Case Western Reserve University

NIH Research Projects · FY 2025 · 2023-12

Project Summary/Abstract: Exploring SGLTs as Theranostic Targets for Cancer Metastasis Cancer requires substantial energy that is commonly provided by increased glucose transport and metabolism. Clinically, this is evaluated using Positron Emission Tomography (PET) imaging of the18F-labeled glucose analog 2-fluoro-2-deoxy-D-glucose ([18F]FDG). [18F]FDG enters cells primarily via facilitative glucose transporters known as GLUTs, which are overexpressed in some human cancers. [18F]FDG cannot be used to probe the sodium-dependent glucose transporters (SGLTs), the other different family of transporters, in cancers. In fact, the role of SGLTs in breast, prostate, lung, head/neck, brain, pancreas, colon, cervix, and ovary is only now starting to be recognized. Given that SGLT expression is even higher in metastasis than in primary tumors, we propose they could be theranostic targets for metastatic disease as SGLTs are able to concentrate glucose and its analogs intracellularly many fold their extracellular concentration. To evaluate this we created a brand new compound called 6FIGA (Fluorescent Iodinated Glucose Analog, modified at the 6 position carbon) which is selectively transported by SGLTs. It can be made with the therapeutic radionuclide 131I to deliver lethal radiation to cancer cells while having a low systemic concentration that minimizes toxicities. Further, SGLT function and therapy planning could be informed by PET imaging using the chemically-identical agent made with the 124I isotope. In addition, we have made it with stable iodine for fluorescent assays and imaging spanning from microscopy to murine in vivo studies. Herein we propose to use this novel trifunctional agent to evaluate SGLTs as a theranostic target for cancer metastasis. Significantly, targeted radionuclide therapy could solve the efficacy problem due to tumor heterogeneity. Radiation originating in SGLT-expressing cells penetrates multiple cell lengths and even kills neighboring cells that lack SGLTs. Our preliminary work shows that 6FIGA is transported selectively by SGLTs in representative metastatic breast and prostate cancer cell lines and an in vivo murine flank human prostate cancer tumor demonstrated 6FIGA concentrating with low off-target uptake. In this exploratory project we will use 6FIGA to explore SGLTs as potential theranostic targets via the aims: 1 Evaluate the time courses and biodistributions of [124I]6FIGA in in vivo metastatic models, measuring tumor and normal tissue concentration time courses and associated dosimetry. We hypothesize that [124I]6FIGA assays SGLT activity and provides different information than [18F]FDG. 2 Conduct proof-of-concept for targeted treatment using [131I]6FIGA radionuclide therapy, assessing overall survival and imaging response to therapy. Correlative studies will be performed, including immunohistochemistry and ex vivo fluorescence imaging, in order to quantify the relation between uptake and protein expression levels. Thus, we will demonstrate the potential of SGLTs as theranostic targets.

NIH Research Projects · FY 2026 · 2023-12

Proper synaptic function relies on a dedicated assembly of pre- and postsynaptic proteins. Neurexin1 is a principal presynaptic organizing protein essential for synaptic function. Mammalian neurexin1 has three isoforms (α, β, and γ). While neurexin1α and β have laminin/neurexin/sex hormone (LNS) domains, 1γ lacks LNS domains. This is important because LNS domains of neurexin1 bind to post-synaptic partners such as neuroligins, which recruit proteins for proper synaptic function. Deleting neurexin1α globally or neurexin1 α and β in hippocampal neurons impairs postsynaptic receptor activation without affecting presynaptic function at CA3-CA1 synapses. Thus, whether and how neurexin1 organizes presynaptic terminals is unclear. Recently, we made the surprising discovery that all three neurexin1 isoforms are covalently attached to a complex glycan, heparan sulfate (HS). We generated a Nrxn1ΔHS mouse line to block HS attachment to all three neurexin1 isoforms and found severe deficits in presynaptic function and synapse ultrastructure. In this proposal, we have generated a Nrxn1-/- mouse line to delete all three isoforms and found similar presynaptic deficits at CA3-CA1 synapses in Nrxn1ΔHS and Nrxn1-/-. These findings now suggest that the neurexin1 protein backbone alone is insufficient for presynaptic function and that additional factors, which act through HS, are involved in presynaptic development. Furthermore, the presynaptic deficit observed in Nrxn1-/- was not found in neurexin1 α and β mutants, raising the exciting possibility that neurexin1γ is sufficient to maintain presynaptic function. Thus, our central hypothesis is that the HS modification has a specific role in neurexin1-mediated presynaptic assembly and function and is essential for the roles of neurexin1γ and a novel HS-binding growth factor pleiotrophin in presynaptic assembly and function. To test this hypothesis, we will examine the specific role of HS in neurexin1-mediated pre- and post-synaptic assembly (Aim 1), the sufficiency of neurexin1γ and its HS in presynaptic assembly and function (Aim 2), and the role of pleiotrophin in presynaptic assembly and function (Aim 3) in mouse brains. To understand the synaptic assembly within an intact brain circuit, we will utilize innovative Expansion Microscopy to visualize synaptic proteins in the higher order at individual synapses. Given the critical role of neurexin1 in synapse development, the expected outcome of this work will provide novel molecular insights into presynaptic development and function. Mutations in neurexin1 and abnormalities in the biosynthesis of HS have been linked to schizophrenia and autism. Therefore, our work will shed light on how mutations in neurexin and heparan sulfate biosynthetic enzymes affect synapse development and function in autism and schizophrenia and may lead to the development of novel therapeutic strategies for these diseases.

NIH Research Projects · FY 2025 · 2023-12

PROJECT SUMMARY Osteosarcoma (OS) is an aggressive malignant primary bone cancer with a high propensity for lung metastasis. Since the development of aggressive chemotherapy and surgery, the 5-year event-free survival rate for non- metastatic patients has attained 70%. In contrast, the outcome for pulmonary metastatic osteosarcoma (pOS) remains poor and the 5-year event-free survival has not improved significantly over the past 3 decades (20%). Therefore, treating pOS effectively remains a challenge. Transforming growth factor-β (TGF-β) is one of the most potent immune suppressive cytokines in tumor microenvironment (TME) and can promote metastasis in solid tumors. TGF-β production is increased in the sera of OS patients. This increase in TGF-β production is correlated with high grade OS and associated with the presence of lung metastases. Therefore, target TGF-β is the new therapeutic approach for the treatment of OS. In this proposal, we will demonstrate the therapeutic effects of a novel inhibitor of TGF-β signaling, TEW-7197, on OS growth in vitro and in vivo and associated immune responses in the OS TME. TEW-7197 (“Vactosertib”) is the first-in-class small molecule inhibitor of the TGF-β type I Receptor (TβRI) kinase and currently in Phase I clinical trial at UH Seidman Cancer Center for Multiple Myeloma, another disease with high TGF-β (#NCT03143985). TEW-7197 is orally available (taken qD or BID) and well tolerated with minimal side effects. Our preliminary data show that blocking TGF-β signaling with TEW- 7197 inhibited OS proliferation in vitro and oral administration of TEW-7197 to OS-bearing mice significantly reduced established pOS in vivo. Our preliminary observation has resulted in an Orphan Drug Designation authorization by the FDA in August 2021 as well as Fast-track IND issuance in January 2023 for the use of TEW- 7197 in OS. More recently, a multi-center phase I/II clinical trial using Vactosertib as monotherapy for the treatment of advanced osteosarcoma in patients age 14 and up been organized and planned for accrual in spring 2023 (#NCT05588648) at 21 sites across US, Europe and Asia. Recently, we have made the additional observation that TGF-β induced c-Myc expression in OS cells and those inductions were completely inhibited by TEW-7197 in OS cells. c-Myc is a major proto-oncogene which is highly amplified in OS. c-Myc overexpression in human OS correlates with aggressive tumor cell invasion and metastasis and worsens overall patient clinical prognosis. Therefore, these new results suggest that c-Myc regulation by TGF-β may be an important therapeutic target in OS. We hypothesize that TGF-β signaling inhibition may be an effective therapeutic strategy against metastatic pOS by modifying tumor-intrinsic signaling (c-Myc regulation) and extrinsic immune-related TME to achieve optimal immune-effector function and maximal clinical response in pOS. To confirm this hypothesis, we will utilize various mouse and human OS cell lines and PDX models in vitro and in vivo. Two specific aims will be pursued to accomplish the overall objective. Specific aim 1 will Determine effects of TGF-β inhibition on c- Myc regulation in OS cells in vitro. Specific aim 2 will Determine whether daily oral administration of TEW-7197 suppresses pOS tumor progression in the syngeneic and humanized mouse models. A fully humanized mouse model will be employed to interrogate TEW-7197 effects on cellular immune responses with preclinical human OS TME in vivo. This research will establish a critical role of TGF-β signaling in pOS progression and provide scientific rationale that targeting TGF-β signaling with orally available small molecule inhibitor should be developed as a viable therapeutic arsenal for pOS patients.

