University Of Connecticut Sch Of Med/Dnt

NIH Research Projects · FY 2025 · 2021-03

Project Summary/Abstract Caspase-4/-5/-11 non-canonical inflammasomes have been known to play pivotal roles in various inflammatory and infectious diseases, such as sepsis. Current studies demonstrate that caspase-4/-5/-11 are activated by directly sensing intracellular microbial infections, such as lipopolysaccharide (LPS, also known as endotoxin), or endogenous products, such as oxidized phospholipids (e.g., OxPAPC), through their N-terminal caspase activation and recruitment domains (CARDs) and C-terminal catalytic domains, respectively. However, the molecular mechanisms of how caspase-4/-5/-11 recognize their ligands and how caspase-4/-5/-11 are activated upon binding ligands remain unknown. In this proposal, we aim to elucidate the structural basis of non-canonical inflammasome signaling by characterizing the interactions between caspase-4/-5/-11 and ligands - LPS and OxPAPC, and determining the high-resolution structures of caspase/-4/-5/-11 in complex with ligands. Our structural findings will provide new therapeutic strategies for sepsis and other non-canonical inflammasome- associated diseases. We propose three specific aims to achieve our goal: 1) Biochemical characterization of the interactions between caspase-4/-5/-11 and LPS-including the identification of the essential structural elements in the LPS molecule and key residues on caspase-4/-5/-11 CARDs that are required for caspase-4/-5/-11 activation; 2) Determine high-resolution structures of caspase-4/-5/-11 CARDs both in their inactive form and in complex with LPS; 3) Characterize the interactions between caspase-4/-11 and OxPAPC, and determine the high resolution structures of caspase-4/-11 catalytic domains in complex with OxPAPC. We will pursue these aims using cutting-edge experimental approaches including biochemical and biophysical characterization, X-ray crystallography, mass spectrometry, electron microscopy, and cellular experiments. The proposed studies will significantly expand our current knowledge on the mechanisms of non-canonical inflammasome signaling, and provide rationale and a structural basis for designing novel strategies to control the activation of the non- canonical inflammasome for better treatment of related diseases.

NIH Research Projects · FY 2025 · 2021-03

Summary The kinetoplastid parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. affect millions of people worldwide, causing Human African Trypanosomiasis (HAT), Chagas’ disease, and various forms of Leishmaniasis, respectively. Since drugs for these neglected tropical diseases are limited, often toxic and difficult to administer, and parasite resistance to existing drugs is on the rise, it is important to develop new chemo- therapeutic strategies. Our research is focused on trypanosome gene expression because the underlying mechanisms deviate substantially from those in the human host. For example, polycistronic transcription of protein coding genes and processing of pre-mRNA by spliced leader trans splicing are parasite-specific steps in mRNA synthesis and maturation. We discovered in T. brucei that the activity of the cyclin-dependent kinase (CDK) CRK9 is required for trans splicing. By generating a cell line that expresses analog-sensitive CRK9 and no wild-type enzyme, we could chemically inhibit the enzyme in specific manner in cultured cells. Surprisingly, we observed an instant splicing block after applying the inhibitor, suggesting that CRK9 carries out essential reversible phosphorylation on the RNA processing machinery. Our preliminary data indicate that one of CRK9’s substrate is the SR protein and known splicing factor TSR1, and that blocking CRK9 activity affects the assembly of the spliceosome, the large and dynamic RNA-protein complex that carries out the splicing reaction. Consequently, we will determine the mechanism of how CRK9 aids or controls the splicing process. Furthermore, CDKs represent a highly druggable enzyme class, and CRK9 forms an unusual trimeric enzyme complex with a deviant L-type cyclin and a kinetoplastid-specific protein, suggesting that CRK9 is a promising target for chemotherapeutic intervention. Therefore, we propose to characterize the enzyme complex and determine a minimal complex that is active and can be expressed recombinantly as prerequisite for future high throughput inhibitor screens. Finally, based on preliminary data, we will test the hypothesis that CRK9 is the target of the compound SCYX-7158 which is currently in clinical trials against HAT.

NIH Research Projects · FY 2025 · 2021-02

Inflammation is designed to destroy, disable, or contain pathogenic invaders, but must be controlled to avoid destruction of key host systems, like the vasculature. When the interaction between immune cells and the vasculature goes awry, it can contribute to vascular lesions in aneurysm, atherosclerosis, and other diseases. Our study of the interactions between innate immune cells and the arterial wall in models of atherosclerosis – a sterile and chronic injury process with a critical inflammatory component – has revealed broad regulation of alternative splicing responses that change the extracellular composition of the inflamed intima and the behavior of recruited immune cells that protect the arterial wall from damage. Guided by these data and novel in vitro CRISPR screens to probe the function of RNA binding proteins (RBP) in the regulation endothelial inflammation, we have discovered a set of RBP responsive to innate immune cell recruitment that are critical in orchestrating the activation of the endothelium through NFkB signaling. Here, we test the hypothesis that one of these RBP, Elavl1, coordinates alternative splicing in the arterial intima in response to innate immune cell recruitment to regulate chronic immune functions (Aim 1). In seeking a deeper understanding of this immune-regulatory system, we made the unexpected discovery that, like Elavl1, many RBP strongly bind to transposable element (TE) sequences inserted within genes and their RNA transcripts (p<0.0001). While most TE are inactive, these vestigial TE sequences account for ~45% of our genome, are found in nearly all genes, and can provide cryptic splice sites in transcripts that depend on RBP activity. Thus, we aim to define the family of TE-binding RBP, to understand their regulation during inflammatory responses, and their impact on splicing patterns and inflammatory responses through binding to TE (Aim 2). The completion of these aims will provide new insight into the contribution of endothelial alternative splicing responses to inflammation in chronic inflammatory states, and the contribution of pervasive TE-derived sequence to transcript regulation through RBP that bind them, providing new avenues to understand and treat chronic inflammation in the cardiovascular system.

