Carolinas Research Day 2021

Carolinas Research Day 2021 Program Book

Via Research Recognition Day VCOM-Carolinas • March 19, 2021

Welcome

Welcome to the sixth 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. 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. The posters in this document show the biomedical, clinical and education-simulation research activities at VCOM-Carolinas and its partner institutions.

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Contents

Abstracts Biomedical Studies....................................................................................................................................................................4

Clinical Studies. .......................................................................................................................................................................18

Educational Studies.................................................................................................................................................................36

Clinical Case-Based Reports..................................................................................................................................................38

Simulation and Technology.....................................................................................................................................................66

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Biomedical Studies

01 An Assessment of Mental Health Outcomes During the Covid-19 Pandemic

Edward Magalhaes, PhD, LPC, Alexis Stoner, PhD, MPH, Savannah Grandy, OMS IV, Joshua Palmer, OMS IV, MPH, Robert Schranze, OMS IV

Edward Via College of Osteopathic Medicine, Carolinas and Virginia Campuses

Methods: This IRB approved research project is a cross-sectional study using a survey methodology approach to describe the impact of the COVID-19 pandemic on mental health outcomes. The survey was an anonymous tool developed by using validated mental health screeners, along with basic demographic information. It included a 9-item Patient Health Questionnaire (PHQ-9) to assess depression, a 7-item Generalized Anxiety Disorder scale (GAD- 7) to assess anxiety, and a 22-item Impact of Event Scale Revised (IES-R) to assess stress. The survey is a state stratified survey distributed to adults 18 years of age or older throughout the following states: Massachusetts, New York, New Jersey, Pennsylvania, Ohio, Virginia, North Carolina, South Carolina, Florida, Alabama, Louisiana, California, and Washington. Distribution was completed using VCOM’s clinical sites, hospital systems, various medical associations, community health and outreach organizations, and social media platforms.

Objective: A total of 1356 participants completed the survey. PHQ-9 levels differed significantly (p<.05) according to age, gender, and education level. Reported GAD-7 levels differed significantly (p<.05) according to age, gender, education level, and marital status. IES-R levels significantly differed (p<.05) according to age, gender, education, and marital status. There was a significant difference between GAD-7 level reported and IES-R level and if someone was a healthcare worker or non-healthcare workers (p=0.02 and p=0.028). IES-R levels significantly differed (p<.05) among types of healthcare workers with over half (54.1%) of those who reported severe levels identified as nurses. There was a significant difference in reported PHQ-9 levels and GAD-7 levels among those who had a prior mental health diagnosis prior to the pandemic (p<.05) with those with higher levels reporting no prior diagnosis. While having a child in school was not a significant contributor to guardian reported differences in GAD-7 levels, anxiety

levels reported did significantly differ according to level of involvement in their children’s school (p=0.037). Where responders lived or what phase of the COVID-19 pandemic did not demonstrate any significance (p>0.05). Conclusion: This study indicates that different risk factors are significantly associated with reported mental health outcomes. Overall, during the COVID-19 pandemic, non-healthcare workers reported higher levels of stress and anxiety compared to healthcare workers. However, nurses were more likely to report stress during this pandemic compared to other health care workers. Clinically, this study helps provide better insight into who might be at greater risk of developing adverse mental health outcomes during future pandemics.

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An Assessment of Mental Health Outcomes During the COVID-19 Pandemic Edward Magalhaes, PhD., LPC; Alexis Stoner, PhD., MPH; Savannah Grandy, OMS-IV; Josh Palmer, MPH, OMS-IV; Robert Schranze, OMS-IV; Edward Via College of Osteopathic Medicine - Carolina Campus (Spartanburg, SC) and Virginia Campus (Blacksburg, VA) Abstract Results Results

BIOM-1

There have been various studies evaluating the psychological and behavioral response to disasters and pandemics among healthcare workers. Most recently, researchers in Wuhan found that a considerable proportion of healthcare workers reported increased depression, anxiety, insomnia, and stress during the peak of the COVID-19 pandemic in China (Lai et al, 2020). Pandemic healthcare workers are often at increased risk for stress and anxiety due to unclear/changing policies, illness stigma, and altered home/work boundaries (Morganstein et al, 2020). This study sought to assess the impact of the COVID-19 pandemic on mental health outcomes (anxiety, depression, and stress) of a sample of individuals 18 and older across 13 states. We utilized an anonymous online survey, which collected demographical information and utilized the GAD-7, PHQ-9, and IES-R to help assess levels of anxiety, depression, and stress among participants. A total of 1356 participants completed the survey, with significant levels (p<.05) of difference seen across age, gender, educational level, and marital status. Prior diagnosis and access to telehealth also showed to have a significant impact (p<.05) on psychological distress. Overall, this study has helped to identify individuals who may be at greater risk for mental health outcomes due to the COVID-19 pandemic. This is a cross-sectional study using a survey methodology approach to describe the impact of the COVID-19 pandemic on mental health. This anonymous survey included a 7-item Generalized Anxiety Disorder scale (GAD-7) to assess anxiety, a 9- item Patient Health Questionnaire (PHQ-9) to assess depression, and a 22-item Impact of Event Scale Revised (IES-R) to assess stress. The survey was distributed to adults 18 years of age or older throughout the following states: Massachusetts, New York, New Jersey, Pennsylvania, Ohio, Virginia, North Carolina, South Carolina, Florida, Alabama, Louisiana, California, and Washington. Distribution was employed using VCOM’s clinical sites, various hospital systems, medical associations, community health and outreach organizations, and social media platforms. Analyses included descriptive statistics, chi square, and the fisher exact to test for significance (p<.05) between groups. Methods

