Pharmacy Undergraduate Research Theses and Honors Research Theses

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Undergraduate Research Theses and Honors Research Theses from the College of Pharmacy

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    The Reactivation/Resurrection of Organophosphorus-Inhibited/Aged Cholinesterases Using Novel Carbamate Quinone Methide Precursors
    (The Ohio State University, 2024-05) Murtha, Megan; Hadad, Christopher
    Organophosphorus (OP) compounds are a class of toxic compounds that are responsible for many toxic exposures and deaths annually. OP compounds inhibit acetylcholinesterase (AChE), an enzyme responsible for the hydrolysis of the neurotransmitter acetylcholine (ACh). OP compounds phosphylate the Ser203 residue of AChE, disabling the hydrolysis of ACh, leading to a cholinergic crisis where death can occur in severe cases. Oximes have historically been used to return the native enzyme from the inhibited enzyme in a process known as reactivation. However, a second step after OP inhibition can occur, known as aging, where spontaneous O-dealkylation occurs. Once aging happens, there are no approved therapeutics to recover the native enzyme. Oximes are also not capable of crossing the blood-brain barrier, and thus are not able to treat OP-inhibited AChE in the central nervous system (CNS) or any symptoms, like seizures, that can arise in the CNS. Previously, our lab has demonstrated effective (>20%) reactivation and resurrection using quinone methide precursors (QMPs) with varying substituents at the 4-position of a phenol framework. One library of such QMPs include dimethoxyindanone-linked QMPs; dimethoxyindanone is a cholinesterase inhibitor with a high affinity for AChE, specifically its peripheral binding site (PAS). These compounds performed significant reactivation of ethyl paraoxon (EP)-inhibited AChE. To expand further on this knowledge, indanone-linked QMP precursors were synthesized with both varying substituents at the 5- and 6-positions and with different lengths of the alkyl chains. In the future, these compounds will undergo cross-electrophile coupling (XEC) to both phenol-based and pyridyl-based QMPs. Our lab has shown that 4-amidophenol QMPs are effective reactivators and resurrectors of OP-inhibited and OP-aged cholinesterases. Due to their strong activity, carbamate QMPs were hypothesized to perform well due to the similarities between amides and carbamates. The synthesis and biochemical screenings of carbamate QMPs are detailed in this report. A total of 33 carbamate QMPs were synthesized with a variety of carbamate side chains and amine leaving groups. These compounds were tested for reactivation potential against OEt-inhibited butyrylcholinesterase (BChE) and AChE as well as resurrection potential against EP-aged AChE. Four (4) compounds showed significant activity in BChE. In AChE, five (5) compounds performed significant reactivation, and of these 5 compounds, two (2) showed significant resurrection activity. Also detailed in this report are biochemical in vitro screenings for reactivation of OP-inhibited BChE using 4-amidophenols. In these studies, 44 compounds were tested against 4 different OP surrogates and results varied significantly between differing inhibited forms. In OiBu-inhibited BChE, 30 of the 44 QMPs synthesized performed significant reactivation, with native enzyme activity as high as 60%. In OEt-inhibited BChE, 10 compounds were significant reactivators, with the top compound reaching 43% native enzyme activity. Overall, this report details the synthesis and screenings of a wide variety of QMPs capable of the reactivation and resurrection of various OP-inhibited and OP-aged forms of AChE and BChE.
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    A New Approach to Mitigating Androgen Receptor Mediated Hepatocellular Carcinoma
    (The Ohio State University, 2024-05) Fukuda, Mayu; Coss, Christopher
    Introduction: Hepatocellular carcinoma (HCC) is a form of liver cancer seen in those with long-term liver disease and is one of the leading causes of cancer mortality globally. Current standard of care has been ineffective at reducing tumor burden and improving patient outcomes, in addition to causing many serious and detrimental side effects. With correlations to prostate cancer (PCa), increased expression and activity of androgen receptor (AR) signaling by AR-full length (AR-FL) and AR splice variants (AR-SVs) have been suggested to promote HCC progression. We investigate a new approach to mitigating AR signaling in HCC through AR degraders, specifically Selective Androgen Receptor Degraders (SARDs) and Proteolysis Targeting Chimeric (PROTAC) molecules. Objective/Hypothesis: To evaluate androgen receptor (AR) antagonism with AR degrader compounds in AR(+) HCC. We hypothesize that AR degraders ARV-110 and UT-155 will exhibit anti-HCC effects by reducing cell growth, tumor development, and transcriptional activity in AR(+) HCC. Methods: To examine AR protein levels following treatment with AR-FL degraders UT-155 and ARV-110, we performed an immunoblot analysis on AR-FL(+) HCC cells. We then conducted cell viability assays to investigate anti-HCC effects on AR-FL(+) HCC cells. To determine if AR degradation blocks AR signaling in AR-FL(+)/AR-SV(+) HCC, we examined AR protein levels and AR-dependent cell viability following treatment of AR degraders in AR-FL(+)/AR-SV(+) HCC cells. Results: We showed