Florida State University

NIH Research Projects · FY 2025 · 2023-09

Abstract Recent technological innovations have significantly increased the efficiency of cryogenic electron microscopy (cryo-EM), causing a proliferation in the number of structures resolved with ever-increasing resolution. As many of the "low hanging fruit" structures have been determined, studies have shifted toward increasingly complex specimens, pushing the capabilities of current processing software and requiring novel methodologies. One class of challenging molecule for cryo-EM is tubular molecules that can have ambiguous symmetries. In particular, membrane remodeling proteins that form tubules are often only locally ordered making them intractable for structure determination. Another challenge for cryo-EM structure determination is the problem of the air/water interface. Often when blotting a sample for cryo-EM preparation, the sample can interact with the air/water interface causing protein denaturation, aggregation, or preferred orientations. We have developed a new technique that we are calling reconstruction of average subtracted tubular regions (RASTR) that has the potential to solve both of these problems. RASTR breaks down tubular cryo-EM samples into individual surfaces which enables structure determination without knowing or applying helical symmetry and classification of tubular samples that are only locally ordered with no long-range order. Here we propose two aims to make RASTR more robust, generalizable, and higher-resolution. These aims will be driven by challenging tubular samples that have proven to be intractable to typical cryo-EM structure determination methods. Finally, a third aim is to extend the RASTR approach to single particles bound to tubular substrates in order to protect them from the air/water interface and prevent preferred orientations. Together the proposed aims give RASTR the potential to become a general platform to enable structure determination of challenging samples.

NIH Research Projects · FY 2025 · 2023-09

Abstract SUMOylation is an essential post-translational modification that adds small ubiquitin-like modifiers (SUMO) to protein lysine residues. SUMOylation regulates many cellular functions, including cell proliferation, DNA repair, and stress response. Deregulation of SUMOylation contributes to genome instability and cancer development. Attachment of single SUMO to proteins often creates scaffolds to nucleate macromolecular interactions. On the other hand, attachment of chains of SUMO (polySUMOylation) often triggers protein ubiquitination and extraction from a macromolecular complex. Recent works demonstrate polySUMO-dependent relocation of damaged DNA, which facilitates damage repair. However, the function of protein polySUMOylation and its regulation during cell cycle remain poorly defined. Our long-term goal is to uncover the molecular mechanisms that control genome stability to provide fundamental knowledge that will help develop treatment strategies for diseases resulting from genome instability, such as cancer. The objective of this project is to investigate how polySUMOylation controls the relocation of two key mitotic regulators during the cell cycle: the RENT (regulator of nucleolar silencing and telophase) critical for mitotic exit, and the CPC (chromosomal passenger complex), essential for chromosome bipolar attachment. We recently found that polySUMOylation induction in yeast cells triggers relocation of these two critical mitotic regulators. Our preliminary data support the central hypothesis that polySUMOylation promotes relocation of some key mitotic regulators for successful anaphase initiation, and activation of polo-like kinase triggers polySUMOylation by phosphorylating a deSUMOylase. Our objective will be attained via the following specific aims: 1) Elucidate the mechanism of polySUMOylation-triggered nucleolar protein delocalization that promotes mitotic exit. 2) Determine how polySUMOylation of CPC subunits promotes CPC translocation. 3) Investigate the temporal control mechanism for polySUMOylation during the cell cycle. To test our hypothesis and achieve our aims, we will combine budding yeast genetics, cell biology, and biochemistry. Successful completion of this research will provide a comprehensive understanding of how polySUMOylation controls subcellular localization of protein complexes in the context of cell cycle. Given the exceptional conservation of both the SUMO system and the cell cycle machinery, principles proved in budding yeast are highly likely to translate to human and other eukaryotes. The results will have an important positive impact on the cell biology field because they will uncover new mechanisms critical for genome stability and unveil new targets for cancer diagnosis and therapy.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Cardiovascular diseases are responsible for more deaths each year than cancer, which is why it is important to study how to keep hearts healthy. Hearts undergo physiological remodeling; this is a structural and functional adjustment that matches contractile capacity to hemodynamic demand. In cardiomyocytes, hormonal and mechanosensitive signaling pathways maintain the balance between normal cell size, hypertrophy, or atrophy. Pathologies develop when the adequate adaptation to a stimulus is mismatched. My long-term goal is to establish an independent research program on understanding how mechanical load affects myocardial structure and function and what are the contributing molecular mechanisms. My recent publication in the Journal of General Physiology shows that changing mechanical stimulus of cardiac myocytes affects the dynamics and post-translational modification of the Z-disc actin-capping protein CapZ. I wish to extend this in a new direction working as an independent investigator. Accordingly, my central hypothesis is that mechanical load of cardiomyocytes regulates protein homeostasis in sarcomeres through the balance between acetylation and ubiquitination of lysine residues. Histone deacetylase 3 (HDAC3) is one known acetylation enzyme of sarcomeric proteins. I focus on the Z-disc proteins CapZ and α-actinin because they both maintain sarcomere integrity and because acetylation sites have been previously found in both proteins. My preliminary data shows that unloading changes the relative abundance of CapZ and α-actinin ubiquitination and acetylation. The goal of the K99 mentored phase is (1) to determine post-translational modifications arising from chemical or mechanical unloading of isolated cardiomyocytes with focus on acetylation and K48-oligo-ubiquitination. The goals of the R00 independent phase are (2) to characterize how HDAC3 activity in cardiomyocytes regulates α-actinin and CapZ deacetylation with varying mechanical load and (3) to determine the changes in post- translational modification of sarcomeric proteins by HDAC3 during left-ventricular unloading in whole hearts. The innovation of this project lies in the combination of cardiomyocyte mechanobiology with post-translational molecular biochemistry to understand how cardiac cells maintain sarcomeric protein balance through the ubiquitin-acetylation pathway in response to mechanical stimuli. The outcomes of this project will expand our knowledge about the signaling pathways responsible for modulating protein homeostasis in cardiomyocytes that may develop new research lines for regulation in hypertrophic cardiomyopathies and sarcopenia. The career development activities in this proposal, in addition to the exceptional mentoring team and research environment at the University of Illinois at Chicago, will support my successful transition to a career as an independent investigator.

