While the anatomy of the four-chambered mammalian heart is well understood, relatively little is known about how the difference in valve leaflet structure, that comprise the tricuspid and bicuspid atrioventricular (AV) valves, impact function of the valves as fluid control elements of the heart. The AV valves act in response to the pressure difference between their respective atria and ventricles. Here we ask the question, “How do transvalvular pressures and shear stresses change the mechanical functioning of tricuspid and bicuspid valves under physiologic conditions?”. To address this, our research group utilizes computational modeling to evaluate the fluid-induced stresses on heart AV valves and determine their impact on function. We have developed a 2D computational representation of fluid-solid interaction of the heart valves during opening. Blood was modelled as a Newtonian fluid, allowing for use of the Navier-Stokes equations. This model implemented the immersed boundary method to solve the interfacing conditions. In all cases, azimuthal symmetry was assumed and the root of the valve leaflet was fixed while the rest of the leaflet deformed elastically. The exit orifice of the bicuspid valve has been noted in literature to be elliptical while the tricuspid is more circular. Given the assumed azimuthal symmetry and continuity, a changing exit condition based on the simplified geometry will contrast the AV valves and differentiate their functional relevance. This interdisciplinary research has relevance on multiple levels. It approaches a physiological question using integration of biological, physical, and engineering perspectives that contributes a more comprehensive understanding than single-discipline perspectives alone.

Group B Streptococcus (GBS) is a gram-positive bacterium which causes nearly 150,000 stillbirths and infant deaths per year globally. About one in five pregnant women carry GBS, a major cause of infant disease. GBS encodes an extracellular enzyme known as Hyaluronidase, which increases the severity of GBS infection and is associated with adverse outcomes such as preterm labor. In this project, we investigated how the GBS Hyaluronidase enzyme suppresses the host pro-inflammatory response in the presence or absence of two crucial extracellular immune response receptors (Toll-Like Receptor(TLR)-2 and TLR4) . While both TLR2 and TLR4 receptors bind to pathogens and pathogen associated molecular patterns (PAMPs), TLR2 is specific to recognizing gram-positive bacteria such as GBS and plays a critical role in initiating the gram-positive pro-inflammatory response. Dendritic cells and macrophages - key components of innate immune defense - were generated from mice that included the wild type (WT) and those lacking either TLR2 or TLR4 or both TLR2/TLR4 . These immune cells were then treated with GBS (GBS WT) or a mutant strain lacking Hyaluronidase enzyme (GBS DhylB). Post GBS infection, host cell supernatants were analyzed for the production of pro- and anti-inflammatory cytokines using multiplex cytokine arrays. Fluorescence microscopy and quantification of bacterial colony forming units were used to determine the extent of bacterial uptake and immune cell death. We expect that GBS Hyaluronidase may suppress pro-inflammatory cytokine production in a TLR2/4 dependent manner in dendritic cells, similar to previous observations with macrophages. Data from this project can elucidate details about the GBS immune response pathway, and may inform the development of therapeutic strategies to prevent Group B Streptococcal infections, stillbirths, preterm births and early onset neonatal sepsis.

Human papillomaviruses (HPV) contribute to approximately 4.5% of cancer cases worldwide. There are currently three vaccines which protect by producing neutralizing antibodies to the structural capsid protein, L1, for each of the most oncogenic HPV types. Despite the high protective efficacy of these vaccines, not enough is known about the antibodies elicited. The aim of this project is to clone and characterize HPV-specific human monoclonal antibodies after vaccination, particularly antibodies reactive to types other than 16 and 18. Plasma cells and memory B cells were isolated from vaccinees at different time points following HPV vaccination. Antibody-encoding transcripts were amplified from pre-screened plasma cells and cloned into expression vectors - one for each antibody light and heavy chain. The expression vectors were co-transfected and the proteins were harvested and purified for one of the antibodies of interest. Antibodies reactive to HPV types 6, 11, 16, 18, 31,33 and 58 were identified. The first antibodies in the process of cloning were reactive to HPV type 11.The findings of this study will allow us to use these monoclonal antibodies as reference standards to determine the quantity of antibodies in serological assays, thus helping us identify the binding affinity and neutralizing capacity of vaccinees’ antibodies. This will aid future studies aimed at answering the question of whether the vaccine doses can be reduced to one, instead of two or three doses.

