Via Research Recognition Day Program VCOM-Carolinas 2025

Via Research Recognition Day VCOM-Carolinas • February 21, 2025

Contents Welcome . .......................................................................................................................... 3 Speaker .............................................................................................................................. 4 Agenda ............................................................................................................................... 5 Posters

Biomedical Research...............................................................................................................6

Clinical Education Research.................................................................................................17

Case Studies..........................................................................................................................50

2025 Research Recognition Day

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Welcome

Welcome to the eleventh 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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David Koerner's career arc from a Division I option quarterback to the pioneering force behind the USPC embodies his unwavering determination and quick decision-making skills. Harnessing the qualities that made him a standout athlete, he has transitioned into a visionary in the field of exercise science, focusing on bridging the gap between academic research and athletic performance. His realization of the disconnect between the academic and athletic worlds fueled his ambition to create a facility where cutting-edge research informs sports performance, rehabilitation, and more. Today, as the driving force of the USPC, David is setting a new standard in the human performance industry, making it the global epicenter for athletes, researchers, and professionals seeking to elevate their game through science. Speaker: David Koerner Co-Founder / Owner at US PerformanceCenter

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Agenda 8:00 am – 8:25 am

Registration

First Floor Reception Area,

Continental Breakfast

Third Floor

8:30 am – 10:50 am

Poster Presentations

Third Floor

8:30 am – 9:40 am

Poster Presentation session 1

9:40 am – 10:50 am

Poster Presentation session 2

11:00 am – 11:30 am

Opening Remarks

Lecture Hall 2

11:30 am – 12:30 pm

Keynote Address: David Koerner, Co-Founder, US Performance Center

Lecture Hall 2

12:30 pm – 1:25 pm

Lunch

First Floor

1:30 pm – 2:00 pm

Alexis M. Stoner, PhD, MPH

Lecture Hall 2

2:00 pm – 2:30 pm

Bidyut Mohanty, PhD

Lecture Hall 2

2:30 pm – 3:00 pm

Tom Lindsey, DO

Lecture Hall 2

3:00 pm – 3:15 pm

Break

3:15 pm – 4:15 pm

DO with Research Distinction

Lecture Hall 2

3:15 pm – 3:30 pm

Anna Deal and Madison Dudick

3:30 pm – 3:45 pm

Nick Minner

3:45 pm – 4:00 pm

Margaret Munz

4:00 pm – 4:15 pm

Robyn Sawyers

4:15 pm – 4:30 pm

Awards Ceremony

Lecture Hall 2

4:30 pm – 4:45 pm

Closing Remarks

Lecture Hall 2

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Biomedical Research

Genome-wide identification of single-stranded DNA sequences that bind to hnRNP K in humans Rachael Baker, B.A., Daniel Ross, B.S., Olivia Lewis, OMS-I, Krishna Patel, OMS-III, Lauren Heirs, OMS-II, Shane Donahue, OMS-II, Tarah Anasseri, OMS-I, Payal Arora, OMS-II, David Eagerton, Ph.D., and Bidyut K. Mohanty, Ph.D. Edward Via College of Osteopathic Medicine, 350 Howard St, Spartanburg, SC 29316

Introduction and Objective

Results

A. The human genome is interspersed with polycytosine rich (polyC-rich) and their complimentary polyguanine rich DNA sequences that can form noncanonical secondary structures. These sequences and secondary structures can affect genome integrity. Regulatory proteins, such as polyC-binding heterogeneous nuclear riboproteins (hnRNPs), can bind to single stranded DNA sequences in a site and structure-specific manner to these DNA sequences. These regulatory proteins are implicated in the pathogenic mechanisms including cancer and other diseases. One of the hnRNPs, namely hnRNP K, shows a binding preference for polyC-rich sequences, and regulates transcription, mRNA stability, splicing, and translation- and promotes cancer. It has been shown that hnRNP K binds to multiple sequences in the mouse genome. Our recent data shows that hnRNP K binds to thousands of polyC-rich DNA sequences in the mouse genome. B. HYPOTHESIS: We predict that hnRNP K binds to polyC-rich DNA sequences in the human genome to promote cancer and other genetic diseases. C. Our OBJECTIVE is to identify the single-stranded DNA sequences within the human genome that bind to hnRNP K and play a role in canc er.

Methods Figure 3. Consensus motif for mouse ssDNA sequences pulled down using hnRNP K (Patel et a l, unpublished).

Discussion Figure 5. ExoChew. (A) A 3% agarose gel showing comparison of human genomic DNA (gDNA), 200 bp sonicated gDNA, and sonicated gDNA that has been treated with T7 Exonuclease. (B) A 6% polyacrylamide gel showing comparison of sonicated human gDNA with and without single stranded binding protein (SSB), and its Exonuclease products with and without SSB.

Discussion and Further Directions

human

Genomic Human DNA

A. Our results indicate the successful transformation of human double-stranded DNA to single-stranded DNA. A pull-down assay was performed using the T7 Exonuclease treated DNA product and His tagged hnRNP K. B. The pull-down products were converted back to double-stranded DNA. The DNA library is currently being sequenced in-house using the Oxford Nanopore MinION MK1D. C. The identified sequences will be analyzed.

Figure 6. Oxford Nanopore MinION MK1D DNA Sequencer.

Figure 1. Map of hnRNP K.

References

1.Patel, K., Lodha, C., Smith, C., Diggins, L., Kolluru, V., Ross, D., Syed, C., Lewis, O., Daley, R. and Mohanty, BK. (2023) ExoChew: An exonuclease technique to generate single-stranded DNA libraries. Biorxiv. doi: https://doi.org/10.1101/2023.10.02.560524. October 02, 2023. 2.Lu J, Gao FH. Role and molecular mechanism of heterogeneous nuclear ribonucleoprotein K in tumor development and progression. Biomed Rep. 2016 Jun;4(6):657-663. doi: 10.3892/br.2016.642. Epub 2016 Mar 29. PMID: 27284403; PMCID: PMC4887935.​

hnRNP K 0

Figure 2. A 6% polyacrylamide gel shift of C9orf72 C6 ((C 4 G 2 ) 6 ) DNA demonstrating the ability of hnRNP K to bind to a human polycytosine-rich DNA sequence.

Acknowledgements

Figure 4. Protocol for isolating and identifying hnRNP K binding DNA sites.

Free DNA

BKM was funded by VCOM REAP grants 1032453 and 1302559.

