Cornell University

NIH Research Projects · FY 2024 · 2021-08

Elastic, Degradable Vascular Grafts with Helical Microfibers When an artery occludes, rerouting blood flow with a vascular graft can save lives or limbs. Autologous grafts are the preferred conduit for replacing small-diameter vessels. However, not all patients have suitable donor vessels, and the wound healing complications associated with the harvest can be severe. Synthetic grafts can be used but the foreign material does not degrade or integrate with the host, leading to graft failure and risk of infection. These grafts function best in large caliber arterial reconstruction but perform poorly in smaller arteries such as in the coronary and below the knee arteries. We will overcome this challenge by using a degradable graft made of elastomeric microfibers arranged in helices mimicking extracellular matrix fibers in the artery. Our preliminary data in rats show these grafts remodel into compliant, elastic vascular conduits resembling the native arteries. The proposed research will address two key questions for clinical translation: 1) will host cells fully populate the long grafts needed for human applications and 2) will the same transformation occur in aged patients and those with systemic diseases such as diabetes? Correspondingly, Specific Aim 1 will investigate host remodeling of 7-inch-long interposition grafts in a sheep carotid artery. Aim 2 will assess graft remodeling in Zucker Diabetic Sprague-Dawley rats, a new polygenetic model of Type 2 diabetes. Aim 3 will evaluate the effectiveness of controlled release of cytokines in the graft to overcome the limitations caused by aging. Upon completion of this project, we expect to have a graft design ready for clinical translation. The knowledge gained will have impact beyond vascular substitutes in the development of other cardiovascular devices.

NIH Research Projects · FY 2025 · 2021-08

SUMMARY/ABSTRACT Organisms are constantly exposed to environmental conditions that challenge the integrity of the genome. Loss of genomic integrity contributes to the development of most cancers. DNA double strand breaks (DSBs) are a dangerous type of DNA damage that can lead to rapid loss of sequence information stored within the genome. Homologous recombination (HR) is one of the primary DSB repair pathways and is predicated on locating an undamaged DNA sequence that matches the damaged DNA sequence elsewhere in the genome. The homologous sequence can then be used to restore the lost DNA sequence information. During normal mitotic growth, HR preferentially repairs DSBs using sequence information stored in the sister chromatid. Aiding in maintenance of allelic variation between genes and preventing unbalanced exchange of genetic information between chromosomes. In contrast during meiosis the homologous chromosome becomes the preferred DNA repair substrate. There is a large amount information on existing pathways that have evolved in S. cerevisiae to promote DNA repair from the homologous chromosome during meiosis. However, little is known about how homologous chromosomes are used for repair in humans. One of the key determinants in chromosome choice during HR, is the organization of the presynaptic complex (PSC). The regulation, formation, and activity of the human PSC is controlled by >45 proteins. However, a basic functional unit of the PSC consists of RAD51 and associated factors (RAD54L) during mitosis, and RAD51, DMC1 and their associated factors (RAD54L, RAD54B, HOP2-MND1) during meiosis. Understanding how these proteins organize into active complexes during HR is a critical step in understanding how human homologous chromosomes are used for HR. Over the course of our studies we will use biochemical and single molecule approaches to understand the mechanism behind RAD51 and DMC1 self-segregation during meiotic PSC formation. We will understand how DMC1 forms a meiotic homology search complex, and with cooperation of accessory proteins, aligns DNA sequences. We will identify how meiotic homology search complexes overcome chromatin. Finally, we will work to understand how conflicts between the two highly related motor protein RAD54L and RAD54B may promote homologous chromosome use during human mitotic HR. In summary, the primary goal of this research proposal will be to use molecular biology, biochemistry, and single molecule approaches to understand how human mitotic and meiotic PSCs organize, and promote DNA sequence alignment during HR. The data we collect from these experiments will be used to build a model for how human homologous chromosome selection may occur during both mitotic and meiotic HR.

NIH Research Projects · FY 2025 · 2021-08

Lung cancer is responsible for more deaths in the United States than any other form of cancer. Unfortunately, many lung cancer patients do not respond to treatments that effectively mobilize cytotoxic T cells against tumors in other cancers (e.g. anti-PD-1/PD-L1 and anti-CTLA4). This lack of response in lung cancer is primarily due to an inability to initiate a robust antitumor immune response. Lung cancer cells secrete the damage-associated molecular pattern protein, High Mobility Group Box 1 (HMGB1) which has a dual function in immunity. Although it can facilitate immune cell infiltration into tumors; its predominant function is to drive the secretion of negative immune regulators including TGF-b and IL-10 and increase expression of programmed death receptor ligand 1 (PD-L1). My preliminary data suggest that monounsaturated fatty acids (MUFA) are required to prevent HMGB1 secretion from lung cancer cells. Therefore, I hypothesize that lung cancer patients with lower concentrations of tumor-associated MUFA will have higher expression of HMGB1 resulting in an immunosuppressive tumor microenvironment (TME). To test this hypothesis, I propose two Specific Aims: 1) Determine the association between MUFA, extracellular HMGB1, and lung cancer in patients; 2) Evaluate the effects of MUFA on secretion of HMGB1 and the activation of cancer-associated fibroblasts in ex vivo tumor models. I will use lipidomic and immunological assays to determine the association between MUFA and secreted HMGB1 in lung cancer patients. Using patient tissue explants, I will measure the effects of pharmacologic inhibition of MUFA on secretion of HMGB1. To study the effects of genetic and pharmacologic inhibition of MUFA on the TME, I will construct vascularized 3-dimensional bioprinted lung tumors constructed using lung cancer cells and lung fibroblasts. This will allow characterization of immune modulating cytokines secreted by cancer-associated fibroblasts, a dominant cell type within lung tumors. The long-term goal of this research is to provide insight into the mechanisms by which tumors orchestrate immune suppression, and enable the development of new strategies to overcome this immunological barrier. This K01 proposal is designed to build upon my training background and track record in basic molecular and cancer biology, and expand my skills as a translational researcher. My scientific advisory committee is composed of accomplished scientists and clinicians with expertise in oncology, lung disease, lipid biochemistry, fibroblast biology and molecular biology. The program outlined in this K01 proposal will propel me into an independent scientific career through rigorous career development activities tailored to my specific research goals.

