University Of California At Davis

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY In numerous flowering plant species, the male reproductive organs (anthers), which nurture pollen grains (containing the sperm, the male germline of plants) and their progenitor cells, accumulate two classes of small RNAs, i.e., the pre-meiotic (21-nt) and meiotic (24-nt) phased, small interfering RNAs (phasiRNAs). Perturbation of the phasiRNA biogenesis pathways in rice or maize causes environmentally-sensitive male sterility. There are many interesting parallels between these plant phasiRNAs and animal PIWI-interacting RNAs (piRNAs), which have also been shown essential for male fertility. However, there is a major gap in our understanding of how the plant reproductive phasiRNAs function to regulate developmental processes important for reproduction. Our long-term goal is to understand mechanistic and functional basis for the dependence on these small RNAs in plant male reproductive development. We and collaborators have described the reproductive phasiRNA pathways in many plant species. We have shown that biogenesis of the 21-nt, pre-meiotic phasiRNAs is initiated in the anther epidermis, but they accumulate in the tapetum, three layers of cells distal, indicating movement across cell layers. Yet, it is largely unknown where the 21-nt phasiRNAs and their biogenesis components localize throughout anther development, as well as what are the targets and thus precise functions of the 21-nt phasiRNAs. Our strong preliminary data has led to our focus on three Specific Aims to characterize 21-nt, pre-meiotic reproductive phasiRNAs in maize, a model for plant reproductive biology, genomics, and genetics. The aims are: (1) Characterize the spatiotemporal localization of key players in pre-meiotic phasiRNA biogenesis, using advanced imaging and single-cell RNA-seq techniques; this aim will generate a spatiotemporal map of 21-nt phasiRNAs, their precursors, microRNA trigger, and biogenesis proteins, in developing maize anthers. (2) Decipher the biogenesis of pre-meiotic phasiRNAs and their developmental roles, via the identification of novel biogenesis factors using biochemical approaches, using publicly available mutants plus CRISPR-Cas9- generated mutants to understand whether the pre-meiotic phasiRNAs are necessary for male reproductive development, and then assign functions to novel components of the pathway. And (3) elucidate the regulatory mechanisms mediated by pre-meiotic phasiRNAs, using established and new molecular approaches to identify the endogenous targets of 21-nt phasiRNAs and examine how they are regulated by the phasiRNAs. The proposed research will have a broad impact on small RNA biology by characterizing the biogenesis and functions of pre-meiotic plant small RNAs in maize anther development. The experiments will provide key insights into where and how they are produced, and what their roles are in anther cell differentiation and male fertility. Comparison of the results of this work with ongoing studies into mammalian piRNAs may shed light on the commonalities in the utilization of small RNA pathways in reproduction and gonad development.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY Signaling by cAMP plays a critical role in synaptic plasticity and memory. The b2-adrenergic receptor (β2AR) forms a unique signaling complex with AMPARs that also includes Gs, adenylyl cyclase (AC), and the cAMP- dependent kinase. Norepinephrine (NE) stimulates via β2AR -cAMP - PKA the phosphorylation of the AMPAR GluA1 subunit on S845, promoting AMPAR trafficking to the postsynaptic membrane. A primary mechanism of long-term potentiation (LTP) of synaptic transmission, which underlies learning and memory, is the accumulation of AMPAR at the postsynaptic site. Members of the Phosphodiesterase 4 family (PDE4A-D) curb cAMP signaling by hydrolyzing cAMP. Sleep deprivation stimulates expression of the PDE4A isomer PDE4A5, which impairs hippocampal long-term potentiation (LTP) and knock down of PDE4A5 rescues learning deficits. Therefore, pharmacological targeting of PDE4A5 may alleviate cognitive deficits in brain disorders. In Aim 1, we found that PDE4A5 binds with its unique ~100 amino acid long N-terminus (NT) to the SH3 domain of PSD-95. PSD-95 anchors AMPARs. For the remainder of my PhD. I propose to define the functional role of PDE4A5 and its association with PSD-95 in AMPAR receptor trafficking. For Aim 2, my postdoctoral position in another lab, I consider expanding this project by employing advanced imaging methods for cAMP signaling and super resolution analysis of AMPAR localization in postsynaptic nanodomains.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Forming and retrieving memories for objects and events is fundamental to human experience. Complementary mechanisms within the medial temporal lobe (MTL) support the formation and retrieval of memories for objects, such that the perirhinal cortex supports memory for features of individual items, and the hippocampus supports recollection of the object within the context it was originally encountered. Although most research on object memory has been conducted using visual stimuli alone, a small body of research has shown that visual objects encoded along with their characteristic sound (e.g., a dog and a bark) are better remembered later on than objects encoded only visually, or with a meaningless or non-characteristic sound. Despite this evidence and the ubiquity of multimodal stimuli within natural environments, our knowledge of the extent of multisensory influences on memory and its underlying mechanisms is very limited. My recent behavioral work showed that the benefits of multisensory encoding are based on improved recollection of the context in which those objects were encoded, suggesting that multisensory processing uniquely engages mechanisms that bind these objects to surrounding information at encoding. However, it remains unclear what features of the encoding context are better retrieved, and what neural mechanisms are modulated by multisensory processing to allow for this benefit. The proposed research addresses this gap in knowledge by collecting the critical evidence to determine the facets of memory for items and surrounding events that are influenced by multisensory processing, and the brain activity patterns and regions that are involved in retrieving such information. We hypothesize that multisensory processing at encoding improves object memory by increasing the likelihood that the object and context will be bound into an episodic memory to support later recognition of that object, rather than by enhancing memory for the individual object itself. Further, we predict that patterns of activation within the hippocampus during retrieval of multimodal objects will also carry information about the environment in which these items were encoded. To rigorously test the type of memory that multisensory processing impacts, there is a need to assess memory for unimodal and multimodal objects that are embedded within rich, naturalistic spatiotemporal context. To this end, we have developed an immersive, naturalistic encoding task within virtual reality (VR) environments, which contain controlled but animated visual and audiovisual objects. This novel approach will allow us to assess whether multisensory processing influences memory for individual objects alone or if these objects are more readily bound to their surroundings to support memory for events within context (Aim 1). Further, analyses of fMRI data will be used to determine which regions of the MTL and cortex are specifically involved in memories of multisensory objects seen in context (Aim 2). The overarching goal of this project is to advance our knowledge of how episodic memories are formed during naturalistic experiences and this work will contribute to our ability to explain and predict real-world behaviors.

