University Of Washington

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY Cancer vaccines are a promising immuno-oncology approach that can elicit highly tumor-specific responses from the immune system, resulting in prolonged anti-cancer protection. Peptide-based vaccines are especially attractive because they have low toxicity risk and can be produced easily at large scale with good storage stability. However, peptide antigens are inherently poorly immunogenic and susceptible to rapid degradation and excretion after administration. Thus, the success of peptide-based cancer vaccines are critically tied to their effective formulation and delivery. The ideal peptide vaccine formulation would replicate each of these critical aspects: recognition and internalization by dendritic cells, activation of these dendritic cells, and antigen delivery to both MHC I (called cross-presentation) and MHC II molecules. In this application, we propose to develop a polymer-based peptide antigen carrier that uniquely addresses all of the aforementioned delivery needs in a well-controlled, scalable system through the integration of targeting ligands, controlled release adjuvants, and cytosolic and lysosomal antigen delivery mechanisms. Our main objectives are to 1) optimize polymer structure and synthesis through material engineering, 2) assess peptide and adjuvant delivery and characterize in vitro and in vivo trafficking and delivery, 3) optimize formulations for vaccine efficacy in a murine melanoma model and 4) evaluate efficacy and safety in a murine breast cancer model for cancer prevention and therapy. Successful completion of these aims will lead to a formulation poised for GMP manufacturing and the IND pathway toward clinical testing.

NIH Research Projects · FY 2025 · 2021-03

The objective of the proposed R01 project is to build on our recent findings that attractive odors from sources of nectar activate conserved olfactory channels in mosquitoes, and the odorant ratios within the scent, along with the inhibition in the antennal lobe (AL), are critical for Aedes aegypti behavior. Feeding on fruits and flowers is a vital behavior for mosquitoes, and mosquito attraction to these sources of nectar is mediated by the ratio of key odorants in the bouquet. Bait-and-kill traps that use fruit syrups have effectively controlled local mosquito populations, but variation in the fruit odor can strongly impact its attractiveness. We still have not identified the odor constituents and ratios that are attractive in the nectar odors – enabling the development of synthetic lures –, nor how this information is detected and processed by the mosquito's olfactory system. Nectar-feeding by female mosquitoes increases their life-span and decreases the gonotrophic cycle, thereby increasing their vectorial capacity, and for adult males, it is the only source of nutrients. Our recent work allowed us to identify the odor constituents that mediate nectar-feeding behaviors in Ae. aegypti and understand how the odor is processed in the brain. Now in this application, we propose to use a combination of behavioral assays, chemical methods combined with calcium imaging in tethered flying mosquitoes, and genetic approaches to study the olfactory basis of nectar-seeking behaviors. Aim 1 will allow us to identify attractive odorants in the scents of diverse plant nectar sources and use heterologous expression systems to de-orphan the cognate odorant receptors (Ors). We also take advantage of the Q-system for genetically characterizing three Ors (Or15, Or49, and Or2) that are essential for representing the proper ratios in nectar odors. In Aim 2, we leverage our existing and new GCaMP expression lines to examine how the ratios of these key odorants are processed in the AL, and how GABAergic inhibition shapes these responses. In Aim 3, we will use our identified odor lures, and artificial lures that vary in their natural ratios of key odorants, to determine their efficacy in bait-and-kill systems. Together, these experiments will test the working hypothesis that nectar odors and their specific odorant ratios activate conserved olfactory channels to be processed similarly by AL circuits to mediate feeding behaviors. While there has been an extensive study of mosquito attraction to blood hosts, we know comparatively less about nectar feeding. Our experiments will identify new odors that can be immediately deployed as attractant lures. Additionally, GABAergic systems are involved in diverse physiological processes in insect vectors, including olfaction, immune response, and arbovirus replication, as well as being potent targets for insecticides. Generating mutants that target the mosquito's olfactory responses or GABAergic pathways could provide additional insight into these diverse processes. Sugar feeding plays an essential role in the vectorial capacity of mosquitoes and the spread of diseases that afflict over a billion people annually. Therefore, unraveling the neural bases of nectar-feeding will enable new gene-targets and tools for their control.

NIH Research Projects · FY 2025 · 2021-03

PROJECT SUMMARY/ABSTRACT HIV/AIDS is a leading cause of morbidity and mortality among children and adolescents, and it is well recognized that HIV care and treatment among children (birth to 9 years) and young adolescents (10-14 years) lags behind that of adults, leading to numerous calls to action. Despite a global consensus toward eliminating new HIV infections in these age groups, assessing progress against targets is impeded, as current estimates do not include substantial amounts of locally-available data. Additionally, new HIV infections in young adolescents have not been quantified. The global HIV burden in these age groups is currently estimated by two groups: the Global Burden of Disease (GBD) study and UNAIDS, both of which currently assume no new HIV infections for those under 15. Both groups use the Spectrum child model, which relies largely on estimated HIV prevalence among adult women, treatment for prevention of mother-to-child transmission, and other assumptions to estimate the HIV burden among children and young adolescents. One of the assumptions is that new HIV infections among those under 15 come from only one source – mother-to-child transmission – assuming no sexual activity and zero HIV incidence in young adolescents. In addition to behavioral factors, evidence shows that the risk of HIV transmission is influenced by biomedical factors (e.g., HIV testing rates, antiretroviral therapy coverage) and structural factors (e.g., national wealth, access to education, health care quality and access). However, there has been no systematic study of the impact of these factors on the trajectory of the HIV epidemic. Given the complexity of the epidemic and the interplay of contributing factors, additional data and analyses are needed to understand the relative impact of these factors. Developing methods to triangulate newly acquired data sources is crucial to enhancing understanding of the drivers of the epidemic, and to generating more robust HIV burden estimates for children and adolescents. Accordingly, the goal of this study is to address the limitations of existing methods by triangulating empirical data sources to produce more accurate and comprehensive estimates of pediatric HIV burden, including an understanding of how biomedical, behavioral, and structural factors impact the burden among children and adolescents. Our proposal advances beyond existing work by providing the first- ever estimates of new HIV infections among young adolescents, and by incorporating these results into an interactive visualization platform to help key stakeholders understand the current and projected future burden of HIV/AIDS among children and adolescents under different scenarios. This project is comprised of four Specific Aims: (1) Incorporate currently unused data sources to estimate mother-to-child transmission and HIV burden among children (0-9 years); (2) Estimate new infections among young adolescents (10-14 years); (3) Quantify the impact of biomedical, behavioral, and structural factors on HIV transmission probabilities and mortality among children and young adolescents; and (4) Forecast future burden of HIV among children and adolescents through 2040 based on current trends in incidence, prevalence, and mortality, and exposure to risk and protective factors.

