Student Presentations
Oral Presentations
The Importance of Understanding Alternative Biological Nitrogen Fixation
Jack Rigsbee, Wake Forest University
In the age of modernized food production, obtaining effective yields via the use of ammonia-containing fertilizers continues to increase in importance. One mechanism by which ammonia (NH3) is produced for fertilizers is the energy expensive Haber-Bosch process. Thus, exploring alternatives such as biological nitrogen fixation could provide environmental benefits. Azotobacter vinelandii is a model for nitrogen fixation, reducing dinitrogen into ammonia by the enzyme nitrogenase. This bacterium is unique because it produces all three known nitrogenases. The primary nitrogenase includes a molybdenum-containing cofactor encoded by the nif operon, while the two “alternative” nitrogenase systems are based on vanadium or iron in their cofactors, encoded by vnf and anf operons, respectively. One essential aspect of these nitrogenases includes assembling and utilizing Fe-S clusters, a process promoted by nifU, vnfU, or anfU gene products. The goals of this project are to genetically modify the A. vinelandii genome to examine systems containing one functional form of these genes and investigate the potential transfer of Fe-S clusters between nitrogenases. Here, we genetically inactivate nitrogenase-encoding genes to observe growth effects in differing metal-containing conditions. Additionally, biochemical formation of Fe-S clusters on NifU was performed in preparation for examination of potential cluster transfer. Overall, we present multiple successfully constructed nitrogenase-mutant strains (and subsequent growth characteristics). These genetic experiments complement in vitro characterization of Fe-S cluster assembly, with and without reducing agents. These results confirm the necessity of specific Fe-S cluster genes when in differing media and set the stage for experimentation to understand cluster transfer capabilities.
Jack Rigsbee, Wake Forest University
In the age of modernized food production, obtaining effective yields via the use of ammonia-containing fertilizers continues to increase in importance. One mechanism by which ammonia (NH3) is produced for fertilizers is the energy expensive Haber-Bosch process. Thus, exploring alternatives such as biological nitrogen fixation could provide environmental benefits. Azotobacter vinelandii is a model for nitrogen fixation, reducing dinitrogen into ammonia by the enzyme nitrogenase. This bacterium is unique because it produces all three known nitrogenases. The primary nitrogenase includes a molybdenum-containing cofactor encoded by the nif operon, while the two “alternative” nitrogenase systems are based on vanadium or iron in their cofactors, encoded by vnf and anf operons, respectively. One essential aspect of these nitrogenases includes assembling and utilizing Fe-S clusters, a process promoted by nifU, vnfU, or anfU gene products. The goals of this project are to genetically modify the A. vinelandii genome to examine systems containing one functional form of these genes and investigate the potential transfer of Fe-S clusters between nitrogenases. Here, we genetically inactivate nitrogenase-encoding genes to observe growth effects in differing metal-containing conditions. Additionally, biochemical formation of Fe-S clusters on NifU was performed in preparation for examination of potential cluster transfer. Overall, we present multiple successfully constructed nitrogenase-mutant strains (and subsequent growth characteristics). These genetic experiments complement in vitro characterization of Fe-S cluster assembly, with and without reducing agents. These results confirm the necessity of specific Fe-S cluster genes when in differing media and set the stage for experimentation to understand cluster transfer capabilities.
Federated Learning for Single Cell Transcriptomics
Nathan Whitener, Wake Forest University
Experimental methods to collect the gene expression data have improved in recent years. These improved methods allow for the analysis of gene expression at a single-cell level, using next-generation sequencing. To date, these experiments generated massive high-dimensional datasets, which are mined for insights into the composition of individual cells in different developmental stages and in different diseases, such as COVID-19 and cancer, for example. We developed several new computational tools, including a deep learning Feed Forward Neural Network (FFNN), to classify single cell gene expression data into unique categories of cell types. In computational experiments on 14 benchmark datasets, ranging in size from 3005 to over 300,000 cells, FFNN performance was high, reaching normalized mutual information scores up to 1.0 and Cohen’s Kappa scores up to 0.86. To train accurate FFNN models, large datasets of single cell gene expression data must be aggregated first and stored in a centralized location. This places a significant burden on the network bandwidth and computer storage because these datasets are acquired in different laboratories around the world. Moreover, centralized data storage and processing are impractical and vulnerable to data privacy issues. Hence, we propose to develop, implement and test a federated deep learning framework. Federated learning is the process of training algorithms from a set of decentralized data locations, this could be computer storage at different laboratories, or even personal computer devices. Hence, the data need not to be aggregated into a centralized location. Instead, the model's parameters are derived locally and used to tune a common model. By removing the need for transfer of data between different storage locations, we will train powerful models without compromising their data privacy. Additionally, the federated learning approach will allow us to overcome data selection biases that frequently occur in single cell studies. As a proof of concept, we test our federated framework in the classification of sex-specific tissue types in scRNA-sequencing data.
Nathan Whitener, Wake Forest University
Experimental methods to collect the gene expression data have improved in recent years. These improved methods allow for the analysis of gene expression at a single-cell level, using next-generation sequencing. To date, these experiments generated massive high-dimensional datasets, which are mined for insights into the composition of individual cells in different developmental stages and in different diseases, such as COVID-19 and cancer, for example. We developed several new computational tools, including a deep learning Feed Forward Neural Network (FFNN), to classify single cell gene expression data into unique categories of cell types. In computational experiments on 14 benchmark datasets, ranging in size from 3005 to over 300,000 cells, FFNN performance was high, reaching normalized mutual information scores up to 1.0 and Cohen’s Kappa scores up to 0.86. To train accurate FFNN models, large datasets of single cell gene expression data must be aggregated first and stored in a centralized location. This places a significant burden on the network bandwidth and computer storage because these datasets are acquired in different laboratories around the world. Moreover, centralized data storage and processing are impractical and vulnerable to data privacy issues. Hence, we propose to develop, implement and test a federated deep learning framework. Federated learning is the process of training algorithms from a set of decentralized data locations, this could be computer storage at different laboratories, or even personal computer devices. Hence, the data need not to be aggregated into a centralized location. Instead, the model's parameters are derived locally and used to tune a common model. By removing the need for transfer of data between different storage locations, we will train powerful models without compromising their data privacy. Additionally, the federated learning approach will allow us to overcome data selection biases that frequently occur in single cell studies. As a proof of concept, we test our federated framework in the classification of sex-specific tissue types in scRNA-sequencing data.
The Removal of Endogenous S-Adenosyl-L-Methionine from DNA Methyltransferase 3A
Joe McPherson, Wake Forest University
In humans, DNA methylation is a crucial step in embryogenesis, neurological function, and many other biological processes. DNA methyltransferase 3A (DNMT3A) is responsible for de novo DNA methylation in mammals and requires S-Adenosyl-L-Methionine (SAM) as the methyl donor. Due to its role in the onset of many cancers, DNMT3A is a potential target for therapeutic approaches that aim to diminish unregulated DNA methylation. Previous work in the Comstock lab revealed that a N-mustard analog of SAM inhibits DNMT3A. To further explore the effectiveness of the analog as a DNMT3A inhibitor in vitro, this research aimed to remove endogenous SAM that is co-purified with DNMT3A. Results presented here demonstrate that the proposed experiment to consume endogenous SAM must overcome challenges surrounding overall enzyme purity and maintaining enzymatic activity.
Joe McPherson, Wake Forest University
In humans, DNA methylation is a crucial step in embryogenesis, neurological function, and many other biological processes. DNA methyltransferase 3A (DNMT3A) is responsible for de novo DNA methylation in mammals and requires S-Adenosyl-L-Methionine (SAM) as the methyl donor. Due to its role in the onset of many cancers, DNMT3A is a potential target for therapeutic approaches that aim to diminish unregulated DNA methylation. Previous work in the Comstock lab revealed that a N-mustard analog of SAM inhibits DNMT3A. To further explore the effectiveness of the analog as a DNMT3A inhibitor in vitro, this research aimed to remove endogenous SAM that is co-purified with DNMT3A. Results presented here demonstrate that the proposed experiment to consume endogenous SAM must overcome challenges surrounding overall enzyme purity and maintaining enzymatic activity.