NIH Research Projects · FY 2025 · 2023-09

ABSTRACT The overarching objective of this Midcareer Investigator Award is to expand the candidate’s capacity to provide excellent mentoring in behavioral diabetes prevention research to diverse early career researchers, including those from underrepresented groups (URG), underrepresented minorities (URM), and women. This mentorship program will leverage the candidate’s intensive research in behavioral interventions for type 2 diabetes prevention within high-risk maternal populations—funded since 2014—which forms a solid foundation for training activities and research opportunities. Mentorship will build upon the candidate’s track record of recruiting and mentoring diverse trainees, and a mentoring philosophy rooted in the science of social belonging, social cognitive theory, emotional intelligence, and strengths-based mentorship. Mentees will develop expertise across four scientific emphasis areas to advance success in behavioral intervention research in diverse and underserved populations: health behaviors for diabetes prevention, health equity, maternal health, and implementation science, with a focus on designing innovative interventions for healthcare system settings. Mentee professional development will emphasize key themes of navigating mentoring relationships, cultural awareness, career planning, and grant writing. The candidate will strengthen expertise in evidence-based mentoring with an emphasis three areas: creating inclusive research environments, mentoring mastery, and leadership. Using a team science approach, mentees will benefit from being embedded within a collaborative, transdisciplinary team of experts in behavioral science, diabetology, epidemiology, perinatology, bioethics, and biostatistics. Mentees will also have access to extensive resources provided by the UC Davis Clinical Translational Science Center as well as established scientific centers devoted to maternal health and healthcare research. The candidate’s active research in behavioral interventions for diabetes prevention, combined with comprehensive, evidence-based, hands-on mentorship, will foster pre-doctoral and postdoctoral mentees’ growth as independent investigators. This award will provide the candidate with the necessary training, resources, and protected time to significantly expand her mentoring practice, with the long-term goal of advancing inclusive excellence in the diabetes research workforce.

NIH Research Projects · FY 2025 · 2023-09

Prenatal Environment And Child Health (PEACH) in ECHO Abstract Few modifiable factors have been identified that improve neurodevelopmental (ND) outcomes. One such factor, supplemental folic acid (SFA), taken before and during the first weeks post-conception, reduced risk for neural tube defects (NTDs) by up to 70% in randomized trials, initiating mandatory food grain fortification and guidelines for SFA intake for people who could or had become pregnant, with subsequent increases in blood folate concentrations and reduced NTD prevalence. While generally regarded as a public health success, the impacts have not been evenly distributed across sub-populations within the US, with Hispanic populations at higher risk for NTDs, more likely to have suboptimal folate status, and less likely to take SFA. Further, the biologic mechanisms behind this protection remain unclear. Prenatal SFA and adequate folate status has since been associated with improved behavioral and language outcomes, higher executive functioning, and reduced risk for attention deficit/hyperactivity disorder (ADHD), autism spectrum disorder (ASD) and associated traits. High folate/SFA has also been shown to attenuate increased risks associated with several contaminant exposures that are more prevalent in groups that are under-represented in research and/or more likely to have sub-optimal folate status and less likely to meet SFA intake recommendations. One example includes plasticizers in personal care products that are more prevalent in Hispanic populations and are linked to increased ND risk which is attenuated with prenatal SFA. Many gaps in understanding and implementation of SFA measures need to be addressed to increase the public health impact of SFA interventions to their fullest potential: 1) How does the timing of SFA initiation and its association with blood folate differ across subpopulations? 2) What other child health outcomes are associated with prenatal SFA and blood folate? 3) How do these associations differ by the timing and dose of SFA intake? 4) How do doses and timing of SFA and folate concentrations linked with improved child outcomes differ across people with different genetic, environmental, and nutritional backgrounds? 5) Does periconceptional SFA/folate status modify associations between environmental contaminants, including novel chemicals, and ND outcomes including behavioral problems and high autistic traits? 6) What biologic pathways are modified in utero by periconceptional SFA and suboptimal folate status? 7) How do those biologic pathways relate to known ND pathways? 8) How does the conceiving partner's preconception folate status relate to placenta epigenetics and the child's outcomes? Our strong team with relevant expertise in reproductive, environmental and nutritional epidemiology, interactions, epigenetics and ND assessment, plus ample experience engaging community partners, will recruit 740 expecting families in Northern California representing diverse racial, ethnic, socioeconomic, genetic and environmental backgrounds to enrich the ECHO cohort with participants traditionally underrepresented in research to address these gaps and produce findings that will substantially inform public health policies for more effective improvement of child developmental outcomes.

NIH Research Projects · FY 2025 · 2023-09

Project Summary Dry eye disease (DED) is an ocular condition affecting about 6.8% of people in the United States, 87% of which have evaporative dry eye disease (EDED). In EDED, abnormalities in the tear film lipids cause tear film instability and increased evaporation. These abnormalities are typically caused by obstruction of the meibomian gland orifices or senescence of the acinar cells of the meibomian gland. Despite the high number of patients with EDED, current therapeutics only provide brief, palliative relief. To design therapeutics specifically for EDED, a better understanding of how the tear film is stabilized is required. To study tear film stability, the tear film of rabbits, a species with a naturally hyper-stable tear film, was compared to humans which identified four nonpolar lipids in rabbit tears that are absent from human tears. One nonpolar lipid (rNPL593) was formulated into a topical therapeutic and used as a treatment in rabbits with induced DED, which increased tear film stability and decreased damage to the cornea compared to untreated rabbits. To explore how rNPL593 increased tear film stability, we developed a novel in vitro experimental platform, which includes synthetic lipids that mimic the tear film lipid layer and the interactions of that layer with the environment and the rest of the tear film. With this model, we observed the spontaneous separation of the nonpolar lipids of the tear film. This spontaneous separation shares many characteristics with an intracellular phenomenon called liquid-liquid phase separation (LLPS). This finding conflicts with the current view of the tear film nonpolar lipid organization which consists of a lamellar arrangement of nonpolar lipids with weak, transient interactions. Instead, we propose that the different nonpolar species are mixed by blinking and that LLPS occurs during the interblink interval. This spontaneous separation destabilizes the tear film eventually leading to its breakup. Additionally, we propose that the alterations in the tear film lipids seen in patients with EDED promotes LLPS, causing their clinical signs. Exciting preliminary data suggests that the addition of rNPL593 to the in vitro model decreases the self-aggregation and phase separation of the nonpolar lipids. In this proposal, we will utilize fluorescence recovery after photobleaching and modulation of the ratios of the nonpolar lipids to further characterize the LLPS phenomenon and the addition of rNPL593 to further interrogate the effect of rNPL593. To determine the clinical efficacy of rNPL593, we will utilize a mouse model of EDED. These mice are deficient in acyl-CoA:wax alcohol acyltransferase 2 (Awat2), which causes alterations in meibomian gland secretions, decreased tear stability and secondary corneal damage, similar to moderate to severe EDED. Preliminary data suggests that Awat2 KO mice treated daily with rNPL593 ameliorated ocular surface disease and meibomian gland obstruction. This project aims to investigate how rNPL593 effects meibomian gland function in Awat2 knockout animals using transillumination meibography, transcriptomics and spatial lipidomics, laying the ground work for future clinical applications for patients with EDED.

