Scientia 2022

SPRING 2022

SCIENTIA THE BAYLOR UNDERGRADUATE RESEARCH JOURNAL OF SCIENCE AND TECHNOLOGY

Editor-in-Chief Isha Thapar Student Editorial Board Faith Abraham, Sanjana Ade, Dafne Andrade, Shivani Ayalasomayajula, Kayla Balkcum, Sinchana Basoor, Rahel Burchardt, Tooba Haris, Tiffany Luan, Kymmia Majedi, Julia Mendes, Arvind Muruganantham, Jessica Ngo, Kate Pogue, Sai Sagireddy, Gabriel Sungcad, Bhaarathi Venkata Faculty Review Board Tamarah Adair, Ph.D.; Sarah Kienle, Ph.D.; Linda Olafsen, Ph.D.; Meredith Palm, Ph.D.; Hugh Riley, Ph.D. Publishing Advisor Rizalia Klausmeyer, Ph.D., Baylor Office of Undergraduate Research Design Team Faith Abraham, Dafne Andrade, Shivani Ayalasomayajula, Sinchana Basoor, Rahel Burchardt, Kymmia Majedi, Julia Mendes, Jessica Ngo, Gabriel Sungcad, Bhaarathi Venkata Funding and Support Baylor Student Government About the Cover The visual on the front cover of Scientia is immunofluorescence staining of microglial activation using Iba-1. This image was provided courtesy of Dr. Lugo's developmental neuroscience lab at Baylor University.

IN THIS ISSUE

3

A Letter From the Scientia Editor-in-Chief

21

Original Research

4

Effects of Outer Membrane Vesicles Produced by Commensal and Pathogenic Strains of Bacteroides fragilis on Viability of Colorectal Cancer Cells Alysia Martinez; Aadil Sheikh; Joseph Taube, Ph.D.

8 12

Review Articles

27

FK506 and Thapsigargin Act with Selective Synergy to Induce Cytotoxicity in TripleNegative Breast Cancer Cell Lines Saawan D. Patel; Joseph Taube, Ph.D.

Harmful Algal Bloom Mitigation Strategies in the Face of Environmental Alterations due to Climate Change and Anthropogenic Nutrient Loading Jonathan D. Norton; Melinda A. Coogan, Ph.D.; Rebecca J. Sheesley, Ph.D.

Tooba Haris; Mary Lauren Benton, Ph.D.

Hope Tucker; Melinda Coogan, Ph.D.

17

Kanali Ghosale; Triniti Taylor; Nicole Wire; Shawn Latendresse, Ph.D.

Analyzing Gene Regulatory Enhancers in Hepatocellular Carcinoma

The Morphological and Behavioral Effects of Rotenone and Paraquat on a Zebrafish (Danio rerio) Model

Less Positive Student-Professor Interactions with Religious Minority Students Predict Lower Intrinsic Motivation and Sense of Belonging

Original Abstracts

36

Intracellular Regulation of Epithelial Na+ Channel (ENaC)

by Ankyrin 3 and Casein Kinase 2 Sinchana Basoor; Antonio G. Soares, Ph.D.; Crystal R. Archer, Ph.D.; James D. Stockand, Ph.D.

37

Impacts of Early Corticosterone Exposure on LPS Later in Life Pranay Sharma; Colin Budries; Donese Cole; Sindhuja Earagolla; Kayla Gilley, M.A; Hailey Rodgers; Elizabeth Vichaya, Ph.D.

Scientia 2022 | 1

URSA Award Winning Abstracts Physics

38

Analysis of K2W7O22 Nanomaterial for a Breathalyzer for Diagnosing Diabetes

38

Observing Anomalous Particle Diffusion within a Dusty Plasma in the PK-4 Experiment

Aman Patel; Weigang Lu, Ph.D.; Blake Birmingham, Ph.D.; Kai Wang, Ph.D.; Danling Wang, Ph.D.; Zhenrong Zhang, Ph.D.

Emerson Gehr; Evelyn Guay; Eva Kostadinova, Ph.D.; Marlene Rosenberg, Ph.D.; Peter Hartmann, Ph.D.; Jorge Carmona-Reyes; Lorin Matthews, Ph.D.; Truell Hyde, Ph.D.

Psychology and Neuroscience

39

Purpose in Emerging Adults: Eschatological Hope as a Predictor for Beyond-the-Self Purpose Yejin (Sally) Lee; Jeanene Jackson; Miranda Wood; Juliette Ratchford, M.A.; Mason Ming, M.A.; Sarah Schnitker, Ph.D.

39

Isolation-Induced Ultrasonic Vocalizations (USVs) using Multiple Methods of the Maternal Potentiation Paradigm Resulted in a Decrease in Calls in Female C57 Mice Doan Tran; Paige Womble; Danielle Santana Coelho, Ph.D.; Katherine Blandin; Srikhar Chilukuri; Siena Condon; D. Nguyen; Joaquin Lugo Ph.D.

2 | Scientia 2022

A Letter From the Scientia Editor-in-Chief Dear Readers, I am excited to publish the 2022 edition of Scientia, and showcase the hard work and commitment shown by Baylor student researchers in their quest to seek solutions to pressing scientific questions.

This edition comes at a very special time, as Baylor was recently recognized as a top-tier research university in December of 2021. Baylor's presitigious Research 1 status stands as a testament to the excellent research being conducted by Baylor faculty members. This also translates to increased research funding and greater opportunities available to undergraduate students to conduct research in Baylor laboratories. I hope that Baylor will continue to attract undergraduate students excited to pursue research in diverse fields and participate in scientific endeavors that solve real-world challenges.

The Baylor administration and faculty are commendably active in supporting and encouraging undergraduate students to pursue research on campus. Various programs and courses engage students through mentorship and internships. I am proud that Scientia adds to this robust research environment by allowing student researchers to share their projects with the wider Baylor community. I also hope that Scientia will play an important role in encouraging budding student researchers by introducing them to a diversity of student-led research projects.

I am grateful to the amazing faculty mentors and the dedicated team of student editors that made the publication of this research journal possible. As a senior, I am humbled to have served as Editorin-Chief for the past two years. I look forward to reading Scientia in the years to come and seeing the amazing research Baylor students continue to conduct.

In closing, I wish to echo President Livingstone’s words upon announcing Baylor's R1 status: “Today is not the end of our journey, but rather a significant step in a trajectory in which we will continue to elevate the quality and impact of our educational and research endeavors.”

-Isha Thapar, Editor-in-Chief

Scientia 2022 | 3

Original Research

Effects of Outer Membrane Vesicles Produced by Commensal and Pathogenic Strains of Bacteroides fragilis on Viability of Colorectal Cancer Cells Alysia Martinez; Aadil Sheikh; Joseph Taube, Ph.D. Department of Biology, Baylor University, Waco, TX

Abstract Colorectal cancer (CRC) is the third most common cancer worldwide and accounts for the second highest number of cancer-related deaths. Patients with Inflammatory Bowel Disease (IBD) are at a significantly increased risk of developing CRC. The development of IBD and CRC are highly correlated with an imbalanced intestinal microbiota composition. The species Bacteroides fragilis can be present in the gut as either a commensal strain, nontoxigenic B. fragilis (NTBF), or as a pathogenic strain, enterotoxigenic B. fragilis (ETBF). Gram-negative bacteria, including B. fragilis, secrete outer membrane vesicles (OMVs), which may pass through the mucosal layer to directly interact or fuse with human intestinal epithelial cells (IECs). The interactions between IECs and OMVs affect homeostasis and pro- or anti-inflammatory host immune responses. Given the association between ETBF with CRC and the lack of an association between NTBF and CRC, we aimed to investigate the morphological and cytotoxic effect of vesicles produced by these bacteria on human colonic epithelial cells.

Introduction Colorectal cancer (CRC) is the third most common cancer worldwide and accounts for the second highest number of cancer-related deaths1. Moreover, patients with Inflammatory Bowel Disease (IBD) are at a significantly increased risk of developing CRC 1,2 primarily resulting from the pro-neoplastic effects of chronic intestinal inflammation1. Both sporadic CRC and IBD-CRC are associated with poor prognoses, with a 5-year survival rate of approximately 50%2. Further, the development of IBD and CRC are highly correlated with an imbalanced intestinal microbiota composition1 and a larger population of bacteria belonging to the Bacteroides genus3. One such species, Bacteroides fragilis, can be present in the gut as either a commensal strain, nontoxigenic B. fragilis (NTBF), or as a pathogenic strain, enterotoxigenic B. fragilis (ETBF), for which there is a significant correlation between presence and active IBD and CRC4.

Communication between the host microbiota, including B. fragilis, and intestinal epithelial cells (IECs) is mediated through a variety of molecular pathways, and may elicit a variety of cellular responses. Given the association between ETBF with CRC and the lack of an association between NTBF and CRC, we aimed to investigate the effect of these bacteria on human colon cells. The two human intestinal cell lines investigated in this study, Caco-2 and HT-29, were isolated from colon adenocarcinomas. One of the most advantageous properties of the Caco-2 cell line is its ability to spontaneously differentiate

4 | Scientia 2022

into a monolayer of cells with many properties typical of absorptive enterocytes with a brush border layer as found in the small intestine. However, HT-29 cells in culture, although essentially undifferentiated, are heterogeneous in that they contain a small proportion of mucus-secreting cells and columnar absorptive cells5. Since B. fragilis are obligate anaerobes, we are unable to co-culture the bacteria directly with human cells. Nevertheless, gram-negative bacteria, including B. fragilis, secrete outer membrane vesicles (OMVs), which may pass through the mucosal layer to directly interact or fuse with human cells. The interactions between IECs and OMVs affect homeostasis and pro- or anti-inflammatory host immune responses6,7. OMVs from Neisseria gonorrhoeae, uropathogenic Escherichia coli and Pseudomonas aeruginosa, have been shown to play a direct role in inducing cell death8. Thus, we opted to assess the effect of OMVs from ETBF and NTBF on the morphology and viability of colonic adenocarcinoma cells.

Materials and Methods B. fragilis (NTBF and ETBF) culture B. fragilis stocks were procured from Dr. Cindy Sears (Johns Hopkins School of Medicine). Glycerol stocks of both NTBF and ETBF were thawed on ice and inoculated into a 15 mL tube of brain heart infusion (BHI) broth with the bacteria

OMV isolation The cultured bacteria were placed into 50 mL tubes and spun down once at 8250xg for 15 minutes at 4 degrees Celsius. Next, the supernatant was filtered through a 0.45 μm vacuum filter (Nalgene™ Rapid-Flow™ Sterile Disposable Filter Units with PES Membrane, Thermo Scientific, Waltham, MA). A BHI plate was then streaked with the filtrate to ensure that no live bacteria remained in the sample. Next, the OMVs were isolated from the filtrate using the ExoBacteria OMV Isolation kit as outlined in the kit instructions. The filtrate was briefly added to the column and allowed to incubate for 30 minutes. Then, the filtrate was removed, allowing for elution of the OMVs out into a 2 mL microcentrifuge tube.

Cell culture Caco-2 cells and HT29-MTX cells were cultured and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Corning, Corning NY). DMEM contains 4.5 g/L glucose, 1 mM sodium pyruvate, 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were cultured at 37°C under a 5% CO2 atmosphere with changes in media every 3-4 days.

MTS Cells were plated with 2000 cells per well in a 96-well plate and allowed to adhere overnight. Outer Membrane Vesicles suspended in PBS were added to the culture medium and incubated for 24 and 48 hours at 37°C, 5% CO2.

Following manufacturer suggested protocol, 20 µL CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS; Promega, Madison, WI, USA) was added and incubated for 1–4 hours at 37°C, 5% CO2. Absorbance was measured at 490 nm using a 96-well plate reader (Fisher Scientific, Hampton, NH, USA).

Brightfield Microscopy Procedures for obtaining the images of ETBF and NTBF B. fragilis OMV treated Caco-2 and HT-29 cells by brightfield microscopy were carried out as described previously. Cells were imaged using the Eclipse Ti2 Brightfield Microscope (Nikon, Tokyo, Japan) at 200x magnification. Images were taken after 48 hours of OMV 10, 250, and 2000 μg/mL exposures.

Results To analyze the effects of ETBF and NTBF OMV treatment on IEC viability, Caco-2 and HT-29 cells were first imaged using brightfield microscopy. Images were taken after 48 hours of treatment with dosages of 10 μg/ml, 250 μg/ml, and 2000 μg/ ml isolated OMVs. These images are displayed in Figure 1 on the next page. The data established a dose-dependent cytotoxic effect induced by both ETBF and NTBF OMV treatments for

both cell lines. At 10 μg/ml, Caco-2 and HT-29 cells exhibit an unaltered epithelial morphology, which is characteristic of their roles in the intestinal lining in vivo. But at higher dosage treatments, cytotoxic effects were evidenced by lowered cell density, which is indicative of decreased viability. However, no dissimilarity could be observed between the cytotoxic effects of OMVs originating from the ETBF versus NTBF strains of B. Fragilis. Moreover, cells were observed undergoing morphogenesis following treatment, characterized by a rounded appearance, which is indicative of cell death upon exposure to 2000 μg/mL of OMVs. In order to quantify the effects of ETBF and NTBF OMV treatments on the viability of IECs, Caco-2 and HT-29 cells were incubated with varying concentrations of isolated OMVs for durations of 24 and 48 hours. They were then assayed for viability using an MTS assay, the results of which are shown in Figure 2 on the next page. Our results indicate that both enterotoxigenic and nontoxigenic B. Fragilis OMV treatments with dosages over 1000 μg/mL induced cytotoxic effects on HT-29 and Caco-2 cell lines across 24- and 48-hour exposure periods. However, there were no detectable differences in cytotoxicity between ETBF and NTBF OMVs for either exposure period. Further, at 24 hours of exposure, HT-29 cells exhibited heightened sensitivity to OMV treatment from both strains relative to the viability of Caco-2 cells. However, while 48-hour exposure time periods demonstrated a heightened cytotoxic effect, this timepoint erased the variation between the two cell lines. The above findings suggest that high doses of B. Fragilis secretory products induce cell death in intestinal epithelial cell lines. This effect was observed to be elevated in the mucus-secreting cell line, HT-29. This is a potential result of the absorptive quality of mucus-secreting and columnar absorptive cells, which may allow for increased uptake and succeeding cell death.

Discussion Dynamic interactions exist in vivo between the intestinal mucosa and intestinal microbes9. Both symbiotic bacteria and invading enteric pathogens first encounter the mucus layer on the intestinal epithelial surface, which provides a protective physical barrier for the host. However, pathogens and symbiotes have developed mechanisms for circumventing this organized mucosal protective system9. For example, bacterial OMVs are one of many modes of communication through which microbes have overcome this physical barrier to impinge upon host IECs at the mucosal interface. While mucoadhesion properties of the intestinal mucus layer have been a topic of research for some time10, this property has been studied primarily as an aid in nutrient uptake. Our study demonstrates that HT-29 cells have a heightened sensitivity to both ETBF and NTBF OMV exposure. This contrast between the non-mucus producing Caco-2 cells and the mucus-secreting absorptive HT-29 cells as well as their respective viabilities after exposure suggests that mucoadhesion may extend as an assisting factor in sensitivity to OMV exposure.

Further, it has been demonstrated that Caco-2 and HT29 cells express dissimilar levels of TLR1, TLR2, TLR3, and TLR411, which may affect their responses to bacterial challenge

Scientia 2022 | 5

Original Research

and allowed to grow to stationary phase overnight. BHI broth was pre-reduced in 50 mL conical tubes with the caps slightly loosened in the anaerobic chamber for 24 hours. For OMV isolation, a single 50 mL tube was inoculated from the 15 mL stationary phase sample, which was sufficient to generate OMVs for downstream analysis.

OMVs, the secretary products of commensal and pathogenic microbes, are sufficient to induce cell death in colonic adenocarcinoma cells. Further studies could elucidate the mode of cell death as well as the cellular responses and mechanistic pathways which are responsible. An understanding of the origins and constituents of the inflammatory pathways exploited by microbes and their secretory products will prove beneficial in our discernment of the origins of and potential therapeutics for colorectal cancer.

HT-29

HT-29

Caco-2

Caco-2

ETBF OMVs

NTBF OMVs

ETBF OMVs

NTBF OMVs

Low 10 µg/mL

Med 250 µg/mL

High 2000 µg/mL Figure 1: The dose-dependent effect of OMVs isolated from enterotoxic and nontoxigenic B. fragilis on Caco-2 and HT-29 cells.

ETBF OMVs 48hr

100

0

1

2

3

log10 Dosage

Caco-2 HT29

100

50

0

1

2

3

log10 Dosage

50

4

ETBF OMVs 24hr

150

0

Caco-2 HT29

100

50

0

NTBF OMVs 48hr

150

Relative Viability

Caco-2 HT29

4

0

0

1

2

log10 Dosage

3

4

NTBF OMVs 24hr

150

Relative Viability

Relative Viability

150

Relative Viability

Original Research

and OMV exposure. Notably, TLR2 and TLR4 are upregulated in HT-29 cells11. This evidence, in conjunction with reports of increased TLR2 and TLR4 expression during intestinal inflammation11, may altogether suggest that inflammation induced cell death, which has been recognized as a mechanism of bacterial cytotoxicity, may be a leading cause of cell death in our study as well as in vivo in colitis-associated cancer.

In conclusion, studying an in vivo model with both cell cultures involves limitations, yet we have demonstrated that

Caco-2 HT29

100

50

0

0

1

2

3

log10 Dosage

4

Figure 2: Viability of Caco-2 and HT-29 cells after 24-and 48-hour treatment with ETBF and NTBF Outer Membrane Vesicles.

6 | Scientia 2022

References Stidham, R. W., & Higgins, P. (2018). Colorectal Cancer in Inflammatory Bowel Disease. Clinics in colon and rectal surgery, 31(3), 168–178. https://doi. org/10.1055/s-0037-1602237 2. Dyson, J. K., & Rutter, M. D. (2012). Colorectal cancer in inflammatory bowel disease:what is the real magnitude of the risk?. World journal of gastroenterology, 18(29), 3839– 3848. https://doi.org/10.3748/wjg.v18.i29.3839 3. Sobhani, I., Tap, J., Roudot-Thoraval, F., Roperch, J. P., Letulle, S., Langella, P., Corthier, G., Tran Van Nhieu, J., & Furet, J. P. (2011). Microbial dysbiosis in colorectal cancer (CRC) patients. PloS one, 6(1), e16393. https://doi. org/10.1371/journal.pone.0016393 4. Haghi, F., Goli, E., Mirzaei, B., & Zeighami, H. (2019). The association between fecal enterotoxigenic B. fragilis with colorectal cancer. BMC cancer, 19(1), 879. https://doi. org/10.1186/s12885-019-6115-1 5. Gagnon, M., Zihler Berner, A., Chervet, N., Chassard, C., & Lacroix, C. (2013). Comparison of the Caco-2, HT-29 and the mucus-secreting HT29-MTX intestinal cell models to investigate Salmonella adhesion and invasion. Journal of microbiological methods, 94(3), 274–279. https://doi. org/10.1016/j.mimet.2013.06.027 6. Ahmadi Badi, S., Khatami, S. H., Irani, S. H., & Siadat, S. D. (2019). Induction Effects of Bacteroides fragilis Derived Outer Membrane Vesicles on Toll Like Receptor 2, Toll Like Receptor 4 Genes Expression and Cytokines Concentration in Human Intestinal Epithelial Cells. Cell journal, 21(1), 57–61. https://doi.org/10.22074/cellj.2019.5750 7. Chu, H., Khosravi, A., Kusumawardhani, I. P., Kwon, A. H., Vasconcelos, A. C., Cunha,L. D., Mayer, A. E., Shen, Y., Wu, W. L., Kambal, A., Targan, S. R., Xavier, R. J., Ernst,P. B., Green, D. R., McGovern, D. P., Virgin, H. W., & Mazmanian, S. K. (2016). Gene-microbiota interactions contribute to the pathogenesis of inflammatory bowel disease.Science (New York, N.Y.), 352(6289), 1116–1120. https://doi.org/10.1126/science.aad9948 8. Deo, P., Chow, S. H., Han, M. L., Speir, M., Huang, C., Schittenhelm, R. B., Dhital, S.,Emery, J., Li, J., Kile, B. T., Vince, J. E., Lawlor, K. E., & Naderer, T. (2020).Mitochondrial dysfunction caused by outer membrane vesicles from Gram-negative bacteria activates intrinsic apoptosis and inflammation. Nature microbiology, 5(11),1418–1427. https://doi.org/10.1038/s41564-020-0773-2 9. Gagnon, M., Zihler Berner, A., Chervet, N., Chassard, C., & Lacroix, C. (2013). Comparison of the Caco-2, HT-29 and the mucus-secreting HT29-MTX intestinal cell models to investigate Salmonella adhesion and invasion. Journal of microbiological methods, 94(3), 274–279. https://doi. org/10.1016/j.mimet.2013.06.027 10. Cecil, J. D., Sirisaengtaksin, N., O’Brien-Simpson, N. M., & Krachler, A. M. (2019). Outer Membrane Vesicle-Host Cell Interactions. Microbiology spectrum, 7(1), 10.1128/ microbiolspec.PSIB-0001-2018. https://doi.org/10.1128/ microbiolspec.PSIB-0001-2018

Scientia 2022 | 7

Original Research

1.

