Ut Southwestern Medical Center

NIH Research Projects · FY 2026 · 2025-04

Targeted therapy of cancer generally involves inhibition of a specific oncogenic pathway that plays a key role as an oncogenic driver in the pathogenesis or maintenance of cancer. Targeted therapy has been most successful in the presence of activating mutations in receptor tyrosine kinase (RTK) pathways that result in accentuated signaling. However, secondary resistance to RTK inhibition is a major therapeutic hurdle. The emergence of secondary resistance implies the persistence of subsets of cancer cells that are not eliminated during the initial treatment. The mechanisms of secondary resistance have been investigated intensively. Both mutational and non-mutational mechanisms of resistance have been describe For example, major mutational mechanisms of resistance to EGFR inhibition in NSCLC include secondary EGFR mutations such as the T790M mutation, and amplification of other RTKs such as MET. These genetic changes are detected months after exposure to tyrosine kinase inhibitors (TKIs). In addition, inhibition of RTK signaling pathways in cancer cells leads to a rapid reprogramming of signaling pathways as the cancer cell tries to restore homeostasis. This adaptive response may protect cells from a loss of RTK signals and play an important role in mediating therapeutic resistance. Our preliminary data indicate that while tyrosine kinase inhibition blocks the kinase activity of RTKs, the RTK does not shut down. Exposure to TKIs triggers a rapid SUMOylation of the RTK and changes its function to an adaptor protein that continues to signal. In this proposal, we focus on mechanisms of resistance to TKIs with a focus on osimertinib treatment in EGFR mutant non-small cell lung cancer (NSCLC) and other activated RTKs to examine the mechanisms and biological significance of RTK SUMOylation in response to TKIs. Preliminary data indicate that osimertinib induces a rapid and biologically SUMOylation of mutant EGFR. EGFR SUMOylation changes its function from a RTK to an adaptor protein. We also detect a rapid SUMOylation of other RTKs following kinase inhibition. RTK SUMOylation constitutes a platform to generate two major facets of the adaptive response, i. e. TNF-NF-κB activation and bypass RTK signaling. Both EGFR SUMOylation and activation can be detected in osimertinib treated tumors. In Specific Aim 1 we determine the mechanisms of RTK SUMOylation in response to TKI treatment. Preliminary data indicate that SUMOylation of the EGFR at K37 is required for both TNF-NF-κB activation and bypass RTK signaling in response to osimertinib and leads to osimertinib resistance. We will also examine additional RTKs in this aim. In Specific Aim 2, we determine the mechanisms of NF-κB and bypass RTK activation in response to tyrosine kinase inhibition. In Specific Aim 3, we determine the biological significance of TKI-induced RTK SUMOylation and its impact on secondary resistance with a particular focus on EGFR mutant NSCLC EGFR in multiple mouse models of NSCLC.

NIH Research Projects · FY 2026 · 2025-04

Project summary – Data-driven mechanistic models of morphology-controlled Ras oncogenic signaling. Cells adapt their shape during differentiation and transformation processes, or when exposed to environmental changes or drug challenges. Accordingly, cell geometry is often considered as a marker for cancer diagnosis and prognosis. However, whether cell geometry itself modulates the oncogenic signaling programs underlying these behaviors is understudied. This proposal provides a training plan for my long-term career to study how cell geometry controls oncogenic signaling that is implicated in cell behaviors such as survival and proliferation. Recent observations in our lab revealed that cell morphology drives key oncogenic processes. For example, bleb formation – hemispherical membrane protrusion – promotes survival signaling in suspended melanoma cells; or the formation of lamellipodia and ruffles promotes a proliferation signaling pathway in Ras-transformed cancers that is independent of the canonical MAP kinase signaling. In both cases, we have some evidence that the narrow spaces of blebs and lamellipodia upregulate signaling events because of scaffolding and diffusion restriction of molecules, which in turn elicits feedback that would not be activated in wider volumes. However, the detailed interplay between constrained morphology and nonlinear signaling effects remains unclear. To address this, experimental approaches must be complemented by computational approaches. My broad research goal is to develop integrated computational platforms that allow us to quantitatively unravel the mechanisms by which cell morphology drives cell signaling. I propose an imaging-guided modeling pipeline that will test the capacity of different formulations of reaction-diffusion systems in realistic geometries to recapitulate the experimentally measured signaling distributions across different cell morphologies. I will drive the calibration of free model parameters and the assessment of the model quality by direct matching of simulated and observed signaling distributions, as developed in my recent paper (PMID: 38177778). Given a calibrated model, I will test the mechanisms by which membrane protrusions – blebs and lamellipodia/ruflles – amplify cell survival and proliferation signals in Ras-mutated cells. In my R00 phase, I aim to develop a novel computational framework to implement 3D live-cell imaging data and study reaction-diffusion systems in dynamic boundaries, where cell morphodynamics govern signaling dynamic. To enhance my background in biophysics, and mathematics, I will pursue additional training in computational modeling, cell biology and systems biology during the K99 phase. The Danuser lab at UT Southwestern is ideal for this training. Dr Gaudenz Danuser is a pioneer in using computation to solve biological questions in cancer biology. Under his supervision, I’ll train to become a computational cancer biophysicist, initiating (K99) and leading (R00) the systems biological tools for studies of morphology-controlled signaling pathways.

NIH Research Projects · FY 2026 · 2025-04

PROJECT SUMMARY Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiovascular disorder. Over 35% of the ~400 mutations identified in HCM patients occur in the motor protein β-myosin. In vitro studies of the R403Q mutant β-myosin revealed hypercontractility of myosin cross-bridge dynamics, including increased ATP turnover and more myosin molecules in the disorganized relaxed sate (DRX) that enables excessive β-myosin contributions to ventricular contractions. When treated with the only FDA-approved HCM-drug mavacamten, the biochemical and structural properties of the isolated mutant β-myosin resembled the WT control. However, direct observation of β-myosin’s conformational alterations due to HCM inducing mutations or mavacamten treatment have yet to be observed in situ. Cryo-electron tomography (cryo-ET) is a powerful imaging technique that enables the visualization of several native cellular macromolecular structures inside intact cells, such as intact myofibrils from cardiomyocytes. However, many of the cryo-ET studies of muscle have been limited to isolated subcellular structures in vitro rather than intact, unperturbed (cardio)myocytes. Additionally, studies often immersed the isolated contractile machinery in conformation inducing buffers before plunge freezing and cryo-ET, possibly further altering the native ratio of myosin active and inactive states. There is a direct need to identify if the isolation procedures utilized to obtain myofibrils result in similar β-myosin structural characteristics found in situ. Further, accurate depiction of native wild type cardiac β-myosin structural features are required to accurately understand how pathological mutations and treatments influence and affect cardiac function and health. To view these contractile proteins within their cellular context, technically difficult high pressure freezing (HPF) and cryo- focused ion beam (cryo-FIB) milling is needed to ensure pristine vitrification of the cells and their organelles. Few labs worldwide have successfully implemented an HFP/cryo-FIB milling/cryo-ET workflow for intact eukaryotic cells and tissues in their native state, including the lab of Dr. Daniela Nicastro, the sponsor of this application. Therefore, I aim to determine the native in situ structure of myofibrils within intact inducible pluripotent stem cell derived human cardiomyocytes using high pressure freezing, followed by cryo-FIB milling to generate thin lamellae that can be imaged by cryo-ET. I will apply this cryo-ET workflow also to characterize structural alterations that occur within sarcomeres due to β-myosin mutations with and without mavacamten treatment. Determining the structural alterations that occur due to mutant β-myosin and mavacamten treatment will provide important insight for future improvement and development of therapeutics able to improve the health of myopathy patients.