NIH Research Projects · FY 2026 · 2021-02

Human pregnancy requires maternal tolerance of the fetus. Some epidemiological evidence suggests that before conception, partner-specific tolerance begins to develop through exposure to semen, which carries paternal antigens that will also be expressed by the fetus. Regulatory T cells (Tregs) play key roles in tolerance during pregnancy, but it is unclear how these cells develop in response to paternal antigens in the female mucosa. Antigen-presenting cells (APCs) are among the first cells to be exposed to paternal antigens. They sense and respond to the local microenvironment, shift maturation status, and can induce either activated or regulatory phenotypes in T cells they encounter. Semen carries a high concentration of extracellular vesicles (EV), which we and others have shown associate with, and induce markers of tolerance in APCs. We hypothesize that these vesicles deliver paternal antigens in the form of MHC molecules, and alter mechanistic pathways to generate tolerogenic APCs, which stimulate the differentiation of Tregs specific for paternal antigens. Furthermore, we predict that in pregnancies complicated by the gestational condition preeclampsia (PE), Tregs activated by antigens present in semen will be less frequent than in healthy pregnancies. In Aim 1, we will investigate how components of semen induce tolerance in APCs from vaginal and cervical tissues. We will utilize multiple methods: our recently developed 28 color APC phenotyping panel, metabolic profiling, and transcriptional analysis to define specific pathways of tolerance induction in subsets of APCs. We will also do functional studies to investigate how exposure of APCs to semen affects the suppressive function of co-cultured T cells. In Aim 2, we will examine where semen EV distribute in the mucosa after vaginal exposure. We will employ two innovative new EV tagging technologies (quantum-dot tagging and barcoded oligonucleotide tagging) to follow the penetration into tissue and in vivo trafficking of semen EV. In Aim 3, we will determine how paternal antigen specific Tregs in the decidua and blood following delivery differ between healthy pregnancies and PE. To do this, we will isolate Tregs activated by semen antigens, and assess the clonality of the activated population by T cell receptor sequencing. We hypothesize that healthy pregnancies will have greater numbers of antigen-reactive Tregs, as well as enhanced expansion of specific clones of Tregs, indicating antigen-specificity, as compared to pregnancies complicated by preeclampsia.

NIH Research Projects · FY 2026 · 2021-02

PROJECT SUMMARY/ABSTRACT Enteric fever, caused by the Salmonella serovars S. Typhi and S. Paratyphi, accounts for nearly 15 million infections and 136,000 deaths each year. We have developed a novel lethal small animal model for enteric fever using humanized mice engrafted with a functional human hematopoietic system. The ability of S. Typhi and S. Paratyphi A to cause lethal infections in humanized mice engrafted with human hematopoietic cells suggests that human macrophages are required for the pathogenesis of enteric fever and may provide a reservoir for persistent infection. A genome-wide analysis of S. Typhi in humanized mice confirms some suspected essential virulence determinants but also reveals unexpected differences between S. Typhi and non-typhoidal Salmonella serovars. Moreover, we have discovered that S. Typhi persists in cultured human macrophages by preventing apoptotic cell death, due to the absence of multiple SPI-2 type III secretion system effectors that play a central role in non-typhoidal Salmonella pathogenesis. NF-κB inhibition selectively kill S. Typhi-infected macrophages, suggesting a novel strategy for the treatment of chronic enteric fever. Recently we have also found significant differences in the responses of enteric fever and non-typhoidal Salmonella to iron deprivation, observed that the gut microbiota antagonizes S. Typhi intestinal colonization, and demonstrated important parallels between S. Paratyphi A and S. Typhi in their interactions with human macrophages and humanized mice. Our central hypothesis is that the virulence of enteric fever Salmonella serovars depends on the evasion of innate immunity and persistence in macrophages. The research plan will examine three specific aims: 1. Avoidance of Macrophage Cell Death by Enteric Fever Salmonella Serovars. Genetic and biochemical approaches will elucidate the molecular mechanisms by which S. Typhi promotes macrophage survival and assess the relevance of macrophage survival to S. Typhi virulence in humanized mice. 2. Interactions of Salmonella Typhi with the Gut Microbiota. The contributions of extracytoplasmic stress responses, microbiota antagonism and avoidance of macrophage cytotoxicity to the ability of S. Typhi to persistently colonize the intestinal tract will be investigated. 3. Salmonella Paratyphi A Virulence. A systematic analysis of S. Paratyphi A virulence determinants and its mechanisms of serum resistance, iron acquisition and macrophage persistence will be performed. The proposed studies will advance our understanding of enteric fever pathogenesis and lead to novel strategies for its prevention and treatment.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY Ambulatory assessment (AA) techniques (e.g., ecological momentary assessment, daily diaries, experiencing sampling) have provided critical tests of theories about the development of alcohol use disorder (AUD) by identifying within-person processes (such as negative or reinforcement, stress exposure, or social context) that can raise the risk for problem drinking and in turn AUD. AA methods are the leading methodological approach in the push towards personalized medicine because it provides a compelling platform for assessment, diagnosis, real-time monitoring, and just-in-time interventions. However, the current utility of AA for personalized models of AUD risk is limited because risky drinking and the risk factors for it (such as changes in moods, stress, or social contexts) change at different scales of time. In other words, even heavy drinkers may only drink a few times a week, but their emotions, stressors and social contexts change multiple times a day. Current AA methods that rely on self-report data have to sample frequently enough to be sensitive to change, long enough to observe sufficient drinking episodes, and to do so while avoiding participant burnout. Passive mobile sensing, which uses sensors (such as GPS, accelerometer, light meter, etc.) available on most smartphones, has been shown in preliminary studies to predict the probability of drinking episodes, but those studies have used relatively small samples. The present career development award aims to develop the candidate’s expertise in passive mobile sensing and the machine learning methods used to analyze passive mobile sensing data. The research proposal will analyze passive mobile sensing data collected in a large sample of regular drinking and marijuana using young adults (age 18 – 22, n = 500; 95.2% who drink), who will be followed using AA over 8 successive weekends as part of a parent R01 (DA 047247). The research goal is to identify passive mobile sensing models of risk factors for drinking (stress, social contexts, sleep, mood, and impulsive states), as well as the drinking episodes themselves. The candidate will develop expertise in these methods and models that will further the development of a research program aimed at developing person specific models of risk for AUD.