11. Furrie, E., Macfarlane, S., Thomson, G., Macfarlane, G. T., Microbiology & Gut Biology Group, & Tayside Tissue & Tumour Bank (2005). Toll-like receptors-2, -3 and -4 expression patterns on human colon and their regulation by mucosal-associated bacteria.Immunology,115(4), 565– 574. https://doi.org/10.1111/j.1365-2567.2005.02200.x

Original Research

Analyzing Gene Regulatory Enhancers in Hepatocellular Carcinoma Tooba Haris; Mary Lauren Benton, Ph.D. Department of Computer Science, Baylor University, Waco, TX

Abstract Genetic mutations occur in nearly one-fourth of hepatocellular carcinoma cases. Many of these mutations influence gene regulatory elements such as enhancers, which aid with proper growth and development of cells and tissues. Mutations in these regions can cause an appearance of traits that were not previously present and contribute to disease processes. Previous studies have shown that combinations of enhancers that regulate the same gene can have redundant elements. This project examined a set of more than 70,000 human enhancer sequences in liver cells with predicted gene targets to find evidence of redundant enhancers.

Further analysis looked at the similarity in patterns of transcription factor binding motifs within enhancers with the same gene target. Multiple methods such as principal component analysis and kernel density plots were used to observe the general trend. The data demonstrated clustering in a specific area of the plot which is indicative of redundant elements. Introducing tissue specificity scores within these enhancer regions will allow us to quantify the number of tissues in which the enhancer appears active. This project provides insight into the role of redundant enhancers in cancer-related diseases.

Introduction Hepatocellular carcinoma (HCC) is the most common type of liver cancer and often has a terminal prognosis.

Approximately 25% of all HCCs appear with genetic mutations (1). Frequent mutations are found in genes TP53, RB1, CCNA2, CCNE1, PTEN, ARID1A, RPS6KA3, or NFE2L2, which alter cell cycle control. Chromosome amplifications that can lead to overexpression in the activation of oncogenic signaling pathways are found in genes CCND1, FGF19, VEGFA, MYC, and MET (1). Studying human enhancer sequences in liver cells with predicted gene targets can provide evidence of a link between altered enhancer activity and gene mutations found in HCC cases.

Enhancers are sequences of DNA that contain binding sites for transcription factors (TFs), which can generate higher levels of transcription. They are rich in motifs that have a high affinity for the binding of TFs (2). Based on a linear DNA sequence, enhancers can be found at various distances from their target genes. This can make identifying enhancers challenging. Active enhancers are brought into proximity of their target genes by forming chromatin loops (3). Studies have shown that groups of enhancers that regulate the same target genes display redundant elements, which have the same characteristics and functions for the same gene. Mutations in these regions can cause an appearance of traits that were not previously present and likely participate in disease processes, including liver cancers such as HCC (2). However, redundancy in enhancer function may help reduce the effects of genetic variants on gene expression levels.

In this study, we performed dimensionality reduction methods and statistical analysis to understand the possible role of enhancers and enhancer redundancy in HCC. We hypothesized

8 | Scientia 2022

that important genes, such as those where genetic mutations can lead to disease, are more likely to have redundant enhancers (as measured by similar transcription factor binding sites).

Materials and Methods Obtaining enhancer sequences This project analyzed a set of over 70,000 human enhancer sequences to look at the similarity of transcription factor binding motifs within enhancers linked to their predicted gene targets.

To obtain these sequences, the H3K27ac and H3K4me3 ChIPseq peaks were downloaded from the Roadmap Epigenomics Consortium (4). H3K27ac peaks were identified as enhancers if the peak was present, and it overlapped an H3K4me3 peak by less than 50% of its length (5).

Dataset generation The enhancer sequences were initially examined using the Pandas library in Python, allowing for manipulation of data for analysis and management of datasets with the offered data structures. Two datasets were merged to obtain collective information about human enhancer sequences. The merged dataset contained approximately 400 motifs present in different enhancer sequences in the original dataset. It specified the number and type of motifs found in each enhancer sequence and included information on the enhancer’s genomic location and attributes. It also identified whether the genes associated with the enhancer sequences are expressed in liver cells. With this information, the genes not expressed in liver cells were filtered out (transcripts per million < 1). With the focus of this

Principal component analysis To demonstrate the impact of gene targets, a principal component analysis (PCA) was run on each dataset to summarize the general patterns of transcription factor binding sites. PCA is a computational dimensionality-reduction method used to reduce the number of variables in the dataset while preserving as much information as possible, making it highly beneficial and efficient for large datasets. Scikit-Learn, a machine learning library in Python, was used to calculate the first and second principal component (PC1 and PC2). PC1 for each enhancer sequence represents the largest component of variance in the motifs present. PC2 for each enhancer sequence represents a second component of variance in the motifs not accounted for in PC1. Using this method we aimed to capture large amount of variance (>80%). We found that PC1 and PC2 explained more than 90% of variance in our data when combined, so we used these two PCs to capture the sequence features and for ease of visualization. To plot these components, seaborn and matplotlib were used. Seaborn is a Python data visualization library based on matplotlib, which is a plotting library in Python.

Density plotting To further analyze the data, a kernel density estimate (KDE) plot was layered on top of a scatterplot for the four datasets.

A KDE plot is a method of visualizing the distribution of data, similar to histograms. It represents the data by producing a continuous density curve, generating a probability distribution by integrating density across a range. The seaborn library was utilized to make the layered plots and used PC1 and PC2 components as the data.

Results Following the generation of the dataset, we quantified the most prevalent motifs observed in the human liver. The top motifs found were MAZ, VEZF1, and ZN467, as seen in the bar graph on the next page (Figure 1). After filtering out genes not expressed in liver cells, similar methods were used to identify the most common genes relating to HCC in the dataset. The genes found were TP53, CCND1, CTNNB1, and IGF2R, which were later used to examine redundant enhancer activity in liver cells.

After running a PCA on each of the datasets, we observed their clustering patterns. All four PCA plots displayed clustering toward the middle left. This common pattern suggests that many enhancers linked to the same gene share the same sets of transcription factor binding motifs and could indicate redundancy among enhancer sequences.

To further investigate this pattern, KDE plots were layered with scatterplots. These scatterplots can be seen in Figure 2 on the next page. The plots for all four genes showed clustering close to 0 for PC1 and PC2. This demonstrates that there is little variation in the TF binding profiles across enhancers linked to the same target gene.

Discussion The most common genes relating to HCC found in the dataset isolated in liver cells are TP53, CCND1, CTNNB1, and IGF2R, with the most frequent mutation found in TP53.

Because this gene helps control the cell cycle, a mutation can cause abnormal cell growth and result in cancer (1).

Additionally, a mutation which amplifies the CCND1 gene leads to over-expression in the activation of various oncogenic signaling pathways (1). An increase in the Wnt-β-catenin pathway is associated with a mutation in the CTNNB1 gene in HCC cases and can lead to heightened oncogenic activity (6).

A mutation in the IGF2R tumor-suppressor gene can result in loss of heterozygosity which can contribute to the development of cancer (7). The most prevalent motifs found in enhancer sequences in relation to TP53 are MAZ and ZN467. The most common motifs found in enhancer sequences relating to CCND1, CTNNB1, and IGF2R are MAZ and VEZF1. These findings of prevalent motifs in common HCC genes are consistent with the preliminary genomic data obtained at the beginning of this project. MAZ participates in transcription termination and polyadenylation, and deregulation of this motif can cause various tissue malignancies (8). VEZF1 is involved in normal and abnormal cellular proliferation and differentiation (9). ZN467 is a transcription factor for several genes and helps recruit histone deacetylase complexes which can terminate transcription (10). These results suggest that genes involved in HCC are associated with multiple enhancer elements and that there may be redundancy in their regulatory landscapes. Such redundancy could impact the effect of genetic variants on gene expression levels.

After running a PCA and creating density plots for the common HCC genes, the clustering patterns were similar across the different plots. This suggests possible evidence of redundant enhancers. Enhancers that are clustered together have similar TF binding profiles and may demonstrate similar functions.

Using these findings, we find enhancer binding sites that appear around the clusters near 0. We hope to determine if there are specific TFs that set apart the outliers on the plot.

Conclusion In this study we used transcription factor binding motifs to identify patterns in groups of enhancers which allowed us to quantify enhancer redundancy which may impact their interactions with disease-relevant genes. Our study is limited to the data available on enhancer annotations with other genes.

We gathered data for multiple studies in the same cell types, however, the experiments were performed in different labs. This may cause variability in our data and reduce the accuracy of our enhancer to gene assignments. The clustered enhancers are candidates for redundancy. In the future, we plan to develop a scoring system to quantify how “redundant” a group of enhancers are using PCA and other dimensionality reduction techniques. A substantial amount of variance was captured by two PCs; we will continue with only two unless adding more is informative for additional genes. If we discover other features that contribute novel information, we can add to or expand

Scientia 2022 | 9

Original Research

project on HCC, four smaller datasets were derived from the original dataset, each focusing on a specific target gene associated with HCC mutations.

relation to their target genes and their significant influence on HCC.

Acknowledgments I would like to thank Dr. Mary Lauren Benton in the Benton Lab for her assistance with this ongoing project.

Top 10 Most Prevalent Motifs in the Human Genome

Top 10 Most Prevalent Motifs in the Human Genome 2500000

2071550

Total Number of Motifs

2000000

1836520

1500000

1335840 1060570

1768710 1281380

1131820

1243140

1045250

954262

1000000

500000 0 MAZ

KLF15

SP2

PATZ1

ZBT17

WT1

VEZF1

ZN341

ZN467

SP3

Motifs

Figure 1: This graph includes the ten most prevalent motifs in the human genome. The x-axis indicates the motifs, and the y-axis is a count of the number of occurrences (frequency) of that motif. The blue bars highlight the three most prevalent motifs found in the human genome.

TP53

CCND1 60

40

40 20

PC2

PC2

20 0

0 -20

-20

-40 -40

-60 -100

-50

0

50

PC1

100

150

200

250

-100

0

IGF2R

100

PC1

200

300

400

60

80

CTNNB1

125

30

100 75

20

50

PC2

PC2

Original Research

our methods. We also hope to introduce tissue specificity scores within these enhancer regions. This will allow us to quantify the number of tissues in which the enhancer appears active.

Studying redundant enhancers and their activity can be applied in the medical field to facilitate the understanding of gene regulatory networks and their influence in controlling disease-associated genes. With the use of bioinformatics tools, this project can confirm the redundant activity of enhancers in

25 0

10 0

-25

-10

-50 -50

0

50

100

PC1

150

200

250

-20

0

20

PC1

40

Figure 2: All four plots show clustering closer to each other which is indicative of similar transcription factor binding profile and similar function. This can provide evidence of redundant enhancers. The x-axis shows PC1, and the y-axis shows PC2. The contours highlight density in particular regions. The outliers in the plots could be due to variance or distance between enhancers.

10 | Scientia 2022

References Aitsebaomo, J. et al. Vezf1/DB1 is an endothelial cellspecific transcription factor that regulates expression of the endothelin-1 promoter. J Biol Chem. 2001 Oct 19;276(42):39197-205. doi: 10.1074/jbc.M105166200. Epub 2001 Aug 14. PMID: 11504723.

2. Bossone SA, Asselin C, Patel AJ, Marcu KB. MAZ, a zinc finger protein, binds to c-MYC and C2 gene sequences regulating transcriptional initiation and termination.

Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7452-6. doi: 10.1073/pnas.89.16.7452. PMID: 1502157; PMCID: PMC49728.

3. Francis, M. et al. Genomic Characterization of the Zinc Transcriptional Regulatory Element Reveals Potential Functional Roles of ZNF658. Biol Trace Elem Res. 2019 Dec;192(2):83-90. doi: 10.1007/s12011-019-1650-9. Epub 2019 Feb 7. PMID: 30734197; PMCID: PMC6685770.

4. Jang, H.S. et al. Clinical significance of loss of heterozygosity for M6P/IGF2R in patients with primary hepatocellular carcinoma. World J Gastroenterol. 2008 Mar 7;14(9):13948. doi: 10.3748/wjg.14.1394. PMID: 18322954; PMCID: PMC2693688.

5. Kvon, E.Z., Waymack, R., Gad, M. et al. Enhancer redundancy in development and disease. Nat Rev Genet 22, 324–336 (2021). https://doi.org/10.1038/s41576-02000311-x 6. Llovet, J.M., Kelley, R.K., Villanueva, A. et al. Hepatocellular carcinoma. Nat Rev Dis Primers 7, 6 (2021). https://doi. org/10.1038/s41572-020-00240-3 7. Roadmap Epigenomics Consortium et al. Integrative analysis of 111 reference human epigenomes. Nature 518, 317–330 (2015).

8. Shlyueva, D. et al. Stampfel, G. & Stark, A. Transcriptional enhancers: from properties to genome-wide predictions.

Nat Rev Genet 15, 272–286 (2014). https://doi.org/10.1038/ nrg3682 9. Tornesello ML. et al. Mutations in TP53, CTNNB1 and PIK3CA genes in hepatocellular carcinoma associated with hepatitis B and hepatitis C virus infections. Genomics.

2013 Aug;102(2):74-83. doi: 10.1016/j.ygeno.2013.04.001.

Epub 2013 Apr 11. PMID: 23583669.

10. Villar, D. et al. Enhancer evolution across 20 mammalian species. Cell 160, 554–566 (2015).

Original Research

1.

Scientia 2022 | 11

Original Research

The Morphological and Behavioral Effects of Rotenone and Paraquat on a Zebrafish (Danio rerio) Model Hope Tucker; Melinda Coogan, Ph.D. Department of Environmental Science, Baylor University, Waco, TX

Abstract Modern research suggests that chemical pollution may have adverse effects on humans. Such instances where this appears to be the case include the development of Parkinson’s disease symptomology among models exposed to the pesticides paraquat and/or rotenone. This experiment tested if paraquat, rotenone, and a combination of paraquat and rotenone would cause notable behavioral and morphological changes in zebrafish (Danio rerio), such as the tendency to avoid the color red or a change in the spinal angle, length, swim bladder area, or ocular distance. This study expected to see a decrease in the tendency to avoid the color red, spinal angle, and length, as well as an increase in swim bladder area and ocular distance. To test this, zebrafish were exposed to paraquat, rotenone, and a combination of paraquat and rotenone through 10 days post-fertilization. On days 11 and 12 post-fertilization, a behavioral assay was conducted. The paraquat test concentrations used were 0, 5, 10, 15, 20, and 25 ppb. The test concentrations for rotenone were 0, 2, 4, 6, 8, and 10 ppb. The equipotent test concentrations for the combination of paraquat and rotenone were 0_0, 2_5, 4_10, 6_15, 8_20, and 10_25, where the former of the two numbers listed represent the concentration of rotenone in ppb and the latter represents the concentration of paraquat in ppb (i.e., 2_5 means 2 ppb of rotenone and 5 ppb of paraquat). The results of this study suggest an additive synergistic effect when paraquat and rotenone are combined in comparison to their individual effects.

Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disorder (Flinn et al, 2008), characterized by the degeneration of dopaminergic neurons in the substantia nigra region of the brain, a reduction in dopamine (DA) levels, degeneration of nigrostriatal DA neurons and a decreased DA biosynthetic capacity (Priyadarshini et al, 2013; Robea et al, 2020). Parkinson’s disease has also been shown to result in the formation of Lewy bodies as a result of α-synuclein aggregates, a presynaptic neuronal protein (Robea et al, 2020). Furthermore, studies have shown a 40-50% reduction of reduced glutathione (GHS), an antioxidant that prevents damage to cellular components caused by reactive oxygen species (ROS) in PD patients (Hauser et al, 2009; Robea et al, 2020). Modern research has found evidence that PD develops as a result of oxidative stress (Robea et al, 2020). Evidence suggests that this occurs because oxidative stress causes mutations in PINK1 (PTEN-induced putative kinase 1) (Robea et al, 2020). PTEN-induced putative kinase 1 is a putative mitochondrial serine/threonine kinase that is necessary for the normal functioning of mitochondria, such as fission and fusion mechanisms, as well as protection of cells against oxidative stress apoptosis (Deas et al., 2009). In addition, PINK1 provides neuroprotection against intrinsic and extrinsic physiological cellular stress (Priyadarshini et al, 2013). While PINK1 aids in mitochondria performance for life-sustaining functions, PINK1 is also linked to being responsible for PARK6associated autosomal recessive PD (ARPD), as PINK1 regulates

12 | Scientia 2022

Parkin activity through phosphorylation (Arkinson & Walden, 202; Priyadarshini et al, 2013). Research suggests that oxidative stress as a consequence of the loss of PINK1 may be a causal factor in the development of PD. Environmental toxins, such as paraquat and rotenone, can cause such oxidative stress and in this manner, may be a factor in the development of PD (Robea et al, 2020).

Paraquat (C12H14Cl2N2) is a chemical used commonly as an herbicide (Robea et al. 2020). Lipid peroxidation, accumulation of α-synuclein, mitochondrial dysfunction, and low levels of GSH were all observed on rodent and zebrafish (Danio rerio) models treated with paraquat in previous studies (Robea et al, 2020). Observations also demonstrated that paraquat can increase the effect of ROS on other molecules (Robea et al, 2020). It was also noted that exposure to paraquat resulted in the formation of Lewy bodies (Robea et al, 2020). These observations have led researchers to hypothesize that paraquat may be a factor in the development of PD. Rotenone (C23H22O6) is a relatively insoluble compound made from plant extracts of the genera Lonchocarpus and Derris that is commonly used as a piscicide (Rotenone, 1993).

Rotenone is an alkaloidal pesticide that has been shown to affect mitochondrial complex I and destroy DA neurons, resulting in PD symptomology (Robea et al, 2020). Furthermore, rotenone was noted to lead to the formation of α-synuclein aggregate and increased oxidant levels (Robea et al, 2020). Not only has

Materials and Methods Zebrafish Protocol for Rotenone in Well Plates (2–5 dpf) The dosing protocol for rotenone from 6–10 dpf used the rotenone stock solution of 250µg rotenone powder in 250 mL DMSO to yield a stock solution of 1000 ppb. Since the rotenone was in powder form, it was kept on a stir plate to ensure that the particles would fully dissolve and would not settle. Six 150 mL beakers were then labeled with corresponding test concentrations of 0 ppb, 2 ppb, 4 ppb, 6 ppb, 8 ppb, and 10 ppb. Using a graduated cylinder, 100 mL of aquarium water was transferred to each of the six labeled beakers. Pipettes were used to remove various volumes of aquarium water from the beakers, and then replaced by the rotenone stock solution. After

the addition of the rotenone stock solution, the stock solution was returned to the stir plate.

