Cornell University

NIH Research Projects · FY 2025 · 2015-09

ABSTRACT Pannexins comprise a unique family of heptameric large-pore channels that are emerging as novel targets for treating common, yet hard to cure diseases such as hypertension and chronic pain. Previous studies indicate that Panx1 is activated through stimulation of structurally unrelated receptors such as G protein- coupled receptors, ligand-gated ion channels, and tumor necrosis factor receptors. However, it remains un- clear what cellular mechanism(s) actually open and close the Panx1 channel downstream of such seemingly unrelated stimuli. Furthermore, Panx2 and 3 are severely understudied and essentially nothing is known about the activation mechanisms of these subtypes. The long-term goal is to elucidate the mechanisms underlying pannexin gating, regulation, and physiological signaling pathways. The specific objectives for this proposal are to identify the physiological pannexin activators and elucidate the subtype-specific activation mechanisms. The central hypothesis is that both Panx1 and 2 are directly activated by naturally occurring signaling mole- cules in living cells and that Panx1 specifically requires posttranslational modifications to be "primed" for its activation. The rationale for the proposed research is that once the direct activation-stimuli and the subtype- specific mechanisms are identified, it will enable us to fill the critical gap in the pannexin-dependent signaling pathway by connecting the upstream cell-stimulation and the downstream ATP-permeable membrane pore formation. To attain the overall objectives, the following three specific aims will be performed:1) Identify the direct pannexin activators for living cells; 2) Elucidate the role of the N-terminal domain (NTD) in pannexin activation; and 3) Uncover the subtype-specific structural features of pannexins. These research aims will be executed by using a combination of a cell-based pannexin activity assay, electrophysiology, functional recon- stitution, and cryo-EM. The research proposed in this application is innovative because it introduces a novel concept that pannexins—including the understudied Panx2—are directly activated by signaling molecules produced downstream of various stimuli in living cells. It is also innovative because it will provide important insights into the structure of the open channel and why Panx2 and 3 behave differently from Panx1. The proposed research is significant because it will provide concrete molecular mechanisms for the missing link in the pannexin signaling function. The proposed research is also expected to provide a strong structural foundation for subtype specific mechanisms of pannexin channels. These results are expected to have pro- found positive impact not only because they provide detailed basic mechanisms, but also because they will open a new door for screening/designing pannexin-specific inhibitors—much-needed molecular tools that have great potentials to serve as novel therapeutics for a variety of currently uncurable diseases.

NIH Research Projects · FY 2025 · 2015-05

Project Summary Cerebral blood flow (CBF) is reduced in Alzheimer’s disease (AD) patients and mouse models by ~20%, but there remains a limited understanding of the mechanisms causing this hypoperfusion or the potential therapeutic benefit of rescuing CBF deficits. Under the previous award, chronic in vivo two-photon excited fluorescence microscopy was used to study CBF in mouse models of AD. While no blood flow disruption in cortical arterioles or venules was observed, blood flow was found to be stalled in ~2% of cortical capillaries in mouse models of AD, as compared to ~0.4% in wild type controls. These capillary stalls appeared early in disease progression, were caused by arrested neutrophils, and had outsized impacts on CBF because they decreased flow speed in up- and down-stream vessels. Antibodies against the neutrophil surface protein Ly6G were serendipitously found to reduce the incidence of capillary stalls immediately, leading to a rescue of two-thirds of the CBF deficit, and, remarkably, to improved memory function within hours. Preliminary data further link this capillary stalling to cellular damage from reactive oxygen species (ROS). In this competitive renewal, the mechanisms underlying neutrophil arrest in capillaries in mouse models of AD and the consequences of improving CBF on AD-related pathology are explored. First, three different hypotheses about the mechanism of neutrophil arrest in capillary segments are tested: a focal constriction of the capillary by a pericyte that prevents neutrophil passage; binding of the neutrophil to increased inflammatory adhesion molecules on endothelial cells; or binding of the neutrophil to basement membrane and adhesion molecules exposed at widened gaps between endothelial cells. Second, the molecular and cellular origin of the ROS that leads to neutrophil arrest is determined using cell type-specific knockouts of ROS producing enzymes. Third, the impact of long-term CBF rescue on the deposition of amyloid- beta (Aβ), a driver of AD pathology, and on neuropathology will be quantified. Critical for this study are recently- developed knock-in mouse models of AD that may better capture the feedback of CBF reductions on expression of amyloid precursor protein (APP), which is cleaved to produce Aβ. Finally, cutting-edge three-photon excited fluorescence microscopy is used to enable imaging of the hippocampus to determine the role of capillary stalling in CBF deficits in one of the first regions of the brain that exhibits AD pathology. The hypothesis that brain hypoperfusion in AD is due to neutrophil arrest in capillaries is both novel and strongly supported by the findings under the previous award. The work proposed in this competitive renewal would uncover the mechanisms underlying that neutrophil arrest, which could suggest therapeutic targets to improve CBF that would be complementary to anti-amyloid and other treatment approaches for AD.

