Weill Medical Coll Of Cornell Univ

NIH Research Projects · FY 2026 · 2023-09

Summary Abstract Metazoan organismal lifespan is determined by a combination of chronological lifespan (the length of time a cell exists in a non-dividing state before dying) and replicative lifespan (the number of times a cell divides before irreversibly arresting; most accurately studied in budding yeast [Saccharomyces cerevisiae]). Many chemical, dietary and genetic interventions can extend lifespan. These are usually first discovered in budding yeast, and subsequently shown to apply in metazoans. However, there is still little understanding of their underlying molecular mechanisms for lifespan extension. A variety of interventions, including medications, genetic manipulations, and calorie restriction (CR), have been demonstrated to extend the lifespan of several species. However, there is a significant knowledge gap as to the identity of the ultimate molecular changes enacted by these antiaging interventions to extend lifespan. We recently showed that overexpression of Gcn4, the yeast counterpart of the metazoan ATF4 protein that induces multiple stress response pathways, extended the yeast replicative lifespan (RLS) (the number of times a cell divides before irreversibly arresting) in a manner dependent on autophagy. This finding inspired us to ask whether autophagy is required for other antiaging interventions to extend the yeast RLS. Our preliminary findings indicate that interventions that extend lifespan in many organisms, including rapamycin, metformin, ribosome depletion, CR, and increased sirtuin activity, extend the yeast RLS in an autophagy-dependent manner. Furthermore, we find that induction of autophagy is sufficient to extend the yeast RLS. Given that autophagy induction is also sufficient to extend lifespan in metazoans, we will use the yeast model to seek ultimate molecular targets of autophagy that promote antiaging.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY / ABSTRACT Ischemic left ventricular systolic dysfunction (iLVSD) is a leading cause of heart failure (HF) and death, for which risk is greatest in patients with multivessel coronary artery disease (CAD). Coronary revascularization (revasc) provides a potential means to improve iLVSD, but response is highly variable. “Viability” imaging (to differentiate infarcted from salvageable myocardium) had been widely touted as a tool to predict revasc response, but its utility has been challenged by recent clinical trials. One reason for observed lack of utility of imaging to predict LV functional recovery after revasc may stem from imaging approaches used to define viability: No prior trial has accounted for contractile dysfunction, hypoperfusion, or non-ischemic substrate in seemingly viable regions – each of which can be uniquely discerned by cardiac MRI (CMR). Our group has conducted single center studies showing transmural extent of infarction to be a powerful predictor of improved LV function after revasc; we have also shown hypoperfusion to predict adverse remodeling and prognosis. Technical research by members of our group has developed a new late gadolinium enhancement (LGE) CMR approach that uses blood suppression (“dark-blood”) to better discriminate between infarct and blood-pool – potentially further enhancing viability as- sessment. It is unknown if differential impact of revasc strategy on LV contractile recovery varies in relation to infarct or perfusion phenotype, if non-ischemic substrate (extracellular volume fraction, epicardial or mid-wall LGE) modifies revasc response, and if LV contractile recovery parallels improved prognosis. To address these critical knowledge gaps, this observational study will leverage the newly initiated STICH3C trial - a prospective multicenter trial comparing percutaneous (PCI) vs. surgical (CABG) revasc for patients with iLVSD and mul- tivessel/left main CAD. Perfusion CMR will be performed pre- (<1 month) and post-revasc (12 months) in 200 STICH3C patients and analyzed via a centralized core lab. Our central hypothesis is that infarct transmurality (LGE) and hypoperfusion will predict LV contractile response (EF) and prognosis (QOL, HF, mortality) after re- vasc, for which treatment effect of CABG vs. PCI on LV recovery will increase in proportion to viable but hy- poperfused myocardium on CMR. Aim 1 will test infarct transmurality and hypoperfusion for prediction of LV recovery (improved ejection fraction, strain) after revasc; Aim 2 will evaluate if non-ischemic tissue substrate modifies likelihood of LV contractile recovery; Aim 3 will test prognostic impact of infarction, hypoperfusion, and non-ischemic substrate after revasc, with focus on residual myocardial tissue properties as predictors of persis- tent LV dysfunction, impaired quality of life, and clinical events (HF readmission, mortality). Our team provides complementary expertise in key project relevant areas - translational CMR research, cardiac surgery, heart fail- ure, and clinical trials - and a track record of productive collaboration. Study findings are well-poised to transform the imaging paradigm through which viability is assessed and inform mechanism for persistent LV dysfunction, so as to improve therapeutic decision-making and clinical outcomes for millions of patients with iLVSD.

NIH Research Projects · FY 2026 · 2023-09

Project Abstract Normal lung aging is associated with multiple structural and functional changes in the respiratory tract. Alveolar macrophages (AM) are long-lived tissue resident innate immune cells of the airways and during steady state conditions, adopt a pro-healing, anti-inflammatory phenotype to maintain lung integrity. AM are key effectors of recognition, initiation, and resolution of the host defense against microbes and play an essential role in mediating host responses to Streptococcus pneumoniae (S. pne). Cell essential and macrophage aged death and the effective clearance of dying cells are processes t hat maintain tissue homeostasis. When efferocytosis is defective, increased tissue injury development of acute respiratory distress syndrome (ARDS) can occur. Despite defects in alveolar phagocytosis being prevalent i n aging, very little is known on how the process of aging and the lung microenvironment contribute to these changes.Our published findings illustrate that an age- associated increase in mitochondrial and endoplasmic reticulum stress during S. pne contributed to dysregulated, overly heightened pro-inflammatory immune responses in AM and lung. To better understand the metabolic factors that might contribute to this phenotype, we examined changes in lipid metabolism in young and aged lung. We observed a molecular reprogramming in response to dysregulated cholesterol homeostasis. Given these findings, we hypothesize that an age-associated increase in lipid metabolism alters innate immune responsiveness and efferocytosis by AMs, thereby contributing to heightened inflammation and prolonged tissue injury in response to S. pne. To test this hypothesis, we have designed three specific aims that will utilize innovative techniques to spatially landscape landscape the mediated utilize the will the resolve single-cell data that will allow us to develop a biologically interpretable of lung pathology from a structural, immunological, and clinical standpoint. This spatial single-cell will enable the pathophysiological characterization of the lung from its macroscopic presentation to single-cell, providing an important basis for the understanding of lipid metabolism on alveolar macrophage process and will provide insights into age-associated changes in lung pathology. In addition, we will metagenomic sequencing of human plasma to distinguish infection and infectious disease, and to assess severity of pneumococcal disease. We firmly believe that fundamental insights gained from this novel assay be applicable to other infection models and will help clarify many of the long-outstanding questions regarding role of aging on specific tissue responses.

