We analyze the International Study of Asthma and Allergies in Childhood (ISAAC) data for Seattle and apply statistical clustering methods to identify asthma phenotypes. This study focuses on Phase One of ISAAC, conducted between 1994 and 1995, in which approximately 3,000 adolescent asthma patients and their parents filled out detailed questionnaires about asthma, rhinitis and eczema symptoms. Asthma is a heterogeneous disease, meaning it has a highly variable clinical presentation, so in order to identify distinct phenotypes, we apply a hierarchical bottom-up clustering method on categorical variables from the questionnaire and verified cluster stability using various linkage methods. The clusters we obtained were distinguished primarily by differences in the severity of respiratory symptoms and the presence of eczema symptoms. After assessing the accuracy of several alternative clustering methods, we conclude by comparing the clusters identified by this analysis to clinically recognized asthma phenotypes. Accurate characterization of asthma phenotypes is important for informing management and treatment strategies for urban adolescents with asthma. Techniques that identify severe phenotypes in population data sets can help target treatment to those who may benefit from high-intensity treatment regimens, careful attention to potential exposures to environmental allergens, and specialist level care.

Type 1 diabetes (T1D) is an autoimmune disease in which the pancreas is unable to produce enough insulin to effectively regulate sugar into the body’s cells. Recent literature suggests that T1D has a prevalence and incidence of 9.5% and 15 per 100,000 people respectively. T1D risk is multifactorial but is heavily dependent on genetics and the environment. Twin studies have shown that although disease risk for the general population is 0.4%, children of diabetic patients are 2 to 9% at risk and identical twins are up to 70% at risk of developing T1D. Genome-wide association studies have shown that many genetic variants associated with T1D risk are found in gene regulatory regions such as enhancer elements. As T1D is an autoimmune disease with a strong genetic component particularly in non-coding, regulatory regions, it follows that thorough genomic profiling of immune cells such as T cells can help identify functional non-coding genetic variants that alter gene regulation for target genes associated with disease risk. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) allows for unbiased identification of cis-regulatory elements (CREs). By utilizing ATAC-seq on various T cell types of both healthy control donors and T1D patients, chromatin accessibility can be collected, and the underlying sequence data can be used to determine allelic differences in transcription factor binding due to T1D-associated genetic variants. I am currently applying ATAC-seq to three T cell subtypes isolated from both donor cohorts. Through these data functional links will be made between non-coding genetic variants and associated target genes to better understand how they impact disease risk in T1D.

Decades of research have demonstrated that RNA molecules can serve as architectural scaffolds, templating the assembly of subcellular compartments in all kingdoms of life. In mammals, architectural RNAs scaffold an array of subnuclear structures that are essential to cellular function including metabolism, DNA repair, and epigenetic programming. Many of these architectural RNAs are causally dysregulated in diseases, including cancer and neurodegenerative disorders. However, the molecular mechanisms of these architectural RNAs remain poorly understood, partially because technologies for identifying the molecules (proteins, DNAs, other RNAs), with which RNAs interact are lacking. To address this challenge, the Shechner Lab has developed a technology termed Oligonucleotide-Directed Biotinylation (ODB), a universal method for elucidating RNA subcellular interactions. ODB applies a powerful method called proximity-biotinylation to individual RNAs. In proximity-biotinylation, a promiscuous biotinylating enzyme (e.g. Horseradish Peroxidase, HRP) is targeted to a subcellular compartment of interest. This enzyme then tags nearby (~10 nm) molecules with biotin, enabling their straightforward isolation and analysis. ODB advances this technology by using RNA-in situ Hybridization (RNA¬–FISH) methods to precisely deploy HRP to individual RNAs. Ongoing work, using a series of model RNA targets, has demonstrated that ODB can reveal the proteins, RNAs, and genomic loci near a target RNA at exceptional depth and precision. My goal is to generalize this ODB protocol, developing methods that can be applied to any RNA target. To examine how RNA abundance influences ODB experimental design, I use pulse-chase, RNA decay experiments to manipulate the expression of the architectural RNA NEAT1, and examine how to adjust parameters of the ODB protocol to compensate for this altered expression. Likewise, I am using imaging-based assays to investigate how ODB's biotinylation radius varies with labeling conditions, increasing the range of ODB’s target radii. Collectively, this establishes general guidelines for adapting ODB to novel RNA targets.

