29th Annual CBB Conference

October 22, 2022

1) Poly (ADP-ribose) Polymerase 1: DNA complexes are dependent on DNA sequence and secondary structure

Fletcher Bain, Jade Miller, and Maria Spies*

 Department of Biochemistry and Molecular Biology, Carver College of Medicine, University of Iowa, Iowa City, IA

Poly (ADP-ribose) polymerase 1 is a key player in DNA repair and transcription regulation.  The role of PARP1 in DNA damage recognition and protein signaling in response make it integral to genome stability.  Beyond this, it regulates gene expression through chromatin remodeling and transcription factor binding.  Many of these activities are dependent upon PARP1 autoPARylation, induced by DNA binding.  PARP1 has long been implicated in G-quadruplex DNA recognition, and recent advancements in detection of these DNA structures in living tissue have shown that we barely understand PARP1 function and activity.  Traditionally PARP1 DNA binding studies have focused on DNA repair substrates, such as blunt end double-strand and nicked DNA.  However recent studies have identified PARP1 to be a potent DNA G-quadruplex binding protein.  The mechanism and function of these interactions are not yet well understood.  Using traditional single-molecule binding techniques, PARP1 is shown to bind to DNA G-quadruplexes in a unique ratio compared to previously studied DNA repair substrates.  Additionally, PARP1 does not bind all DNA G-quadruplexes with the same ratio or affinity.  Following this revelation this study continues to develop techniques to elucidate the binding interaction of PARP1 with 11,000 G-quadruplex containing DNA sequences found in the human genome.  Using a combinatorial method of next generation sequencing and single-molecule total internal reflection fluorescence to identify these characteristics is an ongoing challenge.

 

2) NOVEL ANALOGUES OF THE NATURAL PRODUCT FRAXINELLONE PROTECT AGAINST GLUTAMATE-INDUCED TOXICITY IN PC12 CELLS

Anna E. Bartman¹, Mersad Raeisi², Zetandro Banarjee², Clarence D. Peiris2, Anayah Ferris², Emmanuel Bonsu², David B. Martin²*, and Jonathan A. Doorn¹*

¹Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy University of Iowa, Iowa City, IA 

 ²Department of Chemistry, College of Liberal Arts and Sciences, University of Iowa, Iowa City, IA

Background: Glutamate (Glu) is the major excitatory neurotransmitter in  the central nervous system (CNS) involved in synaptic plasticity, neuronal outgrowth and survival, and memory. Although intracellular Glu concentration is often quantified in the millimolar range, extracellular Glu concentration must remain in the micromolar range. When extracellular levels of Glu rise, aberrant synaptic signaling leads to excitotoxicity which is thought to contribute to many neurodegenerative diseases, including epilepsy and Alzheimer’s disease. In previous reports, limonoids isolated from Dictamnus dasycarpus showed significant neuroprotective activity against Glu excitotoxicity. Of the previously studied limonoid natural products, fraxinellone was one of four compounds that proved to be effective in protecting against Glu excitotoxicity in vitro. With this information, a library of analogues was synthesized from the natural product fraxinellone that proved to be more effective at protecting against Glu toxicity than natural fraxinellone.

Methods: In vitro methods were used to measure the protective properties of the new fraxinellone analogues and to determine their mechanism of protection, which has not been achieved before. PC12 cells were first pre-treated with each compound at a range of 0.05 to 1.0 μM. The compounds were then washed from the cells and 100 μM Glu was added for 24 hours. MTT analysis was then performed to measure cell viability to determine if the compounds were effective at protecting the cells against Glu toxicity. Of the fifteen analogues that were synthesized, six proved to be protective against Glu toxicity, even more than natural fraxinellone. With this information, we then investigated their mechanism of protection by considering potential targets of these compounds, specifically the Nrf2 pathway, through qPCR analysis. We also determined the time course for Glu-mediated ROS production.

Results. Six of the fifteen compounds protected against Glu excitotoxicity. When cells were pre-treated with the compounds that afforded protection before adding the Glu insult, we saw enhancement of gene expression of antioxidant response elements (AREs), a key mechanism of Nrf2 activation. Antioxidant gene expression was not seen for compounds that did not provide protection.

Conclusions: Our findings suggest high levels of Glu are toxic to PC12 cells and induce ROS production in a time-dependent manner. Current findings suggest the compounds protect through Nrf2 activation and induction of an antioxidant response.

 

3) EVALUATING IMPACT OF STIRRING ON THE STABILITY OF PROTEIN PHARMACEUTICALS WITH BOVINE SERUM ALBUMIN (BSA) AS A MODEL PROTEIN

Apurva C. Dusane and M. Reza Nejadnik^*

Department of Pharmaceutics and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA

Production of protein pharmaceuticals is challenging and for the smooth streamlining of this process it is crucial that the produced therapeutic proteins have aggregation levels within limits, thus avoiding any instances of patient immunogenicity or rejection by regulatory authorities. To resolve aggregation related issues, understanding it at the microscopic level becomes vital; aggregation related issues are commonly seen in case of mixing by stirring, and different proteins behave differently when exposed to such mechanical stresses. The goal of our experimental design was to study the impact of the stirring stress on aggregation of protein therapeutics using Bovine Serum Albumin (BSA) as a model drug.

We developed our stirring studies using BSA because it exhibits a particular behavior mainly attributed to its small structure and ability to readily undergo a structural change. We wanted to observe if there was any impact of this differential BSA behavior, influencing protein aggregation. With this view in mind, we performed stirring studies using BSA at different concentration of 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1 and 2 mg/ml. The non-stirred samples were also included across the same BSA concentrations and were supplemented with a second control group that consisted of buffer. Samples were withdrawn from stirring, non-stirring and control groups across all concentration at 0, 30, 60 120 and 180 mins, and were examined using FlowCam and Dynamic Light Scattering (DLS), to assess aggregate formation at nanometer and micrometer size ranges, to interpret concentration-dependent changes in aggregation behavior.

 

4) A GLOBAL SIDE-CHAIN OPTIMIZATION METHOD TO DETERMINE PROTONATION STATES OF TITRATABLE AMINO ACIDS

Rose A. Gogal, and Michael J. Schnieders^*

Department of Biomedical Engineering, College of Engineering, University of Iowa, Iowa City, IA

New techniques for experimental protein structure determination are increasing the number of proteins in the Protein Data Bank every day. However, the resolution of these experimental methods is rarely high enough to determine the placement of protons within an amino acid sidechain. Additionally, determining the exact placement of sidechains requires significant time investment by scientists. In this work, we propose an improvement to our global side-chain optimization method in which the method now predicts the most favorable protonation state of titratable amino acids at a particular pH. We expect this method to improve R and Rfree values of X-ray crystalized structures to better match the X-ray diffraction data and improve the energetics of the protein structure. 