NIH Research Projects · FY 2024 · 2023-09

PROPOSAL SUMMARY Inflammatory bowel disease (IBD) is a highly prevalent intestinal disorder for which there is currently no cure. The current treatment is comprised of anti-TNF therapy which alleviates the symptoms but does not target the cause of the disease. Mutations in the X-linked Inhibitor of apoptosis protein (XIAP) have been identified in IBD patients, suggesting that reduced XIAP activity causes IBD. One of the characteristic manifestations in IBD is excessive death and damage to the intestinal epithelium, which contributes to intestinal inflammation because of the compromised intestinal epithelial barrier against luminal microbiota. We propose to target the root cause of IBD by directly restoring XIAP activity to homeostatic levels in patients with IBD. XIAP is the most potent inhibitor of caspases and apoptosis. Reduced XIAP is associated with increased activation of the inflammasome pathway of innate immune host defense and the upregulation of inflammatory tumor necrosis factor (TNF) and interleukin (IL)-1b cytokines, resulting in hyperinflammation, which is also a characteristic of IBD. We predict that regaining the normal activity of XIAP in IBD would control the excessive cell death of intestinal epithelial cells and restore the healthy function and homeostatic turnover of the intestinal epithelium. The studies we propose here will exploit the pro-apoptotic protein ARTS, which negatively regulates XIAP and promotes its degradation by the Ubiquitin-Proteasome System. We hypothesize that ARTS serves as an important therapeutic target for IBD by boosting the reduced activity of XIAP back to normal. Working with murine and human colonic organoids, we will test the idea that reduced activity of XIAP in cells harboring IBD-associated mutations can be overcome by inhibition of ARTS. Since expression of ARTS is induced in response to stress and DNA-damage, this may also account for reduced XIAP activity in IBD patients without XIAP mutations. Thus, strategies to raise XIAP activity may have broad impact beyond cases in which XIAP mutations are implicated in IBD. Utilizing a complementary approach, we will modulate the activities of ARTS and XIAP using a proprietary panel of small-molecule “ARTS-antagonists” and “XIAP-agonists” which we have identified. We seek to provide proof-of-concept that our “ARTS-antagonists” and “XIAP-agonists” will be able to restore to normal the XIAP function in cells with IBD-associated XIAP mutations as a novel treatment strategy for IBD. We will also test these small molecules in an animal model of IBD. We have two specific aims: (1) Determine the role of ARTS in regulating XIAP-induced apoptosis and inflammation in IBD, and (2) Identify the most potent ARTS-antagonist and XIAP-agonist small molecules that restore XIAP expression and function in IBD models. Our proposal provides a radical new approach for regulating XIAP by its natural antagonist ARTS, whose therapeutic exploitation has not yet been investigated. Our long-term goal is the clinical development of compounds that reverse damage to the intestinal epithelium and promote mucosal healing for an effective and long-lasting treatment of IBD.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY Sepsis-induced immune paralysis (IP) is associated with consequential and often long-term effects including susceptibility to secondary and opportunistic pathogens, and persistent immune disturbances that may culminate in low-grade inflammation and death. Alterations in immune cell metabolism and impaired cell signaling are pathophysiologic features of sepsis-induced IP. There is a strong scientific premise supporting the influential role of fatty acids (FA) and their acyl carnitine (AC) metabolites in cellular energy metabolism, immune signaling, and modulation of cytokine release. These concepts highlight the potential role of metabolites as active contributors to disease pathophysiology rather than solely representing consequences of underlying pathways. To address this paradigm, Lisa Torres, MD, MS, proposes this career development award with the overall objective to characterize metabolic and protein biomarkers of septic IP relative to the most widely accepted surrogate biomarker of IP, CD14+ monocyte HLA-DR expression. With the assistance of a multi-disciplinary mentoring team, Dr. Torres proposes the following Aims in a cohort of critically septic and non-septic patients, stratified by presence or absence of IP: (1) determine the relationship between dysregulated FA metabolism and sepsis-induced IP; (2) characterize the role of immune paralyzed response proteins (IPRPs) as cellular mediators and candidate biomarkers associated with sepsis-induced IP; and (3) explore the effect of IP as a mediating variable between sepsis and patient outcomes. In Aim 1, Dr. Torres will measure fatty acid oxidation (FAO) utilization and perform metabolic tracing with 13C-FAs to assess AC synthesis in PBMCs from recruited patients (N=280). In Aim 2, Dr. Torres will use a THP-1 monocyte-like cell line and patient PBMCs (Aim 1) to determine the impact of IPRP agonists on cellular activation. She will also measure IPRPs in plasma of recruited patients (Aim 1). In Aim 3, Dr. Torres will use causal inference methods to estimate the effect of IP as a mediator on the causal pathway between sepsis and adverse patient outcomes. Dr. Torres's long-term career goal is to become an independent researcher in sepsis translational investigation and molecular epidemiology, engaged in understanding endotypes and mechanisms that drive pathogenesis amongst critically ill patients. In this career development award, her goals are to gain focused training in FA metabolism; become proficient in immune cell signaling pathways of inflammation and inhibition of activation; develop skills to design, recruit and retain a cohort of critically ill subjects to explore the clinical relevance of patient characteristics and sepsis-induced IP on outcomes using causal inference methods; and build expertise in statistical analysis of complex biological data. She will accomplish this through mentoring, coursework, dissemination of research, and hands-on-experience, all necessary for her future success.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Most advanced cancer patients report that religiousness and/or spirituality (R/S) are important to them, yet most also say that the medical system has not met their R/S needs. Support of dying patients' R/S needs may prove especially beneficial to those who are religious, including African American (hereafter, “black”) patients who often rely heavily on religion to cope with cancer. Healthcare chaplains work within medical systems to provide R/S care. Among advanced cancer patients, visits with healthcare chaplains are associated with patients' peaceful acceptance of terminal illness, which is associated with higher rates of advance care planning (ACP), which in turn has proved an effective way to enhance a dying patient's quality of life and receipt of value-concordant end-of-life (EoL) care. Additionally, preliminary data suggest that healthcare chaplain visits are associated with higher rates of completing do-not-resuscitate (DNR) orders among black advanced cancer patients. At present, healthcare chaplains work predominantly in inpatient settings. Thus, advanced cancer patients in outpatient settings have not benefited from the many positive effects of healthcare chaplaincy, including having unmet spiritual needs addressed and the benefits of incorporating spiritual care into EoL decision-making and the cancer care experience. To date, there has not been a randomized controlled trial (RCT) of effects of early integration of healthcare chaplain-led spiritual care on EoL cancer care. We propose here to determine the feasibility of conducting such a trial and to explore potential effects of healthcare chaplain-led spiritual care on spiritual well-being and readiness to engage in ACP among black advanced cancer patients in outpatient settings. Specifically, we propose: Aim #1: Will determine the feasibility of conducting an RCT of effects of early integration of healthcare chaplain-led spiritual care into outpatient oncology care on EoL care. Aim #2: Will explore potential effects of healthcare chaplain-led spiritual care on spiritual well-being and ACP among black advanced cancer patients in outpatient settings. Aim #3: Will explore potential mechanisms by which healthcare chaplain-led spiritual care in outpatient settings affect black advanced cancer patients' EoL care outcomes. Impact: Results will provide evidence of the feasibility of conducting an RCT of the effects of early outpatient (in advance of a terminal inpatient) healthcare chaplain-led spiritual care on spiritual well-being and ACP among black advanced cancer patients. Integration of healthcare chaplain-led spiritual care into outpatient oncology services for advanced cancer patients may be an impactful, scalable way to improve the EoL cancer care experience among black patients with advanced cancer.

