University Of Pennsylvania

NIH Research Projects · FY 2025 · 2017-07

Genomics technology is sufficiently advanced to measure DNA sequence variation and RNA expression in clinical samples. However, the integration of genomic measurements into healthcare is outpacing the training of physicians and scientists, and many are ill-equipped to use this information to improve the health of patients. Thus, it is imperative to train the next generation of physician and scientist leaders in Genomic Medicine. We propose to renewal our post-doctoral training program in Genomic Medicine focuses on translational medicine and informatics and is led by complementary multiple principal investigators with expertise in genomic medical and biomedical informatics. We provide a foundation for training in Genomic Medicine, applicable to both MD and PhD trained fellows, recognizing that each may enter the program with different skill sets and experiences. It essential to provide all fellows with a strong background framework so that they can develop exciting and relevant projects in Genomic Medicine. Trainees enroll in a two-year program, including didactic courses, clinical and laboratories rotations, interactive learning experiences and research training. We require a balanced portfolio of courses, which cover the latest advances in genomics, focused on the role of genomics in disease processes, quantitative sciences, including biomedical informatics and biostatistics, scientific writing and ELSI issues. The 39 trainers were selected based on their expertise in Genomic Medicine and come from multiple departments, which is further enlarged with clinicians experienced in the provision of Genomic Medicine. The trainees are dually mentored in a research project by a balanced team (e.g. clinician and basic scientist) experienced in Genomic Medicine. Trainees participate in clinical rotations designed to give them experiences that range from the generation of massively parallel sequence data to data analysis to reporting back the results to patients. Trainees also participate in interactive activities, including a journal club, research in progress presentations, clinical genetics rounds and retreats. The combination of coursework, experiential clinical rotations, participation in interactive activities, and mentored research experience prepares our physicians and scientists for careers in Genomic Medicine. Over the past five years, we have trained 14 individuals (42% of applicants) in the Genomic Medicine program including eight physicians and six scientists, from both Penn Medicine and the Children’s Hospital of Philadelphia. Our trainees have gone on to independent careers in genomic medicine. We request continued support for the entry of three trainees a year in the Genomic Medicine T32 post-doctoral training program at the University of Pennsylvania.

NIH Research Projects · FY 2024 · 2017-07

Project Summary During pregnancy and lactation, the female skeleton undergoes significant bone loss and bone microstructure deteioration to provide calcium for fetal/infant growth. While weaning induces substantial bone recovery, reproduction-induced bone loss is only partially recovered after weaning. Our objective in the last funding cycle was to uncover protective mechanisms behind the lower fracture risk in women with histories of reproduction and lactation. Using a rat model, we demonstrated that structural adaptation of the maternal skeleton during pregnancy and lactation exerted a protective effect against estrogen deficiency-induced bone loss later in life. Moreover, we discovered enhanced responsiveness to external loading in the rat maternal bone both during lactation and later when subjected to estrogen deficiency by ovariectomy (OVX). During lactation, osteocytes (Ocys) are known to actively remodel their surrounding matrix via perilacunar-canalicular remodeling (PLR). Intriguingly, we also discovered enhanced Ocy PLR in the maternal bone post OVX, which appeared to be a result of “memory” or reactivation of Ocys’ PLR response during lactation. The activated PLR during lactation and post OVX leads to an enlarged lacunar-canalicular system (LCS) and an altered microenvironment of Ocys. Using a multiscale poroelastic model of the LCS, we further demonstrated that the PLR-induced alterations in the Ocy pericellular environment would amplify the mechanical and biochemical signal transduction to Ocys, which could in turn enhance mechanical adaptation of maternal bone to maintain its load-bearing function. These new and exciting findings provide a strong scientific premise for our central hypothesis that Ocy PLR-mediated skeletal adaptation increases Ocys’ mechano-sensing, which in turn enhances the mechano-response of maternal bone during lactation and post OVX. The objective of this renewal continues to be defining maternal bone adaptation mechanisms during challenging physiological events such as lactation and menopause. However, our focus advances from bone micro- and ultra-structural mechanisms (in the last funding cycle) to cellular mechanisms behind maternal bone adaptation and skeletal health (in this renewal). A cutting-edged imaging platform allows us to directly quantify mechano-sensitivity of the Ocy network by measuring in situ Ca2+ oscillations in mechanically loaded bones. In the Aim 1, we will establish the causal role of osteocyte PLR as an important mechanism to regulate bone mechano-sensitivity. In the Aim 2, by employing osteocyte fate mapping in a mouse model, we will for the first time interrogate the mechano-responses between osteocytes with and without exposure to prior lactation or lactation-associated hormonal changes within the same bone. The proposed research project will define a novel and critical function of ostecytes through PLR to regulate the balance between mineral resorption and mechanical integrity of the maternal skeleton during challenging physiological events such as lactation and menopause.

NIH Research Projects · FY 2025 · 2017-07

1 The University of Pennsylvania Training Program in Implementation Science and Mental Health is the only NIMH- 2 funded T32 program in the United States that provides training exclusively in implementation science and mental 3 health. This renewal application requests 5 years of funding to continue to train the next generation of scientists 4 who will study how to improve implementation of assessment, prevention, and intervention practices that can 5 transform mental health delivery across the lifespan. Since 2016, we have prepared 9 postdoctoral fellows, 44% 6 of whom met NIH criteria for underrepresented populations; 89% of whom were female. Those who have 7 graduated have faculty positions across the country. We will continue to provide training to postdoctoral fellows 8 per year and request additional slots for predoctoral trainees. Over the past 35 years, faculty in Penn's Center 9 for Mental Health have collaborated closely with community agencies, including Philadelphia County's 10 behavioral health Medicaid agency, the Philadelphia Departments of Health and Behavioral Health, the School 11 District of Philadelphia, the justice system, the Department of Veteran Affairs, and Penn Medicine. Through these 12 partnerships, we create unique opportunities to embed postdoctoral trainees in the settings in which they hope 13 to conduct implementation research. Penn has emerged as a leader in implementation science, with numerous 14 resources including the Penn Implementation Science Center, founded and directed by Contact PI Beidas, and 15 foundational and advanced coursework in implementation science. The proposed training program includes 16 seven experienced investigators from Penn and two community partners as part of the core executive committee. 17 Additional mentors are available from other schools at Penn including the Wharton School of Business and 18 investigators from other local institutions (Drexel, Temple)to encourage interdisciplinary inquiry and cutting-edge 19 knowledge. Further, a group of national leaders in implementation science (Senior Scholars) interact with 20 trainees regularly. We propose the following aims for our T32: Our trainees will (1) Participate 21 based 22 implementation 23 additional 24 science 25 from c 26 consultant. 27 partner Our T32 brings together trainees and mentors creating a fertile 28 interdisciplinary environment in which to train the next generation of implementation scientists. Successful 29 trainees are future leaders in reducing the research-to-practice gap and scaling up the use of evidence-based 30 practices in community settings. in didactic training, on a personalized needs assessment. Both pre and postdoctoral trainees will take two core courses in (foundational and advanced implementation science); predoctoral trainees will take any courses r equired by their doctoral programs; (2) Receive mentorship from a core implementation and mental health faculty member, an additional content mentor if applicable, and a community partner a state, city, or ommunity organization; and (3) Partner with a practice site to serve as an implementation Trainees will develop and conduct a small implementation research project with their community that will lead to an F, K, or R grant. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page

