Ut Southwestern Medical Center

NIH Research Projects · FY 2026 · 2022-03

Project Summary The programmable CRISPR/Cas gene editing system has great potential for cancer treatment due to the ability to precisely inactivate or repair cancer-related genes. However, delivery of CRISPR to solid tumors for efficient cancer therapy remains limited by the uniquely stiff and fibrotic tumor microenvironment that acts as a barrier to nanoparticle uptake. Here, we propose to directly target tumor tissue mechanics via a multiplexed lipid nanoparticle (LNP) approach involving co-delivery of focal adhesion kinase (FAK) siRNA, Cas9 mRNA, and sgRNA (siFAK + CRISPR LNPs) to enable tumor delivery and enhance gene editing efficacy. We will leverage our recently developed non-viral Selective ORgan Targeting (SORT) LNP platform that enables tissue-specific nucleic acid delivery, protein delivery, and genome editing following intravenous (IV) administration. The proposed approach involves a unique combination of siRNA-mediated gene silencing of FAK, a key modulator of tumor ECM, and CRISPR-mediated gene editing (deletion) of tumor-related genes via a single all-in-one nanoparticle approach. We will further leverage Liver and Lung SORT LNPs for to evaluate gene editing as a strategy for permanent inactivation of programmed death-ligand 1 (PD-L1), supported by the clinical limitations of anti-PD-L1 antibody atezolizumab therapy in hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC). In this grant proposal, we Aim to (1) establish how FAK knockdown enhances SORT LNP-mediated mRNA delivery and CRISPR gene editing, (2) optimize Liver and Lung SORT LNP formulations for multiplexed siRNA + Cas9 mRNA + sgRNA delivery, and (3) evaluate the therapeutic efficacy of Liver and Lung SORT siFAK + Cas9 mRNA + sgPD-L1 delivery in orthotopic and genetically engineered mouse models of cancer. Regulating the mechanical properties of tumor cells/ECM for enhancing the genetic suppression in tumor tissues provides an innovative strategy for treating cancer using CRISPR. We anticipate that this general approach could further synergize with additional types of therapeutics in the future.

NIH Research Projects · FY 2026 · 2022-03

PROJECT SUMMARY Familial Hypercholesterolemia (FH) remains woefully underdiagnosed and undertreated in the US. Recent work from the PI demonstrated the ability to identify blood donors with FH at a similar prevalence as would be expected in the general population. The overall objective in this mixed methods study is to apply implementation strategies to improve treatment of blood donors with FH. The central hypothesis is that the blood donation system can be an effective portal to increase receipt of treatment for individuals with FH. The central hypothesis will be tested by pursuing four specific aims: 1) identify barriers and facilitators to receipt of (low-density lipoprotein cholesterol) LDL-C treatment in blood donors with FH to inform implementation strategy development, 2) develop and produce an implementation strategy bundle to increase LDL-C treatment in blood donors with FH, 3) conduct a Type 2 implementation-effectiveness trial for LDL-C lowering in a community-based blood donation center, and 4) evaluate the implementation bundle using mixed methods. This research will be guided by the Consolidated Framework for Implementation Science Research (CFIR), and will use Implementation Mapping, a theory- and evidence-based process, to develop, implement, and evaluate these treatment enhancing strategies. In the first aim, semi-structured interviews will be used to elicit barriers and facilitators of statin treatment from key stakeholders including: a) blood donors with FH b) family members of donors c) blood donation personnel d) community physicians. Stakeholder feedback will be iteratively integrated to develop, produce, and refine an implementation strategies bundle in the second aim, that will be tested in Aim 3. The third aim consists of a two-arm randomized trial among blood donors with FH comparing a) usual notification of high cholesterol or b) the implementation strategies bundle from Aim 2 with the primary outcome of difference in change in LDL-C levels from baseline to 6 months. The fourth aim will assess the acceptability, appropriateness, and feasibility of the implementation strategy bundle and will inform development of a dissemination package to implement an FH program in other blood donor centers. The research proposed in this application is innovative, in our opinion, because it incorporates the blood donor program, an entirely new approach to identifying and treating FH. This novel portal of enhancing FH treatment involves younger individuals who are less likely to seek regular medical care and complements traditional strategies focusing on patients with medical homes. Ultimately, if successful, this program, informed by implementation science methods, can be rapidly scaled to blood donation centers nationally involving 6.4 million donors annually, having an immediate impact on gaps in FH care.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY/ABSTRACT Shortage of organs for transplantation is one of the largest unmet medical needs. Researchers are currently working on a variety of ways to increase the number of organs available. Recently, the prospect of producing human organs in animals via interspecies blastocyst complementation has raised important attention and may constitute an innovative approach to overcome the worldwide shortage of donor organs. Interspecies blastocyst complementation works by injecting donor pluripotent stem cells (PSCs) from one species into the organogenesis-disabled blastocyst of a different species. The growing host embryo can provide an emptied developmental organ niche for donor cells to occupy. Despite the potential, however, recent work revealed that although donor PSCs can initially engraft to inner cell mass (ICM) of blastocysts from distantly related host species, their chimeric contribution to early post-implantation stages is low. These results suggest the existence of embryonic xenogeneic barriers between evolutionarily distant species. Unlike chimera formation within the same species, a multitude of factors can differ significantly between species, which preclude robust chimerism. Cell competition, a conserved fitness-sensing process during which fitter cells eliminate neighboring less-fit but viable cells, has been proposed as a surveillance mechanism to ensure normal development and tissue homeostasis, and has also been suggested to act as a barrier to interspecies chimerism. During chimera formation, xenogeneic donor PSCs may be treated as unfit or aberrant cells and targeted for elimination. Most recently, we developed an interspecies PSC co-culture strategy and discovered a previously unknown mode of cell competition between species. Interspecies competition between PSCs occurred in primed but not naive pluripotent cells, and between evolutionarily distant species. By comparative transcriptome analysis, we found that genes related to the NF-κB and p53 signaling pathways, among others, were upregulated in less-fit ‘loser’ human cells. Genetic inactivation of TP53, a core component (P65, also known as RELA) and an upstream regulator (MYD88) of the NF-κB complex in human PSCs could overcome their competitive elimination by co- cultured mouse PSCs, thereby improving the survival and chimerism of human cells in early mouse embryos. Based on extensive preliminary results, in this proposal, we will further dissect the mechanisms underlying interspecies primed PSC competition in both loser and winner cells. And, by suppressing interspecies PSC competition, we aim to improve chimerism and provide the proof-of-concept of interspecies blastocyst complementation from donor PSCs of rhesus monkey, our close kin, in distantly related mouse host.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY Naïve lymphocytes exist in a quiescent state until becoming activated by antigen. Their continued survival depends on signals they receive through their antigen receptors and from homeostatic cytokines. How naïve lymphocytes respond to pro-survival signals while continually maintaining quiescence is unclear. This is an important issue: enhanced responses to survival signals can fuel malignant transformation while impaired quiescence can trigger spontaneous immune activation and immune failure. We have recently discovered a new protein complex containing phosphofurin acidic cluster sorting protein-1 (Pacs1) and WD repeat protein 37 (Wdr37) that is required for normal lymphocyte survival and quiescence. Mice lacking Pacs1 or Wdr37 were deficient in circulating B and T cells. Pacs1-Wdr37-deficient B cells exhibited spontaneous proliferation in vivo coupled with increased apoptosis which indicated loss of cellular quiescence. These cells demonstrated increased levels of endoplasmic reticulum stress in vitro and were hypersensitive to oxidative stress. Importantly, Pacs1-Wdr37 deficiency did not impair humoral immune responses. However, it potently suppressed lymphoproliferative diseases resulting from blocked apoptotic pathways. Mechanistically, deletion of Pacs1 or Wdr37 impaired antigen receptor-dependent calcium (Ca2+) release from the endoplasmic reticulum (ER) due to transcriptional downregulation of ER Ca2+ release channels (inositol triphosphate receptors, IP3R). These results lead us to hypothesize that Pacs1-Wdr37 integrates antigen receptor-dependent Ca2+ signaling and cellular stress responses to promote lymphocyte survival and quiescence. We will test this hypothesis by (i) elucidating how disruption of Pacs1-Wdr37 diminishes B cell survival and quiescence; (ii) defining how Pacs1-Wdr37 prevents ER stress and promotes IP3R expression; and (iii) validating Pacs1-Wdr37 disruption as a therapeutic approach to B cell malignancies. Relevance to public health: Signaling networks that permit lymphocyte survival are often co-opted during malignant transformation. There is a need for therapies that subvert pro-survival signaling in diseased lymphocytes while preserving most beneficial immune functions. This proposal will investigate a novel protein complex involved in promoting lymphocyte survival and quiescence while preventing cell stress that is a promising therapeutic target for lymphoid malignancies.

