Now showing items 1-20 of 5111

    • Functional genomics reveals an off-target dependency of drug synergy in gastric cancer therapy [preprint]

      Leylek, Ozen; Honeywell, Megan E; Lee, Michael J; Hemann, Michael T; Ozcan, Gulnihal (2023-11-19)
      The rational combination of anticancer agents is critical to improving patient outcomes in cancer. Nonetheless, most combination regimens in the clinic result from empirical methodologies disregarding insight into the mechanism of action and missing the opportunity to improve therapy outcomes incrementally. Deciphering the genetic dependencies and vulnerabilities responsible for synergistic interactions is crucial for rationally developing effective anticancer drug combinations. Hence, we screened pairwise pharmacological interactions between molecular-targeted agents and conventional chemotherapeutics and examined the genome-scale genetic dependencies in gastric adenocarcinoma cell models. Since this type of cancer is mainly chemoresistant and incurable, clinical situations demand effective combination strategies. Our pairwise combination screen revealed SN38/erlotinib as the drug pair with the most robust synergism. Genome-wide CRISPR screening and a shRNA-based signature assay indicated that the genetic dependency/vulnerability signature of SN38/erlotinib is the same as SN38 alone. Additional investigation revealed that the enhanced cell death with improved death kinetics caused by the SN38/erlotinib combination is surprisingly due to erlotinib's off-target effect that inhibits ABCG2 but not its on-target effect on EGFR. Our results confirm that a genetic dependency signature different from the single-drug application may not be necessary for the synergistic interaction of molecular-targeted agents with conventional chemotherapeutics in gastric adenocarcinoma. The findings also demonstrated the efficacy of functional genomics approaches in unveiling biologically validated mechanisms of pharmacological interactions.
    • Addressing Bottlenecks of Prime Editing Through Improved pegRNA Designs and Rationally Engineered Prime Editor Variants

      Ponnienselvan, Karthikeyan (2023-11-15)
      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.
    • Open science discovery of potent noncovalent SARS-CoV-2 main protease inhibitors

      Boby, Melissa L; Fearon, Daren; Ferla, Matteo; Filep, Mihajlo; Koekemoer, Lizbé; Robinson, Matthew C; Chodera, John D; Lee, Alpha A; London, Nir; von Delft, Annette; et al. (2023-11-10)
      We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.
    • Trends in COVID-19 vaccine administration across visit types in a safety net pediatric practice during the first year of authorization

      Ryan, Grace W; Goulding, Melissa; Beeler, Angela L; Nazarian, Beverly L; Pbert, Lori; Rosal, Milagros C; Lemon, Stephenie C (2023-11-06)
      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.
    • Distinct members of the C. elegans CeMbio reference microbiota exert cryptic virulence and infection protection [preprint]

      Gonzalez, Xavier; Irazoqui, Javier E (2023-11-05)
      Microbiotas are complex microbial communities that colonize specific niches in the host and provide essential organismal functions that are important in health and disease. A key aspect is the ability of each distinct community member to promote or impair host health, alone or in the context of the community, in hosts with varied levels of immune competence. Understanding such interactions is limited by the complexity and experimental accessibility of current systems and models. Recently, a reference twelve-member microbiota for the model organism C. elegans, known as CeMbio, was defined to aid the dissection of conserved host-microbiota interactions. Understanding the physiological impact of the CeMbio bacteria on C. elegans is in its infancy. Here, we show the differential ability of each CeMbio bacterial species to activate innate immunity through the conserved PMK-1/p38 MAPK, ACh/WNT, and HLH-30/TFEB pathways. Using immunodeficient animals, we uncovered several examples of bacterial 'cryptic' virulence, or virulence that was masked by the host defense response. The ability to activate the PMK-1/p38 pathway did not correlate with bacterial virulence in wild type or immunodeficient animals. In contrast, ten out of twelve species activated HLH-30/TFEB, and most showed virulence towards hlh-30-deficient animals. In addition, we identified Pseudomonas lurida as a pathogen in wild type animals, and Acinetobacter guillouiae as avirulent despite activating all three pathways. Moreover, short pre-exposure to A. guillouiae promoted host survival of infection with P. lurida, which was dependent on PMK-1/p38 MAPK and HLH-30/TFEB. These results suggest that the microbiota of C. elegans is rife with "opportunistic" pathogens, and that HLH-30/TFEB is a fundamental and key host protective factor. Furthermore, they support the idea that bacteria like A. guillouiae evolved the ability to induce host innate immunity to improve host fitness when confronted with pathogens, providing new insights into how colonization order impacts host health.
    • Lipofuscin-like autofluorescence within microglia and its impact on studying microglial engulfment

