Undergraduate Research Fellowship Research Articles

Pharmacologic Effects of a Synthetic Macamide Analogue (ZYPC) on A549 and HeLa Cells

Lepidium meyenii L., known as the Maca plant, is acclaimed by the scientific community for its various health benefits in traditional medicine, including prevention of gastritis, depression, and cancer, as well as increasing fertility and improving sexual performance. Macamides (N‐benzylamides) are secondary metabolites derived from Maca that have demonstrated neuroprotective, anti‐oxidative, anti‐inflammatory, endocannabinoid system modulatory, and mild anti‐diabetic properties. The present study investigates the pharmacologic effect of N‐(8Z‐heptadecen‐1‐yl)‐O‐(3‐pyridylmethyl) carbamate (ZYPC), a synthetic macamide analog, on A549 non‐small cell lung cancer and HeLa cervical cancer cells. Both cell lines were treated with different concentrations of ZYPC (1–100 μM). After 24 h, cell viability was measured using a 3‐(4, 5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS) colorimetric assay. ZYPC was found to significantly reduce cell viability in a dose‐dependent manner. The half maximal inhibitory concentration was calculated, using dose‐response non‐linear regression, as 38.94 μM for A549 cells and 38.35 μM for HeLa cells. Subsequently, a caspase 3 colorimetric assay was executed to study ZYPC‐induced reduction of cell viability through activation of apoptosis. HeLa cells were treated with 0, 30, 50, and 100 μM ZYPC and cell lysates were collected after 5 h incubation. A reagent containing DEVD‐pNA, a substrate for caspase 3, was added to the lysates, and incubated for 2 h at 37°C. Unexpectedly, ZYPC was found to have no effect on caspase 3 activity. Then, the involvement of a pro‐survival mechanism was assessed through AKT and phospho‐AKT (p‐AKT) chemiluminescent western blotting. Results obtained from treatment with 0, 10, 30, and 50 μM ZYPC for 24 h were inconclusive for total AKT and p‐AKT levels in both cell lines. Preliminary western blots of poly (ADP‐ribose) polymerase (PARP), a DNA repair enzyme, and its inactive cleaved form indicated constant expression levels of both proteins in A549 cells. Interestingly, there was an apparent increase in cleaved PARP in HeLa cells, which may suggest late‐stage apoptosis. However, further studies are needed to better elucidate the mechanism of anti‐cancer activity exhibited by ZYPC.Support or Funding InformationMCPHS University Summer Undergraduate Research Fellowship Program

‐The Function of CASK in Transcriptional Regulation in Neurons

CASK is a scaffold protein that functions in trafficking and targeting of synaptic proteins in the central nervous system. Emerging literature suggests that CASK localizes inside nucleus and might regulate transcription by interacting with TBR1, a transcription factor essential for brain development. However, these regulatory mechanisms are not well established. We hypothesize that CASK is involved in neuronal activity‐regulated transcription. To test our hypothesis, we used primary cultures of mouse cortical neurons stimulated with KCL depolarization buffer, which is known to activate a robust CREB‐dependent gene expression program and to induce the activation of distal enhancer elements. To measure gene expression genome‐wide, we performed RNAseq in unstimulated or KCL‐treated neurons after knock‐down of CASK expression using specific shRNA. Our results show that CASK knock‐down strongly reduces the number of genes that are regulated by depolarization. These results indicate that CASK is required for gene expression regulation in neuronal cells. To gain further insights in the mechanisms by which CASK regulate transcription, we used RNA in situ hybridization (RNAscope) to test the hypothesis that CASK is not only required for the transcription of coding genes, but also of non‐coding RNAs, termed ‘enhancer RNAs (eRNAs), in proximity of KCL‐regulated genes. We showed that RNAscope is a valid method to measure eRNAs induction after KCl treatment. Ongoing experiments in the lab are assessing the effect of CASK knock‐down on eRNA transcription. Interestingly, we observed by immunoprecipitation that nuclear CASK dimerizes, and that the stability of this homodimer is dependent on the presence of RNA. Finally, we used biochemical fractionation to study CASK localization in the nucleus in both mouse neuronal cultures and brain tissues. These results shed light on how CASK functions in neurons. This study is relevant to human disease because mutations in CASK gene are linked to microcephaly and X‐linked intellectual disability.Support or Funding InformationThis project has been supported by CABRI Undergraduate Research Fellowship

Elucidating the Effects of Distal Charges on the Reduction Potential of the Rieske Protein

The Rieske protein plays a key role in both cellular respiration and photosynthesis, transferring electrons through the respective cytochrome bc1 and b6f complexes of each process. It is characterized by a [2Fe‐2S] cluster and is unique in that one iron is ligated by nitrogens from two solvent‐exposed histidine residues while the other is ligated by the sulfurs of two cysteines. In the electron transport chain (ETC), the Rieske protein oxidizes ubiquinol and reduces cytochrome c, transferring electrons between complexes II and IV.Previous work has demonstrated how hydrogen bonds, solvent accessibility of the ligands, and the presence of nearby charged residues can impact the reduction potential of the protein. However, the full extent of this third element, how far from the cluster these charged residues can reside and still impact the reduction potential, has yet to be explored. Two types of mutants have been created to probe the effects of these distal charges. First, mutations removing positively charged residues were predicted to decrease reduction potential. Likewise, mutations removing negatively charged residues were expected to increase reduction potential. Each mutation either resides a unique distance from the cluster or produces a different change in overall charge, demonstrating the impact of each factor both individually and additively. Lastly, the pKa values of the histidine ligands were also expected to be inversely related to reduction potential.Using pH‐dependent UV‐Visible spectrophotometry, the pKa values of these mutants were determined and correlated to these relationships. While the overall trend of pKa values increased as expected for the negative‐charge mutants, confounding elements also appeared, such as a third pKa sigmoid. Furthermore, E146R, a positive‐charge mutant, also demonstrated increased pKa values, opposite to the hypothesized trend. New mutants that implement a neutral to positive substitutions have been developed to probe the effect of making the protein more positive.Support or Funding InformationMurchison Summer Undergraduate Research Fellowship, Welch Foundation

