Our Landscape, Ourselves: Integrating Process and Traditional food Principles for Wellbeing + Resilience in the Swinomish Tribal Community

Thesis (Master's)--University of Washington, 2016-08

Fungal and Oomycete Diseases

Fungal and oomycete pathogens are the causal agents of many important plant diseases. They affect crops that are staple foods for humans and livestock and are responsible for significant economic losses every year. This in turn generates a global social impact. Although fungi and oomycetes evolved separately, they share similar strategies and weaponry to attack plants. Here we review the challenges to global food security posed by these pathogens, current technologies used for detection and diagnostics, the latest understanding of pathogens' strategies to colonize plants, and current and future control measures. Genomic sequences of several important fungal and oomycete pathogens, as well as many crop plants, are now available and are helping to increase understanding of host–pathogen interactions. Recent developments in this field are discussed.

Development and Application of Predictive Models for Survival, Growth, and Death of Enteric Pathogens in Leafy Greens Supply Chain

Leafy greens are essential part of a healthy diet. Because of their health benefits, production and consumption of leafy greens has increased considerably in the U.S. in the last few decades. However, leafy greens are also associated with a large number of foodborne disease outbreaks in the last few years. The overall goal of this dissertation was to use the current knowledge of predictive models and available data to understand the growth, survival, and death of enteric pathogens in leafy greens at pre- and post-harvest levels. Temperature plays a major role in the growth and death of bacteria in foods. A growth-death model was developed for Salmonella and Listeria monocytogenes in leafy greens for varying temperature conditions typically encountered during supply chain. The developed growth-death models were validated using experimental dynamic time-temperature profiles available in the literature. Furthermore, these growth-death models for Salmonella and Listeria monocytogenes and a similar model for E. coli O157:H7 were used to predict the growth of these pathogens in leafy greens during transportation without temperature control. Refrigeration of leafy greens meets the purposes of increasing their shelf-life and mitigating the bacterial growth, but at the same time, storage of foods at lower temperature increases the storage cost. Nonlinear programming was used to optimize the storage temperature of leafy greens during supply chain while minimizing the storage cost and maintaining the desired levels of sensory quality and microbial safety. Most of the outbreaks associated with consumption of leafy greens contaminated with E. coli O157:H7 have occurred during July-November in the U.S. A dynamic system model consisting of subsystems and inputs (soil, irrigation, cattle, wildlife, and rainfall) simulating a farm in a major leafy greens producing area in California was developed. The model was simulated incorporating the events of planting, irrigation, harvesting, ground preparation for the new crop, contamination of soil and plants, and survival of E. coli O157:H7. The predictions of this system model are in agreement with the seasonality of outbreaks. This dissertation utilized the growth, survival, and death models of enteric pathogens in leafy greens during production and supply chain.

Genomic and biochemical investigation of soybean antioxidant metabolism in response to growth at elevated carbon dioxide and elevated ozone