NIH Research Projects · FY 2025 · 2023-08

Abstract. Extracellular vesicles and particles (EVPs) are a group of heterogeneous nanoparticles that are secreted into the extracellular milieu by many types of cells. EVPs have emerged as important mediators in cell-to-cell communications within organs. Recent studies have suggested that EVPs might be involved in β cell-immune cell interaction and β cell dysfunction and survival in type 1 diabetes (T1D). However, many of these studies were performed using mouse models and not focused on human samples. Hence, there is a unmet need to understand the contribution of human islet/pancreas-associated EVPs in T1D progression. Here, we have assembled an interdisciplinary team, including experts in islet biology (Chen, Liu), EVP biology (Lyden), vascular biology (Rafii), and T1D patient care (Antal), to test the hypothesis that abnormal EVPs of pathogenic human islets and pancreatic tissues disrupt human islet function and survival and trigger T1D progression. We have performed extensive proteomics analyses of EVPs derived from human β cell lines cultured in T1D conditions. In addition, by comparing the plasma membrane protein profiles of human β cell line and primary human islet-derived EVPs with an EVP proteomic database containing 426 human samples, we have identified several plasma membrane protein candidates to isolate β cell-specific EVPs. Here, we will use multiple platforms, including primary human islets from healthy, pre-T1D, and T1D donors, human pluripotent stem cell (hPSC)-derived islet organoids, perfusable microfluidic devices containing vascularized islets, and pancreatic slice culture to systematically examine the role of human islet/pancreas-associated EVPs in islet cell function and survival during T1D progression. In this proposal, we expect to identify membrane-bound EVP protein panels to purify EVPs from human α, β cells, islets, and pancreatic tissue. We will define the EVP protein and RNA profiles and identify signature molecules associated with islet status during T1D progression. Moreover, we will systemically dissect the roles of human islet/pancreas EVPs in human β cell function and survival, as well as their role in the crosstalk between β cells and peri-islet cell niche. Overall, this study will facilitate the identification of novel “biomarkers” for T1D diagnosis and guide the development of new therapeutic strategies to treat and prevent the progression of T1D.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Communication between cells in the brain occurs predominantly via chemical secretion in neuronal specializations called presynaptic terminals where synaptic vesicles (SVs) fuse with the plasma membrane to release neurotransmitter molecules. These membrane fusion events are tightly controlled and modulated by key synaptic proteins such as the neuronal SNARE proteins as well as Munc13-1/UNC-13, Munc18-1/UNC-18, Complexin, and Synaptotagmin. Munc13-1 is major hub for every step in synaptic transmission. Moreover, mutations in the human UNC-13 ortholog are associated with severe neurological and developmental disorders. Importantly, the mechanisms underlying the various Munc13-1 functions are largely unknown. We recently characterized a novel domain at the C-terminus of Munc13-1 termed HC2M that plays a role in SV docking/priming, and mutations in this domain have a devastating impact on synaptic transmission and nervous system function in the model organism C. elegans. Using a combination of genetic, behavioral, imaging, and biochemical methods, we will investigate the role of UNC-13 in driving the assembly of the neuronal SNAREs, a critical first step in SV priming. This project will shed light on some of the enduring mechanistic mysteries underlying synaptic transmission.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Cancer cells exploit multiple metabolic strategies to generate biosynthetic precursors that fuel malignant proliferation. Such metabolic redundancy and plasticity hampers effectiveness of therapies that target cancer cell metabolism and underscores the importance of identifying tumor types most likely to respond to metabolic inhibitors. The goal of this proposal is to test the hypothesis that tumors with impaired metabolic pathways will have limited metabolic plasticity in generating critical biosynthetic intermediates, rendering them susceptible to metabolic inhibition. We focus on the tricarboxylic acid (TCA) cycle, which is a central metabolic hub that supports cell growth and yet is truncated or impaired in several forms of human renal cell cancer (RCC). A subset of RCC tumors arise from germline deficiencies in core TCA cycle enzymes succinate dehydrogenase (SDH) or fumarate hydratase (FH); more commonly, RCC tumors have hyperactive hypoxia-inducible factor (HIF) signaling that blunts TCA cycle metabolism. The goal of this proposal is to determine whether these RCC tumors with altered TCA cycle activity are dependent upon ATP citrate lyase (ACL) as an alternative source of critical metabolic intermediates. Aspartate, synthesized from TCA cycle intermediate oxaloacetate, supports nucleotide and protein synthesis and has emerged as a critical limitation for tumor growth. Nevertheless, how tumors with impaired TCA cycle flux sustain aspartate generation—and whether these compensatory pathways represent a targetable liability—remains largely unknown. My preliminary data demonstrate that SDH/FH-deficient or HIF-active RCC cells have reduced aspartate pools relative to their isogenic controls and that ACL inhibition selectively impairs survival of these cells with defective TCA cycle metabolism. In Aim 1, I will leverage a panel of isogenic RCC lines to test whether genetic and pharmacologic ACL inhibition specifically impairs growth of SDH-/FH-deficient RCC cells in vitro and in vivo. I will exploit genetic tools that supply intracellular aspartate to test the hypothesis that aspartate provision underlies the ACL requirement in SDH-/FH-deficient cells. In Aim 2, I will use RCC cells with hyperactive HIF driven by loss of the von Hippel Lindau tumor suppressor to determine whether cells with suppressed oxidative TCA cycle activity depend on ACL to produce aspartate and enable growth in vitro and in vivo. These studies will shed light not only on a potential metabolic Achilles heel in SDH-/FH-/VHL-null tumors but will also serve as proof of principle that cancer cells with TCA cycle dysfunction engage ACL as an alternative route of synthesizing anabolic precursors. The work and training plan outlined in this proposal will be completed in the laboratory of Dr. Lydia Finley with the co-advisement of Dr. Ross Levine at Memorial Sloan Kettering Cancer Center and will ideally prepare the applicant for further clinical training and a career as an independent physician-scientist.

