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    Structure-Function Studies of Nitrate Reductase Enzymes

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    Nitrate reductase enzymes are a class of molybdenum-based enzymes that undergo a 2-electron redox reaction to reduce nitrate (NO3-) to nitrite (NO2-). This class of enzyme is very important in various geothermal cycles, the most prominent of which is the global nitrogen cycle. There are several types of nitrate reductase based on mechanism and overall structure from organisms such as eukaryotes to bacteria. The focus of this study is the periplasmic nitrate reductase from Campylobacter jejuni and the assimilatory nitrate reductase from Synechococcus elongatus. Both share identical primary coordination spheres at the catalytically essential molybdenum and are predicted to share an overall peptide fold motif. Importantly, for these enzymes, the primary coordination sphere and mechanism are open questions in the community and a focus of this work. Through a series of activity assays and analysis using electron paramagnetic resonance (EPR) spectroscopy, the kinetics, and reactivity of the nitrate reductases were probed in the hope of gaining a better understanding of what affects the reactivity of the Mo center, and what might potentially inhibit the enzyme\u27s activity. Kinetic activity assays using methyl viologen (MV) as an electron donator promoted the turnover of nitrate to nitrate, which allowed for the analysis of active enzymes to obtain the specific activity for both types of nitrate reductase. This assay proved that the synthesis methods of both enzymes were successful in producing active enzymes that could be used in further analysis, and also introduced the possibility that dithionite (DT) is not a good electron donator for these enzymes, but an inhibitor to turnover. Through EPR analysis of NapA, the mechanism behind the conversion of NO3- to NO2- was studied. WT CjNapA underwent turnover and the EPR was obtained before and after, which showed a 6-coordinate structure arising after turnover. This structure aligns with a “Sulfur-shift reaction”, that has been recently proposed in opposition to the previously stated “Oxygen-atom reaction”. This observation gives much more insight into not only the mechanism but also the binding pocket and potential reactivity of these types of enzymes. In addition to understanding the mechanism and reactivity of the enzymes, a new equation to help classify them was formed. This new “Rhombicity” equation was tested for a variety of different molybdenum enzymes, as well as for systems with different transition metals than Mo. This equation helps to classify and quantify the degree of separation of EPR spectra, which allows for better analysis of structure

    The Association Between Aerobic Fitness and Network Connectivity in the Default Mode Network in Healthy Adolescents and Young Adults

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    The beneficial effects of aerobic fitness on psychiatric and cognitive function in older adults have been well demonstrated in existing literature. However, less remains known about the relationship between aerobic fitness and neurocognitive health in emerging adults, who are less likely to suffer from underlying metabolic conditions. Further, few have examined potential sex differences. The transition from adolescence to young adulthood is associated with a reduction in physical activity and accumulating evidence suggests that poor aerobic fitness negatively impacts neurocognition. Therefore, it is crucial to better understand the relationship between aerobic fitness and neurocognitive health during adolescence and young adulthood when interventions may be pivotal. The current study aimed to better characterize the relationships between objectively measured aerobic fitness (VO2 max testing), resting-state functional connectivity in the default mode network (DMN), and neuropsychological performance in healthy emerging adults; sex differences were also examined. Results of the study showed that better aerobic fitness was associated with increased connectivity between the right PCC and left anterior cingulate. The VO2 max*sex interaction was significantly associated with increased connectivity between the right PCC and left precuneus (males displayed a more robust relationship) and increased connectivity between the left PCC and right middle temporal gyrus (females displayed a more robust relationship). Co-activation of the right PCC and left anterior cingulate associated with better aerobic fitness was negatively associated with verbal memory recall for the whole group. Further analysis separated by sex revealed males displayed a negative association between these regions and both verbal learning and memory, while females did not have a significant relationship. Co-activation of the left PCC and right middle temporal gyrus associated with better aerobic fitness was positively associated with verbal memory, with females showing a slightly greater benefit, though no significant sex differences were observed. Lastly, co-activation of the left PCC and right middle temporal gyrus was associated with better inhibition in males. Overall, results from this study contribute to a better understanding of the extent to which aerobic fitness impacts functional connectivity in the DMN of the developing brain and its relationship to neuropsychological performance in a sex-balanced, emerging adult sample. Findings may offer insights into the value of early preventive health behaviors aimed at improving neurocognitive health in youth prior to potential onset of psychiatric or neurologic disorders susceptible to disruptions in DMN connectivity

