Cornell University

NIH Research Projects · FY 2025 · 2022-08

Summary: High-speed imaging of cortical and white matter microvascular flow in AD/ADRD models Although vascular risk factors and cerebrovascular dysfunction are known to be pathogenically linked to AD/ADRD, the mechanisms are not well understood. In AD/ADRD mouse models, several causes of decreased blood flow and regulatory dysfunction that operate at different levels of the microvascular network have been identified. In mice overexpressing mutant genes that cause AD and in mice with genetic and cardiovascular risk factors for neurodegeneration, decreases in cerebral blood flow, impairment of neurovascular coupling, narrowing of capillary lumens by pericytes, and stalling of capillary flow by arrested white blood cells have been reported. However, much remains unknown because current approaches for quantifying microvascular flow are insensitive to events occurring in individual microvessels in a network (i.e. they measure averaged flow across many vessels so miss an event like a capillary stall) or are unable to evaluate network flow and perfusion changes caused by microvascular events (i.e. they measure too few vessels at a time to quantify up- and down-stream flow or regional perfusion changes due to a capillary stall). Flow and perfusion decreases and heterogeneity arising from such events could play an important role in the progression of neurodegenerative disease, as network microdomains with persistent or repeated epochs of network hypoperfusion – “oligemic micropockets” – may be hotspots for brain cell dysfunction, amyloid accumulation, and microinfarcts. Measurement of flow speed in every microvessel across a connected network is needed to investigate how transient microvascular events impact network blood flow and tissue perfusion. This proposal seeks to develop and test a paradigm- shifting approach to 2- and 3-photon (2P and 3P) excited fluorescence imaging to achieve the speed and depth penetration necessary to simultaneously measure flow in ~300 microvessels in the neocortex or ~50 in the deep subcortical white matter (WM) of mice. An adaptive excitation source (AES) generating femtosecond laser pulses “on demand” is synchronized with fast 3D raster scanning and is programmed to fire pulses only where blood vessels reside. Because the maximum laser power that can be delivered to the brain is rate limiting, AES restricts 2P/3P excitation pulses only to blood vessels enabling measurement of the speed, diameter, and signals from additional cell type-specific fluorescent labels from all microvessels in a 300x300x300 µm3 volume in the cortex or in a 200x200x100 µm3 volume in the deep WM (100 Hz volume imaging with 1x1x10 µm3 voxel size) (Aim 1). This innovative imaging capability will be used to explore the collective impact and causal links between selected molecular and cellular mechanisms of CBF abnormality in the cortex of AD mouse models, as well as to test the hypothesis that oligemic micropockets are sites of amyloid deposition (Aim 2). The ability of AES imaging to explore network flow and perfusion in the deep WM of mice enables the examination of the impact of genetic and cardiovascular risk factors associated with AD/ADRD on the WM microvascular network to provide novel insights into how global hypoperfusion induces WM damage at the microscale level (Aim 3).

NIH Research Projects · FY 2025 · 2022-08

Project Summary The study of animal viruses has been critical for understanding basic disease mechanisms, including the pathogenesis of related human viruses. This Mentored Clinical Scientist Research Career Development Award proposal presents a five-year research program to study the mechanisms by which a novel parvovirus successfully replicates in terminally differentiated hepatocytes. Equine parvovirus-Hepatitis (EqPV-H), a novel Copiparvovirus, is known to cause hepatitis and fulminant hepatic necrosis in horses. Preliminary data demonstrate multiple similarities between EqPV-H in horses and parvovirus B19 in humans, including high prevalence in clinically healthy individuals and rare but important associations with fulminant hepatitis. Parvoviruses utilize either the host cell replication machinery or the DNA damage response (DDR) pathway to replicate their viral genomes. The foundation for this proposal is based on studies evaluating the cellular division status of hepatocytes infected with EqPV-H following experimental inoculation in vivo that suggest that cells with viral replication are most commonly in G0 phase of the cell cycle. This proposal will address the following specific aims: Aim 1) Determine the cellular division and DNA damage response (DDR) status of EqPV-H-infected hepatocytes and association with pathology in situ; Aim 2) Evaluate the impact of DNA damage-induced DDR activation and DDR inhibition on EqPV-H replication and hepatotoxicity in vitro; and Aim 3) Determine the role of host cell polymerases in EqPV-H replication in vitro. These aims align with Dr. Jager’s career development goals: 1) Training in cell culture development and optimization; 2) Training in molecular virology, cell biology, and toxicology; and 3) Training in confocal microscopy and spatial transcriptomics. To achieve these goals, Dr. Jager has assembled a highly qualified team of mentors including: his primary mentor, Dr. Gerlinde Van de Walle, a veterinary clinician-basic scientist with expertise in viral pathogenesis and development of novel cell culture systems; co-mentor Dr. Colin Parrish, a skilled molecular virologist with extensive parvovirus research experience; and co-mentor Dr. Robert Weiss with expertise in the DDR pathway. Additionally, the team includes collaborators with expertise in parvoviral replication dynamics, hepatopathology, and equine parvovirus. The candidate plans to submit grants for further federal funding, including an R21 or R01 in the fourth year, with the goal of achieving independence. This K08 award will allow Dr. Jager to develop a sub-specialty in hepatopathology and viral pathogenesis that will help launch his academic career as an independent clinician- scientist.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY/ABSTRACT Post-translational modification by ubiquitin is an essential mechanism to alter protein function in eukaryotes. Ubiquitin, a 76 amino acid protein, is attached to specific proteins via a cascade of ubiquitin activating enzyme E1, conjugating enzyme E2, and ubiquitin ligase E3. Ubiquitination plays an essential role in a broad aspect of cellular processes, including transcription, DNA repair, signal transduction, autophagy, cell cycle, immune response, and membrane trafficking. Aberration in the ubiquitination system leads to a number of human diseases, such as neurodegenerative diseases and cancers. Within the ubiquitination cascade, E3s primarily dictate the specificity of the ubiquitination system and thus are often the focal points of research and attractive therapeutic targets. The Nedd4 family E3s are an essential family of HECT-type E3s, members of which contain an N-terminal C2 domain followed by 2-4 WW domains and the C-terminal HECT domain. Nedd4 E3s recognize substrates carrying a “PPxY” motif through their WW domains. However, most substrates lack such a motif but engage with the ligase through “PPxY” motif-containing adaptors. We recently discovered that in yeast, the ubiquitin E3 ligase adaptor protein Art1 is primed with di-ubiquitination and the attachment of the di-Ub chain to a specific lysine residue in Art1 is warranted for its full activity. In this proposal, we plan to investigate the physiological function of adaptor di-ubiquitination and to elucidate the molecular mechanisms of the modular ubiquitination platform form with Nedd4 E3 ligase and di-ubiquitinated adaptors. Specifically, we will pursue the following aims: Aim 1: To investigate the mechanism of Nedd4 E3 adaptor di-ubiquitination. Aim 2: To determine the role of di-ubiquitinated adaptor-E3 complexes in substrate ubiquitination. Aim 3: To elucidate the molecular architecture of di-ubiquitinated adaptors with the Nedd4 E3 ligases. Uncovering the physiological role of Need4 E3 adaptors di-ubiquitination will be of critical importance to understand the molecular basis of how E3 adaptors specifically recognize substrate proteins and efficiently present the substrates for ubiquitination by HECT E3 ligases. We expect the successful implementation of this proposal will not only make significant contributions to the understanding of the molecular mechanisms underlying the Nedd4 E3 ligase/adaptor mediated ubiquitination, but also shed light on the mechanism of protein quality control governed by targeted ubiquitination.

