Acorn Abstracts
Dami Aiyejuto – The Impact of Pneumolysin from Streptococcus pneumoniae on Paracellular Permeability and Cytokine Secretion by Calu-3 Cell Line
Streptococcus pneumoniae, an opportunistic pathogen, can cause severe pulmonary infections and empyema in individuals with underlying conditions. Its pathogenesis involves adherence to mucosal surfaces, immune system evasion, and virulence factors including Pneumolysin (PLY), a pore-forming toxin. We sought to investigate the hypothesis that pneumolysin contributes to the pathogenesis of S. pneumoniae by increasing epithelial paracellular permeability and inducing cytokine release in vitro. To test this hypothesis, we used the Lactate Dehydrogenase Assay (LDH) to assess cytotoxicity, Transepithelial Electrical Resistance (TEER) to measure paracellular permeability, and Enzyme-Linked Immunosorbent Assay (ELISA) to evaluate cytokine release in a cell culture model. Confluent monolayers of human airway Calu-3 cell line cultured on porous membrane inserts were used to study the role of PLY in epithelial paracellular permeability and inflammatory responses. We cloned the PLY gene, purified the protein, and determined the non-cytotoxic concentrations of rPLY to be utilized in this project. We used TEER technology to assess the integrity of the epithelial junctional complexes. The TEER data showed a significant drop in resistance after treating the cells with various concentrations of rPLY (5,15, and 30 µg/mL) for a 50-minute period. The ELISA data for pro-inflammatory interleukin IL-1β showed an increase in cytokine release in Calu-3 cells treated with 5 µg/mL of pneumolysin over a 24-hour treatment period. Our preliminary data provide insights into the molecular mechanisms underlying PLY-mediated inflammatory response and disruption of lung epithelial barriers. Our findings may have implications for the development of preventative measures and targeted therapy to mitigate lung injury during pneumococcal infections.
Domenica Baez – A Student-Led Hypothesis Driven Tissue Engineering Lab Investigating ECM Stiffness’ Effect on Yap Expression
In the United States, there are about 240,000 new cases of breast cancer identified each year, which highlights the urgent need for novel treatments. Even with its high incidence and lethality, there are still unanswered questions about the pathophysiology of the illness and the best ways to treat it. This presents a unique opportunity for undergraduate students to improve their laboratory skills through novel research projects. With its innovative teaching methodology, the proposed mechanobiology lab allows students to conduct a semester-long research project without pre-established conclusions. By integrating ideas from tissue engineering and molecular biology, this methodology promotes a thorough understanding of experimental design, analysis, and troubleshooting. Students will do hands-on western blot analysis, image analysis, and scientific writing as they investigate the relationship between extracellular matrix (ECM) thickness and the expression of the oncogene Yap in the evolution of breast cancer. In addition to enhancing their learning experience, this project gives them invaluable skills for future healthcare and research employment. Through their involvement in this multidisciplinary study, students strengthen their critical thinking skills and acquire knowledge concerning intricate healthcare issues, ultimately contributing significantly to the progress of breast cancer treatment approaches.
Christian Bell – Detecting Lymphangiogenesis in Response to Atherosclerosis in Danio rerio
As the leading cause of death in the United States and globally, atherosclerosis is a serious health condition best thwarted with preventative medicine. No current treatment can fully reverse this disease, which can lead to life-threatening cardiovascular events. Previous research in humans, mice, and zebrafish (Danio rerio) suggest that the proliferation of lymphatic vasculature around the site of arterial occlusion improves the prognosis of individuals who experienced atherosclerotic injury, but there is limited research if this occurs in other areas of the body, such as the major vessels of the aorta or microvasculature in other somatic tissues. To determine if there is a lymphatic response to atherosclerosis of other vessels, we fed adult zebrafish with a diet known to induce atherosclerosis, then performed immunohistochemical staining for Prox1a to detect lymphatic endothelium and elastin stain to visualize sclerotic vessels on whole cross-sections. Histological analysis suggests an increased lymphangiogenic response in individuals fed a high cholesterol diet compared to a normal diet. Subsequent expression analysis of prox1a via RT-PCR of these tissues further supports this conclusion. Thus, we propose that this acclimation response could potentially be used as a pharmacological target to prevent, treat, and potentially reverse atherosclerosis in humans.