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Biomedical Research

Lighting the way: an economical alternative to feeder cell irradiation for T-cell expansion Michael S. Benavidez Arias, MA, OMS-III, An Nguyen OMS-III, Daniel Ross, BS, NREMT, David H. Eagerton, PhD, F-ABFT and Krit Ritthipichai, DVM, MS, PhD. Edward Via College of Osteopathic Medicine, Dept. of Biomedical Affairs, Spartanburg, South Carolina. Results

Introduction

Background: T-cell therapy has emerged as an effective treatment for blood cancers with an average 80.5% objective response rate (ORR) 1 . A robust T-cell expansion process involves co-culturing T-cells with non proliferating feeder cells combined with anti-CD3 antibody and IL-2. Challenge: Although ionizing irradiation effectively inhibits feeder cell proliferation, the high operating costs limit cell therapy research to well-funded institutions. Rationale: UVC is high energy electromagnetic radiation, causing severe DNA damage. It is widely used for inactivating microorganisms and inducing cell apoptosis 2 . Given UVC generators’ cost -effectiveness, we explored their potential as an alternative to ionizing irradiators for generating non-proliferating feeder cells for T-cell expansion. Hypothesis: We hypothesize that TILs expanded using UVC-irradiated feeder cells demonstrate similar or superior viability, expansion rates, and effector functions compared to those expanded with ionizing irradiated feeder cells.

TILs expanded with UVC-Irradiated PBMCs showed comparable T-Cell expansion

UVC Effectively Suppressed Feeder Cell Proliferation by Inducing DNA Damage

>90% viability

~ 500 folds

>97% T-cells

B.

C.

A.

A.

↑ DNA damage

↑ Cell death

↓ Cell proliferation

B.

Figure 1. The impact of UVC irradiation on DNA and apoptosis. Flow cytometry analysis of phosphorylated -H2AX (DNA damage marker) in CD32hi K562 cells 15, 60, and 120 minutes after UVC irradiation (A) . Line graph demonstrating cell count in CD32hi K562 pre- and post-UVC irradiation from day 0 to 14 (B) . Early (annexin V*) and late apoptosis (Annexin V*7-AAD*) in K562 on day 14 post-UVC irradiation was determined by flow cytometry (C) . Data are presented as mean ± SEM. Two independent experiments; n=5, per group. Two-tailed Student's t-test; *P ≤ 0.05; **P ≤ 0.01; ***p ≤ 0.001 ****p ≤ 0.0001.

C.

~ 50% degranulation

Experimental Design

UVC Irradiated Cells Dampened Glucose Uptake and ATP production While Enhancing Antibody Retention on the Cell Surface ↑ Antibody retention

B.

A.

C.

1. To determine the impact of UVC on feeder cell proliferation apoptosis (Fig. 1)

↓ Glucose uptake

↓ ATP production

Figure 4. Characteristics of Tumor-infiltrating lymphocytes (TILs) expanded with irradiated feeder cells. Bar graphs depicting live cells (left), cell expansion (middle), and T-cell purity (right) (A) . Bar graphs displaying T-cell exhaustion markers in expanded TILs (B) . Bar graphs showing TIL effector function following re-stimulation with anti-human CD3/CD28 antibodies (C) . Data are presented as mean ± SEM. Two independent experiments; n=4, per group. Two-tailed Student's t-test, ns; not significant.

2. To assess antibody retention and cell metabolism of UVC-Irradiated cells (Fig. 2)

Figure 2. An alteration in antibody retention and metabolic function following UVC exposure. Luminescence assay for measuring glucose uptake at 6 and 24 hours post-UVC treatment (A) . Bar graph demonstrating ATP concentrations at 6 and 24 hours after UVC irradiation (B) . Bar graph displaying the percentage of membrane-bound antibodies on cells exposed to UVC for 6 and 24 hours at 25, 50, 75, and 100 minutes after antibody labeling (C) . Data are presented as mean ± SEM. Two independent experiments; n=5, per group. Two-tailed Student's t-test; ***p ≤ 0.001 ****p ≤ 0.0001.

Conclusion

• UVC effectively suppressed feeder cell proliferation, indicated by apoptosis in 95% of irradiated cells. • Cell viability, expansion rates, and effector functions of T cells expanded with UVC-irradiated feeder were comparable to those expanded with ionizing irradiation. • Further investigation into UVC irradiation's scalability and long-term effects on T-cell therapies could revolutionize accessibility and affordability in clinical settings, particularly in resource-limited environments.

3. To investigate the association between antibody retention and energy depletion (Fig. 3)

Glucose depletion Antibody Retention on Cellular Surfaces Was Dose-Dependently Increased Following Energy Depletion

A.

C.

B.

ATP production

Glucose uptake

4. To examine the potential of UVC irradiated feeder cells for T-cell expansion (Fig. 4)

Acknowledgements

References

1. Xiang, X., He, Q., Ou, Y., Wang, W. & Wu, Y. Efficacy and Safety of CAR-Modified T Cell Therapy in Patients with Relapsed or Refractory Multiple Myeloma: A Meta-Analysis of Prospective Clinical Trials. Front Pharmacol 11, 544754 (2020). 2. Sun, W., Jing, Z., Zhao, Z. Dose-Response Behavior of Pathogens and Surrogate Microorganisms across the Ultraviolet-C Spectrum: Inactivation Efficiencies, Action Spectra, and Mechanisms. Environ Sci Technol. 57, 10891 (2023).

• The work is supported by VCOM Seed Grant Program • The authors would like to thank Dr. Bidyut Mohanty for providing anti Gamma H2AX (phospho-Ser139) antibody.

Figure 3. Association between antibody retention and cellular energy depletion. Bar graph demonstrating the effect of cytochalasin B treatment on antibody retention post-antibody labeling (A) . Bar graph showing antibody retention when glucose is depleted (B) . Bar graph depicting antibody retention in rotenone-treated cells (C) . Data are shown as mean ± SEM. Two independent experiments; n=5, per group. Two-tailed Student's t-test; **P ≤ 0.01 ****P ≤ 0.0001. R 2 between 0.7 and 0.9 considered highly correlated.

2025 Research Recognition Day

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Biomedical Research

Circular Dichroism (CD) Spectra Analysis of Secondary Structures formed by Hexanucleotide Repeats in C9orf72 gene Sundeep Bhanot, OMS-2; Levi Diggins, OMS-4; Daniel Ross, BS, NREMT; Rebecca Corallo, OMS-3; Rachel Daley, OMS-3; Krishna Patel, OMS-3; Olivia Lewis, OMS-1; Shane Donahue, OMS-2; Jacob Thaddeus, OMS-2; Lauren Hiers, OMS-2; Christopher Syed, OMS-4; Rachael Baker, BA; David Eagerton, PhD; Bidyut Mohanty, PhD . Edward Via College of Osteopathic Medicine, 350 Howard St, Spartanburg, SC 29316 Introduction Results Discussion