Numerical study of Trailing Vortices behind a Moving Airplane
Justing Patrick, North Carolina Agricultural and Technical State University
Airplane trailing vortices are the circular patterns of rotating air that are left behind a plane. This rotating air plays an important in the lift and drag of the airplane wings, as well as poses major safety issues to other planes next to them. Previous research has studied the trailing vortices analytically (Kuhn and Nielsen, 1972, 10th Aerospace Sciences Meeting) and experimentally (Pino, Parras, Felli,and Fernandez-Feria 2011, Ameri- can Institute of Physics). In this study, we use the vortex sheet method to examine the trailing vortices. The model is using a one-dimensional sheet to model the two-dimensional phenomenon. The model is coded using MATLAB. The parameters that are being examined include the smoothing parameter δ, the number of points on the sheet, and the time step ∆t. We will study the accuracy and efficiency of the model first, then we will examine how these parameters affect the formation and evolution of the vortex sheet.
Justing Patrick, North Carolina Agricultural and Technical State University
Airplane trailing vortices are the circular patterns of rotating air that are left behind a plane. This rotating air plays an important in the lift and drag of the airplane wings, as well as poses major safety issues to other planes next to them. Previous research has studied the trailing vortices analytically (Kuhn and Nielsen, 1972, 10th Aerospace Sciences Meeting) and experimentally (Pino, Parras, Felli,and Fernandez-Feria 2011, Ameri- can Institute of Physics). In this study, we use the vortex sheet method to examine the trailing vortices. The model is using a one-dimensional sheet to model the two-dimensional phenomenon. The model is coded using MATLAB. The parameters that are being examined include the smoothing parameter δ, the number of points on the sheet, and the time step ∆t. We will study the accuracy and efficiency of the model first, then we will examine how these parameters affect the formation and evolution of the vortex sheet.
Dynamics of a Rolling Disk
John Turnage, Wake Forest University
In this talk I will present a study of the optimal time of flight of a falling rolling disk on a surface of revolution. This is a twist on the classic problem of a disk rolling without slipping on the plane, which is a canonical example of a nonholonomic dynamical system. Such systems are characterized by the presence of non-integrable constraints on the velocities, which results in path-dependent behavior. Geometrically, these constraints appear as a distribution on the tangent bundle to the configuration manifold of the system. Optimal control problems on such systems are particularly interesting, since the distribution is non-involutive. Briefly, this means that the distribution defined by the nonholonomic constraints is not closed under the Lie bracket operation on a set of spanning vector-fields. This lack of closure results in a larger attainable set of configurations than would be possible if one only naively considered integral curves along the generalized coordinate curves
John Turnage, Wake Forest University
In this talk I will present a study of the optimal time of flight of a falling rolling disk on a surface of revolution. This is a twist on the classic problem of a disk rolling without slipping on the plane, which is a canonical example of a nonholonomic dynamical system. Such systems are characterized by the presence of non-integrable constraints on the velocities, which results in path-dependent behavior. Geometrically, these constraints appear as a distribution on the tangent bundle to the configuration manifold of the system. Optimal control problems on such systems are particularly interesting, since the distribution is non-involutive. Briefly, this means that the distribution defined by the nonholonomic constraints is not closed under the Lie bracket operation on a set of spanning vector-fields. This lack of closure results in a larger attainable set of configurations than would be possible if one only naively considered integral curves along the generalized coordinate curves
Modeling Sea Level Rise in Florida: An Exploratory Study
Hanna Vaidya, Wake Forest University
Coastal sea level rise is an imminent issue due to potential sea level underestimations, outdated climate reports, and changes to be made to local infrastructure planning. Here in this study, we will focus on sea level rise in the state of Florida. In order to attempt to model local sea level rise, we examine altimetry, temperature and salinity, El Niño Southern Oscillation (ENSO) 3.4, and tide gauge data to fit a model that explains sea level changes in coastal Florida. We analyzed rates and variances of sea surface height anomalies (SSHA) and compare regional and global measurements to local. We modeled sea level changes using both multiple regression and generalized additive (GAM) approaches. The optimal/appropriate model is a GAM with year, global mean sea level (GMSL), regional SSHA, water temperature and salinity, and ENSO as predictors. Future work should be focused on extending the GAM by including other factors such as average monthly winds, atmospheric pressures, and coastal currents
Hanna Vaidya, Wake Forest University
Coastal sea level rise is an imminent issue due to potential sea level underestimations, outdated climate reports, and changes to be made to local infrastructure planning. Here in this study, we will focus on sea level rise in the state of Florida. In order to attempt to model local sea level rise, we examine altimetry, temperature and salinity, El Niño Southern Oscillation (ENSO) 3.4, and tide gauge data to fit a model that explains sea level changes in coastal Florida. We analyzed rates and variances of sea surface height anomalies (SSHA) and compare regional and global measurements to local. We modeled sea level changes using both multiple regression and generalized additive (GAM) approaches. The optimal/appropriate model is a GAM with year, global mean sea level (GMSL), regional SSHA, water temperature and salinity, and ENSO as predictors. Future work should be focused on extending the GAM by including other factors such as average monthly winds, atmospheric pressures, and coastal currents
Optimal intervention strategies to minimize spread of infectious diseases and economic impact on a dynamic small-world network
Minato Hiraoka, Wake Forest University
Danielle DaSilva, Elon University
We investigate the economic impacts of pharmaceutical and non-pharmaceutical interventions on a labor force during an epidemic. Specifically, we study an optimal control problem on a dynamic SIV-type small world network model with the controls corresponding to vaccinations, lockdown orders, and other intervention strategies. The cost functional utilized is a Cobb-Douglas production function measuring labor productivity as well as a functional measuring the cost of treating the disease. Using Pontryagin’s maximum principle we can numerically approximate the optimal control strategy which allows us to determine the optimal level of intervention. These methods illustrate the usefulness of this approach to inform policymakers and better equip society for emerging epidemics
Minato Hiraoka, Wake Forest University
Danielle DaSilva, Elon University
We investigate the economic impacts of pharmaceutical and non-pharmaceutical interventions on a labor force during an epidemic. Specifically, we study an optimal control problem on a dynamic SIV-type small world network model with the controls corresponding to vaccinations, lockdown orders, and other intervention strategies. The cost functional utilized is a Cobb-Douglas production function measuring labor productivity as well as a functional measuring the cost of treating the disease. Using Pontryagin’s maximum principle we can numerically approximate the optimal control strategy which allows us to determine the optimal level of intervention. These methods illustrate the usefulness of this approach to inform policymakers and better equip society for emerging epidemics
Artificial Intelligence-Based Analytics for Impacts of COVID-19 and Online Learning on College Students’ Mental Health
Colin Varady, Wake Forest University
COVID-19, the disease caused by the novel coronavirus (SARS-CoV-2), was first found in Wuhan, China late in the December of 2019. Not long after that the virus spread worldwide and was declared a pandemic by the World Health Organization in March 2020. This caused many changes around the world and in the United States. One of these changes was the shift towards online learning. In this paper, we seek to understand how the COVID-19 pandemic and online learning impact college students’ emotional wellbeing. To do this we use several machine learning statistical models to analyze data collected by the Faculty of Public Administration at the University of Ljubljana, Slovenia in conjunction with an international consortium of universities, other higher education institutions and students’ associations. Our results indicate that learning modality (face-to-face, online synchronous, online asynchronous, etc.) is the main predictor of students’ emotional wellbeing, followed by financial security. Factors such as satisfaction with their university’s and government’s handling of the pandemic are also important predictors
Colin Varady, Wake Forest University
COVID-19, the disease caused by the novel coronavirus (SARS-CoV-2), was first found in Wuhan, China late in the December of 2019. Not long after that the virus spread worldwide and was declared a pandemic by the World Health Organization in March 2020. This caused many changes around the world and in the United States. One of these changes was the shift towards online learning. In this paper, we seek to understand how the COVID-19 pandemic and online learning impact college students’ emotional wellbeing. To do this we use several machine learning statistical models to analyze data collected by the Faculty of Public Administration at the University of Ljubljana, Slovenia in conjunction with an international consortium of universities, other higher education institutions and students’ associations. Our results indicate that learning modality (face-to-face, online synchronous, online asynchronous, etc.) is the main predictor of students’ emotional wellbeing, followed by financial security. Factors such as satisfaction with their university’s and government’s handling of the pandemic are also important predictors
A Detection of Artisanal Scale Mining-Related Objects from Drone Imagery using YOLOv5
Isaias Bahena, Robert Langefeld, Alice Li, Isabella Tian, Wake Forest University
Daniel Langefeld, Wesleyan Christian Academy
The ability to detect illegal mining operations is central to natural resource management and territorial governance. The activity can be difficult to detect and quantify, making methods that can reveal illegal mining in high demand. Mining activity is directly connected to the kinds of objects present at these sites, typically equipment, crops, and changes to geomorphology. As a solution to these challenges, we propose using YOLOv5, a modern deep learning object detection algorithm, to locate these objects within high-resolution (drone+satellite) remote sensing images which were obtained from regions where mining is suspected to be occurring. In this paper, we demonstrate that YOLOv5 has the ability to accurately detect this kind of activity from drone images and provide counts of these objects within each image.
