Cornell University

NIH Research Projects · FY 2025 · 2020-01

Project Summary Testicular germ cell tumors (TGCTs) are exceptionally sensitive to conventional genotoxic chemotherapy. This is likely due to the distinct DNA damage response (DDR) features of TGCTs and the germ cells from which they arise. Unlike somatic cells which often respond to DNA damage by arresting the cell cycle and conducting DNA repair, germ cells as well as long-lived pluripotent stem cells typically avoid the use of error-prone repair mechanisms and favor apoptosis, reducing the risk of genetic alterations in subsequent generations. Similarly, the TGCT precursor lesion, germ cell neoplasia in situ, does not show activation of a DDR, whereas precursor lesions of most somatic cancers express markers of an activated DDR in response to oncogene activation which serves as a barrier to tumor progression. To study TGCTs, our lab has developed the first genetically engineered mouse model of malignant TGCTs by conditional activation of Kras, an oncogene, and inactivation of Pten, a tumor suppressor gene, in germ cells. The malignant teratocarcinomas generated in these mice are composed of pluripotent embryonal carcinoma (EC) and differentiated teratoma tissue. Interestingly, EC cells, both in vivo and cultured in vitro, express stem cell markers, have tumor propagating activity, and are readily killed following chemotherapy treatment. Using cells derived from this model, the experiments proposed here will elucidate the DDR properties of TGCTs and the cells from which they arise, which will inform the mechanisms underlying their exceptional chemosensitivity. Specifically, this proposal aims to: understand how the cells that give rise to TGCTs respond to oncogenic events, apparently avoiding DDR activation (Aim 1), and determine the mechanism underlying the chemosensitivity of the embryonal carcinoma components of TGCTs and the chemoresistance of their differentiated counterparts (Aim 2). Aim 1 will be investigated by generating primordial germ cell-like cells (PGCLCs) and embryonic germ cell-like cells (EGCLCs) from induced pluripotent stem cells (iPSCs) derived from mouse embryonic fibroblasts (MEFs) with conditional Pten and Kras alleles and assessing malignant transformation, the degree of DNA replication stress, the extent of DNA damage, and the nature of the DDR in untransformed and transformed cells. For Aim 2, I will analyze differential gene expression in EC and differentiated cells before and after treatment with genotoxic chemotherapy and investigate the role of differentially regulated pathways in the chemosensitivity phenotype. It is critically important to study this curable cancer because understanding the basis of TGCT chemosensitivity will apply broadly to the development of treatments for the many other cancers that do not respond favorably to conventional chemotherapy. Additionally, understanding the malignant transformation of pluripotent cell types, including embryonic germ cells and iPSCs, will be important for the improvement of stem-cell based therapies, which carry the risk of tumorgenicity.

NIH Research Projects · FY 2026 · 2020-01

Mismatch repair (MMR) factors, which act to remove DNA replication misincorporation errors, also function in genetic recombination and in adaptation to stress. The latter two roles are the current focus of my research efforts. Research Area 1 is centered on crossing over, a process critical in most eukaryotes for the accurate segregation of homologous chromosomes in meiosis to form gametes. Most crossovers in baker’s yeast meiosis result from the biased resolution of double Holliday Junction (dHJ) intermediates in steps involving Exo1 and the Mlh1-Mlh3 MMR endonuclease, a process conserved in higher eukaryotes. Little is known about how seemingly symmetric dHJs are resolved in a biased manner. We developed a model to explain biased dHJ resolution in which the Exo1 protein protects nicks in or near dHJs from being ligated; this protection promotes subsequent resolution by the Mlh1-Mlh3 endonuclease. We are testing it by developing a method in yeast to map meiotic DNA nicks genome-wide in wild-type, exo1, and other mutant backgrounds. The data will be analyzed in combination with Exo1 chromatin localization maps and established maps for meiotic chromatin marks and double-strand break sites to obtain a model for crossover resolution that will be refined through analyses of mutants displaying defects in early to late steps of meiotic recombination. Area 2 focuses on an analysis of baker’s yeast strains containing incompatible MLH1 and PMS1 MMR alleles. Our work supports an incompatibility model in which an elevation in mutation rate contributes to adaptation to stress conditions through the acquisition of beneficial mutations. However, long-term fitness costs associated with an elevated mutation rate must be eliminated by genetic suppression or buffered by mating. We will determine if signatures of adaptation to MMR incompatibility can be directly observed in yeast populations by first screening for mutations in PMS1 which partially or fully restore compatibility with mlh1 alleles containing mutations in different functional domains. We will then utilize information from the 1,010 yeast genomes project to create a model for how Mlh1 and Pms1 incompatibilities arise and are eliminated during adaptation to stress. Area 3 is focused on quality control mechanisms that act in genetic recombination. In the previous cycle we identified several chromatin factors which play roles in homologous recombination template choice and donor template stringency. We hypothesize that one of these factors, the SIR histone deacetylase complex, plays a role in stabilizing repair intermediates during slow to repair events such as break-induced DNA replication (BIR) by forming filaments adjacent to extending recombination intermediates (D-loop). We propose to track in yeast the localization of the SIR complex during BIR and correlate its cellular localization with repair intermediates as measured by chromatin immunoprecipitation and D-loop capture and extension assays. Our work provides an understanding for how defects in genome integrity underlie human infertility, hereditary forms of colon cancer, fungal pathogenesis, and diseases resulting from chromosomal rearrangements.