Formation of i-motif occurs at 288 nm with positive peaks & 262 nm with negative peaks in low pH. G4 formation is favored in neutral to low alkaline pH levels, with max and min peaks seen at 264 nm and 245 nm, respectively. • All oligos demonstrated a dependence on pH levels. As pH levels were increased, the ability to form secondary structures decreased. • Variability in size of tandem repeats also showed differences in CD spectra findings. • Overall, boiling and cooling of oligos demonstrated a reduction in i-motif population. • Boiling and cooling of G4 oligos showed changed in ellipticity along with change in secondary structure formation, with MES buffer showing the greatest change among all buffers. • G4 structures demonstrated the most stability among all buffers when compared to i-motif structures. Continued and more detailed analysis of G4 and i-motif formation among GGGGCC/CCCCGG hexanucleotide repeats has the potential to be very promising. FTD and ALS have a documented clinical course, but little is known about the biomolecular background of the disease states. This area of research can be resourceful in determining potential therapeutic options. Further exploration of the effects of various factors on a greater number of tandem repeats is currently underway. Conclusions

Mutations in the C9orf72 gene are known to be associated with frontotemporal dementia (FTD) & amyotrophic lateral sclerosis (ALS.) This mutation is commonly the expansion of hexanucleotide repeats of GGGGCC/CCCCGG. Normally, 20 copies of this repeat are found, however, are expanded to hundreds to thousands of copies in patients with ALS or FTD. These repeats can form secondary structures known as G-quadruplex (G4) and intercalating motif (i-motif or iM). These secondary structures are influenced by several regulatory factors, including pH, temperature, ions, and various DNA binding proteins. CD spectroscopy was used to find the conditions considered most optimal for formation of secondary structures with the presence of these repeats, particularly sequences containing 2, 4, 6, and 14 tandem repeats of GGGGCC and GGCCCC found in intron 1 of C9orf72.

C9ORF72 G Repeats

Sequence

Nucleotides

Predicted G4s

12

0

C9ORF72 G-2

5'-CCG GGG CCG GGG-3'

24

1

C9ORF72 G-4

5'-CCG GGG CCG GGG CCG GGG CCG GGG-3'

36

1

C9ORF72 G-6

5'-CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG-3'

5'-CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG CCG GGG-3'

84

4

C9ORF72 G-14

Figure 3. Prediction of G4 formation and gel analysis of the oligos

Figure 4. Effects of various pH on G4 formation

C9ORF72 C Repeats

Sequence

Nucleotides Predicted i-motifs

iM Density

12

0

0%

C9ORF72 C-2

5'-CCC CGG CCC CGG-3'

24

1

4.17%

C9ORF72 C-4

5'-CCC CGG CCC CGG CCC CGG CCC CGG-3'

5'-CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG-3'

36

1

2.78%

C9ORF72 C-6

5'-CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CGG CCC CG GCCC CGG-3'

84

3

3.57%

C9ORF72 C-14

Figure 5. Prediction of iM formation and gel analysis of the oligos

Methods

For this study, G4 and i-motif structures were formed with the use of buffers of varying pH. CD spectroscopy was then used to study the presence of these structures.

Figure 6. Effects of various pH on i-motif formation

Figure 1. Hexanucleotide repeats in intron 1.

References

(GGGGCC)x4

(CCCCGG)x4

Figure 7. Effects of boiling on G4 and i-motif formation

Diggins, L., Ross, D., Bhanot, S., Corallo, R., Daley, R., Patel, K., Lewis, O., Donahue, S., Thaddeus, J., Hiers, L., Syed, C., Eagerton, D. and Mohanty, BK (corresponding author) (2024) CD spectra reveal the state of G-quadruplexes and i-motifs in repeated and other DNA sequences. Biophys Rep (NY) 5(1):100187. doi: https://doi.org/10.1016/j.bpr.2024.100187.

Acknowledgements

Figure 8. Effect of single-strand DNA binding protein (SSB) on iM formation

Figure 1. Secondary structure models.

Figure 2. Utilized Buffers.

BKM was funded by VCOM REAP grants 1032453 and 1302559.

2025 Research Recognition Day

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Biomedical Research

THE ROLE OF PERSONALIZED MEDICINE IN CHEMOTHERAPY INVOLVING HISTONE DEACETYLASE INHIBITORS AND COMMON CHEMOTHERAPEUTIC DRUGS USED WITH THEM IN COMBINATION THERAPY. Annabel Crippen, OMS-III 1 , Steven Enkemann, PhD 2 . 1. VCOM Carolinas, Genetics, Spartanburg, SC. 2. Precision Genetics., Greenville, SC.

Results

Introduction

Discussion

Table 2 Enzymes involved in the metabolism of chemotherapeutic agents used in HDAC combination therapy. Each of the chemotherapeutic agents listed above have been investigated for the enzymes that metabolize them where human variation may impact activity. Table 1 The three main HDAC inhibitors and the traditional chemotherapeutic agents they have been combined with to treat cancer. HDAC Inhibitor Romidepsin Vorinostat Belinostat Each HDACi has been used in combination with each of the drugs (or combinations) listed below it. Combination 1 5-azacytidine Tamoxifen Doxorubicin Combination 2 Lenalidomide Carboplatin, Paclitaxel C d y o c x l o o r p u h b o ic s i p n h , a v m in i c d r e is , tine, and prednisone Combination 3 Pralatrexate HDAC inhibitors have been used in multiple combinations with other chemotherapeutic agents.

Combination therapy means many enzymes influence the efficacy and toxicity of a treatment.

Cancer therapy is a dynamic field that is ever evolving. Most drugs currently used to treat cancer target some aspect of rapidly dividing cells. This includes DNA replication pathways, topoisomerases and microtubules vital for replication, and DNA repair pathways. However, these therapies do not differentiate between cancer and normal cells that also replicate, leading to significant side effects. Emerging research suggests that genetic mutations in epigenetic pathways play a role in cancer development by altering regulatory gene expression (Dawson, 2006). This has led to the development of new drugs that target epigenetic pathways and their impact on oncogenesis. Important regulators include histone acetyltransferases (HAT) and histone deacetyltransferases (HDACs). These two molecules function on opposite sides of the balance between de-condensed and condensed chromatin influencing the genes expressed based on a cell's needs (Eckschlager, 2017). HDAC 1, 2 and 3 were found to be overexpressed in multiple different cancers indicating that they could be important therapeutic targets (Eckschlager, 2017). Thus, HDAC inhibitors (HDACis), such as Belinostat, Vorinostat, and Romidepsin have been developed to target HDACs and have shown some potential in reactivating silenced tumor suppressor genes through increased histone acetylation. Romidepsin and belinostat have been approved for the treatment of Peripheral T-Cell Lymphoma (PTCL), while vorinostat is approved for Cutaneous T-Cell Lymphoma (CTCL). However, HDAC inhibitors have shown limited success rates when used as a monotherapy in solid tumors and resistance to HDAC inhibitors has been commonly observed (Suraweera, 2018). These findings have led to HDACis being explored as components of combination therapies, with the idea that they might enhance the therapeutic activity of other chemotherapeutic agents. One drawback of combining drugs for treatment is the increased potential for detrimental side effects. Some of this can be offset by examining the genetics of the treated patient. In order to use personalized medicine for screening patients who might receive combination therapy utilizing an HDAC inhibitor we attempted to identify all the possible enzymes that would need to be genetically evaluated in patient treated with different HDACi combinations.