NIH Research Projects · FY 2025 · 2021-07

Abstract This proposal describes plans to continue a rigorous chemistry-biology interface (CBI) predoctoral training program that is student-centered and designed to provide trainees with core and cross training in chemistry and biological sciences, various abilities to push the frontier of biomedical research at the chemistry-biology interface, and the skills and awareness for diverse career paths. The program, which has been continuously funded by the NIH since 1996, also promotes interdisciplinary collaborative research across the Cornell campus. We request funds to support 10 predoctoral trainees. Each trainee will carry out his or her doctoral thesis research with one or more of 30 faculty mentors affiliated with seven participating units: Chemistry and Chemical Biology, Biological and Biomedical Sciences, Chemical and Biomolecular Engineering, Microbiology and Immunology, Biochemistry and Molecular Cell Biology, Plant Biology, and Nutritional Sciences. Participating faculty have well-funded research programs in chemistry with strong connections to biology or vice versa. Students undergo training in areas that are broadly distributed over chemistry (synthetic organic, bioorganic, bioinorganic, biophysical, natural products, X-ray crystallography, metabolomics, and proteomics) and biology (protein structure and function, enzymology, immunology, signal transduction, chemotaxis, cell biology, host/pathogen interactions, and genomics). The CBI program continues to successfully merge the cultures of chemistry and biology with effective didactic and programmatic initiatives, including the adaptation of evidence-based training strategies and mentor training. CBI trainees take a core set of rigorous courses in both chemistry and biology and undergo responsible conduct of research training as well as rigor and reproducibility training. Trainees attend seminars in their core disciplines, participate together with faculty in a CBI seminar program, and organize an annual CBI symposium that features speakers from diverse career sectors. In addition to research training, our CBI training program puts career awareness and transferable skills training front and center. We design our career awareness and transferable skills workshops to address gaps between career skills needed and skills developed in PhD training that are reported in the literature and obtained through large-scale surveys. We require a sabbatical internship (or employer site visits in special cases) to allow trainees to explore different career options first-hand. These career training activities are designed based on the eight principles of experiential learning. The CBI training activities are accessible to students who are not financially supported by the CBI program and thus have a larger impact on Cornell campus. The program continues to produce a high-caliber cohort of trainees, who are increasingly more diverse due to our continued recruitment efforts, with strong publication records and career outcomes without increasing the time to degree.

NIH Research Projects · FY 2025 · 2021-07

Project Summary Sensory signals encountered under different circumstances may have quite different implications. In the early olfactory system, preliminary evidence suggests that this (non-olfactory) contextual information is integrated into odor representations at a very early stage, potentially even the main olfactory bulb. Recent evidence indicates that the anterior olfactory nucleus (AON), a structure directly adjoining the olfactory bulb, serves to integrate afferent odor information with contextual information from the ventral hippocampus (vHC) and is necessary to solve contextually-dependent olfactory decision-making tasks. The vHC is known to relay task-relevant spatial contextual information to other brain systems. We here hypothesize that direct projections from the vHC to the AON play a dominant role in the integration of contextual and olfactory information, and that the AON embeds this multisensory contextual information into early-stage odor representations. Our preliminary data show that rodents can learn to respond differently to odors based on the spatial context in which they are encountered, and that the expression of such a rule depends on both AON and vHC, whereas a similar but odor-independent task requires vHC but not AON. We propose a multipronged approach to understanding the integration of spatial context into olfactory representations, engaging electrophysiological ensemble recordings and interareal coherence measurements in awake, behaving rodents, the optogenetic manipulation of vHC and AON circuit activities, and a double-labeling strategy for the within-subjects comparison of immediate-early gene (Fos) responses across two experimental conditions separated in time.

NIH Research Projects · FY 2026 · 2021-06

PROJECT SUMMARY / ABSTRACT Enteroendocrine cells (EECs) coordinate a wide variety of signaling networks to maintain metabolic homeostasis. As a rare secretory cell lineage of the gut epithelium, EECs sense and respond to luminal stimuli by releasing a diverse array of hormones that control nutrient sensing, appetite, glycemic regulation, and energy balance. Diet- induced obesity and bariatric surgery have been associated with the dysregulation and restoration of these hormonal pathways, respectively. Moreover, a growing number of pharmacological strategies have emerged that target key EEC signaling pathways to treat metabolic disease. However, despite these advances the molecular mechanisms regulating EEC biology remain incompletely defined. To address this knowledge gap, this proposal aims to determine the role of an EEC-enriched microRNA (miRNA), miR-375, in regulating the effects of dietary and surgical interventions on EEC biology. MiRNAs are short, non-coding RNA molecules that respond to changing environmental contexts and modulate gene expression at the post-transcriptional level. As such, miRNAs are critical regulators of a myriad of biological pathways, including intestinal epithelial development and function. Our lab has previously demonstrated that miR-375 is highly enriched both in intestinal stem cells (ISCs) and along the EEC lineage, and its expression is dramatically reduced by chronic high-fat diet. In addition, our preliminary data demonstrate significant rescue of miR-375 expression in ISCs following bariatric surgery, coinciding with increases in EEC abundance and circulating gut hormone levels. Therefore, I hypothesize that miR-375 exerts context-specific effects on EEC biology during the pathogenesis and amelioration of diet-induced obesity. The proposed studies will test this hypothesis through an interdisciplinary approach using our lab’s established colony of miR-375 knockout (375-KO) mice together with cutting-edge genomic and bioinformatic techniques. In Aim 1, I will assess how the loss of miR-375 exerts diet-specific effects on the distribution of different EEC subtypes by performing high-resolution single-cell RNA-sequencing (scRNA-seq) of small intestinal crypts and villi from wildtype (WT) and 375-KO animals fed either a chronic chow or high-fat diet. In Aim 2, I will determine how miR-375 contributes to surgically-induced EEC adaptations and metabolic improvements through scRNA-seq analyses of crypt and villus samples from diet-induced obese WT and 375- KO mice following bariatric surgery or a control procedure. With these single-cell datasets, I will bioinformatically determine context-dependent changes in overall EEC abundance, subtype distribution (correlated with circulating gut hormone levels), and gene expression (including identification of candidate miR-375 targets). I will also validate these molecular findings in vivo through immunohistochemical assays and metabolic parameters such as body weight and glucose tolerance. Altogether, these findings will further our understanding of EEC regulation and may provide novel therapeutic targets for the treatment of obesity and its comorbidities.

NIH Research Projects · FY 2025 · 2021-06

Uterine cancer is the 4th most frequent malignancy and the 6th cause of cancer-related deaths in women in the US. While the incidence and mortality rates of some cancers, such as lung and colorectal cancers, are declining, they are both increasing for cancers of the uterine corpus. Recent extensive integrated genomic analyses of endometrial carcinomas have provided important insights into the repertoire of molecular aberrations characteristic of these malignancies. They have also identified four major molecular subtypes of endometrial carcinoma, characterized by distinct genetic alterations and clinical behavior. However, utilization of this information is compromised because the cell(s) of origin have not been determined. By analogy with stem cells in other organs and tissues, aberrations in mechanisms governing endometrial epithelial stem cells may lead to a number of pathological conditions, including cancer. Unfortunately, the identity and location of endometrial stem cells remains insufficiently elucidated. A number of recent studies have suggested location of stem cells either in the glandular or luminal compartments of the mouse endometrial epithelium. In humans, such cells are commonly thought to be located in the basalis segment of the endometrial glands. However, according to a recent single cell transcriptome study of secretory phase human endometrium, cells showing characteristics of stem/progenitor cells are located in the upper region of the functionalis. Other studies have suggested that endometrial epithelium can be regenerated by stem cells of stromal/mesenchymal or bone marrow cell origin. Our studies performed in mice conditionally expressing fluorescent reporters in PAX8+ cells support the hypothesis that stem cells involved in the homeostasis of endometrial epithelium are located in the epithelium proper. By using single cell transcriptome analysis, we have identified TROP2 (encoded by Tacstd2) and FOXA2 as reliable markers of luminal and glandular compartments, respectively. Our lineage trajectory predictions, organoid formation and lineage tracing experiments suggest that both luminal and glandular epithelium contain stem/progenitor cells. TROP2 and FOXA2 are also differentially expressed in human endometrial epithelium. Based on previous findings and our preliminary results we hypothesize that endometrial epithelium contains two pools of resident stem/progenitor cells, which may have different propensities for malignant transformation, thereby leading to clinically distinct neoplasms. To test this hypothesis, we will establish cell lineage hierarchy of the mouse endometrial epithelium, test susceptibility of the mouse glandular and luminal epithelium to malignant transformation associated with alterations common for serous and endometrioid carcinomas, and establish the relevance of mouse model findings to human biology.