NIH Research Projects · FY 2024 · 2019-09

Summary Phages, which are the naturally evolved predators of bacteria, may hold the key to combating bacterial pathogens, including the looming threat of multidrug resistant bacteria. Phages are viruses which while harmless to humans and have been successfully engineered as tools to separate, concentrate, and detect their bacterial hosts. Additionally, phages have been used as therapeutic agents to treat patients infected with pathogens resistant to known antibiotics. While the potential benefits of phages are numerous, certain limitations must be addressed in order to fully employ them. The central hypothesis of this proposal is that both top-down and bottom-up approaches can be utilized to design and synthesize novel phages, through a combination of synthetic biology and machine learning. This will result in phage-based tools with increased functionality and customizable host ranges. The rationale for the proposed research is that as the threat of bacterial infections including those with multi-drug resistance continues to grow, phages, which have evolved to efficiently recognize and kill bacteria, will become indispensable tools. Therefore, the ability to rapidly design and engineer new phages for biosensing and therapeutics will be a critical advantage to human health. The proposal contains three specific aims which are supported by preliminary data and cited literature. Aim 1: Site-directed conjugation for advanced phage-based biosensors and therapeutics. Under this aim, phages will be modified with alkyne-containing unnatural amino acids allowing their direct conjugation to 1) azide decorated magnetic nanoparticles, and 2) azide terminated polyethylene glycol. The modifications will allow the development of magnetic phages for bacteria separation and detection, and phages that are more effective therapeutics due to their ability to avoid a patient’s innate immune response, respectively. Aim 2: Decoding phage biorecognition elements using machine learning. In this aim, machine learning will be used to model the binding of phages and their bacterial hosts. The model will enable the prediction of host interactions as well as allow the design and synthesis of novel phage tail fibers which can target specific bacterial isolates. Aim 3: Repurposing phage biorecognition for a broader host ranges. Under the final aim, phage-binding proteins will be replaced with those known to recognize conserved regions of the bacterial LPS, resulting in a phage with a much broader host range. This approach is innovative because it uses top-down characterizations for bottom-up design and synthesis of novel phages. Traditional phage screening methods will be replaced with the rapid synthesis of phages, which are optimized for a particular bacterial isolate. Following the successful completion of the specific aims, the expected outcome is the design and synthesis of phages that can be used to target a selected group of bacteria within Enterobacteriaceae for advanced biosensing and therapeutics. A publically available computer model will allow rapid design of custom phage biorecognition elements which can be added to functionalized phages. These technologies will allow researchers to tip the scales of the co-evolutionary arms race between phage and bacteria.

NIH Research Projects · FY 2026 · 2019-09

Project Summary/Abstract The human intestines are colonized by trillions of microorganisms, termed the gut microbiota, which are thought to rival the number of our own cells. Together, these microbes metabolize small molecules within the intestinal lumen through the activities of bacterial enzymes that carry out biochemical transformations. Growing evidence suggests that these small-molecule metabolites confer major benefits to host physiology. However, the enzymes and biochemical pathways that produce these molecules remain poorly understood. This proposal seeks to develop chemical approaches to understand the metabolic activity of the gut microbiome to better understand metabolite production in the gut and how it contributes to health and disease. The overarching hypothesis guiding this work is that activity-based protein profiling can be used to identify active bile acid metabolizing enzymes within the gut microbiome, which produce secondary bile acids that have critical functions in physiology and disease, and to identify bacterial proteins regulated by bile acids that play important roles in microbial metabolic crosstalk. We will address this hypothesis with the following studies: Develop chemical probes for identifying active bile acid metabolizing enzymes. Building on our systems biochemistry approach, we will develop activity-based probes to target important enzymes in secondary bile acid metabolism within the gut microbiota. These studies will globally identify individual gut bacteria that are actively producing bile acid metabolites, which cannot be addressed with traditional biochemical approaches. Characterize bacterial proteins that utilize or are regulated by bile acids. We will apply these chemical probes to identify bacterial proteins that are bile acid interacting proteins and characterize the roles of bile acids in regulating their activities. These results will advance our understanding of metabolic crosstalk between gut bacterial pathogens and commensal bacteria. Visualize gut microbiota-associated bile salt hydrolase activity in health and disease. We will apply the chemical probes to image active bile acid metabolizing enzymes within the intestinal tissue from mice in both health and disease. These studies will determine the localizations of gut bacterial niches that are actively metabolizing bile acids during gut epithelial damage and inflammation that are affected by maladaptive changes to gut microbial composition. Current technologies based on metagenomics are limited in their ability to report on genes that are present within the microbiome. Our chemical approach will define how activities of enzymes within the gut microbiome carry out metabolism of important small-molecule metabolites that regulate host physiology and pathology. Broadly, our tools will contribute to a deeper understanding of host-gut microbiota interactions and how this complex relationship influences human health and disease.

NIH Research Projects · FY 2025 · 2019-09

PROJECT SUMMARY Most bacteria maintain a cell wall, an essential, mesh-like structure mainly comprising the polysaccharide peptidoglycan (PG). Some of our most powerful antibiotics, the b-lactams (penicillins, carbapenems and cephalosporins) target enzymes required for cell wall synthesis and derive their efficacy from their ability to not only inhibit cell wall biogenesis, but also to actively cause its destruction. Cell wall destruction after exposure to b-lactams is mediated by “autolysins”, a group of enzymes (amidases, lytic transglycosylases and endopeptidase) with the capacity to cut a variety of chemical bonds within the PG mesh. Under normal growth conditions, autolysins engage in important cell wall remodeling functions, such as PG mesh expansion during cell elongation; how these functions are regulated to ensure proper PG maintenance is poorly understood. We have shown that in the diarrheal pathogen Vibrio cholerae, the endopeptidases (EPs) ShyA and ShyC are required for cell elongation during normal growth, but are also key factors mediating cell wall breakdown after exposure to beta lactam antibiotics. How ShyA and ShyC are regulated to ensure proper cell wall maintenance in the absence of antibiotics is unknown. Here, we propose experiments to build a thorough understanding of mechanisms of endopeptidase regulation in V. cholerae on multiple levels. Since M23 EPs are well-conserved throughout Bacteria, our experiments will likely yield insights with broad relevance to other pathogens. Leveraging extensive preliminary screens, we will i) establish how EPs are co-ordinated with PG synthesis, ii) interrogate the functional interaction between EPs and other autolysins, and iii) determine the role of proteolytic processing in EP activity regulation. Taken together, these experiments will provide us with an extensive framework of how bacteria maintain the balance between cell wall synthesis and remodeling, which could ultimately lead to the discovery of new potential targets for antibiotics that modulate autolysin activity.