Metabolism in an environment containing of 21% oxygen has a high risk of oxidative damage due to the formation of reactive oxygen species. Therefore, plants have evolved an antioxidant system consisting of metabolites and enzymes that either directly scavenge ROS or recycle the antioxidant metabolites. Ozone is a temporally dynamic molecule that is both naturally occurring as well as an environmental pollutant that is predicted to increase in concentration in the future as anthropogenic precursor emissions rise. It has been hypothesized that any elevation in ozone concentration will cause increased oxidative stress in plants and therefore enhanced subsequent antioxidant metabolism, but evidence for this response is variable. Along with increasing atmospheric ozone concentrations, atmospheric carbon dioxide concentration is also rising and is predicted to continue rising in the future. The effect of elevated carbon dioxide concentrations on antioxidant metabolism varies among different studies in the literature. Therefore, the question of how antioxidant metabolism will be affected in the most realistic future atmosphere, with increased carbon dioxide concentration and increased ozone concentration, has yet to be answered, and is the subject of my thesis research. First, in order to capture as much of the variability in the antioxidant system as possible, I developed a suite of high-throughput quantitative assays for a variety of antioxidant metabolites and enzymes. I optimized these assays for Glycine max (soybean), one of the most important food crops in the world. These assays provide accurate, rapid and high-throughput measures of both the general and specific antioxidant action of plant tissue extracts. Second, I investigated how growth at either elevated carbon dioxide concentration or chronic elevated ozone concentration altered antioxidant metabolism, and the ability of soybean to respond to an acute oxidative stress in a controlled environment study. I found that growth at chronic elevated ozone concentration increased the antioxidant capacity of leaves, but was unchanged or only slightly increased following an acute oxidative stress, suggesting that growth at chronic elevated ozone concentration primed the antioxidant system. Growth at high carbon dioxide concentration decreased the antioxidant capacity of leaves, increased the response of the existing antioxidant enzymes to an acute oxidative stress, but dampened and delayed the transcriptional response, suggesting an entirely different regulation of the antioxidant system. Third, I tested the findings from the controlled environment study in a field setting by investigating the response of the soybean antioxidant system to growth at elevated carbon dioxide concentration, chronic elevated ozone concentration and the combination of elevated carbon dioxide concentration and elevated ozone concentration. In this study, I confirmed that growth at elevated carbon dioxide concentration decreased specific components of antioxidant metabolism in the field. I also verified that increasing ozone concentration is highly correlated with increases in the metabolic and genomic components of antioxidant metabolism, regardless of carbon dioxide concentration environment, but that the response to increasing ozone concentration was dampened at elevated carbon dioxide concentration. In addition, I found evidence suggesting an up regulation of respiratory metabolism at higher ozone concentration, which would supply energy and carbon for detoxification and repair of cellular damage. These results consistently support the conclusion that growth at elevated carbon dioxide concentration decreases antioxidant metabolism while growth at elevated ozone concentration increases antioxidant metabolism.

Microbial nitrogen dynamics in organic and mineral soil horizons along a latitudinal transect in western Siberia

Soil N availability is constrained by the breakdown of N-containing polymers such as proteins to oligopeptides and amino acids that can be taken up by plants and microorganisms. Excess N is released from microbial cells as ammonium (N mineralization), which in turn can serve as substrate for nitrification. According to stoichiometric theory, N mineralization and nitrification are expected to increase in relation to protein depolymerization with decreasing N limitation, and thus from higher to lower latitudes and from topsoils to subsoils. To test these hypotheses, we compared gross rates of protein depolymerization, N mineralization and nitrification (determined using N-15 pool dilution assays) in organic topsoil, mineral topsoil, and mineral subsoil of seven ecosystems along a latitudinal transect in western Siberia, from tundra (67 degrees N) to steppe (54 degrees N). The investigated ecosystems differed strongly in N transformation rates, with highest protein depolymerization and N mineralization rates in middle and southern taiga. All N transformation rates decreased with soil depth following the decrease in organic matter content. Related to protein depolymerization, N mineralization and nitrification were significantly higher in mineral than in organic horizons, supporting a decrease in microbial N limitation with depth. In contrast, we did not find indications for a decrease in microbial N limitation from arctic to temperate ecosystems along the transect. Our findings thus challenge the perception of ubiquitous N limitation at high latitudes, but suggest a transition from N to C limitation of microorganisms with soil depth, even in high-latitude systems such as tundra and boreal forest.

The broad spectrum of bioactive properties of phenolic extracts: a prospective study in three different plants

Natural resources like plants are currently used all over developed and under developed countries of the world as traditional home remedies and are promising agents for drug discovery as they play crucial role in traditional medicine. The use of plants for medicinal purpose usually varies from country to country and region to region because their use depends on the history, culture, philosophy and personal attitudes of the users (Ahmad et al., 2015). The use of plants and plant products as drugs predates the written human history (Hayta et al., 2014). Plants are a very important resource for traditional drugs and around 80% of the population of the planet use plants for the treatment of many diseases and traditional herbal medicine accounts for 30-50% of the total medicinal consumption in China. In North America, Europe and other well-developed regions over 50% of the population have used traditional preparations at least once (Dos Santos Reinaldo et al., 2015). Medicinal plants have been used over years for multiple purposes, and have increasingly attract the interest of researchers in order to evaluate their contribution to health maintenance and disease’s prevention (Murray, 2004). Recently between 50,000 and 70,000 species of plants are known and are being used in the development of modern drugs. Plants were the main therapeutic agents used by humans from the 19th century, and their role in medicine is always topical (Hayta et al., 2014). The studies of medicinal plants are rapidly increasing due to the search for new active molecules, and to improve the production of plants or bioactive molecules for the pharmaceutical industries (Rates, 2001). Several studies have been reported, but numerous active compounds directly responsible for the observed bioactive properties remain unknown, while in other cases the mechanism of action is not fully understood. According to the WHO 25% of all modern medicines including both western and traditional medicine have been extracted from plants, while 75% of new drugs against infective diseases that have arrived between 1981 and 2002 originated from natural sources, it was reported that the world market for herbal medicines stood at over US $60 billion per year and is growing steadily (Bedoya et al., 2009). Traditional medicine has an important economic impact in the 21st century as it is used worldwide, taking advantage on the low cost, accessibility, flexibility and diversity of medicinal plants (Balunas & Kinghorn, 2005).