NIH Research Projects · FY 2025 · 2017-06

Enter the text here that is the new abstract information for your application. Rapid advances in genomic technologies are transforming our understandings of both human health and human history. These technologies also promise unprecedented power to intervene on the bodies of individuals with or at risk of disease and even to alter the identities or futures of individuals not yet born. Such knowledge and power imply immense responsibility to use them in ways that promote core ethical values, including individual and collective welfare, social justice, protection of the vulnerable, and respect for both the autonomy of persons and the interests of communities. Fulfilling these responsibilities requires a highly trained cadre of interdisciplinary scholars prepared to engage critically and respectfully with the profound ethical, legal and social challenges raised by genomic technologies. Responding to this need, the fundamental goal of the Penn Postdoctoral Training Program in the Ethical, Legal and Social Implications (ELSI) of Genetics and Genomics is to prepare trainees for success as creative, independent investigators in the field of ELSI research. The program, housed within the Department of Medical Ethics and Health Policy at the Perelman School of Medicine, will achieve this goal through three core components: 1) mentored original research leading to high-impact empirical and conceptual scholarly publications; 2) individualized didactic training including and, when appropriate, a Master of Science in Medical Ethics degree; and 3) intensive career development preparation. Trainees will participate as fellows for three years. An outstanding program faculty, consisting of 13 experienced ELSI scholars and mentors with appointments in departments at the Perelman School of Medicine, the School of Arts and Sciences, and the Penn Carey Law School, will serve as preceptors to program trainees. Trainees will present at and participate in the Department of Medical Ethics and Health Policy’s monthly works-in-progress and seminar series, attend a wide range of seminars and conferences across the University focused on genetic medicine and science as well as on ethics, law, economics and health policy, and present at national meetings. Trainees will also gain exposure to genetic medicine through shadowing expert geneticists and counselors drawn from the full spectrum of adult, pediatric and reproductive genetics practice. Finally, trainees will serve as members of one of the UPenn institutional review boards (IRBs) that review ELSI-related research. The program will ensure the fullest possible range of disciplines among enrolled trainees by extensive targeted outreach.

NIH Research Projects · FY 2026 · 2017-06

ABSTRACT Goals: Dr. Perlis requests K24 support to: 1) maintain and expand his mentoring of junior investigators in the area of behavioral sleep medicine (BSM) and in the biopsychosocial approach to the study of insomnia; and 2) expand his program of research on sleep and aging. Background: Dr. Perlis has been involved in patient-oriented research since his late-20’s. He has been the beneficiary of exceptional mentorship and has carried forward this tradition over the entirety of his career. His research, while narrow in focus, extends from basic to patient-oriented to epidemiologic studies. His work includes 175 peer reviewed articles, 38 chapters, and three books. Four of the chapters are in our field’s flagship text book, the Principles and Practice of Sleep Medicine. The work has been highly impactful (H-Index of 68 with over 19,000 citations) and has earned him the SBSM’s Peter Hauri Lifetime Achievement award, nominations for the SRS’s Mary Carskadon Education Award, and the SBSM’s “Champion of BSM” Award. Environment: The University of Pennsylvania is one of the nation’s premier centers for sleep and chronobiology research and sleep medicine / behavioral sleep medicine clinical services. As such, it has much to offer both the PI and his trainees. Development Activities: The core activity for the mentor’s professional development will be the implementation of a 2nd generation of monthly seminar series focused on Sleep and Aging. This series will be co-hosted with Dr. Michael Grander as part of the University of Arizona’s Essential Topics in Behavioral Sleep Medicine Lecture Series. Leading scientists will be invited to discuss their ideas with respect to the effects of aging on sleep. These lectures will be offered as video conferences (open to all at no cost) and will be archived so that they may be accessed into the foreseeable future. During the K24 time frame, Dr. Perlis will also complete the development of an open access sleep health screener and a sleep tracker app. Both will be setup to capture data from thousands of individuals. Both will serve as data archives. Research: The candidate has created a collaborative network to conduct archival analysis with Jr. Investigators using his NIA supported: 1) natural history of insomnia databases and 2) partial reinforcement of medical treatment of insomnia databases (n=3). Additionally, his trainees and collaborators will have access to his programs’ recruitment database (n=5000+), his ISI in psychiatry screening database (n=3,000), a risk benefit network meta-analysis database (PI: J. Cheung), his newly established screener and tracker databases, and three industry databases (two of which will be used to profile sleep in patients with Alzheimer’s and Parkinson’s disease; one of which will be used to profile acute and chronic placebo response in patients with insomnia, with a focus on age effects on placebo responding). The individuals invited to analyze one or more of the 12 databases will have specialty areas apart from the PI, and thus will serve as an opportunity for the PI to both teach and learn. A conference will be held in the 5th year of this award for all collaborators and trainees. Significance: This K24 will support Dr. Perlis’ efforts to promote and mentor patient-oriented research, with a focus on aging effects on sleep continuity.

NIH Research Projects · FY 2025 · 2017-05

PROJECT SUMMARY Almost all brain imaging studies now collect multiple imaging modalities, in an effort to derive measures of both structure and function from diverse imaging sequences. While quantitative data scientists have focused on machine learning approaches for predicting outcomes using multi-modal imaging, rigorous statistical methods for examining the relationship between imaging modalities have lagged behind. At present, the lack of statistical methodologies for assessing inter-modal coupling (IMCo) has left investigators with ad hoc solutions that lack statistical power and are prone to type I error, posing a threat to scientific rigor and reproducibility. In this application, we propose robust methods that leverage subject-specific measurements and use nonlinear modeling to address complex relationships in brain maps or networks, while accounting for important covariates (Aim 1). Furthermore, we will develop powerful approaches for assessing whether effects of interest (e.g., psychopathology, development) are enriched within brain networks (Aim 2). Assessment of this coupling between statistical associations and brain networks will capitalize upon tools from statistical genomics (e.g., gene set enrichment analysis) to provide principled methods for conducting enrichment analyses using high- dimensional, personalized brain networks. Finally, we will use these tools to delineate how trans-diagnostic executive dysfunction in youth with mental illness is related to abnormalities in structure-function coupling within brain networks (Aim 3). To do this, we will leverage three massive data resources: the Philadelphia Neurodevelopmental Cohort (PNC; n=1,601), the Healthy Brain Network (n=3,200), and the Human Connectome-Development (HCP-D; n=1,300) study Taken together, the proposed work builds upon the notable success in the first project period, promising to yield rigorous and generalizable methods for delineating the relationships between complementary measures of brain structure and function.