Isaias Bahena, Robert Langefeld, Alice Li, Isabella Tian, Wake Forest University
Daniel Langefeld, Wesleyan Christian Academy
The ability to detect illegal mining operations is central to natural resource management and territorial governance. The activity can be difficult to detect and quantify, making methods that can reveal illegal mining in high demand. Mining activity is directly connected to the kinds of objects present at these sites, typically equipment, crops, and changes to geomorphology. As a solution to these challenges, we propose using YOLOv5, a modern deep learning object detection algorithm, to locate these objects within high-resolution (drone+satellite) remote sensing images which were obtained from regions where mining is suspected to be occurring. In this paper, we demonstrate that YOLOv5 has the ability to accurately detect this kind of activity from drone images and provide counts of these objects within each image.
Poster Presentations
Are Drosophlia Guttifera Moving from Generalist to Specialist?
Grace Kropelin, Appalachian State University
The evolutionary arms race between insects and their plant/fungal hosts has been intensely studied. Hosts that produce highly toxic compounds typically exclude generalist feeders that are predicted to evolve the ability to detoxify closely related chemical compounds. However, some fly species in the immigrans-tripunctata radiation of Drosophila use both deadly toxic mushroom species (e.g., the death cap and destroying angel mushrooms) as well as edible species as developmental hosts. While these species are classified as generalists, we hypothesize that the one species (Drosophila guttifera) is transitioning from a generalist to a specialist feeding/ developmental strategy. To address this question, we reared larvae from two D. guttifera genotypes on diets with and without a single toxin or a mix of toxins extracted from the death-cap mushroom. We measured several fitness phenotypes (e.g., survival to adult, thorax length, adult weight) to assess whether the larvae exhibited increased performance on diets with the toxins. We also conducted oviposition preference assays in the two lines to determine if females preferred to lay their eggs on toxic mushrooms instead of edible mushrooms or fruits. Our results provide greater context for the associations between this D. guttifera and its host fungi.
Grace Kropelin, Appalachian State University
The evolutionary arms race between insects and their plant/fungal hosts has been intensely studied. Hosts that produce highly toxic compounds typically exclude generalist feeders that are predicted to evolve the ability to detoxify closely related chemical compounds. However, some fly species in the immigrans-tripunctata radiation of Drosophila use both deadly toxic mushroom species (e.g., the death cap and destroying angel mushrooms) as well as edible species as developmental hosts. While these species are classified as generalists, we hypothesize that the one species (Drosophila guttifera) is transitioning from a generalist to a specialist feeding/ developmental strategy. To address this question, we reared larvae from two D. guttifera genotypes on diets with and without a single toxin or a mix of toxins extracted from the death-cap mushroom. We measured several fitness phenotypes (e.g., survival to adult, thorax length, adult weight) to assess whether the larvae exhibited increased performance on diets with the toxins. We also conducted oviposition preference assays in the two lines to determine if females preferred to lay their eggs on toxic mushrooms instead of edible mushrooms or fruits. Our results provide greater context for the associations between this D. guttifera and its host fungi.
Products of Peroxidase-Catalyzed Oxidation of Estrogens
Sarina S. Jackowski, Elon University
Estrogenic carcinogenesis has been widely associated with breast cancer initiation. Studies suggest that mammalian peroxidases catalyze the oxidation of catecholic estrogens to quinone methides, a conjugated organic compound. Quinone methides are proposed to cause toxic DNA modifications. Within these studies, there is a lack of evidence that a quinone is truly the oxidized product of estrogens, as well as a lack of recognition of possible toxic implications of semiquinone intermediates. In this study, oxidized estrogens are examined for their structure and quantity. This can give insight to the oxidative mechanism of mammalian peroxidases when exposed to estrogens, and to the mechanism by which oxidized estrogens may covalently bind to DNA. To analyze these interactions, the oxidation of β-estradiol in the presence of hydrogen peroxide was catalyzed by horseradish peroxidase (HRP) as a standard enzymatic reaction, and then with lactoperoxidase (LPO), an enzyme found in mammary glands. The oxidized compounds were analyzed through thin layer chromatography (TLC) and 1H-NMR. TLC results indicate that both HRP and LPO catalyze the formation of various shared intermediates. 1H-NMR results suggest that HRP forms a catecholic oxidized product, while LPO forms a quinone methide oxidized product. This is consistent with previous studies. Further studies are being conducted to determine the ability of the products to covalently bind to DNA.
Sarina S. Jackowski, Elon University
Estrogenic carcinogenesis has been widely associated with breast cancer initiation. Studies suggest that mammalian peroxidases catalyze the oxidation of catecholic estrogens to quinone methides, a conjugated organic compound. Quinone methides are proposed to cause toxic DNA modifications. Within these studies, there is a lack of evidence that a quinone is truly the oxidized product of estrogens, as well as a lack of recognition of possible toxic implications of semiquinone intermediates. In this study, oxidized estrogens are examined for their structure and quantity. This can give insight to the oxidative mechanism of mammalian peroxidases when exposed to estrogens, and to the mechanism by which oxidized estrogens may covalently bind to DNA. To analyze these interactions, the oxidation of β-estradiol in the presence of hydrogen peroxide was catalyzed by horseradish peroxidase (HRP) as a standard enzymatic reaction, and then with lactoperoxidase (LPO), an enzyme found in mammary glands. The oxidized compounds were analyzed through thin layer chromatography (TLC) and 1H-NMR. TLC results indicate that both HRP and LPO catalyze the formation of various shared intermediates. 1H-NMR results suggest that HRP forms a catecholic oxidized product, while LPO forms a quinone methide oxidized product. This is consistent with previous studies. Further studies are being conducted to determine the ability of the products to covalently bind to DNA.
