Prime editing systems have enabled the incorporation of precise edits within a genome without introducing double strand breaks. With the versatile ability to introduce point mutations, deletions and insertions, prime editors have the ability to correct around 89% of known genetic variants associated with human diseases. However, there are several bottlenecks currently restricting prime editing activity that need to be addressed to further their use as therapeutics. In the first half of this thesis, we address the auto-inhibitory interaction between the PBS and the spacer sequence that affects pegRNA binding efficiency and target recognition. We show that destabilizing this auto-inhibitory interaction by reducing the complementarity between the PBS-spacer region enhances prime editing efficiency. These design parameters were initially fueled by our goal to improve prime editor ribonucleoprotein activity where the auto-inhibitory interaction of the pegRNA is more prominent, but we show that they can be applied to multiple prime editing formats to increase editing rates. In the case of end-protected pegRNAs, we discover that a shorter PBS length with a PBS-target strand melting temperature near 37°C is optimal in mammalian cells. Additionally, we show that a transient cold shock treatment of the cells post PE-pegRNA delivery further increases prime editing outcomes for pegRNAs with optimized PBS lengths. In the first study, we noticed that the prime editor protein had the tendency to aggregate during purification procedures and that the editing rates were still modest in primary cells. MMLV-reverse transcriptase - the prime editor polymerase subunit - requires high intracellular dNTPs levels for efficient polymerization. Prior optimization of the system has been performed in rapidly dividing cell lines like HEK293Ts where dNTP concentration is not a limiting factor. Primary cells that are quiescent or slowly proliferating have tightly regulated intracellular dNTP levels that could limit the reverse transcription process. Therefore, in the second half of this thesis, we address two more bottlenecks of prime editing - solubility of the prime editor protein and the intracellular dNTP concentration. To address that, in the reverse transcriptase domain, we introduced the L435K mutation that improves the solubility of the protein. Additionally, we introduced a V223M mutation that changes the active site of the reverse transcriptase to resemble a lentiviral enzyme that is more efficient in non-dividing cells. We show that this rationally engineered prime editor variant with increased solubility and lower Km to dNTPs, increases editing rates across diverse cell types and in vivo. Finally, we show that targeted SAMHD1 degradation by co-delivery of VPX to increase dNTP concentration in the cell further increases prime editing rates. We believe that addressing these bottlenecks, with the recommendations we describe in this thesis, will contribute to the advancement of prime editor ribonucleoproteins and mRNA for in vivo and ex vivo therapeutics.

We explored patterns of COVID-19 vaccination across pediatric visit types using electronic health record data from 7/1/2021 through 7/25/2022 in a pediatric safety-net clinic. We generated frequencies and descriptive statistics for patient demographic and vaccine administration variables. Analyses were stratified into age subgroups of 5-to-11-year-olds and 12- to-17-year-olds. 1,409 children received at least one dose of the COVID-19 vaccine and 2,197 doses were administered in this first year of vaccine delivery. Most vaccines given were first doses in the series (45%), followed by second doses (38%), and then booster doses (17%). First doses tended to be given at well-child (42%) or nurse visits (48%), while second doses were almost entirely given at nurse visits (87%) and booster doses at well-child visits (58%). Efforts to optimize COVID-19 vaccination could leverage clinic workflow systems to provide reminder prompts for vaccination for scheduling future doses and identify strategies to facilitate vaccination at non-well child visits, particularly for booster doses.

Engulfment of cellular material and proteins is a key function for microglia, a resident macrophage of the central nervous system (CNS). Among the techniques used to measure microglial engulfment, confocal light microscopy has been used the most extensively. Here, we show that autofluorescence (AF) likely due to lipofuscin (lipo-AF) and typically associated with aging, can also be detected within microglial lysosomes in the young mouse brain by light microscopy. This lipo-AF signal accumulates first within microglia and it occurs earliest in white versus gray matter. Importantly, in gray matter, lipo-AF signal can confound the interpretation of antibody-labeled synaptic material within microglia in young adult mice. We further show that there is an age-dependent accumulation of lipo-AF inside and outside of microglia, which is not affected by amyloid plaques. We finally implement a robust and cost-effective strategy to quench AF in mouse, marmoset, and human brain tissue.