NIH Research Projects · FY 2026 · 2022-08

Gene regulation is central to all life, normal and diseased. The long-term goal of this research project is to un- derstand at single bp resolution the molecular organization (architecture) of proteins assembled on the Sac- charomyces (budding yeast) genome. Budding yeast represent an ideal model cellular system due to its simple genome, ease of genetic manipulation, and conservation of transcription and chromatin regulators with human cells. By understanding the precise molecular architecture of epigenomes, we gain a holistic view of genome regulation mechanisms. This project will build on our published set of genome-wide ChIP-exo data that com- prehensively measures the yeast epigenome consisting of over 400 distinct proteins. This expansion will in- volve understanding how epigenomes are reprogrammed by environmental signals. Two broad classes of re- programming will be examined: acute stress responses (e.g., heat shock and oxidative stress) and long-term unfolding of developmental pathways (e.g., starvation responses) brought on by chronic stress. Responses to acute stress reveal molecular architectures that pre-exist in the cell and then re-organize within a few minutes of sensing extracellular signaling. These events are typically transient and so must be captured upon reaching their temporal maxima. In contrast, developmental pathways unfold over hours in yeast and typically rely on de novo synthesis of gene-specific transcription factors. This project will map the precise positional organiza- tion of hundreds of epigenomic components in response to heat shock and oxidative stress, and smaller set of components in response to a much broader array of acute stresses and developmental pathways. This project will also define the functional interdependencies of epigenomic factors, with particular focus on the gene in- duction cofactors Mediator and SAGA. Relevant components of induced transcription will be rapidly depleted, then their impact on Mediator and SAGA binding to promoters examined. Other interdependencies, informed by the organization of epigenomes that will be defined during reprogramming/induction will also be examined. Together these aims will help provide a more thorough understanding of the protein architecture of gene regu- lation that should allow computational prediction of novel gene-environment interactions in diseased tissue.

NIH Research Projects · FY 2026 · 2022-08

Project Summary: Although bacterial ribosomes have been biochemically interrogated for decades, unknown mechanisms of regulation and quality control are regularly uncovered and targeted with new antibiotics. Major differences in translation regulation between Gram-positive and Gram-negative bacteria have come to light in recent years. Although more than 200,000 infections per year in the United States are caused by antibiotic resistant Gram- positive bacteria, major gaps remain in our understanding of how Gram-positives perform ribosome quality control. To address these gaps, my research program will focus on two major areas. 1) We will identify and characterize strategies used by Gram-positive bacteria to detect and rescue stalled ribosomes and investigate the physiological impacts of ribosome stalling. Preliminary data from my laboratory supports a model in which ribosome stalling in Bacillus subtilis and Bacillus anthracis results in frameshifting and premature translation termination. This process is expected to result in toxic truncated proteins and trigger stress responses. We will investigate this model using genetic, structural, and biochemical approaches. 2) We will determine how ribosome flexibility and atypical translation events can be used by the cell to increase coding capacity. We are particularly interested in how frameshifting and stop codon read-through regulates gene expression and how environmental inputs control this type of regulation. We will also use unbiased high throughput genetics to uncover new mechanisms that prevent stalling and that regulate programmed frameshifting and stop codon read-through.

NIH Research Projects · FY 2024 · 2022-07

PROJECT ABSTRACT The candidate, Dr. Elizabeth Moore, seeks the proposed Mentored Research Scientist Development Award (K01) to acquire the necessary training and experience to become an independent translational clinician- scientist focusing on tumor microenvironmental regulation of breast cancer (BC) genome stability and therapeutic resistance. BC therapeutic resistance remains a major hindrance to successful treatment, particularly in the aggressive triple negative (TNBC) subtype. However, it remains unclear how therapeutic response is influenced by the physical and biological characteristics of the tumor microenvironment and which role altered DNA damage response (DDR) mechanisms and metabolic reprogramming play this process. In particular, it remains to be elucidated how fibrotic remodeling of the extracellular matrix (ECM), which is a hallmark feature of a protumorigenic microenvironment, impact BC risk and drug resistance. Given these connections, the overall objective of the proposed studies will be to identify mechanisms of matrix-mediated BC therapeutic resistance. Aim 1 will determine the impact of fibrotic ECM on BC DNA damage response (DDR). Aim 2 will test how fibrotic ECM regulates the DDR and metabolism of tumor cells and their reciprocal interactions. Aim 3 will test the functional links between fibrotic ECM, DDR, and metabolism in vivo. Aims 1 and 2 will utilize translationally relevant, high fidelity 3D tissue culture systems in which features of fibrotic ECM remodeling can be selectively adjusted. Aim 3 will leverage a humanized mouse model of mammary fibrosis and TNBC using human BC cell lines and patient derived xenografts of TNBC. A multidisciplinary approach, including classic molecular techniques, gene expression analyses, metabolomics and Seahorse metabolic analyses, and advanced imaging will be applied to achieve these aims and for the candidate to acquire additional technical skills. Dr. Moore’s mentor is a leading expert in engineering in vitro and in vivo models to study tumor-microenvironment interactions. Co-mentors will provide expertise in the fields of genome stability and the DDR, cellular metabolism and metabolomics, and clinical aspects of breast cancer. Faculty expertise and interdisciplinary collaboration in oncology research is exceptionally strong at Cornell University and further strengthened by the training environment and exceptional core facilities. The planned career development activities, including technical research training, coursework, attendance of seminars and conferences, experience in grant and manuscript preparation, and the refinement of teaching, mentorship, and laboratory management skills will support Dr. Moore’s transition to independence. The incorporation of physical and life science approaches, utilization of highly translational in vitro and in vivo platforms, and the integration of both extracellular and intracellular regulation of BC genome stability will enable Dr. Moore to establish a niche in oncology research. Proposed activities will generate data for a future R01 application and launch Dr. Moore’s faculty career in advancing the field of breast cancer biology with the goal to improve patient prognosis.

NIH Research Projects · FY 2026 · 2022-07

PROJECT SUMMARY/ABSTRACT Pancreatic ductal carcinoma (PDAC) is the most common form of pancreatic cancer and is highly lethal and resistant to therapy. There is a need to explore new, effective, strategies to treat PDAC, given that only ~10% of the patients survive beyond five years. PDAC overutilize extracellular nutrients to sustain their growth. This nutrient dependency, coupled with a low blood supply, limits nutrient availability in the PDAC microenvironment. To achieve therapy and improve patient survival outcome, it is important to understand how PDAC survive in the nutrient-limited condition and the tumor-intrinsic or microenvironmental factors that sustain their survival. In this proposal, we show that PDAC cells rely on cysteine at a far greater extent than other amino acids. Metabolomics profiling revealed that the PDAC cells almost exclusively use cysteine to sustain intracellular glutathione (GSH). While some PDAC cells rapidly generate GSH when starved of cysteine, others maintain their GSH pool when starved of both cysteine and arginine, indicating the use of various mechanisms to sustain GSH and survival in PDAC cells. In addition, we found that under the same cysteine starvation, macrophages produce GSH, which is an important discovery given the high abundance of macrophages in PDAC microenvironment, their arginine catabolic function, and that the macrophage-derived GSH could sustain PDAC. In multiple gene expression datasets of patient tumors, we observed that PDAC express a high level of GSH pathway genes. Based on these data, we hypothesize that GSH is a core nutrient required for PDAC growth, is potentially sustained by tumor-associated macrophages, and that disrupting GSH utilization could improve therapy in PDAC. The aims of this study are 1). to determine the molecular mechanisms driving the dependency of PDAC on GSH – including the epigenetic regulation of GSH pathway, and 2). to determine the role of tumor-associated macrophages as a source and modulator of GSH in PDAC. The overarching goal is to explore whether blocking GSH utilization alone or alongside macrophage activities could be a way to improve PDAC therapy. Aim 1 will be pursued at the K99 phase, while most of Aim 2 will be pursued at the R00 phase. Methods will include gene interference (e.g., CRISPR/Cas9, shRNA, siRNA), pharmacological inhibitors of GSH pathways (including the pentose phosphate pathway), cell culture assays, metabolomics (including stable isotope tracing), dietary mouse models, bioinformatics, promoter analysis/epigenetic methods, RNA sequencing (single cell and bulk), immunohistochemistry, flow cytometry and mass cytometry. The project will receive input from a 5-person mentorship team that have expertise in tumor immunology, metabolism, bioinformatics, and epigenetics. The expected results could a) offer new insights on disrupting GSH pathway to suppress PDAC growth, b) reveal new microenvironmental mechanisms that enable tumor adaptation in nutrient-limited state, and c) reveal new opportunities to overcome resistance to chemotherapy or immunotherapy in PDAC.