Water quality trading: credit stacking and ancillary benefits

Ecosystems can provide many services. Wetlands, for example, can help mitigate water pollution from point sources as well as non-point sources, serve as habitat for wildlife, sequester carbon and serve as a place for recreation. Studies have found that these services can have substantial value to society. The sale of ecosystem credits has been found to be a possible way to finance construction investments in wetlands and easements to farmers to take their land out of production. At the same time, selling one ecosystem service credit may not always be enough to justify the investment. Traditionally market participants have only been allowed to sell a single credit from one piece of land, but recently there have been discussions about the possibility of selling more than one credit from a piece of land because it potentially could lead to more efficient ecosystem service provision. Selling multiple credits is sometimes referred to as credit stacking. This paper is an empirical study of the potential for credit stacking applied to the services provided by wetlands in the Upper Mississippi River Basin, specifically nitrogen, phosphorus and wildlife credits. In the setting of our study where costs are discrete rather than continuous we found that wetlands are a cost-effective way to reduce the nitrogen loads from wastewater treatment plants and that stacking nitrogen, phosphorus and wildlife credits may improve social welfare while leading to a higher level of ecosystem services. However, for credit stacking to be welfare improving we found that there needs to be a substantial demand for the credit that covers the majority of the investment in wetlands, while the credit aggregator has a choice between what ecosystem projects to undertake. If the credit that covers the majority of investment is sold first and is the sole basis of the investment decision and the objective is to improve welfare, a sequential implementation of ecosystem credits is not recommended; it would not lead to an increase in the total amount of ecosystem services provided though it would increase profit for the credit producer.

Separation and identification fresh water leeches of waters resources in Kordistan Province

In the primary studying, known leeches have included into free living and parasitic which the parasitic group, besides of direct impacts like: growth detraction, anemia, making wound in the connecting part with the skin, with making plat for secondary bacterial and molding infections in the toll place, is able to cause to transfer blood flagellate and virus to the fish too. Therefore, by accusing information related to fauna leeches of each area a risk factor from the viewpoint of the possibility of being or accusing transferred diseases by these leeches, one can predict and forehand about them. Freshwater leeches of Iran to present accurate recognition (morphological, molecular) have not been, and there are some limited reports from different parts of the country about them. One of the areas that its leeches have not been identified yet is Kurdistan, By having five latrines and big permanent rivers and 32000 springs and a lot of deep and semi-deep wells and this province is a convenient bed for growing aquatics in the country. Therefore, identifying risk factors for development of aquaculture on water resources is one important factor to access achieving development goals. For recognizing leeches of this province, some samples from 10 stands were token. Samples from under stones, sticking to the fish, turtles, plants and solid substances in the water were separated and after recording their physical characteristics, calming with 10% ethylic alcohol with 10% formalin become fixed and after painting with Carmen acetic acid by standard keys for 7 species of Helobdella stagnalis, Placobdella costata, Hemiclepsis marginata, Erpobdella octoculata, Hirudo medicinalis, Dina lineate lineata have been identified and described. Which Helobdella stagnalis has the highest distribution in the province and the minimum one is Hirudo medicinalis. However, that the data obtained in leeches in Kurdistan is a relatively complete collection in this research, recognizing fauna of these areas needs more studying. The Placobdella costata and Hemiclepsis marginata sticking to the fish were separated among identified species which showed that these are parasites for the fish. The sticking area of those leeches to the skin was accompanied with scales cast, damage to mucous membranes beneath the parasite and bleeding Was associated with Histopathology studying effect includes observing break and disconnection in the leech connecting place to the epithelial layer of epidermis in the skin, destroyed nucleus in skin Epithelial cells with observing necrosis in ulcerative place become of the leech and the sub acute inflammated penetration until acute necrosis with opening in Dermis layer is observable. Kidney of this fish have changes such as: proliferation, like proliferative kidney disease with increasing proliferative glomerular cells and increasing in membranous cells in Capillary corpuscle, observing necrotic cells in haematopoietic tissue of kidney along with increasing in infiltration of leukocyte's cells generally mono nucluars such as lymphocytes and less poly morpho nucluars such as neutrophiles that are symptoms of disorders causing anemia become of nourishing and sucking blood by the leech and creating a chronic kidney infection that originally root is in another place like the skin. Also Hemorrhagic anemia causes losing RBC's is because of using the host blood by the leech. (In this situation, one can see immature RBC red cells in Peripheral blood. To identify potential carriers of the leech to the viruses, after finding them in recorded stands and putting them in 75% ethanol for viruses cause IPN, VHS, IHN, they were tested by PCR that the conclusion of these experiments approved IPN virus in Hemiclepsis marginata and Hirudo medicinalis. This kind of leeches can act like a mechanical carrier and causing spreading the agent of this disease. It is worth mentioning that studying the pathogenicity of this virus for aquaculture sources, mentioned before needs more research. During the study of infected fish with leeches that was done after preparing bloody slides and staining them, no case blood parasites was observed. During a research about infecting fish experimentally to known leeches it become clear that 5 days after being in aquarium including leeches, samples of sticking Hirudo medicinalis leech to the golden carp with scales cast were observed. Including leeches to the fish started with molting the scales in the sticking area in the fish and fish become too uneasy and by rubbing themselves to the malls and things inside the aquarium, tried to separate them. Finally, after around 30 hours, leeches penetrate the skin, feeding from blood and tissue liquids and cause mortality the fish and then they become separated from them. If the corpse of these fish stayed in the aquarium, the Helobdella stagnalis and Erpobdella octoculata would start feeding them.

