Boax Abstracts
Evonn Annor – Patterns in Genomes: A Novel Unbiased Computational Approach for Motif Discovery Through Frequency Analysis
Advancements in genomics and computing power, through initiatives like the
Human Epigenome Project, have propelled bioinformatics research. Yet, we
hypothesize that not all significant patterns have been discovered through this guided process, requiring an unguided computational approach. To address this gap, we introduce a novel unbiased computational tool for analyzing word occurrences within genomic data to identify significant genetic sequences and motifs based on pattern frequency. This tool utilizes alignment-based algorithms to conduct an unbiased preliminary search across chromosome 1 of the Homo sapiens Hg.38 genome. Highlighting the most occurring 10, 20, 50, and 500 motif patterns across chromosome 1. This word search tracked the frequency of the motif patterns across chromosome one and their potential identities. Utilizing established genome browsers such as ENSEMBL and CIS-BPU to identify known motifs and other regulatory features. Additionally, the frequency of identified 10- and 20-character motifs were also compared to random chance to further distinguish whether patterns were statistically significant or occurred by random variation. Results indicate that these patterns have biological significance and can be analyzed across species to identify conserved motif patterns. However, the study acknowledges the need for further validation by expanding the analysis to include the entire genome.
Katherine Dempsey – Expression of Human PNPLA3 Protein in CRISPR-generated Knock-In Mice
Fatty liver disease (FLD) is the leading cause of liver disease in the world and is a disorder characterized by an accumulation of triglycerides (TG) on fat storage organelles called lipid droplets. The strongest genetic risk factor for FLD is a missense variant (I148M) in the patatin-like phospholipase domain-containing protein 3 (PNPLA3). This protein is encoded by an insulin-regulated gene expressed at high levels in liver and adipose tissues. The I148M substitution is well-conserved between mice and humans, and the FLD phenotype has been observed in knock-in mice with this variant. Another missense variant (S453I) is strongly associated with reduced hepatic TG levels in humans but is located in a poorly conserved region of the PNPLA3 protein. Since S453I has not been previously studied in mouse models, CRISPR-Cas9 gene editing technology was used to develop a novel strain of knock-in mice containing human PNPLA3. Genotyping results indicated successful insertion of the human gene, and human PNPLA3 protein expression was verified by immunoblotting analysis. Future studies will be conducted using humanized PNPLA3 mice to study the mechanism by which the S453I variant reduces hepatic TG levels and may lead to a potential therapeutic target for the treatment of FLD.
Jayme James – Apigenin Induces Apoptosis in MDA-MB 231 Cell in vitro
Breast cancer is the most common type of cancer among women. There are around 264,000 new cases each year (Basic Information About Breast Cancer, 2022). Although the first stages of breast cancer have high survival rates, there is a drastic decrease in survival as the cancer becomes metastatic. As the prevalence of breast cancer increases, the desperation for new treatments is also rising. In this project, MDA-MB 231 cells were used to determine the efficacy of apigenin in inducing apoptosis in breast cancer cells. MDA-MB 231 cells are a highly aggressive, triple-negative breast cancer cell line, against which hormone-targeted therapies are futile. Additionally, apigenin is a flavonoid found in plants that has various therapeutic properties, including anti-inflammatory, antioxidant, and antiviral significance. The results of our MTT assay showed a dose-dependent induction of apoptosis. The caspase-3 assay indicated that apigenin induces cell death by activating the caspase pathway. The flow cytometry results revealed that apigenin downregulated the expression of PD-L1 in MDA-MB 231 cells. Furthermore, HEK293T, a human embryonic kidney cell line, was used to test the potential detrimental effects of apigenin on normal cell lines. As a result, apigenin has the potential to be used in in vivo applications.
Anna Wilson – Phallus impudicus: A Study of Secreted Compounds
Each year in the United States around 300,000 women and 2,500 men are diagnosed with invasive breast cancer. In the United States, the most common ways to combat breast cancer are surgery, radiation, and chemotherapies. Although chemotherapy has saved countless lives from cancer, its harmful side effects are well-known. Outside of the United States, in countries such as Japan and China, medicinal mushrooms are considered standard in cancer treatment. Preliminary studies have yielded promising results on certain mushrooms' cancer-fighting and immune-boosting properties. It is believed these properties could be used to lessen the harmful effects of chemotherapy.