NIH Research Projects · FY 2024 · 2014-08

Project summary The piRNA pathway is an evolutionarily conserved mechanism that acts in the germline of metazoans to repress the activity of transposable elements and ensure genome integrity and fertility. At the core of the pathway lies an Argonaute protein of the Piwi clade and the associated small non-coding RNA, piwi-interacting (pi)RNA, which guides the Piwi protein to its targets. In the cytoplasm Piwi proteins act by destroying the mRNA targets using their intrinsic nuclease activity. piRNA also guide nuclear the Piwi to establish repressive chromatin and induce transcriptional repression of genomic targets. We have found that SUMO and an E3 SUMO ligase play an essential role in piRNA-guided transcriptional repression by linking the nuclear Piwi complex to the chromatin modifier. However, individual steps of piRNA-guided chromatin repression remain poorly understood. In this proposal, we will investigate the molecular mechanism of piRNA-guided repression. We will determine how the SUMO ligase is recruited to genomic piRNA targets and explore the role of SUMO in assembly of the repressive piRNA complexes. We will determine functions of Piwi and piRNA in establishment of repressive chromatin and initiation of piRNA biogenesis. In addition to its function in the germline of adult flies, the maternal Piwi-piRNA complex is deposited into the egg and was proposed to activate piRNA biogenesis in the progeny. However, Piwi is indispensable for oogenesis preventing direct interrogation of its embryonic function. To circumvent this problem and study the function of maternal Piwi in embryogenesis we have developed a strategy for depleting maternal Piwi in the embryo. We will apply this strategy to probe the role of Piwi in specification of piRNA clusters in primordial germ cells. How distinct chromatin domains are demarcated is the central question of chromatin biology. In animals, heterochromatin is disassembled in gametes and re-established during embryogenesis. How heterochromatin is assembled de novo during development remains poorly understood. Transposable elements comprise the bulk of heterochromatin, making Piwi-piRNA complexes an ideal tool for finding and marking heterochromatin sequences. During early zygotic development maternal Piwi-piRNA complexes are localized to somatic nuclei of the embryo. We propose that Piwi-bound piRNAs direct de novo establishment of repressive chromatin domains in somatic cells of the early embryo followed by its piRNA-independent propagation and maintenance during later development. We will explore this model by depleting maternal Piwi and analyzing its effect on chromatin. The proposed work will advance our knowledge of RNA-mediated regulation and of transcription chromatin structure in animals. We explore the function of piRNA in transmission of epigenetic information from generation to generation. Our studies promise to shed new light on how distinct chromatin domains are demarked. A detailed mechanistic understanding of Piwi-induced silencing, which prevents transposon activity and DNA damage in germ cells, will facilitate the development of individualized treatment strategies for human sterility.

