2017 Denman Undergraduate Research Forum Accepted Student Abstracts Laboratory/Cellular Health Sciences

Category: Laboratory/Cellular Health Sciences

Title: The nuclear pore complex protein NupA is required for normal localization of the mRNA export factor Gle1 in Aspergillus nidulans

Student Presenter: Leymaan Abdurehman

Faculty Advisor: Osmani, Stephen

Abstract: Mitosis is a fundamental process for the development of multicellular organisms. While significant insights have been obtained into mitotic mechanisms, the disassembly and reassembly of Nuclear Pore Complexes (NPCs) is not well understood. NPCs carry out transport across the nuclear envelope. During mitosis in Aspergillus nidulans, NPCs are disassembled during mitotic entry. Some NPC proteins locate to mitotic structures and operate transport functions in interphase. NupA was identified as a novel NPC protein that co-purified with the conserved NPC protein Nup2 during interphase and mitosis. During mitosis, both proteins locate to mitotic chromatin with unknown functional significance. Deletion of NupA leads to activation of the Spindle Assembly Checkpoint (SAC) as well as late mitotic defects. NupA was found to be important for normal localization of NPC proteins Mad1 and Ndc1. We are interested in determining whether additional NPC proteins localization depends on NupA. As a candidate, we chose Gle1, an essential NPC protein involved in mRNA export. To delete NupA, a gene replacement construct was generated by fusion PCR and transformed into A. nidulans. Since NupA is an essential gene, we analyzed the phenotype of its deletion using the heterokaryon rescue technique. NupA deletion was confirmed through diagnostic PCR with primers flanking the deletion construct. Live cell confocal microscopy indicated that the localization of Gle1-GFP was affected in the absence of NupA. While Gle1-GFP locates exclusively at the nuclear periphery in WT cells, it additionally located in the cytoplasm without NupA after mitosis in G1. Since Gle1 is an important mediator of mRNA export, we speculate that mRNA export might be affected in NupA-deleted cells. Our studies suggest that NupA has important roles in correct localization of NPC proteins particularly after mitosis. We speculate that NupA performs mitotic functions on chromatin that are required for normal localization of NPC proteins. In the future, it will be important to examine whether the localization of other NPC proteins also depend on NupA and Nup2 and if their chromatin location is required for NPC segregation during mitosis. Category: Laboratory/Cellular Health Sciences

Title: Targeting MDSC enhances the anti-tumor effects of checkpoint inhibitor therapy in a mouse model of melanoma

Student Presenter: David Abood

Faculty Advisor: Carson III, William

Abstract: Myeloid derived suppressor cells (MDSC) are a heterogeneous population of early myeloid cells that expand in tumor-bearing hosts. MDSC play a critical role during tumor evasion and have been linked to loss of immune effector cell function through a variety of mechanisms such as the generation of reactive oxygen and nitrogen species (ROS and RNS) and release of immune-suppressive cytokines. Our group discovered that MDSC express a kinase known as Bruton's tyrosine kinase which can be inhibited with the kinase inhibitor ibrutinib which is FDA-approved to treat leukemias. Tumors express immune-checkpoint molecules including programmed death ligand 1 (PD-L1) to escape immune destruction. PD-L1 binds to its receptor, programmed cell death protein 1 (PD-1), which inhibits the function of T cells. Anti-PD-L1 monoclonal antibodies are in clinical use and their administration prevents the engagement of PD-L1 to its receptor, allowing an effective immune response to proceed in upwards of 30% of patients. We hypothesized that inhibition of MDSC with ibrutinib would enhance the activity of a PD-L1 blocking antibody (Ab). We therefore investigated the effect of ibrutinib in combination with an anti-PD-L1 antibody on tumor growth and the levels of MDSC using a mouse model of melanoma. C57BL/6 mice were inoculated with 1x105 B16F1 melanoma cells. Mice were treated with either vehicle control, ibrutinib (12.5 mg/kg/day) via drinking water, anti-PD-L1 antibody (25 µg) injected three times a week, or a combination. Tumors were measured three times a week using digital calipers. Ibrutinib or anti-PD-L1 Ab treatment alone had a modest effect on tumor growth compared to control-treated mice while the combination treatment led to a 32% reduction in tumor volume. Mice treated with ibrutinib, anti-PD-L1 Ab or the combination had average spleen MDSC levels of 6.57%, 5.64%, and 5.46% respectively, compared to the vehicle at 8.58%. These results show that ibrutinib can enhance the anti-tumor effects of anti-PD-L1 Ab in the setting of melanoma. Category: Laboratory/Cellular Health Sciences

Title: Effects of sucrose and omega-3 dietary modification on lipid peroxidation in liver of mouse models of breast cancer and chemotherapy

Student Presenter: Xin Ni Au

Faculty Advisor: Orchard, Tonya

Abstract: Will dietary sucrose and long-chain omega-3 fatty acids (EPA+DHA) affect lipid peroxidation in the liver of mouse models of chemotherapy or breast cancer? Doxorubicin, a widely used chemotherapeutic agent, generates free radicals which may increase lipid peroxidation and oxidative stress. Research suggests that sucrose increases oxidative stress; conversely EPA+DHA limit oxidative stress and lipid peroxidation in the brain of mice treated with doxorubicin. It is unknown how the combination of sucrose and omega-3 fatty acids impacts lipid peroxidation in metabolically important tissues such as the liver in the presence of chemotherapy or mammary tumors. Livers from mouse models of breast cancer (Tumor model: n=32; 8/diet) and chemotherapy (Chemo model: n=40;10/diet) were analyzed for 4-hydroxynonenal (4-HNE), a lipid peroxidation marker, using an enzyme-linked immunoabsorbent assay. In both experiments, 8-9 week old female C57BL/6 mice were ovariectomized and randomized to diets one week later. Tumor model mice were fed low sucrose diets, with either 0% or 2% kcal from EPA+DHA. Two weeks later, mice were injected with metastatic mammary tumor cells or control; after 21 days, tissues were collected. Chemo model mice were fed low sucrose, 0% or 2% kcal EPA+DHA diets, or high sucrose, 0% or 2% kcal EPA+DHA diets. Mice were injected with doxorubicin based chemotherapy or saline two and four weeks later; tissues were collected 11 days after second injection. Mean liver 4-HNE in tumor mice (0.0649ug HNE-BSA/ugProtein;SEM 0.0066) vs. control (0.0613ug HNE-BSA/ugProtein;SEM 0.0066) was not significantly different (p=0.71) and did not differ by diet (p=0.54). Similarly, mean liver 4-HNE in chemo mice (0.0817ug HNE-BSA/ugProtein;SEM 0.0114) vs. control (0.0877ug HNE-BSA/ugProtein;SEM 0.0117) was not significantly different (p=0.72) and did not differ by diet (p=0.42). In conclusion, we found no differences in liver lipid peroxidation in chemotherapy-treated or tumor mice vs. controls when fed varying levels of sucrose and EPA+DHA. Category: Laboratory/Cellular Health Sciences

Title: The role of CCL2 chemoattractant in lumbar myeloid cell trafficking and locomotor recovery following thoracic spinal cord injury

Student Presenter: Kimberly Berry

Faculty Advisor: Basso, Michele

Abstract: Spinal cord injury (SCI) induces inflammation and damage across the cord that hinders neuronal plasticity and locomotor recovery. Previous studies of spinal cord response to injury show increased levels of chemoattractants (CCL2) and cell adhesion molecules (ICAM-1), chemotactic agents that induce the movement of specific cell types. Peripheral myeloid cells invade the lumbar region after thoracic SCI, rendering the lumbar microenvironment refractive to rehabilitation. We hypothesized that removal of CCL2 via genetic knockout (KO) will decrease myeloid cell infiltration into the lumbar spinal cord after SCI, to promote a more permissive microenvironment for rehabilitation interventions compared to typical, wild-type SCI (WT). We also hypothesized that downhill treadmill training 14-21 days post-injury would improve functional recovery in both KO and WT models. We used four groups with SCI at T9: CCL2KO exercised (n=8) or unexercised (n=8), and WT exercised (n=8) or unexercised (n=4). We quantified monocyte infiltration (IBA-1/p2ry12) and ICAM-1 expression via immunohistochemistry to identify co-regulatory features between chemoattractants and cell adhesion molecules in the trafficking of peripheral myeloid cells into the lumbar cord. Locomotion was assessed via Basso Mouse Scale (BMS) and 2D kinematics. Contrary to our hypothesis, the CCL2KO resulted in lower BMS scores than WT with and without training (untrained, p Category: Laboratory/Cellular Health Sciences

Title: Optimization of bioartificial muscle to discover the mechanotransduction pathways of muscle atrophy in microgravity

Student Presenter: Perry Blough

Faculty Advisor: Lee, Peter

Abstract: Of the many physiological changes seen after spaceflight in astronauts, skeletal muscle atrophy is among the most pronounced and damaging to long-duration spaceflight missions. However, the exact mechanism of spaceflight induced skeletal muscle atrophy is not completely understood. Under the funding of NASA and the Ohio Space Grant Consortium, our project is to launch tissue- engineered skeletal muscle constructs termed BioArtificial Muscle (BAM) into space to study the mechanotransduction pathways leading to isolated skeletal muscle atrophy in microgravity. To translate the results of this experiment into medically impactful applications for future spaceflight, it is essential to optimize BAMs to closely resemble in vivo skeletal muscle tissue. Our previous experiments resulted in the lack of differentiation of BAMs. Those that went through differentiation demonstrated weak attachments to the pillars, suggesting there are much more optimal methods to be discovered. Thus, we aim to test for the optimal growth environments of BAMs through variations of cell concentrations, surface area available for BAM attachment, total volume of BAM solution, and BAM length. BAMs will be grown in six-well culture plates and incubated at 37 degrees Celsius. Optimization results will be based upon multiple factors. Firstly, BAM morphology will be examined using immunohistochemical staining for muscle specific tropomyosin. Secondly, we will measure passive and active forces in response to electrical stimulation of the muscle tissue. The optimized conditions will be utilized in all BAMs created for the expected launch and future spaceflight experiments. During spaceflight, we will measure real-time passive and active BAM forces as well as conduct genomic analysis with RNAseq to determine changes in gene expression. Ultimately, the results of this project will contribute to the advancement of medicine and technology that can be utilized in circumventing skeletal muscle atrophy experienced during spaceflight. Category: Laboratory/Cellular Health Sciences

Title: Bioaccessibility of a-tocopherol regardless of form is decreased dose-dependently and only fat- dependent for a-tocopherol acetate

Student Presenter: John Bouranis

Faculty Advisor: Bruno, Richard

Abstract: The bioavailability of unesterified a-tocopherol (a-T-free) in humans is unaffected by fat intake but increases in a fat-dependent manner when ingesting esterified a-tocopheryl acetate (aT-acetate). We hypothesized that vitamin E bioaccessibility would decrease in an a-T-free dose dependent manner, but that a-T-acetate would have lower bioaccessibility, compared with a-T-free, that increases in a fat- dependent manner. We performed simulated digestions under in vitro conditions to investigate dose (15 vs 268 mg) and a-T form (esterified vs unesterified) in relation to increasing fat content provided by non-fat milk (0.2 g), reduced-fat milk (4.9 g), and whole milk (9 g). Simulated digestion of the gastric and intestinal phases was performed and a-T was evaluated by HPLC-ECD from the chyme to determine potential losses during digestion and in the aqueous micelle fraction to calculate bioaccessibility. Recovery of a-T in the chyme was >90% regardless of a-T form, dose, and milk-fat content. For a-T-free, bioaccessibility was 81.8 ± 1% and 36.8 ± 0.3% for low-dose and high-dose digestions, respectively, but were unaffected by milk-fat content. a-T-acetate similarly exhibited 75.4 ± 4.4% bioacessibility during low-dose digestions regardless of milk-fat content, but increased in bioaccessibility from 11.9 ± 2.1% in non-fat milk to 41.2 ± 2.1% in reduced-fat or whole milk treatment when a high-dose was digested. a-T-free and a-T-acetate did not differ in bioaccessibility regardless of fat content (81.8 ± 1.1% vs 75.4 ±4.4%) when low-dose aT was subjected to simulated digestions. However, at a high-dose, bioaccessibility did differ between a-T-acetate and a- T-free in the absence of milk-fat, but did not differ when digested with reduced-fat or whole milk. These findings indicate that a-T-acetate bioaccessibility is affected in a dose-dependent manner, but only when dietary fat is limited. Category: Laboratory/Cellular Health Sciences

Title: Immunomodulatory activities of Pentalinosterol isolated from the roots of Pentalinon andrieuxii

Student Presenter: Grant Brimmer

Faculty Advisor: Satoskar, Abhay

Abstract: The use of natural products as appropriate adjuvants has recently emerged as a promising approach for the development of effective vaccine formulations. Pentalinosterol (PEN) is one of the active compounds from the root of the plant Pentalinon andrieuxii and our previous studies have demonstrated that P. andrieuxii root hexane extract (PARE) and PEN have anti-leishmanial activity against Leishmania mexicana and donovani respectively. The objective of this study was to define the effectiveness of PEN as an immunomodulator that enhances immune responses and can be used as a novel adjuvant. Our in vitro studies revealed that PEN enhances the expression of NF-kB/AP1 transcription factors, promotes genes responsible for the regulation of various immune responses and also up regulates the co-stimulatory molecules on macrophages. Dendritic cells treated with PEN enhanced the IFN- γ production from T cell co-cultures. Further, mice immunized with PEN showed enhanced Th1, Th2 and Th17 cytokine production from their spleenocytes and lymph node cells upon re-stimulating with antigen as well as increased levels of IgG1 and IgG2 in their blood sera. Taken together, our study demonstrates PEN as a potent immunostimulant, which can be used as a promising natural product adjuvant for the development of vaccine formulations.â€‹ Category: Laboratory/Cellular Health Sciences

Title: Insight into why skin cancer patients are at an increased risk of developing internal cancers

Student Presenter: Anna Callahan

Faculty Advisor: Oberyszyn, Tatiana

Abstract: Development of Basal and Squamous Cell Carcinomas, part of the Keratinocyte Carcinoma (KC) group, is associated with ultraviolet light B (UVB) exposure, a known complete carcinogen. Recent evidence has shown that patients with KCs are up to 130% more likely to develop other primary cancers, such as colon cancer. Likewise, many patients with developing intestinal issues tend to first notice symptoms on the skin, suggesting a relationship between the two tissues. However, It is not clear if the internal malignancy precedes the KC or vice versa. Our lab conducted a preliminary study to determine if cutaneous UVB-induced skin cancer development could alter the course of chemically induced colon carcinogenesis. SKH-1 mice were divided into four groups: 1) PBS + water/no UVB, 2) PBS + water/UVB 3), azoxymethane (AOM) + dextran sodium sulfate (DSS)/no UVB, 4) or AOM + DSS/UVB. At the conclusion of the study mice in the AOM+DSS/UVB group developed skin tumors and had the highest incidence of colon tumors, suggesting that internal malignancies could be exacerbated by cutaneous UVB-mediated skin carcinogenesis. We hypothesized that UVB exposure could alter the colon microenvironment leading to the observed increased colon tumor incidence. In fact, the combination of AOM+DSS and UVB exposure was associated with increased inflammation and immune cell infiltration into the colon, as well as alterations in the make-up of the colonic microbiota compared to control mice. These data suggest that cutaneous UVB exposure not only induces skin carcinogenesis but can also enhance AOM+DSS-induced colon cancer by changing the colon microenvironment. Further study is warranted to examine the mechanism by which UVB-mediated skin tumor development affects the colon and if the development of skin cancer could be an indicator of internal malignancy. Category: Laboratory/Cellular Health Sciences