NIH Research Projects · FY 2025 · 2022-07

PROJECT SUMMARY Lymphatic vessel (LV) differentiation, development, and morphogenesis are central in maintaining fluid homeostasis, regulating host immunity, and transporting dietary fat and neuronal waste. All these functions are governed by lymphatic drainage, a transport of interstitial fluid into the lymphatic system through the initial LVs and collecting LVs. The initial LVs show permeable button-like junction morphology and are ready to uptake interstitial fluid; by contrast, the collecting LVs are less permeable with zipper-like junction structure, so that the collecting LVs transport ‘lymph’ to lymph nodes without leaking. Impaired lymphatic drainage contributes to many human diseases, such as lymphedema, immune dysfunction, fibrosis, obesity, cancer, and Alzheimer’s disease. While little is known about why LVs become dysfunctional, clinical studies reveal that inflammation is one of the leading contributors to the lymphatic dysfunction. Although dysfunctional collecting LVs has been extensively studied, how inflammation impacts initial LV development and morphogenesis is unclear, because in our current experimental models, including animal models, we often cannot decouple multifactorial inflammatory factors in the lymphatic endothelium. Since two-dimensional cell culture has failed to recapitulate three-dimensional (3D) tissue architecture of lymphatics, researchers have developed 3D in vitro models of LVs, demonstrating lymphatic sprouting, lymphatic network formation, and LV interactions with other cells. However, these previous models have not created 3D lymphatic structure with specialized LEC junction development enabling controlled fluid drainage through the button-like junctions and physiological inflammatory response. In this proposal, we will use a bioengineered in vitro 3D lymphatic vascular system, exhibiting button-like junction morphogenesis of the LVs and fluid drainage to understand the regulation of LEC junction and drainage by focusing on ROCK1/2 and integrin α5 signaling. In Aim 1, we will examine the roles of ROCKs in LEC junction and drainage. Next, we will scrutinize the mechanisms of ROCKs-mediated junction zippering in LECs. In Aim 2, we will study integrin α5 mediated regulation of LEC junction and lymphatic drainage. We will then determine signal transduction through ROCKs and integrin α5 and evaluate therapeutic efficacy of targeting ROCKs and integrin α5 in lymphatic dysfunction and inflammation models in vivo. In summary, we will use a bioengineered model of 3D lymphatic vessels and fluid transport to provide an understanding of lymphatic drainage in normal and inflammatory conditions.

NIH Research Projects · FY 2025 · 2022-07

Project Summary: This is an R24 proposal to establish a Regional and National Resource in modern Electron- Spin Resonance (ESR) Technology for use by the biomedical community. This resource will build upon 20 years of a very successful National Biomedical Center: ACERT (funded by an NIH P41 grant), in which the new ESR technologies that were developed, driven by biomedical collaborators, were then provided to extensive collaborators and service projects. In its new configuration as an R24 resource, it will focus on user services and collaborations utilizing the already established extensive and unique technologies that have already been developed. We have a well- established administration and operations scheme for this objective. Through outreach efforts we plan to significantly expand the user base both nationally and internationally. We will be providing extensive educational and training capabilities for the expanded user base. This will include a series of workshops, on-line resources, in house training and visits to other labs. The outreach will include tutorial papers in key journals, virtual meetings with the International ESR Society, video lectures on YouTube, and advertisements in leading Journals and Newsletters. The ESR resources we provide are in many ways unique in the world. In addition to our commercial spectrometers for cw and pulse ESR covering the frequency range of 9 to 35 GHz, we have extensive and unique custom-home built spectrometers covering the range of frequencies 9 to 240 GHz. These spectrometers are dedicated to a full range of ESR methods utilized by the biomedical community, especially including techniques for conducting pulse dipolar ESR (PDS), which provides distances between labeled protein and RNA sites from which protein tertiary structure may be inferred. It is applicable to protein complexes in their natural states. ACERT has developed methods that both significantly increase the sensitivity to low sub- micromolar protein concentrations and very small samples, and they greatly increase the throughput, enabling a greater rate of processing of the many samples studied in PDS. While PDS supplies protein structures, the additional techniques of multi-frequency ESR and pulsed 2D-ESR at ambient temperatures supply details of protein dynamics, important in learning about structure/function; this includes the unique ability to study in real time nanosecond to microsecond dynamics, which will address such issues as domain motions in proteins. Our innovative software enables the fitting of the complex 1- and 2-D spectra to extract the details of the complex protein motions, and our recent developments of signal processing software have led to unique packages for greatly increasing the signal-to-noise of weak ESR signals while providing great fidelity, as well as accurately transforming the results of PDS experiments into (complex) distance distributions. These software methods will also enhance the throughput of the Resource.

NIH Research Projects · FY 2025 · 2022-07

Abstract An exciting recent development for high spatial resolution deep tissue imaging is long wavelength three- photon fluorescence microscopy (3PM). Since its first demonstration of imaging subcortical structures in the mouse brain, 3PM has driven rapid progress in deep tissue imaging beyond the depth limit of two-photon fluorescence microscopy (2PM). Long-wavelength 3PM is perhaps the most promising new technology for deep imaging within scattering biological tissues, and has potential impacts in a large number of biomedical fields such as neuroscience, immunology, and cancer biology. On the other hand, there are a number of challenges that must be overcome before 3PM can reach its full potential. Because it is a higher-order nonlinear process, three- photon excitation (3PE) is inherently weaker than two-photon excitation (2PE). The weak signal strength of 3PM is particularly problematic for fast imaging of dynamic cellular process. Furthermore, the laser sources for 3PM are not yet optimized for deep tissue penetration, and the complexity and cost of the excitation source is a major barrier for the applications of 3PM in a typical biomedical research lab. Finally, nearly all 3PM applications today are in the brains. Reaching anatomical frontiers is equally possible in other organs with 3PM, but explicit demonstrations of intravital imaging in novel locations are needed to bring deep imaging capability to other biological systems. The research activity of this proposal will directly address the above challenges for in vivo deep tissue 3PM. We will develop a new generation of 3PM that will improve the performance of existing 3PM by two orders of magnitude and enable multi-color deep tissue imaging with a single excitation wavelength. We will demonstrate the unprecedented imaging capabilities with a low-cost, fiber-based laser system, removing a key barrier for the deployment of 3PM in biology labs. Furthermore, by applying our techniques to a wide variety of biological systems, we will create a valuable knowledge base for the applications of 3PM. Our development of the next generation 3PM parallels the development of 2PM, where the concerted development effort in lasers, microscopes, and biological applications in the 1990s made 2PM ubiquitous in biomedical research labs by the early 2000s. Our vision is to make deep, fast 3PM a routine instrument for a wide variety of biomedical applications just as 2PM does in the shallower regions of biological tissues and organs. The successful completion of this program will enable visualization of dynamic process at the sub-cellular level in intact organs and animal models that are completely beyond the reach of any existing imaging techniques.