NIH Research Projects · FY 2026 · 2014-07

Defining how cells regulate the uptake and efflux of transition metals such as Zn is a key component in elucidating cellular mechanisms of metal homeostasis. Bacterial model systems provide paradigms for understanding regulation mechanisms. In E. coli, the Zn2+-responsive metalloregulator ZntR senses Zn excess and activates Zn efflux systems (e.g., ZntA), while Zur senses Zn sufficiency and represses Zn uptake systems (e.g., ZnuABC), to keep this essential metal at appropriate physiological levels in the cell. Past research has provided significant insights into the structure, function, and mechanism of the protein players in regulating cellular metal concentrations, including metalloregulators, and metal uptake/efflux transporters, etc. Yet, many mechanistic pathways are still poorly understood, especially regarding spatially and temporally coordinated interactions among proteins and/or DNA that can reside at different locations in the cell. The long-term goal here is to understand how metal regulation in the cell can be manipulated for preventive and therapeutic purposes. Toward this goal, the PI has established an internationally recognized and unique research program that applies and develops advanced single-molecule/single-cell imaging approaches to interrogate and understand the mechanisms of bacterial metal regulation both in vitro and in live cells, which are further enhanced by bulk biochemical/biophysical and protein/genetic engineering approaches and by established collaborations with biologists and engineers. The research has led to the discoveries of first-of-their-kind mechanisms of metal- responsive transcriptional regulation and metal efflux. The objective of this renewal is to advance the study and understanding of bacterial metal regulation from single molecules and single cells toward cell communities, comprising three aims that focus on Zn regulation in E. coli: (1) define a “through-DNA” mechanism for Zn uptake- vs-efflux regulation; (2) define the mechanism of ZnuABC for Zn uptake in the cell; and (3) dissect cell-cell interactions in Zn homeostasis within bacterial communities. The research is significant because it will provide novel mechanistic insights into: how metalloregulators can act on each other on DNA, beyond the present paradigm of “set-point” mechanism; the spatiotemporal coordination of multicomponent Zn transporters for Zn uptake; and the cell-cell interactions in Zn homeostasis within a bottom-up cell community; and because these insights will deepen our understanding of cell biology of metals in general, including related processes in human cells, thus providing fundamental knowledge for identifying causes or developing preventions of diseases that involve similar regulation processes or for devising strategies to impair bacterial Zn homeostasis for antimicrobial treatments. The research is innovative because it generates novel mechanistic concepts in metal regulation, uptake/efflux, and emergent behaviors in microbial communities and because it applies novel single- molecule/cell imaging methods as well as microfluidic and optogenetic manipulations.

NIH Research Projects · FY 2026 · 2014-06

PROJECT SUMMARY / ABSTRACT Successful immunity relies on the rapid orchestration of diverse immune cell types that work in a dynamic and cooperative manner to drive location-dependent effector function. Immune cells do not work in isolation, and cooperation between immune cell types requires the sensing of local position-dependent cues that enable immune cells to aggregate into niches to exchange signals in a series of complex feedback loops. These coordinated interactions are critical throughout the developing immune response, from early activation events in inflamed lymph nodes through to effector function and resident memory in inflamed/infected targeted non- lymphoid tissues. A comprehensive understanding of immune responses thus requires the ability to examine the dynamic spatial and temporal exchange of information, in situ, in changing locations within and between tissues. To gain new insight into the biological complexity of immune activation in tissues, this P01 renewal brings immunologists and engineers together into the Programs multi-disciplinary scientific team, adding new concepts, perspectives, and tools to enable immunological questions to be addressed at ever higher spatial and temporal resolution. We aim to integrate dynamic intravital multiphoton microscopy with spatially defined transcriptomics to address fundamental questions about how topographical organization shapes T cell priming and effector function in response to infection. Shared models of infection examined in the lymph nodes and respective skin and lung tissue will enable integration of information across immune response time and space. The goal of this Program Project is to establish new paradigms in tissue immunity by gaining insight into cooperative events that shape immune cell positioning for exchange of signals that regulate effector function in lymphoid and non-lymphoid tissues. By spatially resolving immune response dynamics and transcriptional programs together, we aim to uncover new actionable strategies, based on the principles of immune position/zonation, to overcome poorly immunogenic environments and diminish overt inflammation. Project 1. Spatial Control of Effector T cell Activation. Dr. Deborah Fowell. Project 2. IL-17 Regulates Spatiotemporal Niches in LN. Dr. Mandy McGeachy. Project 3. Spatiotemporal Regulation of T cell Priming. Dr. Brian Rudd. Project 4. Monocytes Support Durable Tissue-Resident T cell Immunity. Dr. Minsoo Kim. Core A. Administrative. Dr. Fowell, Director. Core B. Imaging. Dr. Chris Xu, Director. Drs. Nishimura and Schaffer Co-directors. Core C. Spatial Transcriptomics. Dr. Iwijn De Vlaminck, Director.