Stevie Langstraat – MARS on Mars: Measuring Accommodations in Regolith Soil on Mars – AI Edition
With increasing public demand for space exploration, astrobiology research is a growing scientific field, as colonies on extraterrestrial planets come closer to reality. For these colonies to be successful, efficient nutrient cycling in different environments must be studied. The NASA-affiliated Plant the Moon competition challenges teams across multiple educational divisions to grow crops in simulated Mars soil (regolith) within a given growth period. Students at Southwestern Oklahoma State University have participated in this challenge and have studied interactions between microbes, Mars regolith, mammal fecal matter, and plant growth for food production. With Mars regolith behaving differently than Earth soil, parameters involving plant growth have been tested on top of soil additives. In addition to documenting total plant yield, soil pH and seedling germination rates have been assessed. These characteristics of plant growth have been tested in crops including lettuce, spinach, basil, and radishes, with some of these plants being grown successfully in Mars regolith. A joint research project between computer science and biology will implement artificial intelligence and perform parameter testing on soil additive concentrations and specific growth conditions. Using multiple parameter tests, we anticipate finding the best combination of growth conditions and concentrations for crop yield in extraterrestrial soils.
Giovanna Lopez – The Influence of Age, Size, and Experience on Risk-Taking and Boldness
Experience effects arise when animals adjust their behaviors based on previous experiences. For instance, individuals may become timid and risk-averse following a predator attack. Most studies on experience effects have been conducted in adults, leaving us unsure of how age influences individual responsiveness to prior experiences. Here we examined whether age influences changes in boldness and risk-taking following a predator attack in the mangrove rivulus (Kryptolebias marmoratus) fish. We measured risk-taking and boldness in both juveniles (2 months of age) and adults (6 months of age) before and after a simulated, aerial predation experience. We found significant experience x mass and age x mass interactions on boldness and risk-taking behaviors. Latency to recover from a stressor increased with mass for inexperienced but not experienced individuals. Further, latency to recover decreased with mass in juveniles but increased with mass in adults. Our findings indicate that age and size have more of an impact on an individual's risk taking and boldness compared to prior experience. Further expansion of the study will include a focus on older, slower growing individuals (e.g., 2 years of age) to gain more insight on the interaction between age and experience on individual behavior.
Gabrielle McGill – Small Mammal Presence on Soil and Vegetation in Blackland Prairie Ecosystem
Blackland Prairie ecosystem has been reduced significantly due to human intervention and infrastructure. In order to try and conserve the remaining land it is important to understand how interspecies relationships work. By studying the relationship between the small mammal populations and vegetation, we can better work to conserve Blackland Prairie. This study strives to understand the relationship between small mammals and vegetation by testing soil C:N levels and microbial activity as these factors directly correlate to vegetation species richness.
Mya Moore – Live Cell Imaging of the Actin Cytoskeleton and Fertilization in Nicotine-Induced Polyspermy in Sea Urchins
Polyspermy is typically a lethal phenomenon in which numerous sperm penetrate an egg. To prevent polyspermy, sea urchins form a fertilization envelope, a blockade that forms when a sperm penetrates the egg that blocks excess penetration of sperm. Cytoskeletal elements, such as actin, play critical roles in fertilization envelope development. Eggs exposed to nicotine, however, exhibit polyspermy in a dose-dependent manner and are found to have abnormal actin organization, consequently altering the formation of the fertilization envelope. Our goal was to establish a protocol for live cell imaging of nicotine-induced polyspermy using available reagents and technology at Angelo State University. Our study consisted of spawning sea urchins to collect gametes, subjecting the spawned eggs to a nicotine solution, administering either Hoechst stain or CellMask™ Green Actin Stain to the gametes, and then observation with fluorescent microscopy. Results conclude the pronuclei of gametes were successfully stained with Hoechst. Additionally, live cell imaging was achieved of the actin cytoskeleton in sea urchin eggs exposed to nicotine and not exposed to nicotine. However, fertilization was not observed, as we believe the fluorescent dyes quenched during fertilization. We anticipate that our project will provide insight into visualizing evolutionary adaptations that prevent polyspermy in animals.