Common stinkhorns like Phallus impudicus have been used in the treatment of tumors, specifically breast tumors, for almost a millennium. This project aims to pinpoint a naturally occurring compound within the mushroom P. impudicus that has selective cytotoxicity towards breast cancer cells. P. impudicus was cultured in vitro over a two-week period. Mushroom secretions were captured and analyzed using a fast-protein liquid chromatography system (FPLC). A large protein-containing polysaccharide was isolated. Future studies will focus on viability data collected from 96-well cytotoxicity assays.
Lauren Hinson – The Effect of Environmental pH on Staphylococcus aureus
Staphylococcus aureus is a bacterium that is a part of the normal flora on human skin. Studies of S. aureus have documented its ability to mutate and become resistant to antibiotics such as the Methicillin Resistant strain (MRSA). This is problematic because S. aureus is an opportunistic pathogen for several diseases. The pathogenic nature of S. aureus, along with its ability to become antibiotic resistant, makes it an ideal organism for research. Many studies have been conducted to find a correlation between the environment of the bacteria and its pathogenicity. Environmental pH is an important factor especially to sensitivity of antibiotics. A hypothesis was formulated that environmental pH could alter mutation rates in S. aureus. To further investigate the correlation between pH and mutation, wild-type S. aureus cultures were observed in five distinct pH environments and tested against select antibiotics. The cultures were grown and tested over 8 weeks to determine the influence of pH. Cultures were analyzed phenotypically by measuring antibiotic resistance. Changes in color and antibiotic efficacy occurred after 5 weeks. Though not statistically significant, the alteration in antibiotic efficacy indicates that environmental pH has an effect on S. aureus.
Jill Schoelzel – B12 Limits the Effective Killing of HMEC1 Endothelial Cells by the RTK Inhibitor, Sorafenib
In the early 1990’s, the FDA mandated folic acid supplementation in certain foods such as cereal, bread, and pasta, to help reduce neural tube defects in newborns. Folic acid also plays an important role in many cellular pathways and varying folate levels have been associated with a variety of cancers. The essential Vitamin B12 is closely associated with folic acid in the one-carbon metabolism which results in synthesis of nucleotides and methylation.
Sorafenib belongs to a common class of chemotherapeutic drugs those that inhibit Receptor Tyrosine Kinases. RTK inhibitors are known to target the process called angiogenesis, which is the formation of new blood vessels. This study aimed to determine the effects on Vitamin B12 on sorafenib treated HMEC1 endothelial cells. HMEC1 cells showed significant cell death after 24 hours of 10uM Sorafenib treatment. Vitamin B12 in combination with sorafenib had partial protective effect on cell death. Understanding the interplay between B12, sorafenib, and folic acid may be key to understanding if the one carbon pathway is an important regulator of RTKs effectiveness at preventing angiogenesis in the context of cancer.
Molly Byers Wingard, Lindsey Wells, & Giovanna Lopez – Unseen Opportunity: Do Hermaphrodite Cues Influence Male-Male Aggression in Mangrove Rivulus Fish
Aggressive contests in fish often occur in the presence of an audience, which can affect contest dynamics and outcomes. These so-called “Audience Effects” can vary depending on the sex of the audience members. For example, a female audience can elicit increased use of low-intensity displays and decreased use of high-intensity attacks between the male combatants. Mangrove rivulus (Kryptolebias marmoratus) fish occur naturally as either self-fertilizing, simultaneous hermaphrodites or functional males. Male rivulus’ reproductive success thus depends on their ability to obtain outcrossing opportunities with hermaphrodites. Our study’s aim is to examine if hermaphroditic chemical cues in the absence of visual cues influence the intensity of male-male rivulus contests and if unequal access to these cues provides a competitive advantage. We dosed focal individuals with either DI water (control) or water laced with hermaphroditic cues (treated). Following, we created size-matched pairings that resulted in three combinations of contestants; control-control, control-treated, and treated-treated. We found that untreated, control-control contests were more likely to escalate to high-intensity behaviors compared to treated-treated contests. Further, dosed individuals pitted against untreated individuals did not have a competitive advantage. Our findings show that male rivulus do modulate their aggressive behavior dependent on presence of an audience.
