DigitalCommons@UNMC

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  Multisystem Inflammatory Syndrome in Children - Characteristics, Therapies, and Outcomes

  Reid A. Sindelar, Anji Yetman, and Jean A. Ballweg

  Multisystem Inflammatory Syndrome in Children (MIS-C) is a condition characterized by severe organ system inflammation and shock, presenting approximately 4-6 weeks after infection with the virus SARS-CoV-2. This disease was first identified in April 2020 and appears to be one of the most severe forms of disease associated with COVID-19 in children. A single-center retrospective study was performed on a cohort of patients diagnosed with MIS-C at Children's Hospital and Medical Center in Omaha. A variety of data--including patient demographics, hospital course, treatments, and long-term cardiac outcomes--were analyzed to better understand MIS-C as an emerging disease.

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  Unfolding and Binding Thermodynamics of the PREQ1 DNA Analog

  Grace Sullivan, Irine G. Khutsishvili, Calliste Reiling, and Luis Marky

  The PreQ1 riboswitch is a subset of riboswitches found mainly in eubacteria. It plays an essential role in the biosynthesis of preQ1, a precursor to queuosine (Q). Q is a hypermodified guanine nucleotide that is universally found at the wobble position of certain tRNAs. PreQ1 forms a pseudoknot structure upon binding to preQ1. We use a combination of temperature-dependent UV spectroscopy and differential scanning calorimetry (DSC) to determine the unfolding thermodynamics of a DNA analog (PREQ1) and its control hairpin at two different salt concentrations, 16 mM and 116 mM Na+. Furthermore, we use DSC and fluorescence spectroscopy techniques to determine binding affinities, Kb, for the interaction of preq1 ligand with these DNA analogs. Both oligonucleotides unfold with TMs independent of strand concentration, indicating the formation of hairpin structures. The unfavorable unfolding free energy terms resulted from the typical compensation of an unfavorable enthalpy contributions (disruption of base-pair stacks) and favorable entropy contributions (release of ions and water molecules). The ligand preQ1 yielded a Kb of 5.7 x 105. Supported by NSF Grant (MCB-1912587).

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  Alcohol in dementia: Implications of Alzheimer’s like pathology in alcohol abuse

  Natalie Swanson, Divya Thomas Chemparathy, Shilpa Buch, and Susmita Sil

  Alcohol use is widespread, with 85.6 percent of people ages 18 and older reported drinking alcohol in their lifetime in 2019. Mild to heavy alcohol use is associated with multi-organ dysfunction at the cellular and genetic levels, as well as epigenetic modifications, leading to liver and brain damage and dementia. The role of astrocytes as contributors to Alzheimer’s like pathology associated with cognitive decline in opiate abusers and people with HIV-associated neurological disorders (HAND) has been recently reported from our group. We hypothesize that alcohol could also induce astrocytic amyloidosis. In this study we demonstrated that exposure of human primary astrocytes (HPA) to ethanol resulted in a dose dependent (6.25-200 mM) increase in AD markers-amyloid precursor protein (APP), Aβ 1-42, β-site cleaving enzyme (BACE1), as well as the inflammatory marker IL1βand lncRNA BACE1AS. Next, we demonstrated a time-dependent (0-96h, 12.5 mM) upregulation of AD markers, oxidative stress (4-HNE), alcohol metabolizing enzymes alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH2), and cytochrome P450 2E1 (CYP2E1), as well as proinflammatory cytokines (TNF-α, IL1β, IL6) in HPAs exposed to alcohol. Gene silencing approaches confirmed the regulatory role of lncRNA BACE1-AS in amyloidosis and its interaction with alcohol metabolic pathways leading to neuroinflammation and oxidative stress. Further, in vivo study validated our in vitro findings, demonstrating up-regulation of APP, Aβ1-42, 4-HNE and IL1β in the cortices of ethanol-fed mice (4- weeks, ad libidum) compared to saline controls. This is the first report implicating the role of lncRNA BACE-AS in alcohol-mediated induction of astrocytic amyloidosis, leading to neuroinflammation and oxidative stress, which, in turn, could contribute to cognitive impairments. These findings set the stage for future development of therapeutic strategies aimed at targeting cognitive deficits in alcohol users and abusers.