      Stillman, Jacob M; Mendes Lopes, Francisco; Lin, Jing-Ping; Hu, Kevin; Reich, Daniel S; Schafer, Dorothy P (2023-11-03)
      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.
    • Prevalence and predictors of shared decision-making in goals-of-care clinician-family meetings for critically ill neurologic patients: a multi-center mixed-methods study

      Fleming, Victoria; Prasad, Abhinav; Ge, Connie; Crawford, Sybil; Meraj, Shazeb; Hough, Catherine L; Lo, Bernard; Carson, Shannon S; Steingrub, Jay; White, Douglas B; et al. (2023-10-21)
      Background: Shared decision-making is a joint process where patients, or their surrogates, and clinicians make health choices based on evidence and preferences. We aimed to determine the extent and predictors of shared decision-making for goals-of-care discussions for critically ill neurological patients, which is crucial for patient-goal-concordant care but currently unknown. Methods: We analyzed 72 audio-recorded routine clinician-family meetings during which goals-of-care were discussed from seven US hospitals. These occurred for 67 patients with 72 surrogates and 29 clinicians; one hospital provided 49/72 (68%) of the recordings. Using a previously validated 10-element shared decision-making instrument, we quantified the extent of shared decision-making in each meeting. We measured clinicians' and surrogates' characteristics and prognostic estimates for the patient's hospital survival and 6-month independent function using post-meeting questionnaires. We calculated clinician-family prognostic discordance, defined as ≥ 20% absolute difference between the clinician's and surrogate's estimates. We applied mixed-effects regression to identify independent associations with greater shared decision-making. Results: The median shared decision-making score was 7 (IQR 5-8). Only 6% of meetings contained all 10 shared decision-making elements. The most common elements were "discussing uncertainty"(89%) and "assessing family understanding"(86%); least frequent elements were "assessing the need for input from others"(36%) and "eliciting the context of the decision"(33%). Clinician-family prognostic discordance was present in 60% for hospital survival and 45% for 6-month independent function. Univariate analyses indicated associations between greater shared decision-making and younger clinician age, fewer years in practice, specialty (medical-surgical critical care > internal medicine > neurocritical care > other > trauma surgery), and higher clinician-family prognostic discordance for hospital survival. After adjustment, only higher clinician-family prognostic discordance for hospital survival remained independently associated with greater shared decision-making (p = 0.029). Conclusion: Fewer than 1 in 10 goals-of-care clinician-family meetings for critically ill neurological patients contained all shared decision-making elements. Our findings highlight gaps in shared decision-making. Interventions promoting shared decision-making for high-stakes decisions in these patients may increase patient-value congruent care; future studies should also examine whether they will affect decision quality and surrogates' health outcomes.
    • Identifying vulnerabilities in sugar nucleotide metabolism of cancer cells

      Doshi, Mihir B (2023-10-20)
      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.
    • Investigating effects of environmentally acquired epigenetic factors on the mammalian embryo transcriptome

      Krykbaeva, Marina (2023-10-20)
      The major aim of this work is to shed light on epigenetic effects on embryonic development. To this end, we implemented two experimental paradigms. First, we investigated the effect of maternal diet on the embryonic transcriptome. We used in vitro fertilization to isolate gamete-carried factors and single-embryo RNA-Seq to produce a high-resolution data set in 4-cell, morula, and blastocyst embryos, as well as oocytes. We found that although differential expression was observed in most stages of development, these changes were fairly small in size. Likewise, offspring created using an embryo transfer procedure did not exhibit phenotypic differences as a result of maternal diet. However, alterations in gene expression of mitochondrial respiration and lipid and cholesterol metabolism genes were detected in offspring tissue with a clear sex bias. Second, we compared transcriptomes of embryos produced using three methods of fertilization – natural mating (NM), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI) as well as parthenogenesis. The largest differences were detected in IVF embryos, largely in the categories of translation and ribosome biogenesis. ICSI embryos exhibited a small deviation in differentiation-associated gene expression. Parthenogenesis, an embryo-like system with no paternal contributions, resulted in vast expression changes encompassing ~20% of expressed genes and was further used as a model system to confirm a role for sperm-carried RNAs in regulating embryo gene expression. Lastly, this single-embryo data set was used to characterize stochasticity in gene expression and confirm the presence of both “quiet” and “noisy” genes. Overall, we provide two large-scale data sets comprised of hundreds of embryos, which serves as a systematic approach to investigating the effect of epigenetic factors on the embryonic transcriptome.
    • mRNA Sequence Features Determine the Efficiency of Translation Termination and Association of the Nonsense-Mediated mRNA Decay Machinery with Elongating Ribosomes

