Rutgers Biomedical And Health Sciences

NIH Research Projects · FY 2025 · 2025-09

PROJECT ABSTRACT Tuberculosis (TB) continues to pose a significant global public health challenge, with persistent burden of over 10 million incident cases likely perpetuated by millions of undiagnosed/unreported TB cases contributing to global transmission. We recently observed an unexpected rate of viable Mycobacterium tuberculosis (Mtb) aerosolization among individuals in a high burden TB setting who test negative for Mtb in their sputum. In response to RFA AI-24-049 Halting Tuberculosis (TB) Transmission, this study aims to investigate the role of sputum-negative, Mtb breath-positive individuals in transmission and characterize their underlying clinical spectrum. We will use original breath sampling techniques combined with an Mtb viability assay, highly- sensitive molecular methods, and longitudinal clinical phenotyping with high-resolution imaging to address two specific aims. First, we will assess the association between Mtb breath shedding and transmission. Specifically, we will assess the prevalence of sputum-negative individuals who shed viable Mtb in their breath across high and low TB burden settings. We will then estimate their prevalence in transmission clusters by genotypically linking single nucleotide polymorphisms in exhaled Mtb strains to sequenced Mtb isolates of sputum-positive household members. To estimate infectiousness, we will compare the IGRA positivity rates of child household contacts in sputum-negative, Mtb breath-positive households to those in exclusively sputum-negative, Mtb breath-negative households. Secondly, we aim to examine the extent to which sputum-negative exhaled Mtb reflects underlying pathology, immune activation, and disease progression versus regression over time. We will thus characterize the clinical spectrum of sputum-negative exhaled Mtb through high-resolution CT imaging, immune response markers, and detailed longitudinal evaluations over 12 months. By integrating breath sampling, genotyping, and clinical evaluations, this study will provide critical insights into a potential hidden reservoir of undiagnosed TB transmission. These findings would directly guide comprehensive and early/preventive diagnostic strategies for the missing millions on the undiagnosed TB spectrum, in turn reducing the global burden of TB disease and transmission.

NIH Research Projects · FY 2025 · 2025-09

Among the estimated 10 million people with tuberculosis (TB) in 2022, over 3 million were not diagnosed, underscoring the need for new tools and diagnostic strategies to improve TB case detection, including in difficult to-diagnose populations such as children and people with extrapulmonary or early/subclinical TB. An equally urgent need is for drug susceptibility tests (DST) that can rapidly identify TB drug resistance/susceptibility, including to new drugs, to cure each person with TB and to protect the efficacy of newer drugs for those who may benefit from them in the future. Partially fueled by innovative platforms developed to address the COVID-19 epidemic, the last decade has seen a surge of new TB diagnostic platforms and assays. These new tests, sometimes developed by small companies with limited resources and little or no experience working with Mycobacterium tuberculosis (MTB) and clinical TB, require rigorous and unbiased evaluations. Furthermore, many diagnostic developers do not sufficiently appreciate the challenges presented by sample processing, the (often) need for high test sensitivity, or the acceptable tradeoffs between sensitivity/specific versus assay costs, ease of use and relevant target populations or TB disease states. Thus, the unmet needs of TB diagnostic developers are specific to the type of manufacturer, diagnostic technology, assay format, and intended use. Our proposal brings together a consortium of experienced investigators and a global network of clinical research sites to support TB diagnostic evaluations through a fast-paced and flexible pipeline that includes tech scouting, development mentoring, and clinical testing of early-stage TB diagnostics. With the inclusion of an Analytic Laboratory Core that can assess and help optimize new technologies prior to and after clinical studies our program offers a unique opportunity to accelerate promising TB diagnostics through their optimal development pipeline. Consortium members have almost three decades of leadership experience in diagnostic tech scouting, research, development, clinical trialing, and implementation activities. Fielding a balance of established platforms, fast followers, and cutting- edge technologies, we will perform the following specific aims: 1) identify promising early-stage diagnostics for evaluation and develop for each a stepwise evaluation plan; 2) evaluate the diagnostic accuracy of novel rapid point of care/near care TB diagnostics and determine the impact of relevant patient characteristics on test accuracy; and 3) provide feedback to diagnostic developers, policymakers, and other stakeholders on performance and usability of novel diagnostic tests and potential strategies for use in TB endemic settings.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY All human cells integrate distinct DNA repair pathways and tightly control chromatin architectures to prevent genomic instability. Disruption of these critical maintenance systems or defects in any one pathway result in a mutational burden with profound physiological consequences. DNA repair in humans is predominantly performed by two mutually exclusive pathways mediated by two distinct tumor suppressors – homology directed repair (HDR) by BRCA1 and non-homologous end joining (NHEJ) by 53BP1. These tumor suppressors are examples of intrinsically disordered proteins (IDPs) containing large stretches of low complexity protein sequences. 53BP1 undergoes liquid-liquid phase separation to form biomolecular condensates in vitro and at DNA lesions and my work (during the K99 phase) establishes the essential function of 53BP1 phase separation in NHEJ. In preliminary data, I show the very first evidence that BRCA1 phase separates to form biomolecular condensates and identify a putative prion-like domain for self-assembly. Nuclear organization and chromatin structure are also vital parts of maintaining genomic integrity. DNA damage response requires dynamic rearrangements and chromatin modifications to provoke rapid repair factor recruitment to lesions. Conversely, repair factors and their complexes can also modify chromatin to drive repair programs. For example, recent studies implicate 53BP1 in maintaining heterochromatin. Despite decades of study, it is only in the past several years that condensation of tumor suppressors has been identified. While I have discovered that BRCA1 phase separates, it remains unknown by what mechanism self-assembly occurs and what role it plays in DNA repair through HDR. Further, the contributions of chromatin architecture to repair pathway selection and chromatin organization involving tumor suppressor condensates have not been characterized, leaving gaps in knowledge and possible therapeutic targets. Building on the K99 phase, the goal of this work is to characterize tumor suppressor condensation and how chromatin homeostasis contributes to genomic integrity. In Aim1, my lab will characterize the mechanisms by which BRCA1 phase separates into biomolecular condensates and how these ensembles influence DNA repair and repair pathway choice (HDR vs. NHEJ). In Aim 2, my lab will use super-resolution imaging to directly visualize and define, for the first time, the dynamic activity of chromatin in response to DNA damage and how nucleosome remodeling contribute to 53BP1- and BRCA1-mediated repair programs. Collectively, this work will address fundamental gaps in knowledge regarding the role of phase separations in genome integrity and uncover new paradigms that underlie tumor suppressor activities.