This study indicates that specific risk factors may be significantly associated with reported mental health outcomes during the COVID-19 pandemic. For instance, nurses (who are identified as a target population that should receive early and often mental health screenings), reported to have the highest levels of anxiety among healthcare workers. Similarly, our study aligns with previous research that identified parents who are heavily involved with school to be an at-risk population for increased stress and anxiety during pandemics (Morganstein et al, 2020). This study may provide better insight into who may be at greater risk of developing adverse mental health outcomes during future pandemics. As the pandemic continues to evolve, it is important to allocate mental health resources to populations, such as those discussed above, that may be at a greater risk for adverse mental health outcomes. We feel that our study can be used along with others to further identify these specific populations and the importance of mental health services when access is limited. (p<.05) amongst age, gender, and education level of respondents. In Table 4 , IES-R levels significantly differed (p<.05) according to age, gender, education, and marital status. Additionally, there was a significant difference between healthcare and non-healthcare workers levels with regards to their PHQ-9 and IES-R levels (p=0.02 and p=0.028). IES-R levels significantly differed (p<.05) among types of healthcare workers with over half (54.1%) of those who reported severe levels identifying as nurses. There was a significant difference in reported PHQ-9 and GAD-7 levels among those who had a prior mental health diagnosis before the pandemic (p<.05). While having a child in school was not a significant contributor to differences in GAD-7 levels, anxiety levels significantly differed according to level of involvement in their children’s school (p=0.037). Geographical location had no significance on anxiety, depression, and stress levels. Conclusions A total of 1356 participants completed the survey. As seen in Table 2 , GAD-7 levels differed significantly (p<.05) according to age, gender, education level, and marital status. PHQ-9 levels in Table 3 demonstrate a significant difference

Table 2: GAD-7

Table 1: Demographics

Table 3: PHQ-9

Table 4: IES-R

References

1. Lai, J., Ma, S., Wang, Y., Cai, Z., Hu, J., Wei, N., . . . Hu, S. (2020). Factors Associated With Mental Health Outcomes Among Health Care Workers Exposed to Coronavirus Disease 2019. JAMA Network Open, 3 (3). doi:10.1001/jamanetworkopen.2020.3976 2. Morganstein, J. C., & Ursano, R. J. (2020). Ecological Disasters and Mental Health: Causes, Consequences, and Interventions. Frontiers in Psychiatry, 11 . doi:10.3389/fpsyt.2020.00001 3. Kohrt, B., & Mendenhall, E. (2016). Global mental health: Anthropological perspectives . London: Routledge. 4. Ursano, R., Raphael, B., Weisaeth, L., & Fullerton, C. S. (2017). Textbook of disaster psychiatry . Cambridge: Cambridge University Press.

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Biomedical Studies

02 Predicting the Effects of Oxidative Stress in Disease Phenotypes

Elizabeth Lawson, OMSI, Nicholas Kinney, PhD

Edward Via College of Osteopathic Medicine-Carolinas

Abstract: Glutathione is an important metabolite related to resisting cell damage by reactive oxygen species, as well as other harmful reactants. Maintaining an appropriate GSH/GSSG ratio is key to avoiding cell damage and death considering a wide variety of diseases. Response to oxidative stress, and therefore appropriate glutathione response, is altered in various chronic diseases. Here we use gene expression data (RNAseq) to repurpose a published model of glutathione metabolism and predict effects of oxidative stress in medulloblastoma, the most common pediatric brain malignancy. Future work will expand the investigation to well-established chronic diseases such as diabetes and autism. There is also potential to link the investigation to the ongoing COVID-19 pandemic, as it is hypothesized that healthy glutathione levels could offset inflammatory effects of the virus.

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Predicting the Effects of Oxidative Stress on Disease Phenotypes Elizabeth Lawson OMS-I, B.S., Nicholas Kinney, Ph.D. Edward Via College of Osteopathic Medicine-Carolinas Campus, Spartanburg, SC 29303 .

BIOM-2

Abstract

Results

Conclusions

Figures 3 & 4

Future Directions: Analyze RNAseq databases for the disease phenotypes known to be affected by oxidative stress: 1. Diabetes 2. Down Syndrome 3. Autism 4. COVID-19 5. Breast cancer • Normalize gene expression data heterogeneous experiments: RNAseq data for healthy and disease cohorts. • Explore Human Variome Project and identify relevant RNAseq datasets • Investigate the effects of fasting and input fluctuations in diabetes • Extend the approach to mathematical models for other metabolic pathways.