Patrick Oehler – Characterization of a Coumarin-Substituted 1,2,4-Oxadiazole as a Novel Antifungal Agent
Fungi can pose serious health risks, especially to immunocompromised individuals. This lethal potential is compounded by a recent rise in antifungal resistance brought about by the indiscriminate application of antifungal agents. While antifungal stewardship can slow the development of resistant strains, synthesizing novel agents yet unknown to resistant strains is the only way to truly combat the issue. To this end, we developed a novel coumarin-substituted 1,2,4-oxadiazole. As coumarins and oxadiazoles are heterocycles renowned for their myriad biological activities, ranging from anti-inflammatory to antifungal, this compound was believed to possess antifungal properties. Notably, this product was water-soluble, which could facilitate future development of related medicinal compounds. The antifungal activities of several oxadiazole concentrations against C. albicans were characterized by spectrophotometrically measuring cell concentration to generate growth curves over 48 hours. To distinguish between fungicidal and fungistatic behavior, the concentrations of colony-forming units (CFUs) were measured following the 48-hour incubation period. The R package nls was used to fit logistic curves to the growth curve data, and the resultant parameters will be compared by one-way ANOVA. CFU concentrations were similarly compared by one-way ANOVA. The results showed a decrease in Vmax at the highest oxadiazole concentrations tested.
Reese Ramirez – The Armadillo’s Shell Shows Pregnancy and Lactation Osteopenia
All women lose minerals from their endochondral bones during pregnancy and lactation. Though rare, bone loss during reproduction may be severe, resulting in debilitating osteoporosis. Altering nutrition does not prevent pregnancy and lactation associated bone loss. To support shell mineralization of their neonates, lactating armadillos produce milk with a higher calcium concentration than any other mammal. It follows that armadillo mothers should display substantial osteopenia, or osteoporosis. Our hypothesis is that the adult shell acts as an additional reservoir of calcium, protecting the skeleton from excessive bone loss during pregnancy lactation. We predict the armadillo shell will contain a lower calcium concentration during lactation than during pregnancy. To test our prediction, we measured calcium concentration in the shell, serum, urine, and feces of wild-caught, pregnant armadillos (n=9). We repeated these measurements after the armadillos gave birth (n=7). Shell calcium concentration was lower during lactation than during pregnancy (paired T-test, p<0.05). Serum calcium remained the same, while urine and fecal calcium concentration increased. These results are evidence that armadillos mobilize calcium from the intramembranous bone of the shell to support their developing offspring even when dietary calcium is more than that which is necessary for serum calcium regulation.