NIH Research Projects · FY 2025 · 2019-08

Project summary: The MacCHESS Synchrotron Source for Structural Biology facilitates the utilization of both established and emerging technologies to enable the structural characterization of proteins involved in critically important biological processes with significant consequences for diseases such as cancer, bacterial and viral infections, and neurodegenerative disorders. Recent upgrades to CHESS, including improvements to the storage ring and newly designed beamlines have provided state-of-the-art facilities and enabled greater capabilities since June 2019. MacCHESS will continue to support more than 100 investigator projects, funded by NIH and other government institutions, through two major Technology Operations Cores. These are: 1) Facility for Flexible Crystallography, which will take advantage of unique MacCHESS capabilities to enable the development of new X-ray techniques that may be used to broaden our knowledge of essential biological processes. Examples include continued development of methods for room temperature crystallography toward identifying new structural or ligand/drug binding states, the application of high pressure to crystals, and analysis of macromolecular motions through the study of X-ray diffuse scattering. A high level of support for more routine macromolecular crystallography will also be provided. These methods will help to provide important new insights including the binding interactions and conformational transitions that cell signaling proteins and drug targets undergo which are necessary for their function, and the macromolecular motions essential for the catalytic activities of enzymes that regulate key metabolic processes, DNA transcription and repair, and different aspects of RNA biology. 2) Facility for Biological Small Angle X-ray Scattering (BioSAXS), which will implement state-of-the-art hardware, software, and expertise to support the BioSAXS technique that continues to be in high demand. In addition to determining the shapes of proteins, nucleic acids, and larger protein assemblies in solution, BioSAXS allows researchers to obtain information regarding global conformational changes within macromolecular complexes (e.g., membrane receptors, RNA-splicing complexes, large or multi-subunit enzymes) and the changes in their oligomeric states that have important functional consequences. This core will also provide the necessary equipment and expertise for investigators interested in performing time resolved BioSAXS or BioSAXS studies conducted under high pressure. MacCHESS will provide a strong Administration Core to support these activities and will continue to educate and help to train users and investigators from the biomedical research community new to the field of structural biology, through a Training and Outreach Core. Collectively, these efforts will offer unique opportunities to our users for pursuing some of the most challenging questions in structural biology and for obtaining structure-function information that will ultimately highlight novel therapeutic targets and aid in the development of clinical strategies for dealing with disease.

NIH Research Projects · FY 2025 · 2019-08

PROJECT SUMMARY Food intake is a finely tuned innate behavior controlled by a complex network of sensory, homeostatic, and hedonic mechanisms. In metabolic diseases such as obesity or eating disorders such as anorexia nervosa, the body's normal perception of food and appetite is altered, leading to overeating and weight gain or reduced appetite and weight loss. Understanding the genetic and neural mechanisms regulating food intake and appetite is essential for developing effective treatments for both conditions. While neural circuits that regulate food intake have been extensively studied in rodent models, the complexity of the mammalian brain makes it challenging to explain the underlying molecular mechanisms and circuit dynamics. The overarching goal of this project is to understand the neurogenetic mechanisms by which the brain communicates with the body to regulate food intake and foraging at the neural and molecular levels. We use a genetically tractable model organism, the fly, Drosophila melanogaster, to study the metabolic-state-dependent regulation of food intake and foraging at the levels of genes, cells, and circuits. Previously, we pioneered in vivo functional imaging methods to record the activity of molecularly defined populations of neurons in the fly brain and the enteric nervous system during food ingestion. We also developed high-resolution behavioral assays to capture the foraging and food intake behaviors of individual flies in real time with high temporal resolution. Using these methods, we showed that flies regulate their food ingestion and foraging behaviors by integrating metabolic state and taste/nutritional information in the brain. We further investigated the mechanisms by which a novel class of interneurons (IN1) regulates sugar ingestion. We demonstrated that IN1 neurons regulate sugar ingestion through their bi- directional communication with the fly gastrointestinal tract. Over the next five years, in this R35 MIRA grant, we aim to reveal the molecular pathways that regulate the activity of IN1 neurons in different metabolic states and determine how they become persistently active during sugar ingestion. We will also identify and characterize the functions of central and peripheral neural circuits that interact with IN1 neurons to regulate food intake, foraging, and other innate behaviors. Functional dissection of IN1 circuitry will lead us to the fundamental principles by which the nervous system regulates foraging and food intake. Additionally, we will characterize the molecular and anatomical architecture of the fly enteric nervous system and the functions of enteric neurons in regulating food ingestion and nutrient preference. Understanding the fundamental neural and molecular mechanisms underlying foraging, food intake, and nutrient sensing in flies will help us uncover conserved pathways and principles that regulate these vital behaviors across different species. Once we discover key neurogenetic mechanisms underlying food intake and foraging, we can search for similar processes in more complex mammalian models and in patients suffering from obesity or eating disorders to develop treatment strategies that intervene in the pathogenesis of these life-threatening diseases.

NIH Research Projects · FY 2025 · 2019-08

Project Summary/Abstract There is a growing need for monitoring antimicrobial resistance in animals in order to protect the safety of both people and animals. Capacity for bacterial whole genome sequencing in veterinary institutions supports the mission of FDA to ensure the safety of our nation's food supply and protect public health by enabling real-time monitoring of isolates. In this proposal, we request funding to maintain capacity for our bacterial sequencing services for FDA. The capacity to perform advanced molecular characterization of bacterial isolates in a reliable manner is critical to our ability to respond quickly to suspected microbiological contamination and monitor antimicrobial resistance in animals and feed. Importantly, having this capacity fills a critical surveillance gap by monitoring live animals for antimicrobial resistance. In addition to serving as one of the sequence reference labs, the Goodman laboratory at Cornell has provided analytic support to the program office and mentorship to the other laboratories by providing validation, methods, and Quality Assurance documentation for these procedures. The proposed studies will have important impacts for both human and animal health and will improve the capabilities of Vet-LIRN to perform effective surveillance for food-borne pathogens.

NIH Research Projects · FY 2026 · 2019-01

PROJECT SUMMARY/ABSTRACT Molecular mechanisms regulating and interpreting BMP signaling The highly conserved bone morphogenetic protein (BMP) signaling pathway regulates multiple developmental and homeostatic processes. Malfunction of the pathway can cause a myriad of somatic and hereditary disorders in humans, including skeletal and cardiovascular diseases, and cancer. Thus BMP signaling must be tightly regulated to ensure that signaling happens at the right time, place, level and duration. Due to the vital developmental functions of BMP signaling, it has been proposed that therapeutically targeting specific BMP modulators is a more productive way for treating different diseases caused by defects in BMP signaling. C. elegans provides an excellent model to study the regulation of BMP signaling at single cell resolution during the development of an intact animal. Using a highly specific and sensitive genetic screen, we have identified multiple evolutionarily conserved modulators of the BMP pathway. These modulators include cell surface integral membrane or membrane-anchored proteins, extracellular secreted proteins, as well as transcription factors. Our research goals under this MIRA are to determine mechanistically how different BMP modulators function in regulating BMP signaling, and how BMP signaling is interpreted in specific cellular contexts. We propose to use a multifaceted approach that combines classical molecular genetic studies with cutting- edge imaging, proteomic and metabolomic approaches to dissect the functions of the BMP modulators in C. elegans. Findings from our proposed studies will yield important insights into the complex and intricate mechanisms regulating and interpreting BMP signaling in a multicellular living animal. They may also provide potential therapeutic targets for the different diseases caused by mutations in the BMP pathway.