Abundance, Diversity, and Analysis of Ecologically Important Traits of Southern Appalachian Bumble Bees
Brooke Reutinger, Appalachian State University
Bumble bees are important pollinators that are responsible for the vitality of native plant communities worldwide, and many species have shown population declines in the past twenty years. Despite increased awareness of their plight, scientists are still working to learn basic aspects of their biology. The objective of this project is to understand which environmental factors influence biometrics of ecologically important traits, such as body size and tongue-length, for bumble bees in the Southern Appalachians. We hypothesize that biometrics of these important traits will vary significantly with elevation, latitude, and floral availability, and specifically will be inversely related to higher elevations, northern latitudes, and decreased floral resources due to increased energy expenditures needed in these environments. Furthermore, we hypothesize that the biometrics of medium and short-tongued bees will be less affected by environmental factors than long-tongued bees.
Samples were collected during a Citizen Science Pollinator Inventory that was coordinated in 2019 along the entire length of the Blue Ridge Parkway, NC and VA, in partnership with the National Park Service. Sixty survey sites were randomly selected along the Blue Ridge Parkway. Foraging bees were captured using passive traps that were fluorescently painted to mimic flowers.
Trait biometrics have implications for a number of aspects of bee’s life histories. For example, the larger the bee body size, the longer foraging trips an individual bumble bee can take. Bee foraging distance is directly related to population dynamics, genetics, and life history. Consequences of disturbed foraging distance include reduced pollination for plants, predation, parasitism, nutrient transfer, and seed dispersal. Tongue-length indicates dietary preference, as individuals with longer tongues can access different floral resources. Bumble bee tongue lengths have shown evolutionary sensitivity to abundance of floral resources, with fewer resources resulting in shorter tongues and a more generalized foraging niche. Without having diverse physical traits within bumble bee populations, flower abundance and diversity are at risk of declining. This study examines how the environment influences bumble bee biometrics.
Brooke Reutinger, Appalachian State University
Bumble bees are important pollinators that are responsible for the vitality of native plant communities worldwide, and many species have shown population declines in the past twenty years. Despite increased awareness of their plight, scientists are still working to learn basic aspects of their biology. The objective of this project is to understand which environmental factors influence biometrics of ecologically important traits, such as body size and tongue-length, for bumble bees in the Southern Appalachians. We hypothesize that biometrics of these important traits will vary significantly with elevation, latitude, and floral availability, and specifically will be inversely related to higher elevations, northern latitudes, and decreased floral resources due to increased energy expenditures needed in these environments. Furthermore, we hypothesize that the biometrics of medium and short-tongued bees will be less affected by environmental factors than long-tongued bees.
Samples were collected during a Citizen Science Pollinator Inventory that was coordinated in 2019 along the entire length of the Blue Ridge Parkway, NC and VA, in partnership with the National Park Service. Sixty survey sites were randomly selected along the Blue Ridge Parkway. Foraging bees were captured using passive traps that were fluorescently painted to mimic flowers.
Trait biometrics have implications for a number of aspects of bee’s life histories. For example, the larger the bee body size, the longer foraging trips an individual bumble bee can take. Bee foraging distance is directly related to population dynamics, genetics, and life history. Consequences of disturbed foraging distance include reduced pollination for plants, predation, parasitism, nutrient transfer, and seed dispersal. Tongue-length indicates dietary preference, as individuals with longer tongues can access different floral resources. Bumble bee tongue lengths have shown evolutionary sensitivity to abundance of floral resources, with fewer resources resulting in shorter tongues and a more generalized foraging niche. Without having diverse physical traits within bumble bee populations, flower abundance and diversity are at risk of declining. This study examines how the environment influences bumble bee biometrics.
Basic Characterization and Localization of Important Components of the Human Blood Brain Barrier Proteome in Porcine Cerebrovasculature
Girnar Anand, Wake Forest University
The blood-brain barrier (BBB) is formed by brain capillary endothelial cells (BECs) supported by pericytes and astrocytes. The BBB maintains homeostasis, meets the metabolic demands of the brain and protects the brain against toxic substances circulating in the blood. We aimed to investigate the localization of GLUT-1 (the principal transporter of glucose); LAT-1/S7LCA5 (major transporter of amino acids); Cld-5 (an essential tight junction protein); and PgP (a drug efflux protein)- in an attempt to study and characterize the pig brain and analyze how effective the porcine model is as a translational model to study human cerebrovascular function and disease. While we obtained some positive immunolocalization results for GLUT-1 and PgP, and none for LAT-1 and Cld-5 due to the limited runs of the experiment and limited time of research study- no strong conclusions could be drawn; However, the results do show huge potential and generate reason for further focus and research
Girnar Anand, Wake Forest University
The blood-brain barrier (BBB) is formed by brain capillary endothelial cells (BECs) supported by pericytes and astrocytes. The BBB maintains homeostasis, meets the metabolic demands of the brain and protects the brain against toxic substances circulating in the blood. We aimed to investigate the localization of GLUT-1 (the principal transporter of glucose); LAT-1/S7LCA5 (major transporter of amino acids); Cld-5 (an essential tight junction protein); and PgP (a drug efflux protein)- in an attempt to study and characterize the pig brain and analyze how effective the porcine model is as a translational model to study human cerebrovascular function and disease. While we obtained some positive immunolocalization results for GLUT-1 and PgP, and none for LAT-1 and Cld-5 due to the limited runs of the experiment and limited time of research study- no strong conclusions could be drawn; However, the results do show huge potential and generate reason for further focus and research
A Microwave-Assisted, Two-Step Synthesis of Indolo[3,2-c]quinolines via Fisher Indolization and Oxidative Aromatization
Faisal Yakubu, Winston Salem State University
Herein, we describe a practical two-step synthesis of an Indolo[3,2-c]quinoline scaffold from substituted 4-methoxyquinolines using Fischer indole method and oxidative aromatization in one-pot under Microwave radiation. With this protocol, a biologically active natural product, Isocryptolepine, and its analogues can be readily and efficiently accessed. Also, this method has been efficiently utilized to generate a series of novel pyrrole-containing derivatives of this heterocyclic system.
Faisal Yakubu, Winston Salem State University
Herein, we describe a practical two-step synthesis of an Indolo[3,2-c]quinoline scaffold from substituted 4-methoxyquinolines using Fischer indole method and oxidative aromatization in one-pot under Microwave radiation. With this protocol, a biologically active natural product, Isocryptolepine, and its analogues can be readily and efficiently accessed. Also, this method has been efficiently utilized to generate a series of novel pyrrole-containing derivatives of this heterocyclic system.
Investigation of Electronic Effects of Sn2 Reactions in the Synthesis of 3-Aryl(thio)propanoic acids
Alex Chavez, Winston Salem State University
Aryl thiols are important building blocks in the synthesis of sulfur-containing natural products and pharmaceutically important compounds. We are interested in 3-aryl(thio)-propanoic acids because they are key intermediates for S-heterocycles. This research is focused on the synthesis of 3-aryl(thio)-propanoic acids from aryl thiols via SN2 reactions. The hypothesis is that the electronic withdrawing and donating group on the aromatic ring will affect the reactivity of aryl thiols in SN2 reaction and thus the chemical yields. The scope of aryl thiols will be explored. The results on various electron-donating and electron-withdrawing groups will be presented.
Alex Chavez, Winston Salem State University
Aryl thiols are important building blocks in the synthesis of sulfur-containing natural products and pharmaceutically important compounds. We are interested in 3-aryl(thio)-propanoic acids because they are key intermediates for S-heterocycles. This research is focused on the synthesis of 3-aryl(thio)-propanoic acids from aryl thiols via SN2 reactions. The hypothesis is that the electronic withdrawing and donating group on the aromatic ring will affect the reactivity of aryl thiols in SN2 reaction and thus the chemical yields. The scope of aryl thiols will be explored. The results on various electron-donating and electron-withdrawing groups will be presented.
