Texas A&M University

NIH Research Projects · FY 2025 · 2023-08

PROJECT SUMMARY/ABSTRACT: The impact of synthetic chemistry on society cannot be overstated. Organic chemistry has changed our world in momentous ways, from giving women reproductive rights through the invention of contraceptives, to creating pesticides that allow us to feed the globe. It is axiomatic that innovations in medicine are invariably linked to advancements in organic chemistry as most medicines are synthesized by organic chemists. It has long been recognized that the overall shape of a small molecule is the most fundamental factor that controls its biological effects. It is fortunate that rapid developments in asymmetric synthesis have paved the way for therapeutics to reach the clinic. These triumphs can be attributed to the many innovations in the realms of enantioselective bond forming processes such as asymmetric ion pairing, organocatalysis, C–H activation, Lewis acid/base, BrØnsted acid/base, reductions/oxidations, cross-coupling reactions and many more. Despite these achievements, the state of the art still falls short in many ways from the ideal. Although each of the unique activation modes outlined above allow for high chemo-, diastero- and enantio-selectivities to be achieved, in many cases for the desired bond forming event to occur the substrate must often bear a functional group that is capable of binding or being activated by a chiral catalyst. In particular, the ability to enantioselectively convert inert C–H bonds into carbon- carbon bonds at specific locations without the aid of directing groups is highly desirable because it would further the drug discovery process. To address this challenge, our work has focused on the development of new deprotonation substitution sequences which allow for typically untargetable positions within heterocycles to be directly functionalized. Specifically, we have found that Lewis bases can extract Li cations from strong organolithium reagents allowing highly basic ion pairs to be produced and in turn for typically remote and inert C–H bonds in heterocycles to be deprotonated. Secondly, we have developed a new class of chiral phosphine ligands that enable enantioconvergent cross-couplings with racemic donor reagents. This proposal seeks to merge these concepts by developing asymmetric deprotonation cross-coupling sequences that can allow for typically inaccessible carbon centers to be functionalized. Specific goals of this proposal include: (1) the development of both organolithium reagents and Lewis bases that when combined allow for typically inert C–H bonds in alkaloids to be deprotonated; (2) the development of new chiral phosphine ligands that enable enantioconvergent Negishi cross-coupling reactions with racemic donor reagents; and (3) the development of asymmetric deprotonation cross-coupling sequences that allow for biologically relevant alkaloids to be directly functionalized in a single synthetic operation.

NIH Research Projects · FY 2026 · 2023-07

Although older adults in public housing face serious threats to their heat-related health, current heat assessment and risk prevention frameworks neglect physiological conditions and place-based differences in housing and green space characteristics. Our long-term goal is to develop quantifiable measures and doseresponse relationships between the density and characteristics of urban green infrastructure (GI), defined as green space and natural landscapes, and heat-related health outcomes for older adults, which can inform community planning and public health initiatives that support aging in place. Our objectives are to: 1) assess heat-related health risks for older adults in public housing neighborhoods; 2) determine the effects of GI on micrometeorological conditions and heat stress; and 3) evaluate the extent to which neighborhood GI mitigates heat-related health risks via emotional, cognitive, and social pathways. Our central hypothesis is that neighborhood GI characteristics are associated with a reduced risk of heat-related illness for older adults in public housing. To achieve Aim 1, we will perform heat-related health risk evaluations by combining information on biometeorological heat exposure, group characteristics, and heat coping abilities. Specifically, biometeorological heat exposure will be evaluated based on a novel human heat stress model that accounts for the physiology of older adults. To achieve Aim 2, we will develop 3-dimensional measures of GI characteristics using remote sensing data and street-level imagery and video classifications and identify interand intra-neighborhood GI attributes that relate to micrometeorological parameters and heat stress in older adults. To achieve Aim 3, we will conduct a 2-wave panel survey with multi-stage sampling of older adults in public housing neighborhoods in Houston and Chicago. By comparing baseline measures collected in the spring wave with those during heat conditions in the summer wave, we will assess the sociopsychological pathways through which neighborhood GI is associated with heat-related health and behavioral outcomes, and subjective well-being. The research proposed in this application is innovative because it develops heat-related health risk assessments that integrate a novel age-specific human heat-stress model. It also focuses on GI as a modifiable risk factor and adopts a socioecological perspective to elucidate the extent to which individuals’ interaction with their neighborhood’s GI can moderate heat-related health risks via emotional, cognitive, and social pathways. The proposed research is significant because it is expected to provide strong scientific justification for heat-related health assessment that clarifies the complex transactions between the communitylevel green space and an individual’s health. Ultimately, such knowledge has the potential to inform new public health initiatives that enhance the health and wellbeing of aging populations

NIH Research Projects · FY 2026 · 2023-05

Project Summary The overall goal of this project is to develop a new intracortical electrode array where a flat device is inserted that then deploys microelectrodes at controlled distances within a volume of tissue. This deployable neural interface will overcome two critical limitations associated with recording and stimulation of the cortex: 1) each insertion only leads to placement of electrodes at a point or along a linear path within brain tissue and 2) the recording performance is reduced by adverse tissue response. This work will result in deployable electrode arrays using shape-changing liquid crystal polymer (LCP) substrates with microelectrodes patterned by photolithography. The LCP substrates remain flat during fabrication and processing, then deploy after implantation to predetermined locations that are up to 200 µm away from the implantation site. The central premise of this work is that electrode deployment from a single insertion can enable volumetric placement of microelectrodes in mouse cortex with viable recording and stimulation capability for over four months. The team’s published and preliminary data demonstrate the feasibility of creating deployable electrode arrays using shape-changing liquid crystal polymer (LCP) substrates with microelectrodes patterned by photolithography. To realize this premise, three specific aims are proposed: 1) Fabricate and characterize deployable electrode arrays, 2) Characterize the foreign body response elicited by deploying microelectrode arrays, and 3) Characterize deploying microelectrode arrays by chronic recording and measuring electrochemical performance of the electrodes. The team brings together the necessary expertise in materials and microfabrication (PI Ware) and neural interface design and characterization (Co-I Pancrazio), and histological response to implanted devices (Co-I Capadona).

