Texas A&M Agrilife Research

NIH Research Projects · FY 2024 · 2024-09

PROJECT SUMMARY Persistent organic pollutants such as organophosphate flame retardants (OPFRs) can accumulate in the body and interact with nuclear receptors important in endocrine regulation. Given that OPFR exposure is pervasive in the human population, there is a critical need to determine the impact of long-term exposure to OPFRs on human health. Specifically, the impact of OPFR exposure on metabolic and brain aging remains undetermined. Recent studies showed that perinatal exposure to an OPFR, triphenyl phosphate (TPHP), led to exacerbated high fat diet-induced metabolic syndrome and gut dysbiosis. Notably, gut dysbiosis and the decrease in gut barrier function have been associated with augmentation of systemic inflammation, metabolic dysfunction, neuroinflammation and aging. Preliminary data from the lab showed an age-associated increase in facultative, pro-inflammatory Proteobacteria, an indicator of epithelial cell dysfunction. Consistently, preliminary data showed age-associated increases in gut permeability and systemic inflammation. Furthermore, using untargeted serum metabolomics, tryptophan metabolism was identified as a signature pathway associated with aging; remarkably, indole and indole-3-lactic acid, beneficial metabolites derived from the bacterial tryptophan catabolism pathway, were significantly decreased with age. In addition, there were age-associated decreases in fecal levels of butyric and propionic acids; these microbially produced short-chain fatty acids (SCFA) have been shown to improve glucose homeostasis and insulin sensitivity. Importantly, new preliminary data suggested that acute TPHP exposure in young adult mice exerted deleterious effects on colon epithelial cells, with impaired barrier function and shifts in metabolism that favors colonization of facultative, pathogenic bacteria. Together, these data provide a strong scientific premise for studying long-term effects of TPHP on the microbiome-gut-brain axis in aging. The central hypothesis is that exposure to TPHP induces gut dysbiosis, leading to gut barrier dysfunction, systemic inflammation, and neuroinflammation; these inflammatory pathologies exacerbate aging-associated metabolic and cognitive decline. This exploratory hypothesis will be tested by pursuing the following aim: Define the extent to which TPHP exposure contributes to metabolic and cognitive decline. An aging cohort will be used, starting TPHP exposure from 10 months (M) of age as a mid-life exposure model. Fecal microbiome, metabolome at 10, 15 and 18 M, in vivo metabolic and cognitive function at 13-15 M will be assessed in sub-Aim A, and, using these mice as donor mice, the causality of dysbiotic microbiota induced by TPHP exposure in exacerbating metabolic and brain aging will be mechanistically tested via fecal microbiota transplantation experiments in sub-Aim B. Senescence phenotypes in colon, liver, and brain will be characterized in sub-Aim C. Overall, results will demonstrate the contributions of TPHP-induced gut dysbiosis in driving the host intestinal epithelial cells dysfunction, in the pathogenesis towards pro-inflammatory state and senescence in the host, and provide evidence for TPHP as a new determinant in the exposome in aging.

NIH Research Projects · FY 2025 · 2024-09

Project Summary/Abstract Cells release extracellular vesicles (EVs) that carry signals to alter cell fate or metabolism, promote invasive behavior, or modulate the immune response. EVs show great potential as diagnostic biomarkers for disease progression, especially in inflammation and cancer. EVs also show promise as a platform for targeted drug delivery, given their ability to signal to and be taken up by cells. Furthermore, EVs can be degraded via phagocytosis, providing metabolites to the engulfing cell. Despite the interest in EVs, basic cell biological knowledge about EV biogenesis, targeting, cargo transfer, and in vivo function is lacking, largely due to the challenges of imaging <250 nm vesicles that lack specific markers. We pioneered a novel labeling technique using degron protection assays to specifically label EVs, which allows us to image EVs in vivo using time-lapse microscopy. We take advantage of the optical transparency and simple genetics of the worm model system C. elegans for gene discovery and have established the first molecular pathway describing how EVs bud from the plasma membrane like viruses. We identified conserved proteins that inhibit EV release in worms and human cells and have generated genetic tools and quantitative assays that enable us to screen for proteins that promote EV release. We also used degron labeling to determine how large EVs and cell corpses are cleared by phagocytosis, revealing novel insights into cargo membrane breakdown and phagolysosomal vesiculation for degradation. Our goals are to determine the molecular interactions of the proteins we identified that regulate lipid asymmetry and EV budding, to determine the role of phosphatidylethanolamine lipids in EV budding, to perform genetic screens using sensitized strains to discover novel proteins involved in EV biogenesis, and thereby define the molecular regulation of EV release. We also plan to use our specific labeling technique to track individual EVs to determine how EVs interact with cells and transfer cargo, providing dynamic insights into their functions from developmental signaling to membrane remodeling. We will also use larger EVs and cell corpses to study how membrane-wrapped cargos are processed inside phagosomes, especially the role of autophagy-associated Atg8/LC3 lipidation in cargo membrane breakdown. Breakdown of the EV membrane in endolysosomes may contribute to EV cargo transfer after uptake. This work has the potential to transform EV production and targeting for drug delivery, as well as to identify key players in EV biology that can be targeted to influence viral and metastatic spread, diverse diseases, and homeostasis. Furthermore, defining the mechanisms of phagocytic breakdown is likely to provide insights into immune modulation and inflammation. Finally, this work on lipid asymmetry is likely to reveal novel aspects of lipid regulation during key processes from cell fusion to cell division. Thus, our vision is to discover how EV release is regulated and use this to determine the functional roles of EVs in vivo while also providing mechanistic insights into diverse proteins and lipids that regulate membrane dynamics.