NIH Research Projects · FY 2026 · 2023-08

PROJECT SUMMARY Recombinant interferon-alpha (IFN) remains a highly effective therapy for patients with myeloproliferative neoplasms (MPN). We recently identified that patients with CALR-mutated MPN frequently exhibit normalization of blood counts (i.e. clinical response), but often do not exhibit a decrease in tumor burden (i.e. molecular response), providing an informative model to decipher the mechanisms of therapy-resistance to IFN. Interrogating the molecular impact of IFN on human MPN stem cells may reveal critical insights into mechanisms of therapy-resistance. Thus, we applied our innovative Genotyping of Transcriptomes (GoT) platform – that captures the mutation status and single-cell whole transcriptomes (scRNA-seq) within the same cells – CD34+ cells from serial bone marrow (BM) aspirates from patients with CALR-mutated MPN treated with IFN. Strikingly, we observed that IFN caused major shifts in the differentiation landscapes, distinctly in the mutated and wildtype progenitors: IFN exposure on wildtype cells resulted in a large expansion of lymphoid progenitors, while the mutated cells, in contrast, displayed an expansion of the granulo-monocytic (GM) progenitors (with a less striking expansion of the lymphoid compartment). Our preliminary data indicate that (1) the GM differentiation bias of CALR-mutated stem cells may underlie therapy-resistance, and that (2) the CALR-mutation induced UPR may prime the mutated stem cells toward the GM lineage and play a role in therapy-resistance. To interrogate these hypotheses, we will determine the transcription factor (TF) networks that govern the IFN-induced differentiation shifts by applying a novel single-cell multi-omics platform that captures RNA-seq, chromatin accessibility and somatic genotyping within the same thousands of single cells (GoT-ATAC) to the same IFN-treated cohort (Aim 1a), and by targeting these TF networks in mouse models (Aim 1b). We will define the role of UPR in therapy- resistance in treated CALR-mutated cells through GoT-ATAC and chromatin binding assays (Aim 2a) and by assessing perturbations to the UPR pathways in mouse models (Aim 2b). Finally, we will determine the impact of co-mutations in DNMT3A or ASXL1 in therapy-resistance to IFN in CALR-mutated MPN via application of single-cell multi-omics platforms to clinical samples (Aim 3a) and interrogation of IFN effects on novel mouse models with double mutations (Aim 3b). The project is centered on a conceptually innovative framework in which we superimpose neoplastic and normal hematopoietic development within the same individuals to define how therapy reshapes differentiation topographies, as a function of mutation status and cell identity. This conceptual innovation is enabled by technical innovations in single-cell multi-omics platforms applied to compelling clinical cohorts, coupled with functional assessments in novel mouse models. These studies have the potential to uncover new insights into the mechanisms of molecular resistance to IFN in MPN, resulting in novel therapeutic approaches.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT Over 1 million women have their labor induced in the United States each year, and synthetic oxytocin infusion is the most common method used. However, compared to spontaneous labor, medical induction is resource- intensive, has increased obstetric risks, and is associated with less successful breastfeeding. In contrast to endogenous oxytocin hormone which is released in a pulsatile fashion in the brain, synthetic oxytocin is continuously infused intravenously, resulting in important limitations related to efficacy, safety, and cost. Akin to spontaneous labor contractions, infant suckling of the breast nipple is known to stimulate the pulsatile release of endogenous oxytocin from the posterior pituitary gland. Nipple stimulation therapy via electric breast pump similarly stimulates endogenous oxytocin release, and our preliminary work shows that it is a feasible and acceptable inpatient method that results in a desirable uterine contraction patter n in nulliparas. Our pilot study of 100 randomized nulliparas showed that intrapartum nipple stimulation therapy decreases labor duration and trends toward a significant increase in the rate of spontaneous vaginal delivery compared to synthetic oxytocin infusion. Further, nipple stimulation reduced the dose and duration of synthetic oxytocin even when adjunctive synthetic oxytocin was used. Therefore, nipple stimulation therapy will likely prove to be an efficacious labor induction method that increases the likelihood of spontaneous vaginal delivery, and also have added physiologic benefits. For example, nipple stimulation triggers lactation by inducing the milk ejection reflex, and our preliminary work also shows that nipple stimulation therapy via electric breast pump results in early colostrum production and milk letdown in the majority of women, including first-time mothers. Earlier lactation would alleviate the most common reasons for early breastfeeding discontinuation by improving maternal perception of insufficient milk supply and the severity of weight loss that occurs in infants in the first few days of life as they establish feeding. This in turn would improve the likelihood of sustained breastfeeding for the recommended 6 months, which also has many short- and long-term benefits. Consequently, nipple stimulation therapy during labor has tremendous potential public health and cost benefits, and its success would be particularly important in areas of poverty, including developing countries. We propose a multicenter randomized trial at Yale and Northwestern Universities to compare inpatient nipple stimulation therapy via electric breast pump versus immediate synthetic oxytocin infusion without nipple stimulation for nulliparous women undergoing labor induction. This trial of 988 nulliparous women will provide adequate statistical significance to detect clinically meaningful differences in delivery mode and breastmilk as the sole source of nutrition for newborns. Successful completion of this proposal will provide rigorous data to help us show how this novel and potentially cost-effective method can radically change the way we induce labor and positively impact breastfeeding success and early infant nutrition through lactation.

NIH Research Projects · FY 2025 · 2023-08

Overall Mammalian sperm are stored in the epididymis in a dormant state; they are immotile and unable to fertilize the egg. Upon ejaculation, sperm begin swimming and initiate a process called capacitation, where they become competent to fertilize the oocyte. An initial event in capacitation and activation of motility is the bicarbonate-induced stimulation of soluble adenylyl cyclase (sAC: ADCY10). Men and male mice with the sAC gene knocked out are infertile, and in vivo administration of sAC inhibitors to male mice prevents sperm motility and renders the males temporarily infertile. Thus, sAC is a nonhormonal target, genetically and pharmacologically validated to be essential for male fertility. Besides male-specific sterility, the only other phenotypes of sAC knockout men or mice are dependent upon chronic loss of sAC for extended periods of time. We propose to leverage acutely acting inhibitors, whose effects will be transient, to avoid mechanism-based side effects and limit the inherent perils associated with chronic dosing. The goal of the Weill Cornell Medicine Contraceptive Research Center (WCM-CRC) is to develop acutely acting sAC inhibitors into safe and effective nonhormonal, orally available, on-demand contraceptives which men take only when and as often as needed, shortly before sex. In two Contraceptive Development Research Projects, we propose to improve binding affinity, pharmacokinetics (including oral bioavailability), drug-like characteristics, and safety of sAC inhibitors with the expectation that pharmacokinetic parameters can be optimized to balance efficacy with minimal adverse effects. In Project 1, we focus on a chemical series validated in vivo in a preclinical animal model, and in Project 3, we propose to develop additional leads from structurally distinct scaffolds. In Project 2, we will establish a second, non-rodent animal model for testing contraceptive efficacy; test the in vivo efficacy of optimized sAC inhibitors; and validate sperm motility as a pharmacodynamic biomarker of efficacy for use in early phase clinical trials of an on-demand male contraceptive. A major goal of the WCM-CRC is to identify a clinical lead candidate (along with backups) to advance into Investigational New Drug (IND) enabling studies for a novel oral, nonhormonal contraceptive for men.