Andrew Shropshire & Emily Speer – Analyzing contig54 of Drosophila willistoni
Genome annotation is the process of locating and designating locations of individual genes, allowing researchers to easily view and analyze a gene sequence and its contents¹. The protocols developed by the Genome Education Project (GEP) were merged with other student researcher-written procedures to help us annotate an assigned contig from a newly sequenced genome in Drosophila. Our project is analyzing contig54 in Drosophila willistoni to identify the Drosophila melanogaster orthologs Pcmt, mia, ppan, CG11970, Bdbt, and slmb. To help us identify these orthologs we used gene prediction programs (GenScan, NSCAN, Glimmer, etc.) to align the predictions with the congruent D. melanogaster proteins and the FlyBase databank proteins. Following the analysis of the FlyBank database, we would take the proposed protein and compare it to the entirety of the D. willistoni DNA to perform an exon-by-exon search using the Basic Local Alignment Sequence Tool (BLAST) to compare our null hypothesis (no genetic change from D. melanogaster and D. willistoni) to our analyzing BLAST results. As part of the GEP consortium protocols, our results were verified via the GEP Genome Record Finder followed by the GEP Gene Model Checker as a confirmation of correct annotation before the final proposed exon-by-exon visualized annotation is submitted
Streptococcus pneumoniae, an opportunistic pathogen, can cause severe pulmonary infections and empyema in individuals with underlying conditions. Its pathogenesis involves adherence to mucosal surfaces, immune system evasion, and virulence factors including Pneumolysin (PLY), a pore-forming toxin. We sought to investigate the hypothesis that pneumolysin contributes to the pathogenesis of S. pneumoniae by increasing epithelial paracellular permeability and inducing cytokine release in vitro. To test this hypothesis, we used the Lactate Dehydrogenase Assay (LDH) to assess cytotoxicity, Transepithelial Electrical Resistance (TEER) to measure paracellular permeability, and Enzyme-Linked Immunosorbent Assay (ELISA) to evaluate cytokine release in a cell culture model. Confluent monolayers of human airway Calu-3 cell line cultured on porous membrane inserts were used to study the role of PLY in epithelial paracellular permeability and inflammatory responses. We cloned the PLY gene, purified the protein, and determined the non-cytotoxic concentrations of rPLY to be utilized in this project. We used TEER technology to assess the integrity of the epithelial junctional complexes. The TEER data showed a significant drop in resistance after treating the cells with various concentrations of rPLY (5,15, and 30 µg/mL) for a 50-minute period. The ELISA data for pro-inflammatory interleukin IL-1β showed an increase in cytokine release in Calu-3 cells treated with 5 µg/mL of pneumolysin over a 24-hour treatment period. Our preliminary data provide insights into the molecular mechanisms underlying PLY-mediated inflammatory response and disruption of lung epithelial barriers. Our findings may have implications for the development of preventative measures and targeted therapy to mitigate lung injury during pneumococcal infections.
Domenica Baez – A Student-Led Hypothesis Driven Tissue Engineering Lab Investigating ECM Stiffness’ Effect on Yap Expression
In the United States, there are about 240,000 new cases of breast cancer identified each year, which highlights the urgent need for novel treatments. Even with its high incidence and lethality, there are still unanswered questions about the pathophysiology of the illness and the best ways to treat it. This presents a unique opportunity for undergraduate students to improve their laboratory skills through novel research projects. With its innovative teaching methodology, the proposed mechanobiology lab allows students to conduct a semester-long research project without pre-established conclusions. By integrating ideas from tissue engineering and molecular biology, this methodology promotes a thorough understanding of experimental design, analysis, and troubleshooting. Students will do hands-on western blot analysis, image analysis, and scientific writing as they investigate the relationship between extracellular matrix (ECM) thickness and the expression of the oncogene Yap in the evolution of breast cancer. In addition to enhancing their learning experience, this project gives them invaluable skills for future healthcare and research employment. Through their involvement in this multidisciplinary study, students strengthen their critical thinking skills and acquire knowledge concerning intricate healthcare issues, ultimately contributing significantly to the progress of breast cancer treatment approaches.
Christian Bell – Detecting Lymphangiogenesis in Response to Atherosclerosis in Danio rerio
As the leading cause of death in the United States and globally, atherosclerosis is a serious health condition best thwarted with preventative medicine. No current treatment can fully reverse this disease, which can lead to life-threatening cardiovascular events. Previous research in humans, mice, and zebrafish (Danio rerio) suggest that the proliferation of lymphatic vasculature around the site of arterial occlusion improves the prognosis of individuals who experienced atherosclerotic injury, but there is limited research if this occurs in other areas of the body, such as the major vessels of the aorta or microvasculature in other somatic tissues. To determine if there is a lymphatic response to atherosclerosis of other vessels, we fed adult zebrafish with a diet known to induce atherosclerosis, then performed immunohistochemical staining for Prox1a to detect lymphatic endothelium and elastin stain to visualize sclerotic vessels on whole cross-sections. Histological analysis suggests an increased lymphangiogenic response in individuals fed a high cholesterol diet compared to a normal diet. Subsequent expression analysis of prox1a via RT-PCR of these tissues further supports this conclusion. Thus, we propose that this acclimation response could potentially be used as a pharmacological target to prevent, treat, and potentially reverse atherosclerosis in humans.