NIH Research Projects · FY 2024 · 2024-09

SUMMARY Mitochondrial dysfunction in post-menopausal women due to loss of estradiol (E2) alone or in combination with age-induced accumulation of reactive oxygen species (ROS) may play a central role in development of metabolic dysfunction-associated steatotic liver disease (MASLD). Impaired oxidative phosphorylation (OXPHOS) and increased ROS production are linked to dysfunctional hepatic lipid metabolism and liver steatosis. Despite the impact of age and E2 loss on mitochondrial function in post-menopausal women, there is a lack of targeted therapies to treat menopause-associated hepatocellular mitochondrial dysfunction. Current therapies for menopause, which include hormone replacement therapy (HRT) or selective estrogen receptor modulators (SERMs), have adverse and off-target effects such as increased risk for gynecologic cancer and deep vein thrombosis and stroke. Moreover, none of these treatments specifically target mitochondrial function. New pharmaceuticals targeting hepatocellular mitochondrial function with or without E2 would reduce morbidity due to metabolic disease and improve the quality of life for post-menopausal women. The long-term goal of this research is to develop effective therapies to treat mitochondrial dysfunction in post-menopausal women. The objective of this proposal is to determine if two distinct, targeted nanoparticles can improve mitochondrial function in HepG2 cells cultured in MASLD-like conditions and in aged and E2-deficient female mice. The central hypothesis is that two unique nanoparticles – one that works via the nucleus and one that works directly at the mitochondria via E2 - can be delivered to improve mitochondrial function in aged and E2-deficient hepatocytes whose function is impacted by MASLD. Co-Investigator Dr. Gaharwar has developed a new class of molybdenum disulfide (MoS2) nanoflowers that scavenge ROS, increase transcription factor a mitochondria (TFAM) protein and mitochondrial biogenesis, and yield increased OXPHOS/ATP production. Based on preliminary data using poly(lactic-coglycolic acid)-poly(ethylene glycol)-triphenylphosphine (PGLA-PEG-TPP) nanoparticles covalently bonded to E2 (mito-E2) which demonstrates that mito-E2 colocalizes with mitochondrial (mt) estrogen receptor (ER) beta (β), it is predicted that mito-E2 will target mtERβ and mtER alpha (a) to improve 𝛽 oxidation and ATP production and decrease ROS. The hypothesis will be tested with two Aims: (1) Determine if MoS2 nanoflower and mito-E2 improve mitochondrial function in aged and E2- deficient MASLD-like conditions; (2) Determine the molecular mechanisms of mito-E2 at mtERa and mtER𝛽 in HepG2 cells cultured in MASLD-like conditions. It is expected that each of the nanoparticles will improve mitochondrial function in MASLD-like HepG2 cells and in mouse menopause models through different but complimentary mechanisms. This innovative proposal lays the foundation to develop nanoparticle therapeutics to treat post-menopausal MASLD. Specifically targeting mitochondria will advance precision medicine and improve quality of life in individuals suffering from age-related liver disease.

NIH Research Projects · FY 2025 · 2024-08

Project Summary This new R21 application “Respiratory Distress in Sheep with Hypophosphatasia: Etiology, Functional Consequences and Rescue” will provide novel insights into the role of tissue non-specific alkaline phosphatase (TNSALP) on lung development and function. Hypophosphatasia (HPP) is an inherited disorder of mineral metabolism in patients with loss of function mutations in the tissue-nonspecific alkaline phosphatase gene (ALPL). This disorder is associated with high neonatal morbidity and mortality, impaired musculoskeletal development, and respiratory distress syndrome (RDS), yet the in utero origins of the disease are unknown and the ontogeny of development of lung pathology is poorly understood. The PI’s laboratory developed the first large animal model of HPP in sheep, creating an ALPL gene mutation in exon 10 (c.1077C>G) recapitulating the human HPP phenotype. As observed in human patients, but not in mice, neonatal lambs carrying a loss of function mutation in ALPL commonly have respiratory complications as neonates and have an increased incidence of pneumonia compared to wildtype control lambs throughout life. Preliminary findings in HPP sheep demonstrate severe lung pathology on Day 100 of gestation (GD100), suggesting programming of the postnatal lung phenotype during fetal development, and that the known postnatal respiratory complications result from more than insufficient rib cage mineralization. This sheep HPP model will be used to test the hypothesis that reduced TNSALP activity causes lung-specific developmental abnormalities, as well as deficiencies in diaphragm structure and respiratory function that contribute to RDS by the analyses of WT and HPP fetuses on GD130 and in lambs at 4 months of age. Specific Aim 1 will characterize the respiratory phenotype in WT and HPP sheep throughout development (GD130 and postnatal 4 months of age). Specific Aim 2 will determine the impact of HPP on resting lung function in 4 month old WT and HPP sheep by measuring arterial blood gases (pO2, pCO2), bicarbonate, pH, ammonia and lactate, and bronchoalveolar lavage (BAL). Specific Aim 3 will determine the efficacy of recombinant TNSALP therapy to rescue the developmental respiratory phenotype in HPP sheep using lentiviral GFP control or ALPL-GFP constructs injected into WT or HPP sheep zygotes. This interdisciplinary collaborative team includes a current clinical veterinary physiologist collaborator (an expert on respiratory diseases in ruminants) who will collect arterial blood and bronchoalveolar lavage fluids for assessment of lung pathophysiology. This study will provide the first investigations into the in utero developmental origins of the severe respiratory phenotype in sheep with HPP, and describe contributions of non-skeletal tissues (lung, lung-resident immune cells, diaphragm) to RDS in HPP. These findings will have immediate relevance to the management of RDS in human HPP.

NIH Research Projects · FY 2026 · 2024-08

PROJECT SUMMARY Pregnancy loss is estimated to affect approximately 30% of clinically defined pregnancies in the United States, however the actual number is likely much higher due to spontaneous abortions prior to a woman learning she is pregnant. Similarly, in cattle, almost 50% of pregnancies are estimated to end prematurely. In both species, the majority of pregnancies are lost early in embryonic development and the underlying reason is often unknown or unexplained. One potential mechanism underlying pregnancy loss are alterations to the paternal epigenome. Mammalian spermatozoa exhibit a unique, highly compacted and condensed DNA structure that is strongly dependent on epigenetic mechanisms, including histone hyperacetylation followed by nucleosome eviction. Specifically, 85-99% of sperm nucleosomes are evicted and replaced with protamines, allowing for this remarkable degree of compaction. Human sperm exhibiting abnormal chromatin composition, including excess histone retention or post-translational modifications, are associated with infertility, altered embryogenesis following IVF/ICSI, and pregnancy loss. However, the exact cause of altered embryogenesis as a result of an abnormal paternal epigenome, and ultimately, potential regulatory functions and mechanisms by which paternally contributed histones affect early development, remain largely unknown. In this proposal, sperm from bulls of low fertility that have previously been linked to pregnancy loss will be utilized to understand how sperm chromatin regulates mammalian fertility, embryogenesis, and establishment of a healthy pregnancy. This proposal will test if the paternal contribution of epigenetic information, specifically histones, associated post-translational modifications, and genomic placement, are a critical factor in normal embryo development, placentation, and ultimately successful pregnancy. These studies will utilize previously characterized bulls of high and low fertility to study the paternal histone epigenome and subsequent regulation of the embryo though the following Specific Aims: (1) Establish the baseline paternal histone epigenome in fertile bulls (2) Determine if the paternal histone epigenome is altered in low-fertility bulls and (3) Investigate changes to preimplantation embryos generated from low-fertility bulls. Together, the proposed research will provide important insight into the mechanisms governing early embryonic development, including the effects of an abnormal paternal epigenome on chromatin dynamics and embryonic genome activation. It will additionally provide evidence as to why paternal chromatin results in infertility and altered embryogenesis in cattle and humans. The results of these studies have the potential to ultimately impact reproductive efficiency and help clinical management of patients diagnosed with infertility, poor embryogenesis, and pregnancy loss.