NIH Research Projects · FY 2025 · 2017-04

Abstract Brain aging is commonly accompanied by a number of neuropathologic processes, often co-occurring, that may lead to cognitive decline and dementia. Vascular contributions to cognitive impairment and dementia (VCID) are also extremely common and, due to associations with cardiovascular risk factors, may be mitigated with current therapies. It is clear that effective treatments for AD-related dementias (ADRD) will require early detection of pathologic brain change at prodromal and cognitively normal stages. Imaging methods offer the opportunity to study diverse brain changes present in aging and prodromal AD in ways that were previously impossible. Characterizing these multi-faceted aspects of brain structure, function and pathology not only provides insights into the underlying pathophysiological processes, but also novel predictive in vivo biomarkers. Various studies have shown that relatively early signs of neurodegenerative processes can be detected via AI-based pattern analysis and machine learning (PAML) methods, and that these tools can provide powerful predictive individualized panels of predictors. Our group has been on the frontier of developing PAML methods, and applying them to the new “Imaging-based coordinate SysTem for AGing and NeurodeGenerative diseases” (iSTAGING) consortium, a large-scale effort pursued in the current phase of our grant, which successfully brought together and harmonized over 51,000 MRIs and clinical data from 11 studies and ~34,000 individuals. We aim to capture the heterogeneity of brain change with aging and prodromal AD, by applying our heterogeneity analysis PAML deep learning (DL) methods, which help structuring imaging patterns associated with different brain aging trajectories. Our goal is to enrich the different dimensions of iSTAGING which will reflect various patterns of brain change, hence capturing the underlying heterogeneity in quantifiable and replicable metrics. Although we will include our previously derived measures of rsfMRI networks and of amyloid burden, in the proposed work we will focus on further dissecting neuroanatomical heterogeneity, i.e. on refining the `N' in the AT(N) framework to measure variability in AD neurodegeneration and the contributions of copathologies, and on using these intermediate neuroimaging phenotypes to predict cognitive decline and clinical progression. This will allow us to place each individual into the iSTAGING brain chart and map his/her trajectory, as well to determine predictive indices of brain change and cognitive decline. The current project builds on the foundational work of the previous funding phase, and expands this unique resource to include several studies focusing on longitudinal data, on groups of under-represented socio-economic status, as well as on various co-morbidities including hypertension, diabetes, obesity, smoking and sleep disturbances. The proposed work will also leverage recent developments in deep learning, and will offer advanced methods for harmonization, heterogeneity analysis, and predictive modeling.

NIH Research Projects · FY 2026 · 2017-04

NEURONAL CIRCUITS SUPPORTING LEARNING-DRIVEN CHANGES IN AUDITORY PERCEPTION. Learning to discriminate sounds in noise is fundamental to auditory processing and is critically important for everyday communication and navigation. Both the auditory thalamus and the auditory cortex have been shown as involved in detection of sounds in noise, yet how the microcircuits within intra-cortical, cortico-thalamic and intra- thalamic interactions facilitate detection of signal in noise remains unknown. Our goal is to close this gap in knowledge and determine whether and how multiple microcircuits within the cortico-thalamic loop, including excitatory-inhibitory circuits within the primary auditory cortex; feedback loop between the auditory cortex and the lemniscal and non- lemniscal auditory thalamus; and the cortical feedback via the inhibitory thalamic reticular, contribute to the learning- driven improvement in auditory perception in noise. We will train mice to detect or discriminate between auditory targets in noise using operant conditioning. Detecting and discriminating sounds in noise activates multiple processes, including selective adaptation to background noise and learned amplification of the target representation. We will change noise contrast prior to target presentation to test how contrast adaptation interacts with target detection. We will record the neuronal responses in the cortical and sub-cortical regions during behavior, and use temporally precise, cell specific manipulations to establish the circuit that allows the mouse to learn and carry out this complex task. We will implement a novel generalized linear-non-linear model to continuously estimate gain adaptation in neuronal responses to test whether and how the distinct microcircuits adapt to contrast, detect the target and control neuronal gain. Combined, our results will reveal novel circuit-level mechanisms for hearing in noise across micro-circuits within the cortico-thalamic loop.

NIH Research Projects · FY 2026 · 2017-04

Project Summary Nausea and vomiting promote mammalian survival. Paradoxically, emetic “side effects” are ubiquitously reported for FDA-approved pharmacotherapeutics for obesity, diabetes, and cancer pharmacotherapies and present alongside polymorbidities that contribute to detrimental life-threatening outcomes, such as poor nutrition, quality of life, and patient prognosis. Here, we address two broad unmet clinical needs: 1) All existing FDA-approved glucagon-like peptide-1 (GLP-1)-based therapeutics for the treatment of diabetes and obesity elicit nausea and vomiting in a significant percentage of patients. 2) Despite existing antiemetic treatments available, virtually all patients undergoing chemotherapy continue to exhibit profound debilitating symptoms, such as severe nausea, vomiting, and cachexia. We use modern behavioral and neurogenetic approaches, and appropriate, comparative, preclinical animal models that are critical to produce novel, effective, long-term controls of nausea and vomiting to advance modern metabolic health care. Intestinally derived GIP regulates postprandial glucose through direct action on GIP receptors (GIPR) expressed on pancreatic beta cells. GIP analog efficacy as a monotreatment of diabetes and obesity is at best limited and controversial, however, the expression of CNS GIPRs in regions implicated in nausea/emesis have spawned investigation of central actions of GIP ligands as potential adjunct therapeutics to reduce unwanted adverse events. Specifically, our data support that GIPR and GLP-1R dual agonism provide body weight loss, hypophagia, and glucoregulatory control without nausea and emesis, compared to GLP-1R agonism alone, through activation of the GIP system. The area postrema (AP) and nucleus tractus solitarius (NTS) of the dorsal vagal complex (DVC) play a critical role in ingestive behavior, emesis, and nausea. Widely used emetogenic chemotherapeutics (e.g., cisplatin) and all FDA- approved GLP-1-based ligands activate AP/NTS neurons. Our collective works suggest hindbrain GIPRs block nausea and vomiting induced by GLP-1R and cisplatin chemotherapy in several animal species, suggesting translational broad-spectrum antiemetic potential for GIPR agonists. We have identified cellular phenotypes of AP/NTS GIPR- and GLP-1R- expressing cells, as well as shown the attenuation in AP/NTS neuron activity, and preliminary data). Additionally, we have discovered a molecularly distinct GABA-ergic neuronal DVC population that is modulated by chemotherapy but rescued by GIPR agonism. We hypothesize that there exists an antiemetic system characterized by inhibitory (i.e., GABA-ergic) neurons expressing GIP receptors (GIPR). Here, we will: Aim I: Examine behavioral, anatomical, and transcriptomic mechanisms by which GIPR-GABA+ AP/NTS neurons exhibit antiemetic action. Aim II: Examine GIP antiemetic action in conjunction with established antiemetics using a multi-species approach. Our data in multiple species all indicate that GIP agonism has an antiemetic effect and here we use our unique multi-species approach to define the mechanisms of the GIP system in reducing and/or preventing therapeutic drug-induced nausea and emesis.