Lindsey Wells – Fitness Consequences of Cold Stress in a Self-Fertilizing Hermaphroditic Fish
Temperature stress can lead to physiological disruptions that can result in negative fitness consequences such as reduced fecundity, behavioral impairment, or death. Animals employ various physiological and behavioral strategies to cope with temperature stress such as cold acclimation and reproductive seasonality. The Mangrove Rivulus (Kryptolebias marmoratus) fish, a self-fertilizing hermaphrodite, has demonstrated temperature sex determination in response to temperature stressors and seasonality in reproductive output. However, additional fitness consequences of temperature stress such as changes in development and growth have yet to be explored. Our study aimed to examine how cold-exposure during development affects developmental timing and life-history traits, and to elucidate possible mechanisms underlying seasonal variation in rivulus reproduction. We incubated eggs (zero-day post laying) under temperature conditions that varied in either magnitude (15-20C for 17d) or duration (18C for 5-17d). Control groups were reared at 26C until hatching. Hatchlings were then measured every two months for growth and maturation rates. Our findings indicate that cold exposure at any duration or magnitude impacts latency to hatch, latency to mature, and juvenile mass. Our findings help shed light on how mangrove rivulus respond to cold stress and provide a possible explanation for seasonal variation in rivulus reproductive output.
Michael Knabe – Foraging in 3D: Fruit Fly Positional Foraging in a 3D Arena
Fruit flies (Drosophila melanogaster), exhibit negative geotactic behavior, which can help them escape from predators. We tested the effect of negative geotaxis on foraging in a 3-dimensional setting. We presented the flies with a feeding cube (2.5 cm side length) to test their feeding preference in a 3D arena (10 x 10 x 10 cm) to determine their preference to feed from the top, sides, or bottom of a cube with 9 wells on each side filled with colored sucrose solution. Top, bottom, or sides had either red, yellow, or blue solution. The flies were deprived of food for 24 hours and tested in the dark for one hour. They were then frozen, and the contents of their abdomens were evaluated for color to determine their preferred location for feeding on the cube. There was a preference for the top over the sides and/or the bottom of the cube. The flies also preferred the yellow solution in control trials. The decision about where to feed on the cube seems to be influenced by several variables, including resource position, possible ability to escape or hide from predators, and difference of energetic cost caused by feeding against gravity.
Advancements in genomics and computing power, through initiatives like the
Human Epigenome Project, have propelled bioinformatics research. Yet, we
hypothesize that not all significant patterns have been discovered through this guided process, requiring an unguided computational approach. To address this gap, we introduce a novel unbiased computational tool for analyzing word occurrences within genomic data to identify significant genetic sequences and motifs based on pattern frequency. This tool utilizes alignment-based algorithms to conduct an unbiased preliminary search across chromosome 1 of the Homo sapiens Hg.38 genome. Highlighting the most occurring 10, 20, 50, and 500 motif patterns across chromosome 1. This word search tracked the frequency of the motif patterns across chromosome one and their potential identities. Utilizing established genome browsers such as ENSEMBL and CIS-BPU to identify known motifs and other regulatory features. Additionally, the frequency of identified 10- and 20-character motifs were also compared to random chance to further distinguish whether patterns were statistically significant or occurred by random variation. Results indicate that these patterns have biological significance and can be analyzed across species to identify conserved motif patterns. However, the study acknowledges the need for further validation by expanding the analysis to include the entire genome.
Katherine Dempsey – Expression of Human PNPLA3 Protein in CRISPR-generated Knock-In Mice
Fatty liver disease (FLD) is the leading cause of liver disease in the world and is a disorder characterized by an accumulation of triglycerides (TG) on fat storage organelles called lipid droplets. The strongest genetic risk factor for FLD is a missense variant (I148M) in the patatin-like phospholipase domain-containing protein 3 (PNPLA3). This protein is encoded by an insulin-regulated gene expressed at high levels in liver and adipose tissues. The I148M substitution is well-conserved between mice and humans, and the FLD phenotype has been observed in knock-in mice with this variant. Another missense variant (S453I) is strongly associated with reduced hepatic TG levels in humans but is located in a poorly conserved region of the PNPLA3 protein. Since S453I has not been previously studied in mouse models, CRISPR-Cas9 gene editing technology was used to develop a novel strain of knock-in mice containing human PNPLA3. Genotyping results indicated successful insertion of the human gene, and human PNPLA3 protein expression was verified by immunoblotting analysis. Future studies will be conducted using humanized PNPLA3 mice to study the mechanism by which the S453I variant reduces hepatic TG levels and may lead to a potential therapeutic target for the treatment of FLD.