NIH Research Projects · FY 2025 · 2021-05

PROJECT SUMMARY ABSTRACT Infections by Salmonella present a constant threat to human health in our country and throughout the world. Yet, our progress toward controlling salmonellosis has been largely fruitless; antibiotics are rarely warranted, and, when used, frequently fail due to resistant strains. To control this important foodborne pathogen, it is essential to understand the means by which it colonizes and induces disease. Chemical signals of the intestine, including those produced by both the animal host and the microbiota, can repress Salmonella virulence by reducing its ability to invade the intestinal epithelium. We propose that this signaling defines the fine balance between virulence and growth of the pathogen. We have found that a novel class of chemicals produced by species of the Gammaproteobacteria, termed diffusible signal factors (DSFs), potently represses invasion. DSFs are quorum-sensing molecules that we have found to exist in the large intestine of mice in sufficient concentration to inhibit Salmonella invasion. They therefore represent both a novel instance of inter- species signaling and a means by which Salmonella disease and carriage is modulated by its biological environment. The long-term goal of this work is to identify practical means to inhibit Salmonella invasion in humans and thus to reduce clinical and sub-clinical salmonellosis. Our objectives are to understand how invasion-inhibiting compounds function, and to investigate their efficacy in preventing disease. Our central hypothesis is that the resident microbiota of the large intestine produce chemical signals that repress Salmonella invasion, and that these signals thus dictate the balance between virulence and growth. We aim to test the specific hypotheses that: 1) Intestinal chemical signals (including both DSFs and other microbiota- derived compounds) modulate Salmonella virulence by controlling the proportion of the pathogen population capable of invasion to dictate disease and carriage; 2) Signaling molecules of varying structures bind within a single binding pocket of AraC-type invasion regulators, but utilizing different binding moieties, thus dictating activity and competition among these signals, and; 3) Signals repressive for invasion can be produced in animals using recombinant bacteria to reduce both clinical signs of salmonellosis and intestinal colonization by this pathogen. The work described here is significant and innovative as it has potential to identify a novel means of pathogen control that does not rely upon antibiotics but instead targets attributes essential to colonization and virulence.

NIH Research Projects · FY 2025 · 2021-05

Project Summary Ovarian/extra-uterine high-grade serous carcinoma (HGSC) is the most common and aggressive type of ovarian cancer. It often has no symptoms at early stages and over 80% of patients are diagnosed at advanced, usually incurable, cancer stages, when the tumors have already metastasized. Extensive integrated genomic analysis allowed identification of several clinically distinct subtypes of HGSC. A significant fraction of HGSC arises from the tubal epithelium (TE) located in the distal region of Fallopian tube (aka uterine tube or oviduct). Recent single cell transcriptome analysis of distal TE inferred that HGSC heterogeneity could be connected to diverse cell states present in TE cell lineages. Unfortunately, precise cell lineage-based hierarchy of identified TE cell types has not been yet established. Furthermore, cancer-prone cellular states of TE are insufficiently defined and factors influencing such states remain unclear. Thus, it remains unknown if uneven clinical course of HGSC and development of cellular therapeutic responses may reflect different modes of initiation and progression of this malignancy. Our preliminary studies show that, in addition to known secretory (OVGP1+) and ciliated (FOXJ1+, CD24+) epithelial cells, there are several epithelial cell populations characterized by preferential expression of stem/progenitor cell markers, such as SLC1A3, CD49f (ITGA6), and KLF6. A Monocle cell-lineage trajectory prediction analysis of our single-cell transcriptomic data identified a population of SLC1A3+ stem/progenitor cells that give rise to both secretory and ciliated cells by progressing through transient intermediates, including a KRT5+ cell population. This prediction has been confirmed by lineage tracing of SLC1A3+ cells and ex vivo studies. Cells in a transient state (CD24med CD49f+) form spheres in consecutive rounds of sphere dissociation- regeneration and express KRT5. Under normal homeostatic conditions, KRT5+ cells are largely dormant and minimally contribute to secretory and ciliated cell lineages. However, KRT5+ cells become actively involved in re-epithelialization after mechanical damage. Our preliminary results suggest that stromal charges begin to co- evolve with mutant epithelial cells before the earliest morphologically detectable alterations. Based on previous studies and our preliminary results we hypothesize that cancer-prone TE cell states are determined by levels of epithelial damage and stromal milieu changes. To test this hypothesis we propose (1) to establish the role of specific cell states during homeostatic and posttraumatic regeneration, (2) to determine the impact of epithelial damage on cancer susceptibility of epithelial states and (3) to identify and characterize epithelial and stromal cell lineage dynamics during early stages of TE malignant transformation.