NIH Research Projects · FY 2025 · 2018-09

Project Summary This proposal focuses on using electrochemistry as an unconventional tool to uncover new organic reactions and invent synthetic strategies with the goal of facilitating the preparation of bioactive compounds. Improving the organic synthesis of medicinally active compounds is crucial to modern biomedical research. In this context, oxidation and reduction reactions are among the most important and frequently used processes in organic synthesis. However, manipulating the oxidation states of functional groups in complex settings with high efficiency, precision, and minimal waste remains an important challenge in modern organic chemistry. Given its many distinct characteristics, electrochemistry represents an attractive approach to discovering new reactivities, enabling new synthetic strategies, and meeting the prevailing trends in organic synthesis. Therefore, there exists a clear impetus for the invention of new reaction strategies to improve the scope of synthetic electrochemistry and provide new platforms for reaction discovery and synthetic innovations. In the past funding period, we demonstrated a new catalytic approach that combines electrochemistry and redox-metal catalysis for the oxidative difunctionalization of alkenes to access a diverse array of highly functionalized structures. These promising results led us to envision that electrochemistry will ultimately emerge as a powerful tool for solving a wide range of longstanding synthetic problems. In the new funding cycle, we aim to build upon our previous success but move our research into new grounds. In each of the projects targeted herein, we aim to apply electrochemistry to address a prominent challenge in organic synthesis. The transformations targeted in this grant are either currently unknown or display salient limitations in reaction scope or selectivity. Among the specific reactions that we aim to develop in the context of this grant are: two- and three-component cross-electrophile couplings; asymmetric synthesis and late-stage functionalization of N-containing compounds; and regioselective C–H functionalization of pyridines. In addition, in-depth studies using canonical physical organic and electroanalytical techniques will provide insights into the reaction mechanisms. The development and mechanistic understanding of these proposed transformations will represent significant advances for the field of organic synthesis.

NIH Research Projects · FY 2026 · 2018-09

SUMMARY Meiosis is the specialized cell division that gives rise to haploid gametes for sexual reproduction. During prophase I, homologous chromosomes are physically tethered via the formation of the synaptonemal complex while undergoing DNA double strand break (DSB)-induced recombination. In XY mammals, there is an additional challenge presented by the sex chromosomes, which synapse only at the Pseudoautosomal Region (PAR), leaving vast asynapsed regions that trigger a unique chromosome-wide transcriptional silencing mechanism termed Meiotic Sex Chromosome Inactivation (MSCI). MSCI occurs within the context of the Sex Body (SB), a membrane-less sub-domain of the nucleus that houses the XY. MSCI is a specialized version of the broader process of Meiotic Silencing of Unsynapsed Chromatin (MSUC) that occurs in male and female meiosis when homologs fail to synapse, triggering apoptosis of aberrant germ cells. All of these events are critical to ensure the formation of viable euploid gametes, underscored by the fact that humans show exceptionally high rates of meiotic errors leading to miscarriages and birth defects, with non-disjunction of the sex chromosomes, Klinefelter syndrome, being the most frequent trisomic disorder (1:500 live births). Our labs and others have shown that the kinase ATR is central to many prophase I events, including DSB repair, synapsis, and MSCI. However, these numerous overlapping roles have posed a barrier to understanding the precise mechanistic actions of ATR in meiosis. In the prior funding cycle, we generated novel separation-of-function mouse mutants in ATR regulators that allow us to dissect specific roles for ATR in MSCI with minimal effects on its other meiotic functions. These mice bear mutations in TOPBP1, a key ATR activator that also mediates substrate selectivity, and in RAD9A/B, components of the 911 clamp that helps anchor TOPBP1. Our analysis revealed critical functions of TOPBP1 and 911 in driving ATR functions within the SB, and highlighted essential downstream ATR targets in these processes such as the RNA:DNA helicase Senataxin (SETX). We hypothesize that the ATR-TOPBP1-911 axis plays critical roles in establishing the unique chromatin and transcriptional environment required to initiate, maintain, and terminate MSCI in a temporally restricted manner during meiotic prophase I, and that this function is dependent on downstream ATR targets including SETX. Utilizing our unique mouse models in combination with high-resolution genomic tools and cutting-edge proteomic approaches, we will elucidate the mechanisms by which ATR signaling orchestrates MSCI in male meiosis, and MSUC in male and female meiosis. Finally, based on our findings that ATR signaling is constantly antagonized, indicative of prominent roles for phosphatases during MSCI maintenance and termination, we will determine the mechanisms by which ATR signaling is counteracted by phosphatases to achieve temporally-appropriate shutdown of transcriptional silencing, permitting prophase I completion and enabling the production of high quality gametes.

NIH Research Projects · FY 2025 · 2018-07

Abstract Basic cellular processes essential to mammalian tissue development and adult regeneration, are often controlled by gene expression regulation at the mRNA level. Total mRNA level for each gene depends on two mechanisms: active (or ‘nascent’) transcription and post-transcriptional RNA stability/degradation (e.g. turnover rates). Nascent transcription itself is regulated by multiple steps of recruiting histone-modifying enzymes and opening chromatin, RNA polymerase II (Pol II) initiation and pausing, and the release of Pol II into productive RNA elongation. Diverse mechanisms that regulate total mRNA level may be important in controlling distinct biological processes and pathologies, but this is poorly understood in vivo. Using skin as a model system we will map lineage-specific gene patterns of RNA Pol II activity and nascent transcription in specific cell-types within their natural tissue milieu in the absence of cell isolation. Furthermore, we will investigate changes in these nascent-transcription patterns along an adult tissue stem cell activation and differentiation path, using hair follicle as a lineage prototype. In addition, our work will help dissect relative contributions of active transcription vs post-transcriptional RNA turnover/stability to overall mRNA levels in specific cell types in vivo. Furthermore, we will determine how a known histone repressive mark (H3K9me3), poorly understood in mammalian tissues, acts on nascent transcription and RNA Pol II pausing in vivo. We will uncover for the first time the physiological role of the 3 main H3K9me3 histone methyl transferases in skin development and homeostasis. Finally, we will investigate how H3K9me3 acts on nascent transcription in vivo using our newly developed mouse genetics tools and methodologies. Our work will provide previously unavailable mechanistic insight into gene regulation in mammalian tissue biology using skin as a model system.