NIH Research Projects · FY 2026 · 2025-03

Project Summary/Abstract Essential tremor (ET) is a chronic, progressive neurologic disease affecting 7 million Americans (2.2% of the entire US population). Recent evidence suggests that ET is neurodegenerative. Despite its high prevalence, there are no established biomarkers, serological or imaging, for ET. As a result, the diagnosis is solely based on neurologic history and exam, and there is no method to track underlying disease progression in clinical trials or clinical practice settings. This stands in stark contrast to the options available for Alzheimer’s and Parkinson’s disease (PD). Synaptic vesicle glycoprotein 2A (SV2A) is presynaptically located and ubiquitously expressed in the central nervous system. Positron emission tomography (PET)-derived measurement of brain SV2A has the potential to serve as an in vivo biomarker of synaptic density, and reductions in synaptic density in relevant brain regions have been demonstrated in several neurodegenerative diseases. Considerable data have linked ET to the cerebellum, with evidence of degeneration in the cerebellar cortex. Our preliminary data suggest that there is an in vivo reduction in synaptic density in the cerebellar cortex in ET. In this 5-year proposal, we explore the value of SV2A as a biomarker and diagnostic tool in ET. There are 3 aims. Aim 1a: To measure synaptic density with SV2A PET imaging in 30 ET cases and 30 matched controls. We hypothesize that there will be lower SV2A PET binding in the cerebellar cortex of ET cases compared to controls. Aim 1b: To perform several focused clinical correlational studies to assess the construct validity of the notion that a reduction in cerebellar cortex SV2A PET binding is a valid metric of symptomology in ET. We hypothesize the presence of significant correlations between synaptic density and key clinical features of ET. Aim 1c: To examine synaptic density in the cerebral cortex with SV2A PET in ET vs. controls. We hypothesize that whole brain analyses will uncover lower SV2A PET binding in ET in motor areas (e.g., motor cortex). Aim 2: To assess cerebellar cortex SV2A PET binding as a diagnostic marker for ET by comparing ET to a select number of other related movement disorders with overlapping clinical features: PD (n = 15) and dystonia (n = 15). We hypothesize that a reduction in cerebellar cortex SV2A PET binding, along with its binding in other regions (e.g., substantia nigra), will facilitate imaging-based distinction between these different diagnostic entities. Aim 3: Autoradiographic studies indicate that in vivo SV2A binding correlates with tissue levels of SV2A in postmortem samples. Therefore, SV2A autoradiography will be performed post-mortem in ET (n = 20) and control (n = 20) cerebellar tissue from the New York Brain Bank to determine if a lower postmortem tissue SV2A signal is apparent in ET. We hypothesize that tissue SV2A signal will be lower in ET, serving to independently validate the PET imaging data. This proposal is a highly novel study to rigorously examine whether in-vivo synaptic density can be used to provide important information in ET patients. The particular need we now seek to address, developing a biomarker for ET, is a fundamental unmet need in the field.

NIH Research Projects · FY 2026 · 2025-03

PROJECT SUMMARY Liver transplantation (LT) is the only curative treatment for patients with end-stage liver disease. Despite undergoing a life-saving operation, LT survivors endure complex physical, emotional, and psychological challenges after LT. Predicting who will thrive or languish after LT is challenging. Existing clinical scores have limited predictive value and fail to capture the lived experience of going through this life-changing surgery. This proposal adapts the concept of survivorship from oncology to investigate post-LT recovery and outcomes beyond graft and patient survival. The central hypothesis is that survivorship experiences can be captured best through longitudinal monitoring of patient-reported and clinical characteristics—in turn, this can identify survivorship phenotypes at risk for worse clinical outcomes after LT. Guided by a socioecological model of survivorship, I will: 1) characterize distinct post-LT survivorship phenotypes by mapping trajectories of patient- reported and clinically measured survivorship constructs using group-based trajectory modeling; 2) investigate the relationship between survivorship phenotypes and outcomes including medication non-adherence, quality of life, and health care utilization; 3) adapt a navigator-based intervention into post-LT care and pilot a single- center study exploring the acceptability and feasibility of integrating this navigator intervention into LT care. These data will identify which phenotypes of LT survivors are at risk for worse outcomes and whether a navigator can be integrated into the LT team and successfully direct LT survivors to the necessary resources to overcome challenges. The PI is a clinical researcher and transplant hepatologist at UT Southwestern, with a long-term vision of improving care for LT survivors, including integrating evidence-based interventions to enhance survivorship. The proposed training plan is incorporated with the research aims and builds on her existing knowledge in clinical research whereby she will acquire new, advanced skills in group-based trajectory modeling, longitudinal data analysis, risk prediction, intervention adaptation, and intervention testing. She has assembled an exceptionally talented interdisciplinary team of mentors with complementary expertise: Dr. Amit G. Singal, a world-renowned health services and cancer researcher; Dr. Lisa B. VanWagner, a content expert in LT outcomes including longitudinal data analysis and risk prediction in the LT population; and Dr. Simon Lee, an expert in cancer survivorship and mixed methods. The research studies in this proposal have significant public health impact as they will fill gaps in our understanding of survivorship and clinical care after LT. This award and training plan will provide the PI with the protected time, training, and mentorship, to build an independently funded research career focused on improving survivorship after LT.

NIH Research Projects · FY 2026 · 2025-03

Summary: Neuronal activity-induced gene expression is essential for converting transient stimuli into long-term changes in brain function by modulating synaptic activity, neuron morphology, circuit formation, and behavioral adaptation. Neural plasticity relies on the proper induction of activity-regulated genes (ARGs) through activated Ca2+ signaling cascades and downstream transcription factors and chromatin regulators. We demonstrated for the first time that previous stimulation of ARGs can prime the genes, resulting in faster and more robust expression upon subsequent stimulation, possibly due to epigenetic memory. Intriguingly, it has been reported that cocaine induces gene activation and epigenetic priming in the reward circuit in vivo, which could share similar epigenetic mechanisms as in our experimental systems. The key question is how chromatin regulators prime ARG enhancers at a poised state after a transient neuronal stimulation, leading to stronger response to subsequent stimulation even after an extended period. Mammalian SWI/SNF-like chromatin remodeling BAF complexes, containing core ATPase subunits BRG1/SMARCA4, play significant roles in signaling-induced transcription. Mutations in BAF subunits are associated with neurodevelopmental or psychiatric diseases. We have shown that BRG1 regulates ARG induction by controlling both enhancer chromatin environment and E/P looping. Importantly, we identified a serine phosphorylation site in BRG1, induced by various neuronal stimuli through Ca2+ signaling, which is crucial for ARG enhancer activation. Using mice with BRG1 knock-in phospho- mutations, we demonstrated that BRG1 phosphorylation promotes ARG priming. Cohesin mediated 3D genome reprogramming was identified as a potential priming factor regulated by activity-induced BRG1 phosphorylation. Thus, we hypothesize that neuronal activity-induced BRG1 phosphorylation regulates enhancer priming and epigenetic memory through establishing a cohesin-mediated primed state of chromosome 3D structure. We propose three aims to test this hypothesis. We propose three aims to test this hypothesis. (1) Determine how neuronal activity-induced BRG1 phosphorylation regulates enhancer priming through establishing a cohesin- mediated primed state of chromosome 3D structure. (2) Determine the function of cohesin in BRG1 phosphorylation dependent ARG enhancer priming. (3) Examine the function of BRG1 phosphorylation in regulating cocaine-induced gene activation and priming. Our study will provide significant insights into both the epigenetic mechanism of neural plasticity and the molecular functions of chromatin regulators in neural disorders.