The 6 wells (labeled 1-6), in each of the 6 well plates (labeled A-D), were filled with 10mL of their corresponding concentrations. This process was repeated for each of the eight 6 well plates. Four zebrafish embryos were subsequently transferred into each well using a disposable pipette. The well plates were then partially covered and placed into a temperature and light-controlled incubator. At this point, the temperature and lux of the incubator were recorded.

Zebrafish Protocol for Rotenone in Beakers (6–10 dpf) Concentration dilutions were created among the six labeled 150 mL beakers. Twelve beakers were divided into two groups, A and B, and labeled with their corresponding test concentrations of 0 ppb, 2 ppb, 4 ppb, 6 ppb, 8 ppb, and 10 ppb for each group.

Then, six 500 mL beakers were used to create each dilution. The six 500 mL beakers were filled with 200 mL of aquarium water respectively and then various volumes of aquarium water were removed and replaced with the same volume of rotenone. The rotenone was then returned to the stirring plate. The 200 mL of the solution of the given concentration was then distributed evenly to the A and B beakers labeled with the corresponding concentration, resulting in the A beaker and the B beaker being filled with 100 mL of solution each. This was repeated until all twelve beakers were filled with their assigned concentration of rotenone.

Before transferring zebrafish from the wells to the beakers, they were first fed and photographed. Feeding solution was added to the wells for 10 seconds . The fish were allotted 5 minutes to feed. After the 5 minutes, the zebrafish were transferred into their corresponding beakers. The zebrafish from well plates A and B were transferred to Group A beakers. The zebrafish from well plates C and D were transferred to Group B beakers. Both groups were fed every day until the end of the experimental design. After their daily feeding, the zebrafish were transferred into a new beaker of the same concentration to prevent bacterial infection.

Images were gathered using a light microscope and a microscope camera to record and measure morphological changes that occurred during the span of their exposure. To isolate the fish for photographs, the zebrafish were transferred into 24-well plates and observed under the light microscope.

Pictures were taken and data were recorded. If the zebrafish were especially hyperactive and caused difficulty when capturing images, the fish were exposed to 3-5 drops of a buffered solution of 15-50 mg/L MS-222 Tricaine Methane sulfonate. Utilizing ImageJ, data was recorded including spinal angle, swim bladder area, length, and ocular distance. The image type settings on the software were set to 8 bits, with a magnification of 764 pixels/ mm, as the magnification was consistently at 2.0.

This protocol was repeated each day from 6 dpf – 10 dpf: the test concentrations were remade, the fish were fed and transferred, and photographs were analyzed. Additional observations were recorded, such as the number of unhealthy or dead zebrafish, as well as any observed abnormalities. Fish appearing unhealthy were put into a quarantine beaker, to prevent any possible contamination of the other fish.

Scientia 2022 | 13

Original Research

rotenone been shown to cause damage on a cellular level, but also the larger level of motor skills. As a decrease of DA neurons was observed in zebrafish exposed to rotenone, so was a correlating impairment of motor activity as well as anxious behavior, similar to that observed in PD patients (Robea et al, 2020). In addition, rotenone has the ability to inhibit mitochondrial complex I, as well as promote the generation of Lewy bodies (Robea et al, 2020).

Zebrafish were chosen as the subject for this experiment due to their high fertility, relatively short lifespan of about 3 months, and their transparent characteristic in the embryonic state (Priyadarshini et al. 2013; Singleman & Holtzman, 2014).

Additionally, over 70% of all genes that code for human diseases have functional homologs in zebrafish (Robea et al, 2020).

These qualities make zebrafish an ideal model organism, as the findings from research conducted on the zebrafish can be applied to humans in many instances. Zebrafish are also a useful biological model because they can effectively show any damage to cognitive abilities and brain development during behavioral assays. Poor cognitive abilities may be noted, as the zebrafish naturally interprets the color red as a threat and will thus avoid it. If the zebrafish do not seem to avoid the color red, it can be inferred that the cognitive abilities of the zebrafish were somehow deterred or damaged (Collwill & Creton, 2011).

Another indicator of brain developmental abnormalities in zebrafish is an increase in ocular distance (Lutte et al, 2015).

Since zebrafish express all these characteristics, they are an ideal model for research.

The experiment conducted was designed to test how exposure to paraquat and rotenone may act as a precursor to the development of PD by altering PINK1 and placing dopaminergic neurons under oxidative stress, causing them to undergo apoptosis. In this experiment, we expect to see that exposure to paraquat and rotenone will result in morphological and behavioral effects. The analyzed morphological effects include spinal curvature, decreased length, increased ocular distance, and swelling of the swim bladder. The analyzed behavioral effect includes attraction to the color red, contrasting with typical avoidance behavior observed in zebrafish models.

These endpoints may suggest the potential for PINK1 gene alteration and PD development. Additionally, the relationship between paraquat and rotenone could demonstrate an additive synergistic effect.

Results The data gathered via ImageJ and the behavioral assay were analyzed using data analyses in Microsoft Excel. F tests were conducted between groups and the control for each variable (α=0.05).

The data sets that were found to have p values over 0.05 were analyzed with a t-test with equal variances, while data sets found with p values less than 0.05 were analyzed with a t-test with unequal variances.

The concentrations used in the experiment are shown in Table 1, and results can be seen in the following Figures 1-6.

14 | Scientia 2022

Beaker Rotenone Paraquat Rotenone_Paraquat (ppb) Number (ppb) (ppb) 1 0 0 0_0 2 2 5 2_5 3 4 10 4_10 4 6 15 6_15 5 8 20 8_20 6 10 25 10_25 Table 1: The corresponding concentration of rotenone, paraquat, and the rotenone and paraquat concentration for each beaker number.

4 3.5

Length (mm)

3 2.5 2

*

1.5

*

*

*

1 0.5 0

1

2

4 3 Beaker Number ROT

PQ

5

6

ROT_PQ

Figure 1: Length of the zebrafish in concentrations of rotenone, paraquat, and a combination of rotenone and paraquat from 6-10 dpf, as referenced in Table 1 (* indicates significant difference when compared to control; α=0.05) 0.07 0.06 Swim Bladder area (mm2)

Original Research

Zebrafish Protocol for Rotenone Behavioral Assay (11–12 dpf) During this assay, no stock solutions were used, and no photos were taken. Zebrafish were fed at the beginning of each lab session in the same manner as described in the protocol for 6–10 dpf. Then, two sets of six 150 mL beakers (for a total of twelve beakers) were labeled groups A and B, with the same number sequence as used for test concentrations. Using a graduated cylinder, each beaker was filled with 100 mL of aquarium water. The fish were then transferred to the newly labeled beakers.

Next, six square petri dishes, corresponding to the six test concentrations, were divided into 4 quadrants each, labeled A, B, C, and D. Seventy mL of aquarium water were added to each of the six petri dishes. A large stir bar and a red magnetic ball were obtained. With the use of a pipette with a clipped tip, 8 zebrafish from Group A and B were transferred to the petri dish with the corresponding concentration.

To initiate the behavioral assay, the red magnetic ball was placed in the corner of quadrant A to begin the time sequence.

For every minute, up to 5 minutes, the fish in each quadrant were counted and recorded. At the end of the 5 minutes, the stir bar was used to move the red metal ball to the next corner in a clockwise direction (from A to B, from B to C, and then from C to D) and repeat the behavioral assay. After recording the data from each quadrant, the zebrafish were returned to a new beaker that corresponded to the original dosage, and placed in the incubator.

All protocols were repeated for the combined paraquatrotenone dosing, as well as the paraquat dosing. The paraquat stock solution was made with 5.0 mg paraquat powder in 1L DI to yield a concentration of 5,000 ppb. The test concentrations for paraquat were 0 ppb, 5 ppb, 10 ppb, 15 ppb, 20 ppb, and 25 ppb. The test concentrations for the combined rotenone and paraquat dosing were 0_0 ppb, 2_5 ppb, 4_10 ppb, 6_15 ppb, 8_20 ppb, and 10_25 ppb. The 0_0 concentration consisted of 0 ppb of rotenone and 0 ppb of paraquat. The 2_5 concentration consisted of 2 ppb of rotenone and 5 ppb of paraquat. The 4_10 concentration consisted of 4 ppb of rotenone and 10 ppb of paraquat. The 6_15 concentration consisted of 6 ppb of rotenone and 15 ppb of paraquat. The 8_20 concentration consisted of 8 ppb rotenone and 20 ppb of paraquat. The 10_25 concentration consisted of 10 ppb of rotenone and 25 ppb of paraquat. At the end of the behavioral assay protocols on 13 dpf, the zebrafish were euthanized, lysed, and then frozen.

0.05 0.04

*

0.03 0.02

*

*

*

*

*

0.01 0

1

2

3 4 Beaker Number ROT

PQ

5

6

ROT_PQ

Figure 2: Swim bladder area of the zebrafish in concentrations of rotenone, paraquat, and a combination of rotenone and paraquat from 6-10 dpf, as referenced in Table 1 (* indicates significant difference when compared to control; α=0.05)

0.048

60

0.047 0.046 0.045

% Zebrafish

40

Ocular Distance (mm)

0.08 0.06

0.042 0.041

10

*

0.02

*

*

0

0.04 0

5

10 15 20 Dosing Concentration (ppb)

25

30

0.039

% Zebrafish in Quadrant with Red Ball

0

1

2

3

4

5

Beaker Number

ROT

PQ

6

Ocular Distance (mm) Linear (% Zebrafish in Quadrant with Red Ball) Linear ((Ocular Distance mm))

ROT_PQ

Figure 3: Ocular distance of the zebrafish in concentrations of rotenone, paraquat, and a combination of rotenone and paraquat from 6-10 dpf, as referenced in Table 1 (* indicates significant difference when compared to control; α=0.05)

Figure 6: Correlation analyses of behavioral assay and ocular distance of zebrafish dosed with paraquat (Microsoft Excel; Pearson Correlation) r=-0.2472.

Discussion

190

*

180 170 Spinal Angle (°)

0.043

20

0.04

160 150 140 130 120

1

2

3 4 Beaker Number ROT

PQ

5

6

ROT_PQ

Figure 4: Spinal angle of the zebrafish in concentrations of rotenone, paraquat, and a combination of rotenone and paraquat from 6-10 dpf, as referenced in Table 1 (* indicates significant difference when compared to control; α=0.05) 0.085

0.075 0.07

30

R2 = 0.3643

0.08

25 20 15

R2 = 0.0784

10

0.065 0.06

% Zebrafish

Ocular Distance (mm)

0.044

30

5 0

2

4

6

8

Dosing Concentration (ppb)

10

0

Ocular Distance (mm) % Zebrafish in Quadrant with Red Ball Linear (Ocular Distance) Linear (% Zebrafish in Quadrant with Red Ball)

Figure 5: Correlation analyses of behavioral assay and ocular distance of zebrafish dosed with rotenone (Microsoft Excel; Pearson Correlation) r= -0.2669.

The paraquat dosing protocol resulted in two significant values. One significant value was found for length for the 25 ppb test concentration represented in Figure 1. This rejected our original hypothesis that paraquat dosing would cause a decrease in length. Another significant value was found for swim bladder area in the 25 ppb dosing, represented in Figure 2. This confirmed our hypothesis that exposure to paraquat results in an increase in swim bladder area. For spinal angle and ocular distance measurements, however, no significant values were reported. This rejected the aspect of our hypothesis that paraquat exposure may result in a decreased spinal angle and an increased ocular distance.

The rotenone dosing protocol resulted in only two significant values, both of which were observed for swim bladder area measurements, seen in Figure 2. These significant values occurred at the 2 ppb and 4 ppb test concentrations.

Interestingly, while the significant value at 2 ppb indicates that the swim bladder is significantly greater than the control, the significant value at 4 ppb indicates that the swim bladder is significantly lower than the control. Besides these two points, however, there were no other statistically significant values from the rotenone dosing protocol, suggesting that rotenone may not result in morphological effects.

The combined paraquat-rotenone dosing protocol resulted in a total of ten significant values for length, seen in Figure 1; swim bladder area, seen in Figure 2; ocular distance, seen in Figure 3; and spinal angle, seen in Figure 4. In both length and swim bladder area, significant values were found in the 4_10 ppb, 6_15 ppb, and 8_20 ppb test concentrations, suggesting that exposure to the combination of paraquat and rotenone results in a decrease of length and swim bladder area.

The observed decrease in length confirmed our hypothesis, however, the decrease in swim bladder area contradicted our hypothesis of an increase in swim bladder area. For ocular distance measurements, significant values were recorded for the 6_15 ppb, 8_20 ppb, and 10_25 ppb paraquat-rotenone test concentrations, showing a decrease in ocular distance. This

Scientia 2022 | 15

Original Research

50

0.1

Ocular Distance (mm)

0.12

Original Research

contradicted our hypothesis that exposure to a combination of paraquat and rotenone would cause an increase in ocular distance. For spinal angle measurements, a significant value was recorded for the 8_20 ppb paraquat-rotenone test concentrations. There were noticeably more significant values for the paraquat-rotenone dosing than the individual rotenone dosing or the paraquat dosing. Additionally, a high mortality rate was noted, with all 10_25 ppb dosed zebrafish dying by 7 dpf. By 9 dpf, all but two of the fish (from the 2_5 dosing) had died (excluding the control group). This suggests that rotenone and paraquat have an additive synergistic relationship, as hypothesized.

It should be noted that there may have been some potential errors. Rotenone has a relatively short half-life, resulting in quick degeneration. Rotenone stock solution was remade daily in an attempt to prevent this degeneration. However, it is possible that the compound may have still degenerated too quickly, causing a lower stock concentration. This could provide an explanation as to why there were few to no significant values for the higher test concentrations. It should also be noted that during the rotenone dosing at 6 dpf, there was a brief power outage causing the incubator to drop in temperature. Though unlikely, this could have added stress to the zebrafish- particularly since they are tropical fish with an optimal temperature range of 28.5 ± 0.5°C (Aleström et al. 2019).

Conclusions The data analysis supports the original hypothesis that exposure to rotenone and paraquat may negatively impact the development of the zebrafish from the embryo to the larval stages. The high mortality rates from the combined paraquatrotenone dosing suggested that they have may have a synergistic relationship. The findings of this experiment support the argument that paraquat and rotenone are not only hazardous to wildlife but also might result in neurodegenerative disease pathogenesis in humans. Hence, the widespread use of pesticides must be monitored. These results could be strengthened in the future by utilizing a movement tracking camera, which would allow for the study of the locomotor tendencies of the zebrafish over a longer time period and with greater precision.

Additional future research could also include preventative steps to aid in neuroprotection from oxidative stress inducers, such as paraquat and rotenone, through the application of L-Glutathione Reduced. These future research paths could potentially strengthen the data supporting the hypothesis that paraquat and rotenone at environmentally relevant concentrations may act as precursors to neurodegenerative disease pathogenesis, with debilitating outcomes such as Parkinson’s Disease.

References Aleström, P., D’Angelo, L., Midtlyng, P. J., Schorderet, D. F., Schulte-Merker, S., Sohm, F., & Warner, S. (2020, June).

Zebrafish: Housing and husbandry recommendations.

Laboratory animals. Retrieved December 24, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7301644/

16 | Scientia 2022

Arkinson, C., & Walden, H. (2021, April 23). Parkin function in Parkinson’s disease Models of Parkin-mediated ubiquitination lend insight into the role of pathological mutations. Science Magazine. https://science.sciencemag. org/content/sci/360/6386/267.full.pdf.

Colwill, R. M., & Creton, R. (2011). Imaging escape and avoidance behavior in zebrafish larvae. Reviews in the neurosciences, 22(1), 63–73. https://doi.org/10.1515/ RNS.2011.008 Deas, E., Plun‐Favreau, H., & Wood, N. W. (2009). PINK1 function in health and disease. EMBO Molecular Medicine, 1(3), 152–165. https://doi.org/10.1002/emmm.200900024 Flinn, L., Bretaud, S., Lo, C., Ingham, P. W., & Bandmann, O. (2008). Zebrafish as a new animal model for movement disorders. Journal of Neurochemistry, 106(5), 1991–1997. https://doi.org/10.1111/j.1471-4159.2008.05463.x Hauser, R. A., Lyons, K. E., McClain, T., Carter, S., & Perlmutter, D. (2009). Randomized, double-blind, pilot evaluation of intravenous glutathione in Parkinson’s disease. Movement Disorders, 24(7), 979–983. https://doi.org/10.1002/ mds.22401 Lutte AH, Capiotti KM, da Silva NL, da Silva CS, Kist LW, Bogo MR, Da Silva RS. (2015) Contributions from extracellular sources of adenosine to the ethanol toxicity in zebrafish larvae. Reprod Toxicol. 2015 Jun;53:82-91. doi: 10.1016/j.reprotox.04.2015.001. Epub 2 0 1 5 Apr 13. PMID: 25883026. (PDF) Pesticides Toxicity in Fish with Particular ... https://www.researchgate.net/ publication/272575872_Pesticides_Toxicity_in_Fish_with_ Particular_Reference_to_Insecticides.

Priyadarshini, M., Orosco, L. A., & Panula, P. J. (2013).

Oxidative stress and regulation of Pink1 in Zebrafish (Danio rerio). PLoS ONE, 8(11). https://doi.org/10.1371/journal. pone.0081851 Robea, M.-A., Balmus, I.-M., Ciobica, A., Strungaru, S., Plavan, G., Gorgan, L. D., … Nicoara, M. (2020). Parkinson’s Disease-Induced Zebrafish Models: Focusing on Oxidative Stress Implications and Sleep Processes. Oxidative Medicine and Cellular Longevity, 2020, 1–15. https://doi. org/10.1155/2020/1370837 Rotenone (1993). EXTOXNET: Cornell University. Accessed October 8, 2020. http://pmep.cce.cornell.edu/profiles/ extoxnet/pyrethrins-ziram/rotenone-ext.html.

Singleman, C., & Holtzman, N.G. (2014). Growth and Maturation in the Zebrafish, Danio Rerio: A Staging Tool for Teaching and Research. Zebrafish, 11(4), 396-406. h t t p : / / doi.org/10.1089/zeb.2014.0976

Saawan D. Patel; Joseph Taube, Ph.D. Department of Biology, Baylor University, Waco, TX

Abstract The high prevalence of breast cancer, which affects about 1 in 8 women over the course of their lifetime, has led to many research efforts to discover new treatment options for patients affected by this disease. Triple-negative breast cancer (TNBC) ranks as the deadliest subtype of breast cancer because of the lack of treatment options. The goal of cancer therapy is to kill off cancerous cells while leaving healthy tissue intact. The purpose of this study is to uncover the use of thapsigargin and FK506 as a potential drug combination for TNBC treatment. Our study utilized three cell lines representing two subtypes of breast cancer, TNBC and estrogenreceptor positive (ER+) cells, to determine the degree of specificity of thapsigargin and FK506 in inducing cell death in only the TNBC subtype. Our findings demonstrate that there is synergistic cytotoxicity for co-treatment with thapsigargin and FK506 in TNBC but not in ER+ cells.