NIH Research Projects · FY 2025 · 2024-08

PROJECT SUMMARY Mental health problems among U.S. adolescents have risen precipitously in the past decade, along with social media use, now a staple of adolescent communication and social interaction. Though many believe this is not coincidence, evidence linking the two are mixed at best. A deeper understanding of the developmental connections between social media use and mental health has been severely hindered by a distinct dearth of longitudinal data, scant examination of the dynamic interplay of social media use and mental health among youth from racial and/or ethnic minority groups, and a puzzling omission of the mental health implications of social media content. To fill this critical gap, a longitudinal mixed-methods study is proposed with 250 youth and their primary caregivers from representative racial and ethnic groups. In Phase 1, preliminary data will be collected from 50 adolescents and their primary caregivers to optimize research procedures. In Phase 2, a full-scale longitudinal data collection will be completed with 200 adolescents and their primary caregivers. Data collection in both Phases will focus on intensive 9-day-long periods where the research team will (1) use Ecological Momentary Assessment (EMA) four times per day to measure adolescents’ social media use and mental health in natural settings and (2) continuously capture screenshots of adolescents’ phone screens every 5 seconds to measure an individual’s social media content exposure. These “EMA/Screenshots epochs” will be supplemented with (1) detailed surveys of adolescent social media use, mental health, and other relevant factors from youth-caregiver dyads and (2) interviews with youth. Phase 1 will only involve 1 EMA/Screenshots epoch and a single follow-up survey/interview. Phase 2 will involve 9 EMA/Screenshots epochs (3 years x 3 epochs/year in fall, spring, and summer) and 3 annual surveys/interviews. The Phase 2 design will enable the examination of the bidirectional dynamics between social media use and mental health on daily (Aim 2) and day-to-year (Aim 3) timescales. This highly innovative project will provide urgently needed information regarding the development of youth social media use and mental health across middle to high school ages, their reciprocal influences in both the daily and yearly timescales, and mechanisms of risk for, and resilience against adverse outcomes. This mental health disparities project will provide critical insights and information needed to inform interventions aimed at mitigating the negative impact and maximizing the benefit of social media use for adolescents from different racial and ethnic groups.

NIH Research Projects · FY 2025 · 2024-06

SUMMARY/ABSTRACT This new R01 application entitled “Understanding bone mass in Down Syndrome” is focused on determining the cellular mechanism for the low bone turnover we have identified in people and mice with Down Syndrome (DS), and on characterizing fracture healing responses in different DS mouse models to gain insight into how to better target fracture healing in the DS population with increased propensity to fracture. This proposal will determine the contribution of Regulator of calcineurin 1 (RCAN1) that impacts both NF-κB activity in osteoclasts and Wnt signaling in osteoblasts/osteocytes to the inherently low bone mineral density (BMD) in DS. The proposed experiments will also determine the impact of cessation of the current clinical bone anabolic interventions in the setting of DS as well as define DS fracture healing. Aim 1 will elucidate the pathways through which RCAN1 controls osteoclast and osteoblast differentiation and function. Aim 2 will provide the first direct evidence of fracture healing and repair in the context of low bone accrual and DS. Aim 3 will determine the effects of discontinuation of current pharmaceutical anabolic therapies (anti-sclerostin antibody and intermittent PTH) on bone mass accrual in three preclinical mouse models of DS. The successful completion of this study will lead to a paradigm shift in our understanding of the DS bone phenotype and a new landscape of high-quality research that will provide clarification of the mechanisms that contribute to the low bone mass in DS and more importantly, provide the basis for new directions for the treatment of the fractures and profound osteopenia that affects this population.

NIH Research Projects · FY 2026 · 2024-02

Abstract Influenza A viruses (IAVs) are responsible for seasonal flu and pose a pandemic threat. The overarching goal of our research is to understand the structural and biophysical mechanisms at the molecular level by which nonstructural protein 1 (NS1) of IAVs interferes with host antiviral responses. NS1 is a major virulence factor of IAVs, counteracting host antiviral immune responses. Remarkably, NS1 has a multifaceted strategy to interfere with many host proteins involved in viral RNA (vRNA) sensing and degradation, apoptosis, and interferon production. Furthermore, NS1 is one of the most frequently mutating proteins in the IAV genome. Therefore, studying the evolution of NS1 and its role in immune evasion and modulation is essential for understanding the strain-specific virulence of IAVs. However, to understand the evolutionary development of NS1’s strain-specific functions, it is necessary to examine the interactions between newly acquired mutations and other residues within NS1, which are known as epistatic interactions. Addressing the molecular mechanisms of epistasis is a major challenge in the fields of protein science and evolution. Over the next five years, we will investigate how NS1 interferes with host immune responses in a strain-specific manner. In this proposed research, we will address the questions of how epistatic interactions contribute to the strain-specific interactions between NS1 and host proteins, and how NS1 employs its RNA-binding ability to antagonize vRNA sensors that initiate innate immune responses. To accomplish our goal, we will use an integrated approach including X-ray crystallography, cryo-electron microscopy (EM), NMR and fluorescence spectroscopies, chemical crosslinking using amber-codon suppression, molecular dynamics simulation, and cell-based experiments to parallel our structural and biophysical studies. This research will provide a mechanistic framework for a quantitative understanding of NS1's strain-specific immune evasion functions. As a result, this study is expected to have a positive impact on the development of antiviral agents targeting NS1-host protein interactions.

NIH Research Projects · FY 2026 · 2024-02

PROJECT SUMMARY Copper is an essential micronutrient required for the growth and development of aerobic organisms. Copper serves as a catalytic cofactor for many enzymes involved in various cellular pathways, the most important of which is cytochrome c oxidase required for mitochondrial energy generation. Not surprisingly, mutations that cause systemic or subcellular copper deficiency give rise to various fatal infantile disorders, including Menkes disease and a subset of mitochondrial disorders. Despite decades of work, there are currently no approved treatments for these lethal disorders, which in large part reflects a limited understanding of the mechanisms by which copper is trafficked to mitochondria and the role it plays in mitochondrial metabolism. Filling this knowledge gap will require a multidisciplinary approach that leverages the strengths of different model organisms to understand the mechanisms by which copper is transported, stored, and distributed within cells. Over the last decade, we have taken a multidisciplinary approach to discover new players in copper transport and delivery to mitochondrial cytochrome c oxidase. Through these efforts, we have identified a promising copper-transporting drug, elesclomol, that circumvents disease-causing mutations in the mitochondrial copper acquisition by promoting copper delivery to cytochrome c oxidase and restoring aerobic respiration. Building on this success, we will now focus on identifying critical regulators of mitochondrial copper by leveraging our copper-deficient yeast, zebrafish, and mouse models to decipher the fundamental roles of copper in mitochondrial metabolism. The overarching goals of our research program are to 1) determine the molecular mechanisms of mitochondrial copper acquisition and delivery to cytochrome c oxidase; 2) identify novel roles of copper within the mitochondrial matrix; and 3) develop small molecule adjuvants to enhance the efficacy and safety of elesclomol. To achieve these goals, we will employ genomic, proteomic, and small molecule screens in copper-deficient yeast models to identify endogenous copper-transporting molecules, new copper-dependent mitochondrial metabolic pathways, and small molecules that improve the therapeutic properties of elesclomol. We will translate these discoveries to mammalian model systems to significantly advance our understanding of mitochondrial copper biology.