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  Comparison of Seven Extraction Kits for Detection of SARS-CoV-2 in Wastewater

  Nicholas Sze, Catherine Pratt, Bailey White, Dylan George, and Michael Wiley

  Wastewater-based epidemiology (WBE) has been used to trace outbreaks and monitor populations for pathogenic viruses such as poliovirus. SARS-CoV-2 has been identified as a candidate for WBE as the virus can survive for prolonged periods in wastewater. The use of commercial extraction kits alongside polyethylene glycol separation, electrostatic membrane filtration, and ultracentrifugation have been used to analyze wastewater. However, the most consistent and versatile commercial kit has not been identified. In this study, previously analyzed wastewater was extracted using seven different commercial kits and quantified using cycle threshold (CT) values from PCR. Data was analyzed using ANOVA and pairwise comparisons at 95% confidence. Results reveal the use of ThermoFisher MagMax Microbiome Kit alongside proteinase K to be the most consistent kit when analyzing soil samples. Qiagen’s EnvironWater kit was identified as the kit of choice for liquid samples. ZymoBIOMICS DNA/RNA kit was identified as a versatile kit for analyzing both solids and liquids. Liquids were found to have higher quantities of virus on average, but solids had lower CT values on average. We conclude that isolating solids should be the primary focus when concentrating virus. MagMax Microbiome Kit with proteinase K was identified as the most consistent and accurate kit for analyzing wastewater. The MagMax Microbiome Kit was found to have significantly lower CT values when compared to other kits across all viral concentrations and sample types. The method used to concentrate water is inexpensive and uses less resources compared to previous methods, which allows WBE to be used at a larger scale in more areas. This will be important for monitoring SARS-CoV-2 variants and population health as the pandemic progresses. Further studies should focus on the efficiency of the kits when using a spiked positive control as well as variance between water samples.

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  The CXCR1 Axis: A Putative Therapeutic Cancer Stem Cell-Like Marker in Pancreatic Ductal Adenocarcinoma

  Elizabeth Thomas, Caitlin Molczyk, Paran Goel, Marie Powers, Lubaba A. Zaman, Evie Ehrhorn, Samuel Cohen, and Rakesh K. Singh

  Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest survival rates of all cancers in the United States. Not only is PDAC found at the late stages, but patients also present with or develop chemotherapy resistance at an elevated frequency. Left with limited options for treatment, researchers are investigating new options for these patients. One major area of interest is the sub-population of cells in the tumor called cancer stem cells (CSCs). These cells are known for having high resistance to chemotherapy, along with propagating and re-building the tumor after most non-CSCs have been therapeutically targeted. Previous studies have determined CXCR4, ALDH1, CD24, CD44, and CD133 as markers for CSC-like PDAC cells. In the present study, we investigate the closely related CXCR1 as another possible marker and therapeutic target for PDAC CSCs. CXCR1 is known for its role in inflammation and wound healing. The CXCR1 axis includes the ligands CXCL6 and IL-8, both of which promote the progression of cancer. Previously, Ginesteir et al. has shown targeting the CXCR1 axis in triple negative breast cancer reduced CSC-like phenotypes in vitro and in vivo. Investigations of CXCR1 in PDAC demonstrate IL-8 induces increased tumorsphere formation in vitro (Chen et al.), leading us to investigating CXCR1 in PDAC CSCs. We hypothesize that PDAC cells with high CXCR1 activity also exhibit increased CSC-like characteristics and targeting CXCR1 will reduce those characteristics.

  To investigate the role of CXCR1 in CSC-like phenotype of PDAC, we used the PDAC cell line CD18, along with its gemcitabine resistant (GemR) counterpart. We used the CXCR1/2 antagonist Navarixin at high enough concentrations to inhibit CXCR1. Using the previously found gemcitabine and navarixin IC50 concentrations for each parent cell line, we treated cells for 72 hours. Post-treatment, we analyzed the expression of several known CSC markers, CXCR1, and IL-8 through qRT-PCR and ELISA. We expected to see higher expression and activity of CXCR1 in cells with higher known CSC marker expression. We also anticipated that gemcitabine treatment would induce higher expression of CSC markers, whereas navarixin would exhibit lower expression. From our results, we see the beginning trends of gemcitabine treated cells having increased expression of the CSC markers and navarixin decreasing or not changing the expression levels. These results differ for IL-8, which undergoes an increase in expression when treated with both gemcitabine and navarixin, which may warrant further exploration into the role of ligands in CSC-like phenotypes. One possible explanation for this difference would be the regulation of IL-8 expression based on CXCR1 activity, as IL-8 interacts with CXCR1.