NIH Research Projects · FY 2024 · 2012-04

ABSTRACT The mechanisms discovered through the study of embryogenesis have been fundamental to understanding disease. We use classic chicken embryology and sophisticated mouse genetics to elucidate how basic cellular processes define the shape and function of organs. We are most fascinated by left-right (LR) organ asymmetry, as errors of organ laterality are linked to life-threatening birth defects. The counterclockwise rotation of the gut is an excellent model to study organ laterality. A critical aspect of this rotation is initiation of a leftward tilt directed by the master regulator of LR asymmetry, Pitx2. Failure to do so leads to gut malrotation and catastrophic volvulus in pediatric patients. Whereas rotation forces had long been assumed intrinsic to the gut tube, we instead discovered that gut rotation is driven by asymmetric deformation of the adjacent dorsal mesentery (DM) that suspends the gut, and whose cellular architecture is downstream of Pitx2. A key property of the DM is its exquisite binary organization, with distinct LR compartments that are readily accessible to genetic manipulation. Cellular and extracellular matrix (ECM) changes in each compartment cause the DM to deform and tilt the attached gut tube leftward. This critical bias determines gut chirality and frames a model to explain how LR gene expression is ultimately responsible for changes in cell behavior that initiate asymmetric organogenesis. The DM is also the sole conduit for blood and lymphatic vessels that serve the gut. We discovered that Pitx2-dependent mechanisms directing gut tilting are also crucial for patterning the gut vasculature and provide a mechanism to coordinate these two processes. Whereas most situs-specific organogenesis depends on Pitx2, mechanistic studies downstream have been hampered by a confounding “double-right” isomerism in Pitx2 mutants. Whereas Pitx2 expression in all vertebrates is activated by Nodal, Nodal disappears before asymmetric morphogenesis, leaving unresolved the question of how Pitx2 directs organogenesis. We discovered that Pitx2 expression in the gut is not an extension of previous induction by Nodal. Instead, we demonstrate that gut rotation requires a “second wave” of Pitx2 that is subject to mechanoregulation by the latent TGFb, linking LR gene expression to force translation. In aim 1, we determine the mechanism of Pitx2 dose response during gut, vascular, and lymphatic development and identify two distinct roles for Pitx2 dependent on its repressive threshold on BMP4 signaling. In aim 2, we define the molecular mechanism by which the formin Daam2, a Pitx2 target, directs mesenchymal cell polarity, actomyosin contractile asymmetry, and thereby steers the forces to polarize tilting. In aim 3, we combine cutting-edge physical measurements of tissue properties with quantitative imaging and computational modeling, to elucidate how stiffness and force anisotropies drive gut rotation through mechanical feedback. Together, these studies will significantly advance our understanding of the transcriptional and mechanical control of asymmetric gut and vascular morphogenesis, a critical step toward improved malrotation diagnostics in newborns.

NIH Research Projects · FY 2025 · 2011-06

Project title: Molecular mechanism of piRNA biogenesis Project summary Non-coding RNAs have diverse functions in eukaryotic cells. Use of these non-coding RNAs in therapeutic approaches is a promising but rather unexplored direction in biomedical research. We discovered a new class of small non-coding RNAs, piwi-interacting RNAs (piRNAs), that together with their protein partners, Piwi proteins, recognize and silence endogenous genomic parasites called transposable elements and are involved in regulation of host gene expression. The silencing of transposons is critical in germline cells and the failure of piRNA-mediated repression leads to sterility in both Drosophila and mice. The mechanism of biogenesis of piRNAs appears to be distinct from that of other classes of small non-coding RNAs, microRNA and siRNA. piRNAs are encoded in distinct genomic regions dubbed piRNA clusters that produce long ncRNA transcripts, pre-piRNAs, that are further processed to mature piRNAs, which work as guides to recognize and repress RNA targets. In germ cells of Drosophila, dual-strand piRNA clusters are bound by the Rhi-Del-Cuff (RDC) chromatin complex, which is essential for non-canonical transcription of piRNA precursors and acts as a master regulator of piRNA cluster identity. However, how RDC is recruited to the genome to specify regions for piRNA production remains unknown. Our results suggest that piRNAs that are deposited to the egg by the mother guide recruitment of RDC to mark piRNA-generating loci during embryogenesis, and this mark is maintained during later development. After export from the nucleus, piRNA precursors are further processed and loaded into piwi proteins in a cytoplasmic membraneless organelle called nuage. We identified the scaffold protein of nuage and found that a posttranslational modification, symmetric methylation of arginine, of the cytoplasmic piwi proteins plays an important role in both piRNA biogenesis and nuage assembly. We will capitalize on our findings to understand critical steps of piRNA biogenesis in the nucleus and the cytoplasm. We will attack the problem of cluster specification by studying de novo establishment of piRNA clusters and molecular mechanisms of RDC recruitment and maintenance, and study nuage formation and the role that this compartmentalization plays in piRNA biogenesis. Our studies will help to advance our understanding of the mechanism of transposon silencing, which is important for both fertility and for genomic stability. It will also provide the basis for future use of the piRNA pathway as a tool in research and therapy. Importantly, the significance of the proposed research extends well beyond answering important questions in the non-coding RNA field. Our studies will provide clues to the problems of specification of distinct chromatin domains, decoding of the histone code and formation and function of membraneless cellular compartments. As such, our work will explore fundamental mechanisms that control chromatin organization and cellular compartmentalization.