NIH Research Projects · FY 2026 · 2023-04

PROJECT SUMMARY/ABSTRACT A substantial proportion of individuals with alcohol use disorder (AUD) also meet criteria for posttraumatic stress disorder (PTSD). The co-occurrence of AUD/PTSD is characterized by more severe symptomatology, greater functional impairment, increased suicide risk, and poorer treatment outcomes as compared to either disorder alone. Trauma-focused, cognitive-behavioral interventions delivered alongside interventions for substance use disorders are most effective in reducing PTSD severity and substance use. Cognitive Processing Therapy (CPT) for PTSD and Relapse Prevention (RP) for AUD are two of the most widely used and efficacious behavioral treatments for these conditions. The investigators successfully developed and pilot tested a therapy manual that combines CPT with RP. The preliminary data demonstrate safety, feasibility, high rates of retention (80.0%) and patient satisfaction. Moreover, our data from a recent national survey of frontline mental health providers (N = 76) indicate that CPT is the most commonly used trauma-focused treatment for PTSD and providers are highly interested in an integrative CPT-RP intervention, conferring strong potential for uptake in real-world practice settings. In fact, due to the lack of an available, empirically developed, manualized CPT-RP treatment, 84.0% of frontline providers report attempting on their own to create such a treatment to use with their patients. This may result in highly variable and suboptimal implementation and outcomes. In response to provider input and positive preliminary data, the proposed study directly addresses this critical need by evaluating a new integrative CPT- RP treatment for individuals with co-occurring AUD and PTSD. At present, only one trauma-focused, integrative intervention is available for AUD/PTSD and it uses Prolonged Exposure (PE) to reduce PTSD symptoms. In comparison to PE, CPT is more widely used, often preferred by clinicians, equally as effective in reducing PTSD symptoms, and associated with lower dropout rates. Thus, the new CPT-RP intervention could have wider reach and greater acceptability than exposure-based treatments. Treatment choice is related to improved treatment outcomes, and therefore, there is an immediate need to add to the portfolio of evidence-based, trauma-focused, integrative treatments for AUD/PTSD. The primary objective of this Stage II study is to examine the efficacy of CPT-RP, as compared to RP alone, in reducing (1) alcohol use frequency and quantity and (2) PTSD symptom severity among individuals with current AUD/PTSD. To accomplish this, we will employ a manualized intervention, randomized study design, and standardized repeated dependent measures of clinical outcomes at multiple time points. Putative mechanisms of behavior change will be evaluated via daily monitoring. The proposed study aligns closely with the mission of NIAAA in that it aims to produce maximally efficacious behavioral interventions for AUD and comorbid psychiatric disorders such as PTSD. The findings from this study will provide new information to advance the science of AUD/PTSD comorbidity and innovate clinical practice.

NIH Research Projects · FY 2025 · 2023-04

Project Summary The current cancer treatment bottleneck showcases the need for more innovative approaches in drug discovery efforts. As of recent, immune-checkpoint inhibitors (ICHs) have shown promise in cancer therapeutics, and work by manipulating immunoregulatory pathways involved in tumor detection and elimination. Many cancer types overexpress the PD-L1 receptor to inhibit T cell activation and subsequently evade the immune response. Though proven effective, the PD-1/PD-L1 pathway targeting ICHs are pharmacologically limited by occupancy- based inhibition. A new drug paradigm has emerged through the development of proteolysis targeting chimeras (PROTACs) that are not limited by such a mechanism. PROTACs are heterobifunctional molecules consisting of an E3 ubiquitin (Ub) ligase ligand, a target protein warhead, and a linker connecting the two and work by bringing an E3 Ub ligase and the target protein together through bivalent binding. The formation of a ternary structure through this bivalent interaction drives the polyubiquitination and subsequent degradation of the target via the ubiquitin proteasome system (UPS). Although this drug design has shown great medicinal promise through research findings and clinical trials, there is seldom literature on the use of transmembrane E3 Ub ligase recruiting PROTACs to degrade cancer-promoting membrane proteins such as PD-L1. The proposed project is aimed at investigating the applicability of such PD-L1 PROTACs by recruitment of the transmembrane E3 Ub ligase ZNRF3. In doing so, three specific aims will be investigated: Aim 1. Identifying cyclic peptide binders of the ZNRF3 extracellular domain. Previous research in the Liu lab utilized a cyanobenzothiazole based linker that allows for cyclization of peptides on a phage surface with applications in phage selections against CD44 and SARS-CoV-2 spike protein epitopes. Recombinant bacterial expression of biotinylated ZNRF3 ECD has been developed, and the binding of selected cyclic peptides will be validated through BLI and flow cytometry. Aim 2. Synthesis and in-vitro characterization of PD-L1 PROTACs. Reported binders of PD-L1 will be incorporated in the PROTAC design via polyethylene glycol (PEG) and polyether linkages of varying lengths. The optimal positioning of linkers will be determined through structure-activity relationships (SAR) analysis, binding assays, co-cocrystal structures, and molecular docking studies of ligand-protein interactions of the ZNRF3 ECD and PD-L1. Aim 3. In-vivo characterization of PD-L1 PROTACs. Degradation potency will be evaluated with western-blotting of residual PD-L1 and ZNRF3 dependency on the induced degradation of PD-L1 and mode of action (proteosome vs lysosome) will be assessed. In addition, whole-cell proteomics studies will be conducted to investigate any consequences on normal cellular activities due to PROTAC activity. By accomplishing these aims, new and important conclusions can be made about the efficacy and mode of action of our designed PROTACs that we hope will have a lasting impact on the cancer therapeutics bottleneck and general drug discovery efforts.

NIH Research Projects · FY 2026 · 2023-01

PROJECT SUMMARY In 2021, it is estimated that there will be 149,480 new cases of colon cancer diagnosed in the United States and 52,980 CRC patients will die from this disease in which the incidence and death are only slightly lower in women compared to men. Early diagnosis and improved treatments have decreased the incidence of CRC in the United States; however, global rates of this disease are increasing, particularly in developing countries. Individuals with a family history of colon cancer or inherited genetic mutations have a high risk for developing CRC, however, it is estimated that 60-65% of all cases are “sporadic” with no inherited or genetic risk factors in their background. Some of the risk factors for the sporadic CRCs are shared by many other cancers and this includes age but also other preventable factors such as obesity, sedentary lifestyle, smoking, low fiber/high fat diets, alcohol consumption and high intakes of red meat. Another sub-class of CRC patients are young adults and the incidence of disease among this group has been increasing particularly in high income developed countries. Surgery is a major first option for many colon cancer patients and this can also be accompanied or preceded by radiation and neoadjuvant therapy. High risk patients with stage III and IV colon cancer are treated with various drug combinations including fluorouracil, leucovorin, folinic acid, oxaliplatin and capecitabine which inhibit the risk of disease recurrence. In addition to cytotoxic drug therapies more precision medicine approaches that target specific genes such as EGFR and pathways (angiogenesis) are being developed along with immunotherapies. The proposed research will focus on potential mechanisms and clinical applications of bis-indole derived (CDIMs) agents that bind the orphan nuclear receptor 4A1 (NR4A1, Nur77). NR4A1 is overexpressed in colon cancer patients and regulates expression not only of pro-oncogenic genes and pathways but also PD-L1 and genes associated with T-cell exhaustion that can be identified in tumor infiltrating lymphocytes. Preliminary studies confirm that CDIM/NR4A1 antagonists exhibit both chemotherapeutic activity but also downregulate PD-L1 and reverse T-cell exhaustion and proposed studies in Aim 1 will determine the underlying mechanisms using in vitro cell culture and syngeneic mouse models (Aim 1). In Aim 2 the chemotherapeutic and immunologic effects of CDIM/NR4A1 antagonists will be investigated in an inducible APC knockout mouse model and in mice crossed with whole body and intestinal specific NR4A1 deletion. These studies, coupled with histological analysis of cycling stem cells and single cell analysis of the tumor microenvironment and immune system will confirm the role of NR4A1 as an important emerging drug target for both colon cancer chemo- and immune therapies.