Introduction Breast cancer is the second most common cause of cancer-related deaths in women (Han 2021). There are five main molecular subtypes of breast cancer: luminal A, luminal B, basal-like, HER2-enriched, and normal-like. Luminal A, luminal B, and normal-like are typically estrogen-receptor and/ or progesterone-receptor positive. Human epidermal growth factor receptor 2 (HER2)-enriched breast cancer is HER2 positive. Positivity for these receptors indicates that the cancer cells require either estrogen to stimulate an estrogen receptor (ER) or certain ligands to stimulate HER2 to grow. Because of this dependency, targeting these indicators allows for a point of selectivity when developing treatment options. This targeted approach facilitates the use of novel therapeutics, as drugs that are developed for treating cancers can selectively affect the pathological tissue and leave healthy cells intact. However, most basal-like breast cancers are also categorized as triple-negative breast cancer (TNBC) and lack a dependency on human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), and progesterone receptor (PR). Because of the lack of response to hormone therapy, targeted treatment options are limited for TNBC patients. In addition, TNBC is an aggressive form of cancer, as it is more likely to have spread than other subtypes by the time it is diagnosed, and most likely to have recurrence (Han 2021). Collectively, these aspects help TNBC earn its spot as the deadliest subtype of breast cancer. This paper aims to identify a potential drug combination with enhanced efficacy at targeting TNBC cells.

There are several types of cell death including but not limited to apoptosis, necrosis, and necroptosis (Tang 2019).

The latter of the three is a programmed form of necrosis in which endoplasmic reticular stress induces the release of calcium, mitochondrial swelling, and subsequent plasma membrane rupture. Increased intracellular calcium activates phospholipases, affects cell permeability, and induces membrane swelling. Following this increased calcium level, the mitochondrial permeability transition pore will open, leading to the release of pro-cell death factors such as cytochrome c (Zhu 2018). The endoplasmic reticulum could be a key player in inducing necroptosis. The endoplasmic reticulum is responsible for calcium storage in the cell, and the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA) helps cells maintain low intracellular calcium levels by sequestering calcium into the endoplasmic reticulum (Spitler 2012). Thapsigargin is a calcium ATPase and SERCA inhibitor, which can prevent the sequestering of calcium into the endoplasmic reticulum, thereby inducing necroptosis (Janssen 2009 and Lindner 2020).

FK506, also known as tacrolimus, mediates calcium release by binding to the FK-binding protein on calcium channels on the endoplasmic reticulum, thereby releasing it from the channel.

The release of the FK-binding protein allows for the passage of calcium through the channel from the endoplasmic reticulum to the cytoplasm (Sabatini 1997). Through in vitro cell viability and synergy assays, we demonstrated that a combination of thapsigargin and FK506 results in cytotoxic synergy in the

Scientia 2022 | 17

Original Research

FK506 and Thapsigargin Act with Selective Synergy to Induce Cytotoxicity in Triple-Negative Breast Cancer Cell Lines

Original Research

TNBC cell lines and negligible effects in epithelial-like ER+ cells.

Materials and Methods Cell Lines MDA-MB-231 (TNBC cells), Hs-578t (TNBC cells), and MCF7 (ER+ cells) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Corning Inc., Kennebuck, ME, USA) supplemented with 10% fetal bovine serum (FBS) (EquitechBio Inc., Kerrville, TX, USA) and 1X antibiotics (Penicillin/ Streptomycin, Lonza, Basel, Switzerland) and incubated at 37 degrees Celsius with 5% CO2 (Elenbaas 2001).

Chemical Reagents Thapsigargin was obtained from Selleckchem (Houston, TX, USA), and FK506 and dimethyl sulfoxide (DMSO) was obtained from Sigma Aldrich (St. Louis, MO, USA). These compounds were dissolved in DMSO and kept at 25mM aliquoted stocks at -80 degrees Celsius. The 25mM aliquots of thapsigargin and FK506 were diluted in DMEM appropriately for experimentation.

Cell Viability 2,000 cells were plated per well in a 96-well plate and adhered overnight. The following day, the respective compounds were added to the culture medium. Cells were then incubated for 72 hours. 20µL of CellTiter-Blue® was added to each well and incubated for another 3 hours. CellTiter-Blue® is a reagent that uses the indicator dye resazurin to measure cell metabolism as a proxy for cell viability. Viable cells reduce resazurin to resorufin, a fluorescent compound. Fluorescence was measured using the Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA) at 560nm excitation and 590nm emission.

Synergy Assay MDA-MB-231 cells plated in a 96-well plate were dosed with an increasing concentration of FK506, thapsigargin, and matched-percentage DMSO. After 72 hours, cell viability was measured using CellTiter-Blue® (Promega, Madison, WI, USA). Fluorescence was measured using the Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA) at 560nm excitation and 590nm emission.

Synergy was quantified using the Combenefit program with Loewe modeling and single-agent dose-response shift surface mapping (Di Veroli 2016).

Results The combination of thapsigargin and FK506 is ineffective towards ER+ cells but induces cell death in TNBC After TNBC cell lines Hs-578t and MDA-MB-231 and ER+ cell line MCF7 were dosed with the necroptosis-inducing compound thapsigargin, cell viability assays were performed to measure initial cell viability (Figures 1A-C). All three cell lines exhibited loss of viability upon dosage with thapsigargin alone; however, ER+ cells showed a diminished cytotoxic effect after

18 | Scientia 2022

being concurrently dosed with FK506. Contrasting the effect of FK506 on TNBC cells, concurrent dosage of thapsigargin with FK506 rescues ER+ cells from cell death, as shown by an increase in cell viability as the concentration of FK506 increases (Figure 1A). TNBC cells showed the opposite: FK506 co-treatment decreased cell viability, and the degree of thapsigargin-induced cytotoxicity increases as the concentration of FK506 increases from 3.2µM to 13µM (Figures 1B and 1C). Thus, FK506 is the gatekeeper of cellular fate, inducing both cell death in TNBC cells and better survival in ER+ cells. Taken together, it is apparent that a combination of thapsigargin and FK506 selectively induces cell death more efficiently in TNBC cells than ER+ cells.

FK506 serves as a mediator for thapsigargin-induced cell death Although thapsigargin-induced cell death is enhanced when FK506 is added, it is unknown if FK506 has selective cytotoxic properties against TNBC. To determine whether FK506 is the driver or mediator of the TNBC selective death, we ran a cell viability assay on the cell lines MDA-MB-231, Hs578t, and MCF7. In all three cell lines, FK506 did not cause any change in cell viability as the concentration of FK506 increased (Figure 2A). From this, we deduce that FK506 is the mediator of thapsigargin-induced cell death, as it determines whether there is a protective or increased effect of cell death without causing cell death on its own.

Thapsigargin and FK506 display synergy in TNBC cells Lastly, we tested what relationship thapsigargin and FK506 have. In TNBC, we know that FK506 enhances the cell death mechanism of thapsigargin; however, we do not know whether this relationship is additive or synergistic. In an additive relationship, we would simply expect cell viability of cells dosed with FK506 and thapsigargin alone to subtract directly. In a synergistic relationship, the cytotoxic effects are multiplied.

To determine this, we co-treated cells with increasing concentrations of both compounds. We mapped the response to a Loewe model, where antagonism is denoted by red, additive effects are green, and synergy is blue. After conducting a synergy assay, we determined that there is an increasing synergistic effect as the concentration of FK506 increases when thapsigargin is at 12.5µM, the lowest dose used in this experiment for the TNBC cell line, MDA-MB-231 (Figure 2B).

Discussion This study investigates two compounds, thapsigargin and FK506, that could serve as potential therapeutics against TNBC. Currently, patients diagnosed with TNBC are given limited treatment options because TNBC is difficult to selectively target. We illustrate how thapsigargin-induced cell death can be enhanced in TNBC cells using FK506, a compound with negligible cytotoxic properties at the doses investigated.

Demonstrated by the opposing trends in ER+ versus TNBC cells as the concentration of FK506 increases while concurrently dosed with thapsigargin, our results show a distinct effect on ER+ cells versus TNBC cells. These results convey the possibility of targeted therapy towards TNBC, as the TNBC cells exhibited

metastasis and features a change in migratory properties (Zhang 2018). The MCF7 cells are more epithelial whereas Hs-578t and MDA-MB-231 cells are more mesenchymal. A study performed

by Klettner and Herdegen showed that FK506 is protective in neural cells but not fibroblasts which have a mesenchymal phenotype similar to that of TNBC cells (Klettner 2003). FK506 has other notable neuroprotective effects, similar to that of gracilin A and cyclosporin A (Abbasov 2019 and Shuqi 2009).

An example of such is the protective effect of FK506 against thapsigargin-induced death in SH-SY5Y cells, a model of neurodegenerative disorders like Parkinson’s (Muramoto 2005).

Ultimately, future research comparing the efficacies of these compounds in mesenchymal and epithelial cells is necessary to elucidate their selectivity before moving into in vivo studies.

MCF7 ****

40

Hs-578t 20 0

Thapsigargin [50uM] FK506 uM

Cell Viability (% Alive)

60

Cell Viability (% Alive)

Cell Viability (% Alive)

****

MDA-MB-23

***

+

+

+

+

-

3.2

6.3

13

*

60

****

****

**

80

***

60 40 20 0

Thapsigargin [50uM]

40

FK506 uM

20

+

+

+

+

+

-

3.2

6.3

13

25

0 Thapsigargin [50uM] FK506 uM

+

+

+

+

+

-

3.2

6.3

13

25

Figure 1: (A-C) Cell viability assay using MTS measuring effects of necroptosis inducer, thapsigargin, and FK506 at various concentrations in MCF7 (A), Hs-578t (B), and MDA-MB-231 (C).

Synergy mapped to D-R (LOEWE) FK506 vs Thapsigargin in MDA-MB-231

FK506

MDA-MB-231 MCF7

100 50

100 50

0

-1.0

-0.5

0.0

0.5

log[FK506] μM

1.0

1.5

0 12.5

25

Thapsigargin [uM]

50

100

25

12.5

6.3

3.1

0

Antagonism

150

0 -1.5

Synergy

Hs578t

% Control

Cell Viability (% Alive)

200

FK506 [uM]

Figure 2: (A) Cell viability assay using MTS measuring effects of FK506 in MCF7, Hs-578t, and MDA-MB-231.

(B) Synergy assay with varying concentrations in thapsigargin and FK506 in TNBC line MDA-MB-231.

Scientia 2022 | 19

Original Research

more severe reactions to the drug combination than ER+ cells.

Importantly, the mechanism behind the selective synergy remains to be uncovered. One hypothesis is that ER+ and TNBC cell lines express varying levels of FK-binding proteins which are targets of FK506. Moreover, there are prior reports suggesting that cytotoxicity is due to calcium overload as a result of thapsigargin and FK506 (Alevizopoulos 1997, Cerella 2010, Wang 2019, and Zhu 2018). Because necroptosis can be a calcium-mediated form of cell death, future investigation should account for the extent of calcium release into the cytoplasm as a result of dosage to clarify the relationship between necroptosis and calcium release.

The observed effects may have also stemmed from phenotypic differences amongst the cell lines. The epithelial-tomesenchymal transition is a hallmark feature of breast cancer

Original Research

References Abbasov, M.E., Alvariño, R., Chaheine, C.M. et al. Simplified immunosuppressive and neuroprotective agents based on gracilin A. Nat. Chem. 11, 342–350 (2019). https://doi org/10.1038/s41557-019-0230-0.

Alevizopoulos A, Dusserre Y, Rüegg U, Mermod N. Regulation of the transforming growth factor beta-responsive transcription factor CTF-1 by calcineurin and calcium calmodulin-dependent protein kinase IV. J Biol Chem.

1997 Sep 19;272(38):23597-605. doi: 10.1074 jbc.272.38.23597.PMID: 9295299.

Cerella C, Diederich M, Ghibelli L. The dual role of calcium as messenger and stressor in cell damage, death, and survival Int J Cell Biol. 2010;2010:546163. doi: 10.1155/2010/546163.

Epub 2010 Mar 15. PMID: 20300548; PMCID: PMC2838366.

Di Veroli, G. Y. et al. Combenefit: An interactive platform for the analysis and visualization of drug combinations.

Bioinformatics 32, 2866–2868. https://doi.org/10.1093 bioinformatics/btw230 (2016).

Elenbaas, B. et al. Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells. Genes Dev. 15, 50–65 (2001).

Han, Y., Xu, S., Colditz, G. A., &amp; Toriola, A. T. (2021).

Trends in breast cancer mortality according to molecular subtypes: A population-based study. Journal of Clinical Oncology, 39(15_suppl), 572–572. https://doi.org/10.1200 jco.2021.39.15_suppl.572 Janssen K, Horn S, Niemann MT, Daniel PT, Schulze Osthoff K, Fischer U. Inhibition of the ER Ca2+ pump forces multidrug-resistant cells deficient in Bak and Bax into necrosis. J Cell Sci. 2009 Dec 15;122(Pt 24):4481-91. doi: 10.1242/jcs.055772. Epub 2009 Nov 17. PMID: 19920074.

Klettner, A., Herdegen, T. (2003). The immunophilin ligands FK506 and V-10,367 mediate neuroprotection by the heat shock response. British Journal of Pharmacology, 138(5), 1004–1012. https://doi.org/10.1038/sj.bjp.0705132 Lindner, P., Christensen, S.B., Nissen, P. et al. Cell death induced by the ER stressor thapsigargin involves death receptor 5, a non-autophagic function of MAP1LC3B, and distinct contributions from unfolded protein response components. Cell Commun Signal 18, 12 (2020). https:/ doi.org/10.1186/s12964-019-0499-z.

Muramoto M, Yamazaki T, Morikawa N, Okitsu O, Nagashima T, Oe T, Nishimura S, Kita Y. Protective effect of FK506 against apoptosis of SH-SY5Y cells correlates with regulation of the serum inducible kinase gene.

Biochem Pharmacol. 2005 May 15;69(10):1473-81. doi: 10.1016/j.bcp.2005.02.019. PMID: 15857611.

Sabatini DM, Lai MM, Snyder SH. Neural roles of immunophilins and their ligands. Mol Neurobiol. 1997 Oct;15(2):223–239.

Shuqi Du, et al. Suppression of NF-κB by Cyclosporin A and Tacrolimus (FK506) via Induction of the C/EBP Family: Implication for Unfolded Protein Response. The Journal of Immunology June 1, 2009, 182 (11) 7201-7211; DOI: 10.4049/jimmunol.0801772.

20 | Scientia 2022

Spitler, K.M., Giachini, F.R. and Webb, R.C. (2012), Endoplasmic reticulum stress induces sarco/endoplasmic reticulum calcium ATPase and alters calcium homeostasis in the vasculature. FASEB J, 26: 863.2-863.2. https://doi org/10.1096/fasebj.26.1_supplement.863.2 Tang, D., Kang, R., Berghe, T.V. et al. The molecular machinery of regulated cell death. Cell Res 29, 347–364 (2019). https:/ doi.org/10.1038/s41422-019-0164-5.

Wang Z, Feng J, Yu J, Chen G. FKBP12 mediates necroptosis by initiating RIPK1-RIPK3-MLKL signal transduction in response to TNF receptor 1 ligation. J Cell Sci. 2019 May 20;132(10):jcs227777. doi: 10.1242/jcs.227777. PMID: 31028177.

Zhang, Y., & Weinberg, R. A. (2018). Epithelial to-mesenchymal transition in cancer: complexity and opportunitiers.

Frontiers of medicine, 12(4), 361–373 https://doi.org/10.1007/s11684-018-0656-6.

Zhu P, Hu S, Jin Q, Li D, Tian F, Toan S, Li Y, Zhou H, Chen Y. Ripk3 promotes ER stress-induced necroptosis in cardiac IR injury: A mechanism involving calcium overload XO/ROS/mPTP pathway. Redox Biol. 2018 Jun;16:157-168. doi: 10.1016/j.redox.2018.02.019. Epub 2018 Mar 1. PMID: 29502045; PMCID: PMC5952878.

Kanali Ghosale; Triniti Taylor; Nicole Wire; Shawn Latendresse, Ph.D. Department of Psychology and Neuroscience, Baylor University, Waco, TX

Abstract Student-professor interactions are important for intrinsic motivation and sense of belonging and are crucial for successful social and academic college transitions, especially for ethnic minorities. It was hypothesized that religious minorities experience fewer positive interactions, thus less intrinsic motivation and sense of belonging. Eighty-three college students were recruited and completed the Student-Professor Interaction Scale (SPIS), Psychological Sense of School Membership Scale (PSSM), and SelfRegulation Questionnaire–Learning (SRQ-L). Independent samples t-tests demonstrated that religious minorities felt significantly less connected (t(81) = 2.77, p < .05) and encountered more negative experiences (t(48.86) = –2.17, p < .05). Multiple regressions showed these factors predicted lower intrinsic motivation (R2 = .27, F (1, 81) = 14.98, p < 0.001) and sense of belonging (R2 = .28, F (1, 81) = 15.47, p < 0.001). This demonstrates a difference in student-professor interactions between religious majority and minority students, thus highlighting the need for increased religious awareness at religiously affiliated universities.

Introduction When students enter college, they must adapt to the challenges of adulthood, balance their newfound independence and responsibility (Pittman & Richmond, 2008), and reestablish social connections (Willoughby et al., 2020). This social and academic integration is vital in developing a sense of belonging which ultimately affects university retention rates (Tinto, 1993).

Adjusting to college concerns psychological, educational, and relational functioning. Psychological functioning focuses on a student’s emotions towards their college experience, educational functioning taps into academic integration and success, and relational functioning assesses a student’s social interactions (O’Donnell et al., 2018). Forming interpersonal relationships in college are important, especially those with professors. However, they are often underestimated by students despite research showing students who frequently interacted with professors not only valued their courses more but also developed positive attitudes towards their learning (Thompson, 2001). A possible explanation could be that students are unsure about their professors’ attitudes towards these interactions (Cotten and Wilson, 2006). Thus, it is important to investigate how professors portray themselves and the conversations between students and professors. To assess the quality of student-professor interactions, Cokley and colleagues (2004) developed the Student-Professor Interaction Scale (SPIS) and they found that approachability, respectful interactions, and

availability correlated to students’ academic self-concept (Cokely et al., 2004; Komarraju et al., 2010). Additionally, social interactions not necessarily related to coursework were important for developing students’ motivation to learn and avoid professor disapproval (Cotten & Wilson, 2006).

Sense of belonging is also important and is defined as how an individual perceives their standing from reception into a group, respect, being part of the group, and reinforcement (Goodenow and Grady, 1993). Belongingness is an innate human desire where a minimum quantity and quality (i.e., stability, continuity, mutual affective concern) is actively sought (Baumeister & Leary, 1995). Level of belongingness at the beginning of college is important in predicting a student’s intention to continue studying at a university and a contributing factor is greater faculty interaction (Hausmann et al., 2007). Belongingness at school does not solely rely on belongingness in a single class (Freeman et al., 2007), therefore, this underscores the importance of students having interactions with multiple faculty members. It is also useful in predicting social, psychological, and academic outcomes for students and is linked to positive self-perception of social acceptance, less anxiety and depression, academic competence (Pittman & Richmond, 2008), and motivation (Freeman et al., 2007).

Motivation is another important factor for students to succeed in college. According to the Self-Determination Theory

Scientia 2022 | 21

Original Research

Less Positive Student-Professor Interactions with Religious Minority Students Predict Lower Intrinsic Motivation and Sense of Belonging

Original Research

(SDT; Deci et al., 1991), motivation consists of extrinsic motivation, which originates from external sources and is a compliance to interpersonal or intrapsychic forces, and intrinsic motivation, which is a voluntary choice made to experience the enjoyment from an action (Deci et al., 1991). Schools aim to develop intrinsic motivation in students (Deci et al., 1991) as it has been linked to academic achievement (Taylor et al., 2014), academic satisfaction, and less depression and stress (Huang et al., 2016).