NIH Research Projects · FY 2026 · 2025-03

PROJECT SUMMARY Myoblast fusion, during which mononucleated myoblasts fuse to form multinucleate, contractile muscle fibers, is essential for skeletal muscle development and regeneration. Certain congenital myopathies are characterized by minute myofibers, suggesting that myoblast fusion may be compromised in these conditions. Thus, investigating the mechanisms of myoblast fusion is imperative not only for understanding muscle biology in development and regeneration, but also for developing novel therapeutic approaches for muscle diseases. In the past two decades, the fruit fly Drosophila has been used as a premier genetic and cell biological model to study myoblast fusion in vivo. These studies have uncovered a handful of evolutionarily conserved regulators of myoblast fusion and a novel cellular mechanism. My lab has shown that myoblast fusion is an asymmetric process in which one cell invades its fusion partner using F-actin-propelled membrane protrusions to promote membrane fusion. Similar invasive protrusions have since been observed in the fusion of vertebrate muscle and non-muscle cells, suggesting that these invasive protrusions are used as a conserved and universal mechanism to promote cell-cell fusion. Furthermore, we have discovered a mechanosensory response in the receiving fusion partner and demonstrated that mechanical tension is a driving force for cell-cell fusion. Our work to date has established a biophysical framework for understanding cell-cell fusion – the interplay between the pushing forces and the resisting forces from the two fusion partners at the fusogenic synapse brings the apposing cell membranes into close proximity to facilitate fusogen engagement and membrane fusion. Despite the critical role for actin polymerization in myoblast fusion and the identification of WASP family of actin nucleation-promoting factors (NPFs) required for this process, how the invading fusion partner rapidly generate a large amount of actin filaments at the fusogenic synapse and how these actin filaments are coupled to the cell membrane to effectively propel invasive protrusions remain poorly understood. In this grant, we will take a multifaceted approach, including genetics, cell biology, molecular biology, and biochemistry, to investigate these questions, aiming to obtain a deeper mechanistic understanding of myoblast fusion. We propose the following Specific Aims. In Specific Aim I, we will investigate the role of phase separation in enriching the actin polymerization machinery at the fusogenic synapse. In Specific Aim II, we will investigate the role of Arf GEF Loner and Arf GTPases in upregulating actin polymerization in myoblast fusion. In Specific Aim III, we will investigate the role of APC/C- Fzr and its downstream target Anillin in myoblast fusion.

NIH Research Projects · FY 2026 · 2025-02

Project Summary/Abstract Peripheral pain signals are conveyed to the brain by spinal cord projection neurons. There has been a significant emphasis on one projection neuron population that expresses TACR1, Substance P receptor, in transmitting pain signals to the brain. However, every strategy targeting these spinal cord neurons or the receptor itself using selective, potent antagonists has failed to treat pain in human patients, suggesting that there might be additional, independent ascending channels that also transmit pain signals to the brain. Previously, we identified a novel subset of spinal projection neurons that express another neuropeptide receptor, GPR83. We demonstrated that both TACR1- and GPR83-expressing spinal projection neurons can convey noxious signals to the brain to underlie the affective aspect of pain sensation while differentially responding to innocuous thermal and tactile stimuli. Interestingly, our preliminary data suggest that these newly identified GPR83-expressing neurons are conserved in humans, and Gpr83 is expressed in the human spinal cord dorsal horn more abundantly than Tacr1. Our proposed research will explore an exciting idea that GPR83- expressing spinal projection neurons and the GPR83-mediated neuropeptide signaling pathway can be potential therapeutic targets for treating chronic pain. In particular, we will test the functional redundancy between the two spinal output pain circuits that may ensure animals detect and react to harmful pain signals. In Aim 1, we will first determine the combinatorial, pathophysiological role of these two main subtypes of spinal projection neurons (TACR1- and GPR83-expressing) in chronic pain states by examining changes in intrinsic physiological properties and stimulus-evoked firing activities. We will also test their contribution to chronic pain behaviors by examining the behavioral outcomes of silencing these two pathways simultaneously. In Aim 2, we will determine the role of TACR1 and GPR83 and their neuropeptide ligands in chronic pain by conducting proof-of-principle experiments where we will assess chronic pain behaviors following the knockout of these receptor genes or their ligand genes simultaneously. We will also determine the physiological and circuit mechanisms underlying the TACR1 and GPR83-mediated modulation of chronic pain. Our study will provide insights into the molecular, cellular, and circuit mechanisms underlying the increased nociception in chronic pain states and potentially reveal novel therapeutic targets for treating chronic pain.

NIH Research Projects · FY 2026 · 2025-02

Cholesterol plays a multifaceted role in the regulation of animal cell membranes and an inability to control excessive cholesterol levels has been implicated in many human diseases including atherosclerosis, fatty liver disease and cardiovascular disease. Due to its dual nature (essential but toxic in excess), cellular cholesterol content is tightly regulated. Cellular cholesterol levels are controlled through the feedback regulation of the Scap- SREBP pathway, which is comprised of a collection of membrane proteins in the endoplasmic reticulum (ER). The primary proteins involved in the cholesterol sensing pathway include several integral membrane proteins: Scap, transcription factors named Sterol Regulatory-Element Binding Proteins (SREBPs), and Insigs. When ER cholesterol levels drop below a threshold, Scap transports SREBPs to the Golgi, where proteolytic activation of SREBPs results in cholesterol synthesis and uptake. When ER cholesterol levels rise above a threshold, Insig binds Scap and prevents Scap-SREBP from traveling to the Golgi, terminating further cholesterol synthesis and uptake. The central question of this research proposal is: how does Scap sense changes in ER membrane cholesterol? Understanding the structural and biophysical role of cholesterol’s influence on Scap will uncover the molecular mechanism of feedback regulation of the Scap-SREBP pathway. Preliminary work has yielded a Scap-Insig complex cryo-EM reconstruction enabling the modeling of cholesterol at the Scap-Insig interface. While investigating Scap residues involved in the cholesterol interaction, multiple residues were also identified to play a role in SREBP-transport. Therefore aim 1 proposes to determine the cryo-EM structures of Scap in a SREBP-transporting conformation and in the presence of cholesterol-saturated detergent micelles or reconstituted into cholesterol-rich lipid bilayer nanodiscs. A complete set of structures will enable deciphering of the mechanism of how cholesterol influences Scap to stop transporting SREBPs and instead allow for Insig- binding. Recent moderate-resolution (~4 angstrom) structures of Scap and the Scap-Insig complex suggest a large conformational change in the luminal extramembranous globular domain (L1L7) of Scap between the Insig- free and Insig-bound states. Therefore aim 2 seeks to study how cholesterol influences the movement of the L1L7 domain by developing and implementing a structure guided single-molecule FRET (Fluorescence Resonance Energy Transfer, smFRET) assay to assess the conformations of purified Scap in the presence of cholesterol. Preliminary studies have generated and confirmed the cellular regulation of Scap constructs meant for labeling experiments. To understand the progression of the conformational changes seen in the current structures, movement of the L1L7 domain will be measured at high-speed resolution using smFRET. Together these studies will provide a better structural and biophysical understanding of Scap’s regulation by cholesterol and advance strategies to regulate cholesterol synthesis and prevent cardiovascular diseases.

