VCOM Carolinas Research Day 2023
Via Research Recognition Day VCOM-Carolinas • February 10, 2023
Contents
Welcome ........................................................................................................................... 3 Speaker ............................................................................................................................. 4 Agenda ............................................................................................................................... 5 Abstracts
Biomedical Studies.................................................................................................................. 6
Clinical Case-Based Reports................................................................................................ 15
Clinical Studies. ..................................................................................................................... 42
Educational Studies............................................................................................................... 58
Simulation and Technology................................................................................................... 66
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Welcome
Welcome to the ninth annual Edward Via College of Osteopathic Medicine Via Research Recognition Day on the VCOM-Carolinas Campus. Each year, the Via Research Recognition day is a significant event for VCOM that supports the mission of the College to provide medical education and research that prepares globally minded, community-focused physicians and improves the health of those most in need. The Via Research Recognition Day offers a forum for health professionals and scientists in academic institutions, teaching hospitals and practice sites to present and benefit from new research innovations and programs intended to improve the health of all humans. By attending the sessions with the speakers, participants have the opportunity to learn cutting edge information in the physiological bases of osteopathic manipulative therapy efficacy, new trends in physician-based research networks, and how to develop innovative research projects with high impact for human health. Poster sessions allow participants to learn about the biomedical, clinical and education-simulation research activities at VCOM-Carolinas and its partner institutions.
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Speaker
Karin Peterson received her Ph.D. in microbiology and immunology in 1998 from the University of Missouri Medical School, where she studied autoimmunity and the activation of self-reactive T cells. She then went to Rocky Mountain Laboratories (RML) in 1998 as a postdoctoral fellow in the Laboratory of Persistent Viral Diseases and applied her skills in immunology toward understanding the mechanisms that control the immune response to retrovirus infection. During this time, she became interested in the immune responses to virus infections in the central nervous system (CNS). In 2004, Dr. Peterson accepted a position as an assistant professor at Louisiana State University School of Veterinary Medicine, where she furthered her studies on viral pathogenesis in the CNS and also taught classes in immunology and virology. In 2008, she returned to RML as a tenure-track investigator to study the innate immune responses in the CNS and their role in viral pathogenesis. She was tenured in 2016 and became a senior investigator and chief of the Neuroimmunology Section.
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Agenda
7:30 - 9:00am
First Floor
Registration and Continental Breakfast.............................
8:30 - 10:30am
Third Floor
Poster Presentations..........................................................
10:30am - 12:00pm
Lecture Hall 2
Opening Remarks and Keynote Speaker........................... Karin Peterson, PhD
12:00 – 12:15pm
Student Researcher of the Year Dakota Becker-Greene
12:15 - 1:15pm
First Floor Main Hall
Lunch.................................................................................
1:30 – 2:30pm
Lecture Hall 2
Oral Presentations of Original Research............................ Kirstie Mundok Patrick Ruane Carsten Steinmetz and Kyle Kleiman Oral Presentations of Case Reports.................................. Casey Anders and Chinedum Nkemakolam Danielle Drew and Rayhan Karimi Stephanie McDonald
2:30 - 3:30pm
Lecture Hall 2
3:30 – 3:45pm
Break
3:45 – 4:30pm
Lecture Hall 2
Awards Ceremony and Closing Remarks..........................
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Biomedical Studies
Assessing the Effects of Recovery Capital on Substance Use Treatment Outcomes Trevor C. Doane, OMS III, Brett Cohen, OMS III, James Headid, OMS III, Emma Smith, OMS III, Alexis Stoner, PhD, MA, David Redden PhD Edward Via College of Osteopathic Medicine, Spartanburg, SC
Abstract # BIOM-2
Abstract
Methods
Conclusions
Background: Relapse prevention is both a short term and long term goal of substance abuse recovery and has been shown to be positively associated with recovery capital. Recovery capital is defined as the collection of one’s resources that contribute to the initiation and maintenance of sobriety. Adverse childhood experiences (ACE) are traumatic events that have been well documented as having a correlation with worse health outcomes including higher rates of drug use and addiction. Hypothesis: The aim of this paper is to identify components of recovery capital that deter or encourage positive treatment outcomes and determine if treatment models aimed at increasing recovery capital lead to improved recovery outcomes. Materials and Methods: Demographic information collected before and after program completion by a South Carolina residential recovery center whose treatment model focuses on enabling participants to attain recovery capital were analyzed, and statistical analyses were performed to determine the effects of recovery capital on treatment outcomes. Additionally, self reported data on adverse childhood experiences were collected. Results: Analyses did not provide statistically significant evidence of a difference between the ACE scores of individuals who completed the program and those who did not. There was no statistically significant difference in any individual components of recovery capital between those who completed the program and those who did not although a limitation was that recovery capital as an aggregate was not analyzed. Conclusion: Further work is currently underway to increase the power of the study by enrolling more participants to correct the possibility of a type 2 error; the studied factors may be associated with completing the program but the effect size for those associations are small to medium and a larger sample size is required to detect and declare those associations statistically significant. The study could also indicate that the variables investigated were not associated with completion of the program but other variables yet to be identified