Synthesis of Sulfur-heterocycles via Conjugate Additions of Grignard Reagents to Thiochromones
Nichele Deese, Winston Salem State University
Grignard reagents is one of the most widely used organometallic reagents in carbon-carbon bond formations in Organic Synthesis. We investigated the reactions of Grignard Reagents to thiochromone catalyzed by copper salts. The hypothesis is that copper salts will affect the conjugate addition of Grignard reagents instead of 1,2-addition to the carbonyl group to allow the synthesis of thiochromanone, an important class of organosulfur compounds with rich bioactivities. Excellent yields of 1,4-adducts – thiochromanones can be isolated (up to 90%) under optimal conditions using copper(I) salts with lithium chloride. A large number of commercially available Grignard reagents can undergo conjugate addition to thiochromones to furnish 1,4-adduct in excellent yields. This approach works well with both alkyl and aromatic Grignard reagents, thus providing a unified synthetic approach to privileged 2-substituted thiochroman-4-ones and a potential valuable precursor for further synthetic applications towards many pharmaceutically active molecules. The use of commercially available or easily prepared Grignard reagents will expedite the synthesis of a large library of thiochromanones for further synthetic applications and biological studies.
Nichele Deese, Winston Salem State University
Grignard reagents is one of the most widely used organometallic reagents in carbon-carbon bond formations in Organic Synthesis. We investigated the reactions of Grignard Reagents to thiochromone catalyzed by copper salts. The hypothesis is that copper salts will affect the conjugate addition of Grignard reagents instead of 1,2-addition to the carbonyl group to allow the synthesis of thiochromanone, an important class of organosulfur compounds with rich bioactivities. Excellent yields of 1,4-adducts – thiochromanones can be isolated (up to 90%) under optimal conditions using copper(I) salts with lithium chloride. A large number of commercially available Grignard reagents can undergo conjugate addition to thiochromones to furnish 1,4-adduct in excellent yields. This approach works well with both alkyl and aromatic Grignard reagents, thus providing a unified synthetic approach to privileged 2-substituted thiochroman-4-ones and a potential valuable precursor for further synthetic applications towards many pharmaceutically active molecules. The use of commercially available or easily prepared Grignard reagents will expedite the synthesis of a large library of thiochromanones for further synthetic applications and biological studies.
Acute Impact of Barefoot Running on Running Economy in Recreational Runners: A Conceptual Framework
Allyson H Butts, Gardner-Webb University
Barefoot (BF) running is associated with biomechanical alterations that improve performance by increasing running economy (RE). The purpose of this study was to examine the impact of acute BF running on RE in recreational runners. Participants (n = 20) completed treadmill trials in conventional running shoes (CRS) and BF at 60% and 80% of VO2max. A two-way repeated measures analysis of variance (p < 0.05) measured the effects of shoe condition on RE. Results were expected to reflect the speed-dependent nature of RE, reduced shoe weight in the BF condition, individualized biomechanical and stride adjustments, and the physiological cost of task novelty. Future directions include expanding population, duration, and number of analyzed biomechanical variables.
Allyson H Butts, Gardner-Webb University
Barefoot (BF) running is associated with biomechanical alterations that improve performance by increasing running economy (RE). The purpose of this study was to examine the impact of acute BF running on RE in recreational runners. Participants (n = 20) completed treadmill trials in conventional running shoes (CRS) and BF at 60% and 80% of VO2max. A two-way repeated measures analysis of variance (p < 0.05) measured the effects of shoe condition on RE. Results were expected to reflect the speed-dependent nature of RE, reduced shoe weight in the BF condition, individualized biomechanical and stride adjustments, and the physiological cost of task novelty. Future directions include expanding population, duration, and number of analyzed biomechanical variables.
Oviposition Site Repellents and Deterrents: Are Gravid Sand Flies Afraid of Predatory Mites?
Tricia Levi, University of North Carolina at Greensboro
The vector-borne disease leishmaniasis, which is transmitted via phlebotomine sand flies, affects millions of people worldwide. The most common sand fly control methods involve chemical insecticides that are harmful to the environment, so biological methods of control are preferred. The Wasserberg lab is currently focusing on a “push-pull” method by deterring or repelling flies away from breeding sites and luring them to lethal traps with attractant compounds. I have begun the process of identifying effective deterrents and repellents based on natural enemies of the sand flies. In this study, I tested the hypothesis that gravid sand flies will be repelled or deterred from laying eggs in the presence of predatory mites identified as Stratiolaelaps scimitus womersley. To test the deterrence hypothesis, I evaluated the oviposition activity of gravid female sand flies via separate oviposition bioassays using live and crushed mites. The number of eggs laid by the flies was counted for each replicate. To test the repellence hypothesis, I conducted attraction bioassays with live and crushed mites using free flight cages and sticky traps. The number of flies that landed on the traps was counted for each replicate. The results of the deterrence experiment showed no effect of mite presence on oviposition response. Repellence experiments are on-going and will be presented during the conference.
Tricia Levi, University of North Carolina at Greensboro
The vector-borne disease leishmaniasis, which is transmitted via phlebotomine sand flies, affects millions of people worldwide. The most common sand fly control methods involve chemical insecticides that are harmful to the environment, so biological methods of control are preferred. The Wasserberg lab is currently focusing on a “push-pull” method by deterring or repelling flies away from breeding sites and luring them to lethal traps with attractant compounds. I have begun the process of identifying effective deterrents and repellents based on natural enemies of the sand flies. In this study, I tested the hypothesis that gravid sand flies will be repelled or deterred from laying eggs in the presence of predatory mites identified as Stratiolaelaps scimitus womersley. To test the deterrence hypothesis, I evaluated the oviposition activity of gravid female sand flies via separate oviposition bioassays using live and crushed mites. The number of eggs laid by the flies was counted for each replicate. To test the repellence hypothesis, I conducted attraction bioassays with live and crushed mites using free flight cages and sticky traps. The number of flies that landed on the traps was counted for each replicate. The results of the deterrence experiment showed no effect of mite presence on oviposition response. Repellence experiments are on-going and will be presented during the conference.
Calcium Channel Blockers Mute Eisenia Hortensis Irritation to Noxious Chemicals
Sofia Lopresti, Wake Forest University
Our lab studies chemoreception in the earthworm, Eisenia hortensis. We have developed an assay which involves stimulating the earthworm with an irritating chemical and measuring the amount of exudate it secretes. With this assay, we can determine which chemicals are irritating to earthworms. We do not know the mechanism by which the exudate is produced and secreted, but we know it is triggered by noxious mechanical or chemical stimuli. AITC, a noxious chemical found in mustard and known to stimulate TRPA1 receptors, reliably produces a large amount of exudate. In this experiment, we used our exudate irritation assay to look for mu opioid receptors in earthworms. It has been thought that invertebrates do not have mu-opioid receptors, however some studies on earthworms suggest otherwise. We hypothesized that activating a mu opioid agonist might lessen the effect of the irritating AITC. We tested an over-the-counter mu opioid agonist, loperamide. In humans, loperamide is not significantly absorbed by the gut and has no effect on the central nervous system. Administration of loperamide significantly decreased the amount of exudate secreted in the presence of AITC (p=0.018, Tukey HSD), suggesting that the mu opioid receptor may be playing a role in muting irritation in earthworms. However, naltrexone, a mu opioid receptor antagonist, had no effect on loperamide's ability to lower exudate secretion (p=982, Turkey HSD). Loperamide can also act as a calcium channel blocker so we decided to look at the effects of other calcium channel blockers on the amount of exudate secreted in the presence of AITC. We tested the effect of the calcium channel blockers lanthanum chloride, ruthenium red, and verapamil on exudate secretion in the presence of AITC. When exposed to the calcium channel blockers, earthworms secrete significantly less exudate than when no blocker was applied (p=2.99e-05, one-way ANOVA). The blockers lanthanum chloride (p=0.014, Tukey HSD), ruthenium red (p=0.048, Tukey HSD), and verapamil (p=0.012, Tukey HSD) muted the earthworm’s response to irritation significantly. Taken together our results suggest that earthworms do not possess mu opioid receptors and that calcium plays an important role in the secretion of exudate in response to noxious stimuli.
