Life Sciences

Enhanced MEAGA (Minimum distance-based Enrichment Analysis for Genetic Association)

The goal of the MEAGA (Minimum distance-based Enrichment Analysis for Genetic Association) is to help researchers understand complex traits derived from the correlation between genes. More specifically, this project aims at creating a network of genes with edge weights being their correlation with each other. This will allow researchers to test hypotheses between genetic pathways/functions and the correlation between specific genes. There currently exists a genetic network consisting of genes, but their connections/edge weights are binary (weight of 1 or 0). The goal right now is to convert the existing binary network into a continuous network, so the edges can have continuous values indicating the correlation between genes. The project focuses on graph algorithms such as Prim’s, Dijkstra’s, and Kou’s algorithm to create a genetic network that can create Steiner trees of indicated genes. Thus far, a gene to gene correlation file has been created with different correlation cutoffs of 0.3, 0.5, 0.7, and 0.9. This research can be valuable in identifying associations between genes and certain traits or diseases. This research may help the medical field when identifying the causes of conditions.

Using Human Stem Cell Derived Cardiac Microtissues to Test the Efficacy of a Drug on Treating Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy, or HCM for short, is a hidden disease of the muscle cells of the heart that often goes diagnosed. While HCM can often present little to no symptoms, 5-10% of patients experience “overt dysfunction”, which is the leading cause of sudden cardiac death among young people. HCM is a dysfunction of the cardiac muscle cells (cardiomyocytes) that cause the sarcomere tissue of the heart to thicken as a bicep or quadricep would during exercise, leading to reduced cardiac function. The extracellular regulated kinase (ERK) pathway is a chain of proteins that is heavily involved in the proliferation of cells. We believe that this pathway, when defective, is responsible for HCM. Two steps in this pathway, MEK1 and MEK2, can be blocked by an experimental drug called Trametinib. First we have generated atrial and ventricular specific microtissues using control patient cells. Data shows that chamber specific cells have distinct function and force generating capacity. These tissues will be made for HCM patient cells to test the new therapy. The end goal is that we discover an effective treatment for cardiomyopathy.

Analyzing CRISPR-Cas9 Genomic Engineering through Targeted Deletions

Background: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a technology used to edit genomes at very high precision. It enables precise editing of genomic loci with a RNA-guided CRISPR associated protein 9 (Cas9) nuclease that can cleave the targeted DNA complementary to a guide RNA (gRNA). Precise gene editing via CRISPR-Cas9 has great potential in treating inherited diseases (e.g., cystic fibrosis) or correcting genetic defects.

Objective: To test the specificity and function of the CRISPR-Cas9 in a proof-of-principle experiment, we used two human Emx1 (hEmx1) gene-specific gRNA sequences (3.1+4.1) to guide the Cas9 enzyme for the deletion of the targeted region in human 293AD cells.

Analyzing CRISPR-Cas9 through Genomic Deletions

The Facial Grimace Test in the Detection of Hyperacusis in Mice: Sex Differences

Hyperacusis is an auditory disorder characterized by an increased sensitivity to sounds, which can result reduced auditory pain threshold. Sex differences in the induction of hyperacusis are not well understood. Reports of hyperacusis in humans are higher in men (Paulin et. al., 2016), under the assumption that males have a higher exposure to high-intensity noise resulting in hyperacusis. However, a noise-exposure-induced threshold shift is not necessary for hyperacusis. Due to variability in noise-exposure across life-span and reporting, it is difficult to accurately assess sex differences in the prevalence of hyperacusis in humans. In this study, the same level of noise-exposure was administered to male and female mice, followed by behavioral assays to determine the hyperacusis status of each animal. Preliminary data in the conditioned place-aversion test suggest that there are sex differences in the induction of hyperacusis in mice. To support these results, the facial grimace test was adapted to assess the auditory pain threshold in mice. The facial grimace can be characterized by previously established grimace scales. Data from the facial grimace test in both male and female mice was analyzed in order to determine whether a sex difference was present between mice with and without hyperacusis. This assay confirmed the presence of hyperacusis in male but not female mice. Future studies are needed to investigate the underlying genetic predisposition to the development of noise-induced hyperacusis. Paulin, J., Andersson, L., & Nordin, S. (2016). Characteristics of hyperacusis in the general population. Noise & health, 18(83), 178-184. https://doi.org/10.4103/1463-1741.189244

Evaluating Novel Cancer Drugs and Drug Delivery in Preclinical Studies

Cancer prevention and treatment in the United States has seen many advances over the last few years, however, breast cancer continues to be one of the most widespread diseases, affecting the lives of approximately one in every eight American women. There is a pressing need for a more effective and less invasive treatment for breast cancer, specifically those cancers that originate in the epithelial cells of the milk ducts or lobules, also known as adenocarcinomas. Heat shock protein 90 (HSP90) has become an important target of many drug treatments. At the molecular level, over-expressed signaling proteins that lead to uncontrollable cell growth are stabilized by HSP90. Recent in vitro studies of the specific HSP90 inhibitors, KU757 and KU758, suggest this drug is effective in treating certain types of cancer, such as thyroid cancer and triple negative breast cancer respectively. This study aims to investigate the efficacy of KU757 and KU758 in treating the breast adenocarcinoma cell line called MCF7. Cell proliferation will be measured by MTS assay, and drug efficacy will be analyzed by calculating inhibitory concentration 50 values.