NIH Research Projects · FY 2025 · 2022-09

Overall Program Description Man-made and extreme weather event-related disasters coupled with economic activity and the enhanced vulnerability of communities located in close proximity to potential sources of hazardous chemicals markedly increase risks from catastrophic chemical contamination events. The complexities of chemical exposures and their potential adverse health impacts, the need to rapidly and comprehensively evaluate the potential hazards of exposures to complex mixtures, and the necessity of protecting susceptible populations and life-stages call for novel approaches in the Superfund Research Program. This Center consists of a team of well-established scientists from biomedical, geosciences, data science, and engineering disciplines who share a common goal: to develop, apply, and translate a comprehensive set of tools and models that will aid first responders, impacted communities, and government agencies in characterizing and mitigating the human health consequences of exposure to hazardous mixtures. These will be applicable for both existing contaminated waste sites and disaster-related contamination events. Project 1 will develop novel analytical and computational strategies for exposure assessment of complex mixtures. Project 2 will develop novel tools to rapidly characterize pediatric respiratory health risks from exposure to hazardous volatile organic compounds after environmental disasters. Project 3 will develop and use feto-maternal interface tissue chip models for rapid assessment of preterm birth risks of hazardous substances. Project 4 will continue development of predictive in vitro methods for quantitative evaluation of the complex mixtures and intra- and inter-individual variability in toxicity. Project 5 is responsive to the Superfund remediation mandate by using experimental and computational engineering to develop optimized multi-component sorbents for toxic mixtures. A Disaster Research Response (DR2) Core will be a centralized resource for environmental sampling and assessment before, during, and after disasters. A Data Management and Analysis Core will develop computational and statistical tools for analysis and integration of “big data” in environmental health. A Risk and Geospatial Science Core will provide the Center with data and services for characterizing human health risks and the geographic distribution of hazardous substances during disasters. The Center will engage with community organizations and public health practitioners in Texas to address health concerns of the populations that may be impacted by environmental emergency-related contamination events. We will continue training students and postdoctoral fellows in inter-disciplinary approaches across our scientific areas, decision making and emergency response. The research translation to local, state, national and international stakeholders involves both technology transfer and regular outreach. Finally, the management of this program includes close partnerships with the Texas A&M University administration and the NIEHS-funded Center for Translational Environmental Health Research, and is overseen by the advisors representing academia, federal and state agencies, industry and a non-governmental organization.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Integral membrane proteins reside in the biological membrane where they function and intimately interact with lipid molecules. The membrane environment is dynamic and composed of a rich chemical diversity of lipids. Alongside the complexity of the biological membrane is the growing realization of the important roles of lipid molecules in modulating the structure and function of membrane proteins. Although a small subset of examples exist that provide insight into membrane protein-lipid interactions, how individual lipid molecules influence the structure and function of membrane proteins on the molecular level remains poorly understood. What determines the selectivity of membrane proteins towards lipids? How important is the lipid chemistry such as lipid tail length, stereochemistry, and position of unsaturated double bonds in protein-lipid interactions? Addressing these fundamental questions is hindered not only by identifying the lipids that bind avidly to membrane proteins but also the biophysical characterization of protein-lipid interactions. Herein, this proposal seeks to develop and apply a highly innovative and integrative approaches to better understand how lipids impact the structure and function of membrane proteins. Our first objective is the development of integrative methods, combining lipidomics with native mass spectrometry (MS), to identify specific protein-lipid interactions from natural extracts by using (i) progressive washes of the immobilized membrane proteins and (ii) lipid exchange within empty and membrane protein loaded nanodiscs. In our second objective, native MS technology will be used to biophysically characterize individual lipid binding events to membrane proteins, providing insight into affinity and selectivity. Moreover, MS approaches of membrane proteins in nanodiscs will be employed to glean insight into lipids enriched around membrane proteins. These new methods will identify lipids that avidly associate with the target membrane protein, providing a roadmap for our third objective focused on understanding how tightly bound lipids affect function and structure of membrane proteins. Membrane proteins devoid of any contaminating lipids will be reconstituted into liposomes and nanodiscs in the presence and absence of a tightly bound lipid. Structural and functional studies will lead to visualization of lipid binding sites and structural and functional changes induced by the bound lipids. Observations from structural and functional studies will then be rigorously examined with mutational studies. Taken together, the results and outcomes from our proposed studies are anticipated to have a significant impact in our understanding of membrane protein-lipid interactions and, more generally, how changes in the biological membrane may regulate membrane protein physiological function.

NIH Research Projects · FY 2024 · 2022-09

Project Summary/Abstract Down Syndrome (DS) is a common birth defect caused by trisomy of human chromosome 21 (Hsa21). DS leads to a vast array of clinical abnormalities affecting most systems of the body, including delayed wound healing, and an increased risk of long bone and vertebral fractures. Previously, this laboratory has shown that DS patients have low bone turnover leading to decreased bone mineral density and delayed accrual of peak adult bone mass, and also confirmed the low bone mass phenotype in mouse models of DS. Additionally, it has been recently reported that COX2/PGE2 expression is impaired in DS human dermal fibroblasts, which could contribute to the delayed wound healing and increased risk of infections in the DS population. PGE2 and its receptors are major mediators of inflammation, wound healing, bone formation and bone healing. More specifically, the PGE2 receptor subunit 2 (EP2, Ptger2) and EP4, have been shown to regulate bone formation, and play a crucial role in fracture healing, whereas EP3 and EP4 contribute to macrophage recruitment, the immune response, and lymphangiogenesis in wound healing. However, what is not known is how PGE2 signaling contributes to bone repair and whether low bone accrual in DS impacts bone regeneration. The overarching objective of this proposal is to characterize bone healing in DS mouse models and determine if pharmaceutical treatment at different stages during the regenerative process is able to enhance regeneration in DS. This project will test the hypothesis that bone healing is significantly impaired in DS, and that decreased bone turnover leads to attenuated bone regeneration. This study will utilize the DS mouse models, Dp16 and Ts65Dn, that demonstrate the low bone mass phenotype consistent with the low bone mass observed in DS patients, to investigate de novo bone regeneration after amputation of the terminal phalanx (P3). P3 amputation is a model of mammalian injury that faithfully triggers a well-defined regenerative sequence of events that initiates with inflammation followed by bone resorption, wound closure, and de novo bone formation, allowing the characterization of the entire injury response. Experiments in Aim 1 will seek to characterize P3 regeneration in the DS mouse models compared to WT littermates to test the hypothesis that bone regeneration is impaired in DS mouse models. Aim 2 will investigate the early, or lytic, phase of regeneration and determine whether treatment with a PGE2 receptor (EP3 and EP4) agonist elicits an immune and wound healing response that is sufficient to enhance regeneration in DS. The proposed studies of de novo bone regeneration will help to close the gap in knowledge regarding how DS impacts bone healing and repair, and provide insight into how patient care can be modified to adequately treat bone injuries in the at-risk DS population.

