Engineered heart tissues (EHTs) have emerged as a promising tool for cardiac disease modeling and drug screening, allowing for better study of cardiovascular diseases (CVDs). However, most current EHTs are composed of only a mixture of an extracellular matrix and heart muscle cells, called cardiomyocytes (CMs), without a vascular element. This prevents the study of the impacts of flow and the endothelium on cardiac function, and the role that endothelial cell (EC) dysfunction may play in cardiovascular disease. Endothelial function is closely related to cardiac homeostasis, as risk factors for CVD (smoking, obesity, diabetes, etc.) lead to an increase in pro-inflammatory cytokines, which can trigger EC dysfunction. Thus, this interaction is important to study further. The Zheng lab has developed a perfusable collagen-based EHT model, which incorporates a vascular element. The constructs form a lumen through utilization of needles and collagen, support CMs within the bulk collagen matrix, and the inner lumen of the tube can be endothelialized, serving as an effective in vitro model of cardiac vasculature. This project aims to identify healthy and unhealthy EC flow conditions within the EHTs, hypothesizing that physiologically relevant shear stress will lead to EC alignment and strong barrier properties. . We optimized the fabrication and culture process of the EHTs by fabricating a secondary dish for the EHT constructs while they are under perfusion, in order to avoid contamination risks. We then employed this model to look at EC retention and health at different flow rates, and examined the effects of altered shear stress on EC dysfunction. ECs perfused under physiological shear stress have shown markers of healthy barrier function and alignment. This project establishes a perfusable EHT model that allows us to interrogate EC function under perfusion and, in the future, assess the effect of endothelial dysfunction on cardiac dysfunction.

Dilated cardiomyopathy (DCM) is a leading cause of heart failure around the world. Inherited mutations cause the left ventricle of the heart to enlarge, thinning the heart muscle wall and decreasing the overall function of the heart. In my research project, I will determine if disrupting fibroblast function by knocking out a key developmental signaling factor, p38, can improve, or even reverse, DCM disease characteristics. Specific Aim 1 will be to determine the effects of p38 knockout-induced fibroblast dysfunction on cardiomyocyte function and structural remodeling in late-stage DCM. The rationale is that myocytes in DCM have poor contraction and structurally remodel to longer, thinner morphologies, which occurs in our DCM mouse model around 4 months of age. I expect to see less of these characteristics with the p38 knockout. Specific Aim 2 will assess cardiac fibroblast proliferation and fibrosis in response to disabling cardiac fibroblast function late into the DCM disease process. The rationale is that studying and observing the dynamics of the fibroblast population is critical when understanding the effects of fibroblasts and the p38 knockout model on DCM. In previous studies, the Davis lab identified that cardiac fibroblasts maladaptively respond to inherited DCM mutations in cardiac myocytes, worsening the whole heart. I expect to see less fibroblast proliferation in the p38 model. P38 is essential for fibroblast signaling pathways and functionality, so by knocking it out I will be able to test if fibroblasts are a viable therapeutic target for patients with DCM.

VLA-4 is a surface protein of immune cells that plays an important role in their extravasation into tissues during an immune response. In multiple sclerosis (MS), pathogenic T cells enter the brain and attack nerve cells by using VLA-4 to bind VCAM-1, a cell adhesion molecule on endothelial cells that line blood vessels. Current MS treatments rely on antibodies that bind VLA-4 and block interaction with VCAM-1, preventing a pathogenic immune response. However, antibodies are expensive to manufacture, and their binding cannot be easily regulated to control drug-induced side effects. Aptamers are single-stranded DNA or RNA molecules that fold into sequence-defined structures capable of binding targets with affinities and specificities comparable to antibodies. Being chemically synthesized, they are much cheaper to manufacture and offer no batch-to-batch differences. Unlike antibodies, their binding in vivo is rapidly reversible, which could alleviate some side effects of disease treatments. However, aptamers have limitations in vivo – degradation by nucleases in serum, and rapid clearance into urine. This project designs and assesses modifications to a novel VLA-4 binding aptamer to improve in vivo function, with the goal of developing an alternative for MS treatment. We designed various modifications to the aptamer backbone to prevent nuclease degradation and conjugated the aptamer to a polymer to increase size and reduce clearance. To assess aptamer functionality, an in vitro model of T cell adhesion is used. VCAM-1 coated plates are used to simulate endothelial cells, and VLA-4+ T cells are incubated in the plates to allow adhesion in the presence of modified versions of the aptamer and serum. VLA-4 inhibition by our aptamer designs is assessed by characterizing the extent of cell adhesion inhibition. Successfully designing a modification that significantly improves the in vivo function of aptamers will have broad implications for their clinical translation to in vivo use.

Stimulator of Interferon Genes (STING) signaling contributes to tumor immunity. However, treatments targeting the STING pathway are limited by route of administration, insufficient STING activation, and off-target toxicity. We introduce poly-STING, a copolymerized, mannosylated variant of the diABZI STING agonist-3 known to activate the cGAS-STING signaling pathway, promoting the release of type-1 interferons and pro-inflammatory cytokines leading to tumor immunogenicity. The STING agonist-3 is a non-nucleotide molecule that activates the STING pathway, but it has poor solubility, which limits its usage in-vivo. The developed poly-STING platform improves the drug's solubility, is designed to target immune cells, and provides enzyme-triggered drug release upon delivery, which has been shown to induce improved therapeutic efficacy compared to the free drug. The Pun and Stayton labs seek to investigate modalities for optimization of the cGAS-STING pathway activation and characterize the mechanism of action. Specifically, my project will evaluate STING activation by observing macrophage repolarization from type M2, as the mannose from the poly-STING binds to the CD206 receptors on M2 macrophages. This activates the STING pathway, repolarizing the macrophage to pro-inflammatory type M1. To test effects in vitro, I will culture bone marrow-derived M2 macrophages with various formulations of poly-STING, and repolarization will be measured through flow cytometry and RT-qPCR to quantify expression of macrophage markers. We expect to find higher M1 activity in macrophages treated with poly-STING as opposed to the free drug. Next, I evaluate the therapeutic efficacy of the STING formulations through an in-vivo tumor reduction study using murine models of breast cancer and melanoma, expecting to find longer survival of mice treated with poly-STING. The culmination of this project will result in a polymer-based STING agonist delivery platform that solves the solubility and bioavailability issues associated with the STING-3 agonist, with enhanced efficacy and decreased toxicity after systemic administration.

