University Of Pennsylvania

NIH Research Projects · FY 2024 · 2020-07

Summary Approximately 30% of U.S. adults suffer from tendon and ligament injuries, which frequently occur near insertion sites into bone (i.e., entheses) and do not spontaneously heal. Growth and development studies have demonstrated a critical role for hedgehog (Hh) signaling in driving zonal enthesis formation but it's role in adult enthesis repair is largely unknown. Zonal enthesis formation involves anchoring collagen fibers, synthesizing proteoglycan-rich fibrocartilage, and mineralizing this fibrocartilage. Hh promotes this fibrocartilage formation. Unfortunately, studying this pathway in traditional tendon-to-bone repair has been a challenge since these repair models do not sufficiently anchor collagen fibers to bone, much less produce zones of fibrocartilage. Conversely, ligament reconstructions, where a tendon graft is placed through bone tunnels, can produce zonal attachments. Therefore, ligament reconstruction models, such as the anterior cruciate reconstruction model proposed in this application, can be employed to study the mechanisms that regulate zonal tendon-to-bone repair in the adult. This proposal will address this gap in knowledge by targeting the hedgehog pathway genetically and pharmacologically during tendon-to-bone repair following ACL reconstruction in novel transgenic mouse models. We will define the roles of the hedgehog pathway in specific stages of the repair response from the expansion of the progenitor pool to production of fibrocartilage and bone within zonal tendon-to-bone attachments during the tunnel integration process. By modulating the pathway pharmacologically, we will determine the potential for this pathway to be targeted in a translational fashion that could lead to novel therapies in the future. Our central hypothesis is that the Hh pathway is a critical positive regulator of zonal enthesis formation in the adult and therefore stimulation of the pathway will improve tendon- to-bone repair.

NIH Research Projects · FY 2024 · 2020-07

The broad goal of our proposed studies is to exploit our new insights into the molecular mechanisms and physiological roles of CALHM1 and CALHM3 as components of a novel ion channel in taste perception. We discovered CALHM1 as a membrane protein that expressed throughout the brain and in taste buds that lacks significant homology to other proteins, although five homologs have been identified, and CALHM1 is conserved across species. We identified CALHM1 as a pore-forming subunit of an ion channel with a large pore diameter and gating regulation by voltage and extracellular Ca2+ (Ca2+o). We discovered that CALHM1 is essential for perceptions of sweet, bitter and umami tastes by type II taste bud cells, since CALHM1-knockout mice cannot perceive these tastants. We identified the essential role of CALHM1 by discovering that it is a voltage-gated ATP-permeable ion channel, and that tastant-evoked Na+ action potentials trigger ATP release as a neurotransmitter through CALHM1-associated channels to transduce taste information from the periphery to the central nervous system. We further discovered that CALHM3 is an essential component of the native voltage-gated ATP-release channel, contributing as a pore-forming subunit with CALHM1 to create a heteromeric ATP-release channel in type II cells. Genetic deletion of CALHM3 also eliminates the ability of mice to perceive sweet, bitter and umami substances. The molecular mechanisms and structural bases of ion permeation and gating of CALHM channels are not understood despite their physiological importance. Nor is it understood how integration of CALHM3 into a CALHM1/3 channel so strongly affects voltage-gated activation, a key feature that allows CALHM1/3 channels to respond to action potentials. Temperature notably influences taste perception with physiological and hedonistic implications, but the contribution of peripheral taste-transduction mechanisms to the effects of temperature on the perception and sensation of tastes is largely unknown. We have discovered that temperature strikingly influences CALHM1/3 conductance as well as the electrical excitability of type II cells. We will employ electrophysiology in native taste bud cells and heterologous expression systems, mutagenesis, cryo-EM, and modeling to define the gating mechanisms of CALHM1 and CALHM1/3 channels, how CALHM3 as a pore-forming subunit enhances voltage-gated activation of CALHM1/3 channels, and how CALHM1/3 channels respond to action potentials evoked by tastant stimulation over a wide range of temperatures. Using a novel mouse model in which CALHM1/3 in taste bud cells has been engineered to have distinct temperature sensitivity, we will define how differential effects of temperature on ATP-release channel gating and excitability may provide a mechanism for how a temperature-sensitive channel in the peripheral gustatory system contributes to the influence of temperature on taste sensitivity and perception.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDA) remains one of the most intractable types of cancer, with a 5-year survival rate below 9%. Alarmingly, the incidence of pancreatic cancer is on the rise over the past 20 years, with particularly sharp increases in incidence among young adults. Poor nutrition and obesity are likely culprits, with obesity and diabetes independently conferring increased risk of PDA, but the mechanisms remain unclear. Branched chain amino acids (BCAAs) have emerged as one potentially important link between diet, systemic metabolism, and PDA. We have demonstrated in previous work and preliminary data that BCAAs are avidly consumed by the pancreas, where they contribute prominently to the TCA cycle and to acetyl-CoA pools. We have also recently shown that acetyl-CoA metabolism plays a key role in facilitating pancreatic tumor initiation, leading to the hypothesis that BCAA metabolism is required for efficient pancreatic tumorigenesis. Thus, one major goal of this grant will be to test the role of BCAA catabolism in the pancreas in facilitating acinar cell plasticity and tumor formation. We will use state-of-the-art in vivo isotope tracer approaches to elucidate the fates of BCAAs in the pancreas. We will also employ nutritional, genetic (using two novel mouse models), and pharmacological approaches to define the roles of BCAA catabolism in pancreatic function and tumorigenesis. In contrast to normal pancreatic cells, use of BCAAs as a fuel source is thought to be suppressed as pancreatic cancer develops. Nevertheless, BCAAs are an important biomarker of PDA, with circulating levels elevated years prior to PDA diagnosis, indicating a risk that likely mechanistically differs from that of tumor initiation. BCAAs are also elevated in obesity and diabetes, where they promote insulin secretion and insulin resistance, promoting a hyperinsulinemic state. Insulin itself acts as a growth factor to promote anabolic signaling and metabolism in PDA cells. Our second major goal is thus to evaluate the contribution of systemic BCAA levels and hyperinsulinemia to PDA tumor growth. We will manipulate systemic BCAA levels through nutritional, genetic, and pharmacological approaches to test the impact on tumor growth. We will then query the impact of insulin directly on PDA cells and on cancer-associated fibroblasts (CAFs), along with the impact of reducing hyperinsulinemia on tumor growth. Finally, we will test the potential to target BCAA metabolism through mouse clinical trials. These studies will provide deep insight into the roles of BCAAs in multi-step PDA tumorigenesis and could lead to novel therapeutic strategies for this deadly disease.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY Higher levels of physical activity have been found to improve health and to reduce cognitive decline as adults become older, but more than half of all adults in the United States do not meet their activity goals. Regular physical activity is associated with numerous health benefits and one type of physical activity that is broadly applicable to people of all ages is walking. Programs to increase walking are both broadly feasible and safe, and do not require unique training or specialized equipment. Walking is also measurable, with clearly identifiable individual targets that are associated with health improvements. This study will use a Hybrid Type 1 effectiveness-implementation design to adapt, and test the effectiveness of, two successful social incentive-based interventions to increase physical activity. Social incentive-based interventions can harness and enhance an individual's social network to increase physical activity, and can be more sustainable and scalable than financial incentives. The two interventions are a gamification strategy and financial incentives donated to charity on the participant's behalf. Following an initial phase of community-participatory adaptation of the two proven social-incentive intervention strategies, a three-arm, cluster-randomized trial of 225 families will be conducted to evaluate the two social incentive interventions in the community. The study aims include: 1. To adapt our prior work to improve the implementation of interventions in the community among families that include at least one older adult. 2. To evaluate the effectiveness of two social-incentive interventions among families to increase physical activity relative to control families. 3. To conduct a robust process evaluation that will provide information on the implementation process. 4. To assess the cost of each of the interventions from a societal perspective and compare cost differences between each intervention and control relative to the effectiveness measured by incremental increases in physical activity. 5. To use findings from the effectiveness trial, process evaluation and cost analysis (Aims 2, 3 and 4) to scale-up and disseminate tools and products for use by community organizations locally and nationally. This study focuses on scalable approaches to address health disparities in physical activity by partnering with community organizations in low-income and minority neighborhoods, using behavioral economics to deploy low-cost social incentive interventions, and applying implementation science frameworks from the Consolidated Framework for Intervention Research (CFIR) to improve adoption and dissemination. An effectiveness-implementation study design will maximize contributions to science, public health practice, and reduction of health disparities.