NIH Research Projects · FY 2025 · 2011-06

New Targets for Reproductive Control of Mosquito Vectors Project summary/abstract In this project, we will investigate the actions of specific male seminal fluid molecules in promoting fertility and fitness of the major disease vector of dengue, Zika yellow fever and chikungunya viruses, Aedes aegypti. We will also define and analyze Aedes male and female molecules that associate with and support sperm. Despite the crippling impact of arbovirus infections, conventional mosquito control using insecticides can be operationally difficult and is often ineffective. Strategies to replace or reduce populations through modified male-releases show promise, but their success requires a deep understanding of mosquito reproductive biology. Ae. aegypti males transfer molecules in their seminal fluid to females during mating that induce female behavioral and physiological changes, including stimulation of egg development and oviposition, increased survival, and reluctance to re-mate with subsequent males. Despite the potential of these molecules as vector-control targets, the molecular identity and actions of these molecules are as yet unknown. During the current funding period, we utilized the latest tools to gain novel insights into key aspects of male mating success, sperm biology and female reproductive fitness, including identifying a limited subset of seminal fluid proteins (Sfps) that induce post-mating changes in females, and small-molecules such as juvenile hormone (JHIII) in seminal fluid. We propose two specific aims to (1) identify the role of individual male seminal molecules on female remating, fecundity, fertility, oviposition, lifetime reproductive success and survival and (2) to evaluate the role of male- and female-derived molecules on sperm storage, function, and survival. Our long-term goal is to design and deploy vector control strategies that target these key components of mosquito life history to reduce the tremendous global burden of mosquito-borne diseases.

NIH Research Projects · FY 2026 · 2009-08

PROJECT SUMMARY Sexual reproduction requires precise orchestration of expression of myriad genes in males and females. These genes mediate interactions between the sexes at molecular, cellular, neural and whole- organism levels, including the differential fertility success of sperm from two competing males that mate to a single female. In the current funding period, we used the tractable Drosophila model system to dissect the role of genes expressed in octopaminergic neurons in differential sperm use by a multiply-mated female. In this renewal application, we propose as Aim 1 a set of experiments that identify genes and quantify their effects on mating plug ejection, a proximal phenotype with a key role in sperm competition outcomes. Mating plug ejection timing allows females to exert control over paternity of their future offspring. Early ejection of a mating plug disadvantages the male by decreasing the number of his sperm that can be used; conversely longer retention of the plug gives the male a paternity advantage. Both males and females contribute to the mating plug and its ejection. After completing a GWAS for female and male determinants of mating plug ejection timing and validating the resulting genes, we will perform a grid cross to determine whether variation in mating plug timing is mostly determined by genetic variation in the male, or the female, or an interaction between the two. Follow-up perturbation of those genes will begin to unravel genetic pathways linking male quality or its perception with mating plug ejection. In Aim 2, we will pursue the implications of our recent discovery that mating induces differential exon or promoter use by a large suite of genes in the female brain. Intriguingly, two of these genes, desat1 and foraging, act in mating discrimination or other reproductive- relevant behaviors. Each strongly activates one of four alternative promoters in response to mating. We will survey the transcriptome to quantify how variation in male factors drive post-mating differential transcript use, how the latter varies across female genotypes, and how this regulation occurs. In Aim 3 we extend and generalize models designed to quantify and predict outcomes of pairwise interactions. The classical Bradley-Terry model, widely applied in predicting outcomes of sporting contests between teams that have not yet competed, has a direct analogy in sperm competition “contests.” Assessing fit to models of this class will test whether the males’ fitness can be rank-ordered, and whether those rank orders produce accurate outcome predictions. We will extend these models to the outcomes of the experiments in Aims 1 and 2, and those performed in this project over the years, using the results to assess the overall importance of male x female interactions in each case. Many of the processes that we will study show high levels of evolutionary conservation, implying that our results will expand our understanding of male x female interactions in reproduction that may have relevance to cases of idiopathic human infertility that may involve genetic incompatibility between the partners.

