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Expression, Purification and Characterization of the Human Cytochrome P450 Enzyme 4x1 (CYP4X1) Using Substrate Binding Affinity, Metabolic Assays and Protein Crystallization
No full textThe human cytochrome P450 enzyme 4X1 (CYP4X1) is among the “orphan” cytochrome P450 meaning there is much unknown about its structure, biological function, and regulation in literature study. Scientific work on this protein has primarily been on identifying its physiological functions and expression sites. A recent study found 2 substrates (arachidonic acid and anandamide) catalyzed by the enzyme. Expression in humans appears to be primarily in the brain, prostate, lungs, and neurovascular systems and research has linked the protein as a potential chemotherapy target in brain cancer. So far, only a few inhibitors have been identified. The goal of this research is to characterize CYP4X1. The first step is to express the protein in E. coli JM109 cell using either the pCW or pET28a vector. Protein expression attempts indicated that pET28a vector in JM 109 cells produced a better yield overall. Protein purification was obtained using cobalt and nickel affinity chromatography made possible by a 6-residue histidine tag affixed to the protein’s C-terminal following expression. Protein purification is followed by SDS-PAGE to ascertain purity. Binding affinity assays were obtained for the two previously published substrates, arachidonic acid, and anandamide. Additional compounds that could be ligands based on their structural similarity to arachidonic were also assayed. Purified protein was concentrated and screened with a commercial crystal screen and a crystal hit has been identified with further optimization required
Assessing the Prevalance of Joint-Controlled Mass Wasting in the Southern Appalachian Mountains, U.S.A.
No full textLandslides are common hazards that produce devastating effects worldwide. Within the United States, the Appalachian Mountains are identified as an area of moderate to high susceptibility and incidence for landslides (Mirus et al., 2020; Wieczorek and Morgan, 2008). Given the presence of active seismic zones (eastern Tennessee and Giles County seismic zones) and variable rainfall due to seasonal storms (hurricanes) throughout the Appalachians, ancient earthquake activity or intense rainfall may trigger mass wasting events. Regional bedrock joints may further control the susceptibility of landslides to develop in specific locations during trigger events by providing a pre-existing weakness in the substrate. This relationship is demonstrated by three large rock block slides in the Appalachian Valley and Ridge province downslope of ridges where bedrock units are oriented parallel to regional fractures. To investigate the distribution of joint orientations and landslide occurrence throughout the southern and central Appalachians, I present 3,000 structural measurements of joints collected throughout the southern Valley and Ridge province. The discordance between joint orientations and local strike of bedrock units is used to (a) identify higher- and lower-risk mass wasting zones, (b) focus searches for unmapped, ancient landslides using high resolution digital elevation models, including light detection and ranging (LiDAR) based data and (c) to determine if joint orientations are influenced by regional basement structures. From these techniques, two mass wasting features were identified 60 miles southwest of the Sinking Creek Mountain landslides along the same ridge, within the Giles County seismic zone
Conformational control through co-operative nonconventional C - H-N hydrogen bonds
No full textWe report the design, synthesis, and crystal structure of a conjugated aryleneethynyl molecule, 2-(2-{4,5-dimethoxy-2-[2-(2,3,4-trifluorophenyl)ethynyl]phenyl}ethynyl)-6-[2-(pyridin-2-yl)ethynyl]pyridine, C30H17F3N2O2, that adopts a planar rhombus conformation in the solid state. The molecule crystallizes in the space group P , with Z = 2, and features two intramolecular sp2 -C - H-N hydrogen bonds that co-operatively hold the arylethynyl molecule in a rhombus conformation. The H atoms are activated towards hydrogen bonding since they are situated on a trifluorophenyl ring and the H-N distances are 2.470(16) and 2.646(16)Å, with C - H-N angles of 161.7(2) and 164.7(2)°, respectively. Molecular electrostatic potential calculations support the formation of C - H-N hydrogen bonds to the trifluorophenyl moiety. Hirshfeld surface analysis identifies a self-complementary C - H-O dimeric interaction between adjacent 1,2-dimethoxybenzene segments that is shown to be common in structures containing that moiety
Microvascular Sex- and Age- Dependent Phosphodiesterase Expression
No full textObjective: The cyclic nucleotide second messengers, cAMP and cGMP, are pivotal regulators of vascular functions; their cellular levels are tightly controlled by the cyclic nucleotide hydrolases, phosphodiesterases (PDE). Biologic sex and age are recognized as independent factors impacting the mechanisms mediating both vascular health and dysfunction. This study focused on microvessels isolated from male and female rats before (juvenile) and after (adult) sexual maturity under resting conditions. We tested the hypothesis that sexual dimorphism in microvascular PDE expression would be absent in juvenile rats, but would manifest in adult rats. Methods: Abdominal skeletal muscle arterioles and venules were isolated from age-matched juvenile and adult male and female rats under resting conditions. Transcripts of five PDE families (1–5) associated with coronary and vascular function with a total of ten genes were measured using TaqMan real-time RT-PCR and protein expression of microvessel PDE4 was assessed using immunoblotting and immunofluorescence. Results: Overall expression levels of PDE5A were highest while PDE3 levels were lowest among the five PDE families (p \u3c 0.05) regardless of age or sex. Contrary to our hypothesis, in juveniles, sexual dimorphism