NIH Research Projects · FY 2025 · 2022-02

PROJECT SUMMARY: The metabolism of nutrients has been studied using unfractionated tissues, or in vitro. An unresolved question is how nutrients are metabolized by stem cells in vivo. Our understanding of stem cell metabolism has been limited by the fact that metabolomics typically requires millions of cells, while stem cells are rare. We developed methods to profile the metabolome and to trace stable isotope labeled nutrients in hematopoietic stem cells (HSCs) and other rare cell types purified from tissues. We found that T cell progenitors in the thymus are glucose avid as compared to HSCs, myeloid and B cell restricted progenitors, in contrast to the prevailing view that HSCs are more glycolytic than hematopoietic progenitors. Stable isotope tracing experiments showed that in the bone marrow but not the thymus, glycolysis and the TCA cycle are disconnected. Hematopoietic loss of pyruvate dehydrogenase (PDH), the gatekeeper enzyme that connects glycolysis to the TCA cycle, reduced the number of double positive (DP) T cell progenitors but did not affect HSCs or other hematopoietic cell types. Loss of PDH paradoxically did not impair the TCA cycle in the thymus, but caused accumulation of pyruvate and aberrant redox balance. Cells which do not oxidize glucose in the TCA cycle are classically thought to ferment glucose through glycolysis to lactate via lactate dehydrogenase (LDH). Hematopoietic loss of LDHA, one of the two LDH isoforms, impaired development of erythroid progenitors but not HSCs, T cell progenitors or other restricted hematopoietic progenitors. The cell type specificity in the requirement of LDH and PDH in the hematopoietic system raises the question of why different stem or progenitor cell types choose to use LDH-mediated fermentation or PDH-mediated oxidation in vivo. This application’s objective is to systematically dissect the role of glycolytic as compared to oxidative metabolism in HSCs and restricted progenitors. Our hypothesis is that T cell progenitors require oxidation of glucose via PDH to regulate pyruvate levels and redox homeostasis, in contrast to HSCs, myeloid and B cell progenitors which are metabolically flexible. In Aim 1 we will test the metabolic mechanisms which mediate the effects of PDH on DP cells. In Aim 2 we will determine the cellular and metabolic effects of blocking LDHA/B or PDH alone or in combination in HSCs and restricted progenitors. In Aim 3 we will investigate the role of LDHA/B and PDH in hematopoietic and thymopoietic regeneration. These experiments will identify the contribution of glucose to metabolite pools in HSCs and progenitors in vivo, systematically test the idea that HSCs are glycolytic, and identify mechanisms by which central carbon metabolism regulates hematopoietic differentiation and regeneration. More generally our experiments will address a fundamental metabolic question by testing if stem or progenitor cells in vivo switch between glucose fermentation or oxidation, as is the textbook view, or if some cell types in vivo tolerate the loss of both major glucose catabolic pathways.

NIH Research Projects · FY 2026 · 2022-02

Project Summary Heart failure with preserved ejection fraction (HFpEF) accounts for ≈50% of all patients with heart failure, and is associated with significant morbidity, mortality, and health care expenditures. Yet, no effective treatment for HFpEF has been identified. Commonly coexisting with other metabolic diseases, HFpEF is considered the cardiovascular manifestation of a systemic metabolic disturbance. However, little is known about its underlying mechanisms. Our laboratory recently developed and validated a novel mouse model that faithfully recapitulates most clinical features of human HFpEF. Using this novel mouse model, I discovered significant mitochondrial dysfunction in HFpEF myocardium, which is associated with mitochondrial protein hyperacetylation, a key post- translational modification known to regulate enzymatic activities. This led to our central hypothesis that protein hyperacetylation is a reversible driver of mitochondrial dysfunction and metabolic remodeling in HFpEF and could serve as a meaningful therapeutic target. In this proposal, we aim to (1) Determine the role of Sirtuin 3, the major mitochondrial deacetylase, in regulating mitochondrial function and HFpEF pathogenesis; (2) Identify specific targets through which hyperacetylation impacts mitochondrial function in HFpEF; (3) Determine the therapeutic effect of modulating protein acetylation on HFpEF mitochondrial function and cardiac phenotype. Collectively, these studies will have a meaningful impact on our understanding of HFpEF pathophysiology and potentially unveil novel therapy to effectively treat HFpEF. Furthermore, work proposed here, coupled with a comprehensive training plan, will provide me with the additional knowledge and skills required to launch my career as a successful and fully independent physician-scientist.

NIH Research Projects · FY 2026 · 2022-02

Summary The study of germ cells has shaped our understanding of many basic fundamental processes across different species. Germ cells share a number of features that have long fascinated biologists. These cells undergo meiosis to form haploid gametes, they are exceptionally good at repairing DNA damage, they utilize a number of small RNA pathways to silence transposable elements, and they reprogram their epigenome back to a state that supports totipotency. Here, we proposed to use the Drosophila ovary as a model to continue to gain insights into genome stability, germ cell differentiation, and the cell-specific regulation of mRNA translation and ribosome biogenesis/turnover. Over the last five years, we have adopted and optimized a number of innovative CRISPR-Cas9- and recombineering-based methods for manipulating the Drosophila genome. Using these approaches, we have mutated and/or tagged over 100 genes that exhibit enriched expression in Drosophila gonads. This work provides a solid foundation for our planned efforts over the next five years. We will focus on a number of different but related areas. We will continue to characterize the highly conserved Germ Cell Nuclear Acidic Peptidase (GCNA) gene and its function in protecting the integrity of germ cells across species. We will also continue to characterize how cytoplasmic Rbfox1 controls early germ cell development. Our previous screening efforts have identified a small number of mutations that exhibit germ cell tumor formation or germ cell loss phenotypes. The molecule function of the disrupted genes will be characterized using the tools and methods we have in hand. Lastly, we are in the process of generating a number of innovative tools that will allow us to better assess ribosome biogenesis and turnover during germ cell development and early embryogenesis. We are very excited by this proposed work and believe the successful completion of these projects will have a positive impact on our understanding of germ cell biology and other molecular processes that impact human health.

