University Of Illinois At Urbana-Champaign

NIH Research Projects · FY 2026 · 2022-07

PROJECT SUMMARY/ABSTRACT: Inflammatory bowel diseases (IBD) are becoming more prevalent in the US and represent a major societal health concern. Exposure to psychosocial stressors increases the likelihood of developing IBD in genetically predisposed individuals, implicating a brain-gut axis in the IBD etiological framework. An emerging line of work has established that stress-induced disruptions to the gut microbiota (i.e. dysbiosis) may be the most proximate cause of stress-induced IBD predisposition. This includes data from our laboratory where we showed that a mouse adaptive pathogen (C. rodentium) was more effective at colonizing and inducing colitis in mice colonized by a microbiota from mice exposed to a chronic social defeat stressor. Moreover, our new preliminary data provides evidence stress exacerbates chronic, immune mediated (T-cell) colitis. However, how the gut microbiota and mucosal layer becomes dysregulated in response to stressors and why those changes predispose worsened colitis, is not yet understood. We recently demonstrated that stress induces large shifts in intestinal epithelial cell (IEC) activity that tightly corresponded to changes in gut microbiota function and thinning of the mucus layer. Of those changes observed in IECs, our preliminary data indicate that the reactive-oxygen species (ROS)-generating capacity of IECs may be the most proximate causes of stress-induced dysbiosis and mucosal disruption. Signs of stress in IECs were absent in germ-free (GF) mice at baseline, thus implicating the microbiota in IEC responsiveness. Nevertheless, IECs were still primed to respond differentially to an ex vivo bacterial challenge (evidenced by an increased expression in the ROS-generating enzyme dual oxidase (DUOX2), suggesting that host stress signaling molecules and the gut microbiota are together involved in regulating IEC activity. Intriguingly, the upregulation in DUOX2 and ROS activity in IECs corresponded to expansion of ROS-resistant bacteria that are capable of mucus degradation. These data led us to build a cohesive framework underlying this proposal, whereby stress hormones ‘prime’ IECs to respond to endogenous microbiota signaling/adhesion through heightened ROS generation. This enhanced ROS activity at the mucosal interface creates a unique niche for mucosal associated microbes that are resistant to ROS activity and survive by degrading mucus glycans that normally provide a barrier against both endogenous microbes and pathogens. We hypothesize that this IEC-directed expansion of ROS-resistant, mucus-degrading endogenous microbes is what underlies IBD susceptibility in organisms exposed to chronic, unabated stress.

NIH Research Projects · FY 2025 · 2022-07

Project Summary/Abstract Randomized controlled trials (RCTs) are a cornerstone of evidence-based medicine and are placed high in the “evidence pyramid”. When rigorously designed, conducted, and reported, they provide the most robust evidence on effectiveness of therapeutic interventions. However, they commonly suffer from various types of biases (e.g., selection bias, attrition bias) in study design and execution. In reporting, key methodological characteristics such as randomization and blinding are often omitted, making it difficult to assess the validity and applicability of trial findings. Adherence to reporting guidelines can improve transparency and completeness of reporting for biomedical studies. SPIRIT and CONSORT guidelines help authors report RCT protocols and results publications, respectively. Although endorsed by many high-impact medical journals, adherence to these guidelines remains suboptimal, possibly because journals lack methods for enforcement and verification, which involves a substantial amount of journal staff or editorial time. Furthermore, transparent reporting does not guarantee methodological rigor. We hypothesize that natural language processing (NLP) methods underpinned by SPIRIT/CONSORT guidelines as well as terminological and ontological resources for clinical research can (a) improve compliance by locating key study characteristics in RCT reports and flagging their absence, and (b) support automated rigor assessment and large-scale methodological research by extracting granular machine- readable methodological information from RCT reports. To achieve these goals, we specifically aim to: Aim 1. Create text classification models for assessing transparency and completeness of RCT reports consistent with SPIRIT and CONSORT guidelines. Aim 2. Develop information extraction methods to identify methodological characteristics in RCT reports. Aim 3. Build a web-based compliance tool that generates reports on transparency and guideline adherence of RCT reports. Aim 4. Generate structured transparency reports from published RCT literature for analysis of methodology and reporting quality. The proposed research will develop a set of models, resources, and tools that will assist stakeholders of clinical research in maintaining high reporting standards, synthesizing evidence, and promoting open science practices. They will contribute to improvements throughout the scientific ecosystem, leading to better clinical care and health policy.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY Seasonal influenza epidemic causes 3-5 million infections and 250,000 to 500,000 deaths every year. While seasonal influenza vaccine is available and being constantly updated, its effectiveness is often hampered by the rapid antigenic drift of circulating strains. As a result, a major goal of influenza research is to develop a more effective vaccine. Nevertheless, the poor ability to forecast the evolution of influenza virus poses a huge challenge in influenza vaccine development. Consequently, understanding how the evolutionary trajectories of influenza virus are being shaped can significantly benefit public health. Influenza virus has two surface antigens, namely hemagglutinin (HA) and neuraminidase (NA). While influenza vaccine development has traditionally focused on targeting the HA, NA has received increasing attention as an effective vaccine target in recent years. Evolution of NA is under several biophysical constraints including protein stability, surface expression, and enzymatic activity. These biophysical constraints determine not only the fitness effects of individual mutations, but also how these fitness effects vary in the presence of other mutations (i.e. epistasis). In fact, epistasis has been a main obstacle in evolution forecast since epistasis can lead to opposite fitness effects of the same mutation in different influenza strains. This proposed study will use innovative high- throughput experiments to systematically probe the fitness effects of all possible amino-acid mutations on NA and map epistatic interactions that are involved in the natural evolution of NA. In addition, the molecular mechanisms of epistasis will be characterized by biochemical and structural biology approaches. Statistical modeling will further be applied to quantify the relationships between biophysical constraints of NA and viral fitness. The results will comprehensively reveal the biophysical principles that govern the mutational fitness effects and epistatic interactions in influenza NA, and hence its evolutionary trajectories in natural evolution. This proposed study will therefore promote the construction of a unifying biophysical model to accurately forecast the evolution of influenza virus, which will in turn facilitate the development of next-generation influenza vaccines.