 

5) ALLOSTERIC TUNING OF CASPASE-7: ESTABLISHING THE NEXUS OF STRUCTURE AND CATALYTIC POWER

Kathryn F. Hobbs¹, Jonah Propp², Nicholas R. Vance², Andrew Kalenkiewicz¹, Katie R. Witkin², and M. Ashley Spies^1,2* 

¹Biochemistry and Molecular Biology, Carver College of Medicine, University of Iowa, Iowa City, IA

²Department of Pharmaceutics and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA

Despite the incredible potential of allosteric drugs, less than 1% of FDA approved drugs are known allosteric effectors. This is likely due to an incomplete understanding of the nexus between the distant allosteric site and the active site. The allosteric site in caspase-7 (C7), an apoptotic protease with therapeutic relevance for several human diseases (e.g. Parkinson’s, Alzheimer’s, sepsis), has potential for small molecule targeting. However, numerous drug discovery efforts have failed to provide therapeutically-relevant allosteric inhibitor hits. Here we present the first selective, drug-like inhibitor of C7 based on our previous fragment-based drug discovery efforts. We also elucidate the impact of allosteric binding on the C7 catalytic cycle using an integrated approach involving structural, biochemical, and computational techniques. Our findings suggest allosteric binding disrupts the active site chemistry of C7 and prevents the initiation of the catalytic cycle (acylation) via neutralization of the catalytic dyad, displacement of the substrate from the oxyanion hole, and altered dynamics of the substrate binding loops. This work advances allosteric drug targeting efforts and bolsters understanding of allosteric structure activity relationships (ASARs: patterns linking occupancy at the allosteric pocket with the structure, dynamics, and reactivity at the active site). Future studies may use ASARs to streamline drug screening efforts, particularly computational workflows.

 

6) Dual Targeting of mTOR and YAP/TAZ will synergistically inhibit PI3K-driven sarcomagenesis

Keith Garcia^1†, Ali Khan^1†, Nicole Merritt¹, Colleen Fullenkamp¹, Nicholas P. Scalora¹, Jordan Kohlmeyer¹, Mariah Leidinger¹, Michael Chimenti², and Munir Tanas¹

¹Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, IA

²Iowa Institute of Human Genetics, Carver College of Medicine, University of Iowa, Iowa City, IA

^† these authors contributed equally

Background and Results: Sarcomas are cancers primarily arising from connective tissues. They represent over 50 histological types with few effective targeted therapies. Hyperactivation of PI3K, a serine/threonine kinase crucial for cell survival and proliferation is commonly observed in sarcomas due to deletion of PTEN in approximately 20-30% of sarcomas. Thus, targeting PI3K may serve as an effective potential treatment for these sarcomas. The mTORC1 complex has been well-established as a key downstream effector of PI3K signaling. We have generated additional data suggesting that YAP/TAZ represent key transcriptional effectors of PI3K signaling. mTOR is a regulator for protein synthesis and cell survival while YAP/TAZ, downstream targets of Hippo pathway, are transcriptional coactivators that promote sarcomagenesis and metastasis. How these two pathways coordinately mediate oncogenic PI3K signaling has been poorly studied representing a significant gap in knowledge. Cell viability experiments were done with varying concentration of Verteporfin (YAP/TAZ inhibitor) and Everolimus (mTORC1 inhibitor) to determine the synergistic inhibition in RH30 (human alveolar rhabdomyosarcoma) and A204 (malignant extrarenal rhabdoid tumor). Western blots were performed on sub-confluent RH30 and A204 cells treated with 2.5mM of Verteporfin and 10nM Everolimus. Clonogenic assay was performed on RH30 and A204 cells to look at the effect of mTORC1 inhibition on cell viability. Tissue microarray analysis showed that approximately 46% of samples display loss of PTEN protein expression and 38% of UPS also displayed loss of PTEN expression. Various sarcoma cell lines also exhibit elevated phosphorylation of the PI3K target, Akt, as well as loss of PTEN expression. Synergistic Inhibition was seen in both RH30 and A204 cells at very low concentrations of Everolimus (0.6nM) and Verteporfin (750nM). The western blot analysis showed that upon combinatorial inhibition of YAP/TAZ and mTORC1, phosphorylation of S6 and 4E-BP1 was greatly reduced in comparison to monotherapy controls. Furthermore, inhibition of mTORC1 alone had no significant effect on cell clonogenicity in comparison to vehicle controls.

Conclusions:  This research project will show how mTORC1 and Hippo pathway combinatorically mediate oncogenic PI3K signaling. Previously we have shown that, TAZ/YAP are key downstream transcription factors of PI3K signaling. Furthermore, we also showed that monotherapy of mTORC1 inhibition is ineffective as it leads to cytostatic effect thus proving to be ineffective. Dual inhibition of both the transcriptional and translation apparatus in cells, YAP/TAZ and mTORC1 respectively, may lead to decreased cell viability in PI3K driven sarcomas. YAP/TAZ and mTORC1 may both account for the transformed phenotype in PI3K driven sarcomas. In future, we aim to explore effect of combinatorial inhibition of YAP/TAZ and mTORC1 on clonogenicity, and efficacy in vivo both in xenograft and a sarcoma-prone GEMM model.

Clinical impact: The expected outcome of these experiments is that we will demonstrate that combined YAP/TAZ and mTOR oncogenic activity are critical to the pathogenesis of PI3K activated sarcomas. Since YAP or TAZ are activated in majority of sarcomas, this approach could potentially be applied to multiple histological types of sarcomas.  Furthermore, this will also elucidate the integral role of PI3K in coordinately regulating the oncogenic transcriptional (YAP/TAZ) and protein translation apparatus (mTOR) in sarcomas. Such results will have positive impact by elucidating additional therapeutic strategies for a set of cancers with few effective targeted therapies.