NIH Research Projects · FY 2026 · 2022-06

ABSTRACT Diffusible signal factors (DSFs), long-chain fatty acids with a characteristic cis-2 unsaturation, are produced and used by several genera of gram-negative bacteria as quorum-sensing signals. We have found that the DSF cis-2 hexadecenoic acid (c2-HDA) is extremely potent in inhibiting expression of Salmonella functions necessary for colonization of the intestine and have found this compound to be present in the murine large intestine. As no mammalian source of fatty acids harboring a 2-cis unsaturation has been described, these findings strongly suggest that constituents of the gut microbiota produce and excrete DSFs that inhibit Salmonella virulence. We hypothesize that Salmonella uses the signals of these bacteria to balance its virulence functions, essential but also costly to the fitness and survival of the invading bacteria, with colonization and proliferation of the Salmonella population. Gut microbial metabolites may therefore serve multiple coordinated purposes in pathogens, balancing virulence functions with those required for proliferation within a host and thus affecting pathogen survival in the gut by multiple means. Here we propose to: Aim 1: Use complementary approaches to identify bacteria of the human gut microbiome that produce inhibitory DSFs and characterize their products; Aim 2: Identify the constellation of functions regulated in Salmonella by DSFs and identify mechanisms of this control, and; Aim 3: Using established murine models of Salmonella infection, characterize the biological function and translational relevance of c2-HDA to understand its mechanism of action and to support the eventual development of novel therapeutics, such as live biotherapeutic products, for the control of human salmonellosis.

NIH Research Projects · FY 2025 · 2022-06

Abstract In this proposal, we will develop, manufacture, and perform a multi-site sub-Saharan African clinical validation of KS-COMPLETE — the first true point-of-care sample-to-answer diagnostic system for Kaposi's sarcoma (KS). Our recent large-scale studies in Africa have shown that KS can be diagnosed through quantification of Kaposi's sarcoma herpesvirus (KSHV) DNA in a skin biopsy with high sensitivity and specificity. These efforts have also resulted in the development of TINY — a robust, easy-to-use, infrastructure-free, point- of-care (PoC) technology for KSHV DNA quantification — which is being currently deployed in a multi-site evaluation. The work has also revealed that the key challenge to widespread adoption of skin biopsy-based PoC systems is the time and manual steps required to extract DNA from a skin biopsy — which can be up to 4 hours. KS-COMPLETE will be the first “direct-to-LAMP” diagnostic system for skin punch biopsies. Similar direct-to-LAMP methods have greatly simplified PoC diagnostics for other sample matrices but the solid-phase, collagenous nature of skin has made this a challenge for biopsies. KS-COMPLETE will address this issue with our “SLICER” technology that will automatically process a punch biopsy into smaller “micro-cores” on which we can directly perform DNA quantification in TINY through our “direct-to-LAMP” approach. This approach will reduce the time to result to around 60 minutes, eliminate all the current manual and intensive sample processing steps, and is compatible with cost, robustness, infrastructure, and simplicity requirements for operation in LMICs. Clinical validation of the system will be done through our established network of KS clinical sites in Africa. By the end of the project, we will deliver 12 KS-Complete systems and conduct a multi-site clinical validation. KS is one of the most common cancers in men and women in sub-Saharan Africa. KS is difficult to distinguish from other skin conditions, particularly in Africa where access to trained pathologists is limited and immunohistochemistry is practically non-existent. Early-stage and more accurate diagnosis would confer many clinical benefits. For patients who have KS, it obviates the need for the difficult to obtain, slow, and unreliable histopathology and allows for detection at earlier clinical stages resulting in better clinical outcomes. For patients with mimickers, rapid exclusion of KS allows for timely re-orienting of the diagnostic process and prevents use of potentially toxic chemotherapy. Our direct-to-LAMP diagnostic test could have significant impact beyond the diagnosis of KS as multiple other viral, mycobacterial and fungal-related skin diseases currently diagnosed through traditional pathology could be transitioned to this method and ultimately the point of care.

NIH Research Projects · FY 2026 · 2022-06

ABSTRACT Viruses that successfully evade clearance without killing the host establish chronic infection. However, unresolved low-grade inflammation causes morbidity over time, including development of cancers. How to appropriately modulate immune responses to chronic infection without further damaging the host represents a major clinical challenge. Type-17 immunity is invoked by extracellular bacteria and fungi to control resident microbiota and invading pathogens at barrier surfaces, and to promote tissue repair. Th17 cells have received much attention as drivers of inflammation in chronic autoimmune diseases. However, there are sparse data regarding the role of type-17 responses in response to viral infection. Here we have employed the well-characterized model infection, LCMV clone 13, to test the role of IL-17 during chronic viral infection. IL-17 was increased systemically during the switch to the chronic phase of infection. Using genetic and antibody-mediated blockade of IL-17, our data unexpectedly reveal that IL-17 regulates Th1 and CD8+ T cell activation, exhaustion and immunopathology during LCMV infection. We have identified lymphoid stromal cells known as fibroblastic reticular cells (FRC) as key intermediaries of IL-17 effects in secondary lymphoid tissues. Gene expression analysis and antibody-mediated blockade support a role for excess IFNg in driving T cell exhaustion and immunopathology. These unexpected findings lead to our central hypothesis that IL-17 has an immunoregulatory role during chronic infection by limiting antiviral T cell IFNg-mediated exhaustion and immunopathology. This project is designed to dissect the key elements that we have identified to be required in this unexplored immunoregulatory pathway in chronic infection by probing the source of critical IL-17 (aim 1), LN stromal cells as targets of IL-17 (aim 2) and IFNg-mediated effects on exhaustion and immunopathology in absence of IL-17 signaling (aim 3). Together these data will define a novel and previously unexplored axis operating through IL-17 signaling in stromal cells to regulate IFNg- mediated pathology, revealing new opportunities for future therapeutic intervention in chronically infected people.

