Regulation of Ethanol-Related Behavior and Ethanol Metabolism by the Corazonin Neurons and Corazonin Receptor in Drosophila melanogaster

Impaired ethanol metabolism can lead to various alcohol-related health problems. Key enzymes in ethanol metabolism are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH); however, neuroendocrine pathways that regulate the activities of these enzymes are largely unexplored. Here we identified a neuroendocrine system involving Corazonin (Crz) neuropeptide and its receptor (CrzR) as important physiological regulators of ethanol metabolism in Drosophila. Crz-cell deficient (Crz-CD) flies displayed significantly delayed recovery from ethanol-induced sedation that we refer to as hangover-like phenotype. Newly generated mutant lacking Crz Receptor (CrzR01) and CrzR-knockdown flies showed even more severe hangover-like phenotype, which is causally associated with fast accumulation of acetaldehyde in the CrzR01 mutant following ethanol exposure. Higher levels of acetaldehyde are likely due to 30% reduced ALDH activity in the mutants. Moreover, increased ADH activity was found in the CrzR01 mutant, but not in the Crz-CD flies. Quantitative RT-PCR revealed transcriptional upregulation of Adh gene in the CrzR01. Transgenic inhibition of cyclic AMP-dependent protein kinase (PKA) also results in significantly increased ADH activity and AdhmRNA levels, indicating PKA-dependent transcriptional regulation of Adh by CrzR. Furthermore, inhibition of PKA or cAMP response element binding protein (CREB) in CrzR cells leads to comparable hangover-like phenotype to the CrzR01 mutant. These findings suggest that CrzR-associated signaling pathway is critical for ethanol detoxification via Crz-dependent regulation of ALDH activity and Crz-independent transcriptional regulation of ADH. Our study provides new insights into the neuroendocrine-associated ethanol-related behavior and metabolism

Strategies to Optimize Microbially-Mediated Mitigation of Greenhouse Gas Emissions from Landfill Cover Soils

The overall objective of this project, 'Strategies to Optimize Microbially-Mediated Mitigation of Greenhouse Gas Emissions from Landfill Cover Soils' was to develop effective, efficient, and economic methodologies by which microbial production of nitrous oxide can be minimized while also maximizing microbial consumption of methane in landfill cover soils. A combination of laboratory and field site experiments found that the addition of nitrogen and phenylacetylene stimulated in situ methane oxidation while minimizing nitrous oxide production. Molecular analyses also indicated that methane-oxidizing bacteria may play a significant role in not only removing methane, but in nitrous oxide production as well, although the contribution of ammonia-oxidizing archaea to nitrous oxide production can not be excluded at this time. Future efforts to control both methane and nitrous oxide emissions from landfills as well as from other environments (e.g., agricultural soils) should consider these issues. Finally, a methanotrophic biofiltration system was designed and modeled for the promotion of methanotrophic activity in local methane 'hotspots' such as landfills. Model results as well as economic analyses of these biofilters indicate that the use of methanotrophic biofilters for controlling methane emissions is technically feasible, and provided either the costs of biofilter construction and operation are reduced or the value of CO{sub 2} credits is increased, can also be economically attractive

A Xenobiotic Detoxification Pathway through Transcriptional Regulation in Filamentous Fungi