NIH Research Projects · FY 2026 · 2025-02

Project Summary/Abstract An increasing number of neurodevelopmental disorders are causally linked to variants in genes encoding chromatin proteins that regulate heterochromatin, transcriptionally silenced genomic regions. We recently discovered one such diagnosis, CBX1-related syndrome, characterized by developmental disabilities, hypotonia, and autistic features. This syndrome is caused by heterozygous variants in the CBX1 gene, which encodes heterochromatin protein 1 beta (HP1β), a core structural protein of heterochromatin. The patient-identified variants are all missense within a genomic region encoding a known functional domain of HP1β. The molecular mechanism of CBX1-related syndrome remains to be determined, although our previous publication suggested dominant-negative effects of the mutant HP1β. The predominance of neurological manifestations in individuals with CBX1 variants suggests the important role of HP1β in neuronal gene regulation, but the function of HP1β in brain tissue remains elusive. To investigate the mechanisms underlying CBX1-related syndrome, we created mouse lines with patient-identified CBX1 variants. Heterozygous Cbx1 mutant mice show behavioral abnormalities that suggest delayed neurotransmission. RNA sequencing of Cbx1 mutant mouse brain tissue revealed increased expression of genes regulating synaptic function. These models and preliminary data provide an opportunity to explore the mechanisms linking CBX1 variants to neuropathology. The overall objective of this study is to elucidate the mechanisms of HP1β-mediated heterochromatin regulation and the impact of patient- identified CBX1 variants in mouse models of CBX1-related syndrome. The rationale for this project is that elucidating the impact of the patient-identified CBX1 variants will pave the way toward identifying potential therapeutic approaches for CBX1-related syndrome, because elimination of mutant HP1β could ameliorate cellular dysfunction if its effects were dominant-negative. The central hypothesis is that HP1β variants seen in human patients interfere with neurodevelopment by increasing expression of genes within heterochromatin through a dominant-negative mechanism. This hypothesis will be tested by comparing transcriptomic/epigenomic and neurobehavioral phenotypes among mice with Cbx1 missense and loss-of- function variants and wild-type mice. The knowledge gained from the proposed studies will have a positive impact on the molecular mechanistic understanding of not only CBX1-related syndrome but also neurodevelopmental disorders caused by heterochromatin dysfunction in general. This work will enable me to obtain training in utilizing mouse models of neurodevelopmental disorders, in collaboration with leading experts. The completion of this project will provide me with the skills and experimental data required for an R01 application. The training available through this K02 award represents an essential component toward the attainment of my long-term goal of studying the molecular mechanisms underlying the neurodevelopmental symptoms seen in CBX1-related syndrome and other neurodevelopmental disorders caused by heterochromatin dysfunction.

NIH Research Projects · FY 2026 · 2025-02

PROJECT SUMMARY Most developmental defects remain unexplained by current genetic testing. A key challenge to the prevention and treatment of congenital defects is to interpret the impact of genetic variants (especially those in non-coding regions) and to understand their molecular mechanisms of action. Our long-term goal is to understand how genetic variation contributes to congenital heart disease (CHD). We are motivated by observations that defects in cell fate commitment during development cause CHD. Numerous human and mouse genetic studies have shown that loss-of-function coding mutations in developmental transcription factors (TFs) cause CHD. Since TFs drive gene regulatory networks (GRNs), CHD variants in TFs culminate in the dysregulation of cardiac genes during development. In parallel, non-coding variants have also been linked to CHD. Many of these variants are thought to modify the activity of transcriptional enhancers, especially those that control the expression of developmental TFs and regulatory programs. Together, these studies motivate the need to interpret variant function in the context of developmental regulatory networks. The objective of this proposal is to use cardiac regulatory networks as a platform to understand CHD genes and variants (Figure 1). We will define cardiac development networks (enhancer → TF → genes) and use this network to interpret the functions of coding TF variants and non-coding enhancer variants (both patient-derived and those not yet observed). We hypothesize that this systematic dissection of cardiac GRNs will enable the characterization of variants that impact cardiomyocyte (CM) specification and contribute to CHD. Our rationale is that this knowledge will help define the missing heritability of congenital abnormalities. We propose the following specific aims: (Aim 1) Define the transcription factors and gene regulatory networks of human cardiac development; (Aim 2) Define and characterize genetic variants regulating cardiac gene programs; (Aim 3) Characterize the impact of coding/non- coding variants on molecular and cellular phenotypes. This proposal is innovative because it will use experimentally-derived gene regulatory networks as a platform to interpret CHD genes and coding/non-coding variants. We expect this resource to propel new research horizons. This proposal is significant because it will expand our understanding of how genetic variants impact TF activity, the state of cardiac gene regulatory networks, and lineage specification, which will enable the functional interpretation of known and novel variants in CHD patients.

NIH Research Projects · FY 2026 · 2025-02

Project Summary: Spinal administration of analgesics such as opioids and local anesthetics via an implanted intrathecal drug delivery system have profound efficacy in some of the most severe high impact chronic pain conditions. Despite their profound efficacy, their use is limited primarily because of side effects such as tolerance, psychosis and motor block associated with drugs used in them (opioids, ziconotide, local anesthetics). Novel analgesics that take advantage of spinal pain processing, are safe to use in humans and have minimal motor block and tolerance can be revolutionary in the management of high impact chronic pain conditions. Contulakin-G (CGX) is a snail venom derived peptide that has homology with mammalian neurotensin has been shown to be safe in humans and in a small, pilot Phase1A study demonstrated analgesic effects in patients with spinal cord injury pain, a high impact chronic pain condition. These studies suggested a possibility of a novel, non-opioid, analgesic mechanism that is active in humans. Our preliminary studies in animal models of high impact chronic pain unraveled spinal neurotensin receptor 2 (NTSR2) and subsequent inhibition of voltage-gated calcium channels Cav2.3 and 2.2 as an opioid-independent spinal analgesic mechanism. Importantly, despite profound analgesia, NTSR2 activation was not associated with unwarranted side effects such as rapid tolerance or motor blockade. Despite clear translational relevance of NTSR2-Cav2.3/2.2 pathway, nothing is known about NTSR2 downstream signaling that leads to calcium channel inhibition, particularly so in sensory neurons. Aim 1 will evaluate detailed signaling following NTSR2 activation that leads to calcium channel inhibition in mouse sensory neurons. Aim 2 will assess the anatomical correlation and functional significance of NTSR2-Cav pathway in human sensory neurons and spinal cord. Aim 3 will evaluate in vivo significance of this pathway in models of high impact chronic pain with rigorous independent replication. If successful, proposed studies could unravel the signaling molecules involved in human-tested mechanism for the treatment of high impact chronic pain conditions. Moreover, this information can be utilized for further development of novel analgesics that are biased agonist of or directly engage this signaling pathway.