NIH Research Projects · FY 2025 · 2025-09

Project Summary Cardiometabolic diseases contribute to more than 1 in 4 deaths in U.S. adults, including Asian Americans. Psychosocial stress is a key risk factor leading to elevated risks of cardiometabolic diseases. However, there is limited knowledge of how psychosocial stress affects cardiometabolic health among Asian Americans, especially older foreign-born Asian Americans who disproportionately encounter psychosocial stress due to racism, language barriers, and low socioeconomic status. Notably, a lack of knowledge of key biobehavioral mechanisms through which psychosocial stress affects cardiometabolic health hampers public health efforts to develop effective interventions to promote cardiometabolic health in the understudied Asian American population, which is projected to reach 46 million by 2060. To address these important scientific gaps, the proposed study is to investigate key biobehavioral processes through which daily psychosocial stress affects cardiometabolic health among 300 older foreign-born Chinese Americans. We will use a prospective design and harness the power of ecological momentary assessment and biospecimen sampling to achieve study aims. We propose to: (Aim 1) investigate day-to-day associations among psychosocial stressors (e.g., social isolation), biobehavioral processes (e.g., salivary diurnal cortisol) and blood pressure, and (Aim 2) examine the moderation roles of sociocultural factors (e.g., filial piety, acculturation) on the longer-term effects of psychosocial stressors on cardiometabolic health over two years. Completion of the proposed study will generate new knowledge to facilitate the understanding of the interplay of psychosocial stress and sociocultural factors in determining cardiometabolic health and their underlying mechanisms in older Chinese Americans. As a result, this project will guide the design of future culturally relevant, personalized psychobehavioral interventions that aim to promote cardiometabolic health in this at-risk but underserved population.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Age at onset of breast development (thelarche), a marker of pubertal onset in girls, has decreased dramatically in the past half-century. This trend has important public health implications as earlier puberty in girls is associated with adverse health consequences across the lifecourse, including increased risks of cardiovascular disease and breast and other hormonal cancers. Pubertal onset is triggered by the re-activation of the hypothalamic-pituitary- gonadal (HPG) axis in childhood, a process that is poorly understood. Increased understanding of HPG axis activity and function in early life is crucial for identifying risk factors for early puberty that may be amenable to interventions. The HPG axis is active for a brief period during infancy, known as minipuberty, before it is silenced until pubertal onset. The function of HPG axis activity during minipuberty in girls remains unclear. In this K99/R00 research, I will examine the overarching hypothesis that minipuberty is a critical window of programming with short- and long-term effects on endocrine-sensitive processes including growth, breast development, and pubertal development. To do so, I will use data from 136 girls in the Infant Feeding and Early Development (IFED) study, a prospective cohort of infants previously followed from birth to age 9 months with repeated assessments of serum sex steroid concentrations, anthropometrics, and ultrasound measures of breast bud diameter. In the K99 phase, I will examine longitudinal associations of sex steroid concentrations during minipuberty with breast bud diameter (Aim 1) and velocity of linear growth and weight gain into late infancy (Aim 2). I will also conduct a longitudinal follow-up of the former IFED infants (born 2010-2013) as they transition through pubertal maturation. Combining these unique repeated measures data captured during minipuberty and adolescent puberty, I will evaluate whether sex steroid concentrations during minipuberty are associated with the timing of pubertal development, as assessed by ages at thelarche and menarche, and sex steroid concentrations across the pubertal transition (Aim 3, R00). This research will provide novel insights into the role of minipuberty in shaping breast development and growth into adolescence, which has implications for the etiology, screening and ultimately prevention of early puberty and its long-term sequelae. To achieve these research aims, I will combine prior epidemiologic training with new experiential and didactic training in pediatric and reproductive endocrinology, advanced statistical methods for longitudinal biomarker data, cohort development and implementation, and professional development. My K99 training will take place within the interdisciplinary environment of the Intramural Research Program at the National Institute of Environmental Health Sciences with the support of a mentoring team of outstanding scientists. This career development award will provide me with the necessary experience to launch my independent research program focused on applying innovative epidemiologic approaches to understand how exogenous and endogenous hormonal factors during critical periods of early life affect growth, breast development, and women’s health across the lifecourse.

NIH Research Projects · FY 2025 · 2025-09

Abstract Regenerative medicine has become increasingly important in the aging U.S. society. Development of effective regenerative therapy requires an understanding of normal cell type-specific fate determination processes— precise mechanisms involving signals that come from interactions with neighboring cells. Currently, no reliable salivary regenerative therapies exist due to a lack of such knowledge. Three major types of mammalian salivary gland—sublingual, submandibular (SMG), and parotid (PG)—differ in their physiology, acinar cell types, and disease susceptibility (1). Recent evidence suggests distinct processes for the two types of salivary acini: mucous and serous (2). While these indicate inherent differences, salivary cell type-specific differentiation processes have not been reported. Furthermore, while PG is most susceptible to conditions related to dry mouth, virtually nothing is known about PG development due to difficulties with organ isolation and culture. Thus, there is a critical need to identify cell fate determination processes specific to salivary cell populations that comprise functional organs. In the absence of such knowledge, a mechanistic framework necessary for the subsequent identification of therapeutic targets will likely remain difficult. Important knowledge gaps include when and how cells commit to their fates, and whether their lineages are plastic during development. The SMG around embryonic day (E) 13 shows epithelial morphological plasticity in response to the adjacent mesenchyme (3), which raises a question of whether transcriptional and lineage plasticity underlies this morphological transformation. Notably, my preliminary findings indeed show transcriptional changes indicative of a shift in cell fate—findings I have obtained after successfully establishing explant culture conditions to evaluate morphogenesis of SMG, PG, and recombined tissues ex vivo. The studies proposed here were developed to elucidate salivary differentiation processes with the ultimate goal of developing regenerative therapies targeted toward salivary gland disorders. Based on my preliminary results and findings from other groups (3-7), my central hypothesis is that the salivary mesenchyme guides epithelial cell fate and eventually organ specificity through interactions between epithelial and mesenchymal tissues. The proposed program will define the regulation of the epithelium by the mesenchyme. Further, it will provide the first comprehensive molecular characterization of cellular differentiation processes using single cell RNA-seq of heterotypic epithelial-mesenchymal recombinants. As a clinician scientist, my goal is to conduct research on the specific roles of epithelial-mesenchymal interactions in lineage specificity and translate this knowledge into regenerative/therapeutic interventions. My career development plan has been tailored toward this goal with solid mentorship and training opportunities. In conjunction with institutional support, I am confident that studies/activities outlined in my application will help build upon my existing skillset and facilitate my transition into an independent investigator.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Fibrinogen is a highly-conserved 340 kDa glycoprotein that circulates in the blood of vertebrate species at 2-4 mg/mL. Under homeostatic conditions, fibrinogen is synthesized primarily in the liver as a hexameric homodimer comprised of 2 Aα, 2 Bβ, and 2 γ polypeptide chains. In addition to a well-characterized role in thrombosis and hemostasis, fibrinogen is an acute phase reactant that links the hemostatic system to the inflammatory response. Highly elevated plasma fibrinogen concentrations are observed in multiple settings of injury and infection as well as in cancer, cardiovascular and metabolic diseases, and pregnancy. Fibrinogen contains binding domains for leukocyte integrins, and prior studies have suggested that fibrin(ogen) is a pro- inflammatory molecule that coordinates leukocytes recruitment, adhesion, and activation in multiple settings of tissue injury. However, there are major gaps in our understanding of the role of fibrinogen as an acute phase reactant and pro-inflammatory molecule that must be addressed before fibrinogen can be safely and specifically targeted in humans. Overview of research and goals for the next 5 years: The Poole lab opened in October 2022 with the long-term goal of identifying the role of blood coagulation factors as pro-inflammatory or pro-repair signaling molecules. This proposal builds on the expertise of the Poole lab, resources and assays we utilize to study fibrinogen, and the expertise of our collaborators to address fundamental unanswered questions about the pro-inflammatory properties of fibrinogen in the broad context of injury and inflammation. We pursue key scientific questions such as: 1) How are plasma fibrinogen levels regulated in the acute phase response? Our studies will identify the cellular source of fibrinogen in settings of acute inflammation, chronic metabolic disease, and pregnancy. Furthermore, we will identify the molecular signaling events driving acute phase induction of fibrinogen synthesis. Finally, the mechanisms regulating removal of fibrinogen from circulation (i.e., catabolism) under both homeostatic and pathophysiologic conditions are unknown. We will identify the relevant cell type(s) and receptor(s) that mediate fibrinogen clearance. 2) How is fibrin(ogen) deposited in an injured tissue to enable leukocyte interactions? Extravascular fibrin(ogen) deposits are characteristic of multiple disease states. Our studies will define the mechanism of extravascular fibrin(ogen) accumulation, including the contribution of local synthesis. Furthermore, the fibrinogen molecule is susceptible to multiple post-translational modifications in the setting of disease that can affect its structure and function as a clotting protein. However, the impact of these post-translational modifications on fibrinogen's inflammatory activities are unknown. We will determine the impact of relevant post-translational modifications on leukocyte adhesion and activation. Our group will use novel in vivo and in vitro approaches to address these fundamental questions regarding the inflammatory functions of fibrinogen, thereby enabling the discovery of safe and selective therapies to target fibrinogen in disease.