NIH Research Projects · FY 2024 · 2020-09

Project Summary Many states are aggressively reforming their long-term services and supports systems by constraining the growth of nursing homes and expanding availability of home and community-based services (HCBS) through Medicaid waiver programs, which intend to maximize independent living for individuals at risk for nursing home care. Eligibility criteria for Medicaid HCBS waiver programs include financial and health-related factors, the latter which typically include functional and cognitive deficits. Medicaid HCBS waiver program populations of older adults across the states include individuals living at home with and without diagnosed Alzheimer's disease and related dementia (ADRD) as well as with a wide range of cognitive deficits even without a diagnosis of ADRD. ADRD is associated with many adverse health-related outcomes in population-based studies of community-dwelling older adults; however, whether and how ADRD and cognitive impairment severity are associated with adverse outcomes among older adults receiving services from Medicaid HCBS waiver programs is unknown. Little is known about the strength of informal caregiver support systems and their effects on adverse outcomes for older adults with and without dementia in HCBS programs. Success in meeting self-identified goals of care among older Medicaid HCBS waiver participants, and barriers to achieving these goals, have also not been explored in the context of having ADRD. Moreover, how race and ethnicity might modify effects in associations between ADRD, informal support systems, and health outcomes is unknown in this population. We propose to address these important and interrelated knowledge gaps guided by person-centeredness and health disparities conceptual frameworks. We will study a statewide population enrolled in Connecticut's Home Care Program for Elders (CHCPE), the Medicaid HCBS waiver program for older adults. CHCPE has a racially and ethnically diverse population, and State Medicaid policy decision-makers have expressed strong interest in improving dementia care for CHCPE participants. In Connecticut, a person-centered approach to care planning and implementation guides all Medicaid HCBS waiver program policies and practices. Specific aims guiding this study are to, in the CHCPE participant population: Aim 1: Determine how living with ADRD is associated with health service utilization, including emergency department visits, hospitalizations, and post-acute or long-term admission to nursing homes. Aim 2: Determine whether strength of the informal caregiver support system is associated with utilization of all health services under study, according to ADRD status and racial and ethnic group membership. Aim 3: Determine how living with ADRD, and racial and ethnic group membership, are associated with meeting self-identified goals of care and person-centered outcomes based on their HCBS-related experiences. The study team will disseminate findings to state Medicaid officials and other stakeholders concerned with how best to help CHCPE clients living with ADRD avoid or delay adverse health outcomes and achieve self- identified goals of care. Dissemination activities also will include presentations at annual meetings of relevant national professional and scientific organizations, and publications in relevant peer-reviewed journals.

NIH Research Projects · FY 2025 · 2020-09

The goal of this project is to provide the building blocks for an independent research program focused on the mechanisms by which neural networks incorporate multisensory cues into episodic memories. Discrimination of different contexts composed of distinct constellations of multisensory cues is a hallmark of both episodic memory and spatial navigation, two functions ascribed to the mammalian hippocampus. The Dentate Gyrus of the hippocampus is central to spatial and contextual discrimination; yet the neural mechanisms by which contextual representations are encoded by principal granule cells has remained a significant knowledge gap. Preliminary data based on in vivo two photon imaging indicates a novel elevation of cue-associated activity in the granule cells that correlates with spatial discrimination, both of which are reduced in mice without adult hippocampal neurogenesis. Thus, this project proposes to test the hypothesis that specialized cue cells in the dentate gyrus are critical for anchoring contextual representations and are modulated both by adult neurogenesis and by entorhinal cortical inputs. These studies represent a number of firsts in linking the physiology and behavioral function of the Dentate Gyrus during learning of spatial discrimination by investigating 1) the evolution of spatial and cue-associated activity of granule cells over time in order to support the encoding of contextual representations; 2) the activity of adult born granule cells and their distinct contributions to cue representations; and 3) the activity of the entorhinal cortex afferents to the Dentate Gyrus, and the mechanisms by which multisensory information arriving from the external world generate internal hippocampal representations. To achieve this detailed circuit dissection, I will use an integrative approach that merges in vivo imaging techniques, genetic-based circuit manipulation strategies and computational analysis of multi-neuronal activity. The technical and scientific skills that I will develop during the training period of this project will become the pillars of an independent research career investigating the function and development of the complex neural dynamics which support cognitive functions implicated in neuropsychiatric disorders. This training will be complemented by intense carrier developmental activities and mentorship that will prepare me for the practical aspects of laboratory management, teaching and fund raising. Overall, these studies will provide novel insights into how the Dentate Gyrus local and long-range circuits contribute to cue representations and facilitate contextual discrimination. Since hippocampal damage has been implicated in the cognitive discrimination impairments associated with Alzheimer’s disease and PTSD, constructing a dynamic picture of the Dentate Gyrus, an often overlooked hippocampal region, at cellular and circuit resolutions during the formation of episodic memories may have an important clinical relevance.

NIH Research Projects · FY 2024 · 2020-09

Research from the last decade has identified cellular senescence and alteration of gut microbial composition as primary physiological processes that facilitate aging and a wide range of age-related diseases. Because of their profound impact on health and disease, they represent two promising ideas in developing innovative strategy to improve health and increase longevity. However, the interplay of the microbiome and cellular senescence in age- related metabolic dysfunction is largely unknown. The major goal of this proposed study is to elucidate the causal connection of cellular senescence and the microbiome in older mice under metabolic stress. We showed a high fat diet (HFD) induced senescent cell loads, increased the abundance of pro-inflammatory gut bacteria, and aggravated metabolic function. In contrast, caloric restriction (CR) decreased gene expression associated with senescence in humans and mice. An intermittent fasting (IF) diet that mimics CR improved metabolic function, and increased the abundance of Akkermansia known to have strong anti-inflammation and anti-aging property. Using our novel p21-Cre mouse model, we found that depletion of senescent cells expressing high levels of p21 (p21high) profoundly increased the relative abundance of Akkermansia, and improved metabolic dysfunction in male mice on a HFD. In Aim 1, we will test the hypothesis that cellular senescence modulates the microbiome composition and function. This will be achieved by directly transplanting or genetic clearance of senescent cells in older male and female mice, and determine their impact on the gut microbiome and microbial metabolites (Aim 1a). The microbiome changes will be determined at population level and functional level as these have not been well-defined previously in aging or age-related diseases. Using our novel p21-Cre mouse model, we will further assess if senescence induced alteration of the microbiome is a novel mechanism by which senescent cells influence metabolic function. We will also test the hypothesis that SASP mediates the senescence-induced microbiome changes by inactivating NF-κB in p21high senescent cells (Aim 1b). In Aim 2, we will test the hypothesis that the gut microbiome modulates senescence development. We will examine development of senescence in mice receiving fecal microbiota derived from a HFD (Aim 2a). We will determine the potential suppression of senescent cells by fecal microbiota transplantation of the microbiota derived from IF or mono- colonization of Akkermansia (Aim 2b). Establishment of reciprocal modulation of the microbiome and cellular senescence will deepen our fundamental understanding of the pathophysiology of aging and age-related metabolic diseases, and pave the way to develop robust interventions targeting senescence, microbiome or both to improve health and increase longevity.

