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Item Exploring a Transcriptome-forward Approach for Genetic Evaluation in Thoracic Aortopathy(2023-07-28) Stanley, Katherine; Landis, BenjaminBackground/Objective: Thoracic aortic aneurysm (TAA) is an aortopathy that predisposes to aortic dissection and rupture. Transcriptomic analysis has diagnostic utility in other diseases, but this has not been developed for TAA. We therefore sought to implement the Genome Analysis Toolkit (GATK) as a tool for high-throughput detection of novel variants in RNA sequencing (RNA-seq) data from patients with TAA. Methods: Human proximal aortic tissue samples from 63 TAA patients and 15 controls were used to establish primary smooth muscle cell (SMC) lines in culture. Total RNA was extracted at early passage; RNA-seq was performed using 150 base pair paired-end reads. Variants were called from RNA-seq reads using the GATK’s HaplotypeCaller tool. The MarkDuplicates tool tagged duplicate reads, but these were retained for variant analysis. Singletons were defined as heterozygous variants unique to one sample in the overall group. Results: The RNA-seq pipeline was validated by confirming known mutations in 2 patients, inspecting reads directly in the Integrative Genomics Viewer (IGV), and genotyping for a common single nucleotide polymorphism. Across all 78 samples, the GATK pipeline identified 4,823,462 variant sites, 236,495 of which were in coding regions. Variant sites were filtered for those located within the coding regions of 31 genes curated for known evidence of aortic disease causality. Among the 511 coding variant sites in these genes, 330 single nucleotide variants and 33 insertions/deletions were identified as singletons. Singleton variant calls were evaluated for quality and annotated in order to predict their likelihood of pathogenicity, thus far identifying strong candidate causal variants in FBN1, TGFBR1, TGFB3, GATA4, and HEY2. Conclusion: Application of the GATK RNA-seq pipeline expands our prior IGV analysis, further demonstrating the potential diagnostic utility of transcriptomic analysis of aortic SMCs in TAA. Scientific Impact: Identifying novel aortopathy-associated variants could improve future disease detection and prediction of severity. Presentation recording available online: https://purl.dlib.indiana.edu/iudl/media/059c18mr8hItem Student Perceptions of Two Preclinical Medical School Exam Feedback Approaches(2023-07-28) Gasaway, Elsie; O'Loughlin, Valerie DeanBACKGROUND: In medical school, where learning an abundance of information in a short period of time is required, it is necessary that learners receive valuable feedback after summative assessments (i.e., unit exams). First-year medical students at Indiana University School of Medicine (IUSM) begin their education with a course titled Human Structure (HS), followed by Molecules to Cells and Tissues (MCT). Both courses provided different formats for exam feedback, resulting in anecdotal comments about preference and utility of feedback. This study uses qualitative research methods to examine IUSM-Bloomington students’ perceptions of exam feedback formats with respect to their utility and applicability. METHODS: Five, second-year IUSM-Bloomington medical students participated in a focus group to discuss their utilization and perceived usefulness of HS and MCT exam feedback. A thematic analysis was used to interpret data from the focus group. This study was deemed exempt by the IU-IRB (19409). RESULTS: The thematic analysis revealed that students’ discussions fell into three categories: logistics, utilization, and mentality. These categories were further broken into themes and subthemes, revealing 13 unique codes. Students spent a substantial amount of time discussing logistics of exam feedback. Barriers to utilization of exam feedback included a lack of information provided at the feedback sessions and a lack of time in the schedule available for feedback sessions. Students preferred MCT approach to exam feedback, however they recognized HS course logistics may prevent similar adoption. Students had small suggestions on how to improve feedback in both courses. CONCLUSIONS/IMPLICATIONS: The data suggest students would benefit from small changes in how first-year medical school courses at IUSM provide exam feedback. Improvements could include extending the time of exam review sessions, incorporating a discussion on commonly missed exam concepts, providing answer explanations for incorrect and correct answers, and