Texas A&M Agrilife Research

NIH Research Projects · FY 2026 · 2019-07

PROJECT SUMMARY Generating diverse cell types from a limited number of progenitor cells in development is a significant challenge in multicellular organisms. Through asymmetric cell division (ACD), progenitor cells divide to renew themselves and create new cell types. A key process of ACD is the polarization of progenitor cells. Our research aims to elucidate design principles that govern cell polarization, divisional asymmetry, and the coordination of cell division and cell-fate determination in plant ACD. Stomata are microscopic pores in a plant's epidermis. In the model plant Arabidopsis, stomatal development and patterning provide an excellent platform for studying the molecular mechanisms underlying cell polarity- driven ACD in plants. The PI identified BASL (Breaking of Asymmetry in the Stomatal Lineages) as the first intrinsic polarity protein in Arabidopsis that controls stomatal ACD. BASL functions as a paralog of the conserved PAR proteins in metazoans. Our research, supported by the NIH since 2014, has made significant contributions to the current understanding of the molecular mechanisms underlying stomatal ACD. Specifically, we revealed that BASL functions as a scaffold protein that dynamically assembles specific signaling components required for each step of stomatal ACD. Before ACD, the BIN2 GSK3 (Glycogen synthase kinase)-like kinases are polarized to maintain a high cell-division potential. After ACD, a YODA MAPK (Mitogen-activated Protein Kinase) signaling cascade associated with the polarity module promotes cell-fate differentiation. Furthermore, we identified the BSL1 (bri1 Suppressor 1-like 1) phosphatase that, upon mitosis, becomes polarized to dissociate BIN2 while activating YODA at the plasma membrane. Thus, the BSL1 molecular switch enables the transition from cell division to cell-fate differentiation during stomatal ACD. Additionally, our group has determined that polarization of the peripheral membrane protein BASL requires vesicle trafficking regulated by the PRAF (PH, RCC1, and FYVE) endosomal proteins. PRAFs interact with GNOM, an activator of Arf GTPases that plays pivotal roles in endocytic recycling in plants. The discoveries of BSL1 and PRAF represent major milestones in our recent research and offer profound insights into the current understanding of molecular mechanisms underlying cell polarization and plant ACD. In the next five years, by employing a comprehensive approach combining genetics, biochemistry, advanced cell biology, and proteomics/phosphoproteomics, we aim to (1) understand how regulators of membrane trafficking control targeted vesicle delivery to establish the polarity domain at the plasma membrane, and (2) determine how cell-cycle regulation coordinates with the dynamic assembly of polarity proteins in stomatal ACD. Both directions emerge from our recent discoveries and represent significant areas of inquiry. In addition, we will implement and optimize the usage of TurboID and its variants to enable the spatial and temporal precision of proximity labeling in living plant cells.

NIH Research Projects · FY 2024 · 2019-07

PROJECT SUMMARY Human norovirus (HuNoV) gastroenteritis is a significant public health burden worldwide. The lack of a robust and reproducible HuNoV cell culture system still limits our ability to study fundamental aspects of HuNoV infection. While several recent breakthroughs increased our ability to propagate HuNoVs in vitro (e.g. B cell and human enteroid cultures), these systems are not robust enough, can be time consuming and expensive or hard to replicate. The surrogate models available for HuNoV research do not necessarily reflect essential biological features of HuNoVs and their natural host. In this proposal we will use our novel rhesus enteric caliciviruses (ReCV) model that reflects the biological features and diversity of HuNoVs to identify host determinants of infection. Zoonotic/interspecies transmission of ReCVs and HuNoVs between human and non- human primate hosts suggests evolutionary conservation of shared factors of host susceptibility between the two genera. Here we seek to identify host determinants of enteric calicivirus infection using CRISPR-Cas9 genome wide screening. We recently identified a functional ReCV entry receptor that is necessary for ReCV permissiveness in cell culture. We hypothesize that the ReCV entry receptor is also involved in HuNoV infections. This hypothesis will be tested in the following specific aims. Aim 1. Characterize receptor mediated ReCV infection. Aim 2: Comparative characterization of HuNoV and ReCV infections in enteroid and B cell cultures. In the first aim we will dissect the role of HBGAs in ReCV receptor mediated entry by using ReCV isolates with disctinct HBGA binding, cell lines expressing different HBGAs and/or the receptor, Enterobacter SENG-6 EPS and different synthetic HBGAs. We will also evaluate the role of the different transmembrane isoforms of the receptor in infection and map the ReCV interaction site and importance of glycosylation. In the second aim, we will evaluate the role of ReCV entry receptor in HuNoV infections in enteroid and B cell cultures and identify other cell surface components playing a role in HuNoV infections. We will also evaluate the mechanism of bile or bile salts in promoting HuNoV infections and the role of M cells in infections of polarized cells. Our long-term goal is to identify and characterize viral and host determinants of ReCV and HuNoV infections and to develop novel intervention/prevention strategies. Our proposal uses a novel enteric calicivirus model to understand viral entry and the role of bile and HBGAs in enteric viral infections. Our findings with ReCVs may be directly transferable to HuNoV infection. The major significance of this project is the identification of determinants of susceptibility to enteric calicivirus infection that can lead to improved HuNoV cell culture systems and new intervention/prevention strategies.