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  Analyzing SOD Activity in Lung Tissue of a Murine Model of Marfan Syndrome

  Matthew J. Tiojanco, Andrew Eksi, Kathryn Jespersen, Trevor Meisinger, B. Timothy Baxter, and Wanfen Xiong

  Marfan syndrome is an inherited autosomal dominant disorder caused by a mutation in the Fibrillin-1 gene (FBN1) affecting elastic connective tissue. Marfan syndrome commonly presents with ectopia lentis, aortic dissections, mitral valve prolape, and chronic obstructive pulmonary disease (COPD) in later stages. Patients with Marfan Syndrome are shown to have higher concentrations of reactive oxygen species (ROS) in blood plasma. Increased ROS due to oxidative stress can lead to increased cell damage and death, and have been linked to the formation of aortic dissections5. Superoxide dismutases (SOD) are a class of enzymes that convert harmful oxygen radicals into molecular oxygen and hydrogen peroxide2. Manganese-containing SOD (MnSOD) regulates radical oxygen located in the Mitochondria4. The aim of this study is to explore the role of SOD expression and ROS in relation to COPD found in Marfan syndrome. It is hypothesized that the defect in Fibrillin-1 causes oxidative stress in the lung tissue, which often causes COPD, and it is expected that there would be less SOD activity in tissues from mice with Marfan syndrome. The activity of SOD1 and MnSOD in relation to the oxidative stress that is caused by the deficiency of Fibrillin protein will be determined. To accomplish this, a murine model of Marfan syndrome Fbn1mgR/mgR, mice with a hypomorphic mutation in the Fibrillin-1 gene were compared with homozygous wild type mice. Mice were sacrificed after 1, 4 , and 8 week intervals, and both SOD1 and MnSOD expression was quantified in lung tissue. Results of the study found that there was a significant decrease in SOD1 expression in Marfan mice at the one and four week intervals, but no significant difference in the eight week interval. MnSOD was observed to not have any significant difference in expression in the one and four week Marfan mice, but was expressed at a significantly higher level in the 8 week Marfan mice compared to wild-type controls.

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  Synthesis of Indole-3-Acetic Acid Derivatives and a Urea Carboxylic Acid Derivative by Propylphosphonic Anhydride (T3P)

  Matthias Walters, Rongguo Ren, Yuxiang Dong, and Jonathan Vennerstrom

  The purpose of medicinal chemistry is to efficiently create a variety of compounds with potential for pharmacological efficacy. To promote this diversity, indole-3-acetic acid, a common plant hormone, was used as the starting material for various reactions. The coupling reagent used for these reactions was propylphosphonic anhydride, or T3P, since it has demonstrated efficiency in selective amide formation under mild conditions and it is readily soluble. In the case of multiple viable reaction sites, the intended product will dimerize, as was the case in the synthesis of the compound labeled amide 2 when T3P coupled with both sites of piperazine. N-Hydroxysuccinimide, also referred to as HOSu and NHS, was used to decrease the reactivity of the carboxylic acid—T3P mixed anhydride, so it less readily formed the dimer. This increased the yield of the monomer. Pharmacological efficacy is more probable when synthesizing a chemotype with a known structure-activity relationship, or SAR. Urea carboxylic acid has been found to have antischistosomal activity. In an effort to synthesize a drug candidate with greater likelihood of pharmacological activity, a compound was synthesized from a urea carboxylic acid using T3P by the same method used to synthesize products from indole-3-acetic acid. Five compounds were synthesized using the T3P reagent in an attempt to expand the repository of potential drug candidates. The method for each compound was made largely similar, but it differed in the work-up and purification stages, as the acidity and polarity of the systems varied.

