Cornell University

NIH Research Projects · FY 2025 · 2023-06

Project Abstract Our laboratory has used the phototransduction pathway in retinal rods, a beautifully designed sensory response system, to study how G protein coupled receptors (GPCRs) propagate highly amplified signals. This pathway starts with the absorption of a photon by the GPCR rhodopsin, resulting in its activation of the heterotrimeric G protein transducin by catalyzing GDP-GTP exchange on the transducin-alpha subunit (GT). GTP-bound GT subunits then interact with their effector protein, the cyclic GMP (cGMP) phosphodiesterase-6 (PDE6), a tetrameric enzyme with two catalytic subunits (PDE, PDE) and two subunits (PDE) that bind GT. Binding of GTP-bound GT subunits to PDE6 activates its ability to hydrolyze cGMP to GMP, thus closing cGMP-gated ion channels in retinal rod membranes and sending a signal to the optic nerve. We determined structures for the rhodopsin-transducin complex by cryo-electron microscopy (cryoEM), which together with efforts from other laboratories, led to a detailed picture of how GPCRs activate their G protein partners. However, there is still a great deal to learn about how activated G proteins execute a precise regulation of their effector proteins. Recently, we solved a cryoEM structure for a complex in solution that contains two GTP-bound GT subunits and PDE6, leading to a model describing how transducin activates its biological effector. We will now test important aspects of this model through two broad experimental aims, each comprised of a number of sub-aims: 1) Determine how activated G subunits of the retinal G protein transducin exert a highly tuned regulation of their biological effector PDE6. We will perform: (i) fluorescence read-outs we developed to monitor GT-PDE6 interactions, (ii) studies with a bivalent GT antibody that enables us to form different asymmetric configurations of GT-PDE6 complexes and (iii) site-directed spin probe labeling with electron spin resonance spectroscopy, to test our model for how two GT subunits activate PDE6, as well as (iv) determine if the model is consistent with how RGS9 deactivates signal propagation. 2) Establish a mechanistic basis for how a membrane environment influences the ability of the retinal G protein to activate its biological effector. We will use: (i) fluorescence read-outs to monitor GT-PDE6 interactions to determine how membranes facilitate PDE6 activation by GT, and (ii) FRET to examine the orientation of the PDE subunits on PDE6 in the presence and absence of GTP-bound GT in a membrane environment. We will also: (iii) reconstitute GT- stimulated PDE6 activity in nanodiscs, and (iv) undertake structure determinations of PDE6 alone and bound to GT, to test our model for PDE6 activation in a more physiological setting. The results of these studies will enable us to further develop a comprehensive mechanistic picture for how an activated G protein regulates its biological effector in phototransduction, and how this signal is rapidly terminated when its stimulation has ceased, as well as provide fundamentally important insights into key steps essential for other GPCR-sensory responses.

NIH Research Projects · FY 2024 · 2023-06

Abstract Optical methods provide high-resolution, non-invasive measurement of neural function, ranging from single neurons to entire populations, in the intact brain. Nevertheless, limited penetration depth, spatial scale and temporal resolution remain the main challenges for optical imaging. Laser scanning multiphoton microscopy is the main technology used for cellular-level imaging in scattering brains. Because of the point scanning nature of laser scanning microscopy, the speed of the optical scanner determines the imaging speed, even when there is sufficient signal strength for fast imaging. For inertially dependent scanners like galvanometers and resonant scanners, increasing the scan speed requires decreasing the moment of inertia of the dynamic component, while the need for high spatial resolution requires increasing the aperture of the scan mirror. The balancing of these two conflicting requirements has limited commercially available galvanometer scanners at approximately the same speed for the last 30 to 40 years. The focus of this proposal is to demonstrate a new concept that will improve the scan speed of galvanometer-based optical scanners (galvo-scanners), and increasing the imaging speed of laser scanning microscopy. The proposed new concept leverages the uniaxial nature of galvanometer scanning, i.e., a galvo-scanner scans one spatial dimension only, which leaves the orthogonal dimension free for manipulation. By using a cylindrical lens to focus the beam onto the scan mirror along the axial direction of the galvanometer (i.e., the non-scanning direction) and another cylindrical lens to recollimate the beam after scanning, we can reduce the size of the scan mirror dramatically along the non-scanning direction. The resulting reduction in mirror mass and moment of inertia will increase the scan speed without reducing the scanned field-of-view and spatial resolution. The proposed program dovetails with our effort in developing technologies for brain imaging. We will test and validate the new scanners in laser scanning 2-photon and 3-photon microscopes for in vivo recording of mouse brain activity. The successful completion of this program will create new optical scanners that will improve the imaging speed of all galvo-based laser scanning microscopes. Since the design, test and demonstration are all based on typical multiphoton microscopes, the new concept can be immediately translated to other research labs. Furthermore, we will work with commercial instrument builders to accelerate the research and adoption of the technology developed in this program.

NIH Research Projects · FY 2025 · 2023-05

Robust navigation, which is critical for an animal’s survival, requires the processing of complex sensory information spanning different modalities and time scales. Unlike human-engineered systems, where sensors are passive and modularized and decisions are typically made centrally, biological sensors constantly interact and influence each other, and behavioral decisions are made on different time scales with diverse goals. Further, such decisions are based on actively collected sensory information. Our team’s long-term goal is to elucidate the entire neural circuit dynamics from inter-sensory interaction and multisensory integration in the central brain to the generation of motor commands across different time scales. We use Drosophila melanogaster to take advantage of the genetic tools, rich behaviors, and the available tools to reconstruct the neural circuits with full brain electron microscopy data. We will investigate how visual information, mechanical information (wind), and gyroscopic information (sensing body rotation) are integrated and used to generate motor commands. We aim to test the following hypotheses: (1) Sensory information from one modality affects the processing of different sensory modalities (e.g., gyroscopic sensation may drive neck muscles to effectively change the visual input). (2) Information from multiple sensors is integrated in the central brain using attractor dynamics that unify winner-takes-all and Kalman filter mechanisms. (3) The central brain not only integrates sensory stimuli, but actively drives sensory muscles to maximize task-relevant information, increasing robustness of sensory processing. To test these hypotheses, we combine our team’s diverse and complementary expertise, including two- photon calcium imaging, one photon muscle imaging, whole-cell patch clamping, precise sensory perturbation, high speed behavioral analysis, aerodynamics, single-cell resolution optogenetic perturbation, computational modeling, network theory, and control theory. We will develop a new control theoretical framework supported by anatomical, behavioral, and physiological experiments. We will test predictions of models using behavioral and optogenetic perturbation methods to further refine the theory. The successful completion of this project will put our team in an ideal position to further investigate multiple sensorimotor transformation pathways with different time scales, spanning reflexive response (fast), obstacle avoidance (medium), and voluntary navigational decisions (slow). Overall, our team aims to reveal the computational principles of neural network dynamics underlying robust navigation.