Title: Breakdown product identification of caspase and calpain-dependent proteolysis of BII-Spectrin

Student Presenter: Matt Cefalu

Faculty Advisor: Smith, Sakima

Abstract: βII-Spectrin is a ubiquitous integral protein of the cytoskeleton and is critical in cardiac cell integrity. βII-Spectrin conditional knockout mice demonstrates cardiac phenotypes characterized by bradycardia, heart rate variability, and conduction defects which contribute to calcium handling abnormalities, delayed afterdepolarizations, and arrhythmias. Previous data indicates that βII-Spectrin expression is decreased in human and animal heart failure and congenital forms of human arrhythmias. The mechanism of decreased expression is initiated by proteolysis caused by calcium activation of caspase and calpain-dependent proteases. Decreased expression of βII- Spectrin is a hallmark of various forms of cardiovascular disease, which makes this protein an ideal candidate as a novel biomarker. This study sought to determine the breakdown products of the posttranslational cleavage; which could theoretically be tested for in suspected cases of cardiovascular disease. Methodology includes western immunoblotting of cardiac muscle, skeletal muscle, and urine samples exposed to various concentrations of calcium to activate the proteases. These immunoblots track the progressive breakdown over 24 hours, and identifies the products of this cleavage. Preliminary findings have identified potential breakdown products, but gas chromatography and mass spectroscopy data will be further analyzed to confirm the identity and sequence of the resultant peptides. Using these data, an assay will be created to test for the presence of cardiac specific βII-Spectrin breakdown products, which could signify the early onset of cardiovascular distress. This biomarker could allow earlier diagnosis of cardiovascular disease than current laboratory based methods and expedite the delivery of treatment or preventative care to thousands of patients. Category: Laboratory/Cellular Health Sciences

Title: Regulation of autophagy by Rab9

Student Presenter: Mary Chandler

Faculty Advisor: Strohecker, Anne

Abstract: Autophagy is a highly conserved pathway that sustains survival of an organism by maintaining homeostasis and mitigating damage by degrading and recycling cytoplasmic components. Perturbations in the autophagy pathway have important consequences for aging, neurodegeneration and cancer. A more complete understanding of the pathway and its regulation is essential in order to understand how best to modulate the pathway as a therapeutic strategy. The present study investigates the mechanism of action of a novel negative regulator of autophagy identified by an shRNA screen, the small GTPase RAB9. Silencing Rab9 in murine and human lung cancer cells results in high levels of autophagy, evidenced by increased conversion of the autophagosomal marker LC3 and elimination of the autophagy cargo receptor and substrate p62/SQSTM1. Importantly, this increased activity is independent of nutrient status, suggesting a role for Rab9 in both basal and stress induced autophagy. Knockdown of Rab9 in a human lung cancer cell line H1299 stably expressing a fluorescent reporter that binds phosphatidylinositol 3-phosphate (PI3P), DsRed-FYVE, leads to increased output of the class III PI3K, Vps34. Ongoing studies are analyzing whether this increased kinase activity extends to both the endosomal and autophagy machinery or is directed to the autophagy pathway. This study extends our understanding of the regulation of autophagy by small GTPases and has the potential to identify additional specific targets for human disease treatment. Category: Laboratory/Cellular Health Sciences

Title: Body compositions analysis upon varying fat and carbohydrate consumption

Student Presenter: Ryan Choi

Faculty Advisor: Volek, Jeff

Abstract: Metabolic Syndrome (MetSyn) is a clustered condition that consists of insulin resistance, obesity, high blood pressure and abnormal cholesterol levels. High-fat or ketogenic diets have begun to gain traction in insulin resistant populations, demonstrating a novel ability to improve insulin sensitivity, decrease blood glucose and decrease triglycerides. To date, no well-controlled research studies exist that determine the cardio-metabolic or body fatness effects of full-fat dairy products in the context of carbohydrate intake. Therefore, the current study sought to enhance the understanding of the underlying effects on visceral and hepatic fat as a result of varying quantities of carbohydrate intake and fat consumption. This study utilized a randomized within subjects design. Six adult participants, both male (n=3) and female (n=3) underwent four, four-week, controlled feeding phases: High Carbohydrate + High Cheese (HCHCh), Moderate Carbohydrate + High Cheese (MCHCh), Low Carbohydrate + High Cheese (LCHCh), and High Carbohydrate + Low Cheese (HCLCh). During these feeding phases, participants only consumed the meals and liquids provided to them. Caloric intake was prescribed for weight maintenance following indirect calorimetry for determination of metabolic rates. After completion of each phase, participants underwent a series of body composition assessments, including anthropometric measurements (hip and waist), visceral and total body fat percentage via DXA (Dual- Energy X-Ray Absorptiometry), and visceral and hepatic fat quantification via MRI (Magnetic Resonance Imaging). No significant differences were observed from DXA, MRI, or Anthropometric data. This study suggests that diet rich in full-fat dairy products may not have any deleterious effects on body composition or steatosis. Furthermore, this data helps to further demonstrate that diets high in saturated fat content do not result in obesity and fatty liver disease in humans. Category: Laboratory/Cellular Health Sciences

Title: Optimization of bioartificial muscle to discover the mechanotransduction pathways of muscle atrophy in microgravity

Student Presenter: Michael Chung

Faculty Advisor: Lee, Peter

Title: Identification and characterization of small molecules with inhibitory properties towards Salmonella enterica biofilms

Student Presenter: Bradley Eichar

Faculty Advisor: Gunn, John

Abstract: Salmonella enterica serovar Typhi (S. Typhi) is a human-specific pathogen and the primary causative agent of typhoid fever. S. Typhi infections are responsible for an estimated 21 million new infections each year, resulting in 200,000 deaths. This bacterium persists chronically in the gallbladder of 3-5% of individuals after resolution of acute infection. This chronic carrier state is mediated by S. Typhi biofilms -organized communities of microorganisms that adhere to surfaces resulting in recalcitrance to antibiotics and immune components - on the surface of gallstones. Inhibition or dispersion of biofilms in carriers could help curb the spread of typhoid fever. This study utilized a high-throughput screen of a 4,000 compound ChemBridge™ library for in vitro anti-biofilm activity against Salmonella (S. Typhimurium was used in this assay). This screen initially identified 226 compounds that demonstrated anti-biofilm activity at a threshold of ≥30%. Compounds that were identified initially were re- screened three additional times, and compounds that maintained the ≥30% inhibition threshold were further characterized. Compounds 1, 2, 3, and 4 demonstrated 30.67%, 31.24%, 39.58%, and 36.09% biofilm inhibition, respectively. These candidates were further analyzed using rapid attachment, bacterial viability, EC50, and biofilm dispersion assays to identify those with the greatest therapeutic potential. Overall, the discovery and characterization of compounds with the ability to specifically inhibit or disperse Salmonella biofilm formation is a promising step towards eradication of the Salmonella chronic carriage state. Category: Laboratory/Cellular Health Sciences

Title: The role of endoglin in insulin-mediated angiogenesis

Student Presenter: Fatema Elmasry

Faculty Advisor: Lee, Nam

Abstract: Angiogenesis is essential for tumor survival and progression in that it provides a blood supply for the tumor to increase in size, and TGF-b Smad-dependent signaling is critical in regulating this process. Endoglin, a transmembrane coreceptor of the TGF-β pathway is expressed in endothelial cells including those of the vasculature, and thus contributes to regulation of pro- and anti-angiogenic processes. Recently, our lab discovered novel cross-talk between the insulin and TGF-b pathways. The purpose of this study is to examine the role in endoglin in insulin-mediated Smad signaling. Phosphorylated smad1/5/8 is a proangiogenic transcription factor that cancer tumors need to grow. Immunofluorescence and biochemical methods were used to see cellular changes and differences in protein concentrations. Preliminary data shows that insulin treated cells that contain endoglin have a higher concentration of phosphorylated smad1/5/8. These results can be implicated to type 2 diabetic patients due to their increased insulin levels. There is an increased risk of cancer with type 2 diabetic patients and angiogenesis is needed for cancer survival. Therefore, the findings of this study may provide information to better understand how type 2 diabetic patients develop cancer. Category: Laboratory/Cellular Health Sciences

Title: Hydrogen peroxide susceptibility of mucoid and non-mucoid variants of Pseudomonas aeruginosa

Student Presenter: Anthony English

Faculty Advisor: Wozniak, Daniel

Abstract: Psuedomonas aeruginosa (P.a.) is a Gram-negative opportunistic bacterium that causes serious acute and chronic infections in the hospital-setting. Cystic fibrosis (CF) is a genetic disorder caused by a mutation in the cystic fibrosis transmembrane regulator (CFTR) protein. Consequently, CF patients produce thick and sticky mucus that blocks the airways and creates an ideal environment for chronic bacterial lung infections, most commonly due to P. a. During infection, P.a. can acquire mutations allowing the bacterium to better adapt to the host lung. For example, mutation of the gene, mucA, results in overproduction of the bacterial polysaccharide, alginate, causing the colonizing "non-mucoid" bacteria to switch to a "mucoid" phenotype. Mucoid strains of P.a. are known to be less susceptible to antibiotics and the immune system compared to non-mucoid isolates. However, we recently showed that the mucoid isolate, FRD1, is more susceptible to hydrogen peroxide (a reactive oxygen species produced by immune cells) than an isogenic, non-mucoid variant, FRD2. Given the unexpected finding in the FRD strain background, we wanted to test whether this phenotype is conserved across multiple mucoid/non-mucoid isolates of P.a. As such we utilized a laboratory strain library that replicates mutations found in P.a. isolates derived from Danish CF patients over a period of 40 years. These clinical isolates and laboratory strains were tested for peroxide sensitivity. We found that lab isolates are less susceptible to peroxide compared to clinical isolates and that peroxide sensitivity is independent of mucoid status. One lab strain with a mutation in rpoN (bacterial transcription factor) showed decreased susceptibility to peroxide, suggesting, rpoN may be a negative regulator of an antioxidant (catalase) that provides protection from peroxide. These data support continued investigation of mucoid/non-mucoid P.a. susceptibility to innate immune factors, including peroxide, as we seek to understand the mechanisms of P.a. evasion of host-defenses. Category: Laboratory/Cellular Health Sciences

Title: Factors associated with food safety behaviors in food insecure cancer patients seeking treatment

Student Presenter: Laila Ettefagh

Faculty Advisor: Ilic, Sanja

Abstract: The objective of this study is to determine the risk perception and demographic factors associated with food safety behaviors in cancer patients seeking chemotherapy and radiation treatment. Over 14 million Americans suffer from cancer and are at a greater risk of foodborne disease due to weakened immune systems. However, food safety practices and factors that may lead to risky food safety behaviors are not well understood. This was a cross-sectional study that recruited participants from three cancer-specific hospitals in Ohio. A 173-item questionnaire assessed the food acquisition and preparation practices and attitudes towards different aspects of food safety in the cancer population. It included sociodemographic factors, insecurity status, and disease factors. The data was analyzed in SPSS. Participants (n=120) were mostly breast cancer patients (48.1%), female (84.6%), older than 50 (73.1%), and white non-Hispanic (83.5%). About 38% had a college degree or more and 26.2% had at least one year of college education. A total of 2.9% participants had low food security while 8.7% were marginally food insecure. Low income was associated with risky food acquisition practices, such as buying damaged packages (r=0.318, PPPP Category: Laboratory/Cellular Health Sciences

Title: Novel treatment of immunomodulatory cytokine blockade combined with immune checkpoint inhibition leads to shifted immune response and lower tumor growth rates in a murine pancreatic cancer model

Student Presenter: Daniel Gage

Faculty Advisor: McQuinn, Christopher

Abstract: Pancreatic cancer has one of the lowest 5-year survival rates of all cancers with limited options for chemotherapy, and it is expected to become the second leading cause of cancer death by 2030. Checkpoint immunotherapy with monoclonal antibodies provides promise in the treatment of cancer, but its efficacy has been limited in pancreatic cancer, a malignancy characterized by an immunosuppressive tumor microenvironment and abnormal expression of cytokines such as IL-6. Previous studies have shown that high levels of systemic IL-6 are associated with a significantly lower survival overall. By targeting this cytokine with a monoclonal antibody, it was hypothesized that immune checkpoint inhibition would improve. CTLA4 is one such immune checkpoint molecule, and mice treated with anti-IL-6 and anti-CTLA4 had decreased tumor growth and significantly increased intra-tumoral CD3+ T-cell infiltration. Further analysis of circulating lymphocytes from these mice by FACS indicated a shift in the TH1/TH2, Treg, and TH17 phenotype of mice given the combination therapy. Current studies focus on discerning the mechanism of CD3+ cell infiltration by use of IL-6 chemokine expression. Category: Laboratory/Cellular Health Sciences

Title: Functional characterization of a novel RAF1 mutant in human cancers

Student Presenter: Elijah Gardner

Faculty Advisor: Roychowdhury, Sameek

Abstract: Components of the RAS-RAF-MEK (also known as MAPK/ERK) mitogen signaling pathway are frequently mutated in many types of human cancers. The RAF kinase family signals downstream of RAS to promote cell proliferation and is encoded by three different genes: ARAF, BRAF, and RAF1 (or C-RAF). While activating mutations have been identified in BRAF (e.g. BRAF V600E), mutations in the remaining two RAF isoforms are rare and have been poorly characterized. Here we have report a novel non- synonymous RAF1 mutation, P261R, which was identified in a patient with non-small cell lung cancer. Although sequencing studies have previously detected this mutation in both hepatocellular carcinoma and cholangiocarcinoma, its functional significance remains unknown. Therefore, we propose to elucidate the biologic consequences of RAF1 P261R mutation using cell-based assays. First, we will generate the mutant RAF1 using PCR-based in-situ mutagenesis and then perform stable transfection of established cells with wildtype (wt) and mutant RAF1. To determine the effect of mutant RAF1 on cell proliferation and contact inhibition, we will perform BrdU incorporation and colony formation assays in wt and mutant cells. To determine its effect on apoptosis, we will perform the TUNEL assay. Finally, we will use immunoblotting assays to assess the level and activity of proteins in MAPK/ERK pathway in RAF1 wt and mutant cells. Together, these experiments will help us determine whether RAF1 P261R may be important for oncogenesis and thus identify a new molecular target for therapeutic intervention in human cancers. Category: Laboratory/Cellular Health Sciences

Title: Elucidating the role of MMS22L on uterine epithelium and endometrial cancer.