NIH Research Projects · FY 2025 · 2020-07

Inflammation  is  known  to  cause  bone  destruction  by  excessive  osteoclast  (OC)  activity  in  patients  with  inflammatory  diseases,  such  as  periodontitis.  To  address  the  underlying  causes  of  such  inflammation-­related  bone  loss,  it  is  important  to  understand  how  the  cellular  and  molecular  mechanisms  of  bone  homeostasis  maintained by bone-­forming osteoblasts (OBs) and bone-­resorbing OCs are perturbed by inflammatory stimuli.  Targeting  OC  maturation  rather  than  differentiation  is  of  particular  interest  and  provides  an  added  benefit  of  avoiding  unintentionally  inhibiting  new  bone  formation.  However,  identifying  promising  therapeutic  targets  of  OC  maturation  will  require  greater  understanding  of  its  mechanisms  of  regulation.  Cell  adhesion  is  a  physiologic  process  critical  to  both  OC  maturation  and  its  hallmark  feature,  multinucleation.  In  the  course  of  screening  potential  genes  that  regulate  OC  maturation  in  vitro,  we  identified  a  cell  adhesion-­related  gene,  Pcdh7,  a  protocadherin  member  of  the  cadherin  superfamily.  We  have  now  generated  Pcdh7-­/-­  mice  for  the  purpose  of  further  studying  Pcdh7  in  OC  maturation  and  inflammatory  responses,  and  therefore  propose  the  following  specific  aims:  1.  Investigate  the  role  of  Pcdh7  in  OC  differentiation,  function,  and  inflammatory  bone  loss. We will employ Pcdh7-­/-­ bone marrow (BM) cells to examine expression of known biological markers and  cell  biological  functions,  including  adhesion,  motility,  actin  ring  formation,  ruffled  border  formation,  and  vesicle  trafficking. Pcdh7floxed mice and BM chimeras will be generated for the purpose of more precisely interrogating  OC-­ versus OB-­specific (or other) Pcdh7 functions in the context of bone homeostasis. These mice will also be  employed  to  confirm  the  importance  of  OC-­expressed  Pcdh7  in  the  context  of  inflammatory  bone  loss  and  immune  responses  that  occur  after  LPS  treatment  or  ligature-­induced  periodontitis.  Together,  these  studies  should  elucidate  the  cell-­specific  roles  of  Pcdh7  in  OC  maturation  and  pathologic  bone  loss.  2.  Investigate  mechanisms of Pcdh7 molecular function within OC biology. To investigate how OC-­expressed Pcdh7 protein  regulates  cell  adhesion  and/or  signal  transduction,  we  will  test  a  four-­step  model.  For  each  step,  we  will  test  OC  maturation,  cell  adhesion,  and  activation  of  signaling  pathways,  and  will  employ  both  physiologically-­ activated and hCD3-­inducible retroviral (RV) Pcdh7 constructs. First, we will test whether Pcdh7 mediates cell-­ cell  interactions  that  activate  Pcdh7  intracellular  signaling  by  separately  track  WT  and  Pcdh7-­/-­  OCs  in  mixed  heterotypic  OC  cultures.  Second,  we  will  test  the  effects  of  cytoplasmic  domain  truncation  isoforms  of  Pcdh7  by  assaying  physiologic  expression  in  OCs  and  then  by  RV-­expressing  isoforms  in  OCs.  Third,  we  will  test  whether and, if so, how Pcdh7 mediates intracellular signaling via the oncoprotein SET. Fourth, we will employ  siRNA  and  chemical  inhibitors  to  test  the  relative  contributions  of  Pcdh7-­dependent  activation  of  various  signaling pathways to Pcdh7-­mediated OC adhesion and maturation. Together, these studies will improve our  understanding of the function of Pcdh7 protein generally, and more specifically, how it controls OC maturation.

NIH Research Projects · FY 2024 · 2020-07

PROJECT SUMMARY Interstitial lung diseases (ILDs) are a class of pulmonary diseases pathologically defined by interstitial fibrosis and inflammation. Owing to our limited understanding of the upstream initiators of pathogenesis, ILDs have poor prognoses and limited therapeutics. Mutations in the Alveolar Epithelial Type 2 Cell (AEC2) restricted Surfactant Protein C (SP-C) gene (SFTPC) in a subset of ILD patients supports a growing hypothesis that AEC2 dysfunction is a driver of disease. When we model these disease-related SFTPC mutations in vitro they segregate into two major classes based upon how the mutated SP-C isoform stresses cellular pathways that manage abnormal proteins: mutations that inhibit macroautophagy and mutations that cause endoplasmic reticulum (ER) stress. However, how these AEC2 stress phenotypes, which have also been identified in ILD patients without SFTPC mutations, relate to ILD development remains poorly understood. To understand this relationship, we have generated two unique Sftpc mutation knock-in mouse models, one expressing an SP-C isoform that induces AEC2 macroautophagy dysfunction (SP-CI73T) and the other inducing ER stress (SP-CC121G). Each of these mutations when expressed in the adult mouse lung results in spontaneous alveolitis and lung injury followed by aberrant repair with resultant fibrotic ILD. These models thus provide proof of concept that AEC2 stress is capable of driving spontaneous lung pathology and are robust preclinical platforms. These models also support a second emerging theory of ILD pathogenesis: that in ILD AEC2s, which must act as critical facultative progenitor cells after lung injury by both proliferating and differentiating to repair damaged epithelium, develop dysfunction in their progenitor cell capacity. We discovered that while similar lung pathology develops in each of our Sftpc models, there are divergent AEC2 proliferation phenotypes following lung injury: SP-CI73T AEC2s become hyperpoliferative and SP-CC121G AEC2s become apoptotic and hypoproliferative. Thus, our models support the hypothesis that abnormal AEC2 progenitor cell function plays a central role in ILD development, and also create a platform to develop a mechanistic understanding of how discrete AEC2 stress signatures result in distinct defects in progenitor cell capacity. This proposal has three interrelated aims that seek to understand the molecular and cellular mechanisms that relate AEC2 cell stress, dysfunctional progenitor cell capacity, and ILD. Aim 1 uses bioinformatics and in vivo linage-tracing to characterize the AEC2 stress signaling and progenitor function pathways involved in each model. Aim 2 provides a mechanistic link between cell stress signaling and progenitor cell dysfunction through ex vivo organoid culture and in vivo modeling. In Aim 3 we will generate the first ILD patient-derived iPSC culture model of an ER stress associated SFTPC mutation as a humanized platform to study the pathways identified in Aims 1 and 2. This proposal will also provided the trainee with the diverse and comprehensive training necessary to develop a multimodal independent research program on the role of the epithelium in ILD.