Coronary Vascular Cell Stiffness Influences Coronary Blood Flow

Our laboratory has previously shown that type 2 diabetic (T2DM) coronary resistance microvessels (CRMs) undergo inward hypertrophic remodeling associated with reduced stiffness. Reduced T2DM CRM tissue stiffness is associated with decreased elastic modulus of coronary vascular smooth muscle cells (VSMCs). The goal of this study was to test the hypothesis that reducing or augmenting coronary vascular cell stiffness will increase or decrease coronary blood flow, respectively. Coronary blood flow was measured from hearts isolated from 16‐week‐old control Db/db and diabetic db/db mice and perfused on a Langendorff system at a constant pressure of 80 mmHg. Latrunculin B (1 μM final concentration; n=2 per group) was infused to depolymerize actin and reduce vascular cell stiffness and jasplakinolide (0.2 μM final concentration; n=4 per group) was infused to increase actin polymerization and increase vascular cell stiffness. After drug infusions, hearts were immediately transferred to an adjacent Langendorff apparatus and were perfusion fixed with 4% paraformaldehyde for 2 mins at 1 mL/min. Hearts were then placed in 4% PFA for 24–48 hours and were stored in 70% EtOH until embedding in paraffin and sectioning. Sections were evaluated for F‐ and G‐actin by immunofluorescence. In pooled Db/db and db/db analyses, Latrunculin B caused an increase in coronary blood flow over baseline (34.1 ± 15.0% above baseline, n=7), and jasplakinolide caused a 26.5 ± 4.1% reduction in coronary blood flow from baseline (n=8). Immunohistochemical analysis confirmed a decreased trend in F/G actin in the coronary microvessels of latrunculin B‐perfused hearts (1.00 vs. 0.82 ± 0.08, n=1–4 per group), while F/G actin tended to be increased in the CRMs of jasplakinolide‐perfused hearts (1.00 ± 0.00 vs. 1.50 ± 0.16, p=0.12, n=2–6 per group). Importantly, the F/G actin ratio in jasplakinolide‐treated hearts was not different measured in the myocardium 50–100 μm away from a CRM (1.00 ± 0.06 in vehicle‐perfused hearts vs. 1.01 ± 0.10 in jasplakinolide‐perfused hearts, p=0.96, n=2–6 per group), demonstrating that the drug was efficacious only in the peri‐vascular region. These data support the hypothesis that coronary blood flow increases as vascular cell stiffness is decreased and coronary blood flow decreases as vascular cell stiffness is increased. Collectively, these data suggest that coronary vascular cell stiffness can modulate coronary blood flow.Support or Funding InformationKRM was supported by an American Heart Association Summer Undergraduate Research Fellowship (17UFEL33420025). NIH R00HL116769, R21EB026518, and S10OD023438 to AJT.

Detection of mRNA Encoding Organic Anion Transporters in Zebrafish Eyes

The solute carrier 22 (slc22) family is a group of transmembrane proteins that specialize in the transport of organic ions. A sub‐category of slc22 consists of organic anion transporters (OAT) that transport cyclic nucleotides such as cyclic adenosine monophosphate (cAMP). In the process of light to dark adaptation of fish eyes, cAMP is hypothesized to be a regulatory signal between the photoreceptors and retinal pigment epithelium (RPE) that drives pigment granule aggregation in the latter. We hypothesize that cAMP is exported from the photoreceptors into the sub‐retinal space, and one of the slc22 OAT proteins imports cAMP into the RPE. To test this hypothesis, reverse transcription polymerase chain reaction (RT‐PCR) was used to test for the presence of mRNA encoding seven different OAT proteins (1, 2a, 2b, 2c, 2d, 2e and 3) in whole eyes from zebrafish. Messenger RNA encoding each of the OAT proteins was detected, corroborating results of earlier studies. Experiments are underway to test which of the OAT genes are expressed in isolated RPE. Identification of the OAT proteins in RPE is an important step in illuminating a novel signaling pathway.Support or Funding InformationThis project was supported by Texas State University in the form of an undergraduate research fellowship to MCR and funds from the Office of the Provost awarded to DMG in recognition of her service as Presidential Fellow.

Resveratrol Attenuates Doxorubicin Cytotoxicity and Mitochondrial Dysfunction and Human Proximal Tubular Epithelial Cells