Student-professor interactions play an important role in a student’s success in university, especially among minorities.

Previous studies found that racial minorities experienced more negative interactions and less significant associations to academic motivation compared to their white peers (Cokley et al., 2004). Conversely, caring interactions between professors and Black students at predominantly white institutions (PWIs) predicted higher levels of social and academic engagement (Beasley, 2020), with social engagement contributing to a sense of belonging (Grier-Reed, 2013). These results, however, have not been extended to religious minorities who also experience microaggressions. A microaggression is a form of discrimination that negatively targets certain individuals due to their membership in a group (Nadal et al., 2015), leading to enhanced depressive symptoms (Cheng et al., 2017). Therefore, this study aimed to investigate whether these findings apply to religious minorities. The data was collected from a religiously affiliated university and responses from religious majority students, Christians, were compared to religious minorities. The study assessed the correlation between religious differences and the positivity of student-professor relationships and investigated the subsequent associations with sense of belonging and intrinsic motivation. The study hypothesized that (H1) religious minorities at institutions with strong religious affiliations experience less positive student-professor relationships and that (H2) less positive student-professor relationships would result in less (a) intrinsic motivation and (b) sense of belonging.

Methods The sample consisted of 83 students (62 women, 21 men, Mage = 19.77 years, age range: 18 – 26 years) from Baylor University who were recruited through a web-based survey system. The sample consisted of 45.78%, 25.30%, 10.84%, 9.64%, and 8.43% students identifying as Caucasian, Asian, African American, “Other”, and Hispanic, respectively. There were 11 freshmen, 44 sophomores, 19 juniors, and 9 seniors.

Additionally, 52 students identified as Christians, and 31 students belonged to religious minority groups (i.e. Hindu, Islam, Buddhist, Atheist, Agnostic, No Religion/Spiritual Affiliation, and Other).

Student-Professor Interaction The Student-Professor Interaction Scale (SPIS) (Cockley et al., 2004) is a 40-item questionnaire consisting of 9 subscales of student-professor interactions (i.e. career guidance, off-campus interactions, approachability, validity scale, accessibility, respectful interactions, caring attitude, connectedness, and negative experiences). Items such as, “I feel that one or more professors are supportive of me” are rated on a 7-point Likert

22 | Scientia 2022

scale from 1 (Strongly Disagree) to 7 (Strongly Agree). The alpha reliabilities of the SPIS subscales ranged from 0.50 to 0.93 (respectful interactions a = 0.93, career guidance a = 0.88, approachable a = 0.84, validity scale a = 0.74, caring attitude a = 0.87, off-campus interactions a = 0.50, connectedness a = 0.67, accessibility a = 0.77, negative experiences a = 0.68).

All subscales, excluding the validity subscale, demonstrated construct validity via significant correlations with academic self-concept, intrinsic motivation, and 2 types of extrinsic motivation (identified regulation and introjected regulation).

In this study, items 13, 30, and 33 were reworded to consider religion rather than ethnicity and are as follows: “My professors demonstrate familiarity with my religion” (item 13), “Professors show respect for religious minority students” (item 30), and “My professors seem comfortable interacting with students outside of their religious group” (item 33).

Sense of Belonging The Psychological Sense of School Membership Scale (PSSM) was developed by Goodenow (1993) in youth 10 years and older and was adapted to measure sense of belonging in university students (Alkan, 2016). The adapted scale includes 18 items which are grouped into 3 subscales: acceptance by faculty members, belonging, and acceptance by students. Items such as “People at this university notice when I’m good at something” are rated on a 5-point Likert scale from 1 (Not at all true) to 5 (Completely true). Overall, the modified PSSM scale has high reliability, a = 0.84. The 3 subscales also had good reliability (acceptance by faculty members a = 0.70, belonging a = 0.75, acceptance by students a = 0.76). The PSSM also has significant positive correlations with sense of belonging and satisfaction with university and negative correlations with intention to drop out of university and loneliness (Alkan, 2016).

Motivation The Self-Regulation Questionnaire–Learning (SRQ-L; Black & Deci, 2000) is a 12-item questionnaire consisting of 2 subscales, controlled regulation (extrinsic motivation) and autonomous regulation (intrinsic motivation). Items such as “Because I feel like it’s a good way to improve my understanding of the material” are rated on a 7-point Likert scale from 1 (Not at all true) to 7 (Very true). The alpha reliability of the autonomous and controlled regulation subscale was a = 0.75 and a = 0.67, respectively. The SRQ-L also had positive correlations with the General Causality Orientations Scale (GCOS), specifically, the autonomous subscale correlated with general autonomy orientation on the GCOS (r = 0.44), and the controlled subscale correlated with controlled orientation (r = 0.28) and impersonal orientation (r = 0.34) on the GCOS. Procedures Baylor University students were recruited via convenience and snowball sampling to take an online survey. The survey was distributed to participants as a link through social media and text messaging and completed online through Qualtrics survey system where participants gave their informed consent to participate in the study. Questions about sex, age, ethnicity, current class status, and major were asked. (Continued on page 24).

Subscale

Religious Minority

Religious Majority

SD

M

SD

t

p

Caring Attitudes

5.81

0.90

6.08

0.98

1.26

.213

Off-campus Interactions

3.88

1.63

4.19

1.59

0.83

.407

Career Guidance

5.35

1.12

5.25

1.46

–0.34

.735

Connectedness

4.44

1.16

5.19

1.20

2.77

.007***

Approachability

4.95

1.38

4.97

1.37

0.07

.945

Accessibility

5.21

1.29

5.30

1.23

0.33

.744

Respectful Interactions

5.60

0.90

5.55

0.89

–0.24

.810

Negative Experiences

3.03

1.45

2.37

1.12

–2.17

.035*

1.11

5.70

0.99

0.07

.945

Validity Scale 5.69 * p < 0.05, ** p < 0.01, *** p < 0.001

Table 1: Independent Samples T-tests Comparing Dimensions of Student-Professor Interactions Between Students in Religious Minority and Majority Groups.

M

SD

1

2

3

4

1. Connectedness

4.92

1.24

-

-

-

-

2. Negative Experiences

2.61

1.28

–.53***

-

-

-

3. Intrinsic Motivation

5.61

1.06

.39***

–.50***

-

-

4. Sense of Belonging 64.33 * p < 0.05, ** p < 0.01, *** p < 0.001

5.51

.51***

–.37***

-

-

Measure

Table 2: Summary of Correlations, Means, and Standard Deviations for Variables Connectedness, Negative Experiences, Intrinsic Motivation, and Sense of Belonging.

Variables

B

SE

R2

F

Constant

5.79

0.64

.27

14.98***

Connectedness

0.14

0.10

Negative Experiences –0.34*** * p < 0.05, ** p < 0.01, *** p < 0.001

0.09

Table 3: Results of Multiple Regression Analysis on Level of Connectedness and Negative Experiences Predicting Intrinsic Motivation.

Variables

B

SE

R2

F

Constant

56.28

3.33

.28

15.45***

1.96***

0.50

Connectedness

Negative Experiences –0.61 * p < 0.05, ** p < 0.01, *** p < 0.00

0.48

Table 4: Results of Multiple Regression Analysis on Level of Connectedness and Negative Experiences Predicting Sense of Belonging.

Scientia 2022 | 23

Original Research

M

Original Research

Partipants selected one of the seven options for religious affiliation (Christian, Hindu, Islam, Buddhist, Atheist, Agnostic, No religion/spiritual affiliation, Other). Studentprofessor interactions, sense of belonging, and motivation were assessed with the SPIS, PSSM, and SRQ-L, respectively.

Finally, participants were debriefed about the purpose of the study and thanked for their participation. The study assessed the correlation between 1) religious affiliation and studentprofessor interactions and 2) the relationships between religious minority student-professor interactions and sense of belonging and motivation.

Results Nine independent samples t-tests investigated differences in the dimensions of student-professor relationships between the religious majority and minorities at a religiously affiliated institution (Table 1).

A Levene’s test was conducted on each subscale and homogeneity of variance in the sample was assumed for all but one subscale, negative experiences, where a Welch two-sample t-test was performed instead (caring attitudes F(1, 81) = 0.0047, p > .05; off-campus interactions F(1, 81) = 0.1289, p > .05; career guidance F(1, 81) = 0.99, p > .05; connectedness F(1, 81) = 0.02, p > .05; approachability F(1, 81) = 0.08, p > .05; accessibility F(1, 81) = 1.42, p > .05; respectful interactions F(1, 81) = 0.05, p > .05; negative experiences F(1, 81) = 4.31, p < .05; validity scale F(1, 81) = 0.36, p > .05).

Independent samples t-tests found that religious minority (M = 5.81, SD = 0.9) and majority students (M = 6.08, SD = 0.98) did not experience significantly different levels of caring attitudes from their professors t(81) = 1.26, p > .05. The magnitude of this difference (mean difference = 0.28) was small (Cohen’s d = 0.29). Additionally, the religious minority (M = 3.88, SD = 1.63) and majority students (M = 4.19, SD = 1.59) did not differ in levels of off-campus interactions with their professors, t(81) = 0.83, p > .05. The magnitude of this difference (mean difference = 0.31) was trivial (Cohen’s d = 0.19). Further, religious minority (M = 5.35, SD = 1.12) and majority students (M = 5.25, SD = 1.46) experienced similar amounts of career guidance from their professors, t(81) = –0.34, p > .05. The magnitude of this difference (mean difference = 0.10) was trivial (Cohen’s d = 0.08).

Interestingly, religious minority students (M = 4.44, SD = 1.16) experienced significantly lower levels of connectedness with their professors, t(81) = 2.77, p < .05, compared to religious majority students (M = 5.19, SD = 1.2). The magnitude of this difference (mean difference = 0.75) was moderate (Cohen’s d = 0.63).

Religious minority (M = 4.95, SD = 1.38) and majority students (M = 4.97, SD = 1.37) did not differ in reported levels of approachability of their professors, t(81) = 0.07, p > .05.

The magnitude of this difference (mean difference = 0.02) was trivial (Cohen’s d = 0.02). Also, religious minority (M = 5.21, SD = 1.29) and majority students (M = 5.3, SD = 1.23) reported similar accessibility of their professors, t(81) = 0.33, p > .05. The magnitude of this difference (mean difference = 0.09) was trivial (Cohen’s d = 0.07). Moreover, religious minority (M = 5.6, SD =

24 | Scientia 2022

0.9) and majority students (M = 5.55, SD = 0.89) had similarly respectful interactions with their professors, t(81) = –0.24, p > .05. The magnitude of this difference (mean difference = 0.05) was trivial (Cohen’s d = 0.06).

Interestingly, religious minority students (M = 3.03, SD = 1.45) experienced significantly more negative experiences than religious majority students (M = 2.37, SD = 1.12), t(48.86) = –2.17, p < .05. The magnitude of this difference (mean difference = 0.66) was moderate (Cohen’s d = 0.53). Lastly, religious minority (M = 5.69, SD = 1.11) and majority students (M = 5.7, SD = 0.99) did not differ in the impacts of positive studentprofessor relationships, t(81) = 0.07, p > .05. The magnitude of this difference (mean difference = 0.01) was trivial (Cohen’s d = 0.01). Overall, religious minority students experienced less connectedness and more negative experiences with professors compared to religious majority students.

Since only connectedness (M = 4.92, SD = 1.24) and negative experiences (M = 2.61, SD = 1.28) subscales produced significant differences, correlation and multiple linear regression analyses were only conducted for these subscales in relation to intrinsic motivation (M = 5.61, SD = 1.06).

Analysis suggested a moderate positive correlation between connectedness and intrinsic motivation, r = .39, p < 0.001 and a moderate negative correlation between negative experiences and intrinsic motivation, r = –.50, p < 0.001. There was a large negative correlation between levels of connectedness and levels of negative experiences, r = –.53, p < 0.001 (Table 2). A multiple linear regression found that connectedness and negative experiences significantly predicted intrinsic motivation R2 = .27, F (1, 81) = 14.98, p < 0.001 (Table 3). These results indicate that levels of connectedness and negative experiences predict 27.25% of the variance in intrinsic motivation.

Correlation and multiple linear regression analysis were only conducted for connectedness and negative experiences in relation to sense of belonging (M = 64.33, SD = 5.51) (Table 2).

Analyses suggested a large positive correlation between levels of connectedness and sense of belonging, r = .51, p < 0.001 and a moderate negative correlation between levels of negative experiences and sense of belonging, r = –.37, p < 0.001 (Table 2). A multiple linear regression found that connectedness and negative experiences significantly predicted sense of belonging, R2 = .28, F (1, 81) = 15.47, p < 0.001 (Table 4). The results indicate that connectedness and negative experiences predict 27.89% of the variance in sense of belonging.

Discussion This study supported our first hypothesis that religious minorities experienced less positive student-professor interactions. Particularly, religious minorities reported lower levels of connectedness and more negative experiences than religious majority students. This could mean that differences in religious affiliations may hinder the positivity of studentprofessor interactions. Religion is relatively understudied in research on college students compared to ethnicity where ethnic incongruence is a popular term, referring a situation where two people do not belong to the same ethnic group (Loe & Miranda, 2005). Thijs and colleagues (2012) demonstrated that ethnic

did not account for certain factors that could have influenced the student’s perceived sense of belonging and positivity of student-professor interactions (i.e., time management skills, study skill modifications, membership in a professor’s lab, GPA, post-graduate plans, grade classification differences, gender differences, and membership in Honors programs).

Nevertheless, the results found in this study still highlight the importance of better understanding what affects students’ perceptions of interactions with their professors and future research should take into consideration the aforementioned factors.

Future research should also focus on exploring whether individual religious minority groups experience the same reduction in positive interactions or if this effect is more pronounced in certain groups and the role of connectedness and negative experiences in this relationship. Furthermore, future studies should explore the effect on professors by investigating whether professors belonging to religious minority groups experience more negative interactions when interacting with religious majority students and determine whether they possess a lower sense of belonging and intrinsic motivation.

Overall, the findings of this study are important as student perceptions of interactions with their professors have significant impacts on their academic and mental well-being. Religious minorities at religiously affiliated universities tend to experience less positive student-professor interactions. Therefore, this may lead to lower intrinsic motivation and sense of belonging at the university. With the growing diversity of student bodies, it is important to ensure that religious minorities are treated equally to ensure that their transition to and time during college is conducive for their psychological, social, and academic success.

Acknowledgements The authors would like to thank Dr. Shawn Latendresse for the opportunity to conduct this project and his guidance throughout.

References Alkan, N. (2016). Psychological sense of university membership: An adaptation study of the PSSM scale for Turkish university students. The Journal of Psychology, 150(4), 431-449. https://doi.org/10.1080/00223980.2015.1087373 Baumeister, R. F., & Leary, M. R. (1995). The need to belong: Desire for interpersonal attachments as a fundamental human motivation. Psychological Bulletin, 117(3),497–529. https://doi.org/10.1037/0033-2909.117.3.497 Beasley, S. T. (2020). Student–faculty interactions and psychosociocultural influences as predictors of engagement among Black college students. Journal of Diversity in Higher Education, 1–12. https://doi.org/10.1037dhe0000169 Black, A. E., & Deci, E. L. (2000). The effects of instructors’ autonomy support and students’ autonomous motivation on learning Organic Chemistry: A self-determination theory perspective. Science Education, 84(6), 740-756 https://doi.org/10.1002/1098237X(200011)84:6<740::AID SCE4>3.0.CO;2-3 Bowman, N. A., & Small, J. L. (2012). Exploring a Hidden Form

Scientia 2022 | 25

Original Research

incongruence between ethnic minority Moroccan students and ethnic majority Dutch teachers led to relationship being less close and warm, and involved more conflict and negativity. The authors explained that negative relationships could stem from a lack of mutual cultural understanding and in-group bias.

Different ethnicities and religions hold various expectations and behavior, which can thus lead to misunderstandings between students and teachers. This could be extended to religion where misunderstandings could hinder the ability to form meaningful bonds with faculty and lower connectedness.

Also, individuals may hold generic outgroup attitudes (Tajfel, 1970) and favor individuals in their perceived in-group for no practical reason (Tajfel et al., 1971), thus leading to stereotyping and microaggressions. Therefore, the concept of religious incongruence potentially explains the findings of the study and underscores the importance of religion in these relationships.

The results also supported our second hypothesis as connectedness and negative experiences were able to predict intrinsic motivation and belonging. Teachers that are enthusiastic, passionate, caring, prepared, professional, and respectful (Cokley et al., 2004) create more supportive classrooms (Zumbrunn et al., 2014), and thus could enhance connectedness and reduce negative experiences for students. This supportive environment subsequently could lead to belongingness in the class, students feeling more motivated and engaging more in class, and better academic performances (Beasley, 2020; Zumbrunn et al., 2014). These findings underscore the importance of positive student-professor interactions as their effects also impact a student’s personal attitudes and academic achievements. Additionally, Bowman and Small (2012) found that in religiously affiliated universities with inclusive religious climates, religious minorities experienced greater wellbeing. Thus, with universities becoming more diverse and the importance of positive student-professor relationships, it is crucial to understand the differences in religious beliefs and behaviors and be aware of potential out-group prejudices. By experiencing more positive student-professor interactions, correlating to comparable levels of intrinsic motivation and belongingness, religious minority students may be able to integrate more successfully into their universities socially and academically.

There were several limitations to this study. Firstly, regarding the sample, the sample size was small, and recruitment of participants was via convenience and snowball sampling. While this method created a sample more ethnically and religiously diverse than the Baylor student body, this sample may not be representative in other variables, leading to possible confounds which were not considered. Additionally, different religiously affiliated universities have different religious breakdowns for their student body, thus, the generalizability of the findings from this study may be limited. Secondly, the second hypothesis did not directly compare religious minority and majority groups but rather, the multiple regression analyses considered all 83 participants. Thus, while the analyses demonstrated that less connectedness and more negative experiences could predict lower intrinsic motivation and sense of belonging, no determination was made regarding whether these factors contributed differently for religious majority and minority students. Lastly, while the results were significant, the study

Original Research

of Minority Status: College Students’ Religious Affiliation and Well-Being. Journal of College Student Development, 53(4), 491–509. https://doi.org/10.1353/csd.2012.0050 Cheng, Z., Pagano, L., & Shariff, A. (2017). The development, validation, and clinical implications of the Microaggressions Against Religious Individuals Scale (MARIS). Psychology of Religion and Spirituality, 11(4), 327–338. https://doi org/10.1037/rel0000126 Cokley, K., Komarraju, M., Patel, N., Castillon, J., Rosales, R., Pickett, R., Piedrahita, S., Ravitch, J., & Pang, L. S. (2004). Construction and initial validation of the Student Professor Interaction Scale. The College of Student Affairs Journal, 24(1), 32–50.

Cotten, S. R., & Wilson, B. (2006). Student-faculty interactions: Dynamics and determinants. Higher Education, 51(4), 487–519. https://doi.org/10.1007/s10734-004-1705-4 Deci, E. L., Vallerand, R. J., Pelletier, L. G., & Ryan, R. M. (1991).

Motivation and education: The self-determination perspective. Educational Psychologist, 26(3&4), 325-346 https://doi.org/10.1080/00461520.1991.9653137 Freeman, T. M., Anderman, L. H., & Jensen, J. M. (2007). Sense of belonging in college freshmen at the classroom and campus levels. Journal of Experimental Education, 75(3), 203–220. https://doi.org/10.3200/JEXE.75.3.203-220 Goodenow, C. (1993). The psychological sense of school membership among adolescents: Scale development and educational correlates. Psychology in the Schools, 30(1), 79-90. https://doi.org/10.1002/1521-6807 Goodenow, C., & Grady, K. E. (1993). The relationship of school belonging and friends’ values to academic motivation among urban adolescent students. The Journal of Experimental Education, 62(1), 60–71. https://doi.org/10 1080/00220973.1993.9943831 Grier-Reed, T. (2013). The African American student network: An informal networking group as a therapeutic intervention for Black college students on a predominantly White campus. Journal of Black Psychology, 39(2), 169–184. https://doi.org/10.1177/0095798413478696 Hausmann, L., Schofield, J., & Woods, R. (2007). Sense of belonging as a predictor of intentions to persist among African American and White first-year college students.