NIH Research Projects · FY 2025 · 2025-09

How the process of myelination is regulated during development of the central nervous system (CNS) has been the focus of numerous studies, but less is known about what regulates myelin in the adult CNS. It is now appreciated that myelination is a highly dynamic process throughout life. Myelin plasticity and adaptive myelination are relatively recent concepts describing myelin remodeling in the adult brain in response to stimuli such as learning paradigms or electrical activity. However, there is little knowledge of myelin alterations in the adult spinal cord or of the mechanisms for how myelin is maintained over time in adult brain and spinal cord. Such information is important for determining how changes in levels of myelin contribute to normal adult function and to susceptibility or recovery from demyelinating events. We have recently discovered a novel process of myelin acquisition in the adult mouse spinal cord whereby existing myelin becomes thicker in the normal adult white matter tracks between 2 months of age to 1.5 years of life. In contrast, myelin acquisition does not occur in the corpus callosum of the brain. We also show that loss of the tuberous sclerosis complex 1 (Tsc1) gene in adult oligodendrocytes prevents myelin acquisition in the spinal cord white matter without altering oligodendrocyte differentiation or survival and is necessary for myelin maintenance in the corpus callosum. These findings form the basis of our hypotheses that 1) myelin acquisition in the adult spinal cord is regulated by neuronal activity and occurs in both ascending and descending WM tracts, and 2) adult myelin acquisition in the spinal cord and maintenance in the corpus callosum are regulated by TSC signaling. These findings raise important questions that will be addressed in the aims of the proposal. Aim 1: Determine activity dependence of myelin acquisition in spinal cord WM tracts. This aim will address how myelin acquisition in spinal cord is modulated by neuronal activity and differs according to ascending/ sensory or descending/ motor spinal tracts. Aim 2: Determine the mechanistic basis for how TSC signaling regulates myelin acquisition and maintenance in the CNS. The goal of this aim is to define activation and function of known downstream targets of TSC to determine how TSC signaling contributes to myelin acquisition in the spinal cord and to myelin maintenance in the corpus callosum.

NIH Research Projects · FY 2025 · 2025-09

Extending the limited kidney transplant (KT) life is a priority in an era of severe organ shortage. Among the major causes for premature KT loss are opportunistic viral infections. BK polyoma virus (BKPyV) DNAemia occurs in 15-20% in the first year after KT and has no proven preventive or therapeutic strategy or agent beyond reduction in immunosuppression (IS), which carries the risk of acute rejection (AR). The BEAT-BK adaptive pragmatic trial in adult KT recipients is a randomized, controlled, and multicenter trial will evaluate the herapeutic efficacy of intravenous immunoglobulin (IVIG) for sustained BKPyV DNAemia. IVIG contains natural antibodies to BKPyV that have shown benefit in small retrospective case series (including our own) of KT recipients with BKPyV DNAemia. The adult BEAT-BK trial will enroll 280 eligible KT recipients (mostly adults, a few children) to randomize 1:1 to either structured IS reduction/modification alone (standard of care), or IS reduction/modification + IVIG (study arm). The trial will use a ranked global scores primary outcome (death, allograft loss, reduction in estimated glomerular filtration rate (eGFR) > 10 ml/min/1.73m2 from baseline, AR, lack of viral clearance, reduction in IS load). Children get excluded from most transplant trials and are at high risk of a primary BKPyV infection at KT, for multiple reasons. Due to lack of prior exposure and immunity in many of these children, this primary BKPyV infection can be severe during a period of maximal IS. We therefore propose a planning grant that will develop the infrastructure and documents to conduct the BEAT-BK USA kids trial, to mimic the ongoing adult trial. In a prospective cohort of 120 multi-ethnic pediatric KT recipients across 7 major USA sites, we will perform the following Specific Aims, based on the hypotheses that: A) addition of IVIG (total dose 2 gm/kg over 8 weeks) to standardized IS reduction/modification, will increase the viral clearance rate; and B) will reduce the BKPyV DNAemia relapse rate; and C) will reduce the proportion with a drop in eGFR > 10 ml/min/1.73m2. Eligible subjects will be KT recipients 2 years and older with sustained BKPyV > 1000 viral DNA copies/ml on 2 separate occasions within 3 weeks prior to randomization. All primary endpoints will be evaluated at 12 weeks post-randomization, with further follow up for another 36 weeks, to also measure a series of secondary outcomes. To enhance efficiency, our trial design will replicate the adult trial adaptation of sample size based on accumulating data and pre-specified decision criteria. We will also adopt a pragmatic design where the IS alteration strategy can be tailored specifically to the characteristics of the participants, based on pre-specified guidelines. Our pediatric trial will also uniquely assess the capacity of exploratory novel mechanistic biomarkers to predict the various study outcomes. By study end, we will know if IVIG has true efficacy to clear sustained BKPyV DNAemia without relapse and with maintained allograft function.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY/ABSTRACT Among patients with asthma, a disease that afflicts over 300 million people globally, for over 50% their asthma is not well-controlled by current therapies. β-agonists are a cornerstone medication used to prevent and relieve acute bronchospasm in asthma patients. Unfortunately, excessive use of β-agonists can cause desensitization, internalization, and downregulation of the β2-adrenoceptor (β2AR), leading to tachyphylaxis (loss of clinical efficacy). β-agonists act by binding to the cognate G protein-coupled receptor on human airway smooth muscle (HASM), the β2AR. Activation of the β2AR prompts Gαs to stimulate adenylyl cyclase production of cAMP. Increased intracellular cAMP levels activate protein kinase A (PKA), which phosphorylates mediators of excitation-contraction coupling, leading to HASM cell relaxation and bronchodilation. The regulation of cAMP is canonically considered “switch-like”: the “on-switch” (adenylyl cyclase) is counteracted by phosphodiesterases (PDEs) that degrade cAMP to its inactive form, AMP. Our laboratory recently reported that β-agonist-evoked intracellular cAMP is also negatively regulated by transport of cAMP into the extracellular space via ATP- binding cassette subfamily member C1 (ABCC1). This effect was observed in receptor-dependent (i.e. β- agonist) and receptor-independent (i.e. adenylyl cyclase agonist) manners, suggesting this is a universal response to elevated intracellular cAMP levels. However, the mechanism by which ABCC1 is regulated and the contribution of ABCC1 to β2AR desensitization remain unknown. Our preliminary data indicate that Casein Kinase 2 (CK2), a highly conserved serine/threonine protein kinase, directly phosphorylates and regulates ABCC1 in response to β2AR activation. In addition, the timing of ABCC1 phosphorylation coincides with recruitment of MAPK, an important step in β2AR desensitization and a demonstrated regulator of CK2. Therefore, the overarching hypothesis is that MAPK and CK2 are critical in β2AR-evoked ABCC1 activation, leading to physiological outcomes of increased cAMP efflux and β2AR desensitization. Aim 1 will determine the contribution of CK2 and MAPK in homeostatic and β-agonist-induced ABCC1 activity in cultured HASM cells. In Aim 2, we will assess whether the MAPK-CK2-ABCC1 axis regulates β2AR desensitization and internalization. This research investigates a novel regulatory mechanism of cAMP signaling with the goal of identifying druggable targets that can split β2AR signaling mechanisms and inhibit the loss of clinical efficacy of β-agonists. Further, the proposed training will strengthen the Principal Investigator as an independent researcher in obstructive lung disease and cell signaling.

