Poster Day 2022: Abstracts: Basic Science

Jonathan Brown, Ashlyn Conner, Emily Snyder, and Audrey Vasauskas 

Pulmonary arterial hypertension (PAH) is a fatal vasoconstrictive disease characterized by endothelial dysfunction. While treatments continue to improve, prognosis remains grim.   

We sought to elucidate pathways leading to End-MT and/or endothelial dysfunction in PAH.  We asked what role the stress-response protein FOXO3 might play in the progression of End-MT. Conditioned media from pulmonary artery endothelial cells (PAECs)of the Sugen5416/hypoxia PAH rat model (SPAD) was collected after a 24-hour period. ECs from wild-type (FR6) rats and SPAD were seeded and treated with conditioned media at various time points.   We also verified markers of End-MT in cloned FR6 and SPAD, originally provided by the University of South Alabama.

EndMT was confirmed by the increased expression of smooth muscle actin and transgelin in the SPAD PAH PAECs and the decreased expression of endothelial markers CD31 and vWF as compared to normal PAECs.  Upon treatment with conditioned media from the SPAD PAH PAECs, FOXO3 is elevated in normal PAECs compared to null treated cells at both all time points.  Twenty-four hour treatment with conditioned media revealed no change in the protein levels by western blot nor gene expression by RT-PCR.

Our data indicate PAH (SPAD) PAECs have increased baseline FOXO3.  Normal PAECs exhibit transient increased FOXO3 protein levels when treated with PAH (SPAD) conditioned media. While FOXO3 is primarily activated via phosphorylation as part of the stress response, our data may implicate the accumulation of FOXO3 in PAH (SPAD) PAECs as a protective mechanism in our disease model.

Lucy Page Kelly and Eric Johnson, Ph.D. 

Precancerous diseases, such as IgM monoclonal gammopathy of undetermined significance (IgM-MGUS), have been shown to be caused by mutations in the cytosolic adaptor protein (MYD88), a central player in the interleukin-1 receptor (IL-1R)- and Toll-like receptor (TLR)-induced activation of the nuclear factor-kappa B (NF-kB) pathway. IgM-MGUS has the potential to develop into multiple myeloma (MM), a rare blood cancer. MYD88 is an activator of a proinflammatory pathway, as well as an inducer of cell proliferation.  

In silico analysis was used to compare a MYD88 variant of unknown significance (VUS) to the native protein to determine the potential deleterious effects of the variant. Molecular dynamic simulations (MDS) were performed using the YASARA program with .pdb files encoding the crystal structure of the MYD88 protein with the VUS (L252P).

There was no statistical difference (p < 0.05, t-test) comparing the root mean square deviation (RMSD) data derived from the MDS of the native and VUS proteins.

Although the results of this study lack statistical significance, another predictive program, PredisctSNP, determined that the L252P mutation may have detrimental effects on MYD88. Further research is needed to determine the importance of this particular VUS in the pathway leading to MM.

R.Q. Telese, BS, M.O. Ibiwoye, MD, MS, DTM&H, MPH, PhD, A.A. Vasauskas, PhD, M.J. Hernandez, PhD, FNAOME, J. Lyons, MD and J.D. Foster, PhD, RRT 

Background:

The vascular endothelial cell is a vital component of the blood-brain barrier (BBB) which maintains brain homeostasis between the circulation and brain parenchyma. A rat-specific BBB marker protein, the endothelial barrier antigen (EBA), has been previously identified and an inverse correlation between BBB dysfunction and EBA immunoreactivity is known.   

The purpose of this study was to examine and quantitate the spatial disposition of EBA-immunoreactivity in the rat CNS. 

We applied standard double immunofluorescent protocols with Olympus BX63 cellSense software suite to quantify the distribution of BBB competent microvessels in the forebrain and cerebellum of 40-day-old male rats. EBA-positive microvessels were segmented by global thresholding and quantitated by surface area measurement. Grand mean surface areas from the forebrain and cerebellum sections were analyzed using Welch’s two-sample t-test.

EBA-positive microvessels were detected in all sections examined from the cerebellum and cerebral neocortex. We detected higher density of EBA-positive microvessels in the cerebral gray matter than white matter. Hippocampal microvessels were less arborized than the cerebral neocortex. In the cerebellum, the granule cell layer showed more EBA-positive microvessels than the molecular layer. The cerebellar white matter is less vascularized by EBA-positive microvessels than the molecular and granule cell layers. We found a higher EBA-immunopositive microvessel density in the forebrain than in the cerebellum (P < 0.001).

Significant differences exist in the postnatal expression of EBA between the developing forebrain and cerebellum of healthy rats. Investigations are underway to determine whether these regional differences in EBA expression correlate with pathologies that impact the BBB microvascular structures.