      Mangkalaphiban, Kotchaphorn (2023-10-20)
      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.
    • Non-canonical amino acid incorporation into AAV5 capsid enhances lung transduction in mice

      Chang, Hao; Du, Ailing; Jiang, Jun; Ren, Lingzhi; Liu, Nan; Zhou, Xuntao; Liang, Jialing; Gao, Guangping; Wang, Dan (2023-10-07)
      Gene therapy using recombinant adeno-associated virus (rAAV) relies on safe, efficient, and precise in vivo gene delivery that is largely dependent on the AAV capsid. The proteinaceous capsid is highly amenable to engineering using a variety of approaches, and most resulting capsids carry substitutions or insertions comprised of natural amino acids. Here, we incorporated a non-canonical amino acid (ncAA), Nε-2-azideoethyloxycarbonyl-L-lysine (also known as NAEK), into the AAV5 capsid using genetic code expansion, and serendipitously found that several NAEK-AAV5 vectors transduced various cell lines more efficiently than the parental rAAV5. Furthermore, one NAEK-AAV5 vector showed lung-specific transduction enhancement following systemic or intranasal delivery in mice. Structural modeling suggests that the long side chain of NAEK may impact on the 3-fold protrusion on the capsid surface that plays a key role in tropism, thereby modulating vector transduction. Recent advances in genetic code expansion have generated synthetic proteins carrying an increasing number of ncAAs that possess diverse biological properties. Our study suggests that ncAA incorporation into the AAV capsid may confer novel vector properties, opening a new and complementary avenue to gene therapy vector discovery.
    • Integrating Equity Into Bicycle Infrastructure, Planning, and Programming: A Mixed Methods Exploration of Implementation Among Participants in the Bicycle Friendly Community Program

      Lemon, Stephenie C; Neptune, Amelia; Goulding, Melissa; Pendharkar, Jyothi Ananth; Dugger, Roddrick; Chriqui, Jamie F (2023-10-05)
      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.
    • Surface modification of neurovascular stents: from bench to patient

      Zoppo, Christopher T; Mocco, J; Manning, Nathan W; Bogdanov, Alexei A. Jr.; Gounis, Matthew J (2023-10-04)
      Flow-diverting stents (FDs) for the treatment of cerebrovascular aneurysms are revolutionary. However, these devices require systemic dual antiplatelet therapy (DAPT) to reduce thromboembolic complications. Given the risk of ischemic complications as well as morbidity and contraindications associated with DAPT, demonstrating safety and efficacy for FDs either without DAPT or reducing the duration of DAPT is a priority. The former may be achieved by surface modifications that decrease device thrombogenicity, and the latter by using coatings that expedite endothelial growth. Biomimetics, commonly achieved by grafting hydrophilic and non-interacting polymers to surfaces, can mask the device surface with nature-derived coatings from circulating factors that normally activate coagulation and inflammation. One strategy is to mimic the surfaces of innocuous circulatory system components. Phosphorylcholine and glycan coatings are naturally inspired and present on the surface of all eukaryotic cell membranes. Another strategy involves linking synthetic biocompatible polymer brushes to the surface of a device that disrupts normal interaction with circulating proteins and cells. Finally, drug immobilization can also impart antithrombotic effects that counteract normal foreign body reactions in the circulatory system without systemic effects. Heparin coatings have been explored since the 1960s and used on a variety of blood contacting surfaces. This concept is now being explored for neurovascular devices. Coatings that improve endothelialization are not as clinically mature as anti-thrombogenic coatings. Coronary stents have used an anti-CD34 antibody coating to capture circulating endothelial progenitor cells on the surface, potentially accelerating endothelial integration. Similarly, coatings with CD31 analogs are being explored for neurovascular implants.
    • Phosphaturic mesenchymal tumor: two cases highlighting differences in clinical and radiologic presentation

      Gu, Joey; Ge, Connie; Joshi, Ganesh; Most, Mathew; Tai, Ryan (2023-10-04)
      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.
    • PAM-flexible genome editing with an engineered chimeric Cas9