Environmental factors affecting soil metals near outlet roads in Poznan, Poland: impact of grain size, soil depth, and wind dispersal

We determined the Cd, Cr, Cu, Ni, Pb, and Zn concentrations in soil samples collected along the eight main outlet roads of Poznan. Samples were collected at distances of 1, 5, and 10 m from the roadway edges at depth intervals of 0-20 and 40-60 cm. The metal content was determined in seven grain size fractions. The highest metal concentrations were observed in the smallest fraction (<0.063 mm), which were up to four times higher than those in sand fractions. Soil Pb, Cu, and Zn (and to a lesser extent Ni, Cr, and Cd) all increased in relation to the geochemical background. At most sampling sites, metal concentrations decreased with increasing distance from roadway edges and increasing depth. In some locations, the accumulation of metals in soils appears to be strongly influenced by wind direction. Our survey findings should contribute in predicting the behavior of metals along outlet road, which is important by assessing sources for further migration of heavy metals into the groundwater, plants, and humans.

Development of Imaging Mass Spectrometry Analysis of Lipids in Biological and Clinically Relevant Applications

La spectrométrie de masse mesure la masse des ions selon leur rapport masse sur charge. Cette technique est employée dans plusieurs domaines et peut analyser des mélanges complexes. L’imagerie par spectrométrie de masse (Imaging Mass Spectrometry en anglais, IMS), une branche de la spectrométrie de masse, permet l’analyse des ions sur une surface, tout en conservant l’organisation spatiale des ions détectés. Jusqu’à présent, les échantillons les plus étudiés en IMS sont des sections tissulaires végétales ou animales. Parmi les molécules couramment analysées par l’IMS, les lipides ont suscité beaucoup d'intérêt. Les lipides sont impliqués dans les maladies et le fonctionnement normal des cellules; ils forment la membrane cellulaire et ont plusieurs rôles, comme celui de réguler des événements cellulaires. Considérant l’implication des lipides dans la biologie et la capacité du MALDI IMS à les analyser, nous avons développé des stratégies analytiques pour la manipulation des échantillons et l’analyse de larges ensembles de données lipidiques. La dégradation des lipides est très importante dans l’industrie alimentaire. De la même façon, les lipides des sections tissulaires risquent de se dégrader. Leurs produits de dégradation peuvent donc introduire des artefacts dans l’analyse IMS ainsi que la perte d’espèces lipidiques pouvant nuire à la précision des mesures d’abondance. Puisque les lipides oxydés sont aussi des médiateurs importants dans le développement de plusieurs maladies, leur réelle préservation devient donc critique. Dans les études multi-institutionnelles où les échantillons sont souvent transportés d’un emplacement à l’autre, des protocoles adaptés et validés, et des mesures de dégradation sont nécessaires. Nos principaux résultats sont les suivants : un accroissement en fonction du temps des phospholipides oxydés et des lysophospholipides dans des conditions ambiantes, une diminution de la présence des lipides ayant des acides gras insaturés et un effet inhibitoire sur ses phénomènes de la conservation des sections au froid sous N2. A température et atmosphère ambiantes, les phospholipides sont oxydés sur une échelle de temps typique d’une préparation