Sofia Lopresti, Wake Forest University
Our lab studies chemoreception in the earthworm, Eisenia hortensis. We have developed an assay which involves stimulating the earthworm with an irritating chemical and measuring the amount of exudate it secretes. With this assay, we can determine which chemicals are irritating to earthworms. We do not know the mechanism by which the exudate is produced and secreted, but we know it is triggered by noxious mechanical or chemical stimuli. AITC, a noxious chemical found in mustard and known to stimulate TRPA1 receptors, reliably produces a large amount of exudate. In this experiment, we used our exudate irritation assay to look for mu opioid receptors in earthworms. It has been thought that invertebrates do not have mu-opioid receptors, however some studies on earthworms suggest otherwise. We hypothesized that activating a mu opioid agonist might lessen the effect of the irritating AITC. We tested an over-the-counter mu opioid agonist, loperamide. In humans, loperamide is not significantly absorbed by the gut and has no effect on the central nervous system. Administration of loperamide significantly decreased the amount of exudate secreted in the presence of AITC (p=0.018, Tukey HSD), suggesting that the mu opioid receptor may be playing a role in muting irritation in earthworms. However, naltrexone, a mu opioid receptor antagonist, had no effect on loperamide's ability to lower exudate secretion (p=982, Turkey HSD). Loperamide can also act as a calcium channel blocker so we decided to look at the effects of other calcium channel blockers on the amount of exudate secreted in the presence of AITC. We tested the effect of the calcium channel blockers lanthanum chloride, ruthenium red, and verapamil on exudate secretion in the presence of AITC. When exposed to the calcium channel blockers, earthworms secrete significantly less exudate than when no blocker was applied (p=2.99e-05, one-way ANOVA). The blockers lanthanum chloride (p=0.014, Tukey HSD), ruthenium red (p=0.048, Tukey HSD), and verapamil (p=0.012, Tukey HSD) muted the earthworm’s response to irritation significantly. Taken together our results suggest that earthworms do not possess mu opioid receptors and that calcium plays an important role in the secretion of exudate in response to noxious stimuli.
Investigating the Effects of Carbon Nanodots on Inflammation in Mouse Liver Tissue
Shafaq Ahmed, University of North Carolina at Greensboro
Atherosclerosis is known to be the main cause of cardiovascular disease and is regulated by pro-inflammatory molecules such as macrophage chemoattractant protein-1 (MCP-1), interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6). The liver is known to be the main metabolic organ that is involved in metabolizing abnormal lipid metabolism and complex inflammatory disease. A new class of nanoparticles, Carbon Nanodots (CNDs), have been expressed as potential candidates for bioimaging, biosensing, and drug delivery. There have also been some studies that found them to have anti-inflammatory effects, however the effect of CNDs in the liver have not been investigated. In this study I studied the impact of CNDs on TNF--mediated expressions of pro-inflammatory genes in mouse liver tissues. Liver tissue samples from C57BL/6 mice were used, which were previously treated with either TNF-(25g/kg bw), CNDs (2.5 mg/kg CNDs), both TNF- and CNDs, or neither to serve as the control. Our current results have shown that TNF- increased the expression of pro-inflammatory molecules and decreased the expression of anti-inflammatory molecules. Other experimental data are still in progress, which will allow for a better understanding of the actions of CNDs on liver inflammation in vivo.
Shafaq Ahmed, University of North Carolina at Greensboro
Atherosclerosis is known to be the main cause of cardiovascular disease and is regulated by pro-inflammatory molecules such as macrophage chemoattractant protein-1 (MCP-1), interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6). The liver is known to be the main metabolic organ that is involved in metabolizing abnormal lipid metabolism and complex inflammatory disease. A new class of nanoparticles, Carbon Nanodots (CNDs), have been expressed as potential candidates for bioimaging, biosensing, and drug delivery. There have also been some studies that found them to have anti-inflammatory effects, however the effect of CNDs in the liver have not been investigated. In this study I studied the impact of CNDs on TNF--mediated expressions of pro-inflammatory genes in mouse liver tissues. Liver tissue samples from C57BL/6 mice were used, which were previously treated with either TNF-(25g/kg bw), CNDs (2.5 mg/kg CNDs), both TNF- and CNDs, or neither to serve as the control. Our current results have shown that TNF- increased the expression of pro-inflammatory molecules and decreased the expression of anti-inflammatory molecules. Other experimental data are still in progress, which will allow for a better understanding of the actions of CNDs on liver inflammation in vivo.
The Prevalence of the Female Athlete in NCAA Division I Collegiate Female Athletes
Jasmine Jones, Gardner-Webb University
The Female Athlete Triad is a system of 3 components that afflicts up to 60% of female athletes (McArdle et al., 2015). The components have been defined by the American College of Sports Medicine (ACSM) as low energy availability, menstrual dysfunction, and low bone mineral density. Due to the intense energy expenditure during training and the age at which intense training begins in order to compete at the collegiate level, NCAA Division I female athletes are at a heightened risk for the Female Athlete Triad. This study used the LEAF-Q survey to collect risk assessment levels from each participant. The purpose of this study was to examine the prevalence of the Female Athlete Triad in NCAA Division I female athletes using a large and diverse sample.
Jasmine Jones, Gardner-Webb University
The Female Athlete Triad is a system of 3 components that afflicts up to 60% of female athletes (McArdle et al., 2015). The components have been defined by the American College of Sports Medicine (ACSM) as low energy availability, menstrual dysfunction, and low bone mineral density. Due to the intense energy expenditure during training and the age at which intense training begins in order to compete at the collegiate level, NCAA Division I female athletes are at a heightened risk for the Female Athlete Triad. This study used the LEAF-Q survey to collect risk assessment levels from each participant. The purpose of this study was to examine the prevalence of the Female Athlete Triad in NCAA Division I female athletes using a large and diverse sample.
Microhabitat selection in Sand Fly Larvae; Preference to Different Types of Rearing Material
Nadia Maji, University of North Carolina at Greensboro
Leishmaniasis is a parasitic disease transmitted by sandflies. Leishmaniasis kills between 20,000 and 30,000 people yearly. In the absence of parental care, oviposition (egg laying) site selection by gravid female sand flies is considered to be the most important fitness enhancing decision a sand fly mother would do. Indeed, it was shown that females are attracted to sites containing appropriate larval rearing conditions. However, it is not known if larvae have the capability of adjusting their location within the oviposition sites to locales containing appropriate food. In a previous study, using Phlebotomus papatasi as a model system, it was shown that adult females preferred laying eggs inrearing medium containing 2nd/3rd instar, followed by “expired medium” (rearing medium from which all larvae have matured), and “fresh larval food”, respectively. In my study, I hypothesized that the larvae would show similar preferences to these rearing medium types. Ten first instar larvae were placed in the center of small Nalgene jars, with a plaster-of-Parris base, with 2-3 drops of an aqueous solution of one medium type on one side and water as the control on the other side. The larvae were left for 24 hours in an environmental chamber (26C, RH = 80%)and then the results were recorded. In all cases, larvae appeared to prefer the rearing media over the water control. However, the fresh media appeared be the most attractive. We are currently evaluating the microhabitat selection of 2nd/3rd and fourth instar larvae. My current results (based on 1st instar larvae) suggest that larvae are capable of adjusting the location of their rearing sites by dispersing into sites containing their required nutrients.