Doxorubicin (DOX, Adriamycin), is a cancer chemotherapy employed in the treatment regimen for breast, Hodgkin and Non‐Hodgkin lymphomas, small cell lung cancer and acute lymphoid leukemia. The U.S. FDA issued a Black Box warning for Cardiomyopathy in patients treated with doxorubicin. Other adverse effects include renal impairment. The cellular mechanisms for doxorubicin mediated renal cytotoxicity are not known. Resveratrol (RES) is a phytoalexin found in many fruits including grapes, blueberries, cranberries, dark chocolate and nuts. Antioxidant and anticancer properties have been associated with RES in cell culture and in human clinical studies in cancer patients and in vivo rodent models. Our studies tested the hypothesis that RES protection for DOX renal cytotoxicity, was mediated by maintaining mitochondrial function. All studies were conducted in human noncancerous renal proximal tubular epithelial cells (HK‐2). HK‐2 cells were plated and equilibrated for 48 h. Cells were next pre‐incubated for 1 h with 0 (DMSO) or 5 or 7.5 uM RES followed by a 24 h co‐incubation with 0–5 uM DOX. All results were obtained from 3 independent experiments. Western analysis for protein carbonylation and 4‐hydroxynooneal (4‐HNE) were used as an indicator of oxidative stress. Mitochondrial function was assessed using a Seahorse analyzer system. Prior to examining mitochondrial function, cell number and substrate concentrations were optimized based on the manufacturer’s recommendations. Mitochondrial Stress Test was conducted following the treatments described above and each well was normalized by protein. Viability was assessed using MTT leakage and tyrpan blue exclusion cell counts. RES did not alter cell viability as indicated by comparable MTT values between DMSO and RES groups (p>0.05). DOX was cytotoxic to HK‐2 cells within 24 h. Pretreatment with RES provided protection from DOX to HK‐2 cells. Basal mitochondrial respiration assessed as oxygen consumption rate (OCR) was diminished by 4 uM DOX. Mitophagy mediated by DOX and the effect of RES compared protein expression of LC3BI, LC3BII and the ratio of LC3BII/I. In summary, RES attenuated DOX renal cytotoxicity by maintaining mitochondrial function. Further studies are needed to probe changes in specific mitochondrial proteins mediated by DOX.Support or Funding Information(Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence; M.D. and R.M. supported by WV NASA Undergraduate Research Fellowship: M.D. Supported by MUSOM Summer Research Fellowship).

Determining the Activation Energy of Photoassembly of the Oxygen‐evolving Complex Using an Alternative Redox Mediator

The oxygen‐evolving complex (OEC) is a well‐researched Mn4CaO5 cluster with a cubane‐like structure that is responsible for oxidizing water to fuel photosynthetic electron transfer. It is a cofactor of photosystem II (PSII), which catalyzes light‐induced charge separation and oxidation of the OEC. PSII is comprised of pigment molecules and cofactors that transport electrons to a mobile plastoquinone that is located at the QB site. These reducing equivalents can eventually be used to reduce CO2 into simple sugars. In nature, the OEC is constantly degraded as a result of PSII photodamage and must be reassembled through a light‐dependent process after the affected PSII is repaired. OEC assembly occurs in the presence of light in vivo, but if conducted in situ is significantly enhanced by including a dark incubation after the first light dependent step. This dark‐dependent step is the rate‐limiting step of OEC assembly. Determining the activation energy will provide insight into the mechanism of OEC formation and provide clues on how artificial systems could be engineered.To conduct these studies, subcellular thylakoid membranes were isolated and enriched in PSII and the Mn4CaO5 cluster was removed via an alkaline incubation to generate apo‐PSII. The apo‐PSII enriched membranes were then used to measure the efficiency of OEC assembly in situ at temperatures ranging from 5 – 45 °C using Clark type oximetry. However, when OEC assembly occurs in apo‐PSII‐enriched membranes the rate‐determining step is electron shuttling from the QB site to the terminal electron acceptor {K3[Fe(CN)6]} via the native plastoquinones, thus preventing an accurate determination of the activation energy. This issue was remedied by supplementing with an alternative redox mediator know as phenyl‐p‐benzoquinone (PPBQ), which has been shown to increase the rate of electron shuttling from the QB site to a terminal electron acceptor. OEC photoassembly was successful in the presence of PPBQ and the oxygen production at varying temperatures was used to produce an Arrhenius plot to calculate the activation energy of OEC assembly.Currently, the activation energies have been determined for photoassembled PSII‐enriched membranes in the presence of PPBQ (18 kJ/mol) and native plastoquinones (49 kJ/mol). From these results, we speculate that OEC photoassembly in apo‐PSII membranes with PPBQ allows us to measure the true rate‐limiting step.Support or Funding InformationE.N. was supported by a fellowship from the SURE Program funded through the WV Research Challenge Fund, and administered by the WV HEPC, Division of Science and Research, Award Number DSR.17.09. E.N. had additional support from the WV NASA Space Grant Consortium’s NASA WVSG Undergraduate Fellowship Program and from the DOW Undergraduate Research Fellowship.D.K. and A.S. were supported by the National Science Foundation under Cooperative Agreement No. OIA‐1458952. Amanda Smythers was also funded by the NASA West Virginia Space Grant Consortium, Grant #NNX15AK74A and Training Grant #NNX15AI01H.J.B. had additional support from the WV NASA Space Grant Consortium’s NASA WVSG Undergraduate Fellowship Program and from the DOW Undergraduate Research Fellowship.

Evaluation of In Vitro Models for Pyrrolizidine Alkaloid‐Induced Hepatocellular Injury

ObjectivePyrrolizidine alkaloids (PAs) are toxic chemicals found in many herbal medicines, dietary supplements and even food sources such as honey, bread, and tea. Cases of drug‐induced liver injury due to herbal and dietary supplements are relatively common in Asia and are increasing in the United States. The literature suggests that PAs containing macrocyclic structure are more toxic than those with smaller sidechains. The goal of this project was to evaluate two PAs with macrocyclic structure (monocrotaline and retrorsine) and one PA will a smaller sidechain (retronecine) for their potential to elicit toxicity across two in vitro model systems.MethodsPA toxicity was measured by determining the concentration at which 50% of the cell population was killed (LC50). PAs were dissolved in DMSO and PA‐induced toxicity was measured after 48 hours in two in vitro models: TAMH (transforming growth factor‐α transgenic mouse hepatocytes) and THLE‐2 (transformed human liver epithelial‐2 cells). Statistical analysis of cell viability data was performed using SPSS (Probit regression) to obtain LC50 values.ResultsA decrease in cell viability was observed as PA concentration increased. Interestingly, both cell lines were relatively resistant to PA‐induced toxicity. In many cases only predicted LC50 values for each compound could be generated and are indicated by an asterisk (7.0mM*, 11mM*, and 30.mM* in TAMH for monocrotaline, retrorsine and retronecine, respectively; and 2.2mM, 3.2mM, and >100mM* in THLE‐2 for monocrotaline, retrorsine and retronecine, respectively. Based on the toxicologic parameters, monocrotaline and retrorsine were more toxic (lower LC50 values) than retronecine.ConclusionsWhile PAs have been associated with hepatotoxicity, limited toxicity was seen in both the TAMH and THLE‐2 cell lines. Predicted LC50 values were higher than expected, suggesting TAMH and THLE‐2 cells are not good models for studying PA‐induced liver toxicity. Retronecine, which contains a smaller sidechain, was found to be less toxic than the larger compounds. Future work is currently underway to identify biomarkers responsible for the observed resistance to PA‐induced toxicity as potential targets for preventing hepatocellular injury.Support or Funding InformationThis work was supported by the Pacific University Research Incentive Grant Program and an ASPET Summer Undergraduate Research Fellowship (SURF) award at Pacific University.