NIH Research Projects · FY 2024 · 2023-08

Project Summary/Abstract Genetic disruptions such as pathogenic single-nucleotide polymorphisms (SNPs) in clock genes can perturb circadian rhythms and cause sleep disorders. For example, the tau mutation in the CK1ε gene causes dramatic shortening of the wake-sleep cycle in animals, ~20 hrs instead of normal 24 hrs. The same mutation has been reported as a SNP in humans. Because the circadian clock mechanism is conserved across mammalian species, affected humans would be predicted to have the same altered sleep cycle. Structural and biochemical assays have predicted many potentially pathogenic mutations exist for clock genes. However, we do not yet understand the in vivo significance of these potentially pathogenic mutations. A critical bottleneck in studying the pathogenesis of genetic disruptions is lack of an efficient in vivo system that uses cell models instead of resource-intensive, live animal models. Aim 1. Develop an efficient platform to study mammalian clock mechanisms and identify pathological mutations. To test the hypothesis that functionality of SNPs in clock genes can be studied in a human cell- based platform, we generated endogenous Per1-luc and Per2-luc reporters in U2OS cells where diverse SNPs will be generated and assessed accurately. We have validated the system by confirming consistent knockout phenotypes between our reporter cells and mice, and reproducing similar phenotypes with known mutations. Our system will be further validated by comparing phenotypes of the SNPs between two cell models, U2OS and mPer2Luc MEFs. Aim 2. Elucidate the underlying pathophysiology of critical mutations in CK1δ, CK1ε, Clock and Bmal1 genes. We will use our platform to test hypotheses on quantifiable changes previously proposed for specific mutations in clock genes encoding CK1δ/ε, CLOCK and BMAL1. The majority of these mutations have not yet been validated and quantified in in vivo models. We will focus on these mutations and dozens of SNPs at or near these sites that could be as disruptive as the mutations identified by previous studies. Aim 3. Develop a novel, specific treatment approach for circadian sleep disorders associated with pathological SNPs . Current approaches to treat jet-lag or reset sleep cycles include light therapy, melatonin and a few experimental drugs, none of which are specific to the clock and proven to be effective for circadian disorders. We hypothesize that rapid degradation of limiting clock proteins using PROTACs can counteract the pathogenicity of SNP mutations such that the clock is modulated to compensate the pathogenicity. In summary, as we are now aware that human physiology is greatly impacted by defective clock mechanisms associated with pathological SNPs in clock genes and CRISPR allows efficient genome editing, it is imperative and timely to develop an efficient cell-based platform to study pathogenicity of these clinical SNPs.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY/ABSTRACT Monoclonal antibodies (mAbs) represent an important class of biologic therapeutics that can treat COVID-19, cancer and other infectious diseases. Despite their promising potential, pro- cessing, storage and/or administration of mAbs into patients is challenging because the presence of hydrophobic interfaces during processing and administration (air entrapment in the IV bags or the oil-water interface at the interior of syringes) may promote mAb adsorption to such hy- drophobic interfaces. If mAbs change their native (folded) higher order structures (HOS) upon adsorption to these interfaces, their quality, safety and efficacy will be affected, posing immuno- genicity risks to already susceptible patients. The first step in mitigating these risks is to evaluate the in situ HOS of mAbs (whether folded or unfolded) at hydrophobic interfaces. Determining the in situ structure of mAbs at such interfaces has been a major challenge due to limitations of bulk scale or scattering-based microstructural probing techniques. In this program, we will go beyond such limits and use a combination of a unique molecular probing technique based on NMR spec- troscopy and dynamic surface tensiometry to resolve the details of mAbs HOS and adsorption kinetics at hydrophobic interfaces. In particular, by using high-field spatially and spectrally re- solved NMR spectroscopy that is uniquely available to use through National High Magnetic Field Laboratory, we will assess dynamically 1) the in situ HOS of pure mAbs at hydrophobic interfaces, and 2) nature of their associations with surfactants at interfaces. We will perform tensiometry along with NMR spectroscopy on pure mAbs, isotopically labeled mAbs and mAbs/surfactant combinations at hydrophobic interfaces. We will measure a) dynamic surface tension, b) spa- tially localized chemical shifts in 1D 1H and 2D 1H-13C NMR spectra, c) diffusion coefficients of the mAbs, and d) T2 relaxation of mAbs in the bulk and at the interface under different conditions (e.g., various mAbs and surfactant concentrations, solution pH and ionic strengths). By compar- ing the results of the bulk and interface in terms of metrics (a-d), the team will determine if the native HOS of mAbs has been altered by adsorption to hydrophobic interfaces or their associa- tions with surfactants. The outcome of this study will provide the first mechanistic understanding of mAbs HOS at hydrophobic interfaces. Additionally, the knowledge gained from this research is essential in developing a framework to mitigate mAbs adsorption to hydrophobic interfaces, which can be subsequently utilized to improve efficacious mAb deployment for patients. 1

NIH Research Projects · FY 2025 · 2023-08

Project Summary/Abstract Alkaloid natural products have had an immense impact on the field of therapeutic medicine, and hold promise for the discovery of new neuropharmacological treatments. One area of proposed research in the Smith Lab concerns the development of new psychoplastogenic compounds based on the psychedelic lysergic acid diethylamide (LSD). Although derivatives of LSD have been explored previously over many decades, derivatives bearing various substitutions on the indole ring of the ergoline scaffold have never been explored. All of these derivatives are currently only accessible through total synthesis, and potentially can serve as therapeutic leads for various neuropsychiatric diseases including Parkinson's disease, PTSD, severe depression, and addiction. Our concise synthetic approach to the scaffold of LSD hinges on the utilization and chemical modification of widely available heterocyclic aromatic precursors. This strategy allows for the exploration of chemical space that would otherwise be inaccessible, however therapeutically enabling. Synthetic inspiration drawn from our route to these LSD analogs has led us to investigate the synthesis of marine macrocyclic diamine alkaloids such as the halicyclamines and the sarains. None of these targets have been synthesized previously. Our modular, yet redox-economic platform for assembling these structurally complex compounds hinges on employing heterocyclic starting materials at higher oxidation levels to efficiently access their congeners with lower redox levels. Innovation in this research direction is mainly strategic, demanding a navigation of synthetic assembly that has not been traversed previously. This will also demand tactical advances in chemistry that will undoubtedly shed light on novel reactivity patterns that will be broadly important within the realm of heterocyclic chemistry.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY: While currently available antiretrovirals block viral replication and thus control HIV-1 infection, they do not cure the disease; latent reservoirs of replication-competent virus persist. To eradicate HIV-1 infection, novel antiretro- virals must be developed. These drugs would ideally induce the killing of infected cells once latency is reversed. An attractive direction in developing such antiretrovirals is the inhibition of the HIV-1 Nef protein. By modulating surface-levels of immune receptors, Nef enables infected cells to evade host defense mechanisms. Among the many functions of Nef, surface downregulations of CD4 and major histocompatibility complex class I (MHC-I) are the most prominent and presumably most relevant in antiretroviral drug discovery. By downregulating CD4 from the cell surface, Nef enables CD4-induced epitopes of the viral Env protein to remain concealed, which renders infected cells less sensitive to antibody-dependent cellular cytotoxicity (ADCC). By downregulating MHC-I, Nef disrupts host antigen presentation so that infected cells are protected from killing by cytotoxic T lymphocytes (CTLs). Conceivably, therapeutic inhibition of these Nef functions may restore the activities of ADCC and CTLs, thus facilitating the detection and clearance of infected cells. Crystal structures solved by us showed that Nef-mediated downregulations of CD4 and MHC-I involve a common site on Nef. In each case, however, this site is remodeled by Nef’s association with target-specific, hijacked clathrin adaptor proteins (APs) to uniquely accommodate the intended substrate. Furthermore, when bound to Nef, both the CD4 cytosolic tail and the MHC-I cytosolic tail adopt curved, near-circular postures, which suggests that this multifunctional site of Nef can be targeted by cyclic peptides, a promising new class of therapeutics well-suited to disrupt protein- protein interactions. Supported by promising preliminary data, this project aims to develop small-sized macrocy- clic peptides capable of mimicking the cytosolic tails of CD4 and MHC-I and thus blocking the cellular activities of Nef through inhibition of Nef-mediated protein-protein interactions. High-affinity cyclopeptide Nef inhibitors will be developed through enabling-strategies recently established in our laboratories. Specifically, a powerful phage display platform will be applied to optimize CD4-mimetic cyclopeptide inhibitors that can bind to the Nef/AP2 complex with high affinity. In parallel, a similar workflow will be applied to develop and optimize MHC-I-mimetic cyclopeptides into potent inhibitors that can block recruitment of MHC-I into the Nef/AP1 complex. High-resolu- tion structures of the cyclopeptide-Nef complexes will be obtained to enable structure-based optimization of the Nef inhibitors. Using a panel of cell-based assays, these compounds will be characterized for their efficacy in blocking Nef functions in cells, cell permeability, and cellular toxicity, and this knowledge will be leveraged to guide further derivatization for improved cellular activity. Successful completion of this work should yield cyclic peptide-based Nef inhibitors with high affinity in vitro and significant efficacy in cells, which could ideally be developed into novel antiretrovirals with unique therapeutic potentials.