NIH Research Projects · FY 2025 · 2025-08

Project Summary. In line with NIH’S INCLUDE research plan (NOT-OD-24-044), we propose to develop a multicomponent intervention that combines mobile and wearable technologies to facilitate healthy behaviors (physical activity, diet, sleep) and improve quality of life and nutritional status among children and adolescents with Down Syndrome (DS). Individuals with DS are at higher risk for lifestyle related adverse health outcomes including obesity and sleep apnea, which can lead to reduced well-being in both the child with DS and their caregivers. Mobile technologies (e.g., applications, sensors) are increasingly being used to promote healthy routines at home due to their high ecological validity and scalability. Problematically, there are few wellness apps designed specifically for the DS population. This high-risk/high-reward research application aims to (1) develop ThriveDS, a game-based wellness app for children and adolescents with DS (and their caregivers), and (2) conduct beta- testing of the ThriveDS mobile app to assess its feasibility, acceptability, and usability. Throughout the project, we will involve a group of stakeholders including individuals with DS to guide our research procedures and app development. Content modules, game elements and features for the ThriveDS app will be initially selected based on evidence review and theory-based behavioral change techniques, and finalized following a series of qualitative user research. Focus groups will be conducted with parents of children and adults with DS to understand the users’ needs/requirements from a wellness app and gather feedback on the features, format, and educational content of the Thrive-DS app. Then we will interview experts in relevant fields (e.g., nutrition, physical therapy, and occupational therapy), who will provide insights into key evidence-based mechanisms for DS-specific nutrition, physical activity, and sleep interventions, respectively. We will then employ directed content analysis to synthesize these findings, identify a priority list of elements for the application, and then develop the alpha version (first iteration) of the ThriveDS app. Next, the app will undergo usability testing (n=10) followed by the full development of the app (beta version). We expect that the ThriveDS mobile intervention will use a combination of educational modules/videos, game elements, and personalized, real-time brief intervention prompts based on data from wearable activity trackers (e.g., activity level, sleep patterns). In Aim 2, the app will undergo 3-month beta testing in a cohort of individuals DS (n=27). We will then assess its feasibility (recruitment, retention, usage statistics), acceptability (user satisfaction), and usability (ease of use) via app analytics and questionnaires. We will then interview caregivers (n=15) who participated in the beta testing, which will be analyzed to identify core facilitators and barriers to app usage. We anticipate that our next step will be an inclusive clinical trial in the DS population, addressing component 3 of the INCLUDE Research Plan. We plan to rapidly disseminate our findings and data, thus accelerating development of interventions for co-occurring disorders in DS. We will also disseminate our findings to the local and national DS communities via manuscripts and presentations.

NIH Research Projects · FY 2025 · 2025-08

Summary/Abstract Loss of the Y chromosome (LOY) in hematopoietic cells is a prevalent age-related phenomenon with significant health implications, including increased risks of cancer, neurodegeneration, cardiovascular diseases, and overall mortality. While recent genomic studies have linked LOY to disease risk, the mechanistic connections, particularly in the context of aging, remain unclear. Emerging evidence from cancer research suggests that LOY may contribute to genomic instability, potentially due to defects in DNA repair mechanisms and cell cycle regulation in Y-chromosome-lacking tumor cells. This area of research in aging is relatively unexplored and warrants further investigation. Leveraging the unique aging biobank at the Albert Einstein College of Medicine, where we have determined LOY status, this study aims to rigorously examine the causal relationship between LOY and genomic instability using cutting-edge genomic and molecular techniques. We hypothesize that LOY is associated with increased genome instability, possibly through elevated somatic mutation rates, chromosomal aberrations, or shared genetic determinants. Our study comprises two aims: Aim 1 will assess whether aging men with high LOY exhibit a higher burden of other types of mutations. Using the Einstein Longevity Cohort, we will apply advanced methodologies, including single-molecule and single-cell studies, to evaluate genomic instability in 40 individuals with varying LOY status. In aim 2 we will induce LOY and study its effect on genome stability using induced pluripotent stem cells (hiPSCs) differentiated into T cells. By elucidating the mechanistic basis of the correlation between LOY and genomic instability, this project may offer insights relevant to similar phenomena in women, such as the genetic determinants of aneuploidy or LOX, which have also been reported in aging women. This understanding could facilitate the exploration of molecular pathways, identification of biomarkers, and development of targeted interventions. Ultimately, this novel research may lead to personalized medical approaches and improved health outcomes, particularly in the context of healthy aging.

NIH Research Projects · FY 2025 · 2025-08

PROJECT ABSTRACT “Light” cigarettes were marketed by the tobacco industry – and perceived by consumers – as safer cigarettes for decades before researchers determined that flaws in their filter design increased harm to their users, illustrating the necessity of understanding how cigarette design and perceptions influence smoking behavior and toxicant exposure. Cigarettes using charcoal filters (versus traditional cellulose acetate filters) have been shown to reduce exposure to certain toxicants, but these findings are based on machine-generated estimates of smoking behavior that previously failed to capture the compensatory smoking that occurred with actual human use of “light” cigarettes. Thus, it is unknown if findings demonstrating potential benefits of using charcoal-filtered cigarettes translate to real-world use of these products, given characteristics of the filter (e.g., design, type/amount of charcoal), potential for compensatory smoking, and/or limitations on charcoal’s absorption abilities. Nevertheless, Santa Fe Natural Tobacco Company recently introduced a new charcoal- filtered cigarette under its Natural American Spirit (NAS) brand called 'Sky' and has been promoting it implicitly as a reduced harm product. Given NAS’s continued market growth while other brands are in decline, and a general industry movement towards promoting various lower risk tobacco products, this action could foreshadow greater industry interest in this type of product. Since other brands often mimic NAS’s marketing tactics to promote sales, understanding consumer experiences with Sky could help predict its longer-term commercial success and the likelihood of other companies introducing similar products. Given that use of charcoal-filtered cigarettes is widespread in other countries, the need for ecologically valid research on their use and effects on toxicant exposure, and NAS’s popularity with U.S. consumers, we seek to empirically examine this novel product now, before it gains traction in the U.S. market. We will recruit 210 adults who smoke cigarettes daily to complete a 5-week laboratory-based, open-label, parallel-design randomizing participants, after a 1-week baseline period of smoking their preferred brand, to use one of three types of cigarettes for a 4-week experimental period. Cigarette types are matched on key constituents and characteristics, and will include two charcoal-filtered cigarette varieties (NAS Sky and Tareyton) and a control non-charcoal cigarette (NAS Yellow). Participants will complete weekly sessions to assess primary outcomes of: smoking behaviors (daily cigarette consumption, puffing behavior), product perceptions (subjective ratings, perceptions of harm), and toxicant exposure (carbon monoxide levels, urinary biomarkers of nicotine, volatile organic compounds, and tobacco-specific nitrosamine exposure, and charcoal granule release). Data acquired from this project may be used to inform cancer prevention and control and/or tobacco regulatory efforts.