Stevie Langstraat – MARS on Mars: Measuring Accommodations in Regolith Soil on Mars – AI Edition
With increasing public demand for space exploration, astrobiology research is a growing scientific field, as colonies on extraterrestrial planets come closer to reality. For these colonies to be successful, efficient nutrient cycling in different environments must be studied. The NASA-affiliated Plant the Moon competition challenges teams across multiple educational divisions to grow crops in simulated Mars soil (regolith) within a given growth period. Students at Southwestern Oklahoma State University have participated in this challenge and have studied interactions between microbes, Mars regolith, mammal fecal matter, and plant growth for food production. With Mars regolith behaving differently than Earth soil, parameters involving plant growth have been tested on top of soil additives. In addition to documenting total plant yield, soil pH and seedling germination rates have been assessed. These characteristics of plant growth have been tested in crops including lettuce, spinach, basil, and radishes, with some of these plants being grown successfully in Mars regolith. A joint research project between computer science and biology will implement artificial intelligence and perform parameter testing on soil additive concentrations and specific growth conditions. Using multiple parameter tests, we anticipate finding the best combination of growth conditions and concentrations for crop yield in extraterrestrial soils.
Giovanna Lopez – The Influence of Age, Size, and Experience on Risk-Taking and Boldness
Experience effects arise when animals adjust their behaviors based on previous experiences. For instance, individuals may become timid and risk-averse following a predator attack. Most studies on experience effects have been conducted in adults, leaving us unsure of how age influences individual responsiveness to prior experiences. Here we examined whether age influences changes in boldness and risk-taking following a predator attack in the mangrove rivulus (Kryptolebias marmoratus) fish. We measured risk-taking and boldness in both juveniles (2 months of age) and adults (6 months of age) before and after a simulated, aerial predation experience. We found significant experience x mass and age x mass interactions on boldness and risk-taking behaviors. Latency to recover from a stressor increased with mass for inexperienced but not experienced individuals. Further, latency to recover decreased with mass in juveniles but increased with mass in adults. Our findings indicate that age and size have more of an impact on an individual's risk taking and boldness compared to prior experience. Further expansion of the study will include a focus on older, slower growing individuals (e.g., 2 years of age) to gain more insight on the interaction between age and experience on individual behavior.
Gabrielle McGill – Small Mammal Presence on Soil and Vegetation in Blackland Prairie Ecosystem
Blackland Prairie ecosystem has been reduced significantly due to human intervention and infrastructure. In order to try and conserve the remaining land it is important to understand how interspecies relationships work. By studying the relationship between the small mammal populations and vegetation, we can better work to conserve Blackland Prairie. This study strives to understand the relationship between small mammals and vegetation by testing soil C:N levels and microbial activity as these factors directly correlate to vegetation species richness.
Mya Moore – Live Cell Imaging of the Actin Cytoskeleton and Fertilization in Nicotine-Induced Polyspermy in Sea Urchins
Polyspermy is typically a lethal phenomenon in which numerous sperm penetrate an egg. To prevent polyspermy, sea urchins form a fertilization envelope, a blockade that forms when a sperm penetrates the egg that blocks excess penetration of sperm. Cytoskeletal elements, such as actin, play critical roles in fertilization envelope development. Eggs exposed to nicotine, however, exhibit polyspermy in a dose-dependent manner and are found to have abnormal actin organization, consequently altering the formation of the fertilization envelope. Our goal was to establish a protocol for live cell imaging of nicotine-induced polyspermy using available reagents and technology at Angelo State University. Our study consisted of spawning sea urchins to collect gametes, subjecting the spawned eggs to a nicotine solution, administering either Hoechst stain or CellMask™ Green Actin Stain to the gametes, and then observation with fluorescent microscopy. Results conclude the pronuclei of gametes were successfully stained with Hoechst. Additionally, live cell imaging was achieved of the actin cytoskeleton in sea urchin eggs exposed to nicotine and not exposed to nicotine. However, fertilization was not observed, as we believe the fluorescent dyes quenched during fertilization. We anticipate that our project will provide insight into visualizing evolutionary adaptations that prevent polyspermy in animals.