NIH Research Projects · FY 2024 · 2023-09

Project Summary R-loops are non-B DNA structures that form co-transcriptionally upon reannealing of the nascent transcript to the DNA template strand, resulting in an RNA:DNA hybrid and a displaced single-strand of DNA. R-loops form dynamically over thousands of conserved genic loci in mammalian genomes under normal conditions. However, under conditions associated with dysfunctional RNA processing, “harmful” R-loops are thought to arise and contribute to DNA damage and genome instability phenotypes, resulting in cancer or neurological diseases. What differentiates normal and harmful R-loops remains unclear, and how harmful R-loops lead to DNA damage is not fully understood. Our group recently identified two classes of R-loops: Class I R-loops form during RNA polymerase II (RNAPII) promoter-proximal pausing at an elevated frequency, while Class II R-loops occur throughout gene bodies at moderate frequencies. Importantly, R-loop-associated genome instability phenotypes can be relieved by overexpression of RNase H1, an enzyme that specifically degrades RNA in RNA:DNA hybrids. The observation that RNase H1 primarily binds to promoter-proximal pause regions, and not gene body regions, implicates Class I R-loops as major drivers of genome instability. I hypothesize that Class I R-loops become elevated upon abnormal RNA processing, resulting in long-lasting paused RNAP polymerase II (RNAPII) complexes, transcription-replication conflicts, and DNA double-stranded breaks (DSBs) at promoter regions. To test this hypothesis, I will build upon a cellular model of defective RNA export by depleting THOC5, which is known to trigger R-loop-induced genomic instability and leverage integrative and unbiased genome- wide mapping approaches to directly measure perturbations in R-loop formation, nascent transcription, and DSB formation over time (Aim 1). I will overexpress (OE) RNase H1 in vivo and determine if it can suppress Class I R-loops, reduce paused RNAPII complexes, and lower DSBs (Aim 2). To further clarify the mechanism of genome stabilization by RNase H1 OE, I will investigate the possibility that RNase H1 activity permits the termination of paused RNAPII complexes via the XRN2 (5’-3’ exoribonuclease 2) “torpedo” pathway, thus relieving transcription-replication conflicts (Aim 3). I expect that this work will establish Class I R-loops associated with paused RNAPII complexes as a major class of genome-destabilizing obstacles, clarifying the identity of harmful R-loops and their impact on genomic stability. I also expect to reveal the molecular mechanism underlying the ability of RNase H1 to stabilize the genome, addressing largely ignored gaps in knowledge and highlighting novel roles for XRN2 in genome maintenance at promoter regions. Overall, this will fundamentally advance our understanding of the links between aberrant RNA processing, R-loop metabolism, and genome maintenance in the context of disease relevant processes, such as defects in RNA export associated with intellectual disabilities.

NIH Research Projects · FY 2025 · 2023-09

The mammalian neocortex has a remarkable ability to change over a lifetime, particularly during early development. The development of the cortex, sensory fields and their connections are dependent on the incoming sensory inputs from the sensory receptors in the periphery. This early, spontaneous sensory input, together with sensory experience from the environment shapes the neocortex to generate optimal behavior. We know from studies in humans and rodents that early loss of vision leads to massive changes in the brain; what would normally be visual and posterior parietal cortical areas contains neurons that respond only to somatosensory and auditory stimulation. This reorganized occipital cortex receives ectopic input from thalamic nuclei and cortical fields associated with somatosensory and auditory processing. The current proposal addresses several fundamental questions raised by these previous findings: 1) How does the age of onset of blindness differentially impact cortical connectivity of the medial and lateral divisions of the posterior parietal cortex (PPCL and PPCM)? 2) What are the single-neuron response properties in PPCM and PPCL, and does the age of blindness onset impact these properties? 3) What is the relationship between functional and anatomical changes PPCL and PPCM and the compensatory behaviors mediated by the spared sensory systems? In these experiments, bilateral enucleations in the highly altricial short-tailed opossum (Monodelphis domestica) will be made at two developmental milestones: 1) Prior to the onset of spontaneous activity in the retina, before retinal geniculate axons reach the thalamus, and before thalamocortical axons have innervated the neocortex; 2) When spontaneous activity in the retina is ongoing and retinogeniculate and thalamocortical axons have innervated their targets. Following enucleations, animals will be assessed at two time points allowing us to directly assess the impacts of blindness at important developmental milestones. These data can direct therapeutic interventions to compensate for the loss of vision that targets higher-order cortical function.

NIH Research Projects · FY 2024 · 2023-09

PROJECT SUMMARY/ABSTRACT Over 356,000 cases of out-of-hospital cardiac arrest (OHCA) occur in the US each year, nearly 90% of which are fatal.2 While 99.7% of OHCA in patients over age 60 are cardiac in etiology, a meaningful proportion of OHCA for patients between 20 and 59 years of age are drug-related.3 We previously used data from the NHLBI-funded Postmortem Systematic Investigation of Sudden Cardiac Death (POST SCD)4 to investigate the prevalence of occult drug-related cardiac arrest. We found that 10.3% of OHCA in San Francisco between 2011-2016 were due to occult opioid overdose5 – cases without a history or evidence of drug use that would otherwise have been attributed to a non-overdose cause, such as sudden cardiac death. While it is currently unknown whether naloxone – an opioid receptor competitive antagonist – is beneficial to patients experiencing opioid-associated (OA)-OHCA, a recent American Heart Association scientific statement identified the rigorous evaluation of naloxone’s efficacy in OA-OHCA as an important knowledge gap.1 However, no evidenced-based method to identify OA-OHCA in real-time currently exists, making the targeted-evaluation of naloxone’s efficacy in this group difficult. Our study team leveraged data from the POST SCD study to develop the NAloxone Cardiac ARrest Decision Instrument (NACARDI) – two exam-based criteria that improve the pre-test probability of occult OA-OHCA from 10.3% to 20.6%.6 These criteria were derived from patients in San Francisco County who died of OHCA in the field. The objective of this K38 grant is to answer two outstanding questions: 1) Are the NACARDI criteria valid in OHCA patients who survive to the emergency department (ED), and 2) Are the NACARDI criteria externally generalizable outside of San Francisco? My central hypothesis is that the NACARDI criteria are externally valid and generalizable for identifying patients at risk for occult OA-OHCA. My long-term goal is to use the results of this K38 to develop validated inclusion criteria for a future multi-center NACARDI-enriched clinical trial of naloxone in suspected OA-OHCA. Aim 1 of this proposal will validate the NACARDI criteria in OHCA patients who survived to the ED by calculating screening characteristics for NACARDI in a retrospective cohort of all OHCA patients who were transported to two major hospitals in San Francisco between 2011-2021, using blood and urine toxicology screens to identify OA-OHCA. Aim 2 will refine and externally validate the NACARDI criteria using data from the Canadian Sudden Cardiac Arrest Network (C-SCAN), an EMS- and coroner-based dataset of OHCA patients who died in the field in Toronto, Canada between 2018-2022. Occult opioid overdose will be identified in the C-SCAN dataset through medical examiner adjudication. The NACARDI criteria are innovative because they allow for evidence-based real-time identification of patients at risk for occult OA-OHCA, instead of relying on the current practices of retrospective evaluation via autopsy or post-arrest toxicology testing. The proposed research is significant because identifying patients during a resuscitation who are at risk for occult OA-OHCA is necessary to design an ethical and successful clinical trial of naloxone in this population.