Jayme James – Apigenin Induces Apoptosis in MDA-MB 231 Cell in vitro
Breast cancer is the most common type of cancer among women. There are around 264,000 new cases each year (Basic Information About Breast Cancer, 2022). Although the first stages of breast cancer have high survival rates, there is a drastic decrease in survival as the cancer becomes metastatic. As the prevalence of breast cancer increases, the desperation for new treatments is also rising. In this project, MDA-MB 231 cells were used to determine the efficacy of apigenin in inducing apoptosis in breast cancer cells. MDA-MB 231 cells are a highly aggressive, triple-negative breast cancer cell line, against which hormone-targeted therapies are futile. Additionally, apigenin is a flavonoid found in plants that has various therapeutic properties, including anti-inflammatory, antioxidant, and antiviral significance. The results of our MTT assay showed a dose-dependent induction of apoptosis. The caspase-3 assay indicated that apigenin induces cell death by activating the caspase pathway. The flow cytometry results revealed that apigenin downregulated the expression of PD-L1 in MDA-MB 231 cells. Furthermore, HEK293T, a human embryonic kidney cell line, was used to test the potential detrimental effects of apigenin on normal cell lines. As a result, apigenin has the potential to be used in in vivo applications.
Anna Wilson – Phallus impudicus: A Study of Secreted Compounds
Each year in the United States around 300,000 women and 2,500 men are diagnosed with invasive breast cancer. In the United States, the most common ways to combat breast cancer are surgery, radiation, and chemotherapies. Although chemotherapy has saved countless lives from cancer, its harmful side effects are well-known. Outside of the United States, in countries such as Japan and China, medicinal mushrooms are considered standard in cancer treatment. Preliminary studies have yielded promising results on certain mushrooms' cancer-fighting and immune-boosting properties. It is believed these properties could be used to lessen the harmful effects of chemotherapy.
Common stinkhorns like Phallus impudicus have been used in the treatment of tumors, specifically breast tumors, for almost a millennium. This project aims to pinpoint a naturally occurring compound within the mushroom P. impudicus that has selective cytotoxicity towards breast cancer cells. P. impudicus was cultured in vitro over a two-week period. Mushroom secretions were captured and analyzed using a fast-protein liquid chromatography system (FPLC). A large protein-containing polysaccharide was isolated. Future studies will focus on viability data collected from 96-well cytotoxicity assays.
Lauren Hinson – The Effect of Environmental pH on Staphylococcus aureus
Staphylococcus aureus is a bacterium that is a part of the normal flora on human skin. Studies of S. aureus have documented its ability to mutate and become resistant to antibiotics such as the Methicillin Resistant strain (MRSA). This is problematic because S. aureus is an opportunistic pathogen for several diseases. The pathogenic nature of S. aureus, along with its ability to become antibiotic resistant, makes it an ideal organism for research. Many studies have been conducted to find a correlation between the environment of the bacteria and its pathogenicity. Environmental pH is an important factor especially to sensitivity of antibiotics. A hypothesis was formulated that environmental pH could alter mutation rates in S. aureus. To further investigate the correlation between pH and mutation, wild-type S. aureus cultures were observed in five distinct pH environments and tested against select antibiotics. The cultures were grown and tested over 8 weeks to determine the influence of pH. Cultures were analyzed phenotypically by measuring antibiotic resistance. Changes in color and antibiotic efficacy occurred after 5 weeks. Though not statistically significant, the alteration in antibiotic efficacy indicates that environmental pH has an effect on S. aureus.
Jill Schoelzel – B12 Limits the Effective Killing of HMEC1 Endothelial Cells by the RTK Inhibitor, Sorafenib
In the early 1990’s, the FDA mandated folic acid supplementation in certain foods such as cereal, bread, and pasta, to help reduce neural tube defects in newborns. Folic acid also plays an important role in many cellular pathways and varying folate levels have been associated with a variety of cancers. The essential Vitamin B12 is closely associated with folic acid in the one-carbon metabolism which results in synthesis of nucleotides and methylation.
