Poster Day 2021: Abstracts: Basic Science

Carter Gay, OMS-II and Eric Johnson, PhD

Glucokinase (GCK) is a cytoplasmic enzyme that catalyzes the initial step of glycolysis and serves an important role in coupling insulin release to circulating glucose levels. Mature-Onset Diabetes of the Young (MODY) is a form of diabetes that is associated with mutations in the GCK enzyme. Ten known variants of uncertain significance (VUS) were examined using In Silico and In Vitro methods.

In Vitro analysis was conducted via enzyme assays that were designed to compare the Vmax of the purified variants to the native protein. In Silico analysis was completed via simulations run on the YASARA program. The VUS proteins were examined using .pdb files to compare their structure and stability to the native configuration of GCK. To further strengthen the evidence regarding the nature of each VUS, analysis was run on PredictSNP, a consensus program that provides information from a variety of computational tools and databases.

All VUS proteins, except D132N, demonstrated decreased activity relative to native based on In Vitro analysis. The YASARA simulation data predicted a statistically significant difference only for L122V. PredictSNP predicted a strong likelihood that 8 of the 10 VUS, including L122V, result in deleterious or pathogenic changes to the protein. D132N was predicted to have a neutral effect on structure.

Based on the results, L122V likely results in a deleterious change in protein structure and loss of function in terms of activity. There is evidence to support the other variants result in decreased activity, but more studies are required to support that conclusion.

Susanna Sogge, BS and Michael Ibiwoye, MD, MS, DTM&H, MPH, PhD

Numerous studies suggest that dysfunction of the blood-brain barrier (BBB) is an important step in the neurotoxicity of cadmium. A rat-specific BBB marker protein, the endothelial barrier antigen (EBA), has been previously classified by Sternberger and others.

We hypothesize that acute exposure to cadmium does not affect EBA expression in young rats.

Twenty healthy 40-day-old male Sprague-Dawley rats were randomly divided into two groups: intraperitoneal injection with cadmium chloride (4 mg/kg b.w., n=10) in isotonic saline; intraperitoneal injection with isotonic saline (n=10). All were sacrificed 3 days post-injection. The brains were formalin-fixed, thin sections were processed for immunofluorescent microscopy.

Sections treated as follows: 5% normal donkey serum; mouse anti-EBA (1:500) overnight at 4 °C; PBS wash; donkey anti-mouse Alexa Fluor 488 (1:200) x 1 hour; PBS wash. Mounted in Vectashield antifade with DAPI. Examined with an Olympus BX63 microscope/cellSense software. EBA-immunoreactive microvessels were quantitated by measuring the surface areas per field of view at 40x magnification (0.1 mm^2). Welch’s paired t-test was used to compare grand mean areas of all fields from the control and cadmium-treated brain sections.

We detected a reduction in EBA-positive microvessel surface areas in the forebrain (t = 5.86, df = 1789, p-value < 0.001) and cerebellum (t=73.40, df=1337, p < 0.001) of cadmium-treated rats compared to the normal controls.

EBA is involved in the pathogenesis of cadmium neurotoxicity and could serve as a sensitive marker for studying the impact of cadmium on nascent BBB microvessels in the developing rat central nervous system.

Deniz Bozdag, Gherardo Baudo, Matteo Massaro, Haoran Liu, Hyunho Lee, and Elvin Blanco

Idiopathic Pulmonary Fibrosis (IPF) is a progressive chronic lung disease with a grim prognosis. Currently, drug treatments simply slow disease progression with no impact on mortality rate. IPF is characterized by the over proliferation of fibroblasts along with excessive production of extracellular matrix (ECM) which thickens the alveolar wall, thereby reducing effective gas exchange. Increased TGF-β/SMAD signaling activates fibroblasts and leads to ECM deposition. TGF-β has been shown to induce phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E).