NIH Research Projects · FY 2024 · 2020-09

There is an urgent clinical need to develop new therapeutics to promote bone regeneration. A critical aspect of the bone healing process begins with the expansion of periosteal progenitors that occurs immediately after injury and then the differentiation of these progenitors to bone forming osteoblasts and chondrocytes, yet mechanisms that control skeletal progenitor/stem cell activation, expansion, and differentiation in response to injury are poorly described. Our project will study the role of the R-spondin (ligand) – Lgr (receptor) signaling axis in regulating these progenitors and bone regeneration. R-spondins (roof plate specific spondin) are a family of four secreted matricellular proteins (Rspo1-4) that bind to Leucine-rich repeat-containing G-protein coupled receptors 4/5/6 (Lgrs). Rspo-Lgr interaction potentiate canonical Wnt pathway by preventing the turnover of Wnt Frizzled receptors, and hence determines canonical Wnt signaling levels. While canonical Wnt signaling is known to play an important role in bone regeneration, very little research has explored positive modulators of Wnt signaling. In particular, the requirement of Rspo-Lgr in the context of fracture healing has never been examined due to lack of appropriate models. Our primary goal is to define the requirement of Rspo2/3 and Lgr6 in mesenchymal progenitors in response to bone injury. We have defined three specific aims to address this goal. In Aim1, we will use single and compound Rspo2 and Rspo3 floxed mice crossed with an alphaSMACreERT2 mouse to disrupt the Rspo2/3 genes in mesenchymal progenitors at the time of fracture. Bone healing will be assessed using microCT, histology, molecular analysis, and mechanical testing. Alterations in canonical Wnt signaling and osteogenic potential of Rspo2/3 deficient progenitors will be assessed. In Aim 2, Lgr6 knockout mice will be investigated for their bone healing properties using parameters similar to Aim 1. In Aim 3, Rspo2 will be delivered to bone injury sites and the impact on BMP and Wnt signaling, progenitor activation and differentiation, and bone healing assessed. Completion of this project will identify the requirement of Rspo2/3-Lgr6 interaction in fracture healing and will provide new therapeutic directions for enhancing bone healing.

NIH Research Projects · FY 2024 · 2020-09

Self-Powered Sample Concentrating and CRISPR-based Biosensing for Mobile HIV-1 RNA Detection Abstract HIV/AIDS has become a major public health concern affecting ~37.9 million people worldwide. Early diagnosis of acute HIV infection during seroconversion window will facilitate early intervention. During antiretroviral treatment (ART) of HIV-infected patients, it requires frequent monitoring of HIV viral load to confirm treatment effectiveness, and to identify viral rebound. HIV viral load testing that quantifies HIV viral RNA (circulating HIV virus) in plasma is the most accurate and reliable approach for the ART monitoring and acute HIV detection. However, current standard HIV viral load testing methods rely on expensive equipment and well-trained personnel, limiting their clinical applications in centralized laboratories and hospital environments. Commercially available immunoassay-based point of care (POC) diagnostic technologies, such as OraQuick® HIV Self-Test (HIVST), are not effective to detect acute HIV infections, as well as ART failure. As a consequence, the lack of a simple, rapid, affordable, POC diagnostic tool for HIV RNA detection leaves many individuals unaware of their condition and impedes timely antiretroviral treatment. To fill this gap, we propose to develop a low-cost (~ $ 5), rapid (< 35 min), and sensitive (<1,000 copies/mL), clustered regularly interspaced short palindromic repeats (CRISPR) biosensing platform for HIV viral load testing using finger- prick volume (~50 µL) of whole blood. In the R61 phase (Aims 1-3), we will: i) develop and optimize highly sensitive and specific CRISPR biosensing technology for next-generation nucleic acid-based molecular diagnostics, and ii) design and fabricate a disposable "blood-to-answer", CRISPR biosensing device that integrates self-powered plasma separation, viral RNA enrichment, and CRISPR biosensing detection. In the Phase 33 (Aims 4-5), we will systematically evaluate the performance of our integrated CRISPR biosensing platform, and rigorously validate its feasibility for clinical application by testing HIV clinical samples in the US and Zambia. If successful, such a simple, rapid, affordable, POC detection platform will enable acute HIV diagnosis and viral load testing at home and be appropriate for resource-limited settings where HIV is most prevalent.

NIH Research Projects · FY 2024 · 2020-08

Splicing dysregulation, caused by defects in RNA-binding splice factors including TDP-43, is considered a hallmark and potential driver of neuronal dysfunction and cognitive decline in Alzheimer’s (AD) and Related Dementia (ADRD). Although substantial evidence suggests that genetic causes of splicing dysfunction are not limited to neuronal cells, splicing defects in the BBB endothelium in AD and ADRD have not been examined. Defects in the BBB increase with age and early in the progression of AD and ADRD, where they contribute to disease progression. Through in vivo informatics and in vitro CRISPR screening, we identified TDP-43 and several other AD and ADRD associated splice factors as regulators of post-transcriptional splicing in the endothelium in response to sterile inflammation. Using a novel method to isolate endothelial nuclei from frozen banked human brain tissues, we identified reduced nuclear TDP- 43 levels in endothelial cells of the blood brain barrier (BBB) with age and in AD and ADRD patients. Furthermore, in a novel mouse model, we show that specific deletion of TDP-43 from the brain endothelium leads to leak across the BBB, and activation of the microvasculature and microglial cells. Here, we hypothesize that loss of nuclear TDP-43 in the endothelium contributes to defects in the BBB and microvasculature, and to AD and ADRD by affecting the splicing of pre-mRNA required for the maintenance of a quiescent endothelium. We propose to examine TDP-43 expression and splicing activity in the endothelium in human AD and ADRD, and the effect of loss of nuclear TDP-43 on disease progression in mouse models of AD and the ADRD Frontal temporal lobe dementia (FTLD). Furthermore, since our novel techniques will allow us an unprecedented view of the endothelium in AD and ADRD, we propose to extend our work on TDP-43 to broadly examine splicing alterations in the endothelium in these disease states, and use our established bioinformatics and in vitro screening approaches to determine whether defects in other endothelial splice factors also contribute to BBB dysfunction. The completion of this work will provide new insight into the contributions of post-transcriptional regulation by RNA-binding proteins to BBB defects, microvascular dysfunction and the progression of AD and ADRD.