Despite recent advances in monoclonal antibody (mAb) technology and its rapidly growing market share, therapeutic targets for mAbs are currently limited to membrane proteins which consist of up to 30% of total proteins encoded by the human genome. The other 70% of cytosolic protein targets remain inaccessible inside the cell. Thus, research into intracellular protein delivery is critical to unleash the full potential of protein therapeutics. For example, mAbs can target oncogenes, enzymes, and the complex signaling cascades within the cell, unlocking a completely new domain of protein targets. Current methods for intracellular protein delivery involve either low protein throughput with minimal cell damage/cytotoxicity or high throughput approaches that compromise cell viability. The Gao lab recently developed a highly efficient technology that allows small proteins to be directly delivered into the cytoplasm with minimal damage to the cell, by cholesterol tag. To further this research, we developed a new version of the tag via the covalent linkage of Coomassie Blue dye with 2-hexyldecanoic acid, branched alkyl chains. This new tag could deliver mAbs, specifically immunoglobulin G (IgG), labeled with fluorescent dye. Through this project, I (i) carried out organic synthesis of the new tag, (ii) delivered secondary antibody into HeLa cells, (iii) confirmed protein internalization through fluorescent microscopy, and (iv) delivered anti-Vimentin primary antibodies for live cell imaging of intermediate filament. Ultimately these four aims demonstrate successful intracellular mAb delivery while maintaining its native protein structure. This allows us to utilize this technology to deliver protein therapeutics targeting all kinds of cytosolic proteins including oncogenic proteins such as p53, RAS, and MYC.

T cells rely on cytokines for cell communication and regulating effector function to respond to infections and cancer. Cytokines are sensed by T cells when bound to surface receptors, activating common second messengers that modulate cell differentiation and distinct cell activity. Second messengers allow T cells to mount controlled and effective immune responses; extracellular signal-regulated kinase (ERK) is phosphorylated (pERK) primarily downstream of antigen-T cell receptor engagement to drive proliferation, differentiation, and survival; signal transducer and activator of transcription (STAT) proteins consist of polyfunctional transcription factors phosphorylated (pSTAT) downstream of cytokine signaling, inducing gene expression to regulate cell activity. Different cytokines give rise to distinct responses by activating the same set of second messengers, raising an unresolved question: how can T cells distinguish between different cytokines and mount distinct functional responses? My project aims to investigate the hypothesis that T cells encode information about cytokine identity and magnitude by activating and translating unique combinations of second messengers into distinct gene expression programs. I will stimulate splenocytes with interleukin (IL)-2, IL-7, IL-15, and IL-21 and measure pSTAT and pERK levels using flow cytometry. To analyze differential gene expression, I will analyze an unpublished single-cell RNA sequencing dataset to determine the effects of different cytokines on CD8+ T cell gene expression programs using single-cell sequencing analysis packages. Previously, I found T cells use combinatorial second messenger activity through differential activation of STAT3 and STAT5 in response to IL-2 and IL-21, and expect cells to upregulate effector and memory genes respectively. I expect IL-7 and IL-15 to differentially activate STAT5, respectively upregulating cell survival and memory response genes while both promoting memory cell proliferation. By elucidating relationships between cytokines, second messengers, and gene expression, we can further our understanding of T cell differentiation mechanisms to inform potential therapeutic targets to combat disease.

Solid tumors like liver cancer will grow by promoting angiogenesis, the development of new blood vessels. These vessels are disordered compared to normal vasculature, leading to spatial-temporal differences in blood perfusion to the tumor. Cancer therapies alter tumor vasculature and thus close monitoring of changes in tumor blood flow could predict patient response. Currently, liver tumors are evaluated based on size with computed tomography (CT) or magnetic resonance imaging (MRI). However, contrast-enhanced ultrasound (CEUS) is perhaps better suited to track these blood flow changes than CT or MRI because it uses a vascular agent. With CEUS, blood flow-related parameters such as rise time (RT), mean transit time (MTT), peak intensity (PI) and area under the curve (AUC) – relating to blood velocity, volume, and distribution – can be extracted from CEUS video loops. Quantifying blood flow parameters allows for more sensitive and accurate evaluation of tumor response, but parameter reproducibility needs to be evaluated so that true changes in blood flow can be differentiated from measurement variation. The goal is to establish a standardized liver CEUS imaging and analysis protocol and evaluate the reproducibility of blood flow parameters. I analyzed CEUS scans collected with a standardized method from 80 patients with liver lesions using a MATLAB script to extract the parameters RT, MTT, PI, and AUC. I also performed the same analysis on images from an in-vitro study using the same methodology. I calculated the coefficient of variation (COV) of these parameters between scans to evaluate their reproducibility. The COVs indicate that the quantitative parameters are highly reproducible with agreement between in-vitro and clinical data. This shows that using the standardized methodology, reproducible blood flow parameters can be extracted from image loops and this technique can aid clinicians in the future to decide whether treatment is working.