NIH Research Projects · FY 2024 · 2020-06

PROJECT SUMMARY Brain activity never ceases. When we are asleep, inattentive, or even under general anesthesia, networks of interconnected neurons in the human brain continue to spontaneously generate complex activity patterns. Sensory stimuli perturb this ongoing spontaneous neuronal activity. In order to be consciously detected, the effect of this perturbation needs to be large enough so as to engage thousands of neurons and persist for at least several hundred milliseconds. When we are awake and attentive, the smallest stimuli are sufficient to elicit a large perturbation. Under general anesthesia, however, even the most noxious stimuli do not reach the threshold for conscious perception. Here we address a fundamental question: why are sensory stimuli able to perturb neuronal activity in some states but not in others? We hypothesize that the ability of the sensory stimuli to perturb neuronal activity is related to the property of dynamical systems termed stability. If neuronal dynamics were unstable, the effect of any perturbation would grow over time without bounds and engage ever increasing number of neurons. Conversely, if the dynamics were too stable, then all perturbations will quickly dampen down and fail to reach threshold of perception. Thus, we hypothesize that conscious perception is most likely to occur when the neuronal dynamics are poised precisely between the stable and unstable regimes. We refer to this point as critical. To test the criticality hypothesis, we developed novel mathematical techniques and applied them to neurophysiological recordings in humans and in nonhuman primates. These preliminary findings strongly support the hypothesis. In the proposed project, we will rigorously test the criticality hypothesis using electrocorticography (ECoG) in human subjects implanted with electrodes for epilepsy localization. We will determine how the stability of spontaneous activity varies as a function of sleep and wake, attentiveness and drowsiness, as well as sedation and general anesthesia. We will validate the criticality hypothesis and our ability to estimate stability of neuronal activity by predicting responses to electrical brain stimulation. Using an auditory masked speech detection task, we will also determine whether stability of neuronal dynamics can be used to predict whether a natural stimulus presented at perceptual threshold will be consciously detected. While many other measures of neuronal activity have been previously associated with changes in arousal and perception, at present, it is not possible to apply the existing measures to unequivocally distinguish between activity in the conscious and unconscious brain. Hence, validating this criticality hypothesis would be a major advance. In addition to addressing a fundamental issue in neuroscience, finding an objective and quantifiable measure of sensory responsiveness has profound clinical significance in neurology and in anesthesiology where diagnoses of covert awareness under anesthesia or after brain injury cannot be made reliably with existing technology.

NIH Research Projects · FY 2025 · 2020-06

Summary Up to 80% of lung transplant recipients who survive beyond five years will develop chronic lung allograft dysfunction (CLAD), a heterogenous, progressive condition characterized by the gradual and irreversible functional decline eventually leading to death. Once developed, the majority of CLAD types do not respond well to currently available therapeutic interventions. Early diagnosis of suspected CLAD is therefore crucial to efforts aimed at the delaying disease onset and/or progression, which usually proceed via the aggressive treatment of associated immunological risk factors. Despite recently revised criteria, the current clinical reliance on spirometry to diagnose suspected CLAD suffers from several disadvantages: namely, the global nature of the measurements provided by pulmonary function tests (PFTs), their failure to differentiate between rejection and infection as the cause of functional decline, frequent inter-observer disagreement in interpreting their results and, finally, their inability to improve the targeting of transbronchial biopsy. An imaging modality capable of sensitively and accurately detecting CLAD-onset earlier and with more spatial specificity would provide significant clinical value. In response to this need, the proposed project will use the sensitive, regional measurements of lung function which various hyperpolarized xenon-129 MRI techniques are uniquely capable of providing to develop a set of imaging markers capable of diagnosing suspected CLAD before spirometric measurements reveal a clinically significant functional decline; ideally, these markers will also enable a distinction to be made between obstructive and restrictive forms of CLAD before either becomes symptomatic. The first task of this project will be to use multi-breath HP xenon-129 MR imaging to establish regional specific ventilation (SV) and alveolar oxygen tension (PAO2) as imaging markers for the early diagnosis of obstructive CLAD: increased heterogeneity in these sensitive measures of gas replacement dynamics within the transplanted lung will offer an earlier indication of CLAD-associated functional decline than spirometry. Next, we will use dissolved-phase HP xenon-129 imaging to quantify the efficiency of alveolar gas exchange and transport in order to detect the fibrotic and bloodflow impediments to pulmonary function associated with restrictive CLAD. Finally, we will attempt to radiologically define several novel sub-classifications of CLAD related to known associated risk factors such as ischemia reperfusion injury, respiratory infection, antibody- mediated injury and gastroesophageal reflux.

NIH Research Projects · FY 2025 · 2020-06

PROJECT SUMMARY The number of large-scale multi-site neuroimaging studies has skyrocketed due to growing investments by federal governments and private entities interested in brain development, aging, and pathology. This has led to the accumulation of vast amounts of magnetic resonance imaging (MRI) data. Such data have been acquired with an increasing degree of technical harmonization of scanning protocols, which has been beneficial for reducing inter-site differences. However, extensive evidence from our group and others emphasizes that even under after careful technical harmonization, site effects dwarf biological effects of interest. Over the past five years, there has been an explosion of interest in statistical harmonization methods to address this problem. As part of a highly-successful first project period, our group has pioneered the translation of tools from statistical genomics – such as the ComBat family of methods – to neuroimaging data. These widely adopted methods use empirical Bayes to correct for site effects based on the means, variances, and covariances of imaging features. However, in this context, two key challenges have arisen: missing phenotypes and nonlinear effects. First, as precision medicine turns to neuroimaging, harmonization in the context of diagnostic and prognostic biomarkers has become a central issue. Our group has shown that harmonization methods that aim to preserve disease information in imaging data are critical for scientific rigor and reproducibility. However, such information is not available without knowledge of the phenotype of interest. In these settings, translational investigators are caught in a catch-22: harmonization is not possible without knowing that individual's diagnosis, which is exactly the information targeted for prediction. To address this, here we propose to develop new methods that stochastically impute the phenotype and assess the uncertainty in the predicted harmonization. Second, deep learning has revolutionized the field of predictive modeling due to its sensitivity to complex nonlinear effects. However, its flexibility makes it highly sensitive to diverse technical biases. While there have been several initial forays in using deep learning for harmonization, currently available approaches have critical limitations – such as an inability to address confounding. In this proposal, we will develop novel hybrid deep statistical methods for improved harmonization to mitigate nonlinear scanner effects. We will extend these developments to the setting of longitudinally acquired imaging data, apply these in two large multi-center cohort studies, and release user- friendly software packages for the imaging science community.

