Project description:Increasing atmospheric CO2 concentrations are causing decreased pH over vast expanses of the ocean. This decreasing pH may alter biogeochemical cycling of carbon and nitrogen via the microbial process of nitrification, a key process that couples these cycles in the ocean, but which is often sensitive to acidic conditions. Recent reports indicate a decrease in oceanic nitrification rates under experimentally lowered pH. How composition and abundance of ammonia oxidizing bacteria (AOB) and archaea (AOA) assemblages respond to decreasing oceanic pH, however, is unknown. We sampled microbes from two different acidification experiments and used a combination of qPCR and functional gene microarrays for the ammonia monooxygenase gene (amoA) to assess how acidification alters the structure of ammonia oxidizer assemblages. We show that despite widely different experimental conditions, acidification consistently altered the community composition of AOB by increasing the relative abundance of taxa related to the Nitrosomonas ureae clade. In one experiment this increase was sufficient to cause an increase in the overall abundance of AOB. There were no systematic shifts in the community structure or abundance of AOA in either experiment. These different responses to acidification underscore the important role of microbial community structure in the resiliency of marine ecosystems. amoA gene diversity from two ocean acidification experiments, Monterey Bay experiment (two time points, ambient and acidified) and Vineyard Sound experiment (ambient and acifidied, with and without nutrients) examined with 2 two-color arrays (Cy3 and Cy5): the universal standard 20-mer oligo is printed to the slide with a 70-mer oligo (an archetype). Environmental DNA sequences (fluoresced with Cy3) within 15% of the 70-mer conjugated to a 20-mer oligo (fluoresced with Cy5) complementary to the universal standard will bind to the oligo probes on the array. Signal is the ratio of Cy3 to Cy5.

Project description:Sulfur mustard (SM) is a potent vesicant that targets epithelial cells and tissues. Most vesicant research has been performed using bona fide SM; however, some studies have used simulants, most notably half mustard (2-chloroethyl ethylsulfide; CEES) and nitrogen mustard (mechlorethamine; NM). Although CEES and NM have similarities to SM and can cause vesication, there are distinct differences in the chemical structures and physical properties of these compounds that may impact their toxic effects. Microarray analysis of cultured primary human epidermal keratinocytes (HEK) exposed to each of these vesicants was performed to directly compare the transcriptional responses induced by these vesicants. HEK were exposed in triplicate to concentrations ranging from 0-1000 µM for SM and NM and 0-4000 µM for CEES. Cells were harvested at 1, 2, 4, 8, 16, and 24 h and the RNA isolated for microarray analysis. Transcriptional responses were phenotypically anchored to cell morphology. The dataset was filtered by exposure and timepoint, and an analysis of variance was performed using dose as the factor. The top 500 genes ranked by p-value were analyzed using gene ontology algorithms to identify biological pathways significantly affected by each vesicant. At 2 h post-exposure, p53 signaling, Erk/MAPK signaling, and BMP signaling were significantly affected by all three vesicants. At 4 h post-exposure, p53 signaling , B cell activating factor, and glucocorticoid receptor signaling were significantly affected by all three vesicants. At 8 h post-exposure, there were no significant pathways commonly affected by all three vesicants. These results suggest that, although there are similarities in the transcriptional responses to each of these vesicants, the transcriptional responses appear to differ over time. Thus, extrapolation of results obtained with one vesicant to other vesicants may be complex and may have important implications for the development of vesicant therapeutics.

Project description:Sulfur mustard (SM) is a potent alkylating agent. We are developing medical countermeasures to reduce the injury caused by SM exposure. Screening in the mouse ear vesicant model has identified three effective compounds: dimercaprol (British anti-lewisite), indomethacin, and octyl homovanillamide (OHV). To identify gene expression changes that correlate with compound efficacy we used oligonucleotide microarrays to compare gene expression profiles in vehicle-exposed skin, SM-exposed skin, and skin pretreated with each compound before SM exposure. Mice were topically exposed on the inner surface of the right ear to SM alone or pretreated for 15 min with one of the compounds and then exposed to SM. Left ears were vehicle-exposed. Ear tissue was harvested 24 hr later for ear weight determination (an endpoint indicating compound efficacy). The exposure groups were: methylene chloride (sulfur mustard vehicle); ethanol (drug vehicle); 0.08 mg sulfur mustard; 6.25 mg dimercaprol 15 min before 0.08 mg sulfur mustard; 1.34 mg indomethacin 15 min before 0.08 mg sulfur mustard; 0.6 mg octylhomovanillamide 15 min before 0.08 mg sulfur mustard; 6.25 mg dimercaprol alone; 1.34 mg indomethacin alone; 0.6 mg octylhomovanillamide alone. RNA was extracted from the tissues and used to generate oligonucleotide microarray probes. Principal component analysis of the gene expression data revealed partitioning of the samples based on drug treatment and SM exposure. Vehicle-exposed mouse ears clustered away from the other treatment groups. SM-exposed mouse ears pretreated with dimercaprol or OHV clustered more closely with vehicle-exposed ears, while SM-exposed mouse ears pretreated with indomethacin clustered more closely with SM-exposed ears. This clustering of the samples is supported by the ear weight data, in which the indomethacin group has ear weights closer to the SM-exposed group, whereas the dimercaprol and OHV groups have ear weights closer to the vehicle-exposed group. Correlation coefficients were calculated for each gene based on the correlation between gene expression level and ear weight. These data provide the basis for understanding what gene expression changes are important in the development of effective SM medical countermeasures. Experiment Overall Design: Exposure of mouse ears to sulfur mustard alone, sulfur mustard preceded by drug treatment, or vehicle compounds. Naive controls were also included. Biological replicates of at least n=3 were examined for each exposure condition.

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Effects of mustard invasions on soil microbial assemblages in southern California

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