The effective treatment of individuals with HIV relies on maintaining therapeutic drug concentrations, necessitating accurate measurement of antiretroviral (ARV) drug levels. Current methods, such as liquid chromatography tandem mass spectrometry (LC-MS/MS), are limited by cost and accessibility. Our research addresses this gap by developing the INTEGRase activITY (INTEGRITY) assay for measuring integrase strand transfer inhibitors (INSTIs), a leading class of ARV drugs. This 2-step assay quantifies INSTIs using a DNA strand transfer reaction and quantitative polymerase chain reaction (qPCR). The presence of INSTI drugs disrupts the strand transfer reaction, inhibiting full-length target DNA formation, which is then measured through real-time qPCR. My work focused on optimizing the limit of detection of INTEGRITY by altering the strand transfer reaction conditions and protocol. Specifically, I conducted experiments altering INSTI drug concentrations and optimizing pre-incubation times of integrase with the drug to enhance the LOD. I observed that preliminary incubation of integrase and INSTI drugs for 5 minutes at 37 degrees Celsius improved the LOD of INTEGRITY by an order of magnitude. The simplicity of the INTEGRITY assay, utilizing standard laboratory equipment, holds immense promise for broadening access to routine clinic-based ARV drug level monitoring. This advancement has the potential to significantly enhance HIV care on a global scale by offering a cost-effective and accessible solution for monitoring therapeutic drug concentrations.

Therapeutic ultrasound can induce biological effects that can be utilized for various clinical applications, and its non-invasiveness enables targeted treatments without harming tissue around the target. It can be applied in cancer treatments, where tumors can be primed with ultrasound to improve the delivery of chemotherapy, or even destroyed without the risks of surgery. Such treatment can be further enhanced by microbubbles, which are used clinically as a contrast agent in ultrasound imaging to visualize blood flow. Therapeutic ultrasound can generate microbubble activity known as cavitation that is capable of opening pores in cell membranes or disrupting blood supply to tumors, enabling more efficient drug uptake. My research goal has been to evaluate microbubble activity generated with therapeutic ultrasound and discover ways to optimize this treatment for drug delivery. To monitor microbubble activity during treatment, I use a technique known as passive cavitation detection (PCD) where one ultrasound device transmits sound directed at microbubbles, while the other “passive” device is listening for sound scattered off the microbubbles. I have been developing a PCD setup with a tissue-mimicking phantom that is physiologically similar to tumors for fast and reliable evaluation of ultrasound conditions for cavitation for use in cancer therapy. For this project, I align and control the PCD system with the LabView software, develop several phantoms that mimic cancer tissues for testing microbubble response to treatments, and analyze microbubble signals with a computation software MATLAB to evaluate cavitation activity. In addition to studying ultrasound cavitation, I am currently focusing on the fabrication of a tissue phantom with a cylindrical flow channel acting as a tumor blood vessel. The phantom allows for quick, repeatable experiments and evaluation of tumor vessels with different sizes. The careful study of cavitation activity will lead to more efficient cancer treatments with improved drug uptake.

Microdissected tissues from tumors have shown promise in preserving tumor microenvironment, which is critical for effective preclinical screening evaluation for drug development and cancer precision therapy. The tumor microenvironment’s microvasculature has an essential role beyond nutrient delivery and has been challenging to maintain ex vivo. Microdissection of cancer tissue into thousands of uniformly sized “cuboids” permits simple organization and perfusion of tissue using microfluidics to preserve the microvasculature. Here, we present a promising flow protocol in a 24-well microfluidic platform. The cuboids were cultured in our platform for different periods with different flow rates and without flow. The tissues were then fixed, stained with CD31, and cleared for fluorescence confocal microscopy. We demonstrated that this flow preserves the microvasculature of the tumor microenvironment of microdissected cancer tissues. The development of such a tool can enable medium-high throughput drug screeing and lead to improved cancer diagnostics and therapies for humans.

Advancements in HIV prevention include current pre-exposure prophylaxis strategies (PrEP), which are effective for men, but not for women due to poor partitioning of antiretrovirals (ARVs) to the female reproductive tract. One strategy for sustained delivery of ARVs to the female reproductive tract is the integration of ARV-releasing reservoirs with established intrauterine devices (IUDs). To this end, our lab has investigated reservoirs containing polymer-drug conjugates (drugamers), where the HIV integrase inhibitor raltegravir (RAL) is covalently attached to a polymer through a hydrolysable linker. However, current drugamers release RAL too rapidly to achieve our target of 1-3 years of IUD-mediated delivery. Our current work is directed at redesigning the RAL drugamer linker to extend the duration of release from 30 days to at least one year. We hypothesize that converting the ester linker of our current drugamer design to an acetal carbonate will slow the rate of RAL release, since the rate determining step of acetal carbonate hydrolysis does not involve the particularly acidic hydroxyl of RAL (pKa = 6.6). To date, I have synthesized the required acetal carbonate monomer by forming a carbonate-linked methacrylate through acyl substitution chemistry, and conjugated RAL to this methacrylate through an SN2 reaction. We fully characterized this monomer using NMR spectroscopy and mass spectrometry. Additionally, in a release study in cell media at 37C, I measured the rate of hydrolysis to be approximately 30 times slower than in the current lab monomer. Future directions include polymerizing the monomer and measuring RAL’s rate of release from the drugamer. We plan to attach other antiretrovirals with hydroxyls to the acetal carbonate linker and measure the rate of release from these drugamers as well. These preliminary findings are promising and will inform the design of drugamers for the long-term prevention of HIV.

The drug resistance in tuberculosis (TB) is a rising concern for the diagnosis and treatment of the disease. Being able to detect the presence of drug resistance accurately and rapidly in the patient strain is essential for improving individual treatment outcomes and reducing further transmission of resistant strains, which are more costly and difficult to treat than drug-susceptible strains. However, the current methods come in short in point-of-care (POC) settings, due to problems such as long processing time, high complexity, and necessity for specialized personnel/equipment. Oligonucleotide ligation assay (OLA) provides a high sensitivity and specificity against TB drug resistance, and here, I have developed a novel lateral flow test (LFT) device that incorporates OLA into it, which have shown comparable specificity and sensitivity against traditional protocol of OLA in lab setting followed by LFT. Moreover, the simplicity of the design enables further incorporation of other techniques such as isothermal DNA amplification, for a compact, one-step TB drug resistance diagnostic device for low-resource environment. 