 Cancerous cells have a modified metabolism that supports their demands for increased proliferation. One of the essential molecules in cancer cell metabolism and proliferation is the amino acid aspartate. Aspartate is not only incorporated into proteins, but is also a substrate for nucleotides and other amino acids, including asparagine. Aspartate availability can constrain tumor growth rate, and the consumption of aspartate to generate downstream products can alter aspartate levels. One gene that draws from the aspartate pool is asparagine synthetase (ASNS). ASNS converts aspartate into asparagine, which is used in the production of proteins, but does not increase cell proliferation. Thus we hypothesize that ASNS expression and activity can affect aspartate levels. With this, we aimed to determine if ASNS expression could alter aspartate availability and change sensitivity to aspartate suppressing therapies. Since cancer cells express ASNS to varying degrees, this project sought to determine if ASNS expression could be used to identify those cancers that are most amenable to aspartate suppression therapies. This research sought to better understand the conditions that determine aspartate levels, and how to exploit those conditions to inhibit tumor growth in association with asparagine synthetase.

Calcific Aortic Valve Disease (CAVD) is progressive calcium deposition and collagen buildup on the aortic valve, causing stiffness which impairs normal function ultimately leading to death. CAVD is common in older adults, present in 20-30% of individuals aged over 65, and 48% of patients over 85 years. Our long-term goal is to develop a therapeutic target which can be treated non-invasively as an alternative to surgical valve replacement, the only treatment currently available. This project is part of a larger investigation into the role of Runx2 on aortic valve function in CAVD progression using a unique mouse model developed by the lab. The mouse line has been designed to be genetically predisposed to developing CAVD. The sub-project is comparing variation in aortic valve calcium and collagen deposits, between the diseased model and mice having a Runx2 deletion. Echocardiography imaging comparing the two mice groups showed an improvement in aortic valve function in mice with Runx2 deletion, indicated by improved flow, velocity and pressure. To assess the changes in valve morphology which could contribute to the improvement, we will be using histological staining techniques on sections of the diseased aortic valves. The stain Picrosirius Red will be used to visualize collagen accumulation, and OsteoSense stain for calcium deposits. Sections are imaged using brightfield and fluorescence microscopy respectively. Levels of collagen and calcium will be quantified from the images using image processing programs NIS Elements and ImageJ. Staining analysis has shown a reduction in calcification and collagen deposits in mice with the deletion compared to the control. In the next stages, we plan to quantify the levels of calcification, collagen, and osteocalcin, another bone-like cell marker of cell morphological change, which might contribute to valve impairment. We will also investigate longer term effects of Runx2 deletion.

Surgical palliation/repair of congenital heart defects is associated with significant morbidity and mortality. During the majority of cardiac surgeries, the patient is placed on cardiopulmonary bypass (CPB) in order to minimize ischemic damage while the heart is being operated on. Exposure to CPB results in systemic inflammation and multi-organ dysfunction that is especially severe in neonatal and pediatric patients. Particularly, brain damage resulting in developmental and cognitive disorders have emerged as major concerns in this group of patients due to its negative impact on the patient’s quality of life. Therapeutic intervention is currently limited as the mechanism how the systemic inflammatory response links to brain damage remains unclear. It is hypothesized that CPB-activated immune cells could infiltrate into tissues, releasing intracellular contents that cause tissue injuries. In order to understand how the brain – specifically the neuronal and glial cells – responds to secretory proteins from CPB-activated monocytes, we exposed differentiated neuronal cells (dSY5Y) and glial cells (U118) to conditioned media from either control static or CPB-mimicking sheared monocytes (THP-1). Quantitative polymerase chain reaction was performed to quantify potential changes of various target genes. The data suggested that chemokine C-C-motif ligand 2 (CCL2), chemokine CXC motif ligand 11 (CXCL11), chitinase-3-like protein 1 (CHI3L1), S100 calcium binding protein B (S100B) and interleukin-1-beta (IL1b) were significantly upregulated in both dSY5Y and U118 cells cultured in conditioned media from sheared monocytes compared to the static control. This is the first step to understand the mechanism of interaction between the brain and the activated immune system after CPB so that novel targets could later be identified for therapeutic development. 