Cancer cells exhibit elevated metabolic demands, imposing a need for metabolic reprogramming. The aim of the thesis is to identify a targetable metabolic vulnerability using an approach that leverages the altered pathways in cancer cells to induce the accumulation of inherently toxic metabolites to eliminate cancer cells selectively. Through a systematic analysis of transcriptomics and cancer dependency data, we identified UXS1, a Golgi enzyme responsible for converting UDP-glucuronic acid (UDPGA) to UDP-xylose that is conditionally essential in cells expressing high levels of its upstream enzyme UGDH. Here, we demonstrate that UGDH high cancer cells are dependent on UXS1 to prevent excess buildup of UDPGA, generated by UGDH. Excess UDPGA causes disruption of the structure and function of the Golgi, leading to aberrant protein glycosylation and improper protein trafficking of critical glycoproteins within cancer cells. We find that UGDH expression is elevated in various cancers, including lung adenocarcinoma and breast carcinoma. Furthermore, elevating UGDH expression is beneficial to cancer cells, because UDPGA functions as a substrate in the detoxification of chemotherapeutic agents. Therefore, chemo-resistant cells upregulate UGDH expression, enhancing their susceptibility to UXS1 ablation. Consequently, this study reveals the therapeutic potential of targeting UXS1 in cancer treatment, offering a novel approach to exploit the metabolism of sugar nucleotides in cancer cells.

Translation of mRNA into protein is terminated when the ribosome encounters one of the three stop codons (UAA, UAG, and UGA) at the end of an open reading frame (ORF). Infrequently, stop codons are decoded by a near- cognate tRNA, allowing “readthrough” of the stop codon and synthesis of an extended polypeptide. When termination occurs prematurely, the mRNA is degraded by the nonsense-mediated mRNA decay (NMD) pathway. Premature and normal termination appear to differ in their efficiency, but the exact “rules” of how NMD distinguishes them mechanistically remain to be elucidated. Using ribosome profiling and bioinformatics analyses, this study aims to understand, at a transcriptome-wide level, the cis-acting elements that influence termination efficiency and how premature termination is recognized by Upf1, a key NMD factor. Analyses of yeast and human mRNA sequences in both normal and readthrough- inducing conditions revealed largely conserved roles of identities of the stop codon, the following nucleotide, P-site codon, and 3’-UTR length in readthrough efficiency regulation. The analyses of yeast mRNAs associated with Upf1-bound ribosomes demonstrated that Upf1 binds ribosomes in two distinct complexes across all mRNA ORFs, suggesting that Upf1 associates with the ribosome during translation elongation before premature termination takes place. Together, these results provide insights into the regulation of termination and the early steps of NMD at the transcriptome-wide level.

Introduction: Integrating equity considerations into bicycle infrastructure, planning, and programming is essential to increase bicycling and reduce physical inactivity-related health disparities. However, little is known about communities' experiences with activities that promote equity considerations in bicycle infrastructure, planning, and programming or about barriers and facilitators to such considerations. The objective of this project was to gain in-depth understanding of the experiences, barriers, and facilitators that communities encounter with integrating equity considerations into bicycle infrastructure, planning, and programming. Methods: We administered a web-based survey in 2022 to assess communities' experiences with 31 equity-focused activities in 3 areas: 1) community engagement, education, events, and programming (community engagement); 2) data collection, evaluation, and goal setting (data); and 3) infrastructure, facilities, and physical amenities (infrastructure). Respondents were people who represented communities in the US that participated in the League of American Bicyclists' Bicycle Friendly Community (BFC) Program. We then conducted 6 focus groups with a subset of survey respondents to explore barriers and facilitators to implementing equity-focused activities. Results: Survey respondents (N = 194) had experience with a mean (SD) of 5.9 (5.7) equity-focused activities. Focus group participants (N = 30) identified themes related to community engagement (outreach to and engagement of underrepresented communities, cultural perceptions of bicycling, and funding and support for community rides and programs); data (locally relevant data); and infrastructure (political will, community design, and infrastructure). They described barriers and facilitators for each. Conclusion: Communities are challenged with integrating equity into bicycle infrastructure, planning, and programming. Multicomponent strategies with support from entities such as the BFC program will be required to make progress.