in PDE expression was observed in three genes: arterioles (PDE1A, female \u3e male) and venules (PDE1B and 3A, male \u3e female). In adults, gene expression levels in males were higher than females for five genes in arterioles (PDE1C, 3A, 3B, 4B, 5A) and three genes (PDE3A, 3B, and 5A) in venules. Furthermore, age-related differences were observed in PDE1-5 (in males, adult \u3e juvenile for most genes in arterioles; in females, adult \u3e juvenile for arteriolar PDE3A; juvenile gene expression \u3e adult for two genes in arterioles and three genes in venules). Immunoblotting and immunofluorescence analysis revealed protein expression of microvessel PDE4. Conclusion: This study revealed sexual dimorphism in both juvenile and adult rats, which is inconsistent with our hypothesis. The sex- and age-dependent differences in PDE expression implicate different modulations of cAMP and cGMP pathways for microvessels in health. The implication of these sex- and age-dependent differences, as well as the duration and microdomain of PDE1-5 activities in skeletal muscle microvessels, in both health and disease, require further investigation
Bene\u27fit\u27 assessment in pollination coevolution: mechanistic perspectives on hummingbird bill-flower matching
No full textOne of the reasons why flowering plants became the most diverse group of land plants is their association with animals to reproduce. The earliest examples of this mutualism involved insects foraging for food from plants and, in the process, pollinating them. Vertebrates are latecomers to these mutualisms, but birds, in particular, present a wide variety of nectar-feeding clades that have adapted to solve similar challenges. Such challenges include surviving on small caloric rewards widely scattered across the landscape, matching their foraging strategy to nectar replenishment rate, and efficiently collecting this liquid food from well-protected chambers deep inside flowers. One particular set of convergent traits among plants and their bird pollinators has been especially well studied: the match between the shape and size of bird bills and ornithophilous flowers. Focusing on a highly specialized group, hummingbirds, we examine the expected benefits from bill-flower matching, with a strong focus on the benefits to the hummingbird and how to quantify them. Explanations for the coevolution of bill-flower matching include 1) that the evolution of traits by bird-pollinated plants, such as long and thin corollas, prevents less efficient pollinators (e.g., insects) from accessing the nectar, and 2) that increased matching, as a result of reciprocal adaptation, benefits both the bird (nectar extraction efficiency) and the plant (pollen transfer). In addition to nectar feeding, we discuss how interference and exploitative competition also play a significant role in the evolution and maintenance of trait matching. We present hummingbird-plant interactions as a model system to understand how trait matching evolves and how pollinator behavior can modify expectations based solely on morphological matching, and discuss the implications of this behavioral modulation for the maintenance of specialization. While this perspective piece directly concerns hummingbird-plant interactions, the implications are much broader. Functional trait matching is likely common in coevolutionary interactions (e.g., in predator-prey interactions), yet the physical mechanisms underlying trait matching are understudied and rarely quantified. We summarize existing methods and present novel approaches that can be used to quantify key benefits to interacting partners in a variety of ecological systems
A Mixed-Methods Study of Early Childhood Education and Care in South Korea: Policies and Practices During COVID-19
No full textThis study of South Korea\u27s response to COVID-19 has three purposes. First, it uses document analysis to examine policies, strategies, and resources offered by the South Korean government and public organizations to support young children and families during the first 6 months of the pandemic. Next, it uses open-ended surveys with 30 directors of early childhood institutions to explore institutional-level supports and needs during the pandemic. Finally, it looks at the discrepancies between stated policies outlining the South Korea\u27s response to COVID-19 and the lived experiences of early childhood educators as a route to arriving at recommendations for education policymakers and other stakeholders. To that end, we reviewed government documents (n = 84) containing early childhood education-related responses to Covid-19 established by the Ministry of Education, the Ministry of Health and Welfare, and other relevant government sectors. An online survey with 17 kindergarten and 13 child care center directors was also analyzed. Using content analysis, the findings revealed that the government\u27s policies and guidance for Early Childhood Education and Care (ECEC) as well as the institutional supports for children and families were overall comprehensive in its scope. The analysis, based on the five tenets of the Whole Child approach, also indicated that the government\u27s policy responses and services for ECEC focused mainly on the \u27Safe\u27 and \u27Supported\u27 tenets, while \u27Challenged\u27 was given the least amount of consideration. The survey responses demonstrated different measures taken by kindergartens and child care centers highlighting the separate nature of \u27education\u27 and \u27care\u27 in South Korea, while also indicating limited resources for supporting children\u27s psychological well-being and for children and families in need. This overview provides a foundation for further discussion and research on the impact of Covid-19 on ECEC in South Korea and beyond
Arylethynyl Helices Supported by π-Stacking and Halogen Bonding