Student Presenter: Yannis Hadjiyannis

Faculty Advisor: Leone, Gustavo

Abstract: Targeted gene therapy and DNA repair inhibition have been compelling areas of interest for personalized medicine and cancer treatment. For example, recent work has identified the MMS22L- TONSL DNA repair complex as a novel marker on post-replicative chromatin necessary for cell viability, RAD51 loading, DNA repair, and overall genomic stability. Depletion of MMS22L-TONSL in cancer cells results in decreased proliferation and a reduction in viability in vitro. Furthermore, elevated levels of TONSL have been noted in several cancers, and we have found that deletion of Mms22l (Spr2f- Cre;Mms22LF/F) in cancer sensitized Pten (Spr2f-Cre;PTEN+/F) mice resulted in a reduction in total area of endometrial carcinoma-in-situ when compared to Spr2f-Cre;Mms22l+/+;Pten+/F mice, implicating MMS22L as a novel target for cancer therapy. To validate these findings in more complex systems before advancing to clinical trials, we utilized a transgenic loss-of-function system in 2 month-old and 6 month-old-mice to elucidate the consequence of MMS22L deletion in uterine development and proliferation. Tissue-specific deletion in mouse endometrial epithelial cells using the tissue-specific Cre- recombinase system (Spr2f-Cre) was conducted; given our findings of embryonic lethality for globally deleted Mms22l, the fluctuating expression of Mms22l through the estrus cycle in endometrial epithelial cells, and the dynamic nature of the mouse endometrium. In this study, we demonstrate the impact of Mms22l deletion in adult uterine physiology and reproduction in order to validate the MMS22L-TONSL complex as a novel target for endometrial cancer. Category: Laboratory/Cellular Health Sciences

Title: MYO9B is critical for sensing mechanical strain in bone-forming cells

Student Presenter: Emma Hassell

Faculty Advisor: Lee, Beth

Abstract: Mechanical forces, such as weight bearing exercise, are necessary for growing and maintaining a healthy skeleton. These types of forces stimulate bone-forming cells (osteoblasts) to increase their activity and produce more bone proteins. However, the mechanisms by which cells sense and respond to mechanical forces are not well understood. In this study, we have studied the role of MYO9B, a cytoskeletal regulatory protein, in mechanical sensing. Mice lacking MYO9B are small and do not properly form bone during early stages of development. However, the potential role of MYO9B in sensing mechanical signals has not yet been determined. Therefore, we examined whether osteoblasts deficient in MYO9B are capable of responding to mechanical strain by re-orienting their alignment to minimize stress. To test this, levels of MYO9B were knocked down in osteoblastic cells, and the cells were cyclically stretched along one axis. Unlike normal osteoblasts, cells deficient in MYO9B could not change their orientation in response to strain. This is likely due to the fact that MYO9B-deficient cells do not properly form proteins complexes required for sensing mechanical strain (focal adhesions). Cells lacking MYO9B formed fewer focal adhesions and showed adhesion and spreading defects relative to normal cells. In addition, expression of critical focal adhesion proteins (beta integrins) is altered in MYO9B-deficient cells. These findings are consistent with the poor bone growth observed in mice lacking MYO9B expression and provide a mechanistic pathway by which mechanical strain is sensed in bone. Category: Laboratory/Cellular Health Sciences

Title: Zebrafish as a model organism in the study of organic cation transporters

Student Presenter: Kristen Hong

Faculty Advisor: Sparreboom, Alex

Abstract: Organic Cation Transporter 2 (OCT2) is partially responsible for the uptake of platinum-based anti-cancer drugs, such as Cisplatin. This uptake induces severe neurotoxicity, nephrotoxicity, and ototoxicity and can be life threatening. We found a number of Tyrosine Kinase Inhibitors (TKIs) able to inhibit the function of OCT2 in order to lessen these toxic side effects. This project focuses on exploring the potential utility of zebrafish OCT (zfOct) as a new model to study the effect of TKIs on the toxicity induced by platinum drug. In vitro experiments were performed with Hek293 cells or Hela cells over expressing human OCT2 (hOCT2) or the single zfOct, respectively. The sequence alignment program MAFFT v7, shows 71% identical protein sequences between hOCT2 and zfOct protein, and 86% between human and zebrafish YES1, the tyrosine kinase responsible for OCT2 phosphorylation. Various TKIs proved to inhibit hOCT2 and zfOct uptake of the specific substrate ASP (4-[4-(dimethylamino)styryl]-N- methylpyridinium-iodide) at comparable levels: Dasatinib (63.6% vs 53.6), Nilotinib (66.5% vs 68.8), Sunitinib (70.7% vs 90.6). Uptake of cisplatin was similarly strongly or completely decreased in hOCT2 and zfOct in the presence of these three inhibitors (p-values Category: Laboratory/Cellular Health Sciences

Title: Biodegradable amino-ester nanomaterials for efficient Cas9 mRNA delivery in vitro and in vivo

Student Presenter: Justin Jiang

Faculty Advisor: Dong, Yizhou

Abstract: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR- associated protein 9 (Cas9) system has been widely used as a powerful tool for genome-editing. However, efficient and safe delivery of CRISPR systems is one of the key challenges for their therapeutic applications. In order to address these issues, we designed and synthesized a series of biodegradable lipid-like nanoparticles (LLNs) containing ester groups for Cas9 mRNA delivery. Two lead materials, termed as MPA-A and MPA-Ab, showed tunable rates of biodegradation in cell and animal models. MPA- A with linear ester chains was degraded dramatically faster than MPA-Ab with branched ester chains. Moreover, MPA-A and MPA-Ab demonstrated significantly higher delivery efficiency for Cas9 mRNA than C12-200, a lead material reported previously. Most importantly, both MPA-A and MPA-Ab LLNs exhibited effective and safe delivery of Cas9 mRNA in mouse models. Consequently, these biodegradable lipid-like nanoparticles offer a reliable platform of delivering Cas9 and merit further development as a genome-editing delivery tool for biological and therapeutic applications. Category: Laboratory/Cellular Health Sciences

Title: DNA repair genes polymorphism as indicator for response to high dose melphalan in multiple myeloma autologous stem cell transplant patients

Student Presenter: Jasmine Johnson

Faculty Advisor: Poi, Ming

Abstract: Multiple myeloma (MM) is a cancer of the plasma cells that is characterized by the proliferation of these cells in the bone marrow. MM patients have a five-year survival rate ERCC1, ERCC2, ERCC4, XRCC1, XRCC3, BRCA1, CD3EAP, CDKN1A, and XPC on progression free survival (PFS), 90- day response, and oral mucositis in a cohort of 108 patients. Our results show that XRCC1 rs25487 and XRCC3 rs861539 were associated with PFS in our patient cohort. The hazard ratios (HR) of XRCCC1 rs25487 (AA/AG vs GG) and XRCC3 rs861539 (AA/AG vs GG) were 0.42 (95% CI [0.22, 0.81], p=0.01) and 1.87 (95% CI [0.93, 3.75], p=0.08), respectively. After adjustment with risk index, the HRs of XRCCC1 rs25487 and XRCC3 rs861539 were 0.42 (95% CI [0.21, 0.84], p=0.014) and 2.14 (95% CI [1.02, 4.50], p=0.045), respectively. In addition, five MM cell lines were studied in vitro. MM cell lines MM.1 S-X and LP1 showed resistance to melphalan treatment and induction of both TP53 and XRCC1 mRNA expression but not the other 3 sensitive cell lines. The results of this study indicate the potential utility of XRCC1 rs25487 as a biomarker for melphalan treatment response and support the need to further study in a clinical trial setting. Category: Laboratory/Cellular Health Sciences

Title: Environmental and genetic activation of brain-derived neurotrophic factor modulates T cell immunity to exert an anticancer phenotype

Student Presenter: Ryan Judd

Faculty Advisor: Caligiuri, Michael

Abstract: Recent evidence supports the role of environmental factors on cancer prognosis and progression. To study the effects of the social environment in mice, we use a housing model called an enriched environment (EE) with increased space and more social interaction that is cognitively, socially, and physically stimulating. Previous studies have shown that living in EE confers an anticancer phenotype. However, it is unknown whether changes in T cells play a role in this anticancer effect. Here, we show that CD8 T cells are required to mediate EE's anticancer phenotype in an orthotopic model of melanoma. In IgG control mice, EE tumor mass was decreased by 80% compared to standard environment (SE) (EE=174±67 mg, SE=857±156 mg, p=0.001). In contrast, EE and SE mice administered CD8-depleting antibody showed no significant difference in tumor mass (EE=433±92 mg, SE=764±141 mg, p>0.07). Importantly, within the EE groups, mice administered CD8-depleting antibody had significantly larger tumors than control (p=0.03), indicating that CD8 T cells are at least partially responsible for EE's anticancer effect. In secondary lymphoid tissue (SLT), EE induced early changes in T cell populations, characterized by a decrease in the ratio of CD4 T helper to CD8 cytotoxic T lymphocytes (CD4:CD8 EE=1.51, SE=0.74, p Category: Laboratory/Cellular Health Sciences

Title: Evaluating angiotensin converting enzyme inhibitor and mineralocorticoid receptor antagonist efficacy for beta adrenergic stress-induced cardiac damage in dystrophic mice.

Student Presenter: Feni Kadakia

Faculty Advisor: Rafael-Fortney, Jill

Abstract: Duchenne Muscular Dystrophy (DMD) is an X-linked neuromuscular disorder affecting approximately 1 in 5000 boys. DMD is a result of the absence of the dystrophin protein at striated muscle membranes that subsequently causes progressive muscle degeneration. Heart failure resulting from cardiomyopathy is a leading cause of death in DMD, and the current standard of care is lisinopril, an angiotensin converting enzyme (ACE) inhibitor. Our team has shown both in mouse models and DMD patients that lisinopril combined with spironolactone, a mineralocorticoid receptor antagonist, leads to improved cardiac outcomes at baseline. This study was designed to test whether treatment with lisinopril and spironolactone can prevent beta-adrenergic stress induced cardiac damage in the dystrophic mdx mouse model of DMD. Two groups of male dystrophin-deficient mdx mice, treated (n=10) and untreated (n=10), two groups of mdx females, treated (n=5) and untreated (n=5), along with one group of C57 BL 10 wild type controls (n=15) were enrolled in the study. Mice were treated continuously from 4 weeks-of-age to 21 weeks via water bottles containing drug treatment. The results of this study showed there was not a significant difference in damage between the treated and untreated groups and the treatment combination was not effective in preventing against stress induced damage. This treatment was compared to an additional control group treated with metoprolol, a cardioselective beta-blocker specifically designed to prevent beta-adrenergic stress. The control consisted of 2 groups of 21 week-old male mdx mice, treated for 2 weeks (n=11) or untreated (n=5). Histological sections and composites were made to quantify cardiac damage and data analysis is ongoing. Category: Laboratory/Cellular Health Sciences

Title: Non-phosphorylatable mutant Y705F of Stat3 is embryonic lethal

Student Presenter: Jennifer Heekyung Kim

Faculty Advisor: Majumder, Sarmila

Abstract: The signal transducer and activator of transcription 3 (Stat3) gene mediates vital processes starting from maintenance of pluripotent stem cells to inflammatory responses. Stat3 is activated by phosphorylation at Tyr705(Y705) and Ser727(S727) residues. Previous studies have demonstrated embryonic lethality of Stat3 null mice. Y705 mutations to its non-phosphorylatable form Y705F (tyrosine to phenylalanine) demonstrated dominant negative activity interrupting normal function of Stat3 in vitro. However, there have been no studies on Y705F in vivo. In this study we sought to determine the effect of Y705F-Stat3 in mouse model. Y705F mutation was recombineered into mouse genome. A neo cassette was flanked in front of Y705F with LoxP sites to control the activity of the mutant protein (Stat3neo). Stat3neo mice were bred with Sox2-Cre mice to delete the neo cassette, inducing the knock- in of the mutant Stat3 (Stat3Y705F). Since Stat3 null mice showed embryonic lethality at E8.5, we decided to investigate the viability of Stat3Y705F mice by breeding heterozygous knock-in mice. Out of 52 total mice at weaning age, 14 were wild type and 38 were heterozygous and none were homozygous. Of the 25 embryos that were obtained on E13.5, 4 were wild type and 21 were heterozygous. Out of 10 embryos harvested on E10.5, one was wild type and nine were heterozygous and out of 17 embryos harvested on E8.5, two were wild type and 15 were heterozygous. Homozygous mice were not alive as early as day 8.5 of pregnancy. This significant deviation from the expected Mendelian numbers of mutants, revealed embryonic lethality of Stat3Y705F homozygous mice, underscoring the importance of the Y705 residue in Stat3 function that supports embryogenesis. Furthering our understanding of Stat3's mechanisms will lead to valuable information on Stat3-induced human illness as constitutive activation of Stat3 has been detected in colorectal, skin, liver, and gastric cancer. Category: Laboratory/Cellular Health Sciences

Title: Gene copy number variations and large range of serum protein levels for complement C4 in systemic lupus erythematosus patients

Student Presenter: Emily King

Faculty Advisor: Yu, Chack-Yung

Abstract: Human systemic lupus erythematosus (SLE) is an autoimmune disease characterized by fluctuating serum levels of complement proteins. There are frequent copy number variations (CNV) of complement C4A and C4B genes among different individuals. Our objectives are to investigate how CNV of complement C4 contributes to the great variability of its serum levels, and identify risk factors leading to low complement in SLE. Genomic DNA and sera from 505 SLE patients were used to determine the gene copy numbers of total C4 (C4T), C4A and C4B genes by real-time quantitative PCR, and serum levels of C4 and C3 by radial immunodiffusion (RID). Serum levels of C4 and C3 for each patient over the past five years were retrieved by clinical chart reviews. C4 and C3 levels are shown as mg/100 ml (unit). For statistical analyses, continuous data was compared between groups by Student's t-test, and categorical data by chi-square analyses. C4 protein levels varied from 1-84 units (Median 17). C3 protein levels varied from 8-314 units (Median 86). There was a very strong correlation between C4 and C3 protein levels (R 0.70, P 5.34e-75). The CNV of C4T was 2-9 with a median of 4 (54%), followed by 2-3 (21%). Each C4 gene copy correlated to an increase of 4-6 units of serum C4 protein. The CNV of C4B was 0-4 with a median of 2 (62%). Among the patients, 6.3% consistently had very low levels of C4 ( Category: Laboratory/Cellular Health Sciences

Title: Ibrutinib inhibits hepatocellular carcinoma progression by targeting EGFR signaling pathway

Student Presenter: Eunice Kwak

Faculty Advisor: Ghoshal, Kalpana

Abstract: Hepatocellular carcinoma (HCC) is the fifth most common cancer and the second leading cause of cancer-related deaths worldwide. Despite advancements, the 5-year survival rates among HCC patients remains low at 18%. Sorafenib, the only approved targeted therapy for advanced HCC only marginally prolongs patient survival. This low response rate is attributed to HCC tumors having an intrinsic resistance to sorafenib. Therefore, alternative therapies must be necessary to increase the survival of patients with advanced HCC. In the present study, we explore the therapeutic potential of Ibrutinib (PCI-32765), an FDA approved anti-cancer drug against leukemia that has been identified as an irreversible inhibitor for the Bruton Tyrosine Kinase (BTK). It also exhibited anti-cancer effect against a variety of solid tumors such as lung, breast cancers and in animal models. In this study, we examined anti-proliferative effects of Ibrutinib against HCC cell lines. Our results showed that Ibrutinib inhibited growth of HCC cells grown as monolayers and their ability to form colonies in a dose dependent manner. Additionally, we explored therapeutic potential of Ibrutinib on sorafenib resistant HCC (Huh-7) cell line. Importantly, Ibrutinib reduced the proliferation of both sorafenib sensitive and resistant cells. Elucidation of mechanisms revealed that Ibrutinib inhibited both basal and EGF-induced phosphorylation of epidermal growth factor receptor (EGFR) in HCC cell lines. The reduced EGFR activation was associated with reduced expression of EGFR regulated genes e.g. GJA1, PLD1, MYBL2, JUN, SPRY2. These results demonstrate that EGFR is a target of Ibrutinib in HCC cell lines. In the future, the therapeutic potential of Ibrutinib could be further explored by studying the efficacy of combination therapies with sorafenib against HCC. Overall, these findings support Ibrutinib as potential drug for treating HCC patients. Category: Laboratory/Cellular Health Sciences