NIH Research Projects · FY 2026 · 2020-06

PROJECT SUMMARY The number of large-scale multi-site neuroimaging studies has skyrocketed due to growing investments by federal governments and private entities interested in brain development, aging, and pathology. This has led to the accumulation of vast amounts of magnetic resonance imaging (MRI) data. Such data have been acquired with an increasing degree of technical harmonization of scanning protocols, which has been beneficial for reducing inter-site differences. However, extensive evidence from our group and others emphasizes that even under after careful technical harmonization, site effects dwarf biological effects of interest. Over the past five years, there has been an explosion of interest in statistical harmonization methods to address this problem. As part of a highly-successful first project period, our group has pioneered the translation of tools from statistical genomics – such as the ComBat family of methods – to neuroimaging data. These widely adopted methods use empirical Bayes to correct for site effects based on the means, variances, and covariances of imaging features. However, in this context, two key challenges have arisen: missing phenotypes and nonlinear effects. First, as precision medicine turns to neuroimaging, harmonization in the context of diagnostic and prognostic biomarkers has become a central issue. Our group has shown that harmonization methods that aim to preserve disease information in imaging data are critical for scientific rigor and reproducibility. However, such information is not available without knowledge of the phenotype of interest. In these settings, translational investigators are caught in a catch-22: harmonization is not possible without knowing that individual's diagnosis, which is exactly the information targeted for prediction. To address this, here we propose to develop new methods that stochastically impute the phenotype and assess the uncertainty in the predicted harmonization. Second, deep learning has revolutionized the field of predictive modeling due to its sensitivity to complex nonlinear effects. However, its flexibility makes it highly sensitive to diverse technical biases. While there have been several initial forays in using deep learning for harmonization, currently available approaches have critical limitations – such as an inability to address confounding. In this proposal, we will develop novel hybrid deep statistical methods for improved harmonization to mitigate nonlinear scanner effects. We will extend these developments to the setting of longitudinally acquired imaging data, apply these in two large multi-center cohort studies, and release user- friendly software packages for the imaging science community.

NIH Research Projects · FY 2026 · 2020-06

Project Summary Apical extracellular matrices (aECMs) are glycoprotein and lipid-rich layers that coat exposed surfaces of epithelia to protect them from pathogen infection and other environmental stresses. aECMs also shape and maintain integrity of apical domains (particularly those of narrow tubes) and can form elaborate body decorations. Understanding aECM biology is important because of the widespread roles that these matrices play in animal development and physiology, as well as in human health. Defects in aECMs contribute to medical conditions including deafness, lung disease, kidney disease, and vascular disease. A major challenge in the field has been the difficulty of visualizing these complex matrices, which are often damaged by histochemical fixation and not easily recapitulated within in vitro culture systems. This has hampered studies of how aECM components traffic to the apical domain, assemble at the proper time and place to execute their functions, form layers and three-dimensional substructures, and respond to genetic or environmental perturbations. Such questions can be addressed using a simpler model system. C. elegans has aECMs containing collagens, zona pellucida (ZP) domain proteins, proteoglycans, phospholipids, and other factors similar to those in mammalian ECMs, and it permits easy visualization of endogenous fluorescently-tagged aECM components in live animals, along with facile genetic manipulations. A better understanding of C. elegans aECM can inform the biology of conserved matrix protein families and what could be going wrong in human matrix-related diseases, while also informing design of methods to counter pathogenic nematodes. Important and broadly relevant questions addressed in this work include: 1) What controls aECM assembly at the proper time and place? 2) How are complex three-dimensional aECM structures formed and shaped? 3) What controls aECM protein secretion and endocytosis? 4) How does metabolism influence aECM? These questions will be addressed through genetic mutant analyses combined with light microscopy, electron microscopy, RNA expression profiling, and lipidomics experiments.

NIH Research Projects · FY 2024 · 2020-05

Abstract A major approach in causal inference literature aimed at mitigating bias due to unmeasured confounding is the so- called instrumental variable (IV) design which relies on identifying a variable which (i) influences the treatment process, (ii) has no direct effect on the outcome other than through the treatment, and (iii) is independent of any unmeasured confounder. IV methods are very well developed and widely used in social and health science, although validity of IV inferences may not be reliable if any of required assumptions (i)-(iii) is violated. This proposal aims to develop (a) new IV methods robust to violation of any of (i)-(iii); (b) New negative control methods that can be used to detect and sometimes to nonparametrically account for unmeasured confounding bias; (c) New bracketing methods for partial inference about causal effects in comparative interrupted time series studies. The proposed methods will be used to address current scientific queries in three major substantive public health areas:(1) to understand the health effects of air pollution; (2) to quantify the causal effects of modifiable risk factors for Alzheimer's disease and related disorders; (3) To uncover the mechanism by which a randomized package of interventions produced a substantial reduction of HIV incidence in a recent major cluster randomized trial of treatment as prevention in Botswana, Africa. Our proposal will provide the best available analytical methods to date to resolve confounding concerns in these high impact public health applications and more broadly in observational studies in the health sciences.

NIH Research Projects · FY 2025 · 2020-05

How do the neural circuits controlling movement combine sensory information, memories of past experiences, and internal state information (e.g. thirst, arousal, anger, fear) to produce even simple actions? And how does descending command activity change when a specific action (e.g. a lick in a specific direction) is made for different reasons (e.g. a reflexive vs. memory-guided lick)? Individual motor neurons receive thousands of inputs from across the brain and then integrate that information nonlinearly over dendritic arbors that span hundreds of microns. This complexity makes it difficult to assign a specific causative role to any single upstream circuit. Indeed, past studies have shown that many parallel circuits, from those involving cortex, the superior colliculus, the red nucleus, and simple reflexes in the brainstem, are each involved in driving similar orofacial movements. However, comparatively little is known about how these different pathways normally coordinate their activity with each other during normal behavior. One consequence of this gap in understanding pertains to disease. While many neurological impairments cause localized damage to movement-related brain regions (e.g. motor neuron degeneration in ALS, or the areas surrounding an infarction following a stroke), it is still unclear why some lesions cause permanent deficits while others can be compensated for by changes in the neural activity of other circuits in unaffected parts of the brain. The goal of this project is to discover the logic governing the coordination between different descending motor pathways, to determine how their recruitment depends on brain state (e.g. thirst or arousal), and to measure how such coordination changes following an acute or chronic brain injury. To do this, we will use recently developed large-scale neural recording technologies to interrogate many brain areas— including the motor nuclei themselves. First, using a new method for simultaneously monitoring many areas across dorsal cortex using Ca2+ imaging, I will explore the structure of neural activity contained in corticobulbar (i.e. cortex to medulla) projection neurons preceding both sensory and memory-guided directional lick bouts. Second, I will obtain electrophysiological recordings (using Neuropixels probes) from pyramidal tract neurons in motor cortex, the superior colliculus, and/or the medullar motor circuits themselves. Finally, I will study the acute and chronic effects of shutting down parts of the brain during licking behavior (using both optogenetic- silencing, and a mouse model of ALS to gradually kill motor neurons). These aims will be pursued at Stanford University, working under the co-mentorship of Karl Deisseroth and Surya Ganguli. This exceptional research community has an outstanding track record of both training postdoctoral fellows and successfully placing them in tenure-track faculty positions. My advisory committee will further ensure that I implement my training plan successfully and am able to establish my own lab to study how global brain computations drive specific actions.