NIH Research Projects · FY 2025 · 2020-08

Project Summary This proposal aims to continue operation of the National Resource for Mechanistic Modeling of Cellular Systems, to serve the large community of cell and systems biologists. The Resource encompasses the COPASI and Virtual Cell (VCell) software platforms, which are arguably the most comprehensive and widely used tools for computational modeling of the biophysical mechanisms controlling cell function. VCell supports a number of key biophysical mechanisms, including reaction kinetics, diffusion, flow, membrane transport, lateral membrane diffusion, electrophysiology and rule-based models of multi-state/multimolecular interactions. Simulations can be based on 0D, 1D, 2D or 3D analytical or experimental image-based geometries. Users may choose among multiple available simulation approaches: ordinary differential equations, partial differential equations, stochastic reaction kinetics, network-free simulations, spatial particle-based simulations and spatial hybrid stochastic/deterministic simulations. COPASI enables the simulation and analysis of complex biochemical reaction networks either deterministically or stochastically. It offers a broad range of analysis tools including parameter estimation/optimization, steady state analysis, stoichiometric analysis, sensitivity analysis and metabolic control analysis. VCell and COPASI each boast thousands of active users. From 2020-2024, the Resource has supported research in 437 publications from a wide array of scientific fields, including metabolism, cell signaling, cancer, aging, chemistry, biophysics, and others. The two software technologies were also used to help develop a variety of new experimental methodologies. This support includes 77 NIH- funded grants. The Resource will continue maintaining the software removing bugs and adapting it to new developments in operating systems, as well as expanding their utility by adding other established methodologies for modeling and simulation. Both VCell and COPASI are hosted at the University of Connecticut School of Medicine and benefit from: (1) the common institutional organization under which they operate; (2) a joint website; (3) a common high performance computing facility that serves the computationally intensive needs of users without charges; (4) coordinated training and outreach to the user community in the form of web-based documentation and tutorials, two yearly Computational Cell Biology Workshops and numerous roadshows at national and international meetings. Additionally, VCell and COPASI are leveraged by external systems biology software developers as user-friendly platforms for the wide dissemination of their third party algorithms, software and databases. Finally, the Resource actively engages with the community of researchers defining software standards to assure the reproducibility and reusability of both the software and the models it generates.

NIH Research Projects · FY 2026 · 2020-07

PROJECT SUMMARY/ABSTRACT With the advent of long-read RNA sequencing technology, there is now the opportunity to quantify how disparate alternative mRNA termini choices and internal exon choices are combined in full-length mRNAs. Recent evidence indicates coordinated interactions between alternative termini and exon choices in various organisms and developmental contexts, rather than random combinations. In Drosophila, individual genes have been found to exhibit tight coordination of both alternative first exons and alternative internal exons with alternative 3’UTRs. This project will investigate the role of cell type in coordinating these RNA processing events. A novel long read sequencing approach will be developed (scPL-Seq) that will permit the quantification and mechanistic interrogation of coordinated RNA processing events at the single-cell level during Drosophila neurodevelopment. The regulatory factors that coordinate these RNA processing events in specific cell types will be identified. In Drosophila, we previously demonstrated for the Dscam1 gene that coordination of alternative exon and 3’UTR choice is required for axon growth and neurodevelopment. In human neurons, the functional roles of specific exon-3’UTR combinations are unknown. The scope, regulation, and function of such coordinated events will be investigated in human ESCs differentiated to neurons. Alternative exon to 3’UTR choices will be quantified using a targeted adaptation of direct RNA sequencing (dRNA-target-Seq) that will provide not only connectivity of exonic content in full length mRNAs, but also isoform specific polyA tail lengths and RNA modification information. The factors regulating these coordinated events will be investigated, with an emphasis on neuronal RNA binding proteins. The functional roles of coordinated RNA processing events will be interrogated for specific genes, with an initial focus on transcriptional regulation genes. Preliminary work suggests that exon to 3’UTR coordination occurs for many human genes, including the de novo DNA methyltransferase DNMT3A. The importance of such RNA processing events and their coordination will be investigated in ES derived neurons using loss of function approaches. These pursuits will lead to accessible methods to study regulation of gene expression at the level of full-length mRNAs in diverse systems and single cells. Studying the function of alternative exon to 3’UTR coordinated events in human ES derived neurons will enable future investigations of how disease associated sequence variants affect mRNA processing in the context of full-length transcripts.

NIH Research Projects · FY 2025 · 2020-05

ABSTRACT Hypoxia influences nearly all steps in the metastatic cascade, and is an independent adverse indicator for cancer prognosis. Cellular response to hypoxia is tightly regulated, and is mediated by hypoxia-inducible factors (HIFs), with HIF-1 being the ubiquitously expressed homologue. Canonical response to hypoxia involves stabilization of HIF-1α, which acts as a key transcriptional factor, regulating the expression of more than 1000 gene products indirectly, influencing key steps in cancer progression. However, we discovered that the population response to hypoxia is more complex than the canonically understood response, with a small subpopulation displaying oscillations in HIF-1α stabilization and transcriptional activity in a lactate dependent manner. Lactate is a byproduct of glycolysis, which is itself increased due to HIF-1α activity, and can cause degradation of HIF-1α by chaperone mediated autophagy, driving oscillations. Owing to the centrality of HIF- 1α in transcriptional regulation in hypoxic tumors, oscillations in HIF-1α in a subset of cells could have profound consequences in gene expression, and cancer progression. Our preliminary data show that oscillatory hypoxic input can drive large scale transcriptomic changes, resulting in increased metabolic activity, cell proliferation, as well as altered regulation of pathways related to circadian rhythms, and invasion. These data suggest that possibly emergent oscillations in HIF-1α activity may provide a selective advantage to these cells to escape hypoxia induced stress response. Our preliminary data present a strong rationale to investigate this emergent phenotype in cancer populations, the mechanisms driving these oscillations and the phenotypic consequence of these oscillations. Furthermore, many of the genes responded to oscillating hypoxia as a qualitatively different signal, suggesting presence of regulatory motifs, incoherent feedforward loops (IFFLs) which could distinguish between oscillatory and sustained HIF-1α signal. Using an integrated approach involving computational modeling, bioinformatics, and experimentation, we will systematically identify and validate these IFFLs, as well as the co-factors necessary to form these IFFLs along with HIF-1α. Our aim will not only shed light on the fundamental regulatory mechanisms of decoding of oscillatory signaling in cancer, but also provide a targeting strategy to contain the phenotypic consequences of emergent HIF-1α oscillations. Finally, we will test the consequence of emergent HIF-1α oscillations in vivo in a mouse model of breast cancer tumorigenesis, and test if oscillating HIF-1α confers increased tumorigenicity, proliferation, and survival. Our proposed method will facilitate mechanistically understanding the genesis of oscillations, generation of phenotypic heterogeneity in cancer, as well as understand and target the consequence of this emergent subpopulation in influencing cancer progression.

