University Of Pennsylvania

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY A central goal in HIV/AIDS cure research is preventing or delaying viral rebound following analytical treatment interruption (ATI). It is widely assumed that understanding the viral and host factors involved in in vivo latency reactivation would lead to new, more rational cure strategies; however, the biology and provenance of the rebounding virus remains largely unknown. Here, we propose to leverage four recent discoveries from our groups to gain insight into the mechanisms of HIV-1 recrudescence. Using “gold standard” sampling methods to characterize the latent reservoir before and after treatment interruption (ATI), we discovered that viral isolates derived by quantitative virus outgrowth (QVOA) do not represent or predict the viruses that emerge in the plasma following treatment interruption (1, 2). We also found that rebound viruses, but not QVOA-derived reservoir viruses, were highly resistant to type 1 interferons (IFN-I), indicating potent host innate responses at the site of viral recrudescence (3). Surprisingly, some, but not all, rebound suggesting isolates replicated to high titers in macrophages, a greater biological diversity than previously known. Finally, we discovered that IFN-I resistant rebound viruses have the ability to reseed the reservoir, raising the possibility of long-term clinical consequences of ATI (2, 3). In this application, we will leverage these discoveries to elucidate the viral and host factors that govern HIV-1 rebound. Our hypothesis is that by (i) determining the universality, kinetics and clinical impact of IFN-I resistance during rebound, (ii) defining the viral determinants of IFN-I resistance and the host interferon stimulated genes (ISGs) that place pressure on the rebounding virus, and (iii) tracing the provenance of IFN-I resistant rebound viruses, we will uncover key mechanisms that control HIV-1 reactivation from latency. In Aim 1, we will expand our studies to more diverse ATI trial participants, including women, minorities, acute and early ART initiators and individuals receiving IFN-I modulating therapies, to assess the generality of the IFN-I resistant phenotype of rebound viruses. We will also test to what extent IFN-I resistance persists during prolonged ATI and determine how this influences the rates of IFN-I resistant viruses that reseed the reservoir. In Aim 2, we will elucidate the biological properties of rebound HIV-1, map the viral determinants of IFN-I resistance, and identify the host interferon stimulated genes (ISGs) that place pressure on the recrudescing virus. In Aim 3, we will trace the provenance of rebound virus by testing blood, thoracic duct lymph and lymphatic tissue using regular and modified QVOAs, examine whether long-lived myeloid cells, including brain macrophages and microglia, serve as reservoirs of IFN-I resistant viruses, and explore whether SHIV-infected rhesus macaques recapitulate the IFN-I phenotypes of HIV-1 reservoir and rebound viruses. We expect these studies to improve our understanding of the clinically relevant, rebound competent HIV-1 reservoir, the mechanisms underlying in vivo viral reactivation and the host factors that place pressure on the rebounding virus pool, which should lead to more rational and effective HIV/AIDS cure strategies. .

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY In humans, neutralizing antibodies elicited by HIV-1 coevolve with viral Envs in distinctive patterns, in some cases acquiring substantial breadth. We found that primary HIV-1 Envs, when expressed by simian-human immunodeficiency viruses (SHIVs) in 22 rhesus macaques (RMs), elicited patterns of Env-Ab coevolution strikingly similar to those in humans (Science 371:eabd2638, 2021). This included conserved immunogenetic, structural and chemical solutions to epitope recognition and precise Env-amino acid substitutions, insertions and deletions leading to virus persistence. The structure of one rhesus antibody, capable of neutralizing 49% of a 208-strain panel, revealed a V2-apex mode of recognition like that of human bNAbs PGT145 and PCT64. We subsequently expanded this study to include 150 RMs infected by SHIVs bearing any of 15 different primary HIV-1 Envs; 24 (16%) of these animals developed bNAbs targeting conserved V2 apex, V3 glycan, CD4bs or fusion peptide epitopes. The V2 apex was the most common bNAb epitope targeted in RMs. We concluded that Env-Ab coevolution in RMs recapitulates developmental features of human bNAbs and may serve to guide and accelerate HIV-1 immunogen design for humans. From these preclinical data, we identified HIV-1 Q23.17 Env as the immunogen that most consistently elicited V2 apex bNAbs. Here, we propose to elucidate the Env-Ab coevolutionary pathways by which HIV-1 Q23.17 Env selectively primes, boosts and affinity-matures V2 apex bNAb responses and to translate these findings into an all-SOSIP Env trimer vaccine regimen consisting of a germline-targeted Q23.17 Env prime followed by boosts with lineage-designed Q23.17 Env “imunotypes” capable of affinity-maturing B cells to achieve breadth. Specific aims are: (i) to decipher molecular pathways of Env-Ab coevolution in SHIV.Q23.17 infected RMs that lead to the development of V2 apex bNAbs, including the identification of inferred germline bNAb precursors and lineage intermediates and corresponding Env immunotypes that bind to them; (ii) to use mammalian display saturation mutagenesis to generate Q23.17 Env variants that exhibit enhanced binding affinity to multiple rhesus germline V2 apex bNAb B cell precursors and to engineer these Envs as nanoparticle-delivered SOSIP trimers; (iii) to test the immunogenicity of germline- targeted and lineage-designed Q23.17 Env SOSIP trimers in V2 apex bNAb UCA knockin mice and outbred RMs and to advance the most promising combinations to a proof-of-concept preclinical vaccine trial in RMs; and (iv) to conduct an appropriately powered preclinical vaccine trial in 28 RMs to test the hypothesis that reverse- engineered, B lineage-designed Q23.17 SOSIP Env trimers can prime, boost and affinity mature V2 apex bNAb responses in RMs to an extent that is superior to conventional SOSIP Env immunogens and that protects RMs from heterologous virus challenge. The significance of these studies could be far-reaching: if we can demonstrate consistent induction of bNAbs using germline-targeted, lineage-designed Q23.17 Env SOSIPs in RMs, it would represent a new beachhead for HIV-1 vaccine research that could be translated rapidly into human clinical trials.