NIH Research Projects · FY 2025 · 2023-08

Project Summary. The prevalence of daily cannabis use and Cannabis Use Disorder (CUD) has increased in the United States over the past two decades. Brief, computerized harm reduction interventions that target specific high-risk CUD populations could be an efficient approach to reducing CUD. Distress intolerance (DI), which refers to the tendency to negatively appraise and escape aversive emotional states, is a risk factor associated with stress-related cannabis use motivation and CUD severity/chronicity. Thus, a brief, accessible, low-cost intervention that reduces DI in those with CUD and elevated DI could have a significant public health impact. This proposed Stage I project aims to modify an existing two-session computerized distress tolerance intervention to optimize emotion regulation learning/generalization and test its impact on DI and cannabis use outcomes in a randomized controlled trial. Specifically, the intervention will be condensed to one-session and its imaginal exposure module will be modified to shape emotional engagement with the aim of maximizing within-session habituation, which will be signaled with a novel audio/visual cue (habituation cue). Habituation cues will then be delivered in just-in-time text message reminders triggered by naturalistic distress reported via ecological momentary assessment. After obtaining feedback on the modified Emotional Engagement Distress Tolerance Intervention in a small sample (Specific Aim 1), the intervention’s efficacy compared to a stringent, credible, time-matched health education control intervention will be tested in a randomized controlled trial in 80 cannabis users with CUD and high DI. To measure the intervention’s mechanistic target engagement (Specific Aim 2), multi-method DI assessments will be administered through four-month follow-up. To measure the intervention’s impact on cannabis use (Specific Aim 3a), stress-related cannabis use motivation (lab stress- elicited craving and neurophysiological drug cue reactivity, ecological momentary assessment of stress-elicited cannabis use) will be assessed through the intervention period. Interviewer-assessed cannabis use frequency, CUD severity, and urinary THC metabolite concentration will be measured through 4-month follow-up. Quality of life and anxiety/depression symptoms will also be measured as secondary outcomes through 4-month follow-up (Specific Aim 3b). As an exploratory aim, a wristworn device will be used to measure ambulatory physiology during a portion of the intervention period to evaluate the feasibility of detecting heightened real- world distress based on objective indicators (Specific Aim 4).Our central hypothesis is that, compared to a control intervention, the Emotional Engagement Distress Tolerance Intervention will produce superior reductions in multi-method assessments of DI, stress-related cannabis use motivation, disordered cannabis use, and psychosocial functioning. Successful completion of the proposed aims will (1) justify a subsequent Stage II trial, and (2) inform efforts to integrate the just-in-time habituation reminders with wearable technology in order to increase emotion regulation generalization opportunities and decrease participant report burden.

NIH Research Projects · FY 2025 · 2023-08

The accumulation of particular proteins into long fibrillar aggregates known as amyloids is a common feature of many devastating aging-related pathologies. In type II diabetes mellitus, the main constituent of these aggregates is Islet Amyloid Polypeptide (IAPP, also known as amylin). Like many other amyloidogenic proteins, the aggregation of IAPP has been linked to cellular dysfunction and death. However, the mechanism by which IAPP aggregates form and how this aggregation is linked to cell death remain mysterious. To help reduce this gap, we propose to characterize the oligomeric intermediates of human-IAPP formed in solution, in presence of metals (such as zinc and copper), and in lipid-membrane via three specific aims. 1) In Aim 1, we propose to characterize the intermediates formed by human-IAPP at atomic resolution by NMR spectroscopy. The identified oligomeric intermediates will be tested for cell toxicity and the structural models derived from NMR constraints will be used to evaluate the mechanism and efficiency of amyloid inhibitors. 2) Since a possible genetic link between zinc regulation and type II diabetes has been discovered, we will characterize zinc-IAPP adducts by cell toxicity, NMR and other biophysical experiments in Aim 2. Mutants of oxidized and reduced forms of human-IAPP will be used to probe the metal binding sites, and isothermal titration experiments will be used to measure the metal binding affinities to different amyloid species. In addition, the non-fibril forming and non-toxic rat-IAPP and pramlintide (trade name symlin approved by FDA for use by both type 1 and type 2 diabetic patients) will be used as controls. 3) To gain insight into the lipid-membrane assisted hIAPP aggregation and the mechanism by which hIAPP disrupts the lipid-membrane, we propose to characterize the role of lipid membrane by a variety of biophysical techniques (including high-speed atomic force microscopy), and stabilize hIAPP oligomeric intermediates using lipid-nanodisc technology and solve the high-resolution structure of oligomers by a combination of solid-state and solution NMR techniques. These high-resolution structures will aid in the development of drugs to stop beta-cell death.