transitioning statewide reviews to be campus led.Item HB007 Administration Inhibits LN-229 and Patient-Derived Neurospheroid Glioblastoma Cell Growth With the Degradation of SUMO1 and Cell Cycle Regulator CDK4(2023-07-27) Dougherty, Carson; Wohlford, Reagan; Jung, Sunghan; Hao, ChunhaiBackground and Hypothesis: Glioblastoma is the most common and malignant brain cancer and there is no effective therapy currently available to patients with this malignancy. Small ubiquitin-related modifier 1 (SUMO1) is a key regulator of cancer cell proliferation through its role in its modification of cellular proteins in various human cancers, especially glioblastoma. Degradation of SUMO1 through small molecule degrader, HB007, has been shown to inhibit growth in cancer cell lines and xenografts. Here, we hypothesize that HB007 can inhibit the glioblastoma cell growth through degradation of SUMO1 protein in glioblastoma cells and the cancer stem cell enriched neurospheres. Experimental Design: LN-229 glioblastoma cell viability was measured in response to increasing concentrations of HB007. LN-229 and patient-derived neurospheroid glioblastoma cells were cultured and seeded in 4 different plates at 1000 cells/ml concentrations before being treated with HB007 at increasing concentrations encircling the previously described IC50. Cells were then subjected to a SUMO lysis buffer and analyzed via western blot with antibodies specific to SUMO1, CDK4, and actin. Results: HB007 treated LN-229 cells exhibited an IC50 of 1.470µM. Western blot analysis confirmed the dose dependent reduction in SUMO-1-ylated proteins in HB007 treated cells. A reduction in CDK4 confirmed that cell progression is halted in a dose dependent manner in LN-229 and patient-derived neurospheroid glioblastoma cells when treated with HB007. Specificity of HB007 is towards SUMO1 with no nonspecific degradation of SUMO2/3. Conclusion: The cell growth of LN-229 and patient-derived neurospheroid glioblastoma cells was confirmed, through western blot, to be inhibited in a dose dependent manner by HB007. These results further establish the therapeutic potential of SUMO1 degraders as a novel anticancer drug for glioblastoma therapy. In the future, it is hoped that the bioavailability, potency, and blood brain barrier permeability can be improved to make this drug a potential treatment for patients. Presentation recording available online: https://purl.dlib.indiana.edu/iudl/media/k71n501c5gItem Rate of Fusion Using Novel Synthetic Bone Graft Mixed with Allograft Stem Cell Product in Lumbar Interbody Fusions(2023-07-28) Bartrom, Sam; Smith, MicahBackground: Over 400,000 spine fusions are performed in the United States annually with 75% involving the lumbar region.1 It is the indicated treatment of many chronic orthopedic conditions that fail conservative management. There are numerous surgical approaches; however, common to all is the removal of the intervertebral disc and the insertion of a bone graft which promotes arthrodesis. Iliac crest autografts are regarded as the “gold standard” bone graft material for lumbar fusions. SurGenTec has developed an allogeneic graft, Vimax®, that has illustrated similar qualities to autograft material. This study aims to observe how the mixture of Vimax® and OsteoFlo® will affect the rate of arthrodesis in patients undergoing lumbar fusions. Methods: A retrospective chart review on patients that received a lumbar interbody fusion using Vimax® and Osteoflo® between May 2021 to July 2022 was performed. Demographic information was obtained. Pre-operative radiographs were measured in addition to 2-week, 6-week, 3-month, 6-month, and 1-year post-operative radiographs. Post-operative radiographs were also assigned a Bridwell fusion grade. Results: 96 patients receiving 146 fusions were studied. 23.3% of fusions recorded a Bridwell Grade I (complete fusion) at 3-months post-op and 92.8% at 1-year post-op. The comorbidities of heart disease and hypertension displayed delayed arthrodesis at 6 months and 3 months post-op, respectively. However, both had over 90% Bridwell Grade 1 at 1-year post-op. No significant difference was noted for surgical approach or other comorbidities such as diabetes mellitus, osteoporosis, and renal disease. Segmental lordosis, pelvic tilt, pelvic incidence, and PI-LL mismatch had no significant difference post-op compared to pre-op. Anterior and posterior disc height and foraminal height increased significantly while spondylolisthesis decreased significantly post-operatively. Lumbar lordosis