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Effects of Hormones on Follicular Gata4 and Gata6 Expression
Danielle D. Oberpriller and Shyamal K. Roy Dr.
The GATA gene family consists two sub-groups of six genes namely, gata1-3, and gata4-6 transcription factors.GATA4 increases in developing gonads in the context of sex determination. Both gata4 andgata6 are present primarily in granulosa cells, but also in the theca, germinal epithelium, and corpus lutem. However, little is known about hormones, which may affect GATA4 expression in specific cell types within the ovary. The regulation of both gata4 and gata6 expression seems to be controlled by several factors including hormones, calcium signaling, oxygen levels and transcriptional/translational mechanisms. Deletion of gata4in mice lead to a 75%decrease in litter size while deletion of both gata4 and gata6 results in complete infertility due to inefficient primordial follicle formation, oocyte survival and follicular somatic cell development leading to defective folliculogenesis.
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Explorative Role of miR-216a/217 as a Tumor Suppressor in Pancreatic Cancer
Shreya Repakula, Nagabhishek Sirpu Natesh, Ramakanth C. Venkata, Surinder K. Batra, and Satyanarayana Rachagani
Pancreatic cancer (PC) is a lethal malignancy with a 5-year survival rate of 10.8%. Gemcitabine, in combination with Abraxane and Folfirinox, are current treatments available for metastatic PC, however, all these therapies provided limited patient survival benefit, often resulting in high toxicity. Therefore, there is a need to identify novel therapeutic targets and combination therapies to combat this lethal cancer. MicroRNAs (miRNAs or miRs) have been shown to regulate PC proliferation and metastasis. Recently, miRNA therapies have shown promising results as a single miRNA is predicted to target more than 200 genes, involved in multiple pathways. The objective of this study is to understand the role of miR-216a/217 in PC growth and its progression. We and others have shown that miR-216a/217 was progressively downregulated during PC progression. PC patients with higher miR-216a/217 had better survival. Our In-situ hybridization data showed reduced expression of miR-216a/217 in PC patient samples (TMA - 196 core). Further, over-expression of miR-216a/217 in Capan-1 PC cells in vitro resulted in inhibition of cell proliferation and the epithelial-mesenchymal transition (EMT). In silico analysis have identified protein tyrosine phosphatase type IVA member 1 (PTP4A1) as a direct downstream target of miR-216a/217 in PC. Furthermore, our data indicates that miR-216a/217 inhibits PC metastasis by targeting PTP4A1 and may serve as a prospective therapeutic target in PC.
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Quantifying Serum miRNA using DNA-Gold Nanoparticles: A Modern Approach to Diagnosing Pancreatic Cancer
Matthew Salfity, Prakash Kshirsagar Dr., Maneesh Jain Dr., and Surinder K. Batra Dr.
Pancreatic Cancer (PC) is the third leading cause of cancer-related mortality in America with nearly 50,000 deaths annually. Furthermore, PC’s low 5-year survival rate, 11% overall, is highly dependent on the stage during which it is diagnosed and treated. When confined to the pancreas, the 5-year survival rate has been shown to reach as high as 42%, a nearly four-fold increase. Due to significant mortality differences dependent on PC stage, there is an utmost clinical need to diagnose PC as early as possible to maximize the chance of favorable patient outcomes. Most PC diagnostic methods currently involve imaging techniques including x-rays, CT, MRI, US, PET, ERCP. Issues with these techniques include levels of discomfort for the patient and higher costs and commitment to diagnose PC. For this reason, noninvasive diagnostic tests, such as CA 19-9 and CEA antigen blood tests are widely used to better inform patient outcomes. Current blood tests struggle to show appropriate specificity or sensitivity to reliably diagnose PC, including the limited ability to discriminate between benign and malignant tumors. Lacking validity may lead to patients over or under-investing in PC treatment. In comparison to blood antigens, microRNA (miRNA) is a biomarker that can be much more capable of diagnosing PC. The miRNAs consist of strands of around 20 base pairs in length that are transcribed in the body for gene regulation, controlling the expression of over half of our total genes. Aberrant expression of miRNAs are associated with PC. Current diagnostic tests that utilize miRNA biomarkers (e.g., microarray, qPCR) lack the sensitivity and limit of detection (LOD) required to assay normal versus aberrant miRNA levels since they are designed to detect longer nucleotide chains. Current methods are also cumbersome to use as they require extreme precaution and resources to collect data and avoid contamination. We propose new nanoparticle-based DNA-AuNP technology that will run a real time in situ assay of a patient’s serum sample to more reliably inform PC diagnosis early on.