Hypoxic-Ischemic Encephalopathy (HIE) resulting from a lack of blood and oxygen to the brain is the leading cause of mortality in term newborns. Extracellular vesicles (EVs) serve as critical transporters of biomolecules between cells, with evidence of alleviating inflammation in models after hypoxic ischemia (HI) injury. Therapeutic efficacy of EVs has only been evaluated in males because males are more susceptible to worse outcomes following HIE injury, yet knowledge about EVs and their behavior when administered to females is still needed. In this study, I aimed to address this knowledge gap by systematically comparing the efficacy of male and female neonatal brain-derived EVs (mEVs, fEVs, respectively) applied on male and female neonatal rat ex vivo brain slices. I first confirmed the purity of isolated EVs with protein assays and immunoblots, and utilized an ex vivo oxygen-glucose deprivation (OGD) model of HI injury, applying fEVs and mEVs to sex-matched OGD-exposed brain slices. I evaluated cell viability after 24h of EV exposure, and my results show that fEVs decrease inflammation and cytotoxicity in OGD models. When compared to previous results using mEV treatment, my results suggest that females have a more robust anti-inflammatory response system to injury. Ongoing work to better understand the therapeutic effect of EVs involves further observing morphological shifts in microglia through confocal imaging, as fEV application will likely result in microglia shifting towards anti-inflammatory phenotypes, similar to what was previously observed after mEV application. I am also quantifying expression levels of various inflammatory and reparative genes through reverse transcription quantitative polymerase chain reactions (RT-qPCR). Overall, I have demonstrated in these pilot studies that fEVs have a different therapeutic effect in OGD injury compared to mEVs. This research is intended to open up pathways for more personalized sex-based treatments for various injuries and therapeutics in the future. 

Mutations in ß-myosin heavy chain (MHC) are implicated in familial cardiomyopathies. Such mutations can modify myosin motor function or modulate the number of heads participating in contraction; changes at the protein level in turn affect sarcomere performance and ultimately lead to pathologic cardiac remodeling. Our aim is to better understand the pathogenesis of familial dilated cardiomyopathy (DCM) by studying how the R369Q mutation, which resides on loop 4 of the ß-MHC structure and participates in the interaction with actin, modulates the structure and dynamics of pre-powerstroke myosin. Previously, we have employed molecular dynamics to simulate and study the pre-powerstroke state at atomistic scale. Measurements of root mean square deviation (RMSD) and root mean square fluctuation (RMSF) for each of the six simulations demonstrated that R369Q myosin had a higher average RMSD over the course of the simulation and higher RMSF per residue than did wild type; this suggests that the overall structure of the R369Q ß-MHC is less structurally stable than the wild type. Additionally, we observed a significant loss of several contacts between Switch I and a flexible loop in the Upper 50 kDa Domain in the R369Q structure. Thus, we postulate that the R369Q mutation introduces structural instability into the myosin structure and disrupts the inter-protein communication of structural changes that drives the ATPase cycle. Here, we expand on these results with analysis of a stem cell-derived cardiomyocyte model and Brownian dynamics simulations. Analysis of myofibrils, the contractile organelles of cardiomyocytes, from cultured cells will confirm my previous computational work and give further insight into nucleotide-binding pocket coordination while Brownian dynamics will reveal changes in actomyosin interactions. A multifaceted analysis of the R369Q mutation from computational and experimental approaches will consolidate the mechanism of the mutation, providing a foundation for design of a therapeutic to ameliorate contractile dysfunction.

 Injectable biomaterials can be used for targeted, local delivery of drugs and small protein factors. However, designing a biomaterial that is self-healing, bioresorbable, highly-defined, and allows for the independent release of multiple protein drugs remains a challenge. Interpenetrating network (IPN) hydrogels formed by physical crosslinks provide a solution as they combine the properties of two networks into one material, as well as form gels that are injectable and self-healing. Our project aims to produce the first ever reported fully recombinant protein-based IPN hydrogel that can be used to deliver multiple drugs simultaneously. Recombinant protein-based polymers are advantageous for forming biomaterials due to their bioactivity, biocompatibility, and tunability. Each biopolymer contains coiled-coil proteins linked by a flexible hydrophilic linker that is bisected by a click-like chemistry protein system, known as a polyproteam. These polyproteams allow drugs or protein factors to be covalently ligated to the networks, so that drug delivery is dependent on material erosion instead of diffusion. We characterized the material’s properties using rheometry to measure material stiffness, percent strain at which the material liquifies, and self-healing behavior. Then, we tested the ability of our hydrogel to deliver drugs by ligating epidermal growth factor and insulin growth factor to the hydrogel and measured the release of protein over time through a Western Blot. The bioactivity of the growth factors was confirmed by culturing with human mesenchymal stem cells (hMSCs) and using a Western Blot to measure activation of the ERK signaling pathway. If successful, the IPN hydrogel can be used for clinical applications, as a tool for drug delivery.

Current technology to control 3D cell function takes advantage of bioorthogonal photochemistry to immobilize proteins into materials by using photocages—photoremovable molecular groups that block protein activity. However, diffusion limitations necessitate patterning times ranging from hours to weeks, far longer than the timescales of many biological processes. Protein activation aids signaling events within the extracellular matrix (ECM), leading to downstream changes in cell fate and physiological responses in our bodies. In order to probe and investigate biological systems, hydrogel materials provide an ideal synthetic platform, due to their polymeric, water-swollen characteristics that mimic the native ECM. In this project, I will use light to control the spatial and temporal presentation of biochemical cues through the photoactivation of proteins within hydrogels. We hypothesize that the kinetics of the protein activity between solution and biomaterial studies should correlate, given their dose-dependent response to light exposure—by varying the intensities of light and time intervals of exposure. By characterizing the photoactivatable protein system and controlling protein activity, we intend to use this platform to photoactivate biologically relevant proteins to control signaling that occurs on shorter time scales, applicable to biochemical processes.