NIH Research Projects · FY 2025 · 2021-06

Blood Pressure and Outcomes in Contemporary Left Ventricular Assist Device Recipients PI: Himabindu Vidula, MD, MS University of Rochester Medical Center, Rochester, NY A growing number of advanced heart failure patients are supported by a continuous-flow (CF) left ventricular assist device (LVAD) around the world, but the optimal blood pressure (BP) range for patients on CF-LVAD support has yet to be fully characterized. Previous studies of patients with older LVAD technology have suggested that elevated BP is associated with adverse outcomes, including stroke and mortality. However, the thresholds for maximal and minimal BP for patients supported by contemporary centrifugal flow pumps, such as the HeartMate 3 (HM3) LVAD, are largely based on expert consensus. In addition, limited data exist regarding the lower limit for BP control and BP goals for women, Blacks, and patients with right heart failure (RHF). Finally, the optimal anti-hypertensive medication regimen for LVAD patients is not well defined. A recent study from the Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) suggested that both low and very high BP are associated with increased mortality in CF-LVAD patients, but these retrospective analyses were limited by the availability of BP measurements only at fixed timepoints unrelated to the time of the adverse event. Our preliminary data from the University of Rochester Database, employing time-dependent analysis of 66,618 non-invasive BP measurements in 310 CF-LVAD patients, demonstrate that maintaining mean arterial pressure (MAP) less than 80 mmHg is associated with increased risk of stroke or death during the first year after LVAD implantation. Furthermore, our findings suggest a sex and racial difference in optimal BP thresholds. The association of BP and stroke or death has not been previously studied in a prospective study of HM3 LVAD patients and accordingly BP guidelines in this population are not well defined. We propose a prospective study of 200 newly implanted HM3 LVAD outpatients at 4 different LVAD implantation centers. Following index discharge, patients will measure their BP at home 3 days a week for a 6-month period with a Doppler ultrasound in addition to a standardized protocol of weekly home BP measurements and in-clinic BP assessment every 3 months throughout follow-up. BP will be assessed as a time-dependent covariate for the endpoint of stroke or death. We will evaluate the following specific aims: Specific aim #1 is to validate our findings regarding the association of low BP and the risk of stroke or death and to identify appropriate BP thresholds in HM3 LVAD patients. Specific aim #2 is to determine the optimal BP range in the following subgroups: 1) women as compared to men; 2) Black vs. White; and 3) patients with RHF as compared to patients without RHF. Specific aim #3 is to evaluate the interaction of anti-hypertensive medication use with BP to identify the optimal medical regimen for LVAD patients. Findings from this study, using novel modalities of home BP monitoring with a prespecified follow-up protocol, have important implications for the prevention of death and stroke in LVAD patients and will be used to guide management in this growing population.

NIH Research Projects · FY 2025 · 2021-06

Polygenic risk scores (PRS), that aggregate risk across common variants in the genome, have emerged as a powerful tool towards implementing genomic medicine. Unfortunately, the vast majority of genomic data from which current PRS are estimated is coming only from European ancestry individuals thus prohibiting the implementation of PRS for all individuals across the United States (US) and beyond. Of particular interest are individuals with recent ancestry from multiple continental sources whose genomes are a mosaic of segments of various ancestries. Such a range in genetic ancestry raises unique challenges in PRS method development as the accuracy of existing PRS varies across genomic ancestries. Unlike existing paradigm that largely views genetic ancestry as a confounder in PRS studies, we aim to fully integrate population genetics of the admixture process to yield admixture-PRS that provide improved accuracies for all individuals irrespective of genetic ancestries. We will integrate data of over 230,000 admixed individuals across five medical systems including UCLA, Mt Sinai, Colorado to develop, calibrate and benchmark PRS for admixed individuals.

NIH Research Projects · FY 2025 · 2021-06

PROJECT ABSTRACT Electroencephalography (EEG) is an essential clinical diagnostic and research tool in neurology, neurorehabilitation, cognitive, and behavioral neuroscience. However, in more than 100 years of EEG research, the fundamental EEG technology has remained primitive and game-changing technological innovations have been few and far between. Most current EEG systems rely on gelled silver/silver- chloride or metal electrodes affixed on the scalp with conductive gels or pastes. These devices suffer from the large size of the electrodes, cost, risk of corrosion, preparation, and cleaning. In addition, gels and pastes are necessary to achieve adequate impedance and signal quality, but can be irritating to the skin and dry out over time. Dry (i.e., gel-free) EEG systems can bypass some of the issues of these wet EEG devices, but are still critically limited in terms of subject comfort and signal quality. Finally, MRI-compatible EEG systems for multimodal brain mapping are often highly specialized and expensive. Here, we propose to validate a fully novel, dry EEG system based on MXene materials. MXenes offer high biocompatibility, stability, conductivity, flexibility and low electrochemical impedance. In addition, they can be processed at a low cost, easily integrated into functional neural devices with a variety of geometries and shapes, record brain electrical activity with high fidelity without the need for gels or pastes, and interact weakly with magnetic fields. These properties make MXene ideal to serve as enabling material for the next-generation EEG technologies. In this proposal, we will build on promising pilot data to scale-up and optimize the fabrication and design of MXene EEG electrodes. Specifically, we will aim to outperform the electrodes used in our pilot studies while maintaining fast, cost-effective, and reliable fabrication. Then, we will validate the performance of the best performing MXene electrodes on well-established behavioral tasks associated with readily identifiable EEG spectral characteristics. Finally, we will examine the MRI compatibility of a customized multichannel MXene EEG system for simultaneous EEG/MRI mapping using quantitative and clinician ratings of signal quality, an essential step to propel its widespread adoption in brain research and clinical contexts. By completing this project, we expect to move the field forward by generating a novel dry EEG technology with superior resolution, signal fidelity, and usability compared to current tools. These advantages could pave the way for fundamental innovations in a number of domains including clinical neurology, rehabilitation, and cognitive neuroscience.

NIH Research Projects · FY 2025 · 2021-06

NIH Research Projects · FY 2024 · 2021-06

Project Summary The goal of this proposal is to understand the molecular role of alternative splicing and phosphorylation of ATP- citrate lyase (ACLY) exon 14 and how these events may connect to cellular pathways that promote tumor growth. ACLY is the main source of glucose-derived, nonmitochondrial acetyl-CoA in the cell. Cytosolic acetyl-CoA is an essential building block for fatty acid and cholesterol synthesis, and nuclear acetyl-CoA participates in epigenetic regulation via histone acetylation. Dysregulation of ACLY is associated with cancer and other metabolic diseases, although recent data suggests that splice isoforms of ACLY can also play a major role in disease. Specifically, full-length ACLY, rather than a major splice variant with exon 14 removed, is preferentially expressed in many tumors compared to normal tissue. Moreover, exon 14 has several molecular features, including its location on the protein surface, disordered structure, juxtaposition to putative nuclear localization sequences, and a serine (S481) which is known to be phosphorylated, which make exon 14 a likely player in ACLY regulation. Together, these observations justify my proposed studies to determine the role of exon 14 in ACLY regulation and cancer. To carry out my studies, I have selected cell and mouse models of hepatocellular carcinoma and colorectal cancer, both of which are cancer types that preferentially express full-length ACLY and are known to have increased de novo lipogenesis, to study this phenotype. My strategy involves using fluorescence microscopy and cellular fractionation to study subcellular localization, metabolomics and proteomics to determine metabolic/epigenetic consequences, and colony formation assays and mouse studies to evaluate the tumor-promoting properties of ACLY isoforms and S481 mutants (Aim 1). Furthermore, I will characterize the biochemical and structural underpinnings of these modulations by identifying exon 14-mediated protein-protein interactions with genetically-encoded crosslinkers, performing comparative enzymatic and thermal stability assays, and determining cryo-electron microscopy structures for comparison with the full-length structure of ACLY (Aim 2). Together, these experiments will explain the molecular roles of exon 14, how alternative splicing impacts cellular acetyl-CoA utilization, and how exon 14 inclusion may be supporting tumor growth, which may lead to novel therapeutic strategies.