NIH Research Projects · FY 2025 · 2021-05

The Cornell Center for Reproductive Genomics (CRG) was founded in 2007 with the goal of leveraging state-of- the-art genomics technologies for understanding the biology of the mammalian germ cell. More specifically, our goal has been to understand the genetic, epigenetic, and epitranscriptomic basis for the generation of viable healthy gametes and to explore how alterations in these events could contribute to human infertility. It is well known that disruption of genes required for regulating all aspects of gene expression, including chromatin modifiers, the transcription machinery, and components of post-transcriptional regulatory pathways, leads to the formation of spermatozoa with abnormal head morphology in the mouse, while sperm from men with increased abnormal sperm morphology significantly higher rates of chromosomal aneuploidy, chromatin compaction defects, and altered transcriptome profiles compared to sperm from fertile men. Thus, in this application, we seek to understand how transcriptional, post-transcriptional, and epitranscriptomic regulation of gene expression and chromatin state contributes to the differentiation of haploid germ cells into mature spermatozoa. Three projects are proposed and three cores are proposed. PROJECT I (Danko and Cohen) will focus on the importance of transcriptional regulation of gene expression at the exit from meiosis and entry into spermiogenesis, with a focus on the role of the bromodomain protein, BRDT in facilitating transcriptional shutdown and thus permitting appropriate histone-to-protamine replacement and nuclear compaction. PROJECT II (Grimson, Schimenti, Hwang) will focus on mechanisms and functions of post-transcriptional processing and regulation of mRNAs during spermiogenesis and whether defects in these processes can underlie defects in sperm morphology in patients seeking assisted reproductive technologies. PROJECT III (Jaffrey) will explore the dynamics of N6-methyladenosine (M6A) and N6, 2’-O-dimethyladenosine (m6Am) modifications on RNA through spermatogenesis in mice and in men, and the importance of these epitranscriptomic changes for the production of healthy sperm in mice and men. These studies will be supported by a well-established ADMINISTRATIVE CORE (Cohen) that will facilitate close interactions through regular meetings, trainee events, pilot and seed grants, and our popular “Tri-Repro” Annual Symposium. Our state-of- the-art GENOME INNOVATION CORE (Grenier) will serve as an Innovation Hub for exploring all aspects of gene regulation in reproduction, specializing in a range of next generation sequencing technologies to support the projects. Finally, our OUTREACH CORE (Lin) will provide lab opportunities for nearby, and traditionally underserved, school districts throughout upstate New York, at the same time sending our trainees and faculty out to these communities as role models for young budding scientists. Our center will benefit from the strong research and clinical integration we have established over the past 13 years, by robust and unequivocal institutional support, and by the outstanding scientific environment provided by Cornell University.

NIH Research Projects · FY 2025 · 2021-04

The lysosome transmembrane protein TMEM106B was originally identified as a risk factor for frontotemporal dementia (FTD) with granulin (GRN) mutations and was recently associated with many other neurodegenerative diseases, including Alzheimer’s (AD), Parkinson’s and limbic-predominant age-related TDP-43 encephalopathy (LATE). TMEM106B is also identified as one of the main determinants of brain aging. More interestingly, a D252N mutation in the lumenal domain of TMEM106B was recently found to cause hypomyelinating leukodystrophy (HLD). However, the cellular and physiological functions of TMEM106B remain to be determined. In our preliminary studies, we found that TMEM106B deficiency causes the clustering of lysosomes in the perinuclear region and a block of lysosome transport in the axon initial segment (AIS). TMEM106B deficient mice show autophagy defects and FTD related pathology such as accumulation of phosphorylated TDP-43 during aging. We hypothesize that TMEM106B regulates myelination, autophagy, brain aging and TDP-43 pathology via modulating lysosome trafficking and movement. We plan to determine the mechanisms by which TMEM106B regulates lysosome positioning, lysosome transport along axons and autophagy flow using cell biological and biochemical approaches (Aim1). Our studies have also shown that TMEM106B is highly expressed in oligodendrocytes and TMEM106B deficiency leads to myelination defects in mice. Ablation of TMEM106B leads to trafficking defects of the myelin membrane protein PLP due to increased lysosome clustering in the peri-nuclear region and decreased lysosome exocytosis. We plan to further characterize myelination defects of TMEM106B deficient mice, especially near the AIS region, and determine the effect of the D252N mutation on TMEM106B function and myelination (Aim2). Finally, we found that mice deficient in both TMEM106B and GRN have severe lysosome abnormalities, glial activation, neurodegeneration and accumulation of TDP-43 aggregates. We plan to further dissect how TMEM106B genetically interacts with GRN to regulate TDP-43 pathology and FTD disease progression using mouse models and human patient samples (Aim3). In summary, the proposed studies will shed light on cellular and physiological functions of TMEM106B, the genetic interaction between GRN and TMEM106B and the molecular and cellular mechanisms of many brain disorders with TMEM106B association, including HLD, LATE, AD and, FTD. Our work will also yield novel insights into mechanisms involved in regulating TDP-43 aggregation in AD, FTD and LATE and how myelination defects contribute to neurodegenerative phenotypes in AD, FTD and other brain disorders.

NIH Research Projects · FY 2026 · 2021-04

PROJECT SUMMARY/ABSTRACT The stability of eukaryotic genomes relies on the tight coordination of DNA metabolic processes with DNA repair and cell cycle transitions. Central to this coordination are elaborate signaling networks mediated by DNA damage signaling kinases. Mutations in these kinases are associated with a range of human genetic disorders linked to cancer predisposition, neurological defects, and immunodeficiency. Selective inhibitors of DNA damage signaling kinases are now being used in dozens of clinical trials. However, fundamental questions about how these kinases maintain genome integrity remain unanswered. The Smolka Laboratory investigates DNA damage signaling, with a major focus on the phosphatidylinositol 3′ kinase (PI3K)‐related kinases (PIKKs) and PIKK‐regulated downstream checkpoint kinases. Recent work from our laboratory has strongly influenced the understanding of the action and regulation of PIKKs and checkpoint kinases. In the last funding cycle, we identified mechanistic connections between PIKKs and the DNA repair machinery, revealing a mechanism for suppression of gross chromosomal rearrangements. We also established roles for PIKKs in regulating DNA replication. Using phosphoproteomics, we substantially expanded the map of the PIKK signaling network in budding yeast and mammalian cells, revealing surprising non-canonical motifs targeted by PIKKs. Over the next five years, our research program will uncover novel features, mechanisms and fundamental rules through which PIKK signaling operates and coordinates core processes such as DNA replication. A major focus will be on DNA replication forks, key structures where many spontaneous and drug-induced genomic instabilities arise. The efficacy of many chemotherapy drugs relies on damage amplification through fork stalling and breakage. Understanding how PIKKs control fork transactions will have broad implications for understanding the genesis of genomic instabilities and how PIKK inhibitors can efficiently kill cells undergoing oncogene- induced replication stress. Moreover, we aim to elucidate the relevance of novel motifs for PIKK targeting, which will define a new paradigm for how PIKK signaling propagates and ensures genome stability. We will also tackle a novel frontier in the study of PIKKs related to their spatial organization in cells, which we propose is crucial for understanding the impact of genetic perturbations and mechanisms of drug action. Overall, our work will illuminate how inhibitors of PIKKs, already in clinical trials, affect cell viability and genomic integrity, guiding the design of more effective therapeutic strategies.