TGF-β-stimulated human pulmonary epithelial (A-549) and fibroblasts (MRC5) were used in this study. Western blot was used to examine eiF4E expression in cells. A transwell invasion assay and wound healing assay involving A549 cells were used to examine the effect of siRNA treatment on cell migration.

In the Western Blot analysis, treatment of the eIF4E siRNA showed a decrease in expression of eIF4E in two different cell concentrations at the same treatment dose. In the wound healing assay, eIF4E RNAi significantly decreased TGF-β induced epithelial migration. Cell proliferation studies decrease in MRC-5 proliferation following treatment with eIF4E siRNA. Treatment of A549 cells with siRNA against eIF4E resulted in a reduction in the overall number of migrated cells via a transwell invasion assay.

Our data supports that eIF4E silencing is important for regulating EMT and decreased proliferation and invasion rates. The emerging evidence of eIF4E activation as an important driver of IPF highlights this protein as a potential novel therapeutic target in IPF and other diseases as well.

Danielle M. Glinka, MS and Gordon G. MacGregor, PhD, MBA

Atopic Dermatitis (AD) is a common dermatological disease characterized by erythema, xerosis, and pruritis. It is hypothesized that increased epithelial permeability and decreased tight junction resistance of sweat glands contribute to AD by increasing trans epidermal water loss and decreasing sweat-dependent skin moisturization. We hypothesize that LPS, a glycolipid found on the surface of gram-negative bacteria, promotes epithelial barrier function.

The MDCK cell line is a model epithelium to study the tight and adherens junction regulation and permeability. Transepithelial resistance was measured using an epithelial resistance meter. Epithelial permeability was measured using fluorescent FITC-Dextran size 4KDa. Gene expression of E-cadherin was measured using qPCR, relative to GAPDH expression.

Transepithelial resistance of the MDCK monolayer was 465.3 ± 27.9 (Ω.cm2), which increased to 556.8 ± 14.5 (Ω.cm2) after 3 hours treatment with LPS (1µg/ml), an increase of 20.0 ± 8.3 % (p < 0.05, n = 4). Permeability to FITC-dextran (4kDa) decreased by 15.0 ± 6.7 % (p < 0.05, n = 4) after 3 hours treatment with LPS. Exposure to LPS for 3 hours increased cadherin gene expression 1.9-fold relative to GAPDH.

LPS increased the resistance and decreased permeability across an epithelial monolayer, most likely through increased cadherin expression. Cadherin plays an integral role in intercellular adhesion of keratinocytes and is downregulated in AD. We propose that LPS could play a role in treating the manifestations of AD and suggests a role for the normal skin flora in maintaining epithelial integrity.

Jeff Tomes, BS; Audrey A. Vasauskas, PhD; Caleb Hamilton, PhD; and Jonathan Brown, PhD

Pulmonary arterial hypertension (PAH) is a disease characterized by an increase in pulmonary circulation pressure due to the abnormal differentiation and proliferation of endothelial cells in the intimal layer. PAH leads to constriction of the pulmonary arteries, commonly causing right-side heart failure. Prior studies have demonstrated that Akt activity is elevated in the PAH disease models.

Pulmonary arterial endothelial cells from wild-type (FR6) rats and the Sugen5416/hypoxia PAH model (SPAD) were provided by Dr. Troy Stevens at the University of South Alabama. Both FR6 and SPAD cells were plated in a 6-well plate at concentrations of 7x105 and 5x105 in 2 mL D10F and incubated overnight at 37°C. Antibodies were purchased from Cell Signaling. Western blot analysis was performed with Licor Software. Cells were stimulated with LPS (100 ng/mL) and BMP4 (50 ng/mL) for the indicated times.

The phosphorylation of Akt was increased in SPAD cells stimulated with BMP4 or LPS compared to FR6 cells. Phosphorylation of S6K and GSK3ß were also increased in SPAD cells compared to FR6 cells when stimulated with LPS. Stimulation of FR6 and SPAD with LPS did not result in significant differences in the nuclear location of p65.