Biological processes depend on the intricate functions of proteins, which serve as the driving force in vital reactions. Given the importance of proteins in biological processes, several methods have been developed to control their functions in biomaterial and in vitro. Current methods often rely on secondary fixatives and caustic chemicals, posing risks of functional impairment in target proteins. To address these limitations, my project focuses on modifying cells with non-endogenous machinery, enabling expression of photoactivatable proteins – those with photocaged amino acids installed at essential locations, and using them to optically stimulate protein function. This strategy enables the triggered activation of target proteins with light, mitigating the risks associated with chemical interventions. We predict that the degree of protein activation can be controlled in a dose-dependent manner by manipulating light exposure duration and intensity. Genetic code expansion is used to integrate noncanonical amino acids, site specifically, into proteins expressed in bacteria and mammalian cells. By incorporating these photoactivatable proteins into biomaterials and living cells, our research aims to gain insights into fundamental cell signaling processes through control of targeted proteins. By analyzing biological mechanisms at the molecular level, we can guide cell processes and make informed decisions regarding cellular behavior and fates.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart defect, requiring patients to undergo multiple invasive cardiac procedures, including pulmonary valve replacement (PVR). However, with recent clinical advances, new tools are needed to optimize PVR timing. We believe noninvasively collected cardiopulmonary exercise testing (CPET) data can provide insight into a patient’s need for PVR. Specifically, we hypothesize that patients with a more severe stage of pulmonary valve dysfunction have a limited ability to increase their stroke volume during exercise, an abnormal response that can be assessed by analyzing the behavior of the oxygen pulse (O2-pulse) curve during CPET. A ‘flattening’ of this curve suggests impaired augmentation of stroke volume and potentially a more urgent need for PVR. This research aims to identify metrics that can characterize patterns in O2-pulse. Data were collected from 44 participants with TOF undergoing CPET PVR evaluation and 10 healthy individuals. To find a maximum O2-pulse, we fit a penalized bilinear regression model to this curve. We extracted 8 parameters to mathematically describe the O2-pulse curve, as well as 20 traditional CPET performance metrics. One important parameter that was calculated is the ‘lost area under the curve’ (LAUC), defined as the area under the two calculated regression lines over time subtracted from the area under the curve as determined if the first regression line were to continue on the same slope as is typically expected during a maximal CPET. This value captures both the change in slope and when participants transitioned from a steep increase in O2-pulse to a relatively flattened O2-pulse. The LAUC, among our other identified metrics, can potentially provide insight into the optimal timing of PVR in patients with TOF. Unsupervised machine learning may be a useful tool to characterize patterns in these metrics and search for clinically relevant patient phenotypes.

My research project aims to combat inflammation and fibrosis caused by biomaterials and implants by developing a deeper understanding of the effect of CID on macrophage phenotype. Non-degradable biomaterials can provide long-term stability in the body but can elicit a foreign body response, such as inflammation. The project involves engineered M1 cells which were created and published by the Giachelli and Scatena Lab within the Department of Bioengineering at UW. We have two groups of mice: one control group that has been injected with engineered TLR4 (Toll-Like Receptor 4) cells but not given the CID (Chemically Induced Dimerizers) drug, and another group that has been injected with both the engineered TLR4 cells and given the CID drug. Previous in vitro studies have demonstrated that the engineered TLR4 cells activate the proinflammatory M1 macrophage phenotype when treated with CID. Activation of the proinflammatory M1 phenotype is expected to result in alteration of the healing process, including altering the collagen quantity and structure. At this stage in the project, we have tissue samples from both these groups, and I am currently analyzing these samples using both H&E staining and Picrosirius Red staining thus allowing me to measure healing parameters, like density of collagen. At the same time as data collection, I am using ImageJ to obtain measurements and expect to see the group with CID have a denser collagen structure around the material than the group without CID since CID causes activation of the M1 phenotype. Conducting the analysis on these tissue samples will help address the effect of CID on healing parameters, such as inflammation, and help us develop a better understanding of the roles that M1 and M2 phenotypes play in the healing process.

Bioconjugation, or the covalent linkage between a biomolecule and another chemical group, creates hybrid "conjugates" that exhibit the properties of both biomolecules and exogenous moieties. The N-termini of proteins often fall outside of their final fold, making the N-terminus an optimal site for conjugation while preserving a protein’s native folding and bioactivity. Consequently, N-terminal modification of proteins and peptides has been a long-standing goal in fields like drug delivery, biotherapeutics, and cellular imaging. However, the current techniques for N-terminal protein conjugation are limited by either the introduction of bulky protein assemblies at the conjugation site, the need for multiple costly and complicated steps, or low site selectivity. In this project, we aimed to develop an improved route for N-terminal bioconjugation. We created a generalizable platform for single-step purification and near-scarless N-terminal bioconjugation of proteins by leveraging the chemistry of the atypically split intein VidaL. To evaluate the effectiveness of our platform, we first examined the kinetics and reaction conditions of VidaL bioconjugation, confirming its ability to modify the N-termini of proteins successfully and selectively. Then, we used our platform to conjugate an alkyne, biotin, or FAM-biotin moiety to the N-termini of fluorescent proteins (EGFP and mCherry), a model enzyme (beta-lactamase), and a model growth factor (EGF). Through measuring fluorescence and conducting nitrocefin and proliferation assays, I found that, regardless of the moiety added, bioconjugation did not impact the native function or activity of these proteins. In the future, we expect that this platform's ability to easily N-terminally bioconjugate proteins with minimal impact on their functionality will find use across the growing fields of applied chemical biology.