are. Illicit drug use disorders are common, with a lifetime prevalence of 2-3%, and increase the risk of hospitalization, adverse health effects and death with over 100,000 fatal overdoses occurring in 2021 (Merikangas & McClair, 2012, CDC 2022). However, treating substance abuse remains a challenge as many individuals struggle to maintain sobriety after completing treatment. One factor may be recovery capital defined as the collection of one’s resources that contribute to the initiation and maintenance of sobriety (Cloud and Granfield, 2008.) Things such as employment, housing, financial assets, family support, etc. would all be examples of a person’s recovery capital. Go Forth is a male only residential recovery center in Spartanburg that focuses on enabling participants to accrue recovery capital while in the program through classes as well as program requirements. Our research aims to answer two primary questions. First, does the Go Forth recovery model, which includes life skills training, lead to improved recovery outcomes? Secondly, by focusing on components of recovery capital, can risk factors and protective factors that have a direct impact on treatment outcomes and sobriety maintenance be identified? Introduction
Study Population: The sample population for our study included individuals at a local South Carolina residential recovery center called Go Forth Recovery. The total sample size for our study consisted of 44 program participants. A unique aspect of the Go Forth model is that all residents are male. Therefore, all 44 subjects in our study were male. Go Forth accepted individuals addicted to alcohol, opiates, cocaine, methamphetamine, marijuana and benzodiazepines. Patients were often using more than one substance prior to treatment. Materials: Demographic and recovery capital information was collected for each program participant at the time of admission to Go Forth, and again following program completion. The data forms accessed are incorporated into the Go Forth resident database, and information was transferred to an excel sheet to allow for easier statistical analysis. Any potential identifying information for our subjects in the data was de-identified. Data Collection: The initial demographic and recovery capital information for each participant was collected at the time of arrival at Go Forth as a part of their intake process. This information served as a baseline for each resident prior to the individual going through the treatment process. To collect the post-treatment data, a Go Forth program director contacted former participants and gathered the same types of information that were collected at intake. For our study, we focused primarily on the initial intake data collected. This allowed us to focus on the impact that recovery capital possessed prior to treatment had on treatment outcomes. Statistical Analysis: Analyses began by summarizing continuous variables, such as age and ACE scores, using sample means and variances. Categorical variables, such as completion status of Go Forth participation and race, were summarized using proportions. Inferential procedures examined which characteristics might be associated with completion of the Go Forth program. Of the 44 individuals for which we have data, 34 completed the program, 7 were deceased, and 3 were dismissed. This distribution, specifically the small sample size for dismissed, limited the number of analytic approaches that could be employed. Due to sample sizes, we collapsed deceased and dismissed into one combined category, using Fisher’s Exact test for to test for association between categorical variables and Wilcoxon Rank Sum for testing whether a continuous variable, such as age or ACES scores, differed between groups (completers vs deceased/dismissed). Two sample t-tests were avoided due to the small sample size and normal probability plots indicating potential non-normality. All tests were conducted using SAS 9.4
The present research addresses a gap in literature by investigating a male only residential recovery clinic focused on building recovery capital through life skills training, employment and forging social connections in addition to maintaining sobriety. ACE Scores • There was no statistically significant difference in ACE scores between individuals who completed the program and those who did not. • ACE scores have been well established in the literature as being associated with higher rates of drug abuse and addiction, but the impact of ACE scores on recovery has been less studied (Dube et al 2003, Felitti et al 1998). • These results suggest that GoForth’s unique style of recovery focused on recovery capital may be effective across a range of ACE scores, even higher ACE scores that are typically associated with higher rates of relapse (Derefinko et al 2019) Recovery Capital • No individual element of recovery capital was a predictor of successful completion of the GoForth program. • Grouped assessment of recovery capital was a better predictor of whether an individual would complete the program. However, the difference still did not rise to the level of statistical significance at (p = 0.200) when including a faith home in recovery capital. Limitations • The primary limitation in our study was the small sample size of our population which limited the power of the study. A second limitation is that incarceration rate was not compared between those completing the program and those who did not. Incarceration may be a confounding variable that impacts the attainment of recovery capital. Next Steps • Data collection is ongoing to increase the sample size and study power. When all data is gathered further analysis will be performed examining incarceration status, drug used and comparing completion rates to other facilities.
Results
Table 1. Differences in Recovery Capital Possessed by Graduates and Nongraduates of GoForth Recovery Program
References
Acknowledgements
Analyses did not provide statistically significant evidence of a difference in the distribution of ACES scores between completers and those who did not complete GoForth ( p = 0.550). No individual element of recovery capital was statistically significant although having a family connection (p = 0.566) and access to a faith home (p = 0.417) were the strongest predictors of completing the program. Access to a vehicle was actually a negative predictor of completing the program although this still did not rise to the level of statistical significance (p = 0.515). As expected, the grouped assessment of recovery capital was a better predictor of whether an individual would complete the program. However, the difference still did not rise to the level of statistical significance at (p = 0.200) when including a faith home in recovery capital and (p = .450) when a faith home was not included as part of recovery capital.
The authors would like to thank Nick Wildrick for his help in collecting as well as his dedication to helping others through his work at GoForth Recovery.