Eating a high fat diet enhances sensitivity of rats to serotonin syndrome

Eating a high fat diet can lead to obesity, type 2 diabetes, and dysfunction to neurotransmitter systems. For example, eating high fat chow enhances sensitivity of rats to unconditioned behavioral effects (e.g., lower lip retraction) induced by 8‐OH‐DPAT, a serotonin (5‐HT)1A receptor agonist. It is not known if eating high fat chow also impacts sensitivity of rats to other types of 5‐HT receptor agonists, or other behaviors induced by these drugs, collectively referred to as animal models of 5‐HT syndrome (e.g., flat body posture and forepaw treading). Recently, the FDA approved lorcaserin, a 5‐HT2C receptor agonist for the treatment of obesity. Lorcaserin induces some 5‐HT syndrome behaviors in rats including forepaw treading; however, it is not known if diet impacts sensitivity of rats to these lorcaserin‐induced behaviors. Further, previous research suggests that females might be more sensitive to the effects of 5‐HT receptor agonists, and to the effects of eating a high fat diet. To test the hypothesis that eating high fat chow will enhance the sensitivity of rats to the behavioral effects of 5‐HT2C and 5‐HT1A receptor agonists, 16 male and 16 female rats eating high fat chow (60% kcal from fat) or standard chow (17% kcal from fat) were tested once weekly with cumulative doses of 8‐OH‐DPAT (0.01–1.0 mg/kg, s.c.), WAY 163909 (1.0–32.0 mg/kg, i.p.), or lorcaserin (1.0–32.0 mg/kg, i.p.). 8‐OH‐DPAT induced lower lip retraction, flat body posture, and forepaw treading, while WAY 163909 and lorcaserin only induced forepaw treading. Rats eating high fat chow were more sensitive to lower lip retraction (induced by 8‐OH‐DPAT) and forepaw treading (induced by all three serotonergic drugs), as compared to male rats eating standard chow. These experiments are still ongoing; however, preliminary data and previous reports suggest that female rats will be more sensitive than males to diet‐induced enhancements in 5‐HT syndrome behaviors. Future directions will use selective 5‐HT receptor antagonists to examine the specific receptor subtypes mediating these behavioral effects in male and female rats eating different diets.Support or Funding InformationThe authors have no conflicts to disclose. N.M.B. was awarded summer funding through the American Society for Pharmacology and Experimental Therapeutics (ASPET) Summer Undergraduate Research Fellowship (SURF). N.M.B is also supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under R25GM069621.

Biophysical Studies of a Protein of Unknown Function from Cancer‐Associated Murine Herpesvirus‐68

Members of the Herpesvirus family cause several types of diseases to its mammalian hosts, including infectious mononucleosis, Hodgkin’s lymphoma, and primary effusion lymphomas. Herpesviruses are characterized by their ability to replicate and spread infection in the active lytic phase, but also maintain themselves within a host during the inactive latent phase. Within the gammaherpesvirus subfamily, a conserved protein of unknown function, with a highly conserved domain of unknown function, is expressed during the lytic phase. The protein was observed to be essential to viral replication during the lytic phase of infection when the gene was knocked out of the murine gammaherpesvirus‐68 (MHV68). To characterize its biophysical properties, the protein of unknown function from MHV68 was purified. Analysis of the protein through Circular Dichroism spectroscopy determined the secondary structure to be predominantly alpha‐helical. The thermal denaturation of the protein was pH dependent. Additionally, using a high throughput screen, the thermal stability of the protein was increased in the presence of small organic acids. The goal of the biophysical and structural analyses is to identify the function of the protein in the viral life cycle and to determine if the protein can be targeted to inhibit replication of gammaherpesviruses and their associated diseases.Support or Funding InformationThis work was supported by UWL College of Science and Health, UWL Undergraduate Research and Creativity, and Hardy W. Chan and Sons Chemistry Summer Undergraduate Research Fellowship.