The emergence of induced Pluripotent Stem Cells (iPSCs) have allowed researchers to better study the effects of various diseases and mutations on fetal development. One such way of accomplishing this is the breakthrough of the organoid: a complex, iPSC-derived, 3D structure, that provides biologically relevant models for human systems. Lung Organoids (LO) were developed through this technology. However, the current LO models utilize mature lung phenotypes, which do not consider progenitor stages that may be critical for fetal development and the understanding of diseases that may affect this development in utero. The goal of this project is to provide characterization to the early stages of iPSC LO development: the Embryoid Body (EB) and Anterior Foregut (AFE). Using a previously established protocol, the LOs were fixed with 4% paraformaldehyde (PFA) on day 4 (EB stage) and day 6 (AFE stage), then analyzed with immunofluorescence analysis of the corresponding fetal lung (FL) development markers. 135 day old FL tissue sections were used as a positive control. The markers used to establish characterization were SOX17, a marker for the early endoderm germ layer, OCT4, an iPSC marker for pluripotency, and ECAD, a marker for tissue layer separation and cell migration. We hypothesized that all markers would appear in the EB stage, and the AFE stage would experience an upregulation in SOX17 and downregulation in OCT4 and ECAD. My results confirmed an upregulation of 133% for SOX17 and a downregulation of OCT4 by 58% from the EB to AFE stages. Lastly, as hypothesized, ECAD was present in EBs, but not in AFE. In conclusion, the LO stages proved to be similar to developmental stages of in utero development. Further analysis could help with new disease and mutation models for early development in utero, helping prevent devastating outcomes.

Almost every form of cardiac disease is characterized by fibrosis, or the accumulation of collagen, an extracellular matrix (ECM) protein, secreted by the cardiac fibroblast. The buildup of fibrosis is a major clinical burden, as it contributes to diastolic dysfunction, or the heart’s inability to relax, and arrythmias, or an irregular heartbeat. In previous studies, the Davis lab has found that in chronic injury, the heart likely undergoes minor offenses along with periods of rest which accrue over a lifetime. Even when exposed to repeat injury stimuli, the heart is able to recover, and the cardiac fibroblasts can transcriptionally regress. Yet, what remains unclear is when the heart experiences repetitive stress, which is common with hypertension, will these once-activated cardiac fibroblasts have a more aggressive response? And if so, are the activation cues stored in the primed external environment, or are they intrinsic to the cell? To address this, we developed a fibroblast isolation and injection protocol that will ultimately allow us to isolate discrete populations of fibroblasts and study them in hearts void of injury. Our results found that fibroblasts from donor hearts that were subjected to a myocardial infarction injury were detectable at 4 and 14 days post cardiac injection but had little proliferation. However, there was an increase in host fibroblasts recruited to the graft site, many of which were proliferating, and fibrosis was found within these same regions. These results demonstrate that cardiac fibroblasts from the same strain can be isolated and adoptively transferred to other hearts, without exogenous ECM. We can apply this baseline protocol to further examine fibroblast memory in vivo in a model of intermittent hypertension.

Chimeric antigen receptor (CAR) T-cell therapy is a revolutionary cancer treatment with promising clinical efficacy in treating hematological cancers. Yet, current CAR T cells are designed to target a single antigen and have had little success treating solid-tumors due to tumor-antigen heterogeneity. To address these limitations, we have engineered a universal SpyCatcher003 CAR T-cell system (DB5 CARs) that utilizes synthetic targeting-intermediates conjugated onto CARs to bind multiple cancer-antigens and mediate tumor cell killing. My project aims to utilize a design based approach to engineer and optimize the serum stability of the SpyTag003-peptide to improve T-cell activation for in vivo cancer treatment applications. In preliminary studies, we demonstrate the loading of a synthetic, high-affinity biomaterial folate-SpyTag003 peptide chimera onto DB5 CAR T cells. In addition, folate-SpyTag003 binds with high-affinity and specificity to folate receptor alpha positive (FOLR1⁺) KB tumor cells. CD8⁺ and CD4⁺ DB5 CAR T cells labeled with folate-SpyTag003 chimera showed a robust increase in cytotoxic activity and cytokine expression when incubated with FOLR1⁺ KB cells. Thus, we aim to optimize the SpyTag003-peptide chimera to improve binding to FOLR1 for targeted killing of FOLR1⁺ tumor cells (e.g., ovarian and breast tumors) as an in vivo cancer treatment application. By iteratively improving the design of the folate-SpyTag003 intermediate and DB5 CARs, our approach for universal CAR T-cell therapy could provide safe, cost-effective, and broad-targeting treatments for patients with heterogeneous solid-tumors.

Subtherapeutic drug levels can lead to the failure of antiretroviral therapy (ART) regimens used in Human Immunodeficiency Virus (HIV) treatment and prevention. However, gold-standard HIV drug level monitoring techniques—such as mass spectrometry—require bulky and expensive instruments that are not widely accessible at the point-of-need. Our group developed the REverSe TRanscrIptase Chain Termination (RESTRICT) enzymatic assay to rapidly (30 min) and inexpensively measure tenofovir diphosphate (TFV-DP), a nucleotide analog used in >90% of oral ART regimens and in all approved prevention regimens. However, RESTRICT currently requires trained operators to perform multiple time-sensitive liquid-handling steps. To reduce user intervention and minimize the need for laboratory equipment, we harnessed 3D-printed capillaric microfluidics to self-propel liquids using only surface tension effects encoded in microchannel geometry and surface chemistry. Specifically, we translated the manual tube-based RESTRICT to an automated microfluidic protocol by using autonomous trigger valves to pre-load multiple RESTRICT assay reagents and serpentine channels to control assay timing. Currently, RESTRICT reactions are incubated for 30 minutes at 37˚C, but we decreased the reaction time to 15 minutes and removed the need for an external heating source by incubating at room temperature (25˚C). There was only a 15% decrease in overall signal intensity in the faster, room temperature assays, and measured readout was distinguishable between clinically relevant concentrations of TFV-DP. Our results represent a first step towards integrating RESTRICT reactions and fluorescence readout onto a rapidly fabricated microfluidic chip. We hope to achieve a device that increases the accessibility of HIV drug level monitoring at the point of need without specialized equipment or highly trained operators.