Glutathione is an important metabolite related to resisting cell damage by reactive oxygen species, as well as other harmful reactants. Maintaining an appropriate GSH/GSSG ratio is key to avoiding cell damage and death considering a wide variety of diseases. Response to oxidative stress, and therefore appropriate glutathione response, is altered in various chronic diseases. Here we use gene expression data (RNAseq) to repurpose a published model of glutathione metabolism and predict effects of oxidative stress in medulloblastoma, the most common pediatric brain malignancy. Future work will expand the investigation to well- established chronic diseases such as diabetes and autism. There is also potential to link the investigation to the ongoing COVID-19 pandemic, as it is hypothesized that healthy glutathione levels could offset inflammatory effects of the virus.

Preliminary results suggest differences in response to oxidative stress in medulloblastoma patients compared to healthy controls. In particular, reduced concentration of blood cysteine is seen in the medulloblastoma. Further investigations will pursue this – and similar results – as potential companion diagnostics and prognostics. Blood glutathione is robust to amino acid changes in simulated healthy and medulloblastoma cohorts; specifically, both cohorts demonstrated similar response to fasting conditions and input fluctuations. Future work will investigate the effects of fasting and input fluctuations in more relevant cohorts, i.e., diabetes.

Introduction or Methods

Figure 1 . Glutathione metabolism schematic

References

Figure 2 .

1. Reed, Michael C., et al. "A mathematical model of glutathione metabolism." Theoretical biology and medical modelling 5.1 (2008): 1-16. 2.Lappalainen, Tuuli, et al. "Transcriptome and genome sequencing uncovers functional variation in humans." Nature 501.7468 (2013): 506-511. 3.Heath, Allison P., et al. "EPID-14. GABRIELLA MILLER KIDS FIRST DATA RESOURCE CENTER: COLLABORATIVE PLATFORMS FOR ACCELERATING RESEARCH IN PEDIATRIC CANCERS & STRUCTURAL BIRTH DEFECTS." Neuro-Oncology 22.Supplement_3 (2020): iii321-iii321 4. Pizzorno, J. (2014). Glutathione! IMCJ, 13. Retrieved March 7, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684116/ 5. Silvagno, F., Vernone, A., & Pescarmona, G. P. (n.d.). Antioxidants. doi:https://doi.org/10.3390/antiox9070624

+ 1 2 ,1 + 1 ,2 + 2 For most of the reactions, the original Reed model assumed Michaelis-Menten form with one substrate or random order Michaelis-Menten form with two substrates:

def Vmax(diseaseFPKM):

return 4500*(diseaseFPKM/normalFPKM)

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Biomedical Studies

03 The Long-Range Effect of the Anatomical Variation of the Left Common Carotid Artery Severe Left Internal Carotid Artery Atherosclerosis

Eshaan Zaveri, OMSII, Hunter Peden, MA, OMS II, Zachary Kline, OMSII, Michael Pavy, OMSIII, Artem Boyev, DO, Zoltan Hajdu, MD

Edward Via College of Osteopathic Medicine-Carolinas

Background: One of six deaths in the United States from cardiovascular diseases is due to stroke. With more than 795,000 people suffering from a stroke each year, this disease is the leading cause of long- term disability with a huge economic impact. The prevalence of stroke attributed to extracranial internal carotid artery (ICA) atherosclerosis is about 13.4 per 100,000 persons. We have previously reported that there is a strong correlation between the anatomical variations of the left common carotid artery (LCCA) origin and intimal thickening in this vascular segment. Hypothesis: Based on palpatory findings during anatomical dissections, we hypothesize that anatomical variations of the LCCA origin have a direct effect on the left ICA atherosclerosis. Methods: The carotid arteries of 34 anatomic cadavers were investigated in VCOM anatomy labs between 2018-2020. Of these, 17 cases presented

the anatomic variation of the LCCA originating from the brachiocephalic trunk (BT); the other 17 were arbitrarily selected controls where the LCCA originated from the aortic arch (AA). Measurements of the AA and the primary branches were taken in situ and these were used for 3D reconstruction of the arterial trees using the SimVascular software. Histological samples of ICAs were used for internal measurements and structural evaluation of the walls. Computational fluid dynamics (CFD) calculations were applied to evaluate the flow and shear stress in the ICAs. Results: The CFD calculations on the 3D-reconstructed arterial trees predicted that, beside the previously reported LCCA intimal thickening, serious flow disturbances exist at the level of the left ICA when the LCCA originates from the BT. Initial palpatory assessment indicated hardening of the left ICA walls in these cases. Sectioning of the

paraffin-embedded samples of ICA further indicated severe atherosclerosis on the left side. Internal measurements of the samples from the cases with the anatomical variation revealed a 37% reduction of the arterial lumen compared to the control group. Von Kossa staining showed evidence of severe calcification in all 17 cases with the anatomical variation, while only six ICAs showed low or moderate levels of calcium in the control group. Conclusion: We extended our previous observation regarding the anatomical variations and intimal thickening of the LCCA. An LCCA originating from the BT instead of the AA will have a long-range effect in the left ICA in the form of severe atherosclerosis. Early screening for anatomical variations in the carotid artery could identify a risk factor for ICA atherosclerosis and potentially reduce the incidence of strokes with timely intervention.