Freshwater wetlands are the largest natural source of methane, a powerful greenhouse gas (GHG), to the atmosphere. However, the mechanisms and magnitude of wetland methane (CH₄) emissions are not well understood. There is significant variability of flux estimates between field sites and a lack of agreement between top-down and bottom-up estimations of methane flux, indicating a high level of uncertainty. This study provides the first estimation of aquatic methane flux in the Union Bay Natural Area (UBNA). This site can be categorized as a freshwater wetland and is an urban site that is adjacent to a former landfill. We hypothesize that the flux will be dominated by ebullition (bubble released) emissions and that the total emissions will decrease as depth increases. To measure the total CH₄ flux, we measured diffusive emissions using a flux chamber and LGR portable greenhouse gas analyzer and ebullition emissions with bubble traps that were left in the field site for 48 hours. In addition to these measurements we collected water parameter measurements using an Exo sonde and dissolved gas samples at each diffusive sampling location. With this data, we were able to calculate the measured CH₄ flux and model the flux so that the two values could be compared. This study indicates the spatial variability and depth-dependent patterns of methane flux in the UBNA. There are multiple possible extensions of the research. If more sampling campaigns were conducted throughout the year, the data could be used to describe temporal variability in flux. Additionally, if gas samples are collected from the ventilation systems on the landfill, an isotopic analysis could reveal the percentage of methane emissions that can be attributed to decomposition in the landfill.

The alpine zone (i.e., area above treeline) is Washington's most pristine habitat due to minimal human disturbance. However, plant species richness and the distribution of those species (collectively floristics) are poorly documented by herbarium specimens due to sheer spatial scale, physically challenging access, and a short (10-12-week) growing season. The goal of this project is to generate baseline data for future studies examining climate change impacts on alpine plant communities, improving distribution data for alpine species in Washington, and locating new populations of rare plant species. In 2021 we initiated a 5-year project to conduct comprehensive botanical surveys on 50 alpine peaks in the Cascades Range between the Goat Rocks Wilderness and the Canadian border in Washington. Peaks were selected on the basis of accessibility (i.e., do not require technical climbing to access the summit), elevation (summit area is exposed), latitude (dispersed between the north and south ends of the sampling area) and position relative to the Cascades Range crest (representation of both East Cascades and West Cascades peaks). In 2021 we sampled 11 peaks. Species lists for each peak were generated from historic herbarium specimens, collecting specimens during field work, and generating lists on the basis of sight identification of specimens in the field. We collected at least one specimen of each species, subspecies, or variety of plants encountered in the field. Here I present my analysis of the data examining the frequency distribution of species across sampled peaks, dispersal mechanisms, and flower color, and report notable species range extensions and new rare plant populations.  This analysis can be used as a comparison against future collections in new sites or collections at the same sites in the future to observe the effects of climate change on alpine plant populations.

Mycobacterium abscessus (MABSC) is a species of rapidly growing nontuberculous mycobacteria (NTM) that is most frequently encountered in human NTM infections and is very difficult to treat. Active MABSC disease commonly emerges from pulmonary infections, to which populations with underlying lung disease and depressed immune systems are most susceptible. There is no official standard of care for MABSC infections and current treatment regimens lack efficacy and are met by challenges of intrinsic and acquired resistance mechanisms. Studies evaluating pulmonary disease outcomes report unsatisfactory treatment success rates of approximately 45%. Thus, there is an urgent clinical need for novel antibacterial agents and drug combinations to efficiently and effectively cure MABSC infections. Repurposing FDA-approved drugs can help us economically discover new treatment options with a shorter drug development time, which is critical for antibiotics as the emergence of resistance often outpaces drug development. We approached the repurposing of approved drugs to treat MABSC infections through the whole-cell screening of a drug library of about 2400 FDA-approved compounds and clinical molecules, followed by the selection and characterization of the activity of the most promising hits. Our data revealed a novel antiemetic compound, netupitant, that exhibited a potent anti-MABSC activity (MIC 4-16 µg/ml) and was able to interact synergistically with standard first-line MABSC therapeutics. Netupitant has a good safety profile and accumulates preferentially in lung tissues, making it suitable for treating pulmonary MABSC infections. Additionally, our screen revealed two promising antibiotic drug combinations: eravacycline/clarithromycin and bedaquiline/amikacin that were able to exhibit potent synergistic interactions against clinical MABSC isolates, as determined by checkerboard microdilution assays. Further mechanistic and in vivo studies are needed to evaluate these hits as potential treatment options to improve the current clinical outcomes for MABSC patients.