lower AR-FL protein levels with UT-155 and ARV-110 in AR-FL(+) HCC cells. AR-FL degradation resulted in lower cell viability for higher concentrations of UT-155, but minimal reduction in cell viability with ARV-110 treatment after 24 hours in AR-FL(+) HCC cells. Due to UT-155 also binding outside of the traditional LBD in the AR enabling it to target AR-SVs, we expected reduced cell viability in AR-FL(+)/AR-SV(+) HCC. However, expected effects in AR-FL(+)/AR-SV(+) cells following ARV-110 treatment were unclear given its requirement to interact with the AR-LBD. Anti-HCC effects were seen in AR-FL(+)/AR-SV(+) HCC upon treatment with UT-155 but not with ARV-110. Conclusion: Despite different mechanisms of degradation, AR degraders UT-155 and ARV-110 demonstrated some reduced AR protein levels in AR(+) prostate cancer and HCC cells. However, with UT-155’s overt cytotoxicity, reduced AR protein expression seen in SNU423 AR-FL(+) HCC cells may be due to non-specific effects associated with cell death. As for ARV-110, the reduction in AR protein level with minimal cytotoxicity suggests AR specificity, which is important for avoiding toxicity in non-AR expressing cells. We will continue investigations of AR degraders on invasion potential and AR specificity in future studies.
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    Comparing Immune Reconstitution Following Post-Transplant Cy Myeloablative vs. Reduced Intensity Transplants
    (The Ohio State University, 2024-05) Zhu, Mandi; Choe, Hannah; Coss, Christopher; Swisher, Jeremy
    The incidence of graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (HSCT) remains a significant obstacle to successful outcomes. Although the efficacy of post-transplant cyclophosphamide (PTCy) as prophylaxis to GVHD has showed its ability to target and kill T cells that cause GVHD, PTCy myeloablative treatment has been linked to delayed T cell reconstitution. Reduced-intensity transplants can be utilized since it is associated with lower rates of acute and chronic GVHD, yet similar overall survival compared to Tac/MTX. Therefore, this proposed study aimed to investigate the effectiveness of PTCy reduced intensity (RIC) as prophylaxis for GVHD and compare the immune reconstitution ability with PTCy myeloablative intensity (MAC) and Tac/MTX control group. By utilizing the OMIP-042 protocol for staining antibodies in flow cytometry and FlowJo software for analysis, the results suggested that the monocyte populations were significantly affected after transplant. PTCy MAC patients (with GVHD) had significantly more monocytes compared to Tac/MTX MAC (no GVHD), while Tac/MTX RIC (no GVHD) patients had significantly more monocytes compared to PTCy RIC patients (either with GVHD or no GVHD). PTCy MAC patients (with GVHD) had increased monocyte populations compared to PTCy RIC patients (with GVHD), who also had increased monocyte populations compared to PTCy RIC (no GVHD) patients. There was no significant difference in immune reconstitution between GVHD vs. no GVHD patients. Further studies should be conducted to include patient samples on Day 90 and include more patient samples to detect the immune reconstitution trend. The results of this study could help understand the immune reconstitution in current prophylaxis for GVHD and improve patient outcomes associated with HSCT.
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    Repeated social defeat increases perineuronal nets via microglia and neuronal IL-1 signaling in a region dependent manner
    (The Ohio State University, 2024-05) Britt, Riley; Sheridan, John
    Chronic stress influences development and outcome of mood disorders in humans. Through the release of a myriad of inflammatory signals, chronic stress affects cognitive function, social behavior, and anxiety- and depression-like behaviors. In mice, repeated social defeat (RSD) has been shown to induce anxiety-like behavior and social withdrawal that is associated with a pro-inflammatory immune profile. One such change we documented, using Immunohistochemistry staining (IHC) was in the quantification of perineuronal nets (PNNs), an extracellular structure involved in stabilizing neuronal synapses. These nets showed an increase dependent on stress exposure in specific regions of the brain. Inclusion of PNN material has been found in microglia, suggesting the possibility of microglia mediated PNN formation. Here we administered PLX5622 to deplete microglia, which resulted in the depletion of PNNs in the RSD mouse model, supporting their potential involvement in PNN formation. Another route of PNN mediation was explored through IL-1 receptor-1 knockouts selectively in glutamatergic neurons. The IL-1 receptor is unique to the hippocampus and has been shown to be essential in neuronal sensitization. We showed that the absence of this receptor, and in turn its ability to sensitize neurons, reversed the PNN increase created through RSD in the dentate gyrus. To address PNN involvement in stress resultant behaviors, chondroitinase ABC (chABC) was administered through injection to the hippocampal region of the brain to enzymatically degrade perineuronal nets. The chABC enzyme did not effectively degrade the PNNs in our study, therefore deeming us unable to draw any conclusions about how their depletion impacts stress associated behavioral deficits. Ultimately, the expression of perineuronal nets may play a key role in the neuronal response to social stress.