NIH Research Projects · FY 2026 · 2017-04

Summary: Although memory dysfunction is a frequent and debilitating symptom of traumatic brain injury (TBI), there are currently no effective treatments available for this often persistent deficit. In addition, the neurophysiological basis of this dysfunction remains unknown, hindering rational treatment design. There is mounting evidence that precisely coordinated communications between brain regions are necessary to encode and recall information in the neuronal ensembles that represent episodic, spatial, and working memory. The hippocampus (HC) is the most well-studied region of memory encoding and is considered to be selectively vulnerable in both human and animal TBI. We and others have demonstrated disruptions of oscillations in the HC following TBI, with the loss of theta a notable finding. Rodent studies have demonstrated that restoration of theta using stimulation (neuromodulation) can restore aspects of HC dependent memory. However, the mechanism remains unknown, as does the complex relationship of these neuronal ensembles to oscillations and their correlation with memory deficits after TBI. Without a deeper understanding of how ensemble coding underlying cognition and memory is disrupted post injury, rational design of neuromodulatory and other therapies remains challenging. Therefore, a critical need exists to determine the underlying mechanisms of the disruption of coding in networks underlying memory formation after TBI, and how a reintroduction of theta restores cognitive function. The overall objective of the current application is to determine how the coding of memory in the hippocampus and associated circuitry is disrupted following TBI, and how theta neuromodulation restores function. Our central hypothesis is that TBI disrupts communication within the larger hippocampal network which disrupts oscillatory interactions required for encoding and recall of memory in networks of synchronized neuronal ensembles. This hypothesis is based in part on our preliminary data demonstrating that neurons in the hippocampus synchronize improperly with oscillations following injury, and that prominent interactions between oscillations are lost. We therefore propose to determine whether TBI affects phase precession, theta sequences, and phase amplitude coupling in area CA1 of the hippocampus during overtrained tasks designed for these measures, as well as whether both hippocampi are affected by a unilateral injury. In addition, we will determine the mechanism of learning and memory dysfunction following TBI by examining neuronal ensemble activity across HC-PFC, quantifying ripple features and replay, and correlating these measures with behavioral memory function relying on HC-PFC networks. We will also examine the mechanism of neuromodulatory restoration of spatial/working memory in rats via simultaneous medial septal stimulation and high-density laminar hippocampal/mPFC recordings. Accomplishment of these goals will provide the first detailed analysis of disrupted neuronal coding and oscillatory interactions between brain regions underlying TBI induced memory dysfunction, identify the effects of neuromodulation on these networks, and lead to clinical treatments for this persistent sequelae of TBI.

NIH Research Projects · FY 2026 · 2017-04

PROJECT SUMMARY/ABSTRACT The prevalence and significance of methylation reactions in biology is well established. Methyl groups are appended to a wide array of biological molecules, including numerous small-molecule metabolites and natural products, and various macromolecules, such as proteins, DNA, RNA, carbohydrates, and lipids. In the vast majority of methylation reactions, S-adenosylmethionine (SAM) is the source of the appended methyl group. In classical methyltransferase reactions, strong nucleophiles such as oxygen, nitrogen, and sulfur attack the sp3- hybridized methyl group of SAM in a polar SN2 reaction, affording S-adenosylhomocysteine as a co-product. Carbon atoms can also be methylated by this mechanism, but only if a suitably nucleophilic carbanion can be generated. Relatively recently, it has come to light that SAM can be used to methylate inert carbon or phosphinate phosphorous atoms via pathways involving radical intermediates. These noncanonical SAM- dependent methylations are found in numerous biosynthetic pathways for antibiotic, antifungal, anticancer, and herbicidal natural products, and are catalyzed exclusively by enzymes within the radical S- adenosylmethionine superfamily. Radical SAM methylases currently consist of three classes (Class A, Class B, and Class C) based on structural architecture, cofactor requirement, and mechanism of action. Class A enzymes use a Cys dyad to catalyze methylation of sp2-hybridized carbon centers. Class B enzymes use cobalamin cofactors to catalyze methylation of both sp2- and sp3-hybridized carbon centers. Class C enzymes use two simultaneously bound molecules of SAM to methylate sp2-hybridized carbon centers. In all cases, the appended methyl group derives from a second molecule of SAM. This work will continue our efforts to understand how these radical SAM methylases work, with a particular focus on efforts to determine structures of these enzymes with bound substrates, cofactors, and intermediates. Important systems include RNA methylases that are involved in antibiotic resistance, as well as methylases that are involved in the biosynthesis of important antibiotics, such as thiostrepton A, nosiheptide, and carbapenems, the antibiotics currently of last resort.