 

7) DEVELOPMENT OF 3D PRINTED GENE-ACTIVATED CALCIUM PHOSPHATE CEMENT SCAFFOLDS FOR APPLICATION IN BONE REGENERATION

Noah Z. Laird¹, Esraa Mohamed^{1, ^}, Pornpoj Phruttiwanichakun^{1, ^}, Timothy M. Acri¹, Jaidev L. Chakka¹, Douglas Fredericks², John M. Femino², and Aliasger K. Salem^{1, *}

¹Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA

²Department of Orthopedics and Rehabilitation, Carver College of Medicine, University of Iowa, Iowa City, IA

^{^}These authors contributed equally

The goal of this study is to investigate how modification and gene-activation of 3D printed calcium phosphate cement scaffolds can enhance their ability to regenerate bone upon implantation into large bone injuries and defects. Specifically, modifying the scaffold by altering the method used to harden calcium phosphate cement can impact the surface morphology of the scaffolds, potentially impacting the ability of cells to adhere to the scaffolds and begin the regenerative processes of resorbing the scaffold and producing new bone. Additionally, a gene delivery capability can be granted to the scaffolds by incorporating nucleic acids in such a way that they can transfect local cells and induce expression of therapeutic growth factors. The resulting “gene-activated scaffolds” can assist with the regeneration of bone by inducing production of regenerative growth factors over an extended period. This technique circumvents issues with current approaches that load proteins directly onto the scaffold, namely the issues of low bioavailability of the loaded proteins and off-target effects caused by the large doses required to compensate for this low bioavailability. This work is a proof of concept demonstrating that an optimized 3D printed calcium phosphate cement scaffold can be gene-activated. If these scaffolds are able to induce robust bone formation upon implantation in model bone defects, they may have application as load-bearing implants that can be used to heal large bone defects (critically sized defects) that would otherwise remain unhealed.

 

8) Double-Stranded mRNA Lipid Nanoparticle Vaccines Display Superior Thermostability Compared to Current Single-Stranded mRNA Formulations

Kristopher E. Lukas and Kevin G. Rice*

Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA

Most lipid nanoparticle (LNP) delivery systems have been utilized for the intravenous delivery of siRNA into the liver. Recently, significant advancements have been made to optimize this delivery system for intramuscular use, such as the Moderna COVID-19 vaccine used to deliver single-stranded spike protein mRNA. Despite its overwhelming success, concerns have arisen regarding the thermostability of this formulation during transportation. Therefore, this research aims to optimize the LNP gene delivery system to administer more stable double-stranded mRNA. By utilizing a small-scale PLEXER device, LNPs encapsulating single- and double-stranded Luciferase mRNA were assembled by turbulently mixing 4 lipid components with the mRNA: an ionizable cationic lipid (SM-102), a phospholipid (DSPC), cholesterol, and a pegylated lipid (DMG-PEG 2000). This apparatus allowed us to generate LNPs of comparable particle size (~100 nm) to those found in the existing vaccines as determined through dynamic light scattering measurements. After incubation at room temperature for 12 hours, the mRNA was extracted out of the LNPs. Band intensity from agarose gel analysis indicated that the RNA in the current Moderna COVID-19 vaccine and our single-stranded mRNA formulation degraded by 82% and 55% respectively, whereas our double-stranded mRNA formulation demonstrated no degradation. Furthermore, double-stranded mRNA remained intact after harsher incubation conditions at 37 °C for 12 hours whereas the other two formulations were destroyed. Indeed, single-stranded mRNA consistently degraded over multiple incubation experiments while double-stranded mRNA remained resilient. To discern the nature of this degradation, extractions were repeated in the presence of RNAse OUT, an RNAse inhibitor. Nearly all the RNA in each formulation was recovered, suggesting that the instability is caused by enzymatic degradation by RNAse when not stored under optimal conditions. This early data demonstrates the enhanced stability of double-stranded mRNA as a superior candidate for vaccines over single-stranded mRNA, and these findings could pave the way for significant enhancements in vaccine delivery and strengthen the global vaccine platform.

 

9) APPLICATION OF LYOPHILIZED GENE-DELIVERY FORMULATIONS TO DENTAL IMPLANT SURFACES: NON-CARIOGENIC LYOPROTECTANT PRESERVES TRANSFECTION ACTIVITY OF POLYPLEXES LONG-TERM

Walla I. Malkawi¹, Noah Z Laird¹, Satheesh Elangovan², and Aliasger K. Salem^1*

¹Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA

²Department of Periodontics, College of Dentistry and Dental Clinics, University of Iowa, Iowa City, IA

Titanium is the metal of choice for dental implants because of its biocompatibility and ability to merge with human bone tissue. Despite the great success rate of dental implants, early and late complications occur. Coating titanium dental implant surfaces with polyethyleneimine/plasmid DNA (pDNA) polyplexes improve osseointegration by generating therapeutic protein expression at the implantation site. Lyophilization is an approach for stabilizing polyplexes and extending their shelf life; however, most lyoprotectants are sugars that can aid bacterial growth in the peri-implant environment. In our research, we coated titanium surfaces with polyplex solutions containing varying amounts of lyoprotectants. We used two common lyoprotectants (sucrose and polyvinylpyrrolidone K30) and showed for the first time that sucralose (a sucrose derivative used as an artificial sweetener) might act as a lyoprotectant for polyplex solutions. Human embryonic kidney (HEK) 293T cells were used to quantify the transfection efficiency and cytotoxicity of the polyplex/lyoprotectant formulations coating titanium surfaces. Polyplexes that were lyophilized in the presence of a lyoprotectant displayed both preserved particle size and high transfection efficiencies. Polyplexes lyophilized in 2% sucralose have maintained transfection efficacy for two years. These findings suggest that modifying dental implants with lyophilized polyplexes might improve their success rate in the clinic.

 

10) CATIONIC CaMKIIN-LOADED LIPOSOMES AIMED TO REDUCE CHLORINE-INDUCED AIRWAY OXIDATIVE STRESS

Esraa Mohamed¹, Andrea Adamcakova-Dodd², Isabella Grumbach³, Peter S. Thorne^2,4, and Aliasger K. Salem^1,5*

¹Department of Pharmaceutical Sciences and Experimental Therapeutics, Division of Pharmaceutics and Translational Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA

²Department of Occupational and Environmental Health, College of Public Health, University of Iowa, Iowa City, IA

³Abboud Cardiovascular Research Center, Department of Internal Medicine, Division of Cardiovascular Medicine, Carver College of Medicine, University of Iowa, Iowa City, IA

⁴Human Toxicology Program, Graduate College, University of Iowa, Iowa City, IA

 ⁵Holden Comprehensive Cancer Center, University of Iowa Hospitals and Clinics, Iowa City, IA

Chlorine gas is a powerful oxidizing agent and irritant that humans can be exposed to by accident or through an intentional attack. When inhaled, chlorine rapidly dissolves in the epithelial lining fluid where it generates a variety of reactive oxygen species (ROS) and results in acute lung injury characterized by pneumonitis, edema, cellular proliferation, reduced lung function, and epithelial cell death. This damaging effect has been attributed to oxidative stress and mitochondrial damage. Thus, abrogating oxidative damage is key to preventing lung cell injury.