NIH Research Projects · FY 2025 · 2020-02

This is a competitive renewal application for UH3 DE028520, “Individualized Assessment and Treatment Program for TMD: Coping as a Mechanism.” As per the program announcement (PAR-22-048), ongoing clinical trials supported by NIDCR may be extended if additional time is required to complete the trial. The purpose of this application is to allow us the opportunity to complete the work for which we were initially funded. The current study, “Individualized Assessment and Treatment Program for TMD: Coping as a Mechanism” (U01 DE028520), was funded for the period 2/11/2020 to 1/31/2025. The first year of that study in 2020 saw the country overtaken by the Covid-19 epidemic. In the course of that first grant year we revised all of our procedures to treat patients remotely. We were thus not able to recruit our first subject until 2/01/2021, and recruitment was slowed by the pandemic. Thus, we lost more than a year of time in total. The result is that this study cannot complete all its aims without additional funding during Years 6 and 7 to meet our enrollment, treatment and follow-up goals. The current study is aimed at exploring the extent to which the individualized training of coping skills is an important mechanism of psychosocial treatment. In this renewal we will recruit, and treat 35 patients, and follow the remainder of patients in treatment. As designed, this is a highly innovative behavioral treatment trial. Patients with TMD-related pain of at least 3 months duration all receive a Standard Conservative Treatment (STD) and are randomly assigned to either a Cognitive Behavioral coping skills treatment (STD+CBT), or to an Individualized Assessment and cognitive- behavioral Treatment Program (STD+IATP) for patients with TMD pain. Treatment in IATP is be based on a very detailed functional analysis of the patient’s pain experience, in context, as derived from Experience Sampling (ES). The ES procedure is conducted via smartphone app at a rate of 4 records per day, and is be used to gather information on patients’ pain, momentary cognitions, affects, and coping behaviors, for a 2- week monitoring period prior to the beginning of treatment. Therapists use this information to develop an individual functional analysis of pain and non-pain episodes, and determine what thoughts, feelings and actions are effective for that patient at managing pain and which are not. The information is used to help develop adaptive coping tactics in a 6-session treatment program, offering skills training tailored to specific patient needs. During-treatment ES allows adjustment of the treatment goals and procedures, making the treatment adaptive and able to change with changing circumstances and patient needs. The conventional CBT program promotes coping skills, but is not based on in-vivo assessment of pain. Outcomes include measures of pain, interference, and depressive symptoms out to 12 months. IATP is a step toward a precision behavioral intervention for chronic pain. The results will shed light on mechanisms of treatment for TMD and may have implications for the management of other pain conditions.

NIH Research Projects · FY 2025 · 2019-12

PROJECT SUMMARY The goal of this project is to explore the functional relevance of interactions between brain-derived neurotrophic factor (BDNF) and endogenous cannabinoids (eCB) in regulating activity-dependent synaptic plasticity in the neocortex and hippocampus. Although there is growing evidence for crosstalk between BDNF and eCBs, little is known regarding potential synaptic interactions. We have previously characterized the synaptic effects of eCBs and BDNF in layer 2/3 and layer 5 of somatosensory cortex as well as the CA1 area of hippocampus, and we have recently shown that the presynaptic effects of BDNF at cortical and hippocampal inhibitory synapses are mediated by the BDNF-induced release of eCBs from postsynaptic pyramidal cells. We have also found that BDNF causes release of eCBs at excitatory synapses, and this eCB signaling mitigates the direct facilitatory effects of BDNF at these synapses. We are now poised to explore the functional relevance of these interactions in regulating activity-dependent synaptic plasticity. In particular, we will examine the interactions between endogenous BDNF-induced eCB release and activity-dependent eCB release in regulating the magnitude and direction of plasticity at excitatory and inhibitory synapses. These studies will combine electrophysiology and calcium imaging with pharmacological and genetic approaches to manipulate these signaling systems. We will also examine these signaling interactions using mice engineered to express common human single-nucleotide polymorphisms (SNPs) that affect either endogenous BDNF or anandamide levels. Importantly, we will carry out parallel studies using cultured human induced pluripotent stem cell (iPSC)-derived neurons generated from individuals who carry these same SNPs.

NIH Research Projects · FY 2024 · 2019-09

Abstract Concentrations of ultrafine particles (UFP) are elevated near major roadways and highways. Evidence is strong that living in these areas is associated with substantial respiratory, cardiovascular and other adverse health outcomes. We have contributed to recent evidence of associations between chronic exposure to UFP and cardiovascular disease risk. Indeed, in response to these findings, including ours, there is growing use of in-building air filtration to reduce traffic-related pollution levels in homes and schools near highways, including market-based responses and city ordinances. There is, however, as yet, no empirical evidence that these measures improve health. This proposal builds on preliminary studies in which we conducted randomized crossover trials of in-home air filtration on a smaller scale (N= 20 and 23) and a controlled short term setting (N=77). Our randomized 2-hour exposure study showed that reducing PM with filtration can reduce blood pressure. Accordingly, we propose a blinded randomized crossover efficacy trial (N=240 households consisting of 288 participants) of High Efficiency Particulate Air (HEPA) filtration in near-highway homes that lack mechanical air-handling systems. Households will be randomized to 30 days of either filtration or sham filtration followed by a 30 washout period with a subsequent 30-day period of the alternative assignment. Room air filters that are commercially available will be placed in the bedroom and living room of each home. We will measure UFP and PM2.5 concentrations in 20% of the homes during filtration and sham periods and assess personal exposure in a subset of participants. We will also assess chemical composition of particulate air pollution in 10 homes/year for exploratory purposes that could lead to future lines of research. Our primary health endpoints will be participants’ hsCRP and peripheral blood pressure, measures we have used in multiple observational studies of UFP as well as in our pilot filtration intervention studies. Secondary biological measures that contribute to understanding biological pathways will be IL-6 (inflammation), D-dimer (coagulation), metabolome, central pressure and arterial stiffness. The primary intention to treat analysis will compare outcomes between HEPA filtration to sham filtration. We will have 80% power to detect a difference of 0.6 mg/L in change in hsCRP and a difference in reduction in systolic blood pressure of 3.5 mmHg compared to participants who receive no filtration. Having participants serve as their own controls in the within-subject comparisons of intervention effectiveness increases our statistical power and eliminates the possibility of baseline imbalances in demographic and clinical characteristics. A social science evaluation will inform final adjustments to our approach at the start and also assess participant acceptance and experience with the intervention at the end. Our primary innovation is that this will be the first near highway HEPA intervention trial that is large enough and careful enough to be policy-relevant.