    The Search for the Four-electron Reduced Intermediate in the Cytochrome C Nitrite Reductase (CCNIR) - Catalyzed Reduction of Nitrite

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    Although there are many nitrogen species in the nitrogen cycle, nitrite occupies a centralrole. Nitrite can either reduce to ammonium or dinitrogen or oxidize to nitrate. Ammonium and nitrate can be found in the environment; however, problems arise when the accumulation of these nitrogen species leads to eutrophication in aquatic environments. High ammonium or nitrate levels in lakes or other bodies of water can lead to the overgrowth of algae, which causes the blockage of sunlight to different species below the water surface. Imbalance in the ecosystem is a serious environmental issue that can be addressed by further studying and understanding the nitrogen cycle. Cytochrome c Nitrite Reductase (ccNiR) is a complex multi-heme respiratory enzyme, found in the aquatic bacteria Shewanella oneidensis, that catalyzes the reduction of nitrite to ammonium. Earlier in vitro studies had used the strong reductant methyl viologen monocation radical (MVred) to study the reaction, but under these conditions, no intermediates accumulate, which leaves the mechanistic steps of the catalysis uncharted. In this study, ccNiR-mediated nitrite reduction was effected by a variety of weak reductants in place of MVred. Assays for hydroxylamine and ammonium formation showed that ammonium was still the only significant product under these conditions. However, intermediate species in which partially reduced nitrogenous moieties were bound at the ccNiR active site were now detectable. The kinetics of intermediate and ammonium formation were monitored by conventional and stopped-flow UV-Visible Spectroscopy. The results presented herein pave the way to further characterizing the catalytic intermediates using electron paramagnetic resonance spectroscopy and time resolved X-ray crystallography. To that end, preliminary crystallographic results collected at the European X-Ray free-electron laser facility are also presented

    Information Needs of Korean Immigrants in the United States: Selection and Use of Social Media

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    This mixed-method study investigates the information needs of Korean immigrants on social media and their selection and use of social media for finding information in the United States. The study was designed to answer six research questions: 1) What are the top types of information needs on social media that Korean immigrants fulfill during their everyday lives in the United States?; 2) What types of social media do Korean immigrants most frequently use during their everyday lives in the United States for different types of information needs?; 3) Are there any relationships between the types of social media Korean immigrants select and demographic factors, Information Communication Technology (ICT) experience level, cultural factors, and information needs?; 4) What factors influence the selection of types of social media among Korean immigrants?; 5) How do Korean immigrants use social media for finding information during their everyday lives in the United States?; 6) What factors influence Korean immigrants’ social media use for information seeking in their everyday lives in the United States? Data collection involved questionnaires, diaries, and interviews with 111 Korean immigrant participants completing questionnaires and 16 selected for diaries and interviews. Quantitative and qualitative methods, including descriptive statistic, Chi-square test of independence, and open coding, were used for analysis. In particular, descriptive analysis was utilized to identify education as the top information need on social media in RQ1. In RQ2, it was observed that Social Network Services (SNS) were most commonly used, while YouTube was popular for education, monitoring, and health information. The researcher investigated the relationship between social media types, demographic factors, ICT experience level, cultural factors, and information needs in RQ3 and revealed that there was a relationship between them by Chi-square test. In RQ4, the researcher identified factors influencing social media selection, such as social network influence, cultural preference, information needs, information quantity, and information format, using open coding. In RQ5, it was found that Korean immigrants primarily employed searching a keyword within a social media or clicking or subscribing to feeds/notifications to gather information. In RQ6, factors affecting social media use, such as algorithms, features, relevance of search results, and speed of information delivery, were identified. The study uncovered information needs among Korean immigrants and shed light on their predominant needs on social media. Moreover, the study presented a theoretical model for Korean immigrants\u27 selection and use of social media platforms. It offered practical recommendations to cater to the information needs of Korean immigrants, such as algorithm optimization, improved search functionality, and language support. The study\u27s limitations were acknowledged, and recommendations for future research were provided

    The Effects of Microplastics and Glyphosate on the Alga Chlamydomonas Reinhardtii