NIH Research Projects · FY 2025 · 2023-12

PROJECT ABSTRACT This application is focused development of a new therapeutic for pediatric high-grade glioma (HGG), including a rare subset known as Diffuse Intrinsic Pontine Glioma (DIPG). DIPG is a rare brain cancer primarily affecting children, with approximately 200-300 new annual cases in the United States, less than 2 percent survival at 2 years, and with a limited response to therapeutic radiation (RT). Recently published studies in preclinical glioma models have revealed that RT results in a progressive accumulation of monocyte derived macrophages (MDMs) and activated microglia, and that inhibition of the recruitment of MDMs delays glioma recurrence. There is substantial evidence demonstrating a key role for chemokine (C-C motif) ligand 2 or ‘CCL2’ also known as monocyte chemoattractant protein-1 (MCP-1) in RT-induced inflammation, including the recruitment of MDMs into the brain and tumor microenvironment (TME). These studies not only show that RT induces a transient and selective upregulation of CCL2 within hours of exposure, but also demonstrate disruption of CCL2 signaling during this time frame alone is sufficient to attenuate chronic microglial activation and to allow the recovery of neurogenesis in the weeks following radiation. However, there is a desperate need for new agents that are not only potent inhibitors of CCL2 production and signaling, but that are also safe for administration in combination with RT. The synthetic oleanane triterpenoids (SOTs) evaluated in this application meet these criteria. The research team pursues ground-breaking, high-risk, high-gain studies that address our central hypothesis that chronic, systemic oral administration of a newly developed SOT (CDDO-2P-Im or ‘2P-Im’) will enhance the radiation response of DIPG through mechanisms that include suppression of RT-induced recruitment and activation myeloid cells in the brain, with potential to also limit RT-related toxicity. Preliminary data show 900 nanomoles of 2P-Im/kg of brain at 6 hours after a 1 µmole dose, given either by intraperitoneal injection or by oral gavage in mice, and that 2P-Im inhibits CCL2 production by activated human macrophages at picomolar concentrations in vitro. The project is organized around three specific aims that: (1) define the effect of 2P-Im (both alone and in combination with RT) on in vitro clonogenic survival of DIPG cell lines, including those harboring the histone H3.3 Lys 27-to-methionine (K27M) mutation, and the relationship to direct molecular targets of 2P-Im (AIM 1); (2) determine the dose- and time-dependent effects of 2P-Im on RT-induced recruitment of MDMs and on microglial activation (AIM 2); and (3) demonstrate the in vivo efficacy and activity of 2P-Im (administered in rodent diet) in established, orthotopic PDX models of DIPG, including the capacity of 2P-Im to enhance the radiation response of DIPG xenografts in vivo, (AIM 3). Data generated in this proposal will position 2P-Im for advancement to critical, IND-enabling studies, and as a novel, orally bioavailable therapy for a highly aggressive, refractory rare childhood cancer.

NIH Research Projects · FY 2026 · 2023-09

PROJECT SUMMARY/ABSTRACT Somatic mutations accumulate over time in every healthy cell, and these mutations have long been hypothesized to be an important contributor to aging. However, many unknowns remain about this fundamental process of aging because only recently have ultra-high fidelity sequencing technologies become available that can detect somatic mutations in bulk poly-clonal samples of healthy cells, such as blood. For example, it is entirely unknown how widely aging-related somatic mutation rates (SMRs) vary among individuals in the population, whether SMRs predict mortality and aging-related disease, whether genetic or environmental modifiers may cause outlier SMRs, and whether SMRs are associated with epigenetic aging of the genome. Notably, no study to date has measured SMRs in large numbers of individuals, and no study has measured SMRs with associated individual health, lifestyle, and exposure data. The Women’s Health Initiative and the Cancer Prevention Study-II Nutrition Cohort are longitudinal studies that present a unique opportunity to study the variability, modifiers, and mechanisms of SMRs in the human population. These cohorts’ > 20 years of longitudinal data and blood samples from > 20 years ago enable this proposal’s first large-scale measurements of SMRs in the human population and the first measurements of SMRs in combination with detailed human health data. Here, we will perform ultra-high fidelity profiling of SMRs and mutational patterns in the blood of 3,000 individuals from these cohorts, which is almost an order of magnitude more individuals than all prior SMR studies combined. This study will directly test several fundamental open questions and hypotheses about SMRs, including how widely SMRs and mutational patterns vary among individuals, whether SMRs vary between sexes, whether there are outlier individuals in the population with identifiable causes for their higher or lower SMR, and whether SMR predicts mortality and aging-related disease. Since prior studies suggest that deamination of methylated cytosines is a significant contributor to SMRs, we will also perform the first joint SMR and methylation profiling of the same individuals to assess the mechanistic relationship between genetic and epigenetic aging of the genome. Apart from achieving the first population-level characterization of SMRs and an improved mechanistic understanding of SMRs, this study may establish SMRs as a novel, clinically useful biomarker for healthy aging. As the population ages and the prevalence of aging-related diseases increases, our study may motivate the use of SMRs as a biomarker for predicting aging-related disease risk, potentially enabling early preventive clinical interventions. This study will further establish a scalable approach for studying SMRs—which reflect the cumulative effect of endogenous and exogenous mutagens over the lifespan—in large cohorts, with potential for significant impact on our understanding of human health and aging.