NIH Research Projects · FY 2025 · 2023-07

PROJECT SUMMARY: Currently available antiretroviral therapy is effective in treating HIV infection but cannot eliminate it. Once treat- ment is stopped, viral rebound typically occurs within weeks. Infected individuals must take antiretroviral drugs throughout their lives and, as a consequence, may experience drug resistance and side effects. Novel antiretro- virals that can better treat or even eliminate HIV infection are highly desired. One promising and yet underap- preciated approach is to develop therapeutics that disrupt HIV-induced downregulation of the CD4 receptor and thereby unleash CD4’s remarkable inhibitory power to combat the infection. CD4 is the entry receptor for HIV and plays a vital role in the initial stage of the infection, but its presence later in the virus’ replication cycle strongly inhibits viral replication and sensitizes infected cells to immune-clearance. The potency of CD4 as an inhibitor of the virus is reflected by the stringent, highly concerted mechanisms HIV uses to antagonize CD4. Two viral proteins, Nef and Vpu, are involved in downregulating CD4 from the cell surface and from the endoplasmic reticulum, respectively, leading to degradation of CD4 in either the lysosome (Nef-mediated pathway) or the proteasome (Vpu-mediated pathway). The level of effort here by HIV is arguably unmatched—no other host factors including the well-known restriction factors are antagonized in such a multifaceted manner. This suggests that restoring CD4 levels in infected cells may be significantly damaging to the virus and significantly beneficial to the host. Designing or developing therapeutics to restore CD4 levels, however, is greatly hindered by the lack of high-resolution structural information on the pertinent molecular assemblies, e.g., how Nef and Vpu each recruit CD4 into hijacked host trafficking and degradation machineries. In this project, we will solve such high- resolution structures to gain the knowledge necessary for this drug discovery approach. Our specific aims are: 1) Elucidate how the viral Nef protein hijacks the clathrin adaptor protein AP1 to enable the retention of CD4 in endosomes, thus facilitating the delivery of CD4 to lysosomes for degradation. 2) Reveal how Nef hijacks the host trafficking protein ALIX to channel CD4 into multivesicular bodies and lysosomes for degradation; investigate whether and, if so how, the ALIX-like protein PTPN23 participates in Nef-mediated degradation of CD4 and/or other host factors. 3) Elucidate how the viral Vpu protein targets newly synthesized CD4 in the ER to mediate its polyubiquiti- nation via the β-TrCP/cullin1 complex, thereby redirecting CD4 to the proteasome for degradation. Successful completion of this work should reveal opportunities for the design and/or development of novel ther- apeutics capable of disrupting HIV-induced CD4 degradation, thus restoring CD4 in infected cells to inhibit HIV replication or even eliminate the infection.

NIH Research Projects · FY 2026 · 2023-07

Project Summary This application proposes to compare an optimized, resource-efficient, adaptive obesity treatment against a gold-standard fixed treatment package and assessment only control. The obesity pandemic continues unabated, presaging an onslaught of diabetes. Despite numerous initiatives, gold standard Diabetes Prevention Program (DPP) intensive multicomponent behavioral treatment for overweight and obesity remains too expensive, burdensome, and difficult to scale to suggest that it can be provided to the 2/3 of the population that needs to lose weight. To address this challenge, we strive to optimize less burdensome treatment approaches that can maximize weight loss in the population that has obesity with reduced resource expenditure. In the SMART Weight Loss Management trial, we randomly assigned 400 adults with overweight/obesity to a stepped care weight loss intervention in which first line treatment was either 1) a smartphone app alone (App) or 2) the app plus coaching (App + C). Participants who did not attain adequate weight loss (i.e., averaging >0.5 lb/week) were classified as nonresponders, and re-randomized to be stepped up by a modest or vigorous addition of treatment components. Preliminary Results showed that: 1) More patients achieved clinically meaningful 6 month weight loss with App + C than App; 2) App + C non-responders who adhered to the vigorous step-up (text message and meal replacement) lost as much weight as responders by 12-months. These compelling findings point to a need to test the efficacy of SMARTER stepped-care intervention in a randomized controlled trial. The SMARTer trial is a three-arm, non-inferiority randomized controlled trial that compares the optimized, adaptive SMARTer intervention against gold-standard DPP and Control. The trial will address whether a scalable, stepped-care intervention can stand up to gold-standard DPP by achieving comparable weight loss at less cost. If so, we will emerge with a scalable, effective intervention that tailors to patient response using a stepped-care model. Alongside evaluation of clinical non- inferiority, a comprehensive economic analysis will inform relative affordability and scalability. Hypotheses are that: 1) SMARTer stepped-care will be non-inferior to gold standard DPP in its effect on 6 month weight loss; and 2) The SMARTer intervention will be more cost-effective to implement. We will explore whether extending the SMARTer intervention results in weight loss maintenance at 12 months compared to DPP and Control. Lastly, we will explore mediators and moderators of SMARTer's effect on weight loss to inform future intervention optimization. If successful, findings will support dissemination of a cost-effective obesity population management strategy that facilitates treating obesity with the resources it needs – not more, and not less.

NIH Research Projects · FY 2026 · 2023-06

PROJECT SUMMARY The formation of a percept results from the processing of sensory information across a network of brain regions, each contributing uniquely to perception. Thus, to understand the mechanisms by which the brain encodes sensory information, we must study each component of the network. Olfactory perception is dependent upon a distributed network of processing centers, which are connected in serial and parallel manners. Odor information is organized into receptor-specific channels mapped onto the olfactory bulb. These singular streams of information are broadly projected by mitral/ tufted cells to multiple cortical structures. This direct, parallel input is thought to allow each region to maintain a representation of the stimulus and play a unique role in odor perception. While putative functions have been ascribed to several olfactory cortical regions, the importance of the dorsal tenia tecta for olfactory perception remains a mystery. Here, we propose to examine the connectivity, odor coding, and function of this region. Specific Aim 1 will examine the afferent and efferent connections of the dorsal tenia tecta. Specific Aim 2 will characterize the odor tuning properties of dorsal tenia tecta neurons. Specific Aim 3 will assess the contribution of this region for olfactory perception. Achieving these aims will provide new insight into how odorant features are encoded and mapped within the brain and elucidate the function of this enigmatic brain region for olfactory perception.