NIH Research Projects · FY 2024 · 2020-05

PROGRAM INTRODUCTION SUMMARY: This is the re-submission of a new Program Project (HL146373-01) that we have re-named “Studies of Physiologic and Pathologic Platelet Plug Formation” to more accurately reflect the topics the Program Project addresses. In addition to forming hemostatic plugs at sites of vascular injury, platelets make important contributions to processes such as inflammation, tissue regeneration, host defense, angiogenesis, lymphatic development, and tumor metastasis. Pathologic platelet thrombi are also responsible for much of the morbidity and mortality of arterial vascular disease. There remain large gaps in our understanding of physiologic and pathologic platelet function. Building upon our collective scientific accomplishments, we address these gaps using the cell-biologic, structural-biologic, and computational-biologic methods we have developed. The Program Project we propose consists of four projects and one administrative core unit. All of the projects are based at the Perelman School of Medicine of the University of Pennsylvania. Three projects are based in the Hematology-Oncology Division of the Department of Medicine; the fourth is based in the Division of Hematology of the Department of Pediatrics at The Children's Hospital of Philadelphia. Project 1, re-named “Novel Roles for Phosphoinositide Signaling in α-Granule Biogenesis”, is based on the hypotheses that phosphoinositide synthesis is an essential step in the loading of α-granules with components synthesized in the Golgi and that megakaryocyte phosphoinositides play a previously unrecognized role in the development of congenital megakaryocyte disorders. The objectives of Project 2, entitled “Platelet Integrin Structure and Function”, are to use novel computational and experimental techniques to compare the behavior of αIIbβ3 with that of the other integrins and to identify and quantify the protein-protein interactions responsible for αIIbβ3-mediated fibrin clot contraction. Project 3 is entitled “A Systems Approach to Hemostasis and Thrombosis“. The goals of the studies proposed in this Project are to extend past analyses of platelet thrombus formation and structure from the microvasculature to the macrovasculature, from mice to humans, and from hemostasis to thrombosis. Project 4, entitled “Platelet Factor 4 and Heparin in NETosis and Sepsis”, will test the hypothesis that NETs, neutrophil extracellular traps composed of chromatin released by neutrophils, require partial digestion and release of DNA and histones to be toxic during sepsis. Because infused platelet factor 4, as well as the monoclonal antibody KKO that binds to the complex of platelet factor 4 and heparin, block DNA digestion, both will be protective in sepsis. The four projects are supported by a single core unit that provides for the common administrative needs of the Program.

NIH Research Projects · FY 2025 · 2020-05

Project Summary The Training Program in Rheumatic Diseases is now completing a remarkable 5 years in its current inception training new scientists in the investigation of the pathophysiology, diagnosis, treatment, and outcomes of rheumatic diseases. The Program is a collaborative effort between the Adult and Pediatric Divisions of Rheumatology at The University Pennsylvania and The Children’s Hospital of Philadelphia along with The University of Pennsylvania Biomedical Graduate Studies program, and the Graduate Medical Education programs of the Adult and Pediatric Rheumatology Divisions. The Program offers a rich environment for mentored training in basic laboratory investigation, translational research, clinical epidemiology, and bioinformatics, and spans immunology, muscle and bone biology, and clinical rheumatology. Our multi- disciplinary cohort of trainers come from the Perelman School of Medicine and the School of Veterinary Medicine and represent multiple Departments and Divisions. In recognition of the rapid expansion of technology and methodology that blurs lines between bench and clinical research, the Program has emphasized adding mentors and trainees in bioinformatics, translational science, and other domains to best position ourselves to continue our success in the development of the future scientific leaders in Rheumatology. Post-doctoral trainees are selected from the already highly competitive and select pool of clinical fellows from both the Adult and Pediatric Rheumatology fellowship programs, as well as PhD scientists within the participating Schools investigating aspects of rheumatic disease. Typical appointments are for 2 years, pending appropriate progress by the trainee. Given that our pipeline of meritorious trainees is outstripping current availably in the program, we request to expand this award to support 6 post-doctoral positions. Since the inception of the current T32 award, both the Adult and Pediatric fellowship training programs added and filled additional ACGME-accredited slots, reflecting our ability to attract increasing numbers of talented and qualified trainees eager to take advantage of our unique training and research environment. All trainees benefit from an exceptional set of colloquia, seminars, retreats, and lectures, and trainees are expected to contribute to this environment through their own presentations and publications. Once each trainee’s mentoring team is formed, an Individualized Development Plan is created that is regularly reviewed and revised by the trainee, mentor(s), and PIs of this Program, including assessment of the trainees’ and mentor’s performances. The Program itself is continuously evaluated by direct feedback from trainees and mentors, through annual review by an Internal Advisory Board and an external consultant, and by self-assessment techniques. As a measure of success, in the past 5 years, this Program trained multiple investigators running their own foundation/NIH-funded mentored research programs in Rheumatic Disease and others that are clearly on track to independence upon completing their training.

NIH Research Projects · FY 2026 · 2020-05

SUMMARY Adult hippocampal neurogenesis has garnered significant interest over the past two decades as a robust and unique form of plasticity in a region critical for learning and memory. It has also proven to be fertile ground for understanding fundamental principles of stem cell biology, neuronal development, as well as illustrating the capacity of the mature brain to integrate immature neurons, which has important implications for regeneration and transplantation efforts for neural repair following injury or diseases. Despite considerable progress in understanding the molecular and cellular mechanisms underlying adult neurogenesis, there are still critical outstanding questions in the field that have not been addressed due to the technical limitations of traditional experimental approaches. In the proposed series of studies, we will use several cutting-edge techniques that we have developed or adapted to investigate the developmental origin of adult neurogenesis, its functional impact in the adult brain, and the fidelity of rodent models to human neuronal development. First, we will characterize the origin and properties of embryonic neural precursor cells that give rise to the largely quiescent pool of neural stem cells that maintain neurogenesis throughout life in a rodent model. Building on our recent findings that Hopx-expressing neural progenitors in the embryonic dentate gyrus can generate the constitutive populations in the dentate gyrus before adopting a quiescent state indicative of adult neural stem cells, we will identify the molecular mechanisms regulate this precursor population and its transition into quiescence. These studies will provide novel insight into the intrinsic and extrinsic signaling cues that establish a long-term pool of stem cells in the developing and adult brain. Second, we have developed a 3D organoid model of dentate gyrus development using human induced pluripotent stem cells to investigate the properties of neural progenitors, neurogenesis and fate specification. These studies could lead to the potential identification of human-specific markers of neural stem cells and new granule neurons in the dentate gyrus and mechanistic differences and similarities with rodent models, which would inform the current debate over the extent of postnatal neurogenesis in the human dentate gyrus. Third, we will investigate the functional properties of adult neurogenesis in adult behaving mice using an optogenetic strategy to identify and record electrophysiological activity of single newborn granule cells at different stages of maturation. We will also investigate the circuit- level impact of silencing these cells at the population level. These data would provide novel information to evaluate the hypothesis that adult-born granule cells make a unique contribution to information processing in the hippocampus using techniques with high temporal resolution. Together, these studies combine an array of approaches to answer fundamental questions about the origin, impact, and plasticity of neural stem cells and their progeny in the dentate gyrus using both rodent and human models.