No full textCo-crystallization of a pyridyl-containing arylethynyl (AE) moiety with 1,4-diiodotetrafluorobenzene leads to unique, figure-eight shaped helical motifs within the crystal lattice. A slight twist in the AE backbone allows each AE unit to simultaneously interact with haloarene units that are stacked on top of one another. Left-handed (M) and right-handed (P) helices are interspersed in a regular pattern throughout the crystal. The major driving forces for assembly are 1) halogen bonding between the pyridyl nitrogen atoms and the iodine substituents of the haloarene, with N⋅⋅⋅I distances between 2.81 and 2.84 Å, and 2) π-π stacking of the haloarenes, with distances of approximately 3.57 Å between centroids. Halogen bonding and π-π stacking not only work in concert, but also seem to mutually enhance one another. Calculations suggest that the presence of π-π stacking modestly intensifies the halogen bonding interaction by \u3c0.2 kcal/mol; likewise, halogen bonding to the haloarene enhances the π-π stacking interaction by 0.59 kcal/mol
Simulating nonpoint source pollutant loading in a karst basin: A SWAT modeling application
No full textKarst terrain confounds efforts to parameterize and calibrate The Soil and Water Assessment Tool (SWAT). The purpose of this study was to use SWAT to simulate long-term natural (e.g. climate) and human (e.g. land use) impacts to streamflow, sediment and nutrient loading in a karst catchment. Sub-objectives were to: (1) perform a rigorous model performance assessment of SWAT, and (2) apply SWAT to highlight critical source areas of NPS pollutant loading in a large (HUC-8 spatial scale) karst basin of the central USA. Long-term (25-years) of daily streamflow data collected at seven nested U.S. Geological Survey flow gaging sites were used to calibrate and validate SWAT. Annual estimates of spring flow contributions from dominant springs were disintegrated to daily timeseries\u27 dependent on baseflow separation results calculated from observed streamflow data. Model calibration results of daily streamflow showed percent bias (PBAIS) ranged from 1.1 to 19.3%, and Nash-Sutcliff efficiency (NSE) values ranged from 0.25 to 0.63. Model validation results of daily streamflow showed PBIAS ranged from −5.1 to −32.8%, and NSE values ranged from 0.44 to 0.78. Results highlighted potential critical source areas of pollutant loading from pasture sources. Simulated overland (i.e. not including channel processes) sediment, TN, and TP yields were 108 Mg km yr , 14.1 kg ha yr , 0.68 kg ha yr , respectively, at the HUC-8 spatial scale. The greatest N and P nutrient yields were associated with a waste water treatment plant (WWTP), and WWTP loading contributions persisted over 100 km downstream ultimately accounting for approximately 37% of TP loading contributions at the basin outlet. Results have implications for end users tasked with creating physically realistic models of karst watershed hydrology using SWAT, and point to an ongoing need for improved sub-surface storage and flow routing SWAT modeling routines
SNAPSHOT USA 2019: a coordinated national camera trap survey of the United States
No full textWith the accelerating pace of global change, it is imperative that we obtain rapid inventories of the status and distribution of wildlife for ecological inferences and conservation planning. To address this challenge, we launched the SNAPSHOT USA project, a collaborative survey of terrestrial wildlife populations using camera traps across the United States. For our first annual survey, we compiled data across all 50 states during a 14-week period (17 August–24 November of 2019). We sampled wildlife at 1,509 camera trap sites from 110 camera trap arrays covering 12 different ecoregions across four development zones. This effort resulted in 166,036 unique detections of 83 species of mammals and 17 species of birds. All images were processed through the Smithsonian’s eMammal camera trap data repository and included an expert review phase to ensure taxonomic accuracy of data, resulting in each picture being reviewed at least twice. The results represent a timely and standardized camera trap survey of the United States. All of the 2019 survey data are made available herein. We are currently repeating surveys in fall 2020, opening up the opportunity to other institutions and cooperators to expand coverage of all the urban–wild gradients and ecophysiographic regions of the country. Future data will be available as the database is updated at eMammal.si.edu/snapshot-usa, as will future data paper submissions. These data will be useful for local and macroecological research including the examination of community assembly, effects of environmental and anthropogenic landscape variables, effects of fragmentation and extinction debt dynamics, as well as species-specific population dynamics and conservation action plans. There are no copyright restrictions; please cite this paper when using the data for publication
IDCube lite: Free interactive discovery cube software for multi- and hyperspectral applications
No full textMulti- and hyperspectral imaging modalities encompass a growing number of spectral techniques that find many applications in geospatial, biomedical, machine vision and other fields. The rapidly increasing number of applications requires convenient easy-to-navigate software that can be used by new and experienced users to analyse data, and develop, apply and deploy novel algorithms. Herein, we present our platform, IDCube Lite, an Interactive Discovery Cube that performs essential operations in hyperspectral data analysis to realise the full potential of spectral imaging. The strength of the software lies in its interactive features that enable the users to optimise parameters and obtain visual input for the user in a way not previously accessible with other software packages. The entire software can be operated without any prior programming skills allowing interactive sessions of raw and processed data. IDCube Lite, a free version of the software described in the paper, has many benefits compared to existing packages and offers structural flexibility to discover new, hidden features that allow users to integrate novel computational methods