NIH Research Projects · FY 2025 · 2022-06

Extending imaging depth is one of the grand challenges in optical microscopy, and many creative approaches are under development to mitigate the detrimental impact of the phenomenon of ‘optical scattering’ and enable deeper optical imaging in scattering media. Light propagating in dense tissue undergoes scattering events that scramble the phase of the propagating optical wavefront, and thus disrupts the constructive interference needed to focus/spatially localize the light to a diffraction-limited focal spot. Consequently, microscopic resolution is typically only available in the so-called ‘single-scattering’ (SS) or ‘ballistic’ light regime. OCT is one of the leading modalities in the field of deep microscopy, with maximum imaging depths typically 1–2 mm in scattering tissues. However, the incredible success of OCT has in some ways led to lower motivation than in other optical imaging fields to develop new approaches to address the problem of multiple scattering (MS). This is also a great opportunity – by building upon its already deep imaging capabilities, OCT has the opportunity to once again be at the forefront of research on pushing the imaging depth limits of optical microscopy. We propose an integrated approach that combines (1) long-wavelength OCT (1700 nm window, lower scattering coefficient supporting deeper imaging), (2) spectral-domain OCT (SD-OCT) in the conjugate imaging configuration to enhance the deep OCT signal by 2-3 orders of magnitude relative to the standard imaging configuration, (3) hardware adaptive optics (HAO) to correct tissue-induced aberrations and thereby boost the ballistic signal deep within tissue, and (4) aberration-diverse OCT (AD-OCT) for suppressing MS. Our recently-developed AD-OCT approach combines the advantages of a fiber-based OCT system with the principle behind the highly promising coherent accumulation of single scattering (CASS) method. The CASS method coherently accumulates SS from multiple illumination angles (plane wave illumination in full-field imaging geometry), whereas AD-OCT coherently accumulates SS arising from illuminating the sample with different known aberration states, and leveraging computational adaptive optics (CAO) to circumvent the resolution penalty normally associated with these aberrations. Aim 1 will develop a method to overcome the aberration-diversity saturation limit, implement high- speed GPU-based processing to address the Big Data problem in AD-OCT, and enable real-time feedback at the time of imaging. Aim 2 will quantitatively compare the performance of Gaussian-beam OCT (with and without HAO correction of tissue aberrations) vs. AD-OCT (with HAO correction of tissue aberrations). This will include measurements of the depth-dependent 3D point-spread-function, which will also fill an important knowledge gap in fundamental research on MS in OCT. Aim 3 will demonstrate AD-OCT beyond the current OCT multiple scattering limit in human skin and mouse brain in vivo (we will ‘unlock’ the 2-5 mm depth range). If successful, this proposal will demonstrate the deepest OCT imaging ever performed in human skin and mouse brain, and so is significant from the perspective of fundamental imaging science and the biomedical applications of OCT.

NIH Research Projects · FY 2026 · 2022-06

PROJECT SUMMARY/ABSTRACT The sequencing of many tens of thousands of human genomes has revealed a plethora of sequence differences or variants. Most variants appear to be of no or little functional consequence; however, a small fraction of these variants can alter genome regulation and the susceptibility to and prognosis of particular diseases. Our goal is to develop and use a general and efficient approach to identify and characterize Alzheimer-Disease-associated Variants (ADaVs) that reside in powerful transcription regulatory elements (TREs) called enhancers, which are distributed across vast non-coding regions of the genome and can map considerable distances from the genes that they regulate. The TREs that contain ADaVs are called here ADaV-TREs. Our unique approach relies on our recent demonstration that divergent transcription of enhancer RNAs (eRNAs; most sensitively detected by our PRO-cap assay) is the best mark for precisely defining active enhancers genome-wide (generally to 300 bp or less). We focus on identifying ADaV-TREs associated with AD, the most common cause of dementia, using the exquisitely-controlled differentiation of an induced-pluripotent stem cell line, WTC11, to generate highly homogeneous excitatory neurons and microglia, two of the most relevant cell types in AD. In Aim 1, we use our PRO-cap assay to identify and delimit all TREs in this pair of CNS cell types. These TREs that overlap ADaVs, either rare variants from Whole Genome Sequencing or common variants from Genome Wide Association Studies, provide a highly enriched set of variants that are likely relevant to genome regulation and a particular disease, i.e., AD. In Aim 2, we examine enhancer activity of each ADaV-TRE by high-throughput eSTARR-seq assays relative to the reference (WT) allele. Additionally, we will assay synthetic mutations in these TREs that target and cripple specific TF motifs, and features of core promoter pairs that direct divergent enhancer RNA (eRNA) transcription, using high-throughput mutagenesis and eSTARR-seq assays. In Aim 3, to characterize genome-wide effects of those ADaV-TREs and synthetic mutations showing the most robust alteration in enhancer activity, we will use CRISPR to introduce these perturbations at native loci into WTC11 cells and induce these to differentiate to relevant CNS cell types. We will then characterize the effects of these perturbations using: 1) PRO-seq to measure changes in genome-wide transcription at base-pair resolution; 2) chromatin conformation capture (4C-seq) to examine changes in the 3D enhancer-promoter interaction profiles; 3) ChIP-qPCR to measure alterations in transcription factor (TF) and co-activator binding; and 4) phenotypic assays to reveal disease phenotypes. Our systematic and molecularly-precise analyses will identify TREs that are altered in their regulatory activity and long-range interactions by variants, as well as the TFs and coactivators whose association with TREs are affected. This information will be hypothesis-generating and critical for modeling genome regulation and for dissecting molecular mechanisms of AD.

NIH Research Projects · FY 2025 · 2022-05

Project Summary/Abstract Progression through the cell cycle involves spatiotemporal coordination of cytoskeletal and membrane dynamics with controlled proteolysis events. The anaphase-promoting complex/cyclosome (APC/C) is the main E3 ubiquitin ligase regulating mitosis. Whereas the temporal control of APC/C-mediated ubiquitination is well established, the spatial organization of APC/C function is a key uncharacterized dimension to its activity, access to substrates, and effects on mitosis. We have identified a novel link between phosphoinositides (PIPs), which form a lipid-based code of membrane identity, the microtubule cytoskeleton, and the APC/C that is mediated by PLEKHA5, a pleckstrin homology (PH) domain-containing, PIP-binding protein. We discovered PLEKHA5 as a microtubule- and plasma membrane-localized protein interactor of the APC/C whose depletion by siRNA antagonizes mitotic progression, causing a buildup of APC/C substrates. We propose that PLEKHA5 regulates APC/C subcellular localization and thus controls access to key mitotic substrates. Yet, it is unknown which aspects of PLEKHA5’s molecular properties are required for its cell cycle functions. As well, the localization of the APC/C at different stages of the cell cycle still remains largely a mystery, as is the effect of PLEKHA5 on APC/C localization and function. Further, the role of PLEKHA5 in modulating the composition and E3 ligase activity of the APC/C is unknown. Our long-term research goal is to understand how PIP-sensing proteins read the dynamically changing lipid composition of membranes and transduce this information to regulate the localization and function of important proteins in cell signaling. The objective of this proposal is to understand the molecular events through which PLEKHA5 controls the localization and activity of the APC/C and thus regulates cell cycle progression. The central hypothesis guiding this work is that PLEKHA5 engages the plasma membrane and the microtubule cytoskeleton in a spatiotemporally controlled fashion and recruits the APC/C to these locations to access substrates whose ubiquitination is critical for progression through mitosis. In this proposal, we will first establish molecular mechanisms governing PLEKHA5 regulation of mitotic entry and progression by testing the hypothesis that PLEKHA5 localization and interactions with APC/C are important for its effects on mitosis. In addition, we will determine subcellular localizations of APC/C and the role of PLEKHA5 in controlling APC/C localization and function by developing and applying a suite of “in vivo biochemistry” tools to assess the localization of APC/C, evaluate its colocalization with PLEKHA5, elucidate the effects of PLEKHA5 depletion on APC/C localization, and ascertain how ectopic localization of PLEKHA5 affects its cell cycle functions. Finally, we will determine the direct effects of PLEKHA5 on the composition and in vitro E3 ubiquitin ligase activity of the APC/C. Our studies will establish a new mechanistic framework for understanding how spatial organization of the ubiquitination machinery affects cellular pathways important for health and disease.