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    Biofeedback-Based VR Therapy in Pediatric Patients
    (The Ohio State University, 2024-05) Abdallah, Clare; Olbrecht, Vanessa
    Children and adolescents who need surgery are at a particularly high risk for chronic postoperative pain and opioid consumption. Because of this, our team has been working to develop a non-pharmacologic approach to treating postoperative pain. Biofeedback-based virtual reality is a new therapy intended to reduce pain and anxiety in children and adolescents after they’ve undergone surgery. We have developed ForeVR, a device that measures real-time physiologic data and syncs this with VR breathing games. The aim of this project was to validate the data recorded by the ForeVR device by comparing it to data simultaneously recorded by the Massimo Rad-97, a standard monitoring device commonly used in clinical care. After analyzing the results, we concluded that ForeVR provides breathing data consistent with the Massimo Rad-97, particularly for average respiratory rate and overall respiratory rate time courses. Our next step is to gather more data for validity of ForeVR. Once the accuracy of data capture has been determined, we aim to perform an efficacy trial to assess ForeVR's ability to decrease postoperative pain and anxiety in children and adolescents needing surgery.
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    Cytochrome 2C19 Substrate Characterization of Organophosphate Pesticides
    (The Ohio State University, 2024-05) Revnew, Andre; McElroy, Craig
    Organophosphorus compounds (OPs) are a class of compounds used as pesticides and chemical warfare nerve agents that act by irreversibly inhibiting acetylcholinesterase. Potent OPs such as sarin, an agent used in the Syrian civil war, have been developed as chemical warfare nerve agents. Other compounds have been used in notable terror attempts such as the assassination of Russian dissenter Alexei Navalny. Additionally, OP pesticides are responsible for an estimated 385 million acute poisonings per year (Boedeker et al., 2020). Despite the occurrences of poisonings, little is known about the human metabolism of OPs. To address this, a high throughput fluorogenic assay was developed to evaluate human liver metabolism of OPs by cytochrome P450 enzymes (CYP450). Distinct substrates that are metabolized by different isoforms of CYP450 enzymes to yield fluorescent products were used to assay enzyme activity. A total of 45 OPs were screened for their inhibition characteristics of CYP2C19. It was found that while some agents inhibited the enzyme, others activated the enzyme. This led to further investigation into the metabolism of the OPs by CYP2C19 by evaluating the time-dependency of inhibition or activation. The most potent OP inhibitors were further investigated by determining their IC50. Several compounds were found to have IC50 values in the nanomolar range, indicating potent inhibition. Additionally, several compounds were found to demonstrate time-dependent inhibition characteristics, indicating likely metabolism by CYP2C19. This demonstrates that OP compounds exhibit inhibition of human CYP2C19, and likewise may be metabolized by the enzyme. Our results may guide future development of a therapy to treat organophosphate intoxication.
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    Characterizing Pathologically Relevant Mutations in Human Plastin 3 (PLS3)
    (The Ohio State University, 2020-05) Orchard, Matthew; Kudryashov, Dmitri
    Plastins are a conserved family of proteins that non-covalently crosslink actin filaments into bundled networks aiding in cellular adhesion, division, and motility. Plastins contain a Ca2+-binding head-piece domain, and two actin-binding domains (ABD1 & ABD2). Ca2+-binding to the head-piece domain of plastin inhibits the bundling of F-actin. Of the three human plastin isoforms, plastin-3 (PLS3) is expressed in most solid tissues, and has been linked to diseases such as cancer, spinal muscular atrophy, X-linked osteogenesis imperfecta (OI), and congenital diaphragmatic hernias (CDH). Recently, 8 different mutations resulting in full length PLS3 in patients has been linked to OI (A368D, E249_A250insI-L, A253_L254insN, N446S, & L478P) and CDH (E270K, W499C, & M592V). OI, more generally known as osteoporosis, is characterized by improper bone formation and low bone mineral density resulting in numerous peripheral or vertebral fractures from moderate or no impact. Specific therapies are lacking for the treatment of OI. CDH is a developmental disorder that is characterized by improper formation of the diaphragm, resulting in contents of the abdominal cavity moving up into the thoracic cavity. CDH is often fixed through surgical repair, and is associated with a high level of infant morbidity and mortality related to pulmonary issues. The role of PLS3 in these diseases is not well understood. Since the previously mentioned mutations are present in patients with these diseases, we know that PLS3 plays some underlying role. The learning goals of this project includes characterizing how each mutation affects PLS3 functionality in vitro, PLS3 behavior in cellulo, and further exploring the underlying link between PLS3 and OI/CDH. Each mutant was cloned and purified for in vitro characterization. Differential scanning fluorimetry (DSF) was done to obtain each protein's melting point, which can be used to assess each protein's stability. It was observed that in the osteoporosis-related mutants, there was a slight destabilization observed to PLS3 WT, which was apparent with a decrease in melting temperature across varying degrees (2.6-7.6°C). In the CDH-related mutants, there was no significant change