NIH Research Projects · FY 2025 · 2023-05

Project Summary/Abstract Alphaviruses are human pathogens that represent a global health threat. Mayaro virus (MAYV) and chikungunya virus (CHIKV) are alphaviruses that can cause acute disease with fever, headache, myalgia, chills, and long- term debilitating arthralgia. Adaptation of CHIKV lineages to the urban mosquitoes Aedes aegypti and Aedes albopictus has contributed to its worldwide distribution and led to large outbreaks. MAYV is thought to be restricted to transmission by sylvatic mosquitoes of the genus Haemagogus, but laboratory studies show that MAYV can also be transmitted by Aedes mosquitoes, suggesting the need for improved surveillance and countermeasures. I found that MAYV can infect Aedes mosquitoes from Salvador (Brazil) or Galveston when present at titers found in viremia in humans. This project aims to understand how adaptive mutations and interactions with hosts may lead to the emergence of alphaviruses outbreaks. My central hypothesis is that mutations in the MAYV nsP3 gene can lead to efficient transmission by Aedes mosquitoes through promoting viral-host interactions. I will address this hypothesis through three specific aims. In specific aim 1, I will determine existing and prospective mutations in the genome of MAYV that function in vector competence of Aedes mosquitoes. I have performed next generation sequencing of salivary glands of infected Aedes mosquitoes and found 17 putatively adaptive mutations. I will find and validate MAYV minority variants that arise upon mosquito infection using competition assays, 50% oral infectious dose experiments and dual host models. As a proof-of- principle, I discovered an adaptive mutation in the virus non-structural protein 3 (nsP3). I will then determine at which step of mosquito infection they are important through a series of dissections followed by titrations. In specific aim 2, I will uncover the roles of nsP3 in mosquito infections and vector competence. I will assemble nsP3 chimeras of different MAYV strains to identify regions that are necessary and sufficient for increased fitness in Aedes mosquitoes and study a natural insertion that has evolved at least twice in MAYV strains, suggesting it is adaptive, and its proposed changes in protein phosphorylation. In specific aim 3, I will assess how m6A modifications on MAYV and CHIKV RNA may promote immune evasion and how these modifications in cellular RNAs modulate the immune system. These modifications are thought to have key functions in immune system regulation and immune evasion. I showed that MAYV has m6A modifications on its RNA which are concentrated in the sub genomic RNA and promote viral replication. Completion of this project will have a major impact in the control of alphaviruses by spurring novel surveillance strategies and countermeasures targeting virus-host interactions. This project and career development award aligns well with my current skills and career goals to become an independent principal investigator. It will help me fill gaps in my knowledge of vectors and to develop key administrative and writing skills required to become an independent researcher.

NIH Research Projects · FY 2026 · 2023-04

Abstract Highly regenerative tissues such as blood and skin may utilize blood vessels as dual players in tissue growth functions: one in supplying O2/nutrients and another in regulating tissue stem cell activity via signaling. Correlative evidence suggest that skin endothelial cells may act as signaling niches to adult hair follicle stem cells (HFSCs), regulating their quiescence. To demonstrate this, we need gene targeting of signaling molecules in endothelial cells that in turn would affect stem cell activity. Furthermore, recent work on skin vasculature probed a role of lymphatic vessels but not of blood vessels in adult HFSC activation. Finally, the extent of skin vasculature remodeling and its genetic control during the hair cycle are poorly understood. We propose to use a combined genetic and genomic approach in mice to address all these questions. Our mouse models provide an exciting entry point to address the cross- communication of HFSCs with the neighboring endothelial cells, to probe its physiological relevance, and to place endothelial cells for the first time as bona-fide signaling niches for adult HFSCs. This work will have future broad relevance for human skin regeneration studies and for more in depth understanding of skin vasculature disease.

NIH Research Projects · FY 2026 · 2023-04

Project Summary Skeletal muscle tissues are developed and maintained through the coordinated action of myogenic and non- myogenic cells. Dysregulation of myogenic cell identities and functions are commonly observed in skeletal muscle disease. Facioscapulohumeral muscular dystrophy (FSHD) is the second most common inherited muscular dystrophy and results in progressive muscle weakness without any effective therapies. Numerous cellular etiologies are observed in FSHD, such as loss of myogenic cells, including muscle stem cells and myofibers, and increased fibrogenic, adipogenic, and immune cells. The most common form of FSHD arises from aberrant expression of the DUX4 gene caused by epigenetic de-repression of the D4Z4 locus. DUX4 expression in FSHD individuals is regionally varied and highly sporadic within skeletal muscle tissue. Notably, DUX4 expression is both induced by and has pathogenic mechanisms related to noncoding RNAs (ncRNAs). Noncoding RNAs (including miRNAs, lncRNAs, snoRNAs, and eRNAs) are critical regulators of skeletal muscle cell identities and functions in health and diseases and act through modulation of transcriptional networks. Comprehensive understanding of ncRNA networks and mechanisms is lacking due to a paucity of ncRNA profiling technologies. Conventional single-cell and spatial RNA-sequencing technologies preferentially detect polyadenylated, protein-coding mRNAs, and do not efficiently capture most ncRNAs due to their lack of polyadenylation. In this proposal, we will apply a new RNA mapping technology called STRS-HD that is uniquely capable of efficiently and comprehensively detecting the total transcriptome, including both polyadenylated and non-adenylated transcripts, with single-cell spatial resolution to reveal global ncRNA expression heterogeneity in diverse cell types within skeletal muscles. We will leverage this new spatial total RNA-sequencing method to broadly interrogate noncoding RNAs in healthy skeletal myogenesis and in FSHD pathogenesis. In Aim 1, we will implement this total transcriptomic method to investigate how noncoding RNAs impact cell fate regulation adult skeletal muscle regeneration in mice. We will explore cell type-specific ncRNA expression variation and use spatial transcriptomics to map ncRNA features onto spatially resolved cell-cell communication interactions to provide insights into ncRNA regulation of myogenic cell fates. In Aim 2, we apply these methods to resolve how ncRNAs vary in FSHD pathologies using two mouse models subject to DUX4 anti-sense oligonucleotide therapy. We will integrate spatial total transcriptome maps with histopathology to reveal ncRNA determinants of altered myogenic cell specification and myofiber damage in FSHD. In Aim 3, we will extend the STRS-HD approach to human FSHD biopsies and compare ncRNA features to unaffected familial controls and contrast spatial ncRNA maps to cell-free RNA-sequencing in donor plasma to identify new total RNA biomarkers of FSHD. These new total transcriptomic technologies will be broadly applicable to the study of ncRNAs in developmental and disease biology of skeletal muscle and other tissues.