NIH Research Projects · FY 2026 · 2019-09

ABSTRACT The mechanisms by which mammalian genomes maintain stability through generations and divisions are important, and de-regulation of these cellular programs can lead to somatic mosaicism, disease, and cancer. Many multicellular eukaryotic genomes are replete with repeats that include ancient repeat mobility events, actively mobilizing transposable elements, and large segmentally duplicated regions. Human variation caused by transposable element mobility and structural rearrangements between repeats comprises more than a quarter of variants larger than fifty base pairs in the human population. These structural variants can lead to the insertion of promoter sequences, transcription factor binding sites, and even gene duplication events in mice and humans. Thus, mammalian genomes are comprised largely of repeat sequences, yet the role these sequences play in generating genomic instability, the factors that deter this instability, and the gene regulatory differences due to repeat variation are still poorly understood. Moreover, much of our knowledge on the genomic stability of repeats is derived from studies in yeast or from the use of reporter assays that interrogate discrete repeat sequences and distances. In the past five years, we have developed a robust research program using molecular genomics combined with computational biology to investigate the consequences of repetitive sequences on human and mouse genomes. In the next five years, we will identify repeat mediated rearrangements across large numbers of individuals and diverse species using our established approaches, interrogate how transposons have diversified gene regulation across a half million years of mouse evolution and the epigenetic and transcriptional consequences of this variation, and will identify the guardians of repeat mediated genomic stability and mobility. We will approach the proposed work with a diverse team of computational biologists, tool developers, graduate students, post-baccalaureate researchers, and summer students. The long-term goals of our research are to understand how mammalian genomes guard against variation in the context of repetitive sequences and how repeats can diversify genomes and gene regulation in short evolutionary timespans.

NIH Research Projects · FY 2026 · 2018-08

ABSTRACT Many of the molecular mechanisms underlying well-characterized robust and rapidly inducible transcriptional responses are shared among other systems, so we use hormone-induced transcriptional responses to study gene regulation. We use rapid kinetic regulation and perturbation of transcription cascades, transcription factors, and cofactors to identify key mechanisms, genes, and regulatory elements that are critical for hormone signaling. Transcription factors act as activators or repressors and interface with a constellation of accessory cofactors to regulate distinct steps in the transcription to coordinate gene expression, but the molecular functions of the vast majority of transcription factors remain uncharacterized. We use molecular genomics assays and computational methods to classify transcription factors by their molecular function, as opposed to broad activator and repressor classes, in order to understand the context specificity of gene regulation. We found that the estrogen receptor transcription factor may compete with other transcription factors for limiting cofactors to mediate estrogen-induced repression. We will develop genetic tools to uncouple activation and repression to test various models of repression, such as squelching cofactors from repressed genes. The genes and regulatory elements that are downstream of the first wave of transcriptional response are critical for propagating regulatory cascades. We generate high resolution, genome-wide time course data of regulatory element activity and nascent transcription upon stimulation of differentiation processes. We construct mechanistically interpretable networks to identify effector genes and regulatory elements that are critical for signaling in regulatory cascades. Our research reveals basic principles and rules that govern transcription factor specificity in order to understand how genetics, nutrition, and environmental factors contribute to variation in transcriptional programs that can lead to disease states or ineffective therapies.

NIH Research Projects · FY 2026 · 2018-08

Project Summary / Abstract The pairing of homologous chromosomes is a fundamental process for meiotic recombination but also occurs in non-meiotic cells in a broad range of organisms. Recent studies have revealed that non-meiotic homolog pairing is an actively regulated process and provides an opportunity for interchromosomal interaction. The local pairing status of a particular gene locus differs in a cell type-specific manner and correlates with local chromatin states, suggesting the possibility that non-meiotic homolog pairing may take part in gene regulation. Drosophila male germline stem cells (GSCs) constantly divide asymmetrically to produce one GSC and one differentiating gonialblast (GB). In this system, stem cell-specific genes, including Signal transducer and activator of transcription 92E (Stat92E), are quickly downregulated, providing an excellent model to study preprogrammed changes of gene expression states in vivo. Results from the previous funding periods showed that the homologous regions of Stat92E always closely associate with each other in GSCs (paired) but separate immediately in GBs (unpaired), and the change in pairing states is required for prompt downregulation of Stat92E transcription. It has been shown that GSCs tend to retain old histones H3 and H4, while GBs tend to inherit newly-synthesized histones H3 and H4. When this histone inheritance was compromised by expressing non- phosphorylatable histone H3T3A, the change in Stat92E pairing upon differentiation did not occur, raising the exciting possibility that the pairing states of key genes are epigenetically programmed through the inheritance of old vs. new histones. These observations suggested that the non-meiotic homologous pairing may be a process to reprogram gene activity, and that the alteration of pairing states is an important mechanism that regulates key genes during stem-cell differentiation. In this proposal, we aim to define the role of interchromosomal interaction in non-meiotic stages of germline on cell fate determination and to elucidate the underlying mechanisms. First, we will identify a list of genes differently paired in GSC and GB. Next, we will characterize genome-wide homolog interaction in GSCs and differentiating cells. Lastly, we will investigate mechanisms by which changes in pairing state influence transcription using Stat92E and other identified genes. These experiments will allow us to understand how pairing is developmentally regulated and how it impacts cell fate determination. Homologous allelic pairing also occurs in mammalian stem cells, where alleles of Oct4 transiently pair to share repressive chromatin marks during the transition from pluripotency to lineage commitment. Thus these proposed studies of pairing regulation may elucidate common mechanisms of stem cell differentiation.

NIH Research Projects · FY 2025 · 2018-02

Project Summary The objective of the ENCORE (Encyclopedia of RNA Elements) project is to develop a foundational, functional map of protein-RNA interactions of RNA binding proteins (RBPs) encoded in the human genome, and the RNA elements they bind to across the transcriptome. These RNA elements, when expressed, form the basis of co- and post-transcriptional regulation of human genes. Our strategy consists of developing and integrating a physical map of 400 new RBPs in two different human cell lines with transcriptome-wide measurements of the effects of depleting these RBPs. Over the past 9 years, our group has established highly efficient data production workflows of experimental methods that will enable us to immediately expand these datasets further, which form a crucial and missing link to decipher the mechanisms of post-trancriptional regulation and how these impact genetic variation and disease etiology. When combined with the data we generated over the past 9 years, these efforts will culminate in a comprehensive map of the functional RNA elements recognized by essentially all RBPs expressed in two human cell lines, representing approximately half of the known complement of human RBPs. ENCORE will continue developing and integrating a physical map of hundreds of RBPs in two different human cell lines with transcriptome-wide measurements of the effects of depleting these RBPs. In addition, we will provide training and outreach to establish ENCORE annotations as the standard reference for co- and post-transcriptional research and clinical genomics efforts in the long-term. In summary, the data we will produce in this project will enable a more systematic and comprehensive understanding of the role of RBPs and RNA biology in the contribution to human biology and disease.