NIH Research Projects · FY 2025 · 2023-09

Project summary: Restoration of urinary dysfunction is often in the top 25% priority for rehabilitation in individuals after spinal cord injury (SCI), often ranking higher than restoration of upper limb function. However, current treatments are mostly limited to conservative approaches consisting of intermittent catheterization and behavioral changes such as fluid intake management, but these interventions only provide symptomatic relief and are associated with recurrent urinary tract infections, kidney disease, and high mortality rates. While other pharmaceutical and surgical treatments may be available for some patients with specific complaints such as overactive bladder, the development of novel therapies to improve and restore bladder function after SCI is of clear importance. Vagus nerve stimulation (VNS) paired with rehabilitation is a neuromodulation technology that has demonstrated great potential at restoring motor and sensory function after neurological injury, including incomplete SCI. VNS promotes neuroplastic changes by the timed-release of neuromodulators that can enhance recovery. Here we propose for the first time, a strategy of pairing VNS with bladder function after incomplete SCI to improve symptoms of urinary dysfunction. Our preliminary results support the use of this technology to restore bladder function after SCI. We propose to conduct clinical and preclinical research concurrently to accelerate technology translation. Aim 1 (K99 phase) leverages a separate, ongoing clinical trial (NCT04288245) that has 15 participants, who have the VNS system already implanted and willing to partake in novel therapy approaches. This aim, will demonstrate that VNS therapy paired with bladder function can improve voiding efficiency in humans after SCI. To achieve this, we will modify the interface of the VNS system (ReStore VNS system; FDA-cleared for upper limb rehabilitation after stroke and currently undergoing phase 2 clinical trials for upper limb rehabilitation after SCI) to allow pairing with volitional bladder voiding and conduct a pilot study in currently implanted individuals. Aim 2 optimizes the timing of delivery VNS paired with different stages of the bladder function to improve voiding efficiency in the rat model (sub-Aim 2.1, K99/R00 phase) and characterizes the mechanisms of action behind those changes (sub-Aim 2.2, R00 phase). During Dr. Hernandez-Reynoso’s career, she has extensively investigated the use of electrical stimulation targeted to the peripheral nervous system as a novel treatment of urinary dysfunction, and the development of peripheral and central neural interfaces. As such, she is the ideal candidate to carry this proposal to completion. Results from both aims of this proposal will support the submission of an R01 to conduct an independent clinical trial, and act as a catalyst for the candidate to reach independence as a translational researcher in the neural engineering and bioelectronic medicines field.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Plasma membrane rupture (PMR) in lytic cell death—including pyroptosis, necroptosis, and post-apoptotic secondary necrosis—is a cataclysmic event that releases large-size intracellular molecules known as damage- associated molecular patterns (DAMPs), which in turn propagate the inflammatory response. Lytic cell death plays an important role in host defense against pathogen infections, but its dysregulation is also implicated in many inflammatory diseases and pathological conditions. PMR was thought to be a passive event, until a recent study identified NINJ1 to be responsible for carrying out this process. NINJ1 is a 16-kDa plasma membrane protein previously identified to be mediating cell adhesion through homotypic binding. It has two transmembrane helices and one extracellular amphipathic helix. NINJ1 undergoes oligomerization to induce PMR, and the amphipathic helix seems to play an important role in this process. However, a highly similar protein NINJ2 in the plasma membrane bearing a similar amphipathic helix does not induce PMR. To understand the molecular basis of NINJ1-oligomerization mediated PMR, we use cryogenic electron microscopy (cryoEM) to study the structures of NINJ1 and NINJ2 oligomers. The progress we have made is shedding light on a mechanistic understanding of this process. However, it also points to more hypotheses that need to be tested in order to fully understand this fundamentally important process. PMR is also linked to glycine cytoprotection in a very recent study. It was demonstrated that glycine treatment prevented NINJ1 oligomerization and thus prevented PMR in bone marrow derived macrophages receiving various forms of lytic cell death stimuli. We will include glycine treatment in our test of hypotheses for the search of signal that triggers NINJ1 oligomerization. This further elucidation of the molecular basis of glycine cytoprotection would inform the development of better cell preservation strategies or agents.

NIH Research Projects · FY 2026 · 2023-09

Modified Project Summary/Abstract Section The Positive Peers mobile app is an original platform developed by and for the hardest to reach HIV population, young people with HIV (YPWH). This app holds potential to provide extensive, customizable, self-management tools (i.e., wellness tracker, community forum, chat, frequent original blogs) to young people with HIV anywhere in the US. The Positive Peers app provides health information, health management tools and virtual community support. While the app itself offers a confidential place for young people with HIV to get health information and support, its use is enhanced by the presence of local peer administrators who provide navigation, support and coaching to users. The proposed study seeks to evaluate its effectiveness in improving viral suppression among younger populations, 13-34 years of age who are either newly diagnosed, out of care or not virally suppressed using a randomized control trial design supplemented by an observational cohort of persons who decline to use the app. Clinics in six high priority Ending the HIV Epidemic jurisdictions will train staff as app administrators and utilize the app as a clinic-based tool. Our primary objective is to improve HIV outcomes by offering peer interaction, targeted retention and adherence messaging, and interactive trackers and reminders in one smartphone app. Our specific aims are: Aim 1: Compare the effectiveness of HIV care supported by the PPA to usual care for retention in HIV care and viral suppression of newly diagnosed or re-engaged high priority younger adults with HIV. Aim 2: To identify factors that predict user engagement with primary PPA components and associated effects on retention in care, viral suppression, and HIV related perceived stigma. Aim 3: To determine intervention adoption, usability, fidelity, and cost across study sites. These aims will be addressed in a parallel cohort design randomized controlled trial of 250 newly diagnosed or out of care YPWH from designated high priority sites. Participants will be allocated 1:1 to receive the PPA app upon study entry or to a delayed intervention arm where they will receive the usual care with attention controls for 6 months. This will allow for effectiveness evaluation during the earliest phase of adjustment to the diagnosis while also allowing for longitudinal outcome effects.

NIH Research Projects · FY 2025 · 2023-09

PROGRAM SUMMARY The central hypothesis of this program project is that “Altered squamous epithelial integrity (Prj 1) and inflammatory injury (Prj 2) activate signaling pathways including EPHB2 (Prj 3) that affect precursor cells at the squamocolumnar junction (SCj) transition, esophageal submucosal gland (ESMG), and basal squamous niches, resulting in the alteration of regulatory factors that include Notch, Myc, p63, and SOX9, leading to acinar ductal metaplasia (ADM), multi-layered epithelium (MLE), Barrett's esophagus (BE), and ultimately esophageal adenocarcinoma (EAC).” The Specific Aims of our program are: 1) To elucidate signaling pathways by which mutated VSIG10L alters epithelial integrity leading to MLE and BE like metaplasia on novel mouse models. 2) To define the spatial and temporal pattern of CXCL8 (IL-8) in ESMG following esophageal injury and phenotype the inflammatory infiltrate that leads to the development of acinar ductal metaplasia (ADM) in ESMG associated with BE/EAC. 3)To identify mediators of EPHB2 signaling that lead to c-MYC activation and metaplastic cellular differentiation after injury in the development of BE and its progression to EAC. 4) To define how altered epithelial integrity, inflammatory cells, and alteration of signaling molecules that control differentiation (EPHB2) lead to metaplasia by altering transcription factors. 5) Integrate projects by providing investigators effective support through Core resources with state-of-the-art Biorepository, Bioinformatics, and Administrative Services. These objectives build and synergize on the considerable clinical, basic science, and translational expertise available at our institutions, 1) to focus laboratory research on understanding the genetic susceptibility and molecular changes that influence the development of BE and EAC; and 2) to then translate laboratory discoveries into clinical applications for effective detection, molecular risk stratification, and prevention of progression from BE to EAC.