NIH Research Projects · FY 2025 · 2021-04

PROJECT SUMMARY/ABSTRACT The genetic information encoded in our genome is decoded and implemented via many multi-step processes, including the proper decoding by transcription. Transcription of genes into mRNA by RNA Polymerase II (Pol II) is a complex process that is precisely regulated both temporally and spatially at multiple steps by many large molecular complexes (LMCs). In the past, a number of these LMCs have been identified and their structural and functional role has been studied. Although we have learned a great deal about these LMCs at an individual level, how these LMCs interact and affect one another and Pol II at a more comprehensive level has yet to be achieved. In this project, we are proposing a multi-prong approach to define interactions and structures of LMCs, Pol II, and model transcription factors (TFs) in an unbiased way and, as much as possible, under native conditions. We will also evaluate the function of these specific interactions on the molecular mechanics of transcription and regulation in cells. To this end, we will utilize a novel GFP aptamer-based purification method to identify LMCs and TFs that associate with GFP-tagged Pol II and other critical LMCs. Purifications will be performed rapidly and under native conditions to ensure retention of physiological interactions, and the resulting complexes will be analyzed by both Mass Spectrometry and Cryo-EM to define the composition and structure of these LMCs at the highest depth and resolution possible. Crosslinking with novel protein-protein crosslinkers and subsequent MS analysis (XL-MS) will also be used to capture more transient LMC and TF interactions. In parallel, LMC-APEX2 fusions will be used to biotinylate nearby proteins and identify them by MS analysis following streptavidin purification. Additionally, we will define the location of distinctly modified Pol II complexes or Pol II associated with distinct LMCs at base-pair resolution along transcription units using our new PRO-IP-seq protocol. This information combined with the MS analysis provides a unique and dynamic view of Pol II’s phosphorylation status, composition, associations, and precise positioning along genes, and this information will be critical in deriving molecular models of transcription and its regulation. Previously known and newly identified LMCs and TFs that are deemed to have critical interactions will be perturbed by either RNA aptamer inhibitors or degron- tagging to tease apart their functional roles. The rapid expression RNA aptamers, which interfere with specific LMC interactions, and the rapid degradation of whole LMC subunits with degron technology will allow the detection of the immediate, “primary” roles of those interactions genome-wide using the high-resolution assays such as PRO-seq and ChIP-Exo. These assays will enable us to identify the specific functions of the key LMCs and their interactions at an unprecedented resolution and sensitivity. Overall, we expect to derive a much better and more complete understanding of the transcription cycle and its regulation. This will impact human health by identifying new therapeutic venues and possible lead drugs (RNA Aptamers), as misregulation of transcription has been observed in many disease conditions.

NIH Research Projects · FY 2025 · 2020-11

Project Summary / Abstract Mtb utilizes host-derived lipids to promote pathogenesis and this is a defining feature of this intracellular pathogen. During infection Mtb imports and metabolizes host lipids to support pathogenesis by producing: i) energy, ii) central metabolic intermediates, or iii) polyketide virulence lipids. While the metabolic pathways in Mtb that degrade or process lipids are complex and contain redundant enzymes, the bacterial Mce lipid transporters appear to be specific for dedicated lipid substrates. Aim 1 of this work proposes to employ genetic and biochemical approaches to identify and characterize novel gene/proteins required for fatty acid import in Mtb. While it is understood that Mce1 imports fatty acids, the substrate specificity of this transporter is unknown. Therefore, we intend to define substrates and the biochemical basis of Mce1 substrate specificity. Our preliminary studies indicate that Mtb transports fatty acid precursors of immune signaling lipids via Mce1 and we include here studies to evaluate if scavenging of this immune lipid precursors by Mtb impacts the immune response. Aim 2 proposes to identify and characterize protein subunits that are shared by all the Mce transporters and are required for lipid import in Mtb. We have determined that LucA is required for Mce1- and Mce4-mediated transport and LucA stabilizes these transporter complexes. These studies seek to characterize the basis for this transporter stabilization. Similarly, MceG is required for Mce1- and Mce4-mediated transport and we intend to understand how MceG stabilizes and interacts with Mce1. We will use a genetic approach to silence LucA and MceG in Mtb within chronically infected mice and quantify bacterial fitness to determine the therapeutic potential of drugs that potentially block these proteins.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Following transcription, mRNAs transmit the genetic information that dictates protein production. In eukaryotes, transcription and translation occur at different timings and in distinct subcellular compartments. It is commonly believed that these two fundamental processes are uncoupled and have little “cross-talk”. Recent evidence suggests that translation of aberrant mRNAs leads to genetic compensation, a transcriptional response to rapidly upregulate genes with similar functions. However, the molecular mechanisms by which cytoplasmic translation communicates with nuclear transcription remain elusive. During the course of studying translational control in response to amino acid starvation, we observed transcriptional upregulation of genes undergoing stress-induced ribosome pausing. Relying on a newly developed sequencing method, Ezra-seq, we discovered the existence of endogenous ribosome footprints. We hypothesize that these mRNA fragments serve as novel chromatin modulators by influencing gene expression in a sequence-dependent manner. The goal of this proposal is to characterize the genetic circuit coupling cytoplasmic translation and nuclear transcription. By identifying and mapping chromatin-associated ribosome footprints, we will elucidate the novel type of RNA- mediated epigenetic regulation of gene expression. We propose that the genetic circuit formed by ribosomes contributes to genetic robustness and is a common mechanism for stress adaptation. We will also explore the potential of designing artificial ribosome footprints to achieve controllable gene expression, revealing new routes toward novel therapeutics. The conceptual establishment of ribosome-mediated genetic circuit is transformative in our understanding of the central dogma in molecular biology and opens a new research avenue towards genetic reprogramming.

NIH Research Projects · FY 2024 · 2020-09

Project Summary/Abstract This project evaluates the zoonotic implications of two important possible pathogens of companion animals: E. coli and coronaviruses. E. coli is the most common pathogen tested for antibiotic susceptibility in veterinary diagnostic labs. It is also one of the most concerning for multi-drug resistance; several isolates captured thus far in Vet-LIRN active surveillance are predicted to be pan-resistant to all drugs used in human or animal medicine. Our group has published the first animal host specific E. coli virulence database compatible with tools that can mine whole genome sequencing data. We now propose a comparative evolutionary genomic study to assess the potential of E. coli in dogs to encode novel resistance mechanisms and cause disease in humans. The data will be explored employing the latest developments in bacterial pan- genomic analysis, as well as bacterial GWAS. Such analysis will reveal if there are E. coli loci adapted to dogs, whether such loci have the functional character suggestive of pathogenic potential, and whether dogs may be a reservoir of potential pathogenesis or antibiotic resistance. The present outbreak of coronavirus disease caused by SARS-CoV-2 (COVID-19) is the third documented spillover of an animal coronavirus to humans to have resulted in a major epidemic, within the past two decades. Characterizing the species diversity of coronaviruses from companion animals, represents an important necessary step towards improving our understanding of virus–host interactions and to enhance our preparedness for future outbreaks. We will undertake this characterization in three different host species – horses, cats, and dogs – making use of an extensive set of time series samples (respiratory and feces) that comprise the AHDC’s collection. Sequences of coronavirus genomes will be acquired using two approaches: RNAseq of the respiratory virome and multiplexed amplicon approaches of both respiratory and feces samples; the former will allow us to identify the coronavirus species repertoire of each host, including the identification of any new coronavirus species, the latter will provide the ability to explore aspects of diversity in detail, within and between hosts. Comparative evolutionary genomic analyses of the data will inform on a wide variety of issues related to their zoonotic potential, including for example: (1) Which coronavirus species are more prone to inter-host transmission and is there a directionality to that transmission? (2) Are their host reservoirs for any of the virus species? (3) Which have a history of recombination? (4) Which have a history of molecular adaptation and what viral proteins does that involve? Our final aim is to provide eight other Vet-LIRN sequencing laboratories with the reagents and training to be able to independently sequence and analyze bacterial and viral genomes, including SARS-CoV-2. All three Aims of this project directly support the mission of the FDA to ensure the safety of our nation's food supply and protect public health.