NIH Research Projects · FY 2025 · 2023-06

The goal of this research project is to further the development of a pan-Pneumovirus vaccine and to test our hypothesis that a chimeric Pneumovirus fusion (F) protein vaccine displaying immunodominant epitopes of respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) will induce broad protection against both viruses. RSV and hMPV are widely prevalent agents of childhood viral respiratory infection, causing thousands of deaths and hundreds of thousands of hospitalizations each year. There are currently no approved vaccines to elicit protective antibodies against either virus, and no specific treatment options are available. The F glycoproteins of RSV and hMPV have been well-studied as targets of neutralizing antibodies, and several vaccine candidates for RSV are in clinical trials. We have developed a novel vaccine candidate (RHMS-1) encompassing immunodominant epitopes of both RSV and hMPV F proteins and verified its protective efficacy in mouse and cotton rat models. The rationale for pursuing a chimeric vaccine candidate is based on several factors, including focusing the immune response to only those epitopes that elicit potent neutralizing antibodies rather than less potent or non-neutralizing epitopes to improve protection, reducing vaccine escape compared to previous chimeric vaccines incorporating a single epitope, and the assessment of the first chimeric vaccine candidate beyond the mouse model. Additionally, we will determine immune correlates of protection for hMPV infection in a nonhuman primate model. These critical studies will provide a wealth of immunologic information in highly relevant, pre-clinical models that will guide an evidence-based path toward the optimization of a safe and effective pan-Pneumovirus vaccine. Our research will substantially advance the field by developing a vaccine for protection against the two leading causes of acute lower respiratory tract infection in children. As the pre-fusion RSV F protein has already demonstrated safety and the ability to elicit an effective immune response, we will build upon this success to extend this vaccine for protection against hMPV. In Aim 1, we will computationally stabilize and redesign our vaccine candidate, RHMS-1, using Rosetta to enhance protein stability and immunogenicity, and the best candidates will be rapidly screened in mice as both protein subunit and mRNA-lipid nanoparticle vaccines. In Aim 2, we will conduct structural and epitope analysis of our top vaccine candidate to verify the epitopes on RHMS are similar to RSV F and hMPV F proteins. In Aim 3, we will determine the protective efficacy of the top candidate RHMS vaccine in cotton rat and African Green Monkey models of RSV and hMPV infection. Our proposal is both conceptually and practically innovative as we are designing and testing novel vaccine candidates for protection against two important respiratory pathogens, and we are challenging current paradigms in the field by providing a single antigen for dual-virus protection. Furthermore, the innovation of the team is very high, as this proposal brings together diverse investigators and several state of the art technologies.

NIH Research Projects · FY 2025 · 2023-06

Enhancing robotic head and neck surgical skills using stimulated simulation Abstract We propose stimulated simulation as the next paradigm for accelerating simulator-based training of transoral robotic surgery (TORS) using noninvasive brain stimulation. TORS is a minimally invasive technology that is seeing rapid adoption in the field of otolaryngology - head and neck surgery to resect benign and malignant tumors of the pharynx and larynx as well as treat obstructive sleep apnea. As a relatively novel and complex procedure, surgeons are learning to perform TORS after their residency, mostly during a fellowship year. However, TORS has a steep learning curve, with a minimum of 20-35 cases needed to achieve basic proficiency. Without a structured curriculum or effective simulation platform, most novice surgeons currently reach the 20-35 case benchmark while operating on live patients. Consequently, positive margin (i.e., the tumor is not entirely removed) rates for oropharyngeal cancers in low-volume centers are twice that in high-volume facilities. Commercially available task trainers and virtual reality simulators for robotic surgery have four major deficiencies: (i) they do not prepare trainees to operate in the confined space of the oropharynx and do not provide the procedure-specific skills necessary for TORS, such as tissue manipulation, retraction, or dissection using electrocautery; (ii) they are platform-dependent – training basic robotic skills for the da Vinci platform (Intuitive Surgical, Sunnyvale, CA), though two platforms are currently FDA approved for TORS, and a third one is waiting for approval, (iii) they are costly, limiting their use to the most affluent centers and (iv) they offer no mechanism to accelerate skill acquisition beyond brute-force repeated practice. To address these limitations, we will design, develop, and validate the first Virtual Transoral Robotic Surgical (VTORS) simulator for training TORS procedures and use transcranial direct current stimulation (tDCS), a safe and effective, well-established, commercially available, wearable noninvasive brain stimulation technique that uses very low direct current (<4mA) applied to the scalp to modulate cortical excitability, to accelerate the surgical skill acquisition. We have assembled a highly interdisciplinary team of researchers to accomplish the two aims of the project: (SA1) Design, develop and validate a high-resolution, ultra-low-cost, versatile, and platform-independent Virtual Transoral Robotic Surgical (VTORS) simulator for TORS tongue base resection; and (SA2) Demonstrate the training efficiency with transcranial direct current stimulation (tDCS) (i.e., stimulated simulation) compared with sham stimulation. Based on the latest developments in off-the-shelf virtual reality (VR) and tDCS technology and open- source simulation software, stimulated simulation has the potential to become the preferred training paradigm – simultaneously improving skills and accelerating skill acquisition - for complex psychomotor skills, including surgery, aviation, driving, sports, and music.

NIH Research Projects · FY 2025 · 2023-06

PROJECT SUMMARY The advent of remote technologies such as video conferencing and secure web-portals have revolutionized approaches to health data collection and access to healthcare. For example, these technologies can be deployed in fully remote and hybrid approaches for clinical trials which could help promoting more participation among under-represented groups as well as by bridging geographic distance and reducing participant burden (e.g., less travel time, more flexibility and confidentiality). Remote or online approaches can also increase access to critical services (e.g., mental health counseling, sexually transmitted testing [STI] prevention) that are scarce or geographically dispersed. However, these approaches have yet to be optimized for all possible beneficiaries of technological advances in clinical trials and healthcare, especially targeting under-represented populations that are disproportionately experiencing social and geographic isolation. Groups like Asian American and Pacific Islander (AAPI – including Native Hawaiian), American Indian (AI) and Hispanic/Latino populations are often under-represented in clinical trials, so much so that they often occupy the racial/ethnic category of “other” in research studies. Notably, their under-representation not only leads to this literal “othering,” but also prevents scientists from identifying their unique concerns which in turn diminishing the relevance and effectiveness of technologies and distribution campaigns among them. Sexual and gender minorities (SGM) such as gay, lesbian, bisexual, and transgender populations are also often not strategically sampled for clinical trials, and SGM identities are often not even measured in clinical trials. But many health concerns addressed in clinical trials are over-represented in SGM populations compared to general populations (e.g., HIV, STIs, mental health concerns), thus, making many novel health technologies (vaccines, antibiotics, hormonal therapies) disproportionately relevant to these populations. SGM populations may experience social isolation at increased rates relative to the general population, which can be further exacerbated by medical mistrust and stigmatizing experiences in healthcare and research contexts. To address these concerns, the current proposal seeks to leverage the team’s existing community connections in states across 4 different regions of the U.S. among the aforementioned groups to: (1) (a) co- create and maintain 4 virtual and hybrid advisory groups to address health issues and (b) develop materials for the conduct of remote advisory groups; (2) co-develop and administer a national needs assessment survey for the referenced 4 groups (N = 800) addressing the use of home self-testing technology (up to 3 types) and remote data collection for healthcare research and healthcare provision; and (3) pilot a community driven approach to introducing home self-testing technology and remote data collection to potential participants from these groups to assess feasibility and acceptability.