Table 3 The number of different enzymes that are currently known to influence metabolism and thus efficacy of the drugs used to kill tumor cells with HDACi combination therapy.

HDAC Inhibitor Romidepsin Vorinostat

Belinostat

Combination 1 Combination 2

22

35

27

8

28

49

Combination 3

10

–

–

Drug

Enzymes (HUGO gene symbols) COYAPT3PA14B,3CYP3A5, CYP1A1, CYP2B6, CYP2C19, ABCB1, UGT1A1, UGT2B7, CYP2A6, CYP2C9, CYP3A4 U U G G T T 1 2 A B7 1 , , U U G G T T 2 1 B A 1 3 7 , UGT1A7, UGT1A8, UGT1A9, T 2 1 1, ,UdCCKK1, , CUDCAK, 2N, Th5CCN2T, 3C , MS AP KM1H, DC M1 , PNKT25, CR2R, MT Y1 M, S , CC YY PP 31 AA 52 ,, UC YG PT21BA64, , CUYGPT21CA98, C, YU PG2TC11A91, 0C, YUPG2TD26B, 7C , Y P 3 A 4 , UC YGPT12BB11,5C, YSPU2LCT81,AC1Y, PS1U9LAT 11E, C1Y, CPY2 PA26C, C9Y, CPY2PE11A, F1M, O 1 , FMO3, CES1, SULT2A1, ABCB1, ABCC2, ABCG2 C A T T R P7 1 B , O , G C S T T 1, OCT2, OCTN1, OCTN2, CTR2, ATP7A, CC YY PP 13 BA 17 ,, CC YY PP 21 C9 8A,1C, YAPB3CAB41,, CAYBPC3BA151, , A B C C 1 , A B C C 2 , ABCC10, SLCO1B3 CNBORS 11 ,, CNBORS32,, ANKORS 13 A, N1D, AUKFRS 21 ,CN3 D, PUOFRS,7X, DNHD,UNFQS 3O,1A, B C B 1 , A SO B D CC 1 1, ABCC2, ABCG2, CAT, GPX1,RALBP1, SLC22A16, C C Y Y P P 2 2 B C1 6 8 , C , C Y Y P P 3 2 A C 4 1 , C 9 YP3A5, CYP2C9, CYP2A6, CYP2C8 CA YB PC 3C A1 4, A, CBYCPC32A, A5 ,BCCYCP33, AA 7B ,CCCY1P02, EA1B, CAGB2C, BR1A, LABBPC1B, 1 1 , SLC22A3, SLCO1B3, SLCO1B1 R X hE ABCB1 R R N C M C FPGS, RFC, GGH

Conclusions

Romidepsin Belinostat Vorinostat 5-azacytidine

HDAC inhibitors are in their infancy in the chemotherapy arsenal. Not only is it difficult to identify the best way to use them in treatment they have not been completely studied for how the body metabolizes them in combination therapy. Current research suggests that one might have to investigate human variation in as many as 49 different enzymes in order to identify patients that will likely fail to respond or suffer severe adverse effects during treatment.

Lenalidomide Pralatrexate

Tamoxifen

References & Acknowledgments

Methods

Carboplatin

1. Dawson, Mark A. et al. Cancer Epigenetics: From Mechanism to Therapy. Cell, Volume 150, Issue 1, 12 – 27. 2. Eckschlager T, Plch J, Stiborova M, Hrabeta J. Histone Deacetylase Inhibitors as Anticancer Drugs. Int J Mol Sci. 2017;18(7):1414. Published 2017 Jul 1. doi:10.3390/ijms18071414 3. Suraweera A, O'Byrne KJ, Richard DJ. Combination Therapy With Histone Deacetylase Inhibitors (HDACi) for the Treatment of Cancer: Achieving the Full Therapeutic Potential of HDACi. Front Oncol. 2018 Mar 29. doi:10.3389/fonc.2018.00092 4. Kane M. Belinostat Therapy and UGT1A1 Genotype. 2023 Jul 20. In: Pratt VM, Scott SA, Pirmohamed M, et al., editors. Medical Genetics Summaries [Internet]. 5. Bethesda (MD): National Center for Biotechnology Information (US); 2012-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK593302/

Paclitaxel

The HDACs reviewed were Romidepsin, Belinostat and Vorinostat and the drugs used in combination with HDACs included 5-azacytidine, lenalidomide, pralatrexate, tamoxifen, carboplatin, paclitaxel, doxorubicin, cyclophosphamide, vincristine. These terms were used in combination with terms like chemotherapy, genetics, personalized medicine, and drug metabolism in literature searches using PubMed and Google Scholar databases. Retrieved articles were examined for evidence of the use of HDAC inhibitors in cancer treatment in combination therapy and evidence of enzymes involved in the metabolism, development of side effects, or impacts on the efficacy of treatment.

Doxorubicin

Cyclophosphamide

Thank you to previous students whose papers contributed to this project through providing information on the enzymes for some of the combination drugs.

Vincristine

2025 Research Recognition Day

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Biomedical Research

The Impact of Pharmacogenomics in Antimetabolite Therapy Regimens Using the Antimetabolites Clofarabine, Pentostatin, or Nelarabine

Zachary T. Fitzgerald, B.S. 1 , Steven Enkemann, PhD 2 Edward Via College of Osteopathic Medicine, Spartanburg, SC.