NIH Research Projects · FY 2025 · 2024-01

The long-term goal of the PI Zhang’s laboratory is to decipher intertwined regulatory layers and mechanisms that control RNA silencing and processing, and to eventually manipulate the regulatory components and pathways in biotechnological applications. Here this proposal is to describe how the PI Zhang will investigate unknown mechanisms and regulations of several newly identified components within two master regulatory hubs centered on Serrate (SE) and Suppressor of Gene Silencing 3 (SGS3) for RNA silencing in Arabidopsis. RNA silencing clears endogenous and invasive transcripts, and this process is directed by microRNAs (miRNAs) and small interfering RNAs (siRNAs) in eukaryotes. MicroRNAs are produced from hairpin-structured primary miRNA substrates (pri-miRNAs) by microprocessor that comprises of Dicer-like protein 1 (DCL1), dsRNA binding protein 1 (DRB1), and SE. By contrast, siRNA production entails the initial conversion of single-stranded (ss) RNA to double-stranded (ds) RNA via a complex composed of RNA-dependent RNA polymerase 6 (RDR6) and SGS3 before its entry into DCL4/DRB4 complex. Since levels of miRNAs and siRNAs are critical for their proper functionality in biology, the processing and accumulation of small RNAs need to be tightly controlled. In research area 1, the PI Zhang will study how the components in the SE-centered hub regulate miRNA production. Briefly, the PI Zhang laboratory has recently discovered that RNA secondary structure (RSS) of pri-miRNAs can be remodeled to regulate DCL1/HYL1 activity. Importantly, several RNA helicases (RHs) have been newly recovered from the hub, but how they sensor and re-wire RSS of pri-miRNAs to control miRNA production is unknown. Furthermore, extensive preliminary data also identify key connections between reactive oxygen species (ROS) and microprocessor components that modulate miRNA production. Thus, the PI Zhang will systematically investigate the novel functions and mechanisms of the RHs and catalases from SE hub in miRNA biogenesis in Area 1. In research Area 2, the PI Zhang focuses on the major gap in the siRNA production that is how ssRNA substrates are specified and loaded onto SGS3/RDR6 for dsRNA synthesis. Through a newly developed genetic screening, numerous novel components exemplified by U1-70K and BICE1 among others have been recovered to be engaged in siRNA- mediated RNA silencing. Remarkably, the components interact with RNA polymerases in nucleus and ribosomes in cytoplasm as well as the shared SGS3 protein. In this setting, the PI Zhang would comprehensively study how these factors help SGS3 to fetch the nascent transcripts from chromatin, and to set up the platform for SGS3/RDR6 nearby ribosomes to synthesize dsRNAs. The proposed study will not only reveal new regulatory layers and mechanisms of RNA silencing, but also enable us to explore the highly conserved components to control small RNA production and activities in biological processes, and to eventually to cure physiological disorders that arise from dysfunctions of RNA processing in biotechnology.

NIH Research Projects · FY 2026 · 2024-01

PROJECT SUMMARY Aging is the most prominent nongenetic risk factor for age-associated diseases including late onset Alzheimer’s disease (AD) and related dementias. Accumulating evidence has implicated altered sphingolipid ceramide pathways in aging and neurodegenerative diseases. However, the cellular and molecular basis underlying ceramide aberration and how it contributes to aging-associated cognitive decline and AD pathogenesis remains poorly understood. Our preliminary data revealed aging-dependent accrual of ceramides in astrocytes and, to a less extent, microglia in brain regions known highly susceptible to aging- and AD-related functional deterioration. Lipidomic analysis of young and aged mouse brains uncovered specific upregulation of very long chain (VLC) ceramides with concomitant decreases in corresponding sphingomyelin molecules, suggesting aging-dependent activation of sphingomyelinase. Extracellular amyloid beta deposition induces robust astroglial and microglial activation and ceramide production in AD as well as in a mouse model of amyloidosis at even young ages. Moreover, we found significantly elevated acid sphingomyelinase (aSMase) activity in AD brains compared to age matched controls. Given that elevated astroglial ceramides increase the vulnerability of oligodendroglia to inflammatory cytokine released by microglia, that oligodendroglia/myelin damage and/or dysfunction has increasingly implicated in AD pathology, and that ceramides abnormally accumulate in the microglial lysosomal compartment, we hypothesize that dysregulated ceramide in glial cells is a previously unrecognized active driver in the progression of aging-related cognitive decline and amyloid pathology. The overarching goal of this project is to investigate cellular and molecular pathways leading to disruptions of sphingolipid ceramide homeostasis in physiological aging and amyloidosis conditions and to identify ceramide-mediated pathogenic pathways. Specifically, we will leverage the powerful sphingolipid targeted lipidomics and cell type-specific genetic manipulation of aSMase and VLC ceramide synthase CerS2 to (1) determine the effect of VLC ceramide on astrocyte function and paracrine impact on oligodendroglia and neurons including cellular damage and senescence; (2) investigate the aSMase-ceramide pathway in regulating microglial clearance of myelin debris and amyloid beta and inflammatory activation; and (3) employ novel conditional knockout and knockdown mice to determine the contribution of astroglial and microglial aSMase-ceramide pathway to disease progression in a mouse AD model of amyloidosis. This project shall generate new insights into aging- related dysregulation of the bioactive ceramide and provide a foundation for exploring the potential of interventions targeted at sphingolipids and neuroinflammation.

NIH Research Projects · FY 2024 · 2022-09

Despite tremendous technical advances in drug discovery, de novo development of small molecule drugs is still challenging. High-throughput screening (HTS) with libraries of natural products and other complex molecules remains the bedrock approach. However, HTS is unsatisfactory in many ways: extraordinary cost, poor efficiency, rampant false positives and a complexity of “hits” that hinders hit-to-lead development. Fragment based drug discovery (FBDD) was brilliantly conceived to overcome these limitations, but has arguably not performed as hoped. The limited impact of FBDD is because most fragment “hit” molecules are very weak binders and are undetectable by current assay methods. The enormous potential of FBDD is therefore lost. Here, an approach is to be developed that can reliably detect weak but specific binding with the goal of helping to reinvigorate and enhance early phase small molecule drug discovery. Faithful detection of binding requires that the ligand and protein concentrations be at least on the order of the dissociation constant, which is practically and financially unrealistic for weak binders. The strategy to remove this basic barrier is simple. The water core of the reverse micelle (RM) is used to confine a single protein molecule and fragments at high enough concentrations to overcome the unfavorable binding entropy. NMR spectroscopy then permits site-resolved detection and quantification of binding affinity at reasonable cost. The first application of RM NMR FBDD highlights its potential to greatly expand small drug discovery. A rule- of-three (Ro3) fragment screen of interleukin-1β (IL-1β) shows that 1) weak yet specific binding can be efficiently detected in a structural context; 2) achieving the required high protein and ligand concentrations is economically feasible; 3) a high hit rate is observed; 4) surface coverage is extraordinary and gives unprecedented connectivity potential; 5) highly desired more polar binders are illuminated. The door is now open to more fully realize the tremendous promise of FBDD but critical questions remain: Is the IL-1β surface coverage typical? What is the distribution of fragment hit affinities of Ro3 and rule-of-five (Ro5) libraries more generally? What are the chemical characteristics of useful fragments to choose for an optimal RM NMR screening library? How useful are the very weakly binding hits for lead development? Does the Ro5 library offer a better compromise of hit affinity and surface coverage? What is the most efficient way to carry out RM NMR screening? Is RM NMR screening quantitatively reliable? This project will address these and other technical challenges that stand in the way of creating a strategy that more fully enables the brilliant insights of the FBDD paradigm and unleashes its originally anticipated potential.