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY The mechanism by which metabolism, diet, and olfactory function is linked is not well understood. The rising incidence of diabetes and obesity in our country is epidemic, yet little has been reported as to how chronic metabolic imbalance impacts sensory systems and whether these dysfunctions can be reversed via changes in diet, drug intervention, or selective genome editing. The work in this proposal will bridge gaps in our knowledge concerning how changes in activity of the olfactory bulb (OB) can modify energy homeostasis. To test how changes in OB excitability cause a reduction in body weight and energy metabolism, we will manipulate contribution from a voltage-dependent potassium channel, Kv1.3, exclusively in the major output neurons. Our long-term research goal is to understand how olfaction and metabolism are interrelated - to reveal how olfactory output neurons convey metabolic information. Our proposed aims are based upon the following three hypotheses: (1) Hypothesis 1 = Elimination of Kv1.3 channels in mitral/tufted cells will increase action potential firing frequency and decrease the after-hyperpolarization amplitude, selectively enhance glucose clearance, increase total energy expenditure, and decrease respiratory exchange ratio (increase fat utilization), which will produce a drop in body weight or cause a resistance to diet-induced obesity (DIO). (2) Hypothesis 2 = Odor stimulation will induce specific patterns of c-fos expression within the hypothalamus and other brain regions in mice. DIO will attenuate c-fos activation in control mice with normal Kv1.3 conduction, but not in similarly-fed, but DIO-resistant, mice in which Kv1.3 is selectively edited from mitral/tufted cells. (3) Hypothesis 3 = Restoration of Kv1.3 activity selectively in mitral/tufted cells, but not in the periphery, or decreased excitability will cause a loss in resistance to DIO as measured by body weight, glucose tolerance, and system physiology parameters. Our experiments take a multidisciplinary approach using electrophysiology, genome editing, and metabolic profiling to uncover the importance of relayed olfaction information for energy homeostasis. The knowledge generated from our proposed research defining the impact of olfactory bulb output on metabolic balance can be applied to lessen the health consequences of the rising global problem of obesity and excess food consumption. It is a high priority that we investigate coordination from extra-hypothalamic brain areas to determine their contribution to energy balance – a novel and intellectually challenging view of the olfactory system.

NIH Research Projects · FY 2026 · 2023-04

Nuclear magnetic resonance (NMR) spectroscopy is a unique set of experimental tools for understanding the intricacies of biology, from macromolecular complexes to complex mixtures, from atomic resolution structure to dynamics on timescales of picoseconds to seconds, from chemistry to functional mechanisms and kinetic processes. No other technology has such breadth and potential for basic and applied research and for interfacing with other technologies, such as X-ray crystallography, small angle X-ray scattering, Cryo-EM, mass spectrometry, and many other spectroscopic and analytical tools. Structural characterization serves as the framework for using NMR to understand biological activities, protein-protein and protein interface interactions, functional mechanisms, and kinetic models. Dynamics can be exceptionally well characterized by NMR, which can lead to detailed understanding about how proteins and other macromolecules function, how complexes are formed, and how certain kinetic processes and rates are achieved. The solution NMR spectroscopy of complex mixtures is particularly useful in combination with mass spectrometry for metabolomics and other complex mixtures, whereas solid-state NMR (ssNMR) is uniquely capable of measuring chemical shift and quadrupolar tensors to provide insights into chemical biology. Here, we focus on the frontiers of NMR technology made possible by recent breakthroughs in materials research and instrumentation, and their implementation for a broad user community pursuing fundamental questions at atomic resolution at the forefront of biomedical research. Three Technology Development Projects (TDP) advance the sensitivity of NMR, each featuring novel technologies. TDP1 features the use of high temperature superconductors (HTS) for RF coils, leading to high sensitivity for solution NMR spectroscopy. TDP2 takes advantage of our 600 MHz MAS-DNP NMR instrument, which will provide enhanced sensitivity through the transfer of magnetization from electrons to protons. New and much more robust DNP probes with expanded temperature ranges will be developed. TDP3 uses the 36 T Series Connected Hybrid (36T-SCH) and all-HTS 32 T superconducting (32T-SCM) magnets for ssNMR and solution NMR spectroscopy – the 36T-SCH is the highest-field NMR spectrometer in the world, and the 32T-SCM will be the highest-field spectrometer with low-temperature (4-30 K) capabilities for NMR explorations of biosolids. These platforms will lead to dramatic enhancements in sensitivity and spectacular reductions in signal averaging times. The science will be driven by a select team of ten scientists with Driving Biomedical Projects (DBP), and over 30 Collaborative and Service Projects (CSP) and Technology Partnership Projects (TPP) that span a very broad range of biomedical and biochemical research areas. A major team effort will be placed on training a new generation of NMR users through annual workshops, as well as dissemination through publications and presentations at meetings, a wide variety of scientific organizations, news media, a dedicated website for this Resource, training and educational activities, and posting of training lectures and videos of demonstrations.

NIH Research Projects · FY 2025 · 2023-03

PROJECT SUMMARY/ABSTRACT Despite the increasing attention to the “culturally and linguistically diverse (CLD)” communities in research and clinical practice, monolingual speakers of minority languages living in the United States (US), such as foreign- born populations who speak a language other than English, have been nearly excluded from speech rehabilitation. This is mainly because of the assumption that the target language of intervention is English regardless of the cultural and linguist background of the patients. The goal of the proposed project, framed by the International Classification of Functioning, Disability, and Health ICF), is (1) to address missing personal and environmental factors of the foreign-born populations who are in need of speech rehabilitation and (2) to develop and conduct a small-scale clinical trial that incorporates these missing factors into a speech intervention program. For an initial effort, a total of 32 Korean-speakers with Parkinson’s disease (PD) and their families will participate in the study. To overcome the primary obstacles of delivering speech therapy in Korean, such as the availability of Korean speech rehabilitation programs in US, we employ two recent rehabilitation delivery models: telepractice and group therapy. PD participants will receive speech therapy consisting of 16 sessions of 60 minutes duration delivered over four weeks using a telehealth platform. PD families will attend weekly, 1-hour, family training and support sessions, which will provide family counseling and conversation training. Primary (speech intelligibility, acoustic measures) and secondary (communication participation, health and well-being) outcome measures will be obtained from Pre-, Post-, and Follow-up timepoints. The primary deliverable will be the initial stage for an interdisciplinary speech intervention model for speakers with communication disorders who cannot receive speech therapy in English. Further, health literacy among linguistically and culturally diverse groups will be enhanced. The long-term goal is to expand the number and scope of populations with quality health care access and to develop speech intervention programs that incorporate sophisticated attention to personal and environmental factors specific to the target clinical populations.