These findings indicate that BMP4 and LPS are possible contributing factors in PAH through the activation of Akt. In conclusion, downstream Akt kinases GSK3ß and S6K activity are elevated in SPAD compared to FR6 with stimulation by LPS, but NFĸB nuclear localization appears unchanged.

Shelby Watford, OMS-II; and Eric Johnson, PhD

Mutations in glucokinase (GCK) cause Maturity-onset-diabetes-of-youth type 2 (MODY-2) by producing enzymes with reduced Vmax and increased glucose Km values. This study analyzes variants of unknown significance (VUS) in two regions of the human GCK gene, 258-262, and 412-427.

Bacterial plasmids were generated that express 6X HIS-tagged human GCK proteins. WT and MODY-Associated variant GCK proteins were isolated by affinity chromatography and GCK function was analyzed via activity assay on a UV microplate reader. Vmax and RMSD values were compared using student T-tests and ANOVA. The human GCK variants were also studied using in silico molecular dynamics simulation.

Half of the variants analyzed (G258D, A259V, G261R, V412G, and R422P) showed significant (P<0.05) decreases activity (Vmax) relative to native protein in the enzyme activity assay. One variant, D262N, showed a non-significant increase in activity (P=0.09).

In silico analysis found G258D to be significantly different (P<0.05) when compared to native GCK.

We set out to compare the enzymatic activity of GCK variants to the molecular dynamics simulation results. Enzyme kinetics assays found many variants significantly reduced protein functionality. Additionally, in silico analysis of G258D showed significant alterations of RMSD values indicating that variant-induced changes in GCK protein structural stability may contribute to decreased enzyme functionality. However, most molecular dynamics studies did not significantly show differences in VUS v. native. The discrepancy between the enzyme kinetics findings and current molecular dynamics findings may be resolved with further in silico analyses. 

Arianne M. Serrano, OMS-II; Emily A. Snyder, BS; Jonathan R. Brown, PhD; Audrey A. Vasauskas, PhD; and Caleb L. Hamilton, PhD  

Pulmonary arterial hypertension (PAH) is a progressive disorder of the pulmonary vasculature leading to increased pulmonary vascular resistance and right heart failure. Currently, there are only palliative treatment options available, and prognosis is poor. Endothelial-to-mesenchymal transition (EndoMT) contributes to the vasculature remodeling and formation of plexiform lesions observed in PAH. Increased expression of the fibronectin receptor, integrin 51, has been previously observed in pulmonary artery endothelial cells (PAECs) from the Sugen/hypoxia rat model of PAH. As fibronectin is abundant in plexiform lesions in human PAH, we hypothesized that fibronectin contributes to the pathogenesis of PAH. We hypothesized that fibronectin increases the expression of molecular markers of EndoMT, particularly endothelin-1.

Pulmonary artery endothelial cells from the Sugen/hypoxia rat model of PAH (PAH PAECs) and normoxic control cells (PAECs) were obtained from the University of South Alabama College of Medicine. Cells were treated with either 50g/mL fibronectin or vehicle (equal volume of water) for 3 hours. Whole cell lysates were obtained for Western blot analysis, or cells were fixed with 80% methanol for immunocytochemistry.

Western blot analysis with densitometry showed significantly increased endothelin-1 expression in fibronectin-treated PAECs compared to the vehicle control. Immunofluorescence of endothelin-1 expression was also greater in both fibronectin-treated PAECs.

These results demonstrate that fibronectin treatment increases endothelin-1 expression in PAECs of the Sugen/hypoxia rat model of PAH. This suggests that fibronectin signaling could be a key factor in disease pathogenesis, through increased expression of endothelin-1.