NIH Research Projects · FY 2026 · 2017-03

PROJECT SUMMARY The development of an effective HIV-1 vaccine has proven to be a daunting scientific challenge. Despite decades of research, there are no examples of immunogens that consistently elicit potent broadly neutralizing antibodies (bNAbs). Our scientific premise is that there are three principal obstacles to inducing bNAbs in primates and humans that prior vaccine approaches have largely failed to overcome. These are: i) efficient and consistent priming of multiple HIV-1 bNAb precursor B cells; ii) immunofocused boosting of B cell responses targeting conserved bNAb epitopes and away from off-target epitopes; and iii) affinity-guided maturation of B cell lineages to select for enhanced neutralization breadth and potency. Here, we address each of these requirements. This application is a competitive renewal of an existing HIVRAD award where we hypothesized that a major roadblock to rational HIV-1 vaccine design is the lack of a suitable primate model in which bNAbs can be commonly induced and the molecular, biological and immunological mechanisms responsible for such responses studied in a reproducible and iterative fashion. Overcoming this roadblock was one of the major goals of that grant, and over the past five years we have made substantial progress in reaching this milestone. We did this by developing a novel design strategy for creating simian-human immunodeficiency viruses (SHIV) that bear clinically-relevant primary HIV-1 Envs and that replicate efficiently in rhesus macaques (RMs). We next hypothesized that SHIV- infected RMs could be used to identify HIV-1 Envs that have a propensity for eliciting bNAbs of predetermined epitope specificity, thus allowing for a detailed and reproducible molecular characterization of the coevolutionary pathways of Env and Ab that lead to affinity maturation and breadth. Again, we obtained strong supporting evidence (Science 371:eabd2638, 2021). Here we propose to build on this foundation and to test the hypothesis that elucidation of the molecular pathways of Env-Ab coevolution leading to neutralization breadth in SHIV- infected RMs, combined with biophysical and immunological analyses of key Env-Ab lineage intermediates, can provide a molecular “blueprint” for successful germline-targeted, B cell lineage-based immunogen design. To test this hypothesis, we propose three highly inter-related research projects and three cores: Project 1 - Env-Ab coevolution in SHIV infected RMs leading to V3 glycan bNAbs (Shaw); Project 2 - Optimizing humoral immunity to HIV-1 Env proteins (Kelsoe); Project 3 - Immunogen design to elicit polyclonal bNAb responses to the V3 glycan supersite (Wiehe). These projects will be enabled by Core A – Administrative (Shaw); Core B – Viral and antibody gene sequencing (Hahn); and Core C – Bioinformatics and statistics (Wagh). The significance of the proposed studies is potentially far-reaching: previous studies of HIV-1 SOSIP Env trimer vaccinations have generally elicited only autologous strain-specific Nab responses in outbred animals. If we can demonstrate consistent induction of bNAbs using germline-targeted, lineage-based SOSIP Env trimers as immunogens in RMs, it would represent a major scientific advance and a new beachhead for HIV-1 vaccine research.

NIH Research Projects · FY 2025 · 2017-01

Project Summary/Abstract The meetings of the Androgen Excess-Polycystic Ovary Syndrome (AE-PCOS) Society are the only ones that convene the world's largest group of researchers and clinicians specifically focused on androgen-excess related disorders. Both the Annual and Update meetings bring together diverse participants to discuss their latest research findings, to encourage future research collaborations and to disseminate relevant and accurate health information to the clinical community and general public. The attendees represent several regions of the world and a variety of disciplines including medical, pediatric and reproductive endocrinology, gynecology, internal medicine, psychologists, epidemiologists, clinical nutritionists and physiologists. The two-day Annual Meeting has a well-established format consisting of multiple sessions that include invited scientific lectures, oral and poster presentations, meet-the-professor sessions, all with interactive and/or question and answer opportunities. We provide travel awards for junior investigators and child care awards to encourage the participation of the next generation of researchers and clinicians. The one-day Update Meetings, also held annually, are focused on basic science research and have a specific theme. We have demonstrated participation by females and underrepresented minorities in our meetings, membership and Board of Directors. One of the new highlights is increased collaboration with patient support groups and actively engaging patients and consumers in our meetings. The Society is composed of basic and clinical scientists and clinicians whose major interest is the etiology, diagnosis, and treatment of androgen excess disorders. The AE-PCOS Society has successfully organized 17 Annual Meetings, 4 Update Meetings which are organized as satellite meetings along with the ASRM and Endocrine Society meetings in the USA or stand alone in other international locations. The Society through its meetings and resultant publications (14 guidelines and position statements) has had significant impact on dvelopment of criteria for diagnosis, management and treatment strategies for PCOS.

NIH Research Projects · FY 2025 · 2016-12

Current modalities for preventing dental caries are insufficient, particularly when biofilms rapidly accumulate under cariogenic conditions in susceptible individuals, requiring new approaches. In the previous funding period, we studied the potential of catalytic (peroxidase mimics) iron oxide nanoparticles (IONP) for controlled, pH- dependent activation of hydrogen peroxide as a novel antibiofilm and anticaries treatment. We found that IONP displays selective-biofilm targeting and elimination under cariogenic (acidic and sugar-rich) conditions, while also reducing apatitic demineralization. In vivo studies revealed that IONP are highly effective against caries development without affecting oral tissues and the oral microbiome diversity, confirming therapeutic precision. We also discovered that an FDA-approved IONP formulation, ferumoxytol (FerIONP), displays similar acid pH- activated antibiofilm and anticaries mechanisms in vivo. In search for ways to improve efficacy and applicability to enhance current modalities, we tested the possibility of combining FerIONP with fluoride. We unexpectedly found a remarkable synergy between FerIONP and stannous fluoride (SnF2) that was exceptionally effective in preventing caries in a severe rodent caries model. In this renewal, we propose to further develop this treatment regimen, and then understand its mechanisms of action as well as potential deleterious effects using laboratory, in vivo and human in situ models to facilitate clinical translation and product development. The significance of this work is to develop a more effective and targeted antibiofilm and caries preventive approach for susceptible populations. We hypothesize that FerIONP interacts with SnF2 to modulate both biological and physicochemical properties by increasing localized antibiofilm action and protection against enamel demineralization, potentiating anticaries efficacy without increasing the concentration of agents. We will perform dose-response studies to improve the efficacy of FerIONP/SnF2 and assess local and systemic biological actions in vivo (Aim 1). We will assess enhanced antibiofilm and caries preventive performance at low doses without deleterious effects on oral-gut microbiome or toxicity on oral mucosal tissues and vital organs. We will compare with previous FerIONP regimen (internal control) and currently used antimicrobial fluoride (SnF2). Then, we will investigate the mechanisms of action and biodistribution of FerIONP-SnF2 (Aim2). We will generate specific FerIONP analogues to understand biofilm targeting specificity and their combined effects with SnF2 on enamel structure. We will perform multi-omics to assess the influence on biofilm composition and functional activities as well as biodistribution of FerIONP via radiolabeling. The impact on enamel structure will be determined via physical-chemistry and spectroscopic methods. In Aim 3, we will further elucidate the bioactivity of the improved formulations using the human intra-oral biofilm model with a clinically relevant topical treatment regimen. We envision a viable and novel technology to target virulent biofilms and prevent caries in susceptible individuals under high cariogenic conditions that will motivate product development and clinical efficacy studies.