NIH Research Projects · FY 2025 · 2022-03

Project Summary/Abstract This project aims to develop a complete toolbox for a new technique of mosaic analysis that is compatible with nearly all existing Drosophila melanogaster resources, without the need for further genetic modifications. Mosaic analysis is a powerful approach for studying molecular and cellular mechanisms of human disease. By generating homozygous cells in otherwise heterozygous animals, mosaic techniques allow tissue-specific analysis of pleiotropic genes and have played key roles in many important discoveries in biology. Current mosaic techniques in Drosophila primarily rely on the Flp/FRT site-specific recombination system and require a pair of homologous chromosomes that each contains an FRT sequence near the centromere. However, most existing genetic resources in Drosophila, such as deficiency libraries, transposon-disrupted mutant collections, and strains derived from wild natural populations, do not harbor appropriate FRT sites, preventing their efficient application in mosaic analysis. New mosaic techniques that do not depend on site-specific recombination systems are needed to unleash the full potential of these existing resources. Mosaic analysis by gRNA-induced crossing-over (MAGIC) is a new mosaic technique that does not require site-specific recombination and is compatible with unmodified chromosomes. Although the effectiveness of MAGIC has been demonstrated in the germline, the nervous system, and epithelial tissues, MAGIC reagents are presently only available for a single chromosome arm. This project will first establish an optimized MAGIC toolkit that can be used for mosaic analysis over the entire genome throughout Drosophila tissues. MAGIC screens will also be conducted to identify deficiency lines that are associated with morphological defects in neurons and epithelial cells. Specifically, four aims are proposed: (1) establish an optimized and complete MAGIC toolkit for all Drosophila chromosome arms; (2) generate strains expressing Cas9 in precursor cells of diverse tissues for MAGIC applications; (3) develop anti-CRISPR tools for safe and versatile MAGIC applications in Drosophila; and (4) screen deficiency lines by MAGIC for genes involved in neuronal and epithelial morphogenesis. The Drosophila strains and constructs developed in this project will be donated to the Bloomington Drosophila Stock Center and plasmid depositories, respectively, for easy distribution to the research community. The screen results will be made publicly available for further identification and characterization of responsible genes by interested labs. An advisory committee has been established to provide feedback to this project. Community inputs will be solicited regarding candidate precursor Cas9 lines to generate. The proposed MAGIC tools will allow exploitation of existing genetic resources in systematic gene-function analysis, genome- wide genetic screens, and tissue-specific analysis of natural variants. Attaining the goals of this project will provide the Drosophila community important tools and information that can greatly enhance the value of existing Drosophila resources for understanding human disease.

NIH Research Projects · FY 2025 · 2022-03

Proper growth, septation, and maturation of the cardiac outflow tract (OFT) into valved aortic and pulmonary outlets are essential for oxygenated circulation after birth. 1-2% of live births and up to 30% of pre-term fetal deaths have congenital heart defects, many of which affect the remodeling of the valvuloseptal primordial tissues, called the proximal and distal outflow cushions. Despite much effort uncovering the genetic basis of early OFT cushion formation, this understanding has not explained the clinically relevant phases of growth, condensation and elongation into valves and septa. One reason for this appears to be the domination of conditional and collective signaling mechanisms that are well accessible by genetic approaches. Mechanical forces (shear stress, pressure, tension) are ever present during this complex period of OFT growth and remodeling, but to date no studies have investigated these key interactions, especially for their contributions to OFT defects. We believe that clinically relevant OFT remodeling arise from improper cushion endocardial and/or mesenchymal sensation of and/or response to their local mechanical environment, which in turn drives the incorrect signaling programs. The Butcher lab has pioneered innovative technology 1) to quantify local in vivo mechanical forces within this OFT region and register them with local in situ gene/protein expression, 2) to not-invasively visualize and precisely ablate intracardiac tissues without collateral damage in vivo, and 3) to directly test mechanobiological mechanisms of endocardial cushion growth and remodeling ex vivo. The preliminary data in this proposal present evidence of two mechanoregulated molecular switches that potentiate between OFT cushion proliferation and differentiation, which motivates the novel hypothesis that local mechanosensaton operates molecular switches to control sizing, shape, and stratification of the outflow valves and septa. Aim 1 will implement innovative non- invasive laser photoablations of the formed proximal or distal cushions of the avian OFT to create genetically unbiased clinically relevant outflow tract malformations. We will then quantitatively analyze and register their hemodynamic, morphological and phenotypic changes. We will further apply novel deconvolution integration of sc-Seq and slide-seq to reveal unprecedented spatio-temporal resolution of the cellular course of malformation, and elaborate how known and newly discovered molecular regulatory programs associate with local mechanical stress changes. Aim 2 will test the mechanistic causailty of the mechanotransduction operated molecular switches in the OFT cushion endocardium via shear stress patterns. Aim 3 will test the operation of different mechanobiogical switches in cushion mesenchyme via tension/compression. using high throughput ex vivo organ cultures. The findings from these studies will substantally advance our understanding of mechanoregulation and conditional signaling in outflow tract valuvloseptal maturation, paving the way for strategies to manipulate such signaling programs to reduce or even rescue CHD severity in utero.

NIH Research Projects · FY 2026 · 2022-03

PROJECT SUMMARY: Cold temperature (<15°C) exposure stimulates perivascular beige adipocyte progenitor cells (bAPCs) to generate beige adipocytes. Beige adipocytes act as cellular furnaces to burn blood glucose and free fatty acids to generate heat. Recent studies have shown the metabolic benefits of beige adipocytes, suggesting potential clinical efficacy for obese patients and type 2 diabetics. However, the potential to form cold-induced beige adipocytes declines with age, creating a pivotal challenge to the therapeutic promise for older individuals, many of whom constitute the obesity epidemic. Our studies begin to unravel how aging suppresses beige adipogenic potential and identifies new ways to rejuvenate beige fat cell biogenesis to restore metabolic fitness in aged mammals. Our previous studies have linked cellular senescence, a state of cellular arrest, of bAPCs to the age- associated decline in beige adipose tissue. In an attempt to find additional mechanisms blocking beige fat biogenesis in aged mammals, we found that the expression and signaling of platelet derived growth factor receptor beta (Pdgfrβ) is increased in aged bAPCs. Moreover, ablation of Pdgfrβ within the beige adipose lineage restored beige adipocyte generation and improved metabolic health in aged (not young) mice. Despite beige fat formation in aged Pdgfrβ-deficient mice, lineage-tracing studies revealed that auxiliary source(s) generated beige adipocytes. In agreement, senescence tests demonstrated that Pdgfrβ neither promoted nor reversed cellular senescence. Instead, we found that Pdgfrβ signaling prevents group 2 innate lymphoid cell (ILC2) recruitment and activation within iWAT depots. Mechanistically, we identified that Pdgfrβ elicits signals via Stat1 to suppress the ILC2-inducer, interleukin-33 (IL-33), to control WAT ILC2 activity. Finally, we identify sympathetic tone as a significant regulator of age-induced Pdgfrβ expression. Our aims will elucidate the physiological and cellular role of Pdgfrβ in regulating beige fat biogenesis under aging and obese conditions. We will elucidate the Pdgfrβ-Stat1 signaling mechanism in bAPCs to control ILC2 recruitment via IL-33. We uncover how sympathetic output regulates Pdgfrβ expression to drive the age-dependent beige adipogenic failure. These findings will implicate Pdgfrβ signaling as a central node in the bAPC aging process. Importantly, this application will identify factors that reverse age-dependent beige adipogenic failure with a direct clinical utility to combat excess body fat and metabolic dysfunction to extend lifespan and restore health.