NIH Research Projects · FY 2025 · 2022-06

PROJECT SUMMARY RNA therapeutics hold great promise for the treatment of a number of diseases significantly impacting human health, such as chronic infections, genetic disorders, certain cancers, and presently COVID-19. The leading RNA delivery vehicles approved by the FDA, as well as being considered in several clinical trials, are non-viral lipid- based nanoparticles (LNPs). State-of-the art LNPs comprise standard phospholipids, cholesterol, and ionizable lipids (ILs) that get protonated in acidic conditions. Analogous to enveloped virus, LNPs hijack the endocytic pathway to enter cells. The efficacy of RNA delivery hinges on the ability of LNPs to escape the endosome by fusing with its membrane. However, the factors that control LNPs–endosome fusion remain largely unknown. Enveloped viruses contain proteins that promote fusion by stabilizing the formation of highly curved membrane pores. In LNPs, alternative strategies to bolster fusion include using lipids with non-zero spontaneous curvature that are elusively deemed “fusogenic”. However, understanding membrane fusion requires the consideration of membrane elasticity beyond spontaneous curvature. Specifically, the formation of a fusion pore between two bilayers is dictated by an interplay between the bending modulus and the Gaussian curvature modulus. However, the Gaussian modulus is rarely considered when designing “fusogenic” LNPs, even though bilayer fusion is an occasion for which its value matters the most. The central hypothesis of this work is that raising the Gaussian modulus of LNPs by inclusion of a new class of lipids termed Gaussian curvature lipids (GCLs) has a dramatic effect on the ability of LNPs to fuse with endosomal membranes. Furthermore, we conjecture that membrane fusion, as boosted by GCL integration, is synergistically favored in living systems during active proton pumping and endosome acidification. We combine a team of experts in RNA delivery to cells, membrane protein purification as well as experimental, computational, and theoretical membrane elasticity to test the central hypotheses via two aims. In Aim 1 we will establish the biophysical elastic properties of LNPs to maximize fusion with endosomes. We investigate how fusion takes place at a microscopic level, namely deciphering if the dominant effect is the formation of fusion pores and/or if LNPs feed lipids to endosomal membranes remodeling them and making them more prone to rupture. In Aim 2 we investigate the impact of membrane activity and endosome acidification by measuring in live cells RNA delivery and endosomal fusion of LNPs comprising increasing amounts of GCLs. We will also develop endosome-mimetic vesicular systems reconstituted with endosomal membrane proton pumps (V- ATPase) to elucidate the mechanism of LNP-endosomal membrane fusion during active proton pumping. Our work will raise new physical insights on LNP endosomal escape and establish the desired LNP membrane properties to boost fusion in living systems, resulting in substantially more effective RNA delivery vehicles.

NIH Research Projects · FY 2026 · 2022-06

ABSTRACT The inability of wounds to heal in diabetic patients is the leading cause of lower extremity amputation in the United States. Chronic, localized inflammation is believed to be a causative factor in the slow healing of diabetic wounds, and macrophage cells are implicated as primary mediators of this inflammation. In non-diabetic patients, macrophages are initially in a pro-inflammatory state in wounds and shift over time to an anti-inflammatory phenotype that promotes tissue repair. In diabetic patients, the inflammatory macrophage phenotype persists, resulting in impairment of angiogenesis, granulation tissue formation, and wound contraction required for healing. Systemically administered pharmacological agents that are anti-inflammatory or immunomodulatory do not improve healing in the clinic or in preclinical animal models and, in fact, further impair healing, likely due to off- target effects in other immune or structural cells that facilitate tissue repair. This proposal focuses on the development of drug carriers based on targeted nanomaterials to reroute the delivery of pharmacological agents selectively to inflammatory macrophages in wounds after local administration to eliminate off-target effects. We are particularly focused on inhibiting overactive pathways that generate inflammatory prostaglandins. In our preliminary data, we show that polysaccharide-based nanocarriers can deliver cyclooxygenase 2 inhibitors to wound macrophages to potently diminish inflammatory cytokine expression and expedite wound healing in diabetic mouse models. Aim 1 of this proposal is to optimize formulations that maximize the efficiency of targeted delivery to inflammatory macrophages in wounds using fluorescent and radioisotopically labeled nanocarriers, evaluated in vivo by nuclear imaging and ex vivo by flow cytometry, gamma well counting, and fluorescence microscopy. Aim 2 is to optimize the efficacy and drug release rate of a therapeutic formulation that targets different regulatory pathways of prostaglandin synthesis toward diabetic wound healing. Aim 3 is to evaluate efficacy and off-target effects in multiple murine acute and chronic wound healing models of type 2 diabetes, as well as monocyte-derived macrophages from diabetic patients. Fundamental outcomes of this work will be an understanding of nanomaterial transport in wounds and receptor-mediated mechanisms to target macrophage subpopulations, as well as an understanding of the role of prostaglandin-driven inflammatory processes in macrophages within diabetic wounds. The nanocarrier delivery agents are based on materials already in broad clinical use, which may expedite clinical testing of the resulting therapeutic agents if preclinical results are promising. This work will be undertaken by an interdisciplinary team comprising bioengineers (Andrew Smith Lab) who focus on nanomaterial-based drug delivery and imaging agents, experts in molecular and cellular immunology and mechanisms of diabetic wound healing (Katherine Gallagher Lab), and experts in nuclear imaging and radiopharmacology (Wawrzyniec Dobrucki Lab).

NIH Research Projects · FY 2025 · 2022-06

Project Summary Atopic dermatitis (AD) is a chronic inflammatory skin disease that affects 15-30% of children and approximately 5% of adults in industrialized countries. However, effective treatments to prevent and treat this skin disease are lacking due in part to an incomplete understanding of the disease. The human pathogen Staphylococcus aureus has been linked to AD pathogenesis because more than 90% of AD patients are colonized in the lesional skin with the pathogen. Although S. aureus can produce multiple virulence factors, the mechanism by which S. aureus virulence contributes to AD remains unknown. Using a recently developed mouse model of epicutaneous S. aureus infection that resembles that observed in AD flares, we found that activation of the S. aureus accessory gene regulatory (Agr) quorum-sensing system induces the release of keratinocyte alarmins to trigger skin inflammation. We hypothesize that S. aureus Agr virulence factors play a critical role in skin inflammation associated with AD. We propose three Aims to test our hypotheses: 1) Use a novel AD mouse model to study the role of S. aureus in skin inflammation; 2) Determine the mechanism of S. aureus colonization in the skin and upregulation of Agr expression in lesional skin using the AD-like mouse model; 3) Examine the expression of S. aureus Agr virulence genes in lesional and non-lesional skin of AD patients and the function of AD- associated S. aureus in skin inflammation. These studies will provide critical insight into AD pathogenesis that will help the development of new approaches to treat AD. The candidate is a basic immunologist and Research Investigator at the University of Michigan. Under the guidance of his mentors and advisory team, he will acquire new knowledge and research expertise to test the hypothesis that both S. aureus Agr virulence and skin factors are important for triggering skin inflammation in AD using a newly developed AD model. The research proposed in his K01 application is a new scientific challenge in his career that offers the opportunities to integrate clinical and translational experience with his comprehensive training in basic science. Dr. Matsumoto's career development goals will be supported through close mentorship from an interdisciplinary team, advanced didactic coursework, attendance at professional meetings and workshops, participation in regular seminars, guidance in manuscript preparation and grant proposal development. This training and research activities will provide him with the necessary skills and experience needed to become a successful independent translational investigator.