NIH Research Projects · FY 2026 · 2022-02

Alzheimer’s and related neurodegenerative diseases are a major health problem. The tau protein is known to play a critical role in these disorders. In the disease state tau transitions from a normal, “healthy” three- dimensional shape to one that is capable of self-assembling into pathological aggregates. Remarkably, these aggregates, once formed in a brain cell, appear to exit that cell and gain entry into neighboring or connected cells, where they can serve as disease-causing “templates” to corrupt normal tau protein to an abnormal conformation. Free tau aggregates can bind the cell surface by interacting with specific proteins called heparan sulfate proteoglycans (HSPGs), which are modified in the cell through the attachment of sugar molecules, which themselves are modified by the addition of sulfate groups. These modifications occur during the synthesis of HSPGs, and depend on specific cellular enzymes. One enzyme, NDST1, was previously identified as being very important for enabling HSPGs to be properly modified so as to bind tau protein. Once bound to HSPGs, tau assemblies get into the cell, where they create more aggregates. The mechanisms by which tau can cross the cell membrane are unknown. It is also unknown whether the mechanisms that apply to tau are similar to those that function for other disease-causing proteins. This grant will test the role of NDST isoforms in a mouse model of tau-induced neurodegeneration to see if the pathway implicated by prior studies might be targeted to create new drugs for Alzheimer’s. Additionally, the grant will determine how tau assemblies can cross the cell membrane, and whether mechanisms that apply to tau also apply to other disease-causing proteins.

NIH Research Projects · FY 2025 · 2022-01

Abstract Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of fluid-filled sacs called cysts in both kidneys, but key signals that cause cyst formation are unknown. Multiple downstream cellular pathways are dysregulated during cystogenesis. However, targeting these pathways has limited effects on ADPKD treatment. ADPKD is caused by mutations in genes encoding for polycystin-1 (PC1) and polycystin-2 (PC2). Both polycystins localize to primary cilia. The primary cilium instructs cellular decisions in response to extracellular inputs by compartmentalizing subcellular signaling. However, the role of primary cilium in kidney cystogenesis is inherently complex. Loss of polycystins causes severe cystogenesis, which is mostly cilia- dependent, while loss of cilia by itself causes smaller cysts. These results suggest that a complex interplay between counter-regulatory positive and negative signals in cilia inhibit cyst formation in normal renal tubules and promote cyst growth in ADPKD, respectively. Identifying these signals could have profound impacts on novel therapeutic targets and strategies for ADPKD. However, uncoupling ciliary signals causing cystogenesis from downstream signaling pathways affected during cystogenesis and the difficulty in identification of ciliary signals in absence of cilia have prevented their identification. Here, by studying the ciliary trafficking adapter, tubby family protein Tulp3, we aim to identify and target the key upstream ciliary signals that regulate cystogenesis. We previously showed that Tulp3 functions in ciliary trafficking of membrane proteins without affecting respective protein levels or disrupting cilia by coupling to the intraflagellar transport complex A (IFT-A). We recently showed that embryonic kidney-specific conditional knockouts of Tulp3 developed renal cystogenesis that was less severe than from polycystin loss. Concomitant Tulp3 loss did not inhibit cystogenesis upon PC1 loss, unlike ciliary disruption, but caused early lethality, suggesting accelerated loss of renal function. These results further reinforce the polycystin independent inhibitory role of ciliary proteins in cystogenesis. Other groups have reported suppression of cystogenesis in adult mouse models of PKD from Tulp3 or IFT-A loss. Thus, we hypothesize that Tulp3-regulated ciliary cargoes determine cilia-dependent cyst inhibition during development and PC1/2- inhibited cilia-dependent cyst activation in adults. Here by leveraging our expertise in ciliary trafficking and signaling and using novel mouse models to block trafficking of potential cargo subtypes, we propose to identify ciliary regulators of cystogenesis. In Aim 1, we will determine novel Tulp3 trafficked ciliary cargoes and signaling outputs in cilia relevant to cystogenesis. In Aim 2, we will determine Tulp3 regulated cyst inhibitory ciliary cargo subtypes that genetically synergize with PC1 during murine kidney development. In Aim 3, we will test Tulp3 cargo subtypes as promoters of cystogenesis in adult mouse models of PKD in vivo using genetic epistasis. Our approach will determine dual ciliary drivers impacting cystogenesis in vivo during adult-onset ADPKD and embryonic-onset cystic disease unraveling the molecular nature of cilium-generated signaling in either setting.

NIH Research Projects · FY 2026 · 2022-01

Abstract Glioblastoma (GBM) is the most common primary brain cancer in adults. EGFR is expressed in the majority of GBMs and aberrant EGFR signaling is a major driver of the malignant phenotype. Although the EGFR is considered a prime oncogene in GBM, TCGA analysis indicates that in EGFR amplified GBMs, EGFR ligands are tumor suppressive. Our preliminary data also suggest that ligand-activated EGFR is tumor suppressive. The tumor suppressive effects of ligand-activated EGFR result form an unexpected suppression of invasion. We propose that constitutive and ligand-dependent EGFR wild type signaling triggers distinct signaling pathways. Thus, constitutive EGFR signaling promotes invasion while ligand-activated EGFR signaling turns on proliferation and turns off invasion. We elucidate mechanisms underlying EGFR regulation of invasion and identify BIN3, a protein known to influence the cytoskeleton, as a key suppressor of GBM invasion. We also identify the mechanisms and biological significance of ligand-activated EGFR mediated glioma cell proliferation. We examine the relative contribution of proliferation and invasion to tumor size and prognosis in GBM. An improved understanding of mechanisms that drive GBM invasion is critical to improved treatment. Furthermore, we identify tofacitinib as a drug that can activate the tumor suppressor function of EGFR by increasing EGFR ligand, upregulating BIN3 and suppressing GBM invasion. Tofacitinib is a clinically available and FDA approved drug. Our model holds true for GBMs that express EGFR wild type or the mutant EGFRvIII. In Specific Aim 1: We elucidate the role of RTK transactivation in driving invasion or proliferation. We test the hypothesis that constitutive EGFR signaling promotes EGFR invasiveness whereas ligand-induced EGFR signaling blocks it. Constitutive EGFR signaling leads to activation of Met leading to increased invasiveness. We also identify a TAB1-TAK1-NF- B pathway that drives GBM invasion. Ligand-activated EGFR signaling leads to Axl activation and proliferation and decreased GBM invasiveness. In Specific Aim 2: we uncover mechanisms used by EGFR to suppress invasiveness of GBM cells. We test the hypothesis that BIN3 is a major negative regulator of invasion. Ligand-induced EGFR activity upregulates BIN3 and suppresses invasion. We examine the expression patterns of BIN3, BIN3 partners, EGFR and other RTKs networks in GBM. In Specific Aim 3 we examine the biological effects of constitutive vs. ligand induced EGFR–RTK-BIN3 signaling on GBM invasion in an orthotopic mouse model and examine tofacitinib as a treatment that specifically inhibits GBM invasion in ligand-poor GBMs. .