Title: Analogues of the phyllanthusmin class of natural products: Synthesis, optimization, and antiproliferative properties

Student Presenter: Bernadette Latimer

Faculty Advisor: Fuchs, James

Abstract: The phyllanthusmins consist of a group of arylnaphthalene lignan glycoside-containing natural products possessing potent antiproliferative activity. The most active of these natural compounds, phyllanthusmin D, was isolated from the Vietnamese plant Phyllanthus poilanei and was subsequently used as a lead compound for analogue development and structure-activity relationship (SAR) studies focused on improvement of pharmacokinetic properties. The initial work on the glycone portion of the natural product culminated in a potent series of diphyllin glycosides with a 10,000-fold increase in activity against HT-29 colon carcinoma cells. The aglycone core, however, still remained largely unexplored. With this in mind, analogues have been synthesized through substitution of both the A- and D-rings of the diphyllin system. Optimization of the reaction sequence previously established for the gram scale synthesis of diphyllin was required to facilitate a much smaller reaction scale, amenable to the generation of multiple analogues in milligram quantities. With minor adjustments to this sequence, the desired products could be reproducibly synthesized, eliminating the presence of an undesired intermediate observed in the key cycloaddition reaction. These cycloadducts could then be carried through the rest of the scheme without further modification. The diphyllin analogues produced in this was have been subsequently glycosylated and submitted for biological testing in a variety of cancer cell lines. The antiproliferative activity of these compounds provides important information regarding the structural requirements needed for the potent activity observed for this class of compounds, ultimately helping to improve their pharmacological properties. Category: Laboratory/Cellular Health Sciences

Title: Predictability of lumbar spinal cord microenvironment after thoracic injury based on the gene expression profile of blood neutrophils and monocytes

Student Presenter: Shijia Li

Faculty Advisor: Basso, D. Michele

Abstract: Profound locomotor impairments exist with human spinal cord injury (SCI) even after intensive neuro-rehabilitation. Animal studies show that active recruitment of myeloid cells from the bloodstream into the spinal cord can induce a toxic microenvironment which impedes neuroplasticity and functional recovery. Evidence also showed elevated pro-inflammatory gene expression within infiltrating monocytes in the lumbar cord likely drives inflammation in locomotor networks and impairs recovery. Unfortunately, knowing the real-time microenvironment of the human spinal cord and when it is best to deliver rehabilitation is currently impossible. Therefore, this study aims to predict the lumbar cord microenvironment from gene expression profiles of myeloid cells in blood samples after murine SCI. We hypothesize that changes in gene expression in blood neutrophils and monocytes among four categories - inflammatory, trafficking, extracellular matrix (ECM) remodeling, and growth and repair- will correlate with and predict the lumbar microenvironment. RNA samples collected from 6 naïve and 6 injured mice 24 hours post SCI were processed using Nanostring nCounter technology which analyzes 60 genes in a single run. Inflammatory gene expression was most affected in neutrophils after SCI. For example, chitinase-like-3 was expressed 224% of baseline level. In monocytes, prominent alteration occurred across genes in growth and repair families - transforming growth factor beta-1 was significantly downregulated. Comparing blood and spinal cord samples of the same injured animals, trafficking, ECM remodeling, and growth and repair genes in neutrophils presented strong correlations (R2>0.7), including syntaxin-6 and interleukin-10. However, in monocytes, a relationship was established only among inflammatory genes (R2>0.65), like protein tyrosine phosphatase receptor type C and low density lipoprotein receptor-related protein 1. With the successful identification of relevant gene markers, this study will provide a clinically feasible way to assess the microenvironment of lumbar cord in human subjects at different time points and improve the outcome of rehabilitation. Category: Laboratory/Cellular Health Sciences

Title: Radio- and chemosensitization of human papillomavirus (HPV)-negative head and neck squamous cell carcinomas by the SMAC mimetic LCL161

Student Presenter: Tianyun Li

Faculty Advisor: Williams, Terence

Abstract: The 6th most common cancer worldwide, head and neck squamous cell carcinoma (HNSCC) has a 5-year survival rate of only 40-50%. Furthermore, HNSCC is differentiated between human papillomavirus (HPV) positive and negative types, the latter of which is correlated with worse clinical outcomes. Thus, there is a need for novel therapies to improve outcomes in this disease. One potential novel treatment is LCL161 in combination with radiation (RT) and chemotherapy (e.g. cisplatin, paclitaxel, Abraxane, gemcitabine). LCL161 is part of a new class of targeted drugs, second mitochondria-derived activator of caspase (SMAC) mimetics, that can induce apoptotic cell death by antagonizing inhibitors of apoptosis family proteins (IAPs). The purpose of this study is to investigate the ability of LCL161 to radio- and chemosensitize HPV- HNSCCs in vitro and to validate our findings in vivo. To test the radiosensitizing effect of LCL161 in vitro, we first conducted Western blots on three HPV- and three HPV + HSNCC cell lines to measure levels of IAPs. We hypothesized that a higher expression of IAPs will correlate with an increased radio- and chemosensitization effect by LCL161. Clonogenic assays were then utilized to determine radio- and chemosensitization via LCL161. Lastly, human xenografts were used to validate the radiosensitizing effect of LCL161 on HPV- HNSCC tumors in vivo. We discovered that cIAP1 expression was higher in HPV- HNSCC cells and these HPV- cells were radiosensitized by LCL161 with drug enhancement ratios of 1.21 and 1.23 for Cal27 and UM-SCC-1, respectively. Conversely, HPV+ cell lines were not significantly radiosensitized after treatment with LCL161. The addition of various chemotherapies also chemosensitized the HPV- cells, enhancing the results of LCL161 combined with RT. Finally, we confirmed the ability of LCL161 to radiosensitize HNSCC tumors in vivo with mouse models. Category: Laboratory/Cellular Health Sciences

Title: IL-6 blockade combined with MEK inhibition in pancreatic cancer and cachexia

Student Presenter: Neil Makhijani

Faculty Advisor: Mace, Thomas

Abstract: Patients with pancreatic cancer, as well as many other gastrointestinal (GI) cancers, have the highest incidence of cachexia, which is characterized by a loss of skeletal muscle and adipose tissue mass that cannot be reversed by nutritional intervention. Nearly half of all cancer patients experience cachexia with 20-30% of all cancer-related deaths resulting from cachexia instead of the actual tumor itself. Our group has recently shown that cytokine Interleukin-6 (IL-6) is elevated in patients with pancreatic cancer, which correlates with an overall decrease in survival. Numerous studies have also shown that various factors, including IL-6, can promote communication between the Jak/STAT3 and MEK pathways. In clinical studies, advanced biliary cancer patients treated with a MEK inhibitor (MEKi) as a single-agent had anti-cachectic results, therefore illustrating the promise of MEK inhibitors in GI cancers. Thus, we hypothesized that IL-6 blockade can be utilized to enhance the efficacy of MEK inhibition in reducing pancreatic-induced cancer cachexia. In vivo efficacy studies were conducted with antibodies blocking IL-6 in combination with MEK inhibition. Currently, a pancreatic cancer-induced cachexia model is not available, therefore we tested the combination of IL-6 blockade with MEK inhibition on a classically accepted cancer cachexia model (CD2F1 mice bearing Colon26 tumors). Mice treated with MEKi or the combination of anti-IL-6 plus MEKi resulted in significant tumor inhibition compared to vehicle controls (p Category: Laboratory/Cellular Health Sciences

Title: Fetal bovine serum vs human platelet lysate: In vitro-generation of bone extracellular matrix on poly (propylene fumarate) tissue engineered bone grafts

Student Presenter: Kevin Martin

Faculty Advisor: Dean, David

Abstract: Historically, fetal bovine serum (FBS) has been used in culture media to supply cells with necessary proteins and growth factors for cell proliferation and survival. The FDA is hesitant to approve clinical translation of implants cultured with FBS due to the risk of BSE (Bovine Spongiform Encephalopathy aka "Mad Cow" disease) transmission. Human platelet lysate (HPL) can potentially be used instead of FBS for media formulations. HPL is expected to have a more favorable composition and poses less risk of disease transmission. In this study, HPL was compared to FBS in proliferation and differentiation media used to culture Bone Marrow-derived human Mesenchymal Stem Cells (BM- hMSCs) seeded on resorbable poly(propylene fumarate) (PPF) thin films. One cm2 PPF thin films were washed, sterilized, and seeded with 50,000 BM-hMSCs (RoosterBio, Frederick, MD) each. Samples were cultured in media with 2% FBS or 5% HPL (Cook Regentec, Indianapolis, IN). Proliferation media contained FGF, PDGF, and EGF along with FBS or HPL. A WST-1 metabolic assay was used to quantify proliferation at days 1, 2, and 3 (n=3; total N = 18). Differentiation media contained osteogenic media and BMP 7 along with either FBS or HPL. An alkaline phosphatase (ALP) enzymatic assay (osteoblast differentiation) and an Alizarin Red S stain and assay (ECM mineralization) were conducted on Days 3, 6, 9, 12, 15, and 21 (n=3; total N = 72). ALP and Alizarin Red S reached peak values on days 15 and 21, respectively. The Alizarin Red S results, with scanning electron microscope images at Day 21, indicate that a greater amount of mineralization occurred on samples cultured with HPL (p=0.046). Therefore, HPL appears to be a viable substitute for FBS for the purpose of facilitating attachment, proliferation, and differentiation of BM-hMSCs to osteoblast and promoting ECM deposition on tissue engineered bone grafts. Category: Laboratory/Cellular Health Sciences

Title: Differential resistance of Mycobacterium abscessus variants to acidic and oxidative conditions

Student Presenter: Laurel Mason

Faculty Advisor: Hall-Stoodley, Luanne

Abstract: The aim of this study is to determine if smooth and rough colony morphotypes of Mycobacterium abscessus growing as planktonic cells or as biofilms differ in resistance to acidic pH and hydrogen peroxide. M. abscessus is a highly antibiotic resistant pathogen causing persistent lung infections in Cystic Fibrosis (CF) patients. M. abscessus can survive in macrophages, which control bacteria in phagolysomal vesicles by mechanisms that include low pH, and increased levels of reactive oxygen species (ROS) such as hydrogen peroxide in phagocytic cells. However, little research has investigated differences in resistance to phagocytic defense mechanisms between morphotypic variants. This study will elucidate any differences to gain a better understanding of how M. abscessus may survive and persist in the phagocytic environment within the cell. Using a 96 well plate assay, mCherry transformants of M. abscessus smooth and rough morphotypes were treated with different concentrations of HCl, acetic acid, and hydrogen peroxide over the course of 24 hours. Survival of planktonic or 24-hour biofilm bacteria was assessed by relative fluorescence intensity (RFI) and verified by colony forming unit (CFU) enumeration. Preliminary data suggest that the rough morphotype is more resistant to all of the conditions tested, especially in biofilms. However, acetic acid is effective in controlling M. abscessus, even in biofilms and at low concentrations. Determining differences between both the morphotypic and aggregative forms of M. abscessus will help to target bacterial virulence determinants and improve our understanding of the mechanisms used by this bacterium to survive and persist within the toxic environment of phagocytes that likely contribute to persistent infections in Cystic Fibrosis patients. Category: Laboratory/Cellular Health Sciences

Title: Title: Inhibition of JNK3 as a novel therapeutic target for treatment of Alzheimer's Disease

Student Presenter: Molly McNamara

Faculty Advisor: Yoon, Sung Ok

Abstract: Alzheimer's disease (AD) is an irreversible, progressive disease that slowly destroys cognitive function, ultimately to the point that daily life becomes unmanageable. The number of AD patients is estimated to increase from the current 5.1 million to 16 million in the US by 2050, and the cost of AD is also expected to rise from $236 billion in 2016 to over $1.1 trillion by 2050. In addition, as humans live longer, the incidences of AD is expected to rise rapidly. Currently there is no cure. The goal of the project is to develop a disease-modifying drug that is orally bioavailable by targeting a stress-activated kinase in the brain, c-jun N-terminal kinase 3 (JNK3). Genetic deletion of JNK3 gene from an aggressive AD mouse model, 5XFAD, produced a 90% reduction in pathologic amyloid β42 (Aβ42) levels, inhibition of endoplasmic reticulum (ER) stress, and a significant improvement in cognitive function. One of the mechanisms by which JNK3 influenced generation of Aβ42 includes repeated activation of JNK3 by ER stress or other pathological stimuli that associates with AD pathology. Inhibiting JNK3 activity in advanced AD cases can potentially halt or slow further progression. We tested the hypothesis with a brain penetrating, orally bioavailable, highly-selective, potent pan-JNK inhibitor, SR-3306. For this, 9 month-old Tg2576 mice were administered daily by gavage with 30 mg/Kg of SR3306 or vehicle for 3-5 months, and cognitive tests were performed before tissue harvest. Untreated mice were included as an additional control. Compared to untreated or vehicle treated mice, there was a significant improvement in novel object recognition tests in SR3306 treated mice, suggesting inhibiting JNK3 in AD can be feasible. There was also an overall reduction in ER stress. These data indicate that targeting a linchpin enzyme in neuronal stress pathways, JNK3, is a viable therapeutic option for AD. Category: Laboratory/Cellular Health Sciences

Title: Spinal cord microglial activation in a murine model of neuroma

Student Presenter: Mynor Mendez

Faculty Advisor: Lerch, Jessica

Abstract: A neuroma is an overgrowth of neural tissue associated with increased pain and disorganized nerve fiber fasciculation that often occurs after an injury (e.g. carpul tunnel surgery, amputation). This condition is not well understood, and a strong rodent model has not been established. We performed a sural nerve ligation which induces cellular proliferation and swelling, and expresses the human neuroma marker, semaphorin 3A (Sema3a). In this model, both male and female mice exhibit increased hypersensitivity using the Von Frey test for mechanical allodynia. Since pain hypersensitivity is associated with changes in spinal cord microglial cell activation, we will use IBA1 immunostaining to examine microglial morphological changes associated with activation at different timepoints after injury. Our study will establish the cellular changes that accompany painful neuroma and lead to a deeper understanding of the human condition. Category: Laboratory/Cellular Health Sciences

Title: Müller Glia proliferation in the retina is increased by inhibition of matrix metalloproteinases