NIH Research Projects · FY 2025 · 2022-05

Project Summary/Abstract In biology, there are two fundamental conflicts: the conflicts between organisms and the conflicts between organisms and viruses. During the conflicts, a variety of offensive and defensive weaponry are employed by organisms and viruses. While the molecular mechanisms of the conflicts between organisms and viruses have been extensively studied, significantly less have been carried out with the conflicts between organisms. During the conflicts between organisms, protein toxins are frequently employed as the offensive weapons. Among them, ribotoxins constitutes one of the biggest groups. In fact, colicin E3 was the first ribotoxin to be characterized 50 years ago. It makes a single but precise cut of 16S rRNA in the decoding center of bacterial ribosome, resulting in stalled ribosome and eventual cell death. Over the last half of a century, it is unclear whether there exists a biological system that is able to reverse the ribosomal damage by colicin E3 to allow cell to survive. Employing approaches of bioinformatics, biochemistry, structural biology, and microbiology, we have uncovered a bacterial two-component system, RtcB and PrfH, as the antidote of colicin E3. Specifically, bacterial PrfH recognizes the damaged and stalled ribosome and performs ribosomal rescue. This is followed by RtcB repairing the damaged 30S ribosomal subunit. The sequential events described above are supported by abundant preliminary data from both our in vitro and in vivo studies. In this application, we plan to significantly expand our preliminary studies to systematically characterize the rescue and repair of bacterial ribosome with specific damage in the decoding center with the following three main aims: 1) We will provide insight into bacterial PrfH recognizing and rescuing the damaged and stalled 70S ribosome in vitro; 2) We will biochemically and structurally characterize bacterial RtcB in vitro, with the emphasis of PrfH-coupled RtcB repairing the damaged 30S ribosomal subunit; and 3) We will elucidate in vivo biological functions of RtcB-PrfH using an in vivo attenuated RNA damage system we have developed.

NIH Research Projects · FY 2025 · 2022-04

Project Summary Biofilm, a protective extracellular-polymeric substance that surrounds bacterial colonies, is associated with more than 80% of microbial infections. In the United States, the management cost for biofilm- associated infections reaches 94 billion US dollars and is responsible for 0.5 million deaths annually. In particular, millions of wound patients suffer from biofilm-associated infections that lead to persistent inflammation and edema, and ultimately hinder wound healing. Biofilm bacteria are 1,000 times more resistant to antibiotics than free-floating bacteria. In a clinical setting, it is common to remove biofilm from the wound with debridement or enzymes. However, these methods do not remove biofilm in space deep in the wounded tissue, thus allowing biofilm recurrence. To this end, we recently invented a self-locomotive, antimicrobial micro-robot (SLAM) that can invade and remove biofilm. The SLAM is prepared by activating diatom biosilica doped with MnO2 nanocatalysts (MnO2-diatom) to generate oxygen microbubbles using a 3 % hydrogen peroxide solution. The activated MnO2-diatoms propel themselves to enter the biofilm. Within the biofilm, the activated MnO2-diatoms continue to generate microbubbles that fuse and produce mechanical energy high enough to fracture biofilm. It takes 10 minutes for the activated MnO2-diatoms to remove more than 99.9 % of 0.8 mm-thick P. aeruginosa biofilm with similar depth to full-thickness skin. No adverse toxic effects are observed after cleaning. With this success, our overall goals are to improve biofilm removal from the infected wound using SLAM and, in turn, to promote skin regeneration in the wound. We hypothesize that the activated MnO2-diatoms would detach biofilm from wounds and, in turn, increase access of antibiotics to residual biofilm bacteria. The subsequently enhanced wound disinfection would serve to improve the efficacy of regenerative medicine to skin regeneration in wounds. We will examine this hypothesis by using the vancomycin and a pair of keratinocyte growth factor (KGF)-2 and fibroblast growth factor (FGF)-2 as a model antibiotic and regenerative medicine, respectively. Our specific aims are to: (1) evaluate the efficacy of activated MnO2-diatoms to remove biofilm in wounds, (2) examine if activated MnO2- diatoms improve the efficacy of vancomycin to prevent biofilm re-growth, and (3) investigate the extent that activated MnO2-diatoms increase the KGF2/FGF2 efficacy in stimulating skin regeneration. We will conduct each aim study using the P. aeruginosa or methicillin-resistant S. aureus biofilm-infected excisional wound of male and female CD1 mice. We will assess the biofilm removal and skin regeneration in wounds using a multimodal optical imaging system through collaboration with the Boppart group with expertise in bioimaging. We will also determine the matrix metalloproteinase-9 and tissue inhibitor to metalloproteinase levels in the wound fluid, CD34+/CD45- stem cell mobilization, pro-inflammation and edema, and minimal toxicity of SLAM under guidance by Dr. Neitzel, a dermatologist. Overall, this proposed study will significantly impact efforts to treat non-healing, biofilm-infected wounds using innovative SLAMs. In the end, this study will save wound patients from disability and death.