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Biomedical Studies
Testimonials about the COVID-19 Pandemic in 2022 1 Ghazal Becker OMS-2, 2 Emily Ranta OMS-2, 4 Victoria Reyes MD, 2 Riddhi Shah OMS-2 ,
2 Alexis Stoner, PhD MPH, 3 H. Dean Sutphin, PhD (1) VCOM-LA, (2) VCOM-CC , (3) VCOM-VA, (4) UNITEC
Abstract # BIOM-3
Discussion
Introduction
Results
Question 1: Personal Beliefs and Knowledge About COVID-19 ❖ Decrease in academic opportunities and performance ❖ Mental health impacts ❖ Social limitations “[COVID -19] has prevented me from connecting with my peers at VCOM and has hindered a majority of the learning experiences I had hoped to get out of medical school.” Question 2: Perspectives On The Interventions and Prevention Initiatives Taken In Respondent's Region ❖ Positive view on initiatives and policies overall (international) ❖ Disapprove of the initiatives and policies in place (United States) ❖ Thankful for availability of vaccinations ❖ Negative impact caused by politics on the initiatives taken “ In El Salvador , the pandemic has been well controlled with different health measures that were imposed from the beginning and thanks to the education provided to people about this virus, these measures have been maintained and have allowed the pandemic to be (at a national level) well controlled.” Question 3: Perspectives On The Interventions and Prevention Initiatives Taken In Other Countries “I have limited information or knowledge about COVID -19 prevention initiatives and interventions in other countries aside from what I have read in the news. It is difficult to reach a consensus based on news articles or the data presented within them when it is unclear how widespread testing may or may not be in these other countries.” – US VCOM Auburn Question 4: Perspectives On Social Media and News Outlet Presentation of COVID-19 ❖ Negative view of social media due to it not being scientific and/or spreading false information ❖ Negative view on social media due to political reasons ❖ Neutral view on social media due, seeing positives and negatives Social Media did a good job of presenting both fronts (sides/extremes) in a sensationalized way, but heavily refrained from being a good way to receive or convey scientific information on the issue. – US VCOM Virginia ❖ CDC ❖ Social Media ❖ WHO (international>US) Question 5: Sources Used to Obtain Information ❖ Unfamiliar with other countries’ initiatives ❖ Strict precautions in other countries
The World Health Organization (WHO) declared COVID-19 a pandemic on March 11, 2020. Globally, medical students were required to adapt to this new environment and have been personally affected in several ways. In 2022, students were influenced by social media and informative news outlets. They adapted to restrictions, formed personal beliefs about the pandemic, and gained knowledge about the virus. As medical students with an osteopathic focus, we questioned how our peers in the U.S. and in international settings have been impacted over the course of the pandemic, focusing on their minds, bodies, and spirits. This thematic study focuses on comparing testimonials from students enrolled in four U.S. based medical schools and three international medical schools. These testimonials include medical students' knowledge about the coronavirus and their perspectives on the policies and the initiatives taken. Additionally, testimonials addressed the effect of COVID-19 on students’ personal lives, sources used for updated information, and how students thought social media presented the pandemic. ❖ Subjects for this study were selected from cohorts of medical students enrolled in a course entitled “ Global Seminar for Health and Environment .” ❖ Students were enrolled in this course during the 2022 academic year. ❖ Eligible campuses include: ➢ VCOM-VA (Blacksburg, Virginia) ➢ VCOM-CC (Spartanburg, South Carolina) ➢ VCOM-AU (Auburn, Alabama) ➢ VCOM-LA (Monroe, Louisiana) ➢ El Instituto Tecnológico de Santo Domingo (Santo Domingo, Dominican Republic) ➢ Universidad Evangélica de El Salvador (San Salvador, El Salvador) ➢ Universidad Tecnológica Centroamericana (Tegucigalpa, Honduras) Participants Participants 1. What are your personal beliefs and knowledge of the novel coronavirus and COVID-19? How has it affected you at a personal level? 2. What is your perspective on the interventions and prevention initiatives that have been taken in your country, region, and city? What would you change , if any? 3. What is your perspective for the interventions and prevention initiatives of other countries or regions? What knowledge or information you obtain your information on the novel coronavirus and COVID-19? 4. How has social media and news outlets presented the novel coronavirus/COVID-19 in your country, region, city? How has that affected you ? 5. From what sources do you obtain your information on the novel coronavirus and COVID-19? Survey Questions
Conclusion The COVID 19 pandemic affected medical students in the U.S., as well as internationally. This pandemic has affected student´s education and learning experiences, personal health, family´s health and finances, mental health and limited their social interactions both in 2022. This study was limited by the number of student responses received both internationally and in the United States. We would like future analysis to be focused on student´s response to changing policy structures with increasing vaccination rates and returns to normalcy. This study indicates changes in medical student's perspective in the year of 2022. With respect to personal beliefs and knowledge about COVID-19, the 2022 survey showed medical students believed they had less academic opportunities and more social limitations and mental health impacts. With respect to initiatives and prevention in the respondent's country, the responses demonstrated varying answers of approval and disapproval. International students were more likely to have positive views on their own country’s initiative, while students from the United States were more often displeased or disapproved of the policies in their country. With respect to initiatives and prevention in other countries, across the board the students felt unfamiliar with other countries’ initiatives. Additionally, for those student who did have some idea on other countries’ initiatives, many felt the precautions taken by other countries were too strict. With respect to perspectives on social media and news outlets, both international and United State’s students found social media to be negative and a contributor of false information or political expansion. Finally, with respect to sources used to obtained information, the responses overwhelming used the CDC followed by social media and the WHO.
Acknowledgements
We would like to acknowledge Dr. Stoner, Dr. Sutphin and Ms. Ana Jones for their assistance and guidance as we completed this study. We would also like to acknowledge our fellow medical students among the participating medical schools for their participation in this study. This study was approved by VCOM IRB.
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Biomedical Studies
The Effect of COVID-19 on Preventative Medicine in El Salvador, Honduras, and the Dominican Republic E. Casey Anders, OMS-II. Ami Changela, OMS-II. Angie Wilson, MBA CSSGB CSSBB. Alexis Stoner, PhD MPH. Edward Via College of Osteopathic Medicine, Spartanburg, SC.