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    Microplastics (plastic particles 0.1 μm - 5 mm) are a major ecological problem in terrestrial and aquatic environments. While research on the effects of plastics on organisms has steadily increased, much less is known about the effects of microplastics that have been exposed to a secondary harmful contaminant, such as the herbicide, glyphosate (here referred to as “treated microplastics”). Microplastics that have not been so exposed (“pristine microplastics”) have typically been used in microplastic toxicity studies. A wide range of organisms have been used in studies of the effects of microplastics, but microalgae, which lie at the base of aquatic food webs, continue to be understudied. To understand the interaction between microplastics and contaminants with respect to their effects on microalgae, batch cultures of the chlorophyte alga Chlamydomonas reinhardtii were grown in saturating irradiance (100 µmol quanta m-2 s-1, 16:8 L:D cycle, 18 °C) under nutrient-replete conditions and exposed to treatments of pristine polyethylene microspheres (45-53 µm diameter, 0.1 g L-1 or 1 g L-1), dissolved glyphosate (220 µM or 44 µM), or polyethylene microspheres treated with glyphosate (220 µM or 44 µM), over a 14-day exposure. Growth was monitored using in-vivo Chlorophyll a fluorescence and flow cytometry, while measurements of photosynthetic quantum yield (“Fv/Fm,” using DCMU) and the proportion of dead cells (“mortality,” using the stain SYTOX-Green) were also determined. The main effect of glyphosate was significant and resulted in treated cultures to have significantly lower growth rates and quantum yield than cultures compared to control cultures without glyphosate exposure (µ of 0.99 d-1 versus 1.01 d-1 respectively and Fv/Fm of 0.47 versus 0.58 respectively, p\u3c0.05 in both cases). The main effect of microplastics was also significant and also resulted in decreased quantum yield compared to cultures not exposed to plastics (Fv/Fm of 0.45 versus 0.62 respectively, p\u3c0.05). However, exposure to microplastics actually increased growth rate and reduced mortality compared to cultures not exposed to plastics (µ of 1.09 d-1 versus 0.94 d-1 respectively and dead cells mean of 1.23 % versus 3.01 % respectively, p\u3c0.05). The maximum biomass (“plateau,” measured as chl a fluorescence or cell numbers) achieved by cultures were not different. Glyphosate and microplastics did not interact in terms of significantly modifying each other’s effects. The findings contrast with others in the literature and show that the combined impact of microplastics and other contaminants on an organism is complex and dependent on various factors such as the concentration and characteristics of the contaminants as well as the type of test organism and the parameters being studied. Future work should focus on assessing the effects of different types and forms of plastics in combination with other contaminants

    Monandrous Females Regain Sexual Receptivity and Produce Fewer Offspring After Short Copulations

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    Male fitness is a function of the number of mates they can inseminate, yet males often engage in prolonged copulations rather than seek out new mates. Potential explanations for this behavior include the ejaculate transfer hypothesis, which states that longer copulations function to achieve higher levels of insemination, and the mate-guarding hypothesis which states longer copulations function to prevent the female from mating again. Interestingly, prolonged copulations can occur in monandrous species where these functions would not seem to apply. We investigated prolonged mating in a monandrous-polygynous species, a member of the Enchenopa treehoppers (Hemiptera: Membracidae). These insects form pairs by using plant-borne vibrational signals. Copulation can last up to three hours. We tested a version of the mate-guarding hypothesis by which prolonged copulation functions to reduce female receptivity and promote offspring production. In other words, monandry in this species may be due to male ejaculate substances. If so, females would be more likely to duet with male signals and less likely to produce offspring as mating duration declines. We randomly assigned receptive females to copulation duration treatments of either 10, 30, 45, or 60 minutes before we separated the pairs. We also included a control group where the pairs were not interrupted during copulations. We found that females that experienced copulations lasting ≤30 minutes were more likely to duet with playbacks after mating compared to females that had uninterrupted matings. These control females (copulations lasting 104 minutes, on average) rarely duetted with subsequent playbacks. Further, we found that females that mated for ≤45 minutes were less likely to produce offspring than females in other treatments. Our results suggest that extreme copulation length may function to lower female receptivity and promote offspring production by the transfer of ejaculate substances, effectively causing monandry in some species

    AN EXPERIMENTAL AND NUMERICAL STUDY OF TEMPERATURE UNIFORMITY ENHANCEMENT VIA PASSIVE GEOMETRY MODIFICATIONS IN AIR DILUTION MIXING