NIH Research Projects · FY 2025 · 2021-06

Hematopoietic development is an ordered process in which stem cells give rise to multiple lineages. While early progenitors can be multipotent, lineage-specific progenitors reach a stage where they become exclusively committed to that lineage. For example, B and T cell lineages differentiate from lymphoid-primed progenitors produced in the bone marrow, and exclusive commitment to the B cell lineage occurs as cells transition from the pre-pro-B to the pro-B cell stage. Despite the commitment of pro-B cells to the B lineage, we have made the surprising discovery that conditional knock-out of the ubiquitous multi-functional transcription factor YY1 in pro-B cells, results in the loss of B lineage commitment and the consequent ability to develop into the T cell lineage both in vitro and in vivo. To understand the mechanistic basis for this surprising lineage plasticity, we have developed a new lineage tracing mouse line that will enable us to determine how YY1-null pro-B cells develop into T lineage cells (de-differentiation to more primitive progenitors, or trans-differentiation), assess the potential for YY1- null pro-B cells to develop into other hematopoietic lineages, and determine if YY1-null T cells also exhibit lineage plasticity (Aim 1). Mechanistically, lineage-specific transcription factors bind to DNA and regulate gene expression prior to subsequent large-scale alterations in chromatin structure needed for lineage commitment. Rigorous studies by our laboratory as well as others indicate that despite its ubiquitous expression pattern, YY1 controls long-range chromatin interactions (LRCIs) in a lineage- specific fashion. Our findings support the hypothesis that DNA binding by lineage-specific transcription factors enables YY1 recruitment to distinct genomic loci, thereby enabling YY1 to both generate LRCIs that stabilize lineage-appropriate gene expression, and to generate repressive chromatin marks (H3K27me3) at lineage-inappropriate genes. We will thus, compare the molecular genetic phenotype (gene expression patterns, chromatin accessibility, epigenetic structure, and chromatin folding) of YY1- null pro-B cells developed into DN1, DN2a, DN2b, DN3, DP, CD4+, and CD8+ T cells, compared to wild- type T lineage cells, as well as YY1 conditional knockout T lineage cells (Aim 2). We hypothesize that in the absence of YY1, T lineage development can proceed, but LRCIs needed to stably maintain lineage- specific gene expression, and heterochromatin needed for repression of alternative lineages will fail to fully develop, potentially enabling continuing lineage plasticity. Our experiments may reveal a common mechanism for controlling lineage plasticity, vastly expanding potential applicability of directing YY1-null cells into multiple lineages.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY This proposal’s overall goal is to understand the molecular mechanism of telescripting, a new and major gene expression process that is crucial for full-length transcription of most protein-coding genes and regulates messenger RNAs (mRNAs) isoforms and mRNA length in humans and other complex organisms. mRNAs are processed from nascent RNA polymerase II (PolII) transcripts, which generally includes the removal of introns (splicing) and transcription-terminating 3’-end cleavage and polyadenylation (CPA). Splicing and CPA are specified by splice sites and CPA signals (PASs), respectively. However, numerous PASs indistinguishable from the ultimate, gene ends’ PASs are scattered throughout pre-mRNAs, especially in introns and 3’ untranslated regions (3’UTRs), and can trigger premature CPA (PCPA). PCPA is suppressed by U1 snRNP (U1), human cells’ most abundant small non-coding nuclear RNA-protein particle. For brevity, and to distinguish it from U1’s role in splicing, we call U1 suppression of PCPA, telescripting (as it is necessary for long-distance transcription). Like U1 function in splicing, telescripting also depends on U1 snRNA base-pairing to nascent transcripts, which can be abrogated with U1 antisense oligonucleotides (U1 AMO), causing PCPA. Recent studies revealed that even slight changes in the balance between U1 and PASs has great impact on gene expression and can profoundly alter the mRNAs and proteins cells produce. Such changes occur naturally, for example rapid transcription up-regulation during cell stimulation, and create transient U1 deficit relative to transcription output, causing PCPA that produces shorter mRNA isoforms needed to respond to acute environmental changes. Importantly, U1 AMO recapitulates the same mRNA isoform shifts and U1 over- expression can prevent their production in stimulated cells. U1 AMO also elicits widespread 3'UTR shortening, which occurs in and contributes to cell proliferation and cancer. U1 telescripting’s overarching role in transcriptome regulation impacts transcription, splicing, CPA and thereby all downstream events in the life of mRNAs. It has numerous potential applications in biology and medicine. Realizing them requires detailed understanding of the molecular mechanism by which U1 suppresses PASs and the factors that regulate it, which remain unknown. We have made significant progress towards that, including mapping the transcriptome binding locations of U1 and cleavage and polyadenylation factors (CPAFs), and interpreted them in relation to PCPA locations. We have also captured U1 and CPAFs complexes in cells, determined their compositions and stoichiometries, and determined how cells produce the great U1 abundance required for telescripting. These advances lay the foundation for future studies. I anticipate the mechanistic studies and new information will identify potential points of intervention, including druggable targets, and will advance the prospects of harnessing them for novel therapies.

NIH Research Projects · FY 2025 · 2021-05

Project Summary Osteoporosis and low bone mass are common chronic disorders associated with significant morbidity and substantial healthcare costs. Bone is a dynamic tissue that constantly undergoes coupled remodeling by osteoblasts and osteoclasts. Bone marrow (BM) adipocytes, arising from the same mesenchymal stem cells (MSCs) as osteoblasts, also play a crucial role in bone homeostasis. Therefore, advancing our knowledge on mesenchymal populations in bone and understanding their functions will reveal novel targets that address the unmet clinical need for improved treatments for skeletal diseases. By carrying out large scale single cell transcriptome analysis, we recently computationally defined the hierarchy of BM mesenchymal lineage cells and delineated the in vivo differentiation process of MSCs through multiple intermediate subpopulations. Interestingly, we identified a new subpopulation situated after proliferative mesenchymal progenitors and before classic lipid-laden adipocytes (LiLAs) along the adipogenic differentiation route, and thus named those cells marrow adipogenic lineage precursors (MALPs). These non-proliferative cells express mature adipocyte markers, including Adiponectin (Adipoq), but do not accumulate lipid. In young mice, MALPs, genetically labelled by Adipoq-Cre(ER), exist abundantly as BM stromal cells and capillary pericytes. Morphologically, they display many long cell processes that make contacts among themselves and with surrounding cells, as well as the bone surface, to establish a ubiquitous 3D network inside BM cavity. Cell ablation revealed that these Adipoq+ cells play critical roles in maintaining BM vasculature and in suppressing bone formation. Strikingly, MALPs are rapidly and transiently expanded after focal radiation, implying a reparative role during injury response. One important feature of MALPs is that they highly express many secreted factors, such as VEGFa, RANKL (Tnfsf11), CSF1, Cxcl12 etc, indicating regulatory actions on surrounding cells. These data lead to our central hypothesis that MALPs represent a novel adipose cell type with pivotal roles in regulating their BM environment during skeletal development, homeostasis, aging, and injury repair. Bone marrow adipose tissue (MAT) normally refers to LiLAs, and current MAT research centers on their energy and lipid-related roles. This proposal will expand the concept of MAT to include MALPs, a much more abundant cell population, and its non-lipid-associated actions. Our aims are to: 1) determine the in vivo fate and properties of MALPs; 2) elucidate the role of MALPs in regulating bone marrow vasculature; 3) determine the regulatory actions of MALPs on bone resorption. Innovative approaches, such as single nucleus RNA-sequencing (snRNA-seq), confocal 3D imaging, RNA FISH, genetically modified and reporter animal models, will be used throughout the proposal. Our project will comprehensively characterize a novel mesenchymal subpopulation and its multifaceted regulatory roles in bone. The data we gather here will shed new light on bone, adipose, and vascular biology and identify new targets of intervention on osteoporosis and bone repair.