Abstract # BIOM-4
Introduction
Results
Conclusion
-According to the World Health Organization, the first confirmed case of COVID-19 in Honduras was March 9 2020, in El Salvador was March 16 2020, and in the Dominican Republic was March 2 2020 1 -Current literature suggests that Latin America and Caribbean regions had approximately 15% of COVID-19 cases worldwide 2 -The COVID-19 pandemic strained medical systems and affected healthcare across the world, resulting in patients being turned away from the hospital which resulted in an increased number of preventable deaths 3 -This study sought to describe the impact that the COVID-19 pandemic had on patients seeking medical care for chronic diseases in Honduras, El Salvador, and the Dominican Republic -This is a retrospective descriptive study that utilized a Clinical Rotation Evaluation and Document Organizer (CREDO) database -The data was filtered by country: Honduras, El Salvador, Dominican Republic -The data was filtered by ICD10 codes: I10 (essential primary hypertension), E78.5 (hyperlipidemia), U07.1 (COVID-19, virus identified), U07.2 (COVID-19, virus not identified) -The data was divided into quarters: Quarter 1 is defined from January to March, Quarter 2 is defined from April to June, Quarter 3 is defined from July to September, Quarter 4 is defined from October to December -Quarters with a complete lack of diagnoses were eliminated from comparison -Descriptive analysis was performed to describe change in prevalence and a Wilcoxon test was performed to determine significance Methods -There was a significant difference in diagnosis prevalence for COVID-19 (Wilcoxon test =0.0178) between the three countries, with Honduras having more diagnoses than El Salvador and Dominican Republic (Blue lines in Figures 1, 2, 3) -There was a significant difference in diagnosis prevalence for Hypertension (Wilcoxon test <0.0019) between the three countries, with Honduras having more diagnoses than El Salvador and Dominican Republic (Orange lines in Figures 1, 2, 3) -There was no significant difference in diagnosis prevalence for Hyperlipidemia (Wilcoxon test =0.1305) between the three countries (Grey lines in Figures 1, 2, 3) -There was a significant difference in the number of diagnoses seen between countries (p-value <0.05), but the overall prevalence of each diagnosis within each country did not change Results
-This study found a constant prevalence of diagnosis for Hypertension and Hyperlipidemia from July 2020 through June 2022 -Based on the available data in CREDO, we have deduced that COVID-19 did not have an impact on the number of patients continuing to receive preventative medical care for chronic disease -A major limitation of this study is the amount of data available for each diagnosis. The CREDO system is new, so there aren’t previous records of normal preventative care to show if this is an accurate representation of normal care or if the numbers are decreased due to a new software introduction.
Figure 1. In Honduras, there were significantly more (Wilcoxon test < 0.05) patients diagnosed with Hypertension than COVID-19 or Hyperlipidemia. The overall change in prevalence for COVID-19 was 9.46%, for Hypertension was 8.26%, and for Hyperlipidemia was 0.7%. [n=4758]
Study Area
Figure 2. In El Salvador, there were significantly more (Wilcoxon test < 0.05) patients diagnosed with Hypertension than COVID-19 or Hyperlipidemia. The overall change in prevalence for COVID-19 was 1.94%, for Hypertension was 2.62%, and for Hyperlipidemia was 0.2%. [n=11676]
Figure 4. Map from CREDO showing the locations of the clinics in which the data was pulled from
References
1. World Health Organization. “WHO COVID - 19 Dashboard.” World Health Organization , 2022, covid19.who.int/. 2. Schwalb , Alvaro, et al. “COVID ‐ 19 in Latin America and the Caribbean: Two Years of the Pandemic.” Journal of Internal Medicine , 22 Apr. 2022, www.ncbi.nlm.nih.gov/pmc/articles/PMC9115176/, 10.1111/joim.13499. 3. de Oliveira, Mayra Monteiro, et al. “Repercussions of the COVID -19 Pandemic on Preventive Health Services in Brazil.” Preventive Medicine , vol. 155, Feb. 2022, p. 106914, 10.1016/j.ypmed.2021.106914. Accessed 15 July 2022.
Figure 3. In the Dominican Republic, there were significantly more (Wilcoxon test < 0.05) patients diagnosed with Hypertension than COVID-19 or Hyperlipidemia. The overall change in prevalence for COVID-19 was 0.63%, for Hypertension was 2.03%, and for Hyperlipidemia was 0.26%. [n=44824]
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Biomedical Studies
Identification of Hypoxic Response Intermediates With Relevance to Breast Cancer Survival by Analysis of Hif-1 Alpha Target Genes in Mammary Gland Tumorigenesis Anna C. Deal, OMS-II, Luciana Schwab, Ph.D. Edward Via College of Osteopathic Medicine, Biomedical Department, Spartanburg, S.C.
Abstract # BIOM-5
Introduction
Results
Results
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• Breast cancer (BC) is the second most common type of cancer among women. Breast cancer refers to tumors that originate in various structures of the mammary gland. These are very heterogeneous solid tumors. Several BC subtypes have been identified based on histological characteristics, gene expression, and the presence or absence of specific markers. Each subtype exhibits a different prognosis. • Breast tumors, like many other solid tumors, develop areas of low oxygenation or hypoxia . It has been shown that hypoxia, primarily through the action of the Hypoxia Inducible Factor (HIF), significantly enhances tumor progression and dissemination. Since modulation of HIF by drugs has been difficult to achieve, efforts have been focused on identifying HIF targets that could be affected by pharmaceuticals. • At a cellular level: o Cancer cells adapt to hypoxic stress by activating the HIF-1 pathway o HIF-1 is a heterodimeric transcription factor that regulates the transcription of multiple genes mediating hypoxic response.