IMS normale (~30 minutes). Les phospholipides sont aussi décomposés en lysophospholipides sur une échelle de temps de plusieurs jours. La validation d’une méthode de manipulation d’échantillon est d’autant plus importante lorsqu’il s’agit d’analyser un plus grand nombre d’échantillons. L’athérosclérose est une maladie cardiovasculaire induite par l’accumulation de matériel cellulaire sur la paroi artérielle. Puisque l’athérosclérose est un phénomène en trois dimension (3D), l'IMS 3D en série devient donc utile, d'une part, car elle a la capacité à localiser les molécules sur la longueur totale d’une plaque athéromateuse et, d'autre part, car elle peut identifier des mécanismes moléculaires du développement ou de la rupture des plaques. l'IMS 3D en série fait face à certains défis spécifiques, dont beaucoup se rapportent simplement à la reconstruction en 3D et à l’interprétation de la reconstruction moléculaire en temps réel. En tenant compte de ces objectifs et en utilisant l’IMS des lipides pour l’étude des plaques d’athérosclérose d’une carotide humaine et d’un modèle murin d’athérosclérose, nous avons élaboré des méthodes «open-source» pour la reconstruction des données de l’IMS en 3D. Notre méthodologie fournit un moyen d’obtenir des visualisations de haute qualité et démontre une stratégie pour l’interprétation rapide des données de l’IMS 3D par la segmentation multivariée. L’analyse d’aortes d’un modèle murin a été le point de départ pour le développement des méthodes car ce sont des échantillons mieux contrôlés. En corrélant les données acquises en mode d’ionisation positive et négative, l’IMS en 3D a permis de démontrer une accumulation des phospholipides dans les sinus aortiques. De plus, l’IMS par AgLDI a mis en évidence une localisation différentielle des acides gras libres, du cholestérol, des esters du cholestérol et des triglycérides. La segmentation multivariée des signaux lipidiques suite à l’analyse par IMS d’une carotide humaine démontre une histologie moléculaire corrélée avec le degré de sténose de l’artère. Ces recherches aident à mieux comprendre la complexité biologique de l’athérosclérose et peuvent possiblement prédire le développement de certains cas cliniques. La métastase au foie du cancer colorectal (Colorectal cancer liver metastasis en anglais, CRCLM) est la maladie métastatique du cancer colorectal primaire, un des cancers le plus fréquent au monde. L’évaluation et le pronostic des tumeurs CRCLM sont effectués avec l’histopathologie avec une marge d’erreur. Nous avons utilisé l’IMS des lipides pour identifier les compartiments histologiques du CRCLM et extraire leurs signatures lipidiques. En exploitant ces signatures moléculaires, nous avons pu déterminer un score histopathologique quantitatif et objectif et qui corrèle avec le pronostic. De plus, par la dissection des signatures lipidiques, nous avons identifié des espèces lipidiques individuelles qui sont discriminants des différentes histologies du CRCLM et qui peuvent potentiellement être utilisées comme des biomarqueurs pour la détermination de la réponse à la thérapie. Plus spécifiquement, nous avons trouvé une série de plasmalogènes et sphingolipides qui permettent de distinguer deux différents types de nécrose (infarct-like necrosis et usual necrosis en anglais, ILN et UN, respectivement). L’ILN est associé avec la réponse aux traitements chimiothérapiques, alors que l’UN est associé au fonctionnement normal de la tumeur.