NIH Research Projects · FY 2026 · 2008-07

Project Summary / Abstract The immune system has the powerful ability to swiftly mobilize in response to challenge, migrating between and within tissues to locally deliver inflammatory mediators that can be anti-microbial, anti-tumor or auto destructive. Most infected/inflamed tissues express elevated levels of many chemokines and most chemokine receptors (CRs) can bind to multiple ligands, creating a highly redundant network of signals. The optimization of immune cell navigation through such a complex microenvironment remains unclear. Indeed, current immunotherapies for various cancers have exposed deficiencies in our understanding of how effector T cells (Te) access and position themselves in inflamed tissues, with very inefficient recruitment/retention of CAR T cells into the tumor itself. Such microanatomical positioning driven by CRs and chemoattractants exposes Te to regional antigen and inflammatory signals that likely tune transcriptional programs to boost or restrain effector functions. Indeed, single cell transcriptomics analyses in infected and malignant tissues point to significant functional heterogeneity in tissue Te cells, but lack spatial and temporal information that shape such heterogeneity. We have used an optogenetic strategy to “timestamp” Th1 cells within inflamed tissues and have found an unexpected change in CR expression based on time from tissue entry. This proposal aims to gain new knowledge of the temporal use of CRs that impact Th1 functional (re)programing. Such insight would enable design of targeted therapeutic approaches that harness or manipulate Te within the target tissues of infection, inflammation or malignancy. We have utilized in vivo optogenetic approaches combined with RNAseq to follow changes in CR expression by Te in the inflamed skin. Our data reveal a striking temporal regulation of CR transcription by Th1 cells following tissue entry. Disruption of this sequence of CR expression comprises Th1 function. We hypothesize that CR gene expression modulated in a temporal fashion enables incoming effector T cells to differentially sense chemotactic cues for correct tissue positioning. Moreover, location-specific events of tissue entry, early activation, through to positioning at the infection foci transmit distinct signals that hone the Th1 transcriptional program to optimize effector function. Tested in the following aims: Specific Aim 1. Signals that dynamically regulate CR expression and Th1 intra-tissue programing. Specific Aim 2. Stage-specific CR requirements. Specific Aim 3. Biological impact of temporal regulation of CR expression.

NIH Research Projects · FY 2026 · 2004-09

Project Summary How well organisms age and how long they live are fundamentally influenced by their genetics, life history, and environmental exposures. Gene expression regulators serve as critical nexus points for genome-environment interactions and as conduits for long-term "memory" that can profoundly impact organismal aging trajectories. C. elegans employs conserved gene expression regulatory mechanisms and offers numerous operational advantages, making it a powerful model for the proposed research. The long-term goal of this application is to elucidate the evolutionarily conserved gene regulatory mechanisms that bridge genome-environment interactions to impact healthy aging, particularly how signal communication between tissues and over time impact long-term physiology—discoveries that will have direct translational relevance to human aging research. Building on original discoveries from our laboratory, this application investigates the mechanistic connections between gene expression regulation, stress resilience, and healthy aging in C. elegans. Aim 1 investigates the mechanisms of action of SET-26 and HCF-1, two highly conserved chromatin/gene expression regulators with key roles in longevity that we have studied over the years. This aim will focus on elucidating where and when the ubiquitously expressed SET-26 and HCF-1 function to impact stress resistance and healthy aging. By combining acute depletion of SET-26/HCF-1 with time-course RNA-seq analyses, we aim to uncover both the direct targets of SET-26/HCF-1, and the subsequent molecular responses, which will likely uncover how spatial-/temporal-specific depletion of SET-26/HCF-1 result in systemic effects on healthspan and aging, likely revealing key inter-tissue signal communication. Aim 2 investigates the mechanistic basis of heat hormesis, wherein transient mild heat stress exposure confers stress resilience and promotes healthy aging. We have previously employed a multi-omic strategy to reveal the RNA expression and chromatin dynamics across a heat hormesis regimen and, in combination with functional screens, identified several candidate mediators of the healthy aging effects of heat hormesis. This aim will further delineate how heat hormesis impacts tissue aging, whether it elicits cell-specific molecular responses, and will leverage the candidate mediators as key molecular entry points to further elucidate their spatial-/temporal action in mediating the protective effects of heat hormesis. Since environmental stress exposure is universal across species, mechanistic understanding of stress hormesis will be critical for future development of stress management strategies that promote healthy aging. Taken together, this application takes a two-pronged approach to uncover the fundamental principles of gene regulatory mechanisms that bridge the genome and the environment to promote healthy aging. All the factors we propose to study are evolutionarily highly conserved and will have high relevance to aging biology in other organisms, including humans.