displayed a significant decrease at 2-weeks post-op compared to pre-op, but not at any other timepoint. Conclusions: Vimax® and OsteoFlo® demonstrated effectiveness as an iliac crest autograft substitute.Item Underrepresentation in Cardiovascular Disease Clinical Trials(2023-07-28) Khan, Uzair; Raman, SubhaBackground: When it comes to inequalities in medicine, there are countless avenues through which barriers are placed that lead to worse health outcomes for marginalized groups. One inequality that isn't mentioned nearly as often is how difficult it is for marginalized groups to participate in clinical trials. As such, the results from these studies are most broadly applicable only to white men, the most common demographic to participate, as opposed to people from all races and backgrounds. The project for this summer is the first step in a longitudinal project meant to identify and address the barriers that preclude marginalized groups from participating. Methods: There are three steps to researching this underrepresentation. The first is a literature review. In this step, we searched for any existing literature about the presence of underrepresentation in cardiovascular disease (CVD) clinical trials as well as in other fields. From this literature, the results were analyzed, to ensure the validity of conducting our study, as well as the methodology of the studies, to ensure our own research will follow the established methods of other successful studies. The second is to interview experts in the field, such as others who research this topic as well as cardiologists and community leaders, to determine what barriers and solutions they believe decrease participation of POC patients. The final step is to conduct our own clinical trial that addresses those barriers by applying solutions discerned from the previous steps and creates a successful methodology to properly include underrepresented patients in the future. For this summer, we made it to the second step. Results: The literature review found that there indeed exists a discrepancy in participation of POC patients in clinical trials. Specifically, while the percent of overall nonwhite participation has increased, the number of black participants has remained the same since January of 2001, indicating that while there has been improvement in this inequality, there is still a need to improve recruitment for black participants specifically (Tahhan et al., 2020). Findings from the literature review also indicated potential barriers for underrepresented groups, including mistrust of clinical trials as a whole, time/resources constraints, and just a general lack of awareness of the existence of clinical trials (Clark et al., 2019). While determining solutions is part of the future directions of this study, Clark, et al. seems to suggest that the best solutions will address the interface between patients, physicians, and clinical trial teams on all 3 levels. One final conclusion from the literature review is the fact that there was “no price to pay for achieving diversity” (Batchelor et al., 2021). In looking at studies all over the country from 2 US National Coronary Stent Registries, a higher focus on increasing racial minority groups and women did not negatively influence research site performance, and possibly could lead to lower protocol deviation rates. While the interview portion of the study has yet to be completed, we have interviewed Francine Epperson, a researcher who has investigated barriers to black patient participation in Alzheimer’s clinical trials. Her study concluded that one facilitator to increase black participation is a “return of results to make informed decisions about their health” (Eliacin et al., 2023). Too often, clinical trials take results from participants and apply them elsewhere, with their participants being none the wiser as to what they helped accomplish. By applying and sharing the results of the studies to the communities that are recruited, we establish trust and a continued partnership, something that is desperately needed to help address the underrepresentation in cardiovascular disease clinical trials. Future Directions: The next steps in this project include conducting expert interviews, including cardiologists but also public health and community leaders, to determine barriers to participation and potential solutions. The final step involves consolidating the first two steps into solutions that can be used to conduct a clinical trial that minimizes barriers and increases rates of nonwhite