NIH Research Projects · FY 2024 · 2023-09

Prenatal Environment And Child Health (PEACH) in ECHO Abstract Few modifiable factors have been identified that improve neurodevelopmental (ND) outcomes. One such factor, supplemental folic acid (SFA), taken before and during the first weeks post-conception, reduced risk for neural tube defects (NTDs) by up to 70% in randomized trials, initiating mandatory food grain fortification and guidelines for SFA intake for people who could or had become pregnant, with subsequent increases in blood folate concentrations and reduced NTD prevalence. While generally regarded as a public health success, the impacts have not been evenly distributed across sub-populations within the US, with Hispanic populations at higher risk for NTDs, more likely to have suboptimal folate status, and less likely to take SFA. Further, the biologic mechanisms behind this protection remain unclear. Prenatal SFA and adequate folate status has since been associated with improved behavioral and language outcomes, higher executive functioning, and reduced risk for attention deficit/hyperactivity disorder (ADHD), autism spectrum disorder (ASD) and associated traits. High folate/SFA has also been shown to attenuate increased risks associated with several contaminant exposures that are more prevalent in groups that are under-represented in research and/or more likely to have sub-optimal folate status and less likely to meet SFA intake recommendations. One example includes plasticizers in personal care products that are more prevalent in Hispanic populations and are linked to increased ND risk which is attenuated with prenatal SFA. Many gaps in understanding and implementation of SFA measures need to be addressed to increase the public health impact of SFA interventions to their fullest potential: 1) How does the timing of SFA initiation and its association with blood folate differ across subpopulations? 2) What other child health outcomes are associated with prenatal SFA and blood folate? 3) How do these associations differ by the timing and dose of SFA intake? 4) How do doses and timing of SFA and folate concentrations linked with improved child outcomes differ across people with different genetic, environmental, and nutritional backgrounds? 5) Does periconceptional SFA/folate status modify associations between environmental contaminants, including novel chemicals, and ND outcomes including behavioral problems and high autistic traits? 6) What biologic pathways are modified in utero by periconceptional SFA and suboptimal folate status? 7) How do those biologic pathways relate to known ND pathways? 8) How does the conceiving partner's preconception folate status relate to placenta epigenetics and the child's outcomes? Our strong team with relevant expertise in reproductive, environmental and nutritional epidemiology, interactions, epigenetics and ND assessment, plus ample experience engaging community partners, will recruit 740 expecting families in Northern California representing diverse racial, ethnic, socioeconomic, genetic and environmental backgrounds to enrich the ECHO cohort with participants traditionally underrepresented in research to address these gaps and produce findings that will substantially inform public health policies for more effective improvement of child developmental outcomes.

NIH Research Projects · FY 2025 · 2023-09

Type 1 Diabetes (T1D) is caused by autoimmune destruction of insulin-producing pancreatic beta cells. T1D therapies have largely failed in human clinical trials and thus an urgent need exists for targeting novel immune pathways. We have exciting data showing that a TLR4/MD2 agonistic antibody (TLR4-Ab) permanently reversed T1D in 71%, and had a significant clinical effect in 90%, of acutely diabetic non-obese diabetic (NOD) mice. In our recent Diabetes paper, we showed that TLR4-Ab can mobilize and activate myeloid-derived suppressor cells (MDSC) in vivo, that suppress T cells and ameliorate acute T1D upon adoptive transfer. We showed that TLR4- Ab induces TLR4/MD-2 sequestration in endosomes, unlike the canonical TLR4 agonist LPS (which cannot reverse T1D); however, the mechanism by which TLR4-Ab reverses T1D remains unclear. We have now produced anti-human TLR4 antibodies, allowing us to apply these findings to human T1D. Our combined expertise in autoimmunity/T1D (Ridgway), molecular biophysics/Immunology (Herr) and human T1D (Dolan) is well suited to perform these studies. We will pursue three aims: Specific Aim 1. Mechanism of reversal of T1D by TLR4-Ab-induced MDSCs. TLR4-Ab endosomal sequestration may induce prolonged signaling via the TLR4-mediated TRIF pathway, which protects from T1D (2015, Chervonsky et al.). We show that immobilization of TLR4-Ab on a plate, which prevents endosomal sequestration, eliminated TLR4 signaling. Therefore, we hypothesize that TLR4-Ab-induced TLR4/MD2 endosomal sequestration causes sustained TRIF endosomal signaling that induces APCs to undergo MDSC maturation, inducing immune regulation and reversing T1D. Specific Aim 2. Mechanistic role of Fc structure in TLR4-Ab reversal of T1D and cell suppression. While antibody F(ab) structure determines specificity, the Fc region contributes to antibody function through receptor binding and glycosylation. TLR4-Ab is an IgG3 isotype, which has the longest hinge region and increased glycosylation sites. IgG3 antibodies also form cryoglobulins, impacting avidity and internalization. Our preliminary data show that TLR4-Ab F(ab) and F(ab’)2 fragments elicit decreased NFκB signaling compared to full-length TLR4-Ab. This shows a critical role for Fc structure in TLR4-Ab function. Therefore, we hypothesize that the IgG3 Fc portion of the TLR4-Ab is critical to its tolerizing function and we will test this with immunological and molecular biophysical approaches. Specific Aim 3. Testing therapeutic effects of a novel panel of human anti-TLR4 antibodies on human T1D APCs Since the TLR4/MD2 pathway is strongly evolutionarily conserved, we have developed agonistic human recombinant TLR4-Ab (hTLR4-Ab). We show here that these hTLR4-Abs bind to, and activate TLR4/MD2. Our hypothesis is that hTLR4-Ab treatment will induce MDSCs from myeloid precursors and that these huMDSCs will suppress human T-cell proliferation and activation. These studies will characterize novel innate immune mechanisms by which TLR4-Ab treatment can reverse acute T1D, and our studies on the novel human TLR4 antibodies are the first step in translation to human T1D.