NIH Research Projects · FY 2025 · 2018-07

Project Summary. Catalysis has long been a potent force for advancing biomedical research by enabling the construction of biologically important molecules with ever-increasing speed, efficiency, and versatility. One of the most potent driving forces for progress in the area of catalysis has been the discovery of new catalytic platforms and concepts. The proposed research program is broadly focused on advancing the area of catalysis through the development of novel catalytic platforms, concepts, and methods. In particular, we have developed two generic catalytic strategies with broad applicability over a wide array of reaction types. In the first program, we have helped to pioneer the area of electrophotocatalysis, which combines the power of light and electrical energy within a single catalyst to promote challenging chemical reactions. Especially, we have introduced trisaminocyclopropenium (TAC) ions as a novel class of electrophotocatalyst; these catalysts have proven to be useful for a number of transformations, including C–H bond functionalizations and olefin derivatizations. The current application seeks to leverage electrophotocatalysis to realize other challenging, unprecedented reactions, especially those requiring very high redox potentials or involving multiple redox events. In the second program, we have pioneered the area of catalytic carbonyl-olefin metathesis, using hydrazine catalysis. This foundational capability enables a wide range of potentially advantageous synthetic methods. The current application seeks to make major advances to this program by designing highly reactive, next-generation catalysts that greatly expand the scope of substrates that can be engaged. We also aim to apply this mode of catalysis to useful new chemical methods, and to develop related catalytic platforms using these general principles. These and other investigations into the use of novel ideas in catalysis will continue to serve as a stimulus for advancements in chemical synthesis.

NIH Research Projects · FY 2026 · 2018-04

User Training and Outreach Abstract The two main goals of the user training and outreach component are: (1) provide user training to prepare biomedical researchers to apply NE-CAT technology to their research, and (2) engage in outreach activities to inform the scientific community about the technical capabilities of NE-CAT, promote and enable broader use of NE-CAT technologies, and actively recruit new user research projects to NE-CAT. User training includes online manuals and videos, which are regularly updated, one-on-one, in person training for on-site users, and videoconference-based training for remote users. We also provide enhanced training on all aspects of macromolecular crystallography from sample preparation to structure determination and refinement. We will develop a new system for tracking the training status of each user, enabling support staff to tailor their efforts more effectively. Our training activities extend to the larger user community through annual workshops. We will expand our training by offering a class on practical macromolecular crystallography to both the NE-CAT community and outside groups. NE-CAT maintains a website that is largely targeted to past and present users. We will expand the website to contain more information of interest to potential new users. NE-CAT freely distributes software such as RAPD, NE-CAT’s automated data collection and analysis software, and all hardware designs, many of which have been adopted by other APS beamlines. We will work to broaden our user community through a combination of outreach to new primary investigators and enhanced retention efforts for current primary investigators and postdoctoral scientists.

NIH Research Projects · FY 2026 · 2017-09

Project Summary: Overall Despite the enormous suffering endured by millions of people worldwide, the underlying causes of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) are unknown and effective therapies are lacking. ME/CFS is characterized by debilitating fatigue, musculoskeletal pain, headaches, cognitive difficulties orthostatic intolerance, and sleep disturbances. The absence of simple objective tests prevents many from obtaining an appropriate diagnosis and inhibits drug development because of the lack of biomarkers to monitor the efficacy of experimental therapies. In order to gain fundamental mechanistic insights into ME/CFS, we will leverage the experience, capabilities and varied backgrounds of researchers from four different colleges at Cornell University, Florida Atlantic University, the Hospital for Special Surgery, and an ME/CFS expert physician. We will take advantage of an enormous amount of data already obtained from patients and controls both before and after symptom provocation through exercise, as well as a valuable set of new samples. Three research projects will seek to (1) use cutting-edge multi-omic single cell profiling to examine alterations in cell types, gene expression, and cell-cell interactions that occur in ME/CFS muscle (Project 1), (2) identify tissue injured in ME/CFS following exercise through characterization of RNA released into circulation and (3) identify the RNA and protein cargo of extracellular vesicles in ME/CFS patients that may alter function of target cells (Project 2) and (4) Use genomic and computational methods to better understand the gene regulatory mechanisms that result in immune dysregulation in ME/CFS and systematically identify ME/CFS-specific alterations in signaling across the immune system (Project 3). These three research projects are supported by a Research Core that will act as a resource for genomics technology expertise, reagents, and services and for data management and integrated analysis. Multi-omic analysis and predictive modeling carried out in all three Projects will provide a foundation for future development of therapeutics and diagnostic tests. All Center activities will be coordinated through an Administrative Core, which will foster synergy and integration within the Center, while also being the platform for collaboration with other ME/CFS Collaborative Research Centers, a Patient/Advocate/Caregiver Committee, other ME/CFS researchers, and the Data Management Coordinating Center. The Administrative Core will also be responsible for outreach activities that are designed to increase awareness and understanding of ME/CFS within the research community, health professionals, and the general public, and will administer a pilot project program designed to bring new ideas and researchers into the ME/CFS field.