From their ability to perch perfectly on nearly any surface to their capability of balancing on any perch, birds are phenomenal animals. Avian anatomy includes mysterious features like the lumbosacral organ (LSO) within the spinal cord. Research surrounding the LSO has led to the discovery of a mysterious network of nerve fibers that seem to cross the midline of the spinal cord only in the LSO. The goal of my research was to identify the neuron cell bodies giving rise to these axons and to track their targets. To do so, I used immunohistochemistry (IHC) and tracing. A tracer was injected into the glycogen body, where the surrounding neurons took up the tracer and transported it both retrogradely, toward the cell body, and in the anterograde direction, toward the axon endings. Tracing revealed the structure of the circuit. IHC was subsequently used to test if proteins indicative of sensory nerves were present in the glycogen body, such as CGRP and substance P. These methods, thus far, have indicated the presence of CGRP and substance P in nerve fibers across the glycogen body. These findings suggest the mysterious nerve network in the LSO is a network of sensory neurons abundant in substance P and CGRP, also implicating the glycogen body as a facilitator for the passing of these nerve fibers.

Between 1946 and 1958, the United States conducted 67 nuclear tests in the Marshall Islands, a country in Oceania composed of volcanic islands and coral atolls. The Burke Museum holds biological specimens collected from the Marshall Islands as part of a project sponsored by the Atomic Energy Commission. I am locating, contextualing, and digitizing these specimens in order to democratize access to this biological and cultural information. I am locating as many specimens as possible in the Burke Museum collected during and after this nuclear testing period, beginning in the Herbarium. I am then digitizing these specimens by imaging, databasing, and georeferencing them. There was also an opportunity to test these samples for radiation. I am collaborating with colleagues to contextualize the specimens culturally, and bring attention to the University’s involvement in this part of our nation’s history. Overall, we aim to make this information freely available online so that communities have access to knowledge and resources to inform their decisions. Early investigation suggests that samples exist in at least the ornithological, invertebrate, and botanical (Herbarium) collections, but the total number of specimens remains unknown. This is an opportunity to unite multiple departments in the Burke Museum as we make connections across cultural and biological collections. Ultimately, this research addresses the ethical implications of the University’s scientific past and makes the specimen data of the Burke Museum more accessible.

Bats are the only mammals capable of powered flight and have correspondingly specialized body plans, apparent in the limbs. These specialized morphologies are thought to be the result of adaptations for the demands of flight; the skeletal elements of the bat forelimbs are elongated in order to support flight membranes and increase aerodynamic efficiency, whereas bat hind limbs are relatively short and specialized for hanging and catching prey in flight. Due to a deficient fossil record, the evolution of bat flight is still not fully understood but is hypothesized to be the result of an ancestral transition from gliding to flying. This hypothesis is plausible considering the morphological similarities between bat and glider forelimbs (both elongated) and the contrast between bat and glider hind limbs (shorter versus elongated). In this study, I collected linear measurements of the forelimb and hind limb skeletal elements of bats to add to a dataset of gliding, arboreal, and terrestrial mammals. I then fit evolutionary models to the data to test the hypothesis that A) selective pressures for flight drove the evolution of bat forelimb skeletal elements from glider-like forelimbs and that B) bat hind limbs evolved to become morphologically distinct from those of other mammals. Based on this hypothesis, I predict that A) bat and glider forelimb trait optima will fall progressively farther from arborealist optima and B) bat hind limb trait optima will be located in a unique region of morphospace. Preliminary results show that forelimb long bone lengths have evolved to be progressively longer from arborealists to gliders to flyers, supporting my hypothesis. This research helps address the longstanding question of how bats may have evolved flight from ancestral gliding mammals.