Calcium/calmodulin dependent protein kinase II (CaMKII) is a widely distributed protein that plays a pivotal role in increasing oxidative stress by catalyzing the phosphorylation of proteins that increase inflammatory signaling, cell proliferation, and ion channel activity. CaMKII activity has been reported to be increased under conditions of stress. Therefore, inhibiting its activity using CaMKII inhibitor peptide (CaMKIIN) can be a novel target for mitigating chlorine-induced lung damage. Compared with other CaMKII inhibitors such as KN-62/KN-93, CaMKIIN has a superior potency and specificity. We will test the hypothesis that exposure to chorine will significantly activate CaMKII that in turn results in airway damage and that the administration of CaMKIIN-loaded cationic liposomes (CLs) can reduce oxidative stress and lung damage.

Male and female rats will be exposed to different concentrations of chlorine; then the ROS levels, CaMKII level, mitochondrial damage, and lung damage will be evaluated by DHE staining, Immunofluorescence, Mitotracker® Green stain, AHR and lung histopathology. CaMKIIN-loaded CLs were prepared using a microfluidics technique.  CLs were 148.95±0.35 nm with a surface charge of +19.97 mV±1.63. The positive charge of the liposome should enhance the cellular uptake of CaMKIIN. CaMKIIN-loaded CLs will be administered to the mice after chlorine exposure, and we will evaluate the effect of CaMKIIN on decreasing oxidative stress and lung damage.

This project is the first that address the relation between CaMKII activation and chlorine gas exposure. Defining the relationship between CaMKII activation and chlorine gas exposure will provide us with a potentially potent target that may assist in managing chlorine-induced lung damage. Inhibition of CaMKII suppresses ROS generation and can potentially prevent lung oxidative damage.

 

11) Design and Synthesis of Small Molecules that Target UNC119 for the Treatment of Diabetes Mellitus

Autumn E. Moore¹, Aschleigh Graham¹, Julien A. Sebag², Nikolai Artemyev², and Robert J. Kerns^1*

¹Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA

²Department of Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, Iowa City, IA

Type II diabetes (T2D) is a disease that is characterized by insulin resistance. In skeletal muscle and adipose cells, T2D is induced by impaired GLUT4 translocation. As a result, insulin is unable to bind receptors that trigger a signaling cascade to allow GLUT4 in the vesicles to translocate to the surface of cells for glucose storage. Currently, treatments for diabetes have largely failed to achieve glycemic control or restore insulin-stimulated GLUT4 translocation. As a result, a luciferase-based assay was developed to identify small molecules that could restore GLUT4 translocation in the presence of endogenous insulin in order to prevent hypoglycemia. Small molecule C3, and later C59, were identified as small molecule mediators of GLUT4 translocation in the presence of insulin. In knowing this, we are pursuing the design and synthesis of novel C59 analogs toward the development of a new class of therapeutics that act as insulin sensitizers by increasing GLUT4 translocation while in the presence of insulin.

 

12) USE OF A SMALL MOLECULE (LM11A-31) INHIBITOR OF p75NT RECEPTOR TO ASSESS THE MECHANISM OF PBD, A NEUROTROPHIC NATURAL PRODUCT

Bishnu P Neupane and Williams Florence*

Department of Chemistry, College of Liberal Arts and Sciences, University of Iowa, Iowa City, IA

Progressive nervous system dysfunction due to loss of neural structure and function as well as from neural death is one of the major causes of neurodegenerative diseases (NDDs). Despite of several available therapeutic approaches, there have been no drugs that cause a meaningful improvement in life expectancy for NDDs. The limited efficacy of such drugs reflects poor understanding of mechanism of action and the use of inappropriate targets for treatment development. Therefore, new therapeutic approaches are clearly needed to develop therapeutics for the treatment of NDDs. The design and development of small non-peptide molecules that target specific receptors would be one of the strategies to understand the mechanism of drug-receptor binding and to discover new therapeutics for NDDs.

Phenylbutenoid dimer (PBD), is a small natural product molecule known to promote neuron survival and neurogenesis in neurodegenerative mouse models. It is hypothesized that PBD may operate through modulation of p75 neurotrophic receptor (p75^NTR) signaling. An investigation to explore the neurotophic effect of PBD and its derivatives in PC-12 and SH-SY5Y cells is ongoing in our laboratory. In our study, PBD exhibits significant neurotrophic activity in neuritogenesis assays, and further exhibits a pronounced potentiating effect in combination with NGF protein. We aim to assess the binding mechanism of PBD to p75^NTR in combination with LM11A-31, a known inhibitor of the p75^NTR. LM11A-31 is an orally bioavailable, water soluble, brain penetrating small molecule that downregulates p75NTR degenerative signaling. It is currently undergoing phase 2a clinical trial for Alzheimer’s diseases treatment. We hypothesize that combined treatment of LM11A-31, PBD, and nerve growth factor (NGF) will provide evidence for complementary, competitive, or independent activity of LM11A-31 in relation to PBD action. We have two general goals for our investigations: First to synthesize the LM11A-3, its methylated derivative, and testing them on PC12 cell lines along with PBD. Second, the determination of binding recognition by diazirine photolabile probe. This poster will cover our work to date towards these two goals.

 

13) THERMOELECTRIC MITIGATION OF PSEUDOMONAS AERUGINOSA  BIOFILMS

Parham Parnian and Eric Nuxoll*

Department of Chemical and Biochemical Engineering, College of Engineering, University of Iowa, Iowa City, IA

Current approaches to treat biofilm infections on medical implants involve invasive surgeries, which is costly and life-endangering. Thermal treatment of infections locally, is an alternative treatment and is possible via remote heating of the implant’s surface. However, thermal treatment can cause damage to tissue next to the infected implant. Applying a cooling treatment immediately after the thermal shock can effectively minimize that tissue damage. However, prior studies indicate that significant biofilm viability reduction occurs after the thermal shock is complete, raising the possibility that immediate post-shock cooling could reduce the efficacy of the thermal treatment. This study used thermoelectric technology to test the hypothesis that biofilms can be destroyed in-situ by heating them up and immediately cooling them. Mature Pseudomonas aeruginosa biofilms cultured on thermoelectric devices were thermally shocked at different time and temperature combinations. Immediately after the hot thermal shock, the biofilms were delivered with cold thermal shock at 14 °C for similar duration. Quantifying bacterial population of the biofilms immediately after the shocks showed that post heat shock cooling had no significant effect on the efficacy of the thermal treatment.