NIH Research Projects · FY 2026 · 2021-05

Project Summary. Cocaine is the most widely abused psychostimulant by a wide margin, and it remains a major public health problem in the US. Cocaine use was slowly declining, but in recent years there has been a resurgence in cocaine abuse accompanied by a sharp increase in cocaine-related hospitalizations and deaths. These facts highlight the need for effective medications for cocaine use disorder (CUD) because there are presently no FDAapproved pharmacologic treatments for CUD. Our exciting preliminary results highlight a novel molecular substrate that could be targeted to attenuate or prevent cocaine taking and seeking. Specifically, we show that cocaine exposure alters the expression of KCC2, a K+ -Cl- cotransporter that defines the Cl- gradient in midbrain GABA neurons. Importantly, this cocaine-induced neuroadaptation is associated with circuitry changes in midbrain GABA neurons that promote and elevate further cocaine taking. These findings support the working hypothesis that initial cocaine taking alters midbrain GABAergic circuitry and increases the vulnerability for increased cocaine consumption over time. Thus, KCC2 represents a potential therapeutic target to treat CUD. KCC2 is expressed primarily in the central nervous system, and it is amenable to therapeutic manipulation in humans. KCC2 is highly attractive as a therapeutic target because it is usually constitutively highly active. Therefore, when normal subjects are treated with KCC2 activators, KCC2 activity is already high, such that attempts to increase its activity further do not produce deleterious side effects. Under normal physiological conditions, KCC2 maintains a low intra-neuronal Cl- concentration required for hyperpolarizing, inhibitory GABAergic currents. Our preliminary results indicate that cocaine dose-dependently downregulates KCC2 function in midbrain GABA neurons, thereby altering midbrain GABAergic circuitry. As a consequence of these circuitry changes, downregulation of KCC2 leads to increased cocaine self-administration. That is, cocaine use itself, by downregulating KCC2, perpetuates heavy cocaine self-administration. Our preliminary results indicate that if we prevent KCC2 downregulation or correct KCC2 function, then we decrease cocaine self-administration. The overall goal of this proposal is to characterize the functional state of the midbrain GABAergic circuitry and the disposition of KCC2 function during cocaine self-administration, extinction, and the reinstatement of cocaine seeking (Aims1 & 2). At each phase of the addiction cycle, we will determine the functional state of the midbrain GABAergic circuitry as a causal contributor to cocaine taking or seeking. Finally, we will apply two mechanistically different pharmacotherapies to boost KCC2 function to decrease cocaine self-administration and cocaine-seeking behavior during abstinence (Aim3). These translationally-relevant studies will test potential therapeutic drugs acting to boost KCC2 function to mitigate enhanced cocaine self-administration induced by cocaine itself. The proposed studies of KCC2 as a novel therapeutic target to mitigate CUD are timely, highly significant, and appropriately aimed at the factors underlying the transition to heavy cocaine use.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Advancing causal implementation theory is critical for designing tailored implementation strategies to facilitate clinician behavior change that target specific mechanisms associated with evidence-based practice (EBP) use. Such strategies may be more successful and more efficient than general implementation strategies. The overall goal of this study is to test the generalizability of a conceptual model that posits the causal relationship among variables from organizational and social psychology to predict clinician evidence-based practice (EBP) use. Broadly, the model proposes that organizational factors like climate and culture influence attitudes, norms and self-efficacy, while other organizational factors like workload, resources and organizational friction moderate the pathway between intentions and the use of an EBP. Our recently completed NIMH-funded R21 demonstrated that this model accounted for up to 75% of variance in implementation of three EBPs in community settings. While promising, we were limited by a small sample, and a focus on special education teachers' use of autism interventions. This R01 will test the generalizability of the causal model in a much larger sample from a new group of practitioners, for a different and more widely-used EBP, cognitive- behavioral therapy (CBT). Successful completion would further validate the model, advancing our understanding of the causal pathways in EBP implementation. We will leverage ongoing CBT implementation efforts to recruit approximately 300 mental health clinicians across 40 organizations in two large public health systems (Philadelphia and Texas). After completing training in CBT, clinicians will complete measures of all constructs delineated in the model. Clinicians also will be observed via audio recording delivering CBT with a client on their caseload on two occasions. Following each observation, data on theorized moderators of the intention to behavior gap will be collected via survey. A subset of clinicians who report high intentions and demonstrate low EBP use will be purposively recruited to complete brief semi-structured interviews further assessing reasons for the intention to behavior gap. Our primary dependent variables and implementation outcomes of interest are clinician intentions to use CBT and direct observation of clinician use of CBT. However, as CBT comprises many discrete components that vary in complexity, each discrete component of CBT use will be measured separately. Data will be analyzed via multilevel modeling to test the extent to which intentions and determinants of intention predict each discrete CBT component (Aim 1) and the extent to which organizational and other contextual factors highlighted in the implementation literature predict factors related to intention formation and moderate the association between intentions and CBT use (Aim 2). Results will inform the development of implementation strategies that target modifiable factors explaining substantial variance in intention and in implementation that can be applied broadly across EBPs.

NIH Research Projects · FY 2025 · 2021-05

We are requesting an increase in the yearly direct costs to support the aims of the MERIT renewal. What we propose is a dramatic leap into the future of social neuroscience and lies at the bleeding edge of current technology. The proposed research is innovative, ambitious, and necessary for understanding how primate, and by extension human, brains enable appropriate social behavior in the real world. Our proposal advances understanding of how primate brains generate appropriate social behavior through a tightly-integrated, ambitious set of 4 specific aims combining simultaneous wireless neurophysiological recordings in multiple freely-moving monkeys interacting naturally in varied social contexts, semi-to-fully-automated quantification of social behavior using computer vision, pharmacological manipulations, and reversible perturbations of neural activity. To our knowledge, none of these experiments has ever been attempted in macaques. Our pilot data compellingly demonstrate that we can do it.