      Zhao, Lin; Koseki, Sabrina R T; Silverstein, Rachel A; Amrani, Nadia; Peng, Christina; Kramme, Christian; Savic, Natasha; Pacesa, Martin; Rodríguez, Tomás C; Stan, Teodora; et al. (2023-10-04)
      CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN (R = A or G, Y = C or T) PAM preference, with the N-terminus of Sc + +, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse PAMs and disease-related loci for potential therapeutic applications. In total, the approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning.
    • Measuring The Enduring Imprint Of Structural Racism On American Neighborhoods

      Dyer, Zachary; Alcusky, Matthew J; Galea, Sandro; Ash, Arlene S. (2023-10-03)
      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.
    • Investigating the Role of Chronic Exercise and Autophagy in a Poly(GR) Mouse Model of Frontotemporal Dementia

      Learnard, Heather (2023-09-30)
      Understanding how exercise can attenuate social and cellular deficits seen in frontotemporal dementia (FTD) could provide a unique insight and potential for new therapeutic approaches to help patients suffering from FTD and other devastating FTD associated diseases such as Amyotrophic Lateral Sclerosis (ALS). This project helped study the effect of chronic exercise in a poly(GR)-specific mouse model of FTD, highlighting the specific response exercise has on autophagy and poly(GR) toxicity in this experimental system. The goal of this work was to identify the amount/type of exercise that would be most beneficial to reduce poly(GR) load or other neurotoxicity indicators; as well as, identifying the mechanism underlying autophagy disfunction with GR80 overexpression, and if exercise could also alleviate this disruption. Additionally, to compliment the cortical neuron FTD specific mouse model being used to investigate exercise, we also wanted to make a motor neuron mouse model to better understand poly(GR)’s contribution specifically to ALS. In doing this we used Homeobox Protein 9 (HB9) as a promoter due to its lack of expression in sensory and interneurons, in hopes to study motor neurons only. With a understanding of how poly(GR) impacts both FTD and ALS respectively we can better understand the disease pathology progression and hopefully uncover novel ways to intervene, treat, or prevent these disease more effectively.
    • Multicomponent Pharmacist Intervention Did Not Reduce Clinically Important Medication Errors for Ambulatory Patients Initiating Direct Oral Anticoagulants

      Kapoor, Alok; Patel, Parth; Mbusa, Daniel; Pham, Thu; Cicirale, Carrie; Tran, Wenisa; Beavers, Craig; Javed, Saud; Wagner, Joann; Swain, Dawn; et al. (2023-09-27)
      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.
    • Beyond genome-wide association studies: Investigating the role of noncoding regulatory elements in primary sclerosing cholangitis

      Pratt, Henry E; Wu, Tong; Elhajjajy, Shaimae I; Zhou, Jeffrey Y.; Fitzgerald, Kate; Fazzio, Tom; Weng, Zhiping; Pratt, Daniel S (2023-09-27)
      Background: Genome-wide association studies (GWAS) have identified 30 risk loci for primary sclerosing cholangitis (PSC). Variants within these loci are found predominantly in noncoding regions of DNA making their mechanisms of conferring risk hard to define. Epigenomic studies have shown noncoding variants broadly impact regulatory element activity. The possible association of noncoding PSC variants with regulatory element activity has not been studied. We aimed to (1) determine if the noncoding risk variants in PSC impact regulatory element function and (2) if so, assess the role these regulatory elements have in explaining the genetic risk for PSC. Methods: Available epigenomic datasets were integrated to build a comprehensive atlas of cell type-specific regulatory elements, emphasizing PSC-relevant cell types. RNA-seq and ATAC-seq were performed on peripheral CD4+ T cells from 10 PSC patients and 11 healthy controls. Computational techniques were used to (1) study the enrichment of PSC-risk variants within regulatory elements, (2) correlate risk genotype with differences in regulatory element activity, and (3) identify regulatory elements differentially active and genes differentially expressed between PSC patients and controls. Results: Noncoding PSC-risk variants are strongly enriched within immune-specific enhancers, particularly ones involved in T-cell response to antigenic stimulation. In total, 250 genes and >10,000 regulatory elements were identified that are differentially active between patients and controls. Conclusions: Mechanistic effects are proposed for variants at 6 PSC-risk loci where genotype was linked with differential T-cell regulatory element activity. Regulatory elements are shown to play a key role in PSC pathophysiology.
    • Identifying new players in structural synaptic plasticity through dArc1 interrogation

      Xiao, Cong; M'Angale, P Githure; Wang, Shuhao; Lemieux, Adrienne; Thomson, Travis (2023-09-27)
      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.