Introduction

Results

Discussion

References 1. Nagai S, Takenaka K, Nachagari D, Rose C, Domoney K, Sun D, Sparreboom A, Schuetz JD. Deoxycytidine kinase modulates the impact of the ABC transporter ABCG2 on clofarabine cytotoxicity. Cancer Res. 2011 Mar 1;71(5):1781-91. doi: 10.1158/0008-5472.CAN-10-1919. Epub 2011 Jan 18. PMID: 21245102; PMCID: PMC3531552. 2. Huang M, Inukai T, Miyake K, Tanaka Y, Kagami K, Abe M, Goto H, Minegishi M, Iwamoto S, Sugihara E, Watanabe A, Somazu S, Shinohara T, Oshiro H, Akahane K, Goi K, Sugita K. Clofarabine exerts antileukemic activity against cytarabine-resistant B-cell precursor acute lymphoblastic leukemia with low deoxycytidine kinase expression. Cancer Med. 2018 Apr;7(4):1297-1316. doi: 10.1002/cam4.1323. Epub 2018 Feb 23. PMID: 29473342; PMCID: PMC5911575. 3. Zhang, H., Liu, R., Lou, T., Zhao, P., & Wang, S. (2022). Pentostatin Biosynthesis Pathway Elucidation and Its Application. Fermentation , 8 (9), 459. https://doi.org/10.3390/fermentation8090459 4. Fukuda Y, Schuetz JD. ABC transporters and their role in nucleoside and nucleotide drug resistance. Biochem Pharmacol. 2012 Apr 15;83(8):1073-83. doi: 10.1016/j.bcp.2011.12.042. Epub 2012 Jan 20. PMID: 22285911; PMCID: PMC3319017. 5. Choudhuri S, Klaassen CD. Structure, function, expression, genomic organization, and single nucleotide polymorphisms of human ABCB1 (MDR1), ABCC (MRP), and ABCG2 (BCRP) efflux transporters. Int J Toxicol. 2006 Jul-Aug;25(4):231-59. doi: 10.1080/10915810600746023. PMID: 16815813. Special thank you to the Edward Via College of Osteopathic Medicine – Carolinas library team for aiding in the compilation of source material. Acknowledgements Wn ehl ai lrea bt hi ne emeexcehr at nt hi semi rsebf fye cwt sh iacnhdc tl oh fea pr aabt hi nwea, ypse nl etaods ti na tgi nt o, atno dx i c i t y ag reen ewt iecl lvuanr idaet ri os nt osoi dn , ht huemr ea ni ss lmi ma iyt ei ndf lruees ne ac er cthh ee xepf fl iocrai cnyg ahnodwt o x i c i t y owf etlhl se sa es dl i rvue gr se. nPzoyl my me so rc po hu il sdma sf f ienc tk de ry uegn az cytmi veast iloi kneadnCdKmaentda bAoDl iAs ma s, pAodtdernetsi sailnl yg at lht iesr ignagp tihnekr anpoewulteidc goeuct coouml delse aa dn dt os imd eo reef fpe cetrss.o n a l i z e d treatment approaches, improving both safety and efficacy. ● CDleoof ax ryac by itni dei na en dk iNn ea lsaer a( dbCi nKe) ai sr ec emnet rt aa bl tool i zt he edi rv itah es irma pi leaur tpi ca et hf fwi caayc sy. and exogenous toxicities. ● RG Te sPploenvseel st oi nt pr eaat itemnet sn tt ri enaTt e- Ad LwLi tcho rnr eellaatreadb iwn ei t.hTi nu tmr aocreml l uultaart iaornas- that influence ara-GTP can serve as treatment predictors. ● RtrReaMt1meexnptrreessspioonnslee.vels could also serve as a predictive marker for ● Genetic testing for polymorphisms in dCK, RRM1, ADA, CYP3A4 and pa no as sl oi bg l yt hoetrhaeprys tsoh iomu lpdr ob ve ec po na tdi eu nc tt eedx pb ee rf oi er ne cues ai nngd pt ruer ai nt me ne nu tc l e o s i d e outcomes. Conclusions

Major Take-Aways: ● Tmhoeneofpf ehcotsi vpehnoersysl aot fi ocnl obf ayrda eboi nxey cryetl ii edsi noenkiitns ai snei t(i adlC K ) a n d i t s rimetpeanitrioenffiwcaitchyi.n cells, as efflux via the ABCG2 transporter can • Th ahse br es 2e n2 9l i5n0k8e0d (t Co -v1a2r0i a5bTi)l i pt yo li yn md oC rKp ehxi spmr e isns itohne adnCdKepnrzoymmoattei cr ar ec tgiivoi nt y alonwdetrhaecvtaivriitayn.t rs2231142 in ABCG2 is a missense mutation with ● Pentostatin targets adenosine deaminase (ADA). • Genetic variants like G-to-A at position 22 (Asp8Asn) and C-to-T at pfoousnitdioinn 1A4fr5ic(aLnysA4m9eGrlnic)abnosth reduce ADA activity and are commonly . ● Ra cetdi vuacteedd ai cnttiov iittys oa cf tdi vCeK tcr ai pnhporsepvheant te Nf oerl amr a( ba irna e- Gf rToPm) , rbeedi nugc i n g therapeutic efficacy. • Vs uarrvi ai vnat ls (i rns t4h6i 9s 4e 3n 6z y2 m) aenadr er eadsus oc ec di a et ef fdi cwa ci tyh( iIn2c4rVe ,aAs e1d1 9pGr o, ga nr eds Ps i1o2n2fSr )e. e ● Sthoemiredmi-eatnabdotlriit-epshionshpibhiatteribfoornmusc.leotide reductase (RNR) via • SchoemmeoRthReMr1appyoolyumtcoormphesism leads to higher RRM1 expression and poor Table 1. CYP enzymes implicated in the metabolism of clofarabine, pentostatin, and nelarabine and their excretion. CYP enzyme Key human variants* Function CYP3A4 rs35599367 Degradation of pentostatin CYP2C8 s10509681 and rs11572080 Degradation of pentostatin and clofarabine CYP2A6 rs568811809 Degradation of pentostatin In addition to the enzymes that produce the nucleotide analogs for activity are liver enzymes that promote the degradation and excretion of these drugs.

• Antimetabolites are a diverse class of drugs that have been used extensively for chemotherapy since the 1940’s. • Antimetabolites fall into five categories based on their structural properties. They include purine analogs, pyrimidine analogs, folic acid antagonists, adenosine deaminase inhibitors, and ribonucleotide reductase inhibitors. Clofarabine, Pentostatin, and Nelarabine are relatively new purine analogs. • They disrupt the synthesis of nucleotides and substitute for purine nucleotides interfering with DNA synthesis, RNA synthesis, and ultimately functional protein production. They inhibit key enzymes critical for nucleotide biosynthesis, nucleotide salvage, and DNA repair. • The goal of this research was to provide a comprehensive overview of the key enzymes implicated in the primary and secondary metabolism of the purine nucleoside analogs clofarabine, pentostatin, and nelarabine in order to understand how they differed from other antimetabolites and to identify specific enzymes to assess to improve efficacy and minimize patient toxicity.

Methods

Records identified through database searching: PubMed (n=67) Google Scholar (n=8)

Additional articles identified through genetic variant search tool LitVar2 (n=2)

Records after duplicates removed (n=32)

Records screened based on title and abstract (n=45) Full-text articles assessed for eligibility (n=13) Articles included in analysis (n=10)

Records excluded (n=32) Full-text articles excluded for reasons: ● Ei nnczoymmpal tei tce por ro nc eo st s discussed ● Isnt uc do my oprl ecteel lmu leatra b o l i s m transport mechanism ● Up rnorceel as st eodr pnhoat rcml oasceol yl o g i c tied to pathway of study

rs1065852, rs28371706, rs59421388, rs35742686, rs769258, rs3892097, rs28371725, rs5030656 rs2069514, rs12720461, rs2069526, rs35694136

Degradation of pentostatin and clofarabine

CYP2D6

CYP1A2

Degradation of pentostatin

* Other human variants are known. Those listed have the highest population frequencies.