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Degradation of Airway Secretory Cell Mucin Granules Is Dependent on Lysosome Activity
Erik A. Sillaste, Paul G. Thomes, Elizabeth B. Staab, and John D. Dickinson
Inflammatory airway diseases (e.g. COPD and asthma) are associated with mucous cell metaplasia and mucin hypersecretion, resulting in symptoms such as shortness of breath and cough. However, how secretory cells remove excess mucin granules is poorly understood. Previous research suggests that intracellular degradation pathways, such as autophagy, are involved in the degradation of mucin granules during resolution of mucous cell metaplasia. We thus hypothesized that the elimination of excess mucin granules is dependent on lysosome-mediated degradation in airway secretory cells. Calu-3 cells, an airway epithelial cell line containing abundant mucin granules, were treated with inhibitors of lysosome acidification (Bafilomycin A1) and lysosome enzyme activity (Pepstatin E64d). We found statistically significant increases in the levels of secretory mucin, MUC5AC, by mucin blot, suggesting that the lysosome mediates the elimination of mucin granules. In addition, by immunoblot we observed an increase in the autophagosome markers, LC3-II and SQSTM1, with lysosome inhibition using Bafilomycin A1, indicating an accumulation of autophagosomes and a role for autophagy in the degradation of mucin granules. However, after transfecting Calu-3 cells with a ubiquitin-hemagglutinin tag plasmid to examine the role of the proteasome in the degradation of mucin granules, we observed that our transfection efficiency was low, making it difficult to detect the hemagglutinin epitope by immunoblots. Nevertheless, we found that MUC5AC levels preliminarily increase with the inhibition of the proteasome using MG-132, suggesting a potential role for the proteasome in the degradation of mucin granules. Thus, we can conclude that inhibition of the lysosome increases MUC5AC levels, demonstrating that the lysosome mediates the degradation of mucin granules in airway secretory cells. In addition, while we were not able to conclude that the proteasome is involved in the degradation of mucin granules with certainty, our preliminary data suggests that it is possible that the ubiquitin-proteasome system is involved in the degradation of mucin due to the observed increase in MUC5AC levels with MG-132.
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Appropriateness of Vancomycin Use and Associated Outcomes
Sehajvir Singh, Avi Prajapati, Trevor Van Schooneveld, and Manish Tiwari
Background:
Several studies have documented increased rates of MRSA associated with adverse patient outcomes. Vancomycin remains the primary treatment of choice and use of vancomycin in hospitalized settings is increasing. Inappropriate vancomycin has been shown to lead to the development of vancomycin resistant organisms. Guidelines developed by IDSA have been used to promote appropriate use of vancomycin. However, inappropriate use of vancomycin remains high and its association with patient outcomes has not been well examined. The objective of this study was to examine appropriateness of vancomycin use and associated outcomes based on IDSA guidelines.
Methods:
A retrospective observational study of patients ≥18 years old who had received one dose of intravenous vancomycin in January 2019 was conducted at the University of Nebraska Medical Center. Appropriateness of vancomycin use was evaluated with criteria established through IDSA guidelines. Vancomycin use was considered appropriate in serious infections due to beta-lactam resistant gram-positive organisms, treatment of infections in the setting of beta-lactam allergy, surgical prophylaxis in patients allergic to beta-lactam antibiotics and empiric treatment for serious infections such as septic shock, bacteremia and infective endocarditis, healthcare-associated pneumonia, osteomyelitis and septic arthritis and central nervous system infections. De-escalation of vancomycin after 72 hours was considered appropriate when used empirically. Any deviation from criteria was deemed inappropriate. Variables reviewed included age, gender, length of stay, mortality, intensive care unit (ICU) admission, and length of vancomycin use. Data is expressed as percentage for nominal variables and as mean with standard deviation for continuous variables. Age, gender, mortality and ICU admission were further analyzed by chi-square tests. Length of stay and vancomycin use days were analyzed using unpaired t-test. A P-value < 0.05 was considered significant for the study.
Results:
A total of 595 encounters of vancomycin use were included in the study. Overall, 38.2% of all patients were admitted to the ICU, and hospital mortality was 9.24%. Vancomycin use was shown to be inappropriate in 34.3% of all patient encounters. Slightly higher mortality was noted in the appropriate group, but it was not found to be statistically significant (10.74% in appropriate group vs. 6.37% in inappropriate vancomycin use group; p = 0.10). Mean length of stay in patients between the two groups was not found to be statistically significant (12.60 ± 18.23 days for appropriate group vs. 14.01 ± 21.64 days for inappropriate group; p = 0.40). Significantly higher ICU admission was found for the appropriate group (41.43% in appropriate group vs. 32.35% in the inappropriate group, p = 0.0001). The most common indication for appropriate use of vancomycin was for empiric treatment (47.6%), while surgical prophylaxis in the absence of beta-lactam allergy was the most common indication for the inappropriate vancomycin use group (41.7%).