Fungi are known to utilize transcriptional regulation of genes that encode efflux transporters to detoxify xenobiotics; however, to date it is unknown how fungi transcriptionally regulate and coordinate different phases of detoxification system (phase I, modification; phase II, conjugation; and phase III, secretion). Here we present evidence of an evolutionary convergence between the fungal and mammalian lineages, whereby xenobiotic detoxification genes (phase I coding for cytochrome P450 monooxygenases [CYP450s] and phase III coding for ATP-binding cassette [ABC] efflux transporters) are transcriptionally regulated by structurally unrelated proteins. Following next-generation RNA sequencing (RNA-seq) analyses of a filamentous fungus, Sclerotinia homoeocarpa, the causal agent of dollar spot on turfgrasses, a multidrug resistant (MDR) field strain was found to overexpress phase I and III genes, coding for CYP450s and ABC transporters for xenobiotic detoxification. Furthermore, there was confirmation of a gain-of-function mutation of the fungus-specific transcription factor S. homoeocarpa XDR1 (ShXDR1), which is responsible for constitutive and induced overexpression of the phase I and III genes, resulting in resistance to multiple classes of fungicidal chemicals. This fungal pathogen detoxifies xenobiotics through coordinated transcriptional control of CYP450s, biotransforming xenobiotics with different substrate specificities and ABC transporters, excreting a broad spectrum of xenobiotics or biotransformed metabolites. A Botrytis cinerea strain harboring the mutated ShXDR1 showed increased expression of phase I (BcCYP65) and III (BcatrD) genes, resulting in resistance to fungicides. This indicates the regulatory system is conserved in filamentous fungi. This molecular genetic mechanism for xenobiotic detoxification in fungi holds potential for facilitating discovery of new antifungal drugs and further studies of convergent and divergent evolution of xenobiotic detoxification in eukaryote lineages. IMPORTANCE Emerging multidrug resistance (MDR) in pathogenic filamentous fungi is a significant threat to human health and agricultural production. Understanding mechanisms of MDR is essential to combating fungal pathogens; however, there is still limited information on MDR mechanisms conferred by xenobiotic detoxification. Here, we report for the first time that overexpression of phase I drug-metabolizing monooxygenases (cytochrome P450s) and phase III ATP-binding cassette efflux transporters is regulated by a gain-of-function mutation in the fungus-specific transcription factor in the MDR strains of the filamentous plant-pathogenic fungus Sclerotinia homoeocarpa. This study establishes a novel molecular mechanism of MDR through the xenobiotic detoxification pathway in filamentous fungi, which may facilitate the discovery of new antifungal drugs to control pathogenic fungi

Integrated Laboratory and Fieldwork Exercises for Controlling Greenhouse Gas Emissions from Landfills.

Landfills are large sources of CH4, but a considerable amount of CH4 can be removed in situ by methanotrophs if their activity can be stimulated through the addition of nitrogen. Nitrogen can, however, lead to increased N2O production. To examine the effects of nitrogen and a selective inhibitor on CH4 oxidation and N2O production in situ, 0.5 M of NH4Cl and 0.25 M of KNO3, with and without 0.01% (w/v) phenylacetylene, were applied to test plots at a landfill in Kalamazoo, MI from 2007 November to 2009 July. The addition of NH4+ and NO3- increased N2O production, but had no effect on CH4 concentrations. The simultaneous addition of phenylacetylene reduced N2O production and enhanced CH4 oxidation. PCR analyses showed that methanotrophs, especially those possessing particulate methane monooxygenase, were more abundant than those possessing soluble methane monooxygenase, and, interestingly, archaeal ammonia-oxidizers were more abundant than their bacterial counterpart. Microarray analyses showed NH4+ and NO3- caused the overall methanotrophic diversity to decrease, with a significant reduction in the presence of Type I methanotrophs. The simultaneous addition of phenylacetylene caused methanotrophic diversity to increase, with greater presence of Type I methanotrophs. Also, archaeal amoA gene clone libraries were constructed to examine the long-term effects of the amendment on the AOA community structure. Clone libraries showed that the addition of NH4+ and NO3- increased the presence of Group 1.1b archaeal ammonia-oxidizers, while their presence decreased with the simultaneous addition of phenylacetylene. Several methanotrophs were investigated to examine the relative importance of methanotrophic mediated N2O production. Five out of six Type II methanotrophic strains produced 32 - 342 ppmv of N2O, while two Type I strains did not produce detectable amount of N2O. Collectively, these results suggest that the addition of phenylacetylene with NH4+ and NO3- reduces N2O production by selectively inhibiting archaeal ammonia-oxidizers and/or Type II methanotrophs, but it is currently unknown what the magnitude of N2O production might be from archaeal ammonia-oxidizers. Once the major contributors on N2O production are identified, we may be able to come up with a better strategy to mitigate in situ GHG emissions from a landfill.Ph.D.Environmental EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/84535/1/jdsmail_1.pd