2025 Research Recognition Day

10

Biomedical Research

Unlocking the Promoter Puzzle: G-Quadruplex and I-Motif Secondary DNA Structures in VEGF Gene Regulation and Therapeutic Potential Levi Diggins OMS-IV, Krishna Patel OMS-III, Jake Rodriguez OMS-II, Sundeep Bhanot OMS-II, Daniel Ross, Olivia

Lewis OMS-I, Rachel Daley OMS-III, Rebecca Corallo OMS-III, and Bidyut K Mohanty. Edward via College of Osteopathic Medicine, 350 Howard St. Spartanburg SC

Introduction

Results

Conclusions

References Acknowledgements 6) Del Villar- Guerra, Rafael, et al. “G -Quadruplex Secondary Structure Obtained from Circular Dichroism Spectroscopy.” Angewandte Chemie (International Ed. in English) , U.S. National Library of Medicine, 11 June 2018, www.ncbi.nlm.nih.gov/pmc/articles/PMC5920796/. 7) ER;, Swenson. “Hypoxia and Its Acid - Base Consequences: From Mountains to Malignancy.” Advances in Experimental Medicine and Biology , U.S. National Library of Medicine, 2016, pubmed.ncbi.nlm.nih.gov/27343105/. 8) Lawson, Teegan, et al. “A Structural Perspective on the Regulation of Human Single -Stranded DNA Binding Protein 1 (Hssb1, OBFC2B) Function in DNA Repair.” Computational and Structural Biotechnology Journal , U.S. National Library of Medicine, 28 Mar. 2019, www.ncbi.nlm.nih.gov/pmc/articles/PMC6451162/. Acknoledgements BKM was partially funded by VCOM REAP grants 1032453 and 1302559. 1) Arrigo, A., Aragona, E., & Bandello, F. (2022). VEGF-targeting drugs for the treatment of retinal neovascularization in diabetic retinopathy. Annals of medicine , 54 (1), 1089 – 1111. https://doi.org/10.1080/07853890.2022.2064541 2) Song, D., Liu, P., Shang, K., & Ma, Y. (2022). Application and mechanism of anti-VEGF drugs in age related macular degeneration. Frontiers in bioengineering and biotechnology , 10 , 943915. https://doi.org/10.3389/fbioe.2022.943915 3) Patel, Sonia A, et al. “Molecular Mechanisms and Future Implications of VEGF/VEGFR in Cancer Therapy.” Clinical Cancer Research: An Official Journal of the American Association for Cancer Research , U.S. National Library of Medicine, 4 Jan. 2023, www.ncbi.nlm.nih.gov/pmc/articles/PMC10274152/. 4) Kendrick, S. & Hurley, L. (2010). The role of G-quadruplex/i-motif secondary structures as cis-acting regulatory elements. Pure and Applied Chemistry, 82(8), 1609-1621. https://doi.org/10.1351/PAC-CON 09-09-29 5) Wright, Elisé P, et al. “Identification of Multiple Genomic DNA Sequences Which Form I -Motif Structures at Neutral Ph.” Nucleic Acids Research , U.S. National Library of Medicine, 7 Apr. 2017, www.ncbi.nlm.nih.gov/pmc/articles/PMC5605235/. The promoter-proximal region of VEGF contains polycytosine-rich DNA sequence capable of producing I-motif and a complimentary guanine rich sequence capable of producing G-quadruplex. Our CD analysis created the expected absorption peaks of iM at 280-287 nm and G4 at 260-265 nm, which is consistent with the literature 5,6 . The formation of iM and G4 were favored at different pH. Whereas i-motif displayed an acidic pH requirement, G4 formation was favored at near-neutral pH (Figure 2). This aligns with the hypothesis that hypoxia induced cellular acidity, as seen in PDR and AMD, leads to increased formation of i-motif, supporting a regulatory role of iM and G4 in the pathological and physiological expression of VEGF. Equally, a similar mechanism could be possible in the acidic microenvironments present in tumor cells 7 . Following linearization by boiling, both samples displayed small but significant changes to their pH dependent formation. The formation of both iM and G4 at higher pH (>6.0) displayed a higher-level of formation following linearization (Figure 3), indicating a propensity for structure formation during periods of denaturation such as translation. The emergence of iM and G4 was significantly influenced by the increasing concentration of complementary strand (Figure 4) while keeping the iM-forming sequence at a constant concentration. When the G4-forming sequence was added at 1:1 (orange) and 2:1 ratios (green), iM formation was sustained (Figure 4A). However, at 3:1 and 4:1 ratios, the iM peak decreased, and G4 formation was favored. Conversely, when the G4 forming sequence was held constant with increasing iM sequence, different outcomes were observed, with G-quadruplex formation being favored in all ratios except for 1:1 (orange) (Figure 4B). This indicates the capability of these sequences to coexist and support structure formation in the complementary strand. The formation of iM also displayed a dose-dependent linearization in the presence of SSB protein (Figure 5). SSB represents one of the intracellular proteins 8 . This represents a potential role of protein binding in iM formation and, consequently, in VEGF expression intracellularly. Similarly, this hints at a new target for VEGF targeting therapies. In summary, G4 and iM structures are regulators of gene expression in VEGF and a variety of other oncogenes, tumor suppressor genes and other non-cancer related diseases. The discovery of these structures represent a promising area for future drug development and disease prevention. In the case of VEGF, this could impact treatments for wet age-related macular degeneration, proliferative diabetic retinopathy and cancer.

Vascular endothelial growth factor (VEGF) plays a monumental role in the pathogenesis of wet age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR). In both AMD and PDR, VEGF mRNA and growth factor receptors (VEGFR1, VEGFR2) are upregulated due to retinal hypoxia, ultimately resulting in vascular proliferation that can cause blindness, vitreous hemorrhage, and traction-related retinal detachment. VEGF is a glycopeptide homodimer with several isoforms primarily responsible for pathologic angiogenesis in AMD and PDR 1,2 . Equally, pathological VEGF expression and subsequent angiogenesis plays a key role in tumor growth throughout the body, serving as the key mechanism for vessel development within tumors, which are necessary for uncontrolled proliferation of cancer cells 3 . G-quadruplex (G4) and i-motif (iM) producing (polyG- and polyC-rich) DNA sequences have been discovered within the promoter-proximal region of VEGF. The presence of these structures have also been implicated as key regulators of gene expression. Hypothesis: Cellular influences on G4 and iM formation represent potential sites for the development of dysregulated angiogenesis in eye disease and tumor growth 4 . Elucidating the cellular influences on the development of G4 and iM within the promoter proximal sequence of VEGF represent avenue for future treatment development and risk stratification for disease involving dysregulated angiogenesis, including ophthalmological conditions such as PDR and AMD as well as tumor growth. The goal of this project is to analyze the intracellular factors involved in the development of G4 and iM, including: o Cellular acidity which is influenced by tissue hypoxia as described in the pathogenesis of AMD and PDR. o Protein binding dynamics.