NIH Research Projects · FY 2025 · 2025-08

A hallmark of the host response to Mycobacterium tuberculosis (Mtb) is the development of the inflammatory granuloma, which plays an important role in controlling Mtb while also capable of inducing tissue-damaging lung pathology in chronic tuberculosis (TB), causing necrosis and cavitation. Cavitary lesions, when in communication with the respiratory tree, attain ambient O2 levels to promote bacillary growth to high concentrations, thereby facilitating Mtb transmission. Thus, this process poses a significant obstacle to TB control. Further, the exacerbated pathology can result in permanently compromised lung functions, even after curative therapy. The mechanisms that regulate the development of tissue-damaging immunopathology in chronic TB are incompletely defined. In chronic TB, B cells form in the lungs prominent aggregates that are an integral part of ectopic germinal centers, cellular structures associated with chronic inflammation that can regulate local immune responses. Using B cell-deficient mouse models, we have shown that B cells drive lung inflammation in chronic TB. Compared to B cell-deficient mice, B cell-adequate animals display increased lung infiltration that is associated with enhanced CD4+ T cell expansion and an augmented level of pro-inflammatory interferon (IFN)-g-producing CD4+ T cells; concomitant with diminished lung expression of the anti-inflammatory IL-10. The B cell-deficient mouse CD4+ T cell phenotypes can be reversed by IL-10 signaling blockade. The CD4+ T cell data suggest that in chronic TB, B cells may drive CD4+ T cell expansion and the pro-inflammatory Th1 response, possibly by presenting antigens (Ags) to CD4+ T cells, that latter an understudied B cell function. The IL-10 results suggest that B cells can restrict lung IL-10 expression in chronic TB, and that IL-10 can regulate lung inflammation, possibly by modulating the functions of CD4+ T cells and Ag presenting cells. Our results also suggest a role for cytokines in modulating lung inflammation in chronic TB. Of note, we have shown that necrotic and cavitary lesions in the lungs of humans with chronic TB are decorated with conspicuous B cell aggregates at their periphery, suggesting that B cells may regulate tissue-damaging pathology. This proposal seeks to i) elucidate the mechanisms by which B cells regulate lung inflammation in chronic TB in mice, particularly those operative in the B cell/IL-10/Th1 axis; and ii) characterize the lesion-associated B cell aggregates in the lungs of humans with chronic TB. We will accomplish this by leveraging a variety of knock-out and transgenic mouse strains and the expansive repository of human TB tissues available at the University of Alabama at Birmingham, together with state-of-the-art technologies including multiplex immunofluorescence analysis, gene expression profiling via RNAscope, single-cell RNA-sequencing, and spatial transcriptomics. We believe the results generated by the proposed studies can illuminate how B cells regulate lung inflammation in chronic TB and point toward interventional strategies capable of mitigating TB transmission and the development of undesirable pulmonary function sequalae of chronic TB.

NIH Research Projects · FY 2026 · 2025-08

Project Summary/Abstract Microbes acquire an enormous number of new mutations every day in nature, which enables them to rapidly adapt to environmental changes. At the same time, these microbes are often dependent on interactions between species and strains, especially the exchange of nutrients (cross-feeding). Our ability to predict evolution of mi- crobes in different ecological environments is valuable for treating infectious diseases — for example, anticipating if and how fast a pathogen will evolve resistance to a treatment — as well as for designing personalized medicine based on an individual’s evolving microbiome. The overall goal of our lab’s research is therefore to understand the feedback between these evolutionary and ecological processes in microbial communities. A particular chal- lenge in this field is our ability to quantitatively predict the effects of mutations on fitness, and how those effects vary across ecological environments. The distribution of fitness effects — the set of fitness values for all spon- taneous mutations available to a population — is a key input to predicting how a population will evolve, and its variation across environments can reveal unknown gene functions. However, since we cannot empirically mea- sure the fitness of mutations across all possible environments, it is crucial that we develop general principles for how mutant fitness changes across these environments to predict evolution. We need to know these rules, for example, to predict whether cross-feeding certain nutrients is likely to make a species adapt more slowly or more quickly, and how it changes the genes and molecular pathways under selection. In the next five years, our lab seeks to develop principles by which ecological interactions affect mutant fitness by addressing three questions: 1) Do different kinds of interactions have different effects on mutant fitness? 2) Do different kinds of mutants respond differently to interactions? 3) What mechanisms cause interactions to alter mutant fitness? We will focus on Escherichia coli auxotrophs that allow us to engineer defined cross-feeding interactions, combined with DNA barcoding for generating and tracking high-throughput mutant libraries. We will experimentally measure the effect of cross-feeding different types of nutrients (amino acids, vitamins, carbon intermediates) on mutant fitness, as well as the effect of cross-feeding on different types of mutants (gene knockouts from transposon insertions vs. spontaneous mutations from an evolution experiment). In parallel we will use whole-genome metabolic models to make predictions for these experiments and investigate the underlying mechanisms. In particular, we seek to test how metabolic cheating and changes in nutrient limitation mediate the effect of cross-feeding on mutant fitness. Our long-term goal is to apply the principles we learn from this laboratory system to complex, naturalistic com- munities that involve a range of ecological interactions. Our work on this problem will contribute toward predicting microbial evolution across ecological environments as well as helping discover new functions of genes, especially secondary activities that arise under nutrient conditions not commonly tested in the lab.

NIH Research Projects · FY 2025 · 2025-08

Project Summary/Abstract: My research program investigates how the host metabolism impacts immune responses during infection. While immune evasion strategies by bacterial virulence factors are well recognized, especially during the initiation of infection, the mechanisms by which immune responses are modulated to promote disease tolerance and pathogen persistence are less well understood. Numerous studies have shown that metabolic activities govern the function of immune cells, an emerging field called immunometabolism. Notably, upon stimulation with bacterial components, immune cells typically upregulate glycolysis to fuel inflammation and promote bacterial clearance. However, we and others have recently shown that live bacterial pathogens can manipulate the canonical glycolysis-induced inflammatory signaling to promote persistent infections. Within the next five years, we aim to deepen our mechanistic understanding of how host metabolic activities influence immune cell activation and function. Importantly, we will dissect the metabolic pathways that favor immune suppression versus activation using dietary and pharmacological interventions in our infection models. Such approaches could provide a new framework for treating a range of pathogens at diverse sites of infection, potentially transforming infection control strategies. In addition, the knowledge generated may have broader applications in inflammatory pathologies or metabolic disorders. Our research will be conducted through a multidisciplinary approach, incorporating advanced omics technologies such as next-generation transcriptomics, metabolomics, spatial metabolite imaging and indirect calorimetry, to achieve our objectives.