NIH Research Projects · FY 2024 · 2023-09

Project Summary/Abstract With our expected lifespans increasing, the rapidly expanding aging population is bringing an increased prevalence of dementia, including Alzheimer’s disease (AD) and vascular cognitive impairment (VCI). However, there are still no neuroprotective medicines for treating patients with these conditions. AD and VCI are the most common types of dementia and impose a huge socioeconomic burden as well as devastating impacts on the lives of patients and their caregivers. Both of these forms of dementia are characterized by deterioration of the neurovascular unit that forms the blood-brain barrier (BBB), which in many cases even precedes the onset of cognitive deficits. Unfortunately, however, the mechanisms of BBB deterioration in AD and VCI are unclear. As a result, there are no therapies to protect the BBB. In my thesis work, I have established that the prostaglandin degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is pathologically elevated in both human AD and VCI in human patients. I have also shown that the enzymatic activity of 15-PGDH in the brain is increased in the 5xFAD mouse model of AD, as well as normal aging. Importantly, I have established that pharmacologic and genetic inhibition of 15-PGDH in 5xFAD mice shows robust protection against BBB deterioration and other AD-related pathology, including cognitive deficits, impaired neurogenesis, and axon degeneration, independently of amyloid β pathology. I have also found that of the prostaglandins, prostaglandin D2 (PGD2) is most prominently elevated in the brain by 15-PGDH inhibition in 5xFAD mice. Therefore, I hypothesize that PGD2 is responsible for 15-PGDH inhibition-mediated protection of the BBB, and that this is related to improved endothelial cell barrier function. During the F99 portion of my proposal, I will evaluate whether 15-PGDH inhibition also protects from BBB deterioration in the high fat diet mouse model of VCI. I will utilize innovative 2-photon microscopy in vivo imaging and electron microscopy to determine the trajectory of BBB deterioration, as well as test the protective efficacy of 15-PGDH inhibition. I will also determine whether PGD2 mediates the protective effect both in vivo and in vitro, as previous literature suggests a role of PGD2 in increasing endothelial cell barrier function. During the K00 phase, I will expand my BBB research by investigating the interaction between perivascular macrophages (PVMs) and endothelial cells in the brain in Dr. Chenghua Gu’s laboratory. I will utilize an innovative cre-recombinase system to specifically target PVMs in the brain and investigate altered glucose metabolism in PVMs and transcriptomic profiling in both PVMs and endothelial cells, as a function of AD-related risk factors. Then, I will test how this altered metabolism in PVMs interacts with endothelial cells to initiate BBB deterioration. Successful completion of this study will provide new perspectives of how the BBB deteriorates with aging and dementia-related pathology, which will enable the discovery and development of new neuroprotective approaches for patients suffering from AD and VCI.

NIH Research Projects · FY 2025 · 2023-09

Motor neurons carry electrical signals from the brain through the spinal cord to ultimately generate muscle contraction via the neuromuscular junction (NMJ). The mechanisms involved in initiating and maintaining proper communication between the central nervous system and muscles are incredibly complex, and damage in this communication is the cause of neuromuscular diseases (NMD), such as Amyotrophic Lateral Sclerosis (ALS) and Spinal and Bulbar Muscular Atrophy (SBMA). While these disorders are among the most common NMDs, there is currently no cure or effective treatment for them. The NMD field acknowledges that a substantial number of drugs found to alleviate symptoms in animal models have failed in clinical trials. Even though this highlights the importance of the development of humanized models, a caveat of converting studies into iPSC models is the focus on single cells in detriment of the complex systems of the adult organism. In the NMD field specifically, iPSC investigations have largely focused on addressing motor neuron phenotypes that would prevent their degeneration. Unfortunately, this approach is no longer sufficient, as prolonging motor neuron survival does not assure re-innervation, nor does it guarantee prevention of denervation. Therefore, it is crucial for therapeutic advancement in the NMD field that stem cell research needs to focus not only on identifying cell-specific targets but also on testing those targets on functional NMJ systems that comprise both iPSC-derived motor neurons and skeletal muscles. To achieve this goal, we have recently developed a 2D functional human NMJ system comprised of both iPSC-derived motor neurons and skeletal muscles. Our human iPSC-NMJ model is responsive to optogenetics and we are able to quantitatively measure NMJ function in a multi-electrode array system. Hence, in this R61/R33 IGNITE Phased Innovation Award System, we propose to leverage our newly developed NMJ system to scale functional and morphological assessment (Aims 1 and 2) and validate the system by assaying NMJ-specific dysfunction using two NMDs: SBMA and ALS (Aims 3 and 4). Our lab has previously established an iPSC model for SBMA (R01NS121374-01, K01NS116119-01) and has extensive experience modeling this disease. Additionally, we selected the iPSCs harboring G4C2 hexanucleotide repeat expansion on chromosome 9 within the first intron of C9ORF72, as it represents is the most common genetic contributor to frontotemporal dementia (FTD) and ALS, accounting for ~10% of all cases of those diseases. Thus, successful completion of this R61/R33 will establish and validate a novel model system to facilitate therapeutic discovery for NMDs.

NIH Research Projects · FY 2025 · 2023-09

The function of nuclear DNA is not only determined by its sequence, but also depends on its three dimensional (3D) structure. A particular type of DNA in eukaryotes is called heterochromatin, which refers to as tightly packed DNA structure in the nucleus. Heterochromatin plays a critical role in genome function including DNA structural maintenance, chromosome segregation, epigenetic inheritance, DNA replication, repair and transcription. Recently, increasing evidence suggests the involvement of a new biological process called liquid- liquid phase separation in the formation and function of heterochromatin. Liquid-liquid phase separation, whose concept was borrowed from polymer sciences, is a unique process that involves the formation of membraneless liquid droplets by proteins and nucleotides when their concentration have reached a threshold. These liquid droplets enable the assembly and disassembly of functional protein-based organelles within a cellular compartment following environmental cues. Hence, liquid-liquid phase separation has facilitated our understanding of fundamental cellular information processing, cellular homeostasis, and cellular physiology. Recently, we unexpectedly identified a new player, human 53BP1, in regulating the heterochromatin integrity through liquid-liquid phase separation. 53BP1 was previously known as a critical player in regulating the DNA double strand break repair. However, we discovered that the protective role of 53BP1 in both the structure and the function of heterochromatin is distinct from its canonical activity in DNA double strand break repair. Hence, our studies opened a new research paradigm for this important protein in signaling, biology and cellular physiology. The goal of this proposal is to establish this new research field by addressing several unanswered important questions regarding this new function of 53BP1. These include the molecular basis by which 53BP1 forms the liquid droplets and its significance in biology, genome biology, and cellular physiology. We have used mass spectrometry to identify components in the liquid droplets formed by 53BP1. We will interrogate their functions in this application. By assembling an interdisciplinary team consisting of experts on molecular and cellular biology, proteomics, computational bioinformatics, and next generation sequencing, we will use a combination of cell biological, molecular, biochemical, genetic, morphological, and chemical approaches to answer these questions. Our studies will illustrate a previously uncharacterized function of 53BP1 and a novel interplay between 53BP1 and heterochromatin and determine their impact on genome stability, facilitating our understanding of fundamental cellular information processing, cellular homeostasis, and cellular physiology.