NIH Research Projects · FY 2026 · 2022-09

ABSTRACT/SUMMARY Endometriosis is an inflammatory gynecological disease of reproductive-age women. The two major symptoms are chronic pelvic pain and infertility, which profoundly affect reproductive health and life quality. The pro- inflammatory microenvironment of the endometriotic lesions, endometrium, and the peritoneal cavity are the hallmarks of endometriosis, which leads to chronic pelvic pain and infertility. Emerging evidence suggests that pelvic pain in endometriosis is a combination of visceral inflammatory and neuropathic pain. The long-term goals are to understand the molecular and cellular pathogenesis of endometriosis and identify non-steroidal therapy for endometriosis. Withania somnifera, an ancient medicinal plant, has been used to treat inflammatory disorders, neurological diseases/disorders, neuropathic and inflammatory pain, and cancers. A wide range of therapeutic effects of Withania somnifera is exerted through its active steroidal lactone Withaferin-A. However, no studies have investigated the role of Withania somnifera/Withaferin-A in endometriosis. The proposed timely needed hypothesis-driven novel preclinical research is focused on the therapeutic effects of Withaferin-A in endometriosis. Overarching Hypothesis: Treatment of Withaferin-A suppresses the growth and survival of endometriotic lesions, ameliorates pelvic pain, and restores the endometrial microenvironment to support fertility. Objectives: Determine the therapeutic effects of Withaferin-A on (i) growth and survival of the endometriotic lesions, (ii) pro-inflammatory state of the peritoneal cavity, endometriotic lesions, and pain-brain areas, (iii) pro- inflammatory and hormonal microenvironment of the endometrium, (iv) epithelial and stromal cell-specific gene signatures in the endometriotic lesions and endometrium, (v) ascending and descending pain pathway and central sensitization and the underlying molecular and cellular mechanisms, and (vi) pelvic pain threshold and spontaneous pain. Specific Aim-1 will determine the therapeutic effects of Withaferin-A on endometriotic lesions and endometrium. Specific Aim-2 will determine the therapeutic effects of Withaferin-A on central pain mechanisms in endometriosis. The experimental approaches include the preclinical allograft mouse model of endometriosis, RNA-Seq, electrophysiology of brain neurons, ascending and descending pain pathways, pain behavior, in vivo bioimaging, fluorescence microscopy, and flow cytometry. Innovation and Impact: The new information obtained should fill the gap in the knowledge on therapeutic effects of Withaferin-A in (i) central mechanisms of pelvic pain, gene expression signatures in the pain-brain areas, and their association with chronic pelvic pain in endometriosis; (ii) epithelial and stromal cell-specific gene expression signatures in the endometriotic lesions and their association with growth and survival of endometriotic lesions; and (iii) epithelial and stromal cell-specific gene expression signatures in the endometrium and their association with infertility. (iv) The novel results will establish a molecular therapeutic and preclinical basis to use Withaferin-A as a non- steroidal and non-opioid therapy to treat pelvic pain and infertility in women with endometriosis.

NIH Research Projects · FY 2025 · 2022-08

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea in the hospital and long term health care settings. In addition to the patient toll, the treatment-associated costs of C. difficile infections to the United States healthcare system have been estimated at $5 billion. Although the rate of C. difficile infection in the United States is rising, surprisingly little is known about the mechanisms by which C. difficile spores maintain their extreme resistance properties. C. difficile is believed to be acquired by the host in the form of a dormant spore which contaminates the nosocomial environment. In other organisms, a significant amount of resistant to UV and genotoxic chemicals is provided by the small acid soluble proteins (SASPs). SASPs are thought to nonspecifically bind to DNA and alter its form to prevent the formation of thymidyl-thymidine adducts (thymidine dimers) upon UV exposure. These proteins also provide resistances to reactive oxygen, acids, crosslinking agents and minor resistance to heat. In C. difficile, however, nothing is known about how these important proteins contribute to the C. difficile spore resistance spectrum. C. difficile SspA provides resistance to UV but SspA & SspB play a role in spore formation. The C. difficile ΔsspA ΔsspB double mutant strain does not produce mature spores suggesting that these SASPs regulate, at some level, the sporulation program. This application will analyze the impact of SASPs on spore biology in three aims. Aim 1 investigates how SspA & SspB contributes to spore formation and how the C. difficile SASPs protect the C. difficile genome from UV damage. Aim 2 investigates mechanisms by which the SASPs contribute to spore formation. Aim 3 investigates how the protease that degrades the SASPs during germination is regulated and its spectrum of activity in the spore. Successful completion of these aims will thoroughly characterize the impact of SASPs on spore resistance and lead to the future design of agents that can clean a contaminated environment.

NIH Research Projects · FY 2025 · 2022-06

SUMMARY Transcription activation relies on the cooperative activity of multiple transcription factors (TFs) that bind cis- regulatory elements (CREs) near their target genes. Although techniques like chromatin immunoprecipitation coupled to sequencing helped uncover the genome-wide location of binding sites for many TFs in different spe- cies and tissues, it still remains unclear how TFs cooperate to compete with histones, bind DNA, regulate CRE activity, and drive transcription activation. In this proposal, we will address this knowledge gap by exploiting the circadian regulation of gene expression by the TF CLOCK:BMAL1. Recent reports from our lab and the litera- ture indicate that the circadian regulation of transcription by CLOCK:BMAL1 relies on the interaction between CLOCK:BMAL1 and other TFs rather than on core clock genes only. CLOCK:BMAL1 rhythmic DNA binding promotes the rhythmic removal of nucleosomes at its CREs, strongly suggesting that CLOCK:BMAL1 gener- ates a permissive chromatin landscape that facilitates the rhythmic recruitment of other TFs at its CREs. This cooperation between CLOCK:BMAL1 and other TFs may explain why less than a third of CLOCK:BMAL1 tar- gets are rhythmically expressed under normal condition, and how their rhythmic transcription can be repro- grammed when animals are exposed to a new environment. This proposal builds upon these findings and the proposed experiments will determine if CLOCK:BMAL1 promotes the formation of partially unwrapped nucleo- somes (Aim 1), the modalities of CLOCK:BMAL1 cooperation with other TFs by using a method of single-mole- cule footprinting (Aim 2), and if high-fat diet alters the cooperativity between CLOCK:BMAL1 and other TFs to reprogram CRE transcriptional activity (Aim 3). Results from these experiments are expected to uncover novel and important mechanisms that control the rhythmic expression of thousands of genes, and to provide a new conceptual framework for how biological functions are coordinated between tissues. They are also expected to provide insights into the mechanisms that underlie the role of TF cooperativity in the control of CRE activity that can be widely relevant to fields beyond the circadian field.