NIH Research Projects · FY 2025 · 2022-01

PROJECT SUMMARY A heterozygous missense mutation OtpQ153R/+ has recently been discovered in a cohort of individuals with severe, early-onset obesity. Like many other obesity-associated variants, despite a strong association, a causal relationship has yet been established. Otp encodes a transcription factor that is highly conserved across multiple species. Importantly, mice and humans share the identical amino acid sequence of Otp. To study the functional impact of OtpQ153R/+, we have generated new knock-in mice that carry the same human mutation. Similar to the human subjects, we found that mice heterozygous for OtpQ153R (OtpQ153R/+) survive through adulthood but develop obesity and glucose intolerance. These findings, therefore, strongly support a causal role for OtpQ153R/+ in human obesity. We propose to investigate the mechanisms behind OtpQ153R-induced obesity and glucose deficits. Otp is broadly distributed in the central nervous system. To determine the brain site where Otp deficiency impairs energy and glucose balance, we generated and characterized a floxed Otp allele (Otpflox). Our new preliminary studies show that selective loss of Otp in forebrain Sim1-Cre-expressing neurons reproduces lethality seen in Otp null mice, whereas its haploinsufficiency in these neurons results in obesity. Furthermore, we find that Otp is transiently expressed in a subset of immature POMC neurons in the arcuate nucleus of the hypothalamus (ARH) and is required for the POMC→NPY/AgRP fate switch during development. Selective deletion of Otp in these neurons leads to a significant loss of POMC-derived NPY/AgRP neuron identity. Collectively, our new findings suggest that Otp plays critical roles in two distinct populations of hypothalamic neurons to regulate energy and glucose metabolism. In summary, the overarching goals of these studies are to better understand OtpQ153R-induced pathophysiology and develop mechanism-based therapeutics to mitigate metabolic syndrome in human OtpQ153R/+ patients.

NIH Research Projects · FY 2026 · 2022-01

PPG Title: New Approaches to Reduce Residual Cardiovascular Risk SUMMARY/ABSTRACT In the last 40 years, significant progress has been made in reducing cardiovascular events by lowering plasma LDL-cholesterol (LDL-C). While statins and PCSK9 inhibitors effectively decrease LDL-C levels, significant residual risk of CHD remains even in maximally treated individuals with low plasma levels of LDL-C. Epidemiological and genetic studies suggest that a significant proportion of the residual risk is due to elevated plasma levels of triglyceride-rich ApoB-Containing Lipoproteins (ApoBCLs). The three projects that comprise this Program Project Grant (PPG) will elucidate new molecular mechanisms that regulate the synthesis, secretion, and metabolism of ApoBCLs. Our PPG is comprised of distinguished investigators with a longstanding history of collaboration, five of whom (Goldstein, Brown, Hobbs, Horton, and Cohen) have worked together for 25 years. In Project 1 of this new PPG, Radhakrishnan, Brown, and Goldstein have used an original and innovative screening protocol to identify a cholesterol-mimetic small molecule that binds to Scap with high specificity and blocks activation of SREBPs, the transcription factors that activate genes required for the synthesis of cholesterol, fatty acids (FAs), and triglycerides (TGs). This compound will be used to elucidate the molecular mechanism by which Scap senses sterols, enabling the first description of Scap’s cholesterol binding site at atomic resolution. The current cholesterol mimetic compound and more potent derivatives in development will be used to assess the clinical implications of a Scap inhibitor for reduction of plasma ApoBCLs. In Project 2, Horton, Kim, and Liang have identified a new lipogenic enzyme complex in liver. They will characterize components of the FA synthesis complex and determine how this complex interacts with additional FA modifying proteins and acyl-transferases required to synthesize TGs and ApoBCLs. Completion of the proposed studies will identify new opportunities for therapeutic interventions to reduce the synthesis of FAs, TGs, and VLDL. In Project 3, Hobbs and Cohen used population-based resequencing to identify loss-of-function mutations in angiopoietin-like (ANGPTL) 3 and 8. They showed that mutations in either protein result in lower plasma LDL- cholesterol and TG levels. Their studies will elucidate the mechanisms underlying the ApoBCL lowering effects of ANGPTL3 and ANGPTL8. In the process, they will define a new pathway that promotes clearance of ApoBCLs independently of the LDL receptor. The Research Projects will be supported by three Cores: Administrative, Tissue Culture & Antibody Production, and Mass Spectrometry. Members of this PPG have a long record of collaborative interactions and exceptional productivity. We will continue to focus on bold hypotheses designed to answer critical questions. The investigators take special pride in publishing papers that are characterized by originality and scientific rigor. The successful completion of our projects holds great promise for exposing new therapeutic opportunities for the reduction of plasma ApoBCLs and residual cardiovascular risk.