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THE LONG-RANGE EFFECT OF THE ANATOMICAL VARIATION OF THE LEFT COMMON CAROTID ARTERY: SEVERE LEFT INTERNAL CAROTID ARTERY ATHEROSCLEROSIS Eshaan Zaveri*, OMS-II; Hunter Peden*, MA, OMS-II; Zachary Kline, OMS-III; Michael Pavy, OMS-III; Artem Boyev, DO; Zoltan Hajdu, MD VCOM Carolinas Campus, Spartanburg, SC * equal contribution

Abstract

Results

Conclusions

Figure 1: Left common carotid artery originating from the brachiocephalic trunk (BT) and the carotid bifurcations

Background: One of six deaths in the United States from cardiovascular diseases is due to stroke. With more than 795,000 people suffering from a stroke each year, this disease is the leading cause of long term disability with a huge economic impact. The prevalence of stroke attributed to extracranial internal carotid artery (ICA) atherosclerosis is about 13.4 per 100,000 persons. We have previously reported that there is a strong correlation between the anatomical variations of the left common carotid artery (LCCA) origin and intimal thickening in this vascular segment. Hypothesis: Based on palpatory findings during anatomical dissections, we hypothesize that anatomical variations of the LCCA origin have a direct effect on the left ICA atherosclerosis. Methods: The carotid arteries of 34 anatomic cadavers were investigated in VCOM anatomy labs between 2018-2020. Of these, 17 cases presented the anatomic variation of the LCCA originating from the brachiocephalic trunk (BT); the other 17 were arbitrarily selected controls where the LCCA originated from the aortic arch (AA). Measurements of the AA and the primary branches were taken in situ and these were used for 3D reconstruction of the arterial trees using the SimVascular software. Histological samples of ICAs were used for internal measurements and structural evaluation of the walls. Computational fluid dynamics (CFD) calculations were applied to evaluate the flow and shear stress in the ICAs. Results: The CFD calculations on the 3D-reconstructed arterial trees predicted that, beside the previously reported LCCA intimal thickening, serious flow disturbances exist at the level of the left ICA when the LCCA originates from the BT. Initial palpatory assessment indicated hardening of the left ICA walls in these cases. Sectioning of the paraffin-embedded samples of ICA further indicated severe atherosclerosis on the left side. Internal measurements of the samples from the cases with the anatomical variation revealed a 37% reduction of the arterial lumen compared to the control group. Von Kossa staining showed evidence of severe calcification in all 17 cases with the anatomical variation, while only six ICAs showed low or moderate levels of calcium in the control group. Conclusion: We extended our previous observation regarding the anatomical variations and intimal thickening of the LCCA. An LCCA originating from the BT instead of the AA will have a long-range effect in the left ICA in the form of severe atherosclerosis. Early screening for anatomical variations in the carotid artery could identify a risk factor for ICA atherosclerosis and potentially reduce the incidence of strokes with timely intervention.

➢ In the VCOM Carolinas anatomy lab, 78 total cadavers were evaluated and 17 cases of the anatomical variation were found, where the left common carotid artery originates from the brachiocephalic trunk. This means that between the academic years 2018 – 2020 21.79% of cases presented with the variations, which is more than double of that described in the literature. ➢ In addition to the intimal thickening of the proximal LCCAs originating from the BT, massive atherosclerosis in the LICAs was also found in these cases. ➢ It seems that the mild fluid dynamics perturbation (intimal thickening) due to the anatomic variations of the LCCA are enhanced along the length of the LICA, resulting in calcium deposits in the arterial wall. ➢ Computational fluid dynamics calculations show that the changes in flow and shear stress caused by the anatomical variation of the LCCA origin are enhanced along the carotid tree, having a more severe outcome in the LICA. Overall, we believe that anatomical variation of the aortic arch arteries has much more impact on the hemodynamics than previously thought. Our results demonstrate the long-range negative effect of the anatomical variation of the left common carotid artery origin on the left internal carotid artery. Clinical importance: Based on our findings, around 20% of the US population could have an anatomic variation which enhances the intimal thickening of the LCCA and atherosclerosis of the LICA; atherosclerosis of the carotid arteries is responsible for about half of the cerebrovascular accidents is the US, and therefore, developing a screening method to detect the anatomical variation, and closely monitor the patients who screen positive, would have a huge socio-economic impact on our health care system. Challenges of the study: Our project was based on cadaveric material, meaning the lack of the blood flow with individual characteristics; every patient is different, making it very hard to model their individual cardiovascular parameters; our computational fluid dynamics model suggests that little changes in the flow pattern could significantly alter the changes caused by abnormal branching in both positive and negative direction. Future studies: ➢ we will keep collecting cadaveric samples from the VCOM anatomy labs, and keep improving our model; ➢ we will be performing an in vivo, ultrasonic study to detect potential differences in the flow pattern between the left and right common carotid arteries; ➢ we will investigate existing 3D computerized tomography data complemented by the patient’s history.

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Seventeen cases of the left common carotid artery (B, green arrow) originating from the brachio- cephalic trunk were identified and 17 matching cases of origination from the aortic arch (A, green arrow) were arbitrarily selected. A’ and B’ are magnifications of the highlighted areas of A and B. ICA – internal carotid artery; ECA – external carotid artery; CCA – common carotid artery. Dotted red lines show the level of the histological samples.