NIH Research Projects · FY 2026 · 2016-11

PROJECT SUMMARY In addition to traditional antimicrobials, targeting host defense pathways is an attractive strategy to limit the adverse effect of bacterial infection. One such pathway that has received considerable attention is autophagy, a process where cellular constituents are sequestered in a double-membrane vesicle that is subsequently targeted to the lysosome for degradation and recycling. Autophagy is suggested to be critical for cell autonomous defense because many bacterial pathogens are detected within double-membrane vesicles upon internalization, a process referred to as xenophagy. Therefore, it is possible that drugs that target autophagy will be useful in a wide range of diseases downstream of bacterial infections. In this program, we are studying the contribution of ATG16L1, an autophagy protein that plays a central role in autophagosome formation, in the host response to two model pathogens –Salmonella enterica Typhimurium and Staphylococcus aureus. By studying autophagy in the setting of S. aureus we have discovered that ATG16L1 enable mammalian cells to respond to bacterial infections by producing exosomes, small secreted vesicles that protect the host from infection by neutralizing potent toxins produced by this bacterium. Our studies with Salmonella have discovered that the commonly found ATG16L1 T300A allele impacts the susceptibility of the host towards this pathogen in a non-cell autonomous manner. Thus, the goals of this competitive renewal application are to elucidate the mechanism(s) by which mammalian cells coopt autophagy and pathogen sensing to control exosome biogenesis (Aim 1) and to unravel the molecular details of how ATG16L1 T300A contributes to host-mediated protection from infection by bacterial pathogens. A better understanding of how ATGs participate in non-xenophagy functions can help bridge the gap between cell autonomous defense and complex extracellular mechanisms involved in host-microbe interactions.

NIH Research Projects · FY 2025 · 2016-11

Project Abstract The protozoan parasite Cryptosporidium is one of the most important causes of severe diarrheal disease. In the U.S. this parasite is responsible for half of all waterborne disease outbreaks, some of which have occurred at massive scale. Patients suffering from immunosuppression due to HIV/AIDS, organ transplantation, or cancer are in gravest danger. The global public health impact is even larger: after Rotavirus, Cryptosporidium is the most important diarrheal pathogen in small children. In particular in the context of malnutrition, cryptosporidiosis has a highly significant imprint on childhood mortality. Cryptosporidiosis is also linked to stunting, thus leaving a lasting shadow on the future of children. This parasite has a single host life cycle, asexual and sexual processes occur sequentially in the intestinal epithelium of the same host. Completion of this developmental program is required for continued infection, severe disease, and transmission. We have built robust experimental systems to observe and manipulate the sexual development of Cryptosporidium and we unraveled key elements of the mechanisms that control the underlying gene expression systems. In this application we will define the epigenetic mechanisms that govern transition from asexual to sexual replication, we will unravel how the parasites choses between a male or female fate, and we will use emerging cyro-electron tomography and parasite genetics to understand how the unique ultrastructure of male gametes enables fertilization. The resulting findings will impact on our fundamental understanding of parasite development and on translational efforts to develop drugs and vaccines.

NIH Research Projects · FY 2026 · 2016-09

Summary: Mast cells (MCs) are best known for their roles in IgE-mediated allergic disorders. However, the recent identification of Mas-related G protein coupled receptor-X2 (MRGPRX2) in human cutaneous MCs (MrgprB2; mouse counterpart) as the receptor for antimicrobial peptides, neuropeptides and chemokines has revolutionized the way in which MCs are viewed. Furthermore, the development of MrgprB2-/- mice has been instrumental in delineating the roles of this receptor in non-IgE-mediated disorders. However, it now appears that expression of MrgprB2 is not restricted to cutaneous MCs and that it is also present in lung and gut MCs. We made the surprising observation that in addition to cutaneous disorders MrgprB2 contributes to allergic lung inflammation. MrgprB2 displays only ~62% sequence similarity with human MRGPRX2 and specific inhibitors of the human receptor do not block responses to the mouse receptor. Therefore, studies conducted with mice expressing MrgprB2 may not fully represent disease conditions in humans. To overcome this major hurdle, we utilized CRISPR/Cas9-mediated gene targeting strategy and MC knock-in procedure to replace the endogenous mouse MrgprB2 with functional human MRGPRX2. In aim 1, we will delineate the role of MRGPRX2 on experimental psoriasis and allergic asthma and test the ability of specific inhibitors to modulate these responses. We will delineate how gain- and loss-of-functional variants of MRGPRX2 modulate MC signaling in vitro and disease phenotype in vivo. We will perform spatial transcriptomic analysis to delineate how pharmacologic and genetic modulation of the receptor regulate signaling in MCs and their neighboring immune and non-immune cells in the context of psoriasis and allergic asthma. We found that β-arrestin1, but not β-arrestin2, promotes MRGPRX2 internalization in response to substance P in human skin MCs. By contrast, β-arrestin2 contributes to MrgprB2-mediated NF-κB/ERK phosphorylation and cytokine generation in vitro and experimental allergic lung inflammation in vivo. This difference could reflect difference in β-arrestin utilization by MRGPRX2 and MrgprB2. In aim 2, we will delete β-arrestin1 and β-arrestin2 in mice expressing human MRGPRX2. These mice will be used to determine the effects of these adaptor proteins on MRGPRX2-mediated responses in vitro, disease phenotype in vivo and spatial transcriptomic changes in tissues. Completion of this study may provide a new rationale for the development of novel therapeutic approaches for treating MC-mediated allergic and inflammatory disorders.

NIH Research Projects · FY 2025 · 2016-09

Human Pancreas Analysis Program for Type 1 Diabetes (HPAP-T1D) Abstract Building on the extensive existing infrastructure and scientific collaborations within the Human Pancreas Analysis Program for T1D (2017 – present), we will continue and expand the work and technology of our six cores to advance research in human type 1 diabetes (T1D). Core A will procure human pancreata, immune tissues, and detailed donor medical histories from T1D, non-diabetic but autoantibody-positive (AAb), and control donors; collect immune tissues and isolate islets; and distribute high-quality islets, tissues and cells to the other Cores for further analysis or processing. Core B will perform rigorous physiological phenotyping by perifusion and oxygen consumption assays on the isolated islets, and single islet measurements of intracellular calcium, as well as implement newer technologies to study cell composition. Core C will perform sophisticated immune profiling in spleen and lymph nodes by cell type mapping with differential gene expression and chromatin accessibility analysis, generation of a memory B cell and T cell clonal atlas across different tissues, and specificity analysis on circulating antibodies and selected T and B cell receptors. Core D will perform multiple advanced modalities for the molecular profiling of isolated islets including RNA-seq and DNA methylome analysis of sorted islet cell populations; single cell ATAC-seq and RNA-seq, flow mass cytometry for single cell quantification of more than 30 cell surface and intracellular markers, and spatial transcriptomics. Core E will process all tissues using multiple fixation modalities, cmduct pathology analysis, and biobank remaining pancreatic tissues. Core E will also perform advanced tissue morphometry, including highly multiplexed immunofluorescent imaging using CODEX. Finally, Core F will assemble, annotate, maintain and upgrade an extensive open-access database (PANC-DB) for the program and its member-researchers, and collaborate with HIRN in the sharing of data. The entire program is directed by an Executive Committee consisting of the core leaders and the contact PI, who will interface with HIRN and NIDDK leadership. Taken together, HPAP-T1D will provide physiologic, genomic, genetic, immunologic and histologic analyses of the pancreas and immune- associated tissues in T1D and share these comprehensive, integrative and robustly quality controlled data with researchers world-wide.

