposed to toxicologically relevant MeHg (0.1 and 1.0 µM) continuously for 6 days at two different time points (days 4-10; and/or days 14-20) of early cortical differentiation under conditions below the cytotoxic threshold. Previous analysis at days 21-23 revealed subtle changes in cortical differentiation markers and cellular energetics, but no effect on glutathione levels, a biomarker of acute MeHg toxicity. Therefore, here we assessed the possibility of persistent effects on neurodevelopment at day 38 of differentiation (18 or 28 days after cessation of MeHg exposure). At this time, we observed subtle but significant changes in the population size of different cell types and the fractions of cells present in the different phases of the cell cycle. The observed MeHg-induced changes were developmental-stage and MeHg paradigm-specific. We further assessed overall differential gene expression, quantifying the number of genes showing significantly up- or down-regulated expression and identifying the genes affected at least partially identified biological processes affected. Importantly, the MeHg-induced changes in gene expression were also developmental-stage and MeHg-exposure paradigm specific. Thus, our studies demonstrate for the first time in a human model that MeHg exposure induces persistent changes in gene expression well after cessation of exposure, these changes are cell type- and developmental stage-specific, and this outcome is highly dependent on the concentration and timing of MeHg exposure. We conclude hiPSC-derived developing neuronal cultures provide an excellent human model to study persistent neurodevelopmental toxicity of MeHg in the developing human cortex.

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