NIH Research Projects · FY 2025 · 2023-09

This K01 award resubmission outlines an intensive five-year research career development program focused on multilevel behavioral intervention and dissemination and implementation science research to increase and sustain uptake of the human papillomavirus (HPV) vaccine. Supporting the candidate's career and research goals is a mentoring team comprised of experts in behavioral intervention research, advanced biostatistics methods, and dissemination and implementation science as well as a clinical expert panel. Completion of the candidate's training plan and research study will provide the candidate with the experience, skills, knowledge, mentoring and pilot data needed to become a leading independent investigator in developing, implementing and evaluating multilevel behavioral interventions to reduce the burden of cancer in racial/ethnic minority groups, starting with increasing HPV vaccination rates. The HPV vaccine is a major public health breakthrough and is the optimal primary prevention strategy against HPV-associated cancers. Widespread use of the HPV vaccine is expected to reduce the cancer burden across all racial/ethnic group, as populations of color are disproportionately affected by HPV- associated cancers. Despite CDC recommendations and the public health implications of full vaccination coverage, HPV vaccine uptake among adolescents remains well below the Healthy People 2030 goal of 80%. The National Immunization Survey reported that in 2019, only 54.2% of U.S. adolescents aged 13-17 were up to date with the series. Preliminary data and a review of the literature suggest multilevel interventions that address at least two levels influence show promise in increasing HPV vaccine uptake. The objective of this proposal is to develop, implement and evaluate a multilevel (parent, primary care team, and clinic) intervention aimed at raising adolescent HPV vaccination rates to the Healthy People 2030 goal of 80% for boys and girls using a parallel group cluster randomized trial design. This study will assess the effectiveness of the multilevel intervention on increasing the rates of HPV vaccination among patients aged 9-17 of a primary care network and will identify barriers and facilitators for broad dissemination and implementation. This will be accomplished through the following aims: 1) Refine and finalize a parent, primary care team and clinic ML intervention to increase uptake and completion of the HPV vaccine series among adolescent patients of a primary care network; and 2) Conduct and evaluate the effectiveness and sustainability of the ML intervention. The proposed research is significant because it will contribute to the emerging field of HPV vaccination multilevel intervention research and addresses the need for more scientifically rigorous, evidence-based interventions that can be disseminated and implemented on a large scale. Successful adoption of widespread HPV vaccine coverage programs will reduce racial/ethnic disparities in HPV-associated cancer by ensuring that the next generation of Americans do not experience unnecessary deaths due to HPV-related cancers.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract An urgent need exists for new methods to rapidly prepare complex molecules with the potential to become new drugs. There is a widening gap in both the accessibility of complex core structures that are difficult to exploit and in the availability of core structures that are not already the subject of numerous patents. This gap will be addressed by identifying new synthetic methods that achieve the dual goals of enabling efficient access to useful cores and exploring previously inaccessible "chemical space." The long-term goal of our research program is to understand the reactivity of unstabilized metal carbenes. The objective of this application is to explore the ability of rhodium donor-donor carbenes to engage in a wide variety of useful new applications. The central hypothesis is that appending two "donor" groups to a carbene opens up new avenues of reactivity for organic chemistry. This hypothesis is supported by preliminary results regarding a) the unique ability of donor/donor carbenes to engage in highly enantioselective C–H insertion reactions and b) a new mild and catalytic process for the formation of reactive dienes for cycloaddition reactions! Small molecules comprise the vast majority of treatments for both acute and chronic diseases in both the developed and developing world. Research in this application will lay the groundwork to save lives and enable the next generation of pharmaceutical discovery by focusing on three areas of research. First, we will explore new C–H insertion reactions the enable the synthesis of complex carba- and heterocyclic structures, culminating in an efficient synthesis of polycyclic alkaloid natural products that will be studies for the ability to induce neuroplasticity for the treatment of PTSD, depression and other neurological disorders. Second, we will explore new reactions of rhodium donor-donor carbenes that branch out form C–H insertion into new reactions that access new chemotypes. Finally, we will, for the first time, build a QSSR model that will enable the design, synthesis and exploration of new catalysts that are custom-designed to enhance the reactivity of donor-donor carbenes. The proposed approach is innovative because it is based on a new methodological platform that enables previously inaccessible chemical reactivity. This research is significant because it will change the way synthetic chemists approach targets while at the same time opening up new vistas for discovery of useful molecules for medicine and other fields. Ultimately, the discoveries emerging from our research will represent a vertical step in the assembly of molecular architectures that will translate into new medicines to address our society's most pressing health challenges.

NIH Research Projects · FY 2025 · 2023-09

Overall PROJECT SUMMARY / ABSTRACT The Center for Somatic Cell Genome Editing in Nonhuman Primates will fully support studies that will advance the somatic cell genome editing field, and significantly contribute to the future translation of new and emerging technologies for the treatment of human diseases. The Nonhuman Primate Testing Center brings together a robust team of multidisciplinary investigators with partnerships that capitalize on major strengths at UC Davis including the California National Primate Research Center, Genome Center, and Clinical and Translational Science Center. The program will provide unique state-of-the-art capabilities for investigators to conduct high-quality research in nonhuman primates. The Nonhuman Primate Testing Center leverages a supportive framework that has demonstrated a long-standing commitment to outreach and providing collaborative opportunities to investigators nationwide to advance their translational preclinical research goals, including progression to human clinical trials. Through this powerful and proven infrastructure, the Center for Somatic Cell Genome Editing in Nonhuman Primates will address the following Specific Aims: (1) Provide an administrative and organizational structure to advance somatic cell genome editing studies targeting a range of human diseases in all age groups, (2) Partner with investigators to accelerate their translational research objectives, and (3) Ensure synergism and sharing across the small and large animal testing centers and transparent interactions with the research community and the NIH. Our robust multidisciplinary team is poised to continue our national role and service to the research community by bringing the depth and breadth of opportunities available to support investigators, and safely advance somatic cell genome editing technologies to new treatments for human diseases.

NIH Research Projects · FY 2024 · 2023-09

Project Summary Fidelity of the gene expression program, involving precise temporal and spatial regulation, is critical to cellular function and organismal development. As such, perturbations or disruptions in the mechanisms governing gene expression are often implicated in disease. Neurons appear particularly sensitive to disturbances in gene expression with disruptions in RNA metabolism common in neurological diseases. For example, mutations in various RNA binding proteins (RBPs) involved in nuclear mRNA processing and export are linked to neurodevelopmental disorders (NDDs). One such complex with mutations in multiple subunits tied to NDDs is the THO complex. THO is a highly conserved complex with diverse roles in transcription and mRNA processing, with models suggesting it serves as an interaction hub for coordinating nuclear processing events. Despite these connections to NDDs and centrality to mRNA metabolism, there is still much not known regarding the dynamics of the THO complex and how disruptions in individual components impact gene expression. This knowledge gap necessitates work characterizing the role of the THO complex in gene expression. The central hypothesis of the work proposed here is that THO is critical to coordinating nuclear mRNA metabolism and disruptions in complex function that include NDD-linked mutations lead to altered dynamics of mRNA processing and gene expression outcomes. To address this hypothesis, this project will investigate the dynamics of the THO complex and model impacts of THO mutants in S. cerevisiae. Given the structural and functional conservation of the THO complex, budding yeast is a powerful model system to address this hypothesis utilizing innovative methods that would be extremely time intensive, expensive, and technically challenging in other systems. Specifically, this project employs a novel live cell imaging approach which can track recruitment of RBPs to a transcriptionally active locus over time. Utilizing this technique in combination with other approaches, THO function will be characterized in Aim 1 by temporally characterizing co-transcriptional recruitment of RBPs to a transcriptionally active locus. The outcome of these efforts will be a quantitative framework for recruitment of the THO complex relative to other RBPs. In Aim 2, the impact of complete gene deletions and disease-linked THO mutants on co- transcriptional RBP recruitment dynamics and global gene expression will be assessed. This will clarify the role of THO subunits in mRNP assembly, identify how disruptions in THO subunits shape global gene expression, and functionally characterize a subset of NDD associated point mutants. Completion of these aims will provide models that can be used to generate informed hypotheses for mechanisms by which THO complex mutations contribute to neurological disease. This information is expected to provide a critical foundation for advancing our understanding of clinically identified mutations in RBPs associated with NDDs.