Research in Higher Education, 48(7), 803–839. https://doi org/10.1007/s11162-007-9052-9 Huang, Y., Wei, L., & Wu, J. (2016). Relationship between intrinsic motivation and undergraduate students’ depression and stress: The moderating effect of interpersonal conflict. Psychological Reports, 119(2), 527–538. https:/ doi.org/10.1177/0033294116661512 Komarraju, M., Musulkin, S., & Bhattacharya, G. (2010). Role of student–faculty interactions in developing college students’ academic self-concept, motivation, and achievement. Journal of College Student Development, 51(3), 332–342. https://doi.org/10.1353/csd.0.0137 Loe, S. A., & Miranda, A. H. (2005). An examination of ethnic incongruence in school-based psychological services and diversity-training experiences among school psychologists.

Psychology in the Schools, 42(4), 419–432. https://doi org/10.1002/pits.20062

26 | Scientia 2022

Nadal, K. L., Davidoff, K. C., Davis, L. S., Wong, Y., Marshall, D., & McKenzie, V. (2015). A qualitative approach to intersectional microaggressions: Understanding influences of race, ethnicity, gender, sexuality, and religion. Qualitative Psychology, 2(2), 147–163. https://doi.org/10.1037 qup0000026 O’Donnell, M. B., Shirley, L. A., Park, S. S., Nolen, J. P., Gibbons, A. M., & Rosén, L. A. (2018). The College Adjustment Questionnaire: A measure of students’ educational, relational, and psychological adjustment to the college environment. Journal of College Student Development, 59(1), 116–121. https://doi.org/10.1353 csd.2018.0009 Pittman, L. D., & Richmond, A. (2008). University belonging, friendship quality, and psychological adjustment during the transition to college. The Journal of Experimental Education, 76(4), 343–362. https://doi.org/10.3200 JEXE.76.4.343-362 Tajfel, H. (1970). Aspects of national and ethnic loyalty.

Social Science Information, 9(3), 119–144. https://doi. org/10.1177/053901847000900305 Tajfel, H., Billig, M. G., Bundy, R. P., & Flament, C. (1971). Social categorization and intergroup behaviour. European Journal of Social Psychology, 1(2), 149–178. https://doi.org/10.1002 ejsp.2420010202 Taylor, G., Jungert, T., Mageau, G. A., Schattke, K., Dedic, H., Rosenfield, S., & Koestner, R. (2014). A self-determination theory approach to predicting school achievement over time: The unique role of intrinsic motivation. Contemporary Educational Psychology, 39(4), 342–358. https://doi org/10.1016/j.cedpsych.2014.08.002 Thompson, M. D. (2001). Informal student-faculty interaction: Its relationship to educational gains in science and mathematics among community college students.

Community College Review, 29(1), 35. https://doi org/10.1177/009155210102900103 Thijs, J., Westhof, S., & Koomen, H. (2012). Ethnic incongruence and the student–teacher relationship: The perspective of ethnic majority teachers. Journal of School Psychology, 50(2), 257–273. https://doi.org/10.1016/jsp.2011.09.004 Tinto, V. (1993). Building Community. Liberal Education, 79(4), 16–21.

Willoughby, T., Heffer, T., Dykstra, V. W., Shahid, H., & Braccio, J. (2020). A latent class analysis of adolescents in first-year university: Associations with psychosocial adjustment throughout the emerging adult period and post-university outcomes. Journal of Youth and Adolescence, 49(12), 2459–2475. https://doi.org/10.1007 s10964-020-01318-7 Zumbrunn, S., McKim, C., Buhs, E., & Hawley, L. (2014).

Support, belonging, motivation, and engagement in the college classroom: A mixed method study. Instructional Science, 42(5), 661–684. https://doi.org/10.1007/s11251 014-9310-0

Harmful Algal Bloom Mitigation Strategies in the Face of Environmental Alterations due to Climate Change and Anthropogenic Nutrient Loading Jonathan D. Norton; Melinda A. Coogan, Ph.D.; Rebecca J. Sheesley, Ph.D.

Abstract Algal blooms formed by Microcystis aeruginosa are increasing globally in magnitude, frequency, and range due to the anthropogenic stresses of climate change and nutrient loading. Increased surface temperatures, higher salinities, and more acidic waters along with an excess of available nitrogen and phosphorus from largescale agricultural activity are creating ideal growth conditions for algal blooms. This is concerning to human health, as certain strains of Microcystis produce dangerous cyanotoxins that carry potential lethal consequences if exposed through pathways such as drinking water. Today, most algal bloom management strategies focus on controlling and remediating the immediate effects and consequences of the bloom, which unfortunately are unsustainable and financially inefficient methods of mitigation. This study seeks to propose a multilateral, synthesized, and holistic management approach that integrates multiple spheres of mitigation, including monitoring and detection, best management practices, physical treatment and remediation, and prevention. Information was collected to review the current state of algal bloom mitigation by conducting a literature review about Microcystis aeruginosa ecology, human health effects, the relationship to consequences of climate change, and current available mitigation strategies. Successful algal bloom mitigation will be a combination of contributions between various disciplines that aim to implement sustainable practices designed to control the magnitude, frequency, and duration of harmful algal blooms in the interest of human and environmental health.

Introduction Microcystis aeruginosa is a cyanobacteria that predominantly inhabits freshwater systems but has also been reported in brackish and marine water systems as well (Tanabe et al., 2018). Due to its extensive evolutionary history and diversity, this cyanobacterium has successfully established itself across a global scale (Wilhelm et al., 2020). Cyanobacterium are photosynthetic organisms and thus are crucial to the functionality of many water systems, playing important roles in oxygen production, nutrient cycling, and serving as food sources within ecosystems (Gehringer & Wannicke, 2014).

However, certain species may be classified as toxic strains of cyanobacterium, mainly due to minor genetic variation in gene sequences that result in toxin production (Lee et al., 2020; Wilson et al., 2005). Understandably, the overgrowth of such cyanobacteria like Microcystis aeruginosa can cause severe degradations to the quality of the water within an aquatic system.

These overgrowth phenomena are classified by the term “Harmful Algal Blooms” (HABs). Many algal blooms, such as Microcystis aeruginosa, produce potent cyaonotoxins that are released into the surrounding waters and can be lethal to exposed organisms like fish, macroinvertebrates, and other

vegetation (Olson et al., 2020). These toxins have also been reported to cause serious health complications in exposed humans and land animals as well, as direct ingestion of contaminated water can cause gastrointestinal complications, pneumonia, and death in extreme cases (Olson et al., 2020; Trevino-Garrison et al., 2015; Van der Merwe et al., 2012).

Current research also indicates that microcystin, the cyanotoxin produced by Microcystis aeruginosa, may play a role in the onset of the neurodegenerative disease amyotrophic lateral sclerosis (ALS) (Caller et al., 2009). The inherent dangers that HABs pose to both human and environmental health set the precedent for the research, development, and adaptation of successful plans to mitigate their harmful consequences.

Although algal growth occurs naturally in the environment, environmental stresses as a result of human activity have accelerated their developments by altering the environment in favor of the growth conditions of algae (Wilhelm et al., 2020). The two most important growth determining factors for Microcystis are temperature and nutrient availability in the form of phosphorus and nitrogen (Bui et al., 2018; Davis et al., 2009; Hartnell et al., 2020). Anthropogenic burning of fossil fuels has raised the atmospheric carbon dioxide concentration

Scientia 2022 | 27

Review Article

Department of Environmental Science, Baylor University, Waco, TX

Review Article

from 284 ppm to over 400 million ppm and has increased the average global temperature by over 1°C since the late 19th century (NASA, n.d.). This, in turn, has increased the surface temperatures of freshwater systems (Paerl & Huisman, 2019).

Microcystis growth excels in warm temperature waters due to its peak photosynthetic productivity point of 34.3°C, a much higher temperature tolerance than most aquatic vegetation (Bui et al., 2018). As industrial agricultural practices demand the increased use of synthetic fertilizers, more nutrients are inadvertently entering freshwater systems and causing eutrophic water conditions that are advantageous for the growth of HABs. Total nutrient concentrations and the ratios between nitrogen and phosphorus have also been demonstrated to affect the proportion of toxic and non-toxic strains of Microcystis within the same bloom, indicating that nutrient availability combined with higher temperature might be a mechanism in the success of the more toxic HABs (Davis et al., 2009; Yu et al., 2014). The convoluted nature of HABs and the synergistic effects of climate change and anthropogenic nutrient loading are proving to be significant obstacles to developing successful mitigation and management plans and emphasize the necessity to promptly develop effective and adaptive mitigation plans for the future.

Unfortunately, developing coherent and effective mitigation strategies is often unsuccessful due to scattered research efforts and the lack of motivation and budget to invest in preventative infrastructures. The financial costs and resources associated with producing and implementing HAB mitigation strategies are typically unconvincing when compared to continuing current practices and dealing with remediation when necessary. The prediction and mitigation of HAB toxicity is also sporadically researched, as no single variable accurately correlates or predicts toxin release levels (Hartnell et al., 2020). Thus, the forefront of research regarding HABs must contain new developments in the ability to detect, mitigate, and prevent the growth of HABs in freshwater systems. With poorly developed and inefficient HAB mitigation strategies, the long-term health of global freshwater systems will continue to deteriorate, and the risks associated with contaminated water will create and exacerbate a global public health crisis (Brooks et al., 2016).

The central method of this paper serves to propose an integrative approach to the increasing prevalence of HABs in the face of climate change and anthropogenic nutrient loading.

Without quantitative analysis, this paper will propose integrative HAB mitigation strategies that consider the Best Management Practices (BMP) of various unique regions that suffer from HABs. Since HAB mitigation strategies will have to be uniquely tailored to each case, this paper aims to provide a basic outline and for what mitigation strategies may prove useful in certain cases.

Materials and Methods To evaluate the current state of research regarding the development, implementation, and success of HAB mitigation strategies, a literature search ranging over multiple research topics was conducted to form a broad review of available literature and case studies. To better synthesize recent articles and compare characteristics of current HAB mitigation

28 | Scientia 2022

strategies, the literature was defined by four major categorical topics regarding environmental aspects of algal growth mechanisms, the effect on freshwater systems, and the extent of current human capacity/capability in mitigation. The four categories included were: (1) background information regarding the biotic/abiotic mechanisms that assist in HAB growth/toxin production, (2) impacts on the health of humans, (3) the influence of climate change on the growth of HABs, and (4) available mitigation strategies with empirical tests and supporting evidence. To find relevant articles for each group, each database was searched for all four categories using sets of keywords, using only relatively recent articles in order to assure reliability (< 20 years old). The final result was a literature review that included 27 articles collected from a variety of science databases and encapsulated many aspects regarding the developing issue of increasing HAB prevalence. Given its global prominence and extensively documented research history, Microcystis aeruginosa was chosen as a benchmark algal species to include as a keyword in all searches.

Related Keywords Search Database

Category 1

Category 2

Category 3

Category 4

SCOPUS

Mechanism

Health

Climate Change

Mitigation

PubMed

Microcystis

Microcystis

Microcystis

Microcystis

Google Scholar

Evolution

Toxin/Toxic

Warming

Monitoring

Web of Science

Background

Illness

Anthropogenic

Sensing Prevention Detection

Resulting Papers

3

5

9

10

Table 1: Specifications of scientific databases, relevant keywords, and resulting articles found in the literature.

The keyword categories 1, 2, and 3 provide necessary information to build the foundation on which to develop a comprehensive inspection and synthesis of HAB mitigation strategies. Since there is independent variable (environmental or anthropogenic) that causes HABs, it is important to include many environmental mechanisms, cycles, processes, and relationships when analyzing and evaluating the efficiency of HAB mitigation strategies. Ultimately, the process of proposing integrated mitigation strategies will include evaluating the barriers and most prevalent anthropogenic stress factors causing HABs, assessing and discussing the most important environmental aspects regarding mitigation, and synthesizing strategies in order to reduce gaps in ability and efficiency to improve applicability.

Results and Discussion 1. Barriers of Current Mitigation Strategies A key to understanding the necessity for more integrative HAB mitigation strategies is assessing the multitude of barriers

that impede the success of current strategies. Often, barriers affect multiple aspects of HAB mitigation like the ability to collect data, control public risk, or protect the surrounding environment of freshwater systems. Although HABs exist in a variety of climates, biomes, and environments across the globe, many of the barriers preventing successful HAB mitigation are similar in concept and can be generally classified as either being stress factors to the environment caused by anthropogenic activity or related to the unique competitive evolutionary advantages of the HAB. It is important to consider that often a combination of barriers across anthropogenic activity and natural phenomena prevent mitigation strategies from being successfully developed.

1.2 Evolutionary Advantages of HABs The evolutionary advantages of HABs create significant barriers to successfully integrating mitigation strategies across global freshwater systems. Microcystis, for example, has many characteristics that allow it to outcompete other freshwater vegetative species, including its abilities to tolerate stressful freshwater conditions like high temperatures, high salinity, high pH, and high turbidity (Wilhelm et al., 2020). Benthic vegetative species are often most at risk of being outcompeted, as algal blooms form thick layers of scum at the water’s surface that prevent the diffusion of carbon dioxide and penetration of light into the water that is necessary for photosynthesis (Paerl & Huisman, 2009). The thick accumulation of surface scum also generates heat through trapping and concentrating ultraviolet radiation, therefore exacerbating surface temperature warming further via a positive feedback loop and thus reinforcing an unsuitable habitat for most freshwater vegetation (Bui et al., 2018). However, manipulating the physiochemical variables of freshwater bodies is nearly impossible at large scales yet necessary to reduce the advantage of HABs over benthic vegetation.

Current research is also inconclusive on causal relationships between the waters physiochemical parameters, like temperature, and the tendency for HABs to release toxins into its surroundings. This severely limits the scope and ability to prevent and control the release of HAB toxins, endangering the surrounding environment and human health. Because HABs are well adapted to the conditions of disrupted aquatic systems, mitigation strategies must reduce or eliminate the root causes of the advantageous water conditions in the freshwater system.

2. BMP Aids Current Mitigation Strategies Best Management Practices (BMPs) are very effective strategies that aim to reduce the impact of environmentally strenuous human practices on the surroundings. Current agricultural practices cause excess nutrients from crop fertilizers to infiltrate freshwater systems and intensify vegetative and algal primary productivity (Manuel, 2014; Paerl et al., 2016). Also, the combustion of fossil fuels to power vehicles, generate electricity, and produce materials leads to the climate changes that favor the survival and productivity of HABs (Bui et al., 2018; de Souza et al., 2018). Human changes to natural hydrologic cycles and patterns by damming rivers and increasing impermeable surfaces also disrupts the functionality of global watersheds and greatly worsens freshwater system health (Paerl et al., 2018).

Considering these factors, an analysis of anthropogenic activities and practices near the bloom can be conducted to address areas

Scientia 2022 | 29

Review Article

1.1 Anthropogenic Stress Factors One of the greatest current anthropogenic stress factors on the environment is greenhouse gas emission induced climate change. The consequences of climate change present difficulties for HAB mitigation in a multitude of ways, including the rising of freshwater system surface temperatures, acidification of freshwater systems, reduction in species diversity, and changes in natural weather patterns and hydrology (Chapra et al., 2017; Moore et al., 2008). The varying physiochemical parameters, destruction of natural cycles, and general uncertainty regarding future environmental conditions limits the ability of mitigation strategies to be implemented in affected regions for extended periods of time (Paerl et al., 2016). The consequences of anthropogenic climate change also exacerbate HAB formulation and growth, allowing for the expansion of habitat range and prolonging growth periods during heat waves (de Souza et al., 2018; Paerl & Huisman, 2009).

In addition, the construction of hydrology-alternating infrastructures like dams and wells disrupt natural hydrologic cycles and often result in decreased streamflow velocity, increased residency time, salinization of water, and increased erosion (Van der Veer et al., 1995). These consequences in turn result in thermal and chemical stratification, calm waters, and physiochemical parameters that are beyond tolerable ranges for native vegetation like high salinity, low pH, and high turbidity (Paerl & Huisman, 2009). These hydrologic conditions create a very suitable habitat for algae to quickly grow and suppress native vegetation, leading to the quick and dense formation of algal blooms (Moore et al., 2008).

Although not directly correlated with environmental degradation, differences in human cultures and national policies can also act as anthropogenic mitigation barriers.

Underdeveloped nations may not have the available fiscal or physical resources in order to both invest in freshwater system protection and create sufficient water treatment processes. To sufficiently monitor, test, and treat HABs, significant amounts of time, money, and people must be assigned to the task which can be a taxing burden to the various agencies and institutions involved in such a project. Unfortunately, nations across the world do not adequately fund resources to promote successful environmental protection. For example, the United States EPA receives only 0.2% of the nation’s fiscal budget each year (EPA, n.d.). Underfunding environmental agencies perpetuates the continuation of unsustainable practices, ultimately leaving

severely underfunded environmental protection institutions lagging in the production of effective environmental policies.

Currently, a significant number of surface waters are left unmonitored, freshwater systems become regularly polluted with HABs, and the public is burdened with the cost to repair affected systems each time they occur. In the current scenario, successful and efficient HAB mitigations strategies will be discounted by the human tendency to direct resources to costly remediation efforts, rather than the more effective methods of prevention.

Review Article

that contribute the most to the issue of HAB prevalence and are the most likely to be receptive to improvements in BMPs. 2.1 Reduction of Nutrient Loading Through Surface Runoff Globally, the agricultural industry, including both crop and livestock production, accounts for a significant proportion of nutrient pollution into freshwater systems (Manuel, 2014). The primary mechanism in which these nutrients enter freshwater systems is through unregulated surface runoff. Water that does not percolate into the soil travels along the surface through drainage basins, picking up nutrients by either dissolving the ions or picking up and carrying nutrient-adsorbed soil particles themselves (Chapra et al., 2017). This process, being a nonpoint form of pollution, makes it extremely difficult for humans to accurately and objectively trace nutrients to their sources and can lead to a lack of responsibility among polluters (Paerl et al., 2018). Although there are some BMPs that reduce nutrient use for agricultural operations, in most cases they do not go far enough in reducing environmental impact and continue to degrade the surrounding freshwater systems.

However, there are many improvements that can be made within the global agricultural industry that can reduce nutrient loss while retaining crop productivity. First, many sustainable irrigation methods exist for crop cultivation that both save water and reduce the amount of surface runoff. During drip irrigation, for example, evaporation and surface runoff are reduced by directly applying water to the roots of plant through sub-surface pipelines. Methods such as localized irrigation, where water is distributed parallel to crop rows above ground through pipes under low pressure, are also effective in reducing surface runoff. Physical structures such as dykes and levees surrounding crop fields can be used to restrict surface runoff from exiting the area by retaining it within the field. Vegetation buffers including trees, grasses, and shrubs can also absorb nutrients before leaving the crop field while providing shielding from storm surges.

A much more comprehensive and systematic approach to reduce agricultural environmental impacts would be a shift towards a sustainable agroecology model. This model inherently restricts the use of synthetic fertilizers and promotes sustainable methods of nutrient replenishment to the soil, such as composting and recycling livestock waste (Koneswaren & Nierenberg, 2008). Signature agroecology practices such as notillage, crop rotation, and mulching greatly reduce erosion rates of the soil and greatly sustain the quality of soil, allowing it to absorb and retain water easier (USDA NIFA, n.d.).