NIH Research Projects · FY 2025 · 2021-02

PROJECT SUMMARY Of the greater than 350 Primary Immunodeficiency Diseases (PID) in humans that have been identified to date, the molecular basis of a significant number (~100) of PIDs have yet to be defined. Recently, 9 children (4 now deceased) from 4 independent families were identified with severe PIDs that were linked to mutations in the NCKAP1L gene encoding for Hematopoietic protein-1 (Hem-1), a conserved hematopoietic cell-specific component the WAVE actin regulatory complex (WRC). Affected children presented with severe recurring respiratory and skin infections, failed antibody responses to pneumococcal immunization (characteristic of B cell immunodeficiency), dysregulated cytokine production, and autoimmunity. Although the cellular and molecular functions of Hem-1 orthologues in flies and worms are relatively well characterized, there is a critical knowledge gap regarding the cell specific functions of Hem-1 in the development and functions of primary immune cells. Our longterm goal is to overcome this knowledge gap by dissecting the cell-specific roles of Hem-1 in the development and functions of adaptive and innate immune cells. The objective of this proposal is to disrupt Hem-1 expression in primary murine and human B lymphocytes in a B cell-specific manner to define the roles of Hem-1 in B cell development, protective humoral immunity, and autoimmunity. Our Specific Aims are to utilize inducible B cell specific gene targeting in mice, and CRISPR/Cas9 mediated Hem1 deletion in “humanized mice”, to test our central hypotheses that B cell specific disruption in Hem1 results in: (1) impaired B cell development in part due to reduced homing and retention of developing B cell progenitors in essential lymphoid niches;(2) absent T cell independent antibody responses resulting in crippled protective immunity to influenza A virus and Streptococcus pneumoniae, important community acquired respiratory pathogens; and (3) hyper-responsive B cell signaling and T-bet driven transcriptome, resulting in increased autoantibody production. To demonstrate feasibility, we have generated innovative mouse models to emulate Hem1 PID patients including mice with a non-coding point mutation in Hem1 (Hem1pt/pt), Hem1 null (Hem1-/-) mice, Hem1floxed (Hem1fl/fl) mice, as well as Hem1 deficient ”humanized mice” which contain Hem1 deficient primary human hematopoietic cells. Based on our preliminary results which strongly support our hypotheses, we expect that the results of these studies will be highly significant and will have a high impact because they will define for the first time, the cellular and molecular mechanisms of how loss-of-function variants in NCLAP1L disrupt B cell development, signaling, and protective antibody-mediated immunity resulting in PID and autoimmunity. Because of extensive genetic heterogeneity of the 4 human PID families, limited number of patients, and concurrent infections, the development of these innovative mouse model systems are critical for dissecting the cellular and molecular mechanisms of how mutations in Hem-1 result in PID and autoimmunity, and to provide much needed platforms to develop and test therapies to treat and cure Hem1 deficient children.

NIH Research Projects · FY 2025 · 2021-02

Project Summary/Abstract The putative nucleotide sensor ZBP1 can trigger necroptotic cell death or transcriptional responses, and genetic studies indicate that this pathway is required for host defense against an array of viral pathogens. Furthermore, recent studies show that in some circumstances ZBP1 can become activated under sterile conditions, implicating endogenous cellular products as potential ZBP1 ligands. However, despite long study, the ligand responsible for activating ZBP1 and its interplay with other components of the nucleotide sensing machinery remain controversial, with double-stranded RNA, ribonucleoprotein, and viral Z-form nucleic acid species all suggested as ligand. ZBP1 shares its key nucleotide sensing domain with only one other mammalian protein, ADAR1. ADAR1 inactivates endogenous dsRNA species to limit autoinflammatory pathology, and mutations in ADAR1 in human patients lead to the severe autoimmune disease Acardi- Goutieres syndrome (AGS). We hypothesize that ADAR1 and ZBP1 compete for a common endogenous ligand, whose inactivation by ADAR1 is required to limit ZBP1 activation. In support of this idea, the pathology observed in a newly-developed mouse model of human ADAR1 mutation was fully rescued by ablation of ZBP1. This finding supports our hypothesis, and also implies that autoimmune pathology associated with loss of ADAR1 function is caused by activation of ZBP1-dependent inflammation and cell death. Using these observations as a starting point, the work proposed here will investigate ZBP1 activation and function by pursuing three Aims: First, we will use ADAR1 mutation and additional new mouse models to facilitate isolation and identification of an endogenous ZBP1 ligand. Second, we will assess the contribution of necroptosis as well as ZBP1-mediated inflammatory signaling to the pathology of an animal model of human AGS triggered by ADAR1 mutation, and test the ability of necroptosis inhibitors to ameliorate this pathology. Third, we will investigate the role of other dsRNA sensors, including MDA5 and PKR, to ZBP1 ligand formation and necroptotic pathway activation. Together this work will both reveal key aspects of ZBP1 function, and identify ZBP1-dependent necroptosis as a potentially treatable target to ameliorate AGS.

NIH Research Projects · FY 2025 · 2021-02

In natural vision, objects change appearance over time as they translate, rotate, become occluded or undergo complex transformations, e.g., during biological motion. In these dynamic environments, the visual cortex integrates information over multiple spatial and temporal scales to compute motion trajectories and represent the shape of objects. To understand how form and motion percepts are derived from such dynamic visual input, we will investigate how neuronal responses in area V4—an intermediate stage along the ventral visual pathway—are shaped by the spatiotemporal integration of time-varying visual stimuli. We will test the hypothesis that the spatiotemporal characteristics of V4 neurons are suited for tracking dynamic objects: specifically, V4 motion signals arise from object-tracking over longer spatiotemporal windows than comparable dorsal-stream areas and that V4 signals reflect form transformations at an object level rather than at the level of the local retinal image. We will leverage the percept of long-range apparent motion to probe the role of V4 neurons in motion perception (Aim 1). When a stimulus intermittently skips across the visual field with large spatial and temporal steps, it induces a strong illusory motion percept but neurons in V1 and MT of the dorsal visual stream are strikingly insensitive to the direction of the perceived motion. Psychophysical studies have argued that long-range apparent motion relies not on the dorsal stream but on higher order object tracking processes with large spatiotemporal windows in the ventral visual stream. We will conduct the first neurophysiological investigations in the awake monkey to ascertain the role of V4 in the perception of long- range apparent motion. Next (Aim 2), we will use dynamic stimuli that rotate in the fronto-parallel plane, and translate and rotate in depth, to determine whether V4 neurons encode other common dynamic object transformations (beyond long-range translation), and whether the encoding is based on a sequence of static poses, as in the inferotemporal (IT) cortex, or dynamic transformations. Finally, we will examine the encoding and perception of partially occluded dynamic objects (Aim 3). When an occluded object moves, different parts of the object are revealed over time and integration across time and multiple neuronal receptive fields is required to build an entire object representation. As animals discriminate moving occluded objects, we will study 50-100 neurons with high-density Neuropixels probes. We will use single trial population decoding methods to determine how dynamic stimulus information is integrated across the V4 network to extract object shape and the motion trajectory and how V4 contributes to psychophysical behavior. We anticipate that our results will reveal an important role for V4 in the processing of dynamic stimuli that is complementary to those of MT and IT cortex and will establish the level of internal visual representation operating in V4. Our studies will provide a deeper understanding of the neuronal basis of global motion perception and the tracking of dynamic objects—processes that are impaired in aging populations, especially those with Alzheimer’s disease.