NIH Research Projects · FY 2026 · 2023-08

Abstract A frequent side effect of chemotherapy is infertility and female patients newly diagnosed with cancer or other malignancies are routinely counseled to undergo controlled ovarian hyper-stimulation as a means of fertility preservation. For pre-pubertal girls or women requiring immediate chemotherapy, ovarian hyperstimulation is unavailable and many of these patients opt to cryopreserve ovarian tissue with subsequent auto-transplantation once in remission. The increasing frequency of ovarian tissue cryopreservation (OTC) portends a surge in future demand for auto-transplantation, yet the viability/function of grafts is significantly undermined by post-transplant ischemia. Moreover, the iatrogenic influence of chemotherapeutic agents and/or the inflammatory milieu present following tissue auto- transplantation upsets the inter-follicular homeostasis that governs healthy ovarian physiology. We have developed an approach that utilizes cultured vascular cells to accelerate perfusion of transplanted ovarian tissue. In addition, we have demonstrated the potential for an ovary-specific secreted factor, anti-Müllerian hormone (AMH), to modulate follicular growth and activation. The goals of this proposal are to combine these technologies to enhanced tissue viability and follicular output in the context of auto-transplantation and/or mitigate the negative influence of chemotherapeutic agents on the ovary in situ. The proposal aims to address this burgeoning unmet need by: 1) improving viability and output of ovarian tissue grafts by co- transplanting patient-matched vascular endothelial cells isolated at the time of OTC; 2) manipulating signaling within the graft site at the time of ovarian tissue transplantation to repress premature follicular mobilization; and 3) mitigating the gonadotoxic influence of alkylating chemotherapy via pre-conditioning of ovarian tissue with AMH. In pursuing these goals, the proposal stands to improve outcomes for thousands of women who have undergone or will undergo OTC, while also aiming to forego in future patients the risk, expense, pain, and uncertainty of OTC and auto-transplantation.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Innate immune antiviral pathways are upregulated in Alzheimer’s disease (AD) and are thought to drive AD pathogenesis. The specific mediators of maladaptive antiviral responses in AD are still not understood. Innate immune system utilizes a series of nucleic acid sensors to detect viral genetic material to activate interferon expression. The central DNA sensor in the cell is cyclic GMP-AMP synthase (cGAS). cGAS signaling has emerged as an important player in many neurodegenerative diseases like Parkinson’s disease and amyotrophic lateral sclerosis. The role of cGAS signaling in tau-mediated neurodegeneration remains unstudied. We discover that cGAS signaling is hyperactivated in the brains of mice expressing mutant tau (P301S mice) and human AD patients. Using behavioral assays, electrophysiological readings and single nuclei RNA sequencing, our preliminary results indicate that cGAS activity modifies both the cellular responses to and the functional deficits caused by tauopathy. We observed that partial or complete loss of Cgas rescued tauopathy-associated spatial learning and memory deficits. cGAS is highly enriched in immune cells including microglia. We find that tau induces a cGAS-dependent interferon signature by triggering mitochondrial DNA stress in microglia and show that Cgas deletion alters microglial disease transformation, characterized by reduced expression of interferon genes in disease microglia. It is not known how tau can alter mitochondrial and nuclear dynamics to activate cGAS and how cGAS activation enhances neurotoxic effects of tau. This proposal will investigate the mechanisms and consequences of cGAS and nucleic acid sensing pathways in tauopathy. I aim to dissect the molecular mechanism of cGAS activation in response to tau by focusing on tau dependent cellular processes that induce mitochondrial stress and microglial senescence (Aim 1) and investigate how cGAS mediates maladaptive microglial responses and neuronal damage in tauopathy (Aim 2). Finally, since antiviral responses could be species-specific, there is strong rationale to extend mouse studies of innate nucleic acid sensing to human models. Human and mouse cGAS share only 60% amino acid similarity and have different activation requirements. I will establish human pluripotent stem cell (hPSC)-based platforms to study broader nucleic acid sensing pathways and perform unbiased CRISPR-screens to identify novel regulators of human nucleic acid sensing in tau toxicity (Aim 3). The experiments outlined in this proposal will lead to a better understanding of the role of nucleic acid sensors in tauopathy and AD which might prove translatable to humans.

NIH Research Projects · FY 2025 · 2023-08

This application for an Administrative Supplement proposes a collaborative study to be performed entirely at Weill Cornell Medicine (WCM) in New York, NY. The parent project (R01CA277038) is a collaboration between WCM, GE Research in Niskayuna, NY, and Stony Brook Medicine (SBM) affiliated with the State University of New York in Stony Brook, NY. The parent project addresses the need for reliable, highly sensitive means of detecting metastases to lymph nodes (LNs) and distinguishing them from primary lymphomas and LNs affected by benign conditions. This capability will allow improved staging and treatment of disease. Accordingly, the project builds on encouraging results obtained in prior studies by WCM and SBM using quantitative-ultrasound (QUS) imaging methods to detect metastases in LNs by applying and evaluating these promising methods using a standard clinical scanner to acquire ultrasonic echo-signal data from patients undergoing medically required ultrasonically guided biopsies. The Administrative Supplement will expand the parent study to three additional WCM clinical sites. This strategic expansion will substantially enhance the generalizability of the parent study, which will enable us to characterize how QUS imaging performs among breast-cancer patients from various socio-economic groups, which often experience delays in cancer care. Using patients’ zip code data, we will link individual-level data to the American Community Survey to capture area-level factors such as poverty that are known to impact cancer care delivery. To investigate the potential of this novel technology to reduce delays in breast cancer care, we have assembled a multi-disciplinary team of scientists and clinicians across radiology, engineering, breast surgery, and health services research. The QUS data acquired at WCM will be used to determine whether QUS parameters are different among sub-groups of breast-cancer patients and whether QUS-based classification can be improved by including patient level data. The project will also investigate how QUS technology can reduce barriers to breast cancer care delivery (e.g., by reducing time to treatment initiation).