Nadia Maji, University of North Carolina at Greensboro
Leishmaniasis is a parasitic disease transmitted by sandflies. Leishmaniasis kills between 20,000 and 30,000 people yearly. In the absence of parental care, oviposition (egg laying) site selection by gravid female sand flies is considered to be the most important fitness enhancing decision a sand fly mother would do. Indeed, it was shown that females are attracted to sites containing appropriate larval rearing conditions. However, it is not known if larvae have the capability of adjusting their location within the oviposition sites to locales containing appropriate food. In a previous study, using Phlebotomus papatasi as a model system, it was shown that adult females preferred laying eggs inrearing medium containing 2nd/3rd instar, followed by “expired medium” (rearing medium from which all larvae have matured), and “fresh larval food”, respectively. In my study, I hypothesized that the larvae would show similar preferences to these rearing medium types. Ten first instar larvae were placed in the center of small Nalgene jars, with a plaster-of-Parris base, with 2-3 drops of an aqueous solution of one medium type on one side and water as the control on the other side. The larvae were left for 24 hours in an environmental chamber (26C, RH = 80%)and then the results were recorded. In all cases, larvae appeared to prefer the rearing media over the water control. However, the fresh media appeared be the most attractive. We are currently evaluating the microhabitat selection of 2nd/3rd and fourth instar larvae. My current results (based on 1st instar larvae) suggest that larvae are capable of adjusting the location of their rearing sites by dispersing into sites containing their required nutrients.
Engaging Youth in Substance Misuse Prevention within the State Prevention System: Provider Perspectives
Rebekah Lassiter, Wake Forest University
Youth can be engaged in community and health promotion efforts in a variety of ways (Ozer et al., 2020). Engaging youth meaningfully in developing and implementing substance misuse prevention efforts can improve how youth-targeted prevention is received (Dunne et al., 2017). Partnering with youth has documented benefits for youth and potential to improve prevention efforts; however, engaging youth in prevention is rare and challenging. This study examines the current use, and potential, of youth engagement in substance misuse prevention settings. This project presents findings from 13 semi-structured interviews with prevention providers across one state. Interviewees were asked how they engage youth in their prevention efforts including the benefits and challenges and what they need to increase youth voice in their prevention work. Following both thematic and codebook approaches to qualitative analysis (Terry et al., 2017), four “primary readers” read 3-4 assigned transcripts, wrote a memo capturing overall impressions, and coded text into categories based on predefined categories of interest. Through a series of group discussions, themes were identified and refined and the team organized the themes into findings. The three main findings were: (1) a lack of clarity among providers about youth-targeted prevention and youth-engaged prevention, (2) the need for more training and knowledge-sharing across prevention providers and the youth they work with, and (3) challenges common to all youth engagement work (e.g., how to find and retain diverse youth partners, how best to engage them) as well as specific to substance misuse prevention (rarely seen as a “cool” topic for youth; lack of youth training in substance misuse and prevention models; restrictions on prevention funding constrains implementation of ideas generated by youth). Findings will be discussed in light of creating empowered youth settings.
Rebekah Lassiter, Wake Forest University
Youth can be engaged in community and health promotion efforts in a variety of ways (Ozer et al., 2020). Engaging youth meaningfully in developing and implementing substance misuse prevention efforts can improve how youth-targeted prevention is received (Dunne et al., 2017). Partnering with youth has documented benefits for youth and potential to improve prevention efforts; however, engaging youth in prevention is rare and challenging. This study examines the current use, and potential, of youth engagement in substance misuse prevention settings. This project presents findings from 13 semi-structured interviews with prevention providers across one state. Interviewees were asked how they engage youth in their prevention efforts including the benefits and challenges and what they need to increase youth voice in their prevention work. Following both thematic and codebook approaches to qualitative analysis (Terry et al., 2017), four “primary readers” read 3-4 assigned transcripts, wrote a memo capturing overall impressions, and coded text into categories based on predefined categories of interest. Through a series of group discussions, themes were identified and refined and the team organized the themes into findings. The three main findings were: (1) a lack of clarity among providers about youth-targeted prevention and youth-engaged prevention, (2) the need for more training and knowledge-sharing across prevention providers and the youth they work with, and (3) challenges common to all youth engagement work (e.g., how to find and retain diverse youth partners, how best to engage them) as well as specific to substance misuse prevention (rarely seen as a “cool” topic for youth; lack of youth training in substance misuse and prevention models; restrictions on prevention funding constrains implementation of ideas generated by youth). Findings will be discussed in light of creating empowered youth settings.
Measurements of Phenoloxidase Activity After Immune Priming in Acheta Domesticus
Ian Baranyk, Wingate University
Insects are believed to have an entirely innate immune response. This means their immune system does not need time to reach full strength as it would in organisms with an adaptive immune system such as humans. Recent studies, however, show that the insect immune system may indeed possess some adaptive traits. This is illustrated through the phenomenon of immune priming. Immune priming has been shown in different insects including Bombus terrestris (Sadd et al., 2006). This study will use lipopolysaccharides (LPS) from Eschericha coli to elicit immune responses in the house cricket, Acheta domesticus. This will be measured by examining general immune responses that insects carry out, which are encapsulation, melanization, changes in hemocyte load and phenoloxidase activity. Hemocytes are similar to white blood cells in humans, previous research has shown that during an immune response the load of circulating hemocytes increases (Hillyer, 2016). Melanization occurs alongside encapsulation where cells encapsulate a pathogen and then convert tyrosine into melanin (Hillyer, 2016). Melanization and encapsulation cleaves the inactive form of phenoloxidase, prophenoloxidase into active phenoloxidase (Cornet et al., 2013). Therefore making phenoloxidase levels directly correspond to immune responses. The purpose of this study is to determine if the house cricket has the ability to carry out specific immune responses when exposed to LPS for a second time, this will be done by examining phenoloxidase activity and hemocyte load. Cricket colonies were kept at 30°C for a photoperiod of L:D 10:14. When juveniles emerged into adulthood they were categorized as Day 0 adults. Adults were separated by age in isolated colonies. Three groups of crickets were set up; group 1 was a control group. Group 2 was an experimental control group that was only injected with phosphate buffered saline (PBS). Group 3 was the experimental group that was injected with LPS that has been diluted with PBS. To test for phenoloxidase activity the protocol from Tabunoki et al. (2019) was modified and used for this experiment. Hemolymph was collected from crickets and hemocyte density was counted using a hemocytometer. Preliminary data shows that insects injected with 1mg/mL of LPS in PBS (group 3) have a higher hemocyte count than that of group 1 and 2. It is expected that phenoloxidase levels will be greater in group 3 than groups 1 and 2, as seen in other studies (Tabunoki et al., 2019).
Ian Baranyk, Wingate University
Insects are believed to have an entirely innate immune response. This means their immune system does not need time to reach full strength as it would in organisms with an adaptive immune system such as humans. Recent studies, however, show that the insect immune system may indeed possess some adaptive traits. This is illustrated through the phenomenon of immune priming. Immune priming has been shown in different insects including Bombus terrestris (Sadd et al., 2006). This study will use lipopolysaccharides (LPS) from Eschericha coli to elicit immune responses in the house cricket, Acheta domesticus. This will be measured by examining general immune responses that insects carry out, which are encapsulation, melanization, changes in hemocyte load and phenoloxidase activity. Hemocytes are similar to white blood cells in humans, previous research has shown that during an immune response the load of circulating hemocytes increases (Hillyer, 2016). Melanization occurs alongside encapsulation where cells encapsulate a pathogen and then convert tyrosine into melanin (Hillyer, 2016). Melanization and encapsulation cleaves the inactive form of phenoloxidase, prophenoloxidase into active phenoloxidase (Cornet et al., 2013). Therefore making phenoloxidase levels directly correspond to immune responses. The purpose of this study is to determine if the house cricket has the ability to carry out specific immune responses when exposed to LPS for a second time, this will be done by examining phenoloxidase activity and hemocyte load. Cricket colonies were kept at 30°C for a photoperiod of L:D 10:14. When juveniles emerged into adulthood they were categorized as Day 0 adults. Adults were separated by age in isolated colonies. Three groups of crickets were set up; group 1 was a control group. Group 2 was an experimental control group that was only injected with phosphate buffered saline (PBS). Group 3 was the experimental group that was injected with LPS that has been diluted with PBS. To test for phenoloxidase activity the protocol from Tabunoki et al. (2019) was modified and used for this experiment. Hemolymph was collected from crickets and hemocyte density was counted using a hemocytometer. Preliminary data shows that insects injected with 1mg/mL of LPS in PBS (group 3) have a higher hemocyte count than that of group 1 and 2. It is expected that phenoloxidase levels will be greater in group 3 than groups 1 and 2, as seen in other studies (Tabunoki et al., 2019).
