Life Sciences – UROP Spring Symposium 2021

Research Discipline(s): Life Sciences

Patient specific heart muscle for testing new heart therapies in vitro

Hypertrophic Cardiomyopathy (HCM) is one of the most prevalent heart diseases in the world. In HCM, gene mutations lead to abnormal thickening of the heart muscles and makes it harder for the heart to pump blood. Previous research also indicated that abnormal structure and function of mitochondria is common in HCM. One of the biological pathways in the cell signaling network that contributes to HCM is the ERK1/2 pathway that consists of a series of proteins activation and signals for cell growth. Mutations that cause abnormally high activity of this pathway causes excessive cell growth. One of the proteins in this pathway is called MEK, and lately, there have been several highly-specific inhibitors studied for this protein. This research uses human stem cell-derived cardiomyocytes with MYH7 mutation-caused HCM to study the effect of MEK inhibition in relation to hypertrophic cardiomyopathy. The stem cell was cultured into cardiomyocytes monolayers and treated with 6 different doses of MEK inhibitor PD0325901 (0.1, 0.5, 1.0, 5.0, and 10 µM. Afterwards, we studied the cells shape and sizes by staining the cells with Wheat Germ Agglutinin that binds with cell membrane, and Phalloidin that binds with actin. Another focus in this research is also observing the mitochondria structure and health using mitotracker red dye to get a clear picture of the organelle. We also separated the cell proteins and performed Western blotting to observe the effect of the MEK inhibitor on the ERK1/2 pathway. We hope to find that MEK inhibitors are effective in restoring the mitochondria in cardiomyocytes and improving overall heart cells condition. The results of this study could potentially advance heart-related research and public health.

Cardiac Implications of Bipolar Depression Linked CACNA1C Gene Single Nucleotide Polymorphism

There is a connection that has been observed between cardiac functioning and Bipolar Disorder. This study focuses on understanding the functioning behind the CACNA1C gene mutation and it’s role in the cardiac function of patients with Bipolar Disorder. To produce accurate reactions of adult cardiomyocytes in response to these drugs we first propagated the human-induced pluripotent stem cell cardiomyocytes with Matrix Plus, a substrate that closely mimics the environment found in the human body. Next, we continue this propagation in 96 well plates and generating cardiac muscle tissue on which we tested certain drug therapies with voltage-sensitive dyes to measure their electrophysiological reaction. Next, we performed experiments on the effects certain medications have on the cardiac muscle we have propagated to better understand their effects on the cardiovascular system and the body. We hope to discover a way to create a personalized tool for bipolar patients in order to use our newly obtained knowledge of medications to prescribe them the safest drug possible. If we are able to properly understand the mechanisms behind the CACNA1C gene mutation and its role in cardiac function we could revolutionize the field of personalized medicine and medication screening by making the toxicity screening patient-specific, possibly saving or improving the lives of many bipolar patients whose lives are affected by their medications.

Exploring the CRISPR-Cas9 System through Human EMX1 Gene Deletions

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 Cas9 nuclease that can cleave target DNA that is complementary to a guide RNA (gRNA). Then, the CRISPR-Cas9 system can be used to achieve various goals such as treating inherited diseases (e.g., cystic fibrosis). The objective is to test the specificity and function of the CRISPR-Cas9 system in deleting a targeted sequence in mammalian cells. Mammalian 293AD cells were first transfected with the Cas9-expressing plasmid and human EMX1 ()-specific 3.1+4.1 gRNA sequences. 72 hours after transfection, the DNA was extracted and purified from the cells. The hEMX1 modified region was amplified using a set of primers in the polymerase chain reaction (PCR). The PCR products were run in a 2% agarose gel for gel electrophoresis where the sizes of the DNA fragments were compared to a marker. Results from gel electrophoresis showed that the Cas9 system was successfully able to target the hEMX1 gene when 3.1+4.1 gRNA was present. However, pNTAP posi-tag, a plasmid without the hEMX1 sequence, also showed unexpected cuts which lead to DNA fragments of various sizes. The results show that the CRISPR-Cas9 system was propitious in targeting the hEMX1 DNA sequence. The unexpected cuts made in pNTAP posi-tag will require further experimentation, with one possibility being to extract, purify, and sequence the unanticipated DNA fragments in order to explain the results.