NIH Research Projects · FY 2026 · 2022-01

ABSTRACT Although adipose inflammation is associated with obesity, its role in reprogramming adipocytes and other cells in adipose towards the development of obesity’s metabolic comorbidities including steatosis remain unclear. The complex intracellular (stromal, vascular, immune, and adipocyte) interactions within adipose tissue ultimately regulate its size by balancing adipocyte triacylglyceride (TAG) lipolysis and synthesis. The inflammatory signaling of and between these cell types may also influence adipocyte responses to cAMP modulators. To understand the convergence of these interactions, we previously identified cytokine leukemia inhibitory factor (LIF) as a secretory molecule that increased adipose inflammation and lipolysis. Wild type mice on a high fat diet (HFD) demonstrated 7-fold higher LIF and IL-6 adipose mRNA than matched animals on normal diets. When recombinant LIF was administered to wild-type mice, it caused >50% loss of fat mass through JAK/STAT3- dependent reprogramming of adipose tissue, increasing lipolysis and amplifying inflammation by altering the expression of other cyto/adipokines. JAK inhibitor treatment of rLIF-administered mice suppressed adipose loss through 1) inhibition of adipose inflammation as determined by decreased STAT3 phosphorylation, 2) decreased adipocyte lipolysis, and 3) inhibition of cyto/adipokine changes. To establish the importance of this signaling pathway to adipose inflammation, we selectively silenced LIF receptor (LIFR-α, gene LIFR) or STAT3 in adipocytes and assessed murine development in diet-induced obesity. Both models had decreased adipose inflammation resulting in a 50% increase in adipose mass and a ~75% reduction in total hepatic TAG levels compared to controls, limiting non-alcoholic fatty liver disease (NAFLD) and steatohepatitis in these mice. Conversely, with adipocyte silencing of the JAK/STAT counter-regulator SOCS3 in mice on HFDs, we observed the opposite phenotype with a ~30% reduction in adipose mass compared to controls. We hypothesize that a LIFR-α/JAK/STAT3-dependent Cytokine-Adipose-Hepatic Axis facilitates adipose inflammation, leading to increased lipolysis and altered expression of other cyto/adipokines. The activation of this axis limits adipose expansion, resulting in TAG mobilization from adipose to the liver and ultimately contributing to NAFLD/steatohepatitis. This inflammatory-driven axis also affects adipose responses to systemic metabolic change, sensitizing adipocytes to lipolytic regulation by other cAMP modulators. Finally, we present preliminary data that the IL-6 family of cytokines signal through JAK/STAT3 inducing the expression of adenylyl cyclase 5 (ADCY5) to reprogram adipocytes in regulating lipid mobilization. SA1 will evaluate the contribution of the LIFR-α/JAK/STAT3 signaling cascade in adipocytes to the Cytokine-Adipose-Hepatic Axis. SA2 will use a genetics-based approach to verify that cytokine-mediated reprogramming of adipocytes in promoting lipid mobilization requires ADCY5 function. SA3 will use single cell RNA-Seq techniques in our multiple mouse models to identify non-adipocyte contributors to inflammation-regulated obesity and NAFLD.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY Nonalcoholic fatty liver disease (NAFLD) is becoming a global human health problem. Our long-term goal is to understand molecular mechanisms of NAFLD, and to translate this knowledge into novel therapeutic strategies. Due to the physiologic similarities between humans and mice, and the propensity of mice to develop a disease closely mimicking NAFLD when fed a high fat diet (HFD), mice have provided us with fundamental insights into NAFLD pathogenesis. In humans and mice, genetic variation influences the rate and severity of hepatosteatosis under a given set of environmental conditions. To identify genes that influence the process, we utilized unbiased forward genetic screening and highly automated meiotic mapping to identify mutations that cause NAFLD in mice sensitized by a HFD. Two semi-dominant missense alleles of predicted gene 4951 (Gm4951), named Oily and Carboniferous, were detected in this screen. As distinct from most NAFLD mutants that are associated with obesity, our preliminary data showed that Gm4951 deficient mice had dramatically increased hepatic lipid accumulation without a concomitant increase of body weight on a HFD. Gm4951 was highly expressed in hepatocytes. Knockout of Gm4951 increased lipid content and overexpression of Gm4951 decreased lipid content of primary hepatocytes in vitro, suggesting hepatocyte-intrinsic regulation of lipid content. Gm4951 knockout livers showed decreased expression of lipid oxidation genes. Mass spectrometry analysis of endogenous GM4951 interacting proteins revealed interaction with lipid droplet protein Hydroxysteroid 17β- dehydrogenase 13 (HSD17B13) and lipid oxidation enzymes. Moreover, the transcription of Gm4951 in hepatocytes was activated by interferon gamma (IFN-γ), which effectively decreased lipid content, much as when GM4951 was overexpressed. These results led to our central hypothesis that GM4951 is critical for promoting lipid oxidation, and limits hepatic lipid accumulation. To test this hypothesis, we propose to pursue three Specific Aims. Aim 1 will further investigate the development of NAFLD in GM4951 deficient mice. Aim 2 will determine the precise mechanistic role of GM4951 in regulating lipid metabolism. Aim 3 will study the transcriptional regulation of Gm4951, including the liver-specific expression pattern and the inducible expression by IFN-γ. Understanding how to activate GM4951 and what’s the human homolog of GM4951 would offer approaches to preventing or treating NAFLD. These will be studied in Aim 3 as well. The NAFLD phenotype caused by GM4951 deficiency is fundamentally distinct from the classic obesity-associated NAFLD mouse models. GM4951 is specifically expressed in the liver and operates there to limit lipid accumulation. Thus, finding ways to activate GM4951 will provide a new means of reducing hepatic lipid content. Completion of the proposed work will suggest new therapeutic targets to combat NAFLD.

NIH Research Projects · FY 2025 · 2021-12

Project Summary Abstract Heart disease remains the number one cause of death worldwide, due to the inability of the injured adult heart to regenerate. We seek to delineate the mechanisms that govern development, disease and regeneration of the heart and to build upon this knowledge to restore cardiac function during injury, disease and aging. In contrast to the adult mammalian heart, which lacks regenerative capacity, the neonatal heart can efficiently regenerate following severe injury. To explore the molecular underpinnings of neonatal cardiac regeneration, we have analyzed global changes in gene expression and the epigenome during regeneration of the neonatal mouse heart in comparison to later stage hearts that cannot regenerate. We have also performed single-cell RNA sequencing of cardiomyocytes and the major non-myocyte cell types from neonatal regenerative and non- regenerative hearts with or without injury. Integration of these comprehensive datasets has begun to reveal a “regenerative” chromatin landscape of the heart and the transcriptional activators and repressors of this process. The overarching goal of this project is to build upon this body of information to elucidate the mechanisms that control the responses of the neonatal heart to injury and to harness these mechanisms to promote adult cardiac regeneration and repair. By focusing on regenerative transcriptional circuitry and paracrine signaling mechanisms, we intend to devise new strategies to enhance cardiomyocyte proliferation and survival, angiogenesis and other regenerative processes mediated by various cellular constituents of the heart. Ultimately, the molecular decoding of cardiac regeneration will provide a molecular blueprint for activating endogenous pathways for cardiac repair and facilitate new strategies for restoring function to the injured and aging heart.

NIH Research Projects · FY 2025 · 2021-12

Abstract Physiological Role of De-differentiating Dermal Adipose Tissue Adipose tissue fibrosis is an integral component of dysfunctional fat tissue. This fibrosis exerts detrimental effects on local metabolic responses within adipose tissue, in addition to initiating maladaptive systemic responses. The exact cause(s) of fibrosis in adipose tissue are still a matter of debate and as such, are not well defined. Here, we aim to focus on “dermal adipose tissue fibrosis”, primarily due to its 1) ease of accessibility, 2) our new genetic mouse models that we generated to specifically examine dermal adipose tissue dysfunction and, 3) our initial observation that identified extensive dermal adipocyte differentiation and de-differentiation. We believe that the latter processes are a key aspect of the pathological road to adipose tissue fibrosis. Dermal adipose tissue is skin-associated fat located directly under the reticular dermis. Compared to other well-defined fat pads, dermal adipose tissue displays a high degree of plasticity. Under a variety of physiological conditions, dermal adipose tissue has the capacity to either rapidly and locally expand, or reduce its volume. Our in vivo preliminary studies showed that dermal adipose tissue is negatively associated with collagen production in skin fibroblasts. Importantly, more recent studies in our laboratory identified that the highly dynamic nature of dermal adipocytes allows them to 1) undergo de-differentiation into pre-adipocytes or, 2) convert into alpha-SMA-positive myofibroblasts (when examined in a bleomycin-induced fibrosis model). Here, we propose to examine the following hypothesis: that the adipocyte itself is the major player in preventing adipose tissue fibrosis in response to a metabolic challenge of high fat diet feeding, or bleomycin induction. We will address our hypothesis in three Specific Aims: I) We will retain fat cells in a fully differentiated state, by either ectopically exposing dermal adipocytes to PPARgamma agonists. In parallel, we aim to genetically overexpress PPARgamma then assess the impact on the fibrotic response. In addition to this, we will examine the impact of a complete elimination of adipocytes upon the local fibrotic response in the skin. II) Through lineage tracing, we aim to genetically label and track mature adipocytes as they de-differentiate into pre-adipocytes. Some of these pre- adipocytes can convert to myofibroblasts. We have developed a genetic approach to selectively eliminate myofibroblasts that originate from mature adipocytes. This will allow us, for the first time, to examine the functional relevance of these adipocyte-derived myofibroblasts towards the fibrotic response in adipose tissue. III) With a newly developed “Split Cre” system, we take advantage of a dual promoter system that ensures expression uniquely in the dermal adipocyte. We will manipulate leptin and adiponectin levels locally, then address the impact of local adipokine action in the microenvironment of the dermal adipose tissue on the fibrotic response. While we focus on dermal adipose tissue, results from the proposed studies will also have a profound impact on the understanding of the fibrotic response in other fat depots.