participation. Citations: Batchelor, W. B., Damluji, A. A., Yong, C., Fiuzat, M., Barnett, S. D., Kandzari, D. E., Sherwood, M. W., Epps, K. C., Tehrani, B. N., Allocco, D. J., Meredith, I. T., Lindenfeld, J., O'Connor, C. M., & Mehran, R. (2021). Does study subject diversity influence cardiology research site performance?: Insights from 2 U.S. National Coronary Stent Registries. Am Heart J, 236, 37-48. https://doi.org/10.1016/j.ahj.2021.02.003 Clark, L. T., Watkins, L., Pina, I. L., Elmer, M., Akinboboye, O., Gorham, M., Jamerson, B., McCullough, C., Pierre, C., Polis, A. B., Puckrein, G., & Regnante, J. M. (2019). Increasing Diversity in Clinical Trials: Overcoming Critical Barriers. Curr Probl Cardiol, 44(5), 148-172. https://doi.org/10.1016/j.cpcardiol.2018.11.002 Eliacin, J., Polsinelli, A. J., Epperson, F., Gao, S., Van Heiden, S., Westmoreland, G., Richards, R., Richards, M., Campbell, C., Hendrie, H., Risacher, S. L., Saykin, A. J., & Wang, S. (2023). Barriers and facilitators to participating in Alzheimer's disease biomarker research in black and white older adults. Alzheimer's & Dementia: Translational Research & Clinical Interventions, 9(2). https://doi.org/10.1002/trc2.12399 Tahhan, A. S., Vaduganathan, M., Greene, S. J., Alrohaibani, A., Raad, M., Gafeer, M., Mehran, R., Fonarow, G. C., Douglas, P. S., Bhatt, D. L., & Butler, J. (2020). Enrollment of Older Patients, Women, and Racial/Ethnic Minority Groups in Contemporary Acute Coronary Syndrome Clinical Trials. JAMA Cardiology, 5(6), 714. https://doi.org/10.1001/jamacardio.2020.0359Item USING BOTULINUM TOXIN FOR THE TREATMENT OF GASTROPARESIS (GP) FOR THE SELECTION OF PATIENTS WITH HIGHER CLINICAL SUCCESS, IMPROVED QUALITY OF LIFE (QOL), AND IMPROVED SOCIAL FUNCTIONING POST GASTRIC PERORAL ENDOSCOPIC MYOTOMY (GPOEM)(2023-07-28) Kais, Amr; Al-Haddad, MohammadBACKGROUND While G-POEM remains an effective and exciting treatment for GP patients, predictors of clinical success remain poorly characterized. Botox injection of the pylorus prior to committing to G POEM can help differentiate those who might have a favorable clinical response to this procedure in patients with GP. METHODS To evaluate the utility of Botox injection prior to G-POEM, 124 patients with clinically diagnosed GP who underwent a GPOEM were assessed in this retrospective cohort study. All G-POEM procedures were conducted at a single center and were completed between February 2018 and May 2023. Patients who had received intrapyloric Botox injection (n=79) had QoL and clinical success rates compared to patients who received other treatment options (n=45). Results were assessed at 1-, 3-, 6-, 12-, 24-, 36-, 48-months post G-POEM. RESULTS When assessing symptom severity, the difference between the change in GCSI values for Botox patients and patients receiving other prior therapy was statistically significant at 6 months post GPOEM; +1.27 for Botox patients vs +0.55 for other treatments (p-value of 0.03). At this 6-month checkpoint, 64% of the Botox group achieved clinical success compared to 37.5% for the other treatment group. When comparing QoL, intrapyloric Botox injection has statistically significant improvements in SF-36 total score at 1, 3-, 6-, 12-, and 36-months post G-POEM, while patients receiving other treatments had no statistically significant improvements in their SF-36 total scores. CONCLUSION We hypothesize that clinical improvement on pre GPOEM Botox selects patients with a higher component of pyloric spasm who end up responding more favorably to GPOEM. IMPLICATIONS Intrapyloric Botox injection is technically feasible in almost all patients with GP and does not require special expertise, training, or equipment; therefore, this should be strongly considered prior to referring a patient for G-POEM.Item The Role of SHROOM3 in Congenital Heart Defects(2023-07-28) Hammons, Grant; Aldrich, Ally; Douglas, Rebecca; Guilfoy, Catherine; Jain, Nina J.; McMullan, Ashleigh; Murray, Princess; Rathke, Caelan; Wakulchik, Mark; Voskoboynik, Ellen; Zwierzynski, James B.; Durbin, MatthewBACKGROUND AND HYPOTHESIS: Congenital heart defects (CHD) are the most common, and most frequently fatal birth defects, but most etiology remains unknown. We identified a patient with CHD and implicated a gene called SHROOM3. SHROOM3 binds Dishevelled2 which is the central cytoplasmic component of both canonical and noncanonical Wnt/planar cell polarity (PCP) signaling pathways. PCP drives cell movement and is important to embryogenesis and disruption causes CHD. We