NIH Research Projects · FY 2025 · 2023-09

Abstract In women, breast cancer is the most commonly diagnosed cancer and leading cause of cancer related deaths worldwide, with approximately 2.3 million new cases and 685,000 deaths in 2020. Neoadjuvant chemotherapy (NAC) is commonly applied to reduce the tumor size before surgery for breast neoplasms. Unfortunately, due to the genetic and phenotypic heterogeneity of breast tumors, not all patients respond to conventional NAC. Currently, only about 22% of patients show pathologic complete response (pCR), while the remaining non-pCR patients show either partial response (54% of all patients) or no response to chemotherapy. Early prediction of tumor response to chemotherapy to identify non-responders could 1) reduce unnecessary side effects and costs related to ineffective therapy, and 2) help physicians tailor the treatment plan earlier to achieve better therapeutic outcomes and improve survival. Monitoring tumor response to chemotherapy is currently based on tumor size measured by physical exam, which is subjective, difficult to quantify, and most importantly, temporally delayed compared to underlying biological changes. Quantitative, repeatable and objective methods that could provide an early detection of tumor physiological changes before size changes could significantly improve treatment outcome and the quality of patient care. However, quantitative imaging poses significant technical challenges, which is rarely performed in the clinical setting. Here, we propose to leverage Magnetic Resonance Fingerprinting (MRF), a revolutionary new platform for quantitative MR that was invented by our team, to develop new imaging biomarkers for early assessment of treatment response in women with breast cancer. Our team has developed a breast MRF method to simultaneously generate quantitative 3D T1 and T2 maps in ~6 minutes with excellent reproducibility. We have also expanded our MRF method to simultaneous quantify T1, T2 and ADC maps of the brain with no image distortion. Here, we plan on optimizing this new relaxometry / diffusion MRF method specifically for women with breast cancer (Aim 1). Novel deep learning methods will be developed to provide a fast (<5 minute) and high resolution (1.2 mm isotropic) acquisition for whole-breast coverage along with an efficient post-processing pipeline based on cloud computation (Aim 2). Finally, we will evaluate the developed method for early prediction of treatment response in two patient cohorts with either HER2-positive or triple negative breast cancers (Aim 3). Upon successful completion of this project, the developed MRF technique will provide a practical quantitative breast exam for early prediction of treatment response to NAC and other treatment methods (hormone therapy, antibody-based target therapy, etc.) for women with breast cancer, with the ultimate goal to reduce ineffective treatment in eligible subjects and tailor the treatment methods for optimum therapeutic outcomes.

NIH Research Projects · FY 2025 · 2023-09

Disruption of sleep, circadian rhythm (24-hour sleep-wake cycle) and glucoregulation represent a major problem for individuals undergoing coronary artery bypass graft (CABG) surgery and may result in prolonged length of stay, postoperative convalescence, cardiovascular morbidity, and impaired immune function.3 Cardiovascular disease is the number one cause of mortality4 and CABG and valvular surgeries are the most commonly performed cardiac surgery with an annual rate of over 680,000 in the United States.5 Individuals post-CABG are cared for in the intensive care unit (ICU) environment where exposure to fluorescent lighting, frequent noise, and a fragmented sleep-wake cycle contributes to circadian and glucose disruption.12 Further, the degree to which the circadian rhythm and glucoregulation are altered from baseline in individuals post-CABG is largely unknown. Therefore, the purpose of this descriptive study is to examine sleep and circadian rhythm characteristics and the relationships between sleep, circadian rhythm, and glucoregulation among adults in the intensive care unit postoperative CABG/VR surgery. In Aim 1, we will determine between- and within-person associations of sleep, circadian rhythm and glucoregulation associations among 30 adults post-coronary artery bypass graft with or without valvular surgery. Sleep and circadian characteristics will be described through the timing of behavioral (actigraphy) measures. In Aim 2, we will compare sleep, circadian rhythm characteristics, and glucoregulation among post-coronary artery bypass with or without valvular surgery based on clinical characteristics of operative time, intensive care and hospital length of stay and discharge disposition. Our central hypothesis is that higher sleep and circadian disruption are associated with poorer glucoregulation and that there are sleep, circadian, and glucoregulation among individuals post-CABG/VR. The proposed F31 study and training plan will provide a strong foundation in sleep and circadian rhythm health. Promoting sleep and circadian rhythm stability post-surgery through behavioral modifications may improve glucoregulation by reducing insulin resistance, improving insulin sensitivity, and other short- and long-term postoperative outcomes in a population where such clinical gains may otherwise be difficult to achieve. This study will provide information on the impact on glucose in the setting of sleep and circadian rhythm disruption in coronary artery post-operative outcomes in the ICU where it is critical to optimize glucoregulation. Additionally, these results will provide preliminary evidence to inform future longitudinal and intervention studies to improve glucoregulation and postoperative outcomes in this understudied population.

NIH Research Projects · FY 2024 · 2023-09

Pancreatic ductal adenocarcinoma (PDA) is the 3rd leading cause of cancer death in the U.S. and is generally refractory to chemotherapy. We discovered that the harsh PDA microenvironment primes cancer cells against additional cytotoxic insults (e.g., chemotherapy) and promotes PDA aggressiveness. A better understanding of the molecular underpinnings behind this adaptive program would expose PDAs metabolic vulnerabilities. We identified the RNA binding protein, HuR (ELAVL1), as a major player in the acute pro-survival response. Upon stress, HuR translocates from the nucleus to the cytoplasm with key survival transcripts, like IDH1 (an NADPH generating enzyme). HuR enhances RNA stability and protein translation of target mRNAs, to rapidly adjust the transcriptome in response to stress. Our research highlights two metabolic processes in the HuR adaptive program: a) antioxidant defense and b) mitochondrial performance. HuR silencing in PDA cells produced excessive ROS and NADPH depletion under low glucose or chemotherapy stress. An unbiased RNA seq analysis of antioxidant genes in HuR deficient cells identified IDH1 as the leading antioxidant enzyme under HuR control. RNA binding and RNA stability assays showed that HuR regulates IDH1 post-transcriptionally, and HuR deficient PDA cells had markedly reduced IDH1 mRNA and protein expression. HuR-deficient cells failed to engraft in nude mice, while IDH1 overexpression rescued PDA engraftment. HuR-deficient cells also had dysfunctional mitochondria, reflected by reduced oxygen consumption, ATP, and mitochondrial abundance. Based on this body of work, we hypothesize that PDAs reliance on HuR under low nutrient conditions exposes new therapeutic opportunities. In Aim 1, we establish the survival impact of the HuR- IDH1 axis, by editing out HuR binding sites (CRISPR) in the IDH1 3’UTR. We generated a conditional IDH1 knockout mouse, and will cross it with an established PDA model to validate IDH1 as a therapeutic target. We will test an allosteric modulator of mutant IDH1 (GSK-321) as a novel wild type IDH1 inhibitor in PDA, and launch hit-to-lead optimization to improve drug properties. In Aim 2, we identify specific aspects of mitochondrial biology under HuR control through studies of mitochondrial structure and function in HuR- deficient PDA cells. The importance of the HuR-IDH1 axis on mitochondrial ROS levels will be demonstrated. Additional transcripts will impact HuR’s regulation of mitochondrial performance will be identified. A novel mitochondrial inhibitor, CPI-613, will be combined with HuR or IDH1 inhibition as a new synthetic lethal approach against PDAs adaptive metabolic program. In Aim 3, we will use a diabetic mouse model to show that a hyperglycemic state suppresses the HuR pro-survival network, and sensitizes PDA to chemotherapy. Successful engraftment of HuR-deficient cells in hyperglycemic mice would suggest that HuR is less important under these conditions. Our studies of HuR biology will improve understanding of PDA metabolic tendencies, and reveal therapeutic opportunities relevant to PDAs nutrient deprived microenvironment.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Only 2 in 8 young adults (age 18-30 years) with type 1 diabetes (T1D) achieve glycemic targets (A1C < 7.0%). In well-controlled lab studies sleep deprivation impairs glucose tolerance and insulin sensitivity, a reduces acute insulin response to glucose, impairs body weight regulation (lower leptin, higher ghrelin), and impairs alertness in young adults without chronic conditions and decreases insulin sensitivity in middle-aged adults with T1D. Using cognitive-behavioral approaches to sleep by 1 hour over 6 weeks to 12 months in natural environments is feasible and contributes to improvements in insulin sensitivity, glucose tolerance, and general distress symptoms in young adults without chronic conditions and improved time in glucose range in adolescents with T1D. Sleep duration, regularity, and timing are modifiable targets that may improve glycemia and other important diabetes self-management outcomes in young adults with T1D. Here we leverage our preliminary findings in the proposed study to advance cognitive-behavioral sleep self-management for T1D (K99/R00NR018886). We propose to enroll 248 young adults ages 18-30 years with T1D (50% female, 40% underrepresented) who are not achieving glycemic targets (A1C ≥ 7%). The goals of this study are two-fold: (1) to compare the immediate and short-term effects of a 3-month cognitive-behavioral sleep self-management intervention (CB-sleep) versus enhanced usual care (time-balanced attention control) on sleep health dimensions and glycemia and (2) to determine whether sleep health mediates the associations between the intervention and control condition over 9 months (baseline to 3 months and 6 and 9 months post baseline). We will randomize 1:1 to the CB-sleep or enhanced usual care condition (time balanced attention control). Sleep health dimensions will be rigorously measured using validated tools: regularity (actigraphy and self-report), satisfaction (Patient-Reported Outcomes Measurement Information System Sleep Disturbance), alertness (Epworth Sleepiness), timing, efficiency, and duration (actigraphy and self-report). Glycemia will be determined by A1C (primary outcome) with subgroup analyses of glucose variability/glucose percentage time in range 70-180 mg/dL (via continuous glucose monitors or self-monitored blood glucose six times daily). Data will be analyzed using multivariate techniques, and efficacy will be determined.

