Skin is a densely innervated sensory organ that protects us every day from environmental trauma. As a barrier organ, skin is susceptible to frequent damage that must be promptly and properly healed to prevent infection and restore sensory function. Our lab uses adult zebrafish as a model to study skin injury and repair. Adult zebrafish skin is similar in composition to human skin and transparent, lending itself to high-resolution microscopy. Previous experiments in our lab revealed that dynamic, skin-resident immune cells known as Langerhans cells (LCs) rapidly engulf cellular and axonal debris after injury in the zebrafish skin. Calcium signaling regulates phagocytosis and cell motility in other immune cells, but the role of calcium signaling in LCs is unstudied. Through skin explant assays, various injury paradigms, and confocal fluorescence microscopy, I have established a model for monitoring calcium signaling in LCs. I found that LCs exhibit rapid, transient calcium flashes under homeostatic conditions. However, upon engulfment of large cellular debris generated by precise laser-ablation of skin cells, LCs exhibit an atypical sustained calcium signal lasting an hour on average. To test the requirement of calcium during engulfment by LCs, I treated skin with the drug Thapsigargin to perturb calcium flux. I confirmed that Thapsigargin increases intracellular calcium in LCs and keeps intracellular calcium concentrations elevated for hours after drug addition. During Thapsigargin treatment, I showed that LCs formed phagocytic cups around cellular debris but engulfed fewer laser-ablated corpses compared to controls. Thapsigargin-treated LCs also experienced normal migration to a wound site. My results indicate that calcium flux regulates LC engulfment of large debris, but not through migration. Identifying the molecular mechanisms underlying LC motility and debris removal is ultimately relevant to understanding skin repair and disease states in which the wound healing response is attenuated, such as in chronic wounds.

The Field Programmable Cellular Arrays (FPCA) project at the Molecular Information Systems Lab (MISL) aims to improve current biocomputing systems utilizing spatial organizations of cellular components for logical operations. This can open doors for computation to be done within biological systems where artificial computation has never before been possible. This project encompasses three aims: characterizing the properties of signal propagation within E. coli, constructing biological circuit components for spatial signal processing, and optimizing bioprinting methods for circuits. Signal propagation through molecular signaling is employed to communicate the presence or absence of a signal and truth values to specific cells. We are demonstrating logical states of "1," "0," and the absence of a signal, thereby enabling differentiation between a logical "0" and a lack of signal. Two strains of bacterial cells are capable of performing the logic of a traditional "wire" and a NOR gate. Consequently, by arranging strains in spatially organized layouts, we engineer cellular arrays capable of performing diverse complex logical functions. This research is still in progress and we are in the process of optimizing NOR gate and wire strains. My role explores bioprinting circuits into hydrogels, and I have built a bioprinter with dual extruders to bioprint biological substances into containing slurries. This required designing, printing, and assembling 3D-printed parts. I am now characterizing the behavior of 3D printed materials into various containing slurries. This requires testing the ability of different bioprinting inks to encapsulate bacteria, testing various slurry methodologies, and testing interactions between combinations of these materials over space and time. I am also computationally modeling FPCA circuits at various levels of abstraction. Computational modeling serves to further broader computational goals in this project to compile a logic circuit specification into bioprinter GCODE.

Lake Washington is home to a complicated metapopulation of Oncorhynchus nerka. There is a potentially native population of sockeye spawning in the Sammamish River and tributaries with at least two history variations - typical sockeye that migrate to the ocean and return as adults, and residual sockeye that remain in freshwater and return at a smaller size. Historically, there was also a native, genetically distinct, freshwater-only population of O. nerka called kokanee. Thought to have been eliminated from Lake Washington in the 20th-Century, recent genetic evidence indicates that a small population of native kokanee remains with a single cohort spawning every third year. We are comparing spawning ages for sockeye, residual sockeye, and native kokanee by extracting otoliths (mineralized structures in the ear containing annual rings similar to tree trunks). Otoliths are mounted on slides, viewed under a light microscope, and aged by counting the number of rings present. We expect ocean going sockeye to be age 4 (typical for sockeye); kokanee to be age 3, reflecting the 3-year cycle in kokanee abundance; and residual sockeye to be younger than typical sockeye. Lack of information prevents a clear spawn-age expectation for residuals. A large run of kokanee returned in 2020 and another was expected in the fall of 2023. Surprisingly, a sizable run was present in 2022. We hypothesize that the 2022 run was composed primarily of early-returning 2-year-olds while the 2023 run were typical 3-year-olds. We are comparing the age distributions of kokanee from runs in 2022 and 2023 to test this. Kokanee are remnants of a once abundant population. It is important to have a better understanding of kokanee relative to sockeye and residuals as a basis of conservation; much remains unknown. This study will also help interpret potential differences in spawning ages between similar life history variants.