NIH Research Projects · FY 2025 · 2022-09

One in three youth in the United States are overweight, and 85% of all overweight youth have at least one Metabolic Syndrome (MetS) risk factor that increases their chance of developing cardiovascular disease. Comprehensive school- and evidence-based interventions (EBIs) that focus on students’ physical activity and fruit and vegetable consumption can reduce a school’s prevalence of overweight students by up to 8%. Additionally, multi-level interventions that target school health environments may be more effective than interventions that focus on individuals alone. Currently, there is an absence of multi-level EBIs for middle school students that address both physical activity and healthy eating behaviors and environments. Strong Teens for Healthy Schools Change Club (STHS-CC) is a novel, multi-level, and theory-based civic engagement program that equips and supports middle school students to make a positive food and physical activity environmental change and personally engage in physical activity and healthy eating behaviors to reduce their MetS risk. This study utilizes a local advisory board to engage Cooperative Extension agents, 4-H staff, school administrators, teachers, and students in the refinement of STHS-CC for the middle school setting. Once refined, the local advisory board oversees the delivery and testing of STHS-CC using traditional and collaborative data analysis processes (e.g., Photovoice) in a pilot cluster-randomized controlled trial (n=20 schools; 20-25 participants per school). At the individual level, this study tests STHS-CC’s effectiveness for improving students’ MetS risk factors and positive youth development outcomes (i.e., competence, confidence, character, connection, and caring). At the social level, this study evaluates peers’ support for and participation in physical activity and healthy eating behaviors. At the environmental level, this study evaluates the physical, situational, and policy aspects of schools’ physical activity and nutrition environments. Long-term, the goal of this body of work is to motivate middle school students to improve their physical activity and nutrition behaviors and environments through a student-led EBI that is part of a collection of comprehensive schoolbased health promotion programs. If effective, STHS-CC has the potential to be disseminated through schools, Cooperative Extension networks, and 4-H organizations statewide and nationally.

NIH Research Projects · FY 2025 · 2022-08

Project Summary Single-stranded RNA bacteriophages (ssRNA phages) are small near-icosahedral viruses that use RNA as genetic material to infect bacteria through retractile pili. Recently >15,000 new ssRNA phages have been identified but their hosts and mechanisms of infection remain unknown. Of the steps during the infection cycle of ssRNA phages, how phages package the genomic RNA and recognize its specific host are only known for model ssRNA coliphages such as MS2 and Qβ; and how RNA is ultimately delivered into the cytosol is obscure. From the preliminary data, the PIs find that the previous paradigm set for the infection mechanism of ssRNA phage based on model coliphages can no longer be applied to other ssRNA phages. Host receptors of ssRNA phages, the retractile pili, are usually involved in the virulence of pathogenic bacteria and the sharing of antibiotic-resistant plasmids. This project will focus on phages PP7 and AP205, which infect Pseudomonas aeruginosa and Acinetobacter spp., respectively, via the Type IV pili (T4P). The overall goal is to determine the mechanisms involved in PP7/AP205 packaging, and RNA penetration into the host, a process which involves both host recognition and RNA entry. Specific aims are to reveal the molecular mechanisms for (1) the packaging of PP7/AP205, (2) the interplay between PP7/AP205 and T4P before RNA entry, and (3) the detachment of T4P during RNA entry. This work will not only reveal insights into the infection mechanism of ssRNA phages but also provide guidelines to engineer ssRNA phages for the following purpose: ssRNA phages will be engineered as means to detach pili of pathogenic bacteria, as an alternative strategy for treating multidrug-resistant bacterial infections. Unlike traditional phage therapy by lysing pathogens, virulence and antibiotic resistance spread are inactivated by breaking pili while leaving the cells to grow, without exerting selective pressure on the host to develop further resistance. Such a method also avoids the release of any unwanted cell contents including DNA, proteins, and toxins into the environment which could interfere with other bacteria or affect human cells. In the future, the proposed project will also provide a basis for developing a method for packaging and delivery of a large number of foreign RNAs into bacterial cells. Due to the short life of RNAs inside the cell, they allow transient regulation of the cells and are less likely to exert long-term genetic effects as in the case of DNA plasmids. In addition, RNA delivery with ssRNA phages does not rely on the artificial preparation of cells competent for heat-shock or electroporation, which is hard to perform in situ.

NIH Research Projects · FY 2025 · 2022-06

Project Summary/Abstract The objective of this Food and Drug Administration (FDA) Veterinary Laboratory Response Network (Vet- LIRN) Cooperative Agreement Program is to enable the analyses of animal diagnostic samples, animal foods, and animal drugs when laboratory investigations or surge testing are needed for analyses related to research and testing of microbiological or chemical contamination, either through intentional or unintentional means. This program will increase the ability to conduct investigations via laboratory analysis by supporting new and rapid analytical techniques and providing for well-equipped and staff laboratory facilities. The outcomes will result in increased Vet-LIRN laboratory research and capacity to investigate potential animal foodborne illness outbreaks by fostering training, the use of new technologies, and improving the effectiveness of collaborative partnerships. Funding offered by this program can provide laboratories with the infrastructure to enhance their testing capabilities for both microbiological and chemical methodologies. These cooperative agreements are also intended to expand participation in networks to enhance Federal, State, local, and tribal food safety and security efforts.