Strategies to Optimize Microbially-Mediated Mitigation of Greenhouse Gas Emissions from Landfill Cover Soils

The overall objective of this project, 'Strategies to Optimize Microbially-Mediated Mitigation of Greenhouse Gas Emissions from Landfill Cover Soils' was to develop effective, efficient, and economic methodologies by which microbial production of nitrous oxide can be minimized while also maximizing microbial consumption of methane in landfill cover soils. A combination of laboratory and field site experiments found that the addition of nitrogen and phenylacetylene stimulated in situ methane oxidation while minimizing nitrous oxide production. Molecular analyses also indicated that methane-oxidizing bacteria may play a significant role in not only removing methane, but in nitrous oxide production as well, although the contribution of ammonia-oxidizing archaea to nitrous oxide production can not be excluded at this time. Future efforts to control both methane and nitrous oxide emissions from landfills as well as from other environments (e.g., agricultural soils) should consider these issues. Finally, a methanotrophic biofiltration system was designed and modeled for the promotion of methanotrophic activity in local methane 'hotspots' such as landfills. Model results as well as economic analyses of these biofilters indicate that the use of methanotrophic biofilters for controlling methane emissions is technically feasible, and provided either the costs of biofilter construction and operation are reduced or the value of CO{sub 2} credits is increased, can also be economically attractive

A Data Mining Approach to Predict In Situ Detoxification Potential of Chlorinated Ethenes

Despite advances in physicochemical remediation technologies, in situ bioremediation treatment based on <i>Dehalococcoides mccartyi</i> (<i>Dhc</i>) reductive dechlorination activity remains a cornerstone approach to remedy sites impacted with chlorinated ethenes. Selecting the best remedial strategy is challenging due to uncertainties and complexity associated with biological and geochemical factors influencing <i>Dhc</i> activity. Guidelines based on measurable biogeochemical parameters have been proposed, but contemporary efforts fall short of meaningfully integrating the available information. Extensive groundwater monitoring data sets have been collected for decades, but have not been systematically analyzed and used for developing tools to guide decision-making. In the present study, geochemical and microbial data sets collected from 35 wells at five contaminated sites were used to demonstrate that a data mining prediction model using the classification and regression tree (CART) algorithm can provide improved predictive understanding of a site’s reductive dechlorination potential. The CART model successfully predicted the 3-month-ahead reductive dechlorination potential with 75.8% and 69.5% true positive rate (i.e., sensitivity) for the training set and the test set, respectively. The machine learning algorithm ranked parameters by relative importance for assessing in situ reductive dechlorination potential. The abundance of <i>Dhc</i> 16S rRNA genes, CH₄, Fe²⁺, NO₃^–, NO₂^–, and SO₄^2– concentrations, total organic carbon (TOC) amounts, and oxidation–reduction potential (ORP) displayed significant correlations (<i>p</i> < 0.01) with dechlorination potential, with NO₃^–, NO₂^–, and Fe²⁺ concentrations exhibiting precedence over other parameters. Contrary to prior efforts, the power of data mining approaches lies in the ability to discern synergetic effects between multiple parameters that affect reductive dechlorination activity. Overall, these findings demonstrate that data mining techniques (e.g., machine learning algorithms) effectively utilize groundwater monitoring data to derive predictive understanding of contaminant degradation, and thus have great potential for improving decision-making tools. A major need for realizing the predictive capabilities of data mining approaches is a curated, open-access, up-to-date and comprehensive collection of biogeochemical groundwater monitoring data

Natural Attenuation in Streambed Sediment Receiving Chlorinated Solvents from Underlying Fracture Networks