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Chronic obstructive pulmonary disease (COPD) is a smoking-associated respiratory disease and is the 3rd lead- ing cause of death worldwide. There are increased numbers of dysfunctional airway and alveolar macrophages (AMs) in the lungs of smokers and COPD patients, localizing to areas of injury. Despite this connection, the mechanisms behind how cigarette smoke (CS) elicits AM dysfunction, and how AM dysfunction facilitates the development of small airways disease and emphysema, two defining features of COPD, are poorly understood. This proposal addresses this critical gap in knowledge and tests the hypothesis that AM dysfunction in COPD is mechanistically linked to abnormal iron accumulation in these cells. Using a multicenter prospective COPD (SPIROMICS) cohort, we previously associated increased levels of iron and iron-related proteins in the bron- choalveolar lavage fluid of smokers and COPD patients with adverse clinical COPD outcomes. AMs are the putative source for this lung extracellular iron, as AMs from smokers and COPD patients are iron-overloaded and release iron in culture. We replicated this clinical phenomenon of AM iron accumulation and release using a murine CS-exposure model. We then used single-cell RNA sequencing and discovered novel AM subsets which have a unique iron-related gene expression signature that is consistent with increased iron uptake. These “iron macrophages”, which we designate as FeMacs, have decreased expression of genes associated with phagocytosis and immune activation, suggesting that this CS-induced AM iron accumulation may have functional consequences for AMs, and potentially for the CS-exposed lung. We will test our hypothesis that FeMacs or- chestrate lung injury development in COPD both mechanistically using our murine CS model and translationally using the SPIROMICS cohort. Aim 1 will compare CS-induced small airways damage and emphysema develop- ment between control mice and mice with AMs deficient in nuclear receptor coactivator 4 (Ncoa4ΔCd11c), an iron metabolism defect which mitigates CS-induced iron accumulation. Aim 2 examines Ncoa4ΔCd11c and Ncoa4fl/fl control mice in a CS-Streptococcus pneumoniae infection 2-hit model, thereby determining whether reducing AM iron overload can alleviate CS-induced AM dysfunction and improve response to pathogen. Aim 3 defines the clinical relevance of AM iron accumulation in human COPD and tests AM expression of iron genes and AM iron content as markers of COPD severity. This proposal presents a five-year career development plan that builds on my previous research and integrates the different domains of expertise of my mentorship and advisory teams. It entails a targeted training plan that is tailored towards the development of specific areas related to immunology, macrophage biology, iron biology, and translational research, facilitated by the physical and intel- lectual resources provided by the academic environment at Weill Cornell Medicine. The proposed experiments, didactic training, as well as mentorship team will position me with a unique set of interdisciplinary skills that will enable my transition to independence as a physician-scientist in lung and macrophage biology and iron biology.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY / ABSTRACT Persistent activity in neural circuits supports a variety of brain functions from motor control to navigation to perceptual decision-making. Correlational studies show significant variation in persistent activity patterns during different behaviors, suggesting that individual circuits perform flexible computations that depend on the context of ongoing brain activity and motor functioning. However, establishing the causal significance of this variability is difficult due to technical limitations in existing tools for precisely manipulating circuit dynamics. A tractable system for overcoming this challenge is the zebrafish, a vertebrate model organism with an optically accessible brain that allows simultaneous calcium imaging and optogenetic stimulation with laser microscopy. Research in our lab focuses on the zebrafish oculomotor integrator, a hindbrain circuit involved in adaptive control of gaze position. This circuit generates persistent activity that directly drives easily quantified motor behavior. In prior work, our lab found that different patterns of integrator activity are associated with distinct types of eye movements, but it is unclear how these context-dependent dynamics contribute to oculomotor control and how they relate to the development of sophisticated behavior. In the proposed research, I will conduct simultaneous two-photon imaging and optogenetic stimulation during visuomotor behavior to determine how different patterns of integrator dynamics contribute to different types of eye movements. First, I will collect a comprehensive longitudinal dataset of brain-wide neural activity in the larval and juvenile zebrafish during a broad range of oculomotor behaviors, testing if indeed the number of persistent patterns in the integrator expands with the behavioral repertoire. Then, I will perform real-time closed-loop stimulation of the integrator network at single- cell resolution, steering circuit dynamics along specific patterns of activity to test their causal impact on motor outputs. This research will improve our understanding of flexible control by memory circuits and establish new paradigms for precise manipulation of network dynamics.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Mitochondrial reactive oxygen species (ROS) are strongly implicated in the pathogenesis of diverse aging- associated neurological disorders, including Alzheimer's disease (AD) and frontotemporal dementia. Mitochondria produce ROS during oxidative metabolism and increased production of mitochondrial ROS are causally linked to various processes in AD, including aging, amyloid precursor protein/amyloid-β (APP/Aβ) pathology, tauopathy, and neuroinflammation. Recent work suggests that ROS produced by different mitochondrial sites have distinct roles in cell signaling and disease. However, previous tools to suppress mitochondrial ROS were not site-selective, disrupted respiration, or inhibited ROS only after release rather than blocking production. Thus, the roles of mitochondrial ROS in AD pathogenesis require investigation. Mitochondrial complex III has a large capacity for ROS production and generates ROS toward the cytosol, poising it to regulate intracellular signaling and disease mechanisms. To investigate the effects of complex III- derived ROS, our lab has identified and characterized small molecules that suppress complex III ROS production (S3QELs, “sequels”), but do not block ROS production by other mitochondrial sites or affect other mitochondrial processes. In our preliminary studies using S3QELs, we found that AD-associated neuroimmune factors enhance astrocytic complex III ROS and that complex III ROS promote JAK-STAT3 signaling and gene expression changes linked to disease. In astrocytic-neuronal cultures, S3QELs prevented neuronal dysfunction linked to tauopathy, but did not affect neurons cultured in isolation. In addition, S3QELs reduced neuroimmune and glial alterations in mice expressing mutant human tau. These data implicate complex III ROS in astrocytic signaling and AD-related cascades. I propose to test my central hypotheses that astrocytic complex III ROS are increased by specific disease-related stimuli and modulate astrocytic functions and dementia-linked pathogenic processes through oxidation of distinct cysteine targets, including those related to STAT3. In Aim 1, I will use a genetically-encoded ratiometric H2O2 sensor targeted to specific subcellular compartments in primary mouse and human iPSC-derived astrocytes to define the exact patterns and upstream triggers of astrocytic complex III ROS. I will also use genetic and pharmacological tools to determine the roles of complex III ROS in astrocytic signaling, gene expression, and astrocytic-neuronal interactions. In Aim 2, I will use innovative redox proteomics methods to broadly profile complex III ROS-mediated cysteine oxidation and use targeted and cell-specific genetic manipulations to assess how oxidation of specific cysteine sites alters astrocytic signaling and pathological cascades linked to dementia. The proposed study is likely to elucidate novel oxidative mechanisms that regulate glial signaling and disease cascades and could lead to the development of targeted and effective therapies for aging-related dementias.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT Our overall goal is to develop a fluid mechanics approach to studying tracer transport through tissue for perfusion quantification in magnetic resonance imaging (MRI), which is termed as quantitative transport mapping (QTM). Current/traditional perfusion quantification in MRI and medical imaging in general is based on Kety's method that assumes the same arterial input globally into all voxels in an imaging volume. This global arterial input function (AIF) transgresses the local mass conservation at a voxel and requires the user to choose an arterial region of interest (ROI) with the consequent perfusion value highly dependent on the ROI choice, which is known as the AIF problem. been The AIF problem in Kety's method for perfusion quantification has a major unmet challenge impeding perfusion quantification in MRI.The tracer concentration at the artery entering the voxel is needed to address the AIF problem and can be estimated by following tracer transport through the vascular space according to fluid mechanics, which is the proposed QTM. Accordingly, we propose to develop QTM technology for MRI perfusion quantification, capable of processing all 3 major types of images: dynamic susceptibility contrast (DSC) as in imaging ischemic stroke, multidelay arterial spin labeling (ASL) as in imaging kidney transplant, and dynamic contrast enhanced (DCE) as in imaging breast tumor. We plan to achieve this objective through the following three specific aims: Aim comprising Aim Simverse tracer Aim processing In perfusion 1 Develop vascular Simverses For the brain, kidney and breast, we will develop vascular Simverse datasets of vasculature, flow and permeability distribution, and tracer propagation. 2 Develop compartmentalized quantitative transport mapping. The datasets in the vascular of an organ are used to train DNNs for QTM determination of vasculature, flow and permeability, and propagation from tracer spacetime images. 3 Evaluate quantitative perfusion mapping in patients. The eveloped QTM is evaluated for three major perfusion MRI acquisitions: DSC, multidelay ASL and DCE. summary, the successful outcome of this project will establish the fluid mechanics based QTM for quantification . d as a more effective alternative to Kety's method with preservation of local mass conservation and without the AIF problem.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Preeclampsia (PE) is a leading cause of maternal morbidity and mortality in the United States which has led to an urgent need to accurately predict its risk. It is not known how nonalcoholic fatty liver disease (NAFLD), the liver manifestation of metabolic syndrome and the most prevalent liver disease among women, contributes to PE risk. The long-term objective of this proposal is to define the relationship between NAFLD and PE, to identify strategies to reduce the risk of PE, and to improve short- and long-term maternal outcomes in this population. This project will evaluate the independent association of NAFLD with PE among overweight and obese women. It will also study relevant lipid alterations in women with NAFLD in pregnancy which are associated with endothelial dysfunction that leads to the clinical syndrome of PE, among NAFLD (compared to non-NAFLD) patients during pregnancy. The Specific Aims of this proposal are: (1) To prospectively assess whether NAFLD is an independent risk factor for: i) PE among overweight/obese women and ii) PE with liver injury; (2) To determine whether women with NAFLD in pregnancy have altered levels of bioactive lipids associated with endothelial cell dysfunction, compared with women without NAFLD in pregnancy. This study will be performed in the Mount Sinai Health System, which provides care to a diverse and multiethnic patient population disproportionately affected by both NAFLD and PE. This application will support the candidate's career development into an independent patient-oriented investigator focused on the influence of liver disease in pregnancy on preeclampsia risk and future maternal cardiovascular and liver health, a significant unmet need. The proposed career development plan integrates advanced coursework in translational epidemiology, lipidomics assessment, prospective study design/ analysis, and biostatistics, and experiential learning through the conduct of the proposed research plan, all within a highly supportive research environment. The mentorship team which includes senior investigators with expertise in epidemiologic and translational research in hepatology (Friedman and Terrault), preeclampsia (Roberts), and prospective study design and advanced biostatistical analysis (Sigel), will guide the candidate's research and career development. The superb institutional infrastructure for developing successful clinical investigators and the substantial institutional commitment to the candidate greatly strengthen this application. At the conclusion of this proposal's funding period, Dr. Kushner will be optimally positioned as an independent physician-investigator studying the intersection of liver disease and PE, and their implications on future maternal health.