NIH Research Projects · FY 2025 · 2021-01

Project Summary/Abstract Loss of cardiomyocytes by apoptosis and/or necrosis is a hallmark of cardiac ischemic injury, pathological remodeling, and end-stage heart failure. Although necrosis was traditionally regarded as a passive and unregulated process, emerging evidence has shifted this paradigm and identified several forms of “programmed necrosis”, including death receptor-mediated necrosis (termed “necroptosis”), mitochondria-mediated necrosis, and other regulated necrotic processes. Despite recent progress, how necroptosis is regulated in the heart remain largely unknown and preventing necroptotic cardiomyocyte death is still an important challenge. Our pilot studies identified a new K63-linked polyubiquitination (K63-Ub) dependent necroptosis signaling network that critically regulates cardiac ischemic injury and pathological remodeling. Intriguingly, the K63-deubiquitinase CYLD (cylindromatosis) was markedly up-regulated, whereas the E3 ubiquitin ligase TRAF2 (TNF receptor associated factor-2) was down-regulated, in the heart after ischemia-reperfusion injury. Our preliminary results further identified CYLD and TRAF2 as an activator and a suppressor of myocardial necroptosis, respectively. Importantly, cardiomyocyte-specific ablation of CYLD attenuated ischemic injury and adverse remodeling by inhibiting necroptosis. Acute deletion of TRAF2 in the adult heart caused dilated cardiomyopathy by promoting necroptosis, whereas TRAF2 gene transfer elicited cardioprotection. Mechanistically, we propose a new K63- Ub dependent necroptotic regulatory mechanism whereby CYLD deubiquitinates TRAF2 and TAK1, disrupts TAK1-RIP1 interaction, and promotes the necroptotic complex. As an opposing mechanism, TRAF2 acts as an E3 ligase for TAK1 to inhibit necroptosis signaling. Thus, the reversible K63-Ub (mainly non-proteolytic) constitutes a new paradigm of necroptosis signaling with important biological implications. We hypothesize that the deubiquitinase CYLD, in conjunction with the E3 ligase TRAF2, critically regulate myocardial necroptosis, ischemic injury, and remodeling, thus representing promising therapeutic targets. Aim 1 will investigate the opposing roles of K63-Ub modifying enzymes CYLD and TRAF2 in regulating myocardial necroptosis and ischemic injury in vivo. Aim 2 will define a CYLD/TRAF2 mediated, K63-Ub dependent necroptosis signaling network in cardiomyocytes. Using genetic and molecular strategies, we will investigate the opposing roles of CYLD and TRAF2 in regulating myocardial necroptosis under basal conditions and in the setting of ischemic injury. The proposed studies will provide new insights into the molecular regulation and functional significance of necroptosis in the heart, which has important translational implications, especially given our preliminary results revealing CYLD/TRAF2 as key necroptotic regulators and promising targets for ischemic heart disease.

NIH Research Projects · FY 2025 · 2021-01

PROJECT SUMMARY/ABSTRACT Diarrhea kills more than half a million children each year and is the third largest contributor to lost disability adjusted life years. While rehydration addresses the acute consequences of diarrhea, there are no interventions for diarrhea convalescence during which children are at high risk of malnutrition, lower-respiratory tract infections, and recurring diarrhea episodes. Safe and effective interventions to address the long-term consequences of diarrheal disease are urgently needed. Lactoferrin and lysozyme are milk-derived nutritional supplements that may reduce the duration of diarrheal episodes, treat or prevent underlying enteric infections, improve enteric function, and accelerate nutritional recovery. However, it remains unclear whether their antimicrobial action will translate into significant improvements in the long-term clinical and nutritional outcomes of childhood diarrhea. We propose a factorial, double-blind, placebo-controlled, randomized trial to determine the efficacy and mechanisms of lactoferrin and lysozyme supplementation in minimizing the incidence of diarrhea and promoting nutritional recovery among children recovering from diarrhea and wasting. Kenyan children aged 6-24 months who have been discharged from an inpatient or outpatient hospital stay for diarrhea, and have a mid-upper arm circumference [MUAC] <12.5 cm will be randomized to 16-weeks of lactoferrin, lysozyme, a combination of the two, or placebo. This trial will provide much efficacy, mechanistic, and feasibility data from populations most likely to benefit from these interventions.

NIH Research Projects · FY 2025 · 2021-01

ABSTRACT: Optimization of Lead BKIs for Cryptosporidiosis Therapy The broad, long-term objective of this research is to develop a therapeutic for treatment of Cryptosporidium infection. Cryptosporidium infection causes persistent diarrhea (cryptosporidiosis) that is associated with increased morbidity and mortality in children and immunocompromised individuals. Cryptosporidium is one of the most important pathogens leading to poor outcomes in <2-yo malnourished children in resource poor countries, including >3-fold mortality and strong associations with stunting and impaired neurological development. The only available therapeutic, nitazoxanide, does not work in immunocompromised individuals and has <30% efficacy in malnourished children. New therapeutics for Cryptosporidium are badly needed. We have been developing bumped-kinase inhibitors (BKIs) that selectively target Cryptosporidium calcium- dependent protein kinase 1 (CDPK1) as therapeutics for cryptosporidiosis. In our program, our leads have performed very well, demonstrating efficacy at low oral dosages in mouse, calf, and piglet models of C. parvum and C. hominis, while retaining almost all the favorable safety aspects consistent with a late lead. However, safety issues with BKI leads have stopped us from developing current late leads. We now understand the safety issues associated with late BKI leads and have found these safety issues are not inextricably associated with the structure-activity relationship (SAR) of BKIs’ efficacy against cryptosporidiosis. In this proposal, we will use the efficacy and safety SAR to help drive medicinal chemistry towards a late pre-clinical lead with safety, pharmacokinetic, and efficacy properties that can be developed for treatment in the target population of <2-yo malnourished children and immunocompromised individuals. The Aims are: AIM 1) Establish late leads for cryptosporidiosis therapy, including Subaims 1A) In vitro efficacy and safety testing, 1B) IFN- γ -KO mouse nLuc-C. parvum efficacy testing, 1C) Mouse multidosing, 7d safety testing, 1D) Determine metabolites of BKIs, and, 1E) Design and synthesize new BKIs; AIM 2) Test novel late leads in calf clinical model for cryptosporidiosis; and, AIM 3) Assess advanced late leads for late safety testing, including subaims 3A) Bone safety testing, 3B) Pregnancy/developmental/fetal safety testing; 3C) Rat and dog cardiovascular (CV) safety testing, and, 3D) Pre-GLP safety and polymorph testing. At the end of the grant period, we expect to have a preclinical lead and at least one back up molecule that is ready to move into GLP safety testing, pre-GMP manufacturing scale up, and IND filing to move towards human trials for a BKI for therapy of cryptosporidiosis.