NIH Research Projects · FY 2026 · 2023-04

SUMMARY Although many biomarkers have been identified in AD/ADRD, the most important effect is cognitive function. Links between AD/ADRD symptoms and cerebral blood flow deficits or vascular risk factors such as hypertension are well recognized in patients, but the mechanisms are still under investigation. In mouse models of AD about 2% of capillaries are occluded by an arrested neutrophil and these stalled capillaries have a profound effect on cerebral blood flow. Such capillary stall perfusion deficits (CSPD) could reduce oxygenation and nutrient delivery to neurons and are therefore potential drivers of cognitive dysfunction in AD/ADRD. In AD mouse models, working memory performance is rescued within hours of reducing the incidence of stalled capillaries to increase cerebral blood flow using antibodies against the neutrophil protein Ly6G. CSPD has also been observed in a new non-amyloid ADRD model, hypertensive mice with targeted replacement of the murine ApoE gene with the AD-promoting ApoE4 human allele. In this mouse, rapid rescue of behavior and flow are observed after treatment with the platelet inhibitor prasugrel, suggesting a different cellular cause of CSPD than that observed in the AD models. The rapid time scales of cognitive recovery are too fast for many pathological processes and rule out vascular or neural remodeling. Instead, the speed of memory improvement suggests that changes in the dynamic firing pattern of neurons underlie the rescue and that improved metabolic support by increased cerebral blood flow is a critical factor in determining the functionality of neural circuits. This suggests that slower processes such as protein accumulation and remodeling can be secondary to fast effects linked to improvement of oxygen and metabolite delivery after blood flow increase. This proposal tests the idea that cerebral blood flow recovery leads to corrections in blood oxygenation and in oxygen usage, which then result in metabolic and cellular functional recovery in neurons (Aim 1). Such metabolic changes are hypothesized to underly corrections of aberrant neural activity that ultimately determine behavior. Aim 1 will use gamma oscillations, coordinated neural activity associated with healthy cortical function, as a simultaneous measure of the consequence of the oxygenation changes. In AD mouse models, an imbalance in the activity of inhibitory and excitatory neurons results in reduced fidelity of neural encoding of stimuli. Aim 2 asks if the blood flow improvement also corrects this activity imbalance and improves the precision of stimulus encoding for orientation tuned neurons in visual cortex. Aim 3 tests for normalization of activity in hippocampal circuits involved in the formation and consolidation of memory, directly testing the neural circuits involved in the memory tasks that CSPD reduction improves performance in. Age and sex dependence of these phenomena are investigated in the APP/PS1 model of AD and this study also compares to a new ApoE4-hypertension model and wild type animals with bead injections to mimic capillary stalls. Understanding the mechanism of the rapid improvement in memory function after eliminating CSPD could lead to future therapies that modulate the cognitive symptoms in AD/ADRDs.

NIH Research Projects · FY 2026 · 2023-04

Abstract Adult skin interfollicular epidermis (IFE) renewal is currently described by simple models of relatively homogenous basal stem/progenitor cells. However, long-term IFE renewal is likely orchestrated by the physiological demands of a complex tissue architecture comprising multiple levels of heterogeneity. We began to elucidate the cellular and molecular organization of two spatially distinct IFE domains, their physiological relevance, and the relationship between mouse and human skin. We demonstrate that molecular and cellular states of mouse tail basal microdomains (scales and inter-scales) recapitulate human skin IFE spatial organization in rete ridges and inter-ridges. We begin to uncover a physiological relevance for the skin spatial domains: adaptation to differential UV exposure. We identify multiple IFE populations with distinct behavior in clonal analysis and describe the first in vivo epidermal transit-amplifying (TA) cell. The later uniquely displays a maturation-dependent behavior with a timed-transition from an amplification phase to an extinction phase. This opens-up a new road for investigating molecular mechanisms of timed transitions from a ‘young’ to a ‘mature’ cell state. Using mouse genetics, we develop new tools to label and characterize IFE domains that are most UV exposed and examine in depth: (1) IFE spatial heterogeneity and domain organization in skin and its physiological significance; and (2) the heterogeneity of IFE stem/TA population behavior in skin, how this relates to regeneration capacity of spatial domains, and what are the mechanisms of cell fate transition from a young to a mature TA cell state, and from a stem to a TA cell. We propose that the extraordinary IFE complexity of basal cell states, multiple stem/TA cell populations, and spatial organization may explain the unusual robustness of skin homeostasis in response to constant environmental challenges.

NIH Research Projects · FY 2026 · 2023-04

Project Summary / Abstract It is only recently that the field became aware that certain tissue resident macrophages, including alveolar macrophages and microglial cells, are fetal stem cell derived lineages that behave markedly differently from blood monocyte derived macrophages. With such knowledge we need revisit the role of tissue resident macrophages as HIV-1 reservoirs and their contribution to viral persistence. We have shown that regulatory pathways in infected macrophages, such as pro-survival pathways, can be inhibited by targeting specific lncRNAs, thus driving selective cell death in infected but not uninfected macrophages. Such observations lay the groundwork for eradication of HIV-1 reservoirs, however, biologics, such as lncRNAs, are not as tractable as small molecule inhibitors to progress into therapeutics. We have extensive, documented expertise in macrophage biology in both mouse and human lung, and we have maintained a productive anti-TB drug discovery program based on phenotypic screening for compounds active in infected macrophages. With this expertise we propose the identification and functional characterization of small molecule epigenetic inhibitors capable of modifying host macrophage programming to drive selective induction of cell death in specific myeloid cell lineages, and probing the underlying mechanism(s). Our Specific Aims are: 1. Phenotypic Profiling HIV-1 infected HMDMs and AMs by transcriptional analysis. We will conduct transcriptional profiling on HIV-1 infected HMDMs and AMs to assess the diversity of the cellular responses to infection in both active and latent infection states in the two lineages. 2. Screening small molecule inhibitors of epigenetic programming in experimental infection in HMDMs and AMs, and in HC69.5 microglial cells. We have a library of 735 small molecule epigenetic inhibitors and will screen this compound collection against HIV-1 infected HMDMs, AMs, and against the immortalized human microglial cell line HC69.5, with the emphasis on identifying compounds that drive cell death across the different macrophage lineages and infection models. 3. Progressing hits through mode-of-action studies to identify actionable compounds. We propose analysis of HIV-1/macrophage biology prioritizing compounds that induce cell death in HIV-1 infected macrophages. Finally, to evaluate candidate compounds for their ability to drive cell death and suppress viral persistence we will assess activity through ex vivo drug treatment and cell survival and viral outgrowth from AMs from viremic HIV-1 positive human donors in Malawi.

NIH Research Projects · FY 2026 · 2023-04

Project Summary/Abstract Cancer cells construct a cellular glycocalyx with biochemical and biophysical attributes that protect against attack by effector immune cells. Currently, our mechanistic understanding of how the cancer-cell glycocalyx may physically interfere with any of the multiple pathways and individual steps of effector-cell mediated killing is highly limited. Our overarching hypothesis is that by developing a better physical understanding of the glycocalyx in resistance to immune cell attack, we can better devise new cellular engineering strategies to overcome the glycocalyx barrier. Our project will specifically focus on glycocalyx-mediated protection against attack by Natural Killer (NK) cells, which are attracting significant attention in the field of cancer immunotherapy. NK cells possess natural cytotoxic activity against tumor cells and can be further engineered with a chimeric antigen receptor (CAR) to target a specific tumor antigen. As such, NK cells are exciting candidates for adoptive cell therapy. Cell surface mucins are highly overexpressed in cancer and serve as primary structural elements of the glycocalyx. In this proposal, our aims are to (1) determine how specific molecular properties of mucins govern the glycocalyx structure and thereby mediate cellular resistance to NK-cell attack; (2) identify the specific mechanisms through which mucins physically disrupt NK and CAR-NK attack; and (3) develop NK cellular engineering strategies to overcome the mucin barrier. To complete our aims, we will employ state-of-the-art imaging approaches that our lab has developed for characterizing the nanoscale material structure of the glycocalyx. We also will take advantage of our lab’s expertise and validated tools for engineering the physical structure of the glycocalyx. Combining these imaging and cellular engineering strategies with established techniques in immune cell biology will enable new specific hypotheses regarding the physical functioning of the glycocalyx in protection against immune cell attack to be tested. They will also support the design and testing of engineered NK cells with structure-optimized CARs and glycocalyx-editing enzymes for improved elimination of mucin-bearing cancer cells. Adoptive cell therapy has tremendous promise for treating otherwise recalcitrant cancers. In part due to the technical challenges of manipulating and characterizing the physical structure of the glycocalyx, our physical understanding of the cancer-cell glycocalyx in resistance to adoptive cell therapy is poor. Our project will address this knowledge gap and test new strategies for NK engineering that, if successful, can be further developed for clinical applications.