NIH Research Projects · FY 2025 · 2023-09

Project Summary/Abstract Non-specific/non-pharmacologic therapeutic effects can improve health outcomes, however, little is known about how to maximize these effects, particularly for effects related to patient-clinician interactions. These effects are especially important in treating chronic pain. Preliminary studies indicate that both increased visit length and augmented visit content (designed to elicit a holistic understanding of the individual) enhance non- specific effects related to patient-clinician interactions, but it is unclear which of these factors is more important or if their interaction is synergistic. This proposal’s long-term goal is to advance the science of non-specific therapeutic effects related to patient-clinician interactions to improve health outcomes. Its objective is to quantify the relative effects of, and mechanisms by which, increased visit length and augmented visit content affect patient-clinician interactions and health outcomes in knee osteoarthritis (OA). Knee OA is an ideal condition for studying the effects of visit length and content on patient-clinician interactions and health outcomes because it is a leading cause of chronic pain and disability in older adults, many common treatments have poor safety profiles, and clinical trials of medications for knee OA suggest that non-specific therapeutic effects account for a major component of treatment effects. The central hypothesis is that both increased visit length and augmented visit content will increase the non-specific treatment effects associated with diclofenac gel (a standard topical treatment for knee OA-related pain). Project specific aims are: 1) To determine whether increased visit length and/or augmented visit content increase the non-specific treatment effects of diclofenac gel on knee OA pain and function; 2) To determine how visit length and content affect patient and clinician behaviors and physiology during patient-clinician interactions; and 3) To determine whether patient COMT genotype modifies the effects of increased visit length and augmented visit content on knee pain. The approach is innovative in that there are few randomized trials targeting non-specific effects associated with patient- clinician interactions, and none that directly test the effects of visit length and content on health outcomes or use physiologic measures to assess patient-clinician interactions. The proposed study is significant because it will determine the relative contributions of visit length and content on health outcomes in knee OA and advance understanding of potential clinical, behavioral, physiologic, and genetic mechanisms through which non- specific effects impact health outcomes. This project advances a line of research focused on improving outcomes in knee OA by developing and testing strategies to modify clinical care delivery to maximize non- specific therapeutic effects associated with patient-clinician interactions. It will also help develop new research approaches for studying patient-clinician interactions. Insights gained from this project will also be relevant to other chronic and painful conditions, may help reduce polypharmacy among older adults, and will advance understanding of the roles of non-specific therapeutic effects in health outcomes.

NIH Research Projects · FY 2025 · 2023-08

ABSTRACT The mission of our collaborative Bridges to the Baccalaureate (B2B) program is to develop a diverse pool of biomedical scientists by supporting research-oriented community college students to successfully bridge to UC Davis (UCD) and complete a bachelor’s degree in biomedical sciences in normative time. Our program entails a collaboration between UCD and a nearby community college American River College (ARC). The proposed training plan includes five key elements: (1) Active outreach to STEM-focused trainees from diverse backgrounds at ARC; (2) Trainee exposure to and engagement with research at ARC; (3) An intensive Summer Bridge research experience at UCD prior to transfer; (4) Placement into research labs beginning with the trainees' first quarter at UCD; and (5) A multifaceted retention plan. In addition to research experience, students in the B2B program will also receive administrative, academic, and career-skills support through our coordination with AvenueB and AvenueE, programs at UCD that recruit, support and retain biological science and engineering science-oriented underrepresented, low income, first- generation and women college transfer students (i.e., historically excluded populations). At ARC, a course-based undergraduate research experience (CURE) embedded in the first semester major’s biology class will connect interested students to research opportunities available for units as independent studies. The administration of these independent study experiences will be led by ARC faculty and technicians with UCD faculty and students providing supplemental programming and support. These efforts will expand the opportunities for the diverse student population at ARC to engage in science and research early in their academic journey. At UCD, a Summer Bridge research experience will immerse students in a research environment that mimics key elements of undergraduate research and provides training in robust experimental design, reproducibility, data analysis, responsible conduct in research, an introduction to computation, and career development. Day-to-day mentorship will be provided from staff, faculty and advanced undergraduates and will focus on building our trainees’ sense of community and belonging. Following the summer bridge program, B2B trainees will be placed in faculty research labs under the mentorship of one of the B2B program’s selected faculty mentors and transition to existing training programs (e.g. NIH- MARC) for extra support. Demonstrated success of the partnership between ARC and UCD will lay the groundwork for developing similar collaborations with other community colleges.

NIH Research Projects · FY 2024 · 2023-08

Project Summary Averting future global pandemics requires surveillance programs that can quickly and accurately identify potential new outbreaks worldwide. Mobile health technologies (mHealth) are a promising strategy for collecting data on infectious diseases in remote, underserved settings at the nexus of human and wildlife interactions and zoonotic disease spillover. As the use of mHealth technologies has grown in high income countries, they have also been increasingly investigated in low- and middle-income countries, including Uganda. While potentially promising, these interventions are not one size fits all but instead dependent on a complex interplay between the technology itself and local ethical, cultural, and other norms. Core mHealth technologies such as mobile data collection, cloud storage, and geolocation may be entirely novel for some populations. Differences in understanding of these newly introduced mHealth technologies may pose significant ethical concerns around respect, consent, confidentiality, and beneficence, especially in cultures within Sub Saharan Africa that value a more collectivist social identity. This project will expand research conducted by the parent award which is developing a community-based human and wildlife disease surveillance and outbreak detection system in Southwestern Uganda to investigate bioethical considerations associated with implementation of this platform. Importantly, we will identify key differences in understanding of mHealth concepts between a predominantly oral tradition society as well as a society that places different value on communal vs. individual benefits compared with western societies. Using focus groups, including pertinent subgroups (i.e., community members and leaders, local alternative medicine leaders, park rangers, and poachers) and key representatives from various linguistic, ethnic, and other stakeholder groups, we will develop a locally relevant research ethics framework, including the fundamental concepts of respect, consent, confidentiality, and beneficence. Our study team will then develop novel or select existing, culturally relevant engagement procedures (i.e. familial, community, multimedia, and/or step-wise procedures) which, along with results from the focus groups, will undergo member checking to evaluate accuracy and acceptability. Through a series of bioethics workshops incorporating local western-trained and alternative medicine leaders along with international bioethicists, we will review key outcomes learned through focus group discussions and design a guiding framework for ethically informed implementation of mHealth systems in the Ugandan context. Combined, these products will help local and international partners achieve ethical, sustained, and effective mHealth surveillance systems in similar settings.