in each protein's melting temperature with respect to PLS3 WT. Since the transition point for all of the proteins was well above physiological temperatures, this suggests that each mutant maintains its tertiary structure in the patients, and that the disease pathologies for OI and CDH may be a result of altering PLS3's binding and bundling capabilities. Next, each protein's binding and bundling capabilities were assessed by performing high-speed and low-speed co-sedimentation assays, respectively. None of the mutations affected PLS3's ability to bind to F-actin. However, the mutations have various effects on PLS3's ability to bundle F-actin. The CDH-related mutants bundled actin similarly to PLS3 WT in the absence of Ca2+, but these experiments must be repeated to confirm these results. The osteoporosis-related mutants could be categorized into 3 groups based on their bundling abilities; bundling incompetent (L478P), Ca2+-hypersensitive (N446S and A253_L254insN), and Ca2+-hyposensitive (A368D and E249_A250insI-L). Each OI-related mutant's sensitivity to Ca2+ regulation was confirmed through light-scattering combined with Ca2+ titrations. The osteoporosis-related mutants were expressed into fibroblasts and osteoblasts, which confirmed the biochemical results from the in vitro assays. The Ca2+-hypersensitive mutants and -hyposensitive mutants were localized primarily to the lamellipodia or focal adhesions respectively, while PLS3 WT was distributed between both regions. L478P did not strongly associate with any actin structures and was diffuse throughout the cytoplasm. The importance of Ca2+ in PLS3 localization was confirmed by observing the redistribution of WT and hypersensitive mutants from the leading edge to focal adhesions upon depletion of intracellular Ca2+. The CDH-related mutants were also expressed into cells and illustrate a different association to actin structures compared to PLS3 WT. All of these findings illustrate how mutations perturbing the fine regulation of PLS3 may be the underlying link in these human diseases. This presents PLS3 as a valuable research and therapeutic target in the biomedical field for disease pathologies.
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    (The Ohio State University, 2023-12) Schmitt, Jake; Mitton-Fry, Mark
    Foodborne illness resulting from non-typhoidal Salmonella enterica is a major contributor to global diarrheal diseases , with the potential to induce life-threatening morbidity within afflicted patients. In addition to its widespread impact, S. enterica has developed significant antimicrobial resistance, challenging the efficacy of current antibiotics. Through high-throughput screening (HTS), a promising hit compound was identified as an inhibitor of a unique Fructose-Asparagine (F-Asn) metabolic pathway utilized by S. enterica. This pathway converts F-Asn into Glucose-6-Phosphate (G6P) through a multi-step pathway involving several enzymes, including FraB . Inhibiting FraB leads to cell death due to the accumulation of a toxic intermediate, 6-Phosphofructose-Aspartate (6-P-F-Asp). Building upon the original hit scaffold, an extensive exploration of structure-activity relationships (SAR) has been undertaken in an attempt to enhance the potency of the original hit and create a novel narrow-spectrum antibiotic. The original hit offers a multitude of functionality that can be easily modified to explore the SAR, which allowed for the synthesis of 26 unique derivatives. While the SAR panel may not have yielded significantly more potent compounds, ongoing HTS efforts explore alternative chemical cores to effectively inhibit this crucial metabolic pathway.
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    Vape E-liquid formulation affects craniofacial growth and development
    (The Ohio State University, 2023-05) Richlak, Ethan; Cray, James
    Despite the growing popularity of electronic cigarette products (vape) over the past decade, there is currently a lack of regulation and consistency in the vape industry concerning formulation of the electronic liquids (E-liquids) used in these products. The two main constituents in most E-liquids are the humectants propylene glycol (PG) and vegetable glycerin (VG). It has been shown that PG-based E-liquids generate more carcinogenic carbonyls and increase the concentration of nicotine delivered compared to VG-based E-liquids. The purpose of this study was to test the hypothesis that a VG-based formulation (30:70 PG:VG) would result in fewer negative effects on craniofacial growth and development compared to an evenly concentrated formulation (50:50 PG:VG). Two groups of pregnant mice, the 50:50 PG:VG group and the 30:70 PG:VG group were split into three groups each, nicotine + carrier exposure (vape), carrier only exposure (carrier), and control exposure (control) to produce in utero exposed litters. Somatic and cephalometric measurements of the resulting litters were used to compare the impact of these different E-liquid formulations on craniofacial development. For the 50:50 PG:VG groups, there was significant decreases in cranial base length (Vape vs Carrier, p=0.032, Vape vs Control, p=0.010), weight (Vape vs Control, p=0.002), and posterior facial width (Vape vs. Control, p=0.009). For the 30:70 comparisons, there was a significant decrease in midfacial width (p=0.040) due to vape exposure, but no other discernible differences. The results support the original hypothesis by showing more significant differences resulting from the 50:50 PG:VG formulation compared to the 30:70 PG:VG formulation and suggest greater effects with increased PG concentrations. This insight can be important for decision making regarding the regulation of E-liquid formulations.