hypothesize CHD can result from SHROOM3-loss-of-function due to PCP disruption. PROJECT METHODS: To interrogate SHROOM3’s role in CHD and PCP we utilized an established in vivo SHROOM3-loss-of-function model, Shroom3 gene trap mice (Shroom3gt). We also utilized a loss-of-function model for PCP membrane component VANGL2, (Vangl2+/-). We assayed genetic interaction between Shroom3 and Vangl2 during cardiac development by crossing singly heterozygous null mice to produce compound heterozygous embryos, harvested embryos, and performed histologic analysis for cardiac defects. We also utilized a human in vitro SHROOM3-loss-of-function model, a CRISPR-Cas9 edited SHROOM3 knockout HELA cell line. We assayed cell movement using a scratch assay. RESULTS: Compound heterozygous Shroom3+/gt;Vangl2+/- embryos had a three fold increase in heart defects compared to singly heterozygous Shroom3+/gt;Vangl2+/+ or Shroom3+/+;Vangl2+/- embryos (3 of 19 or 15.7%, versus 1 of 17 or 5.2%, and 1 of 19 or 4.8%, respectively), demonstrating a trend towards genetic interaction between SHROOM3 and VANGL2/PCP during cardiac development. The scratch assays demonstrated cell movement defects due to SHROOM3-loss-of-function consistent with increased cell movement. CONCLUSION AND POTENTIAL IMPACT: We demonstrate SHROOM3 interacts with Wnt/PCP during cardiac development. Further interrogation of SHROOM3’s role in Wnt signaling will provide insight into the mechanisms by which a novel CHD candidate participates in cardiogenesis and will improve CHD diagnosis, management, and therapeutic development. Presentation recording available online: https://purl.dlib.indiana.edu/iudl/media/q47r66rj2cItem Efficacy of Novel Bracing for Treating Sciatica and Cadaveric Dissection to Examine Excursion of the Sciatic Nerve(2023-07-28) Callahan, Kyle; Dellacqua, DaleBackground/Objective: Sciatica affects nearly half of all Americans and can often become debilitating, leading to severe pain that can limit performing activities of daily living. Brace application has not been tried for alleviation of pain. In this study, we seek to find if a novel brace can decrease pain and decrease bothersome level of symptoms for those suffering from sciatica. In addition, this study utilizes a cadaveric dissection to understand how the sciatic nerve stretches and tensions upon lower limb manipulation. Methods: Fourteen patients self-reported pain, functionality, and bothersome levels pre- and post-bracing. Excel’s data analysis tool was utilized to run statistical tests. One cadaver (2 lower limbs) was dissected, revealing the sciatic nerve at the hip and knee, while tibial nerve at the ankle. Excursion was measured utilizing a fixed pin and an initial distance, the leg manipulated, and final distance from pin measured. Ultimately, excursion was deemed final distance minus initial distance from the pin. Results: The brace decreases Visual Analogue Scale (VAS) scores, increases Patient Reported Outcomes, and decreases Sciatica Bothersome Indexes. There was a significant difference in VAS pre- versus post-brace values at initial and 7-day post-visit but not at 21- or 42-day post-visit. Sciatic nerve excursion was greatest at the ankle. Discussion/Conclusion Brace use decreases pain levels, increases functionality, and decreases bothersome level of symptoms. The distal nerve moves more upon manipulation and therefore is more prone to tensioning than the proximal nerve. Dissection data illustrates how the brace positions the limb in a way that promotes “detensioning” of the nerve, alleviating sciatica. More cadaver data is needed. Presentation recording available online: https://purl.dlib.indiana.edu/iudl/media/c58059kh24Item Comparison of Artificial Intelligence and Eyeball Method in the Detection of Fatty Liver Disease(2023-07-26) Catron, Evan J.; Passarelli, Robert P.; Danielle, Wilmes; Wei, Barry; Le, Thi M.U.; Li, Ping; Zhang, Wenjun; Lin, Jingmei; Melcher, Mark L.; Mihaylov, Plamen V.; Kubal, Chandrashekhar A.; Mangus, Robert S.; Ekser, BurcinBackground: Quantification of liver fat content relies on visual microscopic inspection of liver biopsies by pathologists. Their percent macrosteatosis (%MaS) estimation is vital in determining donor liver transplantability; however, the eyeball method may vary between observers. Overestimations of %MaS can potentially lead to the discard of viable donor livers. We hypothesize that artificial intelligence (AI) could be helpful in providing a more objective and accurate measurement of %MaS. Methods: Literature review identified HALO (image analysis) and U-Net (deep-learning) as high-accuracy AI programs capable of calculating %MaS in liver biopsies. We compared (i) an experienced pathologist’s and (ii) a transplant surgeon’s eyeball %MaS estimations from de-novo liver transplant (LT) biopsy samples taken 2h post-reperfusion to (iii) the HALO-calculated %MaS (Fig.1). 