NIH Research Projects · FY 2026 · 2022-02

Abstract. The studies outlined in this proposal focus on the mechanisms by which aggressive breast cancer cells generate large numbers of exosomes with unique cargo, together with a total secretome that significantly enhances their potential for metastatic spread. They are based on exciting developments in the cancer biology field which show that exosomes, a major class of extracellular vesicles (EVs), play important roles in a number of aspects of cancer progression. These include the ability of exosomes to confer tumor cells with the capability to show resistance to chemotherapeutic reagents as well as to immune therapy, together with their roles in promoting metastatic spread. We recently discovered that the down- regulation of SIRT1 by aggressive breast cancer cells has an important influence on the numbers of exosomes that they generate, the nature of the exosome cargo, as well as the composition of their total secretome. This is due to the NAD+-dependent deacetylase/deacylase Sirtuin (SIRT1) playing a key role in maintaining normal lysosomal function through a novel mechanism that ensures the proper expression of a major subunit of the vacuolar ATPae (v-ATPase). We also have recently found that the formation and shedding of exosomes appear to be dependent on the elevations in glutamine metabolism characteristic of breast cancer cells (i.e. their ‘glutamine addiction’). These findings now raise important questions regarding how the dependence of aggressive breast cancer cells on glutamine metabolism influences and/or works together with the down-regulation of SIRT1 expression/activation to regulate lysosomal function and exosome biogenesis, thus producing a secretome that stimulates cancer cell invasiveness and helps drive the metastatic process. The different laboratories participating in this proposal will take advantage of their multi-disciplinary expertise in biochemical and chemical biology approaches in probing cancer cell metabolism and exosome biogenesis, high-resolution imaging, 3D spheroid culture and tumor organoids, and the use of mouse models, in probing three key aspects of the mechanisms driving breast cancer metastasis. These are: 1) Examining the relationship between SIRT1 down-regulation, elevated glutamine metabolism and the generation exosomes with unique cargo by aggressive breast cancer cells. 2) Understanding how SIRT1 down-regulation impacts vacuolar ATPase expression to generate a secretome capable of promoting cancer cell invasiveness. 3) Determining how SIRT1 expression/activity affects exosome production, cell invasiveness and metastatic spread in breast cancer models. The expectation is that these studies will lead to the identification of exciting new treatment strategies for the devastating effects of aggressive breats cancers, and ultimately, for other metastatic diseases.

NIH Research Projects · FY 2025 · 2021-12

Project Summary / Abstract Though CD8+ T cell-based immunotherapies have revolutionized treatment for hematologic cancers and chronic viral infections, T cell exhaustion remains a barrier to fully realizing their therapeutic potential. T cell exhaustion is the hierarchical loss of proliferation, cytokine production, and effector function of CD8+ T cells after chronic antigen stimulation. Not every T cell becomes exhausted to the same degree or at the same rate, but the factors governing heterogeneity in susceptibility to exhaustion are undefined. The Rudd lab was the first to show that a previously-overlooked source of heterogeneity within the naïve CD8+ T cell pool—developmental origin—is deterministic in a CD8+ T cell’s fate after acute infection. We believe that developmental origin is also consequential in chronic infection, for our preliminary data shows that neonatal CD8+ T cells (derived from the fetal liver) are resistant to phenotypic and functional exhaustion, whereas adult cells (derived from adult bone marrow) are more susceptible. We also showed that overexpressing Lin28b, an oncofetal RNA-binding protein that negatively regulates let-7 microRNAs and is only expressed in fetal liver HSCs, is sufficient to convert the adult phenotype to the neonatal one. Our objective is therefore to dissect the developmentally-regulated programs underlying differential responses to chronic stimulation. We will use innovative approaches to test our hypothesis that adult CD8+ T cells are more susceptible to exhaustion than neonatal cells due to age-related differences in let-7/Lin28b expression that program metabolism away from aerobic glycolysis. In Aim 1, we will determine how developmental origin and Lin28b expression impact propensity for CD8+ T cell exhaustion. Results from this aim will make clear how distinct subsets of exhausted cells arise among differently-aged CD8+ T cells, and shed light on whether developmental pathways protect against irreversible exhaustion. In Aim 2, we will determine how Lin28b-mediated metabolic programs underlie differently-aged cells’ susceptibility to become exhausted. These results will provide a mechanistic explanation for how developmental imprinting affects T cell exhaustion dynamics. By investigating the developmentally-distinct CD8+ T cell response to chronic infection, and the role that let-7, Lin28b, and metabolic programing play in said response, this proposal will uncover a previously-unexplored factor in determining T cell exhaustion. Because developmentally-ingrained pathways are common to all T cells, understanding these pathways—and finding strategies to fine-tune them—will have wide- reaching implications for neonatal disease, chronic infection, and cancer alike.

NIH Research Projects · FY 2026 · 2021-12

This application seeks support for a research-emphasis veterinarian embarking on an independent career as a translational veterinarian-scientist. The applicant proposes to study macrophages as drivers of SARS-CoV2- induced lung damage in humans and mouse models, and will develop novel indicator mice to report SARS-CoV2 cellular targets in mice. This multi-disciplinary approach brings together leading experts in lung biology, macrophage biology, virology, mouse genetics and pulmonary pathology. At Cornell University College of Veterinary Medicine, the applicant will perform research in the laboratories of Drs. David Russell and Dr. Hector Aguilar-Carreño (Department of Microbiology and Immunology), with histopathology analyses performed in the laboratory of Dr. Gerald Duhamel (Department of Biomedical Sciences). For specific hands-on training in lung epithelial repair assays, the applicant will work in the laboratory of Dr. Carla Kim (Harvard Medical School/Children’s Hospital Boston). Dr. Russell, primary mentor, is an expert in macrophage biology and their interaction with several lung pathogens, notably M.tuberculosis and HIV. He has a proven track record in training post-doctoral veterinarian-scientists that have gone on to tenure-track positions with federal research funding. Dr. Aguilar-Carreño has published extensively on the entry and egress of enveloped viruses with emphasis on paramyxoviruses, and recently has adapted his well-described mouse challenge platform for antiviral and vaccine discovery, toward SARS-CoV2. Dr. Duhamel is a veterinary anatomic pathologist with a research focus in infectious disease, including lung pathogens in mice. Finally, Dr. Carla Kim is a pioneer in lung epithelial repair and stem cell biology. Adding breadth and depth to the proposed work, the applicant has assembled a team of enthusiastic collaborators (with two practicing MD physicians), including leaders in mouse genetics and lung injury (Drs. Kahn and Morrisey), hyaluronan biology (Dr. Hascall), and human pulmonary pathology (Dr. Borczuk). The research environment at Cornell is exceptional in the disciplines of infectious disease and comparative pathology, while the combined expertise of Drs. Kim and Borczuk adds a strong translational and integrative focus on drivers of alveolar damage. The proposal will probe how resident and recruited macrophages are dysregulated during SARS-CoV2 in mice, whether dependent on direct viral infection of macrophages or on environmental cues. In Aim 1, reporter mice will be generated to identify SARS-CoV2- infected cells during infection and upon asymptomatic recovery. During Aim 2, the nature of macrophage subsets as viral targets will be studied, and their resulting functional deficits determined using assays developed by Dr. Russell. In Aim 3, viral challenge studies in human Ace2-expressing mice will interrogate macrophages as drivers of alveolar damage, and test whether macrophage interventions improve clinical outcomes in mice. The candidate will begin this research as Senior Research Associate in the Department of Microbiology and Immunology (75% research effort), with an expected junior faculty transition in the independent award phase.