NIH Research Projects · FY 2026 · 2017-09

Bioinorganic Chemistry of Nitrogen The long-term goal of the PI’s research program is to understand how biology uses transition metals to control the speciation of redox-active substrates including reactive or “fixed” nitrogen species (RNS). Reactive nitrogen species serve vital roles in biology. For example, nitric oxide (NO) is a cellular signaling agent that regulates vasodilation in mammalian systems. In a separate context, nitrate (NO3–) can substitute for dioxygen (O2) as the terminal electron acceptor during cellular respiration by bacteria that include human pathogens. This proposal describes a continuation of efforts to elucidate mechanisms of the biogeochemical nitrogen cycle via the study of metalloenzymes as well as model complexes that interconvert RNS. A key knowledge gap that will be addressed through proteomics and enzymology concerns the means by which ammonia oxidizing archaea derive chemical energy from the oxidation of hydroxylamine. The operative enzyme and the product of this reaction remain unknown. Activity guided purification and mass spectrometry will furnish the identity of this globally proliferated nitrogen cycle protein for subsequent characterization by spectroscopy, X-ray crystallography, and kinetics. Further work will explore product selectivity in RNS oxidation biochemistry by heme P460 proteins to determine how NO is selected over nitrous oxide (N2O) to differentiate metabolic from detoxification proteins. The PI will continue to collaborate with leading bioinorganic chemists to understand how transition metals prime RNS for oxidation or reduction and how selectivity in these reactions is achieved. These collaborations will leverage the PI’s expertise in X-ray spectroscopic as well as in other inorganic spectroscopies. Key examples of these collaborations involve site-selective spectroscopic probing of metal atoms in the FeMo cofactor of nitrogenase, the means by which multicopper clusters reduce N2O, and studying the electronic structures and reactivities of Lewis-acid stabilized RNS that have been rendered capable of undergoing redox transformations independent of proton transfer.

NIH Research Projects · FY 2026 · 2017-09

PROJECT SUMMARY Transposable Elements (TEs) make up a large fraction of vertebrate genomes, including half of the human genome. The mutagenic properties of TEs are well documented and they are important drivers of genetic variation between and within species. However, how this enormous source of genetic variation has shaped the evolution and biology of species remains poorly understood. Our MIRA project is designed to yield transformative insights into the biological significance of TEs in evolution and disease. Our previous research has focused on the long-term impact of vertebrate TEs in driving genetic innovations. Notably, we showed that TEs have been a recurrent source of raw sequence material co- opted during vertebrate evolution to create new cis-regulatory elements driving changes in gene expression and new protein-coding genes underlying the emergence of novel cellular functions. Most of these events involved ancient TEs long inactive transpositionally. In this next funding cycle, we are turning our attention to young TEs -- those recently or currently mobile. We focus on the developmental impact of young TEs in humans and in zebrafish, a powerful model organism for studies of vertebrate development. By focusing on the functional impact of young retroelements in embryonic development we will uncover the molecular underpinnings of evolutionarily recent biological innovations. Notably, we will investigate the regulatory contribution of TEs in defining features of the human placenta, such as its deep invasion into maternal decidual tissue, and the functional significance of endogenous viral-like particles produced in the early stages of human embryonic development. Our work will also uncover general principles that lead to the cooption of specific TE sequences for cellular function. Specifically, we will test a new provocative model of host-TE interaction in which organismal development becomes dependent on TE- encoded products. We will test this ‘addiction model’ by studying the trans-regulatory activities of Gag (capsid) proteins encoded by endogenous retroviruses in human and zebrafish for which we have obtained preliminary evidence they modulate embryonic developmental processes. The outcomes of this project are expected to shift our view of host-TE interactions from conflicting to mutualistic. Our studies will also yield new mechanistic insights into poorly understood disease processes, such as pregnancy loss by preeclampsia and neurodevelopmental disorders, implicating the dysregulation of young TEs.

NIH Research Projects · FY 2025 · 2017-08

Abstract The central goal of this project is to understand how protein motion gives rise to function. However, visualizing proteins in motion is a problem that is inherently difficult in structural biology as it involves signals from many conformations. To tackle this challenge, we take an interdisciplinary approach. We apply our understanding of the theory of scattering-based structural methods to develop new computational methods for processing and interpretating data representing conformational disorder. We have developed methods to extract dynamic infor- mation from protein crystals that have the potential to animate crystal structures, and we have mapped the structural interconversions of allosteric and flexible enzymes. In the coming years, we will continue to tackle fundamental questions about the molecular mechanisms of protein allostery and catalysis, and to do so, we will expand the scope of our work by integrating cryo-electron microscopy (cryo-EM) with advanced X-ray methods. Our goals are to: capture correlated motions in allosteric networks, apply our expertise with mathematical de- composition to the problem of conformational heterogeneity, and (3) probe protein motions that control catalysis.

NIH Research Projects · FY 2026 · 2017-08

Lysosomal function of progranulin and neurodegeneration Mutations in the granulin (GRN) gene, resulting in haploinsufficiency of the progranulin (PGRN) protein, are a main cause of frontotemporal lobar degeneration (FTLD). PGRN is comprised of 7.5 granulin repeats and processed into individual granulin peptides in the lysosome. PGRN and granulin peptides are critical for proper lysosome activities and regulate many microglia mediated functions. However, how granulin peptides function in the lysosome is still unknown. Using our newly generated antibodies against individual granulins, we have shown that levels of individual granulin peptides can be differentially regulated. One granulin peptide, granulin E, interacts with CD68, a lysosomal membrane protein highly expressed in microglia. We have found that loss of CD68 specifically affects the levels of granulin E but no other granulins and PGRN deficiency leads to changes in CD68 levels and molecular weight, indicating that granulin E and CD68 regulate each other’s homeostasis in the lysosome. We have also discovered that PGRN interacts with alpha-N- acetylgalactosaminidase (NAGA), a lysosomal glycosidase, via the granulin F domain, and regulates NAGA activities. Based on these preliminary data, we hypothesize that individual granulin peptides have unique properties and functions in the lysosome through binding to specific lysosome protein(s). Three specific aims are proposed to further dissect the properties and function of granulin peptides in the lysosome. In Aim1, we will determine the processing and stability of individual granulin peptides, by measuring the levels and half- lives of individual granulins vs full length PGRN in neurons vs microglia, the two main cell types that express PGRN in the brain. Changes in the levels of individual granulins will be assayed during aging and neurodegeneration. The mechanism by which CD68 regulates the levels of granulin E will be elucidated. In Aim2, we will dissect the functions of individual granulins in the lysosome. The mechanisms by which granulin peptides E and F regulate CD68 and NAGA functions will be investigated, respectively. Binding partners for other granulin peptides will be identified through proteomic screens and characterized. In Aim 3, we will investigate the role of granulins in maintaining lysosomal membrane integrity and the transcriptional program involved in microglial activation upon PGRN loss, since we have found that PGRN deficiency leads to impaired lysosomal membrane integrity and enhanced expression of lysosomal proteins and inflammation markers in microglia. The proposed studies will shed light on the physiological functions of granulin peptides in the lysosome and the connection between lysosomal dysfunction and microglial activation. Our work will also facilitate therapeutic development for FTLD and other devastating neurodegenerative diseases.