Phosphaturic mesenchymal tumors are rare, usually benign neoplasms that occur in the soft tissue or bone and are the cause of nearly all cases of tumor-induced osteomalacia. Tumor-induced osteomalacia due to phosphaturic mesenchymal tumor is a challenging diagnosis to make-patients present with variable clinical and radiologic findings and the culprit neoplasm is often small and can occur anywhere head to toe. We present two cases of phosphaturic mesenchymal tumor in the scapular body and plantar foot. In both cases, the patient endured years of debilitating symptoms before a tissue diagnosis was eventually reached. Descriptions of clinical presentation, laboratory workup, surgical resection, and imaging characteristics, with a focus on CT, MRI, and functional imaging, are provided to assist with the diagnosis and management of this rare entity. A brief review of current literature and discussion of the differential diagnoses of phosphaturic mesenchymal tumor is also provided.

A long history of discriminatory policies in the United States has created disparities in neighborhood resources that shape ethnoracial health inequities today. To quantify these differences, we organized publicly available data on forty-two variables at the census tract level within nine domains affected by structural racism: built environment, criminal justice, education, employment, housing, income and poverty, social cohesion, transportation, and wealth. Using data from multiple sources at several levels of geography, we developed scores in each domain, as well as a summary score that we call the Structural Racism Effect Index. We examined correlations with life expectancy and other measures of health for this index and other commonly used area-based indices. The Structural Racism Effect Index was more strongly associated with each health outcome than were the other indices. Its domain and summary scores can be used to describe differences in social risk factors, and they provide powerful new tools to guide policies and investments to advance health equity.

Background: Anticoagulants including direct oral anticoagulants (DOACs) are among the highest-risk medications in the United States. We postulated that routine consultation and follow-up from a clinical pharmacist would reduce clinically important medication errors (CIMEs) among patients beginning or resuming a DOAC in the ambulatory care setting. Objective: To evaluate the effectiveness of a multicomponent intervention for reducing CIMEs. Design: Randomized controlled trial. Participants: Ambulatory patients initiating a DOAC or resuming one after a complication. Intervention: Pharmacist evaluation and monitoring based on the implementation of a recently published checklist. Key elements included evaluation of the appropriateness of DOAC, need for DOAC affordability assistance, three pharmacist-initiated telephone consultations, access to a DOAC hotline, documented hand-off to the patient's continuity provider, and monitoring of follow-up laboratory tests. Control: Coupons and assistance to increase the affordability of DOACs. Main measure: Anticoagulant-related CIMEs (Anticoagulant-CIMEs) and non-anticoagulant-related CIMEs over 90 days from DOAC initiation; CIMEs identified through masked assessment process including two physician adjudication of events presented by a pharmacist distinct from intervention pharmacist who reviewed participant electronic medical records and interview data. Analysis: Incidence and incidence rate ratio (IRR) of CIMEs (intervention vs. control) using multivariable Poisson regression modeling. Key results: A total of 561 patients (281 intervention and 280 control patients) contributed 479 anticoagulant-CIMEs including 31 preventable and ameliorable ADEs and 448 significant anticoagulant medication errors without subsequent documented ADEs (0.95 per 100 person-days). Failure to perform required blood tests and concurrent, inappropriate usage of a DOAC with aspirin or NSAIDs were the most common anticoagulant-related CIMEs despite pharmacist documentation systematically identifying these issues when present. There was no reduction in anticoagulant-related CIMEs among intervention patients (IRR 1.17; 95% CI 0.98-1.42) or non-anticoagulant-related CIMEs (IRR 1.05; 95% CI 0.80-1.37). Conclusion: A multi-component intervention in which clinical pharmacists implemented an evidence-based DOAC Checklist did not reduce CIMEs. Nih trial number: NCT04068727.

The formation, expansion, and pruning of synapses, known as structural synaptic plasticity, is needed for learning and memory, and perturbation of plasticity is associated with many neurological disorders and diseases. Previously, we observed that the Drosophila homolog of Activity-regulated cytoskeleton-associated protein (dArc1), forms a capsid-like structure, associates with its own mRNA, and is transported across synapses. We demonstrated that this transfer is needed for structural synaptic plasticity. To identify mRNAs that are modified by dArc1 in presynaptic neuron and postsynaptic muscle, we disrupted the expression of dArc1 and performed genomic analysis with deep sequencing. We found that dArc1 affects the expression of genes involved in metabolism, phagocytosis, and RNA-splicing. Through immunoprecipitation we also identified potential mRNA cargos of dArc1 capsids. This study suggests that dArc1 acts as a master regulator of plasticity by affecting several distinct and highly conserved cellular processes.