NIH Research Projects · FY 2026 · 2003-04

During prophase I of meiosis, crossovers (COs) ensure that parental homologs remain connected until the first meiotic division, when they must segregate equally into two daughter cells. Errors in CO formation and distribution are largely responsible for the very high rates of aneuploidy in humans. COs are initiated by the formation of DNA double strand breaks (DSBs) in early prophase I, but the number of DSBs are in huge excess relative to the final number of COs. In mammals, most COs are generated through the action of the DNA mismatch repair (MMR) pathway, including the MutSγ (MSH4/MSH5) and MutLγ (MLH1/MLH3) heterodimers. Of the 250 DSBs that form, ~150 load with MutSγ in early prophase I, and only ~ 23 of these will load MutLγ later in prophase I to become the final CO sites. The paring down of MutSγ sites which then load MutLγ must be exquisitely regulated to achieve a specific frequency and distribution of COs, and this lies at the heart of “CO designation”. Recently, we identified Cyclin N-terminal Domain-containing-1 (CNTD1) as a critical factor in determining which MutSγ sites load MutLγ, but the mechanism by which CNTD1 achieves the appropriate frequency and distribution of COs across the genome remains unknown. Studies in our current funding period have shown that CNTD1 interacts with components of the Replication Factor C (RFC) Complex. RFC is a general name for one of four RFC variants which are loaders/unloaders of Proliferating Cell Nuclear Antigen (PCNA). The PI’s lab has shown that PCNA is essential for mouse meiosis, and that post-translational modification (PTM) of PCNA on lysine 164 is critical for this activity. However, nothing more is known about how CNTD1, RFC, and PCNA function in concert to achieve CO designation and activation of MutLγ during meiosis. Recently, we have demonstrated that PCNA interacts in vivo with RNF212B, a protein that functions as an ubiquitin E3 ligase in cell culture, and knockout of which recapitulates the meiotic phenotypes of Cntd1 mutant mice. Thus, we hypothesize that CNTD1 regulates MutLγ activity and CO designation through interactions with RFC and PCNA, collectively forming a “MutLγ Activation Cascade”. We further hypothesize that the function of PCNA these events is dependent on the activity of RNF212B. Studies in this renewal application are aimed at elucidating this important regulatory circuit in a stepwise manner. In Aim 1, we will investigate how CNTD1 recruits RFC and PCNA to CO sites. In Aim 2, we will define the unique roles of each RFC variant in regulating PCNA loading/unloading during prophase I. In Aim 3, we will explore the function of PCNA in driving MutLγ activation, testing two alternative models, and we will determine the importance of PTMs of PCNA for these events. In addition, we will ask how RNF212B functions alongside PCNA to drive its activity. These results will establish CNTD1 as a critical upstream regulator of CO designation through its role in recruiting RFC and PCNA to facilitate MutLγ activation at a subset of MutSγ- defined sites, significantly improving our understanding of the etiology of aneuploidy in human gametes.

NIH Research Projects · FY 2025 · 1995-07

PROJECT SUMMARY Continued support is requested for a “Graduate Program in Comparative Medicine” at Cornell University College of Veterinary Medicine. Six post-doctoral positions are requested to provide training to DVMs seeking a PhD. The Comparative Medicine Program combines the very best that Cornell offers in the form of didactic graduate-level instruction, faculty supervision, and training-related activities. Trainees will follow one of three tracks: track 1 is geared toward a career in basic research, track 2 to a career in translational science, and Track 3 to public health/One Health. Training for each track is structured to ensure the orderly progression of scholars to independence. Research areas available to trainees are intentionally broad and include infectious disease, immunology, epidemiology, cancer biology, cell biology and signal transduction, genomics and genetics, developmental biology, molecular medicine, and neuroscience. The proposed program combines independent, faculty-guided research with formal coursework in experimental design, rigor, and ethics. The program also includes track-specific coursework and training in biostatistics & computational biology. Regular professional enrichment workshops will encompass training in communication, grant writing, and teamwork skills. Biannual workshops will discuss career options and provide career counseling. Graduate research assistantships will provide the first nine months of training support at Cornell's College of Veterinary Medicine. It is expected that trainees will apply for individual fellowships (“K” awards or equivalent) that would support the trainees as they finish their graduate studies and transition to independent careers. However, all trainers are selected to ensure training support continues independent of any fellowship award. We strongly encourage program alums to undertake at least two years of research beyond their Ph.D. degree, preferably in a related discipline and at a different institution, before accepting their initial appointment as an independent investigator. We expect most of our alums to enter careers as faculty members in U.S. veterinary colleges or medical schools or to enter careers in government or industry. The program aims to train veterinary scientists who can meet the national need for trained veterinarians within academia, industry, public health, and government to address problems relating to animal and human health.