NIH Research Projects · FY 2026 · 2022-03

PROJECT SUMMARY / ABSTRACT “Mechanisms of DNA helicases and their regulation” Helicases are a ubiquitous and diverse group of molecular machines that separate the strands of nucleic acids. They are essential actors in many genome maintenance processes in all domains of life, including some viruses. As a result, helicases are biomedically important proteins, and their pathologies are associated with a number of human diseases and cancer. Since uncontrolled unwinding is detrimental to genomic integrity, helicase activity must be tightly regulated in the cell. Furthermore, since many helicases are able to play multiple, distinct roles in a variety of cellular pathways, they must be activated only in the correct contexts. How these different functions are defined and regulated remains poorly understood. In this project, we will investigate the molecular mechanisms by which DNA helicases are regulated. Our studies will focus on the model non-hexameric helicases UvrD, Rep, and XPD, which are critical components of the cellular response to DNA damage in prokaryotes, eukaryotes, and archaea and also serve as prototypical members of the two largest structural superfamilies of helicases. Insights gained on their mechanisms are expected to extend to a number of structurally and functionally homologous systems. Prior work by us and others has shown that these types of helicases have auxiliary domains and/or make secondary contacts with DNA that play regulatory—often, auto-inhibitory—roles. Protein partners to helicases have thus been proposed to activate helicase activity by controlling these mechanisms, thus defining helicase roles in the cell. To gain insights into these mechanisms, our studies will focus on two main research goals: understanding how interactions with DNA and non-canonical DNA structures control helicase activity (Goal 1), and quantifying how encounters with accessory proteins—both protein partners that recruit and activate helicases and proteins that compete for the same DNA substrates—regulate helicases (Goal 2). Our approach for achieving these research goals will integrate advanced single-molecule biophysical techniques—optical tweezers combined with fluorescence microscopy—together with traditional biochemistry and computational biophysics methods. These approaches leverage our group's expertise and that of the assembled collaborators, and have been successfully applied by us in our high-resolution measurements of helicase unwinding and conformational dynamics, their modulation by interactions with accessory proteins, and their connection to atomic-level structural models of helicases,. Beyond providing insights on helicase mechanism and the genome maintenance pathways in which they participate, our studies will advance new biophysical methods for investigating biomolecular dynamics.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, paralytic disorder characterized by the selective loss of motor neurons in the spinal cord and brain. While most cases of ALS are sporadic, toxic gain-of-function mutations in superoxide dismutase 1 (SOD1) are responsible for ~20% of all inherited forms of the disease. Given its causative role in ALS, antisense oligonucleotides (ASOs) and RNA interference (RNAi) have been used to silence the expression of the mutant SOD1 protein. However, owing to their transient lifecycle, ASOs will require a lifetime of costly, invasive administrations, while RNAi is prone to inducing off-target effects. Conversely, while gene-editing technologies, such as CRISPR-Cas9, can be used be used to genetically inactivate mutant SOD1, the implementation of these strategies for gene therapy could prove challenging, as DNA editors can introduce off-target mutations and inadvertently create new, mutant SOD1 protein variants that can compromise their safety. Thus, there remains a crucial need for therapies that can safely and efficiently lower SOD1 for treatment of ALS. An alternative technology that holds little risk for inducing DNA damage within a cell but could still be used to efficiently lower SOD1 are RNA-targeting CRISPR-Cas13 effectors. CRISPR-Cas13 systems possess the programmability and versatility characteristic of DNA-editing CRISPR-Cas nucleases but pose limited risk for inducing genotoxicity since they are unable to cleave DNA. Moreover, Cas13 proteins display favorable specificity compared to gene silencing technologies and many are compact enough to fit within a single adeno- associated virus (AAV) vector, a clinically promising gene delivery vehicle that can mediate long-term, cell-type specific gene expression in the nervous system. Thus, CRISPR-Cas13 has the potential to safely and persistently silence mutant SOD1 following just a single administration of an engineered viral vector. However, it remains unknown whether Cas13 can be harnessed to reduce SOD1 in vivo and treat the disease. The overarching objective of this proposal is to develop a gene therapy for ALS. Specifically, we propose to harness CRISPR-Cas13d technology to lower mutant SOD1 in vivo for treatment of SOD1-linked ALS. In support of the feasibility of this objective, our preliminary studies have demonstrated that Cas13 proteins are more active and specific than a preclinically promising shRNA, that they can be delivered at high efficiencies by AAV9 to spinal cord astrocytes, that they can efficiently lower mutant SOD1 protein throughout the spinal cord, and that they can provide therapeutic benefit. We now aim to optimize the performance of this platform (Specific Aim 1) for the goal of testing its efficacy in mouse models of ALS (Specific Aim 2) and to determine its safety as a gene therapy agent (Specific Aim 3). Thus, by harnessing an innovative technology for transcriptional engineering that can overcome the limitations of traditional gene-silencing, we will develop a new therapy for ALS, a debilitating and currently incurable disorder with few effective treatment options.

NIH Research Projects · FY 2026 · 2022-02

PROJECT SUMMARY Insulin resistance and type 2 diabetes mellitus (T2DM) have presented an enormous burden to public health and economy with increasing prevalence. T2DM is characterized by relative insulin deficiency caused by pancreas β cells dysfunction and insulin resistance in metabolic organs. The liver plays a central role in regulating systemic glucose and lipid homeostasis. Aberrant hepatic insulin action is believed to be a primary driver of insulin resistance, in which insulin fails to adequately suppress hepatic glucose production (HGP), while enhances lipogenesis and triglyceride secretion, a phenomenon referred to as selective insulin resistance. Although downstream signaling cascades mediating insulin's control of glucose and lipid metabolism have been extensively studied, the molecular mechanisms underlying the development of insulin resistance and its differential effect on glucose and lipid metabolism are not well understood. We previously have identified critical functions of lysophosphatidylcholine acyltransferase 3 (Lpcat3), a phospholipid (PL) remodeling enzyme, in lipid metabolism in liver. Loss of Lpcat3 selectively reduces polyunsaturated PL in membranes, leading to decreased membrane fluidity and curvature. Changes in membrane dynamics result in impaired SREBP-1c processing and lipogenesis, and reduced triglyceride secretion in liver. The overall goal of this proposal is to define the roles of hepatic Lpcat3 and PL composition in insulin signaling and systemic lipid and glucose metabolism, and their contribution to the development of insulin resistance. In Aim 1, we will elucidate the mechanisms by which PL composition regulates insulin sensitivity. In Aim 2, we will investigate whether dysregulation of Lpcat3 expression mediates selective insulin resistance in T2DM. In Aim 3, we will test the therapeutic potential of targeting Lpcat3 for hyperglycemia and hypertriglyceridemia in T2DM. The results of this work will advance our understanding of how membrane composition modulates insulin sensitivity and glucose metabolism in liver, and how changes in membrane biophysical properties contribute to the pathogenesis of insulin resistance.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY Influenza A virus continues to be a major global health concern due to antigenic drifts and shifts. Rapid antigenic drifts of circulating human influenza subtypes (H1N1 and H3N2), which are caused by point mutations, can drastically hamper vaccine effectiveness despite annual update of the seasonal influenza vaccine. On the other hand, antigenic shifts, which are caused by genetic reassortment between antigenically distinct strains, can result in devastating pandemic as exemplified by the 1918 Spanish flu. Human infections with different zoonotic subtypes, such as H5N1, H6N1, H7N9, H9N2, and H10N8 have also been reported. As a result, a universal influenza vaccine that can elicit broadly protective antibody responses to diverse influenza strains and subtypes is urgently needed. The discovery of broadly neutralizing antibodies (bnAbs) that target the conserved stem region of influenza hemagglutinin (HA) has raised the possibility of developing a universal influenza vaccine. A number of HA stem bnAbs are encoded by immunoglobulin heavy chain germline gene IGHV6-1. Since these IGHV6-1 HA stem bnAbs can be found in multiple individuals and can cross-react with both group 1 HAs (H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16) and group 2 HAs (H3, H4, H7, H10, H14, and H15), they represent the type of antibody response that needs to be induced by a universal influenza vaccine. This proposed study will use innovative high-throughput experiments to define sequence features in the heavy-chain complementarity-determining region 3, light chain, and somatic hypermutations, that enable an IGHV6-1 antibody to be a cross-group HA stem bnAbs. The underlying molecular mechanisms will be further characterized by structural biology approach. The results will help accurately estimate the germline frequency of IGHV6-1 HA stem bnAbs and understand their affinity maturation pathway, which in turn will benefit the design of a universal influenza vaccine. Furthermore, the experimental framework established in this study will be applicable to characterize any antibody of interest.