NIH Research Projects · FY 2026 · 2019-02

The Molecular Basis of Viral DNA Sensing through the cGAS-STING Pathway The innate immune response is the first line of defense against bacterial or viral infections. Nucleic acids from the pathogens serve as danger signals to the host and induce potent innate immune responses. The cGAS- STING pathway plays a central role in sensing viral DNA. Upon DNA binding, cGAS is activated and catalyzes the synthesis of a second messenger cyclic-GMP-AMP (cGAMP). The engagement of cGAMP by STING initiates three lines of signaling events. First, the transcription factor IRF3 is activated and initiates the induction of type I interferon (IFN-I). Second, the transcription factor NF-kB is activated, which synergizes with IRF3 to promote the transcription of IFN-I genes and mediates the induction of proinflammatory cytokines. Third, the activation of STING induces noncanonical autophagy, which is also important for the clearance of intracellular pathogens. The molecular mechanism of the cGAS-STING pathway has been studied extensively over the last few years. The crystal structure of cGAS bound to dsDNA demonstrates that cGAS is activated by DNA induced oligomerization. The structures of STING bound to cGAMP and small molecule agonists reveal the mechanism of STING activation upon ligand binding. The crystal structures of TBK1 and IRF3 bound to peptides from STING C-terminal tail reveal the molecular bases of TBK1 and IRF3 recruitment and activation. The structures of phosphorylated IRF3 provide insight into the mechanism of IRF3 activation by TBK1. The cryo-EM structure of cGAS bound to the nucleosome core particle reveals the mechanism of cGAS inhibition under resting condition. These comprehensive structural and functional studies significantly advanced our understanding about the cGAS-STING pathway. Despite these extensive studies, many aspects of the cGAS- STING pathway remain poorly understood. As a diffusible second messenger, cGAMP can be exported from infected cells and imported by uninfected cells to induce the expression of IFNs. However, the structural basis of cGAMP export remains to be determined. The exact mechanism of IRF3 activation upon phosphorylation by TBK1 remains poorly defined. The activation of NF-kB plays a critical role in IFN-independent response mediated by STING. However, the mechanism of NF-kB activation via STING in remains to be determined. The proposed research will elucidate the molecular bases of several key events involved in the cGAS-STING pathway with the following aims: 1). Elucidate the structural basis of cGAMP export by ABCC1; 2). Determine the molecular basis of IRF3 activation upon phosphorylation; 3). Investigate the mechanism of NF-kB activation via STING. The proposed studies represent a rigorous and comprehensive investigation into the mechanisms of DNA sensing through the cGAS-STING pathway. It will dramatically advance our understanding of the molecular basis of innate immunity against viral DNA and provide a foundation for innovative approaches to treat viral disease, autoimmune disorder, and cancer.