NIH Research Projects · FY 2026 · 2025-02

Project Summary: Glutamine is a conditionally essential amino acid that has myriad uses in the cell. Aside from direct incorporation into protein, glutamine can be metabolized to generate nucleotides, other amino acids, the Krebs cycle intermediate -ketoglutarate (KG) which is important for energy production and anabolic reactions, and glutathione (GSH) to protect against oxidative stress. We recently determined that mature osteoclasts are characterized by increased abundance of amino acids and nucleotides. Importantly, inhibiting glutamine metabolism reduced nucleotide abundance and inhibited osteoclast differentiation. Unfortunately, the necessity of glutamine metabolism to regulate osteoclast differentiation and bone resorption in vivo has not been investigated. Moreover, how glutamine derived purine nucleotides mechanistically regulate osteoclasts remains enigmatic. In this proposal, we will 1) establish the necessity and sufficiency of glutaminase (GLS) dependent glutamine metabolism to regulate osteoclast differentiation and bone resorption, 2) evaluate the efficacy of the GLS inhibitor Telaglenastat to inhibit bone bone resorption in the ovariectomy mouse model of human osteoporosis, and 3) define the mechanisms by which glutamate oxaloacetate transaminase 2 (GOT2) dependent purine biosynthesis functions downstream of GLS to regulate osteoclast metabolism and differentiation. Our findings will have broad implications in understanding the roel and regulation of metabolism during osteoclast differentiation and activity, bone homeostasis and pathological bone loss.

NIH Research Projects · FY 2026 · 2025-01

Project Summary The myelodysplastic syndromes (MDS) are a heterogeneous group of bone marrow failure syndromes diagnosed in 15,000 patients annually within the United States and collectively carrying a 5-year survival rate of 35%. Hematopoietic stem cells (HSCs) have been identified as the cell of origin for MDS, however their rarity has largely precluded their meaningful study, especially following treatment where hypocellular bone marrow samples yield exceedingly few cells to study. Therefore, little is known about their response to treatments, especially after allogeneic stem cell transplant (alloSCT). In preliminary studies, we have validated a new low- input targeted sequencing workflow using a panel of more than 250 blood cancer associated genes that can detect mutations in as few as 20 sorted cells. The long-term goal of this project is to elucidate the mechanisms necessary for cure of MDS by comparing the response of MDS HSCs to alloSCT in patients who relapse vs. who are cured. The specific aims are as follows: (1) To assess the potential for the detection of MDS HSCs to predict for relapse, and (2) to elucidate the molecular and clonal evolution that MDS HSCs undergo to persist following alloSCT. Specific Aim 1 will apply a novel low-input sequencing protocol we have developed to patient samples following alloSCT to evaluate the variant frequencies of clinically identified MDS driver mutations in HSCs and determine whether their detection correlates with relapse. Specific Aim 2 will use mitochondrial lineage tracing in single-cell DNA- and RNA-sequencing studies to evaluate how both the transcriptional programs and sub- clonal composition of MDS HSCs may evolve during alloSCT to promote relapse.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY Although the bioengineered human corneal endothelial cell (hCEC) monolayer graft have shown vision recovery in animal models, the hCEC monolayer do not integrate with the host cornea due to suboptimal and non-tunable degradation of the hCEC-carrier biomaterial in the anterior chamber, which provide mechanical support to the monolayer. Thus, whether the transplanted hCEC monolayer will integrate with the host cornea following the complete degradation of the hCEC-carrier biomaterial and remain functional thereafter is unknown. To validate the bioengineered hCEC monolayers, there is a clear need to develop a biomaterial that has tunable-degradation rate in-vivo to evaluate the engraftment of the hCECs, and is mechanically strong so that the biomaterial-film/hCEC-monolayer construct does not break during the transplantation. The previous work of the team has established that the extracellular-topography cues can significantly modulate hCEC responses. The preliminary work of the team has developed photodegradable hydrogel (pdGel), which can be degraded in a tunable manner after transplantation, within hours to weeks, using tissue-penetrative light. Accordingly, the objective of this proposal is to develop a nano-topography pdGel-hCEC monolayer graft, evaluate monolayer integration with host cornea by tuning the in-vivo degradation rate of the pdGel, and validate the hCEC-monolayer function in-vivo. It is hypothesized that the nano-patterned pdGel will enable the growth of hCECs as a confluent monolayer, improve the hCEC monolayer function and stability by inducing the deposition of native-like extracellular matrix (ECM), and the tunable photodegradation of the carrier will improve the engraftment of the hCEC monolayer. The rationale for this project is the evaluation of the engraftment of hCEC monolayer with the cornea and the function thereafter will validate the use of bioengineered hCEC grafts for potential treatment of multiple corneal patients with one donor. Towards the overall objective, in the first aim, the hCEC monolayer growth on the pdGel, photodegradation kinetics, the biocompatibility of the degradation products, and the engraftment of the monolayer will be evaluated in-vitro and ex–vivo. In the second aim, using a high-throughput topography platform, the effect of 253 unique pdGel topographies will be evaluated on the hCEC monolayer functions to identify the optimum graft design. In the third aim, the photodegradation rate will be tuned in-vivo using light exposure to evaluate its effect on the hCEC engraftment. The proposed research is expected to be significant because it will validate the bioengineered hCEC-monolayer graft technology using a new photodegradable biomaterial, and it will develop new biomaterial and nano-topography platform that will have significant applications beyond ocular tissue engineering. The proposed research is innovative because it, (1) uses two-photon lithography approach to develop an innovative, high throughput nano-topography platform, and (2) leverages the photo-decomposition liability of the cyanine dye to develop an innovative photodegradable hydrogel for hCEC engraftment.