NIH Research Projects · FY 2026 · 2022-01

PROJECT SUMMARY/ABSTRACT This application requests support for a web-based resource that provides community access to “genomic enzymology” tools to 1) enable identification of uncharacterized proteins for functional discovery, including those that are present in human microbiome and other metagenomic communities; 2) facilitate assignment of function to uncharacterized proteins by providing easy access to genome context/functional linkage; and 3) survey sequence-function space in protein families for identification of candidates for functional screening and improvement of desired biochemical/biophysical properties (useful for systems biology as well as the pharmaceutical and chemical applications). The resource provides three integrated tools: 1) EFI-EST for visualization/analysis of sequence-function space in proteins families using sequence similarity networks (SSNs); 2) EFI-GNT for visualization/analysis of genome context for microbial and fungal proteins in SSNs using genome neighborhood networks and genome neighborhood diagrams to identify functionally linked proteins (e.g., enzymes, transporters, and transcriptional regulators in novel metabolic pathways); and 3) EFI-CGFP for chemically guided functional profiling that maps metagenome and/or meta-transcriptome abundance to clusters in SSNs for identification of important microbiome targets for functional characterization. The resource enables access to the tools without the need for users to have computer programming expertise and/or local access to the necessary computational infrastructure. The resource has been available since 2014; its development and dissemination have been supported by U54GM093342 and P01GM118303. The resource has been accessed by >5,000 domestic and international users and cited in >300 publications and patents, confirming its ease-of- use, popularity, and impact.

NIH Research Projects · FY 2026 · 2022-01

PROJECT ABSTRACT The nervous systems of animals are comprised of neurons connected by a large number of synapses. The resulting neural networks underlie animal behavior and contribute to the storage of learned information in many species. In humans, the miswiring of neural networks likely results in disorders of behavior, learning, and thought. For all these reasons, understanding the development, organization, and disruptions in neural circuits is vital. The goal of connectomics is to produce and study the maps of neuronal connections within nervous systems. Connectomic research requires automated image acquisition of brain tissue images that cover large volumes at high magnification to resolve synapses and methods to generate wiring diagrams from these images. But the connectivity map itself is nevertheless not sufficient to explain the brain functions. Additional information, such as the molecular identity of neurons, needs to be extracted from the same nervous system. The primary goal of this proposal is to develop the heavy metal staining of whole mouse brains and other large brain samples for volumetric electron microscopic mapping of a full connectome (Aim1), generate a library of miniaturized protein binders for correlated light and electron microscopic imaging to bridge connectomics with neuronal cell type studies (Aim2), and expand the use of X-ray microscopy in multiplexed brain imaging (Aim3). These studies will lay the foundation for the development of connectomics and establish new paradigms for multimodal imaging. My career goal is to run an academic lab that develops essential methodologies for brain research. The proposed work that focuses on the most urgent needs of connectomics will become a mainstay for my independent research and allow me to integrate the newly acquired knowledge in neurobiology, biochemistry, and microscopy with my interdisciplinary training in chemistry and materials science. The unique environment at Lichtman lab will put me in a privileged position in order to pursue my career aspirations. I have developed a detailed training plan with my mentor, Prof. Jeff Lichtman, to help me transition to independence. I will meet regularly with him to discuss research progress, strategies for grant writing, student mentorship, and lab management. I will oversee the work of a graduate student to practice my mentorship skills. To broaden my scientific network and establish future collaborations, I will present my work in workshops, connectomics meetings, SfN and ACS annual meetings. As a member of the Harvard Department of Molecular and Cellular Biology, I will have access to leaders in neuroscience, molecular biology, cell biology, and biochemistry, as well as cutting-edge core facilities. The BRAIN Initiative Diversity K99/R00 will provide me the funding required to initiate an ambitious research plan to tackle the outstanding challenges surrounding brain studies.

NIH Research Projects · FY 2026 · 2021-12

PROJECT SUMMARY Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, paralytic and ultimately fatal disease characterized by the selective loss of motor neurons in the spinal cord and brain. The overarching objective of this application to the Optimization Track of the CREATE Bio Program is to refine the safety and efficacy of a gene therapy that we developed for forms of ALS caused by toxic, gain-of-function mutations in superoxide dismutase 1 (SOD1), which account for up to 20% of all familial cases of the disease. Specifically, we have developed an approach to inactivate the production of the mutant SOD1 protein in vivo using CRISPR base editing, a gene- editing modality capable of introducing precise base substitutions in DNA, but without the requirement for a mutagenic DNA break, thereby overcoming a major safety hurdle facing the implementation of traditional gene- editing nucleases. In particular, when delivered to the spinal cord via adeno-associated virus, our SOD1- targeting base-editing platform prolonged survival and markedly slowed the progression of disease in a highly aggressive mouse model of SOD1-linked ALS. Importantly, as opposed to current strategies for silencing SOD1, which target SOD1 mRNA and can have a transient effect that requires a lifetime of redosing or can risk saturating endogenous RNA processing pathways, which could then lead to adverse effects, our approach harnesses a highly precise DNA editing pathway to permanently turn-off the production of mutant SOD1 and involves only a single treatment. Thus, because of its strengths and in vivo efficacy, we now aim to refine this strategy for the ultimate goal of developing a gene therapy for ALS. Specifically, by optimizing its targeting specificity, its editing capabilities, its pharmacokinetics and its safety, which we will ensure via the introduction of a self-inactivating functionality that facilitates its clearance from cells, we will develop a highly optimized SOD1-targeting CRISPR base editing platform that can be used to permanently and effectively treat SOD1-linked ALS. Thus, by capitalizing on: (1) a highly innovative DNA editing technology that has the capabilities to overcome the limitations of gene-silencing and (2) a multidisciplinary research team with complementary expertise in ALS, AAV delivery, gene-editing and immunology, this project will result in not only an optimized therapeutic candidate for ALS, a devastating, debilitating and currently incurable disorder with few effective treatment options, but also lay the foundation for using base editing to safely treat neurological disorders.

NIH Research Projects · FY 2025 · 2021-09

ABSTRACT Rotator cuff tears are common and primarily initiate at the stratified fibrocartilage interface (enthesis) linking tendon to bone. Surgical reattachment of tendon to bone forms a narrow fibrovascular scar rather than regenerates a continuous fibrocartilage enthesis. The resultant sharp boundary between mechanically mismatched tendon and bone leads to strain concentrations and high rates of re-failure at the enthesis. The objective of this proposal is to guide functional regeneration and repair of the structure, composition, and mechanical performance of the injured tendon-to-bone enthesis using an innovative stratified biomaterial. Intraoperative implantation of MSCs at the injury site during surgical repair is an attractive option to accelerate enthesis regeneration. However it is essential to develop a biomaterial carrier to improve retention and regenerative activity of bioactive MSCs across the injury site. We will evaluate the design of an innovative stratified biomaterial to provide mechanical and trophic stimuli to promote MSC retention and enthesis regeneration. We have generated rigorous proof-of-principle data for a collagen biomaterial that contains bone- and tendon-mimetic scaffold compartments linked with a continuous hydrogel interface. We will show the hydrogel interface inhibits strain concentrations that typically form between biomaterials with mismatched mechanical properties under load. Further, the hydrogel interface provides a site to accelerate fibrocartilage- like differentiation and remodeling in response to trophic factors produced in adjacent tendon- and bone- mimetic scaffold compartments. Taken together, we hypothesize inclusion of a continuous hydrogel zone linking tendon- and bone-specific scaffold compartments provides mechanical and trophic advantages to accelerate regenerative potency versus monolithic and conventional stratified biomaterials. To address our hypothesis we will first determine if and how a mechanically-optimized hydrogel insertion both increases mechanical performance and supports fibrocartilage differentiation in vitro (Aim 1). We will subsequently demonstrate trophic factors produced across the stratified biomaterial accelerate enthesis-specific MSC differentiation and matrix remodeling in vitro (Aim 2). We will ultimately evaluate functional repair and regeneration of the rat rotator cuff enthesis using an enthesis biomaterial-MSC construct in vivo (Aim 3). We will use in vitro cyclic strain bioreactor studies to optimize MSC-biomaterial interactions, then a tiered set of in vivo rat rotator cuff injury models to benchmark the quality and kinetics of enthesis regeneration via cellular, tissue morphology, and mechanical metrics. This project will provide essential insight to aid clinical translation of a biomaterial therapy to improve musculoskeletal enthesis regeneration.