NIH Research Projects · FY 2026 · 1976-07

Project Summary The importance of nutritional sciences to improving the public’s health has never been more evident. With two-thirds of the US population overweight or obese, the burden of chronic diseases, including diabetes, is increasing and the economic, social and human costs are significant and growing. With initiatives such as Nutrition for Precision Health, NIH seeks to transform nutritional sciences through innovative research on nutrition, dietary patterns and the effect of nutrition on the microbiome. There is a continuing national need for researchers who take a multifaceted approach to solve the most pressing questions, who understand the translation from basic to clinical levels of inquiry, and who contribute to translation of scientific discovery to evidence-based nutrition policy and practice. The proposed training program in the Division of Nutritional Sciences at Cornell University addresses this need by preparing trainees to produce interdisciplinary science that can drive impact across the translational spectrum from basic sciences to clinical and public health. The training program, with positions for 4 predoctoral trainees per year, is built on Cornell’s nutrition doctoral program, which emphasizes multidisciplinary and integrative scholarship across the biological, physical, behavioral, and social sciences. The 30 trainers participating in this application represent the broad range of disciplines necessary to achieve the goals of the training program and include renowned scientists with expertise spanning from genetics, molecular biology and biochemistry to epidemiology, psychology, and economics. The trainers have active research programs and excellent training records. The proposed training program includes a core curriculum (Grant Writing and Translational Research and Evidence-based Policy and Practice in Nutrition) that is complemented by the WHO/Cochrane/Cornell Summer Institute for Systematic Reviews in Nutrition for Global Policy Making. Trainees also submit an NIH F31 predoctoral application and participate in three enrichment activities including monthly trainee meetings, hosting an annual invited speaker, and organizing an annual half-day symposium. As part of the translational research training, trainees are co-mentored for at least one project in their dissertation. To meet national needs through doctoral training, the training program includes a combined PhD-RD training component for 1 trainee per year that comprises the above program elements and a short translational research or policy experience. The infrastructure to support the proposed training program is well-established, with added strengths from new faculty members with research programs in molecular nutrition, microbiome, proteomics, computational biology and nutrition and health inequalities. Highly successful partnerships with the World Health Organization and Cochrane significantly enhance Cornell’s capabilities in translational science and evidence synthesis. These new and continuing strengths support the program’s objectives to create an unparalleled training experience in the nutritional sciences and prepare the next generation of nutrition scientists.

Other NSERC · FY 2024

Graph Representation Learning, Dynamic Networks, Multi-Layer Networks, Urban Mobility Modelling, Optimizing Public Transit, Network Science, Public Transit, Sustainable Urban Transportation

Other NSERC · FY 2024

Algorithms, Machine Learning, Interpretable ML, Algorithmic Fairness, Theoretical Computer Science

Other NSERC · FY 2024

Continuous Optimization, Nonsmooth, Metric Geometry, Algorithms, Curvature, Computational Methods, Manifolds, Riemannian Geometry, Complexity

Other NSERC · FY 2024

Cold Atoms, Quantum Dynamics, Quantum Many-Body Physics, Superconductivity, Strongly-Correlated Systems, Theoretical Quantum Physics, Density Matrix Renormalization Group, High-Temperature Expansion, Tensor Networks, Path Integrals

Other NSERC · FY 2024

Stochastic Optimization, Online Learning, Ancillary Services, Electricity Markets, Dynamic Pricing, EV Charging Flexibility

Other NSERC · FY 2024

drug delivery, middle ear, inner ear, cell penetrating peptides, liposome

Other NSERC · FY 2024

Quantum Many-Body Physics, Phase Transitions, Non-Equilibrium Phases of Matter, Matrix Product States, Many Body Entanglement

Other NSERC · FY 2024

agroecology, intermediate wheatgrass, competition, sustainability, perennial, crop, farming, disturbance, soil erosion, succession

Other NSERC · FY 2024

Optimal Transportation, Applied Probability, Statistics, Functional Analysis, Numerical Methods, Optimization

Other NSERC · FY 2024

theorem proving, synthetic geometry, computer vision, natural language processing, mathematical reasoning, automated geometry theorem proving, deep learning

Other NSERC · FY 2024

plasma membrane, chemical biology, phospholipids, protein engineering, membrane curvature