Bacteriophage MS2 phage-like particles (PLPs) are artificially constructed viral-like particles. The similarity of the particle to a virus allows the particles to be used as a control system for molecular detection and drug delivery systems. The capsid is made up of single chain coat protein dimers (SCCPD) and a singular maturase, and is able to package mRNA within the protein coat due to the dimerization of coat proteins spontaneously forming the capsid structure in the presence of a packaging signal. In addition, the PLPs also have the ability to display what is being packaged on the surface. Currently, it has been shown that PLPs are able to be purified via His-tag affinity, due to fusion of the His-tag onto the SCCPDs. However, if a peptide is displayed at the SCCPD site, the His-tag must be attached elsewhere on the PLP to ensure the PLP can be purified. To address this, I designed a new PLP by plasmid engineering via site-directed mutagenesis to display a peptide on AB-loops within the SCCPD, whilst packaging the corresponding mRNA within the capsid. I purified the PLP via a His-tag attached to the maturase protein. To verify correct particle formation, I ran SDS-PAGE to observe the density of bands corresponding to SCCPD and maturase. I designed a reverse transcriptase polymerase chain reaction and carried out a nuclease protection assay to verify mRNA packaging. Preliminary data of SDS-PAGE has shown the particle has successfully purified, and is correctly forming due to the observed maturase to SCCPD band density ratio on the gel meeting the expected ratio.

The COVID-19 pandemic has brought to our attention the lack of fast, affordable, and sensitive diagnostic tests available on the market. The majority of commercial diagnostic tools are expensive, despite being quick and sensitive. This conundrum has brought attention to the necessity for developing high-quality tests at an affordable cost, emphasizing the importance of accessible diagnostics that are both rapid and sensitive. The Yager lab has been developing detection tools for infectious diseases, mainly based on isothermal nucleic acid amplification tests, using loop-mediated isothermal amplification (LAMP). The LAMP assay includes a DNA polymerase known as Bst. It was found that assay sensitivity improved when the samples were pretreated with HUDSON, which consists of TCEP (a reducing agent) and EDTA. I am investigating the improvement of Bst DNA polymerase activity with TCEP, using a commercial enzyme kinetics kit (EvaEZ) to quantify the Bst polymerase activity. The EvaEZ assay allows quantitative comparison between conditions by measuring fluorescence intensity indicative of DNA amplification. This was achieved by assessing primer binding, enzymatic extension, and EvaGreen dye intercalation, enabling comparison of the rates of fluorescence generation to evaluate amplification efficiency across positive, negative, and experimental control conditions. This project is still ongoing, and preliminary findings suggest promising results. This study demonstrates the potential for using reducing agents to optimize enzyme efficiency and improve detection sensitivity. This technique can readily improve detection speed and sensitivity both simply and affordably. We are able to be better prepared for the next pandemic.

Capillary microfluidics capitalize on surface tension effects encoded in microchannel geometry and chemistry to transfer liquids without external instruments, making them a user-friendly technology for point-of-care tests. For most applications, hydrophilic surfaces (contact angle < 90˚) are necessary to induce surface tension driven flow. Currently, this is achieved with vacuum plasma chambers that alter surface chemistry. Unfortunately, the hydrophilic properties made with plasma processing are temporary and unstable. Alternatively, an inherently stable hydrophilic 3D-printing resin containing polyethylene glycol diacrylate (PEGDA) and acrylic acid (AA) was recently developed for capillary microfluidics. However, this hydrophilic resin has not been thoroughly validated for inexpensive (<$300) liquid crystal display (LCD) printers. Our objective is to optimize and validate 3D-printing parameters including exposure time, UV power, layer thickness, and lift/retract speed using this hydrophilic PEGDA-AA resin with three LCD 3D printers (AnyCubic Photon Mono X 6K, AnyCubic Photon Mono M5s Pro, and Phrozen Sonic Mini 8K). Validation includes measuring hydrophilic properties as well as the dimensional fidelity of the printed channels compared to the design specifications. Our proof-of-concept prints on the Mono X 6K printer had average contact angle measurements of 42.8° ± 8.77. The percent differences between designed and printed channel lengths, widths, and depths were 31.5 ± 0.23%, 28.9 ± 3.41%, and 2.40 ± 13.9% respectively. By optimizing the print parameters of cost-effective 3D printers with the inherently stable hydrophilic resin, we enable capillary microfluidic technologies for users in low income/resource settings who may not have access to vacuum plasma chambers. Future work will explore additional resin modifications to encourage applications like spatial patterning of hydrophilicity and protein immobilization in microchips. [1]V. Karamzadeh, A. S. Kashani, M. Shen, and D. Juncker, “Digital Manufacturing of Functional Ready‐to‐Use Microfluidic Systems,” Advanced Materials, vol. 35, no. 47