NIH Research Projects · FY 2026 · 2023-03

Viral myocarditis is a heterogeneous disease that is difficult to study and diagnose. Because of the heterogeneous nature of acute viral myocarditis and the difficulty and low diagnostic sensitivity of endomyocardial biopsies (considered the gold standard for diagnosis of viral myocarditis), there is limited knowledge of the molecular pathogenesis of this disease, particularly in infants and neonates where endomyocardial biopsies are less often performed. The specific cells within the heart that respond to viral infection, the nature of their responses, and the spatiotemporal distribution of such responses are not well known. A better understanding of the spatiotemporal response of the heart to viral infection at the cellular and molecular level will provide much needed insight into the pathological processes that drive the active inflammatory process that ensues following viral infection of the heart. The lack of understanding of the molecular pathogenesis of viral myocarditis is in part due to the lack of tools to investigate viral infection in complex native tissues at single cell-resolution. Here we will use innovative spatially resolved transcriptomics, single-cell RNA sequencing (scRNA-seq) tools and bioinformatics, in conjunction with classical virology techniques, and mouse models to study myocarditis in mammalian orthoreovirus (REOV). We have three aims: In Aim 1, we will study the viral and host factors that define the outcome of REOV infection of cardiac tissues. In Aim 2, we will determine the role of pyroptosis in REOV-induced myocarditis. In Aim 3, we will combine our high-resolution single-cell atlas of myocarditis with the principles of liquid biopsies based on cell-free RNA to develop highly specific blood biomarkers of viral myocarditis, thereby addressing an urgent and unmet medical need. We anticipate our studies will provide unprecedented insight into the pathobiology of viral myocarditis. Our experiments will clarify which cell types are infected in complex cardiac tissues and will reveal how infection success depends on both cell state and cellular environment. We will elucidate the role for endothelial cells in viral myocarditis and we will explore whether and how immune cell responses switch from host defense to host injury. We will explore the effects of infected cells on uninfected bystander cells in close physical proximity, and we will map the cellular interactions that mediate this bystander effect. We will also explore the spatial and cell type heterogeneity of innate immune responses within infected and uninfected cardiac tissues. Successful implementation of these studies will lead to new approaches and molecular tools to study viral myocarditis and other viral diseases and may identify novel diagnostic approaches and therapeutic targets for acute viral myocarditis.

NIH Research Projects · FY 2026 · 2023-02

Bloodstream infections (BSI) caused by Pseudomonas aeruginosa have a high fatality rate. They often arise in patients suffering from pneumonia, urinary tract infections, surgical site infections, or patients with severe underlying conditions, including immunosuppression or chemotherapy-induced neutropenia. Systemic P. aeruginosa is particularly difficult to treat due to its robust host accumulation, high virulence, and extensive multidrug resistance (MDR) to conventional antibiotics. As such, BSIs with P. aeruginosa pose a significant threat to public health. Unlike traditional antibiotics, antimicrobial peptides and polymers (AMPs) facilitate bacterial cell death via stochastic bilayer disruption. Despite their potency and promise, AMPs have yet to enjoy broad clinical success, primarily due to their systemic cytotoxicity. One of the few examples of AMPs approved for clinical use is a class of antimicrobial lipopeptides called polymyxins. These compounds are the last resort to treat MDR P. aeruginosa and are limited in their use primarily due to nephrotoxicity concerns. To address the critical selectivity problem that plagues all AMPs, including new synthetic AMPs made in our laboratory (BDT-4G) that are active on polymyxin resistant P. aeruginosa isolates, we will create targeted antibody bactericide conjugate (ABC) prodrugs that actively target P. aeruginosa and release the active antimicrobial only in the presence of host factors secreted at the infection site. This mechanism of action, similar to that used in the field of antibody-drug conjugates, should decrease toxicity due to non-specific exposure while maintaining the antimicrobial potency at the infection site. The antibody targeting P. aeruginosa (Cam-003) should rapidly localize to the bacterial cells upon systemic administration, thus concentrating the conjugated AMP at the P. aeruginosa surface. AMP release from the antibody via host-directed linker cleavage will lead to bacteriolysis. Linker cleavage by host factors instead of bacterial enzymes will minimize the pathogen’s capacity to escape the ABC treatment via mutagenesis. We hypothesize that increasing the residence time at the infection site through antibody targeting will improve ABC potency and minimize cytotoxicity to the host. Developing ABCs as a new class of antibacterial compounds that can eradicate MDR P. aeruginosa will be of immense benefit, particularly for hospitalized and immune-compromised patients. The impact of this effort cannot be overstated, given the current era of accelerated antibiotic resistance.

NIH Research Projects · FY 2026 · 2023-02

For many years Freed has pioneered the development of electron-spin resonance (ESR) methods for the study of proteins and their dynamical effects on membranes. The goal of this proposal is to use those ESR techniques to advance our knowledge of 1) viral membrane fusion and 2) Intrinsic Disordered Protein (IDP)-membrane interactions in conjunction with other biophysical methods. The ongoing COVID19 pandemic has unveiled how limited are our knowledge and methods to combat emerging infectious diseases caused by viral pathogens. A key step in SARS-CoV-2 (“SARS-2”) viral infection is membrane fusion initiated by its fusion peptide (FP) domain in its Spike protein. In fact, membrane fusion is key for all enveloped viruses such as SARS-CoV-1 (“SARS-1”), MERS-CoV, EBOV, influenza and HIV. However, the mechanism of viral membrane fusion is still unclear. We have extensively demonstrated that FP-induced membrane ordering is a prerequisite for viral membrane fusion in all these viruses. We have shown that membrane ordering for some viruses including SARS-1, MERS and EBOV is strongly Ca2+-dependent. Most recently we have shown that SARS-2 FP interacts with membranes more strongly than SARS-1, and it depends very specifically on Ca2+. However, the exact role of Ca2+, as well as the mechanism of membrane ordering are still unclear. The FP only initiates membrane fusion. We have proposed that the transmembrane domain (TMD) of influenza hemagglutinin is important for finalizing membrane fusion. However, this remains to be tested to see if it is applicable to SARS-2. Thus, we plan to continue studies of the mechanism of membrane fusion of SARS-2 as well as other Ca2+-dependent viruses. IDPs lack a stable tertiary structure in solution. After membrane binding, they can either undergo a disorder- to-order transition or still remain in the absence of a stable structure. The viral FPs are such examples. This IDP- membrane interaction localizes IDPs to their target membranes, facilitating interactions with other membrane proteins, and helping to remodel membrane properties. Understanding the structure/function relationships underlying IDP-membrane interactions is a significant challenge because of their highly variable and dynamic nature. We have been using complexin, a key exocytosis regulator related to neurodegeneration, as a model to delineate the IDP-membrane binding mode. We have found that complexins from different organisms have very different modes, which is likely to reflect their different biological functions. We plan to continue to study the membrane-binding C-terminal domain of complexins of several species to determine the mechanisms that govern complexin-membrane binding modes. These findings will be useful for human therapies. ESR is a powerful methodology to study the dynamic and structural properties of SARS FPs and other IDPs, as we have shown. We will employ our well-developed ESR methods to achieve these goals. This will be supplemented by other biophysical methods, including Isothermal Titration Calorimetry and Circular Dichroism.