Nitrated Fatty Acids Reduce Inflammatory Cell Recruitment in Bleomycin‐Induced Lung Injury

Acute lung injury (ALI) leads to infiltration of inflammatory cells and impaired lung function. Matrix Metalloproteinase 9 (MMP9) is a zinc‐dependent endopeptidase that degrades the extra cellular matrix and increases the porosity of the lung. CD11b is a marker for macrophage activation and migration. Nitro‐oleic fatty acid (OANO2), an electrophile that reduces cardiovascular inflammation has not been examined in lung injury. OANO2 modifies cysteine residues via Michael addition altering protein function. The goal of this study were to determine if OANO2 administration would reduce MMP9 expression and cellular infiltration following ALI. An intratracheal bleomycin (ITB) ALI model was used. Two groups of 6 C57BL/6‐J mice were treated intratracheally with bleomycin (3U/kg) or PBS (50uL). Half of the animals received OANO2 (50μg) in the same instillation and 72 hours later. The lungs were collected 7 days post ITB treatment. As previously seen, mice treated with ITB lost a significant amount of body weight; however, the addition of OANO2 mitigated this loss (−2.3 +/− 0.94 vs −0.4 +/− 0.83 g). Histology revealed cellular infiltration, deposition of proteinaceous debris, and airway epithelial damage in ITB mice that was reduced with OANO2 administration. The number of CD11b+ cells (marker for migratory macrophages) and MMP9+ cells was determined by immunohistochemistry. ITB increased number of CD11b+ (69 +/− 17.1 vs 195 +/− 17.1 #cells/HPF). OANO2 treatment reduced CD11b+ cells most in ITB animals (121 +/− 17.1#cells/HPF). ITB increased MMP9+ cell numbers compared to controls (71 +/− 15.7 vs 124 +/− 15.7 #cells/HPF). However, MMP9 expression was increased with OANO2 administration in PBS animals (304 +/− 15.7; 166 +/− 15.7 #cells/HPF), though this increase was not significant in ITB mice. Increased MMP9 expression was confirmed by western blot of whole lung tissue, where both ITB OANO2 and control OANO2 animals displayed high expression. Increased MMP9 expression may result from reduced self‐degradation as a consequence of OANO2 mediated enzyme inhibition. Overall, OANO2 increased MMP9+ cells and decreased CD11b+ cell populations. These findings suggest that treatment with OANO2 may oppose ITB‐mediated pro‐inflammatory cellular activation possibly by inhibition of extracellular peptidases, making it a potential therapeutic agent for acute lung injury.Support or Funding InformationSupported by the Summer Undergraduate Research Fellowship and NIH Grants: 5R25ES020721‐09 (MPI)

Cross Interaction Between OGT and mTOR in T Cells and its Consequences in Autoimmunity

BackgroundSystemic Lupus Erythematosus (SLE) is a chronic autoimmune disease that has been studied extensively, yet an understanding of the mechanisms of this disease remains unclear. It is known that epigenetic mechanisms are involved in the disease. We found O‐GlcNAc Transferase (OGT), an X‐linked gene, overexpressed due to hypomethylation in T cells from lupus patients (J. Autoimmunity, 2013). Furthermore, it was reported that the mechanistic target of rapamycin (mTOR) modifies T cell cytokine patterns and contributes to the TReg defect in patients with lupus. (J. Immunology, 2014).PurposeTo investigate the role OGT has in T cell signaling in order to understand the contribution of its overexpression to lupus. In particular, we investigated the relationship between OGT and the mTOR pathway in T cells to understand their participation in the disease.MethodsHealthy female donors were recruited by advertising. The research was reviewed and approved by the University of South Alabama Review Board for Human Subject Research. Peripheral blood mononuclear cells (PBMC) were purified by density gradient centrifugation, then T cells were isolated by magnetic beads. One fraction was transiently transfected for 48 h with 2μg of OGT siRNA using a nucleofector device (Amaxa). Other fractions were subjected to different treatments. Cells were stimulated after treatments with PMA/Ionomycin for 4h and the whole cell extracts were separated by SDS‐PAGE electrophoresis. Protein expression was analyzed by immunoblotting using specific antibodies. ß actin was used as loading control.ResultsIncreasing the levels of glycosylated proteins with 100 mM Pugnac, a competitive inhibitor of O‐GlcNAc‐β‐N‐acetylglucosaminidase (OGA), or with 5mM Glucosamine, increased the phosphorylation of p70S6K, substrate of mTORC1, which indicates an increase in mTOR activity. On the other side, when OGT is knocked down by siRNA, as expected, the protein expression of OGT levels as well as glycosylated protein levels decreased. Interestingly, the activity of mTOR also decreased. Similar results were observed with 10 nM MHY1485, potent activator of mTOR, or with 50 nM rapamycin and 10 nM Torin‐1, both inhibitors of mTOR, which increased or decreased the phosphorylation of its substrate respectively. However, they were unable to modify the levels of glycosylation.ConclusionsThese results strongly suggest that mTOR activation is OGT‐dependent and is located downstream of OGT in T cells. Overexpression of OGT could be responsible for the increase in mTOR activity in T cells from lupus patients.Support or Funding InformationNIH 5R03 AR067518‐02Summer undergraduate research fellowship (SURF) University of South AlabamaDepartment of Biomedical Science, College of Allied Health, University of South Alabama