 COVID-19 is an infectious viral disease that is caused by the SARS-CoV-2 virus entering host cells through surface spike proteins that bind to surface ACE-2 receptors. Some anti-SARS-CoV-2 antibodies allow SARS-CoV-2 to also have antibody-mediated entry (AME) into immune cells, independent of the ACE-2 receptor. Instead, they often enter via Fcγ receptors. This phenomenon has been correlated with cytokine release syndrome, which occurs when the immune system has a highly inflammatory response to infection and is implicated in severe COVD-19 cases. It has also been shown that other antibodies have demonstrated inhibition of SARS-CoV-2 entry. During binding and viral fusion, all three receptor binding domains (RBD) of the spike protein fold to an upward conformation, which is necessary for binding. Inhibitory anti-SARS-CoV-2 antibodies can impede this process through competitive binding as well as mechanisms such as premature cleavage of the RBD or stabilizing a three-down conformation. We hypothesize that inhibitory antibodies can stop AME from occurring to varying degrees of effectiveness based on inhibitory mechanisms and avidity to the spike protein. To understand the dynamic between AME and inhibitory antibodies, we tested the infection levels of monocytes by SARS-CoV-2 pseudo-virus in the presence of antibodies shown to induce AME combined with varying concentrations of inhibitory anti-SARS-CoV-2 antibodies that have different mechanisms of affecting the spike RBD conformation. ELISA-based assays were also run to quantify antibody avidity to the spike protein. Analysis suggests that high concentrations of multiple neutralizing antibodies inhibit AME and that conformationally inhibiting antibodies are more effective at inhibiting AME than strongly activating ones. Studies of this type can aid in continued development of safe vaccines and therapeutics, as well as help understand how antibodies affect SARS-CoV-2 spike conformational regulation and therefore viral entry.

Alzheimer's disease (AD) is the most common type of dementia with an estimated 1 in 85 people affected worldwide. It is characterized by accumulations of amyloid beta which result in difficulties with memory and other cognitive activities. While there are treatments that show promise, these are only effective in the preclinical phase of the disease where there are no symptoms, which take approximately 20 years to appear. Unfortunately, measuring amyloid beta directly is invasive and costly, so functional magnetic resonance imaging (fMRI) is used to detect changes in the brain caused by amyloid beta accumulation. Moreover, established techniques for using fMRI for detecting AD show limited success. Therefore, the goal of my project is to generate biomarkers of the earliest neural communication changes associated with AD to allow the application of treatment when it is most effective. This will be done through the use of a graph diffusion autoregressive (GDAR) model, recently developed in my lab. The GDAR model is capable of estimating dynamic changes of neural communication, but has only been applied to electrical recordings. I have demonstrated its validity for fMRI data using a healthy control group through analyzing the model’s prediction capabilities and test-retest consistency, which are both strong. Furthermore, I correlated the GDAR model’s outputs with the primary method in the field, functional connectivity analysis, showing only loose correlation and the potential for biomarkers that are novel from what is currently known. Currently, I am applying the model to AD patients, and anticipate its outputs to contrast those of healthy patients through the use of unsupervised clustering techniques. This would allow the model’s outputs to be used as biomarkers for AD and improve the feasibility of its treatment and prevention.

Therapeutic Drug Monitoring (TDM) serves a critical role in optimizing the effectiveness and safety of dapivirine (DPV) vaginal rings as an HIV prevention tool, ultimately leading to improved health outcomes for individuals at risk of HIV infection. Liquid chromatography-tandem mass spectrometry (LC/MS) is a commonly used method for HIV TDM, however, its limited availability leads to delayed results, high cost, and limited utility in clinical practice. The REverse TRanscrIptase Termination (RESTRICT) assay is a rapid and inexpensive test for TDM. DPV has low solubility in aqueous solutions due to its hydrophobic properties. Organic solvents like isopropyl alcohol (IPA), acetonitrile (ACN), and dimethylsulfoxide (DMSO) are typically used for extracting DPV from returned vaginal rings. However, organic solvents often interfere with the performance of enzymatic assays like RESTRICT. In this study, we investigated the compatibility of organic solvents used for DPV extraction with the RESTRICT assays. We tested mixtures of IPA, ACN, and DMSO at varying concentrations of solvent in water. After performing a preliminary experiment with 25%, 50%, and 75% concentrations of IPA in water using RESTRICT, we found similar fluorescence readouts between the different concentrations indicating that IPA and RESTRICT are compatible. The solvent that results in the smallest decrease in signal intensity compared to solvent-free assays will be selected. In the future, we will extract DPV from new and returned vaginal rings and measure drug levels using the RESTRICT assay benchmarking against a conventional laboratory technique like Raman spectrometry. This work represents a first step towards developing a user-friendly test for measuring DPV levels at the point of need.

There is a strong need for rapid and inexpensive tests for monitoring human immunodeficiency virus (HIV) drugs to prevent treatment failure. The REverse TranscrIptase Chain Termination (RESTRICT) Assay provides fast and inexpensive measurement of HIV reverse transcriptase (RT) inhibitors by measuring the extent of DNA synthesis with the HIV RT enzyme in the presence of a patient’s blood. RESTRICT uses an intercalating dye, PicoGreen, to provide fluorescence as a function of the amount of DNA synthesized. However, PicoGreen interrupts DNA synthesis and binds to any double-stranded DNA (dsDNA) present in the sample including background DNA in blood samples, precluding real-time measurements and potentially decreasing the accuracy of results. This project uses RESTRICT in conjunction with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) complexes and reporters to accurately measure RESTRICT results in real-time. Fluorescence is produced by reporters which are made up of a quencher and a fluorophore. When the CRISPR complex detects a specific complementary sequence formed from the single-stranded DNA (ssDNA) template, the Cas enzyme is activated and cleaves the fluorescent reporter. Once cleaved, the fluorophore is released from the quencher thus generating fluorescence. Because the CRISPR complex binds only to specific segments of DNA produced by RESTRICT, we can obtain more specific results without interference from background DNA. In addition, CRISPR complexes and reporters do not interfere with DNA synthesis and can be added at the same time as other RESTRICT reagents, enabling real-time monitoring of RESTRICT reactions. Using a portable fluorescence reader (T8-ISO, Axxin Pty Ltd.), positive control reactions containing RT enzyme provided at least 5X more fluorescence than negative controls without RT. Future work will focus on measuring clinically relevant levels of HIV drugs in aqueous buffers using RESTRICT with CRISPR readout.