 

14) ZWITTERIONIC POLYMER COATINGS FOR DECREASED PROTEIN ADSORPTION

Christopher Ruben, Nolan Burson, Barrin Hendricks, and Jennifer Fiegel*

Department of Chemical and Biochemical Engineering, College of Engineering, University of Iowa, Iowa City, IA

Protein adsorption on drug carrier surfaces occurs when the carriers interact with protein-rich biological fluids. The proteins in these fluids coat the drug carriers altering their surface chemistry and retention time in the body. Zwitterionic polymers are promising coating materials for drug carriers as they create a highly hydrated surface through which proteins cannot penetrate. The goal of this research is to design zwitterionic phosphorylcholine-based polymers and determine how grafting density and molecular weight affect the adsorption of proteins from physiologically-relevant protein mixtures. We have synthesized zwitterionic phosphorylcholine-containing polymers using reversible addition-fragmentation chain transfer (RAFT) thermal polymerization with molecular weights ranging from 10 to 30 kDa. Polymers were attached to gold QCM-D crystals, the polymer coating thickness and surface hydrophobicity measured, and the crystals challenged with protein solutions. There was excellent protein resistance at all molecular weights and coating densities with the 30 kDa performing the best. Choice of solvent also affected the polymer coatings thickness while not affecting the protein adsorption characteristics. This data shows the potential of these polymers to resist protein adsorption. Current studies focus on creating polymer-coated nanoparticles and determining how the polymer coatings alter cellular interactions.

 

15) SECRETED FACTORS FROM ADIPOCYTE LINEAGE CELLS DIFFERENTIALLY IMPACT FIBROBLAST TO MYOFIBROBLAST CONVERSION

Noah Sinclair¹, Jesse Liszewski¹, Mariam Y. El-Hattab¹, Al Klingelhutz², James A. Ankrum¹, and Edward A. Sander^1*

¹Department of Biomedical Engineering, College of Engineering, University of Iowa, Iowa City, IA

²Department of Microbiology and Immunology. Carver College of Medicine, University of Iowa, Iowa City, IA

The conversion of fibroblasts to myofibroblasts is an important process in wound healing. However, overactive myofibroblasts are associated with excessive scarring and tissue fibrosis. Adipose tissues have been identified as active players in the wound healing process. To better understand how adipocyte lineage cells, including adipocyte stem cells, preadipocytes, and adipocytes, might be used therapeutically to reduce scarring, we have been exploring how conditioned medium from these cell types influences fibroblast conversion. We have found that adipocytes secrete a factor that causes fibroblasts to become more contractile, express higher amounts of alpha smooth muscle actin (α-SMA), and secrete higher amounts of collagen than fibroblasts exposed to transforming growth factor beta 1 (TGF-β1) and ascorbic acid. We have determined that this unknown factor is  in the 30-100 kDa range, is inactivated by both lipid depletion and heat inactivation, and is not TGF-β1. Furthermore, this factor results in a transcriptionally distinct myofibroblast phenotype from TGF-β1 activated myofibroblasts, suggesting the possibility that these myofibroblasts could promote better healing.

 

16) YEATS2 AND ZZZ3 ARE UPREGULATED IN SARCOMA CELL LINES SUGGESTING THE ATAC COMPLEX IS A KEY ONCOGENIC DRIVER

Souradip Sinha^1,2, Keith Garcia^1,3, Munir Tanas^1,2,3, *

¹Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, IA

²Molecular Medicine Graduate Program, Carver College of Medicine, University of Iowa, Iowa City, IA

³Cancer Biology Graduate Program, University of Iowa Hospitals and Clinics, University of Iowa, Iowa City, IA

Sarcomas are neoplasms that develop in tissues of bone, fat, muscle, nerve, cartilage, blood vessels, and have few targeted therapies. Epithelioid hemangioendothelioma (EHE) is a vascular sarcoma originating in endothelial cells and is defined by two mutually exclusive fusion proteins, TAZ-CAMTA1 and YAP-TFE3. Our lab has shown that these fusion proteins alter gene expression patterns in sarcoma cell lines by interacting with the Ada2A-Containing (ATAC) histone acetyltransferase complex, specifically YEATS2 and ZZZ3, which are indispensable subunits of ATAC complex.YEATS2 binds to and reads acetyl-H3K27 on enhancers and promoters of actively transcribing genes, and ZZZ3 binds to and reads the H3 tail. Previous literature shows that YEATS2 is critical for histone acetylation in tumorigenesis of non-small cell lung cancer, and both YEATS2 and ZZZ3 are necessary for ATAC complex mediated histone acetylation and gene activation. Recent works also show that YEATS2 is amplified in ovarian cancer, head and neck cancer, esophageal cancer, uterine cancer, and liposarcomas.

To investigate the importance of YEATS2 and ZZZ3 in Sarcomas, RNA-Seq expression data of sarcoma samples from The Cancer Genome Atlas (TCGA) was utilized and Kaplan Meier analysis show that high YEATS2 and ZZZ3 levels correlate with a worse overall survival. Consistent with this, we have observed upregulated YEATS2 and ZZZ3 levels in sarcoma cell lines compared to tissue-matched primary human cell lines via western blot. Knock-down of YEATS2 and ZZZ3 was performed in SKLMS (leiomyosarcoma cell line) and SW872 (liposarcoma cell line). Preliminary data suggest that YEATS2 and ZZZ3 are critical for 2D proliferation and anchorage-independent growth in the SKLMS cell line. We have also observed a decrease in H3K9-acetylation with YEATS2 and ZZZ3 knockdown in the SKLMS cell line, but not in the SW872 cell line. Further studies are required to show the functional importance of YEATS2 and ZZZ3 in these sarcoma cell lines.

Future studies include characterizing the rest of the ATAC complex components which will be achieved by performing an shRNA screen using anchorage-independent growth on poly-HEMA as the readout. Since the ATAC complex binds to enhancers and promoters of selective genes, we will perform ChIP-Seq to determine the specific genomic regions that employ the ATAC complex for their transcriptional activation. Additionally, our long-term goal is to determine key interactors of the ATAC complex, how this epigenetic complex is recruited to specific genomic loci, and how this complex could be therapeutically targeted in sarcomas.

 

17) IMPROVING WOUND INFECTION TREATMENT USING SPRAYABLE, ANTIMICROBIAL HYDROGELS

Riannon Smith¹, Nicole Brogden² and Jennifer Fiegel^1,2*

¹Department of Chemical and Biochemical Engineering, College of Engineering, University of Iowa, Iowa City, IA

²Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA

Burn wounds represent a significant health problem due to high infection rates and pain associated with current treatments; therefore, we aim to develop sprayable, antimicrobial hydrogels to reduce application pain and improve infection treatment. Poloxamers are thermoreversible polymers that are liquid at low temperatures and gel at higher temperatures. This is advantageous for this research because they can be sprayed onto the skin as a cold liquid then transition to a gel as it warms on the skin while providing prolonged drug release in the gel state. Poloxamer hydrogels loaded with ciprofloxacin hydrochloride have been evaluated for desirable physical, antimicrobial, and sprayable properties. Desirable gelation temperatures for formulations are between room and skin temperature (21 - 34°C) in order to spray the formulation as a liquid and transition to gel on skin. Several formulations have been developed that meet this goal. In-line diffusion cells were used to determine the drug concentrations released from the gels, and the concentrations were at or above the minimum inhibitory concentrations for four common wound pathogens for 18 to 21 hours. The antimicrobial-loaded hydrogels have also shown larger zones of bacterial inhibition compared to common topical treatments for burn and chronic wound infections. After analyzing two-dimensional sprays, it was determined the type of nozzle, formulation, and distance largely influence the size and shape of the spray pattern, which is important to determine proper dosing of these topical formulations. These results will help guide future development of sprayable, antimicrobial hydrogels to improve current wound infection treatments.