Cnsk S100a8 ͺ Table 1. Identification of HIF1- α upregulated genes with prognostic value. Each of the 200 genes analyzed in this study was run through the Kaplan-Meier plotter to find the genes that showed a statistically significant correlation between high levels of gene expression and low patient survival (RFS= Relapse-free survival, p<0.05, mRNA). All PAM50 subtypes and all available datasets for mRNA breast cancer were included in the analysis. Cxx1a ͺ Ppic Egln3 ͵ Cd82 Trf
Discussion and Conclusions
References • We have previously shown that mice transplanted with Hif1- α wt MTEC develop larger, faster-growing, and more metastatic tumors than mice transplanted with Hif1- α ko MTEC. Here, we hypothesize that genes upregulated in the wt cells might be the effectors carrying out the hypoxia response and could be more suitable for therapeutic targeting than HIF itself. • By applying the survival analysis tool (Kaplan-Meier plotter), we have successfully narrowed a list of 200 genes upregulated in Hif1- α wt MTEC (microarray results) down to 14. • The 14 genes support our hypothesis for the role of Hif1- α in mammary gland tumorigenesis and their prognostic and predictive value make them relevant to human disease. • By applying the Gene Set Enrichment Analysis tools (Metascape and Enrichr), we have identified pathways and biological processes that we will further study to elucidate the role of these 14 genes in breast cancer. Future directions : • Validate selected genes by RT-PCR, Western Blot, and IHC. • Design the experiments to test the role of the 14 genes in the identified pathways. 1.Microarray data: Krutilina, Raisa I., Hilaire Playa, Danielle L. Brooks, Luciana P. Schwab, Deanna N. Parke, Damilola Oluwalana, Douglas R. Layman, et al. “HIF -Dependent CKB Expression Promotes Breast Cancer Metastasis, Whereas Cyclocreatine Therapy Impairs Cellular Invasion and Improves Chemotherapy Efficacy. ” Cancers 14, no. 1 (2021): 27. 2. K-M Plotter: Győrffy B. Survival analysis across the entire transcriptome identifies biomarkers with the highest prognostic power in breast cancer, Computational and Structural Biotechnology Journal, 2021;19:4101-4109 3. ROC plotter: Fekete J & Gyorffy B: ROCplot.org: Validating predictive biomarkers of chemotherapy/hormonal therapy/anti-HER2 therapy using transcriptomic data of 3,104 breast 5. Enrichr: Chen EY, Tan CM, Kou Y, Duan Q, Wang Z, Meirelles GV, Clark NR, Ma'ayan A. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics. 2013; 128(14). • Steven Enkemann, Ph.D., for engaging in valuable discussions about microarray analysis. • Tiffany Seagroves, Ph.D., for lending advice on the overall project and providing the cells. • VCOM Research Department for providing funding through REAP grant. Acknowledgements cancer patients, Int J Cancer , 2019 Dec 1;145(11):3140-3151 4.Metascape: Zhou et al. Nature Commun. 2019 10(1):1523
Figure 1 . Level of gene expression and correlation with survival and response to treatment. Representative examples of the K-M (top panels) and ROC (bottom panels) plots that were run for each gene. K-M plotter provides prognostic (survival) and ROC plotter predictive (response to therapy) information. All available datasets were used and the response to all chemotherapies was analyzed.
A
B
https://doi.org/10.3389/fonc.2021.559822 .
https://doi.org/10.1038/s41429-021-00451-0
Objective: The goal of this project is to identify HIF1- α target genes with biological significance based on survival and gene ontology analysis. Hypothesis: Since HIF1- α is not easily druggable, we will focus on finding HIF1- α target genes with potential for pharmaceutical targeting.
Figure 2 . Visualization of functional enrichment results . Metascape application was used to find enrichment terms and networks for the 200 genes analyzed in this study. A ) Bar graph of top 20 enriched terms across input gene lists, colored by p-values. B ) Network of enriched terms colored by cluster ID.
A
B
C
Methods
1. Microarray: A list of genes from a microarray study performed in the Polyoma Middle T (PyMT+) transgenic animal model of breast cancer was the starting point of this project. In that study, mammary tumor epithelial cells (MTEC) harboring the Hif1- α wt (wild type) gene were compared to Hif-1 α ko (knockout) cells. 2. Survival Analysis: The Kaplan-Meier (K-M) plotter was used to analyze the relationship between the level of expression of 200 genes from the microarray study and patient survival from all the available datasets for breast cancer mRNA. We focused on genes upregulated in the wt versus the ko cells. 3. ROC Plotter: After identifying 14 genes whose high level of expression correlated with lower patient survival, the ROC plotter was used to investigate the link between gene expression and response to chemotherapy therapy. 4. GSEA: Gene Set Enrich Analysis tools (Metascape and Enrichr) were used to identify pathways and cellular processes related to these 14 genes.
Figure 3. Gene Ontology (G.O.) and KEGG analysis of 200 genes upregulated in Hif-1 α wt MTEC. Enrichr application was used to group genes based on enriched terms and allow for comparisons. A ) G.O. Biological processes. B ) G.O. Molecular process. C ) KEGG.
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Biomedical Studies
SYNERGISTIC EFFECTS OF CALCIPOTRIENE AND RETINOIC ACID ON CELL VIABILITY OF A DYSPLASTIC ORAL KERATINOCYTE CELL LINE
Carsten Steinmetz, OMS-II; Kyle Kleiman, OMS-II; Joseph Metz, DO; Lindsey Richardson, OMS-III; Jaime Foushee, PharmD; Patrick D. Ellis Fisher, PhD; David Eagerton, PhD; Luciana Schwab, PhD Edward Via College of Osteopathic Medicine-Carolinas Campus - Carolinas Campus; Spartanburg, SC
Abstract # BIOM-6
Introduction
Results
Discussion and Conclusions
Context: Dysplastic oral leukoplakia (OL) lesions, or white patches within the oral mucosa, are premalignant lesions that may progress to oral squamous cell carcinoma (OSCC). 1 Data suggest anywhere from 11-36% of OL will transform to OSCC if left untreated, depending on the length of follow up and degree of dysplasia. 2 Several clinical studies have utilized either vitamin A or vitamin D synthetic derivatives in topical formulations for the treatment of dysplastic OL with some success 3-11 but using a combination of the two drugs together has not been examined. In addition, to the best of our knowledge, cell culture studies using oral dysplastic cell lines treated with either vitamin A derivatives, vitamin D derivatives, or a combination of both are lacking. Vitamin A and vitamin D derivatives have distinct unique mechanisms, which when used together could exhibit synergistic effects. Objective: To determine the effect of a drug combination of a vitamin A and a vitamin D derivative on the viability of human dysplastic oral keratinocytes, compared to monotherapy with each agent. It is hypothesized that the combination of these synthetic derivatives would result in a synergistic cytotoxic effect, compared to the use of each drug individually. • A dysplastic oral keratinocyte (DOK) cell line was obtained and cultured according to cell provider instructions. • Cells were plated in 96 well plates, at 1000 cells per well on triplicate wells, and treated with the drugs or vehicle control for 24, 48, and 72 hours. • Cells were treated with: • Vehicle control (DMSO) • 25, 50, 100, 200, or 300 µM of single drug retinoic acid (aka retinoic acid) • 0.5, 1, 10, 50, or 100 µM of single drug calcipotriene • Five different combinations of the two vitamin derivatives together at a fixed ratio of 4:1, retinoic acid:calcipotriene as follows: 12.5+3.1, 25+6.3, 50+12.5, 100+25, and 200+50 µM. • Cell viability was measured utilizing an MTT assay. • The effect of the combination between calcipotriene and retinoic acid on cell viability and synergism was calculated for the 72-hour time point utilizing CompuSyn software. 12-13 Methods
• The combination of retinoic acid and calcipotriene had a more profound effect on cell viability than either drug alone after 48 or 72 hours. • Results from CompuSyn software indicate synergism and the potential for favorable dose reduction for 2 of the 5 studied combinations. • Favorable dose reduction may allow for lower concentrations of each medication to be applied topically to OL lesions, which could limit drug toxicity. • Limitations of the study include the in vitro cell culture design, with continuous application of drug treatments, which may not translate to the intermittent topical application of these agents in a living human model. • Additional studies are necessary to determine the IC50 for calcipotriene. This may allow for the calculation of a drug concentration that will give a more accurate dose-response curve. Other project ideas include the development of an interprofessional OL educational program to increase the early identification of OL lesions.