NIH Research Projects · FY 2026 · 2022-03

Biological clocks play a key role in how organisms adapt to daily (circadian), monthly (circalunar), and annual (circannual) changes in the environment by regulating rhythmic fluctuations in metabolism, hormone and neurotransmitter release, sensory capabilities and behaviors, including sleep. Disruption of circadian rhythms (e.g. shift work, time zone changes (“jet lag”), or social jet lag), can result in significant physiological consequences including sleep and metabolic disorders, as well as increased risk of stroke and cancer. Recent data indicate that the circadian clock is developmentally regulated and that its time-keeping activity is suspended in differentiating tissues to allow clock components to function in a “developmental clock”, that regulates stem/progenitor cell biology and differentiation, among other processes. However, it is not known how this functional shift from circadian to developmental activities is achieved, nor is it known how clock components control normal or malignant stem/progenitor cell behaviors. The mouse mammary gland is a powerful models system for the study of circadian rhythms, development, and stem/progenitor cell biology. Lineage tracing studies have demonstrated that the mammary gland harbors dynamic cell populations in which self-renewing unipotent basal and luminal stem cell lineages give rise to their respective lineages during ductal elongation, and are responsible for breast cancer cellular heterogeneity. We have shown that PER2, a transcription factor in the circadian clock, is differentially expressed during mammary gland development, and is required for branching morphogenesis, suggesting a circadian clock-independent developmental role. Our recent data using Per2-/- mouse mammary glands suggest that PER2 may play a critical role in mammary epithelial cell lineage commitment and homeostasis, and may do so by regulating the expression of EYA2 as well as influencing Wnt/b-catenin and TGF-b signaling. Moreover, we have found that circadian disruption in mice results in down regulation of Per2 expression in mammary epithelial cells and increased EYA2 expression and activation of Wnt/b-catenin. Based on these new results, we hypothesize that PER2 functions to integrate the circadian clock with the developmental clock to control homeostasis of mammary epithelial cell types during ductal elongation and branching morphogenesis. To address this hypothesis three Specific Aims are proposed. Aim 1 will test circadian and circadian disruption dependent changes in stem and progenitor cells in the developing mammary gland. Aim 2 will focus on the role of PER2 in lineage commitment using genetic reporters to trace mammary cell differentiation and examine the effect of PER2 on stem cell homeostasis. Studies in Aim 3 will determine PER2-dependent regulation of EYA2 gene expression and regulation of b-catenin in mammary gland morphogenesis. Upon successful completion of this aim we will have a better understanding of the molecular mechanisms that govern the interaction of PER2 and EYA2 as well as the effect on WNT/ TGFb signaling in branching morphogenesis.

NIH Research Projects · FY 2025 · 2022-02

Project Summary The proposed project serves as a platform to obtain the key training and research experiences in achieving the long-term career goal of becoming a leading academic principal investigator with a primary focus of developing and applying genomic methods to understand the complex genetic components and biological mechanisms of neuropsychiatric disorders. Chromosomal aberrations in the form of large deletions or duplications (copy number variants, CNVs), such as those on 1q21.1, 16p11.2, and 22q11.2, are the strongest known risk factors for neuropsychiatric disorders. For this reason, these large CNVs serve as key points of entry for investigating the molecular etiology. However, it is unknown why each of these CNVs is associated with diverse clinical outcomes. For example, deletions and duplications at 16p11.2 are strong risk factors for schizophrenia (SZ) and autism spectrum disorder (ASD). Duplications produce a greater than 10-fold increase in risk for SZ, but ASD is frequent in carriers of deletions as well as duplications. The large stretches of human-specific segmental duplications (HSDs) that both constitute and mediate the formation of these large “neuropsychiatric” CNVs are inaccessible to current genome sequencing analysis due to their high degree of repetitiveness and complexity. CNV studies to date have not been able to consider the genetic variations inside these hundreds of kilobases to megabases of HSDs. Thus, we do not know the exact “genomic scar” of each CNV in individual carriers including additional smaller-scale rearrangements, gene fusions, and copy number changes of paralogs, the diversity HSD rearrangements across different CNV carriers, and the concomitant functional effects. The major aims here are to (1) develop generalizable genome analysis methods to solve this important problem in psychiatric genetics in established cell lines carrying the 16p11.2 deletions and duplications as the first targets of this new approach, (2) to develop high-throughput genotyping assays so that studying the diversity of HSD rearrangements can be applied to expanded groups of affected individuals where only DNA sample (no cell lines) is available and to future cohort association studies, and (3) to investigate the functional effects of HSD rearrangement diversity on CNV biology using cortical organoid models. To facilitate career development and transition to an independent investigator, the following five training goals will be achieved under the support and guidance of the mentorship team: (1) developing expertise in targeted genome assembly analysis and optical DNA mapping; (2) expanding knowledge in developmental neuroscience and pathophysiology of psychiatric disorders; (3) gaining hands-on expertise in neuronal organoid modelling (4) acquiring expertise in chromatin interaction analysis and single-cell RNA expression analysis of neural organoids; (5) gaining experience in grant writing. The hands-on training will primarily take place at Stanford University with training components conducted at Yale and KU Leuven.

NIH Research Projects · FY 2025 · 2021-12

Project Summary Overnutrition induces insulin resistance, which is a high-risk factor for T2D and NASH. The mechanism underlying insulin resistance-induced hepatic lipogenesis, fibrosis, apoptosis, and inflammation for NASH is unclear. High levels of transforming growth factor-β1 (TGFβ1) in blood and tissues were observed in both human and mice with T2D and NASH. TGF-β1 plays a pivotal role in a diverse range of cellular responses, including extracellular matrix (ECM) synthesis and apoptosis which are essential for tissue homeostasis, but the molecular mechanism by which TGFβ1 regulates glucose metabolism and liver function is incompletely understood. The forkhead transcription factor Foxo1 is a key downstream target of the insulin→ PI3K→protein kinase B (Akt) signaling pathway and the glucagon→cAMP→protein kinase A (PKA) signaling pathway. Akt phosphorylates Foxo1-Ser253, triggering Foxo1 nuclear export and cytoplasmic sequestration for ubiquitination. By contrast, PKA phosphorylates Foxo1-Ser273 to promote Foxo1 nuclear translocation and protein stability in hepatocytes. Upon metabolic stress such as overnutrition, Foxo1 hyperactivation promotes hepatic glucose production (HGP) and hyperglycemia. In this proposal, the PI hypothesizes that hepatic TGFβ1 plays key role in control of Foxo1 via phosphorylation at S273 (Foxo1-pS273), enhancing Foxo1 nuclear activity, and inducing hyperglycemia, liver fibrosis and inflammation, and promoting T2D and NASH. In Aim 1, PI and his team will use genetic approaches to 1) delete the TGFβ1 gene in the liver of mice (L-TGFβ1KO) with or without active Foxo1-S273D/D mutation, or 2) generate liver-specific overexpression TGFβ1 mice (L-TGFβ1OE) with or without inactive Foxo1-S273A/A mutation, investigating whether hepatic TGFβ1 is a key controller for Foxo1-pS273 in promoting HGP, apoptosis, liver fibrosis and inflammation following the NASH diet feeding. Aim2 is to use mouse primary hepatocytes, bone marrow- derived macrophages, and hepatic stellate cells (HSC) to determine the mechanisms by which TGFβ1 promotes macrophage depolarization and HSC activation via Foxo1-pS273 in hepatocytes. The specific- domain interactions of Smad3 and Foxo1 and related target genes expression in hepatocytes will be further investigated. Aim 3 is to investigate whether suppression of systematic or hepatic TGFβ1 signaling is sufficient for the prevention of T2D and NASH in mice, in a Foxo1 dependent manner. Overall, using the genetic, genomic, bioinformatic, and pharmacological approaches, the PI and his team are fully equipped to investigate the new pathophysiological role of hepatic TGF-β1→Foxo1-pS273→TGFβ1 looping system in control of T2D and NASH, which will provide novel insights on mechanism of diabetes and NASH.