NIH Research Projects · FY 2025 · 2016-09

In this competing renewal of R01MH108578, we are seeking to extend findings from the initial study to focus on effects of stress in longitudinal mood and cognitive outcomes of late-life depression (LLD) and to examine stress effects on brain structure and function in LLD. Severe or persistent stressors can result in a number of behavioral and mood changes, including anxiety, dysphoric mood, sleep disruption, altered appetite, and withdrawal from social and pleasurable activities. These stress-related consequences are particularly salient when considering longitudinal outcomes of treated LLD. They may be compounded by an individual's longstanding maladaptive patterns of response to stress, embodied in the construct of neuroticism, which we have shown to be related to poor mood and cognitive LLD outcomes. Moreover, Andreescu et al. (2019) introduced a model of depression recurrence that incorporates the homeostatic disequilibrium hypothesis, which proposes that in geriatric remitted depression, neural networks are in fragile homeostasis that is threatened by stress exposure. Networks of particular importance in stress of LLD outcome are the Default Mode Network (DMN), Salience Network (SN) and Executive Control Network (ECN). The Neurobiology of Late Life Depression (NBOLD) study began enrolling older depressed and never- depressed controls in 2013, enrolling 132 depressed and 44 controls, and currently follows 77 depressed and 22 controls. Subjects are well characterized in terms of mood, cognition, personality and stress (including specific measures obtained during the present COVID pandemic). It is well suited to examine stress effects on longitudinal mood and cognitive outcomes. For the renewal, we will follow current subjects and recruit 75 new subjects, who will be followed for up to 5 years with annual cognitive testing, stress measures and baseline and two-year brain fMRI scan. In preliminary data presented in this application, we show that: 1) In depressed elders, compared with those who developed cognitive decline/dementia in 4 years, those who remained cognitively normal had greater 2-year decrease in neuroticism and increase in Conscientiousness. 2) Worsening in stress over two years is associated with two-year decrease in hippocampal volume and is associated with changes in functional connectivity of key brain regions.. 3) In complex statistical modeling, interactions of changes in neuroticism and changes in the number of stressors recently experienced are associated with cognitive outcome. In this competing renewal, we will examine the following specific aims: 1. To study effects of stressors (obtained on a variety of measures) and neuroticism on longitudinal mood and cognitive outcomes in older adults with history of major depressive disorder (MDD). 2. To study effects of stress and neuroticism on brain structure and function in older adults with MDD history. 3. To explore relationships among variables in Aims 1 and 2 with longitudinal multivariable statistical models.

NIH Research Projects · FY 2025 · 2016-04

Most eukaryotic pre-mRNAs, especially in metazoans, are alternatively spliced to generate multiple mRNAs and proteins. Given the importance of alternative splicing in regulating gene expression and enhancing the diversity of the proteome, it is essential to understand the mechanisms of splicing and how alternative splicing is regulated. In this project, we will study the roles of RNA binding proteins in alternative splicing, with an emphasis on how RNA binding proteins auto- and cross-regulate the splicing their own and other RNA binding protein genes. This work will provide new insight into the mechanisms of RNA processing and how these proteins regulate one another to achieve homoestasis. Many prokaryotes encode CRISPR-Cas systems which are RNA-guided adaptive immune systems that protects prokaryotic organisms against invaders such as viruses and plasmids. Immune memories are encoded as short DNA sequences, called “spacers”, that match invader genomes and are stored as interspersed elements in an array of short repeats (the CRISPR array). The CRISPR arrays are transcribed and processed into guide RNAs which pair with Cas nucleases to recognize and degrade target nucleic acid (interference). New immune memories are formed during “adaptation” when fragments of invader DNA are acquired and integrated into CRISPR arrays for use in future targeting. While a tremendous amount is known about the targeting and degradation of invading nucleic acids, much less is known about the process of adaptation. We plan to further characterize the adaptation process in prokaryotic CRISPR-Cas systems. This work will also provide insight into the mechanisms of adaptation in the immune systems of prokaryotes. In addition to enhancing our understanding of the basic science of prokaryotic immune systems, there is tremendous potential that this work could lead to the development of new tools that can be used for genome editing applications. All of these projects will be addressed using the types of general approaches we have developed such as splicing reporters, single cell RNA-Seq, nanopore sequencing, RNAi or CRISPR screens, and computational genomics. We will also continue to develop additional innovative approaches to address these issues as needed or as opportunities arise due to technical advances in the field.

NIH Research Projects · FY 2025 · 2016-03

Inflammasomes sense an array of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) generated during infection and trauma and represent the first line of defense against infections. In the canonical form, inflammasomes consist of a sensor protein that recognizes PAMPs, an adaptor molecule ASC, and an effector protease, caspase-1. In the noncanonical form, inflammatory caspases related to caspase-1, namely caspase-11 and caspase-4, directly sense cytosolic lipopolysaccharide (LPS) from Gram- negative bacteria and their outer membrane vesicle. Inflammasome signaling culminates in the post-translational activation of IL-1β, IL-18, gasdermin D (a pore-forming protein), and pyroptosis, a lytic and inflammatory form of cell death, and the simultaneous release of DAMPs. Despite the profound implications of inflammasome responses in infections, cancer, and autoimmunity, the regulatory modules that fine-tune the initiation and termination of inflammasome signaling remain mostly unknown. This proposal seeks to comprehensively address this critical knowledge gap in three specific aims by focusing on galectins, a family of β-galactoside-binding proteins. Owing to their capacity to bind to N- or O-glycan termini of various glycoproteins and regulate their membrane localization and signal transduction, galectins have diverse functions in various physiological and pathological processes. Aims 1 and 2 of the proposal will investigate galectins' role in noncanonical inflammasome signaling in murine and human cells and in vivo. Aim 3 will explore how galectins control canonical inflammasome signaling in vitro and in vivo. In summary, the findings from this project would reveal new players in inflammasome signaling with significant implications for human infectious diseases and sepsis.