NIH Research Projects · FY 2025 · 2025-08

Abstract Melanoma is a top cause of invasive cancer in teens and young adults. Teens have low skin protection rates and also increase exposure to natural and artificial ultraviolet radiation as they progress into adulthood. Sunburns are common, and those experienced as a minor and/or repeatedly are strong risk factors for melanoma. Thus, interventions targeting skin cancer risk reduction amongst teens are needed. Yet, teens tend to be resistant to public health recommendations because, as a group, they perceive that they have more immediate priorities than disease prevention and that the consequences of their current health behaviors are in the distant future. Developmentally, they also tend to be experimenters, risk-takers, and highly influenced by peers. Despite the risks of this vulnerable population, prior sun safety interventions for teens and/or their parents have been few, challenging to disseminate, or minimally successful long-term. With smartphones and internet access virtually ubiquitous among US teens, these modalities offer promising directions for cost-effective dissemination of health promotion interventions that reach teens in their usual environments where relevant behaviors occur. Texting, in particular, has been used successfully for young adult sun safety and widely used with teens in other health domains, but only one trial evaluated a sun safety teen texting intervention (in combination with a 5-hour training by a dermatologist). Although texting was acceptable and feasible, there is still a gap in developing and testing a scalable digital intervention. Further, although teens are transitioning to responsibility for their own sun protection, prior studies have failed to address parents’ ongoing influence (e.g., through communication, modeling, facilitating sun protection). For adults, digital interventions through popular social media (e.g., Facebook) have improved health behaviors such as physical activity. Our work shows promising results delivering a sun protection group Facebook intervention to a national sample of parents of teens. Therefore, we will evaluate entirely digital interventions targeting teens (Multimedia Message Service [MMS] individual smartphone intervention) and their parents (Facebook group intervention) in an innovative dyadic approach (Sun Safe Together [SST]). A diverse national sample of parent and teen (13-17 years) dyads will be recruited cost and time-efficiently using machine learning and digital mining of online data. This R34 Planning Grant’s Specific Aims are to collaborate with teens and parents to: 1) supplement and refine a library of sun safety digital messages and 2) pilot test SST to determine recruitment feasibility; acceptability of and engagement with the interactive teen MMS intervention; data collection feasibility; and retention rates. This R34 will prepare us to conduct an R01 to test the efficacy of the intervention components in a national randomized controlled trial of parent-teen dyads in a 2x2 design (parent only, teen only, parent-teen combined sun intervention, or none). No prior study has disentangled the impact of digital interventions on the teen, parent, or both on sun safety. These highly scalable interventions have significant potential for widespread reach and impact.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT: Laminin-211 (Lm211) is an ~800 kDa glycoprotein of neuromuscular basement membranes (BMs) required for BM assembly, structure, and functions. Missense and truncating mutations of the LAMA2 gene cause a congenital muscular dystrophy and neuropathy (LAMA2-Related Dystrophy, MDC1A). The clinical spectrum ranges from mild (ambulatory) to severe (non-ambulatory), depending on the mutation, with corresponding mouse models covering the disease spectrum. The goal of this application is to develop BM structure-altering therapies in two mouse models. Our strategy employs non-replicating adeno-associated virus (AAV) delivery of genes coding for small laminin-binding linker proteins that restore missing Lm211 functions of defective or compensating laminin. The approach is built upon our experience studying BM assembly and structure-function relationships with engineering of novel interactive proteins. Aim I. The dy2J/dy2J mouse, a model for ambulatory dystrophy, bears a mutation within the 2LN domain that prevents polymerization. We found the disease phenotype and pathology are substantially ameliorated by AAV9 delivery of a gene coding for a laminin-binding protein (LNNdG2'). A. To understand dose-benefit relationships, we will compare mice treated at different doses with evaluation of linker and laminin DNA/protein levels, ambulation, strength, histology and seek to better understand molecular mechanisms responding to enablement of laminin polymerization, e.g. effects on apoptosis, myofiber proliferation, and inflammation. B. We will determine the degree of amelioration achievable at later AAV delivery ages. C. We plan to modify the WPRE enhancing sequence and poly A tail to increase expression. Aim II. Most LAMA2-RD patients have little or no Lm2 expression. They remain non-ambulatory with profound muscle weakness, joint-contractures, and life-threatening respiratory impairment accompanied by neuropathy and seizures. The Lama2-/- (dy3K/dy3K) mouse serves as a model. Here the Lm4 subunit is expressed in compensation, mostly as Lm411, and overall trimeric laminin expression is reduced. Preliminary data reveal that muscle and peripheral neuropathy are ameliorated by simultaneous AAV9 expression of two small genes, one coding for LNNdG2' to enable polymerization and the other coding for miniagrin (mag) to link the Lm411 coiled-coil domain to the alpha-dystroglycan receptor. We plan to: A Extend the preliminary analyses to include determination of survival, ambulation, grip-strength, linker/endogenous DNA and protein levels, histology, BM and receptor expression, apoptosis, and regeneration, distinguishing polymerization and receptor anchorage contributions; B Design and evaluate single laminin-binding proteins carrying both needed activities, first in vitro and then in dy3K mice, and C Evaluate mag and dual-function single linker constructs in a myotropic AAV to reduce liver expression/toxicity and dose. Amyelination will be prevented by low-dose AAV9-CBh-LNNdG2'.

NIH Research Projects · FY 2025 · 2025-08

During the K01 period, I will fill critical gaps in my training through meeting five training goals: 1) enhance knowledge in the dyadic functioning of couples with cancer with a focus on young adults (YAs) with cancer and their partners (primary mentor: Sharon Manne, PhD, co-mentor: Katie Devine, PhD); 2) training in the administration and analysis of qualitative dyadic data (co-mentor: Cui Yang, PhD); 3) develop specialized skills in the assessment, analysis and interpretation of social network analysis and ecological momentary assessment data (co-mentor: Cui Yang, PhD); 4) expertise in inflammatory stress-related biomarkers (co-mentor: Anita Kinney, PhD); and 5) conceptual and practical knowledge in analyzing dyadic (among couples) and longitudinal data (co-mentor: Elizabeth Handorf, PhD). These training goals and expert mentorship are designed to support my long-term career goal of becoming an independent investigator identifying and addressing the unique needs and experiences of young adult couples with cancer (YACs), ultimately leading to the development of evidence- based biobehavioral interventions to address YACs unmet needs and enhance their long-term quality of life. Cancer among YACs is a profoundly distressing experience, extracting a significant toll on YACs relationships (e.g., with partners, family, friends). It is well recognized that effective communication within social relationships is critical for fostering health and well-being of couples coping with cancer. Yet, only a few studies are focused on YACs and their dyadic (couple) communication about cancer-related concerns. Even less is known about YACs social networks and interactions within those networks that are most helpful in facilitating well-being. This stalls the development of effective interventions aimed at improving the quality of survivorship for YA survivor and partner. The overall goal of this K01 proposal is to fill a critical gap in the literature by focusing on YACs, exploring their unique needs and experiences, and assessing their dyadic and social network influences on relational, well-being, and stress-related outcomes. This will be achieved through three Specific Aims. First, to adequately capture the YAC experience I will conduct qualitative interviews among 15 YACs to explore how communication within their social relationships facilitates relationship satisfaction and well-being and protects against perceived stress. Second, using social network analysis (SNA) and ecological momentary assessment (EMA) surveys I will characterize YACs social networks and daily social interactions and communication behaviors among 75 YACs. Finally, using SNA and EMA findings I will examine the effects of YACs social network structure and composition, and daily dyadic and social network communication behaviors (e.g., disclosure, responsiveness) on relationship satisfaction, well-being, and physiological responding (salivary biomarkers). My prior training grounded in psycho-oncology, coupled with the skills and knowledge I will acquire during this award period, will equip me to successfully conduct the proposed research and position me to continue my work focused on fostering the long-term health and well-being of this vulnerable and understudied YAC population.