Figure 2: Histological examination of the left internal carotid artery and measurements

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Introduction and Methods

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Introduction Usually, in humans the left common carotid artery (LCCA) originates from the aortic arch (AA). However, variations of the LCCA have been described – most interestingly for us, in about 9% of the cases, the LCCA is branching off the brachiocephalic trunk (BT).

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References

Hematoxylin-eosin (HE) staining was used for general evaluation of the normal (A) and variation (B) samples. For detection of the calcium deposits, von Kossa staining was used (C-normal; D-variation), black is showing calcification. Measurements of the left carotid tree was performed before and after sample collection (E). No statistical significance is detected in BT circumference, left CCA circumference, left CCA length and left ICA internal circumference between the normal (n=17) and variation samples (n=17). However, the left ICA circumference and left ICA wall thickness in the variation samples shows significant (p≤0.05) increase and decrease, respectively, compared to the normal samples.

1. Layton KF, Kallmes DF, Cloft HJ, Lindell EP, Cox VS. Bovine aortic arch variants in humans: clarification of a common misnomer . Am j Neuroradiol . 2006. 27:1541-1542. 2. Benjamin EJ et al. Heart disease and stroke statistics 2018 update: a report from the American Heart Association. Circulation. 2018; 137: e67-e492. 3. Marulanda-Londone, E and Chaturvedi S. Stroke due to large vessel atherosclerosis. Neurology Clinical Practice . 2016; 6: 252-258. 4. Flaherty ML et al. Carotid Artery Stenosis as a Cause of Stroke. Neuroepidemiology . 2013; 40: 36-41. 5. Ross R. Atherosclerosis – an inflammatory disease. The New England Journal of Medicine . 1999; 340 (2): 115- 126. 6. Nakashima Y et al . Early atherosclerosis in humans: role of diffuse intimal thickening and extracellular matrix proteoglycans. Cardiovascular Research . 2008; 79: 14-23. 7. Gimbrone MA et al . Vascular endothelium, hemodynamics, and the pathobiology of atherosclerosis. Cardiovascular Pathology . 2013; 22: 9-15 8. Jakanani GC and Adair W . Frequency in variation of aortic arch anatomy as depicted on multidetector CT. Clinical Radiology . 2010; 65: 481-487

9%

70% Layton et al., Am J Neuroradiol, 2006

The goal of this work was to evaluate the LICA from normal and variation cases by histology and computed fluid dynamics. Material and Methods Based on literature data, intimal thickening is the first step in atherosclerosis and happens upon endothelial activation. It is known that blood vessel endothelium is sensitive to shear stress; this is actually one of the major endothelial activators. Previously we reported that we found a histological and computed fluid dynamics correlation between the intimal thickening and the LCCA originating from the BT. During these dissections, we also observed that the left internal carotid arteries (LICAs) branching off the LCCAs originating from the BTs felt firmer on palpation than those from LCCAs originating from the AA. We hypothesized that the LCCA origin has direct effect on the LICA atherosclerosis. ➢ The aortic arches with the left carotid tree were measured after curricular dissections; ➢ Then, 0.5 mm ring samples were collected 1 cm above the origin of the LICA; these were processed for paraffin embedding and 5 µm sectioning; ➢ After staining the samples with hematoxylin-eosin, ImageJ was used to measure the outer and inner perimeters (circumferences) and measurements were adjusted to counteract the shrinkage during histological processing; ➢ Using the pre- and post-processing measurements, the carotid trees were 3D-reconstructed in SimVascular; the shear stress was calculated by the software at 1 cm above the origin of the LICA; ➢ von Kossa staining was used to detect calcium deposits (as sign of atherosclerosis) in the wall of the LICAs.

Figure 3: Computational fluid dynamics calculations show decreased shear stress in the left internal carotid arteries when the left CCA is originating from the brachiocephalic trunk

variation

normal

Acknowledgements

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This project was funded by the VCOM Research Eureka Accelerator Program awarded to ZH. We would like to thank our student and faculty colleagues for the initial dissections in the anatomy labs.

The average shear stress (measured in dynes/cm 2 ) at the beginning of the left ICA (along the endothelium, 1 cm above the origin) was recorded in both normal (n=17) and variation (n=17) cases. In the variation cases (where the left CCA is originating from the BT), the shear stress is significantly lower (p≤0.05) than in the normal cases (left CCA is originating from the aortic arch).

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Biomedical Studies

04 A Novel Bipyridone Cisplatin Analog Exhibits Enhanced Anticancer Activity in Preclinical Models of Upper Gastrointestinal Cancers

Samhita Bapat, PhD, Vikas Sehdev, PhD, B. Pharm, Noah Kosnik, OMSII, Hannah Fischer, OMSII, Patrick D. Ellis Fisher, PhD, and Byron Bennett, PhD

Edward Via College of Osteopathic Medicine-Carolinas, Long Island University, Idaho State University

Background: Upper Gastrointestinal Cancers (UGCs) respond poorly to conventional chemotherapy due to variable P53 status and overactive mechanisms that mediate drug resistance. Platinum- based compounds like Cisplatin (CDDP) are frequently used for treatment of UGCs. However, clinical use of CDDP is limited due to development of drug resistance and dose limiting side-effects resulting in nausea, vomiting, neutropenia, thrombocytopenia and renal toxicity. Hypothesis/Goal of Study: In this study, we investigated the anticancer potency of a novel CDDP derivative (dichloro [4,40-bis(4,4,4-trifluorobutyl)-2,20- bipyridine] platinum) (DCTF-CDDP) and compared it to CDDP in P53 wild type (P53 WT) and mutant (P53 MT) models of UGCs.