Conclusion:
The study reveals that there was inappropriate use in about one-third of all vancomycin use encounters. The study also found some outcomes such as ICU admission to be statistically significant between the two groups. The study identified common indications for inappropriate use to target further interventions.
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Porcine Models of Pancreatic Cancer: Current Status and Future
Moo Law Eh Soe, Pinaki Mondal, Carlos P. Jara, and Mark A. Carlson
The KRASG12D and TP53R172H mutations are the leading causes of pancreatic cancer, with the third deadliest cancer mortality rate. Majority of the pancreatic cancer research utilizes mouse models. Our lab is currently studying porcine as a suitable model to study pancreatic cancer due to the comparative anatomy and genomic similarities between humans and porcine. Our first approach to develop the porcine pancreatic cancer model was to orthotopically implant the transformed and mutated primary pig pancreatic epithelial cells into the pig pancreas. The second approach was using the Oncopig model with KRASG12D and TP53R172H genetic mutations. The mutated genes were expressed by injecting adenovirus with Cre recombinase to develop tumors. Hematoxylin and Eosin staining, immunohistochemistry, plasmid, and RNA isolation were used to study the porcine pancreas before and after the tumor induction. Results showed that humans and porcine have similar histological characteristics showing comprising islets, ducts, and acinar cells. For the orthotopic model, after two weeks of orthotopic implantation, there were signs of pancreatitis and some evidence of tumor-like cells, but not tumors. Some signs were the formation of acinar to ductal metaplasia, massive immune cell responses, and vimentin expression in the regions of desmoplasia. Our results showed that the Oncopigs did generate pancreatic tumors successfully and are currently being characterized for future use, and the orthotopic model showed promising results. However, implanted tumor cells will be given additional help to allow them to survive from host immune response by over-expressing PD-L1 on the implanted tumor cells surface.
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Dolutegravir reduces migration and invasion abilities of trophoblasts by decreasing HIF-1α expression and MMPs activities
Brianna Speakar, Emma Foster, and Aditya N. Bade
Dolutegravir (DTG) is a first-line antiretroviral drug used in combination therapy for the treatment of human immunodeficiency virus type-1 (HIV-1) infection. Due to roll out of generic DTG-based regimen and rising pretreatment resistance to non-nucleoside reverse transcriptase inhibitors in resource limited countries, 15 million HIV-1 infected people will be treated with DTG by year 2025. This includes women of child-bearing age who remain a significant infected population. However, growing data have suggested that DTG is associated with birth defects. Thus, uncovering an underlying mechanism for DTG-associated adverse fetal development outcomes has gained research interest. To this end, there is a knowledge gap of known adverse events reflecting DTG-associated trophoblasts impairments. We previously reported that DTG inhibits matrix metalloproteinases (MMPs) activities. It is known that activity of MMP-2 and MMP-9 are required for the successful invasion of trophoblasts during early pregnancy and abnormalities in the activities of these proteins can influence impairment in placental implantation, vascular restructuring, and fetal development. We now report that DTG reduces invasion abilities of trophoblasts. Herein, we evaluated concentration dependent effects of DTG on HTR-8 trophoblastic cells. DTG was found to inhibit activities of MMP-2 and 9 under normoxic and hypoxic conditions. Moreover, DTG treatment decreased expression of HIF-1α under both normoxic and hypoxic conditions. Interestingly, decrease in expression of beclin 1 protein was observed, suggesting an effect on autophagy. Further assessments to determine the effects of DTG on trophoblasts functions showed that DTG reduces migration and invasion abilities of HTR-8 cells. In addition, studies performed in pregnant mice validated that DTG decreases HIF-1α expression in placenta. Thus, our proof-of-concept work concludes that DTG can potentially impair placental development by affecting HIF-1α expression and MMPs activities.