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    Delivery of DNA Origami Vaccine using Alginate Microparticles
    (The Ohio State University, 2023-05) Djouadi, Amani; Castro, Carlos
    Deploying the immune system to seek out and destroy cancer cells is a rapidly developing strategy for cancer treatment and prevention. This is accomplished via vaccination against tumor antigens to elicit an antigen-specific anti-cancer immune response. DNA origami nanodevices (DO) have shown enhanced antigen and adjuvant delivery to antigen-presenting cells (APCs) and stimulation of antigen-specific CD8+ T cells. This enhancement is due to DO's unique properties, including size, shape, and programable payload capacity. Despite these features, free DO-based vaccine (DO-VAC) faces a harsh physiological environment within the lymphatic system and the periphery that compromises its integrity and longevity once injected subcutaneously. Previous work demonstrated that alginate-mediated release of vaccines is advantageous to traditional vaccines due to prolonged release of antigen, payload protection, and promoting adequate delivery of payload to lymph nodes. Therefore, it is believed that DO-VAC delivery can be further enhanced by encapsulation in alginate. Encapsulation could address the rapid clearance of DO-VAC, produce a strong persistent immune response, and remove the need for booster shots in clinical settings. Agarose gel and TEM imaging show that DO retained structural stability once released from alginate particles. Based on release studies, alginate encapsulated DO exhibits sustained release properties in release conditions and stability in storage conditions. Encapsulated DO also showed prolonged activity in dendritic cells compared to unencapsulated DO.
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    Optimization of Expression and Purification of Full-Length and Truncated Botulinum Neurotoxin A Light Chain (BoNT/A-LC) for Assay Development
    (The Ohio State University, 2023-05) Wilmer, Melissa; McElroy, Craig
    The research conducted focused on the protein expression and purification of BoNT serotype A Light Chain (BoNT/A-LC), the portion of the BoNT protein specifically involved in SNAP-25 cleavage, in both a truncated and full-length form.1 A variety of conditions were manipulated to determine optimal protein expression conditions: including time after IPTG induction, growth temperature, E. coli strain, and media type. Immobilized metal affinity chromatography (IMAC) eluted using Fast Protein Liquid Chromatography (FPLC) were used to purify the protein based on the affinity of the 6xHis-tag engineered into the protein and then the purified protein was cleaved using TEV protease to remove the 6xHis-tag. Additional methods of purification such as Size Exclusion Chromatography (SEC) and anion exchange were used to further purify the protein. The optimization and purification experiments were analyzed using SDS-PAGE, and fractions corresponding to peaks on the FPLC chromatogram. The empirically determined optimal conditions for expression were BL21(DE3) cells grown in terrific broth (TB) for 22 hours following 1 mM IPTG induction at OD600 of 0.6 with shaking at 20 °C. The pure protein will be used to develop a Fluorescence Resonance Energy Transfer (FRET) Assay that measures BoNT/A-LC cleavage of the SNAP-25 complex using a fluorescence plate reader to obtain the relative fluorescence unit (RFU) values at 470 nm and 527 nm.2 The detection of BoNT/A-LC activity will be calculated by the decrease in the emission ratio (RFU 527/RFU 470) compared to the control wells. This assay will allow for the quantification of the inhibitory potency of novel BoNT/A-LC inhibitors compared to a known inhibitor.