More than 442 million people worldwide have been diagnosed with diabetes, many of which regulate their glucose levels using the pump/catheter system. However, just 2-3 days after the catheter is inserted into the body, the tissue clogs due to the foreign body reaction (FBR), an immune reaction elicited by the body in response to any foreign material injected in the body. At this point, the patient must remove the catheter and insert a new device into fresh skin elsewhere, resulting in excess scar tissue. Our project focuses on preventing the FBR by reducing its triggering event--protein attachment--so that insulin catheters can last longer (2-3 weeks) and can reduce fibrotic accumulation in patients. To combat the frequency of delivery site changes, we have designed a nonfouling zwitterionic polymeric brush coating for the surface of the catheter to reduce protein attachment. For the coating, zwitterionic sulfobetaine methacrylate (SBMA) was surface-polymerized onto the catheter using atom transfer radical polymerization (ATRP). SBMA has been shown to resist protein adsorption down to less than 1ng/cm². The ATRP initiator was plasma-deposited to robustly adhere to the unique geometry of the catheter. In this work, we used a full factorial design of experiment (DOE) to determine significant experimental factors to the polymerization protocol to maximize the amount of SBMA on the surface. The coating was characterized using x-ray photoelectron spectroscopy (XPS) to confirm the presence of SBMA and the radiolabeled protein adsorption assay to measure the amount of protein adsorbed to the coating. We plan to use the results of the DOE screening to further optimize the nonfouling coating and ultimately plan to test this coating on insulin-delivering catheters in a diabetic mouse model to observe sustained lowered blood sugar levels and histologically review the extent of the FBR through collagen accrual.

Cancers are broadly characterized by changes in cell metabolism. Tumor cells typically exhibit functional respiration and inhibition of electron transport chain can impair cancer cell proliferation. However, certain neuroendocrine cancers can arise from loss of function (LOF) mutations in succinate dehydrogenase (SDHx/complex II), which plays a key role in the TCA cycle and in mitochondrial respiration. SDH, which catalyzes the conversion of succinate to fumarate, comprises four subunits: A, B, C and D. LOF mutations in subunits B, C, and D can promote tumorigenesis and mutations in subunit B (SDHB) are particularly associated with malignant and metastatic neoplasms. Interestingly, SDHB impaired cells show an accompanied loss of activity in complex I, implying that unlike the majority of cancer cells, respiration is not essential and may even be antagonistic for SDHB mutant cancer cell proliferation. Indeed, preliminary experiments indicate that inhibition of complex I can restore proliferation to cells treated with an SDH/complex II inhibitor. However, the molecular mechanisms behind this phenomenon are not well understood. We aim to investigate the metabolic mechanisms by which dysfunctional respiration is essential for the proliferation of SDH impaired cells. We hypothesize that inhibition of respiration in these cells can prevent oxidation of NADH to NAD+ at complex I and alter the redox homeostasis in the mitochondria to support proliferation. Specifically, we will test to see if increasing the NADH/NAD+ ratio is the required function of complex I inhibition that rescues cell proliferation in SDH impaired cells. In addition, we will characterize the metabolic consequences of specific alterations SDH, complex I, and mitochondrial redox state. Results from this study should allow us to delineate the importance of metabolic alterations in SDH mutant cancer cells and potentially help identify metabolic vulnerabilities for treatment of SDH impaired cancers.