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Mitochondrial Fusion Promotes Steroidogenesis in MA10 Leydig Cells
Kimber J. Sprout, Michele R. Plewes, and John S. Davis
Infertility affects 1 out of every 6 couples, worldwide, with male infertility playing a primary factor in a third of all cases. Dysregulation of sex hormones is a major cause of infertility. Male hypogonadism is a condition in which the testis does not produce adequate concentrations of testosterone. Males suffering from hypogonadism can be born with the condition or develop it later in life, often from acute injury or infection. Male hypogonadism is treated with testosterone replacement to return testosterone levels to normal. However, one side effect of testosterone treatment is infertility. Understanding mechanisms that regulate the synthesis of sex steroids holds great potential to positively impact reproductive health and overall quality of life. Mitochondria play a key role in the synthesis of all steroid hormones. The first and rate-limiting step in the production of all steroid hormones is the transfer of cholesterol from the outer mitochondrial membrane to the inner membrane. In the current study, we examined the hypothesis that mitochondrial fusion promotes acute steroid synthesis in LH-responsive Leydig cells.
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uBEATS Data Shows Opportunity for E-Learning Module Improvement
Samantha Stuck, Andi Zhang, Peggy Moore, and Dele Davies Dr
UNMC’s uBEATS program (UNO & UNMC Building Excellence in Academics Through STEM) is a series of free e-learning modules about various health science subjects that was launched by the University of Nebraska Medical Center in August of 2020. They were designed to supplement health science topics in schools, especially for students without access to regular education in health sciences. Statistics collected from the uBEATS data dashboard, and from data provided by Dr. Gwen Nugent on uBEATS average assessment scores and student completion rates suggest that engagement is low. Upon review of literature, it was found that uBEATS does have some design flaws that could be linked to disengagement. Several simple additions were discovered that, if implemented into uBEATS’ current curriculum, show promise of boosting motivation and engagement in future iterations of uBEATS.
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The Formation and Application of Polymeric Micro- and Nanoparticles
Simon White, Jason P. Stewart, Stephen M. Curran, D. David Smith, and Joseph A. Vetro
Nano- and microparticles are used in the pharmaceutical industry for sustained release drug delivery systems. For example, polymeric particles are currently used as an FDA-approved drug delivery system for leuprolide acetate to treat prostate cancer1. Our drug of interest is CPDI-02 (formerly known as EP67)—a C5a-derived decapeptide agonist of the C5a Receptor (CD88) that activates mononuclear phagocytes to produce an immune response while potentially minimizing neutrophil-mediated toxicity2. Currently in the Vetro Lab, CPDI-02 is being tested on pigs and mice to treat methicillin-resistant Staphylococcus aureus (MRSA) infections and as the adjuvant for a vaccine for cytomegalovirus (CMV). This investigation explored formulation parameters that impact particle size and loading of CPDI-02 in a traditional oil-in-water (O/W) emulsion. We also explored adapting the formulation using microfluidic chips to generate nano- and microparticles and improve run-to-run consistency in particle size.
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Investigation of CXCR4 Chemokine Receptor Involvement in the Uptake and Penetration of siRNA Nanoparticles in Pancreatic Cancer Cell Lines and 3D Tumor Spheroids
Helen Yoo, Ling Ding, Ekta Kapoor, and David Oupicky
With the urgency of developing novel therapies for pancreatic cancer, the Oupický lab designed a polymer, PAMD-Toc, that incorporates AMD3100, an antagonist of the most widely expressed chemokine receptor in cancer cells, CXCR4. This study seeks to determine if the CXCR4 receptor is involved in the uptake and penetration of siRNA-carrying PAMD-Toc nanoparticles into pancreatic cancer cells and tumors. Findings suggest that the presence of CXCR4 increases the efficacy of PAMD-Toc nanoparticle uptake and tumor penetration, which makes PAMD-Toc nanoparticles a promising siRNA delivery vector for pancreatic cancer gene therapy.
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The Effects of Age and Alcohol on Lipid Metabolism in the Liver
Jessica S. Yoon, Sundararajan Mahalingam, Jacy L. Kubik, Ramesh Bellamkonda, Carol A. Casey, Kusum K. Kharbanda, and Karuna Rasineni
Background: Alcohol-associated liver disease (ALD) encompasses the liver manifestation of chronic alcohol abuse, characterized by different stages of liver damage that progresses from fat accumulation to steatohepatitis, fibrosis and eventually cirrhosis. The severity of liver damage is influenced by age, which is also a predictor for ALD-related mortality. Thus, the purpose of this study was to investigate how aging and alcohol affect lipid metabolism in the liver. Methods: Rats aged 4 months, 8 months, 12 months, and 22 months-old were pair-fed Lieber-DeCarli control or ethanol diet for 6 weeks. Serum and liver were collected for analyses when rats were euthanized. Analyses included histopathology, measurements of non-esterified fatty acid content and hepatic triglyceride content, and gene expression.
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