NIH Research Projects · FY 2026 · 2025-01

Heart failure (HF) and atrial fibrillation (AF) are common and differentially burden Black Americans. Social determinants of health (SDOH) also disproportionately impact Black Americans and contribute to an excess of cardiovascular (CV) events, although many Black Americans survive to late-life free of CV disease. Critical barriers to the development of interventions to prevent HF and AF include the limited knowledge of disease pathobiology and of the molecular pathways by which SDOH and resilience to such adversity influence susceptibility to CV disease. The objective of this proposal is to use proteomics and genomics to identify biologic pathways that are responsible for the development of subclinical and clinical cardiac dysfunction, that mediate the impact of SDOH on HF and AF risk, and that characterize biologic resilience to these outcomes despite adverse SDOH. The central hypothesis is that integrated analysis of proteomic and genomic data in a Black American population will enable discovery of novel inflammation- and fibrosis-related proteins relevant to HF and AF development, and identify biologic pathways underlying SDOH-related risk for and resilience to these CV conditions. The rationale is that identifying proteins with causal effects on cardiac dysfunction and defining molecular mediators of CV resilience to adverse SDOH may yield new therapeutic targets that are especially relevant to Black populations. This project leverages proteomics (~3,000 plasma proteins) measured 20 years apart, existing genomic data, rich CV phenotyping (echo, ambulatory arrhythmia monitoring), and prospectively adjudicated events in the largest prospective CV cohort of Black Americans - the Jackson Heart Study - to address the following aims: (1) Define proteins and protein networks associated with clinical and subclinical cardiac dysfunction; (2) Identify proteins and protein networks associated with atrial fibrillation and subclinical arrhythmia; (3) Identify molecular pathways linking SDOH, and resilience to adverse SDOH, to cardiac function and arrhythmia in Black adults. The unique contribution of this project will be to identify biologic mechanisms responsible for risk of and resilience to HF and AF in Black Americans. This contribution will be significant because it will identify novel therapeutic targets for the prevention of cardiac dysfunction tailored to disease mechanisms impacting Black Americans. These studies will therefore help to reduce the morbidity, mortality, and racial disparity associated with HF and AF. Innovative features include: (a) integration of repeated-measure proteomics and genomics to identify potentially causal proteins for cardiac dysfunction and arrhythmia among Black Americans; (b) focus on an understudied population with excess burden of CVD to interrogate mechanisms by which SDOH influence disease susceptibility; and (c) integration of SDOH and psychosocial assessments to identify protective proteins underlying cardiac resilience in the face of social adversity. Expected outcomes include advancing understanding of the biology of resilience and discovery of therapeutic targets relevant to Black Americans.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY Liver metabolic zonation refers to how liver cells, or hepatocytes, are spatially organized to perform diverse functions such as breaking down toxins or maintaining systemic metabolic homeostasis. This division of labor is crucial for the liver's efficiency but, when disrupted, may underlie various liver diseases, including metabolic disorders and cancer. The lack of genetic tools to specifically target hepatocytes within different liver zones of live mice has hampered our understanding of how zonation impacts disease. In this project, I aim to bridge this gap by employing novel Zone-specific-CreER mouse models developed in our laboratory. These models will enable precise spatial inhibition of key metabolic processes—gluconeogenesis and glycogenolysis—within distinct liver zones: near the portal vein (zone 1), in the middle of the lobule (zone 2), and adjacent to the central vein (zone 3). Gluconeogenesis and glycogenolysis are dysfunctional in Glycogen Storage Disease Type 1a (GSD1a), a condition affecting 1 in 100,000 newborns in the U.S., caused by mutations in the G6PC1 gene. GSD1a manifests through specific and measurable signs including fasting hypoglycemia, lactic acidosis, and hyperuricemia. Patients also develop hepatic adenomas during their teen years, with some tumors progressing to hepatocellular carcinoma (HCC). In mice, G6pc1 deletion from all hepatocytes mimics human GSD1a, including tumorigenesis. Somatic loss of function mutations in G6PC1 are found in >2% of sporadic, adult-onset HCC, and decreased G6PC1 expression is correlated with poor outcomes, implying a role for G6PC1 in HCC even beyond GSD1a. Given that gluconeogenesis and glycogenolysis are highly localized processes, predominantly occurring in zone 1, I hypothesize that specific knockout of G6pc1 in this zone will reproduce GSD1a symptoms. To test this hypothesis, I will undertake two aims. Aim 1 focuses on measuring key metabolic indicators of GSD1a after zone-specific G6pc1 knockout (KO) and assessing compensatory mechanisms of hepatocytes across different liver zones. Aim 2 explores how zonal G6pc1 KO influences hepatocyte competition dynamics, clonal expansion, and tumorigenesis, addressing the unique cancer development pathway seen in GSD1a patients. This study is poised to provide significant insights into the role of metabolic zonation in liver health and disease, aligning closely with the NIDDK's mission to enhance our understanding of liver metabolic diseases. Through detailed genetic, biochemical, and sequencing analyses, I will explore the relationship between liver zonation and disease, potentially leading to novel therapeutic strategies for conditions like GSD1a and beyond. This approach not only advances fundamental liver biology but also offers a new lens through which to examine liver disease pathophysiology.

NIH Research Projects · FY 2026 · 2025-01

Over a 5–10-year period, between 6% and 15% of germline variants undergo reclassification, a process by which the clinical meaning of a given variant is recategorized based on updated evidence. Accurate and definitive variant classification is a critical component of clinical genetic testing, especially as test indications have broadened over time and more genes are being interrogated per test. Reclassification occurs frequently in clinical practice today and has well-demonstrated clinical utility. While consensus exists about the importance of and need for guidance regarding variant reinterpretation, reclassification, and patient recontact, the field of medical genetics is ablaze with debates over the nature and extent of stakeholder responsibilities on these issues. We propose to use deliberative research to construct a framework of principles that can provide practical guidance for ongoing guideline development as genomic science evolves. Our research is directly responsive to numerous calls to action to develop standardized processes and guidelines in reinterpretation, reclassification, and recontact. We will achieve our goals through the following aims: Aim 1: Explore stakeholders’ views (and prime them) regarding key questions related to variant reinterpretation, reclassification, and recontact. Aim 2: Identify underlying values and principles and clarify areas of agreement and disagreement among stakeholder groups. Aim 3: Develop expert consensus on proposed solutions to the key questions. The proposed project will utilize a complementary set of methodologies to provide a comprehensive overview of variant RRR (Aims 1 and 2); a durable framework of practical principles (Aim 2) to guide future guideline as genetic knowledge evolves; and a set of concrete, expert-derived practice recommendations (Aim 3). Generation of a durable framework of principles that can guide ongoing guideline development in medical genetics – rather than a set of recommendations with limited shelf-life- is an important innovative element of the proposed work.

NIH Research Projects · FY 2026 · 2025-01

PROJECT SUMMARY Small cell lung cancer (SCLC) afflicts more than 30,000 patients per year and is rapidly fatal in 94% of cases, with median survival of less than one year. Although untreated SCLC is highly responsive to first-line chemotherapy, benefit is temporary and following relapse, resistance often extends to a broad spectrum of DNA damaging agents. Furthermore, investigational therapies in clinical trials are often confounded by the same cross-resistance that hinders standard second-line agents. Although critically important, cross-resistance is difficult to study experimentally, as it requires a model system that faithfully reproduces clinical outcomes and is adequately powered to capture inter-tumoral molecular heterogeneity. We have generated a panel of 72 patient-derived xenograft models (PDXs) of SCLC from biopsy specimens and circulating tumor cells (CTCs). For both standard chemotherapy and investigational agents, these models faithfully mirror patient responses. However, unlike the patient experience, multiple strategies can be compared for identical tumors. We generated a thorough and quantitative chemosensitivity profile of each PDX model. This profile confirmed that models that are resistant to one regimen are usually resistant to all, demonstrating cross-resistance. Nearly all these cross-resistant models were from previously treated patients. For five SCLC patients we derived PDX models before treatment and again after relapse. The post-relapse models were consistently more resistant, reflecting the changes in the patient’s cancer. These paired models are powerful research tools because they can show directly what made the cancer resistant. In one of these pairs, the relapsed model had a high-level MYC amplification on a circle of extrachromosomal DNA (ecDNA). We demonstrated that the extrachromosomal MYC amplification (ecMYC) was the cause of cross-resistance in that model. We expanded this study to the whole PDX panel and found significant enrichment of ecDNAs with MYC, MYCN or MYCL in cross-resistant models from relapsed patients. These ecMYC/L/N amplifications are the first alterations in the genomes of SCLC tumors to be demonstrated to drive cross-resistance. This important discovery leads to major questions. First, how do ecMYC, ecMYCL or ecMYCN cause resistance? MYC has many cellular functions, and this is likely to be a challenging question, but one major insight is that the cells that have the highs numbers of ecDNAs and are most resistant appear to stop dividing. The second question is what are the other drivers of cross-resistance, in the PDX models that are negative for ecMYC/L/N? In at least one case, an ecDNA with a different gene, XRCC1, seems to cause resistance. Finally, how do we test for this in patients? We propose a way to test for these ecDNAs in the same CTCs that we used to build the PDX models. The goal of this work is to develop a plan of care for patients with relapsed SCLC that is tailored to the driver of resistance in their cancer.