NIH Research Projects · FY 2025 · 2016-09

The Penn Skin Biology and Diseases Resource-based Center (Penn SBDRC) will continue to support and accelerate skin disease research and its translation by providing critical infrastructure, resources, and expertise to skin investigators. The overall goals of the Penn SBDRC are to promote collaboration among skin investigators, especially across disciplines; to draw new investigators with new perspectives to skin research; to foster an environment that supports and cultivates the next generation of skin researchers; to increase access to technology and resources for conducting rigorous, impactful skin research; and to accelerate translation of research findings into innovative therapies for skin disease. To accomplish these goals, the aims of the Center are: Aim 1) To provide cutting-edge approaches and related expertise centered around 3 multi-disciplinary Resource Cores: Cutaneous Phenomics and Transcriptomics (CPAT) Core, Skin Translational Research (STaR) Core, and Data Science and Informatics (DSI) Core. These Cores are highly synergistic, providing high-demand and/or specialized services, valuable tissue/cellular resources, and the expertise to ensure rigorous experimental design, analytical strategies, and accurate interpretations. Aim 2) To establish an Administrative Core that unifies skin investigators and promotes the goals of the SBDRC. The Administrative Core provides oversight and implements the activities of the center. This includes an Enrichment Program that is subdivided into 4 Sub-Cores: The Community Outreach Sub-Core introduces Philadelphia high school students to the excitement of biomedical research and dermatology through the Penn Academy of Skin Health (PASH), a Saturday academy and summer internship program. The Mentoring Sub-Core promotes career success and training through structured mentorship for trainees and junior faculty. The Scientific Enrichment Sub-Core sponsors a seminar series, workshops, and a yearly scientific symposium. The Seed Funding Sub-Core offers funding mechanisms that attract new investigators and support the independence of junior investigators. The Penn SBDRC leverages the excellence of Penn Dermatology’s research and clinical programs as well as that of the entire Penn community to enhance collaboration, bringing new technology to skin research, and providing access to critical intellectual and technical resources that greatly enhance innovation and rigor in the study of skin disease.

NIH Research Projects · FY 2025 · 2016-08

PROJECT SUMMARY This Institutional Training Grant (T32) is a competing renewal of a training program from the Pe- relman School of Medicine (PSOM) at the University of Pennsylvania (Penn) and the Children’s Hospital of Philadelphia (CHOP) to train MD and MD-PhD fellows pursuing careers in bench re- search in microbiology, microbial immunology, and the microbiome. Our program combines the talent and resources available at two independent but linked institutions, Penn and CHOP. Audrey Odom John MD PhD from the Division of Infectious Diseases at the Children’s Hospital of Phila- delphia will serve as Program Director (PD) and Harvey Friedman MD from the Division of Infec- tious Diseases at Penn will serve as Associate Program Director (APD). Our program addresses a critical public health need for a robust infectious diseases physician- scientific workforce. To address this need, our applicant pool will come primarily from the adult and pediatric infectious diseases postdoctoral sub-specialty training programs, but enrollment will be open to all divisions and departments at Penn and CHOP who have MD and MD PhD post- doctoral fellows interested in pursuing basic science research in infectious diseases. Twenty- seven mentors help support the careers of the trainees. There is a high degree of collaboration among the mentors and trainees with shared publications and grants. In addition to mentored research training, our curriculum emphasizes the following: 1) formal course work through Penn Graduate Studies Program; 2) training in responsible conduct of research; 3) training in biostatis- tics and rigor and reproducibility; 4) regular research presentations and feedback; and 5) forums that teach a variety of important career development skills, including scientific writing, public presentations, grant writing, laboratory management, mentoring skills, and becoming knowledge- able about career options. During our initial funding period, three trainees have completed train- ing: two of these have successfully acquired K08 funding and have research-intensive academic positions, and one is in an academic, research-related clinical faculty position. We request con- tinued support for three postdoctoral fellows/year who participate in a program that is designed to be three years in length. This program combines the outstanding CHOP/Penn training environ- ment, an exceptional cadre of trainees and mentors, and substantial institutional resources for research and training in infectious diseases research to promote trainee success.

NIH Research Projects · FY 2025 · 2016-08

ABSTRACT Intrinsic functional connectivity magnetic resonance imaging (fcMRI) is a powerful tool for understanding brain function and development. Despite significant progress in modeling personalized functional networks (PFNs) and leveraging machine learning (ML) to predict brain-behavior relationships, challenges persist, including the heterogeneity of brain development, the difficulty in characterizing diverse PFN patterns, site effects in multi- site datasets, and the limited interpretability of deep learning (DL) models. To address these challenges, this project aims to develop, validate, and disseminate tools for computing and characterizing heterogeneous PFNs, harmonizing multi-site fcMRI data, and building interpretable prediction models. These models will capture PFN patterns linked to variations in brain development and psychopathology in youth. Specifically, we will develop a novel self-supervised DL method to identify distinct biotypes of individual variation in fcMRI data and compute biotype-specific PFNs using a mixture-of-experts approach. We will also develop a robust self- supervised DL method to harmonize multi-site fcMRI data and compute PFNs, employing a test-time adaptation (TTA) strategy. Additionally, we will develop an interpretable DL method to characterize and predict cognition and psychopathology based on PFNs, leveraging a generalized additive model (GAM) framework with a prototype learning mechanism. The tools will be developed and validated using data from the Adolescent Brain Cognitive Development (ABCD) Study, the Philadelphia Neurodevelopmental Cohort (PNC), the Healthy Brain Network (HBN), and the Lifespan Human Connectome Project in Development (HCP-D). The algorithms will be released as a user-friendly toolbox with source code and standalone programs on GitHub and DockerHub. This project will revolutionize our understanding of brain development, enhance our understanding of individual brain development, and pave the way for more effective, personalized treatments in diverse clinical settings.