Chestnut and lemon balm based ingredients as natural preserving agents of the nutritional profile in matured “Serra da Estrela” cheese

Chestnut flowers, lemon balm plants and their decoctions were incorporated into "Serra da Estrela" cheese, to assess their potential to preserve its nutritional properties and provide new foodstuffs. The analyses were carried out after the normal ripening period of 1month and after 6months of storage. The most abundant nutrients were proteins and fats. The most abundant minerals were Ca and Na, while C16:0 and C18:1 were the main fatty acids. Saturated fatty acids were the most abundant, followed by the monounsaturated. Moisture seemed to be lower in the samples with the plants incorporated. The dried plants, when incorporated, seemed to be more efficient as preservers then the decoctions, although these better preserved the proteins. These plants can be regarded as promising natural preservers in foodstuffs cheese, given the preservation of key parameters and the slight impact on the nutritional value.

Distribution of Ra-226, Th-232 and K-40 in soils and sugar cane crops at Corumbatai river basin, São Paulo State, Brazil

The common use of phosphate fertilizers NPK and amendments in sugar cane crops in Brazilian agriculture may increase the Ra-226, Th-232 and K-40 activity concentrations in soils and their availability for plants and human food chain. Thus, the main aim of this study was to evaluate the distribution of Ra-226, Th-232 and K-40 in soils and sugar cane crops in the Corumbatai river basin, São Paulo State, Brazil. The gamma spectrometry was utilized to measure the Ra-226, Th-232 and K-40 activity concentration in all samples. The soil-to-sugar cane transfer factors (TF) were quantified using the ratio between the radionuclide activity concentration in sugar cane and its activity concentration in soil. The results show that, although radionuclides incorporated in phosphate fertilizers and amendments are annually added in the sugar cane crops, if utilized in accordance with the recommended rates, their use does not lead to hazards levels in soils. The soil-to-sugar cane transfer of radionuclides occurred in the following order K-40 > Ra-226 > Th-232. Therefore, under these conditions, radionuclides intake through consumption of sugar is not hazardous to human health. (C) 2009 Elsevier Ltd. All rights reserved.

Shaping the Dicot Fruit: Molecular and Genomic Approaches to Fruit Development

The fruit is one of the most complex and important structures produced by flowering plants, and understanding the development and maturation process of fruits in different angiosperm species with diverse fruit structures is of immense interest. In the work presented here, molecular genetics and genomic analysis are used to explore the processes that form the fruit in two species: The model organism Arabidopsis and the diploid strawberry Fragaria vesca. One important basic question concerns the molecular genetic basis of fruit patterning. A long-standing model of Arabidopsis fruit (the gynoecium) patterning holds that auxin produced at the apex diffuses downward, forming a gradient that provides apical-basal positional information to specify different tissue types along the gynoecium’s length. The proposed gradient, however, has never been observed and the model appears inconsistent with a number of observations. I present a new, alternative model, wherein auxin acts to establish the adaxial-abaxial domains of the carpel primordia, which then ensures proper development of the final gynoecium. A second project utilizes genomics to identify genes that regulate fruit color by analyzing the genome sequences of Fragaria vesca, a species of wild strawberry. Shared and distinct SNPs among three F. vesca accessions were identified, providing a foundation for locating candidate mutations underlying phenotypic variations among different F. vesca accessions. Through systematic analysis of relevant SNP variants, a candidate SNP in FveMYB10 was identified that may underlie the fruit color in the yellow-fruited accessions, which was subsequently confirmed by functional assays. Our lab has previously generated extensive RNA-sequencing data that depict genome-scale gene expression profiles in F. vesca fruit and flower tissues at different developmental stages. To enhance the accessibility of this dataset, the web-based eFP software was adapted for this dataset, allowing visualization of gene expression in any tissues by user-initiated queries. Together, this thesis work proposes a well-supported new model of fruit patterning in Arabidopsis and provides further resources for F. vesca, including genome-wide variant lists and the ability to visualize gene expression. This work will facilitate future work linking traits of economic importance to specific genes and gaining novel insights into fruit patterning and development.