NIH Research Projects · FY 2024 · 2020-09

Abstract Successful regeneration of tissues requires transient increases in stem cell plasticity, proliferation, and differentiation, in order to produce new cells that integrate with preexisting tissues and organs. Pathways governing these critical behaviors have been identified, but how injury signals can trigger stem cell proliferation and differentiation of cells necessary for regeneration remains poorly understood. In most model organisms, regenerative capacity is limited and stem cells are scarce, which has made it difficult to pinpoint the mechanisms regulating stem cell proliferation and differentiation after injury. By contrast, the planarian flatworm Schmidtea mediterranea has abundant stem cells that are activated by injury and fuel continuous regeneration. Like embryonic stem cells, planarian stem cells have the capacity to differentiate into any type of tissue. These pluripotent stem cells can be readily identified, monitored, purified, and thoroughly profiled at the molecular level. We recently made two important discoveries that form the foundation of this proposal. First, injury of any type appears to protect stem cells from lethal radiation, because it halts the cell cycle and fewer stem cells undergo apoptosis. Second, we pioneered a chemical method to selectively remove a single organ, the pharynx. Pharynx regeneration requires the upregulation of the conserved Forkhead transcription factor FoxA in a discrete subset of stem cells immediately after this targeted injury. We find that the extracellular signal-regulated kinase (ERK) is a central driver of these behaviors. ERK promotes differentiation in cultured stem cells, but how it is activated after injury is poorly understood. Together, these findings establish our central hypothesis, which is that injury synchronizes the cell cycle, enabling local cues to channel stem cell differentiation toward discrete cell fates. In Aim 1, we will determine how injury induces cell cycle arrest in stem cells after radiation. We will examine DNA repair and test the function of conserved genes that are upregulated after injury. In Aim 2, we will dissect the mechanisms driving organ-specific regeneration by purification and single-cell sequencing of stem cells proliferating after organ loss. We will identify receptors enriched on these cells, and test their function in organ regeneration to determine if they act upstream of FoxA. In Aim 3, we will identify the upstream receptors that activate MAP kinase signaling in stem cells with combinations of RNAi, pharmacology and biochemistry. This proposal exploits our ability to challenge stem cells with precise insults, providing a lens into the mechanisms that enable flexible stem cell responses during injury and homeostasis. Understanding the molecular mechanisms that govern stem cell behavior in a physiologically-relevant context will inform the design of future strategies for regenerative medicine technologies.

NIH Research Projects · FY 2025 · 2020-09

Modified Project Summary/Abstract Section With urban environments the fastest growing landscapes on the planet, transmission of vector-borne diseases by urban adapted mosquitoes has increased markedly over the past several decades. Urban vectors include Anopheles stephensi, the mosquito responsible for urban malaria across South Asia. Elimination of malaria in South Asia, and preparedness against its further expansion into Africa, hinges on effective action against the disease in cities. We know temperature has strong, non-linear effects on malaria transmission. Although relative humidity also has important effects on malaria epidemiology, its quantitative effects on transmission are vastly understudied and often treated as independent from temperature. Because these relationships are currently not well understood, we have limited capacity to predict the emergence, spread, and control of malaria in urban environments. Our overarching hypothesis is that humidity affects urban malaria transmission by modifying the temperature-transmission relationship. Further, incorporating the effect of humidity will improve predictions of malaria transmission and hot spots of malaria risk in both temporal and spatial models of transmission. Our proposed research will address this knowledge gap through the following specific aims. Aim 1 will investigate the effects of humidity on the temperature-malaria transmission relationship. Comprehensive experiments will be conducted to characterize the effects of both relative humidity and temperature on mosquito and malaria life history traits relevant for transmission. These experiments will be validated over a subset of conditions in India with the local vector and local strains of P. falciparum and P. vivax. These mechanistic relationships will then be integrated into temporal and spatial models of malaria epidemiology in Aims 2 and 3. Aim 2 will formulate and parameterize a temporal coupled human- mosquito transmission model used to predict the seasonal and interannual variation in malaria incidence and vector abundance. Aim 3 will implement a spatial model to predict transmission risk and incidence across urban environments by using meteorological observations with urban land cover data to map environmental suitability for malaria transmission. Suitability maps will then be overlaid with population density and socio- economic factors to predict hotspots for transmission. Two cities in India, Surat and Ahmedabad, experience notable differences in mean annual relative humidity and have maintained extensive surveillance malaria programs over the last two decades. These two cities will provide contrasting opportunities to test the ability of the climate-trait relationships from Aim 1 to improve transmission models of urban malaria. Major outcomes include an improved conceptual framework for the environmental epidemiology of urban malaria based on mosquito biology, and new modeling approaches that apply this knowledge to make predictions of disease transmission. Prediction of upcoming anomalous seasons combined with identification of hotspots will enhance targeted public health intervention.

NIH Research Projects · FY 2024 · 2020-09

Project Summary- Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer (BC), evades hormonal treatment modalities, and patients with TNBC experience high rates of metastasis and have a poor prognosis. Therefore, there is a critical need to find better approaches to treat TNBC. Sirtuin 1 (SIRT1) represents an interesting target in this regard, as SIRT1 has been shown to be implicated in cancer as a tumor suppressor, and in fact, SIRT1 mRNA and protein are significantly downregulated in TNBC. Previous work has shown that decreasing SIRT1 in human TNBC cells promotes the generation of a secretome containing soluble hydrolases and a large number of exosomes, a specific class of extracellular vesicles, with unique cargo. Furthermore, the hydrolases and exosomes produced by TNBC cells depleted of SIRT1 were shown to promote the aggressive phenotype of TNBC cells by promoting cell survival, invasive activity, and metastasis. Thus, it is important to determine whether decreasing SIRT1 expression or activity in vivo in mouse models of breast cancer promotes tumorigenesis and metastasis. Additionally, the mechanism for decreased expression of SIRT1 in TNBC is largely unknown, and a better understanding of SIRT1 regulation will uncover changes in cancer-promoting pathways that make TNBC so aggressive. In this proposal, the effects of decreased SIRT1 expression or activity on tumorigenesis and metastasis will be elucidated (Aim 1), and the transcriptional and post-transcriptional regulatory factors that decrease SIRT1 expression in TNBC will be identified (Aim 2). In Aim 1, three distinct, yet complementary, mouse models of breast cancer will be used to determine the effects of altered SIRT1 expression or pharmacologic modulation of SIRT1 activity on cancer progression in the tumor microenvironment, including tumor growth, invasiveness, and angiogenesis, as well as rate of metastasis. In Aim 2, precision nuclear run-on RNA sequencing (PRO-Seq) and small RNA sequencing will be performed to elucidate transcriptional and post-transcriptional regulators of SIRT1, respectively. For this aim, a system of three human BC cell lines with varying levels of SIRT1 expression will be used. Changes in transcriptional regulation and microRNA expression with PRO-Seq and small RNA-Seq, respectively, will be determined by comparing across these three cell lines. Due to the highly aggressive nature of and difficulty to treat TNBC, it is imperative to develop new therapeutic strategies that slow tumorigenesis and metastasis. Through the outlined six-year training plan in Drs. Richard Cerione and Robert Weiss’s labs, which have expertise in elucidating underlying molecular mechanisms and their effects in vivo through mouse models, by determining the effects of low SIRT1 levels in mouse models of BC and the regulatory factors that mediate this downregulation, this proposal holds promise for clarifying the role of SIRT1 as a tumor suppressor in TNBC.