Serratia marcescens is an opportunistic pathogen that can infect multiple human organs and is responsible for many healthcare-associated infections. It has a mortality risk of up to 58% and early diagnosis is crucial for timely treatment. S. marcescens secretes a unique restriction endonuclease, which has been recognized as a virulent factor and thus can be used as a diagnostic biomarker. To detect this restriction enzyme biomarker, I have designed and investigated a model system using novel restriction endonuclease mediated DNA strand displacement (resDSD), adapted from the enzyme-free DNA strand displacement (DSD) reaction. In a typical DSD circuit, a DNA input “invading” strand invades a duplex DNA substrate, replacing the previous incumbent strand through branch migration to reveal a fluorescence molecule. In contrast, my resDSD circuit employs a restriction endonuclease enzyme input. In my design, the toehold region is concealed and blocked by a strand that the restriction enzyme can cleave. Once cleaved, the toehold region is exposed, allowing an invading strand to hybridize and initiate the DSD cascade. This study represents the first demonstration of the resDSD system. To validate the concept, I used commercially-available restriction endonuclease BamHi instead of S marcescens’ endonuclease. I will also modify E. coli 5-alpha competent strain (c2987h) to secrete BamHi in place of S. marcescens. By investigating this innovative resDSD approach, I aim to establish a reliable method for detecting bacterium such as S. marcescens based on its secretion of the restriction endonuclease. Such a diagnostic tool could contribute to early detection and prompt treatment of infection caused by this opportunistic pathogen or similar pathogens in healthcare settings.

Myotonic dystrophy type 1 (DM1) is a genetic disease that causes many serious health conditions in a variety of tissues including skeletal muscle stiffening and cardiac conduction disorders. This disease affects 1 in 2,300 people worldwide and is the most common form of muscular dystrophy. DM1 is caused by a CTG repeat expansion, which in lay terms means that in a gene, there's a sequence of 10 CTG DNA bases. However, in a specific part of the gene responsible for making messenger RNA (mRNA), the number of CTG repeats increases significantly. This unusual mRNA sequence is linked to the development of the disease. This mutated mRNA (messenger RNA) disables the splicing regulator muscle-blind-like 1 (MBNL1) gene and ultimately causes disease. It does this by sequestering and limiting the MBNL1s critical role in splicing mRNA (figure 1). In my proposed research project, I am focusing on cardiac function when testing adeno-associated viral vector (AAV)-mediated systemic delivery of the MBNL1 gene to increase MBNL1 protein expression in muscle. The lab found that body-wide delivery of AAV vectors with CK8-intron-MBNL1, which expressed MBNL1 only in striated muscle, was toxic in the hearts of mice and caused death (figure 2). Over the last few months, my mentor Matt Karolak and I have learned together methods such as echocardiography and tissue histological techniques to determine whether it is possible to prevent MBNL1 protein production and its damaging effects in the heart while still expressing MBNL1 protein in skeletal muscle for therapeutic disease benefits.