NIH Research Projects · FY 2025 · 2017-08

PROJECT SUMMARY Foodborne illness causes significant morbidity and mortality each year in the United States. Although human foodborne illness is usually associated with the consumption of contaminated food, pets can be direct and indirect sources of bacterial pathogens including Salmonella, Campylobacter, and Listeria. The Food and Drug Administration (FDA) Veterinary Laboratory Investigation Response Network (Vet-LIRN) Veterinary Diagnostic Laboratory Cooperative Agreement Program is designed to promote human and animal health by providing scientific information and building laboratory capacity for routine and emergency response for investigation of outbreaks of disease, information on antimicrobial resistance in pathogens isolated from veterinary patients, and to investigate problems with animal feeds and drugs. Detection of outbreaks requires the ability to collect samples from geographically diverse areas. Texas is home to 29 million people, second only to California in human population, and leads the nation in horse and cattle populations. It is therefore important to understand the dynamics of foodborne diseases among animals and people within the State of Texas. Texas A&M University is home to the oldest, established College of Veterinary Medicine in Texas. The Texas A&M University Veterinary Medical Teaching Hospital Clinical Microbiology Laboratory currently serves as an FDA Vet-LIRN Laboratory. Members of the laboratory have helped to develop Vet-LIRN methods for detection of Salmonella and Campylobacter jejuni, participated in proficiency testing, collected and analyzed samples as part of Vet-LIRN investigations, and provided isolates for the Vet-LIRN ongoing effort to characterize the antimicrobial resistance in pathogens from veterinary patients through whole genome sequencing. The objective of this proposal is to provide infrastructure in Texas to support collection and testing of samples from pets to support the FDA Vet-LIRN mission of protecting human and animal health by prompt recognition of outbreaks and testing of samples associated with outbreaks as well as surveillance assignments as needed by the Vet-LIRN Program Office.

NIH Research Projects · FY 2026 · 2016-04

ABSTRACT Most premature infants experience extrauterine growth restriction for reasons that are unclear and are at in- creased lifelong risk for obesity and type 2 diabetes. Our long-term goal is to identify mechanisms that diminish lean growth and alter metabolic responses to nutrition in preterm infants; these findings will inform the develop- ment of new nutritional strategies to improve outcomes. The objective of this application is to determine if per- sistence of the anabolic resistance to feeding following premature birth impairs lean growth and if specific amino acid supplementation ameliorates lean mass accretion. The central hypothesis is that prematurity limits lean growth by blunting amino acid- and insulin-induced stimulation of protein synthesis and myonuclear accretion in skeletal muscle but can be improved by amino acid supplementation targeted to promote mechanistic target of rapamycin complex 1 (mTORC1)-dependent cellular processes. The hypothesis is based on data from the ap- plicants’ laboratories and supported by the literature. The rationale is that understanding the fundamental mech- anisms by which prematurity alters the anabolic response to nutrition is essential to inform and modify feeding practices for preterm infants to sustain intrauterine growth rates of lean mass after they are born. The hypothesis will be tested by pursuing two specific aims: 1) Determine if the acute protein anabolic resistance to feeding in the preterm is sustained long-term and results in reduced muscle and lean mass accretion; and 2) Determine if supplementation with leucine and/or the arginine precursor, citrulline enhances lean growth by upregulating mTORC1-dependent muscle protein synthesis and myonuclear accretion. When pigs born preterm and term reach ages equivalent to human late-infancy or late-childhood, we will determine body composition, growth rate, energy expenditure, hormone, substrate and metabolite profiles, and skeletal muscle protein synthesis and deg- radation rates, blood flow, amino acid, insulin and eNOS signaling, metabolomic and transcriptomic profiles, and satellite cell abundance and proliferation in response to feeding, pancreatic-substrate clamps, and supplemen- tation with leucine and/or citrulline. The methods are established in the applicants’ laboratories. The approach is innovative because it will use comprehensive approaches that will examine concurrently in vivo responses to preterm birth of the principal processes that regulate muscle growth, i.e., protein synthesis, protein degradation, and myonuclear accretion, and how these processes respond to dietary interventions targeted to promote anab- olism. The proposed studies are unique because they comprehensively examine in a relevant preterm model the mechanisms that underlie the anabolic resistance of the premature which limits lean growth and examine the effectiveness of targeted amino acid supplementation on processes that regulate skeletal muscle growth. The proposed research is significant because it will advance our understanding of how prematurity impacts the anabolic response of skeletal muscle to nutrition. The results will provide novel information to optimize the nutri- tional management of preterm infants to improve their long-term metabolic health and growth.