NIH Research Projects · FY 2025 · 2023-09

Abstract. The rapid progress in genome sequencing has led to significant data collection. Analyzing this data can be transformative in answering the key questions about disease associations and our evolution. However, due to growing privacy concerns about the sensitive information of participants, access to genomic datasets used in studies, such as genome-wide association studies (GWAS), is restricted to only a limited number of large groups. On the other hand, collaborative research over genomic datasets, which will also lead to democratizing genomic data sharing, requires sharing data across collaborators. One way to share such datasets across collaborators is through the IRB process and the use of institutional data use agreements. Currently, due to the sensitivity of data, the GWAS computation can only be carried out after IRB review for all collaborators. In this research, we propose a sandbox environment in which potential collaborators come together and obtain an accurate "preview" of their collaborative research in an efficient, reproducible (verifiable), and privacy- preserving way. Our proposed framework allows each collaborator to share information about their dataset in a privacy-preserving way within the proposed sandbox environment. This will help the researchers (1) rectify their federated datasets from low-quality, biased, or statistically dependent records, (2) generate an accurate preview of their collaborative GWAS results to provide evidence for benefit versus risk tradeoff in IRB approval, and (3) identify what part of the datasets should be shared among the collaborators (once they obtain the full IRB approval). To achieve these goals, we will develop (1) novel algorithms that enable quality control over federated data while preserving ownership and privacy and (2) algorithms that promote reproducibility of GWAS results by developing novel techniques for verifying the correctness of GWAS computation and for sharing the whole research datasets while preserving privacy. Our preliminary results show that the proposed framework accurately provides evidence of reproducibility of GWAS results, identifies low-quality (e.g., statistically dependent) data in federated datasets, and preserves the privacy of individuals in collaborators' datasets. Notably, we show that privacy risk due to the proposed framework is lower than the one accepted by the NIH Genomic Data Sharing Policy. Finally, working together with the IRB from three institutions, we will design a pilot study to explore the efficacy of the proposed framework and its integration into the current IRB process. The outcomes of this research will provide a new strategy for genomic data sharing.

NIH Research Projects · FY 2025 · 2023-09

Limited knowledge of the structures and biological activities of products generated by free radical-induced lipid oxidation in the retina is a barrier to progress in the development of clinically useful new disease markers and mechanistically informed therapeutic interventions. Understand- ing cellular responses to free radical-induced lipid oxidation products is complex owing to their diversity and ability to mimic enzymatically generated receptor agonists. Their unrecognized generation and biological activities almost certainly contribute to the mediocre efficacy of currently available therapeutic measures for age related macular degeneration (AMD). We discovered glutathione (GSH) derivatives produced from oxidatively truncated arachidonyl phospholipids that are structural and functional analogues of cysteinyl (Cys) leukotrienes (LTs). We refer to them as pseudo (ø)LTs. We will test the hypotheses that øLTs contribute to retinal pathology and physiology in a rat model of light-induced retinal degeneration. Pilot studies show that øLTs are present in human retina, are produced in vivo consequent to oxidative insult, and exhibit LT-like biological activities. Therefore, øLTs can elicit cellular responses erroneously presumed to be induced exclusively by CysLTs. Consequently, they are a confounding factor for interpreting previous studies on the involvements of LTs in retinal pathology and physiology. The proposed research will test the hypotheses that øLTs and related GSH derivatives of oxidatively truncated docosahexaenoyl phospholipids contribute to receptor-dependent inflammatory cytokine signal- ing, retinal edema, pathological neovascularization, or physiologically important initiation of retinal pigment epithelium (RPE) autophagy. The potential pathophysiological significance of these glutathionylated products of lipid oxidation lies in the possibility of their ubiquitous generation in high levels, e.g., compared to those of CysLTs. We will test the conclusion of preliminary studies that øLTs are LT receptor agonists and determine if their ability to induce the expression of IL-13 and TGF-b1 promotes LT biosynthesis. Because cross reactivity may confound the interpretation of immunoassays, we quantitate øLTs and LTs by LC-MS/MS. We will determine signaling path- ways, e.g., with RNA-Seq studies, activated by glutathionylated products of free radical-induced lipid oxidation addressing the questions: do they promote inflammation in ocular pathology or initiate autophagy in RPE cells and how do they do it? Molecular level insights into the role of oxidative stress in the pathogenesis of inflammatory eye diseases such as AMD can facilitate development of therapeutic interventions that ameliorate the progression of these common but disabling diseases.

NIH Research Projects · FY 2025 · 2023-09

PROJECT SUMMARY/ABSTRACT Protein post-translational modification (PTM) and alternative splicing enable the limited genome of a cell to dynamically respond to environmental changes by diversifying its protein repertoire. Previous studies have revealed that environmental cues impact alternative splicing through numerous PTMs of RNA-binding proteins associated with the spliceosome. S-nitrosylation, the reversible covalent PTM of a protein cysteine residue by the gaseous signaling molecule nitric oxide (NO) to form an S-nitrosothiol (SNO)-modified protein, has been shown to alter protein function to play profound roles on cellular physiology, including the regulation of gene expression. Specifically, S-nitrosylation is known to directly regulate key transcription factors as well as to modify enzymes that alter the epigenome. Intriguingly, RNA-binding proteins of the heterogenous nuclear ribonucleoprotein (hnRNP) family, including the polypyrimidine tract-binding protein (PTB), are common in proteomic identifications of SNO-modified proteins under multiple conditions and in many cell types. Because PTB is a master regulator of alternative splicing, we are exploring the central hypothesis that NO enables a cell to dynamically regulate RNA splicing through SNO modification of PTB. Our laboratory recently mapped S-nitrosylation to a single cysteine residue in PTB. Dramatic alternations in gene expression when cells are exposed to NO are absent when this cysteine is mutated, strongly supporting a role for SNO in regulating the activity of PTB with transcriptome-wide implications. This proposal will interrogate these findings through three independent aims: Aim 1 will develop a bioinformatic pipeline based on RNA sequencing analyses to identify specific alternative transcripts regulated by SNO-PTB, including those that may have an outsized effect on global gene expression; Aim 2 will elucidate how SNO affects PTB conformation and its association with protein and RNA components of the spliceosome, offering a molecular mechanism for the influence of NO on alternative splicing; and Aim 3 entails the use of a conditional knock-in mutant mouse in which PTB cannot undergo the SNO modification, allowing determination of the physiological role of SNO-PTB by observing the consequences of dysregulated NO signaling. This project will advance our understanding of the role of NO signaling as a crucial mechanism of the cellular response to environmental cues through SNO-modification of a central regulator of alternative splicing, PTB. Heretofore, global effects of NO on cellular function have been attributed to widespread modification of proteins. The role of NO in regulating alternative splicing is previously unappreciated, and provides new perspectives on dynamic regulation of cellular function in health and disease. This study will thus define the role of NO in alternative splicing for the first time, potentially opening new areas of research.