NIH Research Projects · FY 2026 · 2023-01

Adolescents and young adults (AYA) under the age of 25 in the United States (US) are the least likely of all age groups to 1) know their HIV status, 2) initiate PrEP if HIV-negative even when indicated, 3) take medication as prescribed, attend medical appointments, and be virally suppressed if living with HIV. Persistent rates of new infections among US youth indicate that evidence-based and integrated approaches to increase the uptake and use of effective biomedical prevention and treatment products are required. Adolescence and young adulthood are challenging developmental periods, with significant physical, neurocognitive, emotional, and social changes. While multiple vulnerabilities and complex community factors drive the HIV epidemic among youth, these developmental periods are also characterized by opportunities for growth, achievement, and resilience. HIV research networks must be broad in agenda, go beyond traditional clinical sites and individual level interventions and address the multi-faceted, multi-level barriers that have thwarted HIV prevention and care progress among AYA. The newly envisioned Adolescent Medicine Trials Network for HIV Interventions (ATN) is a multicomponent collaborative research enterprise focused on the reduction of new HIV infections among AYA in the US as well as improvements across the HIV continuum of care for youth living with HIV. Such a large-scale, complex clinical research program demands high productivity and strong contributions from each essential component. Our proposed overall network structure is conceptualized as a well-oiled machine with interdependent structures working together seamlessly to achieve the mission of the ATN. The Scientific Leadership Center (SLC) will bring all elements together and ensure that they function collaboratively, effectively, and efficiently with the Operations and Collaboration Center (OCC), NIH, and the overall network. The ATN has identified five high priority research areas to address the substantial and disproportionate gaps in the health outcomes of AYA across the HIV prevention and care continuum. With this application, we include highly innovative and impactful clinical trials led by talented multi-disciplinary teams, addressing priority research areas, spanning all populations of youth, multiple socioecological systems, and a variety of regulatory phases. Drs. Hightow-Weidman and Hosek are uniquely qualified as MPIs to lead the SLC. Both have worked in the field of adolescent HIV prevention and treatment for the entirety of their careers and have worked collaboratively within the ATN for >20 years, giving them insight on how best to make this new iteration successful. Our overall vision for a redesigned and robust ATN is one that not only anticipates and plans for ongoing evolution in the HIV field and an emerging scientific agenda but one with a renewed focus on the most impactful science and priority areas.

NIH Research Projects · FY 2026 · 2023-01

Anxiety psychopathology is highly prevalent in people living with mild cognitive impairment (MCI), Alzheimer's disease and related dementias (ADRD) and their care partners. Recent meta- analyses suggest clinically significant anxiety symptoms in approximately 40% of those with ADRD and approximately 25% in their care partners, as well as increased rates of anxiety in clinical samples of patients with MCI. Moreover, a recent review suggests that elevated anxiety is a marker for and potentially contributes to earlier onset of ADRD symptoms among those with MCI. Despite this, there are no well-established interventions for anxiety in MCI/ADRD or their care partners. Moreover, prior treatment protocols for anxiety are lengthy, excessively rely on intact memory and cognitive abilities, and result in high dropout rates. Brief, mechanism focused interventions offer an efficient, alternative approach to dealing with anxiety in people with MCI/ADRD and their care partners. Anxiety sensitivity (AS) is an extremely well-researched risk mechanism relevant to the genesis and maintenance of anxiety and other forms of psychopathology. AS acts as a broad stress amplification factor as it exacerbates the experience of somatic and emotional sensations, leading to increased distress. As such, individuals with elevated AS are more likely to experience exaggerated responses to a wide array of stressors including cognitive symptoms (e.g., concentration and memory problems). Fortunately, focused interventions have been developed showing that AS can be quickly and effectively reduced. These interventions include psychoeducation but focus heavily on interoceptive exposure (IE) exercises designed to reduce conditioned fear to anxiety-provoking internal stimuli. Across clinical trials, evidence shows such interventions can markedly reduce AS and that these reductions mediate reductions in anxiety symptoms. While AS interventions have been successfully used in a variety of samples, they have not been tested for people with MCI/ADRD. We propose to conduct a fully powered randomized clinical trial (RCT) to test a brief, CBT-based intervention, called cognitive anxiety sensitivity treatment (CAST) for people with MCI/mild AD. We believe the IE component of CAST will be particularly relevant to MCI/mild ADRD where learning may be compromised due to cognitive decline. Moreover, our preliminary data suggest that CAST yields medium to high effect size reductions in AS and anxiety in older adults with MCI. Dyads consisting of MCI/mild AD and their care partners will be randomized to CAST to a Health Education Control (HEC) condition (N = 197) and followed for six months to evaluate change in anxiety and distress, cognitive functioning and quality of life.

NIH Research Projects · FY 2025 · 2023-01

PROJECT SUMMARY Zona incerta (ZI), a brain region located between the ventral thalamus and the hypothalamus, has been revealed to regulate food intake by latest findings. We reported that g-aminobutyric acid (GABA) neurons in the ZI regulate food intake through inhibitory projections to paraventricular thalamus (PVT). In addition to GABA neurons, dopamine (DA) neurons have been found in the ZI for decades. However, little is known about their function in the behavioral regulation. In our pilot studies, we used TH-Cre mice to target ZI DA neurons and found that chemogenetic activation of ZI DA neurons potently increased motivation to obtain food reward. Based on our pilot data, we hypothesize that: 1) ZI DA neurons regulate feeding motivation to control food consumption. 2) ZI DA neurons encode feeding behaviors and respond to metabolic hunger signals. 3) the PVT serves as one of the important postsynaptic areas for ZI DA neurons to exert the functional control on feeding. To test our hypothesis, we will use adeno-associated viral vectors (AAVs) to induce Cre-dependent expression of excitatory hM3Dq, inhibitory hM4Di, or ChR2 selectively in ZI DA neurons of TH-Cre mice. Chemogenetic activation and silencing will be employed to selectively manipulate ZI DA neurons for testing their role in the control of food motivation using progressive ratio (PR) schedule of reinforcement and real-time food consumption and meal pattern using Feeding Experimental Devices (FED). We will also use optogenetics to selectively activate ZI DA projections to determine the role of PVT neurons in feeding regulation induced by ZI DA neurons. In addition to the behavioral studies, we will perform slice electrophysiology in combination with optogenetics to dissect functional ZI DA pathways. In vivo fiber photometry will be applied to examine how ZI DA neurons encode feeding behaviors including food seeking and consumption and slice electrophysiology will be used to study the activity response of ZI DA neurons to metabolic hunger signals. Together, the proposed studies in this application will reveal novel ZI DA pathways in feeding control. The expected outcome from the proposed studies will significantly expand our view of how ZI and central DA signaling regulate feeding motivation and food consumption.