Testing a Novel Drug Agent on Glioblastoma multiforme

Glioblastoma multiforme (GBM) is one of the most aggressive forms of cancer which begins in the brain. In many GBM cases, there are mutations in Epidermal Growth Factor Receptor (EGFR), like EGFRvIII that is the most common variant. Currently, there are no treatments for GBM besides chemotherapy and surgery because a drug would need to cross the Blood-Brain Barrier (BBB) to target GBM. We have developed a novel small molecule, DPI503, that can cross the BBB, binds with EGFR, and promotes its degradation by blocking EGFR dimerization. Preliminary in vitro data show that DPI503 kills U87 glioblastoma cells stably transduced to express EGFRvIII with IC90 of 3 micro M. Therefore, we expect DPI503 to be effective against EGFRvIII expressing tumors. To test this, we will prepare an orthotopic brain tumor model by implanting luciferase-expressing U87-EGFRvIII tumor cells into the brains of SCID mice. The growth of the tumor will be monitored using an IVIS Lumina imaging system after intraperitoneal injection of Luciferin. After confirmation of established tumors, mice will be treated with 4 dose levels of DPI503 (0, 10, 30, 100 mg/kg) via daily oral gavage. We have found that DPI503 treatment is effective against EGFR driven HNSCC and lung flank xenograft model. We expect DPI503 to be effective against the GBM model as well. If single-agent activity is not curative, we would combine radiotherapy with DPI503 treatment in follow-up experiments.

Developing a Novel Drug Agent for EGFR mutant Lung Cancer Patients

Mutant epidermal growth factor receptor(EGFR) spurs on lung cancer. Currently, tyrosine kinase activity inhibitors(TKI) have been used to combat this, however, unfortunately, roughly a year later patients develop resistance to this type of therapy and are left with no other intervention options which have proven to be deadly. So, our goal for the research we have conducted is to find a solution to this problem for patients in this position and give them a chance with a drug agent that is able to function regardless of kinase function. It has been shown by us, and others, that the degradation of EGFR has a very significant effect on whether or not cancer cells remain alive. So, the goal of our research was to test if a drug that causes the degradation of EGFR without anything to do with ATP will improve the odds for patients in these scenarios. In order to explore these ideas, we screened novel drug agent DGD1202 next to osimertinib in various lung cancer lines. We then took these results and were able to see their correlation with EGFR degradation. We currently are still working on this research, and we believe that once completed, a new, safe alternative intervention to lung cancer patients will be available that will greatly improve their odds.

Investigating the role of the Immune Microenvironment in Esophageal Adenocarcinoma

Esophageal adenocarcinoma (EAC) develops from chronic inflammation, termed Barrett’s Esophagus (BE), that then progresses through stages of increasing dysplasia before tumor development at the esophagogastric junction. The impact of the EAC immune microenvironment on patient survival at each stage has yet to be fully understood. This study provides quantitative descriptions of immune populations in EAC and surrounding tissue and its correlation with survival. Core tissue samples showing varying stages of dysplasia to cancer were stained for the T-cell proteins CD3 (general T-cell marker), CD4 (memory T-cells), CD8 (cytotoxic T-cells) or FOXP3 (T-regulatory cells). T-cells remove foreign and cancerous cells from the body. Immune checkpoint proteins normally regulate the immune system and suppress overactivity. In cancer, these proteins promote tumor survival. Immune checkpoint therapy, inhibiting checkpoint suppression, has shown promise in treating certain cancers. Tissue samples were stained for immune checkpoint markers, CTLA4, TIGIT and PD1. Linear regression and correlation matrices compared the overall makeup of the different T-cell and immune checkpoint microenvironment populations during each stage of disease progression and relationships between these populations. Cancer cells were also treated with two cytokines, IL6 and IL8. Previous studies indicate that IL-6 promotes tumor cell survival through chemo-resistance. Results from this study aim to understand if IL-8, which we have shown is upregulated in EAC, acts similarly. The goal of this project is to better understand immune cell interactions with EAC tumors. Our hypothesis is that high concentrations of T-cells in dysplastic or cancerous tissue could indicate a positive patient response to immunotherapy.

Developing a Liquid Biopsy Diagnostic Technique for Head and Neck Squamous Cell Carcinoma

Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer in the United States and has one of the highest recurrence rates. The cancer is frequently caught late, especially in recurring cases, as most diagnostic techniques are highly invasive. Research was conducted to develop a liquid biopsy technique which would use circulating tumor (ct)DNA, also known as cell-free DNA, to track cancer markers in the blood. This technique could be used as both a prognostic and diagnostic technique for patients with HNSCC or those who have a chance of recurrence. We have had success using cell-free DNA extraction procedures to separate the ctDNA from patient blood samples. Now we are looking towards developing a diagnostic technique using this data. This technique would be incredibly useful for monitoring HNSCC patients’ responses to treatment because it would be fast and non-invasive. Particularly, the research is currently focused on HPV positive patients because the HPV ctNDA marker can be easily identified in patient blood. The liquid biopsy technique could also be extended and used for the detection of other types of cancers. This could significantly improve cancer diagnosis times and would help provide frequent and non-invasive tracking of the responses of cancer patients to treatments.