NIH Research Projects · FY 2026 · 2021-12

PROJECT SUMMARY Can germline mutations cause strong resistance to otherwise lethal cancers? Certain germline genotypes might be poorly supportive of tumor vascularization, nutritional demands, or resistance to immune attack, yet compatible with host survival. Of particular interest, some mutations might abet the host response to neo- antigens, or even to self-antigens highly expressed in syngeneic tumors. The identification of resistance mutations could provide new approaches and targets for cancer therapy. At least in human populations, resistance mutations would be very difficult to identify. Human germline genetic variability, stem variability among cancer genomes, and the high frequency of humans who never develop cancer throughout their lives would make mapping novel human resistance alleles all but impossible. In mice, finding such mutations is much easier. Syngeneic tumor lines (with relatively stable genomes) exist for many inbred strains of Mus musculus. The inbred mice themselves have a defined germline reference sequence. Each individual is homozygous at nearly all loci, and almost genetically identical to all others. Over the past several years, we took advantage of this situation to identify genes in which mutations confer cancer resistance. Using the random germline mutagen ENU, we created third generation (G3) germline mutant mice (C57BL/6J strain). A total of 23,751 third-generation (G3) mice from 561 pedigrees, bearing a total of 32,039 non-synonymous coding/splicing changes were enrolled into a screen in which each mouse was injected subcutaneously with 2e5 B16F10 melanoma cells, and anti-PD-1 antibody was administered on days 5, 8, and 11. Tumor volume was measured on days 13 and 20. The G1 male founder of each pedigree was sequenced to identify all non-synonymous coding/splicing mutations induced by mutagenesis, and all G3 descendants were genotyped at all induced mutation sites in advance of screening. Automated meiotic mapping allowed quick detection of even subtle phenotypes and assignment to causative mutations. This screen yielded several mutations causing resistance to transplantable cancers. 14.2% saturation of the autosomal genome was achieved in screening (fraction of autosomal genes with severely damaging or destructive alleles tested in the homozygous state three times or more). Therefore, much remains undiscovered. From what we know already, there is a realistic chance of translating genetic discoveries from this screen to human cancer therapy. This proposal aims to extend screening for cancer resistance, and to further advance mechanistic and translational studies of two resistance mutations, each in a gene with a human orthologue, testing synergy between therapeutic approaches built around each protein target, and laying groundwork for clinical applications.

NIH Research Projects · FY 2026 · 2021-12

Project Summary Episodic memory describes our ability to weave temporally contiguous elements into recollections of rich and coherent experiences. Episodic memory formation is specifically degraded by degenerative conditions such as Alzheimer’s Disease. The activity of ‘time cells’ in the mesial temporal lobe may provide a mechanism for the coding of temporal information that is necessary for the formation of these memories, and we recently published evidence of time cells in the human hippocampus using microelectrode recordings from epilepsy patients. The spike rate of these cells reliably increases at specific moments within a fixed interval, and groups (assemblies) of time cells can represent a ‘temporal space’ analogous to the manner in which hippocampal place cells are held to represent physical space, imposing temporal organization on event representations. In this proposal, we build on preliminary data to investigate the flexible participation of time cells in neuronal assemblies using established methods. We will test how time cells support serial memory by adjudicating between two models, one based on time cell activity (`time cell model’) versus a different proposed mechanism by which serial recall depends on the consistent phase offset of the spiking of hippocampal neurons relative to theta oscillations (‘phase offset model’). This experiment will test key unresolved questions about how time cells contribute to episodic memory. Finally, we will test the impact of cholinergic blockade on time cells and cell assemblies. This novel experiment builds on the preliminary data we present in our proposal showing the effects of scopolamine administration in 10 intracranial EEG subjects, which identify alterations in hippocampal theta and gamma oscillations in the setting of cholinergic blockade. Our experiments include a number of key innovations, including examining time cell activity during serial recall, assembly formation, and especially in the setting of modulation of cholinergic innervation. The data we propose to collect will fill key gaps in understanding related to time cell activity in humans and potentially establish cholinergic modulation in human intracranial EEG subjects as a method to elucidate physiological patterns during mnemonic processing and to model the effects of disorders such as Alzheimer’s Disease in this population.