Student Presenter: Ninoshka Mendonca

Faculty Advisor: Fischer, Andrew

Abstract: Many vision- threatening diseases are the consequence of retinal neuron death. Müller Glia, a glial cell, has the capacity to de-differentiate and proliferate as Müller-Glia derived Progenitor Cells (MGPCs). These MGPCs have the ability to act as a potential source of new neurons. This study focuses on the mechanisms that control the reprogramming of Müller glia into neuronal progenitors and the role Matrix Metalloproteinases play in this process. MMPs are endopeptidases that degrade components of the extracellular matrix as well as cytokines and growth factors. Their role is crucial to neural development. It has been previously seen that intraocular injections of NMDA causes cell death and induces formation of MGPCs in the avian retina. Similarly, FGF2 stimulates formation and proliferation of MGPCs in the absence of damage. Under both damage and non-damage contexts, we investigated MMPs using the drug SB-3CT, an inhibitor of MMP-2 and MMP-9. Using immunohistochemistry, we found that the inhibition of MMPs causes a significant increase in proliferating MGPCs. To identify whether the pro-proliferative effect of SB-3CT was specific to Muller Glia, we investigated the proliferation of other retinal glial types such as microglia and NIRG cells. Data indicated no differences in microglia or NIRG cell proliferation in response to MMP inhibition. These results provide evidence that MMP inhibition exclusively targets Muller Glia. Lastly we investigated if microglia mediates MMP's effects on MGPC proliferation. We ablated microglia using clodronate liposomes and investigated whether the ability of SB-3SCT to stimulate MGPC proliferation was dependent on microglia. We found that microglia do not significantly influence MMPs. In summary, MMPs play a key component in the pathways that regulate MGPC formation. This work contributes to our understanding of the mechanisms that underlie retinal regeneration and can play a role in the development of new therapeutic treatments that target vision restoration. Category: Laboratory/Cellular Health Sciences

Title: The ability of the Aurora-A inhibitor alisertib to potentiate the anti-proliferative effects of VEGFR inhibitors in glioblastoma cells

Student Presenter: Caroline Mifsud

Faculty Advisor: Lehman, Norman

Abstract: Glioblastoma (WHO grade IV glioma) is the most common primary malignant brain tumor in adults, and is generally incurable. The prognosis is poor, with a median survival of only 14.6 months. Aurora-A, a serine-threonine kinase critical for a variety of cellular processes including centrosome duplication, spindle assembly, and mitotic exit, is widely overexpressed in glioblastoma. Additionally, malignant gliomas also possess high levels of vascular endothelial growth factor (VEGF), which is associated with pathological vasculogenesis and angiogenesis. Therefore, drugs that inhibit Aurora-A, in addition to VEGF and its receptor, are of interest in order to inhibit neovascularization and cellular proliferation. It was hypothesized that combining alisertib with a VEGFR inhibitor, such as cabozantinib or vandetanib, would work synergistically to inhibit glioblastoma cell proliferation and induce cell death via apoptosis. The glioblastoma cell lines U87 and U1242 were treated with a range of concentrations of alisertib and either cabozantinib or vandetanib. Glioblastoma cells were seeded at 600 cells/60mm plate. After 72 hours of drug exposure, cell colonies were counted, and the percent survival was graphed relative to the control untreated cells. The results of these experiments were analyzed using Chou- Talalay and Bliss Independence models to test for synergism and determine their statistical significance. Preliminary results of in vitro colony formation assays suggest that alisertib works synergistically to inhibit cellular proliferation with both cabozantinib and/or vandetanib in both cell lines; further in vitro testing is currently in progress. Because of the lack of effective treatments available for glioblastoma, successful completion of this study may provide a basis for clinical trials including these drug combinations. The results of such clinical trials may expand the potential treatment options for this aggressive disease, and improve the lives of glioblastoma patients. Category: Laboratory/Cellular Health Sciences

Title: The association of mannose binding lectin genotype and immune response to Chlamydia pneumonia: The Strong Heart Study

Student Presenter: Alaina Monsey

Faculty Advisor: Anderson, Matthew

Abstract: The incidence of heart disease within American Indians is twice as high as the general US population. The Strong Heart Study (SHS) is an investigative response to the inadequate information on heart disease in American Indians. Previous studies within SHS identified correlations between heart disease and both deficiencies in mannose binding lectin (MBL2), a surface binding protein, and prior infection with Chlamydia pneumoniae. However, a link between MBL2 and C. pneumoniae in contributing to heart disease has not been established. To address this, SHS collected baseline antibody titers (IgA and IgG) to C. pneumoniae and genotypes for common functional MBL2 variants for 694 individuals from thirteen participating tribes. Two common promoter SNPs that determine MBL2 expression (H/L and X/Y) deviated from Hardy-Weinberg equilibrium whereas common coding variants in exon 1 did not. Interestingly, the X/Y promoter SNP correlated significantly with increased C. pneumoniae IgG titer levels. Deviations from the reference sequence for exon 1 were found to be associated with decreased antibody levels to C. pneumoniae. No full MBL2 genotype predicted antibody titer levels. Additional descriptive factors such as elevated age, less education and male gender associated with increased C. pneumoniae IgG antibody titers as well. This study demonstrates that MBL2 genotype influences immune reactivity to C. pneumoniae in the SHS cohort and may contribute both directly and indirectly through pathogen interactions to the progression of CHD among Native Americans. Ultimately, the health community can adopt early prevention strategies to reduce CHD onset and severity based on individuals with specific MBL2 genotypes and microbial infections. Category: Laboratory/Cellular Health Sciences

Title: Fetal bovine serum vs human platelet lysate: In vitro-generation of bone extracellular matrix on poly (propylene fumarate) tissue engineered bone grafts

Student Presenter: Stefani Montelone

Faculty Advisor: Dean, David

Title: Food safety of homemade baby foods: knowledge, practices and behavior of mothers and caregivers in Morogoro, Tanzania

Student Presenter: Kristen Motil

Faculty Advisor: Ilic, Sanja

Abstract: Millions of children, aged 6-24 months, are exposed to foodborne pathogens when homemade, complementary baby foods are introduced into the diet, resulting in diarrhea that often leads to growth faltering episodes or death. Developing effective food safety interventions, appropriate for low-income mothers and caregivers of these children, will reduce the risk of foodborne pathogens in homemade baby food and improve health among Tanzanian youth. The objectives of this study were to determine food safety knowledge among mothers and caregivers of 6-24 month old children in Morogoro, Tanzania and to determine microbial load and prevalence of pathogens in homemade baby foods in these households. Purposive random sampling was used to select 289 participants (150 receiving nutrition intervention, 139 without nutrition intervention). Using purposive sampling design, we recruited mothers and caregivers of children 6-24 months in eight low-income wards of the region. Participants were 18 years or older, had resided in Morogoro for more than six months prior to the study, were able to understand Kiswahili language and give out informed consent to participate in the study. The inclusion criteria did not limit the gender of the caregivers. The data collection tools used was structured questionnaires and structured observations. Samples of some of the baby food were tested for E. coli and L. mono. The group that received nutrition intervention had better practices with regard to breastfeeding (P2 = 13.4) and fed children 3 meals/day (P 2 = 47.1). The group that did not receive intervention often fed only one meal (P 2 = 47.1). The group that received nutrition intervention had a higher level of inadequate food storage practices, and was not aware that contamination may occur during storage. Adequate storage of perishables (P Category: Laboratory/Cellular Health Sciences

Title: In vitro generation, characterization, and reprogramming of monocyte-derived tumor-associated macrophages

Student Presenter: Tiffany Noel

Faculty Advisor: Carson, III, William E.

Abstract: The tumor microenvironment (TME) contains immuno-stimulatory and immuno-suppressive immune cells, including natural killer (NK) cells and myeloid-derived cells, such as pro-inflammatory macrophages (M1), anti-inflammatory macrophages (M2), and tumor-associated macrophages (TAMs). The efficacy of monoclonal antibody (mAb) cancer therapies was shown to depend on the immune system, primarily NK cell anti-tumor activity. After interaction with mAb-coated tumor cells, NK cells produce pro-inflammatory cytokines (e.g. interferon-gamma [IFN-γ]) and kill cancer cells. In breast cancer, TAMs are associated with poor prognosis due to their M2-like characteristics. However, studies suggest that TAMs exhibit plasticity susceptible to stimulatory or suppressive signals within the TME. We hypothesized that TAMs can be redirected to an M1-like phenotype and enhance NK cell anti- tumor effector functions. M1 and TAMs were generated from freshly isolated human monocytes cultured for 7 days in medium supplemented with 10 ng/mL of GM-CSF (M1) or 10 ng/mL of M-CSF, IL-4 and IL-10 and 50% tumor-conditioned media (TAMs). Cell phenotypes were confirmed via surface marker expression (CD14, CD68, CD163, CD206) and gene expression (ILâ€‘6, IL-10, CCL2, CCL22). Coâ€‘cultures were performed with NK cells, monocytes/M1 and/or TAMs in the presence of trastuzumab-coated HER2â€‘positive tumor cells, and supernatants were analyzed by ELISA. Coâ€‘culture experiments demonstrated that TAMs significantly decrease the level of IFN-γ production of NK cells alone (p Category: Laboratory/Cellular Health Sciences

Title: Optimization of bioartificial muscle to discover the mechanotransduction pathways of muscle atrophy in microgravity

Student Presenter: Joshua Ong

Faculty Advisor: Lee, Peter

Title: A modified Chitosan dressing potently inhibits biolfilm forming bacteria

Student Presenter: Priyanka Pandey

Faculty Advisor: Steiner, Shomita

Abstract: Opticell is a dressing which contains properties derived from the shells of crustaceans called chitosan (a deacylated version of chitin) and is used topically on surgical wounds. A modified chitosan dressing (mCSD) with gelling technology is the first application of this advanced biological material for benefitting wound care. Although preliminary clinical studies indicated improved wound closure, the mechanisms for this is unclear. Our aim was to classify the anti-microbial properties of mCSD. Static biofilms (48 hour) of Pseudomonas aeruginosa (PAO1) and Staphylococcus aureus (USA300) were grown on mCSD and examined by scanning electron microscopy (SEM) to evaluate the structural integrity of the biofilm. Along with the SEM imaging, colony forming units were determined to test viability and real time PCR quantitative information was processed to identify gene expression. In addition, the effect of mCSD (intact, denatured, mechanically disrupted, and supernatant alone) on the growth and viability of PAO1 and USA300 were tested. Initial studies of 48 hour static biofilm formation indicated that mCSD serves as a suitable substrate for the growth of primary wound pathogens. Interestingly, when treated with acid or mechanically disrupted, the dressing showed significant inhibitory effects on biofilm formation. However, the base treated dressing did not have a similar outcome. Additionally, bacterial growth curves of PAO1 and USA300 indicated that intact mCSD attenuated the growth kinetics of these pathogens, suggestive of a bacteriostatic effect on both organisms. When exposed to the mCSD supernatant alone, the growth of these cultures was potently inhibited along with their virulence gene expression of both organisms in comparison to untreated cultures. Our observations indicated that i. when altered by acid or mechanical disruption, mCSD was no longer a viable substrate for biofilm adhesion and growth ii. may release soluble factors that aid in the disruption of infection formation in wounds. Category: Laboratory/Cellular Health Sciences

Title: Axon regeneration in the central nervous system: in-vivo and ex-vivo analysis

Student Presenter: Milin Pandya

Faculty Advisor: Shen, Yingjie

Abstract: Extensive damage to a developed spinal cord can be devastating. Nerve cells cannot regenerate effectively following spinal cord injury, leading to impaired sensation and/or movement. The post-injury scar is a major impediment to regrowth. Through signaling with the receptor PTP Sigma, it has been shown that the sugars Chondroitin Sulfate Proteoglycan (CSPG) and Heparin Sulfate Proteoglycan (HSPG) produce opposite effects on neurite growth. CSPG inhibits axon growth at the lesion site, while HSPG promotes it. The first purpose of this experiment is to test whether the injection of additional HSPG at the injury site will alter the signaling of PTP Sigma to favor axon growth and counteract the inhibition of CSPG. Thirteen rats were given a T8 left side lateral hemitransection lesion. Six control rats were treated with collagen at the injury site and seven were injected with the HSPG Glypican-2, infused in collagen. Results from immunohistochemistry show that an increased number of axons from the corticospinal tract (CST), the most difficult to regenerate, are near the injury site border in HSPG-treated rats compared to controls. Furthermore, the presence of the proteins Tau and APP were analyzed via microscopy. The effect of Tau, in particular, on CST axons is lessened in the experimental versus control group, suggesting a decrease in its inhibitory effects. These studies show that Glypican-2 may serve as a promising therapeutic drug for spinal cord injury by promoting axon regeneration into and around the lesion site. In addition, results are currently being collected for the second purpose of the experiment: to construct a similar model in an adult mouse central nervous system cell culture outside of the body. An accurate, reproducible model, which has been a challenge for researchers, will provide the science community with a critical tool to further study regeneration in nerve cells. Category: Laboratory/Cellular Health Sciences

Title: Variants in the gene encoding cholesteryl ester transfer protein and risk for coronary artery disease

Student Presenter: Komal Paradkar

Faculty Advisor: Sadee, Wolfgang

Abstract: Coronary Artery Disease (CAD) continues to be a major killer worldwide, accounting for over 17 million deaths annually. The main cause is atherosclerosis, the accumulation of cholesterol and fatty acids in blood vessels, which leads to adverse cardiac events. Cholesteryl Ester Transfer Protein (CETP) functions in cholesterol transport. CETP has several single nucleotide polymorphisms (SNPs) that are associated with variable levels of cholesterol which may in turn affect risk of atherosclerosis. The focus of this study was to characterize a new candidate variant, rs6499863, a SNP located upstream of the CETP promoter region that we found to be associated with CETP mRNA expression in the Genotype- Tissue Expression project database in both spleen and liver. To test this further, we measured allelic CETP mRNA expression ratios in human autopsy spleen tissues. Any deviation of these ratios from unity (or allelic expression imbalance, AEI) is a powerful indicator of regulatory variants. While rs6499863 alone failed to account for the observed AEI ratios detected at a marker SNP in CETP mRNA, the AEI pattern was consistent with a combined effect together with rs247616, another regulatory variant previously described by our group. Similarly, we found no evidence for an association of rs6499863 alone with CAD in a cohort of 1279 cases and 457 controls. However, this potential enhancer SNP could interact with other regulatory variants and thereby influence risk of CAD, which needs to be studied further. The results suggest that the effect of multiple variants in a single gene locus such as CETP must be considered to understand the genetic influence of the gene on a complex and pervasive disorder such as CAD. The ultimate goal is to find biomarkers that could provide information on risk assessment and facilitate new personalized preventive measures for cardiovascular health. Category: Laboratory/Cellular Health Sciences

Title: RanBP9 abrogation by CRISPR/Cas9 as a novel tool to study DNA damage response in lung cancer