NIH Research Projects · FY 2026 · 2023-02

Changes in the spatial organization of the genome are directly involved in gene regulation during differentiation, cellular stress responses and disease initiation. Existing approaches such as chromosome conformation capture (3C) methods and DNA fluorescent in situ hybridization (DNA-FISH) have provided information on the overall 3D structure of the nucleus at the chromatin level, providing critical insights into how our genomes are regulated. Nonetheless, new technologies are needed to uncover the finer details on the role of nuclear architecture, topological domains, and genomic interactions. Studies of gene regulation would benefit from a technology that fills the niche between the ensemble averaged 3C methods and the single cell, low throughput DNA-FISH approach. We have recently developed a novel optical technology we call “Femto-seq” that does just this – it allows users to obtain DNA sequence information from targeted femtoliter volumes within the nucleus of selected cells. Femto-seq provides a new way to examine genomic contacts near a specific gene locus or any nuclear region of interest (e.g. nuclear bodies). Using 3D localized two-photon excitation, we can photochemically biotinylate any region of the nucleus we can fluorescently label and identify in volumes which can be as small as a ½ of a femtoliter. The process is carried out on a population of cells using a combined two-photon/confocal microscope which images, locates fluorescently labeled regions of interest and then irradiates those regions using 700 nm femtosecond pulses to biotinylate the chromatin by photochemically cross-linking the DNA with a psoralen-biotin compound. Nuclei are isolated and the biotinylated DNA from the targeted region pulled down and sequenced. Because the cells are imaged to locate the regions of interest, they can also be screened for other parameters, allowing for the collection of targeted biotinylated DNA only from user selected cells within the cell population, providing single-cell like genomic information from a sub-set of cells within the population. We have proof-of-concept data from a cell line with a fluorescently labeled transgene we used as our targeted region, and show that we can obtain DNA highly enriched in transgene locus sequences. The goals of this Focused Technology Research and Development R01 project are to (Aim 1) design and construct a dual confocal/two- photon microscope capable of targeting and irradiating user selected regions-of-interest in a population of cells in a high-throughput automated fashion, (Aim 2) create a chromatin isolation pipeline based on novel microfluidic designs to efficiently purify and prepare the DNA for sequencing, and (Aim 3) demonstrate the improvement obtained from aims 1 and 2 in a series of Femto-seq experiments designed to produce quantitative metrics of improvement and to uncover new knowledge on how environmental signals may be relayed through the cytosol and into the nucleus. Femto-seq is a unique new way of investigating the spatial and regulatory relationships between DNA sequences and any microscopically visible region-of-interest in the nucleus.

NIH Research Projects · FY 2025 · 2022-09

Project Summary Functional profiling of microbial communities is critical to understanding their overall effects on host health. Most often, metagenomic shotgun sequencing of microbiome samples is used to assess total functional capacity. Yet, transcriptional responses may vary dramatically between organisms depending on the context, with potentially large effects. Many metabolic functions are only expressed after the organism acutely senses the presence of particular substrates in their environment. Pathogens may only express virulence factors after obtaining a critical quorum of pathogens. Overall, stress responses are critical for survival under changing abiotic and biotic conditions. Being able to comprehensively map out these pathways, which determine the resilience, plasticity, and patho-functions of the microbiome, requires sensitive, robust transcriptional –omics tools. Performing traditional RNAseq analyses on bacterial communities has been the predominant method to gain transcriptional information, but it is hampered by the need for technical workarounds and it provides incomplete information about the transcriptional landscape. Ribosomal RNA needs to be depleted prior to sequencing, it has a poor signal-to-noise ratio arising from varying RNA decay rates, and it is insensitive to the transcription of non-coding RNA that has secondary structure or post-transcriptional modifications. Alternatively, the position of RNA polymerase (RNAP) can be assessed, which provides a real-time readout of transcription. Although so-called nascent transcript sequencing has been performed in E. coli, revealing transcriptional pause sites and other phenomenon elusive when using RNAseq alone, these protocols rely on immunoprecipitation of RNAP and are therefore unsuitable for complex microbial communities where RNAP may be quite diverse and require species-specific antibodies. As a solution, Precision Run-On and SEQuencing (PRO-seq), a method originally created for examining transcription in eukaryotes, may provide an unbiased method to examine transcriptional dynamics on cultured bacteria or in complex microbial communities, such as the human microbiome. Our goal is to test the feasibility of PRO-seq when applied to prokaryotes and to evaluate its ability to capture transcriptional dynamics associated with canonical stress response pathways (heat-shock, oxygen exposure and DNA damage), using a set of quantitative metrics. We aim to validate, and if necessary, modify the protocol so it can be used robustly across species. We plan to develop a computational approach to test the full breadth of transcriptional phenomena that can be observed using this method, such as transcriptional pausing, bidirectional transcription, differences in RNAP function apparent across species, and RNA decay rates, among other aspects. If successful, we expect that PRO-seq will be adopted to study the responses of human-associated microbiota to host diet, inflammatory signals, xenobiotics and to human transcriptional circuitry, more directly.