NIH Research Projects · FY 2026 · 2022-04

PROJECT SUMMARY/ABSTRACT As a naturally existing amber suppression system, the pyrrolysine (Pyl) incorporation machinery has turned into an enormous tool for undergoing amber suppression-based noncanonical amino acid (ncAA) mutagenesis in both prokaryotic and eukaryotic cells. By ectopically expressing tRNAPyl and pyrrolysyl-tRNA synthetase (PylRS) or a PylRS mutant that charges tRNAPyl with an ncAA, about 200 ncAAs have been genetically encoded by the amber codon in various cells. As one of the original pioneers in the research field of engineering the Pyl system for the genetic incorporation of ncAAs, the PI’s group has contributed more than one third of the total encoded ncAAs. These ncAAs contain a large variety of functionalities, allowing a myriad of applications in both academia and industry possible. After more than a decade of engineering the Pyl system for the ncAA incorporation, the field is now able to use the Pyl system-based ncAA mutagenesis technique to conduct grand explorations to address fundamentally important biological questions. In the past, the PI’s lab has devised a variety of ncAA mutagenesis-based approaches for the synthesis of proteins with posttranslational lysine modifications (lysine PTMs). A novel method that allows direct functionalization of ubiquitin and ubiquitin like proteins for their conjugation with other proteins has also been developed in the PI’s lab. With all these methods coming to fruition, the PI’s lab is shifting its research focus to use their developed techniques to study basic and fundamentally important biological questions. Five specific directions will be pursued. The first direction is to use the ncAA mutagenesis technique to produce designer nucleosomes (nucleosomes with defined lysine PTMs) for probing histone lysine sites and PTM types targeted by epigenetic erasers including SIRT6, SIRT7, HDAC1, and LSD1 (HDAC1 and LSD1 in their native complexes). The second direction is to use reconstituted designer nucleosomes as probes to enrich their binding partners from cells whose identities can be determined using mass spectrometry-based proteomic analysis. The third direction is to conduct cryo-EM analysis of designer nucleosomes bound with SIRT6, SIRT7, HDAC1, and LSD1 (HDAC1 and LSD1 in their native complexes) to elucidate the structural basis of the four enzymes in their recognition of targeted lysine sites and PTMs in chromatin. The fourth direction is to synthesize different triubiquitin isoforms and use them as probes to enrich binding partners from cells whose identities will be confirmed with mass spectrometry-based proteomic analysis. The last but not least direction is to synthesize cyclic GMP-AMP synthase (cGAS), which is a frontline sensor in human cells that detect double-stranded DNA from pathogens and triggers innate immune responses, with lysine PTMs and to study the functional roles of lysine PTMs in regulating activity, cellular localization and cellular half- life of cGAS. A long-term goal of the PI’s research is to push applications of the Pyl system-based ncAA mutagenesis technique in combination with other chemical biology techniques to enhance basic biological research.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY Nearly every mammalian cell harbors a timekeeping mechanism, the circadian clock, that drives overt rhythms in gene expression to coordinate the daily activity of biochemical and metabolic pathways. Consistent with the large number of biological functions controlled by the circadian clock, disruption of rhythmic gene expression leads to the development of a wide range of disorders that include metabolic diseases, cardiovascular disorders and cancer. Moreover, most commonly used drugs in the United States directly target the products of rhythmically expressed genes. For these reasons, characterizing the mechanisms underlying rhythmic gene expression is critical to not only understand how clock dysfunction leads to pathological conditions, but also to optimally time pharmacological treatment. Rhythmic gene expression is thought to be primarily regulated by the molecular circadian clock found in every mammalian cell. However, increasing evidences from our lab and others suggest that environmental signals like feeding rhythms generate 24-hour rhythms in gene expression without involving the circadian clock oscillation. In Preliminary Studies, we show that the amplitude of feeding rhythms controls the rhythmic expression of more than 2000 genes in mouse liver. Surprisingly, this effect on gene expression does not seem to directly involve the hepatic circadian clock, which continues to exhibit normal oscillations in core clock gene expression. Rather, our preliminary data suggest that rhythms in gene expression rely on the rhythmic activity of the nutrient-sensing kinase mTOR. This proposal builds upon these new exciting data and the proposed experiments will determine if feeding rhythms regulate rhythmic gene expression by (1) controlling the rhythmic activity of mTOR signaling pathway, and (2) regulating the rhythmic activity of metabolic transcription factors. Results from these experiments are expected to uncover novel and important mechanisms for the regulation of rhythmic gene expression in mammals, and to provide a new conceptual framework for how biological functions are synchronized to environmental cycles and coordinated between tissues. They are also anticipated to lead to the development of novel strategies for advancements in chronotherapy and for the restoration of rhythmic gene expression in humans showing poor circadian rhythms like shift-workers and elders.

NIH Research Projects · FY 2025 · 2021-12

Biotin is an essential vitamin in the human diet. The adequate daily intake for adult humans is in the range of 30-100 micrograms. The biotin market is divided between agricultural and human use with 90% and 10% going to animal and human nutrition respectively. Currently biotin is produced by chemical synthesis at the level of >20 tons/year. Biotin biosynthesis is well-understood and efforts to produce it by fermentation are currently under intense investigation in several biotechnology companies. Biotin functions as a cofactor in all biochemical carboxylation reactions involving bicarbonate as the CO2 donor. The mechanism of biotin mediated carboxylation is now well-understood. Biotin has also found extensive applications in chemical biology where the strong biotin avidin interaction has been extensively used for macromolecular tagging. In contrast to biotin biosynthesis, little is known about biotin catabolism. A few products of biotin catabolism in a soil Pseudomonad grown on biotin as the sole carbon source have been isolated but no genes or enzymes associated with biotin catabolism have yet been identified. This proposal will focus on the isolation of a biotin catabolic strain, the identification of the catabolic operon and the reconstitution of all enzymes involved in biotin catabolism. Mechanistic and structural studies will be carried out on the key enzymes involved in the degradation of the biotin heterocyclic core.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY/ABSTRACT Lipids play a vital role in maintaining cellular function. Altered lipid metabolism is currently considered a hallmark characteristic of many diseases such as malignancies, neurodegenerative diseases, cardiovascular diseases, and diabetes. This has led to a demand for new technologies with comprehensive capabilities for revealing lipid structure and composition. Such technology is essential for the study of lipid structure-function relationships and the development of methods to diagnose and treat pathologies. Recent efforts in mass spectrometry (MS)-based lipidomics, including ion activation methods and chemical derivatization, have expanded the toolbox for lipid analysis. However, there is no single method at present that is capable of resolving all types of lipid structures since lipids are structurally diverse and often contain mixtures of isomers. The lack of efficient and reliable analytical approaches for discerning lipid isomers in biological samples directly leads to the fact that the physiological roles and functions of lipid isomers remain largely unknown. The central vision of my research program is to address the deficiencies in lipid structural analysis technology using the unique microdroplet electrochemical (ME) methods, which take advantage of voltage-controlled electrochemical derivatization of lipid isomers and the dramatically accelerated rates of electrochemical transformations at microdroplet interfaces to achieve structural elucidation. The proposed voltage-triggered ME reactions will be performed in a modified electrospray emitter taking the form of a probe and using standard commercial MS instrumentation. Derivatized products will generate diagnostic ions specific to particular lipid isomers in tandem mass spectra, allowing characterization of detailed structures. During the next five years, my research group aims to develop ME probes for lipid analysis with particular emphasis on isomer identification and quantification so as to realize the promise of ME as a practical research tool for understanding, diagnosing, and treating diseases. A toolbox of ME reactions will be developed to characterize various lipid isomers including lipid class, acyl chain length, double- bond positions, geometries, and sn(stereospecific numbering)-positions, the key information needed for accurate lipid structure annotation. The ME reactions are diverse and can be triggered by voltage changes, so they will be cascaded into a single system (a panoptic ME probe) to identify lipid structures at all levels of isomer specificity in a single experimental run. The ME probe will be used for studying the lipidome of pre-diabetic mouse heart to reveal the initial lipidomic signature in the heart in response to a Western diet and to define the deleterious effects of lipid isomers on the development of cardiac pathology. The expected outcome of this project is to provide a widely applicable approach with enhanced capabilities in lipid structural analysis, which will uncover structure-function relationships in lipid homeostasis and pathology invisible to current lipid profiling.