NIH Research Projects · FY 2025 · 2022-11

Streptococcus pneumoniae is a leading infectious pathogen, causing pneumonia, bacteremia, meningitis, acute otitis media, and nearly one million deaths worldwide each year. S. pneumoniae can be carried in the nasopharynx asymptomatically, which contributes to pathogen spread, as pneumococcal carriage often precedes active infection. Infections occur with increased frequency in high-risk populations, such as individuals with diabetes, asthma, chronic obstructive pulmonary disease, cardiovascular disease, and HIV. Several vaccines are currently in use to prevent pneumococcal infection; however, several factors warrant further research, including limited serotype coverage of current vaccines, limited vaccine efficacy against some vaccine- included serotypes, increased incidence of colonization and infection with non-vaccine serotypes, and widespread drug and multidrug antibiotic resistance among non-vaccine serotypes. This R01 proposal will address these limitations by defining the structural determinants mediating the serotype breadth and protective efficacy of broadly-reactive human mAbs that prevent and treat pneumococcal infection. The scientific premise of this proposal is that mAbs to conserved pneumococcal antigens that are broadly reactive could serve as priority or adjunctive therapies for pneumococcal disease management. This proposal will focus on mAbs to pneumococcal antigens that are highly conserved and are targets of B cells during pneumococcal colonization and infection. Our work will advance the field by generating new therapeutic options for the prevention and treatment of pneumococcal infection for diverse serotypes, including encapsulated and nonencapsulated serotypes, and by identifying protective epitopes on pneumococcal surface proteins. Our innovative hypothesis is that human mAbs targeting conserved pneumococcal surface proteins will exhibit substantial serotype breadth, can treat pneumococcal infection, and that mAb protective efficacy and serotype breadth is correlated to epitope specificity. Our data will provide new findings for the pneumococcal protein vaccine field. In Aim 1, the serotype breadth and protective efficacy of human mAbs targeting conserved protein antigens will be determined in models of both primary and secondary (following influenza virus infection) pneumococcal infection. In Aim 2, we will define the epitopes mediating the protective efficacy of the human mAbs using X-ray crystallography and cryo-EM, which will be critical to the field by informing the development of protein-based pneumococcal vaccines, as we have shown in our preliminary data that the epitope on pneumococcal proteins impacts mAb breadth and protective efficacy. In Aim 3, we will conduct in depth in vitro and in vivo mechanistic studies to assess mAb functions, including opsonophagocytic and agglutination activity, and inhibition of bacterial growth, adhesion, invasion, and biofilm formation. We will also assess the specific immunological pathways important for mAb-mediated bacterial clearance. Overall, our work is both practically and conceptually innovative, and will challenge current treatment paradigms for pneumococcal infection.

NIH Research Projects · FY 2025 · 2022-10

NIH Research Projects · FY 2025 · 2022-09

Emerging adult college students (18−29 years) in the US engage in binge and heavy drinking and experience alcohol-related problems, especially those who are the first in their family to attend college. However, they are hesitant to engage in interventions shown to alleviate stress and alcohol consumption in college students. Using mobile health (mHealth) interventions tailored for emerging adults may address this public health concern. Hence, the long-term goal of this Mentored Patient-Oriented Research Career Development Award (K23) is to launch Dr. Laura Reid Marks’s program of research as an independent clinical scientist with a focus on reducing alcohol-related problems in emerging adults susceptible to academic difficulties This goal will be achieved through a 5-year parallel training and research plan. Training goals include: (1) developing expertise in the theory and practice of T1 translation of the ORBIT model (an NIH model of phased behavioral intervention development); (2) cultivating skills in behavioral economics theory to develop engagement strategies for a behavioral mHealth intervention; (3) implementing novel experimental approaches (i.e., micro- randomized trials; MRTs), to increase mHealth engagement; and (4) building skills to successfully direct a research lab and mentor a lab of students. Training objectives will be met through a comprehensive training plan involving: (1) ongoing individual meetings with mentors (Drs. Naar, Murphy, Nahum-Shani, and Li), to learn from their combined expertise in T1 translation of health intervention, behavioral economics, mHealth, MRT research design, and statistical analyses; (2) courses, workshops, and seminars; (3) conferences and professional development. Skills gained through the training plan will be applied to a project capitalizing on Phases I and II of the ORBIT model in preparation for Phase III. To address Aim 1 (Phase 1), in Years 1−2, we will analyze focus group data collected from emerging adult drinkers at universities to develop and refine strategies drawn from behavioral economics (i.e., episodic future thinking, reciprocity) to increase engagement in a mindfulness application (app) for binge and heavy drinking. To address Aim 2 (Phase I and II), across Years 3−4, we will use a pilot-MRT to test the feasibility, acceptability, and preliminary effect of the Aim 1 engagement strategies to engage binge and heaving drinking participants who fit eligibility (N = 40) in a mindfulness app. Participants will be randomized to one of three conditions (episodic future thinking, reciprocity, or no prompt conditions) daily. A baseline survey, ecological momentary assessments, paradata, and a post-pilot MRT individual exit interview will assess feasibility, acceptability, and the preliminary effect of the engagement strategies delivered as text-based prompts in a smartphone to increase engagement in mHealth mindfulness, reduce stress, and ultimately alcohol consumption. The proposed studies will provide pilot data for Dr. Marks’ first R01 submission to NIAAA, to be submitted in Year 5. The proposal is aligned with the NIH Research Career Development’s goal of ensuring the training of scientists to address U.S. behavioral and biomedical needs.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Abstract The 26S proteasome conducts most regulated protein degradation and eliminates toxic proteins in vivo. The proteasome is an unusually large and complex ATP-dependent protease comprising nearly 70 individual polypeptide subunits. Although the conventional thinking has been that the proteasome is assembled from these subunits in a single, rigid stepwise sequence, recent evidence from our group and others unexpectedly suggests a broader “landscape” of assembly routes may exist in vivo. Although this possibility has not yet been tested, such an assembly landscape would ensure that this essential biological process can continue effectively in the face of assembly roadblocks, and would provide a powerful means to adjust the speed or volume of proteasome biogenesis in response to the cellular environment. There is an increasing interest in harnessing proteasome biogenesis to help treat conditions as diverse as cancer and neurodegenerative disorders. Understanding whether such an assembly landscape exists, and if so, how it is harnessed to ensure rapid and faithful proteasome biogenesis, will be critical to guide development of such assembly-targeted therapies. The goal of this multi-PI application is to test the hypothesis that a proteasome assembly landscape exists in vivo, and that the relative flux through possible routes within this landscape is governed largely by kinetic factors that change in response to the intracellular environment. By combining the PIs’ respective expertise in proteasome biology and in enzyme kinetics and single molecular biophysics, we hope to validate this new paradigm for proteasome biogenesis. The proposed studies, described below, will add a critical new dimension— time—to our understanding of proteasome assembly in vivo. Our experimental approach contains two complementary but independent Aims. In Aim 1, we will utilize a newly established collection of cutting-edge single-molecule and ensemble fluorescence assays to characterize the kinetics of specific proteasome assembly steps. Experiments under this aim are designed to test the hypothesis that the relative flux through two possible assembly routes is primarily under kinetic control, but can be tuned by exogenous factors such as ligands or proteasome-interacting accessory proteins. Aim 2 will employ a suite of newly developed chemical-genetic approaches to assess the relative flux through two possible assembly routes in vivo, and to understand how the flux changes in response to environmental stimuli. Experiments under this Aim will also test in living cells the predictions derived from our in vitro kinetic model of assembly established in Aim 1. The outcomes of these studies will lead to a deeper understanding of proteasome biology and of macromolecular assembly in general, and also promise to illuminate new therapeutic avenues for cancer, neurodegeneration, and other diseases.