Student Presenter: Kareesma Parbhoo

Faculty Advisor: Vincenzo , Coppola

Abstract: The Ran Binding Protein 9 (RanBP9) is involved in the cellular DNA Damage Response (DDR) following genotoxic stress, however, a specific molecular mechanism of RanBP9 in lung cancer is still missing. RanBP9 is an evolutionarily conserved protein with elusive biological functions. We previously demonstrated that reduction of RanBP9 expression reduces or delays the cellular DDR following genotoxic stress such as Ionizing Radiation (IR). In order to completely abrogate RanBP9 expression, we used the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) system, through which I generated two lung cancer cell lines (A549 and H460), in which the endogenous RanBP9 gene is genetically deleted. The clones were screened via western blot to validate the absence of RanBP9. The A549 and H460 knock out cell lines were stably transfected with either plasmids expressing RanBP9 fused to the Green Fluorescent Protein (RanBP9-GFP) or to a protein tag (RanBP9-Myc/DDK) to better understand the biological functions of RanBP9. Using clones expressing RanBP9-GFP, the subcellular localization changes in response to genotoxic stress was visualized to further reveal its role in DDR. The generation of these clones paves the way to biochemical studies aimed to identify the RNA interactors with RanBP9 via RNA immunoprecipitation and RNA sequencing as well as proteins via protein co- immunoprecipitation and proteomics analysis. These experiments will provide a comprehensive representation of the molecular partners interacting with RanBP9 in cellular DDR, particularly in response to genotoxic treatments such as IR and cisplatin. Category: Laboratory/Cellular Health Sciences

Title: Manipulating carbohydrate and cheese intake and the effects on health markers in individuals with metabolic syndrome

Student Presenter: Austin Peterjohn

Faculty Advisor: Volek, Jeff

Abstract: The current dietary guidelines for Americans recommend limiting saturated fat intake. Diets high in saturated fat have been thought to increase total cholesterol levels and increase risk of heart disease. Recent evidence suggests the association between saturated fat intake and risk for heart disease is not so straightforward, especially in the context of carbohydrate intake. Additionally, recent research has begun to elucidate that full-fat dairy consumption, including cheeses, may not result in any untoward cardiometabolic effects. Therefore, the purpose of this investigation was to determine the cardiometabolic effects of increased saturated fat intake when combined with varying levels of carbohydrate intake in individuals with metabolic syndrome. This study utilized a within subjects, randomized controlled feeding design. Male (n=6) and female (n=3) adults with metabolic syndrome completed four feedings phases. Order of completion of the feeding phases was balanced and randomized. The four experimental diets were as follows; high carbohydrate diet (HCLCh) with no cheese, HCD with high cheese (HCHCh), moderate carbohydrate diet (MCD) with high cheese, low carbohydrate diet (LCD) with high cheese. Each diet phase lasted four weeks with a two week washout period between each phase. Calorie intake necessary for weight maintenance per individual was measured using indirect calorimetry with a metabolic cart. Blood pressure, waist and hip circumference, fasted glucose, total cholesterol, HDL, LDL, and blood triglycerides were measured at baseline and after every diet phase. No significant differences between the four diet phases on any of the cardio-metabolic risk factors were found. However, there were trends for decreased fasting blood glucose and decreased triglycerides following the LCD. The current study demonstrated that a high consumption of full-fat dairy does not result in an altered cardio-metabolic risk state. Category: Laboratory/Cellular Health Sciences

Title: Influence of tomato variety on carotenoid accumulation and prostate cancer incidence in the TRAMP mouse model

Student Presenter: Remy Powell

Faculty Advisor: Clinton, Steven

Abstract: Prostate cancer (PCa) is a burdensome disease with approximately 1 in 7 men being diagnosed in his lifetime. The early-onset and long progression of PCa suggests the possibility of prevention based interventions, especially for high risk individuals, prior to traditional treatments. Additionally, there has been an accumulation of evidence for an inverse association between lycopene, the phytochemical which conveys the red color, and tomato consumption and risk of PCa. This relationship has been investigated in epidemiological studies, predominantly capturing red tomato consumption containing trans-lycopene. However, all lycopene is not created equally and the tetra-cis lycopene isomer present in the tangerine tomato is hypothesized to be 50% more bioavailable as preliminary data shows tetra-cis lycopene can lead to 8.5 times greater maximum plasma lycopene concentrations. Furthermore, cis-lycopene is the major conformational form present in prostate tissue. This data suggests that tangerine tomatoes and the cis-lycopene they contain may exhibit more potent anticancer activity. To evaluate this hypothesis, TRAMP mice, a strain genetically modified to develop prostate cancer, were fed either a 10% tangerine or 10% red tomato based diet from 4 weeks until 18 weeks-of-age. The plasma concentrations of lycopene were quantified by HPLC-MS/MS and prostate tissues were processed for hematoxylin and eosin staining followed by histopathologic analysis. Even though the red tomato diet contained more total carotenoids, the plasma carotenoid and total lycopene concentrations from mice fed tangerine tomato diets being considerably higher (5.4 and 2.8-fold, respectively) compared to red tomato diet. Both tomato-based diets resulted in approximately 40% reductions in PCa incidence as compared to those consuming a control diet. Results of these studies may inform future food-based intervention studies designed for cancer prevention. Category: Laboratory/Cellular Health Sciences

Title: Expression of microRNA-155 upregulates metabolic pathways in human natural killer cells to facilitate interferon-gamma production

Student Presenter: Aman Prasad

Faculty Advisor: Chan, Wing

Abstract: Natural Killer (NK) cells are granular innate lymphocytes that orchestrate immune response by releasing interferon gamma (IFN-γ): a critical cytokine heavily involved in intercellular immune and inflammatory processes. Metabolism - the process through which cells maintain energy requirements - has been highlighted as a way through which immune cells regulate such activity. Prior work from our lab has linked microRNA-155 (miR-155) to significant cell proliferation and NK cell mediated IFN-γ release. Furthermore, miR-155 has been shown to upregulate PI3K, which has been associated with cellular metabolism. We therefore hypothesize that miR-155 induced upregulation of IFN-γ occurs through metabolic reprogramming in NK cells. Following miR-155 overexpression in primary human NK cells, Seahorse metabolic flux assays demonstrated that miR- 155(+) cells had significantly higher glycolytic rates than control cells (3.00-fold increase over control). Furthermore, RT-PCR analysis showed that genes involved in glycolysis - such as glut1 (2.00-fold increase over control) and hhk2 (5.50-fold increase over control) - were upregulated following 4 hours of stimulation via IL-15. These data suggest NK cell activation is driven primarily through miR-155 mediated glycolysis. To confirm the role of glycolysis on IFN-γ production, IL-15 stimulated human NK cells were incubated with metabolic blockers inhibiting glycolysis, oxidative phosphorylation, fatty acid oxidation, or glutaminolysis. IL-15 induced IFN-γ production was severely dampened upon inhibition of glycolysis and oxidative phosphorylation (n=3; p=0.005; p=0.001), but not glutaminolysis and fatty acid oxidation (n=3; p=0.15; p=0.72). Upon IL-15 stimulation for 4, 8, 12, and 24 hours, NK cells showed a positive correlation between miR-155 expression and IFN-γ production, with a high regression coefficient (r2=0.96). This indicates that IFN-γ production is dependent on the metabolic profile of NK cells as controlled through miR-155. Thus, our data provides the first indication, to our knowledge, that miR-155 mediates IFN-γ production in NK cells through upregulation of glycolysis and modulation of overall metabolism. Category: Laboratory/Cellular Health Sciences

Title: TrkB agonist promotes the formation of proliferating Müller Glia-derived Progenitor Cells in retina

Student Presenter: Colin Quinn

Faculty Advisor: Fischer, Andy

Abstract: Diseases of the eye can cause death of retinal neurons and result in vision loss. Under such damaging conditions, Müller glia (MG), the primary type of support cell in retina, have potential to reprogram into Müller glia - derived progenitor cells (MGPCs). MGPCs are capable of neurogenesis and can be stimulated to repair damaged retinas. Understanding the signaling cascades responsible for the reprogramming of MG into MGPCs is crucial for the development of treatments. Currently, there are no known treatments to replace lost neurons. In this study, we investigated TrkB signaling in vivo in the reprogramming of MG into MGPCs. TrkB is a receptor for the brain-derived neurotrophic factor (BDNF) signaling pathway, which is involved in neuronal proliferation, survival, and neurogenesis. We examined this by applying intraocular injections of NMDA, an excitotoxin known to cause the death of retinal neurons and stimulate MGPC formation, to post-hatch chicks with TrkB agonist N,N',N''Tris(2-hydroxyethyl)-1,3,5-benzenetricarboxamide (LM 22A4). Using Edu labeling, we examined retinal sections for MG proliferation and found significantly more proliferation in LM 22A4 treated retinas versus control saline treated retinas. Using TUNEL assay to detect dying cells, we found no significant differences between LM 22A4 treated retinas versus control saline treated retinas. In investigation of downstream signaling of TrkB, we probed for activation of MAPK and mTOR pathways and found no significant differences of signaling in LM 22A4 treated retinas. Additionally, we examined whether LM 22A4 was mitogenic to other retinal glia. Using immunohistochemistry, we observed no changes in microglia (CD45+/Edu+) and Non-astrocytic Inner Retinal Glial-like (NIRG) (Nkx2.2+/Edu+) proliferation in LM 22A4 treated retinas. These data suggests that TrkB signaling specifically promotes MG proliferation after retinal damage independent of neuronal survival; thus, TrkB signaling is a target that promotes the regenerative potential of MGPCs. Future studies will investigate mechanisms responsible for this effect. Category: Laboratory/Cellular Health Sciences

Title: iNKT cells enhance CD4-dependent and CD4-independent CD8+ T cell-mediated cytotoxicity and rejection

Student Presenter: Bryce Ringwald

Faculty Advisor: Bumgardner, Ginny

Abstract: Currently, the United States has a shortage of livers suitable for transplantation. Hepatocyte transplantation has great therapeutic promise for patients with metabolic disorders or liver failure. Transplanted hepatocytes elicit both cellular and antibody-mediated immunity that damage and ultimately reject the transplant. Of these two immune responses, cellular immunity is dominant in our mouse model of hepatocyte transplantation and leads to rapid rejection. Cytotoxic CD8+ T cells, critical mediators of cellular immunity, can mature in the presence or absence of CD4+ T helper cells. Despite the role of lymph nodes in stimulating CD4-dependent CD8+ T cells, CD4-independent CD8+ T cells, can be sufficiently activated in the liver, independent of lymph nodes. A unique feature of the liver is a high amount of type I natural killer T cells (iNKTs), an innate immune cell that recognizes foreign and self- expressed glycolipids and is proposed to play a role in directing adaptive immune responses, which includes T and B cells. We hypothesized that, in the liver, iNKTs can help CD4-independent CD8+ T cells (which show activity in the absence of CD4+ T cells and lymphatics) develop into cytotoxic CD8+ T cells that elicit rejection. Using a well-characterized mouse model of hepatocyte transplant, we investigated in vivo cytotoxicity and survival of transplanted hepatocytes. CD4-independent CD8-mediated cytotoxicity and rejection is critically dependent on iNKTs, as both are abrogated in the absence of iNKTs. One mechanism that iNKTs may enhance CD4-independent CD8+ T cells is by enhancing the activation or proliferation of CD8+ T lymphocytes as evidenced e by more activated CD8+ T cells in the presence of iNKTs following transplant into CD4-deficient hosts. We also found that CD4-dependent CD8-mediated cytotoxicity is partially dependent on iNKTs. The implication of these studies is that CD4+ T cells and iNKTs utilize distinct and critical pathways to enhance CD8-mediated effector function. Category: Laboratory/Cellular Health Sciences

Title: Establishing an essay to identify small-molecules that block transmission of the human malaria parasite, Plasmodium falciparum

Student Presenter: Abubakarr Rogers

Faculty Advisor: Drew, Mark

Abstract: Plasmodium falciparum, the most pathogenic human malarial parasite, has multiple life stages in the human host: primarily the asexual blood stages and the sexual blood stages, the latter of which is infectious to female Anopheles mosquitos. Central to efforts to eradicate the parasite, it is important to inhibit gametocytogenesis, the process by which sexually-transmissible gametocytes are formed. This project focuses on establishing a quantitative assay for the process of gametocytogenesis, and gametocytal activity of small-molecules in P. falciparum. In the Drew lab, using reported protocols, the asexual blood-stages of P. falciparum are being cultured and, after 6 days, the hematocrit of the culture is slowly increased to apply stress to the parasites to promote gametocytogenesis. The parasites are then cultured to maintain synchronicity. Currently, the potential transmission inhibiting activity of small- molecule compounds are being tested to determine if they prevent the formation of gametocytes and/or the killing of transmissible gametocytes. Category: Laboratory/Cellular Health Sciences

Title: The Anti-Inflammatory drug alvelestat increases immunoglobulin production in vitro through activation of B-Cell Activating Factor (BAFF)

Student Presenter: John Rowe

Faculty Advisor: Boyaka, Prosper

Abstract: The efficacy of vaccination depends upon the induction of effector B and T cells, as well as memory B and T cell responses capable of providing rapid protection against future exposure to infectious agents. Since most infectious agents enter the host via mucosal surface, new vaccination strategies have focused on the induction of secretory IgA (SIgA), which coats the mucosal surface, in addition to systemic immunity. Previous research in our laboratory has shown that depletion of neutrophils improves the production of SIgA, and thus, demonstrated an inverse relationship between the ability of a vaccine to recruit activated neutrophils and induction of SIgA responses. We investigated whether anti-inflammatory drugs could represent an alternative to neutrophil depletion for induction of SIgA. For this purpose, splenocytes from C57BL/6J mice were cultured in the presence of varying doses of Alvelestat, an anti-inflammatory drug used for the treatment of chronic obstructive pulmonary disease. Flow cytometry analysis after 5 day culture revealed that Alvelestat significantly increased the frequency of cells expressing surface IgA and IgG (p Category: Laboratory/Cellular Health Sciences

Title: miR-21 in resolution of wound inflammation

Student Presenter: david schaeufele

Faculty Advisor: Roy, Sashwati

Abstract: Wound inflammation is a part of the wound healing cascade that aims to restore normal physiological function of injured tissue. Persistent inflammatory resopnse might be detrimental and cause loss of organ function. At a site of injury, cleaning of dead cells by macrophages (efferocytosis) is important to resolve inflammation. miRNAs are small non-coding RNA molecules which regulate gene expression by binding to the 3' UTR of targeted mRNA and have a critical role in inflammation. Our lab has previously reported an increased expression of inducible miR-21 in post-effercytotic peripheral blood monocyte-derived macrophages resulting in a net anti-inflammatory phenotype. However, wound macrophages are considerable different from cultured macrophages and there is no evidence on the role of miR-21 in wound macrophages in vivo. To elucidate the significance of wound macrophage miR- 21 in wound inflammation, an animal model with myeloid specific knock down of miR-21 was developed by crossbreeding mice carrying floxed miR-21 allele (miR-21fl/fl) with LysM-Cre mice. The animals were characterized by genotyping with DNA from tail biopsies by PCR. Bone marrow-derived monocytes (BMDM) were isolated from the femurs of mice and followed by positive selection using magnetic beads conjugated with CD11b antibody. For wound macrophages, PVA sponges were isolated on day 7 by repeated compression followed by CD11b positive selection. RTPCR analysis revealed a significant knowdown (~95%) of miR-21 in BMDM and wound macrophages. Current studies are ongoing to test the significance of the miR-21 in wound inflammation would enable an understanding of the miR-21- dependent mechanisms that are impaired in chronic wounds. Category: Laboratory/Cellular Health Sciences

Title: Biofilm infection impairs wound re-epithelialization

Student Presenter: Aurko Shaw

Faculty Advisor: Roy, Sashwati

Abstract: Microbial biofilms are one of the biggest issues when addressing chronic wounds. It has been shown to elongate the wound healing process. Currently, the understanding of host immunological response to bacterial biofilm is inadequate. Most studies with bacterial biofilm have been done in vitro or ex vivo, which lack pathways for immunological responses. Although these studies have been crucial in studying the microbiological effects, they have lacked the ability to understand the effects of biofilm on host infection under immune surveillance. This study was designed to understand the significance of biofilm on wound healing in vivo. Experiments were conducted on domestic Yorkshire pigs who were subjected to full-thickness burns (2"x2"). The pigs were separated into three groups per the sub-strain of Staphylococcus aureus they would be infected with: ^sarA-, USA300LAC, and ^rexB-. The strains indicate the ability of biofilm formation, as the ^sarA- strain is biofilm deficient, the USA300LAC strain is the wild type (methicillin-resistant S. aureus), and the ^rexB- strain is a biofilm overproducer. Biopsies of the wound were taken at d7, d14, and d35 time points. Biofilm formation was observed and verified using SEM and confocal imaging. Results showed that biofilm formation was the highest in the ^rexB- models, forming thick and dense masses of biofilm colonization on wounds compared to ^sarA- and USA300 models. Interestingly, histological evaluation showed that the biofilm on ^rexB- infected wounds had compromised formation of the epithelial barrier during re-epithelialization of the wounds. Current studies are in progress to understand the mechanisms in which biofilms impact wound re- epithelialization. Category: Laboratory/Cellular Health Sciences

Title: miR155 gene deficient mice fail to mount an efficient Th1 response but control the experimental visceral leishmaniasis caused by L. donovani.