NIH Research Projects · FY 2025 · 2023-08

Project Summary Averting future global pandemics requires surveillance programs that can quickly and accurately identify potential new outbreaks worldwide. Mobile health technologies (mHealth) are a promising strategy for collecting data on infectious diseases in remote, underserved settings at the nexus of human and wildlife interactions and zoonotic disease spillover. As the use of mHealth technologies has grown in urban settings, it has also begun to expand into rural settings but with additional challenges such as reliable availability of data service and geolocation services. Utilizing a One Health approach, this project will develop new geolocation algorithms enabling mHealth technology use in remote communities and develop an integrated mHealth system that allows healthcare providers to monitor outbreaks of disease in both humans and wildlife simultaneously. The goal of this surveillance system is to improve detection of zoonotic spillover and help to answer decades long questions about the ecological scenarios supporting zoonotic disease emergence, enabling forecasting that could help prevent future outbreaks. During the R33 phase of this project we will: (1) refine, expand and evaluate the tiered mHealth surveillance platform for improved longitudinal acute febrile illness case identification; and (2) utilize the system to investigate the epidemiology of zoonotic virus exposures associated with wildlife contact. This project will provide an optimal scenario for rigorously evaluating the benefits of adding an mHealth component to long term zoonotic disease monitoring, benefiting the sustainability of all developed platforms.

NIH Research Projects · FY 2026 · 2023-08

Project Summary Despite many decades of research in early identification of autism, there remain lengthy gaps between parents’ first concerns and formal diagnosis and subsequent access to specialized services. Challenges in reducing this gap include long waitlists, lack of specialized providers in many communities, and the lack of validated screening tools for infants under 18 months of age. Due to methodological challenges in recruiting sufficient numbers of infants with early specific concern for autism in any one geographic area, most studies of early development have focused on infant siblings of children with autism. Telehealth offers the opportunity to expand the scope of early identification studies and conduct the crucial foundational work needed to determine the developmental trajectories and outcomes of infants with early developmental concerns in community settings throughout the United States. We have previously demonstrated the initial feasibility of this approach in our preliminary work developing the Telehealth Evaluation of Development for Infants (TEDI; R21 HD100372 and R21 HD 105161, PI Talbott). Behavioral measures obtained via TEDI are reliable, valid, and highly satisfactory to families. Importantly, we have also found that the majority of infants in our sample have elevated scores on early measures of autism traits, developmental challenges in communication, language, and motor skills, and elevated likelihood of autism relative to general population norms. This preliminary work indicates the need for more thorough examination of this group of infants. We propose to prospectively follow a group of 100 infants ages 6 – 12 months with early parent concerns. We will evaluate them using the TEDI telehealth protocol at four visits each 3 months apart. At 36 months, we will conduct an outcome visit via telehealth to generate clinical best estimate outcomes. The project will address 3 specific aims. In Aim 1, we will determine the proportion and predictors of autism outcomes. Under Aim 2, we seek to characterize the development of a community-based sample of infants later diagnosed with autism by examining differences in developmental trajectories between outcome groups, as well as predictors of developmental outcomes across groups. Finally, in Aim 3, we will identify best practices for supporting family engagement and satisfaction with telehealth-based assessments, and the cultural appropriateness of the TEDI for diverse communities, which will directly support the implementation of telehealth screening and assessment in community settings beyond the COVID-19 pandemic. Successful completion of these aims has the potential to significantly increase families’ access to specialized evaluations and increase the capacity for early identification of infants in need of services. It will also lay the groundwork for future efforts to conduct screening and intervention trials and may ultimately help to increase access to high-quality interventions and improve the developmental outcomes of many more underserved children with autism and other NDD’s.

NIH Research Projects · FY 2025 · 2023-08

Abstract Studies have established the critical roles of a superfamily of phosphodiesterases (PDEs) in hydrolyzing cAMP and its subcellular distribution. We aim to explore the role of PDEs in differential regulation of the cAMP signals at the plasma membrane and in the nucleus. Specifically, we will uncover the regulation of the nuclear cAMP signals under the CNS β2-adrenergic receptor (b2AR) stimulation in hippocampal (HC) neurons during learning and memory. Interestingly, PDE4 inhibitors benefit learning and memory in rodents and humans, indicating that PDE4 may control the βAR-induced cAMP signal in the nucleus. PDE4D isoforms are associated with β2AR to fine-tune subcellular cAMP-PKA signals in fibroblasts and myocytes, and PDE4D5 is associated with an AKAP95/PKA complex in the nucleus. Our preliminary data show that βAR stimulation promotes the nuclear export of PDE4D5 in HC neurons. This relocation of PDE4D5 depends on endosome GRK-phosphorylated b2AR and is critically necessary for delivering cAMP signals into the nucleus. Besides the GRK-phosphorylated b2AR, we have recently characterized another distinct subpopulation of b2AR that are PKA-phosphorylated and located at the PM after agonist stimulation in HC neurons. We hypothesize that two b2AR subpopulations synergistically promote nuclear cAMP signal by arrestin3-dependent export of PDE4D5 from the nucleus. We will characterize the mechanisms underlying the PDE4D5-dependent regulation of nuclear cAMP signaling and gene expression and how the regulation may affect the b2AR signaling in learning and memory (Aim 3). This proposed study will define the role of PDE4D5 in nuclear cAMP signaling, gene expression, and learning and memory, which not only offer new strategies to treat disorders associated with the CNS adrenergic system but also offer an example to study many other Gs-coupled receptors, such as dopamine receptors in the regulation of gene expression.

NIH Research Projects · FY 2025 · 2023-08

Project Summary/ Abstract Improving quantification at high spatial resolution is driving technology developments in nuclear imaging. Positron emission tomography (PET) scanners and single photon emission computed tomography (SPECT) use radiation detectors, which performance can be improved when the image formation process is understood. Optimizing optical mechanisms such as scintillation or prompt photon emission at the core of these detectors is essential to advance the technology and is the focus of this proposal. Due to the complexity of these phenomena and the difficulty to disentangle their components experimentally, research on radiation detector optics relies on simulations integrating high and low energy physics. No simulators currently offer the speed and fidelity necessary to understand image formation from the detector to the system. We propose to develop a radically different AI-based high-fidelity optical modeling framework, allowing multidimensional optical information to be rapidly generated, collected, and processed at the system level. By replacing individual photon tracking with a deep-learning approach, we expect to accelerate simulations by several orders of magnitude in systems involving extensive optical photon tracking, such as large detectors or fast timing detectors. We organize this R01 proposal in three specific aims focusing on implementing this framework in the Geant4/GATE simulators and applying it to time-of-flight (TOF) PET. GATE is a free opensource platform at the forefront of nuclear medicine simulation. We have a track record of developing optical modeling strategies and created the LUT Davis model. This grant will design and implement the optiGAN, a custom generative adversarial network (GAN) that will be trained with high-fidelity simulations based on the LUT Davis model. New light transport features and crystal-photodetector interface models mixing particle and wave optics will be developed and integrated into the optiGAN (Aims 1 and 2). We have extensively studied and developed Cerenkov-based radiation detectors, one of the prompt photon emission mechanisms most pursued to achieve timing resolution below 50 ps and unlock reconstruction-free PET. To develop prompt photon-based PET systems several questions must be solved: how to improve the production and transport of these prompt photons with new materials, how to improve their collection, and how to harness the prompt photon information for fast coincidence timing. These questions motivate the development of innovative detector optics and algorithms for TOF PET, which we will investigate with the optiGAN together with experimental and theoretical work (Aims 2 and 3). The objective of this grant is to enable a leap in detector technology through unprecedented simulation capabilities and new strategies to leverage fast detectors in nuclear imaging scanners. Developing detector technology now that enables the next generation of scanners to respond to clinical and research needs of nuclear medicine is essential, as the integration of these advances requires years before commercialization.