 

18) INVESTIGATING E-CADHERIN MEDIATED FORCE TRANSMISSION

Rebecca L. Splitt, Alicia Salvi, Hannah Campbell, and Kris DeMali*

Department of Biochemistry and Molecular Biology, Carver College of Medicine, University of Iowa, Iowa City, IA

Cells experience both external and internal forces throughout their lifetimes. To withstand these forces, cells trigger energetically costly rearrangements of the actin cytoskeleton and growth adhesion complexes. This process is initiated when Epithelial cadherin (E-cadherin) senses force and signaling is propagated by the recruitment and activation of a master regulator of metabolism, AMP-activated protein kinase (AMPK). This kinase stimulates a signal transduction cascade that results in reinforcement of the actin cytoskeleton and ATP production to pay for the energetic costs of reinforcement. There are still many questions that remain on how cells respond to force. For example, most of the studies in the literature only examine a narrow range of amplitudes of force, while cells experience a wide range of amplitudes of physiological forces. Aim 1 of this study will determine how cells respond to different amplitudes of shear stress. I hypothesize that unique AMPK a isoforms are activated at low and high amplitudes of shear stress. These studies will extend the paradigm of how cells sense and respond to force and provide how diseases associated with defective force transmission and metabolism arise.

 

19) development of a High-Throughput Screening method to investigate interactions between G-protein coupled receptors (GPCRs) and E. coli

Christopher Vidmar¹, Joshua Wilkinson², David Roman², and Jennifer Fiegel¹*

¹Department of Chemical and Biochemical Engineering, College of Engineering, University of Iowa, Iowa City, IA

²Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA

Bacteria display surface molecules that exploit receptors found on epithelial cells to aid in their colonization and survival within the human body. Bacteria can use these receptors as anchor points to avoid clearance from the body, to facilitate internalization into epithelial cells, or to avoid an immune response by hijacking receptor signaling. G-protein coupled receptors (GPCRs) are a transmembrane class of receptors that are ubiquitous within the body and are known to interact with bacterial components. However, identifying these interactions is slow and expensive since traditional techniques investigate only one ligand-receptor pair at a time. We hypothesize that adapting high-throughput screening techniques will allow us to investigate interactions between whole bacterial pathogens and hundreds of GPCRs in parallel. The PRESTO-Tango platform was used as an initial screen to identify interactions between 314 non-olfactory GPCRs with an Escherichia coli (E. coli) strain killed by ciprofloxacin. This initial screening showed 40 potential E. coli­-GPCR interactions. Current work is focused on validating these interactions using PRESTO-Tango to show dose-dependent interactions and to confirm the interactions are due to bacterial surface components or bacterial secreted compounds.

 

20) INVESTIGATION OF MECHANISM OF NEUROTROPHIC HENYLBUTENOID DIMERS

Piyumi Wijesirigunawardana¹,Khyati Gohil² and Florence Williams^1*

¹ Department of Chemistry, College of Liberal Arts and Sciences, University of Iowa, Iowa City, IA

² Department of Chemistry, University of Alberta, Edmonton, AB, Canada

Neurodegenerative diseases are characterized by a loss of nervous system function and increased levels of neuronal death.To address the underlying neurodegeneration and the failure to extend life expectancy requires alternative strategies such as promoting neuronal survival. Neurotrophins are endogenous proteins which exhibit pro-survival, pro-differentiation, and synaptic plasticity by mediating the body’s natural signaling pathways. Unfortunately, the short half-lives of neurotrophins (<1 minute in serum) and inability to cross the blood-brain barrier make them non-viable as broad therapeutic applications in neurodegenerative disease therapy. Therefore, an attractive strategy is the use of small molecules that act as functional analogs to neurotrophins.

One set of functional analogs of neurotrophins are the phenylbutenoid (PBD) dimers. Besides demonstrating neurotrophic activity in a rat pheochromocytoma model cell line (PC 12), as well as primary cultured rat cortical neurons, Matsui et al. showed that oral dosing of olfactory bulbectomized (OBX) mice (a surgical model for neurodegenerative diseases) with cis or trans PBD resulted in significant increases in newly generated neuronal cells in the hippocampal region. Recently reported data in our group further delineates the polygonal behavior of PC-12 cells with neurite projections in the absence and presence of NGF when treated with ±trans PBD. Understanding the mechanism of action of PBD is crucial since it reveals the signaling pathway that initiates a neurotrophic response in neuronal cells. Therefore, the aim of the study is to investigate the mechanism of action of PBD by identifying its cellular recognition partner in a pull-down assay, in which derivatized PBD acts as a bait molecule bound to beads of a solid support. This work constitutes early steps towards therapeutic strategies to combat, and potentially reverse, neuron cell death in neurodegenerative diseases.

 

21) THERMAL MITIGATION OF PSEUDOMONAS AERUGINOSA BIOFILMS GROWN IN VIVO

Konna Zoga¹, Steven Bullard², David A. Stoltz², and Eric Nuxoll¹*

¹Department of Chemical and Biochemical Engineering, College of Engineering, University of Iowa, Iowa City, IA

²Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, IA

Almost 4% of the medical devices implanted in the U.S annually are infected by bacterial biofilms. Current treatment requires multiple, additional surgeries for the replacement of the infected implant, resulting in almost $5 billion of medical expenses annually and impaired quality of life for the patients.  Studies have suggested delivering heat remotely to the infected area as an alternative means of eradicating the biofilms in situ. Thermal treatment of biofilms has been mostly studied with in vitro trials, offering critical information and promising results for the effective mitigation of these infections. However, little is known for the performance of this method in vivo. To better understand how the existing in vitro models relate to the in vivo systems, this project investigated the thermal susceptibility of Pseudomonas aeruginosa (P.aeruginosa) biofilms grown in vivo. Stainless-steel, elliptical coupons were implanted in mice and inoculated, allowing for the growth of biofilm for up to seven days. Upon development, the biofilms were explanted and immersed in heated media, at a few select combinations of temperature and exposure times. These results were compared against biofilms grown in vitro, exposed to the same thermal treatment. Results indicate that the population density of the biofilms in this model is 10⁵ CFU/cm², almost two orders of magnitude lower than the density in our lowest in vitro model while they appear to be more susceptible to the heat treatment. These findings can be the foundations for further investigation and development of a viable, therapeutic approach to medical devices infections.