NIH Research Projects · FY 2025 · 2021-09

This proposal aims to investigate the assembly mechanisms, structures, and functions of polymeric (p) immunoglobulins (Ig) that populate the mucosa. The pIgs are found in vertebrates and together form a structurally diverse group of antibodies. They comprise several Ig heavy chain classes, including mammalian IgA and IgM, which typically contain between two and five Ig monomers and one joining chain (JC); however, potential to assemble with the JC and/or to assemble into polymers of different size varies with vertebrate species and Ig heavy chain class. Following assembly, pIgs are transported to the mucosa by the polymeric Ig receptor (pIgR). In the mucosa, the pIgR ectodomain, called secretory component (SC), remains bound and the complex is referred to as a secretory (S) Ig. SIgA is the predominant mucosal antibody in mammals; it is typically found in dimeric (d) forms; however higher order polymers such as tetramers are functionally relevant. SIgA is associated with unique effector functions compared to monomeric, circulatory antibodies; it can coat, cross-link and agglutinate commensal and pathogenic antigens and also mediate interactions with receptors on host and microbial cells. Despite significance, the structural basis for pIg assembly and SIg functions remained poorly understood through decades of immunological research. In 2020 the cryo-electron microscopy structures of SIgM, SIgA and a dimeric (d) IgA precursor were published revealing unprecedented molecular insights into these crucial complexes and opening the door to new questions and structure-guided experiments. The structures of dIgA and dimeric forms of SIgA revealed two IgAs joined through the JC to form a pseudosymmetric, bent conformation that appears to restrict the positions of antigen-binding fragments (Fabs) and promote access to receptor-binding sites. The SC is asymmetrically bound to one side and is solvent accessible, suggesting it may promote yet uncharacterized interactions with host or microbial factors. These observations raise the questions of how structural differences among pIg are generated (e.g dimer versus tetramer and JC versus no JC), how the bent, asymmetric arrangement of components is induced and maintained, and how it contributes to function. The proposed research program will use structural and biophysical approaches to target these questions. Aim 1 will identify Ig heavy chain residues, structural motifs and/or conformational changes that promote pIg assembly and control pIg polymeric state, while also determining the structural basis for JC- independent pIg assembly and function. Aim 2 will characterize JC-specific mechanisms of pIg assembly and its structural contributions to the pseudosymmetric conformation of dIgA. Aim 3 will characterize the functional significance SC and its capacity to bind microbial ligands. These studies will deliver comprehensive mechanistic models for pIg assembly, generate new pIg structures and report new SIg structure-function relationships. This outcome will improve knowledge of mucosal immunity and provide a foundation for engineering pIg and SIg in order to explore their normal functions and therapeutic potential.

NIH Research Projects · FY 2025 · 2021-09

A wearable alcohol biosensor could represent a tremendous advance towards helping people make informed decisions about their drinking and, ultimately, towards curbing alcohol-related morbidity and mortality. Transdermal sensors, which measure alcohol consumption by assessing the alcohol content of insensible perspiration, offer a uniquely non-invasive, passive, and low-cost method for the continuous assessment of drinking likely to be attractive to a range of populations. But the relationship between transdermal alcohol concentration (TAC) and blood alcohol concentration (BAC) is highly complex, varying across individuals and contexts and involving some degree of lag time. Prior research, which has featured extremely small participant samples and examined old-generation transdermal devices, has been poorly suited to modeling this complexity. Thus, scientists are left with little sense for how to translate data produced by transdermal sensors into estimates of BAC. Importantly, the past decade has seen remarkable technological and analytic developments, offering the potential to tackle the challenge of TAC-BAC translation. In particular, in recent years, machine learning approaches have been developed that are particularly well suited to modeling highly complex and time-lagged relationships within larger datasets. Also during this time period, a new generation of transdermal device has come under development, featuring sleek/compact designs, smartphone integration, and capabilities for sampling TAC at approximately 90 times the rate of older-generation devices. These sensors thus provide a rich source of data for machine learning models and also, for the first time, the potential to produce transdermal BAC estimates in real time. The proposed research leverages machine learning, novel transdermal technology, and large-scale multimodal human testing to translate transdermal sensor data into estimates of BAC. Transdermal sensors will be examined in the context of multimodal research featuring a large and diverse participant sample (N=240) examined both inside and outside the laboratory. The ambulatory arm of the proposed project is aimed at capturing the TAC-BAC relationship across individuals in varying real-world drinking contexts, examining regular drinkers wearing new-generation transdermal sensors in everyday settings while providing prompted breathalyzer readings. This ambulatory research will be complemented by a laboratory study arm, aimed at examining the TAC-BAC relationship among individuals drinking in a controlled setting while alcohol dose and rate of consumption are systematically manipulated. Machine learning algorithms, including deep neural network models, will be used to create estimates of BAC from transdermal sensor data. These estimates will be examined in terms of their accuracy, temporal specificity, and also context-dependence. Thus, results will carry significance for addiction science by translating transdermal sensor data and clarifying the place of these sensors in our arsenal of techniques for assessing, preventing, and treating problem drinking.