NIH Research Projects · FY 2025 · 2025-08

ABSTRACT Systemic lupus erythematosus (SLE, lupus) is a devastating autoimmune disease driven by pathogenic autoantibodies that promote tissue injury. The autoantibodies are produced by dysregulated germinal center (GC) B cells and Age-associated B cells (ABCs) receiving inappropriate differentiation, survival, and proliferation signals from CD4+ T follicular helper (Tfh) cells. The molecular and cellular events that lead to lymphocyte dysregulation during lupus are complex and still not well understood. Emerging data suggest that Natural Killer (NK) cells, best known for their ability to kill infected and malignant cells, play important roles in restricting excessive Tfh cell and B cell responses during infection. In viral infections, depletion of NK cells has been shown to increase Tfh cells, GC B cells, and anti-viral antibodies. Whether NK cells regulate humoral responses during lupus remains unclear. However, circulating NK cells are dysfunctional and significantly reduced in SLE patients with active disease, and this reduction coincides with increases in circulating Tfh cells and B cells and elevated autoantibody titers. We hypothesize that NK cells are important negative regulators of pathogenic humoral responses during lupus, with specific roles in restricting disease-driving Tfh cell and B cell populations. Accordingly, we will dissect the effects of NK cell, Tfh cell, and B cell interactions on autoantibody production and disease progression during lupus. In Aim 1, we will investigate the role of NK cells in restricting Tfh cell, GC B cell and ABC responses at different stages of lupus disease in mice. Aim 2 will assess the direct cellular and molecular interactions that underlie NK cell-mediated regulating of humoral immune responses during lupus. Finally, Aim 3 will test novel therapeutic agents designed to promote NK cell-mediated killing of Tfh cells and B cells for potential therapeutic use in lupus. These studies are expected to critically advance our understanding of how NK cell dysfunction impacts pathogenic Tfh cell and B cell responses in autoimmunity. Ultimately, this information could provide new therapeutic targets to dampen adaptive immune responses in autoimmunity.

NIH Research Projects · FY 2025 · 2025-08

Syphilis is a sexually transmitted, multistage systemic infectious disease with ~6-12M new infections reported globally each year. Treponema pallidum subspecies pallidum (Tp) mother-to-child transmission results in adverse pregnancy outcomes including miscarriage, stillbirth, and congenital syphilis (CS) in newborns. If infected infants do not receive adequate treatment, infection can result in developmental defects, skeletal abnormalities and even development of blindness, deafness etc. CDC has described a substantial increase in CS cases over the years with 3,755 reported in 2022, which showed >11-fold increase just within one decade. The major goal of this project is to establish a mouse model of CS, determine changes in host immune responses, and lay foundation for diagnostics and vaccine development in the future. C4-deficient (C4-D) guinea pigs were previously used to study CS by Dr. Wicher; however, the model has not been adopted by any other laboratory in the last three decades. Under our ‘International Collaboration on an Infectious Diseases Research (ICIDR) project, U01-AI115497 with Dr. Giacani at University of Washington and the late Dr. Arturo Centurion-Lara at Cayetano Heredia University in Peru, we described the function of two outer membrane associated lipoproteins and identified Tp0954 as the first placental cell binding adhesin. We also tested vaccine candidates against syphilis in the rabbit infection model in that project. Rabbits have been used to propagate Tp, and determine vaccines efficacy against syphilis; however, not for assessing CS. Furthermore, rabbits cannot be examined by the existing In Vivo Imaging System (IVIS) to determine colonization at different stages of infection by live imaging. Finally, not many immunological reagents are available to study innate and adaptive immune responses against Tp in the rabbit model. Our collaborator, Dr. Giacani has generated a GFP-expressing infectious SS14 strain of Tp that he generously provided to us to generate preliminary data for this proposal. Using this Tp strain and Balb/c mice, we could detect Tp colonization of different organs in both parents and pups by live imaging using IVIS-200. The results were confirmed by recovery of live, motile spirochetes from organs into medium. We hypothesize that mouse model could be used to study sexual and vertical transmission of Tp, examine by live imaging at different stages and assess inflammatory responses, and induction of protective humoral immunity that could prevent vertical transmission to the later generations of pups. The following aims will test our hypothesis. (1). Determine whether infection of Balb/c mice with Tp triggers immune responses to facilitate vertical and potentially horizontal transmission of this spirochete. (2). Determine whether specific humoral immunity generated during Tp infection prevents/reduces mother-to-pups transmission in the later generations. The major significance of this study is to establish a mouse model for CS, determine host immune responses, conduct longitudinal studies to identify vaccine candidates against syphilis/CS to be tested in the future.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Epilepsy, occurring in 1 percent of the world’s population, is associated with disability, injury, cognitive and neurological dysfunction, depression, loss of productivity, socioeconomic decline and even death. Of this population, 30 percent of epilepsy cases are medically intractable, leaving surgical interventions as the only option for treatment. Whereas open resection, the current surgical standard of treatment, can yield seizure freedom rates as high as 60-80 percent, these are often associated with cognitive dysfunction and focal neurological deficits. Particularly, patients with dominant hemisphere mesial temporal lobe epilepsy (MTLE), the target population for this proposal, are at risk for significant decline in memory and associated disability. It was estimated that there are nearly 200,000 patients with drug-resistant MTLE waiting for treatment. The only option for these patients at present is electrical neuromodulation, which, although effective at reducing seizures, only achieves seizure freedom in ~10% of patients. Patients and their caregivers, however, generally need a 100% decrease in seizures to safely drive a vehicle, hold down a job, enroll in education programs, or live independently. This represents a sizeable treatment gap. To answer this unmet need, we identified gaps in the technology implemented in the current responsive neurostimulation (RNS) device that we aim to address with this proposal. One, the intended design of RNS is to terminate seizures in response to seizure onset, similar to a cardiac pacemaker, which senses and responds to maintain regular cardiac rhythm. In practice, however, RNS does not function this way. Patients with MTLE, on average, have approximately 8 seizures a month. RNS, however, deploys more than 1000 stimulations a day. Two, the FDA-approved use of RNS is to implant the recording and stimulation electrodes in one or two areas of the seizure onset zone (SOZ). This seizure control strategy approaches focal epilepsy as a focus-based disorder, which is outdated. In this proposed study, we plan to design and implement a proof-of-concept epileptic-network closed-loop stimulation (enCLS) device to address the two technology gaps of RNS. This device will adopt a new set of pattern detectors to improve the specificity and timing of ictogenesis detection, coupled with a neurostimulation protocol to stop ictogenesis in the larger epileptic network beyond SOZ. The enCLS device otherwise assumes the same overall physical design, functional usage, intended users, use environment, operating principles, and the safety limits of the current state- of-the-art RNS system. We shall construct a proof-of-concept laboratory enCLS prototype and conduct experiments to inform the design requirements associated with a minimum viable product (MVP) at the end of the funding period. The efficacy of enCLS in seizure prevention, hence meeting the need for seizure freedom, will be compared with RNS. We will leverage the multi-institutional research efforts in partnership with the medical device industry to bring this technology to human use within five years.