NIH Research Projects · FY 2025 · 2021-01

A major research challenge for neurobiology is to understand the neural mechanisms that give rise to an extreme diversity of parallel visual pathways and ultimately the contributions that these pathways make to our perception of motion, form and color. For motion perception the cell types, circuits and synaptic mechanisms that mediate selectivity to the direction of moving stimuli have been intensively studied in the non-primate mammal for decades and over a dozen distinct direction selective pathways are recognized in the mouse retina together with growing evidence for similarly diverse underlying neural mechanisms. The great complexity of the visual pathways found in the mouse is mirrored in the primate, yet surprisingly the abundant direction selective ganglion cells have not been previously identified. The broad long-term objective of this new research program is to elucidate for the first time the cell types, circuits, synaptic organization and underlying cellular mechanisms for direction selectivity in the macaque monkey retina, as an ideal model for human visual processing centered around the fovea. Our proposed research plan arises from a series of discoveries that opens a door to the first detailed study of both the visual physiology and synaptic organization of direction selective circuitry in the macaque retina. In preliminary studies we have identified the primate ON-OFF direction selective ganglion cell as the recursive bistratified type and have developed new methods that permit systematic targeting of this cell type for analysis. The synaptic physiology and directional tuning of this ganglion cell type are the focus of Aim 1 where we test the hypothesis that directional selectivity in the primate is radially aligned with respect to the fovea. Second, we have developed reliable methods for targeting the starburst amacrine cell type, the key retinal interneuron in the direction selective circuit, for both physiological analysis and connectomic circuit reconstruction for the first time. Preliminary data reveal novel features of starburst receptive field structure, directional tuning and connectivity providing the focus for Aim 2 where we test new hypotheses for the cellular origins of direction selectivity and its synaptic transfer to ganglion cells. Finally, we have discovered direction selectivity in the poly-axonal spiking A1 amacrine cell type and evidence for a functional link to ON-OFF direction selective ganglion cells. The focus of Aim 3 therefore is to test the hypotheses that the A1 cells unique axonal component provides synaptic input to both starburst and ON-OFF direction selective ganglion cells, and determine the role of the A1 cells unique dendro-axonal structure in direction selectivity. In sum the broad aim is to characterize the directional tuning properties of these three cell types, and to use connectomics for the first time to determine the underlying synaptic interactions that create direction selectivity in the primate retina. Outcomes will thus have a specific impact on understanding of mechanisms motion processing in human vision and more broadly on growing applications of the primate model for the development of tools and methods for vision restoration.

NIH Research Projects · FY 2025 · 2021-01

PROJECT DESCRIPTION The goal of this international collaborative project, in response to PAR-20-027, is to characterize the genetic architecture of schizophrenia in the Xhosa population of South Africa. The three participating sites have already successfully established the infrastructure necessary to undertake the aims of this proposal. The three sites are University of Washington, Seattle (Mary-Claire King, Jack McClellan, Tom Walsh, MPIs); Columbia University, New York (Ezra Susser, PI); and University of Cape Town, South Africa (Dan Stein, PI). African populations harbor far more genetic variation than out-of-Africa populations, facilitating discovery of associations between genotypes and phenotypes. Our initial study (Gulsuner at al., Science, 2020) was the first large-scale genetic study of schizophrenia in an ancestral African population. We discovered that Xhosa individuals with schizophrenia (cases) are enriched for rare damaging mutations in genes intolerant to such mutations. The effect was particularly strong for damaging mutations in genes involved in synaptic functioning. These results extend understanding of schizophrenia genetics, specifically supporting an oligogenic severe alleles model and a role for rare damaging mutations in genes critical to synaptic signaling and plasticity. For this project, we propose to enroll an additional 1250 cases and 1250 age- and gender-matched controls, all Xhosa-speaking, bringing our total study population to 5425 participants. We will apply new genomic technology to identify previously undetectable classes of mutations likely to be implicated in schizophrenia. The genomic structure of Xhosa cases and controls will be characterized using whole genome sequencing (wgs), both short–read Illumina wgs to identify conventional classes of mutations and (in a subset of participants with existing cell lines) long-read PacBio wgs to identify structural variants of all types, mobile transposable elements, and repeat expansions. In addition, SAX v2, the African-variation-enriched SNP array developed for this project by Affymetrix, will be used to identify copy number variants (CNVs). Africa is the single most informative continent for understanding the human genome and human disorders with worldwide impact. African populations provide the most complete human reference genomes for screening candidate risk alleles for any phenotype. The whole-genome sequencing strategies used in this project allow the comparison of all classes of damaging mutations between cases and controls, including the detection of case-specific copy number variation and repeat expansions, while also providing a resource for human genomics research worldwide.

NIH Research Projects · FY 2025 · 2020-12

ABSTRACT In response to large numbers of senior center clients who suffer untreated depression and the dearth of geriatric mental health providers, we have partnered with senior center stakeholders to simplify Behavioral Activation (BA) to match the skill set of lay volunteers (“Do More, Feel Better”; DMFB). The lay delivery model: 1. makes use of existing volunteer resources that can address the insufficient workforce; and 2. has potential for being an acceptable and sustainable delivery model. However, the capacity of this model to engage the same target (increased activity) and to yield comparable clinical outcomes as professionally-delivered interventions is yet to be determined in a fully-powered trial. This Collaborative R01 proposes fully powered randomized effectiveness trial testing the effect of DMFB in comparison to professionally-delivered BA (MSW BA) on increased activity level (target) and decreased depressive symptoms. The specific aims are to: 1. Test the effectiveness of DMFB, in comparison to MSW BA, for depressed (PHQ- 9>10 and Ham-D>14) older adults (>60) on increasing overall activity level (target) and reducing depression symptoms; and 2. test whether increased activity level predicts greater reduction in depression severity and whether increased activity's impact on depression is non-inferior across conditions. Client participants will be a total of 288 older (>60 years) non-psychotic, non-demented individuals with elevated depressive symptoms from 6 Seattle, 6 New York City, and 6 Tampa area senior centers serving economically and ethnically diverse communities. Eligible clients will be randomized within senior center to either DMFB (n=144) or MSW BA (n=144). Two lay volunteers and 2 MSWs per center will provide the intervention. Our proposal responds to the 2012 IOM report which highlighted the dearth of mental health providers for older adults and the need to develop a workforce of nontraditional providers. DMFB is a streamlined BA intervention that has high potential for sustainability by making use of an untapped volunteer resource and supervision infrastructure within senior centers. Our findings will identify effective interventions for an underserved and difficult to engage population, our partners in aging services would be pleased to implement either delivery format of BA to activate depressed older adults.