NIH Research Projects · FY 2025 · 2023-09

Clinical/Translational (C/T) scientists who conceptualize health problems in new ways and utilize skills that both enhance interdisciplinary work in clinical and community settings and catalyze translational processes are vital to address the nation’s unmet health needs and support fair access to quality care for all populations. Building upon the successes of our existing C/T training programs for predoctoral students, post-doctorates, and junior faculty, and in collaboration with the Workforce Development and Community & Stakeholder Engagement Modules of our Clinical & Translational Science Collaborative, our Clinical and Translational Scientist Training Program for Post-doctorates (CTSTP-Post) will enable a cadre of postdoctoral trainees with varied academic experiences to both deepen their scientific domain expertise and develop knowledge, perspective, and skills necessary to function efficiently and effectively as C/T scientists facing the opportunities and challenges of medicine and health in the 21st century. We will also broaden trainee recruitment by engaging candidates from a wide range of academic institutions and professional backgrounds and fostering a collaborative research environment that supports scientific exchange and mentorship. Strategic partnerships with a range of academic institutions, including those with limited prior involvement in clinical and translational research, will help expand participation in our training program and ultimately strengthen the pipeline of talent entering the C/T science workforce. Through this program, twelve trainees will each complete a two-year appointment. Using a strategic combination of training activities – e.g., mentoring by a successful, experienced C/T scientist, coursework, observerships and other field experiences, workshops, symposiums, seminars/discussions – trainees will acquire deeper understanding of and sharpen their skills in translational processes, interdisciplinary research, team science, communication, systems science and complexity, community and stakeholder engagement, innovation, entrepreneurship and commercialization, dissemination and implementation science, information science and artificial intelligence, differences in health outcomes, professional and leadership development, responsible conduct of research, and methods for enhancing reproducibility. Our program will interact with the proposed CTSTP for Predoctoral Students and K12 Program for Junior Faculty, creating a rich culture of learning and exchange, where perspectives of different disciplines are represented and where trainees share ideas, challenge each other, and grow together. Although each program will be distinct and will have its own Internal Advisory (Steering) Committee and unique components, oversight of all three programs by a single Research Education Advisory Board, a shared mentor pool, strategically selected joint activities, and standardized evaluation approaches and tools will create synergies in training, leadership, and management across programs and assure attainment of program objectives.

NIH Research Projects · FY 2024 · 2023-09

Abstract This R21 proposal, “Repurposing L-NAC to prevent fentanyl-induced respiratory depression” seeks to expand on our evidence that a bolus intravenous injection of the clinically-approved drug, N-acetyl-L-cysteine (L-NAC), reverses the profound respiratory depression elicited by infusion of fentanyl in rats. The clinical effectiveness of opioid analgesics such as fentanyl are compromised by their adverse actions on breathing and arterial blood- gas (ABG) chemistry. Opioid-induced respiratory depression (OIRD) can be reversed by opioid receptor (OR) antagonists but these antagonists also reverse opioid-induced analgesia We are reporting on the efficacies of L- and D-thiolesters such as D-cysteine ethyl ester (D-CYSee) to reverse OIRD while preserving analgesia and our current NIDA funding is allowing us to examine the efficacy of D-CYSee as a reversal agent against fentanyl and analogues in rats (PI: Stephen Lewis, NIH/NIDA U01DA051373: Optimization of Novel Thiolesters as a Therapeutic Strategy for Combating Opioid Overdoses and Abuse) and goats (PI: Matt Hodges, NIH/NIDA 1RF1DA050571: Reversing opioid-induced hypoxemia with thiol-based drugs without compromising analgesia in goats). N-acetyl-L-cysteine (L-NAC), which readily enters central peripheral and cells upon systemic/oral administration, has many beneficial effects in humans/experimental animals and is approved for human use for numerous conditions. There are no reports that L-NAC overcomes OIRD although it is evident that L-NAC (a) provides reducing equivalents to cells, (b) increases intracellular concentrations of L-cysteine/ L-glutathione, and (c) exerts numerous other intracellular actions via multiple enzymatic pathways. We have begun studying the ability of our thiol compounds to overcome the OIRD elicited by continuous intravenous infusion of fentanyl in rats. Such infusions are used widely in adult/pediatric patients but their ability to provide pain relief is greatly compromised by their ability to depress respiration. This project will expand upon our findings that intravenous injection of L-NAC elicits an immediate and sustained reversal of the deleterious adverse effects of continuous fentanyl infusion on breathing and ABG chemistry in anaesthetized rats whereas it did not affect the analgesic effects of the opioid. It appears that continuous infusion of fentanyl somehow sets up a scenario that allows for L-NAC to modulate intracellular signaling cascades that mediate fentanyl-induced OIRD but not analgesia. Our findings raise the possibility that L-NAC could be readily evaluated for potential reversal of OIRD elicited by the infusion of fentanyl in human subjects. The Specific AIMS of this project are: AIM 1 – determine the efficacy of bolus injections of L-NAC to countermand fentanyl-induced OIRD: This will establish how effectively L-NAC reverses the deleterious effects of fentanyl infusion on breathing and ABG (but not analgesia) at early (e.g., 5 min) and prolonged (e.g., 24h) infusion times. AIM 2 – determine the efficacy of co-infusions of L-NAC to countermand fentanyl-induced OIRD: These studies will establish the efficacy of co-infusion L-NAC to reverse the adverse effects of fentanyl on breathing and ABG (but not analgesia) from onset of fentanyl infusion.