NIH Research Projects · FY 2026 · 2017-08

Project Summary Cancer metastasis accounts for over 90% of all cancer deaths. Important abilities of metastatic tumor cells include breaking away from the primary tumor and invading into surrounding tissue before disseminating to secondary tumor sites. Solid tumor stress caused by rapid growth of tumor cells and abnormality of the vascular tissue has long been associated with poor prognosis of cancer. Despite the clinical importance, the basic understanding of tumor mechanics and its relation to tumor invasion is lacking. This is in part due to the lack of in vitro tools that are able to investigate quantitatively tumor mechanics in a physiologically relevant 3D setting. Current material mechanical testing tools such as the rheometer have played important roles in our current understanding of biomaterials and tumor mechanics. However, the conventional rheometer is not easily made compatible with cell culture conditions and results are spatially averaged, masking important single cell and molecular level information. Atomic force microscopy and pipette aspiration are cell culture compatible, but low throughput. The goal of the proposed research is to develop a high throughput microfluidic rheometer for systematic studies of tumor mechanics and invasion in a physiologically realistic 3D setting and compatible with dynamic optical imaging at single cell and spheroid levels. We will deliver a set of principles that govern tumor mechanics and its relation with invasion. We postulate that tumor mechanics is a key predictor for tumor invasiveness. The proposed project is innovative because it represents the first generation of microfluidic rheometers that are capable of full mechanical testing for tumor mechanics studies, and at the same time compatible with tumor invasion experiments. Tools developed here can be easily extended to use for other living materials, and lessons learned here will eventually lead to knowledge important for developing novel diagnostic or/and treatment strategies for cancer.

NIH Research Projects · FY 2026 · 2017-06

The Crane group studies signal transduction systems that respond to or involve photochemistry and redox chemistry. Our overall goal is to understand the behaviors of bacterial chemotaxis and eukaryotic circadian rhythms at the level of molecular reactivity through the study of macromolecular complexes that underlie transmembrane signaling, motility, and gene expression. Chemotaxis has long served as a key system for studying transmembrane signaling, intracellular information transfer, and cell locomotion. Furthermore, many human pathogens that cause diseases, such as cholera, gastric cancer, and Lyme, rely on chemotaxis to establish and sustain infection. The sensory apparatus of chemotaxis displays remarkable sensitivity, dynamic range, and molecular memory. Chemoreceptors, histidine kinases (CheA), and coupling proteins assemble into large molecular arrays, wherein long-range cooperative interactions among components produce highly specific responses that adapt to changing conditions. This proposal continues efforts to understand receptor:kinase assembly, chemoreceptor conformational signaling, and ultimately, CheA regulation. CheA output modulates Nature's consummate nanomachine – the flagella motor. The architecture of the switch complex within the motor will be refined to better understand torque generation and direction switching. A particular focus will be the pathogenic spirochetes, which exhibit asymmetric flagella rotation at their respective cell ends. The second system, circadian clocks, comprises of cell-autonomous timing devices that pace metabolism to the diurnal cycle. Clocks are composed of transcriptional-translational feedback loops (TTFLs) within which repressor proteins inhibit the transcriptional activators of their own genes. Light entrains the clock phase by stimulating photosensors that impinge directly on the TTFLs. In humans, aberrant clock function causes mental illness (sleep disorders, depression, and mania), cell growth deregulation (cancer), and metabolic defects (diabetes and obesity). This project proposes structural and mechanistic investigations of the key light sensor and repressor activities in representative clocks from fungi (Neurospora crassa) and flies (Drosophila melanogaster). A complimentary set of biophysical techniques, including X-ray crystallography, small-angle X-ray scattering, optical spectroscopy, cryo-electron microscopy, and pulse dipolar ESR spectroscopy (PDS), will be applied to accomplish these goals. Biochemical reconstitution that leverages protein engineering to procure key entities will be combined with cellular assays and organismal studies in order to correlate physical properties with biological function. For PDS, new methods for incorporating spin probes that are based on nitroxides, flavins, nucleotides, and metal ions will be deployed for measuring structure and dynamics both in vitro and in vivo. Computational design and molecular dynamics will be used to test and consolidate models. Overall, this program aims to provide a molecular-level understanding for sensing and response through the synergistic application of chemical and biophysical methods.

NIH Research Projects · FY 2025 · 2017-06

Project Summary/Abstract Bacteria and humans have a complex relationship: our abundant commensal organisms provide numerous benefits, whereas pathogenic bacteria impose a large burden of morbidity and mortality. The immune system restricts bacterial growth through nutritional immunity, antimicrobial peptides, lytic enzymes, and phagocytic cells. Potential pathogens respond to these threats by the activation of specific adaptive responses, many of which are critical for virulence. We study stress responses in Bacillus subtilis, a model Gram positive bacterium. One project addresses responses to the changing availability of the essential nutrient metal ions zinc, iron, and manganese. The immune system restricts the growth of pathogens by metal sequestration, both in tissues (e.g. by calprotectin) and after phagocytosis. In addition, phagocytic cells kill cells by metal intoxication. We have demonstrated that metal ion homeostasis relies on specific metal-sensing transcription factors that respond to limitation and excess of iron (Fur and PerR), manganese (MntR), and zinc (Zur and CzrA). We will characterize the genes regulated by these transcription factors, their roles in metal homeostasis, and identify the physiological effects that result from both metal ion limitation and intoxication. This work will build upon our recent identification of the major efflux systems for both iron and manganese. The insights from these studies will be directly relevant to the similar stress responses present in human pathogens. The immune system also restricts the growth of pathogens by production of antibacterial peptides and lytic enzymes, both of which affect the integrity of the cell envelope. The cell envelope is also a target for many of our most important antibiotics. In a second project, we have defined several distinct cell envelope stress responses in B. subtilis, with a focus on those regulated by alternative sigma factors. We have identified an array of mutants with alterations in stress response pathways and in key central metabolic pathways that have elevated sensitivity to cell wall antibiotics, including the critically important beta-lactams. In addition, we explore newly discovered antibiotic synergies with possible implications for clinical approaches. Selection of antibiotic resistant suppressors provides a powerful approach for delineating the basis of antibiotic synergies, and the roles of specific stress response pathways. These pathways are central to cell envelope homeostasis generally, in addition to their role in sensing and responding to antibiotic- induced stress, and are implicated in the emergence of antibiotic tolerance and resistance in pathogens.