In addition, environmental landscape and ecosystem restoration can reduce the load of nutrient entering freshwater systems through surface runoff. Wetlands, for example, have the unique ability to treat water in the form of a filtration process, reducing water turbidity, salinity, and most importantly, nutrient loads (Vermont DEC, n.d.). Although a majority of natural wetland ecosystems have been altered or destroyed through anthropogenic development, careful and methodical assessment and planning can implement constructed wetlands, designed solely for the purpose of improving water quality before entering nearby streams. Although not every region and environment can construct and successfully maintain a wetland

30 | Scientia 2022

ecosystem, there are variations within wetland species and operation that can survive in tropical, boreal, and temperate regions (Encyclopedia Britannica, n.d.). Similarly, the riparian zones of freshwater rivers act as buffers and water filtration systems that improve water quality before entering the river (Décamps et al., 2009). Identical to wetlands, a significant portion of riparian zones have been altered and destroyed due to human development and urbanization, severely increasing the risk of nutrient and waste pollution. Although these methods can be costly, environmental restoration can serve as a critical buffer between freshwater systems and non-point nutrient pollution. Environmental restoration projects can be implemented synchronously with improvement of agricultural BMPs to greatly reduce the global risk of nutrient loading into freshwater systems.

2.2 Reduction of Carbon Dioxide and Greenhouse Gas Emissions As discussed previously, the alterations to the environment caused by anthropogenic climate change largely favor the development and productivity of HABs over other freshwater vegetative species. Research indicates that the reduction of greenhouse gas emissions through the burning of fossil fuels is vital to slow and eventually alleviate the consequences of global climate change (Gehringer & Wannicke, 2014).

Although a daunting task, it is essential that greater awareness of anthropogenic activities that contribute to greenhouse gas emissions as well as methods of reduction be addressed as quickly as possible.

To begin a global emission reduction effort, total emission goals, international agreements, and both domestic and international policies must be in agreeance regarding the scrutiny and responsibility carried by each nation to reach emissions goals. The Paris Climate Accord and the Kyoto Protocol are both examples of international efforts to reduce global emissions with fair and just expectations, however such agreements can quickly deteriorate due to political conflicts or changes in governmental administrations. One of the most critical and plausible solutions to reducing greenhouse gas emissions is through the subsidization of and transition to renewable energy sources. Energy harvested through solar, wind, geothermal, and hydropower sources do not emit any greenhouse gases in the immediate process of energy production. These technologies can be implemented to produce energy, power transportation vehicles, and sustain modern infrastructure all while reducing the emissions of greenhouse gases at local and individual levels (Gielen et al., 2019).

Another significant source of greenhouse gas production lies in the global agricultural industry, as the transportation of food, manufacturing of fertilizers and pesticides, operation of mechanical vehicles, and upkeep of livestock such as cattle contribute a significant portion to total global greenhouse gas emissions (Koneswaran & Nierenberg, 2008). However, sustainable agriculture inherently assesses harmful practices in modern agriculture and seeks to find alternative methods to reduce environmental impact. The agroecology model, for example, promotes local agricultural operations in order to reduce transportation, restricts the use of manufactured pesticides and fertilizers, and relies on manual labor as opposed

to fossil fueled mechanical tools. A transition to sustainable farming along with domestic and international policies subsidizing a transformation of the current energy system to a renewable one would greatly reduce the global greenhouse gas emission rate, and therefore begin to alleviate and strip the suitable environments and competitive advantages of HABs in global freshwater systems.

3. Analysis of Mitigation Strategies – Finding What Works After identifying the potential barriers, BMPs, and environmental issues related to the increase of HAB prevalence, the process of developing mitigation strategies can be fully inclusive and conscious of the environment, circumstances, and goals of each case of HAB presence. Since HABs are related to various systems in the environment, it is important to emphasize that modern mitigation strategies must be continuously supplemented by relevant data across multiple fields to better understand and address causing factors of each HAB case. No successful strategy will be based upon the information collected in one field, but will instead be an interdisciplinary effort that analyzes the development of HABS as a function of larger environmental systems. Through this coalition, efforts can be prioritized to improve upon the methods in which strategies are developed, including detection, treatment, and prevention.

3.1 Improving Detection Ability and Efficiency One of the forefronts in the field of HAB detection lies in the technology of remote sensing, specifically through the integration of satellite imaging in order to collect geospatial data. The ability for humans to remotely collect data on specific regions and designated areas of study greatly expands the range of area that can be sufficiently monitored given a certain capability. Geospatial data is widely available on the internet through programs such as Google Earth and databases like ESRI, both providing datasets that can be synthesized and mosaicked to create larger, more detailed, and higher quality data through software such as ArcGIS. Remote satellite imaging can assess both small and large freshwater systems, providing real time analysis and reports of study sites at a relatively high measure of accuracy (Van der Merwe & Price, 2015).

There are several methods in which satellite remote sensing can detect the presence of HABs in freshwater system, although the main and most promising method currently used is spectrometry. Algae are biologically unique aquatic vegetation that carry distinctive spectral characteristics that can be found using spectrometry analysis on water bodies (Zhang & Zhang, 2014). In current capacities, remote satellite mass spectrometry is capable of distinguishing HABs from other types of freshwater vegetation and therefore determine locations of high density, specifying areas of concern within water bodies for direct treatment (Shen et al., 2012). Spatial analysis can also serve as a form of reconnaissance, acquiring large data sets over extensive areas in order to direct attention to areas that exhibit signs of HAB infection.

It is important to consider the drawbacks of satellite remote sensing however, such as resulting data inaccuracies due to obstructions such as clouds, birds, tree canopies, and shadows.

All of these factors may potentially reduce the reliability of spectrometry data, which is concerning considering how common these factors are in the environment. However, geospatial data collection can also be collected through specialized technologies such as small unarmed aircraft systems (sUAS) in order to fill such drawbacks and preserve

Scientia 2022 | 31

Review Article

2.3 Proper Protection and Maintenance of Freshwater Systems Although limited, there are some effective methods in which the promotion of water body activity can help alleviate and disrupt the establishment and development of HABs in freshwater systems. For example, small to medium sized freshwater bodies can be skimmed and sifted in order to remove algal colonies and layers of scum from the surface of the water (Paerl et al., 2016). Despite this method being a temporary restoration, it provides a rather quick and efficient way to halt the establishment of HABs in freshwater systems. Also, surface waters can be artificially mixed in order to prevent the development of HAB favorable thermoclines and chemoclines (Van der Weer et al.,1995). Reducing the residency time of freshwater systems such as lakes and reservoirs by flushing and drainage also promotes water activity and restricts successful HAB establishment (Chapra et al., 2017).

The responsibility to regulate point sources of pollution is typically designated to the state. This often entails enforcing certain standards regarding acceptable amounts of total pollution, such as total maximum daily loads (Paerl et al., 2016).

However, a much more effective strategy would be to divert nutrient contaminated water away from freshwater altogether, disposing of the waste in a separate, more sustainable method.

Specifically, bodies of freshwater systems that are utilized for drinking water should be regulated and protected strenuously from point source nutrient pollution. HAB development near drinking water intake stations are primary causes of drinking water contamination and serve as severe threats to public mixed in order to prevent the development of HAB favorable thermoclines and chemoclines (Van der Weer et al.,1995).

Reducing the residency time of freshwater systems such as lakes and reservoirs by flushing and drainage also promotes water activity and restricts successful HAB establishment (Chapra et al., 2017).

The responsibility to regulate point sources of pollution is typically designated to the state. This often entails enforcing certain standards regarding acceptable amounts of total pollution, such as total maximum daily loads (Paerl et al., 2016).

However, a much more effective strategy would be to divert nutrient contaminated water away from freshwater altogether, disposing of the waste in a separate, more sustainable method.

Specifically, bodies of freshwater systems that are utilized for drinking water should be regulated and protected strenuously from point source nutrient pollution. HAB development near drinking water intake stations are primary causes of drinking water contamination and serve as severe threats to public health, as cyanotoxins are difficult to filter out in modern water treatment practices (Chapra et al., 2017). However, with proper management and protection of freshwater systems and bodies, the mechanisms and preferences of HAB development can

begin to be reduced and alleviated, allowing for opportunities of efficient and successful mitigation strategies to be implemented.

Review Article

data accuracy. Advantages of sUASs include exceptionally high resolution, low cost of operation, and the ability to fully survey freshwater systems to generate complete and comprehensive datasets (Van der Merwe & Price, 2015). Various forms of remote sensing can provide complementary datasets that can be combined and amalgamated in order to produce high resolution, highly accurate data regarding HAB detection and geospatial analysis within freshwater systems.

With the ability to perform risk assessment on freshwater systems and detect the presence of HABs through geospatial analysis, field data collection will become much more focused and effective in regard to determining physiochemical parameters, environmental quality, and cyanotoxin prevalence.

Such biochemical markers can then be analyzed in the lab in order to produce information regarding the specific species of HAB prevalent, the proportion between toxic and nontoxic colonies, and if any toxins have contaminated sources of drinking water (Stauffer et al., 2019). For example, recent developments in the designing of genetic primers have significantly increased the ability to detect and distinguish the genes of toxic strains of Microcystis from those of the non-toxic strain of Microcystis in water samples (Lee et al., 2020). Such improvements in lab testing significantly reduce uncertainty regarding whether an algal bloom might serve as a toxic hazard and can serve as a significant tool in protecting public health by quickly restricting access to water that contains the genes of toxic HAB strands. The ability to better detect and assess the initial states of HAB infested freshwater systems is an important step to provide necessary information on which to base treatment and restoration efforts upon, which in turn will reduce the magnitude, extent, and frequency of HABs. 3.2 Improving Treatment of HAB Affected Systems Currently, HABs are responsible for infrastructure and environmental damages that have accumulated to nearly 1 billion dollars in the United States within the past few decades (Anderson et al., 2000). This amount alone emphasizes the economic need to develop mitigation strategies for HABs. Currently, the price of inaction to prevent HABs is reflected in the financial costs and laborious effort associated with repeatedly restoring affected systems. Although not ideal, this management approach has led to the development of unique and moderately effective methods of which to treat and alleviate affected surface waters.

One of the most practical methods is through the physical manipulation of the water’s surface, using various devices such as pumps and nets to skim and remove any HAB colonies from the affected water body (Paerl et al., 2016). This temporarily disrupts the growth and density of the HAB. However, it provides no solution to achieving a rebalance of the water systems physiochemical properties (Yu et al., 2017). On the other hand, chemical treatments can provide the means in which to improve and rebound unhealthy water quality, such as introducing chemicals to alter pH, decrease salinity, or decrease turbidity. These treatments are often fairly dangerous though, as introducing synthetic chemicals into freshwater systems can adversely affect the health of vegetation, fish, or macroinvertebrates that depend on the system (Paerl et al., 2016

32 | Scientia 2022

Paerl et al., 2018). Similarly, water treated with chemicals to alleviate HABs may become unpotable for humans, causing a risk to public health and creating a financial burden by requiring the water to be purified after treatment.

In light of these concerns, emerging research is beginning to investigate natural methods that can eliminate HABS that maximize the success of the treatment while minimizing risk and damage to the environment. The use of modified clays, for example, have shown significant results in the ability to both eliminate HABs in freshwater systems and prevent subsequent reoccurrences, as the physical and geochemical properties of the clays allow for absorption of algal cells, nutrient contamination, and the transfiguration of physiochemical parameters back to their equilibrium states (Yu et al., 2017). The benefits of clay treatment include straightforward application, relatively low cost, and the lack of any negative effects on either the freshwater systems organisms or the quality of the water.

Although the discussed remediation methods are moderately effective in alleviating the short-term effects of HAB contamination, their costs will accumulate over time and their success rates will be challenged as the severity and frequency of HABs increase due to climate change. It is this reason that the importance of prevention and mitigation must be emphasized in the issue of rising HAB prevalence, as the health of the environment and the public cannot sustainably rely on restoration efforts that will constantly face new challenges due to environmental alterations caused by climate change.

3.3 Improving Prevention Strategies to Avoid Reoccurrence Improving the ability to prevent and reduce the severity of HABs in freshwater systems is a relatively daunting task, as HABs occur naturally and algal vegetation is often vital in the success of freshwater systems. However, with the extent of human reliance on freshwater, it is important to impose practices and regulations that adequately protect freshwater systems from HAB establishment and growth. The most critical prevention strategy that can be applied at a global level is the implementation of BMPs regarding agricultural production and the emission of greenhouse gases, as discussed in section 2.

Without the necessary nutrients, habitat, and climate that offer HABs unique evolutionary advantages over native vegetation, the risk of developing eutrophied systems that lead to HABs is dramatically reduced. Finally, improving public education on the extent of HAB impact on global freshwater systems can generate the necessary awareness in order to promote transitions to sustainable lifestyles. Also, citizen scientists can be recruited and trained in order to increase the ability for research institutions to monitor and collect data on as many freshwater systems as possible, improving the ability to detect HABs early.

Integrative HAB Mitigation

Form Data Collection Methods

Drinking Water Sources Water Sources Commercial Fishing Zones Effluent Discharge Sites

Water Sample Collection Remote Sensing Biodiversity Monitoring

Collect Field Data Consistency Physical and Chemical Parameters Inclusive of Weather Variation

Analyze Data Statistical Methods Trends and Correlations Toxin Concentration

Determine Key Factors Nutrient Loading High Surface Temperature Altered Hydrology

Implement BMPs

Remediate and Restore

Reduce Fertilizer Use Riparian Zone Conservation Decrease CO2 Emissions

Modified Clay Application Residency Time Reduction Surface Skimming

Enforce Protective Policies

Mandate Monitoring Programs Subsidize Green

Energy Research & Development

Figure 1: Example model displaying the developmental process of integrative HAB mitigation plans that can be adapted to individual bloom cases.

Conclusion and Future Research As humans continue to alter the environment through carbon emissions and excess nutrient loading, it will become increasingly difficult to predict and prevent the development of HABs in global freshwater systems. The unique evolutionary advantages of HABs that thrive in human altered ecosystems will continue to increase the frequency, duration, and magnitude of HABs and generate higher risk for public health and the stability of the environment. However, through a synthesis of BMPs, freshwater protection procedures and policies, and data analyzation methods, mitigation strategies can be developed and executed to reduce the impact of HABs on global freshwater systems. The ecosystem variations of climate, geology, species diversity, human impact, and water physio-chemistry will require unique mitigation strategies for each freshwater system affected. The most emphasized aspect of any HAB mitigation strategy should be prevention by means of limiting nutrient loading and carbon emissions. For long term mitigation to be successful, environmental policy implementation on both the local and international scale must be concrete and sustainable, unapologetically protecting the health of freshwater systems in the face of environmental alterations.

To continue to refine HAB mitigation strategies, future research must be directed towards observing the changes in HAB growth and toxicity in reaction to changing nutrient concentrations. To better target and suppress practices that contribute to HAB development, controlled experiments may be necessary to measure the effect of external environmental inputs, such as high levels of carbon dioxide, warmer temperatures, high/low nutrient levels, and changes in hydrology on the growth of HABs. To improve the collection of HAB related data, research must also continue to improve upon detection capabilities, like improvements in toxic genetic marker detection or developments of remote sensing strategies apart

from spectrometry. Physical treatment of freshwater systems must also be improved upon, and the method of clay treatments specifically must be thoroughly tested and applied to a variety of scenarios in order to assure efficiency regarding restoration and overall safety. Most importantly, these refinements and improvements in research must be prioritized and reached in a decisive manner. The risk of environmental damage and adverse effects to human health far outweigh the costs of successful and integrative HAB mitigation strategies; therefore, the lens in which HAB mitigation is to be viewed through must be directed towards the long-term preservation of freshwater systems, the environment, and the development of a sustainable society with minimal environmental impacts.

References Anderson, D. M., Hoagland, P., Kaoru, Y., & White, A. W. (2000). Estimated annual economic impacts from harmful algal blooms (HABs) in the United States. Woods Hole Oceanographic Institution. https://doi.org/10.1575/1912/96 Brooks, B. W., Lazorchak, J. M., Howard, M. D. A., Johnson, M.V. V., Morton, S. L., Perkins, D. A. K., Reavie, E. D., Scott, G. I., Smith, S. A., & Steevens, J. A. (2016). Are harmful algal blooms becoming the greatest inland water quality threat to public health and aquatic ecosystems?: Harmful algal blooms: The greatest water quality threat? Environmental Toxicology and Chemistry, 35(1), 6–13. https://doi. org/10.1002/etc.3220 Bui, T., Dao, T.-S., Vo, T.-G., & Lürling, M. (2018). Warming affects growth rates and microcystin production in tropical bloom-forming Microcystis strains. Toxins, 10(3), 123. https://doi.org/10.3390/toxins10030123 Caller, T. A., Doolin, J. W., Haney, J. F., Murby, A. J., West, K. G., Farrar, H. E., Ball, A., Harris, B. T., & Stommel, E. W. (2009). A cluster of amyotrophic lateral sclerosis in New

Scientia 2022 | 33

Review Article

Identify Risk Area

Review Article

Hampshire: A possible role for toxic cyanobacteria blooms. Amyotrophic Lateral Sclerosis: Official Publication of the World Federation of Neurology Research Group on Motor Neuron Diseases, 10 Suppl 2, 101–108. https://doi. org/10.3109/17482960903278485 Chapra, S. C., Boehlert, B., Fant, C., Bierman, V. J., Henderson, J., Mills, D., Mas, D. M. L., Rennels, L., Jantarasami, L., Martinich, J., Strzepek, K. M., & Paerl, H. W. (2017).

Climate change impacts on harmful algal blooms in U.S. freshwaters: A screening-level assessment. Environmental Science & Technology, 51(16), 8933–8943. https://doi. org/10.1021/acs.est.7b01498 Davis, T. W., Berry, D. L., Boyer, G. L., & Gobler, C. J. (2009).

The effects of temperature and nutrients on the growth and dynamics of toxic and non-toxic strains of Microcystis during cyanobacteria blooms. Harmful Algae, 8(5), 715– 725. https://doi.org/10.1016/j.hal.2009.02.004 de Souza, M. S., Muelbert, J. H., Costa, L. D. F., Klering, E. V., & Yunes, J. S. (2018). Environmental Variability and Cyanobacterial Blooms in a Subtropical Coastal Lagoon: Searching for a Sign of Climate Change Effects.

Frontiers in Microbiology, 9, 1727. https://doi.org/10.3389/ fmicb.2018.01727 Décamps, H., Naiman, R. J., & McClain, M. E. (2009). Riparian Zones. In G. E. Likens (Ed.), Encyclopedia of Inland Waters (pp. 396–403). Academic Press. https://doi.org/10.1016/ B978-012370626-3.00053-3 Encyclopedia Britannica. (n.d.). Wetland—Geographic distribution of wetlands. Encyclopedia Britannica. Retrieved April 11, 2021, from https://www.britannica.com/science/ wetland Gehringer, M. M., & Wannicke, N. (2014). Climate change and regulation of hepatotoxin production in Cyanobacteria.

FEMS Microbiology Ecology, 88(1), 1–25. https://doi. org/10.1111/1574-6941.12291 Gielen, D., Boshell, F., Saygin, D., Bazilian, M. D., Wagner, N., & Gorini, R. (2019). The role of renewable energy in the global energy transformation. Energy Strategy Reviews, 24, 38–50. https://doi.org/10.1016/j.esr.2019.01.006 Hartnell, D. M., Chapman, I. J., Taylor, N. G. H., Esteban, G. F., Turner, A. D., & Franklin, D. J. (2020). Cyanobacterial abundance and Microcystin profiles in two southern British lakes: The importance of abiotic and biotic interactions.