NIH Research Projects · FY 2026 · 2021-11

Project Summary: The Flavivirus genus (referred to as flaviviruses) consists of numerous emerging and re-emerging global pathogens of critical human significance. Endemic and emerging flaviviruses like dengue virus (DENV), Powassan virus (POWV), Zika virus (ZIKV), West Nile virus, Japanese Encephalitis virus and Yellow fever virus continue to spread and cause significant human disease. We have used RNA structural data from a conserved 3’ untranslated region (UTR) pseudoknot called xrRNA1 to develop an attenuation approach in a highly conserved structural region of the flavivirus 3’UTR for vaccine development. This approach allows us to 1) swap out flavivirus structural genes in our clone to rapidly develop chimeric, attenuated flavivirus vaccines for mosquito-borne flaviviruses and 2) provides a conserved site for attenuation for tick-borne flaviviruses like POWV. Based on our preliminary data, we hypothesize that xrRNA1-mutant, attenuated flavivirus vaccines will be safe, immunogenic, and provide protection from challenge in murine models of flavivirus disease. The objective of the proposed studies is to complete pre-clinical development of the attenuated flavivirus vaccine approaches. We will complete our proposed work in three aims that will evaluate immunologic and virologic outcomes following virus challenge after vaccination with candidate ZIKV vaccine (Aim 1), DENV vaccine (Aim 2), and POWV vaccine (Aim 3). We have recently published our data showing attenuation and immunogenicity of mutant xrRNA1 ZIKV (X1) in pregnant and non-pregnant mice. In this proposal, we will first evaluate the efficacy of ZIKV X1 vaccine in pregnant and non-pregnant mice challenged with ZIKV and DENV. These studies will allow us to evaluate ZIKV vaccine efficacy during pregnancy and evaluate the role of ZIKV vaccination in DENV disease enhancement. Next, we will use the attenuated, ZIKV vaccine platform developed in our laboratory using xrRNA1 structural data, insert chimeric pre-membrane and envelope structural genes from DENV1-4 and evaluate the attenuation, immunogenicity and efficacy of monovalent and quadrivalent DENV1-4 vaccine candidates. Given the complexity of DENV infection, we will evaluate disease enhancement and immunodominance in our quadrivalent vaccines along with efficacy. Third, we will expand our attenuation strategy in the X1 structure to tick-borne flaviviruses by utilizing our recently defined secondary structure of the POWV 3’UTR. Using POWV mutant vaccine candidates with targeted mutations in the X1 structure, we will characterize attenuation, immunogenicity, and efficacy of a POWV vaccine approach in a murine model of disease. The proposed studies will begin to translate our structural understanding of xrRNAs in the flavivirus 3’UTR into potential vaccine candidates. Moreover, this project will initiate studies focused on developing a platform for vaccine development for emerging flavivirus infections.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Defects in the carefully orchestrated processes of retinal development, homeostasis and retinal immune surveillance lead or contribute to a wide range of diseases. It is now clear that genetics not only play a role in these processes but may also modulate diabetic retinopathy. Our short-term goal is to identify and characterize gene/protein defects and molecular pathways that lead to abnormal retinal development/homeostasis, altered retinal immune surveillance and modulation of diabetic retinopathy. The long-term goal is to leverage our research discoveries to understand retinal disease processes, and to identify novel therapeutic opportunities. We propose that a high-throughput and unbiased strategy provides an ideal approach to discovery of gene/phenotype associations in this setting. In collaboration with Nobel laureate Bruce Beutler, we will employ a robust state-of-the-science and unbiased forward genetics approach, in which thousands of new random mutations are generated and mice demonstrating retinal anomalies are identified by screening using fundus photographs and OCT. Our approach has significant advantages compared to other existing protocols. Most importantly, ours is the first and only protocol in which all mice have been pre-genotyped at all mutant loci. In addition, the large scale of our system and the large pedigree size will also add to the discovery power. Together, these advantages will allow us to identify and pursue novel gene/phenotype associations related to retinal development, homeostasis and disease. We have identified over 43 gene-phenotype associations after covering just 8% of the mouse genome. Of these, 12 genes have weak associations to the retina in the literature, and another 20 genes have not been reported in association to the retina. This is strong evidence that expanding our screening to include the remaining 92% of the mouse genome will yield many more gene-phenotype associations related to retina development, homeostasis and immune surveillance. Of note, our proposal starts by selecting a few of the most promising genes we have already identified for further study. We will harness the power of CRISPR/Cas9 gene editing, single cell RNA sequencing, co-immunoprecipitation experiments with highly sensitive mass spectrometry and proteomics analysis, our recently published light injury model and other techniques to explore the mechanisms of these associations. We will also apply the streptozotocin model of diabetic retinopathy to our OCT retinal imaging pipeline to identify genes that can modulate early diabetic retinopathy. This proposed research will advance our knowledge of retinal health and disease, and we anticipate that it will lead to the identification of new therapeutic avenues.

NIH Research Projects · FY 2025 · 2021-09

PROJECT SUMMARY People and other animals learn many of their complex and socially oriented behaviors by imitating more experienced individuals in their environment. Vocal imitation is one of the more striking and readily quantifiable examples of this type of learning, but the genetic basis of this complex trait is still poorly understood. The goal of this research is to determine the genetic basis of vocal imitation abilities by establishing the first mutagenesis screen in a vocal learning species and the genetic tools for independently testing the function of the identified genes by developing novel transgenic models using germline gene targeting technologies. Humans are the only primate and one of only a handful of mammalian species to have evolved the facility for vocal imitation. Aside from humans, songbirds, and in particular zebra finches, are the best studied vocal learning species and they provide the only practical platform for systematically identifying the genes involved in this important social behavior. Like speech, zebra finch song is a culturally transmitted behavior learned via imitation. Moreover, functional, genetic and molecular parallels underscore the use of zebra finch for identifying genes essential for vocal imitation. We hypothesize that a forward genetic dominant screen, followed by the detailed genetic mapping and manipulations developed through this proposal, will identify convergent and divergent genetic signatures for this polygenic trait. Establishing a forward genetic screen and the genetic tools for verifying gene function in zebra finches will provide a novel, comprehensive, and broadly impactful approach for trying to understand the genetic basis of vocal and social communication.

NIH Research Projects · FY 2025 · 2021-09

Biomolecular condensates concentrate select groups of macromolecules into discrete foci in eukaryotic cells in the absence of a surrounding membrane. Condensates are found throughout eukaryotic cells, and function in processes ranging from signal transduction to RNA metabolism to gene expression. Aberrant condensates have been implicated in neurodegeneration, cancer and skin diseases. Our understanding of eukaryotic cell organization was transformed by the discovery that many condensates appear to form through liquid-liquid phase separation (LLPS) of multivalent macromolecules. My lab played an important part in this discovery by establishing key principles, including the essential role of multivalent interactions in promoting biological LLPS, the regulation of LLPS by covalent modifications, and the ability of LLPS to increase enzymatic activity. In recent years, we showed that and how LLPS can produce membrane-associated clusters that increase the specific activity of signaling molecules. We also showed that intrinsically disordered regions of proteins (IDRs) can undergo LLPS to produce liquid droplets that harden to solids over time, likely due to formation of amyloid filaments, and that misregulated hardening may contribute to neurodegeneration. Further, we proposed the first model to explain condensate composition based on a scaffold/client framework. Most recently, we showed that chromatin has an intrinsic propensity to undergo LLPS, providing a new view of eukaryotic genome organization. Here, we propose a broad program to address leading questions in the biomolecular condensate field. We will use microfluidics to assess LLPS of thousands of IDRs in a single experiment, leading to a predictive model for the sequence determinants of LLPS and amyloid formation by IDRs. This work will deepen our biophysical understanding of these processes, predict which IDRs are likely to contribute to specific biological processes (e.g. transcription) through self-assembly, and reveal how IDR mutations lead to disease through amyloid formation. We will also examine how individual components of yeast P bodies impact the LLPS threshold and composition of the compartments and how RNA helicase and RNA decapping activities are modulated within them. This work will lead to a new model of condensate composition based on the patterns of interaction between components, and will explain how composition and encapsulation can control enzymatic activities in native condensates. Finally, we will learn how internucleosome spacing and diverse histone post-translational modifications control chromatin LLPS to generate biochemically and functionally distinct genomic regions, examine whether NUT carcinoma is caused by defective LLPS, and develop a new approach to drug design based on targeting small molecules to condensates. Together, the work will reveal new principles of biological phase separation, explain how phase separation can be used to control RNA metabolism and genome organization and function, and provide insights into the mechanisms and potential treatments of neurodegeneration and cancer.