NIH Research Projects · FY 2025 · 2023-09

Project Summary The hallmark of pluripotent stem cells (PSCs) is their capability to self-renew and differentiate, which is governed by the core pluripotency circuitry consisting of pluripotency factors OCT4, SOX2, and NANOG. Enhancers are fundamental in regulating the spatial and temporal expression of pluripotency genes and lineage specific genes during cellular differentiation and embryogenesis. Enhancer-regulating epigenetic modifiers play critical roles in normal physiological processes and human pathogenesis. Epigenetic marks such as H3K4me1 and H3K27ac are widely believed to regulate the activity and higher-order chromatin structure of enhancers by directly remodeling local chromatin and/or recruiting reader proteins. However, recent discoveries of catalytic- independent functions of multiple histone modifiers suggest that these enzymes govern enhancers and stem cell differentiation via a non-catalytic manner. Despite the importance of epigenetic modifiers in mammalian development and human diseases, how they sustain stem cell identity and impact human health is poorly understood. I previously demonstrated that while enhancer activation does not require the catalytic activity of histone methyltransferase MLL4 in PSCs, it is regulated by the functional antagonism between MLL4 and histone demethylase LSD1. Using state-of-the-art unbiased approaches, my lab recently unveiled novel mechanisms underlying the role of enhancer-regulating epigenetic modifiers in PSCs, providing insight into elucidating gene regulation and cell fate transition. Here, I propose to build two research areas in my laboratory focused on enhancer-regulating epigenetic modifiers. The first research area will focus on identifying catalytic-independent functions of LSD1 in governing gene expression and cellular differentiation. The second research area will focus on determining how MLL4 and its interactors modulate enhancer activity and cell fate transition. I anticipate that accomplishing the proposed studies will reveal novel mechanisms underlying enhancer regulation, decipher how stem cell self-renewal and differentiation are governed, and pave the way for understanding the pathogenesis of diseases driven by enhancer malfunction.

NIH Research Projects · FY 2026 · 2023-09

PROJECT SUMMARY/ABSTRACT Preterm (<37 completed weeks of gestation) birth (PTB) causes the majority of neonatal mortality and morbidity. Around 50% of PTBs are associated with preterm premature (i.e., pre-labor) weakening and rupture of the fetal membranes (FM: amnion-chorion-decidua parietalis) (pPROM). Infection/inflammation and decidual bleeding are major drivers of pPROM. Development of therapies to prevent pPROM and PTB are confounded by gaps in understanding how pregnancy is maintained in the face of inflammatory and bleeding challenges at the FM. The proposed research builds on our previous work examining the mechanisms by which inflammatory stimuli increase risk for pPROM by weakening the FM, and our demonstration that progesterone (P4) prevents inflammation- induced FM weakening. Using our ex-vivo FM explant model, we found that inflammatory stimuli weaken FM by inducing granulocyte-macrophage colony-stimulating factor (GM-CSF) production by decidual stromal (DS) cells. Our studies suggest GM-CSF is a critical intermediate in both inflammation- and bleeding-induced FM weakening. Importantly, we found that P4 prevents GM-CSF production by DS cells. Recently we found that GM-CSF, in addition initiating a cascade of events which cause FM weakening, induces P4 production within the FM. Based on those data, we hypothesize that a locally-acting, paracrine, negative-feedback system exists within the FM, whereby GM-CSF produced in response to localized inflammatory and bleeding stimuli induces P4 production by adjacent chorion cytotrophoblast (CTB) cells. The P4, in turn, inhibits GM-CSF production by the DS cells and GM-CSF- induced FM weakening. This hypothesis is supported by our recent finding that blocking P4 production or action each independently weakens the FM. Thus, locally produced and locally acting P4 is essential for maintaining the structural integrity of the FM. This novel and groundbreaking hypothesis will be tested by achieving two Specific Aims: 1) identify the signaling pathway by which GM-CSF increases P4 production by CTB cells and whether this P4 production declines in association with FM weakening at term and pPROM, and 2) determine the mechanism by which P4 exerts anti-inflammatory activity in DS cells. Achieving the Specific Aims will provide more substantive understanding of P4 function at the human maternal-fetal interface to maintain human pregnancy and prevent pPROM. Understanding this process is important for development of effective therapies to prevent, or at least decrease, the risk for pPROM-induced preterm birth.

NIH Research Projects · FY 2024 · 2023-08

Penicillin-binding proteins (PBPs) are a proven β-lactam drug target, yet resistance to β-lactam antibiotics, such as carbapenems and cephalosporins, has resulted in a global health problem. There are a number of resistance mechanisms of which β-lactam degrading β-lactamases is one of the main culprits. Our goal is to overcome the resistance mechanisms often associated with β-lactams by studying and developing a different type of PBP inhibitor, the pyrazolidinone. The pyrazolidinones YU253434 and YU253911 contain a siderophore moiety to facilitate iron-mediated uptake. We have found that these two pyrazolidinones cannot be hydrolyzed by Classes A, C, and D β-lactamases and are only slowly hydrolyzed by (Class B) metallo β-lactamases. YU253434 and YU253911 also compared favorably to aztreonam, ceftazidime, meropenem, ceftolozane/tazobactam, and ceftazidime/avibactam when microbiologically tested against panels of Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii (all four are ESKAPE pathogens). YU253911 also significantly lowered colony- forming units in a mouse thigh-infection model with an MDR P. aeruginosa strain. The pyrazolidinones target and inhibit PBP3, and we have delineated their binding modes to P. aeruginosa PBP3 crystallographically. Overall, we found that these pyrazolidinones have several favorable attributes, yet further improvements are needed in terms of PBP3 IC50, uptake, and ability to overcome the known PBP3 F533L resistance mutation. We propose these improvements in the following two specific Aims. Specific Aim 1. To improve the pyrazolidinone affinity via structure-based modifications targeting the R2 group. Based on the crystal structure, we hypothesize that hydrophobic substituents added to pyrazolidinones in the siderophore-linker will interact with the hydrophobic bridge residues F533 and V333 in PBP3 and thereby improve affinity. Additionally, these hydrophobic interactions are designed to counteract the F533L resistance mutation. We will test the compounds microbiologically against panels of well-studied K. pneumoniae, A. baumannii, Escherichia coli, and P. aeruginosa, measure PBP inhibition both wt and F533L P. aeruginosa PBP3, and probe their binding mode crystallographically, biophysically, and using molecular dynamics simulations. Specific Aim 2. To improve the iron–independent and iron-mediated uptake of pyrazolidinones via adding an amine-containing moiety to the siderophore-linker (Aim 2a) and by incorporating a siderophore with an electron-withdrawing –Cl group adjacent to the hydroxyl groups to improve iron affinity (Aim 2b). This high-risk, high-reward proposal aims to develop a more potent non-β-lactam PBP-targeting pyrazolidinone that could lead to a novel therapeutic strategy to combat antibiotic resistance.

NIH Research Projects · FY 2025 · 2023-08

Abstract: The proposed research investigates how genomic polymorphism of the TNFRSF13B locus predicts and potentially governs the immune response to and outcomes of transplantation. The investigators (de Mattos Barbosa et al., 2021) recently found that non-synonymous mutations of TNFRSF13B occur 5-fold more frequently in kidney transplant recipients that develop antibody-mediated rejection than in recipients with persistently healthy grafts. The working hypothesis of this application is that TNFRSF13B polymorphism shapes B cell responses to allotransplantation in ways that determine pathogenicity of the responses. Since affinity-maturation, kinetics, self-non-self discrimination and persistence of elicited antibody production have been connected with TNFRSF13B function, these characteristics will be evaluated in allo-specific B cells isolated from kidney transplant recipients. Because TNFRSF13B is among the most polymorphic genes in humans and other mammals, the proposed research will draw on diverse pools of kidney transplants already enrolled in the Michigan Genomics Initiative, and in the NIH APOLLO study to connect genotypes with transplantation outcomes. The results obtained with these cohorts will be verified by analysis of genotypes and outcomes of subjects in two major NIH studies, DeKAF and GEN03. The large number of kidney transplant recipients enrolled in the aforementioned studies will provide a vast pool from which the phenotype and implications for transplantation of the most common allelic TNFRSF13B variants can be identified. The functional properties of the most important TNFRSF13B alleles in turn will be confirmed and the mechanism ascertained by engineering tnfrsf13B mutations in mice and testing B cell functions and outcomes of heterotopic cardiac allotransplants.