Methods and Results: For this study, we evaluated the effect of CDDP or its derivative (DCTF- CDDP) on AGS (P53 WT) and FLO-1 (P53 MT) UGC cell lines. After treatment with CDDP or DCTF-CDDP; MTT cell viability, clonogenic cell survival, cell cycle, Annexin V staining, and western blot analyses were carried out to measure cell viability, cell survival, cell cycle progression, cell death, and expression of apoptotic proteins, respectively, in AGS and FLO-1 UGC cell lines. Our in vitro data indicate that DCTF-CDDP is significantly more potent at inhibiting cell viability and cell survival of P53 wild type and mutant UGC cells. The cell cycle, cell death, and pro-apoptotic protein expression analyses further indicate that DCTF-CDDP is much more effective in suppressing cell cycle

progression and inducing apoptotic markers in P53 wild type and mutant UGC cells. Conclusions: Overall, our study suggests that DCTF-CDDP could be an effective CDDP derivative that can be used to achieve better therapeutic outcome at lower doses and toxic side effects. Future studies characterizing the anticancer and toxicological effects in vivo are warranted to further develop this promising analogue.

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A NOVEL BIPYRIDONE CISPLATIN ANALOG EXHIBITS ENHANCED ANTICANCER ACTIVITY IN PRECLINICAL MODELS OF UPPER GASTROINTESTINAL CANCERS Samhita Bapat 1 , Vikas Sehdev 2 , Noah Kosnik 2 , Hannah Fischer 2 , Patrick D. Ellis Fisher 2 , and Byron Bennett 3 1 Division of Pharmaceutical Sciences, A & M College of Pharmacy and Health Sciences, Long Island University, Brooklyn, NY 2 Department of Biomedical Sciences, Edward Via College of Osteopathic Medicine, Spartanburg, SC 29303 3 Chemistry Department, Idaho State University, Pocatello, ID

Introduction Upper Gastrointestinal Cancers (UGCs) respond poorly to conventional chemotherapy due to variable P53 status and overactive mechanisms that mediate drug resistance. 1 Platinum-based compounds like Cisplatin (CDDP) are frequently used for treatment of UGCs. 2 However, clinical use of CDDP is limited due to development of drug resistance and dose limiting side-effects resulting in nausea, vomiting, neutropenia, thrombocytopenia and renal toxicity. In this study, we investigated the anticancer potency of a novel CDDP derivative (dichloro [4,40-bis(4,4,4- trifluorobutyl)-2,20-bipyridine] platinum) (DCTF-CDDP) and compared it to CDDP in P53 wild type (P53 WT) and mutant (P53 MT) models of UGCs. Goal of the Study For this study, we evaluated the effect of CDDP or its derivative (DCTF-CDDP) on AGS (P53 WT) and FLO-1 (P53 MT) UGC cell lines. After treatment with CDDP or DCTF-CDDP; MTT cell viability, clonogenic cell survival, cell cycle, Annexin V staining, and western blot analyses were carried out to measure cell viability, cell survival, cell cycle progression, cell death, and expression of apoptotic proteins, respectively, in AGS and FLO-1 UGC cell lines. Our in vitro data indicate that DCTF-CDDP is significantly more potent at inhibiting cell viability and cell survival of P53 wild type and mutant UGC cells. The cell cycle, cell death, and pro-apoptotic protein expression analyses further indicate that DCTF-CDDP is much more effective in suppressing cell cycle progression and inducing apoptotic markers in P53 wild type and mutant UGC cells. Methods and Results

Results

Figure. 2: DCTF-CDDP treatment is significantly more effective than CDDP at inducing SubG1 phase and altering cell cycle progression in UGC cells:

Figure 4. DCTF-CDDP is significantly more effective than CDDP at inducing apoptotic markers in P53 wild type and mutant UGC cells:

Figure 1: DCTF-CDDP is significantly more potent than CDDP against P53 wild type and mutant UGC cells: .

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Conclusions Overall, our study suggests that DCTF-CDDP could be an effective CDDP derivative that can be used to achieve better therapeutic outcome at lower doses and toxic side effects. Future studies characterizing the anticancer and toxicological effects in vivo are warranted to further develop this promising analogue. Fig. 3: (A & B) UGC cells were treated with CDDP or DCTF-CDDP for 8 hrs. and protein expression was evaluated. The data indicate that, compared to CDDP, treatment with DCTF-CDDP mediated enhanced induction of apoptotic proteins in UGC cells. Veh, Vehicle; CDDP, Cisplatin; and DCTF- CDDP, Cisplatin Derivative.

Fig 2: (A & B) The cell cycle data indicate that, compared to CDDP, DCTF-CDDP treatment mediates higher induction of SubG1 phase and suppression of G1 and G2-M phases, respectively, in UGC cells. Veh, Vehicle; CDDP, Cisplatin; and DCTF-CDDP, Cisplatin Derivative. Figure 3. DCTF-CDDP treatment is significantly more effective than CDDP at inducing apoptosis in P53 wild type and mutant UGC cells:

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References

1.Hohenberger P, Gretschel S. Gastric cancer. Lancet. 2003;362:305-315. 2.Grunberger B, Raderer M, Schmidinger M, Hejna M. Palliative chemotherapy for recurrent and metastatic esophageal cancer. Anticancer Res 2007;27:2705 – 14.