NIH Research Projects · FY 2025 · 2021-09

Project Summary In response to the RFA for a Cellular Cancer Biology Imaging Program we propose a program focused on imaging and molecularly probing the cell biological events that drive the formation of new metastatic tumors. Specifically, we will address two questions: 1) How does the intersection of shifts in cell-intrinsic and cell- extrinsic signals associated with shifts in expression of the membrane adaptor protein Caveolin-1 affect the metastatic propensity of pediatric sarcoma (Research Testbed Unit 1)? 2) What are the effects of cell-intrinsic and cell-extrinsic variation in lipid metabolism on melanoma metastasis patterns (Research Testbed Unit 2)? Answers to both questions depend on technology to capture the molecular, metabolic, and morphological states of individual metastatic cells as they colonize the distant site: In the Technology Development Unit-1 we will develop a multi-modal, multi-scale live imaging platform to investigate the effects of intersecting microenvironmental variation across an organism and cell intrinsic heterogeneity on metastatic spreading. The platform will leverage the exquisite optical and physiological properties of the zebrafish embryos to ‘watch’ at once how cells form human tumor xenografts spread to multiple distant sites where they form metastatic tumors. The microscope will allow seamless switching between a high-throughput screening mode observing the metastatic patterns in tens to hundreds of embryos in one experiment and a high-resolution imaging mode with fully isotropic resolution of 300 nm in XYZ that allows detailed analysis of the molecular, metabolic, morphologic, and proliferation/survival states of individual cells within an emerging metastatic niche. In the Technology Development Unit-2 we will develop a multi-scale imaging platform to investigate by hyper-spectral analysis the molecular, metabolic, morphological, and functional states of metastatic cells across entire mouse organs. The platform will leverage advances in tissue clearing, fully automated high-speed and high-resolution light-sheet fluorescence imaging, and computer vision, to integrate a mesoscopic imaging mode for fast acquisition of volumes of up to 20 x 20 x 20 mm at a ~5-10 micron isotropic resolution with a nanoscopic imaging mode providing 300 nm XYZ-resolution throughout a 300 micron field of view anywhere in the organ. Biological features can thus be rapidly identified and immediately interrogated with high subcellular resolution. We will then develop physically and chemically accelerated 60-plex cyclic immunofluorescence assays to comprehensively characterize the molecular, metabolic and architectural states of colonizing cells and their surroundings in the metastatic niche in thick (~200 microns) tissue sections. To accurately describe metastatic heterogeneity, the entire system, including sample handling, labeling, and imaging, will be fully automated and operated in a high-throughput fashion. Our goal with this system is to enable comprehensive profiling of heterogeneous cell metastatic cell behavior in 100’s of intact tissue specimens. Together, these platforms will generate versatile imaging tools for a new era of in situ cancer cell biology.

NIH Research Projects · FY 2025 · 2021-09

Project Summary/Abstract: Tuberculosis (TB) kills 1.5 million people per year. Efforts to reduce this number have been hindered by the lack effective diagnostics and a protective vaccine underpinned by gaps in the understanding of the immune response in TB disease. While cellular immunity is important, the humoral immune response to infection by Mycobacterium tuberculosis (Mtb) is poorly understood. Antibodies, specifically IgG, are critical components of the adaptive immune response which have been indispensable in our understanding of infectious diseases and vaccine development. Antibodies function through the combination of recognizing antigens by the Fab domain and the recruitment of immune effector responses via the Fc domain. Variability in the Fc domain by isotype, subclass, and post translational glycosylation impact engagement with Fc receptors on immune cells that alter function in clinically significant manners. We have published that the antibody Fc domain diverges between individuals with latent infection who appear healthy and able to restrict bacteria compared to active TB disease which is permissive to Mtb replication. These distinctions are linked to differential Mtb burden in an in vitro primary human monocyte derived macrophage model of infection. How exactly antibodies might function in this context and its physiological relevance are questions that this proposal begins to address. The specific aims are 1: Determine how the Mtb antigenic repertoire impacts polyclonal IgG functions, 2: Identify the macrophage pathways by which the IgG Fc modulates Mtb survival, 3: Examine the in vivo effect of polyclonal IgG on chronic Mtb infection. The scientific objective of this proposal is to determine how polyclonal IgG contributes to restrictive and permissive host states for Mtb. The central hypothesis is that polyclonal IgG from individuals with active TB disease induces a host state permissive to bacterial growth. The overall goal is to understand fundamental mechanisms of humoral immunity in TB through antibodies to inform diagnostic, therapeutic and vaccine design.

NIH Research Projects · FY 2024 · 2021-09

SUMMARY Lipid and cholesterol-rich Western diet is a major risk factor for many non-communicable diseases, including cardiovascular disease, obesity, diabetes, metabolic syndrome, and inflammatory bowel disease (IBD). A common lipid derivative associated with the pathogenesis of these inflammatory and metabolic diseases is low-density lipoprotein (LDL) which is scavenged by its receptor (LDLR) expressed in almost all tissue. Despite the known function of LDL in atherosclerosis, its role in other diseases is poorly understood. Inflammation is a common trigger for atherosclerosis and other non-communicable diseases. However, whether native LDL, which is the most abundant physiological form of LDL, is involved in the inflammatory response is unknown. The goal of this study is to define a role of native LDL in inflammatory response, so the association of LDL with many human diseases can be explained. This critically important objective was stemmed from our preliminary studies in which we observed that mice having high blood LDL are highly susceptible to experimental colitis. Interestingly, mice defective in LDLR (Ldlr-/-) were relatively protective against colitis. Reduced colitis susceptibility of Ldlr-/- mice was associated with suppressed inflammation and decreased activation of inflammatory signaling pathways such as NF- kB and MAPK, pointing to an uncharacterized function of LDL/LDLR in inflammation. Indeed, we observed that native LDL stimulates macrophages in vitro to produce inflammatory molecules. Given that no study yet documented a role of native LDL in innate immune signaling and inflammatory responses, our observation underscored a novel mechanism of pathogenesis of inflammatory disorders associated with high blood LDL. We, therefore, hypothesize that endocytosis of LDL through LDLR induces inflammatory responses in myeloid cells causing inflammation, and such an inflammatory pathway imparts a major contribution in inflammatory disorders like colitis. This hypothesis will be tested through two specific aims: Aim 1. To dissect the pathway involved in LDL-mediated induction of inflammatory responses; Aim 2. To define the role of native LDL and its receptor in intestinal inflammation. Using biochemical and molecular biology techniques, we will explore signaling events and mechanisms involved in LDL/LDLR-mediated activation of NF-B and MAPK pathways. We will use Ldlr-/- mice and mouse models of colitis to investigate the in vivo relevance of LDL/LDLR in inflammatory disorders. Overall, this study will explore a novel biological function of native LDL which will help elucidate the pathogenic mechanism of diseases associated with high blood LDL. Furthermore, this study will decipher a yet unknown role of blood LDL in colitis pathogenesis. The findings of this study will open the opportunity to treat IBD and other non- communicable diseases by targeting LDL synthesis or LDLR downstream signaling pathways.