Locating TATA Binding Protein in the RNA Polymerase I Pre‐Initiation Complex

Transcription of eukaryotic DNA into RNA is carried by three main RNA polymerases (Pols I–III). Pol I synthesizes ribosomal DNA (rDNA) into ribosomal RNA (rRNA), which has been directly linked to the rate of protein synthesis, cell growth and proliferation. The efficiency of Pols’ function is controlled by general transcription factors, which enable the enzyme to interact with promoter DNA at the initiation stage. In Saccharomyces cerevisiae, Pol I transcription initiation relies on four main factors, one of which is TATA Binding Protein (TBP). TBP is an essential universal transcription factor used by all three eukaryotic Pols. As its name eludes, TBP typically binds to TATA motifs found in promoter DNA. Interestingly, unlike Pol II and III, Pol I promoters are TATA‐less. Thus, unlike the location of TBPs in other PICs, the location and function of TBP within the Pol I PIC remains unclear.Current Cryo‐EM structures of CF‐DNA‐Pol I initiation complex leaves little room for TBP to bind to its “typical” Pols II/III position. Therefore, we hypothesize that TBP must occupy a unique location, putatively more upstream, within the Pol I PIC. Combining available structural data with previous crosslinking data of CF and TBP, we modeled the interaction between the two proteins with varying distance constraints. In addition, we analyzed TBPs location on the rDNA promoter by site‐directed hydroxyl radical cleavage experiment, known as FeBABE, where DNA phosphothioate modification probes were constructed based on our predicted models. Our results from these combinational methods elucidated that TBP locates more upstream within Pol I PIC.Support or Funding InformationThis work was a part of SUNY Upstate Medical School’s Summer Undergraduate Research Fellowship 2019 and was supported by National Cancer Institute (NCI), National Institute for Dental and Craniofacial Research (NIDCR), and Carol Baldwin Research Foundation.FeBABE site‐directed hydroxyl radical cleavage.A and B. Sites of FeBABE incorporation into rDNA promoter in clusters of 5 include the “typical” location of TBP (1), upstream of the CE (2), 5′ of CE (3), and within the CE (4). C. Western blot showed FeBABE specific cleavage products. Cleavages were observed when FeBABE was incorporated at all four positions. Most intense cleavages were observed when FeBABE is incorporated upstream of the CE. D. Quantitation of TBP cleavages.Figure 1

Investigation into the Metabolic Limitations of Alpha Conotoxins as Drugs in the Gastrointestinal Tract

Peptides isolated from cone snails, known as conotoxins, have potential in treating neuropathic pain. Peptides often lack efficacy as drugs due to poor pharmacokinetic profiles despite displaying high potency and selectivity. Conotoxins present significant sequence diversity, but little information exists about their pharmacokinetic properties. We hypothesize that this diversity will correlate with dynamic pharmacokinetic profiles, with some peptides exhibiting increased stability. In this study, we aimed to gain a basic understanding of the stability of the conotoxins α‐IMI and α‐GI in the stomach and intestines. Both have different primary sequences despite serving as antagonists of the acetylcholine receptor, which is a potential target in cancer, chemotherapy‐induced allodynia and possibly pain associated with Crohn’s disease. In order to study these peptides, novel mass spectrometry (LC‐MS/MS) methods were developed incorporating multiple reaction monitoring scans for each peptide in simulated gastric and intestinal fluids. The quantitation range of our method was 0.5–5 μg/mL, which is 20 times more sensitive than traditional methods. To compare gastrointestinal metabolic stability, each peptide was incubated in simulated gastric fluid and intestinal fluid and their stability was measured using our LC‐MS/MS method at times ranging from 0–90 minutes. Both peptides displayed a high amount of stability in gastric fluid, as no significant loss of either peptide was determined after 90 minutes. In intestinal fluid IMI degraded rapidly (t1/2= ~6 minutes), whereas α‐GI displayed far more stability in the same environment (t1/2= ~257 minutes). α‐GI incorporates a prominent helix in its structure which is likely responsible for its increased stabilization in comparison to IMI. Considering the differences in the metabolic stability of these peptides, further study of conotoxin sequences may result in the identification of stable molecular scaffolds. In the future, we plan to pinpoint what particular features are responsible for metabolic stability or susceptibility across more conotoxins, such as differences in sequence, structure and rigidity and plan to use this information to produce analogs with increased stability within the gastrointestinal tract.Support or Funding InformationSource of Research Support: This work was supported by the Pacific University Research Incentive Grant Program and an ASPET Summer Undergraduate Research Fellowship (SURF) award at Pacific University.

Ppargc1a Regulates Prostaglandin Signaling to Control Ciliogenesis and Renal Multiciliated Cell Fate Choice During Development

Cilia are microtubule‐based organelles that function in a multitude of physiological contexts to perform chemosensing, mechanosensing, or fluid propulsion. The process of ciliogenesis is highly regulated and disruptions result in disease states termed ciliopathies. The genetic and molecular events that lead to dysfunctional ciliated cells are still not fully understood. Here, we show novel roles for peroxisome proliferator‐activated receptor gamma 1 alpha (ppargc1a) during ciliogenesis in mono‐ and multiciliated cells (MCCs) as well as discernment of renal tubule MCC fate choice during embryogenesis. We discovered that ppargc1a performs both roles by affecting prostaglandin levels, where cilia formation and renal MCC fate were restored with prostaglandin E2 (PGE2) treatment. Genetic disruption of ppargc1a specifically reduced expression of the prostanoid biosynthesis gene prostaglandin‐endoperoxide synthase 1 (ptgs1), and suboptimal knockdown of both genes revealed a synergistic effect. Further, ptgs1 overexpression rescued ciliogenesis and renal MCCs in ppargc1a deficient embryos. These findings position Ppargc1a as an essential genetic regulator of prostaglandin synthesis during ciliated cell ontogeny.Support or Funding InformationNational Institutes of Health (R01DK100237 to R.A.W.), a 2019 University of Notre Dame Advanced Diagnostics and Therapeutics Graduate Fellowship Award (to J.M.C.), and a 2019 University of Notre Dame College of Science Summer Undergraduate Research Fellowship Award (to A.A.)