NIH Research Projects · FY 2025 · 2021-09

SUMMARY Mitochondria operate as a central hub for many metabolic processes by sensing and responding to the cellular environment to maintain homeostasis. Consequently, their disruption is a key factor in the onset and progression of many human conditions, including metabolic disorders, neurodegenerative diseases, and cancer. Mitochondrial homeostasis is primarily maintained through the recycling of damaged mitochondria by targeted autophagy, termed mitophagy. Mitophagy is tissue-specific and occurs in response to both cellular stress and differentiation cues. Differentiation-cued mitophagy is often termed programmed mitophagy and has recently gained attention for its contribution to epigenetic status, cell fate decisions, metabolic adaptation and differentiation. Although these and other effects have been attributed to mitophagy, little is known about the upstream signaling pathways that induce mitophagy to meet specific cellular needs. Distinct morphological differences in mitochondria exist during the post-natal stages of mammary gland development. This suggests that mitophagy plays an important to the development of this tissue. Identifying the mechanism by which mitochondrial homeostasis is maintained during mammary gland development will provide much needed insight into the broader role of mitochondrial adaptation in normal development and disease. We have shown that Singleminded-2s (SIM2s; expressed from Sim2), is differentially expressed during mammary gland development and is a key regulator of functional mammary gland differentiation. Our recent results utilizing mammary gland- specific over- and under-expressing Sim2s transgenic mice show that SIM2s is required for functional lactation, and does so, in part, through direct interaction with the PRKN mitophagy complex. Based on these new results, we hypothesize that mitophagy-dependent mitochondrial adaptation is essential for mammary gland functional differentiation and that SIM2s is required to maintain mitochondrial homeostasis. To address this hypothesis we propose two Specific Aims. In Aim 1, we will determine the mitophagy-driven metabolic transition required for mammary epithelial cell differentiation by crossing the mito-QC mouse model with MMTV-Sim2s and Sim2fl/fl mice to assess mitophagy and mitochondrial architecture and metabolic adaptation. In Aim 2, we will define the physical basis for, and functional outcomes of, interactions between SIM2s, ATM, PINK1/PRKN, and LC3 in mitophagy and mammary gland differentiation. Successful completion of this proposal will provide insight into heretofore unknown mechanisms of mitochondrial adaptation under physiological conditions. We expect results from these studies will help define the mechanism of mitochondrial adaptation in mammary gland development, lactation, and cancer.

NIH Research Projects · FY 2025 · 2021-08

ABSTRACT Parkinson disease (PD) is the second most common neurodegenerative disease that affects nearly 5 millions of people around the world. PD is associated with intracellular aggregation of α-synuclein (α-Syn), a small protein that is involved in the regulation of lipid metabolism and synaptic vesicle trafficking. A growing body of evidence suggests that alterations in lipid profile, which were observed in both brain and plasma, can be directly linked to the onset of α-Syn aggregation. We hypothesize that the toxicity of α-Syn aggregates is determined by their structure, which in turn is controlled by the lipid composition of neuronal membranes. Our group pioneered development of a label-free, non-invasive and non-destructive approach that can be used to determine the structural organization of individual α-Syn aggregates. We will use this innovative biophysical imaging approach to unravel protein secondary structure of α-Syn oligomers grown in the presence of different lipids. We will investigate toxicity of these oligomers on primary dopaminergic neurons from midbrain, striatal, and cortical areas of the mouse brains. This will reveal the relationship between the structure of α-Syn oligomers and their toxicity. The proposed work also aims to determine the extent to which α-Syn oligomers with different structures exert toxic effects to the specific subsets of neurons.

NIH Research Projects · FY 2024 · 2020-09

Abstract Texas Food Defense and Animal Food Product Testing for Microbiological, Chemical and Radiological Hazards, Genome Sequencing, and Special Project Tracks The Office of the Texas State Chemist (OTSC) is comprised of the state agency authorized to regulate animal food (Texas Feed and Fertilizer Control Service (FFCS) and an ISO 17025:2017 accredited laboratory (Agricultural Analytical Service (AAS)), which analyzes regulatory samples for FFCS, the Food and Drug Administration and the United States Department of Agriculture. OTSC provides regulatory oversight for 5000 firms that manufacture feed with locations in Texas, the United States (US) and abroad and distribution of 23 million tons of feed in Texas. OTSC serves a large animal population including approximately 900,000 dairy cows, 5 million cattle on feed, 12.6 million cows and calves, 21 million layers, and 115 million broilers along with 12 million dogs and cats, 500,000 equids, and 1.5 million sheep and goats. AAS laboratory personnel include 12 analysts, four laboratory attendants, three PhD research scientists/lead chemists, one laboratory quality manager and one laboratory associate director. The laboratory is organized into four teams that specialize in microbiology, chromatography/mass spectrometry, elemental analysis, and radiochemistry. FFCS regulatory personnel include 14 field investigators, a manager of accounting, operations manager for registration and labeling, an associate director of compliance and an associate director of field operations. In this project. OTSC will strengthen food defense capability in the areas of microbiology, chemistry, and radiochemistry; expand surveillance of microbiological and chemical hazards, expand involvement in whole genome sequencing, develop and validate new methods and participate in all laboratory flexible funding model program activities and meet reporting requirements. As an outcome of this project, OTSC will expanded capacity and capabilities of the Texas animal food testing laboratory in support of an integrated food safety system in the disciplines of microbiology, chemistry and radiochemistry in food defense, animal food microbiological and chemical product testing, whole genome sequencing, capability/capacity development, and special project tracks

NIH Research Projects · FY 2024 · 2020-09

Abstract: Texas Animal Feed Regulatory Program Standards Maintenance and Preventive Controls Initiative The Office of the Texas State Chemist (OTSC) is the designated state agency that oversees the regulation of animal feed including sample analysis. Specifically, OTSC is comprised of the Texas Feed and Fertilizer Control Service (FFCS) which is the regulatory agency and the Agricultural Analytical Service (AAS) which analyzes regulatory samples for FFCS and the Food and Drug Administration. During the past 5 years, OTSC has fully implemented the eleven standards, achieved ISO 17025:2017 accreditation, created a document management system and is utilizing the system for AFRPS implementation. During the next 5 years, OTSC will more fully integrate AFRPS through a number of activities including exploring how the PCAF regulations could be incorporated into Texas Commercial Feed Rules, building a more robust facility risk ranking matrix and analysis platform, expanding audits to focus on PCAF and CGMP inspections, utilize feed- related illnesses or death data in the risk ranking matrix, continue to evaluate industry compliance through the enforcement matrix, expand outreach content and audience and incorporate Laboratory Flexible Funding Model program samples into the OTSC annual plan of work. OTSC will expand the number of qualified field investigators who can perform PCAF inspections by seven who will be supported by a Preventive Control Qualified Field Inspection Trainer and Auditor. An expanded educational outreach program will include development of new content including probability charts for hazards by feed type identified in the FDA Guidance #245, model plans, and annual updates of the status of the Texas feed industry involving risks associated with these hazards. Two new courses will be developed and delivered in a face-to-face and online format to the Texas feed industry and beyond and are titled: Food Safety Plan Development and Verification and the second titled Auditing a Food Safety System. These courses will include the new content listed above and class room exercises enabling participants to complete or revise their food safety plan, verify its ability to manage risk including verification of control measures including prerequisite programs and preventive controls. OTSC field investigators, equipped with this new information, will practice educating while regulating during non-FDA inspections of commercial feed facilities. These activities will be entered into a feedback loop to continually improve the training based on new information and participant feedback.