NIH Research Projects · FY 2026 · 2014-09

Protein Kinases C (PKC) define a family of lipid-activated kinases that are key effectors of phosphoinositide signaling – a major intracellular signaling pathway of eukaryotic cells. Consistent with this central activity, dysregulation of PKC signaling is implicated in cancer progression, cardiac disease, diabetes, and Alzheimer’s disease. Because of the fundamental role of PKC in signal transduction, the development of modulators of PKC activity – both for therapeutic and basic research purposes – is widely recognized as one of the major challenges in the field. Addressing these challenges requires an atomic-level description of PKC control – in particular, how lipids and exogenous agonists regulate PKC activity. This requirement defines a major gap in understanding of PKC regulatory mechanisms. This research proposal builds upon two key discoveries made by our laboratory. First, an atomistic resolution of how DAG is recognized and captured in membranes by the conserved homology 1 (C1) domains of PKC that eluded the field for 30 years. Second, the discovery that novel PKC isoforms have an unexplored lipid-sensing function that adds a novel facet to control of PKC activity. This renewal application proposes to apply an integrated research strategy consisting of solution NMR spectroscopy, x-ray crystallography, atomistic molecular dynamics simulations, and imaging experiments in yeast and mammalian cells to address the following Specific Aims: (1) determine the role of phosphoinositides in PKC localization and activation, and (2) determine the molecular basis of PKC activation by novel tool compounds and drugs that target the C1 domains. These studies will deliver functional, structural, thermodynamic, and kinetic information to understand the determinants of potency and isoform selectivity of exogenous PKC agonists and provide key information regarding how PKCs execute coincidence detection of DAG and phosphoinositides. Insights into the PKC regulatory mechanisms obtained from the proposed studies will be impactful in that these will extend the already large problem of PKC regulation into previously unexplored areas. The atomistic information regarding C1 interactions with exogenous ligands and regulatory lipids will be directly applicable to five other large families of essential signaling proteins that rely on C1 domains for regulation of their DAG-dependent activities. This knowledge will also facilitate a more efficient structure-based design of pharmacological agents that modulate PKC activity by direct targeting of its C1 domains.

NIH Research Projects · FY 2025 · 2004-08

Project Summary Veterinarians are broadly trained health professionals uniquely qualified to participate in biomedical research, having an understanding of health and disease at the organismal level with an appreciation of comparative biology1,2.The “One Health” concept, integrating discoveries in both human and veterinary medicine, has received increased attention with new and emerging zoonoses, as well as increasingly common chronic conditions (e.g., cardiovascular disease and diabetes mellitus), and increased concerns with the effects of environmental and dietary toxins on fetal and early stages of development. As translational research with animal models continue to expand to approximately half of all current NIH research projects1, veterinarians, particularly those with research training, make increasingly valuable contributions to biomedical research.3 Reports by National Academy of Sciences National Research Council Committees concluded that there is a critical shortage of such veterinarians4,5. The Texas A&M University summer Veterinary Student Research Training Program (VMSRTP) is evolving and expanding during our next T35 renewal period. In addition to our previous goal to introduce veterinary students to the biomedical research environment to entice trainees to discover research as an exciting career option, we will now expand our program recruitment efforts to include veterinary students who have previous research experience. Providing these experienced students with a robust summer biomedical research experience with experienced federally funded mentors and enhanced training in responsible conduct of research methods and scientific communication skills will likely enhance their individual research successes, and further establish their interest and commitment to veterinary careers in biomedical research. In this competitive renewal, the program will be led by a new Program Director, a new Program Coordinator, a reconfigured Advisory Committee, and a more focused research mentor base with 27 experienced mentors with strong federal extramural biomedical funding. The summer program includes: 1) biomedical research in a mentor’s laboratory; (2) at least 12 hours formal training in responsible conduct of research; (3) weekly lunchtime (hour-long) training sessions in scientific communication via oral and poster presentations, abstract and manuscript preparation, as well as training in critical evaluation and presentation of primary journal articles; (4) oral research presentations in the annual CVM research symposium; and 5) research poster presentations at the National Veterinary Scholars Symposium. The program has exceptional institutional support and a broad array of state-of-the-art facilities, including preclinical GLP labs, a national biodefense lab, primate center, strong institutional collaborations, and a recently funded DHHS Biodefense vaccine center. A major obstacle to the efforts of academic institutions to produce more research veterinarians is the difficulty of providing adequate salary sources to introduce trainees to research. The training positions provided by this T35 would assist recruiting efforts, increase numbers of veterinarians in research, and help alleviate this national shortage.