Toxins, 12(8), 503. https://doi.org/10.3390/toxins12080503 Koneswaran, G., & Nierenberg, D. (2008). Global Farm Animal Production and Global Warming: Impacting and Mitigating Climate Change. Environmental Health Perspectives, 116(5), 578–582. https://doi.org/10.1289/ehp.11034 Lee, J., Choi, J., Fatka, M., Swanner, E., Ikuma, K., Liang, X., Leung, T., & Howe, A. (2020). Improved detection of mcyA genes and their phylogenetic origins in harmful algal blooms. Water Research, 176, 115730. https://doi. org/10.1016/j.watres.2020.115730 Manuel, J. (2014). Nutrient Pollution: A Persistent Threat to Waterways. Environmental Health Perspectives, 122(11), A304–A309. https://doi.org/10.1289/ehp.122-A304 Moore, S. K., Trainer, V. L., Mantua, N. J., Parker, M. S., Laws, E. A., Backer, L. C., & Fleming, L. E. (2008). Impacts of

34 | Scientia 2022

climate variability and future climate change on harmful algal blooms and human health. Environmental Health, 7(Suppl 2), S4. https://doi.org/10.1186/1476-069X-7-S2-S4 NASA. (n.d.). NASA: The relentless rise of carbon dioxide. Climate Change: Vital Signs of the Planet. Retrieved March 15, 2021, from https://climate.nasa.gov/climate_resources/24/ graphic-the-relentless-rise-of-carbon-dioxide Olson, N. E., Cooke, M. E., Shi, J. H., Birbeck, J. A., Westrick, J. A., & Ault, A. P. (2020). Harmful algal bloom toxins in aerosol generated from inland lake water. Environmental Science & Technology, 54(8), 4769–4780. https://doi. org/10.1021/acs.est.9b07727 Paerl, H. W., Gardner, W. S., Havens, K. E., Joyner, A. R., McCarthy, M. J., Newell, S. E., Qin, B., & Scott, J. T. (2016).

Mitigating cyanobacterial harmful algal blooms in aquatic ecosystems impacted by climate change and anthropogenic nutrients. Harmful Algae, 54, 213–222. https://doi. org/10.1016/j.hal.2015.09.009 Paerl, H. W., & Huisman, J. (2009). Climate change: A catalyst for global expansion of harmful cyanobacterial blooms.

Environmental Microbiology Reports, 1(1), 27–37. https:// doi.org/10.1111/j.1758-2229.2008.00004.x Paerl, H. W., Otten, T. G., & Kudela, R. (2018). Mitigating the expansion of harmful algal blooms across the freshwaterto-marine continuum. Environmental Science & Technology, 52(10), 5519–5529. https://doi.org/10.1021/acs.est.7b05950 Shen, L., Xu, H., & Guo, X. (2012). Satellite remote sensing of Harmful Algal Blooms (HABs) and a potential synthesized framework. Sensors, 12(6), 7778–7803.

Stauffer, B. A., Bowers, H. A., Buckley, E., Davis, T. W., Johengen, T. H., Kudela, R., McManus, M. A., Purcell, H., Smith, G. J., Vander Woude, A., & Tamburri, M. N. (2019). Considerations in harmful algal bloom research and monitoring: Perspectives from a consensus-building workshop and technology testing. Frontiers in Marine Science, 6, 399. https://doi.org/10.3389/fmars.2019.00399 Tanabe, Y., Hodoki, Y., Sano, T., Tada, K., & Watanabe, M. M. (2018). Adaptation of the Freshwater Bloom-Forming Cyanobacterium Microcystis aeruginosa to Brackish Water Is Driven by Recent Horizontal Transfer of Sucrose Genes.

Frontiers in Microbiology, 9, 1150. https://doi.org/10.3389/ fmicb.2018.01150 Trevino-Garrison, I., DeMent, J., Ahmed, F., Haines-Lieber, P., Langer, T., Ménager, H., Neff, J., van der Merwe, D., & Carney, E. (2015). Human illnesses and animal deaths associated with freshwater Harmful Algal Blooms— Kansas. Toxins, 7(2), 353–366. https://doi.org/10.3390/ toxins7020353 US EPA, O. (2013, February 8). EPA’s Budget and Spending [Overviews and Factsheets]. US EPA. https://www.epa.gov/ planandbudget/budget USDA. (n.d.). Organic Agriculture | National Institute of Food and Agriculture. Retrieved April 12, 2021, from https://nifa. usda.gov/topic/organic-agriculture Van der Merwe, D., & Price, K. (2015). Harmful algal bloom characterization at ultra-high spatial and temporal resolution using small unmanned aircraft systems. Toxins, 7(4), 1065–1078. https://doi.org/10.3390/toxins7041065

Review Article

Van der Merwe, D., Sebbag, L., Nietfeld, J. C., Aubel, M. T., Foss, A., & Carney, E. (2012). Investigation of a Microcystis aeruginosa cyanobacterial freshwater harmful algal bloom associated with acute Microcystin toxicosis in a dog.

Journal of Veterinary Diagnostic Investigation, 24(4), 679– 687. https://doi.org/10.1177/1040638712445768 Van der Veer, B., Koedood, J., & Visser, P. M. (1995). Artificial mixing: A therapy measure combating cyanobacteria in Lake Nieuwe Meer. Water Science and Technology, 31(8), 245–248. https://doi.org/10.1016/0273-1223(95)00377-Y Vermont DEC. (n.d.). Wetland Functions and Values: Surface and Ground Water Protection | Department of Environmental Conservation. Retrieved April 12, 2021, from https://dec. vermont.gov/watershed/wetlands/functions/water-quality Wilhelm, S. W., Bullerjahn, G. S., & McKay, R. M. L. (2020). The complicated and confusing ecology of Microcystis blooms.

MBio, 11(3), e00529-20, /mbio/11/3/mBio.00529-20.atom. https://doi.org/10.1128/mBio.00529-20 Wilson, A. E., Sarnelle, O., Neilan, B. A., Salmon, T. P., Gehringer, M. M., & Hay, M. E. (2005). Genetic variation of the bloomforming cyanobacterium Microcystis aeruginosa within and among lakes: Implications for Harmful Algal Blooms.

Applied and Environmental Microbiology, 71(10), 6126– 6133. https://doi.org/10.1128/AEM.71.10.6126-6133.2005 Yu, G., Jiang, Y., Song, G., Tan, W., Zhu, M., & Li, R. (2014).

Variation of Microcystis and microcystins coupling nitrogen and phosphorus nutrients in Lake Erhai, a drinking-water source in Southwest Plateau, China. Environmental Science and Pollution Research, 21(16), 9887–9898. https://doi. org/10.1007/s11356-014-2937-1 Yu, Z., Song, X., Cao, X., & Liu, Y. (2017). Mitigation of harmful algal blooms using modified clays: Theory, mechanisms, and applications. Harmful Algae, 69, 48–64. https://doi. org/10.1016/j.hal.2017.09.004 Zhang, C., & Zhang, J. (2014). Current Techniques for Detecting and Monitoring Algal Toxins and Causative Harmful Algal Blooms. Journal of Environmental Analytical Chemistry, 02(01). https://doi.org/10.4172/2380-2391.1000123

Scientia 2022 | 35

Intracellular Regulation of Epithelial Na+ Channel (ENaC) by Ankyrin 3 and Casein Kinase 2 Sinchana Basoor; Antonio G. Soares, Ph.D.; Crystal R. Archer, Ph.D.; James D. Stockand, Ph.D. Department of Cellular and Integrative Physiology, University of Texas Health Science Center San Antonio, TX

Original Abstracts

Hypertension affects roughly half of the adults in the United States and costs the nation nearly $130 billion every year. Defects in renal sodium transport may contribute to hypertension. The epithelial Na channel (ENaC), specific to the distal nephron, reabsorbs Na from urine and is regulated by several hormones, including aldosterone and vasopressin. While the function of ENaC is understood, its intracellular regulation remains unclear. Previous research has shown that binding motifs for ankyrin-3 (Ank3) overlap with a casein kinase 2 (CK2) phosphorylation site in β-ENaC’s C terminus (βC). CK2 is activated by protein kinase A (PKA), a component of the vasopressin receptor signaling pathway. We hypothesize that CK2 phosphorylation mediates ENaC translocation to the plasma membrane via Ank3, a cytoskeletal transport protein, thereby increasing ENaC activity. To understand this, we tested the interactions of βC with Ank3 and CK2 using biophysical methods. We also examined the physiological implications of CK2 activation via GsDREADD (stimulatory G protein designer receptors exclusively activated by designer drugs) mice, which serves as a model for the Gs protein-cAMP vasopressin signaling pathway selectively activated by CNO. Metabolic cage, immunofluorescence, and patch clamping studies were conducted on Gs-DREADD mice. Urine sodium excretion was reduced after treatment with CNO compared to wild-type mice. Immunofluorescence microscopy showed colocalization of ENaC and Gs-DREADD in the distal tubule. Patch clamping experiments showed that CNO-induced activation of Gs-DREADD led to increased ENaC activity. These results are consistent with a model in which vasopressin increases ENaC trafficking via interactions between CK2 and Ank3. These interactions with ENaC provide potential inhibition targets for blood pressure control.

36 | Scientia 2022

Impacts of Early Corticosterone Exposure on LPS Later in Life Pranay Sharma; Colin Budries; Donese Cole; Sindhuja Earagolla; Kayla Gilley, M.A; Hailey Rodgers; Elizabeth Vichaya, Ph.D. Department of Neuroscience, Baylor University, Waco, TX

Depression is a prevalent disorder affecting over 200 million people worldwide. It is a complex and multifactorial disease involving many risk factors. Depression is twice as common among women, and early life stress (ELS) is associated with increased risk of depression. Stress exerts many physiological effects, including the activation of the sympathetic nervous system and hypothalamicpituitary-adrenal (HPA) axis. Murine models of ELS indicate that sex, timing, and stressor severity play a role in whether ELS enhances depression or promotes resiliency. To clarify the role of early HPA axis activation, we used maternal corticosterone administration. Corticosterone in drinking water transfers from the dam to pups via maternal milk. We hypothesized that maternal corticosterone from postnatal days 2-8 would increase inflammation-induced depression-like behavior in early adulthood, and female mice would be more sensitive to these effects. Maternally treated pups were administered the saline-control, or 0.5 mg/ kg liposaccharide in early adulthood and were tested the next day in locomotor activity, forced swim (FST), tail suspension (TST), and Y-maze spontaneous alterations tasks to explore depressive-like behavior and cognitive effects. In an independent sample, we confirmed this liposaccharide dose induces neuroinflammation, through increased mRNA expression of Il1b, Tnf, and Itgam within the brain. Lipopolysaccharide reduced activity in pups (8-9 weeks). We observed an LPS by corticosterone by sex interaction (p=.039) such that LPS-injected females exposed to corticosterone showed reduced immobility while male mice showed a nonsignificant elevation, potentially indicative of a protective effect in female mice. Further research is needed to explore this protective effect.

Scientia 2022 | 37

Original Abstracts

Keywords: Depression; Hypothalamic-pituitary-adrenal axis; Early Life Stress; Corticosterone, Liposaccharide

Physics

Analysis of K2W7O22 Nanomaterial for a Breathalyzer for Diagnosing Diabetes Aman Patel; Weigang Lu, Ph.D.; Blake Birmingham, Ph.D.; Kai Wang, Ph.D.; Danling Wang, Ph.D.; Zhenrong Zhang, Ph.D. Nanomaterial K2W7O22 (KWO) is a ferroelectric semiconductor that has been shown to be sensitive to acetone at room temperature.

This property makes KWO an effective component for an acetone breathalyzer, with acetone being a biomarker for monitoring diabetes. Understanding the mechanism of the interaction of acetone and the KWO nanomaterial can help in optimizing the interaction to increase the sensitivity of the material. The study of the material using laser spectroscopy reveals that the KWO nanomaterial displays photoluminescence (PL) at 630 nm wavelength in a saturated acetone environment. The PL signal is enhanced when the sample is continuously radiated with a 532 nm laser. The PL signal is also observed in the presence of ethanol and acetone mixed with oxygen gas. The PL signal was centered at the same wavelength for each environment, but the sample was most sensitive in saturated acetone. The emission of K-doped tungsten oxide observed at 630 nm using the 532 nm laser excitation suggests that this emission is due to the presence of defect states in the band gap of the nanomaterial. The strongly enhanced emission of the PL is attributed to the electron transfer between acetone and the KWO nanomaterial.

Observing Anomalous Particle Diffusion within a Dusty Plasma in the PK-4 Experiment

URSA Award Winners

Emerson Gehr; Evelyn Guay; Eva Kostadinova, Ph.D.; Marlene Rosenberg, Ph.D.; Peter Hartmann, Ph.D.; Jorge Carmona-Reyes; Lorin Matthews, Ph.D.; Truell Hyde, Ph.D. This study investigates the diffusion of particles within dusty plasma clouds using video data from the PlasmaKristal-4 apparatus taken onboard the International Space Station. The analysis is focused on nine experiments where gas pressure was varied in the range (28-70) Pa and currents in the range (0.35-1.0) mA. We used particle tracking software to obtain the trajectories of the particles in each video, which were then used to calculate mean squared displacement and to construct velocity distribution functions. We analyzed the differences in diffusion of the particles in each video and found trends associated with the changes in pressure and current. The average velocity of all the particles in each video were subtracted from their trajectories to minimize error and eliminate drift. We generated velocity auto-correlation functions to make sure the drift was subtracted correctly. The slope of average mean squared displacement of the particles as a function of time implies the particles in all cases have super-diffusive motion, and that this super-diffusion feature is enhanced with pressure. The physical explanation of this phenomena is related to the formation of stringlike dust structures within the dust cloud.

38 | Scientia 2022

Psychology and Neuroscience

Purpose in Emerging Adults: Eschatological Hope as a Predictor for Beyond-the-Self Purpose Yejin (Sally) Lee; Jeanene Jackson; Miranda Wood; Juliette Ratchford, M.A.; Mason Ming, M.A.; Sarah Schnitker, Ph.D. Identifying a purpose in life has been associated with greater life satisfaction and well-being across various developmental stages (Bronk et al., 2009). Previous studies have also shown that religion can help cultivate a sense of purpose and meaning (Mariano & Damon, 2008; Tirri & Quinn, 2009; Liang & Ketcham, 2017). Although research has shown links between religion and developing a sense of purpose, less research has been conducted on what factors of religion affect what specific purposes an individual will develop.

This is particularly of interest in emerging adults, where the development of purpose is found as an important aspect of healthy identity development as they enter adulthood (Bronk, 2012). The purpose of this study was to investigate whether eschatological hope would predict beyond-the-self purpose. Two hundred and twenty-five students at a Christian university participated and a bivariate linear regression analysis was conducted. Eschatological hope accounted for a significant amount of variance in beyondthe-self purpose, R2 = .07, F(1, 223) = 15.75, p = .001. Findings from this study can help inform the specific mechanisms underlying the role that religion has on developing a sense of purpose in addition to which specific purposes are likely to be fostered.

Isolation-Induced Ultrasonic Vocalizations (USVs) using Multiple Methods of the Maternal Potentiation Paradigm Resulted in a Decrease in Calls in Female C57 Mice Doan Tran; Paige Womble; Danielle Santana Coelho, Ph.D.; Katherine Blandin; Srikhar Chilukuri; Siena Condon; D. Nguyen; Joaquin Lugo Ph.D.

Scientia 2022 | 39

URSA Award Winners

Ultrasonic vocalizations (USVs) are a tool widely used to study communication behaviors. In mouse pups, USVs are induced through isolation from the dam and littermates. Maternal potentiation (MP) is a type of USV paradigm used to study communication as well as the social dynamic between pups and the dam. Mice’s calling rates have been shown to increase after brief reunion with the dam.

Nevertheless, methodologies used in previous studies are unclear. In this study we utilized three different protocols on postnatal day (PD) 9 in C57 Bl/6J mice. In protocol 1, the pup was first isolated from its home cage for 5 minutes, and USVs were recorded. It was then reunited into the home cage and allowed maternal retrieval. After 5 minutes, it was reisolated with USVs recorded. Protocol 2 followed protocol 1’s procedures, but USVs were recorded for only 2 minutes. Lastly, protocol 3 was the same as 2 but utilized only one dam per cage. Our results showed no significant differences in the number of calls between before and after reunion in protocol 1 and 2 for females and male pups (p > 0.05). In protocol 3, there was also no significant difference in males (p > 0.05). However, female pups performed significantly fewer calls after reunion (p <0.05). Our data suggests that we were not able to replicate the findings showed in the scarce literature of maternal potentiation in mice. The development of a new protocol is necessary to find a more significant potentiation in male pups.

Scientia's Mission Scientia shall provide a professional platform upon which undergraduates of Baylor University are able to publish personally conducted and outstanding research in the areas of biological sciences, physical sciences, mathematics, and technology.

Accepted Formats Research Articles (maximum 4500 words including captions and references) presenting major findings performed by current undergraduate level students enrolled at Baylor University. Research articles must include an abstract, introduction, materials and methods, up to six figures or tables, results, and discussion.

Review Articles (maximum 6000 words including captions and references) synthesizing developments of interdisciplinary significance written by current undergraduate level students enrolled at Baylor University. Review articles must include an abstract and an introduction outlining the topic of discussion.

Abstracts (maximum 500 words) proposing research topics currently being investigated by current undergraduate level students enrolled at Baylor University.

Submitting to Scientia To find out more or to submit to Scientia for publication, please visit www.baylorscientia.org To read previous editions of Scientia online, please visit https://www.baylorscientia.org/previous-issues For more information, please email Scientia's Editorial Board at baylor.scientia@gmail.com

40 | Scientia 2022

Want to become involved in research at Baylor University and beyond?

Learn about Baylor Undergraduate Research in Science and Technology (BURST)!

BURST is the student organization for Baylor undergraduate students interested in scientific research.

Mission To increase awareness of undergraduate research within the Baylor campus, we aim to provide opportunities for undergraduates to optimize their research experiences, and educate them in the proper habits and techniques of research in scientific fields.

Journal Clubs Members participate in peer-led Journal Clubs of a variety of fields. Each Journal Club reads through and discusses a selection of research articles.

Lab Tours A tour of the lab in the Baylor Sciences Building (BSB) allows members to see various research environments across campus. Members have the opportunity to ask questions, visualize the research techniques they have learned about, and occasionally gain hands-on experience with lab equipment.

Scientia Scientia is the Baylor Undergraduate Research Journal of Science and Technology. First published in the Spring of 2014, Scientia is a yearly publication produced by BURST members and supported by the Baylor College of Arts and Sciences, and funded by Baylor Student Government.

Conferences (HoT) Members who are currently doing research are encouraged to attend a variety of conferences, where they can present their findings to the scientific community in a professional environment. BURST works closely with URSA during URSA Scholars' Week, and recently launched the inaugural Heart of Texas (HoT) Undergraduate Research Conference.

Service in the STEM Fields BURST organizes opportunities for members to actively engage in spreading interest for the sciences and technology in Waco.

STEM Lectures Each semester, BURST organizes lectures featuring research experts from Baylor and beyond.

Academic Workshops BURST organizes a workshop once a semester to teach its members fundamental laboratory skills. We also have workshops dedicated to advanced lab techniques, Resume and CV, Internship Applications, Cover Letters, How to Create a Research Presentation, and much more!

Social Events Throughout the semester, BURST offers several opportunities to just hang out with other members!

Research Internship Day BURST hosts an annual BURST Research Internship Day to increase awareness of the many research internship opportunities for undergraduate students at Baylor.

For Prospective Members Are you interested in joining BURST? Please contact us at burst@baylor.edu and find out more at https://www.baylor.edu/burst/

Scientia 2022 | 41