NIH Research Projects · FY 2024 · 2021-09

Integral membrane proteins reside in the biological membrane where they function and intimately interact with lipid molecules. The environment of the biological membrane is dynamic and composed of a rich chemical diversity of lipid molecules. Alongside the complexity of the biological membrane is the growing realization of the important roles of lipid molecules in the folding, structure, and function of membrane proteins. In fact, there is often density in maps of structures determined by X-crystallography and cryoEM that are ascribed to lipids but their identity remains largely unknown. Although a handful of examples exist which provide insight into membrane protein-lipid interactions, how individual lipid molecules influence the structure and function of membrane proteins on the molecular level largely remains poorly understood. What determines the selectivity of membrane proteins towards lipids? How important is the lipid chemistry, such as lipid tail length, stereochemistry and position of unsaturated double bonds, in protein-lipid interactions? Do membrane proteins recruit, through allostery, their own microenvironment? Here, this proposal seeks to address these fundamental questions by developing new tools and reagents to probe membrane protein-lipid interactions using the ammonia channel (AmtB) from E. coli in complex with its regulatory protein GlnK as a model membrane protein system. More specifically, native Mass Spectrometry (MS) technology, whereby non-covalent interactions are preserved in the mass spectrometer, will be employed in combination with new MS approaches pioneered in the Laganowsky group that, unlike other biophysical methods, allow individual lipid binding events to membrane protein complexes to be resolved and interrogated. The proposed studies build off the foundation of previous work where native MS technology is integrated with other biophysical techniques, such as Surface Plasmon Resonance (SPR) and X-ray crystallography, to address fundamental questions regarding membrane protein-lipid interactions. More specifically, proposed studies are aimed at unravelling cooperativity for a considerable number (up to 20) of individual lipid binding events to AmtB by the (i) use of charge-reducing molecules and (ii) synthesis of new detergents engineered for native MS applications. Next, proposed studies pushing the technological limits of MS technology aimed at deducing allostery within heterogeneous lipid binding events to AmtB. Here, these studies move beyond previous work on mixtures of two different lipid headgroups to more complex lipid mixtures, composed of three to four different lipid species, binding to AmtB. Novel approach is proposed to deduce the position-dependent effects of bound lipids on AmtB by using a combination of protein engineering and covalent labeling strategies. Taken together, the results and outcomes from our proposed studies are anticipated to have a significant impact in our understanding of membrane protein-lipid interactions and, more generally, to our understanding of membrane protein biology, especially how changes in the biological membrane may regulate membrane protein physiological function.