Sorafenib belongs to a common class of chemotherapeutic drugs those that inhibit Receptor Tyrosine Kinases. RTK inhibitors are known to target the process called angiogenesis, which is the formation of new blood vessels. This study aimed to determine the effects on Vitamin B12 on sorafenib treated HMEC1 endothelial cells. HMEC1 cells showed significant cell death after 24 hours of 10uM Sorafenib treatment. Vitamin B12 in combination with sorafenib had partial protective effect on cell death. Understanding the interplay between B12, sorafenib, and folic acid may be key to understanding if the one carbon pathway is an important regulator of RTKs effectiveness at preventing angiogenesis in the context of cancer.
Molly Byers Wingard, Lindsey Wells, & Giovanna Lopez – Unseen Opportunity: Do Hermaphrodite Cues Influence Male-Male Aggression in Mangrove Rivulus Fish
Aggressive contests in fish often occur in the presence of an audience, which can affect contest dynamics and outcomes. These so-called “Audience Effects” can vary depending on the sex of the audience members. For example, a female audience can elicit increased use of low-intensity displays and decreased use of high-intensity attacks between the male combatants. Mangrove rivulus (Kryptolebias marmoratus) fish occur naturally as either self-fertilizing, simultaneous hermaphrodites or functional males. Male rivulus’ reproductive success thus depends on their ability to obtain outcrossing opportunities with hermaphrodites. Our study’s aim is to examine if hermaphroditic chemical cues in the absence of visual cues influence the intensity of male-male rivulus contests and if unequal access to these cues provides a competitive advantage. We dosed focal individuals with either DI water (control) or water laced with hermaphroditic cues (treated). Following, we created size-matched pairings that resulted in three combinations of contestants; control-control, control-treated, and treated-treated. We found that untreated, control-control contests were more likely to escalate to high-intensity behaviors compared to treated-treated contests. Further, dosed individuals pitted against untreated individuals did not have a competitive advantage. Our findings show that male rivulus do modulate their aggressive behavior dependent on presence of an audience.
Lindsey Wells – Fitness Consequences of Cold Stress in a Self-Fertilizing Hermaphroditic Fish
Temperature stress can lead to physiological disruptions that can result in negative fitness consequences such as reduced fecundity, behavioral impairment, or death. Animals employ various physiological and behavioral strategies to cope with temperature stress such as cold acclimation and reproductive seasonality. The Mangrove Rivulus (Kryptolebias marmoratus) fish, a self-fertilizing hermaphrodite, has demonstrated temperature sex determination in response to temperature stressors and seasonality in reproductive output. However, additional fitness consequences of temperature stress such as changes in development and growth have yet to be explored. Our study aimed to examine how cold-exposure during development affects developmental timing and life-history traits, and to elucidate possible mechanisms underlying seasonal variation in rivulus reproduction. We incubated eggs (zero-day post laying) under temperature conditions that varied in either magnitude (15-20C for 17d) or duration (18C for 5-17d). Control groups were reared at 26C until hatching. Hatchlings were then measured every two months for growth and maturation rates. Our findings indicate that cold exposure at any duration or magnitude impacts latency to hatch, latency to mature, and juvenile mass. Our findings help shed light on how mangrove rivulus respond to cold stress and provide a possible explanation for seasonal variation in rivulus reproductive output.
Michael Knabe – Foraging in 3D: Fruit Fly Positional Foraging in a 3D Arena
Fruit flies (Drosophila melanogaster), exhibit negative geotactic behavior, which can help them escape from predators. We tested the effect of negative geotaxis on foraging in a 3-dimensional setting. We presented the flies with a feeding cube (2.5 cm side length) to test their feeding preference in a 3D arena (10 x 10 x 10 cm) to determine their preference to feed from the top, sides, or bottom of a cube with 9 wells on each side filled with colored sucrose solution. Top, bottom, or sides had either red, yellow, or blue solution. The flies were deprived of food for 24 hours and tested in the dark for one hour. They were then frozen, and the contents of their abdomens were evaluated for color to determine their preferred location for feeding on the cube. There was a preference for the top over the sides and/or the bottom of the cube. The flies also preferred the yellow solution in control trials. The decision about where to feed on the cube seems to be influenced by several variables, including resource position, possible ability to escape or hide from predators, and difference of energetic cost caused by feeding against gravity.