NIH Research Projects · FY 2025 · 2020-08

Project Summary The preparation of complex organic molecules is vital to the development of next-generation therapeutics. However, the synthesis of molecules bearing certain fluorinated and chlorinated functional groups, such as fluoroalkenes, remains challenging. A major barrier to installing these motifs within complex molecules is that many fluorinated and chlorinated building blocks are gases at room temperatures, which complicates high- throughput reaction optimization. In a similar vein, the signaling molecules hydrogen sulfide, nitric oxide, and carbon monoxide, possess therapeutic potential that is difficult to unlock due to their gaseous nature. Molecular donors for these molecules often result in the co-generation of toxic byproducts upon gasotransmitter release. We propose an interdisciplinary solution to these and other challenges relevant to medicinal chemistry: the use of porous materials, such as metal-organic frameworks (MOFs) and porous organic polymers (POPs). Supported by NIGMS, we have achieved several breakthroughs related to the synthesis and therapeutic delivery of bioactive molecules. First, we demonstrated the first example of fluorinated gas storage within MOFs, enabling their safe handling as solid reagents to streamline reaction development (publication in Science). Second, in a similar vein, we have shown that MOFs can be used to safely deliver hydrogen sulfide for the treatment of ischemia-reperfusion injury, which can occur during a heart attack or stroke. We have developed MOF-based catalysts for several industrially important reactions, including amide bond formation and halogen exchange. We have also employed electroactive molecules to achieve the selective electroreduction of (hetero)aryl halides without precious metal catalysts. We have also developed several methods for the synthesis of porous materials, including high-concentration solvothermal, mechanochemical, and ionothermal approaches. In the next research period, we will pursue three interconnected projects. First, we will employ our gas- releasing MOFs to develop several methods for the installation of vinyl fluorides and chlorides, as they are stable bioisosteres for ubiquitous amides in drug-like molecules. To achieve this goal, transition metal catalysis, electrosynthesis, and photoredox catalysis will be employed; the latter will require the development of new MOFs for gas storage that do not strongly absorb visible light. We will also develop novel methods for chemical ligation and stapling of peptides. We will also continue to add gases to our growing library of materials, further expanding the scope of synthetic transformations we can develop. Second, we will move beyond gas storage through weak non-covalent interactions to slow down gas release under ambient conditions and achieve stimuli-responsive gas release. We will demonstrate this concept using hydrogen sulfide delivery from MOFs before extending it to other biomedically relevant gases. Last, we will leverage the unique properties of isolated metal-halide sites in MOFs to design new catalysts for nucleophilic aromatic substitution and C-H functionalization reactions, leveraging confinement effects to tune reaction selectivity.

NIH Research Projects · FY 2026 · 2020-08

PROJECT SUMMARY Lipids serve as important mediators of host-microbe interactions yet many mechanisms that support signaling through these interactions have yet to be characterized. Of special interest are classes of lipids that have well- defined signaling pathways in host systems but have less well understood synthesis pathways in microbial systems. My research program has focused on defining how a class of lipids, the sphingolipids, can mediate interkingdom host-microbe interactions through the functions of these lipids as structural components of membranes and as chemical signals. To understand the importance of sphingolipids to host-microbe interactions, we have developed techniques to trace sphingolipid transfer through these interactions. These efforts have alerted to us to under characterized pathways in microbial sphingolipid metabolism that we have been able to profile and manipulate to deepen our understanding of the importance of microbial sphingolipid metabolism to host-microbe interactions. We have integrated techniques in microbial genetics, biochemistry, flow cytometry, high-throughput sequencing, and liquid chromatography-mass spectrometry to define host- microbe sphingolipid transfer and characterize key microbial sphingolipid metabolism genes. Our innovations in investigating sphingolipid-dependent host-microbe systems have the potential to accelerate discovery in a range of host-microbe systems and for a wide range of metabolites. To capitalize on this potential, we have used our workflows to investigate new ways that cholesterol mediates host-microbe interactions and propose to expand to other classes of bioactive lipids. The goals for the next five years of the research program are to use techniques developed from our investigations into sphingolipid-dependent host-microbe interactions to accelerate knowledge of how the important lipid classes of sphingolipids, cholesterol, and fatty acids mediate host-microbe interactions. For sphingolipid-dependent host-microbe interactions, we plan to further define genes contributing to bacterial sphingolipid metabolism and determine how this gene activity may generate bioactive signaling molecules with relevance to prominent host cellular receptors. For cholesterol-dependent host-microbe interactions, we plan to further define microbial metabolites produced from exogenous cholesterol and expand on investigations into how microbial cholesterol sulfotransferase activity regulates microbial and host phenotypes. For fatty-acid dependent microbe interactions, we plan to define the microbes that interact with distinct saturated and unsaturated fatty acids as well as the potential suite of bioactive metabolites produced from these interactions. Together, these efforts will deepen our knowledge on how lipids mediate host-microbe interactions at a level that can inform microbiome-centric therapeutic interventions.

NIH Research Projects · FY 2026 · 2020-07

PROJECT SUMMARY / ABSTRACT Maintaining stable gene transcription patterns is critical for cellular programming. Likewise, orderly switching from one transcription pattern to another, termed reprogramming, is necessary for development, as well as for numerous other biological processes. Dysregulated reprogramming can have catastrophic consequences, with outcomes ranging from developmental disease to cancer. Notably, epigenetic abnormalities, failure to differentiate, and inappropriate cellular programming are intricately linked to carcinogenesis. Our objective is to define the function of H2A.Z (a variant form of histone H2A) in regulating cellular programming. We will utilize cultured mouse cells, human cancer cells, and developing zebrafish embryos, combined with a series of next- generation genome-wide sequencing approaches, to functionally test how H2A.Z patterns regulate several aspects of cellular reprogramming, including transcriptional activation. Our prior studies have uncovered considerable diversity in H2A.Z function, including regulation of chromatin accessibility, transcription factor binding patterns, bivalent chromatin establishment, and nucleosome remodeler function. One interesting possibility is that H2A.Z interacting proteins influence its genomic localization and mediate its function. To test this hypothesis, we will genetically manipulate the regulators of H2A.Z localization in zebrafish embryos, and cultured mouse cells, and then assess outcomes via genomics and proteomics studies. Successful completion of this project will define the role of H2A.Z in controlling gene expression patterns and in cellular programming. We propose that this broader concept, where changes in epigenetic marks underlie cellular reprogramming, might be a general principle in biology, with relevance to developmental biology, stem cell function, and carcinogenesis.