Figure 2. Cell viability with calcipotriene or retinoic acid monotherapy
Figure 3. Cell viability with calcipotriene and retinoic acid combination therapy
Images
Figure 4. Oral leukoplakia classifications 14
Verrucous Non homogenous OL
Nodular Non Homogenous OL
Homogenous OL
Speckled Non homogenous OL
Table 1. Quantitative assessment of the inhibitory effect of calcipotriene and retinoic acid combination therapy using the combination index (CI) method
References
1. Warnakulasuriya S, Johnson NW, van der Waal I. Nomenclature and classification of potentially malignant disorders of the oral mucosa. J. Oral Pathol Med . 2007;36:575-80. 2. Lee JJ, Hong WK, Hittelman WN, et al. Predicting Cancer Development in Oral Leukoplakia: Ten Years of Translational Research. Clin Cancer Res . 2000;6(5): 1702-10. Additional references:
Figure 1. Experimental design
• The half-maximal inhibitory concentration (IC50) was able to be determined for retinoic acid, but not for calcipotriene. • Results show that the addition of the two drugs together had a more profound inhibitory effect on cell viability: the IC50 for retinoic acid monotherapy was 90 µM, while IC50 for retinoic acid + 0.25x calcipotriene was 50 µM at 72 hours. • Furthermore, analysis of the experimental data in combination with simulation values from the CompuSyn software indicated a slight synergism for 2 of the 5 combinations tested here and a favorable dose reduction of 1.4-fold for retinoic acid.
Treat with retinoic acid, calcipotriene, or combination therapy
Culture dysplastic cell line
Measure cell viability using MTT assay
Acknowledgements
The authors would like to thank VCOM’s Research Department for providing financial support for this project.
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Biomedical Studies
ONGOING CHANGES IN THE USE OF QUATERNARY AMMONIUM COMPOUND DISINFECTANTS IN DENTAL CLINICS SINCE COVID-19 Jessica E. Marino, Harthe D. Blankenship, Kaitlyn F. Braffitt, Theresa J. McCann, Terry C. Hrubec Edward Via College of Osteopathic Medicine – Carolinas Campus – Spartanburg, SC
Pri This
Location of Practice
BIOM-7
It’s d large Cus The form plac an ic Sma mul T from butt If yo title mak drag Sma with Wan inste righ Cha prop by d
• There was no difference between the location of practice with respect to number of practices, changes in disinfectant, and percent increases in spraying and wiping.
It was hypothesized that there would be an increase in the number of QAC disinfectant products being used to combat the increased number of cases of COVID-19 in healthcare facilities. dialkyldimethylammonium compounds (DADMACs). These active ingredients have been identified in over 200 common disinfectant products. Due to their ability to inactivate enveloped viruses, the United States Environmental Protection Agency (EPA) added QACs to their list of disinfectants to combat the COVID-19 pandemic. While long considered safe, recent studies in rodent models show that QACs cause neural tube defects at three closure sites and increase embryonic death. Additionally, exposure causes infertility in both males and females with abnormal sperm morphology and decreased sperm concentrations and decreased ovulation of eggs and implantation of embryos. Other studies found that QACs increase proinflammatory cytokines, decrease mitochondrial function, and disrupt cholesterol homeostasis. Similar changes were seen in people. Inflammation was increased, mitochondrial function was decreased, and cholesterol synthesis was disrupted in a dose-dependent manner. The concentration of QACs present in the blood were at a level shown to cause adverse effects in animal and cell culture models. Due to the potential toxicity of these chemicals, we wanted to determine if exposure to QACs has increased due to the pandemic. Quaternary ammonium compounds (QACs) are among some of the active ingredients in common disinfecting products. QACs consist of chemicals like benzalkyl dimethylammonium compounds (BACs), alkyltrimethylammonium compounds (ATMACs), and • States were ranked by 28-health metrics from the Kaiser Family Foundation. Those that fell within ± 3 states from the median were then ranked. The 8 states that were consistently closest to the median were selected. Those 8 states include AZ, DE, FL, IL, MI, PA, SC, and VA. • A survey asking both closed and open-ended questions was created within Question Pro to determine what changes, if any, occurred with disinfection protocols since the COVID-19 pandemic. • Dental clinics listed on the state’s Department of Health were contacted via phone to fill the survey out, either through the phone or by email. • The survey questions focus on the type of disinfectant used, changes in frequency or concentration, and changes in cleaning method (mop, wipe, etc.). • Statistical analysis was conducted using a Chi Square with p < 0.05. • This survey is ongoing and interim results were collected.