 

22) RAPID IDENTIFICATION and production of metabolites, AND ITS OPTIMIZATION USING HUMAN CYTOCHROME P450 2D6 (CYP2D6) ENGINEERED IN Thermothelomyces heterothallica (formerly Myceliopthora thermophila), (C1)

Shuvendu Das^{1, 2}, Reid Hogan¹, Ronen Tchelet³, Mark Emalfarb³, and Mark Arnold^{1, 2}*

¹Center for Biocatalysis and Bioprocessing, Office of Vice President for Research, University of Iowa, Coralville, IA

²Department of Chemistry, College of Liberal Arts and Sciences, University of Iowa, Iowa City, IA

³Dyadic International, Inc. 140 Intracoastal Pointe Drive, Suite # 404, Jupiter, Fl

Our long-term research objective is to create a “microbial human liver” (MHL) capable of producing human relevant metabolites from putative therapeutic agents. The envisioned engineered microbial system will mimic the drug metabolism of the human liver. This microbial system will have the potential to produce high density of cells during fermentation, thereby enabling prep-scale synthesis of meaningful drug metabolites. Such as microbial system will enable (i) preparation of drug metabolites for Metabolite in Safety Test (MIST) studies and use as reference standards, (ii) studies of drug-drug interactions, and (iii) straightforward identification of human metabolites of drugs for registration. As all putative drugs must be characterized with respect to metabolites produced in humans, our MHL cells can reduce the use of animal derived data for such characterization. Prep-scale synthesis of metabolites is generally necessary to provide enough material for MIST analyses that generally requires classical organic synthesis which can be cumbersome, difficult, low yielding and time consuming. Alternatively, human or animal microsomes can be used to generate mg amounts of metabolites, but this approach is both costly and time consuming. Engineered MHL cells promise to produce high activities of cells for synthesizing clinically meaningful drug metabolites. The concept is simple and involves adding MHL cells engineered to provide human CYP activity to drug-candidate of interest. Incubate for 3 h and analyze the resulting metabolites. There is no need for cumbersome microsomal preparations. This presentation will summarize our initial findings that illustrate feasibility of this approach. In this work, human CYP2D6 activity is engineered into C1 as a representative CYP biocatalyst. The presence of the targeted hCYP2D6 activity was determined by monitoring the O-demethylation of dextromethorphan to dextrorphan over a period of 2-3 hours. This conversion mimics the human liver by producing a known major metabolite of the FDA approved drug, dextromethorphan. Our findings demonstrate the feasibility of the MHL approach and justifies further development of more complex MHL cellular systems. In addition, this is the first report of expressing hCYP biocatalytic activity in non-yeast filamentous fungal expression system.

 

23) SWSAP1-SWS1 functions to stimulate RAD51-mediated repair following alkylation damage

Sarah Hengel ^1,2, Katherine Oppenheimer^,1,2 and Kara Bernstein^1,2

¹Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA

²UPMC Hillman Cancer Center, Pittsburgh, PA, correspondence SAH202@pitt.edu

Chemicals in our environment from consumer industrial products, tobacco smoke, pesticides and byproducts of water treatment processing can cause alkylation-induced DNA lesions. DNA lesions can cause replicative stress which can lead to genome instability and cancer. The RAD51 protein plays essential roles performing homology search and strand invasion activities for faithful repair using a sister chromatid or homologous chromosome as a repair template during replication stress. RAD51 activities are regulated by mediators including BRCA2, PALB2, the RAD51 paralogs. The RAD51 paralogs include the proteins RAD51B, RAD51C, RAD51D, XRCC2, and SWSAP1. Mutations in RAD51, and its mediators, are found in breast and ovarian cancers, and Fanconi anemia-like syndromes. Here, we focused on an uncharacterized RAD51 regulator, SWSAP1 and its binding partner SWS1 known as the human Shu complex. The Shu complex is an evolutionarily conserved regulator of alkylation-induced DNA damage.  We found that the depletion of the yeast and human Shu complex components in S. cerecevsiaa and RPE-1 cells respectively, results in sensitivity to alkylating agent MMS. However, the function of the human Shu complex in alkylation-lesion processing and the repair mechanisms utilized remains unknown.  We find that recombinant SWSAP1-SWS1 form a heterodimeric protein complex by quantitative size exclusion analysis.  We show that like other RAD51 mediators, SWSAP1-SWS1 binds to human RAD51 by native page and enhances RAD51 ssDNA binding and extension using FRET-based biophysical assays. We show that SWSAP1-SWS1 is a bonafide RAD51 recombination mediator that stimulates RAD51-mediated D-loop formation on RPA-coated ssDNA. Like other RAD51 mediators, SWSAP1 mutations have been found in breast and prostate cancers as well as uterine/endometrial cancers. To determine if SWSAP1 and SWS1 protein interaction is important for its function, we analyzed 13 SWSAP1 mutants for altered protein interactions with SWS1 by yeast-2-hybrid.  Finally, direct observation of HaloTag-(JF503)-SWSAP1 molecules from nuclear extracts shows co-localization with Cy3-RAD51 on ssDNA using a combined optical tweezers and confocal fluorescence microscope (C-Trap, Lumicks). Together, these findings show for the first time that like BRCA2, the human Shu complex components SWSAP1-SWS1 are capable of stimulating RAD51-mediated repair in the context of RPA coated ssDNA. Therefore, SWSAP1 mutations in breast and ovarian tumor samples may play a functional role in cancer cells. These studies provide foundational insight into Shu complex function following alkylation damage and their subsequent repair.

Dr. Sarah R. Hengel is looking for a post doc and lab technician to join her lab starting September 2023 in the Biology Department at Tufts in Medford MA. Her lab focuses on understanding mechanisms of genome maintenance during replication stress. The factors she studies are mutated or upregulated in cancers so translational as well as basic science projects are available! 