Nemaline Myopathy (NM) is a severe genetic muscle disorder defined by muscle weakness and the presence of nemaline rods (rod-shaped intracellular aggregates). This disease is associated with multiple clinical subtypes that result from pathogenic genetic variants across 12 different genes, including skeletal 𝛼-actin (ACTA1). NM-associate mutation H40Y impacts the ACTA1 monomer structure such that it disrupts polymerization. Without efficient and accurate polymerization, ACTA1 monomers form altered protofilaments which do not properly support the cross-bridge cycle and result in contractile dysfunction. The variation and complexity of NM pathology coupled with the rarity of this disease have served as significant barriers to the development of any treatments for NM. Here we show the effects of top performing therapeutic small molecules simulated in the presence of NM-associated mutation H40Y on the structural and mechanical properties of ACTA1. Using Molecular Dynamics simulation data, we have quantified differences between H40Y and wildtype ACTA1. Furthermore, we searched for and designed a target small molecule to fix mutant actin polymerization and mechanical instability. Our results demonstrate how our lead designed small molecule alters the dynamics of the H40Y ACTA1 pentamer when simulated docked in its intended binding pocket. We anticipate that our best small molecule candidate will be tested in vitro for its ability to impact actin polymerization in polymerization assays produced from induced pluripotent stem cells bearing the H40Y mutation. Furthermore, following successful in vitro validation the small molecule may be extensively studied as a potential novel therapy for NM.

In the United States, 1.5 million individuals suffer a fracture due to bone disease each year. It is well documented that mechanical load affects bone development, but our understanding of the cellular mechanisms behind bone development under load is limited. Current human induced pluripotent stem cell (hiPSC) derived bone tissue models have more relevant human physiology compared to traditional animal models. However, there is a lack of dynamically loaded hiPSC bone tissue and diseased hiPSC bone tissue models in vitro. We propose a novel, three-dimensional bone tissue model as a platform for musculoskeletal disease modeling that allows for compressive loading that will enhance maturity as well as induce diseased bone phenotypes. We improved upon existing poly-L-lactide solvent cast scaffold techniques by incorporating a polyvinyl alcohol mold and an annealing step that increases the uniformity of the scaffolds and allows for higher throughput fabrication. Osteoblasts were derived from hiPSCs using established differentiation protocols and seeded into the 3D, porous, poly-L-lactide scaffold to generate in vitro bone tissue that generates significant extracellular calcium. We propose an arduino-powered, 3D-printed loading device that can apply physiologically relevant dynamic loads to the scaffold and hypothesize improved bone tissue maturity in comparison to 2D cultures and unloaded 3D scaffolds. By screening for markers of early bone development such as type I collagen, markers of later development such as osteocalcin, and assays for extracellular calcium, we can track the maturity and development of bone tissue. We expect that 3D bone growth with static loading will reveal diseased bone phenotypes such as decreased calcium deposition and immature bone, whereas dynamic loading will promote bone growth and lead to mature bone. Ultimately, this model will improve our ability to investigate the effects of mechanical loading in developing and diseased bone.

Cardiomyopathy is currently the leading cause of death for patients with Duchenne muscular dystrophy (DMD), a severe neuromuscular disease affecting young boys. With no current cure, gene therapy is a promising solution, but supplementation with drug therapies is likely inevitable to fully address the pathology seen in older patients. The use of human-induced pluripotent stem cell (hiPSC) models for drug studies is beneficial due to the direct relevance to human physiology and the potential development of personalized care. Dystrophic hiPSC cardiomyocytes have been shown to exhibit calcium reuptake delays, higher resting calcium levels, and frequent arrythmias. The Mack Lab previously conducted a preliminary drug screen on healthy and DMD-affected cardiomyocytes and found that certain L-type calcium channel blockers (CCBs) indicated a cardioprotective effect. These drug compounds (namely Nitrendipine and Nimodipine) have been shown to alleviate cardiac fibrosis in patients through vasodilation. In this project, I am validating the beneficial aspects of the drug compounds. I initially hypothesized that treatment of DMD hiPSC cardiomyocytes with L-type CCBs will rescue resting calcium levels and normalize relaxation kinetics. To assess this, I cultured mature hiPSC cardiomyocytes on a Microelectrode Array (MEA) system capable of maintaining physiological conditions while measuring properties of cardiac electrophysiology. To enhance cellular maturity, I treated hiPSC cardiomyocytes with MicroRNA Maturation Cocktail (MiMaC) and cultured long-term prior to MEA assessment. Using the MEA, I have found that the QT interval for DMD hiPSC cardiomyocytes was significantly longer than isogenic controls. In current experiments, I am using this platform to validate the effect of L-type CCB compounds of interest in relation to DMD cardiomyopathy. The development of this novel platform may not only have broader implications for DMD drug discoveries and targeted therapies, but it can potentially serve as a powerful preclinical model for other neuromuscular disorders.