Whitney Vaughan OMS-II, Patrick G. Dickinson OMS-II, and Gordon G. MacGregor PhD, MBA 

The proposed therapeutic effects of infrared sauna use include increased wound healing, benefits in cardiovascular health, and treatment of depression. Recently, there has been much interest in the modulation of the endocrine system by infrared wavelengths. Far infrared radiation is thought to modulate mitochondria, increasing the synthesis of ATP and the production of melatonin. The physiological effects of melatonin are many and include detoxification of free radicals, increased bone formation, a role in psychiatric disorders, and oncostatic effects.  

We offered research subjects therapy in a TheraSauna infrared sauna for 45 minutes. Blood pressure, a sweat sample and body weight were measured. A sample of saliva was collected before and after exposure to 45 minutes of infrared sauna therapy. Melatonin levels were measured in the saliva using a specific ELISA assay.

We measured endocrine metabolites in saliva samples of subjects taken before use and after 45 minutes in the infrared sauna. The infrared sauna therapy increased saliva melatonin levels by 64%, from 8.8 ± 4.3 pg/ml to 14.4 ± 4.4 pg/ml (p = 0.02, n = 5) in 45 min.

We hypothesize that the infrared sauna exposure increased melatonin secretion into the blood, either by the classic pineal gland secretion, or from local extrapineal sources of melatonin which include the epidermis. We propose that this sauna-induced melatonin may have therapeutic potential in neurodegenerative conditions such as Alzheimer’s disease or Parkinson’s disease, as well as in treatment of depression or post-COVID conditions.

Whitney Vaughan OMS-II, Patrick G. Dickinson OMS-II, and Gordon G. MacGregor PhD, MBA

Galen (129-216 AD) proposed that sweating could decrease body edema from any cause. Sweat is known to be high in sodium, potassium, and urea. These electrolytes and solute levels increase in cardiac and renal failure. The easy availability of the infrared sauna as a tool to induce sweating implores the re-evaluation of this as a therapeutic strategy for fluid and electrolyte loss in conditions of volume overload.  

 Research subjects were exposed to a TheraSauna Infrared sauna for 45 minutes. Blood pressure, a sweat sample, a saliva sample, and body weight were measured. Sweat was collected by filter paper from the thigh and center of upper back. Electrolytes in sweat were measured using ion-selective electrodes. Urea was measured using a colimetric assay.

Subjects lost an average of 576 ± 169 ml of sweat during the sauna use. There was a large amount of sodium loss, 676 ± 59 mg, which corresponded to about 1.7 g of salt lost. Potassium lost in the sweat was 213 ± 109 mg. Urea loss in sweat is significant and was measured as 398 ± 169 mg. The concentration of urea in the sweat was 64 ± 28 mg/dl, which is about 3-8 fold more than plasma levels.

The infrared sauna is an effective tool for sweat, salt, and urea loss and for removing fluid from the interstitial and extracellular fluid compartments. In conditions of fluid overload and edema, such as heart and renal failure, this may provide an effective supportive therapy.

Griffin Morehead, Eric Johnson 

The goal of this project was to investigate a single nucleotide polymorphism (SNP), Cys297Phe, in the MUTYH gene on the p arm of chromosome 1. This gene codes for MUTYH glycosylase, which repairs oxidative damage to DNA, particularly in the stomach, colon, and areas of rapid proliferation. Mutations can cause MUTYH-associated polyposis, an autosomal recessive condition correlated with an increased risk of colorectal or gastric cancers. This variant of unknown significance (VUS) disrupts the binding site of an iron-sulfur cluster, impacting the protein’s ability to bind DNA.  

I-TASSER by Zhang Lab was used to generate a model of the protein. YASARA by YASARA Biosciences, a modeling and dynamics simulation program, was used to assess changes in the alpha carbon Root Mean Squared Deviation (RMSD).

The results of the RMSD comparison, as well as a combined Dynamic Cross Correlation Matrix (DCCM), indicated a significant change in the protein structure over time. The DCCM results also showed a correlation between the variant in the iron-sulfur cluster binding site and the active site of the enzyme. 

The RMSD deviation suggested the VUS significantly altered the protein’s energetics and dynamics in intracellular environments. DCCM data analysis suggested variation in the iron-sulfur cluster binding site at position 297 is negatively correlated with active site binding at position 131. YASARA simulation showed the SNP at position 297 caused significant changes from the reference. FASTA analysis using PredictSNP estimated Cys297Phe is deleterious and pathogenic with 87% confidence.

Sarah Taylor, Eric Johnson 

Glucokinase is a major diver of metabolism in the liver and it is regulated by glucokinase regulator protein (GKRP). Several single amino acid substitutions in the protein sequence of GKRP have been linked to metabolic disorders such as those seen in patients with diabetes. In this study the variant of unknown significance (VUS) Q91K of GKRP is evaluated for its pathogenic potential.  