Exploring the Function and Regulation of Arabidopsis EIN2 in Ethylene Signaling

Ethylene is an essential plant hormone involved in nearly all stages of plant growth and development. EIN2 (ETHYLENE INSENSITIVE2) is a master positive regulator in the ethylene signaling pathway, consisting of an N-terminal domain and a C-terminal domain. The EIN2 N-terminal domain localizes to the endoplasmic reticulum (ER) membrane and shows sequence similarity to Nramp metal ion transporters. The cytosolic C-terminal domain is unique to plants and signals downstream. There have been several major gaps in our knowledge of EIN2 function. It was unknown how the ethylene signal gets relayed from the known upstream component CTR1 (CONSTITUTIVE RESPONSE1) a Ser/Thr kinase at the ER, to EIN2. How the ethylene signal was transduced from EIN2 to the next downstream component transcription factor EIN3 (ETHYLENE INSENSITIVE3) in the nucleus was also unknown. The N-terminal domain of EIN2 shows homology to Nramp metal ion transporters and whether EIN2 can also function as a metal transporter has been a question plaguing the ethylene field for almost two decades. Here, EIN2 was found to interact with the CTR1 protein kinase, leading to the discovery that CTR1 phosphorylates the C-terminal domain of EIN2 in Arabidopsis thaliana. Using tags at the termini of EIN2, it was deduced that in the presence of ethylene, the EIN2 C-terminal domain is cleaved and translocates into the nucleus, where it could somehow activate downstream ethylene responses. The EIN2 C-terminal domain interacts with nuclear proteins, RTE3 and EER5, which are components of the TREX-2 mRNA export complex, although the role of these interactions remains unclear. The EIN2 N-terminal domain was found to be capable of divalent metal transport when expressed in E. coli and S. cerevisiae leading to the hypothesis that metal transport plays a role in ethylene signaling. This hypothesis was tested using a novel missense allele, ein2 G36E, substituting a highly conserved residue that is required for metal transport in Nramp proteins. This G36E substitution did not disrupt metal ion transport of EIN2, but the ethylene insensitive phenotype of this mutant indicates that the EIN2 N-terminal domain is important for positively regulating the C-terminal domain. The defect of the ein2 G36E mutant does not prevent proper expression or subcellular localization, but might affect protein modifications. The ein2 G36E allele is partially dominant, mostly likely displaying haploinsufficiency. Overexpression of the EIN2 N-terminal domain in the ein2 G36E mutant did not rescue ethylene insensitivity, suggesting the N-terminal domain functions in cis to regulate the C-terminal domain. These findings advance our knowledge of EIN2, which is critical to understanding ethylene signaling.

Assessment of the Occurrence and Potential Risks of Pharmaceuticals and their Metabolites in Fish and Water Using Liquid Chromatography Mass Spectrometry

A comprehensive method for the analysis of 11 target pharmaceuticals representing multiple therapeutic classes was developed for biological tissues (fish) and water. Water samples were extracted using solid phase extraction (SPE), while fish tissue homogenates were extracted using accelerated solvent extraction (ASE) followed by mixed-mode cation exchange SPE cleanup and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Among the 11 target pharmaceuticals analyzed, trimethoprim, caffeine, sulfamethoxazole, diphenhydramine, diltiazem, carbamazepine, erythromycin and fluoxetine were consistently detected in reclaimed water. On the other hand, caffeine, diphenhydramine and carbamazepine were consistently detected in fish and surface water samples. In order to understand the uptake and depuration of pharmaceuticals as well as bioconcentration factors (BCFs) under the worst-case conditions, mosquito fish were exposed to reclaimed water under static-renewal for 7 days, followed by a 14-day depuration phase in clean water. Characterization of the exposure media revealed the presence of 26 pharmaceuticals while 5 pharmaceuticals including caffeine, diphenhydramine, diltiazem, carbamazepine, and ibuprofen were present in the organisms as early as 5 h from the start of the exposure. Liquid chromatography ultra-high resolution Orbitrap mass spectrometry was explored as a tool to identify and quantify phase II pharmaceutical metabolites in reclaimed water. The resulting data confirmed the presence of acetyl-sulfamethoxazole and sulfamethoxazole glucuronide in reclaimed water. To my knowledge, this is the first known report of sulfamethoxazole glucuronide surviving intact through wastewater treatment plants and occurring in environmental water samples. Finally, five bioaccumulative pharmaceuticals including caffeine, carbamazepine, diltiazem, diphenhydramine and ibuprofen detected in reclaimed water were investigated regarding the acute and chronic risks to aquatic organisms. The results indicated a low potential risk of carbamazepine even under the worst case exposure scenario. Given the dilution factors that affect environmental releases, the risk of exposure to carbamazepine will be even more reduced.