Atherosclerosis is the build-up of cholesterol, fats and other substances in artery walls. These builds-ups (plaques) can cause vessel blockages, leading to heart attacks and strokes. Removal of cholesterol from plaques is facilitated by the physiologic process of reverse cholesterol transport (RCT). In RCT, apolipoprotein AI (apoAI) protein removes cholesterol from vascular tissue, forming high-density lipoprotein (HDL) that is transported to the liver for excretion. Gene-therapy strategies that use vectors to increase apoAI expression in arteries slow and reverse atherosclerosis in rabbits. However, because apoAI has multiple activities including reducing inflammation, it is unclear whether vector-derived apoAI reduces atherosclerosis by contributing to RCT. To determine whether apoAI gene therapy contributes to RCT, I am developing a method that can track HDL that contains vector-derived apoAI in vivo. If we can detect vector-derived apoAI/HDL in treated arteries as well as plasma and liver, we will conclude that vector-derived apoAI contributes to RCT. I am first developing methods that purify and detect endogenous apoAI/HDL; these methods will be applied to detect vector-derived apoAI/HDL (the vector-derived apoAI will have a molecular tag). I used density-gradient ultracentrifugation to isolate HDL from pulverized rabbit tissue and plasma, then immunoblotting to detect apoAI/HDL. I detected abundant apoAI/HDL in plasma, but not in aorta and liver. Immunoblots of artery extracts (before ultracentrifugation) detect apoAI, suggesting that apoAI is lost during ultracentrifugation. I am testing whether other methods can purify apoAI/HDL from aorta and liver (e.g. affinity chromatography to isolate apoAI from tissue extracts, followed by size-exclusion chromatography to isolate apoAI/HDL). After I develop a method to isolate apoAI/HDL from tissue, we will treat rabbit arteries with a tagged apoAI vector and measure tagged apoAI/HDL in arteries, plasma, and liver. Verifying how vector-derived apoAI works will help us to improve its atheroprotective effects.

Human Herpesvirus-8 (HHV-8) causes Kaposi’s sarcoma (KS)-- a cancer of cells that line lymph or blood vessels. Individuals whose T cells have been compromised by HIV are particularly at risk for KS. The disease remains endemic in many parts of sub-Saharan Africa, making KS a leading cause of cancer death in Uganda. While HHV-8 has been known to cause KS since 1994, and T-cells that can recognize HHV-8 are likely to be critical for control of KS, there is little known about the specific parts of the virus recognized by T-cells. Our goal is to ultimately determine the antigenic targets of HHV-8-specific T-cells. Because recovery of live T-cells from biopsies is challenging, we are re-creating “artificial T-cells” with candidate T-cell receptor (TCR) sequences obtained from KS lesion biopsies from Uganda. These candidate TCRs will be queried for reactivity to HHV-8. To do this, we are generating a set of every known HHV-8 protein by moving cloned DNA expressing these proteins into a specific plasmid that is useful for these T-cell studies. To create artificial T-cells, we are cloning candidate TCRs into lentiviral vectors which allow us to force the TCRs to be expressed in these cells and fluoresce green if the cells recognize a viral protein through their TCRs. These artificial T-cells are then screened against every HHV-8 protein to find which protein they recognize. Currently, flow cytometry results demonstrate adequate expression of candidate TCRs by artificial T-cells, and the HHV-8 protein set is >90% complete. Ultimately, we hope to determine HHV-8 proteins that are recognized by HHV-8-specific T-cells in order to better understand which parts of the virus are targeted by these T-cells and to design T cell or vaccine therapies to treat persons with KS.