Observation of Insect Immunity in Response to Secondary Exposure of LPS in the House Cricket, Acheta domesticus
Niki Apostolou, Wingate University
Most insects including the house crickets, Acheta domesticus, have an innate response from their maternal immune system from birth. These responses include melanization, encapsulation, changes in hemocyte count, and phenoloxidase activity. After birth and into adulthood, if they are exposed to any type of foreign pathogen their immune system will take measures to combat them. However, there is a costly response that will be up-regulated. Due to the strong up-regulation of their immune response, safety mechanisms can be activated against exposures and infections. Previous study of the social insect Bombus terrestris has shown specificity in protection through immune defenses for selective pressures over evolutionary time (Sadd et al., 2006). Therefore, the aim of our study is to investigate if adult house crickets, Acheta domesticus, have the ability to perform a specific immune priming during a secondary exposure with lipopolysaccharides (LPS, an immune elicitor) of Escherichia coli through the means of measuring immune function by encapsulation activity and hemocyte count. Crickets were maintained in a colony until adult emergence. Newly emerged adults were designated as Day 0 and then maintained in isolated colonies by age (Day 1, 2, 3, etc. - 24, 48, 96, etc. hours respectively after adult emergence). The crickets were separated into three groups: group 1 - control, group 2 - experimental control injected with Phosphate Buffered Saline (PBS) only, and group 3 - experimental group with LPS in PBS. To evaluate immune function by encapsulation activity and hemocyte count. Encapsulation activity was measured using a monofilament that was inserted into the abdomen of Day 6 crickets from each group. Monofilaments were removed 24 hours after insertion, photographed, and the mean grayscale values were analyzed using ImageJ. Hemolymph was collected and hemocytes counted using a hemocytometer. Preliminary data shows that crickets possess adaptive immune traits. A previous study involving immune priming in scorpions showed that individuals injected with LPS in PBS did show higher survival than the control group as an indication that immune priming played a significant role (Galvez et al., 2020). After secondary exposure, it is expected that the crickets in group 3 will have a greater encapsulation and hemocyte load compared to groups 1 and 2 (Tabunoki et al., 2019).
Niki Apostolou, Wingate University
Most insects including the house crickets, Acheta domesticus, have an innate response from their maternal immune system from birth. These responses include melanization, encapsulation, changes in hemocyte count, and phenoloxidase activity. After birth and into adulthood, if they are exposed to any type of foreign pathogen their immune system will take measures to combat them. However, there is a costly response that will be up-regulated. Due to the strong up-regulation of their immune response, safety mechanisms can be activated against exposures and infections. Previous study of the social insect Bombus terrestris has shown specificity in protection through immune defenses for selective pressures over evolutionary time (Sadd et al., 2006). Therefore, the aim of our study is to investigate if adult house crickets, Acheta domesticus, have the ability to perform a specific immune priming during a secondary exposure with lipopolysaccharides (LPS, an immune elicitor) of Escherichia coli through the means of measuring immune function by encapsulation activity and hemocyte count. Crickets were maintained in a colony until adult emergence. Newly emerged adults were designated as Day 0 and then maintained in isolated colonies by age (Day 1, 2, 3, etc. - 24, 48, 96, etc. hours respectively after adult emergence). The crickets were separated into three groups: group 1 - control, group 2 - experimental control injected with Phosphate Buffered Saline (PBS) only, and group 3 - experimental group with LPS in PBS. To evaluate immune function by encapsulation activity and hemocyte count. Encapsulation activity was measured using a monofilament that was inserted into the abdomen of Day 6 crickets from each group. Monofilaments were removed 24 hours after insertion, photographed, and the mean grayscale values were analyzed using ImageJ. Hemolymph was collected and hemocytes counted using a hemocytometer. Preliminary data shows that crickets possess adaptive immune traits. A previous study involving immune priming in scorpions showed that individuals injected with LPS in PBS did show higher survival than the control group as an indication that immune priming played a significant role (Galvez et al., 2020). After secondary exposure, it is expected that the crickets in group 3 will have a greater encapsulation and hemocyte load compared to groups 1 and 2 (Tabunoki et al., 2019).
Effects of Potassium Perchlorate on Developing Medaka Primordial Germ Cells
Beh Reh, University of North Carolina at Greensboro
Perchlorate is a chemical compound commonly used in military artillery and equipment. It has been detected in drinking water, air, soil, and breast milk. Human exposure can occur in the theatre of war and areas adjacent to military training grounds. High concentration of perchlorate has been found to affect reproduction in vertebrates including fish. However, whether environmental concentrations of perchlorate can affect primordial germ cells (PGCs) is not clearly understood. In the present study, we examined the effects of 0, 10, 100, and 1000 μg/L potassium perchlorate exposure on the development of medaka embryos and their PGCs. Perchlorate exposure delayed hatching time, reduced heartbeat, inhibited migration of PGCs, increased developmental deformities in the larvae. The 10000 and 20000 μg/L concentration of perchlorate was lethal to embryos, whereas co-treatment of embryos with vitamin C (1 mg/L) completely blocked perchlorate-induced mortality. RNAseq analysis showed genes involved in proteolysis, metabolic processes, peptides activity, hydrolase activity, and hormone activity were significantly altered. Among the component, extracellular, intracellular, sarcoplasmic and 6-phosphofructokinase and membrane-bounded processes were affected. The results suggest that perchlorate affects development of medaka larvae and vitamin C can ameliorate perchlorate-induced embryo mortality. Additionally, perchlorate alters global transcriptional network in PGCs suggesting its influence on germ cell function and fertility later in life.
Beh Reh, University of North Carolina at Greensboro
Perchlorate is a chemical compound commonly used in military artillery and equipment. It has been detected in drinking water, air, soil, and breast milk. Human exposure can occur in the theatre of war and areas adjacent to military training grounds. High concentration of perchlorate has been found to affect reproduction in vertebrates including fish. However, whether environmental concentrations of perchlorate can affect primordial germ cells (PGCs) is not clearly understood. In the present study, we examined the effects of 0, 10, 100, and 1000 μg/L potassium perchlorate exposure on the development of medaka embryos and their PGCs. Perchlorate exposure delayed hatching time, reduced heartbeat, inhibited migration of PGCs, increased developmental deformities in the larvae. The 10000 and 20000 μg/L concentration of perchlorate was lethal to embryos, whereas co-treatment of embryos with vitamin C (1 mg/L) completely blocked perchlorate-induced mortality. RNAseq analysis showed genes involved in proteolysis, metabolic processes, peptides activity, hydrolase activity, and hormone activity were significantly altered. Among the component, extracellular, intracellular, sarcoplasmic and 6-phosphofructokinase and membrane-bounded processes were affected. The results suggest that perchlorate affects development of medaka larvae and vitamin C can ameliorate perchlorate-induced embryo mortality. Additionally, perchlorate alters global transcriptional network in PGCs suggesting its influence on germ cell function and fertility later in life.