Chimeric Antigen Receptor T (CAR T) Cells are receptor proteins that can be modified to allow T cells to target specific antigens. CAR T therapy has shown promise in preclinical experiments in patients with solid tumors, such as glioblastoma, however limitations in distribution of CAR T cells in the brain limit the effectiveness of this treatment. Most commonly, CAR T cells are administered intraventricularly through a surgically implanted device and allowed to diffuse throughout the cerebrospinal fluid filled cavities and pathways to reach the tumor. However, there is little evidence supporting the effectiveness of current methods of administration, potentially due to a lack of target engagement. We hypothesize that the glymphatic system of the brain could be used to optimize delivery of CAR T cells to solid tumors. The glymphatic system is a network of perivascular pathways that facilitates the anatomically distinct movement of cerebrospinal fluid (CSF) into the interstitium of the brain, helps distribute solutes such as glucose, lipids, and neurotransmitters, and serves as a solute clearance system in the brain. Physiologically, glymphatic function is mediated by different factors such as arterial pulsation, vasomotion, and heart rate – which can be manipulated with anesthetics, pharmaceuticals, or even sleep. We proposed exploration of the difference in parenchymal distribution of CAR T cells when injected in the cisterna magna versus intraventricularly in mice. Non-tumor bearing mice were injected via the cisterna magna or intraventricularly with fluorescently labelled CAR T cells. We observed that at 1-, 4-, and 24-hours post-injection, CAR T cells were localized in the sub-ventricular regions similarly, regardless of injection site. In follow-up experiments, we will employ the same technique in tumor bearing mice, with and without pharmacological intervention to define the effect of glymphatic function on distribution and effectiveness of CAR T cells.

The human proteome consists of tens of thousands of proteins produced from sequences translated from the human genome. Further, each of these proteins can be modified post-translationally to create an even larger set of unique proteoforms. With such a massive catalog, a tool that could accurately and inexpensively fingerprint proteins with single-molecule resolution in real-time would have a transformative impact on biology, medicine, and healthcare. To develop such an approach, we are utilizing nanopore sensor technology. Nanopores function by electrically-examining proteins at the molecular scale. As a protein molecule passes through the nanopore—a minuscule orifice in a thin membrane—it modifies the ionic current. Each protein induces a unique alteration in the current, producing a distinctive signal pattern, or 'squiggle'. These squiggles effectively act as molecular fingerprints, potentially enabling us to identify and classify different proteins based on their specific current changes as they traverse the nanopore. In this project, we are tasked with building a machine learning model to classify proteins based on their squiggle templates when passed through a nanopore. We found that a classification model based on a standard Convolutional Neural Network (CNN) performed well on simulated data of eight synthetic protein designs, but failed to generalize properly to experimental test data. In contrast, a 1-Nearest Neighbor model significantly outperformed the neural network architecture on the synthetic protein test data. We plan to assess this model's performance on fingerprinting the human proteome through a simulated dataset, with the ultimate goal of sharing our findings with other labs that specialize in developing precision medicine, targeted drugs, and technologies for understanding protein structure, function, and interaction.

The Manduca sexta hawkmoth, a proficient pollinator, employs its antennae to efficiently navigate its surroundings. With their antennas highly developed olfactory sense as well as their wide range of odor recognition using their sensory receptor cells, moth antennae are an ideal candidate for developing reliable biosensors. In contrast, commonly used artificial sensors are inefficient and inaccurate in chemical detection. Furthermore, their manufacture is challenging due to their inconvenient design for the user. To evaluate the antenna's effectiveness as a biosensor model, we assessed neural activity in the moth antenna by means of an electroantennogram (EAG). To do this, we attached the removed antenna to a circuit to amplify and measure voltage variations across the antennal nerves during odor stimulation. We then placed the circuit into a wind tunnel and administered a selection of odorants over a determined cycle of durations ranging from 0.2, to 10 seconds. The odorants included a floral mixture from Datura flowers and a certain compound in the mixture (linalool) known to be attractive to moths, as well as volatile organic chemicals (VOCs) replicating both healthy and COVID breath. Our findings show strong initial spikes of electrical activity in the receptor cells correlating to odorant release​, favoring the shorter durations and both the floral mixture and linalool. Prolonged exposure (5 and 10 second durations) to odorants caused continuous increased activity in the antennae, with a more pronounced response observed in the COVID VOCs and linalool. These results demonstrate that moth antennas are a suitable model for the construction of highly accurate and efficient biosensors, and support the feasibility of implementing them in devices aimed at detecting and identifying substances of interest. Future work will explore additional COVID-associated compounds and apply data to an algorithm for machine learning software to enhance capabilities for disease diagnosis.