NIH Research Projects · FY 2025 · 2020-04

Project Summary HIV-1 infection is typically well controlled with combination antiretroviral therapy (ART). However, viral reservoirs persist in lymphoid tissues leading to rapid rebound viremia following ART discontinuation. The mechanisms that underlie viral persistence include latency, proliferation or clonal outgrowth of cells harboring intact viral genomes, and immune responses that are inadequate to access or kill infected cells. After almost a decade of intensive research to cure HIV infection, there has been little success in eliminating or even reducing HIV-1 reservoirs, indicating that barriers to achieving this goal are formidable. It has become clear that basic questions and mechanisms of how viral persistence is maintained need to be addressed in relevant animal models that can reveal vulnerabilities in these reservoirs and inform hypothesis-driven interventions to impact their size and durability. The Hoxie lab has described a unique nonhuman primate model in which a 2 amino acid deletion in the SIVmac239 envelope cytoplasmic tail, disrupting a highly conserved cellular trafficking signal, produces a virus termed ∆GY that is highly replication fit during acute infection but is rapidly controlled to undetectable levels in plasma by cellular immune responses in the absence of neutralizing antibodies. Viral reservoirs are clearly present years after infection, as demonstrated by anti-CD8 cell depletion studies, and have been detected and quantified by state of the art assays in PBMCs and lymphoid tissue. This proposal will use the ∆GY model of elite cellular control to test the hypothesis that an intervention with a potent and long- lasting neutralizing antibody, with or without the latency reversing-like activity of CD8 cell depletion, thus exerting both cellular and humoral immune attack on the viral reservoir, will accelerate the decay of and/or eliminate replication competent viruses. Four Specific Aims are proposed: 1) to define and quantify viral reservoirs during elite immunologic control of ∆GY, characterizing relevant cell types, transcriptional activity, integration sites, and mechanisms that underlie persistence; 2) determine if long term expression of eCD4-Ig, a novel engineered antibody-like molecule with potent neutralizing and non-neutralizing functions against SIVmac239 and ∆GY, with or without CD8 cell depletion to activate virus production, can synergize with host cellular immune responses to reduce reservoirs; 3) extend findings from Aims 1 and 2 to SIVmac239 infection in which viral control prior to eCD4-Ig and CD8 cell depletion is exerted through ART rather than cellular immune control; and 4) create novel SHIV and HIV-1 isolates that contain mutations analogous to the ∆GY mutation for future studies to explore interventions that can build on the findings of this proposal to reduce or eliminate persisting HIV-1 reservoirs. If viral reservoirs in the ∆GY model can be reduced or eliminated, this study will provide a proof of concept that this goal is feasible, and inform immunological interventions during ART-suppression animal models and in humans.

NIH Research Projects · FY 2026 · 2020-04

Project Summary Changes in the physical properties of tissues are often associated with disease, not simply as consequences of pathology but as drivers of it. Cells build structures that are strong enough to resist the mechanical stresses that are generated by external forces such as gravity and impact, as well as those that emerge from the same molecular structures and cellular assemblies that evolved to generate biological movement and force. This MIRA renewal application combines two physical studies related to cell and tissue function. One study continues to be focused on the mechanical properties of purified biopolymer networks derived from the cytoskeleton and the extracellular matrix, intact cells, and whole tissues, and expands on previous work that defines of the elastic properties of these materials to also consider their dissipative features. Mechanical energy dissipation in biological material can arise from the viscoelasticity of the protein networks, poroelasticity from water/cytosol flow through the network pores, or random motor-driven movements of network strands. We have built new instruments to measure poroelastic relaxation and visualize fluid flow at the micron scale and collaborate with theorists to provide models beyond the classic Darcy theory that are needed for these non-linear systems with anomalous Poisson's ratios. The second related set of studies is focused on the cell's nucleus. This is a new direction initiated in the current MIRA period showing that the nucleus is highly deformable by small but long- lived stresses, because most of the work of deformation is dissipated rather than elastically stored. The dissipation depends on ATP hydrolysis and the function of a specific nuclear motor: the BRG1 ATPase of a chromatin remodeling complex. We plan to characterize the functions of this motor complex in molecular detail and determine if the motor itself responds to force, as do some cytoskeletal motors. We will extend our current studies of nuclei in karyoplasts to cell types other than fibroblasts and focus on explaining how the stiffness of the nucleus is altered when cells are cultured under different mechanical conditions. We will continue work with theorists to explain the nonlinear elastic response of semi-flexible polymer networks and determine how they are altered by particles within the network mesh (e.g. ribosomes or vesicles in the actin network, protein condensates in the nucleus, cells within the ECM of tissue) and thereby show how these physical models help explain nucleus, cell, and tissue mechanics. We also continue studies to show how important viscoelastic properties of the substrate, especially dissipation, are to cell phenotypes and to develop new materials by which to study them.

NIH Research Projects · FY 2026 · 2020-04

SUMMARY Heart failure (HF) is a leading cause of death worldwide, and the leading cause of hospital admissions in patients over 65 in the US. Novel therapies, addressing novel pathways, are direly needed. The role of metabolism in cardiac pathology has long been of interest. We have focused for several years on the role of branched chain amino acids (BCAAs: leucine, valine, isoleucine), which have been noted for decades to be elevated in heart failure, and often to predict adverse outcomes. Preclinical studies from numerous labs including ours has provided compelling evidence that systemic activation of BCAA catabolism is beneficial in heart failure, and multiple pharmaceutical companies are actively developing novel agents to do so in humans. Despite these efforts, mechanistically how activation of BCAA catabolism protects from heart failure remains surprisingly unknown. During the current funding period we have made substantial headway in addressing this question, and we have come to surprising conclusions, most notably: (1) that the cardiac benefits of promoting BCAA catabolism are not direct, but rather occur secondary to BCAA oxidation in other tissue(s), and (2) that BCAA catabolism promotes vasodilation. These data and additional preliminary data thus lead us to hypothesize that: Systemic activation of BCAA catabolism activates BCAA catabolism in smooth muscle cells (SMCs), leading to SMC relaxation, cardiac vasodilation, improved myocardial blood flow (MBF), and cardioprotection. We will test this hypothesis by: Aim 1: Identify how BCAA catabolism in SMCs inhibits vasoconstriction. Aim 2: Test if BCAA catabolism in smooth muscle cells promotes cardiac vasodilation and MBF. Aim 3: Test the role of smooth muscle BCAA catabolism in cardioprotection. Aim 4: Test if systemic activation of BCAA catabolism promotes cardiac MBF in humans. These highly focused studies will elucidate the role of BCAA catabolism in cardio-protection. Deep understanding of BCAA catabolism in the context of heart failure may lead to novel therapeutics.