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  Synthesis of Indole-3-Acetic Acid Derivatives and a Urea Carboxylic Acid Derivative by Propylphosphonic Anhydride (T3P)

  Matthias Walters, Rongguo Ren, Yuxiang Dong, and Jonathan L. Vennerstrom

  The purpose of medicinal chemistry is to efficiently create a variety of compounds with potential for pharmacological efficacy. To promote this diversity, indole-3-acetic acid, a common plant hormone, was used as the starting material for various reactions. The coupling reagent used for these reactions was propylphosphonic anhydride, or T3P, since it has demonstrated efficiency in selective amide formation under mild conditions and it is readily soluble. In the case of multiple viable reaction sites, the intended product will dimerize, as was the case in the synthesis of the compound labeled amide 2 when T3P coupled with both sites of piperazine. N-Hydroxysuccinimide, also referred to as HOSu and NHS, was used to decrease the reactivity of the carboxylic acid—T3P mixed anhydride, so it less readily formed the dimer. This increased the yield of the monomer. Pharmacological efficacy is more probable when synthesizing a chemotype with a known structure-activity relationship, or SAR. Urea carboxylic acid has been found to have antischistosomal activity. In an effort to synthesize a drug candidate with greater likelihood of pharmacological activity, a compound was synthesized from a urea carboxylic acid using T3P by the same method used to synthesize products from indole-3-acetic acid. Five compounds were synthesized using the T3P reagent in an attempt to expand the repository of potential drug candidates. The method for each compound was made largely similar, but it differed in the work-up and purification stages, as the acidity and polarity of the systems varied.

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  Asking the Right Questions: Screening for Second-Hand Tobacco Exposure in Pediatric Primary Care.

  Emma A. Weis, Arwa Nasir, Dave Finken, Chris Youngman, and Kerri Foulk

  Cigarette smoking is the leading cause of preventable death in the United States, and increases the risk of heart disease, stroke, and lung cancer. Second-hand smoke (SHS) exposure increases the risk of many acute and chronic childhood diseases including respiratory infections, asthma, cancer, perinatal mortality, delayed growth and development, and SIDS. Screening for SHS exposure in children is recommended during well child visits and aims at identifying the risk, providing an opportunity to educate the family on the harmful effects of tobacco, and offering resources to help quit smoking or reduce the child's exposure. Smoking cessation interventions and support have been shown to be effective in initiation of smoking cessation efforts, successful quitting, and prevention of relapse. The aims of this study are to optimize the screening process for tobacco exposure in children by asking most effective screening question and implementing universal screening in all health care visits, as well as to prevent SHS exposure by offering education and smoking cessation resources for families who are interested in smoking cessation. The number of parents/caregivers with a documented answer to the question increased significantly after the intervention and continued to increase in the maintenance phase. However, the number of parents who gave positive answers (yes for smoking) remained roughly the same from pre to the post implementation period, resulting in a decrease in the proportion of positive answers in the total sample. In addition, more than 1,100 families asked for resources to help with smoking cessation.

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  In vitro comparison of Ethanol Metabolism in Precision Cut Liver Slices from C57Bl/6, Balb/c, DBA/2J and 129S1/SvlmJ Mice and with the Aldeyra Product ADX-629

  Duncan Works, Mariah Tessin B.S., Michael J. Duryee M.S., Ted R. Mikuls M.D., and Geoffrey M. Thiele Ph.D.

  Alcoholic liver disease (ALD) is common consequence of excessive alcohol consumption [1]. When the liver is damaged by the intake of alcohol, repair mechanisms are deployed, which results in fibrosis or scarring of the liver. Development of this disease is due to the byproducts of ethanol metabolism. These byproducts include acetaldehyde from the metabolism of ethanol and malondialdehyde from the breakdown of cell membranes during injury. An Aldeyra product, ADX-629, is a small molecule that acts as a reactive aldehyde species (RASP) inhibitor. ADX-629 covalently binds free aldehydes, thus diminishing excessive RASP levels. To determine the aldehyde scavenging abilities of ADX-629 in attenuating fatty liver disease, precision cut liver slices (PCLS) were exposed to varying concentrations of ADX-629 as well as 25mM of ethanol. PCLS, which provide a novel in vitro/ex vivo experimental model, were then measured for triglyceride levels and supernatants were analyzed for acetaldehyde levels. It was found that ADX-629 reduced the acetaldehyde levels released from PCLS while also decreasing triglyceride levels. ADX-629 offers promising clinical uses such as in the prevention of fatty liver formation in patients with non-alcoholic steatohepatitis and in the treatment of alcoholic patients by preventing oxidative stress caused by the breakdown of ethanol thereby, preventing ALD.