NIH Research Projects · FY 2024 · 2021-09

Cornell University aims to increase the number of minoritized faculty in the biological, biomedical, and health sciences through establishing an NIH FIRST Program at Cornell University. Cornell FIRST will support the hiring and retention of 10 new assistant professors from groups underrepresented in their fields, while transforming institutional climate into a culture of inclusive excellence. The strength of Cornell’s program is its foundational roots as a complex private institution with a public mission, with its founding based on support for diversity, a culture of interdisciplinary research, and a track record of catalyzing change at different scales that were institutionalized. Given Cornell’s success in establishing programs for the effective development and support of early-career faculty, particularly those underrepresented in their fields, Cornell is in an excellent position to test the hypothesis that FIRST Cohort faculty will be successful in an environment that supports advocacy through sponsorship, consistent and individual-centered mentoring, and evidence-based professional development. We further hypothesize that Cornell’s institutional culture and scientific excellence will be enhanced with the hiring of a FIRST Cohort of diverse faculty. Cornell’s FIRST program features interdisciplinary hiring of faculty underrepresented in their fields, across six colleges and 20 departments, with a focus on retention, career development, and evaluation. Cornell proposes 1) to hire a diverse cohort of 10 new faculty into 3 research clusters, taking advantage of Cornell’s existing interdisciplinary field system approach where faculty are organized by research interest rather than by department, within broad areas of quantitative biomedical sciences, infection biology, and health equity; 2) foster sustainable institutional culture change using novel combinations of institutional policies that impact hiring, mentoring, promotion and tenure, salary equity, and other initiatives aimed at enhancing compositional diversity, retention, and success; 3) enhance faculty development, retention, progression, and promotion building on Cornell’s track record of successfully developing and implementing cutting edge programs that effectively support faculty through their career, particularly those underrepresented in their fields; and 4) to evaluate and learn from our hiring, climate, and faculty development approaches by identifying which strategies and activities are most effective and sustainable at an institutional scale assessing our progress to ensure that they are developed and implemented in an effective manner, and effectively interact with the FIRST CEC. We expect that the Cornell FIRST program will successfully hire, retain, and support 10 new faculty underrepresented in their fields, while fostering sustainable institutional culture change to support inclusive excellence. Cornell FIRST will increase faculty diversity in the biological, biomedical, and health sciences while contributing to the diversity of academy, and future generations of the STEM workforce.

NIH Research Projects · FY 2024 · 2021-09

Summary The ability to determine the three-dimensional location of fluorescently labeled biomolecules in cells with 10 to 70 nm resolution has led to an explosion of discoveries in biology. Super-resolution optical microscopy has led to recent dramatic breakthroughs in our understanding of the organization of molecules in a wide variety of protein assemblies and has led to discoveries of new supramolecular architectures present in organelles. The spatial resolution typically achieved by super-resolution optical microscopy remains, frustratingly, considerably larger than most biomolecules. The goal of this technology development proposal is to create a technology for localizing individual biomolecules with angstrom precision. We propose a technology for localizing molecules using spin labels. The proposed work will employ a magnetic resonance force microscope, in which an attonewton-sensitivity cantilever with a 100 nanometer diameter magnetic tip is operated near a sample surface in high vacuum at cryogenic temperatures. The magnet-tipped cantilever serves two roles. It acts as a force-gradient detector, enabling the observation of magnetic resonance from individual electron spins as a shift of the cantilever's mechanical resonance frequency. It furthermore provides a source of magnetic field gradient, 5 gauss/angstrom or larger, that makes possible the three dimensional magnetic resonance imaging of individual electron spin labels with angstrom spatial resolution. Proof- of-concept data has been acquired demonstrating the ability to detect magnetic resonance from 100's of nitroxide spin labels and to spatially resolve electron spin density at a resolution 100 times smaller than the diameter of the magnetic tip. We present a stepwise technology development plan — backed by theory, simulations, and preliminary data — for achieving the detection of individual nitroxide spin labels and imaging their locations in three dimensions with angstrom precision. Proposed innovations include achieving near-unity spin polarization by operating at high magnetic field and low temperature using novel cryogenic chip-scale microwave sources, employing better inter- ferometric cantilever position detectors and spin modulation schemes to evade sample-related noise, harnessing synchronized cantilever and spin excitation pulse sequences to achieve high fidelity spin modulation, developing robust Bayesian image collection and reconstruction protocols, and fabricating improved cantilevers and magnetic tips for increased per-spin sensitivity. The technology will be validated using well characterized nucleic-acid rulers, biomolecules, protein complexes, and antibodies. Proof-of-concept experiments will be carried out to demonstrate the applicability of the technology to flash frozen biological samples and the ability to carry out correlative fluo- rescent localization experiments. Taken together the proposed work represents a new technology for localizing an individual (spin-labeled and fluorescently labeled) biomolecule in a flash-frozen cell with angstrom precision.

NIH Research Projects · FY 2024 · 2021-08

Treponema pallidum (Tp), Borrelia burgdorferi (Bb), Leptospira interrogans (Li) and Treponema denticola (Td) are spirochete bacteria that cause syphilis, Lyme disease, leptospirosis, and are associated with periodontal diseases in humans, respectively. These organisms cause substantial morbidity and mortality in the United States and throughout the world. Owing to the prevalence of Lyme disease and emergence of antibiotic resistance in Tp and Td, our long-term goal is to develop novel drugs that specifically treat diseases caused by spirochetes. Spirochetes are highly invasive bacteria, and their unique mode of motility plays an essential role in their ability to penetrate and invade host tissues and organs. The flagella of spirochetes reside within the periplasm and are thereby shielded from the immune system. A key component of bacterial flagella termed the hook joins the flagella filament to the membrane-imbedded rotary motor. The hook consists of multiple FlgE proteins, and in contrast to other bacterial flagella, spirochete FlgE proteins are covalently cross-linked to one another. This cross-link involves formation of a novel lysinoalanine (Lal) amino acid. The central hypothesis is that the FlgE proteins are covalently cross-linked to strengthen the hook for optimal motility and virulence. It is proposed that understanding the structure of the cross-link, its chemical synthesis and its role in virulence will lead to the development of drugs that inhibit cross-linking for treating spirochetal diseases. Specific Aim 1. Investigate the effect of FlgE cross-linking on the infectivity of Bb. Mutants of Td and Bb that are unable to cross-link their hook proteins are also altered in shape and deficient in translational motility. To determine the importance of cross-linking for Bb virulence, we will produce a virulent strain impaired in FlgE cross-linking and evaluate its ability to swim and sustain infections in both mice and ticks. Specific Aim 2. Develop small molecule inhibitors of FlgE cross-linking. The chemistry of LA formation is biologically unprecedented. Based on mechanistic and structural studies we have established cross-linking assays with recombinant FlgE proteins from Td and Bb for large-scale inhibitor screens. With these assays we have discovered an inhibitor of FlgE cross-linking and Bb motility. We will further characterize the action of this compound and continue to identify and characterize additional classes of inhibitors to be used for studying pathogenesis in hosts and eventually as lead compounds for therapeutics. Specific Aim 3. Determine the effects of FlgE cross-linking on the structure and stability of the flagella hook. To test whether the FlgE cross-links stabilize the hook to resist the high mechanical stress it likely experiences in the periplasmic space, we will analyze the physical properties of cross-linked and non-cross- linked hooks. In addition, the requirement of cross-linking will be tested by chemically restoring function in absence of Lal.