(E, Z, E)‐GGPP: A Potential Novel Bactericidal Compound

Mycobacterium tuberculosis (Mtb), a pathogenic Actinobacterium, has infected one quarter of the world’s population, according to the World Health Organization. The standard treatment of Mtb is a regimen of antibiotics, which has resulted in a mutated strain, XDRTb, that is resistant to every known antibiotic. The key to this antibiotic resistance is thought to be the fastidious cell wall. Most bacteria, including Mtb, utilize an isoprenoid glycosyl carrier for transfer of proteoglycan structures to the extracellular side of the membrane; however, unique to Mtb is the flexibility of its isoprenoid metabolism. Mtb synthesizes a carrier composed of 10 stereospecific isoprene units that is produced by elongation of the two‐unit (E)‐GPP by the (Z)‐FPP synthase, Rv1086, to produce (Z,E)‐FPP which then has 35 carbons added in a cis‐orientation by Rv2361c, resulting in (Z8,E)‐decaprenyl diphosphate (DPP). Although this pathway is well characterized, multiple additional isoprenoid synthases are encoded in the Mtb genome, and previous in vitro studies have indicated Rv2361c can utilize multiple alternate substrates. While attempting to understand the apparent flexibility of DPP metabolism, it was observed that heterologous co‐expression of either of the encoded (E,E,E)‐GGPP synthases, Rv0562 or Rv3383c, with Rv1086 in the heterologous host, Escherichia coli (E. coli), results in complete cell death. Computational modeling of both Rv0562 and Rv3383c suggest that both active sites may accept (Z,E)‐FPP as a substrate, and if this is true, the product would be the novel compound (E,Z,E)‐GGPP. Protein purifications of Rv0562 and Rv3383c individually combined with synthetic (Z,E)‐FPP have resulted in the synthesis of the potentially novel terpenoid product, (E,Z,E)‐GGPP. Subsequent coupled assays of Rv0562 or Rv3383c and Rv1086 resulted in higher product formation than the synthetic substrate, suggesting complex formation. Purification of the proposed novel product will be confirmed spectroscopically and has implications for synthesis of a new antibiotic.Support or Funding InformationUniversity of Wisconsin ‐ Parkside College of Natural and Health Sciences’ Summer Undergraduate Research Fellowship and the SC Johnson Integrated Sciences Laboratory

Understanding the Interplay between Human Epidermal Growth Factor Receptor 2 and Neuraminidase 1 on the Regulation of Fatty Acid Binding Protein 5 in Breast Cancer Cells

Human epidermal growth factor receptor 2 (HER2), a member of the epidermal growth factor receptor (EGFR) family, accounts for 15–20% of breast cancer cases. HER2 is a glycosylated protein with sialic acid groups attached on the surface of the protein. Sialylated proteins can be regulated by enzymes called Neuraminidases 1 (Neu1) that catalyze the removal of sialic acid residues from glycoproteins. One of the oncogenes activated by the downstream signaling effector of the HER2 pathway is a protein called fatty acid binding protein 5 (FABP5). Elevated expression of FABP5 is correlated with poor prognosis in several carcinomas and overexpression of FABP5 prevents retinoic acid‐mediated growth suppression of mammary carcinoma cells. The objective of this study is to examine whether reducing Neu1 activity or its expression decreases HER2 activation and subsequently the downregulation of FABP5 in HER2 positive breast cancer cell line, MDA‐MB‐453. Using sialic acid assay, we quantitated the levels of sialic acid in MDA‐MB‐453 cells treated with oseltamivir phosphate, an inhibitor of Neu1 activity. Oseltamivir phosphate reduced the concentration of sialic acid in a dose‐dependent manner. Through western blot analysis, we demonstrated that suppressing the activity of Neu1 with ostelamivir phosphate in MDA‐MB‐453 cells reduced heregulin‐mediated induction of the active phosphorylated HER2 and its downstream target, FABP5. Furthermore, silencing Neu1 using siRNA reduced the expression of FABP5 protein expression in MDA‐MB‐453. Taken together, this data demonstrated that inhibition of Neu1 prevented heregulin‐induced HER2 activation, and in turn, regulated the expression of FABP5 in MDA‐MB‐453 cell line. We propose that these studies will contribute to the understanding of how Neu1 and HER2 communicate to modulate the expression of FABP5, Thus, Neu1 can be used as clinical target to improve the efficacy or sensitivity of retinoic acid in HER2+ breast cancer patients.Support or Funding InformationFunding was obtained through the Summer Undergraduate Research Fellowship, Office of Undergraduate Research (I.M) and the Pat Capps Covey College of Allied Health Professions Start Up Fund (P.T.) from the University of South Alabama.

Breathing and Respiratory Muscle Function in Senescent Wild‐type and Ts65Dn Male Mice

Down Syndrome (DS) results from trisomy 21 and is the most common cause of mental retardation in the US. People with DS display reduced muscle tone and cardiorespiratory complications that may contribute to breathing disturbances. Ts65Dn (Ts) mice, a model of DS, exhibit increased apnea frequency at 3 months of age (7±9 vs. 36±28; wild‐type (WT) vs. Ts) that persists at 6 (19± 7 vs. 77±58) and 12 months (5±6 vs. 64±77; p<0.001 for age, strain, and age by strain). People with DS have increased life expectancy and it is unknown how breathing changes across the lifespan in this population. We tested the hypothesis that ~20‐month‐old Ts mice would have altered ventilation compared to age‐matched WT mice. To test this hypothesis, unrestrained barometric plethysmography was used to quantify breathing frequency (breaths/min), tidal volume (VT; mL), VT/inspiratory time (VT/Ti; mL/sec), minute ventilation (VE; mL/min) and ventilatory equivalent for CO2 (VE/VCO2) in 18 to 22‐month‐old male WT (n=12) and Ts (n=13) mice. Data Sciences International Ponemah software was used to analyze flow tracings during exposure to air (20.93% O2, balanced N2). Data are expressed as mean±S.D., WT vs. Ts. A main effect of strain was uncovered using MANOVA (p=0.033). Breathing frequency (171±33 vs. 136±41 breaths/min; p=0.025) and VE (74.6±25.7 vs. 50.3±15.5 mL/min; p=0.010) approached the a priori cutoff (Bonferroni correction, p<0.01). VT (0.439±0.112 vs. 0.377±0.075 mL/breath; p=0.123) and the ratio of VE/VCO2 (45.5 ±22.0 vs. 42.4±19.8; p=0.715) were not different. VT/Ti (3.65±1.03 vs. 2.49±0.759 mL/sec; p=0.004) was reduced in Ts vs. WT, which suggests Ts mice have a reduced ventilatory drive. Since people with DS demonstrate evidence of muscle weakness, we examined diaphragm (DIA) and sternohyoid (SH; representative pharyngeal dilator) muscle function in WT (DIA n=10, SH n=9) and Ts (DIA n=7, SH n=7) mice. Twitch (2.2±1.1 vs 2.3±1.0 N/cm2; p=0.835) and tetanic (11.2±7.3 vs. 11.6±4.8 N/cm2; p=0.900) force, and the force frequency‐relationship (p=.766) for SH muscle were equivalent between groups. Similarly, no differences were observed for DIA twitch (6.0±2.7 vs. 5.2±2.1 N/cm2; p=0.438) and tetanic (20.7±6.0 vs. 19.9±2.8 N/cm2; p=0.758) force, or the force‐frequency relationship (p=0.339). Diaphragm function was preserved in aged Ts mice which is in line with similar VT between groups. These data reveal the increased propensity for apnea in Ts mice is independent of upper airway muscle weakness and may alternatively be of neural origin. Current studies are examining motor control of the upper airway in Ts mice.Support or Funding InformationThis work was funded by 1R15HD076379 (LRD), 3R15HD076379 (LRD). Additional support was granted by the Le Moyne College McDevitt Center Undergraduate Research Fellowship in Natural Science (MS‐N, MR), and the Le Moyne College Biological Sciences Department.