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    Pilot Student-Led Point-of-Care Testing Program
    (The Ohio State University, 2023-05) Ruzga, Melanie; Woods, Zach
    The College of Pharmacy Point of Care(POC) Testing Training course was implemented to encourage and educate students to engage with communities through health screening events. This training and outreach help to provide improved access to POC services such as glucose testing meters and counseling. The goal of this research is to analyze the data collected from screening events which occurred during the first two years of the POC program and then propose other services that should be incorporated into future screening events. This is a retrospective study evaluating patients' self-reported health conditions and measured blood glucose. This data was drawn from questions answered on 169 unique Blood Glucose Release Forms collected during screenings, which occurred at seven sites throughout Columbus, Ohio between October 2020 to April 2022. Data was summarized then analyzed to investigate the opportunity for additional screening services that will best support the Columbus population currently being reached by these screening events. Patient forms were submitted with varying degrees of completion. With 167 responses, the average age was 36.3 years old. 76 patients were 20-30 and 35 were 50-65. With 159 responses, 73 reported male. With 142 responses, 38 reported their race as Black/African American. With 121 responses for minutes of exercise/week, 12 patients reported 0 minutes, 32 reported 30 minutes and 19 reported 60. With 101 responses for previously known health conditions, 34 reported high blood pressure (BP) and 12 diabetes/high blood sugar. With 157 measured blood glucose levels, 119 had normal values, 26 elevated, 12 high, and 0 considered low. The collected results help to define the population that participates in the program's screening events. Based on risk factors defined by the American Heart Association, the addition of blood pressure (BP) screenings and heart health education may be the most impactful addition to benefit this program's patient population. Next steps for the POC service is to develop training for student pharmacists on this new service with the end goal of improving health outcomes for the greater Columbus community.
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    Quantification of Resveratrol in Red Wine using Liquid Chromatography—Surface-Enhanced Raman Spectroscopy (LC-SERS)
    (The Ohio State University, 2022-12) Wang, Kristen; Schultz, Zachary
    Resveratrol is a stilbenol molecule found in red wine that may have many health benefiting properties, such as preventing inflammation in blood vessels. This metabolite is found naturally in grapes and is one of the reasons why red wine is promoted as healthy enough to drink a glass every day. This project aimed to quantify the amount of resveratrol in a sample of red wine using LC-SERS. SERS is a non-destructive quantitative method that measures the inelastic scattering of light, where the signal is enhanced by adsorbing the sample on a metal nanostructure. For this project, a thermally evaporated silver substrate placed in a homemade flow cell for SERS detection. Red wine was separated through by using reverse phase chromatography before flowing into the flow cell where the SERS signal was obtained. A calibration curve of resveratrol was made by flowing different concentrations of resveratrol though the flow cell. Once that was achieved, the resveratrol was injected into the HPLC to determine the retention time. By separating resveratrol from the other components of red wine, the SERS signal can be compared to the reference spectrum for identification and intensity provides quantitative information. Data analysis took place on MATLAB, using airPLS as a background subtraction method and Pearson correlation to compare sample spectra with a reference spectrum.
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    Quantitative Assays of Mismatch Repair Activity
    (The Ohio State University, 2021-05) Yu, Tiffany; Fishel, Richard
    Defects in the human mismatch repair (MMR) genes are the cause of Lynch syndrome as well as 10-40% of sporadic colorectal, gastric, endometrial, ovarian, and upper urinary tract tumors. The MMR system recognizes and repairs polymerase misincorporation errors and functions as a sensor in DNA damage signaling. MMR-deficient cells have increased mutations from unrepaired errors which drive tumorigenesis, and the lack of a DNA damage response results in resistance to several common cancer chemotherapeutic drugs. Single-molecule methods have proven to be insightful to the MMR process, however, evaluating whether the mismatch repair proteins used in experimentation match wildtype activity is vital for accurate results in single-molecule studies. This project will develop an assay to evaluate MMR activity on a variety of mismatched substrates. Several constructs will be built as part of the project to test the efficiency of the assay and MMR proteins through ssDNA and dsDNA hybridization. Wild-type cell extracts will be isolated and mixed with the mismatched constructs to evaluate wild-type activity and optimized conditions. Cell extracts without MMR activity will be mixed with MMR proteins and assayed for complementation using gel electrophoresis.
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    Preliminary in silico Docking Study with Selected FDA Approved Drugs for Neurological Disorders
    (The Ohio State University, 2021-05) Slemp, Khayla; Carcache de Blanco, Esperanza
    The current study focused on five FDA approved drugs that are prescribed to treat various neurological conditions. The drugs selected for the study include Memantine, Levodopa, Tetrabenazine, Gabapentin, Zolpidem. In silico docking methods were used to assign Ki values to the FDA approved drugs with the 10 different targets. The in silico study evaluated if these five drugs would bind to 10 targets that are not known to bind to the five drugs tested. The targets selected include 5HT5 receptor, 5HT4 receptor, α7 nicotinic acetylcholine receptor, α4β2 nicotinic acetylcholine receptor, D4 receptor, monoamine oxidase A (MAO-A), monoamine oxidase B (MAO-B), GABAA, GABAB, and calcium-sensing receptor (CaSR). The results from the study concluded that all drugs in this study have an interaction with at least one of these 10 targets. These off-target interactions could be useful in discovering if they are the result of the drug's side effects, or if they are part of the drug's mechanism of action. The data from this study could also be useful for repurposing the drugs to treat other conditions. Further research needs to confirm if any of the significant interactions are real, and what type of change they cause in the body.