NIH Research Projects · FY 2024 · 2015-09

Abstract: Overall. Computation plays a critical role in biomolecular applications of nuclear magnetic resonance spectroscopy (NMR), such as structural biology, metabolic studies, disease diagnosis, and drug discovery. Powerful software packages from a variety of sources facilitate computation in bio-NMR, but challenges include the difficulty of disseminating and maintaining a diverse set of software for a diverse set of computer platforms, communication between software packages, and the lack of persistence of software. The aim of this proposal is support continued operation of a Center for NMR Data Processing and Analysis that will simplify dissemination, support, and use of a broad range of widely-used NMR data processing and analysis software packages. Central components of the project include the use of virtualization technology to provide a “zero configuration” software environment that requires no configuration on the part of users. It will run on almost any computer platform and simplify both dissemination and maintenance. An archive will ensure software persistence that is essential for reproducible research. The Center will establish a publically accessible website for discovery, evaluation, and access to a diverse set of NMR software, and will develop tools for interoperation among software packages. In addition to a single, unified downloadable package, the platform will be made available for remote access, as a Platform as a Service (PaaS). By facilitating the deployment, utilization, interoperation, and persistence of advanced software for biomolecular NMR, the resource will advance the application of biomolecular NMR for a wide range of challenging applications in biomedicine, and help ensure the reproducibility of bio-NMR studies. !

NIH Research Projects · FY 2025 · 2013-09

Abstract BACE1 is an enzyme required for Aβ generation, and chemical inhibition of BACE1 has been explored for reducing Aβ generation. Five brain-penetrable BACE1 inhibitory dugs were recently tested in human trials. Unfortunately, despite a noted reduction in Aβ plaque loads, these clinical trials were terminated, largely due to neuronal and synaptic toxicity. This setback indicates the importance to known more BACE1 biology and to develop safer BACE1 inhibitors for AD treatment, especially considering the growing evidence from anti- Aβ trials that reducing Aβ plaques early can lead to reduction of other AD pathologies. In this proposal, we aim to test a novel hypothesis that increasing inhibition of BACE1 in glial cells, along with boosting microglial phagocytic functions, will reduce AD pathologies while minimizing synaptic side effects. This hypothesis is supported by our recent observations that Bace1 deletion in microglia facilitates the transition of homeostatic microglia to more phagocytic stage-1 disease-associated microglia (DAM-1). Consistently, deletion of microglial Bace1 in 5xFAD mice reduces amyloid deposition. If Bace1 in astrocytes is deleted, the expression of genes, such as clusterin (Clu), CXCL14 and ApoE, important for removal of amyloid plaques by astrocytes, is increased. These results suggest that BACE1 inhibition in glial cells has a beneficial effect on the clearance of amyloid plaques. However, many questions remain to be answered. For example, it is not clear whether Bace1 deletion will affect tau pathology. We do not yet know how BACE1 inhibition will enhance microglia to remove tau pathology. Considering the known role of chemoattractant molecule CXCL14 in mediating migration of immune cells and reduced levels of CXCL14 in AD brains, we will ask whether the expected elevated levels of CXCL14, in response to the inhibition by BACE1, will promote phagocytic function of microglia. This question will be address by utilizing an inducible transgenic mouse model that has already been generated. In this proposal, we will explore whether deletion of BACE1 in PS19 mouse microglia will reduce tau pathology in Aim 1. In Aim 2, we will ask whether a low dose inhibition of BACE1 in AD triple-transgenic model coupled with deletion of Bace1 in microglia will reduce both amyloid and tau pathologies effectively. In Aim 3, we will investigate whether enhancing expression of CXCL14 will promote microglia migration and phagocytosis. These studies are centered on improving microglia function and the knowledge gained from this study will help to develop a therapeutic approach that will favor BACE1 inhibition in glial cells coupled with molecular boosters of microglia phagocytic function for AD treatment.

NIH Research Projects · FY 2025 · 2012-06

ABSTRACT Our major goal is to demonstrate the efficacy of local delivery of anti-senescence drugs when used for immunomodulation at the local cellular level for bone healing. In the immune system, macrophages play a major role in switching inflammation on or off during healing, and they balance activities of bone-forming and bone-resorbing cells. We recently reported that a thin layer of biomimetic calcium phosphate could be used in a novel way as a transient barrier layer (TBL) to sequentially deliver two drugs in a step-wise fashion to switch macrophage phenotype from pro-inflammatory to pro-regenerative. We discovered that macrophages are able to make holes through the TBL and thereby control delivery timing to a drug below the TBL without release of drugs into the media. We now propose to apply this TBL technology to improve macrophage transitions impaired by age as a means to improve bone healing in the elderly. In aging, as well as in metabolic disorders, the immune response is affected by senescent cells that no longer replicate, but have a senescence- associated secretory phenotype (SASP) that produces high levels of proinflammatory molecules. Certain chemotherapy drugs, known as senolytics, however, kill senescent cells and other drugs, called anti-SASP drugs, block their pro-inflammatory cell signaling. Both approaches have been shown to enhance bone density in older mice when given systemically over months, yet both types of anti-senescence drugs have unwanted side effects, and neither approach has been investigated in bone healing contexts. We now propose using the TBL on a bone graft substitute to control local delivery timing of either a senolytic or an anti-SASP drug in a bone microenvironment to maximize calvarial bone repair in old mice. Our preliminary studies support our hypothesis that delivery timing is critical to improve macrophage switching, osteoblast differentiation, and scaffold resorption, and restrict osteoclast maturation all required for optimal bone repair. Aim 1 is a mechanistic in vitro study to understand how appropriately-timed delivery of senolytic ABT-263 or anti-SASP Rux restores osteogenic communication between osteoblasts, macrophages and osteoclasts from old mice compared to cells from old humans relative to young controls. We will broadly capture effects of these drugs on in vitro cell cross-talk by RNAseq. These studies will establish commonalities between human and mouse cell culture responses to the drugs and establish our model for eventual translation to human subjects. The in vivo studies of Aim 2 (with ABT-263) and Aim 3 (with Rux) will determine the appropriate dose and timing of anti- senescence drugs in old mice to suppress senescent cell activity and increase osteoprogenitor activity/decrease osteoclast activity guided by timely macrophage transitions. Our results will reveal the fundamentals of timed, local delivery of drugs to modulate macrophage transitions in injured old mice with heightened inflammation, as well as accelerating the safe and effective implementation of anti-senescence drugs. This will have dramatic implications for healing in the elderly as well as for obese and diabetic patients.