NIH Research Projects · FY 2025 · 2020-03

Project Summary/Abstract: Traumatic brain injury (TBI) is one the leading causes of mortality and morbidity affecting humanity, and a recognized risk factor for late-life neurodegenerative disorders. The absence of validated biomarkers in the neurotrauma field is a barrier to drug development in this area, and as a consequence there are currently no disease-modifying therapies that limit the burden of TBI. TBI is a complex disease process, and there is a need to identify and measure subtypes of injury, in order to develop precision medicine approaches where specific pathobiological processes are targeted by mechanistically appropriate therapies. Traumatic axonal injury (TAI) is a common pathologic consequence of TBI, and underlies some of the most disabling consequences of injury, including cognitive and affective problems. Recent breakthroughs in pre-clinical models indicate that novel therapeutic interventions are effective in promoting resilience of injured axons and improving neurologic outcome after experimental TBI. Translation of such promising therapies into successful clinical trials will require prognostic biomarkers that can measure TAI in individual patients, so they can be selected for early phase studies of axono-protective therapies, as well pharmacodynamic biomarkers than can measure the biologic efficacy of such treatments. Currently, the best biomarker for TAI is fractional anisotropy (FA) and mean diffusivity (MD) of white matter tracts, measured using diffusion tensor imaging (DTI) MRI. This technique, while robust, is poorly suited for dynamic longitudinal assessments, and measures the end-result of axonal degeneration, rather than an early step in the neurodegenerative process. Recently, the ability to assay axonal proteins in peripheral blood has made it potentially feasible to assess of TAI rapidly, inexpensively, and longitudinally. The axonal protein that holds the most promise as a marker of axonal degeneration is neurofilament light chain (NF-L). We hypothesize that NF-L is a prognostic biomarker of TAI. Our project has 3 specific aims: Specific aim 1. We will determine reference intervals (RIs) for NF-L according to Clinical Laboratory Standards Institute (CLSI) guidelines, using commercially available assays (Quanterix, LLC, Lexington, MA). Specific aim 2. We will measure NF-L in existing serum samples from participants enrolled in a multi-center observational study (TRACK-TBI) who also have MRIs at 2 weeks and 6 months after injury. The relationship between NF-L elevations and neuroimaging measures of TAI (DTI measure of FA at the 2-week scan) and axonal degeneration (white matter volume at 6 months after injury) will be assessed. Specific aim 3. We will extend the follow-up period of a subset of TRACK-TBI participants from 1 year to 5 years after injury, to assess the relationship between persistent NF-L elevations and neurodegeneration. The existing clinical, imaging, and biomarker data in these subjects will be leveraged to identify risk factors, co- morbidities, and prognostic biomarkers of long-term TBI-associated degeneration.

NIH Research Projects · FY 2024 · 2020-02

PROJECT DESCRIPTION The overall goal of this application is to characterize a novel protein quality control (PQC) system in mammalian cells and to elucidate its role in tumorigenesis. Oncogenic transformation is a progressive process during which normal cells acquire a set of traits to overcome various constraints that govern their proliferation. Here we will test the notion that a heightened ability to remove misfolded proteins may be a new characteristic of tumor cells. Protein folding is a challenging process in normal unstressed cells, and even more so in incipient and established neoplastic cells, which frequently encounter high oxidative stresses that damage proteins. However, PQC systems that remove misfolded proteins in mammalian cells and the role of these systems in tumorigenesis are not well understood. Our lab recently found that many mammalian tripartite motif (TRIM) proteins can specifically recognize misfolded proteins and mark them for proteasomal degradation, and that certain TRIM can also directly activate the proteasome. Moreover, we observed that the capacity to remove misfolded proteins is markedly increased in cancer cells due to the up-regulation of TRIMs. This higher degradation power mitigates oxidative stress associated with oncogenic growth and permits oncogenic growth. These findings indicate TRIMs as versatile regulators of protein quality, connect the clearance of misfolded proteins to antioxidant defense, and suggest a previously unrecognized characteristic of tumor cells. Our central hypothesis is that TRIM proteins constitute a major PQC system in mammalian cells that are critical for antioxidant defense and oncogenic transformation. We propose three specific aims. First, TRIM proteins exist in a large number including over 70 in humans. To gain a comprehensive view of the TRIM system, we will systematically investigate the role of all human TRIMs in proteasomal degradation of misfolded proteins and define the molecular basis for their different potency. Second, we will investigate how the accumulation of misfolded proteins causes high oxidative stress, and how TRIMs ameliorate this stress through the clearance of misfolded proteins. Third, we will determine the role of the TRIM system in cancer progression using cell and animal models. Moreover, our results suggest that the removal of misfolded protein is highly sensitive to proteasome inhibition, which may provide an explanation for proteasome-inhibitor-based therapies for cancer. We will test the notion that increasing production of misfolded proteins, combined with proteasome blockage, may be highly effective in killing cancer cells. Collectively, these aims will address critical issues pertaining to protein homeostasis and oncogenic transformation, and will likely provide valuable information for the development of effective therapies.

NIH Research Projects · FY 2026 · 2020-01

Cryptosporidium causes self-limiting diarrheal disease in immune competent individuals and life-threatening illness in young children as well as patients with defects in T cell function. Current drugs are poorly efficacious, and a vaccine would be a game changer for the pediatric disease. However, there is a major gap in our understanding of what types of protective immunity would be required for successful vaccination. Based on clinical and experimental studies, we know CD4+ T cells and the production of IFN-γ are important for parasite control but the actual events that restrict Cryptosporidium are unknown. The advent of parasite molecular genetics has transformed the ability to modify Cryptosporidium and in the last funding period these laboratories have generated the tools needed to answer fundamental questions about the mechanisms of resistance to this organism. Remarkably, while IFN-γ is important for parasite control, in vaccination experiments we also found significant protective CD4+ T cell dependent memory even in the absence of IFN-γ and our studies implicate IL- 22 in resistance to Cryptosporidium. Based on this foundation, the vision of this proposal is to leverage our advances in parasite biology, mouse models, and immunological tools to understand: 1) the relationship between the IFN-γ and IL-22- mediated pathways 2) Utilize the differential activities of STAT1 and STAT3 in enterocytes to understand the basic mechanisms that promote parasite clearance 3) Use of novel genetically attenuated parasite strains to understand the parasite and host factors required for long term CD4+ and CD8+-T cell mediated protective immunity. The studies propose here will deliver information on the innate and adaptive pathways that promote T cell mediated control of Cryptosporidium and how EC responses limit parasite growth. This information is relevant to understanding the type of long-lived cellular immunity required for vaccine protection from Cryptosporidium, and insight into how to best target this response to the gut to reduce both parasite burden and damage to the tissues. We expect the mechanisms discovered in the course of this program to be of broad interest to the understanding of numerous diseases of the intestinal tract of infectious, inflammatory and malignant etiology.