NIH Research Projects · FY 2024 · 2021-09

Project Summary To sense the environment, cells rely on membrane-embedded receptors. The receptor tyrosine kinase (RTK) family of signaling proteins is large, diverse, and centrally important both to human development diseases and cancers. Evidence so far supports a model that signal passage through RTKs is initiated by a structural change in the extracellular domain and then conducted through the transmembrane (TMD) and juxtamembrane (JMD) domains to the cytoplasmic kinase domain. The receptors usually are activated in the dimer form. Numerous RTK mutations confer diseases, e.g. single point mutations in ~30% of residues of the TMD of the fibroblast growth factor receptor 3 (FGFR3) are pathogenic, while mutations of tropomyosin receptor kinase A can lead to cancers. Understanding the structural interactions of the FGFR3 and TrkA signaling TMD and JMD therefore is crucial for fundamental biology and for future development of therapies that may target these pathways. Atomistically resolved TMD+JMD dimer structures are the major objective of this project. Application of traditional computational and crystallographic methods is hindered by the fluid nature of the membrane environment. Our goal is to develop novel efficient computational methods that guide and maximally leverage NMR, FRET, and in-cell experimental data and apply these methods to capture the FGFR3 and TrkA TMD and TMD+JMD dimer structures for the wild type and mutated pathogenic forms. In Aim 1, we will combine our novel highly mobile membrane mimetic model, capable of spontaneously capturing candidate TMD dimer structures, with a novel minimally biased way of applying a reduced number of computational restraints based on experimental distance measurements. The resulting TMD dimer structures will be validated by comparing computed and experimentally measured parameters. These structures will reveal the role mutations play in RTK dynamics. In Aim 2, we will use our computational-experimental approach to determine the role that juxtamembrane domains play in RTK signaling. The resolved structures of the mutated dimers will facilitate understanding of the pathology and mechanisms of receptor activation. Our novel computational approaches combined with extended expertise of co-investigators and collaborators in NMR, FRET, RTK signaling, and membrane-associated phenomena, uniquely position us to develop and apply this methodology. We will also develop an open-source, user friendly workflow plugin for a widely-used software suite that will allow efficient use of the proposed protocols by the scientific community. Completion of the specific aims will increase our ability to efficiently gain structural information on RTKs and will open new research avenues for investigating mechanisms of transmembrane signaling in health and disease leading to development of new treatments.

NIH Research Projects · FY 2025 · 2021-09

Project Abstract A large portion of important drugs and pharmaceutical intermediates either originate from plants or are synthesized from petrochemical-based products. Several natural products of significantly high value to human life (e.g., antibiotics, pharmaceuticals like Artemisinin, anticancer agents like Taxol (paclitaxel) among others) have been produced by organisms that are not optimal for industrial production. Although some of these natural products can be chemically synthesized, the complex structures of several of these compounds makes chemical synthesis difficult and commercially infeasible. As a result, there is increasing need to develop sustainable and readily tractable technological platforms to synthesize these drugs and pharmaceutical intermediates. The central objective of this proposal is to develop a sustainable technological platform that harnesses light energy and biocatalysis to synthesize molecules of significant relevance to human life. We envision doing this by establishing cyanobacterial endosymbionts within yeasts cells, such that the endosymbiotic cyanobacteria provide ATP and assimilated carbon (generated from photosynthesis) to the yeast cells, which utilize it to produce biologically important natural products. This platform will allow us to couple the remarkable biosynthetic and biocatalytic potential of yeast to the photosynthetic ability of cyanobacteria to develop a sustainable and simple bioproduction platform to produce natural products of significant value to human life (vide infra). Premise: (i) Saccharomyces cerevisiae has been recently harnessed to produce high titers of biologically important molecules such as amorphadiene and artemisinic acid, (precursors to Artemisinin) and taxadiene (a key precursor to Taxol), (ii) we had previously developed model endosymbiosis between S. cerevisiae /E. coli to study mitochondrial evolution are currently engineering yeast/cyanobacteria endosymbiosis (preliminary data in Specific Aim 1) and (iii) our preliminary data on engineering model yeast/cyanobacteria endosymbiosis. In this proposal, we will focus on three key areas: (i) We have engineered experimental platform to establish endosymbiosis between model cyanobacteria, Synechococcus elongatus, and model budding yeast, S. cerevisiae. We will expand this platform by engineering novel cyanobacterial mutants as putative endosymbionts. We will extensively characterize the engineered yeast/cyanobacteria endosymbiosis to develop strategies to improve their stability, growth rate and homogeneity. (ii) We will create a metabolite-driven synthetic communication system to control endosymbiosis and optimize our platform for metabolic engineering. (iii) We will utilize our photosynthetic endosymbiotic platform to produce key precursors of FDA approved compounds, Artemisinin and Taxol. These studies will be the first step towards our long-term goal of developing a photosynthetic and genetically tractable endosymbiotic platform for the bioproduction of biologically important novel natural products as well as molecules like biopolymers and biofuels.

NIH Research Projects · FY 2025 · 2021-09

PROJECT ABSTRACT: The ability to measure and quantify the composition and abundance of various metabolites in biological samples, also referred to as metabolomics, provides a unique window into the complex biological processes at different scales. So far, the field of metabolomics has mainly been driven by technologies based on mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. These technologies, although powerful, only measure metabolite profiles in homogenized biological extracts, e.g., biofluids or dissected tissues, thus losing the spatial information of the underlying metabolic processes. As spatial heterogeneity is a hallmark of metabolism, especially in complex biological systems such as animals and humans, obtaining spatially resolved metabolomics has been a dream of many biomedical scientists and engineers. In recent years, MS imaging (MSI) has emerged as a tool of choice for imaging metabolomics, which allows for the generation of spatially localized metabolite profiles from tissue sections. One major limitation of MSI is that it requires post-mortem or invasive tissue sampling, thus unable to probe metabolism at the most physiologically relevant states. This has limited its translation to human studies. MR spectroscopic imaging (MRSI) is another alternative for imaging metabolomics. It combines the powers of MRI and NMR spectroscopy to produce spatially resolved tissue metabolite profiles, noninvasively. However, MRSI is highly limited in its poor spatial resolutions. Furthermore, most MRSI studies only target a single nucleus (e.g., 1H), thus limited in the number of molecular species measured. The overall goal of the proposed research is to develop a research program that will pave a path towards in vivo imaging of tissue metabolomics. Specifically, we aim to develop an unprecedented high-resolution multinuclear MRSI technology that can simultaneously map a large number of metabolites in vivo, synergizing advancements in ultrahigh- field MRI instrumentation, fast data acquisition, and machine learning driven computational imaging techniques. We also propose a novel multimodal MRSI and MSI imaging framework for validating our multinuclear MRSI technology and integrating two complementary biochemical imaging modalities for tissue metabolic profiling. Novel computational approaches will be developed to analyze the high- dimensional metabolomic data. Success of the proposed research will establish a new paradigm for generating and analyzing imaging metabolomics data. This paradigm will transform metabolomics into a powerful noninvasive and tissue specific technology (from an invasive and nonspatial-specific one) for studying metabolism in living animals and humans. These advances will enable new means to unravel the metabolic basis of normal physiological functions and different diseases, inspiring developments of new biomarkers, novel treatments, disease prognosis and management strategies.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY Human antibody repertoire is highly diverse due to VDJ recombination and somatic hypermutation. VDJ recombination is a somatic recombination process that assembles the variable region of an antibody from a diverse set of gene segments, known as variable (V), diversity (D), and joining (J) genes. During the course of an immune response, antibodies will increase affinity to their antigens through somatic hypermutation. The huge diversity of antibodies enables human immune system to confer protection against various pathogens by recognizing a wide range of antigens and epitopes. Detailed molecular characterization of antibody-antigen interaction is crucial to vaccine and therapeutic development, as well as the fundamental understanding of the human immune system. The binding specificity and epitope of an antibody are determined by its structure, which in turn is determined by its amino acid sequence. As a result, information on the binding specificity and epitope of an antibody are encoded in its amino acid sequence. However, accurately predicting the epitopes of antibodies from their sequences is an extremely difficult task because our understanding of antibody sequence-function relationship is far from comprehensive. This proposal aims to develop a library-to-library screening approach to characterize antibody-antigen interaction in a high-throughput manner, with a focus on influenza A hemagglutinin (HA) as a proof-of-concept. Specifically, we will determine the HA-binding specificity and conformational epitope of hundreds of thousands of antibodies in a single experiment. Subsequently, antibody sequence features that are associated with different epitopes on HA will be systematically identified. We further aim to use these antibody features to identify HA-binding antibodies from publicly available antibody repertoire sequencing datasets as well as predict their epitopes on HA. While this proposed project focuses on influenza HA, our approach can be easily extended to any antigen of interest. This proposal will open up the possibility for antibody sequence-based epitope prediction and provides new perspectives to the understanding of human antibody repertoire.