NIH Research Projects · FY 2026 · 2023-08

In the United States, more than a third of patients are referred to a specialist each year, and specialist visits constitute more than half of outpatient visits. Even though all physicians highly value communication between primary care providers (PCPs) and specialists, both PCPs and specialists cite the lack of effective information transfer as one of the most significant problems in the referral process. Therefore, it is critical to investigate a new method to improve communication during care transitions. With their ubiquitous use, it is recognized that electronic health records (EHRs) should ensure a seamless flow of information across healthcare systems to improve the referral process. But, a lack of accessible and relevant information in the referral process remains a pressing problem. Recently, emerging deep learning (DL) and natural language processing (NLP) methods have been successfully applied in extracting pertinent information from EHRs and generating text summarization to improve care quality and patient outcomes. However, existing technologies cannot be applied to process heterogeneous data from EHRs and create high-quality clinical summaries for communicating a reason for referral. Responding to PA-20-185, this project will develop and validate a novel informatics framework to collect and synthesize longitudinal, multimodal EHR data for automatic referral form generation and summarization. While the referring provider and specialist can be any type of provider for any condition, the focus in this application has been on headache for primary care, because it is an extremely common symptom and affects people of all ages, races, and socioeconomic statuses. More importantly, relevant information needed for headache referrals has been defined in local and national evidence-based practice guidelines. Therefore, a health information technology solution to make these data accessible will empower communication between PCPs and specialists, which can improve the care of millions of patients suffering from disabling headache disorders. Based on our preliminary data and our experience with an interdisciplinary team of data scientists and physicians, we plan to execute specific aims: 1) Convert text-based guidelines into a standards-based algorithm for electronic implementation; 2) develop models to automatically populate data from EHR and clinical notes to fill the referral form; 3) create a framework to summarize the longitudinal clinical notes to fill out the referral form; and 4) develop and validate the headache referral system with a user-centered design approach. The research proposed in this project is novel and innovative because it will produce and rigorously test new solutions to improve the communication between health professonals to ensure that safe, high-quality care is provided and care continuity is maintained. The success of this project will (1) fill important gaps in our knowledge of understanding the types of information exchange that will optimize patient care during transitions and (2) provide evidence-based solutions to enable the exchange.

NIH Research Projects · FY 2025 · 2023-08

1 2 functions, 3 have 4 spatial resolution and sensitivity. Such specifications impact both neurologic and neuro-oncologic diseases. In 5 the former, they allow detecting, quantitating, and tracking small changes of PET signal in minute brain regions 6 such as brain nuclei that have been implicated in many neurologic diseases. In the latter, they improve the 7 accuracy of tumor target volume definition in radiotherapy and surgical resection, thus treatment outcome. 8 The only commercial brain-dedicated PET is the HRRT, a 2 decade old technology that has been discontinued. 9 Therefore, there is compelling need to develop the next generation brain-dedicated PET with ultra-high 10 specifications to improve diagnostics that can institute therapies earlier in the evolution of the disease. 11 This proposal brings together two highly collaborative teams from Weill Cornell Medicine (WCM) and the 12 Institute for Instrumentation in Molecular Imaging (i3M), with an industrial partner, Oncovision, to build an 13 ultra-high performance brain-dedicated PET, UHB-PET. UHB-PET will exhibit: (i) volumetric spatial 14 resolution of ~0.5mm3 across the gantry, that is >4x better than that of the best brain-dedicated PET being 15 developed; (j) effective sensitivity >26x that of the brain PET with the highest spatial resolution being developed. 16 Our intensive experimental and Monte Carlo simulation results prove that our goals are highly achievable, 17 which we will attain as follows: Specific 18 maximize 19 FOV 20 and 21 Quantitative machine 22 learning to accurately determine the 3D position of 511keV 's interaction within the semi-monolithic slab, infer the 23 attenuation-corrected PET without CT scans, and minimize image noise, thus reduce the administered dose), (d) 24 use a preconditioned fixed-point image reconstruction approach to suppress the noise in sub-millimeter size 25 pixels, (e) adopt motion tracking tool we previously developed to correct for inter- and intra- head motion during 26 dynamic PET imaging, and (f) adopt methods from our previous work to accurately image-derive the input function 27 for kinetic modeling. Specific Aim 3: Assessment of image noise, target lesion visibility and quantitative accuracy 28 attained by the scanner in a characteristic set of specific neurology and neuro-oncology human studies. 29 The ultimate goal is a fully operational ultra-high performance dedicated brain PET scanner with accurate 30 quantitative capabilities for diagnosing and monitoring treatment in brain diseases. . Positron Emission Tomography (PET) is a powerful quantitative tool for studying metabolic and biochemical pharmacology, and pathology in living brains. In the past 3 decades, a myriad of brain PET tracers, been developed.In parallel, PET underwent dramatic advancement in technology that enabled much higher Aim 1 : In 2.5 years we will build UHB-PET with trapezoidal-shape (to sensitivity) semi-monolithic LYSO slabs coupled to high-performance SiPM readout, 26.7cm axial and ~28cm diameter, high 200psec detector timing resolution, isotropic spatial resolution <0.8mm FWHM 0.72mm Depth-of-interaction (DOI) resolution. Specific AIM2 : In parallel with AIM1, we will develop a Image Reconstruction tool. We will (a) incorporate accurate physics modeling, (b) use