Tushar Shah, MD; and Heong Jin Ahn, OMS-II

Neonatal brain injury resulting from oxygen deprivation around the time of birth (Hypoxic Ischemic Encephalopathy, HIE) is a leading cause of neonatal mortality and disability worldwide. Therapeutic hypothermia (HT) is the only therapy shown to improve outcomes in HIE, but neuroprotection is not universal, with 50% of cooled babies developing adverse neurodevelopmental outcomes. This is likely due to an incomplete understanding of the mechanisms of HT in a clinically heterogenous population.

Differential gene expression through transcriptome analysis (RNA-seq) would reveal the influence of gene expression on the therapeutic response with HT. The Vannucci model (unilateral carotid ligation followed by hypoxia) was used to induce HIE in term-equivalent rat pups. Pups were randomly assigned to three treatment groups: Sham (control), normothermia (NT)/HIE, and HT treatment. They were then harvested at 24 hours and 5 days after injury.

Using next generation sequencing, a comparison of gene expression between groups was performed. Genes with an adjusted p-value < 0.05 (Wald test) were called as differentially expressed genes. Significantly differentially expressed genes were clustered by their gene ontology and the enrichment of gene ontology terms was tested using Fisher exact test. 296 unique genes were upregulated and 482 unique genes were downregulated at 24 hours when comparing NT vs. HT, affecting the neuropeptide signaling and inflammatory response pathways on gene ontology analysis.

Further analysis of this data will identify unique inflammatory pathways instrumental in the therapeutic response to HT and assist in developing a personalized neuroprotective approach in the treatment of a multifactorial disease such as HIE.

Emily Snyder, BS, MT(AAB); and Audrey Vasauskas, PhD

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by pulmonary vasoconstriction leading to high circulation pressures in the pulmonary vasculature and subsequent right heart failure. Serotonin (5-HT), an inflammatory marker, is produced in pulmonary artery endothelial cells (PAECs) and has been shown to play a role in the differentiation of smooth muscle cells (SMC) in PAH. Data from our laboratory and others indicate endothelial to mesenchymal transition (EndMT) contributes to PAH, transforming cells to constrictive SMC-phenotype. We hypothesize that 5-HT may play a role in the transformation of PAECs in PAH leading to the constrictive vascular phenotype characteristic of the disease.

PAECs from Sugen/hypoxia treated PAH rats (PAH) or control PAECs were gifted from the University of South Alabama. Cells were treated with Dexamethasone (1mM) or vehicle control and incubated at 37C for 24 hours. RNA was isolated and cDNA made. Comparative real-time PCR was performed using FAM-labeled Htr2A, Htr2B, and Tph1 probes.

PCR analysis indicate serotonin transporter receptor expression is decreased in PAH PAECs.

These data may indicate serotonin (5-HT) signaling abnormalities in PAH cells compared to control cells. Studies are underway to determine specific expression aberrations and downstream effects in PAECs.

Justin D. Anderson, Cameron Lee, Gracie Giang, April Jones, Zhongyu Liu, Kennedy Parker, Kim Benner, Heather Searcy, and Jennifer S. Guimbellot

Cystic Fibrosis (CF) is a genetic multisystem disorder requiring the use of a wide range of acute and chronic medications. We hypothesized that while pharmacogenomics (PGx) variants likely exist in the CF population at similar frequencies to the general population, their higher drug exposure rate increases risk for adverse drug events (ADEs).

Patients with CF (n=82) donated DNA samples for PGx variant analysis after consenting for the study. Two years of medication encounter data was available and gathered from the electronic medical record for 82 patients. The open-source command-line program, Pharmacogenomics Clinical Annotation Tool (PharmCAT), was used to provide clinical annotations for each patient.

CF cohort was 52.4% female with mean age of 22.1 years (Range: 3-66 years old) and were 93.9% Caucasian. CF patients had a mean 20.34 medication events (Range: 6-69) each year. Events were categorized as chronic (45.7%), prolonged acute (4.2%), and acute (50.2%). Antibiotics comprised 26.5% of the medication exposures. 97.6% of patients had at least 1 actionable PGx variant; over 70% of patients had ≥3. Approximately 61% of patients had an actionable variant in the CYP2C19 gene and 22% of these patients experienced an ADE correlated with their genotype.