250 patients had undergone LT at Indiana University between 2020-2021, and 211 had sufficient data for inclusion. Each biopsy was digitized into 5 random non-overlapping tiles at 20x magnification (a total of 1,055 images). We used HALO software for analysis and set the minimum vacuole area to 10μm² to avoid the inclusion of microsteatosis. Microsteatosis was excluded by the pathologist and the surgeon by the eyeball method using the same 1,055 images. Each %MaS estimation was compared with early allograft dysfunction (EAD). EAD is defined by the presence of at least one of the following: INR >1.6 on postoperative day (POD) 7, total bilirubin >10mg/dL on POD7, or AST/ALT >2000IU/L within the first 7 days following LT. Results: Of 211 LTs, 42 (19.9%) had EAD. The mean %MaS estimation of pathologist and transplant surgeon were 6.3% (SD: 11.9%) and 3.2% (SD: 6.4%), respectively. HALO yielded a significantly lower mean %MaS of 2.6% (SD: 2.6%) than the pathologist’s eyeball method (p<0.001). The mean %MaS calculated by HALO was higher in EAD patients than in non-EAD (p=0.032), but this difference did not reach statistical significance in the pathologist’s estimation (p=0.069). Conclusions: Although mean %MaS measurements from all parties were mild (<10%), human eyeball estimations of %MaS were significantly higher than HALO’s %MaS. The HALO-calculated %MaS differed significantly between the EAD and non-EAD LTs which might suggest a possible correlation between the AI’s steatosis analysis and EAD outcomes. However, pathologic variables other than %MaS (necrosis or cholestasis) should be included in future analyses to determine whether %MaS is the dominant parameter predicting EAD. AI is a promising tool to quantify liver steatosis and will help pathologists and transplant surgeons predict liver transplant viability.Item Investigating Post-Operative Refractive Outcomes in Patients Undergoing Cataract Surgery to Assess the Potential Impact of a Concurrent Diagnosis of Dry Eye Disease (DED)(2023-07-27) Almalahi, Ateik; Schroeder, BarbaraBackground: Cataract surgery is one of the most performed surgical procedures in the world. A cataract is defined as opaqueness in the interior of the ocular lens2. The exact etiology of cataracts is multifactorial ranging from environmental conditions to biochemical changes induced by aging3. The Intraocular lens (IOL) power calculation is an essential part of the pre-operative planning for cataract surgery as it determines the specific IOL that should be utilized for a patient. Keratometry measurements are required for this IOL power calculation, however, Dry Eye disease (DED) has been shown to cause inaccurate keratometry measurments4,5. Objective: We hypothesize that patients with DED undergoing cataract surgery will have a larger deviation from the predicted spherical equivalent (SE) post-operatively. Methods and Materials: Patients who were over the age of 18 and underwent cataract surgery were included. Patients who had a diagnosis of glaucoma, Herpes Simplex Keratitis, punctual plugs, undergone Laser-Assisted in Situ Keratomileusis (LASIK) surgery, Radial Keratometry, or any form of corneal scarring were excluded from this study as they can negatively impact keratometry measurements. A total of 197 eyes from 118 patients were included in the analysis. Results: The DED sample had a statistically significant larger SE deviation from the predicted SE compared to the healthy sample (p=0.037). The DED sample also had a statistically significant larger percentage of patients with an SE deviation of 0.50 D or greater (p=0.002). Finally, the DED had a statistically significant older age than the healthy group (p=0.028). Conclusion: The significant difference in age between the healthy sample and the DED sample confirms the significant correlation between age and prevalence of DED the literature has described6. The statistically significant increased post-operative SE deviation from predicted SE within the DED sample can be the result of the increased variability in the tear film associated with DED. This can lead to inaccurate keratometry measurements, thus leading to incorrect IOL power calculations.