Identifying Prospective Genes That Function During DNA Repair in the Bdelloid Rotifer Adineta vaga

The bdelloid rotifer Adineta vaga is a microinvertebrate with a highly efficient DNA repair system that allows individuals to recover from DNA damage caused by desiccation or ionizing radiation. The mechanism behind this remarkable DNA repair in A. vaga is not yet understood. Previous transcriptome sequencing in our lab has identified over 250 genes that are upregulated in response to DNA damage induced by ionizing radiation. These upregulated genes are the strongest candidates for having a role in DNA repair. The objective of this project is to characterize the functions of candidate genes involved in DNA repair in bdelloids and, thereby, establish potential pathways that comprise this mechanism. Three approaches were used to begin to investigate the functions of these candidate DNA repair genes in bdelloids. First, we used real‐time PCR to quantify gene expression of several candidate DNA repair genes during recovery from desiccation. Genes that were upregulated relative to the non‐desiccated control were identified as most likely to have a role in DNA repair as opposed to radiation resistance. Second, we constructed a yeast library of A. vaga proteins for use in yeast two‐hybrid screens against candidate DNA repair proteins. To produce the library, cDNAs representing the full complement of A. vaga proteins were cloned into plasmids that were then transformed into yeast. Yeast strains that each expressed a different candidate DNA repair protein would then be mated with the library to screen for protein‐protein interactions, which would provide evidence for related cellular functions. Third, we developed the molecular tools for using CRISPR/Cas9 genome editing to inactivate candidate genes in A. vaga. In vivo gene knockouts would provide mutant rotifers that could be studied to assess the roles of these genes in DNA repair. Single guide RNAs (sgRNAs) for several genes were created and incubated with the Cas9 enzyme to form sgRNA‐Cas9 complexes. These sgRNA‐Cas9 complexes were shown to successfully cleave their target gene sequences in vitro. To test this method in vivo, these sgRNA‐Cas9 complexes were transfected into A. vaga embryos via electroporation. Genomic DNA was purified from treated rotifers and used in T7 endonuclease I assays to identify subsequent mutations in the target genes. Once perfected, these methods of investigating the roles of candidate genes stand to elucidate the mechanism behind the extraordinary DNA repair in A. vaga.Support or Funding InformationFunding was provided by a Summer Undergraduate Research Fellowship from the Arkansas Department of Higher Education to JTW, and a Hendrix College Faculty Project Grant to AMS.

Histidine Reactive Chemicals Modify the Thermus thermophilus CuA Redox Center

The electron transport chain is a series of redox protein complexes that couple the shuttling of electrons with the pumping of protons, in order to create the electrochemical gradient that drives ATP synthesis. The first redox center of the terminal complex from the Thermus thermophilus bacteria has been isolated as TtCuA and has a binuclear copper center bridged by two cysteines and ligated by two histidines and two weak axial ligands. H157 of TtCuA, a solvent exposed ligating histidine, has demonstrated modification by diethyl pyrocarbonate (DEPC), an exogenous chemical modifier, that is facilitated by the residue’s fast proton exchange with the solvent. The H157‐DEPC adduct is reversible over 48 hours at room temperature, as supported by mass spectrometry, electrochemistry, and visible CD spectroscopy. This is one of two modifications, out of 12, that becomes removed. Due to the lability of the H157‐DEPC bond, it is possible that the H157 Nɛ is primed for proton pumping in the electron transport chain.In order to model a possible in vivo modification, TtCuA was exposed to 4‐hydroxynonenal (HNE), an endogenous lipid peroxidation product associated with multiple metabolic and neurodegenerative diseases. Exposure of TtCuA to 20 equivalents of HNE appears to reduce the protein over 48 hours and four modifications are observed via mass spectrometry. Exposure of the Pseudomonas aeruginosa azurin protein, a mononuclear blue‐copper denitrification protein with similar ligand groups to TtCuA, is not reduced by HNE, despite modification. Azurin also lacks a ligating histidine that can be modified by DEPC. In conclusion, the binuclear copper center of TtCuA has hyperreactivity in comparison to a similarly ligated mononuclear copper center.Support or Funding InformationMurchison Undergraduate Research Fellowship, Trinity University Chemistry Department, Mach Research Fellowship, Welch Foundation