Changes in Disinfectant Practices Due to COVID-19
Number of Practices
Increase Spray (%)
Increase Wipe (%)
Change in Disinfectant
Percentage
Urban
15
12
46.6% 66.6%
Completed Surveys
6.6%
Rural
20
15
50% 70%
Changes in Disinfection Protocols
75%
Suburban
23
18
47.8% 52.2%
Disinfection Occurring More Often
73.6%
• Although cleaning products containing QACs were widely used before the pandemic, there was an increase in usage after the start of COVID-19. • Out of the practices that completed the survey, the percent of QAC products increased from 92% to 98% after COVID 19. • The number of calls made and the number of responses (blue bars) for a response rate of 7.3%. This is above the national average for phone surveys. • Of the 93 surveys started, 84 were fully completed (teal bars). 58 of these used the same cleaning procedures throughout the entire practice. 16 practices used an outside cleaning service which terminated the survey.
Changes in Disinfectant Type 24.4% Changes in Disinfectant Concentration 22.5%
Survey Calls and Responses
0 10 20 30 40 50 60 70 80 90 100
1000 1200 1400
1276
84
58
0 200 400 600 800
Number
Number
16
93
Thank you to all the practices who responded to our survey and to all the healthcare personnel who have worked during the COVID-19 pandemic. This project was in part funded by VCOM and is IRB exempt. • Hora, P. I., Pati, S. G., McNamara, P. J., & Arnold, W. A. (2020). Increased Use of Quaternary Ammonium Compounds during the SARS CoV-2 Pandemic and Beyond: Consideration of Environmental Implications. Environmental Science & Technology Letters , acs.estlett.0c00437. https://doi.org/10.1021/acs.estlett.0c00437 • Hrubec, T. C., Melin, V. E., Shea, C. S., Ferguson, E. E., Garofola, C., Repine, C. M., Chapman, T. W., Patel, H. R., Razvi, R. M., Sugrue, J. E., Potineni, H., Magnin-Bissel, G., & Hunt, P. A. (2017). Ambient and Dosed Exposure to Quaternary Ammonium Disinfectants Causes Neural Tube Defects in Rodents. Birth defects research , 109 (14), 1166 – 1178. https://doi.org/10.1002/bdr2.1064 • Hrubec, T. C., Seguin, R. P., Xu, L., Cortopassi, G. A., Datta, S., Hanlon, A. L., Lozano, A. J., McDonald, V. A., Healy, C. A., Anderson, T. C., Musse, N. A., & Williams, R. T. (2021). Altered toxicological endpoints in humans from common quaternary ammonium compound disinfectant exposure. Toxicology reports , 8 , 646 – 656. https://doi.org/10.1016/j.toxrep.2021.03.006 • Melin, V. E., Potineni, H., Hunt, P., Griswold, J., Siems, B., Werre, S. R., & Hrubec, T. C. (2014). Exposure to common quaternary ammonium disinfectants decreases fertility in mice. Reproductive toxicology (Elmsford, N.Y.) , 50 , 163 – 170. https://doi.org/10.1016/j.reprotox.2014.07.071 • Melin, V. E., Melin, T. E., Dessify, B. J., Nguyen, C. T., Shea, C. S., & Hrubec, T. C. (2016). Quaternary ammonium disinfectants cause subfertility in mice by targeting both male and female reproductive processes. Reproductive toxicology (Elmsford, N.Y.) , 59 , 159 – 166. https://doi.org/10.1016/j.reprotox.2015.10.006 • These results imply higher QAC exposure to dental practice personnel. • The main routes of exposures are inhalation of aerosols during spraying, and orally from touching disinfected surfaces with hand to mouth transfer. • QAC disinfectants have been a cornerstone in preventing the spread of SARS-CoV-2; however, based on their toxicity the disinfectants should be used judiciously and with appropriate PPE. • COVID-19 resulted in increased disinfection protocols to combat COVID-19. • Interim results found an increase in both the use of products containing QACs and the frequency of cleaning.
Calls Made
Responses
Completed Surveys
Outside Cleaning Service Used
Same Procedures throughout Practice
Class of Disinfectants Used Before and After COVID-19
96%
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
90%
8%
2%
2%
2%
QAC
QAC + Alcohol
Bleach
Before COVID-19 Current
• The frequency of
Percent Increase in Method of Cleaning Since COVID-19
cleaning throughout dental practices was increased due to COVID The cleaning methods that showed the largest increase were spraying and wiping.