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Stroke is a devastating disease and leading cause of death and disability in the United States. Ischemic stroke results in massive activation of numerous immune cells that can infiltrate the brain following blood-brain-barrier breakdown. The gut microbiota has previously been identified as a significant factor affecting outcome and severity of ischemic stroke in clinical studies and animal models. However, mechanisms underlying the modulatory role of microbiota on immune cells following stroke remain unclear. Dendritic cells (DCs) act as the bridge between innate and adaptive immunity, with their ability to sample material from the intestinal lumen and shape T-cell responses. Antibiotic-induced alteration of microbiota in mice results in stroke neuroprotection in mice following middle cerebral artery occlusion model of ischemic stroke compared to control mice carrying conventional microbiota, which are similarly treated but carry antibiotic-resistant microbiota resulting in microbiota similar to that of naïve mice. This effect is attributed to the greater capacity of intestinal and mesenteric lymph node dendritic cells of mice carrying “altered” microbiota to induce T-regulatory cells (Tregs) in the small intestine which subsequently suppress destructive pro-inflammatory IL-17+ γδ T cells that traffic to the brain following stroke. Using our in vitro model to simulate intestinal DC-T cell interactions, we show that priming naïve DCs with isolated contents from the small intestine (SIC) of mice carrying “altered” or “conventional” microbiota and subsequent co-culture with CD4 cells similarly induces greater proportions of Tregs following SIC from mice carrying “altered” microbiota compared to SIC from mice with “conventional” microbiota. This proposal seeks to elucidate the mechanism by which altering microbiota may result in changes in pattern-recognition receptors or toll-like receptor ligands that are responsible for a DC-tolerizing phenotype and Treg induction observed with microbiota alteration in mice. Using a variety of in vitro and in vivo approaches, I aim to identify the DC receptors and signaling machinery responsible for sensing these luminal contents and producing a tolerogenic phenotype, determine DC-produced signals/cytokines necessary for intestinal Treg induction, and establish the role of pro- inflammatory IL-6 in stroke neuroprotection vs poor stroke outcome in mice carrying “altered” or “conventional” microbiota. In summary, I seek to understand how intestinal DC receptor ligands that are microbiota-dependent can act as regulators of intestinal immunity and stroke outcome, as well as identify potential therapeutic targets.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY Kidney transplantation is a life-saving procedure for patients with end-stage renal disease. Tacrolimus is utilized to prevent rejection of the kidney transplant but has a narrow therapeutic window with subtherapeutic tacrolimus levels associated with acute rejection and supratherapeutic levels associated with nephrotoxicity and neurotoxicity. Importantly, kidney transplant recipients with tacrolimus trough variability, i.e. marked intra-patient variation in tacrolimus trough levels, are at increased risk for acute rejection and kidney transplant loss. The factors predicting intra-patient tacrolimus trough variability, however, are not well understood. Our preliminary studies support a relationship between gut bacterial metabolism of tacrolimus and tacrolimus trough variability. The overall objective of this study is to define the relationship between the gut microbiota and tacrolimus trough variability in kidney transplant recipients. Our central hypothesis is that specific gut microbial species are associated with metabolism of tacrolimus and intra-patient tacrolimus trough variability. The hypothesis is based upon and inspired by our pilot studies: (1) Faecalibacterium, Blautia, and other commensal organisms directly metabolize tacrolimus into M1, a lesser active tacrolimus metabolite (2) M1 production is present in the fecal specimens of kidney transplant recipients (3) blood M1 is detected in kidney transplant recipients after oral administration of tacrolimus (Guo et al., Drug Metabo Dispos 47(3):194-202, 2019; Guo et al., Transplant Direct 6(10):e601, 2020). In this study, we will recruit 80 kidney transplant recipients for serial collection of fecal specimens during the first 3 months following transplantation and will profile the gut microbiome using metagenomic sequencing. As in vivo biomarkers of bacterial tacrolimus metabolism, we will profile blood M1 (the bacterial tacrolimus metabolite) levels and quantitative fecal M1 production to assess their relationships with intra-patient tacrolimus trough variability as well as acute rejection and de novo donor specific antibody development against the kidney transplant. In Aim 1, we will identify the gut bacterial species associated with tacrolimus metabolism. In Aim 2, we will determine the gut bacterial and blood profiles associated with intra-patient tacrolimus trough variability. Significance. Our study will enable development of gut-based and blood-based biomarkers to identify kidney transplant recipients at high risk for tacrolimus trough variability. Our study will provide the framework for providing improved precision delivery of immunosuppressive therapies in kidney transplant recipients.

NIH Research Projects · FY 2024 · 2023-08

PROJECT SUMMARY As we get older, we learn to modulate our behaviors to optimize reward outcomes. These adaptive choices are orchestrated by current sensory conditions, internal cognitive states, and future expectations. In adolescence, rewards circuits that link peripheral detection of sensory stimuli to central circuits involved in decision-making and motivational states continue to grow, remodeling the microcircuit connectivity within the medial prefrontal cortex (mPFC). This development may explain why adolescents demonstrate increased impulsivity and diminished behavioral flexibility, and fail to optimize reward outcomes. However, the developmental changes within the reward circuits that inform differences in reward learning during adolescence are poorly understood. The mPFC is a key area for emotional regulation, decision making, and reward-seeking. The reward- modulating properties of mPFC are derived from inputs from the ventral tegmental area (VTA). During adolescence, VTA inputs into mPFC are still developing, and the functional impact of these developmental changes is unknown. One influential theory suggests that increased dopaminergic (DA) signaling in adolescence drives heightened reward sensitivity. However, additional mechanisms such as changes in local mPFC connectivity and changes in reward information sent to the mPFC are likely at play. In this proposal, we use a combination of chemogenetics, optogenetics, anatomical, and neural calcium imaging, to test how the developing adolescent mPFC (Aim 1) and VTA projections to mPFC (Aim 2) contribute to adolescent reward behaviors and influence reward optimization strategies (Aim 3). This work reframes the role of neuronal subtypes, and probes if their role in a given behavior is shaped by the age of the microcircuit, asking the question, do cells carry the same information in adolescents as they do in adulthood? In addition to the value of this work from a basic science perspective, this study is likely to produce testable hypotheses that will tackle why certain psychiatric disorders such as impulse control and feeding disorders tend to emerge during adolescence. Training in calcium imaging, neuronal activity data analysis and advanced anatomical techniques will be provided by the mentor Dr. Conor Liston, with additional expertise in 2-photon imaging provided by the consultants Drs. Rajasethupathy and De Marco Garcia. Dr. Sullivan will serve as a consultant on developmental behavioral neuroscience, providing feedback on experimental design and outcomes. Dr. Bravo Rivera will provide an additional behavioral neuroscientific perspective and will be instrumental in providing additional career development training to the applicant. Together the mentor and the External Advisory Committee will facilitate the transition of Dr. Manzano Nieves into an independent research career focused on uncovering how postnatal development alters brain circuits to bias behavior and create psychiatric vulnerabilities.