Single Molecule Dynamics of RNAs in Stress Granules

Stress granules (SGs) are condensates of RNAs and proteins formed in the cytoplasm of the cell when exposed to acute stress. Though recent studies have concluded that SGs appear to play an important role in tumor progression, gene expression, and neurodegeneration, the exact function of SGs is not completely understood. Furthermore, the dynamics of the mRNAs near the surface of the SGs is an area of active research and may provide important clues about the granules’ function, for example, how they can govern the exchange of materials in the cell. To study the dynamics of the mRNAs near the granules’ boundaries, we developed an algorithm which delineates the boundaries in highly inclined and laminated optical sheet (HILO) microscopy images. The simple but fast and robust algorithm we developed to be used with ImageJ is capable of finding the SGs’ boundaries with diffraction limited accuracy and works even on highly inhomogeneous cell images and for granules of arbitrary shape, size, and internal structure, which is an advancement compared to existing methods. After identifying the mRNA molecules in the same field of view using single molecule microscopy, we will use our approach to compare the mRNAs’ locations and SGs’ boundaries over time to study the dynamics of mRNAs on the SG surface. We are now applying this algorithm to study the dynamics of the mRNAs in the cell in order to better understand the interaction of mRNAs with SGs.

Identifying neuronal components necessary for cool temperature sensing

Temperature sensing is necessary for homeostatic regulation, probing the environment for pleasant or aversive cues, and must be reliable across a broad range of temperatures. Under normal conditions, cool temperatures are not painful. However, patients suffering from chronic pain perceive normally innocuous cool temperatures as an unbearably painful sensation known as cold allodynia. How innocuous cool temperatures are signaled in the spinal cord has not been well studied. This study aims to uncover the basic neuronal components necessary for cool temperature transmission in the dorsal horn of the spinal cord. To accomplish this, an intersectional genetic strategy was implemented to ablate several different cell-type markers in the dorsal horn, then tested for temperature sensing impairments. Interestingly, Calbindin 1 (Calb1) neurons, which are mostly local interneurons, were necessary for the detection of cool but not cold stimuli. Calb1 is a heterogeneous population. Using In Situ Hybridization and immunohistochemistry, the Calb1 population was characterized based on markers for excitatory/inhibitory neurons, laminae layer, and somatosensory neurons for pain and itch. By understanding these different subpopulations, the role Calbindin 1 neurons play in transmitting cool and cold sensations can be further investigated. This study provides insight into the neural basis of cool temperature transmission in the spinal cord, which may lead to treatments for patients suffering from cold allodynia.

Characterization of TRPM8-expressing sensory neurons

The cation channel TRPM8 has been implicated in cold detection, yet no identification of distinct subpopulations of TRPM8-expressing sensory neurons responsible for detecting different levels of cold stimuli (noxious vs. innocuous) has been made. The current study takes advantage of Designer Receptors Exclusively Activated by Designer Drugs (DREADD), light-sensitive channelrhodopsin-2 ion channels (ChR2), thermal behavioral assays, immunostaining, and RNAscope technology to investigate the role of different subpopulations of TRPM8 sensory neurons in dorsal root ganglia of mice. First, we want to study the function of TRPM8 sensory neurons in vivo by expressing an inhibitory Gi DREADD selectively in TRPM8 sensory neurons in the dorsal root ganglia (DRG) of mice. Upon injection of the DREADD agonist clozapine-N-oxide (CNO), the DREADD-expressing TRPM8 sensory neurons could be selectively inhibited to infer neuronal function following cold-plate behavioral assays. Sensory neurons in the DRG can be broadly divided into three groups based on cell body diameter: small, medium and large. From our RNAscope results, “small” TRPM8 neurons expressed significantly higher levels of the trpm8 gene than “medium to large” TRPM8 neurons, potentially providing a basis for differentiation between the two subpopulations. Thermal-related behavioral experiments are still ongoing. We hypothesize that the two subpopulations of TRPM8 sensory neurons serve distinct functions in sensing cold stimuli of different intensities.

Role of STING pathway in photosensitivity and interferon-kappa responses in keratinocytes