NIH Research Projects · FY 2024 · 2021-09

The ongoing pandemic of COVID-19 is designated by World Health Organization (WHO) as a Public Health Emergency of International Concern. Similarities among ACE2 receptors predict that there are several animals could function as reservoirs for the virus. Recent studies by us and others identified felid animals, including domestic cats, tigers and lions as highly susceptible to SARS-CoV-2 infection. These findings cause great concerns on the potential for human to animal and animal to human transmission, along with the virus mutations that appear as the virus goes back and forth between species. One goal of this study is to design and prepare novel reagents and assays for detection and surveillance in animals. A second goal is to develop a feline animal model. Together, these data will be incorporated into models for understanding the risk of animal infection for veterinarians, other animal care professionals, and the general public. Specific Aims are: 1). To generate and characterize specific reagents for use in COVID-19 research and diagnostics; 2). To develop diagnostic assays for detecting COVID-19 virus infection in animals; 3). To establish a feline model to study SARS-CoV-2 pathogenesis; 4). To apply novel diagnostic assays in the surveillance of pets and zoo animal populations. Outcomes of this study will generate a panel of SARS-CoV- 2-specific antibody reagents, diagnostic standards, and assays for rapid detection of SARS-CoV-2 infection in all species of animals. The diagnostic assays will be applied to COVID-19 surveillance networks, which will identify important animal reservoirs. The novel serological assay can also serve as a DIVA test to differentiate between the vaccinated and infected animals (and humans). The feline pathogenesis studies will improve our understanding of viral pathogenic mechanisms, host immune responses, and provide the source of samples for early detection and test validation.

NIH Research Projects · FY 2025 · 2021-09

Project Abstract Adequate diet quality has the potential to promote childhood cognitive health and have a lasting impact on children’s ability for learning and achievement. My laboratory has identified that lutein, a plant pigment or carotenoid found in rich quantities in dark green vegetables, is uniquely suitable for supporting childhood cognition and achievement. Lutein is the predominant carotenoid in neural tissue, serving roles as an antioxidant across neural membranes. Further, lutein, along with two other carotenoids (i.e., zeaxanthin and mesozeaxanthin) accumulate in the macular as macular pigment, which is known to strongly correlate with brain lutein. My work has linked macular pigment optical density (MPOD) – a noninvasive measure of retinal and brain lutein – to greater childhood cognitive function. However, the cognitive implications of lutein and zeaxanthin intake in children have not been directly investigated. This proposal aims to establish a causal relationship between lutein intake, cognitive function, and academic performance. The central hypothesis is that lutein consumption will benefit cognitive function and academic achievement in preadolescents. I also anticipate that gains in cognitive outcomes will be mediated by the improvement in MPOD. These hypotheses will be tested by conducting a randomized placebo-controlled double-blind trial to examine the effects of 9-month carotenoid supplementation on MPOD, cognition (attention and memory), and achievement among 8-10-year-old children (N=288, 144/group). The active supplement will comprise of 10mg lutein + 2mg zeaxanthin. This work will provide the evidence-base for recommendations to improve dietary practices for optimal childhood cognitive function and achievement. The proposed research is relevant to human health and the NIH mission because it will provide novel data supporting evidence-based recommendations to improve dietary practices for optimal cognitive function and achievement in childhood.

NIH Research Projects · FY 2025 · 2021-09

Cerebral microbleeds (CMBs) and microhemorrhages (CMHs) result from blood leakage across the blood- brain interface (BBI). Subsequent millimeter-sized blood clots lead to inflammation, cellular injury, and neuro- degeneration. Such CMBs are associated with deterioration of BBI integrity with aging, disease, traumatic brain injury, and the sequelae of strokes, which impact >795,000 people in the United States every year. Notably, CMBs and hemorrhagic/ischemic stroke occurrence is not random, but rather clusters in early day or evening. Understanding of the circadian dynamics of the BBI with respect to vulnerability to blood leakage is limited. It has been difficult to study in vivo or via on-chip models and there is no drug treatment. In addressing this gap, this proposal responds to FOA RFA-HL-20-021. The purpose of this FOA is to support high risk/high reward research on the blood component of the Blood-Brain Barrier and the associated Interface to facilitate the development of a more complete neurovascular-blood model for translational applications with direct relevance to humans. It is an R61/R33 Exploratory /Developmental Phased Award. Because knowledge of the circadian dynamics in BBI vulnerability to blood leakage is limited, we aim to create a new biomimetic brain transport model with mimicry of the coagulation system and circadian rhythm. We will develop an innovative microfluidic platform to examine interactions of coagulation factors and circadian oscillations of both 1) the blood/vascular components and 2) dynamic vascular pressure across the BBI over the circadian cycle. We propose to reproduce circadian dynamics of the BBI by culturing human endothelial cells containing a clock-gene reporter on the ‘vascular’ side with polarized astrocytes, neurons, and microglia in the ‘brain’ compartment. This project assembles the expertise needed to facilitate the creation of enhanced platforms that more closely model the human BBI. Contributions of team members will be: Han–Microfluidics and biotransport analysis; Kong– engineering of BBI; Gillette–Assemble/validate a human iPSC circadian reporter-in-chip and assess rhythms and fluxes, and consultation from Flick on blood coagulation factors and Obrietan on the circadian reporter transgene. This grant will be separated into 2 phases: Focus in YR 1-2 (R61) will be on establishing tools and in YR 3-5 (R33) on utilizing those tools to achieve our research goals. This will enable us to replicate dynamics of the BBI in human brain and to probe it in the context of the oscillatory circadian cycle that drives integrative physiology and behavior, including sleep and wakefulness. By targeting both sides of the BBI and their intersection, we will gain insights into the emerging view that the BBI is plastic, changing with time-of-day, loss of sleep, the stress of infection, and aging. This has significant implications for the role of the circadian clock in blood coagulation in the brain and neurovascular function. The outcome will contribute to developing therapeutic opportunities that target the temporal occurrence of adverse cerebrovascular events, including hypertension, cognitive disorders and dementias, and gait syndromes.