NIH Research Projects · FY 2025 · 2021-08

Project Summary/Abstract Inflammation plays a critical role in secondary tissue damage after spinal cord injury (SCI), however, there is no widely accepted therapeutic for mitigating destructive inflammatory events in the injured spinal cord. L-selectin is an adhesion and signaling receptor on immune cells that has been recently shown to be a critical mediator of long-term neurological deficits following SCI. Disrupting L-selectin function with diclofenac, an FDA-approved non-steroidal anti-inflammatory drug (NSAID) that induces L-selectin “shedding”, improves tissue sparing and long-term recovery when administered by 3 hours post-SCI. L-selectin shedding, therefore, represents a potential therapeutic strategy to mitigate damage associated with acute inflammation. However, the specific mechanisms through which L-selectin attenuates secondary tissue damage remain unclear. L-selectin has been shown to promote destructive effector functions in neutrophils, the most abundant immune cell type in human blood and first to invade the injured spinal cord in large numbers. The hypothesis of this proposal is that L- selectin shedding reduces the pathogenic activities of neutrophils and associated secondary tissue damage after SCI. The objectives of this work are to determine the effect of L-selectin shedding on the activation of neutrophil effector functions, further elucidate the role of neutrophils in secondary tissue damage after SCI, and determine if intravenous delivery extends the therapeutic window for diclofenac. Specific Aim 1 will test the hypothesis that L-selectin shedding reduces the activation of cytotoxic neutrophil effector functions in the presence of myelin. Myelin can serve as an abundant ligand for L-selectin and may exacerbate cytotoxic effector functions in neutrophils. Using mice that cannot shed L-selectin (L(E) mice) and WT mice treated with diclofenac, the effect of L-selectin shedding on neutrophil effector functions will be quantified in vitro in response to myelin exposure as well as in the acutely injured spinal cord. Specific Aim 2 will test the hypothesis that neutrophils are the primary immune cell type whose destructive functions are mitigated by L-selectin shedding. Early neutrophil depletion will be investigated in L(E) mice and in WT mice treated with diclofenac to determine the extent to which L-selectin shedding reduces secondary damage and neurological deficits by attenuating pathogenic neutrophil activities. Specific Aim 3 will test the hypothesis that intravenous delivery of diclofenac can induce rapid shedding of L-selectin on neutrophils in the blood and extend the window of opportunity. Long-term neurological recovery and tissue sparing will be assessed following delayed intravenous administration of diclofenac in WT mice. Diclofenac treatment will also be assessed in L(E) mice to confirm that the therapeutic mechanisms of action is through L-selectin shedding. The collective results will help uncover the roles of L- selectin shedding and neutrophils in secondary damage after SCI and validate L-selectin shedding as a therapeutic target to improve long-term neurological recovery. The findings from this proposal will also be applicable to attenuating damaging inflammation observed in other central nervous system injuries or disorders.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY Nearly 3 million antibiotic resistant infections occur per year in the United States. This problem is especially acute in gram-negative bacteria, where the outer membrane (OM) which surrounds the aqueous periplasm acts as a permeability barrier capable of excluding many antibiotics. We are interested in the OM of Enterobacterales (e.g., Escherichia, Salmonella, Klebsiella), which are adapted to an enteric environment rich in toxic molecules, such as bile salts, necessitating an especially strong OM. It has become clear that the permeability of the OM can be altered by the physiological state of the cell. Specifically, stresses such as nutrient limitation can result in strengthening of the OM permeability barrier. Elucidation of the pathways responsible for this strengthening will lead to new targets for the development of small molecules that can weaken the OM permeability barrier. We have found enterobacterial common antigen (ECA), a conserved component of the Enterobacterales OM and periplasm, to be important for OM impermeability under stress. Two forms of ECA (phospholipid-linked ECA (ECAPG), and cyclic ECA (ECACYC)) have different roles related to OM permeability; however, their precise functions remain unknown, in part, because many steps in their biogenesis are poorly understood. Our long-term goal is to understand the biogenesis of ECA to facilitate functional studies and identify potential antimicrobial targets. Specifically, this project aims to elucidate, in Escherichia coli K12, the regulation of and unknown steps in biogenesis of the forms of ECA contributing to antibiotic resistance. Biochemical reactions are required for these forms of ECA to be produced and yet the genes responsible for these steps and the regulation of these steps are largely unknown. The central hypothesis is that ECAPG and ECACYC can be differentiate through their unique biosynthetic genes and regulatory roles. This hypothesis will be addressed with the following aims: identify the genes and substrate necessary for ECA to become a phospholipid head group forming ECAPG using genetic interactions with other biosynthesis pathways (Aim 1); elucidate factors and mechanisms involved in ECACYC biogenesis using an antibiotic sensitivity suppression phenotype we discovered (Aim 2); and uncover the mechanisms of the two novel pathways of ECA regulation we discovered (Aim 3). These conceptually innovative aims will be approached through a blend of high-throughput genomics, genetic screens and selections, and biochemical techniques. Completion of this project will identify genes and residues important for biogenesis of ECAPG and ECACYC, which represent targets for development of small molecules weakening the OM. In addition, this will allow genetic analyses of ECA function, providing insights into Enterobacterales biology.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY Comorbidity in the anxiety disorders is common and strongly associated with poor outcomes, lower rates of remission, increased disability and higher rates of relapse. Yet despite its clinical relevance, clinicians are unsure how to treat comorbid anxiety patients, in part because it is unknown whether comorbid cases are marked by distinct pathophysiology, or whether they should simply be conceptualized as the 'sum of their parts' (i.e., multiple, separate clinical entities). Characterizing comorbid individuals as having multiple, independent, disease processes occludes understanding of synergistic, pathophysiological processes that may uniquely characterize highly comorbid patients. The current project focuses on a neural profile that corresponds to increased comorbidity load across the anxiety disorders and is routed in aberrant brain connectivity. This neural profile is comprised of increased alarm (heightened amygdala) and reduced motivated attention (attenuated late positive potential, LPP; an EEG measure of elaborated stimulus processing) to negative images, and is referred to as HARM-A (Heightened Alarm, Reduced Motivated Attention). Controlling for separate effects of alarm and motivated attention, preliminary data suggest that higher HARM-A is associated with (a) greater internalizing psychopathology; (b) higher rates of past comorbidity (controlling for current comorbidity) and (c) increased dysphoria 12-24 months later (controlling for baseline dysphoria). Those with higher HARM-A also showed aberrant connectivity between key nodes involved in threat detection and appraisal (amygdala-anterior cingulate cortex), as well as a stronger link between stress and negative affect. Therefore, HARM-A might underlie the worse outcome in comorbid anxiety cases, and may do so by increasing risk for negative affect generation following stressful events. The current project extends this preliminary work by examining negative emotion processing in 180 individuals, recruited to insure dimensionality on current and past internalizing symptoms. Participants will undergo 3 multi-level assessments (fMRI, EEG, clinical interview, self-report measures) over 24 months; at each assessment, participants will also complete 10 days of experience sampling assessments of stressful events and negative affect. The project will test a bidirectional model of HARM-A, in which HARM-A predicts increased future comorbidity load and higher scores on latent, transdiagnostic, internalizing psychopathology, which will in turn predict increased HARM-A (i.e., a mutually reinforcing “spiral”). It will also assess the neurocircuitry that supports higher HARM-A and will use experience sampling data to test whether HARM-A predicts stronger linkage between stress exposure and subsequent negative affect. Finding that HARM-A is prospectively and reciprocally associated with worse internalizing psychopathology, delineating its neurocircuitry and elucidating its role in the linkage between stressors and negative affect would deliver a mechanistic explanation for greater disease burden in comorbid anxiety and provide a path forward for more etiologically tractable understanding of anxiety-related disorders, and for the development of targeted treatments.

NIH Research Projects · FY 2025 · 2021-02

The primary focus of this research proposal is the discovery and elucidation of novel biochemical pathways for the biosynthesis and metabolism of complex and simple carbohydrates in the human gut microbiome. The total number of genes contained within the distinct bacterial species that inhabit the human gut exceeds the number of human genes by more than two orders of magnitude. The metabolic diversity within these bacteria contributes significantly to the maintenance of human health and physiology. Unfortunately, a significant fraction of the enzymes and metabolic pathways contained within the bacterial species localized in the human gut have an uncertain, unknown, or incorrect functional annotation. This uncertainty demonstrates that a substantial fraction of the metabolic potential found within the human gut microbiome remains to be properly characterized. The experimental approach for the discovery and elucidation of novel biochemical pathways for the metabolism of complex carbohydrates will employ the concerted and synergistic utilization of computational biology, bioinformatics, three-dimensional protein structure determination, metabolomics, and physical screening of focused compound libraries. This investigation will further be directed towards a complete understanding of the assembly and biosynthesis of the diverse capsular polysaccharides in the human pathogen Campylobacter jejuni, the leading cause of human gastroenteritis world-wide. The capsular polysaccharides are important for the invasion and colonization of the host organism and the monosaccharides that comprise the CPS in various strains of C. jejuni are unusual and complex. This endeavor will focus on the elucidation of the molecular pathways for the biosynthesis of the unusual array of monosaccharide building blocks and the associated molecular logic for the directed assembly of unique polysaccharide sequences by a series of sugar transferase enzymes. The determination of the substrate and reaction diversity contained within these newly discovered enzyme-catalyzed reactions will provide unique insights into the molecular mechanisms for the evolution and development of novel enzymatic activities and will provide potential targets for therapeutic intervention.