NIH Research Projects · FY 2025 · 2021-05

Globoid cell leukodystrophy (GLD), or Krabbe, is a fatal pediatric neurodegenerative disease caused by mutations in GALC. It is so-named due to the appearance of globoid cell macrophages. The major hurdle to curing GLD is treatment of central nervous system (CNS) pathology. Hematopoietic stem cell transplant (HSCT) is the only treatment, but is not curative, and must be administered presymptomatically in early infancy. HSCT is thought to work by therapeutic engraftment of donor macrophages and replacement of globoid cells, but in the brain, does so inefficiently. Despite being pathognomonic for GLD, little is known about globoid cells in the brain - their function, origin, and formation. It is unknown if globoid cells arise from embryonically-derived tissue resident microglia or HSC-derived infiltrating macrophages, the degree to which they are pathogenic, and if their replacement is key to GLD treatment. This is a critical knowledge gap that has limited the advancement of more effective GLD therapies and is the focus of this proposal. Our central hypothesis is that globoid cells are unique reactive microglia and that robust replacement by “true” microglia is sufficient to treat GLD CNS neuropathology. We are experts in the study of brain macrophages by direct CNS transplantation in mice. The twitcher (GALCKO) mouse is a widely accepted model of GLD. We created new methods to 1) distinguish microglia, infiltrating macrophages, and transplanted donor macrophages from each other and 2) replace host brain macrophages with directly injected cells at high efficiency without HSCT, including by engineering the first small molecule inhibitor-resistant variant of CSF1R, a survival receptor for brain macrophages. In this proposal, we will apply these new methods in the GALCKO model to determine the role of brain macrophages in GLD pathogenesis and treatment. In Aim 1, we will define the origin and transcriptomic identity of all reactive brain macrophages, including globoid cells, in GALCKO, including after HSCT. This knowledge promises to reveal new therapeutic targets for GLD. In Aim 2, we will test the hypothesis that direct replacement of GALCKO microglia with healthy surrogates eliminates globoid cells and drives the neurotherapeutic effects of HSCT. If true, this approach has great translational potential to maximize engraftment efficiency and broaden the therapeutic window for cell therapy. Finally in Aim 3, we will determine if HSC-derived cells are effective microglial surrogates, given the distinct functions of non-microglial macrophages in the CNS. This will guide future work to enhance the efficacy of cell therapies for brain diseases. Completion of these aims will fill longstanding knowledge gaps about the role of microglia and other macrophages in the pathogenesis and treatment of pediatric neurodegenerative diseases.

NIH Research Projects · FY 2025 · 2021-05

SUMMARY: Heart Failure with Preserved Ejection Fraction (HFpEF) is on pace to become the dominant form of heart failure, yet we have no treatments to offer patients. Our preliminary data suggest that abnormalities in skeletal muscle oxidative phosphorylation capacity (SM OxPhos) may contribute to exertional intolerance. SM OxPhos is a complex metric, incorporating both (a) intramuscular perfusion and (b) mitochondrial oxidative reserve capacity, suggesting that both need to be measured to understand the mechanism underlying SM OxPhos impairment. Our group has developed novel MRI sequences which can evaluate both measures. Moreover, we have identified a unique metabolite signature in skeletal muscle biopsy samples from HFpEF patients: a reduction in NAD+ and Propionyl-CoA, indicating metabolic perturbations that may lead to energetic deficits and impair mitochondrial reserve. The goal of this proposal is to investigate the relationship between SM OxPhos and submaximal exercise endurance in HFpEF, with the scientific premise that improvements in SM OxPhos will translate into improvements in exercise endurance. We focus on submaximal exercise endurance as this better reflects the level of exertion reached by HFpEF patients during daily activities. In Aim 1: We will test three interventions in 53 subjects with HFpEF in a cross-over trial: (1) Potassium nitrate (KNO3), which predominantly targets exercise intramuscular perfusion; (2) The combination of KNO3 with nicotinamide riboside (NR) and Propionyl-L-Carnitine (PLC), which targets both intramuscular perfusion and mitochondrial oxidative reserve capacity by replenishing the identified metabolite deficiencies; and (3) Potassium chloride (active control). We hypothesize that while both KNO3 and combination therapy will improve submaximal exercise endurance, combination therapy will lead to greater overall increases. We will also assess the impact of our interventions on SM OxPhos, and the relationship between SM OxPhos and submaximal exercise endurance. In Aim 2: we will test a new diagnostic strategy to identify the mechanism underlying a specific HFpEF patient’s impaired SM OxPhos: the response to supplemental oxygen (100% O2). The lack of SM OxPhos response to oxygen suggests that an impairment in mitochondrial reserve is preventing the utilization of additional oxygen. We hypothesize that these patients will derive greater benefit from combination therapy (KNO3+NR+PLC) by addressing mitochondrial reserve in addition to increasing intramuscular perfusion. Our proposal will comprehensively assess the relationship between SM OxPhos and submaximal exercise endurance using complimentary techniques. It will test novel therapeutics, with the primary goal of improving submaximal exercise endurance and also identify which patients should be treated with which therapy. This proposal has the potential to change the landscape of HFpEF therapeutics, giving us a mechanistically rational strategy to offer relief to these otherwise limited patients.

NIH Research Projects · FY 2026 · 2021-05

The accuracy of chromosome segregation during cell division is essential for genome stability. At the heart of this process are kinetochores—molecular machines that link chromosomes to microtubules and drive their movement. Despite substantial progress in defining individual kinetochore components, critical gaps remain in understanding how these proteins and their multimolecular assemblies mediate versatile motility behaviors. These goals are exceptionally challenging due to the dynamic, force-generating nature of microtubules and the need to interrogate complex functions such as frictional sliding and adaptive coupling at microtubule ends in real time. Our laboratory addresses these challenges by reconstituting human kinetochore proteins in vitro and investigating their force-dependent behaviors using state-of-the-art single-molecule and ensemble assays based on quantitative fluorescence, versatile optical trapping, and mechanistic modeling. This proposal is driven by two recent discoveries from our lab that offer new conceptual footholds for dissecting kinetochore assembly and force regulation at the molecular level. First, we uncovered a novel sliding clutch mechanism by which the Ndc80 complex—a central microtubule-binding component of the kinetochore—moves along the microtubule wall under force. Our leading model posits that Ndc80 modulates its sliding friction to resist plus- end-directed forces and prevent microtubule-end detachment, while enabling low-friction minus-end-directed sliding under forces that segregate chromosomes. We will now identify the molecular and structural determinants of this force-sensitive sliding by analyzing frictional behaviors of Ndc80 variants and other kinetochore-associated microtubule-binding proteins at both single-molecule and ensemble levels. Second, using real-time single-molecule fluorescence, we discovered a kinetic maturation process that stabilizes Ndc80 recruitment to its unstructured kinetochore receptor, CENP-T. Through a novel method to recreate CENP-T multimers, we demonstrated that clustering enhances this maturation process in vitro—likely reflecting effects from the dense molecular environment of the native kinetochore. We will investigate the molecular basis and regulation of maturation rate and assess whether other interactions between Ndc80 and its kinetochore receptors are governed by similar mechanisms, by traditional one-step binding, or by yet unidentified processes. Building on these advances, we will begin to increase the complexity of our reconstructions as a foundation for long-term exploration of higher-order kinetochore assemblies. These assemblies—comprising multimerized recombinant proteins, DNA origami scaffolds, native particles, and mitotic cell lysates—will better approximate cellular conditions and enable us to examine how distinct components contribute to emergent properties such as slip-clutch motility and velocity stabilization. Together, these efforts will advance a rigorous molecular framework for understanding how force and non-traditional kinetic pathways regulate kinetochore assembly and function, shaping future investigations of aneuploidy and related disease mechanisms.