NIH Research Projects · FY 2025 · 2016-08

Project Summary Periodontitis is a prevalent inflammatory disease that causes destruction of the tooth-supporting tissues (periodontium). The maintenance of homeostatic mechanisms is essential for protection against inflammatory damage in the periodontium. In this context, the function of immune cells needs to be tailored according to specific environmental challenges; for instance, the ability to mount a robust immune response needs to be followed by timely resolution of inflammation and restoration of tissue integrity. This adaptation is known as functional immune plasticity and results from an intimate crosstalk of immune cells with tissue-derived factors, which, in turn, are regulated by and reflect changes of the tissue microenvironment. The functional characterization of a novel endogenous homeostatic molecule, derived from periodontal tissue-resident cells and designated developmental endothelial locus-1 (Del-1), has significantly contributed as a prototype paradigm to the emerging concept that tissues have a “regulatory say” over the host immune response. This project investigates the overarching hypothesis that Del-1 acts as a local endogenous regulator of functional immune plasticity by not only regulating periodontal inflammation but also by promoting resolution thereof, and hence periodontal homeostasis. The proposal comprises three specific aims and focuses on relevant animal model-based mechanistic and intervention studies, including mice with lineage-specific deletions or overexpression of Del-1. In Aim 1, it is proposed that Del-1 promotes the resolution of periodontal inflammation. Aim 2 investigates the mechanism(s) by which Del-1 promotes homeostatic immunity. Specifically, it is proposed that Del-1 modulates macrophage plasticity via two complementary mechanisms; inhibition of inflammatory signaling and promotion of a pro-resolution reprogramming of macrophages, both of which may impact the function of other immune cells, such as T cells. Aim 3 is to determine the importance of the location of Del-1 expression in the regulation of periodontal inflammation and bone loss. The concept to establish here is that the cellular source and location of homeostatic molecule expression is functionally important in that it allows it to perform distinct functions in an appropriate context. This application therefore offers a fundamentally new insight into the local mechanisms that govern and tailor the function of the immune system. The proposed research is likely to reveal hitherto unknown mechanisms of immune system plasticity relevant to the pathogenesis of oral diseases; these mechanisms can be harnessed to develop innovative approaches to inhibit destructive inflammation and restore tissue integrity.

NIH Research Projects · FY 2026 · 2016-08

Project Summary Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma accounting for about 32,000 new cases per year and leading to death in over 40% of cases. This proposal seeks to investigate the biological significance of KLHL6, a gene mutated in mature B-cell cancers, with DLBCL displaying the highest rate of mutations. KLHL6 assembles into a functional CULLIN-RING Ubiquitin ligase (CRL) complex and cancer-associated mutations inhibit KLHL6 interaction to CULLIN3, resulting in loss of activity to transfer ubiquitin chains. In this proposal, we investigate KLHL6 as a master regulator and tumor suppressor of the NOTCH signaling. An investigation of the cell autonomous and drug resistance in murine model of DLBCL as well as patient derived DLBLC xenotransplants will be pursed. Building up on our data, the central hypothesis of this proposal is that deregulation of the KLHL6 function is crucial to lymphomagenesis and impacts therapy. Thus, we aim in modeling loss of Khll6 in a mouse model of DLBCL (Aim1) and we will study how impairment of the NOTCH pathway impacts the therapeutic efficacy of B-cell receptor inhibition in DLBCL (Aim2). Overall, this proposal investigates the mechanisms of DLBCL pathogenesis and treatment. The clinical success of proteasome inhibitors, bortezomib, and E3 ubiquitin ligase glues for the treatment of hematologic diseases has made the Ubiquitin pathway a bona fide target for cancer therapeutics. Thus, defining how novel E3 ligases function at a molecular level and investigating their role in inflammation is critical in order to develop more specific therapeutic avenues.

NIH Research Projects · FY 2026 · 2016-07

ABSTRACT In mammals, spinal cord injury frequently leads to irreversible damage mainly due to the very limited capacity of injured central nervous system (CNS) axons to reconnect with their preinjury targets. Functional regeneration requires injured CNS axons to extend over long distances and reconnect with their original synaptic targets, however even in animal models current treatment strategies produce only modest levels of recovery. Despite enormous progress over the past decades, our knowledge and understanding of the fundamental molecular pathways and mechanisms that contribute to the process of spinal cord regeneration has left many fundamental questions unanswered. For example, are growth rates of regenerating axons uniform, are they preprogramed and invariable or are they modulated as they extend towards and into the injury site? And if so, what mechanisms and genes regulate and tune regenerating growth rates? In contrast to mammals, non-mammalian vertebrates including zebrafish have retained a remarkable capacity for spontaneous CNS regeneration. We have developed a laser-based axotomy approach to study spinal cord regeneration in larval zebrafish at single axon resolution in otherwise intact animals. From a candidate screen we identified the Cadherin EGF LAG receptor celsr3 to play a critical role in CNS regeneration. Our preliminary data reveal that in wild type animals regenerating M-ell axons switch to 3 fold higher growth rates once they cross the injury site. Celsr3 mutant M-cell axons respond to injury and grow across the injury site at growth rates indistinguishable from wildtype siblings, but then fail to increase their growth rates and frequently stall prematurely at about 25% of pre-injury length. Thus, our preliminary results identified a genetic entry point into the fundamental yet understudied question of whether and if so through which molecular mechanisms regenerating spinal cord axons regulate their growth rates along their regenerative path as their environment changes. Finally, we find that Celsr3 is also required for optic nerve regeneration but is dispensable for peripheral nerve regeneration, strongly suggesting that Celsr3 plays a selective role in CNS axon regeneration. The experiments in this proposal will (1) determine cellular and molecular mechanism by which Celsr3 growth rates selectively of regenerating CNS axons; (2) identify the molecular signaling cascade through which celsr3 promotes regeneration; and (3) Identify additional entry points into pathways that promote spontaneous spinal cord regeneration. Combined, our results are expected to make significant contributions to fundamental mechanisms that promote spontaneous spinal cord regeneration in vivo, and lay the foundation for a comprehensive analysis of spontaneous spinal cord regeneration. Although spontaneous spinal cord regeneration is largely absent in mammals, mechanisms of spontaneous spinal cord regeneration might be masked and thus undetectable by the presence and dominance of growth inhibitory mechanism. Our studies therefore complement studies in mammalian models that focus predominantly on strategies to overcome growth inhibition.

NIH Research Projects · FY 2025 · 2016-07

As many as 100 million people in the US have non-alcoholic fatty liver disease (NAFLD), which can lead to hepatic injury and fibrosis, characteristics of non-alcoholic steatohepatitis (NASH), and in turn can progress to cirrhosis and hepatocellular carcinoma cancer (HCC). To date there are no FDA-approved therapy for NAFLD or NASH. The mTOR pathway is a critical nutrient sensing pathway in many cell types, including hepatocytes. mTORC1 has thus been studied as a target to modulate lipid homeostasis in the liver, but its role remains unclear, with multiple excellent studies lead to seemingly opposing conclusions. We have now uncovered a highly specific branch of mTORC1 signaling in the liver, regulated by the FLCN protein, the inhibition of which leads to coordinated activation of lipid catabolic pathways and strong suppression of de novo lipogenesis (DNL), thereby potently protecting from both NAFLD and ensuing NASH. We thus hypothesize that FLCN represents a uniquely attractive therapeutic target to treat these diseases. We propose experiments to: 1. Understand how, mechanistically, the FLCN arm of mTORC1 signaling suppresses DNL 2. Understand how, mechanistically, the FLCN and canonical arms of mTORC1 signaling feedback on each other 3. Formally test the validity of FLCN as a therapeutic target to treat the NAFLD/NASH/HCC spectrum.