Fig. 1: UGC cell lines were treated with various concentrations (0.1-20 µM) of CDDP or DCTF-CDDP. (A & B) The cell viability assay data indicate that a 72 hr. treatment with DCTF-CDDP is significantly more potent and efficacious than that of CDDP. (C & D) The cell survival assay data shows that DCTF-CDDP is more effective than CDDP at suppressing the long term survival of UGC cells. Veh, Vehicle; CDDP, Cisplatin; and DCTF-CDDP, Cisplatin Derivative

Fig 2: (A & B) The FITC-Annexin V flow cytometry data shows that, compared to CDDP, 24 hr. treatment with DCTF-CDDP significantly enhances the percentage of cells in intermediate to late stages of apoptosis . Veh, Vehicle; CDDP, Cisplatin; and DCTF-CDDP, Cisplatin Derivative.

This study is supported by and carried out at the Department of Biomedical Sciences, Edward Via College of Osteopathic Medicine, Gibbs Cancer Center and Department of Chemistry, Idaho State University

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2 0 2 1 R e s e a r c h R e c o g n i t i o n D a y

Biomedical Studies

05 Exposure to the Environmental Endocrine Disruptor Tolylfluanid During Cerebral Brain Development Significantly Alters Cortical Neuron Gene Expression

Amar Patel, Ken Nguyen, Rachel Winstead, Olivia Mattner, Rebecca Beaudry, Katherine Baumgarner, Bradley Baumgarner, PhD2, and Stephan Brown, MD, PhD

Edward Via College of Osteopathic Medicine-Carolinas, Gibbs Cancer Institute, University of South Carolina Upstate

Introduction: Tolylfluanid (TF), (the active ingredient in agricultural pesticides) is an endocrine disruptor that has been shown to increase anaerobic glycolysis and reduce oxygen consumption in human cells. Both gene expression and environmental input are essential for normal brain development, and disruption of either can fundamentally alter neural outcomes [1]. Brain formation begins with the development of neural progenitor cells, which are derived from the ectodermal germ layer [2]. The ensuing period of fetal development extends through the end of gestation. During this time there is rapid growth and elaboration of both cortical and subcortical structures, including the rudiments of the major fiber pathways (Kostovic and Jovanov-Milosevic 2006). Similar to pluripotent stem cells, neural progenitors rely almost exclusively on anaerobic glycolysis to meet their energy needs [4]. The differentiation of neural progenitor cells into functional cortical neurons coincides with a transition from anaerobic glycolysis to oxidative phosphorylation [4-5]. Neural differentiation increases the expression of genes associated with

mitochondrial metabolism while simultaneously decreasing the expression of glycolysis-related genes [5]. TF is an environmental EDC that has been shown to reduce glucose oxidation in mammalian cells [10]. Chen et al. [10] discovered that TF reduced glucose oxidation in human adipocytes by directly inhibiting the mitochondrial pyruvate carrier (MPC). A previous investigation by Vacanti et al. [11] revealed that pharmacological inhibition (similar to TF) or genetic deletion of MPC significantly reduced glucose oxidation in human skeletal muscle cells, which increased mitochondrial fatty acid glutamine oxidation [11]. 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. Hypothesis: TF deceases glucose oxidation in developing neurons causing metabolic stress. This constant stress alters neuron metabolism leading to gene expression alterations.

We propose to determine whether TF exposure during central nervous system development could significantly alter fully differentiated human brain organoids. The central nervous system forms in the third week of fetal development and is derived from neuroectodermal germ tissue (1). In the brain, most of the glucose is oxidized to produce the vast amounts of ATP required to maintain cellular processes (2). The availability of energy to neuronal progenitor cells could limit the brain’s size and activity (3). Conclusions: 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 neurons and cortical) differently than other neural cell types. Metabolic studies showed reduces the glycolytic rate of fully differentiated cortical neurons under basal and compensatory (anaerobic) conditions when compared to vehicle treated cells.

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EXPOSURE TO THE ENVIRONMENTAL ENDOCRINE DISRUPTOR TOLYLFLUANID DURING CEREBRAL BRAIN DEVELOPMENT SIGNIFICANTLY ALTERS CORTICAL NEURON GENE EXPRESSION Amar Patel 1 , Ken Nguyen 1 , Rachel Winstead 1 , Olivia Mattner 1 , Rebecca Beaudry 1 , Katherine Baumgarner 1 , Bradley Baumgarner, PhD 2 and Stephan Brown MD, PhD 1 1 Gibbs/VCOM CAROLINAS, Spartanburg, SC 29303 2 USC Upstate, Spartanburg, SC 29303 Abstract Results Conclusions

BIOM-5

Table 2 .