NIH Research Projects · FY 2025 · 2020-12

PROJECT SUMMARY Patients with adult congenital heart disease (ACHD) live with a life-long chronic illness that has the potential to adversely affect quality of life and cause early mortality. They experience varied levels of physical and cognitive disability and face complex social and emotional challenges, in part because of impaired psychosocial development related to their disease. They report poor health-related quality of life and feel ill- equipped to effectively cope with the impact of illness and navigate difficult health care decisions. Palliative care offers great opportunity to strengthen quality of life and improve the medical care of patients with ACHD, but evidence-based palliative care is not yet incorporated in ACHD care. Resilience, harnessing personal resources to sustain physical and emotional well-being in the face of stress, is a promising target for palliative care intervention because it promotes self-efficacy and improves quality of life. The long-term goal of this award is to promote Dr. Steiner’s development into an independent physician scientist working to improve palliative care for patients with ACHD. Dr. Steiner will evaluate resilience as it relates to health-related psychosocial outcomes and adapt and test a successful resilience intervention for patients with ACHD. The “Promoting Resilience in Stress Management” (PRISM) intervention has demonstrated efficacy in improving quality of life and alleviating psychological distress among adolescents and young adults with cancer and has been successfully adapted for patients with other chronic illnesses and their parents. In Aim 1 of this proposal, Dr. Steiner will conduct a prospective cohort study to determine the association between resilience and specific patient-centered psychosocial outcomes, namely quality of life and psychological distress, in patients with ACHD. In Aim 2, she will use semi-structured qualitative interviews to understand patients’ knowledge of and perspectives regarding resilience. She will assess the acceptability of PRISM and identify ways to refine its content for this patient population. Aims 1 and 2 will inform Aim 3, in which she will conduct a pilot randomized trial to evaluate the feasibility and preliminary efficacy of the PRISM intervention to enhance resilience in patients with ACHD. To support her career development, Dr. Steiner proposes an integrated curriculum with the following learning objectives: 1) research in psychological function and behavioral interventions, 2) advanced qualitative research methods, 3) clinical trial design, implementation, and statistical analysis, and 4) grant and manuscript preparation. The proposed project activities will take place within the robust intellectual environment offered by the University of Washington, an institution with a strong commitment to the development of investigators’ academic research careers, and she will have available the vast resources of the Division of Cardiology and the Cambia Center for Palliative Care Excellence. In this rapidly-growing population of patients with ACHD, identifying ways to maximize quality of life and incorporate palliative care is essential and will ultimately encourage healthcare engagement and enhance patient care.

NIH Research Projects · FY 2025 · 2020-12

Project Summary Pain is the number one reason patients seek health care and greater than 20% of the US population is affected by chronic pain. Existing therapeutics have limited efficacy and a narrow therapeutic period, while also evoking deleterious side effects. It is therefore vital to understand pain processing in order to develop novel therapeutics to address this pressing health care crisis. According to the International Association for the Study of Pain (IASP) pain is “an unpleasant sensory and emotional experience associated with actual or potential tissue damage”. Intrinsic to the emotional suffering elicited by pain perception is the attribution of a negative valence to nociceptive stimuli. Dysregulation of aversive motivational circuits may underlie much of the suffering associated with chronic pain conditions. Hedonic valence is a measurement of the intrinsic attractiveness (positive) or averseness (negative) of a stimulus. Pain typically has a negative valence, which is normally advantageous, driving self-protective behavior. Perversely, humans can sometimes assign a positive valence to nociceptive stimuli; think pleasure from spicy foods. This implies that the neural circuits that assign negative valence to nociceptive stimuli are malleable and that pain and aversion can be decoupled. Critically, there have been limited tools to investigate how valence is assigned to nociceptive stimuli by the nervous system. Here, we propose to investigate the effects on nociceptive processing by Analgesic Screen 1 (AS1), a small molecule we discovered that reverses the hedonic motivation (movement toward or away from) of nociceptive stimuli including heat and the noxious chemical allyl isothiocyanate (AITC) in larval zebrafish, rendering these highly aversive stimuli attractive or rewarding in a dose dependent manner. Remarkably, AS1 can tune the valence of nociceptive stimuli, transforming the valence from aversive to neutral to attractive. AS1 has no previously identified target or function. We hypothesize that AS1 potentiates the activity of the dopamine reward system via D1 receptor activation by promoting release of dopamine in the presence of nociceptive stimuli. Experiments in this proposal will make use of the unique advantages of the zebrafish and mouse model systems to test these hypotheses and when completed, we will have characterized the effects of AS1 on nociception and identified upon which neural circuits AS1 acts to invert the valence of nociceptive stimuli from aversive to attractive. In Aim 1, we propose to determine the effects of AS1 on aversion evoked by nociceptive and other aversive stimuli and how these stimuli alter neuronal activity in the CNS in the presence or absence of AS1 in zebrafish. In Aim 2, we will use a comprehensive genetic approach to assess the role of D1 receptor dependent dopaminergic signaling on aversion elicited by nociceptive stimuli in the presence or absence of AS1 in zebrafish. In Aim 3, we will ascertain the effect of AS1 on nociception, place aversion, whether these effects are dependent on D1 receptor activation and where AS1 effects neuronal activity in the CNS in response to nociceptive stimuli using the mouse model system.

NIH Research Projects · FY 2025 · 2020-12

ABSTRACT Our U01 project supports NIAID’s mission to better understand, treat, and prevent infectious diseases by focusing on pre-erythrocytic malaria vaccine development. Vaccines that efficiently stop the Plasmodium sporozoite (spz) or liver stage can provide complete protection against malarial disease and will enable eradication efforts. There are currently no FDA-approved malaria vaccines for use in humans although repeated dosing with intravenously-administered attenuated spz has shown sterile protection against challenge in multiple Phase 1-2 clinical trials. Recently, CD8+ T cells that reside in the liver, namely liver resident memory T cells or TRM cells, have been identified as key cell types in protection against liver stage infection. Vaccine strategies that increase liver TRM cells and can be readily adapted to clinical use are therefore critically needed. Such vaccines could bolster CD8+ T cell immunity and may result in T cell-focused vaccines that achieve durable, high-grade protection for persons in endemic and non-endemic regions. Our laboratory has developed a two-dose vaccine that uses a DNA prime followed by an attenuated spz boost or ‘trapping dose’ that increases liver TRM cells and achieves sterile protection. This project aims to improve upon spz-based trapping by developing an orally-administered nanoparticle-based trapping vaccine. The University of Washington will collaborate with Johns Hopkins University to develop this more easily manufactured, more easily deliverable, and less expensive vaccine. In Project 1, we will define a threshold of Pf antigen-specific TRM cells needed to achieve protection using DNA prime/sporozoite trapping. In Project 2, we will optimize nanoparticles for liver- specific delivery and expression profile in hepatocytes using a variety of nanoparticle compositions, sizes, surface characteristics, and formulation strategies. In Project 3, we will evaluate the optimized nanoparticles in prime-and-trap vaccination in mice and non-human primates for safety, tolerability, immunogenicity, and efficacy. If successful, this project will deliver an optimized prime-and-oral trap vaccine rationally designed to elicit complete protection against the Plasmodium liver stage.