NIH Research Projects · FY 2025 · 2021-05

SUMMARY Modulation of the cytoplasmic concentration of Ca2+ ([Ca2+]i) by inositol trisphosphate (InsP3)-triggered release of Ca2+ from the endoplasmic reticulum (ER) is a ubiquitous signaling system that regulates numerous cell physiological processes. InsP3-mediated [Ca2+]i signals are manifested as repetitive spikes or oscillations, and they can be highly localized or propagate to provide signals to discrete parts of the cell. At the heart of this complex signaling system is the InsP3R ion channel. We have provided rigorous understanding of the ion- channel properties of the InsP3R, by studying the channel using powerful quantitative single-channel patch- clamp electrophysiology of native ER membranes, a technique that we pioneered; how those properties are regulated by physiological agonists and protein interactions; and how changes in these properties are reflected in physiological outcomes. An important physiological target of InsP3R-mediated Ca2+ signals are mitochondria. InsP3R channels play a fundamental role in the regulation of cell metabolism, primarily by supplying released Ca2+ to mitochondria to stimulate TCA-cycle dehydrogenases to promote oxidative phosphorylation (OXPHOS) and ATP production. We discovered that low-level constitutive InsP3R-mediated Ca2+ release to mitochondria is essential for maintaining basal levels of OXPHOS and ATP production in most cell types, and that cancer cells have a particular reliance on this pathway for their survival. The primary pathway for mitochondrial Ca2+ uptake is the mitochondrial Ca2+ uniporter (MCU), a Ca2+-selective ion channel in the inner mitochondrial membrane (IMM). As for the InsP3R, we have employed biochemical and powerful biophysical approaches to understand the ion-channel properties of MCU, including patch-clamp electrophysiology of MCU Ca2+ currents in individual mitoplasts. Our overarching effort has been to quantitatively understand the molecular physiologies of the InsP3R and MCU channels whose integrated activities control cellular physiology and life and death decisions. Recently, cryo-electron microscopic (cryo-EM) structures of both the InsP3R and MCU have been solved. Because of our exertise in the biophysics and molecular physiology of these intracellular ion channels, we are uniquely positioned to exploit this new information to address important questions regarding the molecular mechanisms of ion permeation and channel gating and their regulation of both Ca2+ ion channels. Our goals are to understanding the molecular mechanisms of InsP3R channel gating regulation, to gain fundamental new insights into the molecular mechanisms of MCU channel ion permeation and gating regulation, including by interacting mitochondrial proteins, and to exploit the information gained from the first two goals to provide quantitative insights into ER-to-mitochondrial Ca2+ transfer. Because of the fundamental reliance of cancer cells on this signaling system and its role in familial Alzheimer's disease, we expect that these studies will provide new and critical quantitative insights into a signaling pathway that is important in many cell physiological processes.

NIH Research Projects · FY 2025 · 2021-05

Project Summary/Abstract Protein-protein interactions (PPIs) are involved in a diverse array of critical biological processes, including cell proliferation, growth, differentiation, and apoptosis. To date, there is no general way to modulate collagen protein-protein interactions, and many fundamental aspects of biomolecular recognition are still unknown. Specifically, numerous interactions between collagen triple helices and proteins are not fully characterized or understood. We have recently shown that collagen aza-peptides in which at least one alpha-carbon atom has been substituted with nitrogen, have additional interstrand hydrogen bonds in their collagen triple helix, resulting in hyperstability and more efficient self-assembly. As a result, even short collagen aza-peptides reliably self-assemble into thermostable triple helices, making them a promising choice for novel triple helix mimics. With this project, we propose to use minimal aza-peptides to create linear and cyclic collagen peptide triple helix mimics. Our specific aims are as follows: 1) Synthesize and characterize hyperstable synthetic mimics of collagen peptides; 2) Synthesize and characterize cyclic collagen peptide triple helix mimics (CCP-mimics); 3) Characterize protein interactions our synthetic collagen peptides. Aza-peptides will be synthesized using solid-phase peptide synthesis (SPPS) in order to introduce aza-amino acids into the collagen backbone at precise locations and optimize thermodynamic stability of the linear and cyclic peptides. The modular nature of our new collagen peptide systems will provide highly tunable platforms into which any biologically relevant protein binding sequence can be integrated. Our collagen peptide mimics will serve as new chemical tools for understanding the fundamental biology and biochemistry of the collagen-protein interactome. The proposed studies could ultimately serve as the fundamental science leading to future biomedical treatments for pathologies in which precisely modulating collagen-protein interactions could significantly enhance patient outcome.