NIH Research Projects · FY 2026 · 2019-12

Summary: Mast cells (MCs) are tissue-resident immune cells that are best known for mediating IgE/FcεRI-mediated hypersensitivity and atopic disorders. However, recent excitement in MC research has been the realization that a diverse group of cationic amphipathic peptides and FDA-approved drugs activate human MCs via a novel G protein coupled receptor (GPCR) known as MRGPRX2 (mouse counterpart, MrgprB2). This receptor is implicated in host defense, immediate drug hypersensitivity reactions (IDHRs), neurogenic inflammation/pain and a variety of cutaneous disorders. Not surprisingly, there is an intense interest in developing MRGPRX2 inhibitors for treating MC-mediated disorders. Although several small molecule inhibitors have been developed, a phase 2 clinical trial with a promising lead was recently paused because of toxicity issues, emphasizing the need for additional approach. This proposal is focused on two sets of proteins that directly interact with GPCRs (G proteins and GPCR kinase 2, GRK2) that are traditionally viewed as displaying opposing effects on receptor function. We will test the novel hypothesis that G proteins and GRK2 interact with MRGPRX2 to promote MC-mediated IDHRs, anaphylaxis and cutaneous disorders. In aim 1, we will knockdown the expression of Gαi and Gαq family of G proteins in human MCs and determine the impact of this deletion on MRGPRX2-mediated responses in vitro. We will use retrovirus to express MRGPRX2 in marrow-derived MCs (BMMCs) in the absence of G proteins. These BMMCs will be engrafted into MC-deficient mice. This approach will be used to determine the role of specific G proteins on MRGPRX2-mediated IDHRs, anaphylaxis, rosacea and psoriasis in vivo. We will express MRGPRX2 variants with defective coupling to different G proteins in BMMCs and these cells will be engrafted into MC-deficient to mice. This strategy will be used to determine the impact of disrupting MRGPRX2/G protein interaction on disease phenotype in vivo. In aim 2, we will cross MRGPRX2-KI mice with Cpa3Cre/GRK2flox mice to determine the role of GRK2 on functional regulation of MRGPRX2 in MCs. We will disrupt MRGPRX2’s interaction with GRK2 and determine the impact of this disruption on MC function. We have identified specific Ser residues in MRGPRX2 that undergo GRK2- mediated phosphorylation in agonist-stimulated MCs. We will generate phosphorylation-deficient mutants and determine the effects of these mutations on MC cell function in vitro and in vivo. Completion of this study may provide a new rationale for the development of novel therapeutic approaches for treating MC-mediated disorders.

NIH Research Projects · FY 2025 · 2019-12

Project Summary Triple-negative breast cancer (TNBC) accounts for ~10% of all the breast cancer cases, but its survival rate is lower due to the lack of effective targeted treatments. This underscores the importance of finding new treatments for therapy-resistant TNBC, which is further complicated by the disease heterogeneity. Unfortunately, success of targeted therapies in TNBC has been limited, partly due to the lack of a detailed and mechanistic understanding of the drivers of each disease subgroup. An important contributor to the TNBC pathobiology is Notch signaling. Hyperactive Notch signaling promotes tumor growth, increases chemotherapy resistance, decreases survival, and increases the chance of metastases. Although the biomarkers of the Notch-active TNBC subgroup and drugs to target Notch signaling have been recently developed, treating patients with Notch inhibitory agents has been ineffective to date, partly due to the limited understanding of how the Notch signaling controls these fundamental processes. An important direct consequence of Notch signaling is to activate key TNBC genes, including MYC, CCND1 and SOX9. MYC is one of the most important proto-oncogenes promoting tumor growth and survival. CCND1 controls cell division among other cellular processes. SOX9 increases metastatic potential. Despite their importance, existing drugs fail to directly target these proteins. We propose to leverage the regulatory relationships between Notch and its target genes to selectively and efficiently target them. In order to achieve this goal, we first need to understand the mechanisms by which Notch regulates MYC, CCND1, and SOX9 in TNBC. We propose to use cutting-edge functional genomics and chromatin conformation assays to elucidate their Notch-directed regulatory mechanisms at population and single-cell resolutions. To develop more potent therapeutic options, we plan to use the latest single-cell resolution technologies to discover how drug-resistant cells circumvent the effect of Notch inhibitory drugs and maintain the expression of these critical Notch targets. We plan to use this knowledge in the future to tailor therapeutic strategies for individual TNBC patients with activated Notch signaling, and in the process, hope to improve the survival of patients with this aggressive and difficult to treat form of breast cancer. !

NIH Research Projects · FY 2025 · 2019-09

Extended-release naltrexone (XR-NTX) reduces overdose risk and is filling a niche for opioid addicted patients that do not want agonist maintenance or cannot access it. However transitioning to naltrexone requires detoxification, which is a major hurdle. Methadone or buprenorphine tapers are effective but require a 7 to 14-day opioid-free interval before starting naltrexone, leaving ample time to relapse. Non-opioid detoxification with an alpha-2 adrenergic receptor agonist may shorten the time, and lofexidine was recently approved for this indication. It is safer than clonidine however like clonidine, it does not reduce the subjective effects of withdrawal and patients do not like it. A medication that better targets these symptoms may improve outcomes and increase the proportion that transition to XR-NTX. Pregabalin may be such a medication. It potentiates the activity of glutamic acid decarboxylase, inhibits calcium influx and release of excitatory neurotransmitters, raises GABA levels, and is approved for neuropathic pain, fibromyalgia, adjunctive therapy for adults with partial onset seizures and in Europe, for anxiety. It was not controlled in Russia for several years but was placed on their equivalent of our Schedule V due to reports that opioid addicted persons were using it to reduce withdrawal and abuse. Based on this information, Krupitsky and colleagues randomized 34 consenting, heroin-addicted inpatients under double-blind conditions to pregabalin or clonidine-based detoxification protocols. More pregabalin than clonidine patients completed detoxification (p = 0.01) and pregabalin patients had better retention than clonidine patients (p = 0.001) with no differences in adverse events. Here we propose to see if pregabalin can be combined with lofexidine to better reduce the subjective effects of opioid withdrawal than lofexidine, and increase the proportion that transition to XR-NTX. Such a dosing combination could lower the detoxification hurdle for patients who are interested in antagonist treatment or who are in settings where it is unavailable or difficult to access. This work will require two phases, and both fit into the UG3/UH3 announcement. In UG3 we will study pregabalin/lofexidine combinations to identify one that reduces withdrawal-related subjective effects without generating more serious adverse events than lofexidine alone. In UH3 we will test that combination in an adequately powered trial to determine if it increases the number of patients that complete detoxification and transition to XR-NTX. Hypotheses are that we will identify a dosing combination that is safe and reduces opioid withdrawal to a greater degree than lofexidine alone, and that this lofexidine/pregabalin combination will result in more patients completing detoxification and transitioning to XR-NTX. The ultimate goal is to generate data to support new or modified indications(s) and/or inclusion of new recommendations in product prescribing information to improve detoxification outcome and increase the proportion that transition to XR-NTX