NIH Research Projects · FY 2026 · 2025-01

Clear cell renal cell carcinoma (ccRCC), which accounts for approximately 85% of all renal cancers, is resistant to a variety of cancer therapies and is highly lethal. Metastasis poses a significant challenge in the treatment of kidney cancer, with lung metastasis accounting for approximately 40-50% of all kidney cancer metastases. While the 5-year average survival rate for kidney cancer without metastasis is approximately 90%, the survival rate for distant metastasis (such as lung, bone and brain) is only 10-15%, emphasizing the urgent need to understand the biology underlying kidney cancer lung metastasis. Here we perform a genome-wide Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) screen and identify that Hepatic leukemia factor (HLF) controls lung metastasis in ccRCC. Most of the reports about HLF focused on leukemia studies and its function in solid cancer has never been characterized. This proposal, for the first time, will characterize the role of HLF in ccRCC lung metastasis and examine its underlying molecular mechanism. In Specific Aim 1, we will characterize the functional significance of HLF on kidney cancer lung metastasis by using gain-of-fuction and loss-of-fucntion studies in ccRCC orthotopic xenograft tumors, patient derived xenograft (PDX) models, immune- competent mouse models and humanized mouse models. In Specific Aim 2, we will delineate the molecular mechanism by which HLF loss promotes kidney cancer lung metastasis by integrated ChIP-Seq and RNA-Seq analysis. In particular, we will delineate the molecular mechanism of HLF-LPXN on regulating ccRCC migration at the single cell level. Successful completion of these aims will provide mechanistic insight into signaling pathways controlling lung metastasis in ccRCC and set the foundation for therapeutic intervention targeting the HLF signaling axis in ccRCC.

NIH Research Projects · FY 2026 · 2025-01

Project Summary/Abstract Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the leading genetic cause of kidney disease, with an estimated economic burden of seven billion dollars annually. The only treatment to slow disease progression has risk of severe side effects. Thus, there is significant need to advance our knowledge of ADPKD pathogenesis to identify novel ways to slow cyst growth. We have discovered that sterol regulatory element binding proteins (SREBPs) may attenuate cyst growth. SREBP1/2 are the master transcriptional regulators of lipogenesis genes. Our preliminary studies show that reducing SREBP1/2 activity accelerates cyst growth in mouse models of PKD. Conversely activating SREBP1/2 signaling markedly slows cyst growth in mouse models of ADPKD and prolongs survival. These results are unexpected, considering that higher fatty acid (FA) synthesis or sphingolipids have been shown to aggravate PKD. However, in addition to lipids, SREBP1/2 also enhance cholesterol synthesis with SREBP2 being preferentially active at loci of genes in the cholesterol biosynthesis pathway. Indeed, we have discovered that cholesterol is reduced in mouse and human kidneys with PKD. We have also found that reduction of only Srebp2 also aggravates cyst growth, implicating the cholesterol pathway. Thus, while lipids such as FA and sphingolipids are deleterious, cholesterol species may in fact be beneficial in ADPKD. Based on these intriguing preliminary observations, we hypothesize that cholesterol plays a cyst- attenuating role in ADPKD. We will test this hypothesis in the following specific aims: 1)Determine if reducing intracellular cholesterol by inhibiting de novo synthesis or uptake aggravates ADPKD; 2) Determine if enhancing intracellular production of cholesterol slows ADPKD; 3) Identify cholesterol client proteins in ADPKD. In summary our studies will advance our understanding of metabolism in ADPKD and open new avenues of exploration for future therapeutic development targeting the cholesterol pathway.

NIH Research Projects · FY 2026 · 2025-01

Project Summary/Abstract Cytoskeletal proteins perform essential roles in cell biology, and they generally evolve under stringent sequence conservation across phyla for these functions. One cytoskeletal family known as the actin-related protein (Arp) superfamily evolved before the last common ancestor of eukaryotes and plays many fundamental roles in the cytoplasm and nucleus, including nucleating actin, transporting cargo, repairing DNA damage, and chromatin remodeling. Most studies of Arps are motivated by their ubiquity and stringent conservation. However, we have discovered that the Arp superfamily displays unexpected diversification via the rapid expansion of paralogs and accelerated amino-acid substitutions in Drosophila and mammals. Interestingly, this genetic diversification across phyla has acquired tissue-specific expression and is often enriched in the male germline, a striking contrast to the ubiquitously expressed canonical Arps. Arp diversification is largely unexplored relative to studies of almost universally conserved Arps, most likely due to the lack of deep evolutionary roots. However, rapid sequence divergence suggests functional innovation that provides a fitness advantage and raises questions regarding how Arp diversification has specialized for tissue-specific roles. All Arps share the canonical actin fold, and accumulating evidence indicates that recent Arp adaptation has repurposed the actin fold for critical functions in fertility and development and may play unique roles in actin biology. We will gain molecular insight into this unexpected diversification of Arps in Drosophila and mammals to reveal how it specializes cytoskeletal networks for specific cellular contexts. Here, we outline our research program over the next five years, focusing on three distinct projects investigating Arp diversification in insects and mammals. We will determine how evolution alters the canonical Arp2/3 complex, an essential actin nucleator found in most eukaryotes, and tailors actin polymerization kinetics, stability, and structure for tissue- and species-specific actin networks. Additionally, we will investigate a young, non-canonical Arp that arose in flies and plays a role in fertility. Studying this Drosophila Arp provides a unique opportunity to genetically dissect how evolution modified the actin fold for novel functions without comprising actin’s essential roles. Lastly, we will focus on several non-canonical, rapidly evolving Arps in mammals that form a complex, localize to actin in the testis, and are critical in shaping the sperm head. We will identify how these divergent Arps adapted for complex assembly and gain structural insight, which will shed light on how mutations in these Arps lead to human infertility. Overall, this proposal uses an interdisciplinary approach, including cell biology, genetics, and biochemistry, to understand from a molecular to an organismal level how Arps across phyla evolve and functionally innovate for specialized roles. Our long-term goal is to extend our analyses to other cytoskeletal families that display unique evolutionary diversification to broadly study the roles of cytoskeletal proteins beyond core “housekeeping functions” and reveal how these proteins unexpectedly adapt and fulfill roles their canonical counterparts fail to accomplish.