NIH Research Projects · FY 2026 · 2017-05

The increasing recognition of RNA’s importance provides strong motivation for understanding how its dynamic structures contribute to biology at the molecular scale. Much of RNA’s versatility is derived from its intrinsic conformational flexibility and capacity to interact with a broad range of cellular partners. Assessment of RNA’s structures is challenged by the variations that supports these multiple roles. This program supports the continued application of tightly coupled experimental and computational tools to characterize ensembles of RNA structures. Recent advances enhance the resolution or interpretation of solution-based measurements, revealing subtle but important changes in rigid elements, like duplexes, or the full range of structures assumed by highly flexible unpaired strands. This project now extends studies of RNA from isolated motifs to biologically functional elements and aims to resolve their atomically detailed workings. A distinct focus is on the structural variation of single-stranded motifs. The capture or release of these highly flexible regions is a potent signal exploited in regulating the translation of mRNAs into proteins, via interactions with ions, small ligands or RNA motifs such as duplexes or loops in helix-junction-helix motifs. Because of their importance in controlling gene expression, these motifs offer new targets for therapeutic intervention or strategies for stabilizing existing RNA-based drugs or vaccines. Finally, this project will continue successful efforts to characterize the interaction of flexible nucleic acid motifs with proteins, laying the groundwork for understanding how large nucleic acid-protein complexes function.

NIH Research Projects · FY 2026 · 2016-05

PROJECT SUMMARY/ABSTRACT Since this training program was first funded 34y ago, the specific problems that confront maternal and child nutrition (MCN) have not gone away, but rather crucial challenges continue to exist and new ones have emerged. As a result, there remains a compelling need to understand not only how to prevent nutritional problems in women of childbearing age and young children but also how to mitigate the effects of these problems on the later health of both women and their children. The proposed program is significant because, in the short term, it will fill a major gap in training in MCN and, in the long term, individuals trained by this program should fill a major gap in producing and interpreting integrative research on MCN. We propose to train 4 (3 predoctoral and 1 postdoctoral) trainees by leveraging the resources of the renowned graduate program in the Division of Nutritional Sciences at Cornell University, one of the largest academic units devoted to human nutrition in the country. The proposed training program will be led by a multi-PD group composed of four faculty members in the Division of Nutritional Sciences at Cornell University: Drs K.M. Rasmussen, L. Bellows, J. Finkelstein, and J. Hoddinott. Program oversight will be provided by an External Advisory Committee. Trainees will be mentored by a total of 14 trainers, who have active research programs as well as an exemplary training record. The co-trainers have differing yet complementary skills in a wide range of disciplines related to MCN as well as a long history of collaboration with one another in both training and research. Trainees will complete a core curriculum consisting of 3 graduate-level courses: Topics in Maternal and Child Nutrition, Grant Writing, and Translational Research and Evidence-based Policy and Practice in Nutrition. These courses will be supplemented by the highly valued weekly meeting of the MCN Research Forum as well as training in the responsible conduct of research and rigor and reproducibility in research. Use of an Individual Development Plan along with our innovative Annual Collaborative Project will further enhance the trainees’ career and leadership development as well as their resilience in the face of a changing environment for research. Excellent facilities are available in the Division for the proposed program, including well-equipped laboratories, animal facilities, an outpatient metabolic unit, a clinical chemistry laboratory, and extensive support for statistical computing. Trainees will also have access to shared research facilities throughout the campus and academic centers and institutes that focus on subjects that range from vertebrate genomics to food and nutrition policy. As a result of the proposed program,pre- and postdoctoral trainees will be able to identify the most important problems in MCN, address them with their research, and effectively translate the results of their research into novel policies and appropriate actions.

NIH Research Projects · FY 2024 · 2015-09

PROJECT SUMMARY Double faulting on match point is intensely disappointing. Yet it is also a performance error that could help improve your future serve. ‘Limbic’ structures such as the lateral habenula (LHb), ventral pallidum (VP) and ventral tegmental area (VTA) have classically been associated with hedonic functions. But this emphasis might result from behaviorist traditions that train lab animals with rewards and punishments. A more general function of the limbic system may be to impose valence on any prediction error, including mistakes that occur during motor performance. If this is the case, then decades of progress on how the brain processes reward can generalize to motor sequence tasks such as speech, sport, and musical performance. In past work, we discovered that when a male songbird unexpectedly sings the right note, its VTA dopamine (DA) neurons are activated in the same way as when a thirsty primate unexpectedly receives juice. And following song errors, its DA neurons are suppressed as when a primate experiences disappointing reward omission. We also found that when males sing to females, these performance evaluation signals are turned off and DA neurons are instead activated by female calls. These discoveries have important implications for motor learning circuits that motivate the proposed work. Frist, to determine how performance quality is evaluated in circuits upstream of VTA, we will anatomically identify inputs to VTA, perform lesions to test which are necessary for song learning, record VTA responses to microstimulation of distinct inputs, and conduct neural recordings to identify auditory error and/or timing signals important for error computation (Aim 1). Second, in past work we identified the VP as a hub for auditory, motor, and error processing during singing. In pilot experiments we are identifying LHb and subthalamic nucleus (STN) as novel targets of VP that also project to VTA. To dissect VP’s role in performance evaluation, we will anatomically define VP inputs and outputs, and will record STN-, LHb-, and motor cortex-projecting VP neurons during singing (Aim 2). Finally, our past discovery that DA error signals are turned off when males sing to females is unprecedented. To determine if DA signal gating is behaviorally relevant, we first test if males can learn from experimentally controlled errors with females present (Aim 3.1). To test if error signals are gated off globally, we will record neurons in VTA-projecting areas (auditory cortex, VP, STN, LHb) as we control both perceived error and female presence (Aim 3.2). Altogether, these studies will identify the neural correlates of the internal evaluation systems that construct motor sequences. A major impediment to understanding pathological activity patterns observed in BG-related diseases is a limited understanding of signal propagation through the healthy circuit. The proposed work aims to understand the functions of DA-BG signals and how they are processed at successive stages of the circuit. At stake in this issue is the potential to tailor therapies, such as neural circuit re-programming and deep brain stimulation for movement disorders, based on detailed knowledge of normal brain physiology.