NIH Research Projects · FY 2025 · 2025-08

Seizures and alcohol poisoning cause significant mortality due to respiratory arrest. Sudden unexpected death in epilepsy (SUDEP) is the leading cause of epilepsy-related death and causes more years of potential life lost than any other neurological disorder except stroke. Respiratory arrest plays a critical role in SUDEP. Alcohol-related poisonings are the leading cause of acute alcohol-related mortality with an incidence higher than alcohol-related motor vehicle accidents. Respiratory arrest is a central feature of fatal alcohol poisoning. The long-term objective of this proposal is to determine the convergent mechanisms by which seizures and alcohol poisoning affect breathing to enable the rational development of novel treatment strategies. Seizures and alcohol poisoning increase adenosine signaling. Excessive adenosine suppresses breathing. Adenosine inhibits serotonergic and noradrenergic neurons which contribute to respiratory regulation. Our preliminary data indicate that seizures cause adenosine surging in the serotonergic raphe nuclei and that adenosine contributes to the respiratory effects of seizures and alcohol poisoning. These observations form the scientific premise of our overarching hypothesis that adenosinergic inhibition of serotonergic and/or noradrenergic neurons contributes to the effect of seizures and alcohol poisoning on breathing. Our approach utilizes G-protein-coupled receptor-activation-based (GRAB) sensors, a cutting-edge fiber photometry technique, in concert with well-established transgenic manipulations in rodent models. The relationship between respiratory disruption, adenosine signaling, and blood gasses is complicated by the fact that both hypercapnia and hypoxia increase adenosine signaling. In Aim 1, GRAB sensor fiber photometry will be used in artificially ventilated mice to differentiate between (a) adenosine signaling resulting directly from the seizure or alcohol poisoning and (b) adenosine surging that is mediated indirectly by hypercapnia/hypoxia due to respiratory disruption unrelated to adenosine signaling. In Aim 2, GRAB sensor fiber photometry will be used to determine the spatial and temporal characteristics of alterations in adenosinergic, serotonergic and noradrenergic signaling during seizures and alcohol poisoning. In Aim 3, a Cre-LoxP strategy will be used to delete A1 or A2A adenosine receptors in serotonergic and noradrenergic neurons to determine the location and subtype of adenosine receptors that mediate the effect of seizures and alcohol poisoning on breathing. Career Development Summary: This proposal will allow the applicant to reach his career goal of establishing an independent disease-oriented neuroscience research program by initiating a line of inquiry that: (1) topically expands on his prior work, (2) will give him extensive training on a state-of-the-art imaging technique with many other applications, (3) can be taken with the applicant to his next position, and (4) may have a transformative effect on our understanding and treatment of the respiratory sequelae of seizures and alcohol poisoning.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Cryptococcus (Cn) species have a global impact on human health as an environmental, opportunistic fungal pathogen that most commonly presents as pulmonary disease or meningoencephalitis (CM) causing an estimated 180,000 deaths annually in a range of patient populations. Despite knowing that T-cells are required for immune protection, little is known about the molecular mechanisms driving susceptibility to cryptococcosis. Our long-term goal is to identify genetic and immunological defects leading to susceptibility to Cn to inform pathobiology and personalized care. To address this goal, whole exome sequencing was performed on a cohort of previously healthy patients without known immunosuppression to identifying rare alleles associated with cryptococcosis and predicted to have deleterious functional consequences. For the first time, gene enrichment pathway analysis identified MTOR signaling deficits as enriched in this population. The goal of this proposal is to determine whether identified MTOR variants cause dysfunction in MTOR signaling associated with increased susceptibility to cryptococcal disease. Our central hypothesis is that MTOR deficiency predisposes patients to cryptococcosis and suggests functional MTOR signaling is required to clear Cn. This will be accomplished by: (Aim 1) Characterizing the immunologic consequence of MTOR deficiency on susceptibility to Cn using established murine models of cryptococcosis; and (Aim 2) Determining how patient MTOR variants alter cell signaling and function in patient lymphocytes. The results from this proposal will begin to elucidate for the first time mechanistically how human genetic variants in a single gene (MTOR) are associated with specific susceptibility to cryptococcosis in previously healthy individuals with no known immunologic defects. Thus, these results will provide novel insights into human disease susceptibility to Cn and reveal the protective immune responses affected by MTOR deficiency. Ultimately, expanding our definition for who is at risk for cryptococcosis and suggesting improved treatment opportunities for cryptococcosis patients identified to have aberrant MTOR signaling through pharmacologic modulation of cell-specific immunity. The applicants career goal is to become an independent scientist in the field of fungal immunology with a focus on understanding the mechanism by which specific host genetic defects lead to susceptibility of previously healthy patients to cryptococcosis. To meet this goal, the applicant will begin the process of transition to an independent scientist by beginning to apply for tenure-track faculty positions in fall 2024 cycle. Further, the applicant proposes a career development plan that will allow her to gain more experience in host genetic susceptibility/fungal immunology, grant writing, and leadership skills through practical experience, formal course work, and mentoring. Highly accomplished collaborators specialized in similar areas as the proposed research project will mentor the applicants career development and provide expertise on different aspects of the project.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Genetically engineered immune cells are an exciting and promising frontier for treating a wide range of complex diseases. However, hyperinflammatory toxicities such as cytokine release syndrome plague clinical trials, stymieing their widespread clinical adoption. Macrophages are innate immune cells that fulfill many roles in tissue repair, regeneration, and homeostasis and are important regulators of inflammation. However, they are significantly under-utilized as engineered immune cell therapies because significant knowledge gaps exist in understanding how to engineer synthetic gene circuits that work robustly in human macrophages. Tools do not yet exist for determining how macrophages should be biologically manipulated to activate desired effector functions (biological design principles). Tools also do not exist for determining what gene circuit architectures are needed to robustly induce desired gene circuit behaviors (gene circuit design principles). The overall goal for this proposal is to create a human macrophage design toolkit for engineering therapeutic macrophages. Our published and preliminary data demonstrate that we have developed tools that enable us to discover cell signaling interventions that can control macrophage effector functions (biological design principles) and gene circuit architectures that can exert robust behaviors in human macrophages (gene circuit design principles). Here we will apply both these approaches to elucidate biological and gene circuit design principles that can be used to engineer therapeutic macrophages that can suppress inflammatory cytokine secretion or induce anti- inflammatory cytokine secretion in inflamed tissues. We will elucidate biological design principles using an interpretable machine learning approach that we previous developed. This approach combines biochemical screening with predictive network modeling and machine learning to discover network mechanisms causally regulating cell phenotypes. We will elucidate gene circuit design principles using a recently developed ultra-high- throughput genetic screening approach (CLASSIC). This approach synthesizes and screens large, barcoded gene circuit libraries to associate gene circuit architectures with gene circuit behaviors. With these design principles we will engineer gene circuits for controlling IL-1β or IL-10 secretion in inflamed tissue contexts and validate these synthetic gene circuits in human monocyte-derived macrophages and THP-1 cells. In its entirety, this Trailblazer R21 project is a first step towards addressing the unmet need for design principles for engineering therapeutic macrophages. We envision that insights gained by this project will help establish engineered macrophages as a platform technology for treating a wide range of complex human diseases.