NIH Research Projects · FY 2025 · 2022-09

The specialization of somatic cell types for unique functions is arguably the most important driver of physiological complexity in animals. Key innovations in subcellular structure, including the development of a specialized secretory vesicle, increased the evolvability of cells and provided new opportunities for cellular innovation during the diversification of animals. For example, the emergence of cells with the capacity to secrete gel-forming mucus enabled segregation of internal and external tissue compartments facilitating the evolution of organ systems. Despite the value of novel cell function as a source for the evolution of animal complexity, the genomic mechanisms promoting the origin and diversification of new cell types remain poorly understood. Recent advances in sequencing technologies have provided a window into the genomic and transcriptomic environments of numerous cell types from diverse organisms. While these studies have hypothesized roles for both newly evolved genes and newly constructed regulatory relationships as critical elements of cell identity, understanding how new genes get wired into gene regulatory networks (GRNs) to drive the origin of new cell types remains a key gap in our knowledge of animal development. One challenge limiting progress in this area is that it is still not feasible to manipulate gene expression in many animal models, hampering our ability to translate observations of gene expression into functional relationships. A powerful system for modeling GRN evolution must have a novel trait with a measurable phenotype, an identified network of genes controlling the trait, and a genetically tractable organism for experimental testing. The novel and diverse seminal fluid proteins of Drosophila fit all these characteristics and studies in this system have revealed how novel effector genes can rapidly acquire essential functions affecting both physiology and behavior. Cnidocytes – the explosive, venom- rich piercing cells that give jellyfish their sting – offer many of the same benefits as Drosophila for modeling GRN evolution. Unique in both form and function, cnidocytes comprise a diverse lineage of cell types found only in cnidarians (corals, sea anemones, and jellyfish). Many of the regulatory genes necessary for cnidocyte development are already known to be novel and unique to this cell type, providing an unparalleled opportunity to study how new transcription factors become indispensable for the origin of new cell types. The proposed research will achieve three goals; it will: (1) construct the network of genes controlling the unique morphologies of the four types of cnidocyte in the sea anemone Nematostella vectensis, (2) reveal the step-wise assembly of a unique GRN subcircuit through comparisons of closely related cnidarians, and (3) develop a technique for redirecting cells to acquire novel secretory functions. By constructing the GRN that promotes morphogenesis in diverse cnidocyte types, we can pinpoint the genes necessary to drive autonomous development of the piercing apparatus in new cell types. Thus, this research provides a framework for adapting cnidocytes for other novel functions that could contribute to new delivery mechanisms for topical drugs.

NIH Research Projects · FY 2025 · 2022-09

Adverse pregnancy outcomes can be the consequence of defects in several factors, such fetal or maternal genetics, environmental exposures, uterine dysfunction, preeclampsia, nutrition, infection, inflammation, and placental insufficiency. Development of a healthy placenta from trophectoderm precursors enables proper nutrient, gas and waste exchange between the fetus and mother. Additionally, maternal and placenta-intrinsic inflammation at the interface must be controlled to protect the fetus. This project addresses how genomic instability (GIN) during oogenesis and embryogenesis can cause sexually dimorphic pregnancy outcomes, and how the placenta may be especially susceptible to this condition. Research into this underappreciated cause of adverse pregnancy outcomes is motivated by findings that female mouse embryos are dramatically more prone to lethality when bearing certain GIN-causing mutations of DNA replication or repair genes. In one such model that will be utilized in this project, the female-biased lethality was due to increased susceptibility to inflammation, whereas male embryos were protected by the anti-inflammatory effects of testosterone. Preliminary data implicate the placenta as the sensitive tissue underlying the embryonic death. Remarkably, this sex-biased lethality occurred only if the dam also had a GIN genotype. The goals of this project are to understand the tissue(s), cells, and mechanisms driving GIN-induced lethal inflammation. This will be accomplished using the power of mouse genetics, genomics, and embryo manipulation. Aim 1 will test whether the placenta, the embryo, or both, are responsible for female-biased lethality. We hypothesize that the highly polyploid trophoblast giant cells may be especially sensitive to compromised DNA replication and GIN, triggering innate inflammation. Aim 2 addresses why oocytes must come from high GIN mothers for the sex bias to occur in fetuses bearing the mutant lethal genotype. Preliminary experiments implicate that such dams produce oocytes with compromised mitochondria, and this hypothesis will be tested using mitochondrial augmentation and -omics analyses. Aim 3 seeks to identify the molecular basis of lethal embryonic inflammation, with a focus on triggers of innate immunity. A combination of genetic and molecular assays will be used to test the hypothesis that nuclear GIN leads to mitochondrial RNA and DNA leakage, activating a pathway(s) that stimulates transcription of inflammation-driving interferon genes. Overall, if successful, the results will be relevant for interpreting and addressing individual cases of recurrent pregnancy loss that may have a basis in intrinsic inflammation during gestation, and provide insights into underappreciated mechanisms causing adverse pregnancy outcomes including miscarriage and intrauterine growth retardation.

NIH Research Projects · FY 2024 · 2022-09

PROJECT SUMMARY Abnormal metabolism is a hallmark of cancer that helps cancer cells to grow, undergo malignant transformation, and survive under stressful conditions such as nutrient deprivation. Cancer cells are exposed to many cellular stresses during tumorigenesis, which must be overcome for the propagation of malignancy. In cancer, the abnormal activation of many signaling networks serves to disconnect the control of growth, metabolism, and survival, and recent efforts have sought to therapeutically target cancer metabolism. The phosphatidylinositol 3- kinase (PI3K)-AKT (protein kinase B) signaling pathway is the most activated in human cancer and has a wide range of effects on cellular metabolism. We have recently identified the Cdc42/Rac guanine nucleotide exchange factor (GEF) dedicator of cytokinesis 7 (Dock7) as a novel signaling node that supports sustained basal AKT activation and mechanistic target of rapamycin (mTOR) activity as determined by its downstream target S6 kinase (S6K) during stressful conditions to maintain signaling activity required for cell survival and transformation. We find that Dock7 is required for multiple cancer cell lines to resist anoikis and exhibit anchorage-independent growth. While we observe relatively low levels of AKT phosphorylation compared to stimulation by growth factors, Dock7-dependent signaling is critical for the survival of cancer cells during nutrient deprivation. I hypothesize that under cellular stress Dock7 serves as a scaffold for AKT, sustaining its phosphorylation and organizing signaling partners for mTOR signaling required for stress survival. This project will investigate the role of this novel Dock7/AKT/mTOR signaling activity in providing a survival benefit to cancer cells under cellular stress. I propose to study the impact of Dock7-dependent signaling activity on AKT/mTOR signaling, cell survival under stress, and critical characteristics of malignant progression and aggression. In Aim 1, I will investigate the functional activities of the Dock-homology region 2 (DHR2) domain of Dock7, which is responsible for GEF activity, in basal AKT phosphorylation for cancer cell stress survival and malignant transformation. In Aim 2, I will next identify the novel role of the DHR1 domain in Dock7-dependent AKT phosphorylation, cancer cell stress survival, and malignant transformation. Then, in Aim 3, I will identify the subcellular location of this Dock7 signaling complex under stress conditions and determine the individual roles of DHR1, DHR2, and activated Cdc42 in Dock7 localization. The work in this proposal will provide biochemical characterization of Dock7 signaling activity that will lead to a mechanistic understanding of Dock7-dependent AKT/mTOR activation in cancer cell stress survival. These findings will not only contribute to the understanding of cancer aggression and metabolism but may also identify new therapeutic targets for cancer treatment.