NIH Research Projects · FY 2026 · 2021-05

ABSTRACT Exosomes are small lipid-encapsulated vesicles secreted by cells to the extracellular milieu. They carry bioactive molecules, such as signaling proteins and microRNAs, that potently influence the behavior and function of the recipient cells. Studies in recent years have implicated the exosomes in a wide variety of pathophysiological processes including neural degeneration, viral propagation, tumor metastasis and immune suppression. Furthermore, exosomes are considered “treasure troves” for diagnostic biomarkers, and being actively developed as nano-carriers for therapeutic applications. Despite the great interest across many fields, our basic cell biological understanding of exosomes is lacking. The biogenesis of exosomes begins with the invagination of endosomal membrane to form intraluminal vesicles (ILVs). These endosomes, known as multivesicular endosomes (MVEs), are then transported to the cell periphery, where they release the ILVs as exosomes. While the biogenesis of MVEs is largely mediated by the ESCRT complex, the molecular mechanism that mediates the targeting of the MVEs to the plasma membrane for exosome secretion remains elusive. In addition, how the biogenesis and intracellular trafficking of the exosomes are regulated by signaling molecules is largely unknown. In this application, we propose to study the molecular interplays among the Rab family of small GTPases, phosphoinositides, and the exocyst complex, and understand how they function in concert in directing the MVEs to the plasma membrane for exosome secretion. In addition, we will investigate how oncogenic signaling proteins, particularly Mitogen Activated Protein Kinases (MAPK), through phosphorylating key components of the exosome secretion machinery, promotes the release of exosomes, which in turn suppress cytotoxic T cells for immune evasion and tumor progression. A multidisciplinary approach that combines biochemistry, cell biology, tumor biology, and immunobiology will be taken in our study. Our work will not only elucidate the fundamental molecular mechanisms of exosome secretion, but also contribute to the understanding of immune suppression and tumor progression, with future implications for biomarker development and cancer therapeutic applications.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY/ABSTRACT Dr. Colin Ellis is an Assistant Professor of Neurology at the University of Pennsylvania with clinical expertise in both epilepsy and genetics. His long-term goal as a physician-scientist is to translate genetic discoveries into clinically meaningful tools that improve the care of people with epilepsy across the lifespan. Epilepsy is a highly heritable disorder and often runs in families. Recent progress identifying the genes that cause epilepsy applies mostly to severe pediatric epilepsies with de novo (not inherited) mutations. Familial epilepsy presumably also has a genetic basis, yet most families with epilepsy do not have an identifiable single gene mutation. This proposal tests the hypothesis that familial epilepsy is caused by the aggregate effects of many variants (risk alleles) distributed across the genome. Experiments will apply polygenic risk analysis to a unique cohort of over 1,000 multiplex epilepsy families. The specific aims are (1) determine the genome-wide contribution of polygenic risk to familial epilepsies, (2) identify novel and candidate biological pathways that contribute to familial epilepsy, and (3) quantify the transmission of polygenic epilepsy risk within families using a novel analytic method. The results of this project will contribute to understanding the genetic basis of familial epilepsy, the biological pathways that are potential treatment targets, and the future development of clinical tools that use patients’ genetic data to improve the diagnosis and management of epilepsy. The proposal builds on Dr. Ellis’s prior research on the heritability of phenotypic traits within familial epilepsies, and extends that research into the analysis of genomic data. His training plan will develop his expertise in genomic research methods, statistical genetics, and bioinformatics. He will also gain training and skills in clinical trial design and implementation, positioning himself for the future clinical applications of this and other genetic research in his dedicated neurogenetics clinics. Dr. Ellis’s team of mentors and advisors at the University of Pennsylvania (Drs. Hakon Hakonarson, Laura Almasy, Kai Wang, and Brian Litt) provide specific expertise in his areas of skill development and strong track records of mentorship. He will also leverage his existing relationships with external advisors through the international Epi4K Consortium (Drs. Samuel Berkovic, Michael Epstein, and Ruth Ottman) who are among the world leaders in his field of epilepsy genetics research. Dr. Ellis’s clinical background in epilepsy and genetics, with the additional support of this career development award and his unique mentorship opportunities, will position him to succeed as an independent physician- scientist focused on using genetic discoveries to improve the lives of people with epilepsy.

NIH Research Projects · FY 2025 · 2021-05

Project Summary Alzheimer’s disease (AD) is a major public health crisis with no available cure. Given recent failures of many AD clinical trials, there is an urgent need for developing effective strategies to identify new AD targets for disease modeling and new candidates for drug repurposing and development. We propose here a research project to develop transformative big data analytic approaches in the fields of translational bioinformatics, machine learning and deep learning to advance drug repurposing for AD. Our overarching goal is to develop innovative machine learning and deep learning approaches as well as informatics tools and pipelines that leverage big data in relevant biomedical domains. These big data include large-scale genetic, multi-omics, imaging, cognitive and other phenotypic data from landmark AD studies, functional interaction data among drugs, proteins and diseases, pharmacologic perturbation data, electronic health record data, and MarketScan data. Our proposed computational research is aimed at developing novel translational informatics approaches to analyze various types of molecular, clinical and other relevant data to identify individual drugs or drug combinations with favorable efficacy and toxicity profiles as candidates for repositioning against AD or AD- related dementia (ADRD). To achieve our goal, we have four Aims. Aim 1 is to develop network-based multi- omics data integration methods to identify genes and pathways as novel targets for AD drug repositioning research. Aim 2 is to develop informatics strategies to prioritize and evaluate promising candidate targets via examining their associations with AD biomarkers and phenotypes. Aim 3 is to develop knowledge-driven drug repurposing methods using network reinforcement and drug scoring to identify AD candidate drugs. Aim 4 is to prioritize and evaluate the identified candidate drugs for repurposing against AD/ADRD using pharmacologic perturbation, EHR and MarketScan data. Successful completion of these aims will produce novel translational big data analytic methods and tools to improve our understanding of the genetic, molecular and neurobiological mechanisms of AD, facilitate the identification of novel promising targets and drugs for repurposing, and ultimately have a translational impact on disease treatment and prevention. These advances are fundamental to the NIA NAPA goal of effectively treating or preventing AD/ADRD by 2025. The resulting methods and tools are also expected to impact biomedical research in general and benefit public health outcomes.

NIH Research Projects · FY 2025 · 2021-04

ABSTRACT Age-related neurodegenerative diseases pose an immense biomedical challenge. Plastic changes in the brain underpin aging-related cognitive decline and neurodegeneration but little is known about the neuroprotective pathways that forestall these processes in healthy aging brains. In mammals, glia composition and properties display age-related dynamics including a shift to a more neuroprotective function as the brain ages. In Drosophila, glia are also implicated in regulating brain health and lifespan, underscoring a deep evolutionary conservation of glia function. The goal of this proposal is to determine how glia contribute to healthy brain aging and longevity using Harpegnathos saltator ants, a powerful model system to study the molecular and epigenetic regulation of aging. Adult Harpegnathos workers can become queens (called “gamergates”) via a phenotypic transition that results in a 5-fold extension of lifespan. We performed single-cell RNA-seq before and after the transition of workers to long-lived gamergates and found remarkable plasticity in the glia. Specifically, we found that ensheathing glia cells were substantially expanded in gamergate brains. Interestingly, gamergates retained high levels of ensheathing glia as they aged, whereas worker brains were rapidly depleted of these cells over the course of their life. Ensheathing glia cells respond to damage and provide general housekeeping and neuroprotective functions in Drosophila but they are not known to contribute to healthy brain aging and longevity. Our data suggest the hypothesis that an expanded ensheathing glia compartment contributes to the prolonged lifespan of gamergates. In Aim 1, we will determine the molecular and cellular changes that accompany the ensheathing glia dynamics during differential aging in worker and gamergates. In Aim 2, we will investigate the role of a specific receptor that is expressed in ensheathing glia cells and might directly regulate their expansion in response to the expression of a reproductive gene in gamergates. In Aim 3, we will utilize primary ant neuronal cultures and genetic manipulations in Drosophila to determine the causal link between ensheathing glia and longevity and its mechanism. Together, our work will elucidate 1) new molecular pathways that control glia plasticity, 2) a new biological role for glia plasticity, and 3) mechanisms for the regulation of healthy brain aging by glia.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT The proposed K01 project will use multimodal magnetic resonance imaging (MRI) and machine learning (ML) to elucidate the neurocognitive processes underlying treatment failure in young adults with opioid use disorder (OUD). Young adults are at particularly high risk of OUD and fatal opioid overdose. The monthly injectable extended-release opioid antagonist naltrexone (XR-NTX) is a highly effective OUD treatment and is particularly well suited for young adults. However, XR-NTX adherence and relapse show considerable individual variability, and the behavioral and clinical factors associated with such variability remain inconclusive. Previous research has demonstrated the potential for multimodal MRI and ML techniques to elucidate the neurocognitive factors that contribute to treatment response beyond behavioral and clinical measures. This project will take advantage of the cutting-edge MRI and ML methods to model brain structures and functions that predict XR-NTX treatment outcomes in young adults with OUD. The study will evaluate 18–34 year-old OUD patients before and during the first three months of XR-NTX treatment, a period associated with the highest rate of dropout from treatment. The primary outcome will be opioid relapse confirmed by weekly urine toxicology and self-report. The secondary outcome will be non-adherence defined as failure to complete the first three injections. The study will focus on five baseline measures of brain structures and functions that are potentially predictive of treatment response: 1) grey matter volume; 2) functional connectivity with the ventral striatum; 3) reactivity to opioid cues; 4) inhibitory control; and 5) self-evaluation. ML techniques will be used to reveal the patterns of brain structures/functions that are associated with each outcome variable. Based on literature and preliminary findings, we anticipate that combining MRI with behavioral and clinical assessments will better account for individual variability in XR-NTX treatment outcomes in young adults with OUD, than using the behavioral and clinical variables alone. The data will unveil novel brain mechanisms that contribute to the risk of treatment failure in this critical population. The project will also serve as a training vehicle for Dr. Zhenhao Shi to improve his clinical and computational skills and facilitate his independent career development. Specifically, it will enable Dr. Shi to achieve five training goals: 1) to advance his knowledge in the methodology of clinical research; 2) to gain hands-on experience in leading clinical projects; 3) to master ML and multivariate methodologies; 4) to apply multimodal MRI techniques to translational and clinical research; and 5) to advance his general independent research skills including leadership, networking, collaboration, scientific writing and grantsmanship. Through a combination of didactic and hands-on activities, the project will fulfill Dr. Shi's training needs and enable his transition to a successful and independent research career in applying advanced computational approaches to the neuroscience research of substance use disorders and their treatments.