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    A Substituted Indole Scaffold as a Class of Allosteric HIV-1 Integrase Inhibitor
    (The Ohio State University, 2019-12) Bockbrader, Ross H.; Fuchs, James R.
    Although a number of drugs have been developed and approved to combat HIV over the past 40 years, mutations of the virus and its protein targets have necessitated the continued discovery and design of even more novel compounds today. In the Fuchs lab, we are approaching this problem by targeting an allosteric binding site on HIV-1 integrase, an integral enzyme that is responsible for integrating the viral genome into the host's DNA. This allosteric site is the same binding site as that used by the cofactor LEDGF/p75, a host cell protein that promotes the activity of integrase. Binding of small molecule drugs at this allosteric binding site along the CCD dimer interface of integrase results in the inactivation of the protein due to the formation of aberrant integrase multimers. This hyper-multimerization occurs as a result of interaction of the CTD subunit of another integrase protein with the CCD dimer interface. We are currently synthesizing new inhibitors based on a well-studied quinoline core scaffold and scaffold hoping to an indole core scaffold. The indole core scaffold was chosen due to the existence of well-established synthetic routes and the ease of functionalization of the indole core. In addition, the tilt of aromatic ring in the allosteric binding pocket has been hypothesized to improve binding of the compounds in the presence of known mutations, namely the A128T mutation. Inhibitors of this type are also structurally unique and may lead to the development of new intellectual property. The ultimate goal of these studies is to develop compounds with greater intregrase inhibitory activity through binding to the LEDGF/p75 site than previously studied compounds by optimizing functional groups around the indole core.
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    (The Ohio State University, 2019-05) Ho, Aimee; Fuchs, James
    Natural products isolated from plants of the Phyllanthus genus, specifically members of the arylnaphthalene subclass of lignan lactones, show promise as a potential treatment option for ovarian cancer. These compounds are worth examining further because several structurally related synthetic analogues have displayed highly potent cytotoxic activities against ovarian cancer cell lines in the nanomolar range. Our lead compound, PHY34, exerts its cytotoxic effects by inhibiting autophagy in ovarian cancer cells which is followed by apoptotic cell death. Potent experimental agents with attractive mechanisms of action, including autophagy modulators, are needed to continue to fight cancer, potentially overcoming the problem of chemoresistance and increasing the chances of survival for patients. In order to further assess and obtain structure-activity relationship data for this class of compounds, several new analogues were synthesized by modifying the D-ring on the diphyllin core of the phyllanthusmins. The diphyllin precursors, representing the core ring system, were synthesized using a five-step method with 3,4-dimethoxybenzaldehyde as the precursor. At that stage, the sugar moiety could then put on at the C7 position to complete the synthesis of the derivatives. A total of six analogues with D-ring modifications were made, and characterization data was collected for both the diester and diphyllin precursors to confirm the structural assignments. This included collection of both proton and carbon NMR spectra for each compound for a total of twelve proton NMR spectras and twelve carbon NMR spectras as well as the determination of their accurate masses using mass spectrometry. Biological data for four of these compounds were also obtained to assess their potency against two ovarian cancer cell lines.
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    Novel Selective Estrogen Receptor Modulator (SERM) OSU-ERβ-12 Demonstrates Anti-fibrotic Efficacy
    (The Ohio State University, 2020-12) Klein, Abigail; Coss, Christopher
    Purpose: Based on demonstrated ERβ-selective transcriptional activity in a cellular model, it was hypothesized that in vivo, efficacious doses of the novel ERβ agonist OSU-ERβ-12 could be determined that have minimal activity in ERα-dependent tissues. These doses were hypothesized to have an anti-fibrotic effect in a Non-Alcoholic Steatohepatitis (NASH) liver disease model. Design: We used uterotrophic stimulation in an estrogen-naïve female mouse model to assess ERα activity. Briefly, groups of mice were administered doses of the ERβ selective agonist OSU-ERβ-12 and compared to a control ERβ-selective ligand, LY500307. The ERα effects of each ligand were characterized using bodyweight-normalized uterine tissue weights. Then doses of OSU-ERβ-12 were given to mice treated with 6 weeks of CCl4 to establish efficacy of OSU-ERβ-12 as an antifibrotic compound. Liver sections were stained with picrosirius red and the staining was then quantified using computer analysis. Results: OSU-ERβ-12 demonstrated ERβ-selective activity at doses <30 mg/kg, with 10 mg/kg being the highest selective dose. This dose was also efficacious at reducing the amount of fibrotic tissue picrosirius red staining seen in a liver section that was challenged with CCl4. LY500307 showed increased toxicity and decreased efficacy as compared to OSU-ERβ-12. Conclusions: Consistent with cellular activity data, uterotrophic stimulation data and the CCl4 challenge data suggest that 10 mg/kg doses of OSU-ERβ-12 are both ERβ-selective and efficacious in a CCl4 model of liver disease.