G-quadruplex and i-motif formation at VEGF promoter by CD analysis

Figure 2A: VEGF-C Strand with i-Motif formation at absorption peak ~280-285 (nm).

Figure 2B: VEGF-G Strand with G4 formation at absorption peak ~260-263 (nm).

Figure 3A: VEGF-C Strand with i-Motif formation at absorption peak ~280-285 (nm) following linearization by boiling.

Figure 3B: VEGF-G Strand with G4 formation at absorption peak ~260-263 (nm) following linearization by boiling.

o Complimentary double-stranded DNAbinding dynamics. o Structure formation following linearization by boiling.

Methods

For this work, iM and G4 were formed in-vitro using buffers with varying pH, which preferentially promoted the corresponding structure formation. The presence of these structures were then studied using Circular Dichroism (CD), a spectroscopic method that measures the absorption of right and left circularly polarized light in optically active molecules including DNA and proteins. Once the absorption peaks of G4 and iM were standardized, the formation of these structures in various conditions could be analyzed. The formation of G4 and iM was studied in the presence of competing guanine and cytosine-rich sequences as well as E. coli derived single strand binding protein (SSB) and following linearization by boiling. VEGF C and VEGF G oligodeoxynucleotides (DNA oligos) were purchased from IDT and Sigma Aldrich. The oligos were dissolved in water to a concentration of 100 picomoles/microliter. Appropriate amounts of oligos were mixed with appropriate buffers to carry out CD measurements. Buffers included: Sodium cacodylate pH 5.5 and 7.4, Tris-acetate pH 6.0, MES pH6.5, Tris-KCL pH 7.4. E. coli SSB was purchased from MCLAB with a concentration of 5 mg/ml.

Figure 4B: VEGF-G Strand competition with VEGF-C strand at varying concentrations. 1:1 ratio (Orange).

Figure 4A: VEGF-C Strand competition with VEGF-G strand at varying concentrations. 1:1 ratio (Orange).

Figure 1A: VEGF gene promoter with rich cytosine and guanine strands

Figure 1B: G-quadruplex and i-Motif structure

Figure 5: VEGF- C strand i-motif formation competition with SSB protein binding.

2025 Research Recognition Day

11

Biomedical Research

The Discovery of DNA Sequences Binding to a Cancer-Associated Protein by a Novel Technique Krishna Patel 1 , Daniel Ross 1 , Chirag Lodha 1 , Christopher Smith 1 , Levi Diggins 1 , Venkata Kolluru 1 , Olivia Lewis 1 , Christopher Syed 1 , Rachel Daley 1 , Rebecca Corallo 1 , Jacob Thaddeus 1 , Sundeep Bhanot 1 , Lauren Hiers 1 , Shane Donahue 1 , Rachael Baker 1 , Olivia McLean 1 , David Eagerton 1 , Ramu Anandakrishnan 2 , and Bidyut K Mohanty 1 1 Edward Via College of Osteopathic Medicine - Carolinas, Spartanburg, SC; 2 Edward Via College of Osteopathic Medicine - Virginia, Blacksburg, VA

Discussion

Introduction

Results

The ExoChew method demonstrates high efficiency in generating ssDNA libraries for DNA-protein interactions and DNA structural studies. Genome-wide sequencing data show that ExoChew achieves coverage (98.26%) comparable to sonicated double-stranded DNA (98.33%) with minimal regional biases and uniform read distribution. Using ExoChew, sequencing analysis successfully identified the consensus motif for hnRNP K binding in mouse DNA. This highlights its applicability in detecting protein-DNA interactions with high sensitivity . Further clustering analysis of polypyrimidine sequences revealed regions enriched in hnRNP K binding motifs. These findings validate ExoChew’s ability to map regulatory sequence patterns and binding sites. Localization of hnRNP K motifs relative to annotated genes demonstrates their association with functional regulatory regions, such as promoters and enhancers. This strengthens the clinical and biological significance of ExoChew in elucidating mechanisms of transcriptional regulation and its potential in cancer research. Conclusions • Efficient Library Preparation: ExoChew generates ssDNA libraries with genome coverage comparable to conventional dsDNA, providing uniform sequencing read distribution. • Motif Identification: ExoChew enables the detection of biologically relevant sequence motifs, such as those for hnRNP K binding, facilitating high-resolution mapping of protein-DNA interactions. • Functional Genomics Utility: Clustering analysis of polypyrimidine sequences uncovers regulatory sequence patterns linked to hnRNP K binding, with implications for gene regulation. • Clinical Relevance: ExoChew provides a scalable platform to study protein-ssDNA interactions in cancer biology, uncovering potential therapeutic targets for oncogenic pathways of hnRNP K.

• Genomic instability, a driver of cancer and various other genetic disorders, arises from errors in or during DNA replication, repair, and transcription. Single-stranded DNA (ssDNA) and secondary structures like G-quadruplexes and i-motifs (generated from polyguanine- and polycytosine-rich ssDNAs) play vital roles in gene regulation but can compromise genome integrity when mismanaged. • Proteins such as hnRNP K bind polycytosine-rich ssDNAs and I-motifs to regulate transcription and genomic stability. Overexpression of hnRNP K in cancers, including breast-, colorectal-, and pancreatic cancers, promotes tumor progression, metastasis, and chemoresistance. Despite its significance, studying protein-ssDNA interactions remains limited by inefficient ssDNA generation methods prone to reannealing and poor scalability. • HYPOTHESIS : We hypothesized that our novel ExoChew would produce high- quality ssDNA libraries suitable for investigating genome wide protein-ssDNA interactions and DNA secondary structures, advancing understanding of genomic instability in cancer and genetic disorders. • RESEARCH GOAL: Develop ExoChew , a novel enzymatic method for generating genome-wide ssDNA libraries, addressing inefficiencies of traditional approaches. • CLINICAL IMPORTANCE: ExoChew enables large-scale protein-ssDNA interactions , providing insights into cancer progression, genomic instability, and potential therapeutic targets.

Figure 3. PAGE gel showing ExoChew products of mouse genomic DNA and SSB binding. Genomic DNA was sonicated followed by ExoChew using T7 exonuclease or E. coli exonuclease III that generates single-stranded DNA. The products of sonication and ExoChew were incubated with SSB and analyzed by PAGE.