NIH Research Projects · FY 2025 · 2021-01

Injury to the cervical spinal cord results in an immediate and permanent loss of hand function due to massive disruption of neural circuitry. There are currently no effective therapies that can improve neurological outcomes for individuals living with spinal cord injury (SCI). New neurons can be provided to injury sites via transplantation of neural progenitor cells (NPCs). These cells differentiate into diverse subtypes of neurons that support the establishment of new synaptic connections with neurons in the injured host nervous system. Multiple studies have reported modest gains in forelimb function following NPC transplantation in preclinical cervical SCI models; however, the underlying mechanisms by which engrafted neurons promote functional recovery are unclear. In order to develop more effective neural replacement strategies that can robustly and reproducibly improve recovery of hand function, it is critical to develop a fundamental understanding of how engrafted neurons synaptically and functionally integrate into injured forelimb motor circuitry. To address this gap in knowledge, we will utilize a mouse model of SCI to characterize the contributions of NPC grafts to forelimb functional recovery. First, we will determine how varying the phenotypes of transplanted neurons influences graft synaptic integration into the injured nervous system. We have shown that NPC grafts can be enriched for distinct classes of neurons. We will use this strategy to determine how manipulating graft cellular composition influences integration with injured motor circuits. Second, we will determine whether graft neuron activity is sufficient to functionally modulate forelimb motor circuits. Through chemogenetic modulation of engrafted neurons, we will interrogate the functional contributions of graft activity to electrophysiological responses in the forelimb muscles. We will also determine whether graft activity is acutely required for the execution of skilled forelimb motor function. Finally, we will examine whether activity-based rehabilitation can increase synaptic integration of grafts into forelimb motor circuits and improve motor functional recovery. This study will establish a new and critical framework regarding the ability of engrafted neurons to integrate into and functionally modulate injured forelimb motor circuitry. Moreover, this work will highlight the critical importance of graft cellular composition in restoring functional outcomes. Our strategy to functionally interrogate NPC grafts has applications for broader work focused on restoring locomotor or autonomic outcomes after spinal cord injury. Results of this study will reveal new biological mechanisms by which neural grafts can provide therapeutic benefits. These findings will constitute a critical body of knowledge that will accelerate research efforts to develop more therapeutically effective human cells for clinical treatment of spinal cord injury.

NIH Research Projects · FY 2025 · 2021-01

The directed movement of eukaryotic cells away from a source of a chemorepellent appears to play a major role in development and the resolution of inflammation, but very little is known about eukaryotic chemorepellents and how they direct cell motility. Chemorepulsion of Dictyostelium cells from a secreted protein called AprA is a model where one can combine biochemistry and genetics to elucidate chemorepulsion. Using available mutants and a preliminary genetic screen, we identified the AprA receptor, identified several components of the AprA signal transduction pathway, and found that AprA chemorepulsion involves a fundamentally different mechanism from chemoattraction. AprA has predicted structural similarity to the human secreted protein dipeptidyl peptidase IV (DPPIV), and shares functional properties with DPPIV. We found that DPPIV is a chemorepellent for human and mouse neutrophils, found that the G protein-coupled receptor PAR2 mediates DPPIV chemorepulsion, and found that small molecule PAR2 agonists act as neutrophil chemorepellents. Preliminary studies indicate that there are strong similarities between the AprA and PAR2 agonist chemorepulsion mechanisms. Acute respiratory distress syndrome (ARDS) is an untreatable disease involving damage to the lungs causing neutrophils to enter the lungs. The neutrophils further damage the lungs, causing more neutrophil entry in a positive feedback loop, and this results in the death of ~40% of the ~200,000 ARDS patients per year in the US. An exciting insight into a possible therapeutic approach is that when DPPIV or PAR2 agonists are aspirated into the lungs, they induce neutrophils to leave the lungs in two mouse models of ARDS. The key roadblock to moving this into the clinic is that we need to know what the PAR2 agonist chemorepulsion mechanism is to anticipate potential drug interactions and side effects. To gain insights into a fundamental mechanism, and ways to induce neutrophils to leave a tissue, we propose to elucidate the AprA and PAR2 agonist chemorepulsion mechanisms using the power of Dictyostelium to lead the work. We will rigorously test newly identified Dictyostelium chemorepulsion pathway components, and determine where they function in the pathway. We will complete the Dictyostelium genetic screens to gain further insight into chemorepulsion, and then use this information to guide an examination of possibly similar mechanisms in neutrophils. While examining human neutrophil chemorepulsion, we observed a significant difference between male and female neutrophils, and we will delineate the extent and molecular mechanism underlying this difference. Together, the proposed work will elucidate a poorly understood fundamental mechanism, elucidate an unexpected sex difference in the innate immune system, and help elucidate how PAR2 agonists could be used clinically to drive excessive neutrophils out of a tissue, with possibly different treatments for men and women.

NIH Research Projects · FY 2025 · 2020-12

ABSTRACT In response to PAR-18-747, Addressing the Challenges of the Opioid Epidemic in Minority Health and Health Disparities Research in the U.S. (R01), this study seeks to determine the role of staff capability and quality to improve treatment access, engagement, and medication- assisted treatment (MAT) maintenance dosage, i.e., methadone, buprenorphine and naltrexone for African-American and Hispanic populations in opioid treatment programs (OTP) nationwide. The study will rely on the National Drug Abuse Treatment System Survey (NDATSS), which is a unique, nationally representative longitudinal data (2005-2017); the study also will collect two waves of new data in 2023 and 2025 to assess the effect of standards of care on OTP process measures [wait time, retention, and adequate MAT dosages). The study seeks to improve measurement of standards of care using a gold standard instrument - the Organizational Assessment (COA360) and assess its predictive validity on process measures in 2023 and 2025. Findings will inform treatment programs’ use of quality-of-care strategies to improve OTP wait time, retention and adequate dosage, and with these efforts reduce racial/ethnic disparities in opioid use disorder treatment. Policymakers will be able to make decisions about how to allocate scarce resources needed to address the opioid epidemic.

NIH Research Projects · FY 2026 · 2020-09

PROJECT SUMMARY/ABSTRACT Over the past decade, single-cell technologies have enabled a new era of high-resolution interrogation of cell- type diversity, greatly enhancing our understanding of the role that cell types play in development and disease. Recent advances in single-cell multiomics and spatial transcriptomics technologies, combined with the wealth of existing bulk-tissue sequencing and genetic data, offer unprecedented opportunities for integrative molecular analyses across diverse dimensions and modalities. In our pursuit of these opportunities, we are hampered by the lack of rigorous statistical methods and scalable computational algorithms. The research program of my lab centers around developing statistical/computational methods and bioinformatics tools to: (i) detect structural variants by both bulk and single-cell sequencing under various experimental designs; (ii) assess intratumor heterogeneity and reconstruct tumor phylogeny by multi-regional and/or longitudinal sequencing of both bulk tumor specimens and single cells; (iii) profile genomic, transcriptomic, and epigenomic heterogeneity at the cellular level using single-cell omics approaches; (iv) identify cell-type- and cell-state-specific gene expression and transcriptional regulation by single-cell multiomics and decode spatiotemporal cellular heterogeneity in both time and space; and (v) investigate interconnections between mammalian DNA excision repair, DNA damage checkpoints, and circadian clock. Our long-term vision is to introduce problems arising from new biomedical data to the statistics community and to provide data-driven statistical methods and open-source tools to biomedical researchers for better data analysis and experimental design. Specifically, in the next five years, our proposed program of research will focus on the following interconnected objectives: (i) causal inference using population- scale single-cell omics data; (ii) prioritizing trait-relevant cell types/states by single-cell multomics and GWAS summary statistics; (iii) spatiotemporal interrogation of circadian rhythmicity by single-cell multiomics and spatial transcriptomics; and (iv) profiling enhancer RNAs by single-cell multiomics data. We will apply our methods to both publicly available and in-house-generated datasets, using a combination of computational and experimental approaches for validation. Our methods will be released as freely available, open-source packages and modules, accompanied by comprehensive tutorials and workflows designed to be accessible and valuable to the biomedical research community.