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    Antibiotic Efficacy in Treating Variant Pseudomonas aeruginosa and Staphylococcus aureus Biofilms
    (The Ohio State University, 2019-05) Li, Anthony; Stoodley, Paul
    This study investigates antibiotic efficacy against planktonic vs biofilm bacteria in vitro, using eight different antibiotics: carbenicillin, ciprofloxacin, colistin, gentamicin, meropenem, rifampicin, tobramycin, and vancomycin. The main aim is to see which antibiotic can best clear the biofilm, which antibiotics correlate to the appearance of variant colonies, and which antibiotics can eradicate variant colonies. Biofilms are resilient toward chemical and physical challenges due to the secretion of extracellular polymeric substance and high cellular density. This makes biofilm infections difficult to treat. Some examples of biofilm infections are osteomyelitis and cystic fibrosis pneumonia most commonly caused by S. aureus and P. aeruginosa respectively. This study uses bioluminescent bacteria, S. aureus SAP231 and P. aeruginosa Xen41 as the test subjects for antibiotic efficacy. There are four major components to this study: first, the minimum inhibitory concentration of planktonic cells against antibiotics were found. Second, the antibiotic delivery mechanism was tested to see if mode of antibiotic delivery has an effect on biofilm clearance. Third, antibiotics and combinations of antibiotics are tested against biofilms. Lastly, the variant colonies are quantified to see which antibiotics correlate to variant colony formation. The results show that biofilm infections are more difficult to treat than planktonic bacteria, and the use of multi-combinatory antibiotics are effective at treating biofilm. A combination of antibiotic with vancomycin was very effective in reducing biofilm and variant colonies in S. aureus. A combination of antibiotic with ciprofloxacin was very effective in reducing biofilm and variant colonies in P. aeruginosa.
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    Characterization of microRNAs as Post-Transcriptional Modulators of DNA Topoisomerase IIα in Etoposide (VP-16) Resistant K562 Cells.
    (The Ohio State University, 2019-05) Kania, Evan; Yalowich, Jack; Elton, Terry
    The enzyme DNA topoisomerase IIα (TOP2α) induces covalent complexes with DNA and produces transient double-strand DNA breaks crucial for processes such as replication and normal chromosomal dysjunction at mitosis. TOP2α is an important target for clinically effective anticancer agents, such as etoposide (VP-16), since these drugs stabilize the otherwise short-lived enzyme-DNA covalent complexes, thereby inducing cytotoxic DNA damage. However, the efficacy of these agents is limited by chemoresistance. Our lab has characterized acquired resistance to VP-16 in human leukemia K562 cells. The cloned resistant cell line, K/VP.5, contains reduced levels of TOP2α compared to parental K562 cells. The goal of this project is to test the hypothesis that TOP2α levels are decreased in K/VP.5 cells, in part, through miRNA-mediated mechanisms. Pooled miRNA qPCR profiling experiments were performed to investigate the expression levels of ~500 miRNAs in K562 and K/VP.5 cells. hsa-miR-9-3p and -5p (miR-9-3p and -5p) were overexpressed in K/VP.5 cells compared to K562 cells. The TOP2α 3ʹ-UTR harbors putative miRNA recognition elements (MRE) for these miRNAs. Therefore, these miRNAs were chosen for further study. To assess post-transcriptional regulation of TOP2α by miRNAs, a dual luciferase reporter plasmid harboring the entire 3ʹ-UTR of TOP2α mRNA (998 bp) was constructed (psiTOP2α/UTR). Transfection with psiTOP2α/UTR demonstrated decreased luciferase expression in K/VP.5 compared with K562 cells, suggesting altered post-transcriptional regulation in resistant cells. K562 cells that were co-transfected with psiTOP2α/UTR and miR-9-3p or -5p mimic resulted in a statistically significant decrease in luciferase expression only for miR-9-5p. Mutating the putative miR-9-5p seed sequence prevented the decrease in luciferase activity, demonstrating a direct interaction of this miRNA with the MRE of TOP2α. Immunoblotting for TOP2α in K562 cells transfected with miR-9-3p or -5p mimic resulted in decreased TOP2α protein compared to mock transfected K562 cells. Conversely, immunoblotting for TOP2α in K/VP.5 cells transfected with miR-9-3p or -5p inhibitor resulted in an increase of TOP2α protein, strongly suggesting a role for both miRNAs in acquired resistance to VP-16. Our findings indicate that miR-9-3p and -5p regulate TOP2α expression levels. In addition, results presented here contribute to the elucidation of chemoresistance mechanisms and have the potential for circumvention of drug resistance by modulation of miRNA concentrations.