Library Type ‘‹ ƒ–‡† †• ͻͺǤ͵͵ š‘ Š‡™ •• ȋ ‘”™ƒ”† ƒ† ‡˜‡”•‡Ȍ ͻͺǤʹ͸ Figure 2. Comparison of DNA sequencing of sonication products and T7 exonuclease products of E. coli genome. Coverage (%)

Total number of reads 220,913 Number of pyrimidines in sequence

Mouse gDNA ExoChew products ↓ 6-HIS-hnRNP K ↓ Protein-ssDNA complex pulled down ↓ Extraction of DNA ↓ Conversion of ssDNA to dsDNA ↓ Sequencing

Polypyrimidine Sequence (PPS) (CT)(CT)CT)+ (CT)(CT)CT)+ (CT)(CT)CT)+ (CT)(CT)CT)+

Gap Length

1 1 2 2 1 1 2 2 5

12 15 12 15 12 15 12 15 12

131,265 88,951 163,534 121,680

CCC+ CCC+ CCC+ CCC+ CCC+

4,381 2,282 6,290 3,220

Methods

14,350

• Polypyrimidine sequence (PPS) = Sequence of 3 or more pyrimidines with no intervening purines, i.e. (C/T)(C/T)(C/T)+ • Gaps = intervening nucleotides between adjacent PPS • Gap Size = number of nucleotides in the Gaps • Cluster of PPS = series of PPS with Gap Size <= N (1 or 2) and Cluster Length >= L (12 or 15) • Cluster Length = number of Pyrimidines in the PPSs in the cluster. Does not include nucleotides in the Gaps. Table 1. Number of DNA fragments containing various pyrimidine sequences.

1 References

1.Patel, K., Lodha, C., Smith, C., Diggins, L., Kolluru, V., Ross, D., Syed, C., Lewis, O., Daley, R. and Mohanty, BK. (2023) ExoChew: An exonuclease technique to generate single-stranded DNA libraries. Biorxiv. doi: https://doi.org/10.1101/2023.10.02.560524. October 02, 2023. 2.Lu J, Gao FH. Role and molecular mechanism of heterogeneous nuclear ribonucleoprotein K in tumor development and progression. Biomed Rep. 2016 Jun;4(6):657-663. doi: 10.3892/br.2016.642. Epub 2016 Mar 29. PMID: 27284403; PMCID: PMC4887935.

Figure 4. Consensus motif of mouse ssDNA sequences pulled down using hnRNP K.

Figure 5. Location of consensus motif in sequencing reads relative to annotated genes.

Acknowledgments

Figure 1. The ExoChew technique and its uses.

The work was partially funded by VCOM’s REAP grant # 1032453 to BKM.

2025 Research Recognition Day

12

Biomedical Research

Common Connective Tissue Progenitor Cells are the Alternative Solution to the Failing Mesenchymal Stem Cells in Regenerative Medicine Angelo D. Monaco, OMS-I; Korinna Toth, OMS-I; Zoltan Hajdu, MD Edward Via College of Osteopathic Medicine, Carolinas Campus, Spartanburg, SC Introduction Results Results

Figure 4: In vivo CCTPCs

• The stromal mesenchymal stem cells (MSCs) were originally observed and described by Friedenstein: plastic adherent, spindle-shaped cells of the bone marrow, which can differentiate into osteogenic, fibrogenic and adipogenic lineages. • The in vitro differentiation potential were reported by numerous labs way broader then initially described. This gave the original momentum for developing stem cell therapies. However, the human in vivo results came out very disappointing. • In our lab, we have identified the hematopoietic-derived common connective tissue progenitor cells (CCTPCs), which differentiate into multiple connective tissues across the body. Objective: to compare the in vitro and in vivo behavior of MSCs and CCTPCs for their potential use in regenerative medicine. Cell Cultures: • Murine and human bone marrow mononuclear cells were analyzed for viability and CD45 (hematopoietic) expression then plated in T25 flasks until the first cell attachments were observed (approx. 2 weeks). • The free-floating cells were replated while the adhered cells were fed and passaged. This was repeated for 12 times. • Cells were continuously analyzed: pictures of the cultures (for morphology) and immunohistochemistry (for phenotyping) were performed at each manipulation. Mouse bone marrow transplants: • Lethally irradiated wild type mice (Ly=5.1) were tail vein injected with hematopoietic stem cells isolated from EGFP/Ly-5.2 transgenic mice. • Bone marrow and other tissue samples were collected from the chimeric mice starting 1 month and up to 2 years post transplant. Methods

Figure 1: Identification of MSCs and CCTPCs in cultured bone marrow cells. A B C

B

D

A

C

CD45 CD34 CD133

One-year post-transplantation virtually all bone marrow cells are GFP + (A, green). The mouse mitral valve (B – low mag, C-high mag) shows fusiform-shaped CCTPCs (red: CD45, green: GFP). Adult human mitral valve showing CCTPCs (red: CD45, green: CD133). Blue: YoPro1 (nuclei).

Immunohistochemistry on cultured, cytocentrifuged human bone marrow mononuclear cells. A – CD45 - MSCs (adherent), B – CD45 - (arrows) and CD45 + free floating cells, C – CD45 + /CD34 + /CD133 + CCTPCs (arrows). Green on A,B: YoPro1 (nuclei). Red on B: CD45 (hematopoietic). Figure 2: Morphology of the cultured bone marrow cells. A B C D 3 days After a month of culturing the bone marrow cells, the initially adhering cells (MSCs) are virtually the same as the initially free floating cells (CCTPCs) (A, D). B,C – CD45 labeling of the CCTPCs as they take a fusiform shape in culture. Figure 3: In vitro differentiation potential of the MSCs and CCTPCs. Hsp47 Alkaline Phosphatase Alcian Blue Oil Red O MSCs CD45 + /CD133 + /C D34 + Both cell populations have the same in vitro differentiation potential toward fibrogenic, osteogenic, chondrogenic and adipogenic lineages. MSCs 1 day

Discussion

• The human bone marrow contains only about 1% of MSCs, which is not enough for autologous stem cell therapies. • The CD45 + hematopoietic bone marrow cells give rise to the MSCs. • Only CD45 + hematopoietic cells contribute to different tissues in vivo, we never seen bone marrow-derived parenchymal cells.

CCTPCs

Conclusion

MSCs are only an artificial cell population, the CCTPCs are their real alternative to use in regenerative medicine.

References

This project was supported by the NIH COBRE SC Biocraft Pilot Project (ZH), the VCOM Research Eureka Accelerator Program (ZH). The authors wish to express special thanks to Kayla Afkinich for critical reading of our abstract. Acknowledgements 1. Friedenstein et al.: Heterotopic bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation. 6(2): 230-247. 1968. 2. Cagliani et al.: Immunomodulation by mesenchymal stromal cells and their clinical applications. J Stem Cell Regen Biol. 3:1-26. 2017. 3. Jackson et al.: Adult mesenchymal stem cells: Differentiation potential and therapeutic applications. J Postgrad Med. 53:121-127. 2007. 4. Ogawa et al.: Hematopoietic stem cells are pluripotent and not just “hematopoietic”. Blood Cells Mol Dis. 51(1): 3 -8. 2013. 5. Bucala et al.: Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair. Mol Med. 1(1): 71-81. 1994.

2025 Research Recognition Day

13