PGx variants in CF patients mirror those of the general population their high medication burden increases their likelihood of experiencing an ADE. Almost every patient in this cohort had at least one actionable variant. A significant portion experienced predictable ADEs. Implementation of PGx guidelines should be considered in the CF population.

Erika Haviland, OMS-II and Lawrence LeClaire, PhD

Metastatic breast cells require complex signaling to an array of regulatory proteins to control cell migration. The coordination of these signaling proteins is necessary to regulate the actin cytoskeleton and allow a metastatic cell to migrate from a primary tumor in response to growth factors. The microRNA miR-141-3p has been shown to regulate the expression of key signaling proteins responsible for cancer metastasis in several tissues. Here, we lay the groundwork to investigate the role of miR-141-3p on cell migration and invasion in established human breast cancer cell lines.

MDA-MB-231 metastatic breast cancer and MCF-10a normal breast human cell lines were cultured by standardized procedures.  Scratch wound and invasion assays were used to measure migration and invasion rates of cells. TaqMan RT-PCR and real time PCR was used to quantify miR-141-3p in cells.

Using scratch wound assays and phase contrast microscopy, we found that metastatic MDA-MB-231cells migrated at rates of more than 4 times that of normal breast tissue lines MCF-10a. In Boyden chamber assays, MDA-MB-231 cells were more than 5X more invasive than normal cells. Next, using real-time PCR, we quantified miR-141-3p in both cell lines. Consistently, MCF-10a expressed miR-141-3p at more than 29.3% that of MDA-MB-231 cells.

Together, these data show that miR-141-3p levels correlate with migration and invasion rates for invasive and normal cells. Future experiments will use this system as a testbed for manipulating miR-141-3p expression to link microRNAs to cell signaling and cancer metastasis.

Marcus J. Kwon, Caleb L. Hamilton, Jonathan R. Brown, Emily A. Snyder, and Audrey A. Vasauskas

Pulmonary arterial hypertension (PAH) is a fatal vasoconstrictive disease characterized by proliferative inflammation, endothelial dysfunction, and metabolic abnormalities in the small pulmonary arteries. While treatments are improving, prognosis remains grim. Immunophilin FKBP51 regulates glucocorticoid receptor (GR), binds to Hsp90, and stabilizes microtubules (MT) in normal pulmonary artery endothelial cells (nPAECs). Prior work indicates increased metabolism of PAH pulmonary artery endothelial cells (pPAECs) and MT abnormalities.

pPAECs and nPAECs were gifted from the University of South Alabama. Cells were treated for 24 h with dexamethasone (DEX) and ICC performed for α-tubulin and FKBP51 (novel mAb). siRNA was used to knockdown FKBP51 (confirmed by RT-PCR), and ICC for GR and α-tubulin was performed. Colocalization was quantified by ImageJ.

ICC revealed colocalization of FKBP51 with MTs in both pPAECs (64%) and nPAECs (65%). This is the first report showing clear MT staining pattern of FKBP51. pPAECs exhibited dense α-tubulin and FKBP51 staining compared to nPAECs. Upon 24 h DEX, FKBP51 is observed with diffuse cytoplasmic and MT-like staining pattern in pPAECs, whereas nPAECs showed a perinuclear staining pattern with reduced cytosolic organization. siRNA to FKBP51 in pPAECs disrupted GR localization in pPAECs compared to nPAECs.

pPAECs exhibit aberrant metabolism compared to nPAECs and are mechanistically poorly understood. Our data suggests MT-stabilizing FKBP51 may contribute to metabolic changes through regulation of MT dynamics affecting GR trafficking, potentially leading to novel treatment approaches as targeting MT has proven efficacious in treating other proliferative inflammatory diseases.