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    This work studies the effect of passive geometries on the temperature uniformity downstream of gas turbines dilution zones. The experimental setup simulates the non-reacting hot and cold air mixing in a dilution chamber, with inlet Reynolds number in the range of 40,000 – 95,000. Several CFD models were investigated and validated against experimental results. The proposed passive geometries include dilution jet extender parts that are installed on the large dilution holes (5mm to 25mm). The second proposed geometry is converging nozzle parts, called jet area modifiers, installed on the inner side of the large dilution holes. The converging nozzle parts have lengths of 5mm, 15mm, and 25mm and area ratios of 0.5 and 0.75. The presented research investigates the effect of different factors affecting temperature uniformity. Results indicate that temperature uniformity is the strongest factor affecting the usefulness efficiency, followed by the jet extender length. The Reynolds number does not have a significant effect on the usefulness efficiency. Jet extenders offer improvement on the temperature uniformity index by 0.9% - 14.9% depending on the temperature ratio and extender length. The added pressure drop varies between 5% and 28% depending on extender length. Jet area modifiers result in an improvement in the temperature uniformity index between 2% and 29%, and pressure drop of 8%-36% compared to the baseline extender case. The realizable k-ε model showed good agreement with experimental data and performed well against k-ω (SST) and Reynolds Stress Turbulence (RST) models

    Permeation Characteristics of Neutral and Charged Solutes through Covalent Organic Framework (COF) Nanofiltration Membranes

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    Membrane technology is an important part of sustainable development in water treatment processes and can provide solutions to water scarcity through desalination, water contamination, ecosystem rehabilitation, along with applications in biomedical engineering for separation processes. Nanofiltration membranes with Covalent Organic Frameworks (COFs) have unique structure and properties with high porosity, large surface area, and ordered pore structure, and have emerged as a promising energy-efficient water purification technology. Moreover, these properties provide opportunities for efficient organic carbon separation. Using neutral solutes, polyethylene glycol and sugars at a range of molecular weights, comparison of the molecular structure of the compound and its effects on rejection can be observed. The neutral charge allows the characterization of the (COF) membrane and its efficiency with negligible effects on rejection rate from electro-repulsion forces. In this research, we will investigate the molecular weight cut off performance of the COF membranes with neutral particles, and use transport models to analyze the data to estimate membrane pore size, molecular structure, and separation mechanisms

    A Self-Boosting Signal Type in an Insect?

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    Sexual mimicry, whereby individuals of one sex mimic the behaviors, signals, or morphology of the other sex, is widespread in animals. We were interested in the function of a recently reported form of sexual mimicry in Enchenopa treehoppers. These treehoppers communicate with plant-borne vibrational signals. In pair formation, males produce advertisement signals and females respond with their own, establishing a duet that continues until the male finds the female. In addition to their advertisement signals, males often also produce another signal type, which mimics female duetting signals. We asked about the function of these female-mimic signals by testing the following hypotheses: that they function to inhibit competing males; to increase the likelihood of female response; or to give the males producing them a motivational self-boost . We found that female-mimic signals did not inhibit other males from signaling, nor did they increase the likelihood of female response. However, males that produced a female mimic signal subsequently showed higher signaling effort than those that didn’t, in spite of being in lower body condition. These results give tentative support to the self-boost hypothesis and suggest a novel function for sexual mimicry. Sexual mimicry is usually thought to function to affect the behavior of receivers. Here we show low body condition males producing a female mimic can provide positive motivational feedback to themselves as a self-boost. However, it may sometimes function to affect the behavior of signalers

    The Motility of Phage Resistant Mucoid Escherichia coli

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    Lytic bacteriophages are viruses that infect and kill bacteria and have the potential to be used as alternatives to antibiotics. However, bacteria can evolve resistance to phage which could render this treatment method ineffective. We are investigating the resistance mechanism of mucoidy and its effect on motility. To do this, we isolated 27 E. coli mucoid mutants using laboratory selection experiments with phage U136B. We then quantified these mutants’ phage resistance using efficiency of plaquing (EOP) assays and their bacterial motility using swim agar plates. We found that all 27 mucoid E. coli mutants are resistant to phage U136B, however we did find that some mucoid mutants were only partially phage resistant. We also discovered that all 27 mucoid mutants are less motile than the wild type bacteria, but some mutants were more motile than others. In future work, we will confirm motility with phase contrast microscopy and test if mucoid mutations influence biofilm production. Our results suggest the evolution of mucoidy could coincide with reduced motility, which would be a beneficial side effect of phage therapy

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