NIH Research Projects · FY 2025 · 2021-04

Project Summary/Abstract Kidney transplant extends life, improves quality of life, and reduces healthcare costs. Unfortunately, the waiting list exceeds 94,000 people while only approximately 14,000 deceased donor kidney transplants (DDKT) occur annually and many patients wait >5 years for a DDKT. For the elderly and some other patient groups, it is common to die waiting. Yet, nearly 600 kidneys from donors infected hepatitis C virus (HCV) were discarded in 2018 (50.1% of the total number of kidneys from HCV-viremic donors); hundreds more kidneys are never procured because of the perception that no center will accept them. Early successes of pilot clinical trials and single-center series of transplanting kidneys from HCV-viremic donors have demonstrated the potential for this practice to increase the number of lifesaving kidney transplants by more than 1,000 kidney transplants each year. However, the dominant system for assessing kidney quality also applies a lower quality score to any kidney from an HCV-viremic donor, thereby promoting organ discard. Also, early experiences from uncontrolled studies without well-matched comparator groups has led to reports of unexpected complications and/or higher than anticipated rates of treatment failures that underscore the need for a formal multi-center clinical trial. Recent reports have highlighted a series of post-transplant complications that necessitate evaluation in a large multi-center trial, for example: a) fibrosing cholestatic HCV in several HCV-negative recipients of an HCV-viremic donor; b) increased incidence of CMV viremia in recipients of HCV-viremic kidneys; and c) membranoproliferative glomerulonephritis. While these complications are rare, they underscore the view from transplant leaders, including the American Society of Transplantation, the American Association for the Study of Liver Diseases, and the Infectious Disease Society of America that this practice is considered `experimental' and is best performed under IRB-approved protocols with rigorous informed consent and assurances of access to HCV treatment. Furthermore, despite increased transplantation of kidneys from HCV- viremic donors into HCV-negative patients, there remain persistent knowledge gaps that need to be addressed for this practice to be accepted as routine clinical care from the perspective of patients, providers, and payers. This multi-center trial seeks to provide significant knowledge gaps that remain by addressing these specific aims: a) estimate HCV cure rates in HCV-negative recipients of HCV-viremic kidneys with a narrow confidence interval; b) determine whether consenting to receiving an HCV-viremic kidney improves survival; c) evaluate 1- year renal function of HCV-viremic kidneys compared to matched comparators; d) assess whether HCV- negative recipients of HCV-viremic kidneys have increased risks of CMV infection; and e) determine if the prevalence of chronic kidney disease pathology is similar in HCV-viremic vs HCV-negative kidney donors. The overarching goal is to determine if kidneys from HCV-viremic donors can safely be transplanted into HCV- negative patients with end-stage renal disease.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT The epidemic of obesity and type 2 diabetes has increased prevalence of associated cardiometabolic diseases including elevated serum triglycerides (TGs) and non-alcoholic fatty liver disease (NAFLD). There is resurgent interest in drugs targeting the hepatic nuclear receptor PPARα. Such fibrate drugs are already used in hypertriglyceridemia, and there is new appreciation that TGs independently cause atherosclerotic cardiovascular disease such that lowering TGs reduces risk. Furthermore, PPAR agonists may be among the first drugs approved for NAFLD. Adverse clinical outcomes in NAFLD like cirrhotic liver failure, hepatocellular carcinoma, and liver-related death are all closely linked to hepatic fibrosis. Our preliminary data in a NAFLD mouse model show unexpectedly that PPARα deficiency and PPARα agonist treatment both fail to change hepatic steatosis, but markedly affect fibrosis (increasing and decreasing it, respectively). PPARα functions in hepatocytes to bind regulatory DNA and affect expression of key genes in lipid metabolism. The related nuclear receptor HNF4α is not a drug target, yet regulates similar genes and binds similar regulatory DNA. We hypothesize that the interplay of PPARα and HNF4α in DNA binding affects hepatocyte gene regulation, relevant to the pathogenesis and therapeutics of NAFLD. Our experiments probe genome-wide nuclear receptor binding sites and gene regulation, in normal and steatotic livers, basally and in response to drugs. Aim 1 defines the interdependency of PPARα and HNF4α in liver gene regulation, using mouse models deficient in either or both. Aim 2 extends these studies of PPARα and HNF4α to NAFLD, in both mouse models and human biospecimens. Aim 3 deploys the powerful tools of genetics to characterize PPARα and HNF4α interplay in sequence-specific DNA binding. By comparing inbred mouse strains, natural polymorphisms affecting binding motifs will reveal mechanisms for selective and common DNA binding by PPARα and HNF4α. In human liver samples, we will test the hypothesis that that non- coding genetic variants in PPARα/HNF4α genomic binding sites underlie some differences among people in TG levels, NAFLD, and response of these to PPARα agonist drugs. Beyond this potential clinical relevance, these studies use innovative genomic and genetic approaches to address key unanswered questions in the biology of PPARα, including its interplay with related nuclear receptors.