NIH Research Projects · FY 2024 · 2022-09

Most U.S. children living in out of home care (OOHC) have debilitating impairments regulating emotions and behavior and the success of treatment efforts for these children depends principally on caregivers’ capacity to provide developmentally enriching, therapeutic care. While living in OOHC, the adults who care for these children during the critical hours outside of formal therapy play central roles in their treatment. Yet, OOHC caregivers receive little education about how to meet the unique relational needs of the children they serve and lack a clear understanding of their own therapeutic role in each child’s rehabilitation. In addition, the most commonly-used training programs for caregivers in OOHC cover an eclectic range of topics without a specific focus on relational skills, and few have empirical support. Ultimately, in order for OOHC services to optimize children’s rehabilitation and mitigate the long-term sequelae of developmental trauma, it is imperative to provide opportunities for caregivers to develop skills for eliciting developmentally enriching interactions (DIs) during their ordinary care routines. Toward that goal, we propose two specific aims. Aim #1: Produce a video- based Developmental Interaction Workshop Series (DIWS) that enables caregivers to repeatedly observe and practice specific forms of DI and to create opportunities to increase their frequency during daily care routines. The DIWS will include two 4-hour sessions for caregivers and supervisors, as well as two 90-minute sessions for supervisors. A beta version will be implemented in one Residential Care (RC) agency, revised as needed, and then fully implemented in three RC agencies. Aim #2: Evaluate the DIWS using mixed methods (staff and child surveys, staff interviews, and ethnography) in four RC agencies to document preliminary evidence of its impact, acceptability, and feasibility. We expect the DIWS to lead (1) caregivers to become more capable, motivated, and purposeful about eliciting DIs in their caregiving role, and (2) caregivers and children to perceive a greater prevalence of DIs during routine daily activities. We will also identify individual, organizational, and implementation-related factors related to uptake. The DIWS will provide a developmentally-informed framework for understanding and enhancing the child-adult relationships in OOHC. Mixed-method evaluation results will provide the foundation for future RCTs of the program’s efficacy, inform program improvements, and facilitate its wider dissemination.

NIH Research Projects · FY 2025 · 2022-09

Project Summary/Abstract Gene regulation is central to all life, normal and diseased. The long-term goal of this research program is to un- derstand the molecular mechanisms governing the regulation of all genes in yeast and human systems. This basic knowledge will help produce better diagnostics and treatment options for people. Regulation of the hu- man genome is very complex. Therefore, this research program is focused initially on the simpler yeast Saccha- romyces to experimentally dissect mechanisms of gene regulation that are fundamental and common to all eu- karyotic life. Concepts developed in yeast are ultimately tested in human cells, thereby accelerating discovery. This research program has developed an ultra-high-resolution assay called ChIP-exo to map the bound locations of essentially any protein throughout any genome at base-pair resolution. Using this strategy, a com- prehensive first-of-its-kind epigenome map of the protein-DNA architecture of yeast cells was established. This is now being established in human cells. The epigenome is defined here as the compilation of all molecular in- teractions with DNA and RNA, beyond base-pairing. The next phase of this research is to understand the func- tional interactions among the protein components of the epigenome. This will be achieved in part through dele- tion, mutation, and/or rapid depletion of protein components of the epigenome, particularly those involved in inducible and constitutive transcription. The former is gene-specific and can be hyper-expressed in response to specific signaling events. The latter is general to most genes and typically occurs at low levels. Importantly, the research program here is defining the protein architecture that specifies inducible versus constitutive promot- ers. Once protein components of this architecture are experimentally removed (e.g., sequence-specific tran- scription factors or their cofactors), then the impact of this removal will be measured on chromatin organiza- tion, loading of the core transcription machinery and subsequent transcription. A parallel strategy will be em- ployed in human tissue culture cells to assess conserved paradigms. This research will also continue with its previous biochemical reconstitution of chromatin organization across entire genomes using purified proteins, but now adding in components of the transcription machinery and their regulatory factors. A biochemical system will provide greater control over the experimental parame- ters and therefore provide greater insight into molecular mechanisms of gene control. It is now clear that in- duced genes coalesce in 3D space within the nucleus. However, it remains unclear which genes coalesce into which hubs. Therefore, 3D mapping technologies like SPRITE will be adopted to measure gene clustering. This will provide insight into how multiple genes become coordinately induced by regulatory signals. Taken to- gether, the product of this research program will be a detailed molecular understanding of transcription and its regulation.

NIH Research Projects · FY 2025 · 2022-09

Biodegradable metallo-elastomer Biodegradable elastomers are useful in many biomedical applications. Elastomers are crosslinked network polymers. The crosslinks can be made of covalent bonds or weak bonds such as a physical bond. The former produces thermosets, which usually have high elasticity but cannot be processed after crosslinking. The latter produces thermoplastics, which usually have lower elasticity but are easier to process. Metal coordination bond has medium bond strength in between covalent bonds and weak physical bonds. We will invent a series of biodegradable metallo-elastomers where the crosslink is formed by metal coordination bonds. An advantage of this approach is that one polymeric ligand can bind many different metal ions, thereby producing variant elastomers, each with unique properties. Furthermore, metal ions have inherent bioactivities, an area underexplored in biomaterials. Our preliminary study demonstrates that the materials can be highly elastic; matching or exceeding the elasticity of elastomers crosslinked by covalent bonds. Furthermore, the resultant elastomers contain very small amounts of metal ions and exhibit no noticeable toxicity. On the contrary, they are more biocompatible than polycaprolactone (PCL), used in many FDA-approved medical implants. Many transition metal ions have inherent bioactivity. Enzymes further enhance and specify these activities by providing amino acid ligands and binding pockets. Copper ion (Cu2+) is one of the first angiogenic factors discovered and is known to upregulate angiogenic growth factors. In redox enzymes such as superoxide dismutase, Cu2+ provides the critical redox activity to break down the superoxide radical. This research will elucidate the structure-function relationship of metallo-elastomers in two specific aims: the first will explore the pro-angiogenic properties of Cu2+, the second will study the anti-ROS activities of Cu2+. Taking advantage of the elasticity of these polymers, we will test the polymers created in this proposal in models of skin wound healing. Aim 1 will investigate the angiogenic properties of Cu metallo-elastomers and their potential in improving the survival of skin flaps. Aim 2 will investigate the capability of Cu metallo-elastomer to decompose reactive oxygen species using a polymer bearing basic resemblance to the active site of superoxide dismutase. These materials will potentially increase the integration of skin grafts. Upon completion of this project, we expect to have built a basic framework on how metallo-elastomers interact with biological systems. We will better understand how altering the basic structure of the elastomer will impact its function. Furthermore, we will appreciate the effectiveness of these elastomers in increasing the survival and integration of skin grafts and skin flaps. The knowledge gained will fundamentally impact biomaterial design and practically impact host integration of medical implants.