NIH Research Projects · FY 2026 · 2020-06

Reproductive health is a cornerstone of human well-being, extending beyond fertility to impact longevity, endocrine health, and quality of life. Addressing the complexities of reproductive disorders—including infertility, endometriosis, and reproductive aging—requires a collaborative and translational approach that bridges basic science with clinical insight. The Tri-State Regional Symposium on Reproductive Health Sciences (Tri- Repro) was established in 2018 to meet this need by fostering an interdisciplinary community that spans four premier institutions: Cornell University’s Center for Reproductive Sciences (CoRe), the Magee-Women’s Research Institute (MWRI) at the University of Pittsburgh, the Center for Women’s Health and Reproductive Medicine (CWHRM) at the University of Pennsylvania, and RU Repro Group at Rutgers University. Tri-Repro is a trainee-led, annually rotating symposium held in the Northeastern US that emphasizes both cutting-edge reproductive science and professional development. The symposium is free and open to attendees from institutions across the country, creating an accessible and informal environment. Central to its success are three innovations: (1) full organizational leadership by a Trainee Organizing Committee, (2) a no- cost, regional format that facilitates wide participation and networking, and (3) a rotating host structure that promotes institutional ownership and thematic diversity. The annual two-day symposium features five Keynote Speakers, including the Stuart Moss Memorial Lecturer, and additional talks selected from trainee and early- career faculty abstract submissions. Activities include a poster session, networking reception, and a career development forum addressing entrepreneurship, FemTech, clinical translation, alternative career tracks and funding strategies. This R13 renewal application supports four primary goals to support Tri-Repro: Goal 1: To maintain a small, local conference model to support networking and research exchange among reproductive scientists and clinicians across the Northeast. Goal 2: To prioritize trainee career development through leadership roles in meeting organization, scientific programming, and interactive forums with renowned leaders across academia, industry, and funding agencies. Goal 3: To facilitate cross-institutional collaborations, as well as interactions between basic and clinical researchers, through in-person engagement in an intimate, informal setting. Goal 4: Expand the focus on translational science and entrepreneurship to better prepare trainees and early- career investigators for impactful, real-world applications of their work. In summary, Tri-Repro is a high-impact, low-cost model for scientific engagement that complements national meetings by cultivating a supportive community of reproductive scientists and clinician-investigators. By fostering regional collaboration and prioritizing trainee development, Tri-Repro directly supports the NIH mission to cultivate future leaders and improve reproductive health through translational innovation.

NIH Research Projects · FY 2026 · 2020-04

Project Summary / Abstract A hallmark of eukaryotic cells is their compartmentalization. The evolution of membrane-bound organelles enabled separation, concentration, and specialization of functions critical for cell behavior. Yet this compartmentalization created challenges for cells, which must control the flow of material into and out of these organelles. Our lab's long-term goals are to elucidate how cells exert control over the flow of proteins and lipids within the endomembrane system. We seek to understand the Golgi complex, which serves as the nexus of many different trafficking pathways in eukaryotic cells. We have focused many of our investigations on the Golgi- localized GTPases, their regulators, and their effectors to determine how they govern protein trafficking through this enigmatic organelle. Research projects in our lab use multiple approaches, including protein biochemistry, live-cell imaging, in vitro reconstitution, and structural biology to discover fundamental mechanisms. Our work has resulted in significant advances in our understanding of how the function of the Golgi is regulated: we uncovered an extensive network of cross-talk interactions between GTPase pathways regulating Golgi trafficking, we established the pathways and substrates regulated by the Rab-activating TRAPP complexes, we identified new effectors of Arf GTPases, and we determined the structural and mechanistic basis for regulated activation of several Golgi GTPases of the Arf and Rab families. Our work has revealed how the structures of each of these essential regulators are significantly different, even those that share common domains or subunits, and the structures explain their function and regulation. Our work has underscored the importance of membranes as more than just locations where GTPase signaling reactions occur. Organelle membranes are platforms for hundreds of biochemical reactions and interactions in cells, so we need a fundamental biochemical understanding of how proteins behave on the surface of membranes in order to understand the mechanisms driving cell biology. There are over 170 Ras-related GTPases in human cells, and most of their signaling reactions occur on the surface of organelle membranes. Although many of these GTPases have been studied in great detail, the role of the membrane surface in controlling, regulating, and coordinating GTPase signaling reactions remains poorly understood. Our goals for the next five years are to address several key questions in the field using a multidisciplinary approach. We will use cryoEM and functional experiments to visualize and understand how interactions with organelle membranes and crosstalk regulators influence the structure and function of GTPase signaling reactions. We will determine how the activation of Arf GTPases is coupled to their insertion into membranes. We will use new screening approaches to identify new GTPase-interacting proteins and we will experimentally characterize the functions and roles of these proteins as potential regulators or effectors. Our planned work has broad significance for understanding signaling, trafficking, organelle homeostasis, and membrane biology.

NIH Research Projects · FY 2025 · 2020-04

Project Summary The proposed research will uncover new radical-based methodologies that facilitate the synthesis of complex bioactive compounds. Organic radicals are highly reactive species with unique chemoselectivities that complement canonical two-electron chemistry. Recently, the emergence of new reaction strategies that leverage single-electron redox events and harness radical intermediates for the selective functionalization of organic molecules has provided chemists with useful tools for solving contemporary synthetic problems. However, the highly reactive nature of many organic radicals has made it difficult to impart catalyst-control over the regio- and stereoselectivity of these fleeting intermediates, especially when complex reaction systems are concerned. In the past funding period, we developed new catalytic strategies that leverage the unique redox features of low-valent Ti complexes to achieve redox-neutral and net-reductive transformations, and through catalyst innovation, we demonstrated that free radical-mediated reactions can be made highly enantioselective. These promising results prompted us to continue to invent novel catalytic and reaction strategies that can effectively harness radical intermediates and provide powerful tools for solving a range of longstanding synthetic problems. In the new funding cycle, we will build on our previous success while moving our research into important new areas of inquiry. In the proposed work, we aim to advance new approaches that employ radical-based catalysts or reagents to address prominent challenges in organic synthesis. The transformations targeted in each project area are either currently unknown or significantly limited in reaction scope or selectivity. Some of the specific reactions that we aim to develop are: site-selective oxidation of alcohols, enantioselective oxidation of amides and ethers, oxidative synthesis of stereogenic- at-phosphorous(V) compounds, site-selective amination, halogenation, and desaturation of alkanes, and deconstructive functionalization of alcohols. In-depth studies using canonical physical organic and electroanalytical techniques will provide insights into the reaction mechanisms that will be used to guide optimization. Ultimately, the realization of these proposed transformations will represent a significant advance for the field of organic synthesis and support innovation in the synthesis of biomedically important molecules.