The glucokinase regulator protein was investigated through many bioinformatics modalities. The first of which was YASARA, a protein modeling program. It was used for visualizing the molecular structure and evaluating the dynamics of the native and variant glucokinase regulator proteins. After data collection from the simulations using YASARA, the root mean square deviation (RMSD) calculation was obtained and plotted on a graph to compare native and variant proteins. A two sample T-test assuming unequal variances was done as well. The VUS was further analyzed using the variant prediction programs PredictSNP and EVE. 

Using YASARA to simulate fluctuations in conformation of the variant and native proteins, and utilizing the corresponding t-test, it was observed that there was no statistical difference between the two structures. PredictSNP calculates the pathogenicity of the VUS as neutral with an 83% confidence. Using the program EVE, Evolutionary model of Variant Effect, Q91K had an EVE score of 0.208 which is considered benign. 

The single amino acid substitution Q91K of GKRP is likely benign and should be tolerated for patients. As such it is not expected to produce disease in patients. 

Philip Breton, Eric Johnson

Galactose-1-phosphate uridyltransferase (GALT) breaks down galactose to glucose for metabolism and its dysfunction is implicated in classical galactosemia. There are many different variants of GALT with different clinical presentations of classical galactosemia depending on the residual activity of each GALT enzyme. The purpose of this experiment is to compare the effectiveness of each variant by comparing the relative Vmax’s of each variant. 

In this experiment 12 different variants were assayed to compare their Vmax’s with their respective wild type. The variants were run in multiple batches and the Vmax’s were normalized with the Wild type to compare across all batches.

All of the variants showed decreased Vmax’s compared to their respective wild types except for 3 which were not statistically significant. 3 of the variants showed comparable Vmax’s to their wild types, 2 were greater than 50%, the remaining 7 showed <25% Vmax compared to their respective Vmax’s. 

The seven with the lowest relative Vmax and the greatest dysfunction were all in between residues 45 to 55. According to ConSurf this is a more conserved section of the protein. Patients with variants in this region would most likely show the more severe phenotypes of classical galactosemia.  

Markia Pettway, Eric Johnson PhD

The goal of this project is to evaluate the potential pathogenicity of a human variant of unknown significance (VUS), A134T, in the Kirsten RAS, K-RAS, protein. K-RAS proteins function by binding GTP to activate an intracellular signaling cascade involving protein kinases promoting cellular division. K-RAS proteins are expressed all throughout the body, especially in colon, lung, and pancreatic tissues, where its mutated forms contribute to highly pathogenic forms of cancer. The A134T VUS is of particular interest because of its hypothesized potential to interrupt the standard and highly conserved protein structure of K-RAS by replacing Alanine, a non-polar amino acid, with Threonine, a polar amino acid, in the amino acid sequence. 

Initially I-TASSER by Zhang Lab was used to produce protein structure models of the K-RAS protein with and without the variant. Then YASARA was used to evaluate changes in atomic movement of the protein segment over time in the native and VUS protein. 

Preliminary results did not indicate a notable difference in total protein organization in the intracellular environment based on a comparison of the VUS to the Wild Type in Root Mean Square Deviation (RMSD). The RMSD data was initially unexpected because Consurf, a software program calculating residue conservation, shows that the A134 residue is highly conserved and would likely affect protein structure if changed. 

However, additional Consurf information revealed that A134T is in fact tolerated supporting the RMSD data that the mutation in question would not provoke major deviations from original protein structure. 

Kelley Lynn Turner, Smit Bhimami, Erika DeHaviland, and Lawrence LeClaire 

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 signaling proteins responsible for cancer metastasis. Here, we examine the effect of micro-RNA expression on the MAP kinase, MAP4k4, which controls cytoskeletal assembly and migration in cells. These data lay the groundwork to develop early detection and treatments for breast cancer patients.
 

MDA-MB-231 metastatic breast cells and MCF-10a normal breast cell lines were cultured by standard procedures.  Assays were used to measure migration and invasion rates of cells. Real time PCR was used to quantify miR-141-3p in cells. MAP4k4 expression was analyzed by western blot analysis.

We found that MDA-MB-231 cells migrated more than 4-fold faster and 5-fold more invasive than controls. Using real-time PCR, we showed that MCF-10a expressed miR-141-3p more than 29.3% more than MDA-MB-231 cells. We compared the expression of MAP4k4 in each cell line. MD-MB-231 cells express MAP4k4 8-fold more than controls. Over expression of MAP4k4 migration rates in cells compared to controls.  

Together, these data suggest that miR-141-3p controls expression of MAP4k4 and correlates with migration and invasion rates for invasive and normal cells. Detection of circulating miR-141-3p may play a role in early detection methods and can be used to control cancer cell metastasis.