Patrick Oehler – Characterization of a Coumarin-Substituted 1,2,4-Oxadiazole as a Novel Antifungal Agent
Fungi can pose serious health risks, especially to immunocompromised individuals. This lethal potential is compounded by a recent rise in antifungal resistance brought about by the indiscriminate application of antifungal agents. While antifungal stewardship can slow the development of resistant strains, synthesizing novel agents yet unknown to resistant strains is the only way to truly combat the issue. To this end, we developed a novel coumarin-substituted 1,2,4-oxadiazole. As coumarins and oxadiazoles are heterocycles renowned for their myriad biological activities, ranging from anti-inflammatory to antifungal, this compound was believed to possess antifungal properties. Notably, this product was water-soluble, which could facilitate future development of related medicinal compounds. The antifungal activities of several oxadiazole concentrations against C. albicans were characterized by spectrophotometrically measuring cell concentration to generate growth curves over 48 hours. To distinguish between fungicidal and fungistatic behavior, the concentrations of colony-forming units (CFUs) were measured following the 48-hour incubation period. The R package nls was used to fit logistic curves to the growth curve data, and the resultant parameters will be compared by one-way ANOVA. CFU concentrations were similarly compared by one-way ANOVA. The results showed a decrease in Vmax at the highest oxadiazole concentrations tested.
Reese Ramirez – The Armadillo’s Shell Shows Pregnancy and Lactation Osteopenia
All women lose minerals from their endochondral bones during pregnancy and lactation. Though rare, bone loss during reproduction may be severe, resulting in debilitating osteoporosis. Altering nutrition does not prevent pregnancy and lactation associated bone loss. To support shell mineralization of their neonates, lactating armadillos produce milk with a higher calcium concentration than any other mammal. It follows that armadillo mothers should display substantial osteopenia, or osteoporosis. Our hypothesis is that the adult shell acts as an additional reservoir of calcium, protecting the skeleton from excessive bone loss during pregnancy lactation. We predict the armadillo shell will contain a lower calcium concentration during lactation than during pregnancy. To test our prediction, we measured calcium concentration in the shell, serum, urine, and feces of wild-caught, pregnant armadillos (n=9). We repeated these measurements after the armadillos gave birth (n=7). Shell calcium concentration was lower during lactation than during pregnancy (paired T-test, p<0.05). Serum calcium remained the same, while urine and fecal calcium concentration increased. These results are evidence that armadillos mobilize calcium from the intramembranous bone of the shell to support their developing offspring even when dietary calcium is more than that which is necessary for serum calcium regulation.
Andrew Shropshire & Emily Speer – Analyzing contig54 of Drosophila willistoni
Genome annotation is the process of locating and designating locations of individual genes, allowing researchers to easily view and analyze a gene sequence and its contents¹. The protocols developed by the Genome Education Project (GEP) were merged with other student researcher-written procedures to help us annotate an assigned contig from a newly sequenced genome in Drosophila. Our project is analyzing contig54 in Drosophila willistoni to identify the Drosophila melanogaster orthologs Pcmt, mia, ppan, CG11970, Bdbt, and slmb. To help us identify these orthologs we used gene prediction programs (GenScan, NSCAN, Glimmer, etc.) to align the predictions with the congruent D. melanogaster proteins and the FlyBase databank proteins. Following the analysis of the FlyBank database, we would take the proposed protein and compare it to the entirety of the D. willistoni DNA to perform an exon-by-exon search using the Basic Local Alignment Sequence Tool (BLAST) to compare our null hypothesis (no genetic change from D. melanogaster and D. willistoni) to our analyzing BLAST results. As part of the GEP consortium protocols, our results were verified via the GEP Genome Record Finder followed by the GEP Gene Model Checker as a confirmation of correct annotation before the final proposed exon-by-exon visualized annotation is submitted