Student Presenter: Rachel Sperling

Faculty Advisor: Satoskar, Abhay

Abstract: Micro RNAs are becoming increasingly investigated in the study of immune regulation during disease because of their impact on gene expression. Of particular interest is micro RNA 155 (miR155) which is vital for T cell mediated immune responses and Th1 associated IFN- γ production. Since IFN-γ is required for a protective host response against infection with Leishmania, we investigated the role of miR155 during the course of L. donovani infection. Mir155 KO and WT C57BL/6 mice were infected with L. donovani amastigotes and disease progression was monitored at 15, 40, 60, 90 and 120 days post infection. L. donovani infected miR155 KO mice displayed significantly higher parasitic loads in their livers and spleens compared to WT controls, but eventually resolved their infection. As expected, Leishmania antigen (LmAg) stimulated spleen cells from miR155 KO mice produced significantly less IFN-γ compared to WT controls. Interestingly, infected livers from miR155 KO mice expressed significantly higher gene transcript levels of IFN-γ and NOS2 suggesting that distinct tissue-specific host immune mechanisms are involved in the infected spleens and livers of miR155 mice. Furthermore, miR155 KO mice contained more PD-L1 expressing inflammatory monocytes in their livers and spleens compared to WT controls. Blockade of inflammatory monocyte recruitment in miR155 KO mice using a CCR2 antagonist also significantly reduced parasitic loads. In conclusion, our results reveal that although miR155 is involved in controlling parasite loads in the liver and spleen during L. donovani infection, it is not required for eventual disease resolution. Furthermore, our data shows that miR155 may play distinct roles in regulating IFN-γ production in spleen versus liver during L. donovani infection. Category: Laboratory/Cellular Health Sciences

Title: Efficacy of a novel oncolytic virus RAPTOR for treatment of breast cancer brain metastases in vitro and in vivo

Student Presenter: Alex Sprague

Faculty Advisor: Kaur, Balveen

Abstract: Breast cancer is among the leading causes of brain metastases (BCBM), with median survival of treated patients ranging from 2 to 25 months. Oncolytic viral therapy (OVT) is a promising biological therapy for the treatment of cancers, allowing researchers to delete harmful viral genes and insert therapeutic transgenes. These viruses efficiently lyse target cells while sparing non-target cells. Phosphatase and tensin homolog on chromosome ten (PTEN), a tumor suppressor, is downregulated in BCBM, resulting in neoplastic tumor outgrowth. A variant of PTEN, PTEN-Long, which is able to be secreted from and re-enter cells, has been combined with OVT to generate a novel virus called Reengineered And PTEN-Long Overexpressing Recombinant (RAPTOR). In vitro, RAPTOR infected human and mouse breast cancer cell lines show decreased Akt activity by western blot, and RAPTOR exhibits similar lytic efficacy and replication kinetics in these cell lines compared to parental HSVQ virus. In vivo, BCBM bearing FVB/N mice treated with RAPTOR showed dramatically improved survival over HSVQ treated mice (n=10 mice per group, p=0.044), as well as an increase in infiltrating and activated immune cells both 3 and 7 days following virus treatment. Moreover, 90% of RAPTOR treated animals were long term survivors versus 40% of HSVQ treated. We propose that the reintroduction of PTEN by RAPTOR alters the gene expression profile of the tumor and its microenvironment, restoring the ability of immune cells to infiltrate and destroy the tumor. We are actively investigating the role of activated macrophages, microglia, NK, and T-cells on the enhanced survival of RAPTOR treated immunocompetent mice. RAPTOR is a revolutionary treatment for BCBM, killing cancer by both lysing cells and recruiting immune effector cells, and further studies could lead to an understanding of how this treatment is able to attack cancer on multiple fronts. Category: Laboratory/Cellular Health Sciences

Title: Isolation of the competing effects of fluid mechanical forces on angiogenesis

Student Presenter: Griffin Spychalski

Faculty Advisor: Song, Jonathan

Abstract: Blood vessels comprise a hierarchical network that transports oxygen and nutrients throughout the body. Expansion of this network occurs through angiogenesis, where endothelial cells that line blood vessels sprout and branch to support tissue growth. Angiogenesis is necessary to repair injured tissue, while excessive angiogenesis is characteristic of rapidly growing solid tumors. In vivo, blood vessels are continuously exposed to fluid mechanical forces due to blood flow, including shear stress tangential to the vessel wall. Previous research has produced conflicting results regarding the angiogenic role of shear stress, suggesting both inhibitory or triggering effects due to challenges in isolating shear stress from additional fluid forces. To detach the role of shear stress in regulating angiogenesis from the effects of additional fluid forces, we implemented a microfluidic in vitro model of a branching vessel. The microfluidic platform was fabricated using soft lithography, and features an inlet channel lined with mouse aortic endothelial cells that bifurcates around a type I collagen-filled compartment. Shear stress due to fluid flow was imparted along the endothelium over 48h using a programmable syringe pump. To assess the effects of shear stress on angiogenesis, the change in sprouting area in the downstream channels was assessed under static and perfused conditions after 48h. Comparing the static conditions (0 dyn/cm2 shear stress) to the perfused conditions (5 dyn/cm2 shear stress), significantly less sprouting resulted under 5 dyn/cm2 shear stress (pin vivo, and explain new growth in vessels with stagnating flow. Taken together, our results will inform future investigations into the mechanisms of mechanical signal transduction within endothelial cells, which can lead to new targets for angiogenesis-based cancer therapies. Category: Laboratory/Cellular Health Sciences

Title: Transforming growth factor-B signaling regulates the proliferation of Müller Glia-derived progenitor cells in the retina

Student Presenter: Natalie Squires

Faculty Advisor: Fischer, Andy

Abstract: Müller glia cells in the retina can be stimulated to form Müller glia-derived progenitor cells (MGPCs) that can regenerate neurons. The cell signaling pathways involved in the formation of MGPCs are beginning to be revealed, and a better understanding will help to develop novel strategies for treating retinal diseases in humans. The goal of this study was to investigate whether Transforming Growth Factor-β (TGFβ)-signaling influences the ability of Müller glia to reprogram into proliferating MGPCs. Using the chick model system, MGPC formation was stimulated by intraocular injections of NMDA, which causes neuronal cell death. Immunohistochemistry was used to detect nuclear Smad2/3, as readout of TGFβ-signaling, in Müller glia after retinal damage. Using PCR, we found that the TGFβ2 ligand was highly expressed in the retina 2 days following NMDA treatment when MGPCs are known to form. Therefore, we applied intraocular injections of recombinant TGFβ2 following damage. We found that activation of TGFβ-signaling inhibited the formation of MGPCs and significantly decreased expression of the stem cell markers Pax6 and Klf6. We utilized small molecule inhibitors to TGFβ-signaling: SB-4316542, a TGFβ receptor inhibitor, and SIS3, which inhibits activation of SMAD transcription factors. Inhibition of TGFβ- signaling following NMDA treatment significantly increased the number of proliferating MGPCs. The growth factor FGF2 can stimulate MGPC formation in the absence of damage. Co-application of FGF2 and TGFβ inhibitor SIS3 significantly increased the number of proliferating MGPCs. Taken together, these results suggest that manipulation of the TGFβ-signaling pathway influences the reprogramming of Müller glia into proliferating MGPCs. Further studies on the factors that stimulate or inhibit the formation of neurogenic MGPCs will help develop novel approaches for treating diseases of the retina. Category: Laboratory/Cellular Health Sciences

Title: Retinoic acid signaling stimulates the proliferation of Müller glia-derived progenitor cells in the avian retina

Student Presenter: Lilianna Suarez

Faculty Advisor: Fischer, Andrew

Abstract: By the age of 65, one in three people will have a vision impairing disease. There are currently no therapeutic treatments for vision loss. Unlike the human retina, lower vertebrate retinas can regenerate their entire retina following damage. In the avian retina, Müller glia-derived progenitor cells (MGPCs) have the ability to dedifferentiation, proliferate, and subsequently differentiate into neurons. A number of signaling cascades have been implicated in governing these processes. In development, the retinoic acid pathway promotes proliferation and neuronal differentiation. As regeneration usually recapitulates development, we investigated whether retinoic acid influences the formation and proliferation of MGPCs in the avian retina. Preliminary results show the retinoic acid binding protein, CRABP, is expressed in MGPCs following NMDA-excitotoxic damage. We found that activation of retinoic acid signaling in a damaged retina using a retinoic acid homolog, TTNBP, increased the number of proliferating MGPCs. This was associated with an increase in the expression of Pax6, a transcription factor that is characteristic of retinal progenitors. Conversely, the inhibition of retinoic acid signaling using BMS493, a competitive inhibitor to the retinoic acid receptor, decreased the number of proliferating MGPCs. In FGF2-treated retinas, and in the absence of damage, the formation of MGPCs was increased by activation and decreased by inhibition of retinoic acid signaling. There was a corresponding increase in Pax6 expression following retinoic acid activation and a decrease in expression following inhibition of retinoic acid-signaling. These findings implicate retinoic acid signaling as an important therapeutic target to drive the formation and proliferation of MGPCs. Category: Laboratory/Cellular Health Sciences

Title: Defined media for differentiation of human mesenchymal stem cells to the bone lineage for use in a tissue engineered graft

Student Presenter: Briana Swan

Faculty Advisor: Dean, David

Abstract: Differentiation of human mesenchymal stem cells (hMSCs) with off-the-shelf osteogenic media is not optimized to differentiate and bring about the formation of bone extracellular matrix (ECM). We wish to optimize media to speed cell differentiation and deposition of ECM for the preparation of bone substitute materials. The Osteo Engineering Laboratory at The Ohio State University, in collaboration with Trailhead Biosystems and the Ohio Alive team at Case Western Reserve University, have developed a custom defined media regimen to drive differentiation of hMSCs without reaching an end-stage osteocyte fate, as osteocytes would express antigens that risk rejection of our bone graft. This study compares the effectiveness of the Ohio Alive bone tissue engineering defined media against current off- the-shelf media from RoosterBio (Frederick, MD), Lonza (Walkersville, MD), and HyClone (Logan, UT). Our bone marrow-derived hMSCs (BM-hMSCs) were cultured on 1x1 cm coupons of cast poly(propylene fumarate). After three days of proliferation, samples were placed in one of the four media studied. Alizarin Red S assay and stain were conducted on days 0, 1, 2, 3, and 6 (n=3) to observe calcification, an indicator of bone ECM formation. Statistical comparisons showed all four media were not significantly different with respect to calcium deposition by day 6 (p > 0.06). This suggests that either they are equally effective in mineralization by this time, or that more time must be allotted to determine a difference amongst these four types of media. With further testing, including a larger sample size and RNA analysis of biogenic markers, this experiment will show which media best drives these cells to deposit bone ECM. Category: Laboratory/Cellular Health Sciences

Title: Gender differences in blood pressure which affect murine stroke models

Student Presenter: Spencer Talentino

Faculty Advisor: Nimjee, Shahid

Abstract: 13 million annual global deaths can be attributed to occlusive arterial thrombosis leading primarily to stroke (87% ischemic) and myocardial infarction.1 The leading cause of ischemic stroke is carotid stenosis which occurs more frequently in men. While men experience a stroke rate of ~1.25 compared to women, women account for over 60% of stroke mortality by an unknown mechanism.1 The current standard of care to treat stroke uses intravenous recombinant tissue plasminogen activator (rTPA) and carries significant side effects.2 Patients treated routinely experience restenosis and symptomatic intracranial hemorrhage (ICH), which limits use to ~4.5 hours after symptom onset, resulting in 90% of patients unable to receive rTPA safely.2 Initially, this project intended to study ICH interventions, but revealed significant gender differences, which warrant further study. This project compared physiologic parameters of male and female mice including electrocardiography, heart rate, and blood pressure. Mice underwent middle cerebral artery occlusion (MCAO)3 and puncture, resulting in intracranial hemorrhage (ICH). After ICH, parameters were recorded while rTPA or vehicle was infused intravenously. 90 minutes after ICH injury, high resolution magnetic resonance imaging was performed. After MRI, monitoring was recorded before sacrifice and tissue harvest. Male mice had significantly higher blood pressure compared to females at baseline (p4 Recent studies also show that effectively lowering blood pressure immediately after intravenous thrombolysis protects against secondary ICH.5 Baseline blood pressure differences between genders may play a significant role in ischemic stroke outcomes and, more importantly, their response in blood pressure to pharmacological interventions. Category: Laboratory/Cellular Health Sciences

Title: The effect of multiple mild traumatic brain injures in adolescence on sleep in adulthood

Student Presenter: Kyra Ungerleider

Faculty Advisor: Weil, Zachary

Abstract: Every year, approximately 1.7 million people in the United States sustain a traumatic brain injury. Almost 90% of those injuries are considered mild TBIs (mTBI) and many go unreported and untreated. When injuries occur during early development they can interfere with critical processes such as myelination and synaptic pruning, and perhaps consequently, TBIs can be particularly devastating in children. Further, children that suffer traumatic brain injuries are at a much higher risk for future neurotrauma. Patients that suffer traumatic brain injuries report a large range of functional consequences including problems with cognition, executive function and sleep. The goal of this study is to determine if multiple mild traumatic brain injuries during adolescence lead to disrupted sleep cycles in adulthood and if this effect is prevented by an enriched environment, a manipulation that has been shown to protect the brain from TBI. Mice were divided into four groups in a full factorial design: sham and standard housing, injury and standard housing, sham and enriched housing, and injury and enriched housing. At three weeks of age, the mice received 3 mTBI's over the course of a week (MoWeFri). They were then allowed to age normally in enriched or standard conditions until adulthood. At 11 weeks of age, sleep quantity and quality was assessed using wireless biotelemetry, and injury severity was determined via silver staining. Injured mice showed unilateral axonal damage in the corpus callosum 9 weeks after injury. These mice had reduced REM and NREM spectral power following sleep deprivation and they failed to enhance REM sleep in response to environmental enrichment. These long lasting changes in sleep could be related to the cognitive and depressive symptoms reported by mTBI patients. Category: Laboratory/Cellular Health Sciences