NIH Research Projects · FY 2025 · 2020-11

ABSTRACT Oral pre-exposure prophylaxis (PrEP), composed of tenofovir disoproxil fumarate and emtricitabine, is effective for preventing HIV acquisition, but PrEP efficacy is highly dependent on drug adherence. In several trials and implementation studies, PrEP clients have difficulties maintaining adequate adherence and persistence, and monitoring their PrEP use is challenging. PrEP providers have relied on self-reported adherence, which is often inaccurate and unreliable. The lack of an objective PrEP adherence monitoring tool has led to inefficient counseling and poor supportive care. To address these issues, we completed a randomized pharmacokinetic study to determine drug levels during controlled directly-observed PrEP. Longer-term metabolites, such as tenofovir-diphosphate (TFV-DP) (~17-day half-life), provide a more accurate picture of long-term PrEP adherence. We recently developed a novel enzymatic assay that semi-quantitatively measures the concentration of TFV-DP by measuring inhibition of reverse transcriptase, which is the cellular target of oral PrEP drugs. In this proposal, our primary objectives are optimizing the REverSe TRanscrIptase Chain Termination (RESTRICT) assay to measure drug concentrations of PrEP clients, to establish validation for CLIA criteria when implemented in a near-patient clinical lab, and to evaluate the feasibility and acceptability of using the RESTRICT assay for drug level measurement among PrEP clients and providers. We will test our central hypotheses with three specific aims: (1) to calibrate and optimize the RESTRICT assay for measuring long-term TFV-DP drug concentrations, compared to gold-standard liquid chromatography tandem mass spectrometry (LC-MS/MS) measurement; (2) to validate the RESTRICT assay for meeting established CLIA criteria to enable clinical reporting of an objective near-patient measure for monitoring long-term TFV-DP drug concentrations; (3) to evaluate the feasibility and acceptability of near-patient TFV-DP testing among PrEP clients and providers at a major PrEP clinic in Seattle. Our proposed study will be the first to validate a rapid, near-patient long-term objective measure of oral PrEP adherence. This study will also provide crucial data on the feasibility and acceptability of a novel approach for improving PrEP delivery and monitoring to prevent HIV transmission. The results of this study will develop a new tool that may help improve PrEP delivery in the US and worldwide.

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT The transition of care from acute-care hospital to skilled nursing facility (SNF) is a poorly coordinated process that exposes some of our most vulnerable patients to a high risk of complications, emotional distress, and hospital readmissions. Preventable, medication-related problems are a common result of this poorly coordinated care and are a major driver of the adverse outcomes that patients experience following hospital discharge. The standard discharge processes currently required by The Joint Commission include hospital staff completing a paper-based discharge summary and medication reconciliation form. Despite this process, up to 75% of patients admitted to SNFs have at least one medication discrepancy between the SNF admitting orders and the hospital discharge plan. In 2014, a Washington State collaborative led by the University of Washington and including stakeholders from over 50 SNFs and hospitals ─ the Improving Nursing Facility Outcomes using Real-Time Metrics (INFORM) collaborative ─ was formed to address this and other SNF-related quality of care issues. To reduce the likelihood of medication-related problems during care transitions, INFORM stakeholders developed the Pharmacy Integrated Transitions (PIT) program, which improves the standard transition process by adding: 1) a structured, synchronous “warm-handoff” between clinical teams at the hospital and the SNF, incorporating patients and their families or caregivers, by using commonly available teleconferencing; and 2) a coordinating transitional pharmacist to reconcile, adjust, and monitor medications during and after discharge from the hospital. To determine the effectiveness of the PIT program across a heterogenous healthcare delivery systems, we propose a cluster randomized trial comparing the PIT program with The Joint Commission care transition process for patients transitioning from four diverse hospitals within one health system to a network of 16 SNFs. This system-level randomized controlled trial will test the effectiveness of the PIT program in reducing medication- related problems (MRPs) and improving patient-, family-, and caregiver-reported measures of the quality of care transition. This study aims to: 1) compare the effectiveness of the Pharmacy Integrated Transitions (PIT) program and the Joint Commission transition process on MRPs, and 2) compare patient and caregiver-reported measures of quality of communication and care coordination between the PIT program and the Joint Commission transition process. This study addresses a critical evidence gap that impacts the care of millions of people and is primed for broader national uptake, as most hospitals and SNFs already employ the necessary key personnel, resources, and technology, requiring only redeployment and training.

NIH Research Projects · FY 2024 · 2020-09

SUMARY Circadian rhythms depend on the molecular transcription/translation negative feedback loop (TTL) operating in clock neurons, and on the network properties of these neurons. Among the properties that could be recruited by the circadian clock are changes in the identity of pre/post synaptic partners and/or strength of the connectivity between clock neurons, a property collectively termed as circadian structural plasticity. Our central hypothesis is that circadian structural plasticity within the central circadian clocks of mammals and Drosophila are part of the time-encoding mechanisms. We will employ mouse and fly genetics combined with state-of-the- art quantitative 3D light and electron microscopy techniques to address the extent of structural plasticity within specific neurons of the mouse suprachiasmatic nucleus (SCN) and the Drosophila circadian network. Specific aim 1 will assess how widespread structural plasticity is in the Drosophila circadian network as well as which are the functional consequences of those structural changes. We will explore the extent of circadian neuronal remodeling of subsets of PDF and non-PDF pacemaker neurons using CM and SBEM (sub-aims 1A i and ii). We will examine time-of-day dependent functional connectivity changes among clock neurons through chemogenetic GCamP6-reporting (sub-aim 1B). Sub-aim 1C will examine the behavioral consequences of impairing structural remodeling; sub-aim 1D will further investigate the molecular mechanisms underlying circadian structural plasticity. Specific aim 2 will examine the degree of circadian structural remodeling in SCN VIPergic neurons, which are an essential component of the timekeeping mechanism, through virally mediated sparse-labeling (CM) (sub- aim 2A), or serial block-face scanning electron microscopy (SBEM) with a marker that enables the analysis of dendritic ultrastructure (sub-aim 2C). Finally, we will assess if circadian oscillations in VIP neuronal processes rely on the TTL by repeating experiments in 1A in VIP-specific Bmal1-/- mice (sub-aim 2B). Specific aim 3 will explore if connectivity of VIPergic neurons changes throughout the 24-h cycle. Using GFP reconstitution across synaptic partners (GRASP), we will investigate if these connections change with circadian time through immunocytochemistry and CM analysis in fixed tissue (sub-aim 3A) as well as ex vivo in SCN slices (sub- aim 3B). We will also determine whether GRASP-detected rhythms depend on the canonical TTL by repeating experiments in 2A and 2B in VIP- or SCN astrocyte-specific Bmal1-/- mice (sub-aim 3C). Our experiments test predictions of the hypothesis that circadian structural plasticity represents a defining feature of central neuronal circadian pacemakers. Support for this hypothesis would provide a critical new perspective to understand how these pacemakers encode time at the network level. Furthermore, the experiments we propose represent a unique opportunity for research capacity building in Argentina, where the foreign principal investigator is located, and where students and postdocs will be trained in techniques that are still not fully developed in that country.