NIH Research Projects · FY 2026 · 2024-12

Microtubules (MTs) are essential dynamic polymers required for chromosome segregation and intracellular organization, and are the direct targets of anti-cancer chemotherapeutics like taxol and the Vinca alkaloids. The dynamic properties of MTs are central to their function, and they derive from the structural and biochemical properties of individual tubulin subunits and how they interact within the MT lattice. It is increasingly appreciated that tubulin subunits adopt distinct conformations as part of the GTPase-dependent polymerization dynamics, and that regulatory proteins selectively recognize subsets of these conformations to control MT elongation, stability, and switching. Research in the laboratory uses a combination of structure (X-ray crystallography and cryo-EM), quantitative biochemistry, time-lapse observation of microtubule dynamics, and computational simulations. Mechanism-specific, site-directed tubulin mutants are used to integrate the different sources of information and also to test hypotheses and create new tools. In the present proposal, we will focus on complementary lines of investigation to provide new insights into the physical origins and regulatory mechanism of MT dynamics. We will reveal the allosteric logic of the tubulin conformation cycle and provide fresh biochemical insight by studying newly identified mutations linked to microtubule stability. We will use single-molecule observations of tubulins interacting with microtubule ends to define and quantify biochemical mechanisms and generate new hypotheses. We will reveal how relatively divergent α-tubulin isotypes and/or primitive tubulins alter MT dynamics and structure. Finally, we will define how specialized TOG domains recognize tubulin and/or MTs to regulate MT dynamics. This work will advance understanding of fundamental mechanisms of MT dynamics, recognition, and regulation, and it will begin to reveal critical points of allosteric connection within αβ-tubulin. Longer-term goal of this research are: to build a quantitative molecular understanding of how allostery and the tubulin conformation cycle dictate MT stability, dynamics, and mechanochemistry, to define mechanisms by which regulatory factors recognize tubulin and control MT dynamics, and to place this knowledge more firmly into a functional context.

NIH Research Projects · FY 2026 · 2024-12

Project Summary Red Blood Cell Transfusions (RBCT) are necessary and life-saving in premature and critically ill infants, who experience severe anemia due to both physiologic and iatrogenic factors. Recently, we have elucidated the connection between anemia and necrotizing enterocolitis (NEC); specifically, the “leaky gut” presentation characterized by monocytic infiltration, RBC transfusion-associated activation of infiltrated monocytes, and the resulting intestinal mucosal injury. Neurocognitive development is often impaired in patients with NEC and, a large clinical trial confirmed that RBC transfusion in extremely premature infants may contribute to these neurodevelopmental outcomes. This finding is supported by several preclinical studies. However, the underlying mechanism(s) by which anemia and RBC transfusion directly or indirectly correlate with neurocognitive impairment through the development of an inflammatory cascade is unclear. Critical evaluation of the association between anemia/RBC transfusion and anemia-transfusion-associated brain inflammation (ATBI) and their effects on neurodevelopment are necessary to improve clinical practice and develop therapeutic strategies for prevention or amelioration. Our preliminary studies using our established pre-clinical murine model of anemia/RBC transfusion demonstrate that anemia is associated with a “leaky gut” that leads to endotoxin in the bloodstream, causing early brain inflammation by activation of microglia. Then, RBC transfusion potentiates this early brain inflammation by recruitment of NEC-causing monocytes (raised from the intestine) in the brain. Consistent with this finding, the brain inflammatory response could be dampened either by depletion or inhibition of NEC-causing monocytes. Thus, here we will propose a novel hypothesis that anemic neonates are uniquely predisposed to early brain inflammation, and RBCT exacerbates this brain inflammation leading to altered neurodevelopment. We propose this finding in anemic premature infants due to infiltration of inflammatory activated monocytes, with a phenotype similar to monocytes found in the anemic-RBC transfused intestine via the ‘gut-brain axis’. To test our central hypothesis, we will pursue the following specific aims: Aim 1: Elucidate the mechanisms by which severe anemia and RBCT induce/exacerbate monocyte infiltration and cause inflammation in the neonatal brain. Aim 2: Investigate the role of RBCT-NEC causing monocytes in ATBI. Aim 3: Determine the effect of phlebotomy-induced anemia and neonatal ATBI on neurocognitive impairment in adulthood. Our studies will have therapeutic implications for treating anemia/RBC transfusion-associated brain inflammation, as the elimination of the NEC-causing inflammatory monocytes could contribute to the amelioration of neurodevelopment deficits.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY The dentate gyrus (DG), a part of the hippocampus (HPC), plays a pivotal role in encoding and retrieving episodic memory. A mounting body of research implicates HPC abnormalities, including those in the DG, in the pathophysiology of psychosis. This includes observed structural and functional changes in the HPC of individuals suffering from psychosis, such as those diagnosed with schizophrenia (ScZ). Pertinently, studies have identified decreased neurogenesis and changes in the morphology and functionality of DG granule cells in both animal models of psychosis and postmortem brain analyses of ScZ patients. ScZ is a complex mental disorder marked by a mix of symptoms: hallucinations, delusions, disorganized thinking, and impaired social functioning. The onset of ScZ varies, but there's a known age dependence with symptoms typically appearing from late adolescence to early adulthood, generally between the late teens and early thirties. Emerging evidence proposes a potential correlation between age-dependent dysfunction in the dentate gyrus (DG) and psychosis, although the exact nature of this relationship remains unclear. In this proposal, our objective is to reverse-translate discoveries from post-mortem human brain analyses. We specifically target DG excitatory neurons, aiming to modulate them using the chemogenetic technique known as DREADD (Designer Receptors Exclusively Activated by Designer Drugs) to gradually inhibit DG activity in the HPC in an age-dependent fashion. Concurrently, we will longitudinally monitor HPC activity using a chronic high- density in vivo recording strategy. Our hypothesis is that a reduction in DG activity in the HPC will instigate abnormal hyper-excitable neuronal activity in downstream CA3 / CA1 regions during adolescence, propagating into the entorhinal cortex (EC), which will subsequently disrupt normal brain activities, including oscillatory patterns and neuronal spiking activities, post adolescence. Preliminary data show the emergence of large, spontaneous, irregular hypersynchronous activity in the HPC, which evolves over time and persists into adulthood in mice. This spontaneous activity is characterized by large transient local-field potential (LFP) deflections coupled with large population spiking activity that has a temporal structure lasting for 50-100 milliseconds. Notably, these hyper-synchronous events tend to occur during quiet wake or slow-wave sleep periods, but not during active running states. Our aim is to consolidate two working hypotheses in this grant period: (i) how gradual suppression of DG triggers the hyper-synchronous events in HPC; (ii) the how the hyper-synchronous events impact normal sharp-wave ripples (SWRs) and behavior. We anticipate that our modeling and investigation of the early onset of ScZ, extrapolated from post-mortem brain analyses of human patients, will illuminate the neurological processes occurring in the adolescent brain during quiet / sleep periods, and aim to eventually submit a full five-year research proposal.