Title: miR-21 in resolution of wound inflammation

Student Presenter: Tessia Vasquez

Faculty Advisor: Roy, Sashwati

Title: Expression and functional importance of discoidin domain receptors in dentoalveolar tissues

Student Presenter: Carolyn Wang

Faculty Advisor: Foster, Brian

Abstract: Collagen type I is an essential extracellular matrix (ECM) structural protein in dentoalveolar tissues, including dentin and cementum, and the rapidly remodeling periodontal ligament (PDL) and alveolar bone. Discoidin domain receptors (DDR1 and DDR2) regulate collagen fibrillogenesis and deposition in the ECM, and their extracellular domains have been shown to control mineral deposition. The roles of DDRs in dentoalveolar development and function have not been previously reported. In these studies, we aimed to analyze Ddr1 and Ddr2 mRNA expression during dentoalveolar development, and test the functional importance of DDR1 using a mouse model. We analyzed spatiotemporal expression of Ddr1 and Ddr2 in mice from 8 to 60 days postnatal (dpn) by in situ hybridization (ISH). Tissues from wild-type (WT) and Ddr1 knock-out (Ddr1-/-) mice were harvested at 4-6 months and analyzed via radiography, microcomputed tomography, histology, and histomorphometry. Expression of Ddr1 was identified in the epithelial tissues including the enamel organ and gingiva, but expression was not detected in any ectomesenchymal cells of the pulp, dentin, or periodontium. Conversely, Ddr2 expression was identified in odontoblasts and osteoblasts. Radiography revealed no major differences in Ddr1-/- vs. WT dentoalveolar structures; however, Ddr1-/- mandibles featured defective condyles and abnormal subchondral bone structure. Histomorphometry measurements of oral, dental, and periodontal tissues did not identify significant anatomic differences, including gingival epithelium. These studies indicate distinct expression patterns for DDR1 and DDR2, suggesting dissimilar functions in dentoalveolar tissues. While oral and dentoalveolar tissues appear functional in the absence of DDR1, ongoing studies will analyze mineralization in bones and teeth in these mice. Category: Laboratory/Cellular Health Sciences

Title: The efficacy of multi-kinase inhibitor CP1 in acute myeloid leukemia therapy

Student Presenter: Ziyue Wang

Faculty Advisor: Elgamal, Ola

Abstract: Acute myeloid leukemia (AML) is the most common form of adult acute leukemia in the United States. Annually 20,000 patients are diagnosed with AML, which is therapeutically challenging owing to its heterogeneity. Fms like tyrosine kinase 3 (FLT-3) mutations occur in up to one third of AML patients and is associated with poor prognosis. FLT3-internal tandem duplication (FLT3-ITD) or FLT3-tyrosine kinase domain (TKD) mutations constitutively activate the FLT3 proliferative pathway. Several selective FLT3 inhibitors have been developed to target FLT-ITD, however patients often relapse due to the acquisition of a FLT3-TKD. The use of a multi-kinase inhibitor could potentially overcome the resistance due to the TKD mutation. Compound 1 (CP1) is a novel, multi-kinase inhibitor which reduces phosphorylated levels of FLT3 as well as members of SRC family kinases, which are known to be upregulated in some types of AML. In this study, we investigate the efficacy of CP1 in human AML cell lines and determine the effect on downstream targets. Using an MTS proliferation assay we show that CP1 has an IC50 of Category: Laboratory/Cellular Health Sciences

Title: Regulation of macrophage cell death protein Mcl-1 during infection by Mycobacterium tuberculosis

Student Presenter: Ashlee Weaver

Faculty Advisor: Schlesinger, Larry

Abstract: Tuberculosis (TB) is one of the most threatening infectious diseases in the world today. In 2015, there were 1.8 million deaths resulting from infection with Mycobacterium tuberculosis (M.tb), the causative agent of TB. Following inhalation of M.tb, the bacterium infects alveolar macrophages in the lung, where it proliferates. In order to survive in the alveoli, M.tb manipulates the host cell immune response. Peroxisome proliferator-activated receptor gamma (PPARg), a master transcriptional regulator present in macrophages, is regulated by M.tb and is important for M.tb growth. To identify PPARg effectors during infection, we performed NanoString gene expression analysis of human monocyte-derived macrophages (MDMs) that had been transfected with scrambled control or PPARg specific siRNA (to knockdown PPARg), and then infected with M.tb. The results showed that PPARg knockdown leads to decreased Mcl-1 expression and increased Bax expression. These proteins regulate apoptosis, which is a cell death method used by the host to control infection. M.tb limits apoptosis to survive in its host. We decided to focus our studies on Mcl-1, which inhibits apoptosis. We hypothesized that M.tb increases Mcl-1 expression via PPARg during infection. The NanoString data were validated with qRT-PCR analysis of MDMs infected with M.tb with or without PPARg knockdown. Western blot analysis was also conducted to determine if M.tb increases Mcl-1 protein, and if this occurs through PPARg. These studies yielded confirmatory results for Mcl-1. The goal of our ongoing studies is to determine if M.tb represses apoptosis via PPARg regulation of Mcl-1 and similar genes. We are currently conducting Mcl-1 promoter reporter assays in the presence or absence of PPARg agonists to observe Mcl-1 promoter-driven expression of luciferase. The results of our study will help demonstrate how M.tb manipulates its host cell to survive and cause infection. Category: Laboratory/Cellular Health Sciences

Title: Lipoxygenases mediate production of endogenous PPAR agonists for Mcl-1 during infection by Mycobacterium tuberculosis

Student Presenter: Kiersten Woodyard

Faculty Advisor: Schlesinger, Larry

Abstract: Tuberculosis (TB) is one of the deadliest infectious diseases, with over one third of the world population infected with Mycobacterium tuberculosis (M.tb), the causative intracellular pathogen of macrophages. M.tb has been found by the Schlesinger lab to upregulate the transcriptional regulator PPARg in alveolar macrophages. It is through PPARg that M.tb suppresses immune responses and limits apoptosis, which is a type of regulated cell death. PPARg upregulates Mcl-1, which suppresses apoptosis furthering M.tb survival. Previously, the upstream PPARg endogenous agonist for Mcl-1 production was unknown, leading to my project in which I investigated potential endogenous PPARg agonists during M.tb infection. Cyclooxygenase-2 (COX-2) mediates production of the PPARg agonist PGJ2 from Arachidonic Acid (AA) and lipoxygenases (LOXs) mediate the production of PPARg agonists 15-HETE and 13-HODE from AA. It is unknown if these agonists are produced at high enough concentrations to activate PPARg inside the cell. We hypothesized that COX-2 and/or LOXs are important for PPARg activity through production of these agonists, and thereby increase Mcl-1 expression. We infected human monocyte-derived macrophages with M.tb, chemically inhibited COX-2 or LOXs, and probed for Mcl-1 as a read-out of PPARg activity by assessing changes in Mcl-1 protein levels in cell lysates by Western Blot. We found that Mcl-1 protein did not significantly change with COX-2 inhibition, while Mcl- 1 protein did significantly decrease when LOXs were inhibited. The results provide evidence that LOXs are important for PPARg activity with 15-HETE and 13-HODE as potential endogenous PPARg agonists during M.tb infection, which had previously been completely unknown. Furthering our understanding of how M.tb suppresses host cell immune responses will hopefully contribute to novel clinical host- directed therapies. Category: Laboratory/Cellular Health Sciences

Title: Endurance performance and body composition responses to 12 weeks of Keto-adaptation and physical training

Student Presenter: Tyler Young

Faculty Advisor: Volek, Jeff

Abstract: Nutrition is critical to mission success and survival of military personnel. Favorable body composition can impact relative strength and power to weight ratio. Ketogenic diets have been shown to enhance endurance performance and induce favorable changes in body composition. Therefore, the purpose of this study is to assess the extent to which a ketogenic diet (KD) and training intervention will affect endurance performance and body composition in military personnel. A personalized (KD) intervention guided by daily blood ketone measures was implemented in five healthy participants engaged in physical training three times per week. Healthy adults, 21±0.82yrs, participated in a twelve week KD and physical training intervention with emphasis on increased strength and power. Baseline and post-intervention body composition, hepatic, and heart fat measures were assessed prior to via dual energy x-ray absorptiometry (DXA), Cohen's effect size (d) and paired t-test were used to analyze results. Results are reported as mean±sd. Average blood ketones were maintained at 1.35mmol. Total body fat percentage and body weight were decreased from pre to post (25.7±2.2% to 20.0±3.1%, d=2.1 | p=0.02) and (83.5±8.2kg to 75.2±5.4kg, d=1.2 | p=0.01), respectively. Total average fat mass and lean body mass were decreased (20.8±2.9kg to 14.4±1.32kg, d=2.92 | p=0.01) and (59.9±5.6kg to 57.7±6.1kg, d=0.34 | p=0.006), respectively. Visceral adipose volume was unchanged (406.0±224.2cc to 390.7±85.3cc, d=0.09 | p=0.93). VO2max was unchanged (45.1±6.9ml/kg/min to 49.5±7.3ml/kg/min, d=0.62 | p=0.3). These preliminary data support an improvement in body composition and fat mass loss. Visceral adipose tissue and VO2max were unaffected by 12wk KD intervention in five healthy individuals. Ongoing work will continue to explore these responses in a larger cohort. Category: Laboratory/Cellular Health Sciences

Title: Evaluating the accuracy of a low cost diagnostic test for susceptible and drug resistant tuberculosis

Student Presenter: Annie Zhang

Faculty Advisor: Kwiek, Jesse

Abstract: A person dies of tuberculosis (TB) every 21 seconds. While acid fast bacilli (AFB) smear is the most common TB diagnostic method in Malawi, it is labor intensive, subjective, has less-than-ideal sensitivity, and cannot assess TB drug susceptibility. TB color plates (called CX-test), which simultaneously diagnose TB and TB drug-susceptibility to isoniazid (INH), rifampicin (RIF) and ciprofloxacin in ~14 days, are a low cost, simple, highly sensitive alternative TB diagnostic. TB CX-test has been tested extensively in research laboratories for its capacity to discern between susceptible and multidrug resistant TB (the latter defined as resistant to INH and RIF), but its performance in the field is not well characterized. In this pilot study, we enrolled 96 individuals suspected of TB, 10 percent of whom were HIV positive, at a rural hospital in Malawi. Participants provided basic demographic data and submitted sputum samples for TB testing using AFB staining and the TB CX-test. Discordant samples were resolved by the WHO approved GeneXpert TB diagnosis test. Sensitivity, specificity, positive- and negative predictive values for the TB CX-test were calculated. Our results showed that 12.5% of participants had a positive TB diagnosis, with only one specimen presenting discordant results between the AFB smear and TB CX-test with the GeneXpert tiebreaker confirming the TB CX-test results. The TB CX-test was highly sensitive (100%) and specific (100%). At a range of TB prevalence values, PVP and NPV were calculated to be 100%. The TB CX-test detected a small number of drug resistant cases (3%), with the TB CX-test detecting RIF resistance when the GeneXpert did not. Our findings demonstrate that the TB CX-test accurately identifies TB and may be an effective, low cost TB diagnostic method with the potential to simultaneously diagnose TB and test for drug sensitivity. Category: Laboratory/Cellular Health Sciences

Title: Impact of RAB9 silencing on autophagy signaling

Student Presenter: Bofei Zhang

Faculty Advisor: Strohecker, Anne

Abstract: Autophagy is an intracellular process that maintains homeostasis and enables survival of the cell under nutrient starvation by digestion and recycling of cell components and elimination of toxic materials and pathogens. Autophagy has become an intriguing therapeutic target as deregulation of the pathway has important consequences for aging, neurodegenerative diseases and cancer. A more complete understanding of the molecular mechanisms that modulate autophagy is necessary in order to determine the best way to target the pathway therapeutically. The small GTPase RAB9 was identified as a negative regulator of autophagy in a previously conducted shRNA screen although the mechanism by which it functions is unknown. The mechanistic Target of Rapamycin (mTOR) is the master negative regulator of autophagy, functioning at multiple steps in the pathway. The present studies will determine the TOR dependence of Rab9-mediated inhibition of autophagy. We will use immunoblot to assess TOR activation and autophagy in human lung cancer cell lines stably expressing control shRNAs or those targeting two distinct regions of Rab9 in the presence or absence of the mTORC1/2 inhibitor Torin2. In parallel, we will examine the impact of Rab9 silencing on localization of the transcription factor TFEB, which drives many genes related to autophagy and lysosome biogenesis, via immunofluorescence. TFEB localization is controlled in part by mTOR phosphorylation, which prevents its nuclear localization. This work will elucidate what if any portion of Rab9-mediated autophagy suppression is mTOR dependent in human lung cancer. Partial TOR dependence would suggest that Rab9 regulates autophagy via novel mechanism(s). This work will expand our understanding of autophagy regulation and reveal new targets of intervention for autophagy modulating therapies Category: Laboratory/Cellular Health Sciences

Title: Lisinopril and spironolactone as a treatment option for physically active Duchenne muscular dystrophy mdx mice

Student Presenter: Jonathan Zins

Faculty Advisor: Rafael-Fortney, Jill

Abstract: Duchenne muscular dystrophy (DMD) is a fatal X-linked neuromuscular disease that affects approximately 1:5000 newborn males. DMD is caused by a mutation in the gene encoding the dystrophin protein, loss of which causes muscle membrane fragility and progressive muscle degeneration. Delayed motor development is seen at age 2-5, leading to a complete loss of ambulation in the early teens, and death near age 25. Previous research from our laboratory demonstrated the use of FDA approved mineralocorticoid receptor (MR) antagonists (spironolactone) in combination with angiotensin converting enzyme inhibitors (lisinopril) in a mouse model of DMD resulted in decreased pathology and improved muscle function in both cardiac and skeletal muscles. In previous studies, mice experienced little to no physical exercise; however, young DMD patients are very active. We hypothesized that the combined treatment of lisinopril with spironolactone at an early stage of disease in physically active mdx mice will protect muscle fibers from degeneration. To this end, 12 mice were treated with a combination of lisinopril and spironolactone starting at 4 weeks of age and 12 mice were left untreated. These mice underwent treadmill exercise from 6 weeks until 10 weeks of age. 12 mdx mice and 5 C57/BL10 mice were left sedentary and untreated as controls. Once sacrificed, skeletal muscles were analyzed for evidence of necrosis, fibrosis, and cell regeneration via immunofluorescence and hematoxylin and eosin staining. We found that the amount of damage in the tibialis anterior muscle showed no statistical difference between treated and untreated exercise groups. This suggests that in physically active mice, drug dosages may need to be increased or used in combination with other therapies to attain the same effect seen earlier in sedentary mice. Future studies will optimize dosages to overcome the damaging effects of exercise.