 

24) DETECTION OF G-QUADRUPLEX DNA STRUCTURES AT SINGLE-MOLECULE LEVEL USING G-QUADRUPLEX BINDING PROTEIN, G4P

Paras Gaur, Fletcher E Bain and Maria Spies*

Department of Biochemistry and Molecular Biology, Carver College of Medicine, University of Iowa, Iowa City, IA

DNA replication is an important event in all living cells. To maintain genomic integrity, the DNA must accurately and fully replicate before the cell divides, but frequently the replication process is hindered either because of DNA lesions or due to the presence of non-canonical DNA structures which acts as an obstacle to the replication fork progression. One of these DNA structures is G-quadruplexes (G4), which are formed by Hoogsteen guanine-guanine pairing within guanine-rich nucleic acids. Some G4s interfere with vital cellular processes such as DNA replication, DNA repair and RNA transcription. Other G4s play important non-pathological roles in regulating these processes. Several bioinformatics studies predicted that over 700,000 sequences in human genome have potential to form G4 structures, among which approximately 10,000 form G4s in chromatin of living cells and are most found in promoters of oncogenes and tumor suppressors. Several reagents have been developed to visualize G4 in cells. None of these reagents, however, has been successfully used in in vitro studies and their specificity for different types of G4s is poorly characterized. Recently, Zhen et al synthesized a small artificial protein called G4P based on the G4 recognition motif from DHX36 (RHAU) helicase. G4P was reported to bind the G4 structures in cells and in vitro, and to display better selectivity towards G4s than previously published BG4 antibody. G4P applicability to single-molecule studies and its specificity for different types of G4 remains unclear. Here, we have a purified the G4P and characterized it for G4 binding using single-molecule Total Internal Reflection Microscopy. We found that G4P binds to various G4 structures with a broad range of affinities. Among tested sequences, the telomeric G4 displayed weakest affinity and the shortest life time of the bound state, thus making it a poor reagent for visualizing human telomeres. To improve the stability of G4P-G4 complexes, we are constructing and analyzing several distinct G4P derivatives, namely G4Px2, G4PFJ, and G4PFJx2. The latter constructs have a G4 binding motif from human FANCJ helicase, which is known to bind telomeric G4 with high affinity. In the future, we have will use these constructs to perform G4-Chip analysis in human cells and in vitro (using a synthetic DNA library) to understand G4s with different sequences, and structures and how they affect DNA replication and repair machineries.

 

Posters can be posted on Friday October 21st from 4-5pm (Pharmacy Building Atrium).  Please remove posters immediately following conference. 

Session 1 (12:00-12:45pm)- odd numbered posters will be presented 

Session 2 (12:45-1:30pm)-even numbered posters will be presented

CBB will keep award winning poster (or get a copy) to be displayed for the year at the CBB

 

 

Examples of Previous posters and preparation Instructions

1) ALLOSTERIC TUNING OF CASPASE-7: ESTABLISHING THE NEXUS OF STRUCTURE AND CATALYTIC POWER

Kathryn F. Hobbs¹, Jonah Propp², Nicholas R. Vance², Andrew Kalenkiewicz¹, Katie R. Witkin², M. Ashley Spies^1,2*

¹ Biochemistry and Molecular Biology, Carver College of Medicine, University of Iowa, Iowa City, IA ² Division of Medicinal and Natural Products Chemistry, Department of Pharmaceutics and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA

2) DEVELOPMENT OF 3D PRINTED GENE-ACTIVATED CALCIUM PHOSPHATE CEMENT SCAFFOLDS FOR APPLICATION IN BONE REGENERATION

Noah Z. Laird¹, Esraa Mohamed^{1, @}, Pornpoj Phruttiwanichakun^{1, @}, Timothy M. Acri¹, Jaidev L. Chakka¹, Douglas Fredericks², John M. Femino², Aliasger K. Salem^{1, *}.

¹Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA, ²Department of Orthopedics and Rehabilitation, University of Iowa, Iowa City, IA^@These authors contributed equally. ^*Corresponding author.

3) CATIONIC CaMKIIN-LOADED LIPOSOMES AIMED TO REDUCE CHLORINE-INDUCED AIRWAY OXIDATIVE STRESS

Esraa Mohamed¹, Andrea Adamcakova-Dodd^2, Isabella Grumbach³, Peter S. Thorne^2,4, Aliasger K. Salem^1,5*.

¹Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, University of Iowa, Iowa City, IA 52242 

²Department of Occupational and Environmental Health, College of Public Health, University of Iowa, Iowa City, IA 52242

³Abboud Cardiovascular Research Center, Division of Cardiovascular Medicine, Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, IA 52242

⁴Human Toxicology Program, Graduate College, University of Iowa
 

⁵Holden Comprehensive Cancer Center, University of Iowa Hospitals & Clinics, Iowa City, IA 52242

Background: Glutamate (Glu) is the major excitatory neurotransmitter in  the central nervous system (CNS) involved in synaptic plasticity, neuronal outgrowth and survival, and memory. Although intracellular Glu concentration is often quantified in the millimolar range, extracellular Glu concentration must remain in the micromolar range. When extracellular levels of Glu rise, aberrant synaptic signaling leads to excitotoxicity which is thought to contribute to many neurodegenerative diseases, including epilepsy and Alzheimer’s disease. In previous reports, limonoids isolated from Dictamnus dasycarpus showed significant neuroprotective activity against Glu excitotoxicity. Of the previously studied limonoid natural products, fraxinellone was one of four compounds that proved to be effective in protecting against Glu excitotoxicity in vitro. With this information, a library of analogues was synthesized from the natural product fraxinellone that proved to be more effective at protecting against Glu toxicity than natural fraxinellone.

Methods: In vitro methods were used to measure the protective properties of the new fraxinellone analogues and to determine their mechanism of protection, which has not been achieved before. PC12 cells were first pre-treated with each compound at a range of 0.05 to 1.0 μM. The compounds were then washed from the cells and 100 μM Glu was added for 24 hours. MTT analysis was then performed to measure cell viability to determine if the compounds were effective at protecting the cells against Glu toxicity. Of the fifteen analogues that were synthesized, six proved to be protective against Glu toxicity, even more than natural fraxinellone. With this information, we then investigated their mechanism of protection by considering potential targets of these compounds, specifically the Nrf2 pathway, through qPCR analysis. We also determined the time course for Glu-mediated ROS production.

Results. Six of the fifteen compounds protected against Glu excitotoxicity. When cells were pre-treated with the compounds that afforded protection before adding the Glu insult, we saw enhancement of gene expression of antioxidant response elements (AREs), a key mechanism of Nrf2 activation. Antioxidant gene expression was not seen for compounds that did not provide protection.

Conclusions: Our findings suggest high levels of Glu are toxic to PC12 cells and induce ROS production in a time-dependent manner. Current findings suggest the compounds protect through Nrf2 activation and induction of an antioxidant response.

 

POSTER PREPARATION

(1) The poster board has a 44" H x 68" W area for mounting the posters.
(2) Lettering and illustrations on the poster pages must be large enough to be read by attendees from a distance of 3 feet or more.
(3) Each poster must have a title, authors and affiliation section at the beginning. Material should be displayed in logical sequence (e.g., introduction, objectives, results, conclusion).
(4) Authors are responsible for mounting their own poster material on poster boards which will be provided at the conference. The Center will provide magnets for the poster boards.