Following allogenic hematopoietic cell transplantation (allo-HCT), patients are at risk of developing graft-versus-host disease (GvHD) in which donor T-cells attack the recipient’s healthy tissues, including in the gut leading to inflammation, diarrhea, and sometimes death. Low gut bacterial diversity in the host has been associated with GvHD severity. Anaerobic bacteria in the Lachnospiraceae family, specifically Blautia species, have been associated with reduced GvHD related mortality. Using family and genus specific qPCR assays, we quantified the bacterial concentrations of Lachnospiraceae and Blautia species pre- and post-transplant in patients. We extracted DNA from stool samples of 306 HCT patients. Samples were collected pre-transplant and post-transplant at day 30 and day 60. GvHD gut stage was graded 0-1 (none-mild), 2-4 (moderate-severe). Quantitative PCR assays were designed using primers targeting specific regions of the bacterial 16S rRNA gene. Amplicon specificity was confirmed using post-run melt curve analysis. There was a significantly higher (p=0.016) concentration of Lachnospiraceae in patients presenting none-mild GvHD (6.78x10⁷ copies per swab) versus moderate-severe gut GvHD (1.84x10⁷) at day 60. A similar association for Lachnospiraceae was trending (p=0.088) at day 30. In addition, there was significantly higher concentrations of Blautia species in patients with none-mild (3.33x10⁶ at d30, 1.05x10⁷ at d60) versus moderate-severe gut GvHD (1.35x10⁵ at d30, 1.41x10⁶ at d60) at both day 30 (p=0.026) and day 60 (p=0.014) post allo-HCT. There were lower concentrations of both Lachnospiraceae (1.89x10⁷) and Blautia (2.30x10⁶), regardless of GvHD stage, at day 30 versus pre-transplant (p<0.0001) or day 60 (p<0.0001), likely reflecting the impact of antibiotic treatment during neutropenia immediately following allo-HCT. Higher concentrations of Lachnospiraceae and Blautia in the gut following transplant were associated with less severe gut GvHD; these bacteria could be markers or drivers of less severe GvHD.

Opiate overdose deaths in the US continue to increase at an alarming rate, yet we still only have a partial understanding of the neural mechanisms that regulate opiate addiction. The paraventricular thalamus (PVT) and nucleus accumbens (NAc) are regions of the brain that have both been implicated in addiction to drugs of abuse. Prior studies show that the somatic effects of opioid withdrawal are mediated by excitatory input from the PVT to the NAc. Recently, cannabinoid compounds such as tetrahydrocannabinol (THC), the principle psychoactive constituent of marijuana, has been shown to alleviate effects of opioid withdrawal. Our goal for this project was to determine how cannabinoids regulate the PVT-NAc circuit to modulate these opiate withdrawal symptoms. This study had two principal aims: 1. Understanding the physiological mechanisms by which cannabinoids can modulate PVT-NAc circuits, and 2. Using in-vivo photometry and optogenetics to determine whether we can treat the behavioral effects of opiate withdrawal by using cannabinoid compounds to inhibit the PVT-NAc circuit pathway (n=10 animals, age 12-24 weeks). We first demonstrated that a genetically defined population of anterior PVT (aPVT) neurons expressing the neuropeptide Neurotensin (NTS) sends excitatory projections to the NAc, which are regulated by cannabinoid signaling. Using in-vivo fiber photometry to record activity of aPVT to NAc projections, I showed that rewarding stimuli inhibits the circuit, whereas painful stimuli (such as heat from a hotplate) activate the circuit. Administration of morphine was sufficient to block the pain-induced activation of the aPVT-NAc circuit, and caused robust analgesia. Furthermore, following chronic morphine administration, I used naloxone to precipitate opiate withdrawal, and demonstrated that enhancing cannabinoid signaling was capable of reducing some of the symptoms of opiate withdrawal. Future studies will aim to understand the causal relationship between cannabinoid modulation of the aPVT-NAc circuit and the development of opiate withdrawal.