NIH Research Projects · FY 2025 · 2022-09

Optical recordings of activity are critical to probe neural systems because they provide high-resolution, non-invasive measurements, ranging from single neurons to entire populations in intact nervous systems, and are readily combined with genetic methods to provide cell type-specific recordings. Nevertheless, the limited penetration depth, spatial scale and temporal resolution remain major challenges for optical imaging. Cellular resolution imaging in scattering brains is typically achieved with multiphoton microscopy (MPM). Because of the nonlinear excitation process, the development of multiphoton imaging depends critically on ultrafast technologies, particularly femtosecond sources. From the first demonstrations of second harmonic generation (SHG) and 2-photon fluorescence (ruby laser), the first 2-photon imaging (mode-locked femtosecond laser), to the deepest 3-photon imaging so far (long wavelength optical parametric amplifiers), advances in multiphoton imaging have been largely propelled by the innovations in laser technologies. This research proposal aims to continue this trend. We will develop and disseminate a new generation of ultrafast lasers and multiphoton imaging tools that will enable deep, fast, and large-scale imaging of structure and function with cellular and subcellular resolution. To approach the fundamental limits defined by the “photon budget”, we will develop an adaptive excitation source (AES) at 1300 nm for deep tissue 3-photon microscopy (3PM). By feeding the structural information of the sample to the laser source, the AES generates on-demand pulses only within regions of interest (ROIs) and transforms a conventional multiphoton microscope into a “random-access” microscope for the ROIs. We will integrate the AES with high speed scanners and optimize the photon budget and scanning systems. We will further test and validate the performance of the new imaging technology in three proof-of-concept experiments in animal models. The research involves close interactions between the PI (Chris Xu) and Co-investigators (Alex Kwan, Frank Wise, Nilay Yapici, and Rafael Yuste). Furthermore, we will work with industry partners to explore commercialization of the technology, which will provide a direct path to broad dissemination. The combination of 1300 nm AES and 3PM will transform our ability to image deep and fast and will have a broad impact on neuroscience where high resolution, high speed imaging deep within an intact brain is required.

NIH Research Projects · FY 2025 · 2022-08

PROJECT SUMMARY Psychedelics are compounds that produce an atypical state of consciousness characterized by altered perception, cognition, and mood. Among psychedelics, psilocybin has gained attention recently because early clinical trials indicated potential antidepressant effects, leading to a ‘breakthrough therapy’ designation from the FDA to test psilocybin for major depressive disorder. However, despite the promise, the biological mechanisms underpinning psilocybin’s potential therapeutic action are poorly understood. Our lab employs subcellular- resolution two-photon microscopy to visualize dendritic structure and function in head-fixed mice. The goal of this project is to characterize how a single dose of psilocybin may alter dendritic architecture in the medial frontal cortex of the mouse and the associated cellular mechanisms. The hypothesis is that psilocybin promotes spine formation by activating specific serotonin receptor subtypes and exerts differential effects on distinct subtypes of pyramidal neurons. To test the hypothesis, we propose a series of experiments that combine subcellular-resolution optical imaging, conditional knockouts, and causal perturbations in mice. The results will answer crucial questions regarding psilocybin’s ability to promote structural plasticity in vivo and delineate receptors and cellular factors that underlie the plasticity-promoting actions. We expect the mechanistic insights will be important as the field evaluates psychedelics as a potential treatment option for neuropsychiatric disorders and searches for novel antidepressants.

NIH Research Projects · FY 2026 · 2022-08

PROJECT SUMMARY/ABSTRACT Infertility occurs in approximately 15% of women of reproductive age in the United States. Approximately half of the cases involve impaired ovulation, the cause of which is often elusive. The aim of this project is to achieve new understanding of ovulatory defects that will serve as a foundation for effective treatment of infertility. It is known that obesity negatively impacts female fertility and ovulation, but the underlying mechanism remains to be elucidated. Recent findings highlight the crucial role of preovulatory ovarian vascular remodeling in successful ovulation: the ovulatory luteinizing hormone (LH) surge induces a series of vascular remodeling processes in the ovary, including changes in vascular permeability, vessel contraction and formation of new blood vessels. CCAAT/enhancer binding proteins alpha and beta (C/EBPα and C/EBPβ, jointly abbreviated C/EBPα/β) are rapidly induced in granulosa cells by the LH surge and function as important regulators of ovulation. Based on our preliminary data, which show the profound effects of C/EBPα/β deficiency on ovarian vascular remodeling in mice, we hypothesize that C/EBPα and C/EBPβ are key mediators by which the LH surge controls vascular remodeling in preovulatory ovaries. Given obesity’s negative impact on vascular function in general and on ovarian blood flow in women in particular, we also propose the novel concept that ovarian vascular remodeling is a key mediator between obesity and ovulation failure. This proposal first seeks to use murine models of ovulation failure and disrupted ovarian vascular remodeling to identify their regulatory mechanisms downstream of the LH surge, then addresses the knowledge deficit around obesity’s impact on ovarian vasculature. To achieve these goals we will apply 3-dimensional quantitative intravital imaging to a transgenic mouse line with ovary-specific ablation of C/EBPα/β to first define the specific vascular remodeling events regulated by C/EBPα/β in preovulatory ovaries, then determine in a diet-induced obese mouse model the impact of obesity on the activity of C/EBPα/β and vascular remodeling during ovulation. We will further seek understanding of cell type-specific mechanisms regulating preovulatory vascular remodeling and ovulation using single-cell, next-generation sequencing technologies; these approaches will also reveal whether epigenetic mechanisms regulating chromatin accessibility play a key role in preovulatory vascular remodeling and ovulation, and whether C/EBPα/β mediate this interaction. Successful completion of the proposed studies will advance our understanding of ovulation regulation and have a major impact by elucidating links among obesity, epigenetic regulation, and ovarian function, thus enabling improved treatment of many cases of female infertility.

NIH Research Projects · FY 2025 · 2022-08

Abstract Microbes in host microbiomes, human infections and the natural environment often live in spatially structured aggregates and interact antagonistically with each other. Toxin-mediated antagonistic interactions are widespread in the gut, skin, and other human microbiomes, and protect these communities against external invasion. Recent results suggest that spatial structure can strongly affect the evolutionary dynamics of microbial populations, and, in turn, microbial interactions can feedback on the formation of spatial structure. For example, we found that mechanical interactions among dividing cells in growing yeast colonies reduce the power of natural selection by reducing the rates at which lower fitness strains go extinct and fitter ones expand in these populations. Despite spatial structure and microbial interactions have a strong impact on the evolutionary dynamics of microbes relevant for human health, most of what we know about microbial evolutionary dynamics comes from experiments with well-mixed liquid cultures with limited interactions among cells. To fill this gap, my group is interested in understanding quantitatively how spatial structure, mechanics and biological interactions impact the adaptive evolutionary dynamics of microbial populations. We approach this question via experimental evolution, synthetic biology, and mathematical modeling. In preliminary experiments, we found that evolving yeast colonies selecting for faster expansion on agar surfaces results in notable changes in cell shape: cells evolved from an ellipsoidally shaped ancestor to being elongated and almost rod- like, changing the way cells interact mechanically when growing and dividing. We hypothesize that an elongated cell shape is advantageous for faster expansion because it reduces cell packing, and that this adaptive change is associated with changes in the way genotypes cluster in space leading to increased genetic drift, the temporal change in allele frequencies due to chance events. Recently, we showed that a toxin-producing microbe can only invade a landscape occupied by a weaker toxin-producer if its inoculum is larger than a critical size, and that adaptive evolution can alter the dynamics of antagonism. We will experimentally investigate the dependence of the critical inoculum size on the strength of the interaction, and we will study how spatial structure controls the fate of mutations that confer resistance to the toxin produced by either the invader or resident strain. Finally, we will investigate how antagonistic interactions among microbes affect the dynamics of invasion in the gut of the nematode Caenorhabditis elegans: these experiments will help us translate results obtained in simple laboratory settings to the more complicated but more realistic dynamics of invasion of a host microbiome.