Contaminant discharge from fractured bedrock formations remains a remediation challenge. We applied an integrated approach to assess the natural attenuation potential of sediment that forms the transition zone between upwelling groundwater from a chlorinated solvent-contaminated fractured bedrock aquifer and the receiving surface water. In situ measurements demonstrated that reductive dechlorination in the sediment attenuated chlorinated compounds before reaching the water column. Microcosms established with creek sediment or in situ incubated Bio-Sep beads degraded C₁–C₃ chlorinated solvents to less-chlorinated or innocuous products. Quantitative PCR and 16S rRNA gene amplicon sequencing revealed the abundance and spatial distribution of known dechlorinator biomarker genes within the creek sediment and demonstrated that multiple dechlorinator populations degrading chlorinated C₁–C₃ alkanes and alkenes co-inhabit the sediment. Phylogenetic classification of bacterial and archaeal sequences indicated a relatively uniform distribution over spatial (300 m horizontally) scale, but <i>Dehalococcoides</i> and <i>Dehalobacter</i> were more abundant in deeper sediment, where 5.7 ± 0.4 × 10⁵ and 5.4 ± 0.9 × 10⁶ 16S rRNA gene copies per g of sediment, respectively, were measured. The microbiological and hydrogeological characterization demonstrated that microbial processes at the fractured bedrock–sediment interface were crucial for preventing contaminants reaching the water column, emphasizing the relevance of this critical zone environment for contaminant attenuation

Natural Attenuation in Streambed Sediment Receiving Chlorinated Solvents from Underlying Fracture Networks

Contaminant discharge from fractured bedrock formations remains a remediation challenge. We applied an integrated approach to assess the natural attenuation potential of sediment that forms the transition zone between upwelling groundwater from a chlorinated solvent-contaminated fractured bedrock aquifer and the receiving surface water. In situ measurements demonstrated that reductive dechlorination in the sediment attenuated chlorinated compounds before reaching the water column. Microcosms established with creek sediment or in situ incubated Bio-Sep beads degraded C₁–C₃ chlorinated solvents to less-chlorinated or innocuous products. Quantitative PCR and 16S rRNA gene amplicon sequencing revealed the abundance and spatial distribution of known dechlorinator biomarker genes within the creek sediment and demonstrated that multiple dechlorinator populations degrading chlorinated C₁–C₃ alkanes and alkenes co-inhabit the sediment. Phylogenetic classification of bacterial and archaeal sequences indicated a relatively uniform distribution over spatial (300 m horizontally) scale, but <i>Dehalococcoides</i> and <i>Dehalobacter</i> were more abundant in deeper sediment, where 5.7 ± 0.4 × 10⁵ and 5.4 ± 0.9 × 10⁶ 16S rRNA gene copies per g of sediment, respectively, were measured. The microbiological and hydrogeological characterization demonstrated that microbial processes at the fractured bedrock–sediment interface were crucial for preventing contaminants reaching the water column, emphasizing the relevance of this critical zone environment for contaminant attenuation

Environmental Fate of the Next Generation Refrigerant 2,3,3,3-Tetrafluoropropene (HFO-1234yf)

The hydrofluoroolefin 2,3,3,3-tetrafluoropropene (HFO-1234yf) has been introduced to replace 1,1,1,2-tetrafluoroethane (HFC-134a) as refrigerant in mobile, including vehicle, air conditioning systems because of its lower global warming potential. HFO-1234yf is volatile at ambient temperatures; however, high production volumes and widespread handling are expected to release this fluorocarbon into terrestrial and aquatic environments, including groundwater. Laboratory experiments explored HFO-1234yf degradation by (i) microbial processes under oxic and anoxic conditions, (ii) abiotic processes mediated by reactive mineral phases and zerovalent iron (Fe⁰, ZVI), and (iii) cobalamin-catalyzed biomimetic transformation. These investigations demonstrated that HFO-1234yf was recalcitrant to microbial (co)­metabolism and no transformation was observed in incubations with ZVI, makinawite (FeS), sulfate green rust (GR_SO4), magnetite (Fe₃O₄), and manganese oxide (MnO₂). Sequential reductive defluorination of HFO-1234yf to 3,3,3-trifluoropropene and 3,3-dichloropropene with concomitant stoichiometric release of fluoride occurred in incubations with reduced cobalamins (e.g., vitamin B₁₂) indicating that biomolecules can transform HFO-1234yf at circumneutral pH and at ambient temperature. Taken together, these findings suggest that HFO-1234yf recalcitrance in aquifers should be expected; however, HFO-1234yf is not inert and a biomolecule may mediate reductive transformation in low redox environments, albeit at low rates