NIH Research Projects · FY 2026 · 2023-08

The human fetal brain consumes up to 60% of the body’s oxygen and energy consumption, despite making up ~13% of body mass. When the demand for oxygen in the placenta and developing brain exceeds its supply, hypoxia is induced, followed by changes to mitochondrial respiration, protein translation, and oxidative stress. Oxidative stress and epigenetic mechanisms within the placental-brain axis act at the interface of genetic and environmental risk factors in autism spectrum disorders. Using placental samples from a prospective high-risk cohort, we recently identified and named a novel gene NHIP (neuronal hypoxia inducible, placenta associated) and demonstrated its epigenetic, genetic, and transcriptional association with autism. NHIP is transiently expressed in response to hypoxia and neuronal differentiation, two examples of elevated oxidative stress. NHIP encodes a previously undiscovered micropeptide that localizes to the nucleus and is predicted to be neuroprotective, based on the lower expression of NHIP in placenta and brain samples from autism compared to control. The predicted structure of the NHIP peptide is an amphipathic helix that has similarity to a 9aaTAD motif found in transcriptional activation domains of many DNA binding proteins. We propose to test the hypothesis that NHIP acts as a competitive inhibitor of multi-protein complexes, thereby protecting developing and differentiating neurons following transient waves of hypoxia. Because NHIP is an “undiscovered protein” whose function had not been described before our recent study, this proposal will focus on the major research questions that are critical for determining the therapeutic relevance of NHIP. Specifically, what is the function of NHIP in neurons and brain, how is it regulated in response to hypoxia, and is it protective of neuronal oxidative stress? We propose three specific aims using well-characterized in vitro and in vivo models, including an inducible human neuronal cell line (LUHMES) engineered for NHIP transcript or peptide loss, human brain extracts with known NHIP genotype and expression levels, and mouse brain following NHIP peptide administration and/or hypoxia. Aim 1 will determine the molecular mechanisms of NHIP function and examine both protein-specific and global cellular impacts of NHIP loss. Aim 2 will determine how NHIP is transcriptionally responsive to hypoxia-induced oxidative stress by identifying the transcription factors and their genetic and epigenetic requirements for binding to the NHIP promoter and enhancer. Aim 3 will determine if exogenously delivered NHIP/NHIP protects neurons and embryonic neural precursor cells from hypoxia-induced oxidative stress. Together, the results from these proposed studies will provide the first functional characterization of NHIP, an understudied micropeptide that is associated with resilience to autism spectrum disorders. The potential impact of these results will be a potential therapeutic small molecule that could be used in early intervention therapy for autism and other neurodevelopmental or hypoxia-related disorders.

NIH Research Projects · FY 2025 · 2023-08

Chloropicrin (CP) and phosgene (PG) are widely available chemical threat agents, yet the mechanisms of in vivo acute toxicity and long-term pathophysiologic impacts are not well understood. CP, which is in current use as a soil fumigant to sterilize fields before planting high value crops and is widely available. CP is known to cause a biphasic death response characterized by lung edema that occurs either in the first 24 hrs or after 8-10 days. This suggests immune cell mediated and tissue repair responses are key to determining outcomes. However our new data also suggests that there is conducting airway and olfactory epithelial injury in the acute phase of toxicity. The cellular targets and the LC50 for mice is not firmly established. The pathogenesis is likely through tissue damage from binding of CP/PG or their metabolites to sulfhydryl (SH) groups in proteins impacting cell viability and potentially modulated by elements of xenobiotic metabolism in various cellular compartments, as well as instigation of an influx of immune cells into the lung, including both macrophages and neutrophils. Our team is well positioned to address the mechanism of action of these chemical threat agents due to our strong research backgrounds in lung injury and repair (Van Winkle), inhalation exposure science of toxic chemicals (Bein) and relation of tissue inflammation to biological responses (Vogel). The central hypotheses are that PG is more potent than CP in inducing toxicity in mice and that adequate repair is dependent on macrophages with functional CYP19A1, the estrogen synthesis enzyme. The hypotheses will be addressed in three Specific Aims that will 1) Define the dose response and acute injury pattern 2). Define the temporal pattern of lung injury and repair and 3) Test the hypothesis that macrophage estrogen synthesis is important for lung tissue repair following CP or PG exposure. These studies will advance our understanding of how acute injury, local metabolism and target cell type and estrogen synthesizing macrophages contribute tooutcomes following in vivo exposures to CP or PG. This will advance our understanding of tissue specific responses, a research area that is, of necessity, best investigated in animal models and which sets the stage for medical interventions.

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY Adoptive cell transfer (ACT) using ex vivo expanded anti-tumor T-cells has garnered significant interest due to successes in treating melanoma and other cancers. This is a highly personalized therapy, in which autologous T-cells that can target the tumors are required. However, finding cells that specifically target tumors remains a major hurdle for the widespread application of T-cell based ACT therapies. The current methods of lymphocyte enrichment result in modest increases in tumoricidal T-cells with little control over the clonal heterogeneity. A technology that overcomes these challenges would significantly lower the barriers (e.g., reduce cost, reduce off-target effects) for broad dissemination of ACT therapies. The primary goal of this project is to develop a separation technology to enrich a population of lymphocytes with tumoricidal T-cells based on their capacity to recognize autologous tumor antigens. The premise of our microfluidic technology is that tumoricidal T-cells can be separated from a bulk leukocyte population when exposed to tumor-derived peptide-major histocompatibility complex I under optimal flow conditions. The specific aims are to: 1) Develop a microfluidic device to enrich a population of lymphocytes with antigen specific T-cells, and 2) Demonstrate the capacity of the microfluidic platform to enrich patient-derived Peripheral Blood Mononuclear Cells with tumoricidal T-cells using patient- matched tumor cells. Accomplishing our primary goal will create a potentially disruptive technology that could pave way for wide-spread application of T-cell based ACT therapies, and the agnostic feature (i.e., no a priori knowledge of tumor antigen(s) is required) of the technology would make it broadly applicable for a personalized medicine approach to a range of cancers.