Cutaneous Lupus Erythematosus (CLE) is an autoimmune disorder marked by scarring skin lesions, often triggered by exposure to ultraviolet (UV) light resulting in a significant loss of quality of life for CLE patients. Better understanding of the molecular mechanisms driving CLE is needed as currently, the lack of Food and Drug Administration (FDA)-approved therapies for CLE presents a fundamental challenge in the treatment of CLE patients. The outermost layer of the skin is known as the epidermis, which consists of mostly keratinocytes. Interferon kappa (IFN-?), a member of the type I IFN family, is constitutively expressed in keratinocytes. IFNK overexpression in lesional lupus skin predisposes CLE patients to inflammation and photosensitivity, thus it is an intriguing target for novel therapeutics. Previously, we had observed a significant delay in IFNK expression relative to IFN-ß, another member of the type I IFN family, following stimulation with poly-IC, an activator of the antiviral TLR3 signaling pathway, or UVB. In addition, upregulation of IFNK was dependent on STING, an endoplasmic reticulum adaptor protein. We thus sought to further understand the regulation of different IFNs in keratinocytes. Using CRISPR-Cas9, we generated knockout keratinocytes for IFNB expression. Here, we reveal that in the absence of IFN-ß, IFNK expression is significantly reduced in keratinocytes treated with poly-IC or UVB. Elimination of mediators downstream of the type I IFN receptor, such as STAT1, also abrogated IFNK but not IFNB expression. Further, IFNK, but not IFNB, expression remained dependent on STING signaling upon UVB-irradiation. Given this information, therefore, we suggest that early triggering of IFNB is required to drive a STING-mediated upregulation of IFNK. Currently, we are examining whether this regulation is aberrant in CLE vs. healthy control keratinocytes, which would provide a mechanism for dysfunctional overexpression of IFNK in CLE skin. If so, this pathway would serve as a target for reducing inflammation and photosensitivity in CLE patients.

Targeting Candida albicans Virulence

Fungal pathogens like Candida albicans can cause devastating human disease. Treatment of candidemia is complicated by the high rate of resistance to common antifungal therapies and the toxicity of many antifungal compounds due to the conservation between essential mammalian and fungal proteins. As the number of immunocompromised and hospitalized patients vulnerable to fungal infections increases, it is essential to discover new targets and approaches for targeting these deadly fungal pathogens. An attractive new approach for antimicrobial development is to target virulence factors; these are non-essential processes that are required for the organism to cause disease in human hosts. This approach expands the potential target space while reducing the selective pressure towards resistance, as these targets are not essential for viability. In C. albicans, the key virulence factor is a morphogenetic switch from yeast to filaments. We have developed a high-throughput image analysis pipeline that can readily distinguish between yeast and filamentous growth in C. albicans and identify cytotoxic molecules. Based on this clear phenotypic assay, we have screened compounds for their ability to inhibit this important virulence factor or cause fungistatic or fungicidal effects. Also, to avoid host cell toxicity, the compounds screened are compounds used as treatments for other medicinal purposes. We have begun to use these compounds to screen for resistant mutants of C. albicans, and in the future we will use these resistant mutants to leverage the tractable genetic systems of C. albicans to determine mechanism of action, thus allowing for targeted development of new antifungal therapies. Overall, this approach will build a platform for rapidly developing new molecules for antifungal therapeutics.

Design of cancer-treating proteins

One of the most elusive diseases known to man in terms of treatments, cures, and diversity is cancer. Despite how far the medical field has come, there are still no cures for any type of cancer and treatment options vary drastically in terms of success depending on the type and stage the disease is caught at. When looking to find better treatments or even possible cures for cancer, the most important factor to consider is how proteins produced by cancer cells differ from normally produced proteins and how these proteins interact with one another. In addition knowing things like a cancer protein’s binding sites and what could interfere with and inhibit the function of cancer proteins is critical when coming up with possible treatments and cures. By analyzing these protein-protein interactions and performing a de novo protein design, it is possible and feasible to find proteins that can target cancer-related proteins and slow or stop the spread of the disease in the body.

Design of Novel Protein to Inhibit PD-1/PD-L1 Protein-Protein Interaction for Cancer Immunotherapy

Cancer is the second leading cause of death in the United States and around the world. All cancer is a result of gene mutations, which can cause the formation of abnormally functioning proteins that change a cell’s behavior from normal to cancerous. Contrary to prior cancer treatments focusing on treatments not native to the human body, this research study aims to harness the natural immune response. This project involves the design of a novel protein sequence using a computational protein design program called UniDesign. This novel protein will be designed to inhibit the PD-1/PD-L1 protein-protein interaction, which is responsible for preventing T-cells from destroying other cells. In cancer patients, the new protein could be used to inhibit the PD-1/PD-L1 pathway, which would allow T-cells to destroy cancer cells. Thus, this treatment utilizes the native immune response as a source of cancer therapy. In order to determine the effectiveness of the novel protein at inhibition, the binding affinity of the new protein sequence will be compared to that of the original PD-1/PD-L1 PPI. As such, the protein developed in this project will have the potential to target the PD-1/PD-L1 PPI to ultimately treat cancer in patients.

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