NIH Research Projects · FY 2024 · 2020-09

Cross-species transmission of pathogens is a major threat to public health worldwide and accounts for 75% of emerging human infectious diseases. Bats act as asymptomatic reservoir hosts for numerous zoonotic viruses, that are lethal in humans and for which no vaccines or specific therapeutics exist, indicating that the chiropteran immune system can control these viruses. Although there has been a recent growing interest in the peculiarities of bat innate immunity, their humoral immune response remains unexplored at the molecular level despite its known participation to fighting off pathogens. Previous studies revealed unusually fast bat B cell proliferation, compared to other mammals, and an exceptional immunoglobulin combinatorial diversity, suggesting a possible way these mammals successfully cope with an astounding diversity of viruses. We propose that the long co-evolution of bats with viruses could have led to the presence of highly specific immunoglobulin variable heavy chain segments playing a role in successfully controlling pathogens and that bat antibodies represent an untapped source of viral inhibitors. The proposed project will illuminate the role of bat humoral immunity in controlling pathogen infections, identify novel therapeutics against zoonotic viruses and guide the computational design of next-generation protein inhibitors of viral entry. This work will generate tools to combat emerging and re-emerging zoonotic viruses, including some pathogens that have not yet emerged or been discovered, and will be key to assist pandemic preparedness effort.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY In 2019, abuse of prescription and illicit opioids resulted in an estimated over 47,000 deaths in the United States. The transition from therapeutic use to destructive opioid use disorder occurs through the maladaptive activation of mesocorticolimbic circuits. Despite decades of research linking these pathways with opioids, surprisingly little is understood about how opioids modulate the brain in vivo in space and time in freely moving animals. This is, in part, driven by the inability to detect and monitor opioids at sub-second timescales. Together, these issues highlight the need for significant advancements for “in vivo precision pharmacology” as indicated specifically in this RFA-DA-20-019 NIDA program announcement. Recent developments using photoactivatable opioid compounds (optopharmacology) together with new optofluidic hardware devices show exciting promise for finally understanding the temporal characteristics of opioid signaling. However, further advances in opioid detection and activation are necessary for fully decoding how opioids modulate neural circuits in vivo. Here we address this challenge head on with a multi-disciplinary team of biochemists, neuroscientists and bioengineers. We will utilize a series of cutting-edge approaches to: 1) develop novel opioid sensors for in vivo, sub-second measures of fentanyl, morphine, and methadone, 2) demonstrate the utility of optopharmacological approaches for dissecting opioid action, and 3) apply the sensors and optopharmacological approaches to perform in vivo precision pharmacological experiments to modulate pain and reward circuits related to drug abuse.

NIH Research Projects · FY 2025 · 2020-09

Project Summary Diabetes mellitus is a major health problem; nearly half of adults in the U.S. have either diabetes or pre- diabetes. The link between adiposity and the development of Type 2 diabetes (T2DM) is well characterized, but less is known about the impact of environmental factors on risk of T2DM. Research increasingly implicates traffic-related air pollutants (TRAP) with increased risk of T2DM—especially in vulnerable urban populations, but studies thus far have wide ranges of results or have substantial methodological limitations. Other community-scale environmental factors, including aspects of the built and natural environment are also potential risk or protective factors for T2DM and may act through interactions with physical activity, diet and visceral adiposity. This study will incorporate state-of-the-art environmental exposure assessment with detailed health measures and data on potential confounders, including genetic susceptibility, to study these relationships---in a comprehensive framework—focusing on a fast-growing population at disproportionate risk of T2DM risk, through the Hispanic Community Health Study/Study of Latinos (HCHS/SOL) cohort. HCHS/SOL provides a longitudinal assessment of glycemic control along with a broad range of clinical, anthropometric, and psychosocial factors, and begin a third comprehensive clinical exam in early 2020. This proposal adds a multi-dimensional environmental assessment to the HCHS/SOL cohort, effectively leveraging the planned examination and other funded ancillary studies. Through air pollution monitoring and modeling, accelerometry data, GPS logging, genetic cluster analysis, and advanced geostatistical approaches, the proposal takes advantage of extraordinary set of available resources to measure physical activity (amount and location), individual TRAP exposure, built environment features, genetic susceptibility, and health measures concurrently. The objective of “SOLAir” is a series of hypothesis-driven, policy-relevant analyses, to understand the environmental influences on T2DM, applying a theoretical framework that includes the interplay between environmental factors and physical activity. This proposal will address the following aims: 1) to assess whether long-term exposures to traffic-related air pollutants (TRAP) increase risk of pre-diabetes and T2DM among Latinos; 2) to determine how environmental factors interact with physical activity to influence T2DM risk; and 3) to examine whether environmental factors contribute differently to sub-types of T2DM identified by genetic clusters, phenotypic characteristics, and metabolomic features.

NIH Research Projects · FY 2024 · 2020-09

Project Summary/Abstract Detection of intracellular DNA by the cGAS-STING pathway mediates host defense against DNA viruses, contributes to specific human autoimmune diseases, and can be harnessed to promote anti-tumor immunity. The presence of billions of base pairs of genomic DNA in all nucleated cells raises the question of how cGAS is not constitutively activated. A widely accepted explanation for this is the sequestration of cGAS in the cytosol, which is thought to prevent cGAS from accessing nuclear DNA. We have found that endogenous cGAS is predominantly a nuclear protein, regardless of cell cycle phase or cGAS activation status. We show that nuclear cGAS is tethered tightly by a salt-resistant interaction. This tight tethering is independent of the domains required for cGAS activation, and it requires intact nuclear chromatin. We identify the evolutionarily conserved tethering surface on cGAS and we show that mutation of single amino acids within this surface renders cGAS massively and constitutively active against self-DNA. Thus, tight nuclear tethering maintains the resting state of cGAS and prevents autoreactivity, revealing a fundamental mechanism that controls the resting state of cGAS. In this grant application, we have developed innovative new tools to define the cGAS nuclear tethering compartment, quantitate changes in cGAS disposition during DNA virus infection, and explore cGAS- associated nucleic acids before and after its activation in the context of infection and autoimmune disease. Our studies will uncover fundamental new aspects of cGAS biology, will identify new targets for therapeutic modulation of cGAS activity, and will provide new insights into the principles of self/non-self discrimination by intracellular nucleic acid sensors.