NIH Research Projects · FY 2026 · 2020-06

The ability to deliver pathogen-resistance genes into mosquito populations has long been sought as a potential alternative for disrupting dengue or malaria transmission where funds and infrastructure are the limiting factors in effective mosquito control. While effective gene drive transgenes based on CRISPR/Cas9 have been developed for model organism Drosophila and for malaria mosquitoes, Aedes aegypti, the most medically important vector of dengue, yellow fever and chikungunya viruses, lags behind for reasons that remain largely unexplored and unknown. In this project, D. melanogaster and A. aegypti will be employed to evaluate novel hypotheses regarding how genome structure and DNA repair influence both homing gene drive and transgene removal based on single strand annealing. Following from previous work, multigeneration cage experiments will be performed on this transgene removal strategy in the context of an active gene drive in both flies and mosquitoes, followed by a wave of transgene removal (Aim 1). Next, the role of local microhomology, nuclease characterisics and DNA repair protein recruitment will be examined on both the rates of both homing gene drive and transgene removal in A. aegypti, where gene drive has lagged behind (Aim 2). Finally, the role of chromosomal position on both homing gene drive and transgene removal will be tested in the context of both synthetic targets and new haplolethal target genes (Aim 3). This innovative approach takes advantage of naturally occurring processes that are conserved throughout eukaryota to completely eliminate all transgenic sequences following potential field releases. Thus, it is anticipated that this project will have a substantial impact on National and International conversations concerning gene drive technology as a whole, and will raise expectations for what is possible in any future trial to generate pathogen-resistant mosquitoes.

NIH Research Projects · FY 2026 · 2019-07

ABSTRACT The Aryl Hydrocarbon Receptor (AhR) is a member of the Per-Arnt-Sim (PAS) domain protein family that regulates adaptive and toxic responses to a variety of chemical pollutants, including polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons, most notably 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We observed that the newly identified endogenous AhR agonist, cinnabarinic acid (CA) does not upregulate prototypical AhR target gene, Cyp1a1 and is not involved in xenobiotic regulation. On contrary, CA induced expression of a novel AhR target gene, stanniocalcin 2 (Stc2) in liver. Our studies indicate that in response to CA-treatment, AhR interacted dichotomously to xenobiotic response elements (XREs, 5'-GCGTG- 3') present within the Stc2 promoter regulating its expression. Additionally, this proposal capitalizes on our recent observation that CA treatment protected against non-alcoholic fatty liver disease in an AhR-dependent manner. The in vitro model that mimics fatty liver disease, showed significant role of STC2 in CA-specific AhR- driven hepatoprotection. Moreover, we recently constructed hepatocyte-specific Stc2 conditional knockout mice (Stc2-hKO) which showed exacerbated hepatic steatosis and metabolic deterioration. The studies proposed in this renewal application are logical extension of our current award and hypothesize that the CA- specific AhR-regulated hepatic STC2 signaling plays critical role in attenuation of lipogenesis resulting in protection against non-alcoholic fatty liver disease. Using the Stc2-hKO mice, Specific Aim 1 of this application will investigate role of in vivo STC2 signaling in AhR-mediated protection against steatohepatitis in response to CA treatment. Specific Aim 2 will utilize lipidomics and single-nuclei transcriptomics to characterize CA-specific STC2 mediated hepatoprotective pathways involved in the mitigation of lipogenesis. Specific Aim 3 will investigate AhR-mediated regulation of Stc2 promoter in non-alcoholic fatty liver disease. The current application will unravel mechanism of transcription regulation of Stc2 by CA-specific AhR activation and characterize its significance against non-alcoholic fatty liver disease.

NIH Research Projects · FY 2024 · 2019-07

PROJECT SUMMARY Nearly 70% of US adults are overweight or obese, and the consequences can include increased risk for several types of cancer, diabetes, and cardiovascular disease. Compared to their urban counterparts, rural residents tend to have higher rates of cancer, obesity, physical inactivity, and poor diet. Rural residents also have higher rates of poverty and lower rates of health insurance, and face unique challenges accessing healthy foods, and/or physical activity opportunities, which contribute to these rural health disparities. Civic engagement for built environment change (CEBEC) integrates resident-led community assessments with environmental change initiatives aiming at improving population health. In several pilot studies, engaging and empowering residents to identify solutions to improve community health have demonstrated successful implementation. Evidence demonstrating positive change in behaviors and health outcomes using the CEBEC approach are limited in scope and rigor but show notable and encouraging outcomes (e.g. increased physical activity). This study proposes to evaluate a CEBEC intervention, the multilevel Change Club (CC) project, which would be implemented in eight rural locations. The objective of the CC is to reduce rural risk factors for obesity, cancer, and other chronic diseases through CEBEC physical activity and healthy eating projects. The CC provides a menu of effective interventions; step-wise planning strategy; behavior change strategies and goal setting; assessment and engagement tools; and ongoing support via conference calls, webinars, and discussion boards to rural resident CCs, which typically include about 12-16 residents. For Aim 1, we will evaluate individual-level health and behavioral outcomes in eight rural towns. We will measure outcomes in CC members, local social network members (SNM) of CC members, and a sample of town residents recruited through mass mailings and community events. The primary outcome is Simple 7 composite cardiovascular risk score, which includes blood pressure, glucose and cholesterol, BMI, diet, smoking, and physical activity; additional outcomes are knowledge, attitudes, beliefs, and self-efficacy related to healthy eating and exercise, including use of community resources for healthy eating, physical activity, and health care; and environmental factors. Objective measures, including BMI, biochemical measures, accelerometry, dermal carotenoids, and blood pressure, will be collected with CC members and a sample of SNM and town residents. Aim 2 includes a mixed methods process evaluation examining unintended consequences; implementation barriers and facilitators, including costs; and the effect of community/built environment/policy, social/collective, and individual-level factors on intervention-specific outcomes. We will compare costs across sites and explore cost- effectiveness of CC interventions relative to change in Simple 7 score. This project is an innovative opportunity to evaluate multilevel rural CEBEC interventions focused on chronic disease risk factor behaviors. Outcome and process data will provide critical insight into the viability of CEBEC interventions for future dissemination.