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
19. •
76%
60%
31%
2%
Spray
Wipe
Mop
Soak
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Biomedical Studies
The Effects of Tolylfluanid on Dopaminergic Cell Development and Dopamine Production Rayhan Karimi 1 , Shreya Shah 1 , Madison Battersby 1 , Yassine Lahlou 1 , Katherine Baumgarner 1 , Bradley Baumgarner, PhD 2 and Stephan Brown MD, PhD 1 1 VCOM CAROLINAS, Spartanburg, SC 29303 2 USC Upstate, Spartanburg, SC 29303
Abstract # BIOM-8
Abstract
Results
Conclusions
Cerebral organoids from iPSCs were grown and treated with varying concentrations of TF (0, 1 μM, 2 μM and 5 μM) to analyze global gene expression. Lund human mesencephalic (LUHMES) cells are human embryonic neuronal precursor cells that can be maintained as proliferating cells due to the expression of a tetracycline-regulatable (Tet-off) v-myc transgene. LUHMES cells were used as the model for dopamine production and cell viability of dopaminergic cell after TF treatment. Results Our results showed decreased proliferation and development of several regions of the brain. The expressivity of SATB2, vimentin, proliferation marker Ki-67 showed a reduction in growth of the superficial layers of neuron and subventricular structures as well as a decrease in development of the early ventricles. In contrast, SOX6 and TTF1 suggested increased growth in ventricle and dopaminergic neurons. Overall, the data showed developmental deviation from the early brain in TF (5 μM) treated cerebral organoids. Ultimately, our results demonstrated that TF causes global suppression of proliferation and development of maturing central nervous system. Tolylfluanid (TF), (the active ingredient in agricultural pesticides/fungicides) is an endocrine disruptor that has been shown to increase anaerobic glycolysis and reduce oxygen consumption in human cells. TF is an environmental endocrine disrupting chemical (EDC) that decreases glucose oxidation in developing neurons causing metabolic stress by inhibiting the entry of pyruvate into the mitochondria. This constant stress alters neuron metabolism leading to gene expression alterations. Both gene expression and environmental input are essential for normal brain development and can fundamentally alter neural outcomes including the development of dopaminergic neurons. Chen et al. discovered that TF reduced glucose oxidation in human adipocytes by directly inhibiting the mitochondrial pyruvate carrier (MPC). A previous investigation by Vacanti et al. revealed that pharmacological inhibition (i.e. TF) or genetic deletion of MPC significantly reduced glucose oxidation in human skeletal muscle cells, which increased mitochondrial fatty acid glutamine oxidation. Therefore, it appears that by blocking mitochondrial pyruvate uptake, TF can increase the utilization of other oxidative fuels. However, the potential impact of TF exposure on cellular glycolytic capacity has yet to be fully investigated. Introduction or Methods
Continued
Results reveal that treating induced-pluripotent stem cell derived cerebral organoids with TF throughout differentiation significantly altered organoid development and particularly effected subpopulations of neurons (ie. Dopaminergic and cortical neurons) differently than other neural cell types. Metabolic studies showed a reduced glycolytic rate of fully differentiated cortical neurons under basal and compensatory (anaerobic) conditions when compared to vehicle treated cells (not shown). Major findings revealed that treating induced-pluripotent stem cell derived cerebral organoids with TF throughout differentiation significantly altered organoid development and particularly affected subpopulations of neurons (ie. dopaminergic neurons and cortical) differently than other neural cell types. Metabolic studies showed a reduction in the glycolytic rate of fully differentiated cortical neurons under basal and compensatory (anaerobic) conditions when compared to vehicle treated cells. Chronic exposure to TF caused an increase in dopamine production with no significant impact on cell viability.
DAPI
MitoRed
Merge
Control
DAPI
MitoRed
Merge
TF-treated
DAPI
TTF
Merge
Control
DAPI
TTF
Merge
TF-treated
TF had a minimal effect on Mitochondrial biogenesis but increased dopaminergic neuron development compared to control.
Vimentin
DAPI
Pax-6
Merge
Table 2 .
Seahorse XF Cell Mito Stress Test Bar Charts
Basal
Proton Leak
40.0
10.0
8.0
Control
30.0
6.0
TF-treated LUHMES cells. SeaHorse assay results for OCRs of Tet-treated LUHMES, in real time under basal conditions and in response to mitochondrial inhibitors. TF-treated DAPI Metabolic Profile of Tet-treated Vimentin DAPI Sox-6 DAPI Sox-6
Pax-6 20.0
Merge
4.0
10.0
OCR (pmol/min)
2.0
OCR (pmol/min)
0.0
0.0
Maximal Respiration
Spare RespiratoryCapacity
80.0
40.0
60.0
30.0
40.0
20.0
Ki67 20.0
Merge
10.0
OCR (pmol/min)
OCR (pmol/min)
0.0
0.0
ATP Production
Non-mitochondrial Oxygen Consumption
10.0 15.0 20.0 25.0 30.0 OCR (pmol/min)
25.0
20.0
References
Ki67 10.0 15.0
Merge
5.0 OCR (pmol/min)
0.0 5.0
0.0
Coupling Efficiency (%)
Spare RespiratoryCapacity (%)
64.0% 66.0% 68.0% 70.0% 72.0% 74.0% 76.0% OCR (pmol/min)
250.0%
1. Buckingham M, Meilhac S, Zaffran S. Building the mammalian heart from two sources of myocardial cells. Nat Rev Genet. 2005; 6:826 – 835. 2. Parameswaran M, Tam PP. Regionalisation of cell fate and morphogenetic movement of the mesoderm during mouse gastrulation. Dev Genet. 1995; 17:16 – 28. 3. Garry DJ, Olson EN. A common progenitor at the heart of development. Cell. 2006; 127:1101 – 1104. 4. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009; 324:1029 – 1033. 5. Chung S, Dzeja PP, Faustino RS, et al. Mitochondrial oxidative metabolism is required for the cardiac differentiation of stem cells. Nat Clin Pract Cardiovasc Med. 2007;4(Suppl 1):S60 – S67.
150.0% 200.0%
100.0%
0.0% 50.0%
OCR (pmol/min)
Dopamine Production
Table 3 .
Viability
Table 1 : Preliminary Data
5 μ M TF
Control
.5 μ M TF
Acknowledgements
Dopamine production from exogenousl-DOPA and its storage Tet-LUHMES cells. Time course of intracellular (A) and extracellular (B) dopamine levels in Tet-LUHMES cells (open circles with dotted line) and Untreated-LUHMES cells (filled circles with solid line) after 1 μ ml-DOPA incubation for various durations (0 – 48 hr). *p < 0.01 compared with the Untreated group and also with time 0 by Newman – Keuls test.
I want to thank VCOM Research and the REAP Grant initiative for funding this project. I also want to thank the students that have participated in this research project.
Cerebral organoids exposed to TF showed a marked decrease in cortical neuron development. Red color represents cortical neuron marker.
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