Tolylfluanid (TF), (the active ingredient in agricultural pesticides) is an endocrine disruptor that has been shown to increase anaerobic glycolysis and reduce oxygen consumption in human cells. Both gene expression and environmental input are essential for normal brain development, and disruption of either can fundamentally alter neural outcomes [1]. Brain formation begins with the development of neural progenitor cells, which are derived from the ectodermal germ layer [2]. The ensuing period of fetal development extends through the end of gestation. During this time there is rapid growth and elaboration of both cortical and subcortical structures, including the rudiments of the major fiber pathways (Kostovic and Jovanov-Milosevic 2006). Similar to pluripotent stem cells, neural progenitors rely almost exclusively on anaerobic glycolysis to meet their energy needs [4]. The differentiation of neural progenitor cells into functional cortical neurons coincides with a transition from anaerobic glycolysis to oxidative phosphorylation [4-5]. Neural differentiation increases the expression of genes associated with mitochondrial metabolism while simultaneously decreasing the expression of glycolysis-related genes [5]. TF is an environmental EDC that has been shown to reduce glucose oxidation in mammalian cells 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. Organoids were also cryo-sectioned to determine protein expression and morphological discrepancies between the treated and non- treated groups. 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. Gene expression data via qPCR currently in being optimized. Introduction or Methods

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 reduces the glycolytic rate of fully differentiated cortical neurons under basal and compensatory (anaerobic) conditions when compared to vehicle treated cells (not shown). RNAseq analysis shows altered dopaminergic neuron development which correlated with the immunohistochemical data showing increase in TTF labeled neuron. These neurons further develop into dopamine producing neurons which brings to question, how does TF effect dopamine production? The data shown in this presentation suggest TF is mediating metabolic stress causing structural cellular changes as well as gene alterations. Further evidence of metabolic manipulation leading to altered developmental patterns. These environmental factors play a significant role in effecting normal human physiology and can ultimately alter biological thresholds for disease.

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

Control

Vimentin

DAPI

Pax-6

Merge

TF-treated

Sox-6

DAPI

Ki67

Merge

References

Sox-6

DAPI

Ki67

Merge

TF-treated

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.

TF-treated neurons showed a decrease in vimentin (neuronal scaffolding protein) and cell proliferation

Table 3 .

Gene Ontology

Table 1 .

5 μ M TF

Control

.5 μ M TF

RNA seq data from the TF-treated cerebral organoids showed altered gene expression in genes related to aging and dopaminergic neuron differentiation. Further analysis of the expression data linked many of the gene altered by TF are linked to forebrain development. Ͳ

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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2 0 2 1 R e s e a r c h R e c o g n i t i o n D a y

Biomedical Studies

06 Effects of Adipor1 Agonists on Insulin-Independent Glucose Transport Pathways in Skeletal Muscle Cells

Joseph Metz, OMS-III, Marwan Mahmoud, OMS-II, Patrick Ellis Fisher, PhD

Edward Via College of Osteopathic Medicine-Carolinas, Gibbs Cancer Institute

Abstract: As of 2015, the CDC estimates that nearly 30.3 million Americans were living with Diabetes mellitus and another 84.1 million Americans were classified as pre-diabetic. With such alarming up- trends over previous years, further elucidation of insulin-independent pathways for glucose metabolism is critical. Adiponectin is a protein-derived hormone that is produced and secreted from adipocytes that appears to help regulate glucose metabolism. A systematic review demonstrated that higher levels of circulating adiponectin are associated with a lower risk of developing type 2 diabetes and levels were inversely related to insulin resistance (1). Developing adiponectin receptor agonists could be an important turning point in the treatment of type 2 diabetes. We are examining the effects of treating cultured human skeletal muscle cells (myocytes and myotubes) with the plant lignan matairesinol and its gut microbial metabolites enterolactone and enterodiol. Previous work has shown the binding and activation of the

adiponectin receptor 1 (AdipoR1) by matairesinol (2). We hypothesize that matairesinol, along with its metabolites, can stimulate the AdipoR1 receptor and induce the translocation of insulin-responsive glucose transporter type 4 (GLUT4) to the plasma membrane, resulting in increased glucose uptake and a potential restoration of insulin sensitivity via downstream pathways. In the present study, we show that matairesinol treatment induces increased glucose uptake in human skeletal muscle tubes, similar to insulin and adiponectin. We have grown primary skeletal myocytes and differentiated them into polynucleated myotubes. Treating these with insulin, adiponectin, or our test compounds, we examined glucose uptake in muscle cells using a luciferase-based glucose uptake assay. We are currently working to determine the mechanism of activation, hypothesized to be via the AMP-activated Protein Kinase (AMPK) signaling pathway.

Clinically, these studies could lay a foundation for future work creating small molecule compounds or agonistic antibodies that could be used to decrease the use of insulin in type 2 diabetic patients. Our results could inspire new first-line medications for diabetic patients, due to the potential cardiovascular benefits associated with AdipoR1 activation and mechanistically solving the pathophysiological problem in type 2 diabetes, insulin insensitivity. 1. Shanshan L, Shin HJ, Ding EL, et al. Adiponectin levels and risk of type 2 diabetes: a systematic review and metanalysis. JAMA. 2009;302(2):179-188 2. Sun Y, Zang Z, Zhong L, et al. Identification of adiponectin receptor agonist utilizing a fluorescence polarization based high throughput assay. PLoS ONE. 2013;8(5):e63354

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